Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 8 uppsatser.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

A thesis submitted in partial fulfilment of tine requirements for the Degree of Master of Technology: Biomedical Technology, Durban University of Technology, 2007. / In 2004, it was reported that KwaZulu-Natal had the greatest number of HIV infected people, approximately 1.8 million people, of whom an estimated 450 000 were in need of antiretroviral drug therapy based on their Cluster of Differentiation 4 (CD4) counts and clinical status. Studies on the success of antiretroviral drugs in improving the quality of life in HIV infected individuals have been extensively performed and published. However, there are no published data on the effect that immune boosters have in improving the quality of life in such persons. Considering the side effects, toxicity, multi-drug regimens and drug resistance problems associated with antiretroviral therapy, alternative or supplementary therapies may play an important role in improving the quality of life in HIV infected people. Such therapy might help in situations where some patients who qualify for antiretroviral treatment are unable to access them because of several reasons such as long waiting lists, travelling costs, unwilling to take antiretroviral drugs, etc. Some patients have reservations in taking antiretroviral drugs. The stigma associated with the disease may be a major factor. The aim of this study was to investigate the change in the immune status of HIV infected patients that were on the Inochi New Medicine immune booster, as well as, to assess the safety and efficacy of this immune booster in improving the patients’ quality of life. / M

Wang, Xiaoan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 145-166). / Abstracts in English and Chinese. / Table of Contents --- p.I / Acknowledgements --- p.VI / Publications --- p.VII / Abstract (in English) --- p.VIII / Abstract (in Chinese) --- p.X / List of Figures --- p.XII / List of Tables --- p.XVI / List of Abbreviations --- p.XVII / Chapter Chapter One. --- Introduction --- p.1 / Chapter 1.1 --- Overview of oseltamivir --- p.1 / Chapter 1.1.1 --- General description of oseltamivir --- p.1 / Chapter 1.1.2 --- Pharmacological activities of oseltamivir --- p.3 / Chapter 1.1.3 --- Pharmacokinetics of oseltamivir --- p.3 / Chapter 1.1.3.1 --- Absorption of oseltamivir --- p.4 / Chapter 1.1.3.2 --- Distribution of oseltamivir --- p.5 / Chapter 1.1.3.3 --- Metabolism of oseltamivir --- p.6 / Chapter 1.1.3.4 --- Elimination of oseltamivir --- p.8 / Chapter 1.1.4 --- Side effects and toxicities of oseltamivir --- p.9 / Chapter 1.2 --- Overview of Chinese medicine formulae CMF1 (Yinqiaosan and Sangjuyin) --- p.9 / Chapter 1.2.1 --- Background and clinical use of CMF1 --- p.9 / Chapter 1.2.2 --- Quality control of CMF1 by manufacturer --- p.11 / Chapter 1.2.3 --- Major active components of CMF1 --- p.12 / Chapter 1.3 --- Previous studies on herb-drug interactions between O and CMF1 --- p.18 / Chapter 1.4 --- Rationale of the current study --- p.19 / Chapter 1.5 --- objectives --- p.19 / Chapter Chapter Two. --- Identification and quantification of major marker compounds in Yinqiaosan and Sangiuyin products --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and methods --- p.23 / Chapter 2.2.1 --- Chemicals --- p.23 / Chapter 2.2.2 --- Instruments --- p.24 / Chapter 2.2.3 --- Chromatographic conditions --- p.24 / Chapter 2.2.4 --- Preparation of standard solutions --- p.25 / Chapter 2.2.5 --- Calibration curves --- p.26 / Chapter 2.2.6 --- Validation of the assay method --- p.26 / Chapter 2.2.7 --- Sample preparations for Yinqiaosan and Sangjuyin products --- p.27 / Chapter 2.2.7.1 --- Sample extraction from Yinqiaosan or Sangjuyin granules --- p.27 / Chapter 2.2.7.2 --- Sample extraction from Yinqiaosan or Sangjuyin tablets --- p.27 / Chapter 2.2.7.3 --- Sample extraction recoveries --- p.27 / Chapter 2.3 --- Results and discussions --- p.28 / Chapter 2.3.1 --- Chromatography --- p.28 / Chapter 2.3.2 --- Linearity and sensitivity --- p.33 / Chapter 2.3.3 --- Accuracy and precision --- p.33 / Chapter 2.3.4 --- Stability --- p.36 / Chapter 2.3.5 --- Contents of identified active components in commercial available Yinqiaosan or Sangjuyin products and CMF1 --- p.36 / Chapter 2.3.6 --- Sample extraction recovery --- p.40 / Chapter 2.4 --- Conclusion --- p.43 / Chapter Chapter Three. --- Effect of CMF1/CMF1 components on the metabolism of oseltamivir and related mechanistic studies --- p.44 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and methods --- p.47 / Chapter 3.2.1 --- Materials --- p.47 / Chapter 3.2.2 --- "Verification of metabolism of O in rat GI tract, plasma and liver microsome" --- p.48 / Chapter 3.2.3 --- Inhibition of