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ADVANCES IN CLINICAL CHEMISTRY VOLUME 53

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Advances in CLINICAL CHEMISTRY Edited by GREGORY S. MAKOWSKI Clinical Laboratory Partners Newington, CT Hartford Hospital Hartford, CT

VOLUME 53

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands This book is printed on acid-free paper. ⬁ Copyright ß 2011, Elsevier Inc. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://www.elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-385855-9 ISSN: 0065-2423 For information on all Academic Press publications visit our website at www.elsevierdirect.com Printed and bound in USA 11 12 13 10 9 8 7 6 5

4 3 2 1

CONTENTS CONTRIBUTORS

................................................................................

ix

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xiii

Use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Multiplex Genotyping KLAUS MEYER AND PER MAGNE UELAND 1. 2. 3. 4. 5. 6.

Abstract ... ................................................................................... Introduction ................................................................................. Genotyping Using Mass Spectrometry ................................................... MALDI-TOF-MS .......................................................................... Multiplex Genotyping by MALDI-TOF-MS ............................................ Conclusions .................................................................................. References. ...................................................................................

1 2 3 3 8 19 20

Rheumatic Heart Disease: Mediation by Complex Immune Events L. GUILHERME, K. F. KO¨HLER, AND J. KALIL 1. 2. 3. 4. 5. 6. 7.

Abstract ... ................................................................................... Rheumatic Heart Disease .................................................................. Epidemiology ................................................................................ Genetic Susceptibility....................................................................... Immune Mechanisms Involved in RHD ................................................. Rheumatic Heart Disease Lesions ........................................................ Conclusions .................................................................................. Acknowledgments........................................................................... References. ...................................................................................

31 32 32 32 39 42 46 47 47

Dipeptidyl Peptidase in Autoimmune Pathophysiology KEI OHNUMA, OSAMU HOSONO, NAM H. DANG, AND CHIKAO MORIMOTO 1. 2. 3. 4.

Abstract ... ................................................................................... Introduction ................................................................................. Molecular Aspects of CD26/DPPIV Role in Immunity ................................ Cellular Aspects of CD26/DPPIV Role in Immunity ................................... v

52 52 53 60

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CONTENTS

5. Clinical Aspects of CD26 Role in Immune Disorders................................... 6. Summary and Perspective .................................................................. Acknowledgments ........................................................................... References ....................................................................................

63 71 71 72

Biochemistry of HELLP Syndrome CHIARA BENEDETTO, LUCA MAROZIO, ANNALISA TANCREDI, ELISA PICARDO, PAOLA NARDOLILLO, ANNA MARIA TAVELLA, AND LOREDANA SALTON 1. 2. 3. 4. 5. 6.

Abstract....................................................................................... Introduction.................................................................................. Pathogenesis of HELLP and Preeclampsia: The Role of Placenta .................... Inflammatory Response in HELLP Syndrome........................................... HELLP Syndrome, Complement Pathway, and the Coagulation System ............ Conclusion ................................................................................... References ....................................................................................

85 86 89 92 95 98 98

Homocysteine in Pregnancy MICHELLE M. MURPHY AND JOAN D. FERNANDEZ-BALLART 1. 2. 3. 4. 5. 6. 7. 8.

Abstract....................................................................................... Introduction.................................................................................. Homocysteine Metabolism ................................................................. Prophylactic Folic Acid, Folate Status, and Homocysteine ............................ Choline and Homocysteine ................................................................. Nonnutritional Factors Associated with tHcy ........................................... Health Benefits of tHcy Lowering ......................................................... Pregnancy .................................................................................... Acknowledgments ........................................................................... References ....................................................................................

105 106 106 108 109 110 110 111 129 129

Diabetes and Tuberculosis: Analysis of a Paradox DIBYAJYOTI BANERJEE, RAJASRI BHATTACHARYYA, DEEPAK KAUL, AND PRIYA SHARMA 1. 2. 3. 4. 5.

Abstract....................................................................................... Introduction.................................................................................. Definition of the Problem .................................................................. Analysis of Previous Experimental Results to Solve the Paradox ..................... Probable Difference in Glycation Patterns of Macrophage Proteins and Its Precursors ................................................................. 6. In Silico Analysis of the Possibility of Glycation-Induced Inhibition of NADPH Oxidase ......................................................................... 7. Solving the Paradox ......................................................................... Acknowledgment ............................................................................ References ....................................................................................

139 140 140 142 142 143 146 150 150

CONTENTS

vii

EGCG, Green Tea Polyphenols and their Synthetic Analogs and Prodrugs for Human Cancer Prevention and Treatment DI CHEN, SHENG BIAO WAN, HUANJIE YANG, JIAN YUAN, TAK HANG CHAN, AND Q. PING DOU 1. 2. 3. 4. 5. 6. 7.

Abstract ... ................................................................................... Introduction ................................................................................. The Role of Tea Polyphenols in Carcinogenesis ........................................ In Vitro and In Vivo Preclinical Studies on Tea Polyphenols .......................... Tea Polyphenols and Their Molecular Targets in Cancer Cells ....................... Tea Polyphenol Derivatives in Cancer Studies .. ........................................ Conclusions .................................................................................. Acknowledgments........................................................................... References. ...................................................................................

156 156 157 158 162 166 169 170 170

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

179


CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

DIBYAJYOTI BANERJEE (139), Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India CHIARA BENEDETTO (85), Department of Obstetrics and Gynaecology, University of Torino, Torino, Italy RAJASRI BHATTACHARYYA (139), Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India TAK HANG CHAN (155), Department of Chemistry, McGill University, Montreal, Quebec, Canada DI CHEN (155), The Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute; and Departments of Oncology, Pathology, and Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan, USA NAM H. DANG (51), Division of Hematology/Oncology, University of Florida, Gainesville, Florida, USA Q. PING DOU (155), The Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute; and Departments of Oncology, Pathology, and Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan, USA JOAN D. FERNANDEZ-BALLART (105), Unit of Preventive Medicine and Public Health, Faculty of Medicine and Health Sciences, IISPV, Universitat Rovira i Virgili, Tarragona; and CIBER Fisiopatologıá de la Obesidad y Nutricioń (CB06/03), Instituto de Salud Carlos III, Madrid, Spain

ix

x

CONTRIBUTORS

L. GUILHERME (31), Heart Institute (InCor), School of Medicine; and Immunology Investigation Institute, National Institute for Science and Technology, University of Saõ Paulo, Saõ Paulo, Brazil OSAMU HOSONO (51), Division of Rheumatology and Allergy, Research Hospital, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan K.F. KO¨HLER (31), Heart Institute (InCor), School of Medicine; and Immunology Investigation Institute, National Institute for Science and Technology, University of Saõ Paulo, Saõ Paulo, Brazil J. KALIL (31), Heart Institute (InCor), School of Medicine; Immunology Investigation Institute, National Institute for Science and Technology; and Clinical Immunology and Allergy Division, School of Medicine, University of Saõ Paulo, Saõ Paulo, Brazil DEEPAK KAUL (139), Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India LUCA MAROZIO (85), Department of Obstetrics and Gynaecology, University of Torino, Torino, Italy KLAUS MEYER (1), Bevital AS, c/o Section for Pharmacology, University of Bergen, Bergen, Norway CHIKAO MORIMOTO (51), Division of Rheumatology and Allergy, Research Hospital; and Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan MICHELLE M. MURPHY (105), Unit of Preventive Medicine and Public Health, Faculty of Medicine and Health Sciences, IISPV, Universitat Rovira i Virgili, Tarragona; and CIBER Fisiopatologıá de la Obesidad y Nutricioń (CB06/03), Instituto de Salud Carlos III, Madrid, Spain PAOLA NARDOLILLO (85), Department of Obstetrics and Gynaecology, University of Torino, Torino, Italy KEI OHNUMA (51), Division of Rheumatology and Allergy, Research Hospital, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan

CONTRIBUTORS

xi

ELISA PICARDO (85), Department of Obstetrics and Gynaecology, University of Torino, Torino, Italy LOREDANA SALTON (85), Department of Obstetrics and Gynaecology, University of Torino, Torino, Italy PRIYA SHARMA (139), Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India ANNALISA TANCREDI (85), Department of Obstetrics and Gynaecology, University of Torino, Torino, Italy ANNA MARIA TAVELLA (85), Department of Obstetrics and Gynaecology, University of Torino, Torino, Italy PER MAGNE UELAND (1), Bevital AS, c/o Section for Pharmacology, University of Bergen, Bergen, Norway SHENG BIAO WAN (155), Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China HUANJIE YANG (155), The Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute; Departments of Oncology, Pathology, and Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan, USA; and Department of Life Science and Engineering, Harbin Institute of Technology, Harbin, China JIAN YUAN (155), Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China


PREFACE I am pleased to present Volume 53 of Advances in Clinical Chemistry series for the year 2011. In the first volume for this year, a number of diverse topics are reviewed. The first review explores the powerful role of matrix-assisted laser desorption/ ionization time of flight mass spectrometry for multiplex genotyping. The versatility of this method provides flexible assay design and high accuracy with excellent sample throughput. The second paper reviews the autoimmune molecular mimicry of streptococcus pyogenes antigens in rheumatic heart disease. The pathophysiology of autoimmune disease is continued with a review of the role of dipeptidyl peptidase (CD26). The use of this interesting molecule as a target for therapeutic intervention of selected immune disorders is discussed. The biochemistry of HELLP syndrome in pregnancy is subsequently presented with a focus on placental involvement and maternal systemic responses. The next is a comprehensive review of homocysteine in pregnancy, with emphasis on its biological and pharmacologic regulation. The next manuscript investigates the importance of diabetes mellitus as a predisposing factor for infection with mycobacterium tuberculosis. The role of NADPH oxidase, a key enzyme in respiratory burst, is explored with respect to this disease process. The volume concludes with a fascinating review on the role of tea polyphenols in carcinogenesis, including DNA methylation and oncogene expression. I thank each and every contributor of Volume 53 and thank colleagues who contributed to the peer review process. I also extend my appreciation to my Elsevier liaison, Gayathri Venkatasamy, for continued editorial assistance I hope the first volume for 2011 will be enjoyed by our readership. As always, your comments and suggestions for up-to-date review articles for the Advances in Clinical Chemistry series are always appreciated. In keeping with the tradition of the series, I would like to dedicate Volume 53 to the newest member of our family, Kirk. GREGORY S MAKOWSKI

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ADVANCES IN CLINICAL CHEMISTRY, VOL. 53

USE OF MATRIX-ASSISTED LASER DESORPTION/ IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY FOR MULTIPLEX GENOTYPING Klaus Meyer1 and Per Magne Ueland Bevital AS, c/o Section for Pharmacology, University of Bergen, Bergen, Norway

1. 2. 3. 4.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genotyping Using Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MALDI-TOF-MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Principle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Analysis of Nucleic Acids by MALDI-TOF-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Multiplex Genotyping by MALDI-TOF-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. SNP-Genotyping Using Primer Extension Assays . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. SNP-Genotyping Using Nonprimer Extension Assays. . . . . . . . . . . . . . . . . . . . . . 5.3. Genotyping of Other Polymorphic Variants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Molecular SNP-Haplotyping. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Quantification of Mixed DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 3 3 3 4 8 8 12 14 16 17 19 20

1. Abstract After completion of the human genome project, the focus of geneticists has shifted to elucidation of gene function and genetic diversity to understand the mechanisms of complex diseases or variation of patient response in drug treatment. In the past decade, many different genotyping techniques have been described for the detection of single-nucleotide polymorphisms (SNPs) and other common polymorphic variants. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is among the most 1

Corresponding author: Klaus Meyer, e-mail: [email protected] 1

0065-2423/11 $35.00 DOI: 10.1016/S0065-2423(11)53001-X

Copyright 2011, Elsevier Inc. All rights reserved.

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MEYER AND UELAND

powerful and widely used genotyping technologies. The method offers great flexibility in assay design and enables highly accurate genotyping at high sample throughput. Different strategies for allele discrimination and quantification have been combined with MALDI (hybridization, ligation, cleavage, and primer extension). Approaches based on primer extension have become the most popular applications. This combination enables rapid and reliable multiplexing of SNPs and other common variants, and makes MALDI-TOF-MS well suited for large-scale studies in fine-mapping and verification of genome-wide scans. In contrast to standard genotyping, more demanding approaches have enabled genotyping of DNA pools, molecular haplotyping or the detection of free circulating DNA for prenatal or cancer diagnostics. In addition, MALDI can also be used in novel applications as DNA methylation analysis, expression profiling, and resequencing. This review gives an introduction to multiplex genotyping by MALDI-MS and will focus on the latest developments of this technology.

2. Introduction Understanding the basis of human genetic variations is a major task in medical research. Various diseases have been related to changes in gene sequences caused by different types of polymorphisms and mutations, which can alter the structure and function of a protein. Among these variants, attention has been focused in the recent years on single-nucleotide polymorphisms (SNPs). These biallelic single-base changes have a frequency of at least 1% in a given population and are found in the human genome at a frequency of about one in 1000 base [1]. Public databases as the general catalog of genetic polymorphism maintained by NCBI [2,3] actually consist of over 143 million reported SNPs, of which 19 million reference SNP clusters have been validated (dbSNP build 132; http://www.ncbi.nlm.nih.gov/SNP/snp_summary.cgi). Only a small portion of SNPs changes the coding sequence of a protein or affects gene regulation and thus represents a potential genetic risk factor. The majority of SNPs does not change the amino acid sequence and can serve as markers for disease causing genes in genome-wide association studies. The international HapMap [4] has to date registered more than 3.1 million so-called tag-SNPs, which tag common haplotype blocks and help to reduce the number of genotypes screened in genome-wide association studies. The number of registered tag-SNPs will further increase with completion of the 1000 Genomes Projects (www.1000genomes.org), which is aimed to achieve a nearly complete catalog of common human genetic variants. Due to their biallelic nature, SNPs are easy to determine and their interpretation can be highly automated. Various methods for genotyping of SNPs have been developed in the past few years and validated with respect to accuracy and sensitivity, flexibility of the assay design, throughput, and costs per genotype [5–8]. Methods based on

GENOTYPING BY MALDI-MS

3

mass spectrometry provide very reliable platforms for genotyping [9]. Among these, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) belongs to the most powerful techniques for high-throughput multiplexing of SNPs [7,10–16] and has also been used for the detection of other common variations, such as restriction fragment length polymorphisms (RFLPs) [17], short tandem repeats (STRs) [18,19], insertions and deletions [20], and copy number variations (CNVs) [21]. The ability of sensitive and (semi-) quantitative allele determination has enabled pooled DNA analysis [22,23] and noninvasive prenatal fetal diagnosis [24].

3. Genotyping Using Mass Spectrometry A characteristic feature of mass spectrometry is that the detection is based on an intrinsic physical property (mass to charge ratio), which contributes to highly accurate data. Traditional methods use indirect methods like fluorescence or radioactive reporter tags, which have to be labeled to the target molecule. While the number of different tags is limited and thus only supports low-level multiplex applications per reaction, MS provides multiple data points per experiment and enables high-level multiplex genotyping. In addition, latest mass spectrometers provide sensitive analysis with limits down to the femtomole and attomole range [25,26]. The breakthrough of MS for the analysis of large biomolecules was introduced by the development of new gentle ionization methods, which prevent the decay of the molecules during the ionization process. Nowadays, two ionization methods are commonly used for biomolecules: electrospray ionization (ESI) [27] and MALDI [28]. ESI is a much softer ionization method than MALDI and allows the characterization of very large biomolecules of up to 108 Da [29]. In combination with ion trap, QTOF or FT-ICR MS, the technique has been successfully used for the analysis of nucleotides, especially intact PCR products, plasmid, or phage DNA [9]. However, MALDI has become the technique of choice for of MS-based multiplex genotyping [30]. The method is less dependent on highly purified samples than ESI, and the direct combination of MALDI with TOF analyzers enables robust and rapid genotyping with throughputs of several thousand samples per day [31].

4. MALDI-TOF-MS 4.1. PRINCIPLE The MALDI process can be separated into three fundamental steps of analyte/matrix preparation, desorption of the upper matrix monolayers, and ionization of the analyte molecules. Successful MALDI involves incorporation

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of analyte molecules between the matrix molecules, either in the bulk or within the crystal surface, and separation of the analyte molecules from each other. There is no single MALDI matrix or protocol that is suited to all analytical questions, and a proper choice of both is crucial for the analytical outcome [32–34]. In addition, various factors as the analyte-to-matrix ratio, type of solvent and additives, amount of salts, and characteristic of the target surface influence the crystalline morphology and, thus, the quality of mass spectra. During the MALDI analyses, a short UV- or IR- laser pulse of a few nanoseconds irradiates the crystals and leads to desorption/ionization of the matrix and analyte molecules into the gas phase (Fig. 1). The wavelength of the laser should be close to the absorption maximum of the matrix molecules. The processes of material desorption and ionization of the matrix and analyte molecules are intertwined and take place on a micrometer geometrical and nanosecond time scale. While the mechanisms of desorption have been partly understood by comparison of experimental data with simulations [35,36], no unifying theory exists that explains the formation of the gas-phase ions [37–39]. This might be caused by the large variety of factors including type of analyte molecules, matrices, preparation, and experimental conditions, which influence the process of ionization. Various analyzers as orthogonal TOF, FT-ICR, QIT, QTOF, QIT-TOF [29], or Orbitrap [40] have been coupled to MALDI, but axial TOF (including reflectron TOF, TOF/TOF) has become the most common type for MALDI spectrometers. Axial TOF analyzers, as illustrated in Fig. 1, are ideally suited to MALDI sources, as only a very short laser pulse is needed and the ions fly along a flight tube, hit a detector, and are registered by a digital oscilloscope [41]. Ions are typically accelerated by 20 kV into the flight tube, and as all ions have the same kinetic energy E ¼ zeU ¼ 1/2mv2, they travel with different velocities v and arrive the detector at time t ¼ L(m/2E)1/2 where L is the length of the flight tube. For MALDI-TOF-MS instruments, there are two primary sources of error related to the flight time. The first is caused by the initial velocity distribution of MALDI ions and is compensated in current TOF instruments mainly by pulsed-ion extraction (also called delayed extraction) instead of using a static acceleration field [42]. The second source of errors is introduced by energy dispersion due to nonflat sample morphology and is compensated by a oneor two-stage reflectron analyzer. Here, ions with higher energy travel a longer way through the reflector than ions with lower energy and the ion packets can be refocused on a second detector. Modern research-grade instruments are equipped with both delayed extraction and reflectron TOF-MS [43]. 4.2. ANALYSIS OF NUCLEIC ACIDS BY MALDI-TOF-MS Even though modern MALDI-TOF instruments are well suited for highlevel multiplexing applications in genomics, determination of nucleic acids by MALDI is generally difficult when compared to peptides. Assay

5

GENOTYPING BY MALDI-MS

L a s e r

Oscilloscope

Start t~m1/2

Source

Flight tube

m+3 DE

m+2

Reflectron

m+1 D

U≈20 kV D

Lin

Ref

FIG. 1. MALDI-TOF-MS. MALDI-TOF instruments basically consist of three major components: a vacuum chamber housing the source and the flight tube, a pulsed UV laser, and a fast oscilloscope. The sample placed on a stage inside the source is irradiated by a short UV-laser pulse of a few nanoseconds. The matrix/analyte molecules are desorbed/ionized, and accelerated by an electric field of about U ¼ 20 kV into a field-free drift path inside the flight tube. The oscilloscope is started by the laser pulse for measurement of the flight time. As all molecules are accelerated to the same kinetic energy E ¼ zeU ¼ 1/2mv2, ions of different masses (m1 < m2 < m3) are separated from each other during the flight and hit the detector DLin after t ¼ L(m/2E)1/2 (L ¼ length of the flight path). Typically 20–100 laser shots are accumulated for a MALDI spectrum, and the detector signals are registered by the oscilloscope. Linear time-of-flight MS (registration at DLin) has become the standard mode for most genotyping applications by MALDI-TOF-MS. Mass resolution can be optimized by application of delayed ion extraction (DE). Reflectron TOF MS instruments can further increase mass resolution (at the expense of signal intensity) by reflecting the ions at the end of the flight tube back on a second detector DRef.

development requires accurate evaluation of the instrumental aspects, matrix, purification, sample preparation and analysis, and primer design in order to achieve highest degree of multiplexing at sufficient signal quality. 4.2.1. Instrumental Aspects The mass range, the mass resolution, and the mass accuracy are central parameters of a mass analyzer that define the limits of an MS-based assay. Common MALDI-TOF-MS instruments use UV lasers, which allow the identification of oligonucleotides, for example, sequencing ladder products, in a mass range of up to 30,000 Da (ca. 100 nucleotides) [44]. This upper mass

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limit is caused by size-dependent fragmentation of DNA during desorption, which results in loss of signal intensity toward higher masses. RNAs are more stable than DNA in MALDI because the additional 20 -hydroxyl group stabilizes the glycosidic bond and reduces depurination and fragmentation of the entire oligomer [45]. Large DNA fragments such as PCR products of up to 1400 bp can be detected using infrared MALDI [46,47], which is known to be considerably softer than UV-MALDI. Further increase of the mass range requires new types of detectors, as the sensitivity of microchannel plate detectors, commonly used in TOF instruments, decreases with molecule velocity. Cryogenic detectors have shown to decrease the detection limit by several orders of magnitudes [48]. Although MALDI-TOF-MS allows the principal detection of small PCR products 95% (15-plex) [70]. The first and most simple approach was a single-base extension assay called PinPoint developed by Haff and Smirnov in 1997 [71], which terminates the extension by conventional ddNTPs on the polymorphic site (Fig. 2). Allele discrimination is based on the mass difference between the four terminators (9 Da for ddA/ddT; 15 Da for ddT/ddC; 16 Da for ddA/ ddG; 24 Da for ddC/ddA; 25 Da for ddT/ddG; and 40 Da for ddC/ddG). The level of multiplexing could be increased to 20 by careful assay design with ‘‘mass-tuning’’ of the extension primers [72]. Typical mass spectra of a 20plex PinPoint assay are illustrated in Fig. 3. Sample purification is based either on reversed-phase columns or on ion-exchange resins. However, the PinPoint suffers from ambiguous identification of A/T heterozygotes in routine analyses due to the low mass difference of 9 Da between the extension products for masses > 5000 Da. Thus, this approach has mainly been used in assays, which did not analyze A/T transversions (lowest frequency of ca. 7% of all single nucleotide changes [73]), or if a short primer length below 5000 Da was compatible with assay design. A simple way to increase the mass difference for A/T, but also other heterozygotes, is the usage of ddNTPs/dNTPs mixtures or mass-modified terminators. The PROBE assay was introduced by Little and coworkers in 1997 [74,75] and uses one ddNTP and three dNTPs for extension reaction (Fig. 2). This multibase extension approach enables clear distinction between all reaction products, in the way that mixtures are optimized to always result in at least one nucleotide (ca. 300 Da) mass difference between two allele-specific

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Singlebase extension

Multibase extension

PinPoint, iPLEX

PROBE, MassEXTEND

Primer

ddNTP

ddNTPs

x

dNTPs

x

PCR product

GOOD

VSET Photocleavage

*

Alkylation

* x

x

FIG. 2. SNP-genotyping by primer extension assays. Primer extension can be performed as single- or multibase extension reactions. All types of reactions require purified PCR products. Single-base extension by the PinPoint and iPLEX assays represent the simplest approaches. Here, a primer is annealed next to the mutation site (X) and extended by a ddNTP. While the PinPoint uses natural ddNTPs and suffers from insufficient allele separation of A/T heterozygotes and mass interferences by salt adducts, the iPLEX reaction is performed using massmodified terminators, which enable unambiguous allele identification. The GOOD assay represents the most advanced single-base extension assay and includes charge-tagging and backbone neutralization. Primers are modified by photocleavable o-nitrobenzyl moieties for primer shortening and charge-tagging. Extension reaction uses synthetic alpha-thio ddNTPs. After extension primers are cleaved by UV-light and are alkylated in the last step to avoid cationization by salt adducts. Multibase extension assays use mixtures of ddNTPs and dNTPs (gray tones) and have the advantage of larger mass differences between signals of different alleles or primers and salt adducts. The PROBE and the MassEXTEND use three dNTPs and one ddNTP for allele determination. Dependent on the nucleotide sequence, primers can be extended by several bases before reactions are terminated by a ddNTP. The length of extension products is reduced in average by usage of three ddNTPs and one dNTP as demonstrated by the VSET assay.

extension products. However, this method can also lead to misinterpretation of genotyping results due to polymerase-pausing artifacts, which can have the same mass as ‘‘real’’ termination products. This weakness can be minimized by the use of three ddNTPs and one dNTP, which results in mainly short extensions as demonstrated by the VSET assay [52]. Another drawback of the PROBE assay is the use of magnetic beads for primer immobilization, which restricts throughput and increases cost and was the reason that the PROBE, but also other heterogeneous assays as the Solid Phase Capture Single-Base Extension [55] and the GenoSNIP [76], were replaced by homogenous techniques. However, heterogeneous assays produce highly purified

11

GENOTYPING BY MALDI-MS 1

2

3

4 5

67

89

10 11 12

131415 16

18

20

19 S 17

Relative intensity

S

Wild type Heterozygous Homozygous

8 11

5 1 4

2

1415

12

9

6 3 7

S

19 9

17

10

18

13

20

16

S

5000

6000

7000

8000

9000

10,000

m/z FIG. 3. MALDI-TOF spectra of a primer extension assay. Typical MALDI-TOF-MS spectra of a primer extension reaction are illustrated. The upper panel shows the spectrum of a 20-plex primer mixture before extension reaction, which spans a mass range from 4500 to nearly 10,000 Da. Primers are numbered from 1 to 20 and standards for mass calibration are labeled (S). The lower panel shows the spectrum after reaction (PinPoint), and wild type, heterozygous, and homozygous mutant genotypes are assigned. Peaks of residual reaction primers are connected to the upper spectrum.

extension products and, thus, have demonstrated the highest level of multiplexing so far with 50 SNPs per reaction [77]. A very popular homogenous multiplexing assay for SNP-genotyping by MALDI is the MassEXTEND assay by Sequenom [58,78]. It is an improvement of the PROBE and uses ion-exchange resins for desalting. In addition, the assay uses sample dilution with deionized water for optimization of the crystallization and desorption/ionization process, as the homogenous assay design cannot completely remove contaminations from buffers and reaction components. The MassEXTEND is routinely performed with up to 12 simultaneous SNPs, but higher multiplexing levels can be obtained by careful design of the extension primers. The assay is performed with microarrayed MALDI targets and nanoliter dispensing devices. The reduction of the processed sample volume lowers assay costs and makes the MassEXTEND a very cost-effective choice for 2–10 plexes when processed in 384 plates.

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For higher multiplexing of SNPs, Sequenom’s iPLEX represents a more cost-effective approach and routinely genotypes between 25 and 29 loci per reaction [70,79]. In contrast to the MassEXTEND, this assay is a single-base extension format and uses exclusively mass-modified ddNTPs. The assay creates mass differences large enough to differentiate between all bases compared to natural ddNTPs (ddA/ddT: 56 vs. 9 Da; ddC/ddT: 80 vs. 16 Da; and ddG/ddT: 40 vs. 25 Da). In contrast to the MassEXTEND, the iPLEX produces smaller mass gaps between primers and extension products and, therefore, enables a higher multiplexing level of up to 40 SNPs per reaction. While the iPLEX assay is the result of advanced desalting techniques and new types of terminators, other approaches aim to handle the difficulties of oligonucleotides detection at the level of primer synthesis. These approaches use neutralization of the sugar-phosphate backbone to avoid purification from salts [80], charge-tagging to increase signal–noise ratio [81], and primer cleavage to utilize the lower mass range [76]. These concepts have been merged and realized in different versions of a homogenous approach called ‘‘GOOD assay’’ by Sauer et al. [82,83]. Common to other assay formats, the target region is amplified by PCR and the mutation site identified by primer extension. The extension primer carries a charge-tag close to the 30 end and a phosphorthioate bridge on the 50 side of the charge tag. Primer extension is performed using alpha-thio ddNTPs. Digestion by 50 -specific phosphodiesterase cleaves the primer down to the first phosphorthiodate, which results in a short oligonucleotide including a charge tag and the SNP-specific nucleotide. Finally, alkylation by methyl iodide of the phosphorthiodate bridge neutralizes the backbone and prevents cationization by sodium or potassium. Recently, the assay protocol has been improved by less-toxic reagents for alkylation and photocleavable o-nitrobenzyl moieties for primer shortening and charge-tagging [84]. So far, the GOOD assay has not become as popular as the MassEXTEND or iPLEX due to relatively low multiplexing level of 30 kb [112]. Cosmid/fosmid libraries were constructed from fragmented isolated DNA covering 10% of the genome and individual clones were genotyped if tested positive in a pool of 96 clones. 5.5. QUANTIFICATION OF MIXED DNA MALDI-MS has been shown to enable quantification of proteins and peptides [26]. For oligonucleotides, the strong heterogeneity of the 3-HPA crystal morphology makes quantification a demanding challenge, although the more homogenous structure of nucleic acids, consisting of four similarly building units, provides some compensation. The key to achieve high reproducibility and, thus accurate quantification of nucleic acids, is to analyze a large fraction of the sample/matrix by rastering the laser over many sample spots and/or shrinking the preparation to a ‘‘single’’ crystal using miniaturization. Especially, Sequenom’s silicon chip based MassARRAY technique has been proven to minimize sample heterozygosity so that the highest impact on accuracy and reproducibility is caused by DNA preparation. Quantitative MALDI analyses in genomics are based on the calculation of the ratio of allele-specific primer extension products, which enables the relative and absolute quantification of allele frequencies. A popular genotyping application using relative allele frequencies is screening of pooled DNA to validate

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SNPs in association studies and to characterize their allele frequency in various ethnic groups [113–115]. Pooling decreases the number of necessary genotypes in large-scale studies and represents a shortcut to identify associations between genetic loci and phenotypes. Only SNPs that show significant difference between two pools of, that is, case and controls are selected for individual genotyping. As a drawback in pooling, rare alleles may be missed and individual information as genotypes and haplotypes is lost. Ross et al. were the first who described the relative quantification of allele-specific extension products in mixed DNA by MALDI and found that frequencies down to 5% could be accessed routinely [22]. Further studies demonstrated a limit of detection of about 2% and a standard deviation of 2–3% for allele frequencies between 10% and 90% [23,116,117]. Detailed analysis has shown that this deviation is mainly caused by unbalanced amplification during PCR. When allele frequencies determined from the pooled samples were compared to the real frequencies calculated, a deviation of 150 kDa

Intralesional T cell clones Clone 1 TCR: BV3 BJ2S1 AV5 AJ34 M5(1–20), M5(81–96), M5(83–103), M5 (121–140), M5(163–177), M5(183–201) Clone 2 TCR: BV13 BJ2S7 AV2 AV3 LMM 28(1647–1664), LMM 28B(1660–1677), LMM 32(1699–1716), Mi v derived proteins, 35kDa/pI 8.84

The data extracted from Refs. [16,54,55] are examples of antigen recognition of both streptococcal and human proteins mediated by molecular mimicry, T cell degeneracy and epitope spreading mechanisms. LMM—light meromyosin peptides: LMM28-SLQSLLKDTQIQLDDAVR; LMM28B-DDAVRANDDLKENIAIVE; LMM32-RSRKLAEQELIETSERVQ; M5 peptides sequences recognized:1-20-VTRGTISDPQRAKEALDKY; 81–96-DKLKQQRDTLSTQKET; 121–140-ESKENEKALNELLEKTVKDK; 163–177-ETIGTLKKILDETVK; 183–201-LDETVKDKLAKEQKSJQNI; underlined—shared sequences.

TABLE 4 CROSS-REACTIVE HUMAN T CELL CLONES RECOGNIZED N-TERMINAL REGION STREPTOCOCCAL M PROTEIN PEPTIDES AND CARDIAC PROTEINS Amino acid residues–M protein N-terminal portion M5a (1–20) M5a (81–96) M5a (83–103)

TVTRGTISDPQRAKEALDKY DKLKQQRDTLSTQKET LKQQRDTLSTQKETLEREVQN

M5a (163–177) M5b (151–167) M5b (176–193) M5b (111–129)

ETIGTLKKILDETVK TIGTLKKILDETVKDKIA IGTLKKILDETVKDKLAK TRQELANKQQESKENEKAL

a

Heart-tissue derived proteins, cardiac myosin synthetic peptides, and other cardiac proteins Myoc-30–43 kDa; LMM-4 (1334–1351) Va-150 kDa; 90–150 kDa; 30–65 kDa Va- 30 > 150 kDa; LMM (1413–1430); LMM (1439–1456); Vimentin; intracellular proteins (PDIA3 and HSPA5) Va- 150–10 kDa; 59–56 kDa/7.76 pI; Myoc-24–44 kDa; LMM (1707–1624) Human purified myosin; Tropomyosin; LMM (1740–1752); S2(43–67) peptides Human purified myosin; Tropomyosin; LMM (1740–1752); S2(43–67) peptides Human purified myosin; Laminin; S2(201–225) peptide

M5 peptides sequences from Manjula et al. [58]. and bfrom Miller et al. [59]. All peptides presented cross-reactivity with human heart-tissue proteins [53,54,56,57,60]; bold and/or underlined typed peptide sequences correspond to the identical amino acid residues. Va, valvular-derived proteins; Myoc, myocardium-derived proteins; LMM and S2, synthetic cardiac myosin peptides from LMM and S2 fragments of the human cardiac myosin beta chain [54,57]. M5 arecognized by intralesional and bperipheral T cell clones.

RHEUMATIC HEART DISEASE

45

CD4þ infiltrating T cells are predominant in the heart tissue rheumatic lesions [63,64], and the first evidence of the molecular mimicry between streptococcus and heart tissue was obtained through an analysis of these hearttissue infiltrating T cells. Three immunodominant regions of the M protein (residues 1–25, 81–103, and 163–177), heart tissue proteins (myocardium and valve-derived proteins as well as vimentin), and synthetic peptides of the beta chain of cardiac myosin-light meromyosin region (LMM) were recognized by cross-reactivity by intralesional T cell clones [53,56,60] (Table 4). In the same way, peripheral T-cell clones recognized human purified myosin, tropomyosin, laminin, and LMM and S2 peptides (Table 4) [56]. Using a proteomics approach, we showed that T cells recognize vimentin, reinforcing the role of this protein as a putative autoantigen involved in the rheumatic lesions. In addition, we identified myocardial and valvular autoantigens that were recognized by heart-infiltrating and peripheral T cells from RF/RHD patients. Novel heart-tissue proteins were identified, including disulfide isomerase ER-60 precursor (PDIA3) protein and a 78-kDa glucose-regulated protein precursor (HSPA5). The role of PDIA3 in RHD pathogenesis and other autoimmune diseases is not clear (Table 4) [65]. The fact that several cardiac proteins cross-react with streptococcal M peptides suggests that cross-reactivity might occur first through mimicry that results in the recognition of other human proteins, especially valvular proteins, and eventually through epitope spreading and degeneracy mechanisms as mentioned above (Table 3). By using a molecular approach, we evaluated the Vb chains usage of TCR and the degree of clonality of heart-tissue infiltrating T cells [55] (Table 3). In RHD, the autoreactive T lymphocytes that infiltrate both myocardium and valves were identified in oligoclonal expansions, as determined by the analysis of their TCRs [55]. We observed several expanded T cell populations with an oligoclonal profile in the heart tissue of chronic and acute RHD patients; these oligoclonal T cell populations are in contrast with the peripheral blood, which contains polyclonal TCR-BV families. A high number of T cell oligoclonal expansions were found in the valvular tissue, indicating that specific and cross-reactive T cells migrate to the valves [55], and upon specific cytokine stimulation, they expanded at the site of the lesions. Cytokines are important secondary signals following an infection because they trigger effective immune responses in most individuals and probably deleterious responses in patients with autoimmune disease. Three subsets of T helper cytokines are currently described. Antigen-activated CD4þ T cells polarize to the Th1, Th2, or Th17 subsets, depending on the cytokine secreted. Th1 is involved with the cellular immune response and produces IL-2, IFNg, and TNF-a. Th2 cells mediate humoral and allergic immune responses and produce IL-4, IL-5, and IL-13. Th17 has more recently been described as a type of proinflammatory response mediated by IL-17. The cytokines TGF-b, IL-6, and IL-23 are the factors that induce the Th17 lineage.

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In both myocardium and valvular tissue, the infiltrating mononuclear cells secrete the inflammatory cytokines IFNg and TNF-a. Mononuclear cells secreting IL-10 and IL-4 (regulatory cytokines) were also found in the myocardium tissue; however, in the valvular tissue, only a few cells secrete IL-4, suggesting that these low numbers of IL-4 producing cells may contribute to the progression of valvular RHD lesions [2].

7. Conclusions The knowledge acquired during the last 25 years favors the molecular mimicry mechanism as one of the most important leading autoimmune reactions in RHD. Degeneracy of T cell antigen recognition and epitope spreading mechanisms are fundamental for the autoimmune process mediated by both T and B cells. The imbalance of cytokines may lead to Th1-mediated tissue damage. The description of intralesional T cell reactivity has demonstrated the role of CD4þ T cells as effectors of the autoimmune lesions and explained how these cells expand when driven by streptococcal cross-reactivity in heart tissue. The pattern of cytokine production in the heart lesions favors a Th1-mediated disease. Many proteins and cardiac myosin epitopes were identified as putative cross-reactive autoantigens. Diagram 1 illustrates the major known events leading to RHD. In addition, we should consider the possibility of new events, mainly those related to the innate immune response (the first line of combat to the streptococcal infection), as effectors of RF/RHD reactions. Several genes with inflammatory function have been shown to be associated with the predisposition of the development of the disease (Table 2) and their function was described in the section of genetic susceptibility. Another issue that


47

DIAGRAM 1. Major events leading RHD an autoimmune disease, mediated by molecular mimicry between S. pyogenes and heart-tissue proteins.

should be addressed is the factors that may drive migration of inflammatory T cells to the site of RHD lesions. ACKNOWLEDGMENTS We acknowledge all of the people at the Heart Institute (InCor) School of Medicine from the University of Sao Paulo who contributed to the scientific data published elsewhere and described in this review. This work was supported by grants from ‘‘Fundaçao de Amparo a` Pesquisa do Estado de Sao Paulo (FAPESP)’’ and ‘‘Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq).’’

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[47] P.V. Lehmann, T. Forsthuber, A. Miller, E.E. Sercarz, Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen, Nature 359 (1992) 155–157. [48] R.S. Fujinami, M.B. Oldstone, Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity, Science 230 (1985) 1043–1045. [49] K.W. Wucherpfenning, J.L. Strominger, Molecular mimicry in the T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein, Cell. 80 (1995) 695–705. [50] D. Mason, A very high level of crossreactivity is a essential feature of the T-cell receptor, Immunol. Today 19 (1998) 395–404. [51] D. Mason, Antigen cross-reactivity: essential in the function of TCRs, The Immunologist 6 (1998) 220–222. [52] M. El-Demellawy, R. El-Ridi, N.I. Guirguis, M.A. Alim, A. Kotby, M. Kotb, Preferential recognition of myocardial antigens by T lymphocytes from rheumatic heart disease patients, Infect. Immun. 65 (1997) 2197–2205. [53] L. Guilherme, S.E. Oshiro, K.C. Fae, et al., T cell reactivty against streptococcal antigens in the periphery mirrors reactivity of heart infiltrating T lymphocytes in rheumatic heart disease patients, Infect. Immun. 69 (2001) 5345–5535. [54] K.C. Fae´, J. Kalil, A. Toubert, L. Guilherme, Heart infiltrating T cell clones from a rheumatic heart disease patient display a common TCR usage and a degenerate antigen recognition pattern, Mol. Immunol. 40 (2004) 1129–1135. [55] L. Guilherme, N. Dulphy, C. Douay, et al., Molecular evidence for antigen-driven immune responses in cardiac lesions of rheumatic heart disease patients, Int. Immunol. 12 (2000) 1063–1074. [56] L. Guilherme, E. Cunha-Neto, V. Coelho, et al., Human heart-infiltrating T-cell clones from rheumatic heart disease patients recognized both streptococcal and cardiac proteins, Circulation 92 (1995) 415–420. [57] K.C. Fae, D.D. Silva, S.E. Oshiro, et al., Mimicry in recognition of cardiac myosin peptides by heart-intralesional T cell clones from rheumatic heart disease, J. Immunol. 176 (2006) 5662–5670. [58] B.N. Manjula, A.S. Acharya, M.S. Mische, T. Fairwell, V.A. Fischetti, The complete amino acid sequence of a biologically active 197-residue fragment of M protein isolated from type 5 group A streptococci, J. Biol. Chem. 259 (1984) 3686–3693. [59] L.C. Miller, E.D. Gray, E.H. Beachey, M.A. Kehoe, Antigenic variation among group A streptococcal M proteins: nucleotide sequence of the serotype 5 M protein gene and its relationship with genes encoding types 6 and 24 M proteins, J. Biol. Chem. 263 (1988) 5668–5673. [60] N.M. Ellis, Y. Li, W. Hildebrand, V.A. Fischetti, M.W. Cunningham, T cell mimicry and epitope specificity of cross-reactive T cell clones from rheumatic heart disease, J. Immunol. 175 (2005) 5448–5456. [61] J.E. Galvin, M.E. Hemric, K. Ward, M. Cunningham, Cytotoxic monoclonal antibody from rheumatic carditis reacts with human endothelium: implications in rheumatic heart disease, J. Clin. Invest. 106 (2000) 217–224. [62] S. Roberts, S. Kosanke, T.S. Dunn, et al., Pathogenic mechanism in rheumatic carditis: focus on valvular endothelium, J. Infect. Dis. 183 (2001) 507–511. [63] V. Raizada, RCJr Williams, P. Chopra, et al., Tissue distribution of lymphocytes in rheumatic heart valves as defined by monoclonal anti-T cells antibodies, Am. J. Med. 74 (1983) 225–237. [64] E. Kemeny, T. Grieve, R. Marcus, et al., Identification of mononuclear cells and T cell subsets in rheumatic valvulitis, Clin. Immunol. Immunopathol. 52 (1989) 225–237. [65] K.C. Fae´, D. Diefenbach da Silva, A.M. Bilate, et al., PDIA3, HSPA5 and vimentin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients, J. Autoimmun. 31 (2008) 136–141.


DIPEPTIDYL PEPTIDASE IN AUTOIMMUNE PATHOPHYSIOLOGY Kei Ohnuma,* Osamu Hosono,* Nam H. Dang,† and Chikao Morimoto*,‡ *Division of Rheumatology and Allergy, Research Hospital, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan † Division of Hematology/Oncology, University of Florida, Gainesville, Florida, USA ‡ Division of clinical immunology, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan

1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Molecular Aspects of CD26/DPPIV Role in Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Structure of DPPIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. CD26-Binding Proteins and Their Impact on Immunity . . . . . . . . . . . . . . . . . . . . 3.3. Characterization of DPPIV Enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Cellular Aspects of CD26/DPPIV Role in Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. CD26 and T Cell Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. CD26 and Other Immune Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Clinical Aspects of CD26 Role in Immune Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Serum CD26/DPP4 Activity in Immune Disorders . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Rheumatoid Arthritis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Multiple Sclerosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Inflammatory Bowel Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Type I Diabetes Mellitus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Transplantation: Graft Rejection and GVHD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Other Immune-Mediated Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Summary and Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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51 0065-2423/11 $35.00 DOI: 10.1016/S0065-2423(11)53003-3


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1. Abstract CD26 is a 110-kDa surface glycoprotein with intrinsic dipeptidyl peptidase IV (DPPIV) activity that is expressed on various cell types and has many biological functions. An important aspect of CD26 biology is its peptidase activity and its functional and physical association with molecules with key roles in human immunological programs. CD26 role in immune regulation has been extensively characterized, with recent findings elucidating its linkage with signaling pathways and structures involved in T cell activation as well as antigen-presenting cell–T cell interaction, being a marker of disease behavior clinically as well as playing an important role in autoimmune pathogenesis and development. Through the use of various experimental approaches and agents to influence CD26/DPPIV expression and activity, such as anti-CD26 antibodies, CD26/DPPIV chemical inhibitors, siRNAs to inhibit CD26 expression, overexpressing CD26 transfectants, soluble CD26 molecules and proteomic approach, we have shown that CD26 interacts with structures with essential cellular functions in T cell responses. We will review emerging data that suggest CD26 may be an appropriate therapeutic target for the treatment of selected immune disorders.

2. Introduction CD26 is a 110-kDa surface glycoprotein with intrinsic dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) activity expressed on numerous cell types and has many biological functions. Following its initial description in 1966 as glycylprolyl-b-naphthylamidase [1], the enzyme peptidase was later named DPPIV and was found to be the same as CD26, a 110-kDa extracellular membranebound glycoprotein that is expressed on many tissues [2]. Originally characterized as a T cell differentiation antigen, CD26 is preferentially expressed on a specific population of T lymphocytes, the subset of CD4þ CD45ROþ memory T cells, and is upregulated following T cell activation [3]. Besides being a marker of T cell activation, CD26 is also associated with T cell signal transduction processes as a costimulatory molecule [4–6], and is involved in various T cell functions such as cell migration and cytokine production [3,7,8]. The amino acid sequence of human CD26 shares 85% homology with the rat DPPIV enzyme and the mouse thymocyte activation molecule (THAM), the mouse homolog of human CD26 [2,9]. While CD26 is expressed in various organs including brain, endothelium, heart, intestine, kidney, liver, lung, skeletal muscle, pancreas, placenta, and lymphocytes [3,10–13], CD26 knockout (CD26-KO) mice with C57BL/6 background display an apparently normal phenotype [14]. However, the percentage of

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CD4þ T cells is lower in the spleen lymphocyte population of CD26-KO mice than CD26-positive wild-type mice [14]. After immunization of mice with poke-weed mitogen (PWM) in vivo, serum levels of total IgG, IgG1, IgG2a, and IgE were markedly decreased in CD26-KO mice than those in wild-type mice. Moreover, IL-4 and IL-2 levels in sera of CD26-KO mice were decreased, and production of interferon-gamma (IFN-g) was delayed in response to PWM immunization. In clinical settings, altered levels of CD26/ DPPIV expression and DPPIV enzyme activity have been demonstrated in several immune-mediated conditions, including rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel diseases (IBD), systemic lupus erythematosus (SLE), and organ transplantation [15–21]. These results indicate that CD26/DPPIV mediates immune function and disease pathogenesis via its involvement in the development, maturation, and migration of T cells, cytokine secretion, T cell-dependent antibody production, and immunoglobulin isotype switching of B cells [22–24]. We describe herein the immunological aspects of CD26/DPPIV and its association with clinically observed autoimmune diseases.

3. Molecular Aspects of CD26/DPPIV Role in Immunity 3.1. STRUCTURE OF DPPIV Human CD26 is a 110-kDa cell-surface glycoprotein that belongs to the serine protease family, which is characterized by the Ser-Asp-His catalytic triad in the C-terminal region. The primary structure of the enzyme was first determined by cloning and sequencing of the cDNA of rat DPPIV in 1989 [25]. For human DPPIV, the complete cDNA and derived amino acid sequences were published by our group and others in 1992 [2,26,27]. The human DPPIV gene is located on chromosome 2 locus 2q24.3, spans 70 kb, and contains 26 exons [10,28]. The nucleotides encoding the sequence around the active site serine at 630 residue (Gly-X-Ser-X-Gly) are split between two exons, distinguishing the genomic organization of DPPIV from that of the classical serine proteases [10,29]. CD26/DPPIV is a member of a complex gene family, many of which also cleave structurally related peptides [30]. These include prolyl endopeptidase (PEP), acylaminoacyl peptidase (ACPH), CD26/DPPIV, fibroblast activation protein a (FAPa), DPP8, DPP9, DPP10, and DPPX [30,31]. FAPa forms a heterodimer with CD26/ DPPIV, and these heterodimers are found on fibroblast and melanocytes [32–34]. The amino acid sequence of human FAPa shows 52% identity with DPPIV, and the FAPa gene is located in the 2q23 region, which is close to the DPPIV locus [35,36]. As shown in Fig. 1, human CD26 is composed of 766

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Extracellular domain (AA29−766)

DPPIV

Binding proteins : (Known-binding site) ADA ECM (fibronectin, collagen) HIVgp 120 Streptokinase Plasminogen Caveolin-1 (Unknown-binding site) Extracellular domain: HIV Tat CXCR4 receptor M6P/IGFII receptor

C-terminal catalytic regions (AA511−766) Catalytic triad (S630, D708, H740)

Cysteine-rich region (AA290−510)

Membrane/cytoplamic domian: CARMA1 Glycosylated region (AA49−289)

Flexible segment (AA29−48) Membrane domain (AA7−28) Cell membrane Cytoplasmic domain (AA1−6)

Cytoplasm

FIG. 1. Domain structure of CD26/DPPIV. Schematic representation of CD26/DPPIV is shown in a single molecule for convenience. A schema of DPPIV substrate is shown at Cterminal catalytic region in chain of circles. Please see the text in detail for primary structure and interacting proteins.

amino acids, including a short cytoplasmic domain of six amino acids, a transmembrane region of 24 amino acids, and an extracellular domain with dipeptidyl peptidase activity which selectively removes the N-terminal dipeptide from peptides with proline or alanine at the penultimate position [2]. The N-terminus of human CD26/DPPIV protein contains the consensus sequence (DW(V/L)YEEE), with the first two Glu (E) amino acids in this sequence being necessary for enzyme activity, while the C-terminus is also highly conserved (an a/b-hydrolase domain) [37,38]. By analysis of three-dimensional crystal structure of human DPPIV, the catalytic site (Ser630-Asp708His740) is determined to be located in a large cavity (also called a central tunnel), formed between the a/b-hydrolase domain and eight-bladed b-propeller domain (Fig. 2) [39], which contains the consensus sequence (DW (V/L)YEE) that is conserved in S9B protease [37,39]. Single amino acid point mutation in the b-propeller motif identified Glu205 and Glu206 as essential for DPPIV enzyme activity [37], and the central tunnel and a/b-hydrolase domains both participate in DPPIV inhibitor binding (shown in CPK coloring of Fig. 2) [40,41]. The amino acids lining the opening to the catalytic site pocket control substrate specificity [42]. Single amino acid point mutation at


C

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C

N

N Cell membrane

Cytosol FIG. 2. Three-dimensional crystal structure of CD26/DPPIV. CD26/DPPIV forms a homodimer at the cell surface (one subunit shown in green, the other in magenta). The a/b-hydrase domain is closest to the membrane and contains the catalytic triad S630, D708, and H740, in which the inhibitor Val-pyrrolidide is shown in CPK coloring. In both subunits, S–S bridge or carbohydrates are shown in yellow or blue, respectively. Figure is reprinted by permission from Nature Publishing Group [39], Figure 1a, copyright 2003.

His750 residue is important for dimerization [43]. The carbohydrate chains (blue chains in Fig. 2) account for about 20% of the total molecular weight and cause much heterogeneity, with at least eight active DPPIV forms with pI values between 3.4 and 5.2 being isolated by 2D-PAGE from total cell lysates of mitogen-activated human lymphocytes [44].

3.2. CD26-BINDING PROTEINS AND THEIR IMPACT ON IMMUNITY Adenosine deaminase (ADA) interacts with ADA-binding protein, now identified as CD26/DPPIV [45]. Deficiency of ADA in humans causes profound lymphopenia and results in severe combined immunodeficiency disease (SCID), associated with the intracellular accumulation of toxic metabolites of purine metabolism [46]. The residues Leu294 and Leu-Val-Ala-Arg343 of human CD26/DPPIV have been identified as essential for the binding of

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ADA [47,48]. Of note is that ADA–DPPIV binding was not observed in mice and rats [45,49,50]. A possible function of CD26/DPPIV-bound ADA is the modulation of the local extracellular adenosine concentration that provides negative signals to the interior of T cells through cell-surface adenosine receptors [51]. Indeed, Jurkat cells transfected with mutant CD26/DPPIV defective in ADA binding are much more sensitive to the inhibitory effect of adenosine on T cell proliferation than wild-type CD26/DPPIV transfectants [52]. Naturally occurring ADA mutants can also elucidate the role of the CD26/DPPIV–ADA interaction. An enzymatically inactive, DPPIV-binding ADA mutant was demonstrated in a patient with early onset SCID, with the other ADA allele not being expressed in this patient [53,54]. Furthermore, an adult who expresses a mutant ADA with catalytic activity but defective CD26/DPPIV-binding capacity and who has a nonsense mutation in his second ADA allele was found to be healthy [51,55]. These findings suggest that the catalytic function of ADA is more important in preserving intact immune function than its interaction with CD26/DDPIV. On the other hand, the interaction of ADA with CD26/DPPIV may provide a costimulatory signal in T cell activation mediated by the CD3/T cell receptor complex, independent of the enzymatic activity of ADA [51,56]. However, a direct involvement of CD26/DPPIV-bound ADA in T cell activation has been contested [48]. Lymphocyte–epithelial cell adhesion experiments revealed that the adhesion of a number of T cell lines was greatly dependent on CD26. Triggering the ADA-binding site by exogenous ADA or monoclonal antibodies (mAbs) significantly reduced this adhesion [57]. Extracellular matrix (ECM) has been reported to be associated with CD26/DPPIV [58–60], particularly involving collagen and fibronectin. The collagen-binding site on CD26/DPPIV is situated in a cysteine-rich region between residues 23 and 491 [61]. Meanwhile, the fibronectin-binding site on CD26/DPPIV is identified by nitrocellulose-binding assays using 125I-labeled CD26, and the fibronectin-binding site is located between residues 469 and 479, distinct from the site of DPPIV activity [60]. CD26 interaction with ECM may promote cell migration, and CD26/DPPIV can also serve as a functional collagen receptor for CD4þ T cell activation [62,63]. However, controversies do exist regarding the binding of CD26/DPPIV to the ECM proteins and its role as a cell adhesion molecule, based on such studies as the report showing that CD26/DPPIV on T lymphocytes is not involved in T cell adhesion to endothelial cells or fibroblasts [64,65]. On the other hand, numerous studies suggest that the interaction between CD26/DPPIV and ECM has an important role in the biology CD26-positive tumors [66]. CD45, also called common leukocyte antigen, is a protein tyrosine (Tyr) phosphatase [67]. Various isoforms of CD45 exist, as naive T lymphocytes express large CD45 isoforms and are usually positive for CD45RA, while


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activated and memory T lymphocytes express the smallest CD45 isoform CD45RO, which lacks exons RA, RB, and RC. Able to facilitate T cell activation, CD45RO is a T cell membrane-linked Tyr phosphatase that interacts with CD26 along with the cytoplasmic domain 2 of CD45 in lipid rafts, which are cholesterol-rich microdomains in cell membranes, leading to modification of cellular signaling events in peripheral blood T cells [68–70]. Treatment with the anti-CD26 antibody 1 F7 increased CD26 recruitment to lipid rafts of nonactivated peripheral blood T cells, resulting in enhanced Tyr phosphorylation of c-Cbl, Zap70, Erk1/2, p56lck, and TCR-. However, the CD26–CD45RA interaction in cord blood T lymphocytes occurs outside lipid rafts, and this nonraft association of CD26–CD45 is associated with decreased CD26-mediated T cell activation [71]. Mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) binds CD26 via M6P residues in the carbohydrate moiety of CD26 to mediate CD26 internalization, a specific interaction that is important for CD26-mediated T cell activation and migration [72,73]. Following T cell activation, CD26 mannose 6 phosphorylation increases, a process which results in CD26 internalization. Published reports have shown that CD26 cross-linking induces colocalization with M6P/IGFII, and competition with exogenous M6P prevents cross-linking-mediated internalization of CD26, hence inhibiting CD26-mediated T cell activation. Treatment of CD26 with either a glycosidase or a phosphatase completely abrogates CD26–M6P/ IGFIIR binding, showing that both glycosylation and phosphorylation of CD26 are required for M6P/IGFIIR binding [72]. CXCR4, a chemokine receptor, interacts with CD26/DPPIV on T and B cell lines [74]. Binding of the CXCR4 ligand stromal cell-derived factor 1 alpha (SDF-1a; CXCL12) to its receptor causes cointernalization of CXCR4 and CD26/DPPIV, and the association of CXCR4 and CD26/DPPIV may provide a mechanism for regulating SDF-1a activity in the local milieu. We recently demonstrated that the presence of CD26 enhances SDF-1a-mediated invasion of T cell lines [75], a process regulated in part by the PI-3K (phosphoinositide 3-kinase) and MEK1 pathways, as indicated by the increased phosphorylation of p44/42 MAP kinase and Akt in the presence of SDF-1a. CXCR4, as well as CCR5, is identified as a major coreceptor for HIV-1 [76–78]. The expression of CCR5 by CD45ROþ memory T cells, most of which also express high levels of CD26/DPPIV, can partly explain the early reduction of CD4þCD26þ T cells and the defective response to recall antigens in HIV-1 infection [79,80]. Cells with a degree of expression of CD26/ DPPIV that is comparable to that of activated T cells are more sensitive to X4 HIV-1 infection (CXCR4-mediated entry, or formerly designated T-tropic HIV-1) and X4 HIV-1-mediated apoptosis than cells that express low levels of CD26/DPPIV [81,82]. However, overexpression of CD26/

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DPPIV suppresses X4 HIV-1 entry [81]. Since chemokine receptors are exploited by HIV-1 for cell entry, several chemokines provide a certain degree of protection against HIV-1 infection [83]. This protection is the result of the occupation by the chemokine of its receptor, followed by internalization of the chemokine–receptor complex. HIV-1 infection in a T cell line was impaired by DPPIV inhibitors, pointing to a role of DPPIV in the control of chemokine–receptor interaction [84]. The C3 region of the HIV-1 envelope glycoprotein gp120 binds to CD26/ DPPIV on lymphocytes, inhibiting the association of CD26 to ADA [85]. The interaction between CD26/DPPIV and gp120 does not require DPPIV activity but is CD4 or CXCR4 dependent [74,86]. Indeed, the C3 region of gp120 is not exposed to the surface [87], and the interaction with CD4 or CXCR4 could unmask this hidden region, allowing binding to CD26/ DPPIV. Franco et al. investigated proliferation and cytokine secretion following ADA costimulation of cells from HIV-infected patients [88]. In this study, an effect of ADA on T cell proliferation was found in HIV-1-infected patients and correlated positively with the CD4þ percentage and the nadir CD4 count and negatively with viral load, demonstrating that the response depends on the immunological status of the patient. The robust ADAinduced increase in cytokine production was markedly reduced in T cells from HIV-1-infected subjects. Moreover, it was found that gp120 was responsible for the impairment of the ADA–CD26 interaction and the subsequent ADA-induced effect on both costimulation and cytokine production. The cationic HIV transactivator protein Tat is reported to bind to CD26/ DPPIV and inhibit its enzymatic activity [89]. Tat is immunosuppressive in vitro, and some investigators have attributed this immunosuppressive effect of Tat to a specific interaction with CD26/DPPIV [90,91]. Other investigators, however, could not confirm a specific Tat–CD26/DPPIV interaction and indicated that the cationic Tat might interact nonspecifically with negatively charged regions in proteins [92,93]. The inhibition of CD26/ DPPIV by Tat was indeed found to be dependent on the sialylation status of CD26/DPPIV and on the salt concentration [94]. Plasminogen type 2 (Pg2) glycoforms a, b, g, d, and E on synovial fibroblasts isolated from RA patients bind through the sialic acids of their O-glycan to the residues 313–319 of CD26/DPPIV [95]. The binding of Pg2 g, d, and E is followed by activation by the urinary-type plasminogen activator (u-PA) located in the vicinity and results in intracellular calcium mobilization [96]. It has been reported that streptokinase (SK), a protein secreted by streptococci which facilitates the development of focal infection in association with Pg, can bind to CD26/DPPIV expressed by rheumatoid synovial fibroblasts [97]. While SK is a very potent immunogenic protein, its functional role on


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T cells, particularly with regard to its association with CD26, remains to be elucidated. Regarding CD26 physical structure, its central tunnel, DPPIV enzyme pocket, and dimerization site are all necessary for binding to caveolin-1, which is found to be the costimulatory ligand for human CD26 [98,99]. This process is followed by T cell costimulation via CARMA1 interaction with the cytoplasmic/membrane domains of CD26 [100], as described below. 3.3. CHARACTERIZATION OF DPPIV ENZYME DPPIV is a serine protease that cleaves dipeptides from the N-terminus of peptides [2]. In biological samples, DPPIV activity is determined by means of chromogenic or fluorogenic substrate. For example, the release of paranitroaniline from Gly-Pro-para-nitroanilide is monitored spectrophotometrically as a function of time [101,102]. It is recommended to measure both activity and antigen levels in parallel since the specific activity of DPPIV was found to be altered in certain pathological conditions [102,103]. Commercial assay kits for enzyme activity or antigen levels of CD26 are available. The enzymatic activity of CD26 appears to be very important in enhancing cellular responses to external stimuli. For example, Jurkat cells transfected with wild-type CD26 consistently demonstrate greater activation than parental CD26 negative Jurkat or cells transfected with CD26 mutated at the DPPIV enzymatic site [104]. Moreover, we have shown that exogenous recombinant soluble CD26 (rsCD26) with DPPIV enzyme activity enhances the proliferative response of peripheral blood lymphocytes (PBLs) to stimulation with the soluble antigen tetanus toxoid (TT) [105]. This enhancing effect of rsCD26 was not observed with mutant rsCD26 lacking DPPIV enzyme activity [105]. Although the precise mechanism underlying the enhancing effect of DPPIV enzyme activity has not yet been elucidated, one possible explanation for the role of DPPIV enzyme activity in T cell activation is its binding activity to the putative ligand caveolin-1 [99,100]. Possible substrates of CD26/DPPIV include several critical cytokines and chemokines [22]. Activity of RANTES (regulated on activation, normal T cell expressed and secreted; CCL5) is altered by DPPV-mediated cleavage, as CD26/DPPIV-processed RANTES affects important activities such as those implicated in monocyte chemotaxis and HIV-1 infection [106,107]. Other important chemokines that appear to be substrates of DPPIV enzymatic activity include eotaxin (CCL11), macrophage-derived chemokine (MDC; CCL22), interferon-inducible chemokines (CXCL10), and other chemokines involved in the inhibition of HIV infection [107]. In addition, recent work showed that CD26 plays an important role in the mobilization of hematopoietic stem cell (HSC) and hematopoietic progenitor cells (HPCs)

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induced by granulocyte colony-stimulating factor (G-CSF) [108]. One of the substrates of CD26/DPPIV is CXCL12 (SDF-1 a), an important chemokine that serves as a chemoattractant for HSC/HPC [109,110]. It has been shown that CXCL12 can be selectively truncated in vitro by CD26/DPPIV, and the truncated molecule lacks the ability to induce migration of hematopoietic cells isolated from mouse bone marrow. Furthermore, treatment of mice with CD26/DPPIV inhibitors during the process of G-CSF mobilization results in a significant increase in the number of mobilized HPC [108,109]. An exciting development regarding DPPIV involves its role in glucose metabolism, as inhibition of endogenous glucagon-like peptide 1 (GLP-1) degradation by reducing DPPIV activity is an alternative strategy for improving the incretin action of GLP-1 in vivo and regulating glucose levels (reviewed in Ref. [31]). The first oral selective DPPIV inhibitor was approved by the Food and Drug Administration (FDA) in October 2006, and additional DPPIV inhibitors are under review by FDA for clinical use [31,111]. More selective small molecule inhibitors of DPPIV are currently being investigated in clinical trials for the treatment of impaired glucose tolerance and type 2 diabetes [31,112].