metabolism of O by CMFl/CMFl components --- p.49 / Chapter 3.2.3.1 --- In vitro inhibition of metabolism of O in rat plasma --- p.49 / Chapter 3.2.3.2 --- In vitro inhibition of metabolism of O in rat liver microsome (RLM) --- p.49 / Chapter 3.2.4 --- Mechanistic study of enzyme inhibition of O in recombinant human Carboxylesterase 1 (hCE 1) --- p.50 / Chapter 3.2.5 --- Sample preparation and LC/MS/MS analysis --- p.50 / Chapter 3.2.6 --- Data analyses --- p.52 / Chapter 3.3 --- Results --- p.53 / Chapter 3.3.1 --- "Verification of metabolism of O in rat GI tract, plasma and liver microsome" --- p.53 / Chapter 3.3.2 --- Inhibition of metabolism of O by CMF1/CMF1 components --- p.53 / Chapter 3.3.2.1 --- Enzyme inhibition of metabolism of O by CMFl/CMF1 components in rat plasma --- p.53 / Chapter 3.3.2.2 --- Enzyme inhibition of metabolism of O by CMF1/CMF1 components in rat liver microsome (RLM) --- p.58 / Chapter 3.3.2.3 --- Selection of potent enzyme inhibitor from CMF1 --- p.60 / Chapter 3.3.4. --- Mechanistic study of enzyme inhibition of O in recombinant human Carboxylesterase 1 (hCE 1) --- p.61 / Chapter 3.4 --- Discussions --- p.63 / Chapter 3.5 --- Conclusion --- p.74 / Chapter Chapter Four. --- Effect of CMFl/CMFl components on the absorption of oseltamivir and related mechanistic studies --- p.75 / Chapter 4.1 --- Introduction --- p.75 / Chapter 4.2 --- Materials and methods --- p.79 / Chapter 4.2.1 --- Materials --- p.79 / Chapter 4.2.2 --- PAMPA permeation model --- p.80 / Chapter 4.2.2.1 --- Permeation of O and OC in PAMPA --- p.80 / Chapter 4.2.2.2 --- Sample preparation and LC/MS/MS analysis --- p.81 / Chapter 4.2.2.3 --- Data analysis --- p.81 / Chapter 4.2.3 --- Absorption of O in presence of CMF/CMFl components in Caco-2 and MDCK cell monolayer models --- p.82 / Chapter 4.2.3.1 --- Cell culture --- p.82 / Chapter 4.2.3.2 --- Preparation of loading solutions to the cell models --- p.83 / Chapter 4.2.3.3 --- Stability of O in transport buffer --- p.84 / Chapter 4.2.3.4 --- Cytotoxicity tests of O and CMFl/CMFl components --- p.84 / Chapter 4.2.3.5 --- Transport study in Caco-2 and MDCK monolayer model --- p.85 / Chapter 4.2.3.6 --- Sample preparation and LC/MS/MS analysis --- p.86 / Chapter 4.2.3.7 --- Data analysis --- p.87 / Chapter 4.2.4 --- Absorption of O in presence of CMF 1 in rat in situ single pass intestinal perfusion model --- p.88 / Chapter 4.2.4.1 --- Preparation of perfusion solutions --- p.88 / Chapter 4.2.4.2 --- Stabilities of O and arctigenin in perfusate --- p.88 / Chapter 4.2.4.3 --- Rat in situ single pass intestinal perfusion of O in presence and absence of CMFl and relevant inhibitors --- p.89 / Chapter 4.2.4.4 --- Sample preparation and LC/MS/MS analysis --- p.90 / Chapter 4.2.4.5 --- Data analysis --- p.90 / Chapter 4.3 --- Resul ts --- p.91 / Chapter 4.3.1 --- Permeation of O and OC in PAMPA --- p.91 / Chapter 4.3.2 --- Absorption of O in presence of CMF/CMF1 components in Caco-2 and MDCK cell monolayer models --- p.92 / Chapter 4.3.2.1 --- Stabilities of O in transport buffer --- p.92 / Chapter 4.3.2.2 --- Cytotoxicity tests of O and CMF1/CMF1 components in transport buffer --- p.93 / Chapter 4.3.2.3 --- Proof of O as a substrate of P-gp by Caco-2 cell model --- p.95 / Chapter 4.3.2.4 --- Effect of CMF 1 on the absorption transport of o in Caco-2 cell mode --- p.98 / Chapter 4.3.2.5 --- Effect of CMF1 components on the absorption transport of o in Caco-2 cell model --- p.102 / Chapter 4.3.2.6 --- Effect of arctigenin on bi-directional transport of o in Caco- 2 cell model --- p.106 / Chapter 4.3.2.7 --- Proof of O as a substrate of P-gp by MDCK transfected cell lines --- p.108 / Chapter 4.3.2.8 --- Bi-directional transport of O in MDCK-MDR1 cell model --- p.111 / Chapter 4.3.2.9 --- Effect of CMF 1 on the absorption transport of O in MDCK-MDR1 cell model --- p.112 / Chapter 4.3.3 --- Absorption of O in presence of CMF1 in rat in situ single pass intestinal perfusion model --- p.113 / Chapter 4.3.3.1 --- Stabilities of O and arctigenin in the perfusion buffer --- p.113 / Chapter 4.3.3.2 --- Intestinal absorption of O in presence and absence of CMF1 in rat in situ intestinal perfusion model --- p.114 / Chapter 4.4 --- Discussions --- p.116 / Chapter 4.5 --- Conclusion --- p.124 / Chapter Chapter Five. --- Preliminary evaluation of antiviral activity of CMFl/CMFl components --- p.125 / Chapter 5.1 --- Introduction --- p.125 / Chapter 5.2 --- Materials and methods --- p.128 / Chapter 5.2.1 --- Materials and animals --- p.128 / Chapter 5.2.2 --- Animal treatment --- p.129 / Chapter 5.2.3 --- Plasma sample collection and preparation --- p.130 / Chapter 5.2.4 --- Evaluation of antiviral activities of CMFl/ CMFl components --- p.130 / Chapter 5.2.4.1 --- Plaque reduction assay --- p.131 / Chapter 5.2.4.2 --- Optimization of plasma sample dilution ratio --- p.131 / Chapter 5.2.5 --- Data analyses --- p.133 / Chapter 5.3 --- Results and discussions --- p.135 / Chapter 5.3.1 --- Ex vivo evaluation of antiviral activity of CMF1 --- p.135 / Chapter 5.3.2 --- In vitro evaluation of antiviral activity of CMF1 major marker compounds --- p.139 / Chapter 5.4 --- Conclusion --- p.141 / Chapter Chapter Six. --- Overall conclusion --- p.142 / References --- p.145