4. Cellular Aspects of CD26/DPPIV Role in Immunity 4.1. CD26 AND T CELL FUNCTION While CD26 expression is enhanced following activation of resting T cells, CD4þCD26high T cells respond maximally to recall antigens such as TT [3]. Cross-linking of CD26 and CD3 with solid-phase immobilized mAbs can induce T cell costimulation and IL-2 production by CD26þT cells [2,3]. In addition, anti-CD26 antibody treatment of T cells enhances Tyr phosphorylation of signaling molecules such as CD3 and p56lck [7,113]. High CD26 cell-surface expression is correlated with the production of TH1-type cytokines, such as IFN-g, and CD26 expression is induced by stimuli that favor the development of the TH1 response [114–116]. CD26þT-helper cells stimulate antibody synthesis in B cells and activate MHC-restricted cytotoxic T cells [3,117]. Moreover, previous reports showed that CD26þ T cells exhibit strong migratory ability through endothelial cells, and are present at high levels in the rheumatoid synovium and the synovial fluids [118–121]. CD26 may have, therefore, an important role in T cell biology and overall immune function. The question of whether the enzymatic activity of CD26/DPPIV is required for its T cell activation has been addressed by many investigators. In human immunology, some groups have shown that the enzyme activity of


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CD26/DPPIV is not required for T cell activation by the use of deletion mutants and by the use of DPPIV inhibitors [122–124]. However, DPPIV inhibitors suppress in vitro T cell proliferation and cytokine production following antibody-mediated, alloantigenic, and/or mitogenic stimulation [125–127]. It has been reported that the in vitro inhibition of DPPIV enzyme activity in T cells induces intracellular signals leading to the suppression of the production of activating cytokines and an enhanced production of transforming growth factor (TGF)-b1 [115]. Furthermore, we have shown that DPPIV activity is required for CD26-mediated T cell costimulation in vitro [104]. Jurkat T cell line (derived from a human T cell leukemia) transfected with CD26 shows greater activation than CD26-negative Jurkat cells or Jurkat cells transfected with CD26 mutated at the DPPIV enzymatic site (serine residue at 630 to alanine) [104]. When stimulated by anti-CD3 and CD26 antibodies or the combination of anti-CD3 and phorbol esters, the wild-type CD26 (DPPIV containing)-transfected Jurkat cells showed more IL-2 production than the mutant CD26 (DPPIV deficient) or vector-only control transfected cells. In addition, DPPIV activity can change T cell response to a variety of external stimuli through CD26 and/or the CD3/ TCR complex to regulate IL-2 production [104]. More recently, we have demonstrated that exogenous soluble CD26/DPPIV enhances T cell memory response to recall antigen via upregulation of the costimulatory molecule CD86 on monocytes [105]. Subsequently, we identified caveolin-1 on monocytes as a costimulatory ligand for CD26, with its binding to CD26 being dependent on the DPPIV enzyme pocket [98,99]. CD26 ligation by caveolin-1 on antigen-presenting cell (APC) recruits CD26-interacting CARMA1 to lipid rafts, resulting in the formation of a CARMA1–Bcl10–MALT1–IKK complex, and this membrane-associated Bcl10 complex then activates IKK through ubiquitination of NEMO [100]. Finally, we have shown that anergy is induced in human peripheral T cells using CD26-blocking by its ligand caveolin-1-Ig fusion protein [128]. Finally, we have shown that anergy is induced in human peripheral T cells following blocking of this specific interaction with CD26 by the use of a caveolin-1-Ig fusion protein [125]. These findings clearly show that CD26-mediated T cell costimulation is associated with T cell immunity in man. Similar questions regarding the role of the enzyme activity of CD26 on mouse immunology has been raised. CD26-KO mice develop normally and have no obvious abnormal immune phenotype in the absence of an inflammatory stimulant [14,129], although the possibility that other DPPIV-like enzymes besides CD26 influence mouse T cell immunity has been demonstrated [130]. An alternative approach has been attempted in vivo using DPPIV inhibitors. Mice treated with selective DPPIV inhibitors have shown intact T cell recall responses and exhibit normal in vivo cytotoxic T cell responses [131]. However,

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DPPIV inhibitors suppress in vitro murine T cell proliferation and cytokine production [132]. Moreover, treatment with other DPPIV inhibitors reduces NOD mouse CD4þ splenic T cell migration, leading to the preservation of islet transplants [133,134]. Taken together, the direct role of DPPIV enzyme activity on T cell function remains to be settled, due to different findings from studies that have varying experimental conditions. 4.2. CD26 AND OTHER IMMUNE CELLS It has been reported that 0–5% of freshly isolated CD20þ B cells do express the CD26 antigen [135]. Following stimulation with PMA or Streptococcus aureus protein, the fraction of CD26-positive cells increased significantly. Interestingly, induction of CD26 expression on B cells from patients with combined variable immunodeficiency occurs in a manner similar to the B cells from healthy donors. Meanwhile, CD26 is not expressed or is found only at low levels on monocytes of healthy adult [136]. Flow cytometric analysis of dendritic APCs generated from peripheral blood of normal donors in the presence of granulocyte/macrophage colony-stimulating factor and IL-4 revealed intermediate levels of CD26 during a 2-week culture period [137]. While these findings suggest that CD26 may also be an activation marker for B cells and APC, its precise role in these cells remains to be elucidated. Meanwhile, only a small fraction of peripheral NK cells was found to express CD26. Using K562 as target cells, Madueno et al. analyzed the lytic capacity of NK cells stimulated with different anti-CD26 mAbs or following separation into CD26þ and CD26 subsets, and observed no detectable difference in the amount of chromium released by the target cells [138]. These investigators also evaluated redirected lysis through CD16 by arming both CD26þ and CD26 effector cells with an anti-CD16 antibody, and demonstrated that CD26 cells exhibited significantly less CD16-dependent lysis than CD26þ cells. These results indicate that CD26 is related to CD16dependent lysis but not to NK cytolysis, which may be mediated through protein Tyr phosphorylation. However, Shingu et al. found that NK cell cytotoxicity against breast adenocarcinoma cells was decreased in CD26mutant rats (DPPIV-deficient and reduced CD26 surface expression), suggesting that DPPIV activity was associated with NK cytotoxicity [139]. We recently identified caveolin-1 in APC as a binding protein for CD26 and demonstrated that CD26 on activated memory T cells directly faces caveolin-1 on TT-loaded monocytes in the contact area, which was revealed as the immunological synapse for T cell–APC interaction [99]. Moreover, we showed that residues 201–211 of CD26 along with the serine catalytic site at residue 630, which together constitute a pocket structure for CD26/DPPIV, contribute to binding to caveolin-1 scaffolding domain [99]. More recently,


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we demonstrated that caveolin-1 binds to Tollip (Toll-interacting protein) and IRAK-1 (interleukin-1 receptor-associated serine/threonine kinase 1) in the membrane of TT-loaded monocytes and that following exogenous CD26 stimulation, Tollip and IRAK-1 disengage from caveolin-1, with IRAK-1 being subsequently phosphorylated to upregulate CD86 expression [98]. It is conceivable that the interaction of CD26 with caveolin-1 on antigen-loaded monocytes results in CD86 upregulation, therefore enhancing the subsequent interaction of CD86 and CD28 on T cells to induce antigen-specific T cell proliferation and activation.

5. Clinical Aspects of CD26 Role in Immune Disorders 5.1. SERUM CD26/DPP4 ACTIVITY IN IMMUNE DISORDERS It has been reported that the signaling capacity of soluble DPP4 mediates immune function [105,140]. Many clinicians reported the altered level of serum DPP4 enzyme activity in various immune-mediated diseases (Table 1), including bronchail asthma [141], allogeneic kidney transplantation [20], atopic dermatitis [142,143], Graves’ disease [144], IBD [16,145], MS [146], psoriasis [143,147], RA [15,103,148,149], systemic sclerosis (SSc) [151], SLE [17,103,150], ANCA (antineutrophil cytoplasmic antibodies)associated vasculitides [152,153]. Changes in serum DPP enzyme activity is originally attributed to DPP4, while the substrates used to assess enzyme TABLE 1 ALTERED CIRCULATING DPP4 ENZYME ACTIVITY AND MEMBRANE CD26/DPP4 EXPRESSION IN INFLAMMATORY DISEASES Disease

Circulating

Membrane

Asthma Allotransplant (kidney) Atopic dermatitis Graves’ disease (Basedow) IBD (CD, UC) Multiple sclerosis Psoriasis Rheumatoid arthritis Systmeic lupus erythematosus Systemic sclerosis ANCA-associated vasculitides

# # " " # " # # # # #

ND # # " " " ND " ND ND ND

Reference [141] [20] [142,143] [144] [16,145] [146] [143,147] [15,103,148,149] [17,103,150] [151] [152,153]

ND, not determined; #, decrease DPP4 activity or expression; ", increase DPP4 activity or expression.

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activity, predominantly H-Ala-Pro and H-Gly-Pro, can also be cleaved by DPP8 and DPP9. Therefore, it is possible that the changes in enzyme activity observed are due to members of the DPP4 family other than DPP4 alone. The possible involvement of DPP in driving diverse disease pathologies has resulted in the investigation into the effect of DPP inhibitors in various preclinical models, as described below. 5.2. RHEUMATOID ARTHRITIS RA is a chronic, inflammatory autoimmune disease and is characterized by progressive invasion of synovial fibroblasts into the articular cartilage and erosion of the underlying bone, followed by joint destruction [154]. Several reports on RA patients have shown that levels of DPPIV enzymatic activity in plasma and surface expression of CD26/DPPIV on peripheral blood mononuclear cells can be dependent on disease severity and treatment [15,103,118,120,121,148,155]. Moreover, Mavropoulos et al. found that antiTNF-a therapy increases DPPIV activity and decreases autoantibodies to the chaperone protein Bip (GRP78) and phosphoglucose isomerase in 15 patients with RA [155]. A number of proinflammatory peptides are supposed to be involved in the pathogenesis of RA, and have their biological activity controlled by limited proteolysis executed by DPPIV and DPPIV-like hydrolytic activity [24,156]. Several investigators have demonstrated that SDF-la (CXCL12), a substrate of DPPIV, in synovial fluid of RA patients is a crucial mediator controlling the influx of lymphocytes and monocytes/macrophages into the inflamed synovium and thus triggering joint destruction [19,157]. Furthermore, Cordera et al. studied IL-12, IL-15, soluble CD26, and ADA serum levels from 35 patients with active and inactive RA as well as those of control patients [149]. Patients’ sera had higher IL-12 and IL-15 levels, and the level of soluble CD26 was inversely correlated with the number of swollen joints. These findings suggest that these cytokines and CD26 are associated with the inflammation and immune activity in RA. However, expression of CD26/DPPIV on CD3þ T cells in synovial fluid of RA patients has been reported to be decreased, compared to that of healthy adult patients with osteoarthritis [19,118,121]. We reported the presence of CD26þ T cells infiltrating the rheumatoid synovium as demonstrated by immunohistochemical studies, and noted high expression of caveolin-1 in the rheumatoid synovium vasculature and synoviocytes [158]. These data suggest that the CD26–caveolin-1 upregulation of CD86 on activated monocytes leads to antigen-specific T cell activation in RA, and that CD26þ T cells is associated with the migratory phenotype of activated elements tending to infiltrate the rheumatoid synovium. Using CD26-KO mice, Busso et al. uncovered potential molecular targets for DPPIV in antigen- and collagen-induced arthritis models [148]. They


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demonstrated a decrease in plasma DPP activity during arthritis that could be attributed solely to DPPIV [148]. Furthermore, damage severity, as indicated by synovial thickness, knee-joint inflammation, and histological grading, was more severe in CD26-KO mice than in wild-type control mice. In this study, SDF-1a concentrations were higher in CD26-KO mice with arthritis, and the authors proposed that in CD26-KO mice, SDF-1a levels are not regulated and this may contribute to the increase in disease severity [148]. Since DPPIV enzyme activity in CD26-KO mice is still detected at a decreased level compared to that found in wild-type mice (20% of wild-type level) [130], and DPPIV-like enzyme activity such as DP8 or DP9 is detected in CD26-KO mice [130], data obtained from CD26-KO mice need further investigations for clarification. Using a type II collagen-induced or alkyldiamine-induced rat model of arthritis, Tanaka et al. determined the in vivo efficacy of DPP inhibitors in normal and DPPIV-deficient Fischer rats [159]. The inhibitors alanylpyrrolidine-boronic acid (Ala-boroPro), Lys-[Z (NO2)]-thiazolidide (LZNT), and Ala-Pro-nitrobenzoylhydroxylamine were dissolved in saline and administered orally or subcutaneously using prophylactic and therapeutic dosage regimes. Treatment with any of the three inhibitors reduced hind paw swelling, an indicator of disease severity. The same group has also demonstrated the effectiveness of other DPPIV inhibitors, TMC-2A and TSL-225, in arthritis rat models induced by alkyldiamine [160]. These data suggest that inhibition of DPPIV enzyme activity may be a novel therapeutic approach in patients with RA, a concept that awaits validation by clinical studies. 5.3. MULTIPLE SCLEROSIS MS is a well-characterized CD4þ T cell-mediated autoimmune disease resulting in central nervous system (CNS) inflammation, demyelination, and neuronal injury [161]. CD26 expression on T cell surface is higher in blood and cerebrospinal fluids of patients with MS [162]. Krakauer et al. have demonstrated that the number of circulating CD4þ CD45ROþ CD26high T cells in patients with MS correlated with disease severity, and these CD4þ CD45ROþ CD26high T cells express surface molecules in a pattern consistent with the hypothesis that these cells represent Th1 cells with CNS-homing potential in MS [163]. Reinhold et al. have shown that T cell clones from MS patients express significantly higher levels of DPP4 compared to resting peripheral blood T cells [164]. Moreover, addition of the DPPIV inhibitor LZNT or Lys[Z(NO2)]-pyrrolidide (LZNP) to myelin basic protein-stimulated T cell clones inhibited production of IL-4, IFN-g, and TNF-a, suggesting that inhibition of DP activity in vivo may suppress the inflammatory response associated with MS [164].

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An association of DPPIV and MS has been extensively studied in experimental autoimmune encephalomyelitis (EAE), an animal model of MS that is induced by inoculation with CNS antigens to illicit an immune responses and paralytic disease. Preller et al. addressed the role of DPPIV in the EAE Th1 immune response using CD26-KO mice [165]. Similar to the results from an experimental arthritis model obtained by Busso et al. [148], EAE disease severity was significantly greater in CD26-KO compared to wild-type mice [165]. Concentrations of IFN-g and TNF-a were also elevated in CD26-KO mice and latent TGF-b was lower in comparison to wild-type controls, implicating a direct role in the production of these cytokines for DPPIV. The authors proposed that synthetic and natural ligand inhibitors of DPPIV could bind to the active site of the enzyme, leading to a reduction in proinflammatory cytokines, an increase in TGF-b, and decreased T cell proliferation, whereas this regulatory pathway might be absent in CD26-KO mice [165]. However, the authors did not address the potential involvement of DPP8 and DPP9, which are all active in CD26-KO mice and might also play a role in regulating inflammatory responses [130]. In further studies, LZNP was administered in EAE model mice [146]. In this system for T cell-mediated autoimmune inflammatory disease involving the CNS, the severity and incidence of the acute phase was significantly reduced, and the onset of clinical symptoms was variably delayed. These data suggest that the inhibition of T cell proliferation and effector functions by DPPIV inhibition contributes to the observed protective effect. 5.4. INFLAMMATORY BOWEL DISEASES Crohn’s disease (CD) and ulcerative colitis (UC) are categorized as IBD, being characterized by chronic remittent or progressive inflammatory conditions that may affect the entire gastrointestinal tract and the colonic mucosa, respectively, and are associated with an increased risk for colon cancer [166]. Sera from IBD patients contain lower levels of circulating DPPIV activity, while membrane expression of CD26/DPPIV on T cells isolated from IBD patients is higher than that from healthy controls [16,167,168]. These clinical observations indicate that CD26/DPPIV might play a significant role in perpetuating the inflammatory response associated with IBD. Abbott et al. investigated the effect of DPPIV deficiency in mice during experimental colitis, hypothesizing that CD26-KO mice would have higher concentrations of the intestinotrophic growth factor GLP-2, which can affect the level of protection and repair during dextran sulfate sodium (DSS)induced colitis [169]. The study showed that disease severity was greater on day 14 and crypt area was reduced on day 9 of DSS-induced colitis in CD26KO mice. These results support the hypothesis advocated by Preller et al.


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that the absence of DPPIV on the cell surface might exaggerate inflammatory responses [165]. However, there were no differences in markers of intestinal growth, including cell proliferation, crypt depth, and colon weight, in CD26KO mice compared to that in wild-type controls, suggesting that the biological activity of GLP-2 was not increased in these animals. Assessment of circulating DP enzyme activity indicated that there was no difference between wild-type and CD26-KO mice, suggesting that the residual DPP enzyme activity might have been responsible for GLP-2 cleavage and explaining why no differences in these parameters were observed [169]. The effects of synthetic inhibitors of DPPIV in an animal model of IBD have been reported by Bank et al. [170]. In this study, using the DSS-induced colitis model, BALB/c mice were treated with LZNP and an aminopeptidase N inhibitor, either alone or in combination. Significant decreases in disease activity and increased concentrations of TGF-b and FOXP3 were recorded in mice receiving the combination treatment compared to vehicle treatment. However, treatment with LZNP alone also seemed to reduce disease severity. More recently, the same group demonstrated the efficacy of two DPP inhibitors, Ile-Thia and Ile-Pyrr-(2-CN)*TFA, in wild-type mice with DSSinduced colitis. Both these inhibitors significantly reduced disease activity after 6 days of DSS ingestion [171]. Furthermore, histological indicators such as crypt depth and area were improved in mice treated with either Ile-Thia or Ile-Pyrr-(2-CN)*TFA. These studies provide further evidence of the antiinflammatory effects of DPP inhibitors. These findings suggest that the presence of DPPIV on the cell surface is critical for the immunomodulatory effects of DPPIV inhibitors and that the interaction between the inhibitor and DPPIV is essential for a reduced inflammatory response. 5.5. TYPE I DIABETES MELLITUS Type 1 diabetes is a chronic autoimmune disease, during which the pancreatic b-cells are selectively destroyed, and is thought to be a TH1 cellmediated disease that involves CD8þ T cells and innate immune cells [172]. Although DPPIV inhibitors have been extensively studied in treatment of type 2 diabetes as described above [112], little is known about their potential for treatment of type 1 diabetes. Infusion of GLP-1, a substrate of DPPIV, was shown to reduce glycemic excursions in type 1 diabetic patients, and this result was attributed to reduced glucagon levels and delayed gastric emptying [173,174]. In preclinical studies, the DPPIV inhibitor isoleucine thiazolidide was shown to improve glucose tolerance in both streptozotocin (STZ)induced and BioBreeding diabetic rats, associated with increased b-cell survival and possibly islet neogenesis [175,176]. Additionally, it has been shown that the DPPIV inhibitor MK0431 prolonged islet graft survival in

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STZ-induced diabetic mice, and that MK0431 pretreatment resulted in the prolongation of islet graft survival in an autoimmune type 1 diabetes model, the NOD mouse, through a mechanism that includes modulation of CD4þ T cell migration [133,134]. However, the results reported in these studies suggest that direct inhibitor effects on DPPIV, and not increased levels of active GIP and GLP-1, are mainly responsible for the improvements in graft retention of pancreatic b-cells. Therefore, further investigation will be needed to reveal a role of DPPIV in the insulitis of type 1 diabetes. 5.6. TRANSPLANTATION: GRAFT REJECTION AND GVHD Transplantation is the most effective treatment for end-stage organ failure, but organ survival is limited by immunological responses between recipient and donor, including graft rejection, graft-versus-host disease (GVHD), and side effects of immunosuppressive agents [177]. T cells are central to the process of transplant rejection through allorecognition of foreign antigens leading to their activation, and T cells are also involved in the orchestration of an effector response that results in organ damage. Schwemmle et al. have investigated the dynamics of CD26 cellular expression and circulating DPP IV activity in 56 kidney transplant patients [20]. These authors showed that CD26 expression on PBLs decreased significantly from baseline preop for up to 18 months, and that serum DPP IV enzymatic activity is significantly depressed for at least 12 months. These novel indicators potentially reflect an immunosuppressed state in recipients of well-functioning renal allografts. In a rat heart transplant model, the role of CD26/DPPIV in the immune cascade triggered by organ transplantation and leading to acute rejection of cardiac allografts in rat recipients has been analyzed [178]. Transplantation of heart resulted in an early increase in cellular CD26 expression, followed by a rise in DPPIV serum activity, which peaked at day 6, prior to the time of actual graft loss. Moreover, the specific DPPIV inhibitor prodipine abrogated acute rejection and prolonged cardiac allograft survival to 14 days. Prodipine treatment also prevented the early peak of cellular CD26 expression and thoroughly suppressed systemic DPPIV activity. These results demonstrate the role of CD26/DPPIV in alloantigen-mediated immune regulation in vivo and provide further evidence that CD26/DPPIV plays an important role in the mechanism of allograft rejection. Furthermore, in rat or mouse lung transplantation models, specific inhibition of DPPIV has been shown to have a protective effect of the grafted lungs [179,180]. GVHD remains a major cause of morbidity and mortality in allogeneic stem cell transplantation (alloSCT). In GVHD, mature donor T cells that accompany the stem cell graft attack recipient tissues, especially the skin, liver, gastrointestinal tract, and lung. Therefore, all patients undergoing


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alloSCT receive GVHD prophylaxis to impair T cells function; however, treatment to prevent GVHD can be deleterious since mature donor T cells play a critical role in mediating reconstitution of the adaptive immune system, especially in adults with diminished thymic function [181]. Recipients of alloSCT are thus at great risk for infections, particularly when prolonged immunosuppression is required for treatment of GVHD. Some approaches (e.g., B7-blocking, OX40-blocking, and T cell depletion by specific antibodies) to promote the positive effects of alloSCT without GVHD have been developed recently in the clinical settings (reviewed in Ref. [182]). Although the role of CD26/DPPIV in GVHD needs to be studied in more detail, treatment with a murine antibody against human CD26 was reported to have an effect of controlling GVHD in patients with steroid-resistant acute GVHD following alloSCT [183,184]. Moreover, we have previously examined the expression of CD26 as well as the reconstitution of CD26-mediated T cell costimulation via the CD3 and CD2 pathways at various times in patients with CD6-positive T cell depleted allogeneic bone marrow transplantation (alloBMT) [21]. In this study, we found that the percentage of CD26- and CD3-positive cells, as well as the levels of expression of both antigens, was lower than in normal controls during the first 4 months after CD6-depleted alloBMT. Subsequently, the level of lymphocytes expressing CD3 and CD26 and the quantitative surface expression of CD3 and CD26 were not significantly different between patients and normal controls. Functional studies showed that CD26-mediated T cell proliferation via the CD3 pathway was considerably improved and almost reached normal levels by 1 year, whereas recovery of CD26-mediated T cell proliferation via the CD2 pathway was delayed for at least 2 years after CD6-depleted alloBMT. As CD26 involvement in the regulation of human thymocyte activation is restricted preferentially to the CD3 pathway, unlike its involvement with both CD3 and CD2 pathways of peripheral T cells, our results suggest that the different effects of CD26-mediated costimulation via the CD3 and CD2 pathways after CD6-depleted alloBMT may be a reflection of peripheral T cell immaturity in those individuals. Taken together, it may be possible that the full therapeutic potential of alloSCT will be realized by approaches that aim to minimize GVHD by targeting CD26-mediated T cell regulation. 5.7. OTHER IMMUNE-MEDIATED DISORDERS CD26/DPPIV levels in human serum (soluble CD26) have been examined in patients with various medical conditions, such as autoimmune diseases, infectious diseases, and psychiatric disorders [185]. To determine the role of soluble CD26 in the pathophysiology of patients with SLE, we measured levels of soluble CD26 and its specific DPPIV activity in serum [17]. Serum

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levels of soluble CD26 and its specific DPPIV activity were significantly decreased in patients with SLE, and were inversely correlated with SLE disease activity index score, but not with clinical variables or clinical subsets of SLE [17]. More recently, Lam et al. reported that CD26 expression on invariant natural killer cells of SLE patients is decreased significantly than that of healthy controls [186]. However, Stollar et al. have demonstrated that a remarkable elevation of serum DPPIV activity occurred along with the development of lymphadenopathy and an enhancement in antinative DNA antibody production in MRL/Mp-lpr/lpr mice [187]. The pathology of a number of ANCA-associated vasculitides, including microscopic angiitis, Wegener’s granulomatosis, and Churg–Strauss syndrome, is characterized by a cell-mediated response involving activated CD4þ and CD8þ T cells. Soluble DPPIV activity in plasma was decreased in an inverse correlation with disease activity and increased again during remission [152,153]. SSc is a systemic disorder which typically results in fibrosis of the skin and multiple internal organ systems [188]. The basic mechanism appears to involve endothelial cell injury, the overproduction of ECM proteins including collagen and fibronectin, and the aberrant activation of the immune system. As described earlier, CD26/DPPIV has a binding site for fibronectin and collagen proteins, while also interacting with these ECM proteins [58–60]. Moreover, it is thought that CD26 possesses a significant degree of endopeptidase activity, in addition to its exopeptidase activity, and CD26 is able to cleave several types of collagen, which are rich in proline, hydroxyproline, and alanine [189]. Therefore, the level of DPPIV activity may be associated with excessive collagen deposition in SSc. Tamaki et al. have examined the circulating concentrations of sCD26 in 56 patients with SSc, and showed that the serum DPPIV levels in SSc patients were significantly lower than those of the control subjects [151]. The authors hypothesized that lower serum DPPIV levels could thereby contribute to the abnormal accumulation of collagen and other matrix protein in SSc. However, Thieliz et al. have shown that the DPPIV chemical inhibitors LZNT and LZNP suppress proliferation in human skin fibroblasts and keloid-derived skin fibroblasts in vitro, and that these inhibitors abrogate TGF-b1-induced profibrotic effects in vitro and, as shown in a mouse model of cutaneous fibrosis, in vivo [190]. CD4þ T cells in patients with AIDS develop an intrinsic defect in their ability to recognize and respond to recall antigens prior to a detectable reduction in the total number of CD4þ T cells [191,192]. The response to recall antigens is clearly a property of CD4þ CD26þ T cells, since this is the only helper population known to proliferate in response to soluble antigens, and to induce both MHC-restricted cytotoxic T cells capable of killing virusinfected target cells and immunoglobulin-secreting B cells [3].


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In this regard, a selective decrease in CD26þ T cells has been reported in HIV-1-infected individuals prior to a general decrease in CD4þ T cells [193,194]. Moreover, Tat, a regulatory protein encoded by the HIV-1 genome which has been shown to suppress the response of human peripheral T cells to soluble antigens [195,196], can bind to CD26 and partially inhibit DPPIV enzyme activity [89]. We have shown that the DPPIV enzyme activity of plasma soluble CD26 was low in HIV-1-infected individuals, and was inversely correlated with HIV-1 RNA, and that the in vitro addition of rsCD26 could enhance purified protein derivative-induced lymphocyte proliferation [102]. These results suggest that the specific DPPIV enzyme activity of plasma soluble CD26 in HIV-1-infected individuals contributes to the immunopathogenesis of HIV infection. Taken together, the above findings suggest that CD26/DPPIV plays an important role in the pathophysiology of autoimmune diseases and other immune-mediated disorders. Moreover, in vivo rsCD26 supplementation in immunodeficient patients with decreased soluble CD26/DPPIV may be useful in restoring the decreased immune response.

6. Summary and Perspective In this review, we discuss the role of CD26 in immune regulation, focusing particularly on recent findings elucidating its linkage with signaling pathways and structures involved in T-lymphocyte activation as well as APC–T cell interaction. A key aspect of CD26 biology is the identification of a natural ligand for CD26, which had been previously called a ‘‘phantom molecule’’ since its putative costimulatory ligand had not been characterized. Our recent novel findings that identified a costimulatory ligand for CD26 therefore contribute to the area of T cell costimulation and signal transduction. As discussed above, CD26 appears to have a pivotal function in the biology and treatment of autoimmune diseases or GVHD, and current laboratory studies and early human clinical trials are being conducted to examine novel approaches to these conditions based on our recent knowledge of CD26. Taken together with emerging findings regarding the various molecular aspects of CD26 biology, accumulating evidence from clinical observations are suggesting that CD26 may be an appropriate novel target for the treatment of immune disorders. ACKNOWLEDGMENTS This work was supported by Grant-in-Aid of The Ministry of Education, Culture, Sports, Science and Technology (K. O. and C. M.); Ministry of Health, Labour, and Welfare, Japan (C. M.); and by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (C. M.).

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BIOCHEMISTRY OF HELLP SYNDROME Chiara Benedetto,1 Luca Marozio, Annalisa Tancredi, Elisa Picardo, Paola Nardolillo, Anna Maria Tavella, and Loredana Salton Department of Obstetrics and Gynaecology, University of Torino, Torino, Italy

1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Clinical Features of HELLP Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Laboratory Findings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Maternal and Perinatal Outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Pathogenesis of HELLP and Preeclampsia: The Role of Placenta . . . . . . . . . . . . . . . . 4. Inflammatory Response in HELLP Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. HELLP Syndrome, Complement Pathway, and the Coagulation System . . . . . . . . . 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Abstract The HELLP syndrome is a serious complication of pregnancy characterized by hemolysis (H), elevated liver (EL) enzymes, and low platelet (LP) count that occurs in 0.2–0.6% of all pregnancies and in 10–20% of cases with severe preeclampsia and frequently leads to adverse maternal and perinatal outcome. The exact pathobiology of HELLP syndrome has not been clearly defined. As it is considered a form or a complication of severe preeclampsia, it likely has its origin in aberrant placental development and function resulting in ischemia-producing oxidative stress. However, there is still a debate on whether HELLP must be considered a severe form of preeclampsia or a separate disease entity. It can be described as a placenta-induced disease, as is preeclampsia itself, but with a more acute and predominant inflammatory 1

Corresponding author: Chiara Benedetto, e-mail: [email protected] 85

0065-2423/11 $35.00 DOI: 10.1016/S0065-2423(11)53004-5


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process typically targeting the liver and with a greater activation of the coagulation system. This occurs during a disordered immunologic process and may be due to a genetic predisposition. In this review, we discuss the main biochemical characteristics of HELLP syndrome, particularly focusing on molecular aspects of placental involvement and maternal systemic responses.

2. Introduction The HELLP syndrome is a serious complication of pregnancy characterized by hemolysis (H), elevated liver (EL) enzymes, and low platelet (LP) count occurring in 0.2–0.6% of all pregnancies and in 10–20% of cases with severe preeclampsia [1]. In about 70% of the cases, the HELLP syndrome develops before delivery with a peak frequency between the 27th and 37th gestational weeks; 10% occur before the 27th week, and 20% beyond the 37th gestational week [2,3]. In some cases, it develops in the postpartum period, usually within the first 48h in women who have had proteinuria and hypertension prior to delivery [2]. 2.1. CLINICAL FEATURES OF HELLP SYNDROME The majority of women with the HELLP syndrome have had hypertension and proteinuria, which may be absent in 10–20% of the cases [4]. The more common symptom at presentation is right upper abdominal quadrant or epigastric pain, nausea, and vomiting being less frequent [4]. Up to 30–60% of women have headache; about 20% have visual disturbances and other features of severe preeclampsia or impending eclampsia [4]. The clinical symptoms often precede the laboratory findings. In some cases, however, the HELLP syndrome may present with nonspecific viral syndrome-like symptoms or malaise [4]. The syndrome, which is considered a complication of preeclampsia, is characterized by prominent endothelial cell damage within the liver. Hypovolemia is suggested with a decrease in the liver blood flow on Doppler examination in patients with preeclampsia, who have subsequently developed HELLP syndrome [5]. Hepatic ischemia may cause infarction, subcapsular haematomas, and intraparenchymatous hemorrhage, resulting in hepatic rupture, a rare but severe and life-threatening complication [6]. Recurrent episodes of hepatic haematoma and rupture in subsequent pregnancies have been reported, suggesting that there may be a specific predisposition to this condition [7]. On liver biopsy, periportal hemorrhage, focal parenchymatous necrosis, and macrovesicular steatosis may be observed in

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up to one-third of patients. Fibrin and hyaline deposits are seen by immunofluorescence at the level of liver sinusoids. However, there is little correlation between histological findings and clinical presentation [8]. Hemolysis, one of the major characteristics of the disorder seen on a microangiopathic blood smear, reflects the damage of the vascular endothelium. Red cell fragmentation represents the extent of small vessel involvement, and schizocytes and Burr cells are usually present [9]. Polychromatic red cells are also seen in blood smears, and increased reticulocyte counts reflect the compensatory release of immature red cells into peripheral blood. Decreased PLT count in the HELLP syndrome is due to their increased consumption. Platelets are activated and adhere to damaged vascular endothelial cells, resulting in increased platelet turnover with shorter lifespan [9]. 2.2. LABORATORY FINDINGS Hemolysis causes increased serum lactate dehydrogenase (LDH) levels and decreased hemoglobin concentrations [10]. Free hemoglobin is converted to unconjugated bilirubin in the spleen or may be bound by haptoglobin. The hemoglobin–haptoglobin complex is cleared quickly by the liver, leading to decreased haptoglobin plasma levels [10,11]. Low haptoglobin concentration is the preferred marker of hemolysis [12]. Thus, the diagnosis of hemolysis is supported by high LDH concentration and the presence of unconjugated bilirubin, but the demonstration of low or undetectable haptoglobin concentration is a more specific indicator. Intravascular hemolysis is diagnosed by abnormal peripheral blood smear, increased serum bilirubin ( 20.5 mol/L or 1.2 mg/100 mL), and elevated LDH levels (> 600 units/L (U/L)) [13,14]. However, according to Smulian et al., the threshold of normal LDH values may be much lower than 600 U/L depending on the laboratory method adopted [15]. Elevation of liver enzymes may reflect the haemolytic process as well as liver involvement. Enhanced aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels are mostly due to liver injury [10]. Visser and Wallenburg used ALT > 30 U/L to define abnormality (2 SD above mean) [16], while Sibai suggests a cutoff value for ALT > 70 U/L [4]. Thrombocytopenia (platelets < 100,000/ml) is obligatory in the HELLP syndrome. Reduced platelet count in pregnancy may be also caused by gestational thrombocytopenia, immune thrombocytopenic purpura, and preeclampsia [17], but in those cases, hemolysis and liver damage are usually absent. Sometimes, the differential diagnosis of HELLP from ‘‘acute fatty liver of pregnancy’’ (AFLP) may be very difficult. AFLP is a rare but life-threatening complication mainly of the third trimester. The patient usually presents with

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a 1- to 2-week history of malaise, nausea, vomiting, epigastric or right upper quadrant pain, headache, or jaundice, more rarely with hepatic encephalopathy or coma. Signs of preterm labor and/or fetal demise may be present. Other findings may include hypertension, proteinuria, low-grade fever, ascites, and bleeding from severe coagulopathy [18]. Common laboratory findings in AFLP are hemoconcentration, elevated white blood count, and normal or reduced platelet count. The production of antithrombin, fibrinogen, and coagulation factors by the liver is significantly reduced, often leading to disseminated intravascular coagulation. Serum electrolytes will reveal evidence of metabolic acidosis with elevated creatinine and uric acid values [19]. Blood sugar is usually low, but may be high in association with pancreatitis. Liver enzymes (AST, ALT, alkaline phosphatase) and bilirubin will be elevated. The increase in bilirubin is mainly of the conjugated form, with levels usually higher than 5 mg/dL. Ammonia levels increase in the late stage of the disease [20]. Ultrasonography, CT, and MRI are not sufficiently sensitive to confirm or exclude the diagnosis, and the gold standard for confirming the diagnosis of AFLP is the liver biopsy with special stains for fat, such as red oil: histopathological findings reveal swollen, pale hepatocytes with central nuclei. However, liver biopsy is rarely used in the clinical practice [18]. Prompt delivery, within 24 h from the diagnosis and after maternal stabilization, is a reasonable approach. In general, most patients with AFLP will start to improve 2–3 days after delivery. However, in some cases, deterioration in liver and renal function, encephalopathy, and coagulopathy may continue, requiring life-supporting treatments. In rare cases, liver transplantation will be required [21].

2.3. MATERNAL AND PERINATAL OUTCOME The HELLP syndrome is associated with both maternal and neonatal complications. Spontaneous rupture of a subcapsular liver haematoma in pregnancy is a rare but life-threatening complication that occurs in less than 1% of the cases with the HELLP syndrome. Rupture most often occurs in the right liver lobe [4, 22–24]. More common and serious maternal complications are abruptio placentae, disseminated intravascular coagulation, and subsequent severe postpartum bleeding, retinopathy, cerebral bleeding, and stroke [25–32]. In a large retrospective cohort study comprising 442 pregnancies complicated by the HELLP syndrome, the maternal mortality was 1.1% [2], which is in accordance with other reports [4,33,34]. Isler et al. found cerebral hemorrhage or stroke to be the primary cause of death in 26% and the most contributing factor in another 45% of the deaths [35]. Maternal mortality rate in hepatic rupture ranges from 18% to 86% [8].


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Perinatal mortality and morbidity are considerably high in the HELLP syndrome and are primarily dependent on the gestational age at onset [36,37]. The perinatal mortality rate related to the HELLP syndrome is between 7.4% and 34% [4,38,39]. Neonates delivered before completion of 32 weeks’ gestation have the highest risk of perinatal death [36,37].

3. Pathogenesis of HELLP and Preeclampsia: The Role of Placenta The exact pathobiology of HELLP syndrome has not been clearly defined. As it is considered a form or a complication of severe preeclampsia, it likely has its origin in aberrant placental development and function resulting in ischemia-producing oxidative stress. An abnormal interaction between maternal and placental tissue at the time of trophoblast implantation is thought to play a key role in the pathogenesis of preeclampsia. In preeclamptic pregnancies, trophoblast invasion of the spiral arteries at the time of placentation is confined to the inner layer of the myometrium so that a low resistance, high flow uteroplacental circulation typical of normal pregnancy cannot be established, and placental ischemia will develop [40,41]. The insufficient trophoblast invasion is believed to be the consequence of the abnormal interaction between decidual immune cells and paternally derived antigens on trophoblasts’ surface [42]. Owing to generalized vasoconstriction, microthrombi formation in small vessels, and plasma volume reduction, blood flow is impaired in every organ and tissue. The maternal vascular endothelium is an early target of placental ischemia, and its activation is responsible for the generalization of damage [43,44]. The clinical manifestations of preeclampsia begin with the loss of vascular refractoriness to vasoconstrictors typical of normal pregnancy, with a subsequent increase in peripheral resistances. The mechanisms underlying the impaired vascular reactivity in preeclampsia are not fully understood, but they might be attributed to the systemic endothelial dysfunction [43,45,46]. The link between abnormal trophoblast invasion, placental ischemia, and maternal vascular endothelium activation is still unclear. Oxidative stress following immune rejection of the trophoblast in the decidua seems to play a key role. The features of endothelial damage in PE are similar to those observed in other diseases in which the impact of oxidative stress is known, such as atherosclerosis, diabetes, septic shock, and ischemia–reperfusion syndrome. In PE, oxidative agents are released into the intervillous spaces by activated leukocytes and the ischemic placenta itself. Several markers of oxidative stress significantly increase during PE, even before the clinical onset of the disease, and endogenous antioxidants sharply decrease [47–49].

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Moreover, the placental release and the activity of angiogenic and endotheliumprotecting agents, such as the ‘‘vascular endothelial growth factor’’ and the ‘‘placental growth factor’’ are deeply impaired [50–53]. HELLP syndrome has many features in common with preeclampsia and can be described as a placenta-induced disease, as is preeclampsia itself, but with a more acute and predominant inflammatory process typically targeting the liver and with a greater activation of the coagulation system. This occurs during a disordered immunologic process and may be due to a genetic predisposition [8,54,55]. There is still a debate on whether HELLP must be considered a severe form of preeclampsia or a separate disease entity. It is well documented that the placenta is a prerequisite for the development of both preeclampsia and HELLP syndrome, and it has been thought that differences in placental gene expression may account for clinical and molecular differences between the two syndromes. In a recent study, Buimer et al. investigated differences in gene expression between placental tissue obtained from normotensive pregnant women and women with preeclampsia or HELLP syndrome [56]. In their study, first, comparison of serial analysis of gene expression profiles of 28 weeks’ control placenta (from idiopathic preterm delivery) to a HELLP/preeclampsia matched for gestational age identified 404 differentially expressed transcripts. Second, using semiquantitative real-time PCR, the expression levels of 37 of these transcripts were analyzed in placentas from normal pregnant women and from patients with HELLP or preeclampsia. Third, nearest centroid classification method determined the HELLP-specific molecular signature consisting of the upregulated expression of genes encoding the vascular endothelial growth factor receptor (Flt1), leptin, pappalysin2, and WW domain containing transcription regulator 1 (WWTR1), combined with downregulated expression of the genes encoding cadherin-associated protein (CTNNAL), glutathione S-transferase-p1 (GSTP1), and calgranulin A (S100A8). Four of these seven genes (Flt1, GSTP1, leptin, and pappalysin2) have been previously associated with preeclampsia, although not specifically to HELLP syndrome [57–60]. The altered expression of WWTR1, CTNNAL1, and S100A8 has not been associated with placental function or dysfunction previously. The authors found that this set of seven genes expression discriminates HELLP placenta from control and preeclamptic placenta with a 24% misclassification rate (95% CI 8.3–41.9), independent from known risk factors like parity and ethnicity. Although it is not known the exact role of the placental expression of these genes in the pathogenesis of preeclampsia and HELLP syndrome, this finding might suggest that HELLP is not a variant of preeclampsia but a separate disease entity. It might be suggested that the abnormal placentation in HELLP syndrome may trigger a specific expression of placental genes different from those activated in preeclampsia, leading to enhanced local and


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systemic inflammatory response and endothelium activation, with a particular involvement of the liver and the coagulation system probably due to maternal predisposition. At this regard, it has been recently observed that the allelic and carrier frequencies of the BclI polymorphism of the glucocorticoid receptor (GR) gene were significantly higher in women with HELLP syndrome compared to healthy pregnant women (p ¼ 0.004, OR 2.89) and to those with severe preeclampsia (p ¼ 0.013, OR 2.56) [61]. Moreover, the BclI carrier status had a significant impact on clinical laboratory parameters of women with HELLP syndrome, as the AST, LDH, and ALP levels were significantly higher, whereas the platelet count tended to be lower in BclI carriers than in noncarriers. There were no significant differences in carrier and allelic frequencies of the N363S and ER22/23EK polymorphisms of GR gene between groups [61]. Previous studies have demonstrated that the BclI polymorphism of GR gene results in increased glucocorticoid sensitivity in vivo and is associated with cardiovascular risk factors and with autoimmune diseases [62,63]. The finding suggests that BclI polymorphism of the GR gene may play a role in the pathogenesis of HELLP syndrome and may account for clinical characteristics of the syndrome, such as EL enzymes, LP count, and sensitivity to corticosteroids. Since preeclampsia and HELLP syndrome develop exclusively in human, it seems particularly interesting that alignment analysis of DNA sequences obtained from database indicated the absence of the BclI site in six animal vertebral species [61]. The difference between HELLP and preeclampsia is underlined also by clinical aspects. For example, women with preeclampsia and no HELLP proved to have more often a profile consistent with the metabolic syndrome. Moreover, preeclamptic patients have a fourfold higher prevalence of thrombophilia as compared to those who had experienced HELLP [64]. Certain maternal features known as risk factors for preeclampsia, such as obesity, have not been associated with the HELLP syndrome [65]. Furthermore, women with preeclampsia differ from those with HELLP by the presence of a smaller placenta with more infarcts, and this data support the view of a longer subclinical disease period preceding preeclampsia as compared to HELLP [66]. Sep et al. postulate that preeclampsia differs from HELLP by a more gradual course in early pregnancy due to unfavorable constitutional conditions for placental growth and development [64]. Eventually, intervillous hypoxia results in the placental release of toxic substances pushing the subclinical condition into the well-known clinical symptomatology. Conversely, an abnormal immune response to the placental allograft with nonappreciable negative impact on placental function is thought to characterize the subclinical phase of HELLP. The acute course and appearance of

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HELLP, with episodic exacerbations together with the sensitivity to corticosteroids, suggest a key role for an immune-mediated and prominent inflammatory response [67].

4. Inflammatory Response in HELLP Syndrome The maternal signs of HELLP syndrome as hypertension, proteinuria, intravascular coagulation, LPs, and hemolysis can all be explained by a systemic inflammatory activity involving a maternal endothelial cell dysfunction. In many cases, the severity of HELLP syndrome fluctuates, resulting in a pattern of exacerbations and remissions. During exacerbations, systemic endothelial activation produces abnormalities due to thrombotic microangiopathic hemolysis, with periportal or focal parenchymal necrosis of hepatocytes [13]. This leads to increased plasma levels of AST and glutathione-Stransferase alpha 1-1 (GSTA1-1), a very sensitive marker for hepatocellular damage [2,68]. Van Runnard Heimel et al. [55] recently observed that during a HELLP exacerbation, plasma levels of C reactive protein were significantly higher than those in normal pregnancy or in preeclampsia and decreased during remission. In HELLP patients, plasma levels of IL-8 and tumor necrosis factor-a (TNF-a) were below the detection limit, as in normal pregnancy and preeclampsia. Plasma levels of IL-1b, IL-10, and sIL-6R did not differ between groups, neither during exacerbations nor during remissions of HELLP. Significant differences were found in IL-6 and IL-1Ra levels. During a HELLP exacerbation, plasma levels of both cytokines were significantly increased as compared with preeclampsia and normal pregnancy. During HELLP exacerbation, median GSTA1-1 levels were significantly higher as compared with preeclampsia and normal pregnancy. The authors concluded that these findings confirm that the development of HELLP syndrome is associated with a further intensified inflammatory response. Moreover, they observed that prednisolone therapy in patients with HELLP syndrome abolished the rise in plasma levels of the cytokine IL-6 during exacerbation. Since the activated vascular endothelium is an important source for circulating IL-6, it may be speculated that corticosteroids act by stabilizing the endothelium in patients with HELLP as previously reported [69–71]. These observations are in agreement with the results of several randomized controlled trials [12] showing a rapid recovery of the platelet count during corticosteroid therapy in patients with HELLP syndrome. This effect may be the result of the decrease of endothelial activation following corticosteroids administration. In the study of van Runnard Heimel et al. [55], prednisolone had no beneficial effect on the liver damage commonly seen in HELLP exacerbation, as reflected by an unaltered course


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of plasma levels of AST, ALT, and GSTA1-1 during prednisolone therapy. A possible explanation of this finding is that prednisolone does not seem to impede the formation of microthrombi, the main cause of hepatic damage. However, a major pathogenic mechanism for liver disease in HELLP syndrome is Fas (APO-1, CD95)-mediated apoptosis of hepatocytes [54]. Fasligand was found to be produced in the placenta. Extracts of placenta were cytotoxic for human hepatocytes and cytotoxic activity increased as HELLP syndrome developed [72]. It is possible that corticosteroid is not able to prevent the cytotoxic activity of placental microparticles on the liver. The role of the placenta in triggering the inflammatory response typical of HELLP syndrome has been recently investigated by Tranquilli et al. [73]. They analyzed the expression of 96 genes involved in inflammatory response in the placenta from women with HELLP syndrome and from healthy women at term and evaluated some cytokines probably involved in important steps of the inflammatory response such as transforming growth factor (TGF)-b, interleukin (IL)-6 Ra, IL-10, IL-16, and CCL-18 and CXCL5 chemokines. Macroarray analysis identified 14 genes encoding differentially expressed cytokines. Gene expression measurement (HELLP vs. healthy) revealed a significant upregulation for IL-10, IL-6 receptor, and TGF-b3 in HELLP placenta, while the expression of CCL18, CXCL5, and IL-16 was significantly downregulated. IL-10 has a powerful anti inflammatory effect. It is important in regulating immune function, by inhibiting macrophages, reducing antigen-specific T-cells proliferation, and diminishing the antigenpresenting capacity of monocytes via downregulation of class II major histocompatibility complex expression [74]. It has also been reported that IL-10 has an important inhibitory role in regulation of T-cell responses and acute inflammation, and it downregulates matrix metalloprotease (MMP)-9 [74]. Its overexpression in HELLP placenta may be regarded as a compensatory mechanism against excessive inflammatory response. In HELLP placenta, the expression of IL-6 receptor (IL-6 Ra) is increased. IL-6, a cytokine normally produced at the maternal–fetal interface, stimulates MMP-2 and MMP-9. IL-6 Ra produces a signal transduction occurring through two pathways: the Ras/mitogen-activated protein kinase (MAPK) pathway and the Jak/Stat pathway, thus making it an important control in the inflammatory response. It is known that dysregulation of IL-6-type cytokine signaling contributes to the onset and maintenance of several inflammatory and autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, osteoporosis, and multiple sclerosis [75]. The TGF-b family is involved in cellular proliferation and differentiation, extracellular matrix modification, tissue remodeling, and angiogenesis. It has been observed that TGF-b3 is overexpressed in preeclamptic placentas, and its inhibition restores the invasive capability of extravillous

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trophoblasts [76]. In HELLP placenta, TGF-b3 was 2.5-fold upregulated as compared to placenta from normal pregnancy, and this suggests an involvement in pathogenic mechanisms of HELLP syndrome. Chemokines are implicated in angiogenesis, cell recruitment, and lymphoid trafficking and can modulate innate and adaptive immune response [77]. The chemokines decrease observed in HELLP placenta may be involved in the derangement of immune activity control at the maternal–fetal interface. Finally, since IL-16 has been linked with modulation of Th2 cell-mediated inflammation in response to allergens [78], the decrease of IL-16 expression in HELLP placenta suggests a role of the cytokine in the immune and inflammatory features of the syndrome. In preeclampsia, there is exacerbation of physiological changes associated with pregnancy such as insulin resistance, altered immune responses, and inflammatory pathway activation. These exaggerated responses seen in preeclampsia are reminiscent of metabolic syndrome and also are evident in gestational diabetes mellitus. Many features of the insulin resistance syndrome have been associated with this condition. These include hypertension, hyperinsulinemia, glucose intolerance, obesity, and lipid abnormalities. Other accompanying abnormalities may include elevated levels of leptin, TNF-a, tissue plasminogen activator, plasminogen activator inhibitor-1 (PAI-1), and testosterone. The documentation of these features before the onset of preeclampsia suggests that insulin resistance or associated abnormalities may have a role in this disorder. Furthermore, the recognition that features of the insulin resistance syndrome persist many years after pregnancy among women with preeclampsia raises the possibility that these women may have increased risk for future cardiovascular disease. These observations suggest that interventions to reduce insulin resistance may reduce the risk of both hypertension in pregnancy and later life cardiovascular complications [79]. The link between insulin resistance and preeclampsia is not clear but novel findings providing some insight have been reported recently. Inositol phosphoglycan P-type (P-IPG) in preeclampsia has been extensively investigated and increased production has been demonstrated. This molecule acts as a second messenger of insulin, enhances the metabolic effects of insulin, and is associated with insulin resistance. An increase in urinary release of P-IPG during pregnancy may herald the onset of preeclampsia. The overexpression of P-IPG during preeclampsia may be a counter-regulatory mechanism to insulin resistance since these molecules mimic insulin action. Further knowledge about the nature of the metabolic syndrome during preeclampsia and the degree of association between its components will help to inform future research efforts. To date, there are no studies specifically aimed to investigate the link between insulin resistance and HELLP syndrome [80].


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5. HELLP Syndrome, Complement Pathway, and the Coagulation System Evidence from the literature supports a role for the complement system in the pathogenesis of preeclampsia/HELLP. Increased levels of the complement alternative pathway (CAP) activation fragment Bb early in pregnancy were associated with the subsequent development of preeclampsia [81]. Complement activation was shown to induce dysregulation of angiogenic factors and to cause fetal rejection and growth restriction in a murine model of spontaneous miscarriage and intrauterine growth restriction [82]. Complement activation products, particularly C5a, stimulate monocytes to produce sVEGFR-1 (sFlt-1) and thereby sequester VEGF [80], and it is known that increased release of sFlt-1 from ischaemic placenta is one of the key pathogenic mechanisms in the development of preeclampsia/HELLP syndrome and in the endothelial activation typical of the diseases [50]. Moreover, it has been observed that some SNPs polymorphisms of VEGF, particularly the VEGF 460TT and the þ 405CC genotypes, may increase the risk for HELLP syndrome [83]. HELLP syndrome shares several clinical and biological features with thrombotic microangiopathy (TMA). TMA is characterized by the occurrence of thrombi in the microvasculature of several organs, leading to thrombocytopenia, mechanical haemolytic anemia, and end-organs failure [84]. TMA may be classified into three main subtypes: ADAMTS-13 deficiency-related TMA, complement dysregulation-related TMA, and indeterminate TMA [85]. Previous studies have shown that HELLP is not associated with a deficiency in ADAMTS-13 activity [86], which is a disintegrin and metalloprotease that cleaves the high molecular weight von Willebrand factor oligomers secreted by endothelial cells, resulting in unusually large multimers that can aggregate to form microvascular platelet thrombi [87]. In a recent study, Fakhouri et al. [88] evaluated the plasma concentration of factor H (FH), factor I, C3, C4, and factor B in 11 patients with HELLP syndrome. They also analyzed the expression of membrane cofactor protein (MCP) and the coding sequences for complement factor H (CFH) and complement factor I (CFI). They identified four patients with a mutation in one of the genes coding for proteins involved in the regulation of the alternative pathway of complement and linked with the C3b activity. Two patients had low C3 and factor B levels, clearly indicating a dysfunctional regulation of the CAP. Although obtained in a very small number of subjects, these findings suggest that an abnormal genetic control of the CAP may be a risk factor for the occurrence of HELLP syndrome and open new fields of investigation.

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Previous reports indicate that HELLP syndrome appears in a more severe form in patients’ carrier of antiphospholipid antibodies, and in those patients it may be refractory to standard treatment leading more frequently to the development of hepatic infarcts [89–91]. The role of the complement system in antiphospholipid antibodies syndrome (APS) has been investigated in murine models [92]. Passive transfer of human antiphospholipid antibodies (aPL) resulted in complement activation and generation of split products that induced placental injury and led to fetal loss/growth restriction [82,93]. The complement activation by aPL in other vascular areas may cause inflammation and thrombosis. Mice deficient in C3 or C5 are less susceptible to aPL-induced thrombosis and endothelial cell activation. Inhibiting C5 activation prevents aPL-induced thrombocytopenia, and mice treated with inhibitors of complement activation are protected from fetal loss [92]. The complement and coagulation system are linked through both direct and indirect interactions, and the vascular endothelium plays a key role in this interaction. The complement increases blood clotting properties. C3a and C5a induce platelet activation and aggregation, upregulation in PAI-1, and tissue factor expression [94,95]. Complement also has thrombogenic properties by decreasing protein S activity [96]. C3a and C5a contribute to the regulation of the cytokine response, modulating production and secretion of TNF-a and IL-6 by macrophages [95], which may enhance TF expression and platelet production and activity. A damage of the endothelial cell lining may activate the complement system and clotting, and the thrombotic tendency of each organ varies depending on the local anticoagulant and procoagulant characteristics. This may explain the tissue specificity of the liver for HELLP syndrome, but the role of complement activation in the pathogenesis of HELLP remains to be established. The coagulation system seems to play a key role in HELLP syndrome. Microthrombi formation, due to endothelial dysfunction and probably to complement system activation, is essential in determining the clinical features of the disease. The fibrinolytic system is necessary for clot dissolution, and in a recent study, Guven et al. investigated the role of fibrinolytic and antifibrinolytic activities in the pathophysiology of HELLP syndrome [97]. They measured the levels of plasma tissue-type plasminogen activator (tPA), thrombin-activatable fibrinolysis inhibitor (TAFI), PAI-1, thrombin– antithrombin complex (TAT), and thrombomodulin (TM) in normal pregnant women, nonpregnant women, and women with HELLP syndrome. Compared to the control groups, the mean tPA, PAI-1, TAFI, TAT, and TM levels were significantly increased in HELLP patients, suggesting an involvement of fibrinolytic system in HELLP, possibly as a response to the activation of coagulation system.


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Moreover, components of fibrinolytic system may be involved in the control of trophoblast invasion and spiral artery remodeling at the time of placentation. It is known that MMPs and their specific inhibitors are involved in this process [98]. The invasive behavior of trophoblast cells correlates with MMP-9 expression, and tissue inhibitor of MMPs (TIMPs) inhibits their invasive capacity. MMP-2 and MMP-9 cooperate closely with the urokinase-type plasminogen activator (uPA) system, mutually activating their components [98]. Synergism of these proteolytic factors in placental development has been shown in animal experiments [99]. PAI-1, expressed in villous and extravillous trophoblasts, as well in maternal decidual tissue, is suggested to be closely involved in control of trophoblast invasion [100]. Recently, Pildner von Steinburg et al. observed that in patients with HELLP syndrome, mRNA placental expression of MMP-2 and TIMP-2 was decreased, and mRNA expression of MMP-9 and uPA receptor was undetectable [101]. These findings, although preliminary, suggest a decrease in matrix remodeling in placentae from patients with HELLP syndrome. Preeclampsia is characterized by an imbalance between two cyclooxygenase metabolites of arachidonic acid, thromboxane, and prostacyclin (PGI2) that favors thromboxane. Because of the biologic actions of these two eicosanoids, this imbalance might explain major clinical symptoms of preeclampsia, such as hypertension, platelet aggregation, and reduced uteroplacental blood flow. In the maternal circulation, this imbalance is primarily manifested by decreased production of PGI2 by endothelial cells. Platelet thromboxane synthesis is only increased in severe preeclampsia. In the placenta and leukocytes, the imbalance is exacerbated by increased production of thromboxane coupled with decreased production of PGI2 in both mild and severe preeclampsia. Longitudinal measurements of urinary metabolites of thromboxane and PGI2 reveal that the thromboxane/PGI2 imbalance predates the onset of clinical symptoms of preeclampsia. The imbalance between thromboxane and PGI2 is most likely caused by oxidative stress, which is manifest in preeclampsia by increased lipid peroxidation and decreased antioxidant protection. Oxidative stress may drive this imbalance because lipid peroxides activate the cyclooxygenase enzyme to increase thromboxane synthesis, but at the same time they inhibit PGI2 synthase to decrease PGI2 synthesis [102]. Furthermore, the endothelial cell dysfunction typical of preeclampsia leads to alterations in the release of vasodilator substances such as nitric oxide (NO), PGI2, and endothelium-derived hyperpolarizing factor, and thereby reductions of the NO-cGMP, PGI2-cAMP, and hyperpolarizing factor vascular relaxation pathways. These alterations may also increase the release of or the vascular reactivity to endothelium-derived contracting factors such as endothelin, thromboxane, and angiotensin II. These contracting factors could increase intracellular Ca2þ concentrations ([Ca2þ]i) and stimulate

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Ca2þ-dependent contraction pathways in vascular smooth muscle. The contracting factors could also increase the activity of vascular protein kinases such as protein kinase C, leading to increased myofilament force sensitivity to [Ca2þ]i and enhancement of smooth muscle contraction. The decreased endothelium-dependent mechanisms of vascular relaxation and the enhanced mechanisms of vascular smooth muscle contraction represent plausible causes of the increased vascular resistance and arterial pressure associated with preeclampsia. It is reasonable to think that these mechanisms are involved in HELLP syndrome, but they are not specific features of HELLP [46].

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HOMOCYSTEINE IN PREGNANCY Michelle M. Murphy*,†,1 and Joan D. Fernandez-Ballart*,† *Unit of Preventive Medicine and Public Health, Faculty of Medicine and Health Sciences, IISPV, Universitat Rovira i Virgili, Tarragona, Spain † ´ n (CB06/03), CIBER Fisiopatologıá de la Obesidad y Nutricio Instituto de Salud Carlos III, Madrid, Spain

1. 2. 3. 4. 5. 6. 7. 8.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Homocysteine Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prophylactic Folic Acid, Folate Status, and Homocysteine . . . . . . . . . . . . . . . . . . . . . . Choline and Homocysteine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nonnutritional Factors Associated with tHcy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Health Benefits of tHcy Lowering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Changes in tHcy During Normal Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Animal/In Vitro Studies and Homocysteine and Reproduction . . . . . . . . . . . . 8.3. Prospective Pregnancy Studies in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4. Elevated tHcy and Subfertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5. Early Pregnancy Loss/Miscarriage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6. Embryo, Placental, and Fetal Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7. Gestational Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8. Other Adverse Pregnancy Outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9. DNA-Methylation and Imprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.10. Long-Term Effects of Exposure of Developing Fetus to Homocysteine . . . . 8.11. Future Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Abstract The aim of this review is to evaluate the evidence for and against fasting plasma total homocysteine (tHcy) as a biomarker/risk factor of impaired reproductive function before and during pregnancy. Apart from nutritional 1

Corresponding author: Michelle M. Murphy, e-mail: [email protected] 105

0065-2423/11 $35.00 DOI: 10.1016/S0065-2423(11)53005-7


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MURPHY AND FERNANDEZ-BALLART

and lifestyle factors, tHcy is also influenced by physiological factors specific to pregnancy such as hemodilution, increased glomerular filtration rate, and endocrinological changes. These lead to a considerable reduction under normal circumstances in tHcy by midpregnancy. Stimulating excess endogenous homocysteine production before and during pregnancy in animal experiments and adding exogenous homocysteine to cell cultures result in the impairment of reproductive and developmental processes from preconception throughout pregnancy and during subsequent development of the offspring. Different studies have confirmed that elevated tHcy is a risk factor for subfertility, congenital developmental defects, preeclampsia, and intrauterine growth retardation. There is conflicting evidence that elevated tHcy is a risk factor for miscarriage, gestational diabetes, premature rupture of the membranes, placental abruption, and offspring with Down syndrome. Prospective, sufficiently powered, studies from preconception/early pregnancy are required to determine whether tHcy is a risk factor for these pregnancy complications.

2. Introduction The investigation of the biological and pharmacological regulation of circulating homocysteine has received considerable interest due to the evidence that associates elevated fasting plasma total homocysteine (tHcy) with increased morbidity and mortality from the earliest stages of life until old age. While this review will focus on pregnancy, regulation of homocysteine metabolism and morbidity associated with alterations in metabolism outside of pregnancy will also be briefly considered because subclinical pathological mechanisms present from before conception may have a considerable influence on pregnancy outcome. Elevated tHcy is often caused by suboptimal status in any of the key micronutrients that regulate it. Thus, it is not easily discerned whether the underlying cause of elevated tHcy or elevated tHcy itself is what contributes to the morbidity and mortality associated with this condition. The evidence for an association between elevated tHcy and pregnancy complications from in vitro, animal, and human studies is reviewed here.

3. Homocysteine Metabolism Homocysteine is formed following transmethylation of the essential sulfurcontaining amino acid, methionine [1]. Homocysteine metabolism is closely regulated by genetic–nutrient interactions and largely dependent on dietary supply of the B-group vitamins: folate, cobalamin, pyridoxine, and riboflavin (to a lesser extent); choline and betaine (Fig. 1) [2]. Suboptimal status or

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HOMOCYSTEINE IN PREGNANCY Glycine THF Serine

Remethylation

5-MTHF Dimethylglycine

BHMT

Choline

Betaine

Transmethylation

MTHFR

Methionine

MTR

MTRR [B2]

B2

5,10-MTHF

B12

Folate cycle

Methionine cycle

S-adenosyl-methionine

S-adenosyl-homocysteine

Homocysteine

Liver and kidney

PO

Serine

N

Homocysteine thiolactone

Cystathionine CgS

As

B6

N tR

Transsulphuration

m

CbS

B6

Cysteine FIG. 1. Simplified diagram of homocysteine metabolism (adapted from Ref. [2]). The roles of key micronutrients (methionine, folate, vitamins B12, B2 and B6, choline, and betaine) are highlighted. THF, tetrahydrofolate; 5,10-MTHF, 5,10-methylenetetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate; MTHFR, 5,10-methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; BHMT, betaine–homocysteine methyltransferase; CbS, cystathionine-b-synthase; CgL, cystathionine-g-lysase; PON, paraoxonase; and mtRNAs, methionyl-tRNA-synthase.

deficiency in any of these micronutrients can lead to disturbance of homocysteine metabolism. In healthy humans, 56% of transmethylated methionine is catabolized through the transsulfuration pathway to cysteine and the remainder is remethylated to methionine [3]. The former pathway is initiated when the pyridoxal phosphate-dependent enzyme, cystathionine-b-synthase (CbS), catalyzes the condensation of homocysteine with serine to form cystathionine. Methyl groups for the remethylation route are provided from the folate cycle during the conversion of 5-methyltetrahydrofolate to tetrahydrofolate. Cobalamin is required to bind the methyl group to methionine synthase (MTR) for which it is a cofactor. Functionality of this enzyme is maintained by the flavoprotein, methionine synthase reductase (MTRR) [4]. The transfer of the methyl group to homocysteine results in the remethylation of homocysteine to methionine. An alternative remethylation route occurs in the liver and kidney when betaine (dietary or the product of choline catabolism) is converted to dimethylglycine by betaine–homocysteine methyltransferase. Both low dietary folate and cobalamin intake lead to elevated tHcy [5]. The extent of the

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contribution of the other nutrients involved in remethylation has been shown to vary in situations of different folate status. When folate stores are replete, cobalamin intake becomes a limiting factor for homocysteine remethylation [6]. When daily folate requirements are not met, high choline intake is associated with lower tHcy [7]. Choline and betaine intake were inversely associated with tHcy in the prefortification with folic acid era in the USA. Postfortification, this association is no longer present [8]. Genetic polymorphisms that affect the role of the enzymes implicated in any of the homocysteine metabolic pathways can lead to increased requirements for the micronutrients involved and eventually to increased tHcy if these requirements are not met. The 677 C!T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene [9] is relatively common in Caucasian, Hispanic, and Chinese ethnic groups [10]. MTHFR plays a key role in the folate cycle. Its flavin adenine dinucleotide (FAD)-binding site accepts and transfers the electrons required for the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. The reduced affinity of the protein for its FAD-binding site leads to increased susceptibility to FAD dissociation and to reduced enzyme activity in the mutant form compared to the wild type [11]. The resulting reduction in 5-methyltetrahydrofolate production limits the supply of methyl groups from the folate cycle for homocysteine remethylation to methionine and enhances the risk of elevated tHcy in situations of low, although not deficient, folate status [12]. However, tHcy is lower in the presence of optimal folate status in all genotypes compared to their counterparts with suboptimal folate status. The enhanced affinity of MTHFR for FAD in the presence of bound folate to the enzyme [11] may maintain/enhance methyltetrahydrofolate’s role in homocysteine remethylation. However, riboflavin (precursor of FAD) status has also been shown to interact with this polymorphism. tHcy is increased when riboflavin deficiency is induced in pregnant mice with mild Mthfr deficiency [13]. An inverse association between riboflavin status and tHcy has been reported in men, in the presence of the mutant MTHFR 677T allele [14–17], and riboflavin supplementation was shown to lower tHcy in men with the 677TT genotype [18].

4. Prophylactic Folic Acid, Folate Status, and Homocysteine The folate cycle remethylation route has received considerable attention in the field of pregnancy research, given the role of prophylactic folic acid in the prevention of neural tube defects (NTDs) [19–21] and the gradual implementation of mandatory and voluntary fortification of flour/staple foods with folic acid in different countries around the world. This policy was pioneered in the USA and the primary aim was to reduce the incidence of NTDs by

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effectively increasing folic acid intake in the target population (women of fertile age) without harming nontarget groups by exposing them to excessive doses of folic acid [22]. Inevitably, the individual effects of exposure to prophylactic folic acid vary depending on genetic constitution and underlying folate status. As expected, however, global folate status in fortified populations has improved and average tHcy is now lower [23]. NTD rates in the USA and Canada have fallen by about 50% compared to prefortification rates [24]. Moderately elevated tHcy has been associated with NTD-affected pregnancies [25] and other pregnancy complications such as recurrent preeclampsia [26,27], intrauterine growth retardation (IUGR) [28], and preterm birth [29]. Elevated amniotic liquid homocysteine concentration has been associated with NTDs [30] and congenital heart defects [31]. Therefore, the benefit of mandatory fortification with folic acid in reducing the risk of pregnancy complications may extend beyond that of NTD prevention.

5. Choline and Homocysteine Recently, more attention has been given to the role of, and importance of, dietary choline in homocysteine metabolism during pregnancy. Choline contains three methyl groups and is required for phospholipid synthesis and neurotransmitter function. Its metabolism is closely interrelated with that of folate. Rats made folate deficient were reported to have a greater dependence on choline for homocysteine remethylation [32]. It has been suggested that the remethylation of homocysteine may also be more dependent on the choline– betaine pathway in situations of inadequate folate intake in women [7] and MTHFR 677TT Mexican–American men with deficient folate status [16]. Inhibition of choline uptake during pregnancy was associated with growth retardation and NTDs in rats [33]. Feeding diets that only met an eighth of choline requirements to Mthfrþ/ mice before and during pregnancy was associated with ventricular septal defects in the offspring [13]. High periconception dietary intake of methionine, betaine, and choline has been associated with less risk of an NTD-affected pregnancy in a study of Californian women [34]. Dietary data relating to the 3 months before conception was collected retrospectively from the mothers on average 4.6–4.9 months after giving birth. Surprisingly, in another Californian study, high maternal serum choline during midpregnancy was associated with increased risk of cleft lip palate (CLP) in the offspring [35]. However, the authors were unable to provide a biological explanation for this observation. They reported that cases with CLP offspring had lower serum methionine and cobalamin than controls. However, it is not clear whether this concurred with high choline levels in the same patients and whether high choline levels were of dietary or endogenous origin. The latter

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might represent a compensatory mechanism to maintain homocysteine remethylation to methionine. Alternatively, high midpregnancy choline may be marking a separate biological process, unrelated to choline status at the time of neural tube formation. Estradiol injection in rats has been shown to stimulate choline synthesis [36]. Plasma choline gradually increases during pregnancy [37–39], possibly to ensure sufficient polyunsaturated fatty acid supply to the fetus [37]. Thus, endocrinologically driven choline synthesis during midpregnancy might confound the association between dietary choline supply and plasma choline concentrations.

6. Nonnutritional Factors Associated with tHcy The previously mentioned genetic-nutritional control of homocysteine regulation has a major influence on tHcy. However, lifestyle and physiological/ endocrinological factors have also been associated with tHcy in large-scale population studies. Age [40] and sex [40,41] are strong determinants. tHcy is also positively associated with serum creatinine [42–44] and inversely associated with bone mineral density in women [42]. Independent associations between smoking, alcohol consumption and caffeine intake [42,44], exercise [42,45], serum insulin [46], and obesity in women [47] have also been reported. Female hormones have been inversely correlated with tHcy outside of pregnancy in both synthetic and endogenous forms. tHcy was reduced following 17 b-estradiol administration in male rats [48] and men [49]. In a randomized control trial, postmenopausal women on hormone replacement therapy were reported to have lower tHcy following 5–7 years of treatment than controls that had received no substitution treatment [50]. Some of the observations concerning endogenous hormonal fluctuations have not involved a direct measurement of circulating hormone levels. However, the coincidence of key endocrinologically driven changes in the woman’s reproductive cycle with changes in tHcy suggests that they are associated. From puberty, tHcy is lower in girls than in boys and remains lower in women throughout their fertile life than in men [41]. tHcy has also been reported to be lower in the luteal than in the follicular phase of the menstrual cycle [51,52] and in fertile than in menopausal women [41].

7. Health Benefits of tHcy Lowering Elevated tHcy is an independent graded risk factor for vascular disease [53], and even moderately elevated tHcy has been associated with increased risk of cardiovascular disease (CVD), osteoporosis, cognitive decline, and history of


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pregnancy complications [42]. Adult tHcy in the range of 5–15 mmol/L is considered normal [54], although reducing it by 1 mmol/L within this range has been associated with reduced CVD risk [53]. Hyperhomocysteinemia has been classified as moderate (15–30 mmol/L), intermediate (30–100 mmol/L), and severe (> 100 mmol/L) [55] outside of pregnancy. Reference ranges, from large-scale studies, during pregnancy are unavailable to date. Considerable effort in the field of homocysteine research in recent years has focused on the investigation of the health benefits of lowering moderately elevated tHcy. However, secondary prevention of disease progression with tHcy-lowering treatment has proved ineffective in studies of recurrence of cardiovascular events reported so far [56–58]. It remains to be shown whether tHcy lowering is an effective measure in the primary prevention of the lesions that it has been associated with. Preconception supplementation with 400 mg/day of folic acid was not shown to reduce the risk of miscarriage in a Canadian study [59]. However, primary prevention of adverse pregnancy outcome with interventions that lead to lower tHcy has been largely successful. Apart from optimal folate status, elevated cobalamin status has also been shown to offer a considerable degree of protection against NTDs [60]. While this inevitably leads to lowering of tHcy, it is not clear how this contributes, if at all, to the observed protection. Reported regular multivitamin or prenatal supplement use (of unknown composition) from before pregnancy throughout the periconception period was associated with a 45% reduction in risk of preeclampsia [61]. Another prospective cohort study reported a 63% reduction in risk of preeclampsia in women who reported taking high-dose folic acid supplements (1 mg/day) for varying lengths of time from preconception until the beginning of the second trimester of pregnancy [62]. The results from both of these studies need to be confirmed in randomized controlled intervention trials.

8. Pregnancy 8.1. CHANGES IN THCY DURING NORMAL PREGNANCY Pregnancy tHcy is affected by the same underlying nutrient-genetic and lifestyle factors as in the nonpregnant state. However, the considerable variation in tHcy that occurs throughout pregnancy may be as a result of a physiological adaptation to this condition. Longitudinal studies have shown that maternal tHcy is substantially reduced during the first two trimesters of pregnancy compared to preconception concentrations [63,64]. The decrease is only partially explained by folic acid supplement use [63]. Physiological factors such as hemodilution, albumin decline, and enhanced

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renal function all contribute to the decline in tHcy [63–65]. Pregnancy hormones are also likely to have an important influence, given that the decrease in tHcy that occurs between preconception (geometric mean [SD]: 8.2 [1.3] mmol/L) and midpregnancy (5.2 [1.3] mmol/L) cannot be explained by folic acid supplementation or by these physiological effects of pregnancy alone [63]. By 6.5–8 weeks of pregnancy, before the effects of hemodilution and placental hormone production and in the absence of folic acid supplement use, tHcy was already on average 11.5% lower than at preconception. This decrease could be associated with hCG production during early pregnancy, although to the best of our knowledge this has not been investigated. A study of patients receiving ovarian stimulation treatment reported reduced tHcy compared to day 2 (median [min, max]: 9.1 [5.0, 75.3] mmol/L) of the menstrual cycle following hCG administration (8.4 [4.3, 71.6] mmol/L) [66]. However, from day 2 and prior to treatment with hCG, patients had also received follicle-stimulating hormone for a number of days. tHcy has been previously reported to be lower in the luteal than in the follicular phase of the menstrual cycle [51,52]. Subsequently, other pregnancy hormones may also further reduce tHcy until midpregnancy. The decrease that occurs during pregnancy is significantly correlated with the coinciding increase in estradiol [67]. During the last trimester of pregnancy, tHcy returns to similar concentrations observed at preconception in mothers that do not use folic acid containing supplements during the second and third trimesters [68]. This late pregnancy increase in tHcy occurs despite the continuing influence of hemodilution and increased glomerular filtration rate. Other researchers have also observed the increase in tHcy toward the end of pregnancy and attributed it to low folate status [69]. While low folate status will inevitably contribute to the rise in tHcy, late pregnancy tHcy also rises, though not to the same extent, in folic acid supplemented mothers [68]. Either the supplementation regime is not sufficient to maintain adequate folate status or the rise is independent of folate status. The transsulfuration and remethylation pathways have been reported to dominate homocysteine metabolism to different extents depending on the time of pregnancy. A study of methionine kinetics during human pregnancy reported a higher rate of transsulfuration during the first trimester compared to the last and higher remethylation and transmethylation rates in the last trimester compared to the first [70]. These differences were suggested to be due to the dependence of the fetus on maternal cysteine supply in early pregnancy and to the increased methionine requirements of the rapidly developing fetus in late pregnancy. A study of amniotic fluid samples from pregnant mothers collected between 13 and 18 weeks gestation showed that methionine concentrations gradually decreased and that homocysteine concentrations gradually tended to increase during this time frame [71]. Regardless of the cause, tHcy increases within the normal range during late


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pregnancy with normal evolution and outcome. An in vitro study has shown that incubating human myometrium with homocysteine increased its contractility [72]. It remains to be investigated whether the increase in homocysteine during late pregnancy contributes to the onset of uterine contractions in preparation for birth. 8.2. ANIMAL/IN VITRO STUDIES AND HOMOCYSTEINE AND REPRODUCTION There is varied and conflicting evidence in the literature regarding the capacity of moderately elevated tHcy to cause the diseases with which it has been associated. A number of animal and in vitro experiments have been designed to investigate the effects of elevated tHcy from before conception and throughout pregnancy on reproduction and on development in the offspring. These studies have provided valuable information on the biological and clinical effects of provoked endogenous tHcy excess and of exposure to pharmacological doses of homocysteine. However, the challenge remains to interpret the relevance of the evidence provided from acute exposure to supraphysiological homocysteine concentrations in controlled experiments, in the context of chronic exposure to concentrations in the physiological range, and in a free-living environment in humans. The difference in tHcy between the Mthfrþ/ and wild types of the MTHFR knockout mouse is suggested to be analogous to that observed between MTHFR 677TT and 677CC individuals [73]. Inducing folate deficiency and hyperhomocysteinemia (threefold increase in mean tHcy compared to folate replete animals) in mice before mating was associated with fetal resorptions, decreased fetal weight, and developmental delays [74]. Mthfrþ/ female mice fed folic acid-deficient diets before and throughout pregnancy had a lower number of corpora lutea in the ovaries. Pregnancies were affected by different embryonic defects, greater fetal loss, IUGR, and severe placental defects [75,76]. The mean maternal tHcy, during pregnancy, was 70 mmol/L compared to 50 mmol/L in the 677TT model on folic aciddeficient and control diets, respectively [76]. Similarly, inducing choline or riboflavin deficiency in Mthfrþ/ mice was associated with hyperhomocysteinemia during pregnancy (mean tHcy: 40 and 90 mmol/L, respectively, compared to Mthfrþ/ fed on control diets, 38 mmol/L) [13]. There was a greater incidence of embryonic delays and reduced embryonic growth in the offspring of Mthfrþ/ mice that had been fed diets containing a sixth of riboflavin requirements before and during pregnancy. Riboflavin-deficient diets were also associated with reduced left ventricular wall thickness in the offspring. Both riboflavin and choline deficiency (induced by feeding oneeighth of daily choline requirements) were associated with ventricular septal

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defects. Despite being exposed to higher tHcy concentrations in the study of induced choline deficiency than in the study of induced riboflavin deficiency, overall the offspring from the latter experiment had more developmental problems. These results suggest that the combined exposure to the enzyme defect and insufficient riboflavin was more harmful than the elevation in tHcy produced by choline deficiency in animals with the same enzyme defects but adequate riboflavin supply. Nevertheless, the tHcy ranges reported in all of these animal studies would correspond with intermediate hyperhomocysteinemia in nonpregnant humans. While reference ranges have yet to be defined for human pregnancy, such concentrations are considerably higher than those reported in previous pregnancy studies with both normal and pathological outcomes [26,27,39,63,64,68–70]. Although the consequences of the virtual eradication of a single micronutrient from the diet can clearly be attributed to deficiency of that single vitamin under experimental conditions, it is difficult to reproduce free-living conditions in humans in which variation in supply of multiple micronutrients and in lifestyle habits that also affect tHcy may occur at any given moment. The incorporation of homocysteine into protein and tRNA is prevented by the production of the highly reactive thioester, homocysteine thiolactone (HTL) by methionyl-tRNA synthetase during the amino acid editing process of protein synthesis [77,78]. HTL has been reported to be capable of damaging proteins through homocysteinylation of protein lysine residues [79]. High intracellular HTL concentrations (0.25 pmol/105 cells) have been reported in cells cultured in medium containing homocysteine but lacking in cobalamin and folate [80]. However, reported serum HTL concentrations ranged from being undetectable, in half of the healthy adult men studied, to 0.38 nM [81]. Incubation with either HTL or homocysteine during gestational days (GD) 0–2 caused NTDs and congenital heart defects in chick embryos [82]. Later incubation (GD 8.5) caused growth retardation, blisters, and somite development abnormalities in mouse embryos [83]. No NTDs were observed either in this study or in a separate study of mouse embryos in which incubation was with homocysteine (1.3 mM) or HTL (0.65 mM) on GD 8 for 44 h [84]. Incubation of cultured human placental trophoblasts with HTL caused apoptosis [85]. Homozygous MTHFR knockout mice have reduced survival or delayed development and cerebellar abnormalities. Older heterozygous and homozygous knockouts have abnormal aortic lipid deposition and also elevated tHcy and altered S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH) levels. These latter abnormalities were associated with DNA hypomethylation in the brain and ovaries [73]. Inducing hyperhomocysteinemia in pregnant rats (26 mM compared to 6 mM in controls by the end of pregnancy) by including 1 g/kg body weight of methionine in their daily drinking water was associated with


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increased lipid peroxidation (indicative of oxidative stress) and apoptosis in the offsprings’ brains [86]. The authors suggested that the apoptosis may have been caused by the oxidative stress and that the fetal brain is more susceptible to oxidative stress due to its less developed antioxidant defense mechanisms. No information is provided regarding the diet composition. On the assumption that daily folate, cobalamin, and other micronutrient requirements were met, this report provides evidence of a potential association between elevated homocysteine and impaired brain development that is not marking folate or cobalamin deficiency. Other studies have shown that fetal exposure to hyperhomocysteinemia during pregnancy was associated with lasting effects in the offspring. Hyperhomocysteinemia induced by feeding pregnant dams methyl-deficient diets was associated with impaired motor and cognitive development in rat pups [87]. Feeding methyl rich diets to the newborn rat pups, as a secondary preventive measure, did not reverse the cognitive impairment associated with regions of the brain in which homocysteine had accumulated during development. Homocysteine was shown to have accumulated in neurons and astrocytes of the hippocampus, cerebellum, striatum, and subventricular zone of the brain. tHcy concentrations in the rat pups were considered to be comparable with mild hyperhomocysteinemia (13 4 mmol/L). Fetal exposure to hyperhomocysteinemia from early pregnancy, reaching a sustained tHcy concentration of 33 3.9 mmol/L, was also associated with impaired cognitive function in the offspring of rats [88]. The hyperhomocysteinemia was induced and maintained by feeding with a very high methionine content diet. The IUGR and impaired postnatal growth observed in the offspring of rats fed methyl donor-deficient diets from preconception throughout pregnancy and lactation were proposed to be due to dysfunction of the gastric ghrelin cell system caused by the remodeling of gastric cell organization [89]. Maternal hyperhomocysteinemia (mean tHcy: 16.4 1.5 mmol/L), induced by feeding rats drinking water containing homocysteine from preconception throughout pregnancy, was associated with a lower number of live births in the intervention than the control group (mean tHcy: 4.7 1.7 mmol/L) and with impaired ossification in the offspring [90]. This effect was despite ensuring that dietary folic acid and pyridoxine supplies were sufficient to meet pregnancy requirements. Whereas the involvement of folate metabolism and homocysteine in agingrelated diseases, several developmental abnormalities, and pregnancy complications has given rise to a large amount of scientific work, the role of these biochemical factors in the earlier stages of mammalian reproduction and the possible preventive effects of folate supplementation on fertility have, until recently, been much less investigated.

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8.3. PROSPECTIVE PREGNANCY STUDIES IN HUMANS The advantage of prospective studies is their enhanced reliability because they are less vulnerable to bias due to the effect of disease on tHcy (‘‘reverse causality’’) and because they control for confounding from established risk factors. Cohorts of women followed from preconception and broadly representative of the population are generally very difficult to collect by virtue of the fact that they require synchronized planning of the pregnancy by the mother and the investigating team. Preconception blood samples were collected from a cohort of 423 Chinese textile industry workers that were planning on becoming pregnant [29,91]. The time between sample collection and the index pregnancy varied by up to 1 year. Information regarding the association between preconception tHcy and gestation length and outcome was obtained from this cohort. Another longitudinal study from preconception throughout pregnancy was carried out in Spain (PREC study, 1992–1996 [63]). Couples planning a pregnancy were invited to do so with the investigating team and blood sample collection was carefully timed to be during the periovulationary phase of the menstrual cycle (conception occurred during this cycle or a maximum of 2 cycles later), at 6–8, 20, and 32 weeks of pregnancy, during labor and from the cord. Although this study was from a relatively small cohort of 93 mothers of medium–high socioeconomic status, its strength is that each woman acted as her own baseline, nonpregnant control and blood samples representative of each phase of pregnancy were collected [63,65,66,92]. Another prospective pregnancy study included the collection of a blood sample during the first trimester, enabling the investigation of early pregnancy tHcy with pregnancy evolution and outcome [69]. The large-scale Child Health and Development nested case–control study collected prenatal blood samples from a cohort of 19,044 pregnant women that received prenatal care in Northern California (1959–1966) and from 12,094 of the offspring at follow-up between 1981 and 1997. This study investigated the association between pregnancy tHcy and schizophrenia in the offspring [93]. The role of homocysteine on pregnancy outcome and development from preconception through to adulthood has been investigated to differing extents throughout the life cycle. Although no study to date has investigated homocysteine and health throughout the life cycle from preconception to old age in the same subjects, different studies have led to evidence covering this topic at all stages of development (Table 1).

8.4. ELEVATED THCY AND SUBFERTILITY In a study of 156 subfertile couples, spermatozoa homocysteine concentration was higher in men with male factor subfertility (58.8 pmol/million cells) and idiopathic subfertility (20.9 pmol/million cells) than in fertile men


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(12.4 pmol/million cells). Follicular fluid homocysteine concentration was higher in women with endometriosis (18.8 nmol/mg protein) compared to patients with unexplained infertility (9.2 nmol/mg protein) and fertile women (12.4 nmol/mg protein) [94]. Both seminal and follicular fluid homocysteine concentrations were reported to be negatively correlated with embryo quality. Increasing tHcy by 1 mmol/L in ejaculate and follicular fluid was associated with 19% and 58% reductions, respectively, in embryo quality. A limitation of this study is the lack of information provided on the suitability of the controls, on lifestyle factors known to be associated with subfertility such as smoking, and on the validity of the regression models used (explicative capacity of the model, significance of the model, number of patients included in the model, nonexistence of colinearity between independent variables, etc.). In a separate study, hyperhomocysteinemic (tHcy > 17 mmol/L) in vitro fertilization (IVF) patients were randomly assigned to tHcy-lowering treated and untreated groups. Increased implantation and pregnancy rates were observed in the treated group in which tHcy was reduced to 18.3mmol/L) and/or postmethionine load hcy > 61.5mmol/L compared to those with normal tHcy (< P95 for each of these measurements based on healthy subjects) in a study in the Netherlands. tHcy was inversely correlated with vascular element perimeter in this study [98]. Pregnancy loss was not attributable to conventional causes such as chromosomal rearrangements, severe uterine anomalies, antiphospholipid antibodies, and thyroid dysfunction in any of these patients. Low folate status at the time of miscarriage diagnosis was associated with an increased risk of miscarriage in a case–control study in Sweden [99]. However, tHcy was not measured in this study. Elevated tHcy was associated with increased risk of miscarriage ( 9.9 mmol/L: twofold; 12.3 mmol/L: fourfold; 15.3 mmol/L: sevenfold) in a case–control study in France that compared 743 women that had miscarried between gestational weeks 8 and 9 with 743 controls that had an elected termination of pregnancy for the first time [100]. Cases and controls were matched for age, number of pregnancies, and time elapsed since miscarriage. In a separate study in Syria, in which pregnancy was confirmed by hCG presence or by ultrasound scan, miscarriage cases (N ¼ 43) were shown to have lower cobalamin status than pregnant controls (N ¼ 32) [101]. Blood samples were collected at the time of miscarriage diagnosis and controls were matched with cases by gestational age. Although tHcy tended to be higher in cases than in controls, it was not significantly so. Despite the observed trends, it is not clear that there were sufficient controls, and although not significantly so, more cases were smokers than controls. Pregnant mothers with tHcy P90 (based on pregnant control reference concentrations) were twice as likely to have a miscarriage (OR: 2.1 [95% CI: 1.2, 3.6]) in a study of 103 miscarriage cases and 1077 controls [102]. However, although tHcy was determined during the first 20 weeks of pregnancy, it was not known if the miscarriage had occurred before or after the blood sample was collected. Elevated tHcy ( P95) in both mothers (14.0 mmol/L) and fathers (19.6 mmol/L) was associated with fivefold and sevenfold increases in risks of idiopathic recurrent pregnancy loss compared to controls in an Indian study [103]. However, no information was provided regarding the time that had passed between sample collection and the last pregnancy loss. A study of the potential causes of repeated pregnancy loss in data collected from 1020 women in Tennessee at least 6 weeks after a miscarriage event showed that among other possible unconventional causes of miscarriage, tHcy was elevated (>14mmol/L) in 14% of the women [104]. However, the relevance of this finding is not clear, given that there were no controls in this study. Poor pyridoxine status in baseline blood samples (collected before ending contraception with the intention to become pregnant) from 364 women in Anhui, China, was associated with greater risk of miscarriage in a prospective study in which each of conception, pregnancy

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(lasting at least 6 weeks after the onset of the last menstrual period), and early pregnancy loss (pregnancy lasting for less than 6 weeks) were confirmed by urinary hCG detection [91]. However, neither being in the highest tHcy quartile nor having tHcy 12.4 mmol/L was associated with increased risk of pregnancy loss. 8.6. EMBRYO, PLACENTAL, AND FETAL DEVELOPMENT 8.6.1. Neural Tube Defects Apart from the different studies that have associated low maternal folate and/or cobalamin status with risk of an NTD-affected pregnancy, other studies have also reported higher tHcy in mothers with a history of NTDs in their offspring than in control mothers with previous pregnancies unaffected by NTDs [105–107]. Early pregnancy mean tHcy was higher in pregnancies that went on to be affected by NTDs [8.6 2.8 mmol/L] compared to normal pregnancies [7.9 2.5 mmol/L] [25]. Higher amniotic fluid homocysteine concentration was reported in NTD case pregnancies (2.6 1.6 mmol/L) than in controls (1.5 0.4 mmol/L) [30]. Another study reported a higher proportion of amniotic fluid homocysteine concentrations > 1.85 mmol/L (P90 in controls) in pregnancies affected by NTDs compared to unaffected control pregnancies [108]. Analysis of blood samples collected prior to elective termination of an NTD-affected pregnancy showed that tHcy was higher in these pregnancies (mean [min–max] 6.3 [4.8–8.2] mmol/L) than in unaffected pregnancies (mean [min–max] 5.8 [4.6–7.2] mmol/L) [109]. Children with NTDs have also been reported to have higher tHcy (median [range]: 10.0 [4.8–24.3] mmol/L) than control children (4.5 [1.99.6] mmol/L) [110]. 8.6.2. Congenital Heart Defects Elevated maternal tHcy has also been associated with congenital heart defects in the offspring. Median tHcy (3–6 months after giving birth) was higher in mothers (11.9 mmol/L) of children with congenital heart defects than in control mothers (9.4 mmol/L) of unaffected children [111]. Case mothers with tHcy above P90 in controls (13.0 mmol/L) were five times more likely to have a child with a congenital heart defect. This observation was confirmed in subsequent studies in which postpregnancy blood samples were also collected from case and control mothers. Case mothers selected from a birth defects registry and controls (mothers of live births, free of defects) selected from a birth certificate registry were studied [112]. Elevated tHcy (greater than P70 in control mothers) was associated with a greater risk of previously affected pregnancies. Based on tHcy in control mothers, there was a dose–response relationship between increasing tHcy from P70


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(8.59 mmol/L) upward in cases and risk of previously affected pregnancy. Case mothers with tHcy at P70 were four times and those with tHcy at P95 (10.72 mmol/L) were 12 times more likely to have a history of affected pregnancy. This study also reported that more case mothers were smokers than controls. Subsequently, it was reported from the same study that a history of smoking at periconception combined with elevated postpregnancy tHcy (> P75 in control mothers; > 8.59 mmol/L) was associated with a 12-fold increase in risk of history of an affected pregnancy [113]. Another study also confirmed that mothers with hyperhomocysteinemia (tHcy > 14.3 mmol/L), determined 17 months after pregnancy, were almost three times more likely to have had a pregnancy affected by congenital heart defects than normohomocysteinemic mothers [114]. In each of these studies, tHcy was determined after the diagnosis of the complication and, in some cases, a considerable time after the affected pregnancy was over. One study reported that elevated amniotic fluid homocysteine concentration was associated with increased risk of carrying a fetus with an isolated nonsyndromic congenital heart defect [31]. 8.6.3. Placental Development and Function MRNA for both methionine synthase and for 5,10-MTHFR is highly expressed in the human placenta during both early and late pregnancy while expression for CbS and betaine–homocysteine methyltransferase is low and undetectable, respectively [115]. This suggests a high folate requirement for placental homocysteine metabolism. It has recently been shown that homocysteine can cross the human placenta from mother to fetus [116]. The authors propose that this is potentially harmful to placental function and fetal development for various reasons. With regard to placental function, the presence of homocysteine in the syncitiotrophoblast membrane of the placenta could potentially lead to alterations in placental metabolism, vascular function, and induction of apoptosis. Development might be impaired in a fetus deprived of essential amino acids due to competition with homocysteine for transport. However, different studies of uncomplicated pregnancies in healthy women have reported that maternal tHcy at birth is higher than umbilical cord tHcy. Mean maternal tHcy at birth was reported to be 5.4 1.4 mmol/L compared to 4.5 1.8 mmol/L in 35 mother–cord pairs [117]. The authors reported a descending concentration gradient from maternal vein to umbilical vein to umbilical artery. Two subsequent studies confirmed that maternal tHcy at birth is higher than in the cord in larger groups of mother–cord pairs. Mean maternal tHcy at birth was reported to be 8.3 2.9 mmol/L compared to 7.9 2.9 mmol/L in 201 mother–cord pairs in an Irish study [118]. In this study, maternal tHcy was found to be the greatest predictor of cord tHcy. In the PREC study, we observed that maternal tHcy at birth was higher than

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cord in mother–cord pairs that were both supplemented (mean [95% CI]: 6.6 [8.0, 9.1] mmol/L compared to 5.4 [7.7, 9.4] mmol/L in 37 mother–cord pairs) and unsupplemented (8.6 [5.9, 7.3] mmol/L compared to 6.8 [6.0, 7.6] mmol/L in 47 mother–cord pairs) with folic acid during late pregnancy [68]. 8.6.4. Placental Vascularization and Preeclampsia Optimum placental development depends on trophoblast invasion of maternal uterine vessels with resulting changes in their anatomical characteristics from narrow muscular to wide nonmuscular vessels. This enables high flux and low resistance utero-placental blood flow that facilitates the transfer of nutrients across the placenta. Conceivably, in women with underlying undiagnosed pathological vascular conditions, these anatomical adaptations to pregnancy may be impaired. Preeclampsia is a pregnancy-specific disorder that develops after 20 weeks gestation [119]. The placenta appears to be fundamental in the development of preeclampsia, and abnormal vascularization of the placenta is often present [120]. A number of studies have reported that pregnancy tHcy is higher in preeclampsia patients than in controls before the onset of the pregnancy complication and that having elevated tHcy during the first half of pregnancy increases the risk of preeclampsia [26–28,100,121] (Table 2). However, this was not confirmed in a Finnish study in which timing of sample collection was also prior to preeclampsia diagnosis [122]. The number of preeclampsia cases was less in this study. While two of the studies that found an association between elevated early pregnancy tHcy and risk of preeclampsia reported that smoking habits did not differ between cases and controls [28,120], smoking was not reported in the Finnish study. The conclusions regarding the association between tHcy and preeclampsia differ among some studies in which preeclampsia was diagnosed before sample collection for tHcy determination. The main difference between the studies that reported similar tHcy in preeclampsia cases and controls or that found no increased risk associated with elevated tHcy was the timing in blood sample collection. Seven studies in which blood samples were collected after 30 weeks gestation reported elevated tHcy in cases compared to controls [64,123–128]. Three studies that investigated the association between tHcy determined in blood samples collected between 22 and 26 weeks gestation reported no difference between cases and controls [125,129,130]. Interestingly, Hogg et al. [125] reported no difference at 26 weeks but did report a difference at 37 weeks in the same group of patients. Thus, the timing of sample collection appears to be an important factor. The association may be masked when tHcy concentrations are at their lowest during midpregnancy, possibly due to hormonal influences. The weight of the evidence suggests that the association between elevated tHcy and preeclampsia is detectable in early and late pregnancy blood samples. Other studies have also


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reported a dose–response relationship with higher tHcy in cases of severe preeclampsia than mild [126,128]. Some studies have combined the use of tHcy determination with Doppler imaging to investigate their predictive capacity of preeclampsia and IUGR (pregnancy complications associated with impaired placental function). Doppler imaging analysis can be used to obtain information on placental vascularization through the observation of uterine artery waveforms and the registration of the pulsatility index of blood flow (indicative of degree of resistance). No difference in median midpregnancy tHcy (22–24 weeks) was observed between 275 patients with abnormal Dopplers (5.6 mmol/L) and 408 controls (5.4 mmol/L) in a UK study [129]. However, in a prospective study in which tHcy was determined in blood samples collected between 15 and 19 weeks of pregnancy, the combination of abnormal Doppler results with tHcy > P95 (6.3 mmol/L) had higher predictive capacity of a preeclampsia outcome than either elevated tHcy or Doppler alone [28]. The former study provided no information on folic acid supplement use. In the latter, folic acid users after 12 weeks of pregnancy were excluded. However, it is not stated what percentage used supplements during the first trimester. It is not stated in either study whether the blood samples were processed according to the guidelines to prevent artifacts in tHcy determinations [131]. 8.6.5. Fetal Size and Intrauterine Growth Retardation Different studies have investigated the association between elevated maternal tHcy and fetal growth. There is evidence for and against a negative association between maternal tHcy and birth weight. In a large study of 5883 Norwegian women, those with tHcy in the top quartile (10.7–78 mmol/L) were more likely to have previously had a child with very low birth weight compared to those in the lowest quartile (3.6–7.5 mmol/L) [OR: 2.01; 95% CI:1.23, 3.27] [132]. Preconception tHcy 12.4 mmol/L was not associated with increased risk of low birth weight in a study of 423 Chinese mothers [29]. Preconception samples were collected at a routine medical checkup in women that had obtained the necessary governmental approval to become pregnant. A case–control study of 483 mothers that gave birth to children with IUGR (birth weight < P10) and 468 control mothers reported that increasing postpartum (up to 48 h) maternal tHcy by 5 mmol/L was associated with less probability of having had a pregnancy affected by IUGR [133]. tHcy in this study varied from 1.42 to 18.32 mmol/L. In our prospective study in Spanish women, moderately elevated maternal tHcy ( 7.1 mmol/L) at 8 weeks gestation was associated with a threefold increased risk of reduced birth weight in the offspring [68]. In another study, amniotic fluid homocysteine concentrations were higher in pregnancies with a small for gestational age outcome (N ¼ 39; 1.31 [1.64] mM) than in those that resulted in adequate size for gestational age at birth (N ¼ 393; 1.02 [0.55] mM) [134]. In a separate study,

TABLE 2 ASSOCIATION OF PREGNANCY THCY WITH PREECLAMPSIA RISKa Plasma tHcy (mol/L) mean (SD)/P50; [N] Design (year)

Location

Time of sample (gestation week)

Case

Control

OR (95% CI)b

tHcy cutoff (mol/L)

Befored (1999) [120] (2001) [26] (2001) [122] (2003) [27] (2006) [28]

USA Ireland Finland Ireland Turkey

15–22 (16.5 1.5) 15.3 4.0; 14.9 3.4 16 15.9 3.6; 15.6 3.4 16.2 3.3; 16.3 3.2

[52] 9.8 (3.3) [56] 7.0 (1.6) [34] 8.4 (2.4) [71] P50: 7.1 [32]

[56] 8.4 (1.9) [112] 6.9 (1.8) [68] 7.1(1.5)[142] P50: 5.0 [324]

3.2 (1.1–9.2) 2.8 (1.4–5.9) 1.6 (0.6–4.4) 4.1(1.4–12.6) 13.8 (3.1–26.9)

5.5 > 10 > 7.7 7.8 6.3

(2008) [102]

Canada

4–20 weeks

[65]

[1707]

2.7 (1.4–5.0)i

7.05–5.71j

Labor Labor 26 37

8.7 (3.1) [20] 9.7 (5.2) [20] 5.2 (1.3) [16] 6.6 (2.1) [46]

5.0 (1.1) [20] 7.2 (2.3) [32] 4.6 (1.4) [409] 5.3 (1.7) [409]

– – – –

(2004) [129] (2004) [64] (2004) [126]

USA USA USA (African American) UK New Mexico Turkey

22–24 30–33 33

P50: 5.5 [586] 3.2 (0.2) [24] 6.1 (1.6) [25]

(2007) [127] (2009) [130] (2009) [128]

Norway Canada China

Term 24–26 ‘‘3rd trimester’’

P50: 5.4 [80] 4.4 (0.6) [15] 10.5 (2.5) [12] 11.2 (3.6) [46] P50: 8.2 [47] 3.4 (0.9) [113] 8.2 (0.8) [24] 10.9 (2.5) [62] 12.6 (1.4) [38]

P50: 6.4 [51] 3.7 (0.9) [443] 6.0 (0.6) [30]

Folate statusc

Smokingc

BMIc

–e NSg – NS Sup users exclh Suppk

Yes – – – NS

Higherf – – – NS

–

–

– – – –

NS NS NS

– – –

NS NS –

– – –

– – –

– – –

– – –

– – –

1.5 (1.0–2.3) 0.7 (0.3–1.3) –

– > 11.0 –

NS Supp ?n

– Lowerm NS

Higher Higher –

d

After (1997) [123]l (1998) [124] (2000) [125]

a Only studies reporting pregnancy tHcy concentrations, gestational age at the time of blood sampling, and with samples collected from complication-free control pregnant patients of similar gestational age at time of sampling as cases are shown. b Risk of preeclampsia in cases compared to controls. c Reported/considered in the study. d Timing of sample collection with respect to diagnosis of preeclampsia. e Information not provided. f Higher in cases. g No significant difference between cases and controls. h Supplement users excluded. i Relative risk. j P90 was calculated in tHcy adjusted for gestational age in weeks for each 2-week interval from 4–5 to 18–20 weeks of pregnancy. tHcy was lower in samples taken later in pregnancy. k Used folic acid supplements. l Time of sample collection: cases ¼ 35 4 weeks, controls ¼ 40 1 week. m Fewer controls were smokers than cases. n States that ‘‘none were known to be supplement users’’; however, neither were patients specifically asked nor was folate status compared between cases and controls.

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maternal tHcy was reported not to differ between 12 pregnancies affected by IUGR (at 30.1 3.5 gestational weeks) and 8 unaffected control pregnancies (at 25.6 6.1 gestational weeks). However, 44.7 14.8% of total albumin was cysteinylated compared to 20.9 6.1% in control pregnancies [135]. 8.7. GESTATIONAL DIABETES tHcy has been shown to vary among different categories of response to glucose loading and tolerance tests performed at 24–28 weeks gestation. In the studies reported to date, tHcy was determined during the same gestational time frame as the glucose metabolism test. tHcy was reported to be higher in women that were diagnosed as glucose intolerant following an oral glucose load of 50 g, compared to women with normal responses [136]. tHcy was also reported to be higher in women that were diagnosed with gestational diabetes following a glucose tolerance test (response to oral load of 100 g of glucose) [137,138]. In the majority of these studies, women with abnormal glucose metabolism were older than the controls that had a normal response to the glucose tolerance tests and in some, their BMI was also higher. Smokers were excluded in some studies, but others do not provide any information regarding this factor. In the normotensive group of control women from a preeclampsia study, glucose intolerant patients had lower tHcy compared to normal controls and there was no difference between tHcy in gestational diabetes patients and controls [139]. Age is not compared between groups and smoking is not considered in this latter study. 8.8. OTHER ADVERSE PREGNANCY OUTCOMES The association between elevated maternal tHcy and presence of or history of a pregnancy affected by premature rupture of membranes (PROM), placental abruption, preterm delivery, and Down syndrome has also been investigated. In a study of PROM, no difference was observed in tHcy measured during weeks 32–35 of pregnancy between cases and controls and having tHcy > P95 (concentration unspecified) did not significantly increase the risk of PROM [140]. It was also reported in this study that cases were more likely to smoke and to have BMI ( 12.4 mmol/L at preconception [91] and above the median or in the highest quartile during the second–third trimester of pregnancy was associated with increased risk of preterm birth [145]. The conclusions regarding the association between elevated maternal tHcy and Down syndrome are conflicting. A prospective case–control study, in which prenatal blood samples were collected, reported no difference in tHcy in 48 cases with Down syndrome affected pregnancies and 192 controls [146]. However, there is considerable diversity in study design, selection of cases and controls, timing of blood sample collection, and adjustment of the analysis for confounding variables among the studies reported to date. The evidence available has been reviewed recently [147]. A number of studies investigating the possible contribution of polymorphisms affecting folate/ cobalamin and homocysteine metabolism to Down syndrome have also been considered in the same review.

8.9. DNA-METHYLATION AND IMPRINTING DNA methylation in the earliest stages of development has a direct influence on gene imprinting in the offspring and is dependent on methyl donor status, of which tHcy is a biomarker. DNA methylation capacity leads to hyper- or hypomethylation of DNA. The balance between each of these situations plays an important role in gene imprinting. An inverse correlation between cord tHcy and LINE1-DNA methylation (indicative of global DNA methylation level) was recently reported in humans [148].

8.10. LONG-TERM EFFECTS OF EXPOSURE OF DEVELOPING FETUS TO HOMOCYSTEINE Apart from congenital defects that are diagnosed in utero or at birth in the offspring of mothers with elevated tHcy during pregnancy, fetal exposure to elevated tHcy may also increase the risk of developing chronic disease in later life. Conceivably, fetuses exposed to elevated tHcy in utero that do not achieve optimal growth may be at increased risk of developing CVD as adults

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[149]. Long-term prospective studies from pregnancy throughout adulthood are required to test this hypothesis. There is conflicting evidence in the literature regarding the association between elevated tHcy in adults and risk of schizophrenia. However, it has been suggested that schizophrenia may actually arise due to anomalies in neurodevelopment rather than neurodegeneration [150]. Results from a nested case–control study in which prenatal samples were collected during the years 1956–1965 and the offspring followed up into adult life suggest that exposure to elevated tHcy in utero may also increase the risk of developing schizophrenia [93]. Fetal exposure to elevated homocysteine (third trimester tHcy 12.1 mmol/L) in utero was associated with a twofold increase in schizophrenia in adult offspring in this study of 63 adult cases and 122 controls. One of the mechanisms proposed was excessive stimulation of the N-methyl-D-aspartate (NMDA) receptor by homocysteine as previously observed in a study of rats [151]. Homocysteine has neuroexcitatory (agonistic at the glutamate binding site) and neuroprotective (antagonistic at the glycine coagonist site) functions at the NMDA receptor. However, in situations of homocysteine excess, overstimulation of the NMDA may occur. Dosedependent neurotoxicity was observed from homocysteine concentrations as low as 10 mM in the presence of 50 mM glycine concentrations. While the in vitro experiments were carried out at glycine concentrations that were double those reported for cerebral spinal fluid (CSF) in healthy newborns [median (95% CI): 12.1 (5.3, 24.4 mmol/L)] [152], the proposed mechanism might be feasible if glycine concentrations were higher in fetuses under unusual circumstances. High cerebral fluid glycine concentrations may occur if the blood–brain barrier is disrupted in cases of stroke or head injury in adults [153,154]. Other studies have reported that perinatal administration of NMDA receptor antagonists to rats lead to apoptopic neurodegeneration in the frontal cortex and long-lasting deficits in behavioral and cognitive function in the offspring [155]. 8.11. FUTURE RESEARCH The advance in knowledge in the field of tHcy and human pregnancy would be greatly enhanced by prospective studies, ideally, from preconception throughout pregnancy and indeed childhood. Such studies are highly time consuming and expensive to run. However, if the collection of preconception blood samples and lifestyle data is not feasible, collection could be started during the first trimester at the latest. Lifestyle habits, especially toxic habits, often change after the first prenatal check up, especially in the case of unplanned pregnancies. There is considerable variation in study designs and quality and quantity of data regarding lifestyle variables. These


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factors may contribute to the conflict in the literature in this field. Further studies are required to establish whether elevated tHcy is associated with increased risk of miscarriage, PROM, placental vasculopathy, gestational diabetes, and Down syndrome. With regard to preeclampsia and IUGR, the mechanism linking elevated tHcy and these complications needs to be elucidated. ACKNOWLEDGMENTS Research grants: MICINN SAF2005-05096; The Spanish Ministry of Health (Instituto de Salud Carlos III, Thematic Network G03/140, and RTIC RD06/0045/0009), FEDER (Fondo Europeo de Desarrollo Regional); Public Health Division, Department of Health, Catalonian Autonomous Government; Centre Catala` de la Nutricio´, Institut d’Estudis Catalans.

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DIABETES AND TUBERCULOSIS: ANALYSIS OF A PARADOX Dibyajyoti Banerjee,1 Rajasri Bhattacharyya, Deepak Kaul, and Priya Sharma Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

1. 2. 3. 4. 5.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Definition of the Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analysis of Previous Experimental Results to Solve the Paradox. . . . . . . . . . . . . . . . . Probable Difference in Glycation Patterns of Macrophage Proteins and Its Precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. In Silico Analysis of the Possibility of Glycation-Induced Inhibition of NADPH Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Solving the Paradox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

139 140 140 142 142 143 146 150

1. Abstract Diabetes mellitus is a known predisposition factor for tuberculosis. However, the cause of such interrelationship is incompletely understood. Diabetes mellitus is characterized by hyperglycemia. Interestingly, glucose has been shown to stimulate NADPH oxidase, the key enzyme involved with respiratory burst in monocytes and macrophages. This glucose-induced genesis of reactive oxygen species via respiratory burst is contrary to the survival strategy of Mycobacterium tuberculosis as an intracellular pathogen in phagocytic cells. To address this paradox, diabetes and tuberculosis are reviewed at the molecular level. 1

Corresponding author: Dibyajyoti Banerjee, e-mail: [email protected] 139

0065-2423/11 $35.00 DOI: 10.1016/S0065-2423(11)53006-9


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2. Introduction NADPH oxidase activity is responsible for the killing of intracellular pathogens. Glucose is known to stimulate NADPH oxidase of phagocytic cells. As such, the induction of NADPH oxidase activity within phagocytic cells in diabetic subjects would be expected to enhance the destruction of intracellular pathogens such as Mycobacterium tuberculosis. However, it is well known that diabetic individuals have increased risk of infection in general and tuberculosis specifically. To evaluate this paradox, we analyzed published data and performed in silico analysis to more closely investigate this contradictory phenomenon. Literature analysis provided insight about possible increased extent of glycation of macrophage NADPH oxidase subunits compared to precursor cells. In silico analysis provided evidence for possible glycation-induced inhibition of NADPH oxidase. Based on these data, we hypothesize that in the chronic hyperglycemic state there is a potential for glycation-induced inhibition of macrophage NADPH oxidase, which may explain the increased occurrence of tuberculosis in diabetic individuals.

3. Definition of the Problem The NADPH oxidase of phagocytic cells is an electron transport system that catalyzes the one electron reduction of oxygen to superoxide, a process essential for host defense against invading microorganisms [1]. Although existence of various isoforms of NADPH oxidase is described, in the phagocytic cells, NOX-2 isoform (NADPH oxidase isoform 2) of the enzyme predominates. NOX-2 requires the assembly of at least five additional components for its activation [2]. They include membrane-bound p22phox, which helps to stabilize the NADPH oxidase proteins and dock cytosolic factors along with cytosolic proteins p47phox, p67phox, the small GTPase Rac, and p40phox. Cell stimulation via protein kinase C (PKC) pathway leads to phosphorylation and translocation of p47phox to the membrane. Because p67phox is bound to p47phox, this process also translocates p67phox. At the membrane, p67phox directly interacts and activates NOX-2 [2]. The membrane-bound phagosome is derived from plasma membrane by incorporation of cytosolic factors upon cell stimulation. It contains NADPH oxidase components and phagosome coat protein coronin 1A/tryptophan aspartate coat (TACO) protein. On phosphorylation, NADPH oxidase is activated and TACO is dissociated from phagosome, which then fuses with the lysosome, an event critical for killing of intracellular pathogen [3]. The importance of NADPH oxidase in host defense has been demonstrated by the

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recurrent and life threatening infections that occur in patients with chronic granulomatous disease, a hereditary disorder with defective NADPH oxidase activity [1,4]. Glucose is known to stimulate NADPH oxidase activity of phagocytic cells [5–8]. High glucose-induced oxidative stress is known to stimulate NADPH oxidase in mononuclear cells via PKC stimulation [7]. It is also proved to induce p22phox component of NADPH oxidase of monocytes [6]. Moreover, augmentation of the neutrophil respiratory burst is noted through the action of advanced glycation end products [8]. THP-1 cells, a human monocytic cell line, also show glucose-induced stimulation of PKC activity, which is expected to stimulate its NADPH oxidase activity [5,7]. Diabetes mellitus, a common disease of the modern world, is characterized by persistent hyperglycemia and glycation [9]. It is also known to have circulating mononuclear cells with increased PKC activity and increased NADPH oxidase gene expression [10]. In diabetic animal model, the peritoneal macrophages are reported to show enhanced NADPH oxidase activity compared to normal controls [11]. Therefore, it is expected that in the diabetic state, glucose-induced NADPH oxidase activity of phagocytic cells will better kill the intracellular pathogens as NADPH oxidase activity is directly proportional to the killing of intracellular bacteria via generation of superoxide inside the phagosome. But contrary to this, it is well known that diabetes mellitus is associated with increased infections in general [12] and tuberculosis in particular [13]. Diabetic mice infected with M. tuberculosis by high-dose (105 colony-forming units [cfu]) intravenous injection are reported to have increased mortality and higher bacterial lung burden compared with euglycemic mice. Insulin treatment of diabetic mice, a standard therapy to normalize the blood sugar, appeared to restore resistance to M. tuberculosis infection in one study [14]. The immunological basis of increased susceptibility of tuberculosis in diabetes mellitus is not clearly understood to date [15]. It has been proven that mycobacterium–macrophage interactions are critical for pathogenesis of tuberculosis, and M. tuberculosis resides inside the stable phagosome of host macrophages as intracellular pathogen [4,16]. It has also been conclusively shown that NOX-2 is critical for efficient innate immune response to mycobacteria [17]. Importance of NADPH oxidase generated reactive oxygen species (ROS) in the inflammatory response against mycobacteria is also proved beyond any doubt [18–20]. So the coincidence of known increased NADPH oxidase activity of phagocytic cells (particularly macrophages) in association with high-glucose conditions and prevalent infections especially tuberculosis in diabetic individuals with persistent hyperglycemia is a paradox.

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4. Analysis of Previous Experimental Results to Solve the Paradox Mouse peritoneal macrophages isolated from streptozotocin-induced diabetic mice are reported to show increased NADPH oxidase activity [11]. Another experiment using the same mouse model demonstrated significant decrease in the phagocytic activity of resident peritoneal macrophages 12 weeks after induction of diabetes compared with age-matched control mice [21]. NADPH oxidase activation is an integral part of cellular phagocytosis function and inhibition of a component of NADPH oxidase impairs phagocytic function of the cell [22]. This means that there is concomitant increase and decrease of NADPH oxidase activity of macrophages at hyperglycemic state. There must be a rational basis for the above phenomenon. Moreover, infections with pathogen-like M. tuberculosis which resides inside macrophages [4,16] are well known to be more with chronic hyperglycemic state [23], a phenomenon which cannot be explained if glucose-induced activation of macrophage NADPH oxidase activity is a universal fact. It is also reported that phagocytosis function of macrophage correlates inversely with advanced glycation end products in a mice model of chronic diabetes [21], which increases the possibility of glycation-induced inhibition of macrophage NADPH oxidase. Moreover, epidemiological research proved a strong positive association between occurrence of high-glycated hemoglobin (a marker of protein glycation and chronicity of diabetes) and pulmonary tuberculosis [24]. In this connection, it is also worth mentioning that murine macrophage cell line is shown to produce advanced glycation end products when infected with M. tuberculosis [25]. Thus, it is logical to appreciate that at hyperglycemic state, M. tuberculosis macrophage interaction will generate more advanced glycation end product compared to physiological condition.

5. Probable Difference in Glycation Patterns of Macrophage Proteins and Its Precursors Tissue macrophages are derived from circulating monocytes. They differ in several vital properties from their precursor cells like oxygen consumption, protein synthesis, transcription profile, etc. [26,27]. In an in vitro macrophage culture system, it is shown previously that cells in the monolayer retain more glucose compared to the cells in suspension and release carbon dioxide at a slower rate [28]. It indicates that the mature macrophage which adheres as monolayer may concentrate more glucose compared to its precursors due to slow rate of glucose metabolism. Recent proteomic analysis has confirmed


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that alveolar macrophages contain fewer amounts of glycolytic enzymes compared to its precursors [29]. Although the hexose monophosphate (HMP) shunt pathway is more active in macrophages compared to monocytes [30], in the context of tuberculosis that will be unable to lower intracellular glucose, as mycobacterial factors are known to inhibit HMP shunt pathway [31]. Human peritoneal macrophages are also shown to consume more glucose compared to its precursor monocytes [32]. Consequently, macrophages are also reported to have more glucose transporters compared to its precursors [33]. Therefore, it is evident that glucose concentration inside macrophage is more compared to its precursor in general and particularly if infected with M. tuberculosis. This increases the chance of enhanced protein glycation in macrophages compared to monocytes particularly at chronic hyperglycemic state. There is no reason to believe that macrophage NADPH oxidase glycation pattern can escape this possibility. Moreover, the macrophages are known to express GLUT5, a fructose transporter, significantly more compared to its precursors [33]. In diabetic condition, blood fructose is elevated significantly compared to nondiabetic state [34] and reduction of plasma fructose is observed to be beneficial in mice model of diabetes [35]. So, in diabetic state, the macrophages are expected to accumulate fructose much more (in addition to glucose) compared to its precursors and it is well known that fructose causes glycation much faster than glucose [36]. Therefore, in chronic diabetic state, macrophage proteins are expected to be more glycated when compared to the proteins present in the monocytes. Glycation is known to inhibit many enzymes [37]. Subsequently, we have analyzed the chance of glycationinduced inhibition of NADPH oxidase subunits using tools of computational biology. 6. In Silico Analysis of the Possibility of Glycation-Induced Inhibition of NADPH Oxidase To analyze the glycation and phosphorylation sites, five proteins—coronin 1A, which coats the phagosome [3], and p47phox, p67phox, p40phox, and p22phox, which are components of NADPH oxidase [2]—are selected. Sequences are taken from Swissprot database (www.expasy.ch). The accession numbers for the proteins are as follows—coronin 1A: P31146; p47phox: P14598; p67phox: P19878; p40phox: Q15080; and p22phox: P13498. The structures of p47phox both activated and inactivated (PDB code, 1NG2, 1OV3 [38]) and p40phox (1OEY [39]) are taken from Protein Data Bank (PDB) [40]. The glycation of the e amino group of lysine (Lys) residues is predicted by using NetGlycate 1.0 server [41]. The phosphorylation sites are identified by

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TABLE 1 PREDICTED GLYCATION SITES OF THE PROTEINS Name of the protein

Swissprot accession no.

Glycation sitesa

Coronin 1A

P31146

K10, K20, K45, K66, K72, K198, K233, K287, K324, K400

P47phox

P14598

P67phox

P19878

P40phox

Q15080

P22phox

P13498

38K, 52K, 55K, 119K, 146K, 169K, 235K, 282K, 385K 20K, 23K, 51K, 86K, 110K, 117K, 133K, 138K, 139K, 168K, 170K, 201K, 232K, 256K, 271K, 286K, 345K, 418K, 444K 32K, 78K, 98K, 156K, 192K, 228K, 253K, 323K 58K, 60K, 71K, 78K

Phosphorylation sitesa,b

Reference of the phosphorylation sites

S8, T67, T131, S194, S243, S291, S311, S315, S391, T412 S303, S304, S315, S320, S328 T233

[42]

T154, S315

[44]

132T, 147T

[45]

[38] [43]

a The one-letter amino acid codes for Lys, Ser, and Thr along with the sequence numbers are noted. b Phosphorylated Ser/Thr residues which are sequentially located within four residues before or after the predicted glycated Lys residues are in bold.

literature survey and the references are mentioned in Table 1. Interface area between p47phox and p22phox is calculated by ProFace [46] and molecular diagrams are drawn using pymol (http://www.pymol.org) [47]. Components of NADPH oxidase and phagosome coat protein coronin1A/TACO are activated by phosphorylation [2,3]. Here, we have checked the possibility of hindrance or masking of phosphorylation sites of the above proteins by glycation at its predicted sites. The glycation and phosphorylation sites of the relevant proteins are given in Table 1. In coronin 1A, out of 10 potential phosphorylation sites, three serine (Ser) and one threonine (Thr) residues are sequentially positioned within four residues before and after the predicted glycated Lys residues. It is obvious that residues which are sequentially nearer are also structurally closer. So, if the Lys residues become glycated, phosphorylation of Ser/Thr residues which are closer to those Lys residues will be affected. As a result, coronin1A/TACO will not be phosphorylated and not be dissociated from the phagosomes [3]. In p47phox, the phosphorylated Ser residues are located at the polybasic region [38]. But none of the Lys residues in this region are predicted to be glycated (Fig. 1A)

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A

B

SH3 SH3

Lys295

Lys282

Lys235 Lys169 Ser304 Ser303

Lys235

Ser328

Ser320

Lys317

Poly Pro peptide

Ser315

C Ser315 9.09 Lys323

FIG. 1. Ribbon diagram of p47phox and p40phox along with phosphorylation and predicted glycation sites. Inactivated (1A) and activated (1B) structures of p47phox are presented. In 1A, the polybasic region containing the phosphorylation sites is in yellow and the SH3 domain is in green color. Predicted glycated residues are in green while two Lys residues in the polybasic region are in cyan color ball-and-stick mode. The phosphorylation sites are in white color. All the phosphorylation and predicted glycation sites and two Lys residues in the polybasic region are labeled. In 1B, the complex structure of the –SH3 domains of activated p47phox (in blue color) with the binding region of p22phox (in magenta color) is shown. The interface region of SH3 domain is in green color. Lys235, a predicted glycation site which is present at the interface is shown in green color using stick mode. In 1C, the structure of p40phox along with one predicted glycation site, Lys323, and one phosphorylation site, Ser315, in ball-and-stick mode are shown. The distance between the amino group of Lys323 and Ser315 is shown by black dotted lines along with the distance. In all the figures, the residues are labeled by three-letter amino acid codes followed by residue numbers.

and all the predicted Lys residues for glycation are not located near to this region. Further, none of the phosphorylated Ser residues are in contact to the predicted glycated Lys residues in the three-dimensional structure. Lys235 of SH 3 domain of p47phox is located at the interface of p47phox and p22phox complex (Fig. 1B). So glycation of Lys235 will affect the complex formation between p47phox and p22phox. In p67phox, the important phosphorylation site Thr233 [43] is positioned adjacent to the predicted glycated Lys232. For the

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activation of p40phox, Thr154 and Ser315 are phosphorylated [44]. Lys156, positioned after two residues of Thr154, is predicted to be glycated, but there is no prediction of glycation of Lys residue near Ser315. However, E amino group of Lys323 which is sequentially eight residues after Ser315 is predicted to be glycated and the distance between the E amino group of Lys323 and the ˚ (Fig. 1C). So, if this amino side chain hydroxyl group of Ser315 is 9.09 A group becomes glycated, phosphorylation of the side chain hydroxyl group may be hindered. p22phox is present in the membrane which is predicted to be glycated at multiple sites and also known to be activated by phosphorylation [45], but the predicted glycation sites of this protein are not closer to the known phosphorylation sites. The computational study clearly demonstrates that there is enough chance of glycation-induced inhibition of NADPH oxidase by multiple ways. Moreover, phagosome coat protein coronin1A/TACO also shows multiple glycation possibility and probability of glycation-induced masking of its potential phosphorylation sites. The effect of glycation on TACO may not affect the NADPH oxidase activity directly but glycation-induced hindering of the phosphorylation sites may result in formation of a stable phagosome preventing its fusion with lysosome. In the stable phagosome, an invading microorganism like M. tuberculosis may reside minimizing the chance of it getting cleared by the host immune system.

7. Solving the Paradox It is known that the survival factor of M. tuberculosis as intracellular pathogen is due to its ability to nullify intracellular oxidative stress in its microenvironment. As phagosome lysosome fusion is arrested by mycobacterial factors, lysosomal myeloperoxidase is not expected to generate toxic radicals in the context of tuberculosis [3,16]. Consequently, mycobacterial factors inhibit phagosome acidification and thus not providing the acidic pH of myeloperoxide action to produce reactive hypo halite radicals [48,49]. Moreover, it is observed that the bacteria evolved a number of approaches to detoxify ROS and RNS including superoxide dismutase (SOD), Kat G (catalase/peroxidase), alkyl hydroperoxide reductases, thioredoxin reductases, methionine sulfoxide reductases, etc. It also contains forms of hemoglobins, that is, truncated hemoglobin which directly detoxifies NO [50]. It has been hypothesized that M. tuberculosis cell wall components can be part of its constitutive defense against ROS. Moreover, cyclopropanation of mycolic acid of pathogenic mycobacteria is also known as a potential ROS scavenger [51]. Therefore, the virulence of mycobacterial infection is defined by its effective mechanisms of not allowing the host to produce


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toxic oxidizing radicals and, if some ROS are produced, to detoxify it by plenty of antioxidants available within its system. If glucose-induced significant stimulation of macrophage NADPH oxidase activity occurs universally, increased association of chronic diabetes and tuberculosis cannot be a reality. Published experimental data clearly shows that glucose stimulates NADPH oxidase activity of monocytes and glucoseinduced oxidative stress causes gene expression of monocyte NADPH oxidase components. So, when monocytes are just converted into macrophage at hyperglycemic state, it may have increased NADPH oxidase activity. But, in chronic hyperglycemia, when the macrophages are exposed to higher concentration of glucose and fructose for long, due to reasons stated above, there may be more glycation of macrophage NADPH oxidase subunits compared to the enzyme subunits of monocytes. The in silico study indicates that enhanced glycation of NADPH oxidase subunits can inhibit the enzyme activity. Thus, although high glucose can be stimulatory for monocyte NADPH oxidase but probably chronic high-glucose exposure can be inhibitory for macrophage NADPH oxidase activity. Hyperglycemia-induced generation of ROS via stimulation of NADPH oxidase from circulating monocytes contributes in development of oxidative stress in diabetes [6]. Increased oxidative stress impairs antigen processing and presentation to T cells [52]. So, the stimulation of monocyte NADPH oxidase at hyperglycemic state will downregulate the T cell mediated adaptive immune response, which also can contribute significantly to cause tuberculosis in diabetes. This idea is further supported by the fact that diabetic mice display a delayed adaptive immune response in response to tuberculosis [53]. In this connection, it is worth mentioning that depressed immunological function observed in diabetes is currently thought to be main culprit for increased association of tuberculosis in diabetic state [54]. Insulin stimulates superoxide production both in monocytes and in macrophages probably via NADPH oxidase stimulation [55]. Overt diabetes mellitus is gradually getting recognized as a state of absolute deficiency of insulin due to loss of beta cell mass, which is proportional to the duration and severity of hyperglycemia [56–58]. Therefore, in long-standing diabetes in presence of glycation-induced inhibition of NADPH oxidase, the scope of insulin-induced stimulation of the macrophage NOX-2 enzyme may be reduced due to unavailability of the hormone and that can also contribute to cause tuberculosis. Macrophage culture with high glucose resulted in inhibition of cholesterol efflux and cholesterol accumulation and that is also confirmed in an in vivo study using type II diabetes mice model [59]. Moreover, high-glucose condition stimulates cholesterol biosynthesis in macrophages, which is inhibited by insulin [60]. In long-standing diabetes, there are both hyperglycemia and

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deficiency of insulin, which can synergistically cause cholesterol accumulation inside macrophages. This intramacrophage cholesterol accumulation in long-standing diabetes may also play an important role for tuberculosis development, as the role of cholesterol is critical in entry of M. tuberculosis in macrophages [61]. Nitric oxide (NO) is a free radical generated in M. tuberculosis-infected macrophages to help fight infection [62]. In diabetes mellitus, there is impaired NO synthesis due to hyperglycemia-induced accumulation of endogenous inhibitor of NO synthase (NOS) pathway [63]. It is also known that reactive nitrogen species (RNS) play an essential role in host defense against M. tuberculosis in the mouse model of tuberculosis. It is evident by the increased susceptibility of mice deficient in the inducible isoform of NOS2 [64]. In contrast, the role of ROS alone in protection against tuberculosis is less clear, and mice defective in the ROS-generating phagocyte NADPH oxidase are relatively resistant [64]. Therefore, in diabetic state, the macrophage NOS pathway may be inhibited by endogenous inhibitor and that can also account for increased incidence of tuberculosis in diabetes. In addition, NO produced by inducible NOS can combine with superoxide to generate additional products with enhanced toxicity, such as peroxynitrite [65]. In uncontrolled diabetes due to glycation-induced inhibition of macrophage NOX-2 and hyperglycemia-induced accumulation of endogenous inhibitor of NOS resulting in reduced NO formation in macrophage may synergistically account for increased incidence of tuberculosis in chronic diabetes mellitus by reduced formation of peroxynitrite in macrophage. In contrast to normal individuals, significant antibody glycation has been observed in diabetic mellitus. In vitro antibody glycation substantially lowers the affinity of an antibody for its antigen and significantly increases the rate of dissociation of the antigen–antibody complex [66]. This phenomenon, if occurs in vivo, can also account for increased incidence of tuberculosis in diabetes. Moreover, enhanced nonenzymatic glycosylation (glycation) of immunoglobulin G is known to impair complement fixation [67]. In this regard, opposing view is also recorded in the literature [68]. However, oxidative stress in diabetes, which is contributed substantially by ROS from circulating monocytes via hyperglycemia-induced stimulation of its NOX-2, may also contribute to accelerated protein glycation (including macrophage NOX-2 glycation or antibody glycation). This is due to generation of enhanced thiobarbituric acid reactive substances along with significant glutathione depletion in many tissues which are known to accelerate glycation process [69–71]. All these factors (Fig. 2) in combination justify the association of sustained hyperglycemia with tuberculosis in spite of hyperglycemia-induced stimulation of phagocyte NADPH oxidase.


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Prolonged hyperglycemia Insulin

Glucose and

fructose

Endogenous inhibitor of NOS Antibody glycation

Macrophages accumulate relatively more glucose and fructose and metabolizes the sugars in slow rate

NOS in macrophage

NO

Monocyte NOX-2

Reduction of Relatively increased chance of humoral immune macrophage protein glycation response and complement fixation NOX-2 glycation in macrophage

Macrophages accumulate cholesterol

O2−

Decrease macrophage O2− production O2−

Oxidative stress Peroxinitrite Increased susceptibility for TB

FIG. 2. Flowchart showing probable mechanisms of increased association of tuberculosis in chronic uncontrolled diabetes mellitus. The flowchart shows that in long-standing hyperglycemia, there is reduction of plasma insulin with elevation of plasma glucose and fructose, which may result in more glycation of macrophage NOX-2 subunits compared to enzyme subunits of monocytes causing inhibition of macrophage NOX-2 resulting in less generation of superoxide in Mycobacterium tuberculosis-infected macrophage. Increased sugars can also cause antibody glycation affecting the humoral immunity of the host. Hyperglycemia-induced stimulation of monocytes NOX-2 can cause oxidative stress, a phenomenon known to downregulate T cellmediated adaptive immune response. Hyperglycemic condition generates endogenous inhibitor of nitric oxide synthase, which can inhibit macrophage NOS that can reduce NO formation in tuberculosis-infected macrophage. In chronic hyperglycemia, simultaneous inhibition of macrophage NOX-2 and NOS2 can reduce formation of peroxynitrite in tuberculosis-infected macrophage causing diminished mycobacterial killing. Reduction in insulin and hyperglycemia together can cause more cholesterol accumulation in macrophage, a factor important for entry of Mycobacterium tuberculosis in macrophage. For details, please refer text.

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ACKNOWLEDGMENT DB and RB acknowledge Department of Science and Technology (DST), Ministry of Science and Technology, Government of India, New Delhi for financial support.

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EGCG, GREEN TEA POLYPHENOLS AND THEIR SYNTHETIC ANALOGS AND PRODRUGS FOR HUMAN CANCER PREVENTION AND TREATMENT Di Chen,*,† Sheng Biao Wan,‡ Huanjie Yang,*,†,} Jian Yuan,‡ Tak Hang Chan,¶ and Q. Ping Dou*,†,1 *The Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, School of Medicine, Wayne State University, Detroit, Michigan, USA † Departments of Oncology, Pathology, and Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan, USA ‡ Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China } Department of Life Science and Engineering, Harbin Institute of Technology, Harbin, China ¶ Department of Chemistry, McGill University, Montreal, Quebec, Canada

1. 2. 3. 4. 5.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of Tea Polyphenols in Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vitro and In Vivo Preclinical Studies on Tea Polyphenols . . . . . . . . . . . . . . . . . . . . . Tea Polyphenols and Their Molecular Targets in Cancer Cells. . . . . . . . . . . . . . . . . . . 5.1. Green Tea and ()-EGCG Protect DNA from Methylation and Damage . . 5.2. Tea Polyphenols and Inhibition of Oncogene Expression . . . . . . . . . . . . . . . . . . . 5.3. Tea Polyphenols Inhibit Proteasome Activity in Cancer Cells . . . . . . . . . . . . . . 5.4. Other Molecular Targets of Tea Polyphenols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Tea Polyphenol Derivatives in Cancer Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Q. Ping Dou, e-mail: [email protected] 155

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1. Abstract Cancer-preventive effects of tea polyphenols, especially epigallocatechin3-gallate (EGCG), have been demonstrated by epidemiological, preclinical, and clinical studies. Green tea polyphenols such as EGCG have the potential to affect multiple biological pathways, including gene expression, growth factor-mediated pathways, the mitogen-activated protein kinase-dependent pathway, and the ubiquitin/proteasome degradation pathway. Therefore, identification of the molecular targets of EGCG should greatly facilitate a better understanding of the mechanisms underlying its anticancer and cancer-preventive activities. Performing structure–activity relationship (SAR) studies could also greatly enhance the discovery of novel tea polyphenol analogs as potential anticancer and cancer-preventive agents. In this chapter, we review the relevant literature as it relates to the effects of natural and synthetic green tea polyphenols and EGCG analogs on human cancer cells and their potential molecular targets as well as their antitumor effects. We also discuss the implications of green tea polyphenols in cancer prevention.

2. Introduction The history of tea consumption dates back around 5000 years ago in ancient China. Today, tea is the most popular beverage consumed by twothirds of the world’s population. Green tea, black tea, and oolong tea are all derived from the Camellia sinensis plant and contain an assortment of compounds, the most significant of which are polyphenols. The differences among green, black, and oolong teas are derived from their fermentation processes. Green tea does not readily undergo fermentation, but black tea is completely fermented, while oolong tea contains partially fermented leaves. Among all teas consumed in the world, green tea is the most studied for health benefits [1], and tea polyphenols are considered to contribute to the preventive effects on various pathological disorders, including cancers. The major components of tea are catechins which contain a benzopyran skeleton with a phenyl group substituted at the 2-position and a hydroxyl (or ester) function at the 3-position. Variations to the catechin structure include the stereochemistry of the 2,3-substituents and the number of hydroxyl groups in the B- and D-ring. Belonging to the flavan-3-ol class of flavonoids, the most abundant catechins found in tea leaves include ()-epigallocatechin-3-gallate [()-EGCG], ()-epigallocatechin [()-EGC], ()-epicatechin-3-gallate [()-ECG], ()-epicatechin [()-EC], ()-catechin-3-gallate [()-CG], and ()-gallocatechin-3-gallate [()-GCG] (Fig. 1).

EGCG, GREEN TEA POLYPHENOLS AND THEIR SYNTHETIC ANALOGS

R1

OAc OAc

R2 HO

AcO

O

O

R3

OAc O

O OH

157

OH

O

OAc

OAc

O

OAc

OH OH 1 (EGCG): R1 = R2 = R3 = OH

OAc EGCG peracetate

2 (ECG): R1 = H, R2 = OH, R3 = H 3 (Afzelichin): R2 = OH, R1 = R3 = H FIG. 1. Chemical structures of major tea polyphenols and synthetic prodrug of EGCG (EGCG peracetate or Pro-EGCG).

Although the cancer-preventive effects of green tea and tea polyphenols have been reviewed previously [1–6], the current review critically analyzes the existing data relating to the chemopreventive effects of tea polyphenols, and specifically their molecular targets involved in this process. Furthermore, the derivatives and synthetic analogs and prodrugs of tea polyphenols along with their antitumor effects reported recently by our groups and other scientists are also summarized. We sincerely apologize to those authors whose work is not cited in this short review due to space limitation.

3. The Role of Tea Polyphenols in Carcinogenesis Cancer development initiates (called the initiation stage) from changes in gene modifications such as an irreversible genetic alteration which can occur via simple mutations, transversions, transitions, and/or small deletions in DNA [7]. Alternatively, genetic alterations may result from changes in gene function, without alteration of the DNA sequence, including histone modification, transcriptional activity, and DNA methylation [8]. The second stage of carcinogenesis is promotion, involving reversible changes in the expression of the genome mediated through promoter–receptor interactions. The final stage of carcinogenesis involves progression that is characterized by karyotypic instability and malignant growth and enhanced by formation and propagation of genetic errors that occur due to increased cellular proliferation [7,9].

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Chemoprevention aims to decrease the occurrence of cancer by the administration of natural or synthetic compounds [4]. Numerous studies have demonstrated the chemopreventive potential of green tea and tea polyphenols [10–13]. Dietary cancer chemopreventive agents exert their effects by modulating multiple cell signaling pathways involved in the carcinogenic process [14]. Furthermore, various studies indicate that diet-derived compounds are capable of prolonging one or more stages of the carcinogenic process [1,4,15]. For example, green tea polyphenols (GTPs) affect multiple important biological pathways and carcinogenesis. It is generally accepted that many of the cancer chemopreventive effects of green tea are mediated by its most abundant polyphenol, ()-EGCG. In the following sections, we will summarize the biological role of green tea and ()-EGCG as well as its synthetic analogs and prodrugs from both preclinical and clinical studies, with particular emphasis on the molecular targets involved in cancer prevention. 4. In Vitro and In Vivo Preclinical Studies on Tea Polyphenols The antitumor effects of tea polyphenol and ()-EGCG were discovered from various cancer cell lines, animal models, and clinical studies. For example, in over 20 tumor cell lines, as listed in Table 1, tea polyphenols and ()-EGCG showed significant growth-inhibitory and antitumor effects. Tea inhibited carcinogenesis in various animal models bearing lung, skin, esophagus, and liver cancers [65–67]. Green tea infusion (e.g., 1.25 g of tea leaves brewed in 100 ml water), as the sole source of fluid intake in A/J mice, significantly decreased N-nitrosodiethylamine (NDEA)-induced lung tumor incidence (by 36–44%) and tumor multiplicity (by 44–60%) [68]. The inhibition of skin carcinogenesis by tea was also investigated. It was found that oral administration of GTPs reduced UVB-induced skin cancer incidence by 35%, tumor multiplicity by 63%, and tumor growth by 55% [16]. Furthermore, the inhibitory effect of GTPs was associated with reduced expression of the matrix metalloproteinases (MMP)-2, MMP-9, and CD31, which are important factors in tumor growth, metastasis, and angiogenesis [16]. In addition, topical application of GTPs (6 mg/animal) in DMBA-initiated mice prior to promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) or mezerein (MEZ) resulted in significant protection against skin tumor formation in terms of tumor incidence (by 32–60%), multiplicity (by 49–63%), and tumor volume/mouse (by 73–90%) [69]. The inhibitory effects of green tea on hepatocarcinogenesis were tested in mice, given pentachlorophenol (PCP) as a carcinogen following treatment with the initiator diethylnitrosamine (DEN). It was found that in mice that were pretreated with a green tea


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TABLE 1 A SUMMARY OF THE BIOLOGICAL EFFECTS OF TEA POLYPHENOLS AND ()-EGCG ON VARIOUS CANCER CELL LINES AND TUMORS Agents from tea Green tea Tea polyphenol Green and black tea Tea polyphenol and ()-EGCG Tea polyphenol and ()-EGCG ()-EGCG Tea polyphenol and ()-EGCG Tea polyphenol and ()-EGCG Tea polyphenol and ()-EGCG Tea polyphenol and ()-EGCG Tea polyphenol and ()-EGCG Tea polyphenol and ()-EGCG ()-EGCG ()-EGCG ()-EGCG ()-EGCG ()-EGCG ()-EGCG Green tea extracts and ()-EGCG ()-EGCG ()-EGCG Tea polyphenol and ()-EGCG

Tumors and tumor cells

References

Epidemiological Skin tumor Esophageal tumor Cervical cancer cells

[11–13] [16] [17] [18–20]

Ovarian carcinoma

[21,22]

Human epidermoid carcinoma A431 cells Human breast cancer cells and tumors, Mouse mammary epithelial cells Oral squamous carcinoma cells, Oral cancer cells, Oral epithelial cells growth in vitro, IFN-g-stimulated human oral cancer cells Human drug-resistant lung cancer cells, Human lung cancer cells Human hepatocellular carcinoma cells, Phase IIa liver cancer Bladder cancer cells

[23] [24–28] [29–32]

[33–37] [38–40] [41,42]

Prostate cancer

[43–49]

Head and neck tumor Mouse embryonic fibroblast cells Human osteogenic sarcoma (HOS) cells Laryngeal squamous carcinoma cells Nasopharyngeal carcinoma cells Renal cell carcinoma Intestinal tumor, Colorectal cancer

[50,51] [52] [53] [54] [55] [56,57] [58–60]

Hypopharyngeal carcinoma cells Pancreatic cancer cells Human gastric cancer cells

[61] [62] [63,64]

infusion and continuously exposed to the same infusion during the process of initiation and promotion, the incidence of hepatocellular tumors was decreased by 40% [70]. Decaffeinated green tea or decaffeinated black tea extracts as the fluid intake were also given to N-nitrosomethylbenzylamine (NMBzA)-induced esophageal tumor model of Sprague–Dawley rats, and this resulted in reduced esophageal tumor incidence and multiplicity by 70% in both groups [17].

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Several groups have reported that ()-EGCG plays a critical role in preventing the growth of cervical cancer associated with apoptosis induction. Moreover, the growth-inhibitory effects of ()-EGCG in cervical cancer cells may be associated with the inhibition of telomerase [18–20]. ()-EGCG may also augment the chemotherapeutic effects by inhibiting the endothelin axis and downstream signaling pathways associated with overcoming drug resistance in ovarian carcinoma [21–23]. In our previous study, ()-EGCG and its prodrug, ()-EGCG peracetate or Pro-EGCG (Fig. 1), were found to potently inhibit human breast cancer cells and tumor growth [24]. The suppression of cell proliferation and gene expression by combinatorial synergy of ()-EGCG, resveratrol, and g-tocotrienol in estrogen receptor-positive MCF-7 breast cancer cells was also demonstrated [25]. Another investigation revealed that ()-EGCG inhibited growth in the mouse viral mammary epithelial carcinogenesis model RIII/MG and induced apoptosis, suggesting the clinical relevance of EGCG as a chemopreventive agent [26]. Various in vitro studies demonstrated that tea polyphenols or pure ()EGCG inhibited oral squamous carcinoma cells and oral epithelial cell growth [29–31,71]. ()-EGCG suppressed indoleamine 2,3-dioxygenase, an immunomodulatory protein by blocking the g-interferon-induced JAKPKC-d-STAT1 signaling in human oral cancer cells [32]. A number of studies revealed that ()-EGCG exerted inhibitory effects in a variety of cancer cells by targeting multiple cellular molecules. ()-EGCG was found to inhibit the growth rate and induce G2-M arrest in lung cancer cells in vitro and in vivo [33–36,72,73]. ()-EGCG was also reported to inhibit human hepatocellular carcinoma cells [38–40,74]. ()-EGCG was found to downregulate N-cadherin and suppresses migration of bladder carcinoma cells [75]. Additionally, ()-EGCG was found to inhibit transitional cell carcinoma of the bladder cell line T24 [41]. Preclinical evidence showed that green tea extract (GTE) induced ornithine decarboxylase and activated extracellular signalregulated kinase in bladder carcinoma ECV304 cells [42]. In an in vivo study, it was suggested that ()-EGCG and its prodrug ()-EGCG peracetate could augment the anticancer effects in androgen-independent prostate cancer [43]. Preclinical studies have been demonstrated that tea polyphenol and ()EGCG could inhibit cell proliferation and induce cell death in a variety of cancer cells including head and neck tumor [50], lung cancer [76], prostate cancer [44–47], breast cancer [25], mouse embryonic fibroblast cell tumor [52], human osteogenic sarcoma [53], human epidermoid carcinoma [23], laryngeal squamous carcinoma cells [54], nasopharyngeal carcinoma [55], renal cell carcinoma [56,57], intestinal tumor [57,58], colorectal cancer [59,60], hypopharyngeal carcinoma [61], and pancreatic cancer cells [62]. Several studies also revealed the effects of ()-EGCG on gastric cancer


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[63,64]. The apoptotic effect of ()-EGCG on the human gastric cancer cell line MKN45 was observed via activation of the mitochondrial pathway [63]. Regarding human prostate cancer, epidemiological studies show that green tea consumption may reduce the incidence of prostate cancer. Kurahashi et al. investigated 49,920 men aged 40–69 years, who completed a questionnaire that included their green tea consumption habit and followed up the subjects more than 10 years [77]. In this group, 404 men were diagnosed with prostate cancer, of whom 114 had advanced cases, 271 were localized cancers, and 19 were of an undetermined stage. The results showed that green tea consumption was not associated with localized prostate cancer but was associated with a dose-dependent decrease in the risk of advanced prostate cancer [77]. A clinical trial was performed to determine chemopreventive effect of green tea catechins (GTCs) on human prostate cancer development. Sixty volunteers with high-grade prostate intraepithelial neoplasia (HG-PIN), who would develop prostate cancer within 1 year, were given three GTC capsules, 200 mg each (total 600 mg/day) or placebo [44]. After 1 year treatment with GTCs, only one person was diagnosed with prostate cancer among the 30 GTCs-treated men (incidence, 3%), whereas nine cancers were found among the 30 placebo-treated men (incidence, 30%) [44]. The results demonstrate that GTCs are safe and very effective for treating premalignant lesions and preventing prostate cancer development [44]. A Phase II trial was conducted by the North Central Cancer Treatment Group to explore green tea’s antineoplastic effects in patients with androgenindependent prostate carcinoma [78]. Forty-two patients, who were asymptomatic and had progressive prostate specific antigen (PSA) elevation with hormone therapy, were administrated orally with 6 g of green tea per day [78]. Patients were monitored monthly for response and toxicity. The results showed that only one patient had shown tumor response defined by 50% decrease in PSA from baseline (229–105 ng/dl) but this decrease was not sustained beyond 2 months. The overall response rate to GTCs was only 2% [78]. Green tea toxicity was observed in 69% of patients and included nausea, emesis, insomnia, fatigue, diarrhea, abdominal pain, and confusion. This clinical trial demonstrated that green tea has limited antineoplastic activity among patients with androgen-independent prostate carcinoma [78]. In conclusion, many studies using tea polyphenol and ()-EGCG in various cancer cell lines support their cancer-preventive effects (Table 1). Furthermore, results from Phase IIa chemoprevention trial of GTPs carried out in high-risk individuals have demonstrated the relative safety of GTP consumption in human subjects [38]. The established antitumor and cancerpreventative activities of tea polyphenol and ()-EGCG also encourage investigators to further elucidate their essential molecular targets.

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5. Tea Polyphenols and Their Molecular Targets in Cancer Cells Tea polyphenols possess broad inhibitory activity against carcinogenesis and are effective when administered during its initiation, promotion, or progression. However, the molecular mechanisms of tea polyphenols responsible for their inhibitory actions are not fully understood. Some of the molecular mechanisms proposed include protection of DNA from damage and/or methylation in normal cells, inhibition of tumor proteasome activity, inhibition of oncogene gene expression, induction of apoptosis, cell-cycle regulation, and inhibition of cell proliferation and tumor promotion-related events, which are discussed further below. 5.1. GREEN TEA AND ()-EGCG PROTECT DNA FROM METHYLATION AND DAMAGE Epigenetics is the study of reversible, heritable changes in gene function that occur without a change in the sequence of nuclear DNA [8]. Epigenetic mechanisms control eukaryotic development beyond DNA-stored information, but can also contribute to carcinogenesis by altering chromatin structure, histone acetylation, transcriptional activity, and DNA methylation. Epigenetic silencing by hypermethylation of tumor suppressor or DNA repair-related genes occurs most frequently during the early stages of the carcinogenesis [79]. Moreover, the epigenetic change may result in additional changes in gene constructions. It was reported that silencing of the O6methylguanine-DNA methyltransferase gene (MGMT) resulted in cells with the ability to acquire a specific type of genetic mutation in p53 and subsequently an inability to repair DNA guanosine adducts [80]. ()-EGCG was reported to inhibit the activity of DNA methyltransferase (DNMT), resulting in CpG demethylation and reactivation of methylationsilenced genes in human esophageal cancer KYSE 510 cells such as p16INK4a, retinoic acid receptor ß (RARß), MGMT, and human mutL homologue 1 (hMLH1) [81]. Caudal-related homeobox transcription factor 2 (Cdx2), a tumor suppressor gene is frequently inactivated by promoter hypermethylation in gastric carcinoma and colorectal cancer cells. Green tea decreased the Cdx2 methylation frequency in a dose-dependent manner, showing 10/25 (40%), 7/18 (39%), 2/8 (25%), and 0/6 (0%) of Cdx2 methylation frequency in tested groups of people who consumed three or less, four to six, seven to nine and ten cups or more a day, respectively [82]. Ultraviolet radiation (UVR) can induce DNA damage, which is one of the mechanisms of tumor formation. It was found that preincubation with ()-EGCG significantly decreased DNA damage induced by UVR in human skin fibroblasts, lung fibroblasts, and epidermal keratinocytes cell


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lines [83]. Furthermore, it was observed that the peripheral blood cells from individuals who consumed green tea showed lower levels of DNA damage compared to that from controls [83]. In a Phase II random controlled tea intervention trial, 143 heavy smokers, aged 18–79 years old, were randomly grouped to drink green tea, black tea, or water for 4 months. Assessment of urinary 8-OHdG, an indicator of oxidative DNA damage after adjustment for baseline measurements and other potential confounders, revealed a significant decrease in urinary 8-OHdG (31%) after 4 months of drinking decaffeinated green tea (P ¼ 0.002) [84], suggesting that regular green tea drinking might protect smokers from oxidative damage. 5.2. TEA POLYPHENOLS AND INHIBITION OF ONCOGENE EXPRESSION It has been shown that various genes associated with carcinogenesis are influenced by tea polyphenols. Tea polyphenols or ()-EGCG were found to reduce expression levels of cyclin D1 and bcl-2 and increase expression of p53 and p27, which resulted in apoptosis induction in several tumors including hepatocellular carcinoma, lung carcinogenesis, and nasopharyngeal carcinoma [35,37,55,74]. It has been reported that expression of MMP-9 gene, which is involved in cancer cell growth and metastasis, is inhibited by EGCG in macrophagedifferentiated HL-60 myeloid leukemia cells [85]. Treatment with EGCG dramatically inhibited MMP-9 protein secretion by human myeloid leukemia HL-60 cells with IC50 value of 3.2 mM. EGCG also diminished MMP-9 gene expression and inhibited mRNA levels in the HL-60 cells [85]. Overexpression of the HER-2/neu receptor (HER-2) and epidermal growth factor receptor (EGFR) is frequently observed in patients with breast carcinoma and also in patients with head and neck squamous cell carcinoma (HNSCC). Masuda et al. showed that EGCG could significantly inhibit the phosphorylation of HER-2 in breast and HNSCC cell lines and also induce inhibition of Stat3 activation, inhibition of c-fos, and cyclin D1 promoter activity, and decreased cellular levels of the cyclin D1 and Bcl-XL proteins [51]. It has been reported that ()-EGCG and GTPs inhibited TNF-a gene expression in the cells, mediated through inhibition of NF-B activation [86]. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 has been shown to be overexpressed in breast and lung tumors, and characterized as an early marker of lung cancer [87]. EGCG could potently inhibit the hnRNP A2/B1 gene expression with IC50 29 mM which resulted in growth inhibition of human lung cancer cells [72]. It was reported that EGCG suppressed growth of human lung cancer cells through inhibition of the Ras-GTPase-activating protein SH3

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domain-binding protein 1 (G3BP1) [35], which is a member of enzymes associated with the heterogeneous nuclear RNA-binding proteins and is an element of the oncogenic Ras signal transduction pathway [88]. Inhibition of oncogene expression by green tea and EGCG also tested in animal studies. Hu et al. reported that high level of expression of c-myc, c-raf, and c-H-ras oncogenes in lung tissue was induced in mice by treatment with tobacco-specific nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK). After feeding with 2% tea extract for 4–8 weeks, 50%, 20%, and 50% inhibition of c-myc, c-raf, and c-H-ras gene expressions, respectively, were observed in lung tissue of the NNK-treated mice [89]. EGFR upregulation has been shown to be associated with a number of mechanisms relating to tumor development [90]. MMP-2 overexpression has been reported to be associated with the invasive and malignant phenotypes of many cancer cell lines in vitro and in vivo [91,92]. EGCG could downregulate mRNA expression and protein levels of EGFR in breast cancer cells [27]. Treatment of human breast cancer cells with EGCG also reduced the activity, protein expression, and mRNA expression level of MMP-2 [28]. 5.3. TEA POLYPHENOLS INHIBIT PROTEASOME ACTIVITY IN CANCER CELLS The ubiquitin/proteasome system is responsible for the degradation of regulatory proteins that are involved in critical cellular processes such as cell cycle and apoptosis [93,94]. The eukaryotic proteasome contains at least three known catalytic activities: chymotrypsin-like, trypsin-like, and caspaselike or peptidyl–glutamyl peptide-hydrolyzing (PGPH)-like activities [95]. The proteasomal activity is required for tumor cell proliferation and drug resistance development [96]. Therefore, targeting the proteasome-mediated degradation pathway has been considered as an important approach for cancer therapy and prevention. The association of proteasome inhibition and apoptosis induction has been observed in studies of ours and other groups [97,98]. Indeed, clinical trials have demonstrated that the proteasome inhibitor Bortezomib (Velcade, PS-341) has antitumor activity in multiple myeloma and other type of cancers [48,99,100]. We reported that ()-EGCG potently and specifically inhibited the chymotrypsin-like activity of the proteasome in vitro (IC50 ¼ 86–194 nM), and that ()-EGCG could induce tumor cell growth arrest in G1 phase of the cell cycle [101]. For the first time, we reported that an ester bond within ()-EGCG played a critical role in its inhibitory activity of the proteasome [101]. In addition, synthetic ()-EGCG amides and ()-EGCG analogs with modifications in the A-ring, C-ring, or ester bond inhibited the chymotrypsin-like activity of purified 20S proteasome with altered potencies,


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induced growth arrest in the G1 phase of the cell cycle in leukemia Jurkat T cells, and suppressed colony formation of human prostate cancer LNCaP cells [102]. ()-EGCG is the most potent polyphenol in green tea, but it is unstable under neutral or alkaline conditions (i.e., physiologic pH). In an effort to discover more stable polyphenol proteasome inhibitors, we synthesized several ()-EGCG analogs with –OH groups eliminated from the B-and/or D-rings. In addition, we also synthesized their putative prodrugs with –OH groups protected by acetate (Fig. 1) that can be removed by cellular cytosolic esterases. We first examined the structure–activity relationship (SAR) of these unprotected and protected compounds with respect to their proteasome-inhibitory potentials. We found that decreasing the number of –OH groups from either the B- or D-ring leads to diminished proteasome-inhibitory activity in vitro. However, in cultured tumor cells, the acetate protected analogs were capable of potently inhibiting the proteasomal chymotrypsinlike activity by as much as 97% [103]. Furthermore, we found that, compared to ()-EGCG, protected analogs exhibited greater potency to inhibiting proliferation and inducing apoptosis in human leukemic, prostate, breast, and simian virus 40-transformed cells [104]. Importantly, the protected analogs were nontoxic to human normal and nontransformed cells [104]. 5.4. OTHER MOLECULAR TARGETS OF TEA POLYPHENOLS In several reviews, multiple molecular targets of GTPs were summarized [6,105,106], such as mitogen-activated protein kinases (MAPKs), EGFRmediated pathways, and the insulin-like growth factor (IGF)-I-mediated signal transduction pathway. Herein, we emphasize some important published findings in which cancer-specific protein targets were modulated by tea polyphenols. ()-EGCG could suppress heregulin-b1-induced fatty acid synthase expression in human breast cancer cells by inhibiting phosphatidylinositol 3-Kinase (PI3K)/Akt and MAP kinase cascade signaling [107]. ()-EGCG also inhibited the endothelin axis and decreased ETAR-dependent activation of the p42/p44 and p38 MAPKs and phosphatidylinositol 3-kinase pathway in ovarian carcinoma [18]. Adhami et al. reported that after oral administration of GTPs for 24 weeks, the progression and invasion of prostate cancer was inhibited in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model [49]. The chemoprevention effect of tea polyphenols in the TRAMP animal models was associated with decreased IGF-I level in the prostate tissue of the mice [49]. These findings were supported by another study, in which tea polyphenols could inhibit IGF-I-mediated Akt phosphorylation [108].

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The data from both in vitro and in vivo preclinical studies indicated that IGF-I signal transduction pathway is one of the critical molecular targets for tea polyphenols. Other studies revealed that tea polyphenols could target the MAPKs pathway. Opare Kennedy et al. found that GTE possessed growth-inhibitory effects toward Ehrlich ascites tumor cells associated with a cellular thioldependent activation of MAP kinases [109]. A poor prognosis of small-cell lung carcinoma (SCLC) is particularly due to the development of drug resistance. Sadava et al. found that EGCG could induce apoptosis in both drug-sensitive (H69) and drug-resistant (H69VP) SCLC cells with similar IC50 values ( 70 mM). Treatment of both cell lines with EGCG at the concentration of IC50 values for 24 h also resulted in 50–60% reduced telomerase activity [34]. Taken together, as seen with other natural compounds, tea polyphenols play potent-inhibitory roles in different stages of carcinogenesis and toward multiple targets throughout various tumor types. We have listed some of the major molecular targets of tea polyphenols in this review, which are associated with their anticancer and cancer-preventative effects.

6. Tea Polyphenol Derivatives in Cancer Studies The low absorption, poor membrane-permeability, metabolic transformations, and unstability of tea polyphenols tend to restrict their clinical use. The hydroxyl groups of ()-EGCG are subject to be modified through biotransformation reactions, including methylation, glucuronidation, and sulfation, resulting in reduced biological activities in vivo [110–112]. Many efforts have been made by chemical modification of tea polyphenols to improve their bioavailability [113,114]. In 2001, we reported the first chemical synthesis of epigallocatechin gallate (1) in an enantioselective manner providing separately the natural ()-EGCG as well as its enantiomer [115]. This was followed by the syntheses of ECG (2), Afzelechin (as the gallate 3) [116], and a number of analogs (Fig. 1) [103,117,118]. Structure–activity studies revealed that EGCG showed the optimal activity toward proteasome inhibition among the natural GTPs and the synthetic analogs (4a–4e) (Fig. 2) [101,117,119]. Decreasing the number of –OH groups from either the A-, B-, or D-ring of EGCG was found to diminished proteasome-inhibitory activity in vitro (Fig. 2) [116,118,120]. The carbonyl function of EGCG and analogs is essential for their proteasome-inhibitory activity [101]. The ester oxygen at C-3 can be replaced by the NH isostere with only minor

EGCG, GREEN TEA POLYPHENOLS AND THEIR SYNTHETIC ANALOGS R1 HO

O

R3

O

AcO

OR2

R2

R4

R3

O OH

OR1

R1

R2

R

O

HO

OH O

O R4

O

OAc

OAc

O

R5 R6

167

OH

OH

O

OAc

OR3 OR4

OAc

4a: R = R1 = R2 = R3 = H, R4 = R5 = R6 = OH 4b: R = R1 = R2 = H, R3 = R4 = R5 = R6 = OH

5a: R1 = R2 = OAc, R3 = R4 = H 5b: R2 = OAc, R1 = R3 = R4 = H

6a: R2 = R3 = R4 = H, R1 = CO(CH2)2CH3 6b: R1 = R3 = R4 = H, R2 = CO(CH2)2CH3

4c: R1 = H, R2 = R3 = R4 = R5 = R6 = OH, R = Ph

5c: R1 = R2 = R3 = R4 = H

6c: R1 = R2 = R4 = H, R3 = CO(CH2)2CH3

4d: R = R4 = R6 = H, R1 = R2 = R3 = R5 = OH

5d: R3 = OAc, R1 = R2 = R4 = H

6d: R1 = R2 = R3 = H, R4 = CO(CH2)2CH3

4e: R = R4 = R6 = H, R1 = R2 = R3 = OH, R5 = NH2

5e: R1 = H, R2 = R3 = OAc, R4 = Ph

7a: R2 = R3 = R4 = H, R1 = CO(CH2)6CH3 7b: R1 = R3 = R4 = H, R2 = CO(CH2)6CH3 7c: R1 = R2 = R4 = H, R3 = CO(CH2)6CH3 7d: R1 = R2 = R3 = H, R4 = CO(CH2)6CH3 8a: R2 = R3 = R4 = H, R1 = CO(CH2)14CH3 8b: R1 = R3 = R4 = H, R2 = CO(CH2)14CH3 8c: R1 = R2 = R4 = H, R3 = CO(CH2)14CH3 8d: R1 = R2 = R3 = H, R4 = CO(CH2)14CH3

FIG. 2. Chemical structures of EGCG analogues 4a–8d.

attenuation of activity toward purified proteasome, but enhanced potency toward cellular proteasome. This most observation is most likely due to increased stability [119]. In order to increase the stability of ()-EGCG, we appended peracetate protective groups to its reactive hydroxyl groups and results showed that the EGCG peracetate could be converted into ()-EGCG when incubated with cell extracts. Interestingly, a series of bioassays show that EGCG peracetate has no inhibitory activity against a purified 20S proteasome, but exhibits increased proteasome-inhibitory activity in intact leukemic cells over natural ()-EGCG, indicating an intercellular conversion taking place. Therefore, our results indicate that EGCG peracetate (also named Pro-EGCG) (Fig. 1) may function as a prodrug of the GTP proteasome inhibitor ()-EGCG [113]. In vivo studies using mice also showed a significant inhibition of breast tumor growth by Pro-EGCG, compared with ()-EGCG, associated with increased proteasome inhibition and apoptosis induction in tumor tissues [104]. The peracetates of ()-EGCG analogs (5a–5e), with –OH groups eliminated from the B- and/or D-rings, showed similar effects as those of the EGCG peracetate (Fig. 2) [103]. These studies disclosed that the prodrugs of ()-EGCG and its analogs not only increased their stability but also improved their bioavailability. In a follow-up study, ()-(EGCG) monoesters modified with butanoyl (6a–6d) (EGCG-C4), octanoyl (7a–7d) (EGCG-C8), palmitoyl groups

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(8a–8d) (EGCG-C16) (Fig. 2) were synthesized by a lipase-catalyzed transesterification method, and their antitumor activities were investigated in vitro and in vivo. EGCG-C16 suppressed tumor growth in vivo in colorectal tumorbearing mice in comparison to an untreated control, vector control (DMSO), and EGCG [121]. It was suggested that O-methylation of the catechins by catechol-Omethyltransferase (COMT), an enzyme ubiquitously present in humans, may reduce the cancer-preventive effects of the catechins [122]. We also synthesized nine different methylated catechins (9a–9i) (Fig. 3) which are metabolites or potential metabolites of tea catechins in biomethylation [123]. It was found that the addition of a methyl group on the B- or D-ring of ()EGCG or ()-ECG led to decreased proteasome inhibition and, as the number of methyl groups increased, the inhibitory potencies further decreased [124]. Metabolic O-methylation of EGCG may indeed reduce the effectiveness of EGCG as it relates to its anticancer activity [125], lending support of the human study [122]. Another study disclosed that 3-O-(3,4,5-trimethoxybenzoyl)-()-epicatechin (10) (TMECG) (Fig. 3) showed significant antiproliferative activity against

FIG. 3. Chemical structures of EGCG analogues 9a–13.


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several cancer cell lines, especially melanoma [126]. In an anticancer screening, 3-O-acyl and alkyl-()-epicatechin derivatives (11a–11c) (Fig. 3) exhibited superior anticancer activity compared to ()-ECG. Furthermore, several compounds that were modified aliphatic chains with moderate sizes (C8–C12) showed strong anticancer activity (IC50 ¼ 6.4–31.2 mM) [127]. More recently, we synthesized several novel fluoro-substituted ()-EGCG analogs, namely F-EGCG analogs (12a, 12b) (Fig. 3), as well as their prodrug forms with all –OH groups protected by acetate. The prodrug form of one F-EGCG analog exhibited greater potency than the previously reported peracetate of ()-EGCG to inhibit proteasomal activity, suppress cell proliferation, and induce apoptosis in human leukemia Jurkat T cells. These results demonstrate the potential of these compounds to be developed into novel anticancer and cancer-preventive agents [128,129]. Several studies have revealed the reversal effect of tea polyphenols and ()-EGCG on multidrug resistance in human carcinoma cells [130]. In our recent study, it was found that permethyl ()-EGCG and its analogs exhibited promising P-gp modulating activity in a P-gp overexpressing breast cancer cell line (LCC6MDR). One micromolar of permethyl ()EGCG (13) (Fig. 3) could sensitize LCC6MDR cells toward paclitaxel by 18.2-fold. These results currently await publication.

7. Conclusions Tea polyphenols are potent bioactive compounds that possess anticarcinogenic activities. They interfere with the initiation, development, and progression of cancer by modulating critical processes of cellular proliferation, differentiation, apoptosis, angiogenesis, and metastasis. Although tea has been consumed for centuries, it has only recently been studied extensively as a health-promoting beverage that may act to prevent a number of diseases including cancer. Various studies showed that tea polyphenols potently induce apoptotic cell death and cell-cycle arrest in tumor cells but not in their normal cell counterparts, and that GTPs affect multiple biological pathways. Various animal studies have revealed that treatment with green tea inhibits tumor incidence and multiplicity in different organ sites such as skin, lung, liver, stomach, mammary gland, and colon. Recently, Phases I and II clinical trials have been conducted to explore the anticancer effects of green tea in humans. Studies focusing on the purified tea polyphenol compound ()-EGCG should continue to provide researchers an improved understanding of the pharmacokinetics of tea polyphenols such as absorption, distribution, their role in anticancer reactions, metabolism and molecular mechanisms. Although structural modifications of ()-EGCG have

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shown promising results toward their anticancer effects, work should continue on optimizing and evaluating additional analogs of GTPs in an effort to discover more potent, stable, and specific tea polyphenol analogs as potential novel anticancer agents. A major challenge of cancer prevention is to integrate new molecular findings into clinical practice. Identification of more molecular targets or biomarkers for tea polyphenols is paramount to cancer prevention and treatment by green tea/synthetic ()-EGCG analogs, and will greatly assist in a better understanding of its anticancer mechanisms. ACKNOWLEDGMENTS This work is supported in part by research grants from the National Cancer Institute– National Institutes of Health (to Q. P. D.; 1R01CA120009 and 3R01CA120009-04S1), and the Areas of Excellence Scheme established under the University Grants Committee of the Hong Kong Special Administrative Region, China (to T. H. C.; Project No. AoE/P-10/01).

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INDEX A

T‐cell function cross‐linking, 60 human immunology, 60–61 Jurkat cells, 61 mouse immunology, 61–62 toll‐interacting protein, 63 Churg–Strauss syndrome, 70 Cleft lip palate (CLP), 109–110 CLP. See Cleft lip palate

Acute fatty liver of pregnancy (AFLP) description, 87–88 laboratory findings, 88 Acute rheumatic fever (ARF) chromosomal location, HLA genes, 36 incidence, 32 non‐HLA class II genes, 34–35 recurrence, 32 S. pyogenes infection, 39 AFLP. See Acute fatty liver of pregnancy ARF. See Acute rheumatic fever

D Diabetes and tuberculosis definition glucose, 141 mycobacterium–macrophage interactions, 141 NADPH oxidase, phagocytic cells, 140 experimental results, analysis, 142 glycation patterns and precursors hexose monophosphate (HMP), 143 tissue macrophages, 142 in silico analysis, glycation‐induced inhibition computational study, 146 coronin1A/TACO, 144 glycation sites, 144 lysine residues, 144–145 p47phox and p40phox, 145 proteins, 143 serine and threonine residues, 145–146 NADPH oxidase activity, 140 paradox, solving antibody glycation, 148 insulin, 147 macrophage culture, 147–148 M. tuberculosis, 146 NADPH oxidase, 147 nitric oxide (NO), 148 sustained hyperglycemia, 148–149

C CD26/DPPIV caveolin‐1, 62 characterization hematopoietic progenitor cells (HPCs), 59–60 Jurkat cells, 59 paranitroaniline, 59 impact, immunity adenosine deaminase (ADA), 55–56 caveolin‐1, 59 CD45/common leukocyte antigen, 56–57 extracellular matrix, 56 glycoprotein 120, 58 HIV‐1 infection, 57–58 M6P/IGFIIR, 57 streptokinase (SK), 58–59 Tat, transactivator protein, 58 NK cells, 62 structure amino acid point mutation, 54–55 carbohydrate chains, 55 domain, 54 fibroblast activation protein (FAP), 53–54 nucleotides, 53 three‐dimensional crystal, 55 179

180

INDEX

Dipeptidyl peptidase (DPP), autoimmune pathophysiology CD26, immune disorders inflammatory bowel disease (IBD), 66–67 multiple sclerosis, 65–66 rheumatoid arthritis (RA), 64–65 serum CD26/DPP4 activity, 63–64 systemic sclerosis (SSc), 70 transduction process, 52 transplantation, graft rejection and GVHD, 68–69 type 1 diabetes, 67–68 cellular aspects, CD26/DPPIV CD26 and T cell function, 60–62 immune cells, 62–63 molecular aspects, CD26/DPPIV CD26‐binding proteins, 55–59 DPPIV structure, 53–55 enzyme characterization, 59–60 poke‐weed mitogen (PWM) immunization, 53 DPP. See Dipeptidyl peptidase, autoimmune pathophysiology E EGCG. See Epigallocatechin‐3‐gallate Electrospray ionization (ESI), 3 Embryo, placental and fetal development congenital heart defects elevated maternal tHcy, 120 hyperhomocysteinemia, 121 fetal size and intrauterine growth retardation, 125–126 NTD, 120 placental development and function mother–cord pairs, 121–122 MRNA, 121 placental vascularization and preeclampsia associated risk, 122–124 Doppler imaging, 125 trophoblast invasion, 122 Epigallocatechin‐3‐gallate (EGCG) analogues, chemical structures, 167, 168 antitumor effects, 158 apoptosis induction, 166 biological effects, 159 cervical cancer, 160 DNA protection, methylation and damage controlled tea intervention trial, 163 epigenetics, 162

ultraviolet radiation (UVR), 162–163 gastric cancer, 160–161 metabolic O‐methylation, 168 MMP‐9 gene expression, 163 monoesters, 167–168 oncogene expression, 164 peracetate, 157, 160, 167 proteasome inhibition, 164–165 structure–activity studies, 166 ESI. See Electrospray ionization G ‘‘GOOD assay’’, 10, 12, 17 Graft‐versus‐host disease (GVHD) CD26/DPPIV, 69 morbidity and mortality, alloSCT, 68–69 Green tea catechins (GTCs), 161 Green tea polyphenols (GTPs) molecular targets, 165 oral administration, 158, 165 phase IIa chemoprevention trial, 161 GTCs. See Green tea catechins GTPs. See Green tea polyphenols GVHD. See Graft‐versus‐host disease H HELLP syndrome clinical features hemolysis, 87 hypovolemia, 86–87 symptom, 86 complement pathway and coagulation system antiphospholipid antibodies (aPL), 96 C3a and C5a, 96 contracting factors, 98 fibrinolytic system, 96–97 MMPs, 97 preeclampsia, 95 prostacyclin (PGI2), 97 TMA, 95 development period, 86 inflammatory response chemokines, 94 exacerbations and remissions, 92 macroarray analysis, 93 P‐IPG, 94 prednisolone therapy, 92–93

INDEX TGF‐ , 93–94 laboratory findings AFLP, 87–88 lactate dehydrogenase (LDH) levels, 87 thrombocytopenia, 87 maternal and perinatal outcome mortality, 89 rupture, liver, 88 pathogenesis and preeclampsia, placenta BclI polymorphism, 91 centroid classification method, 90 intervillous hypoxia, 91–92 oxidative stress, 89–90 trophoblast invasion, 89 Hemolysis (H), elevated liver (EL) enzymes, and low platelet (LP) syndrome. See HELLP syndrome Homocysteine and choline CLP, 109–110 phospholipid synthesis and neurotransmitter function, 109 plasma, 110 health benefits, tHcy cardiovascular events, 111 elevated levels, 110–111 metabolism genetic–nutrient interactions, 106 methyl groups, 107 micronutrients role, 106–107 MTHFR, 108 polymorphisms, genetic, 108 transmethylation, 106, 107 non‐nutritional factors, tHcy, 110 pregnancy adverse outcomes, 126–127 animal/in vitro studies, 113–115 DNA‐methylation and imprinting, 127 early loss/miscarriage, 117, 119–120 elevated tHcy and subfertility, 116–117 embryo, placental and fetal development, 120–126 gestational diabetes, 126 long‐term effects, 127–128 prospective studies, humans, 116 tHcy changes, normal, 111–113 prophylactic folic acid and folate status NTD, 108–109 pregnancy complications, 109 tHcy, 106

181

Homocysteine thiolactone (HTL), 114 HTL. See Homocysteine thiolactone Hyperhomocysteinemia classification, 111 congenital heart defects, 121 fetal exposure, 115 pregnant rats, 114–115 I Immune disorders and CD26 graft rejection and GVHD allogeneic stem cell transplantation (alloSCT), 68–69 prodipine treatment, 68 T‐cell proliferation, 69 transplantation, 68 HIV‐1 infection, 70–71 inflammatory bowel disease (IBD) animal model, 67 deficiency, 66–67 membrane expression, 66 multiple sclerosis (MS), 65–66 rheumatoid arthritis (RA) CD26‐KO mice, 64–65 description, 64 inhibitors, 65 serum CD26/DPP4 activity altered levels, 63 DPP inhibitors, 64 signaling capacity, 63 systemic sclerosis (SSc), 70 type 1 diabetes mellitus, 67–68 Inositol phosphoglycan P‐type (P‐IPG), 94 Intrauterine growth retardation (IUGR) maternal tHcy, 125–126 tHcy and Doppler imaging, 125 ‘‘Invader squared assay’’, 14 IUGR. See Intrauterine growth retardation M MALDI‐TOF‐MS. See Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry MassEXTEND assay, genotyping allele determination, 10 noninvasive detection, CNVs, 19 and SABER assay, 19 SNP‐genotyping, 11–12

182 Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) assay costs, 20 genotyping, 3 molecular mass determination, 19 multiplex genotyping molecular SNP‐haplotyping, 16–17 nonprimer extension assays, 12–14 polymorphic variants, 14–16 quantification, mixed DNA, 17–19 SNP, primer extension assays, 8–12 nucleic acids, analysis assay development, 4–5 instrumental aspects, 5–6 matrices, 6 preparation and analysis, 7–8 sample purification, 6–7 principle error, sources, 4 matrix and analyte molecules, 4 steps, 3–4 UV/IR laser pulse, 4, 5 SNPs, 2–3 Methylenetetrahydrofolate reductase (MTHFR) folate cycle, 108 homozygous knockout mice, 114 Mthfrþ/‐mice, 113 Miscarriage, pregnancy biochemical determinations, 117 chorionic villous vascularization, 117, 119 urinary hCG detection, 119–120 Molecular mimicry, RHD antibody‐mediated, 40 S. pyogenes, 39 T‐cell mediated autoreactivity, 42 cross‐reactivity, 41–42 monoclonal antibodies, 40 S. pyogenes, 40–41 streptococcal and self‐antigens, 43 types, 39 MTHFR. See Methylenetetrahydrofolate reductase Multiplex genotyping, MALDI‐TOF‐MS molecular SNP‐haplotyping diploid organisms, 16 drawback, 17

INDEX nonprimer extension assays, SNP amplicons, 14 hybridization, ligation/clevage reactions, 12–13 oligonucleotide ligation, 14 peptide nucleic acid (PNA), 13 polymorphic variants alternative splicing, 15–16 copy number variations (CNVs), 16 MALDI‐based assay formats, 14 restriction fragment mass polymorphism (RFMP), 15 single base indels, 15 quantification, mixed DNA allele frequencies, 17–18 heterogeneity, 17 pooling, 18 SABER assay, 19 SNP, primer extension assays amplification, 8 assay development, 9 ‘‘GOOD assay’’, 12 heterogeneous assays, 10–11 magnetic beads, immobilization, 10 MassEXTEND, 11–12 mass spectra, 20‐plex PinPoint assay, 9, 11 PROBE assay, 9–10 N NADPH oxidase activity glucose, 141 induction, phagocytic cells, 140 mouse peritoneal macrophages, 143 Neural tube defects (NTDs) embryo, placental and fetal development, 120 inhibition, choline uptake, 109 prevention, 108–109 NTDs. See Neural tube defects Nucleic acid analysis, MALDI‐TOF‐MS assay development, 4–5 instrumental aspects parameters, mass analyzer, 5 reflectron, 6 matrices, 6 preparation and analysis, sample acquisition time, 8 droplet procedure, 7

INDEX SpectroCHIPs, 8 sample purification biotin‐linked terminators, 7 negative charge, sugar‐phosphate backbone, 6–7 P PinPoint assay mass spectra, 9 single base extension, 10 P‐IPG. See Inositol phosphoglycan P‐type Pregnancy adverse outcomes placenta vasculopathy, 127 PROM, 126 animal/in vitro studies apoptosis, 114, 115 elevated tHcy, effects, 113 HTL, 114 hyperhomocysteinemia, 114–115 Mthfrþ/‐, 113 DNA‐methylation and imprinting, 127 early loss/miscarriage, 117–120 elevated tHcy and subfertility homocysteine concentration, 116–117 IVF, 117 embryo, placental and fetal development congenital heart defects, 120–121 fetal size and intrauterine growth retardation, 125–126 neural tube defects, 120 placental development and function, 121–122 placental vascularization and preeclampsia, 122–125 gestational diabetes, 126 long‐term effects in utero, 127–128 NMDA receptor, 128 prospective studies, humans advantage, 116 biological fluids, homocysteine, 116, 118 blood sample collection, 116 tHcy changes, normal amniotic fluid samples, study, 112 in vitro study, 113

183 nutrient‐genetic and lifestyle factors, 111 ovarian stimulation treatment, 112 R

RHD. See Rheumatic heart disease Rheumatic heart disease (RHD) ARF, 32 epidemiology, 32 genetic susceptibility alleles, HLA class II genes, 33 chromosomal location, HLA genes, 36 cytokine gene polymorphisms, 37–38 ficolins, 38 immune response activation, 32 mannan‐binding lectin (MBL), 38 MHC class II molecules, 37 non‐HLA class II genes, 34–35 IL‐4þ cells, 32 lesions Aschoff bodies, 42 cross‐reactivity, 44, 45 cytokines, 45–46 myocardium and valvular tissue, 46 molecular mimicry, immune mechanism antibody‐mediated, 40 S. pyogenes, 39 T‐cell‐mediated, 40–42 types, 39 S SABER. See Single allele base extension reaction SEQureDX technology, 19 Single allele base extension reaction (SABER), 19 Single‐nucleotide polymorphisms (SNPs) CNVs and, 16 genotyping methods, 2–3 nonprimer extension assays, 12–14 primer extension assays, 8–12 haplotyping, 16–17 identification, 15 multiplexing, 19 polymorphism, 37 tag‐SNPs, 2 SNPs. See Single‐nucleotide polymorphisms

184

INDEX T

T‐cell receptor (TCR) degeneracy bacterial and self‐antigens, RF/RHD, 41 demonstration, 42 mechanisms, 43 monoclonal antibodies, 40 TCR. See T‐cell receptor Tea polyphenols black and oolong teas, 156 carcinogenesis chemoprevention, 158 stages, 157 chemical structures, 157 chemopreventive effects, 157 components, 156 derivatives, cancer biotransformation reactions, 166 EGCG analogues, 167 monoesters, 167–168 O‐methylation, 168 peracetate protective groups, 167 reversal effects, 169 in vitro and in vivo preclinical studies biological effects, 159 (‐)‐EGCG, 160–161 esophageal tumor, 159 growth‐inhibitory and antitumor effects, 158 GTCs, 161 prostate cancer, 161 molecular targets, cancer cells DNA protection, methylation and damage, 162–163 MAPKs, 165

oncogene expression, inhibition, 163–164 proteasome activity, 164–165 small‐cell lung carcinoma (SCLC), 166 TRAMP model, 165–166 THAM. See Thymocyte activation molecule tHcy. See Total homocysteine Thrombotic microangiopathy (TMA), 95 Thymocyte activation molecule (THAM), 52 TMA. See Thrombotic microangiopathy Total homocysteine (tHcy) choline intake, 108 congenital heart defects, 120–121 elevated levels and subfertility, 116–117 gestational diabetes, 126 health benefits, 110–111 increased morbidity and mortality, 106 mean maternal, birth, 121–122 miscarriage cases, 117, 119 nonnutritional factors, 110 normal pregnancy, changes, 111–113 NTD‐affected pregnancy, 109 pregnancy and preeclampasia risk, 123–124 riboflavin deficiency, 108 TRAMP. See Transgenic adenocarcinoma of the mouse prostate Transgenic adenocarcinoma of the mouse prostate (TRAMP), 165 U Ubiquitin/proteasome system, 164 W Wegener’s granulomatosis, 70

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