This dissertation is divided into two main sections describing major portions of my Ph.D. research: (1) development of two enzymatic assays for identifying inhibitors of SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and the associated proofreading exonuclease complexes, two key enzymatic activities of SARS-CoV-2, the virus responsible for the COVID-19 pandemic and (2) the design and implementation of four novel single-color fluorescent DNA sequencing by synthesis (SBS) methods, including the synthesis of many of the key nucleotide analogues required for these studies. In response to the COVID-19 pandemic, the first part of my research is focused on the discovery of potential therapeutics for combating coronavirus infections. Chapter 1 describes the identification of several polymerase and exonuclease inhibitors for SARS-CoV-2 using novel mass spectrometry-based molecular assays. SARS-CoV-2 has an exonuclease complex, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RdRp and the exonuclease could overcome this deficiency. Chapter 1 reports the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of identified exonuclease inhibitors, RNAs terminated with the active forms of the prodrugs like Sofosbuvir, Remdesivir and Favipiravir were largely protected from excision by the exonuclease, while in the absence of exonuclease inhibitors, there was rapid excision. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment. Chapters 2-6 describe the single-color DNA SBS studies. Chapter 2 provides essential background on the structure of DNA, the DNA polymerase reaction, and several key DNA sequencing technologies, with an emphasis on the design of nucleotide analogues for the DNA SBS approach. Chapter 3 delineates a one-color fluorescent DNA SBS method based on a set of nucleotide reversible terminators (NRTs) comprising two orthogonal cleavable linkers, one fluorescent dye and one anchor. Chapter 4 describes a one-color hybrid DNA sequencing approach using a set of dideoxynucleotide analogues bearing two orthogonal cleavable linkers, one fluorophore and one anchor as well as a set of unlabeled NRTs. By introducing a pH responsive fluorophore into the design of nucleotide analogues, Chapter 5 demonstrates a novel type of single-color DNA SBS method using a set of NRTs comprising one pH-responsive fluorescent dye or one non-responsive fluorescent dye tethered with one cleavable linker. Chapter 6 presents another option for the single-color DNA sequencing technique using a set of deoxynucleotide analogues comprising the above pH responsive or non-responsive dyes tethered with a cleavable linker, along with a set of unlabeled NRTs. The one-color SBS approaches have the potential for higher sensitivity, miniaturization and cost effectiveness compared with four-color SBS methods. Finally, Chapter 7 summarizes the SARS-CoV-2 antiviral drug discovery and one-color sequencing techniques and discusses potential follow-up research on these projects.

Thesis (M.Tech.: Biomedical Technology)-Dept. of Biomedical Technology, Durban University of Technology, 2007 xxiii, 244 leaves / In 2004, it was reported that KwaZulu-Natal had the greatest number of HIV infected people, approximately 1.8 million people, of whom an estimated 450 000 were in need of antiretroviral drug therapy based on their Cluster of Differentiation 4 (CD4) counts and clinical status. Studies on the success of antiretroviral drugs in improving the quality of life in HIV infected individuals have been extensively performed and published. However, there are no published data on the effect that immune boosters have in improving the quality of life in such persons. Considering the side effects, toxicity, multi-drug regimens and drug resistance problems associated with antiretroviral therapy, alternative or supplementary therapies may play an important role in improving the quality of life in HIV infected people. Such therapy might help in situations where some patients who qualify for antiretroviral treatment are unable to access them because of several reasons such as long waiting lists, travelling costs, unwilling to take antiretroviral drugs, etc. Some patients have reservations in taking antiretroviral drugs. The stigma associated with the disease may be a major factor. The aim of this study was to investigate the change in the immune status of HIV infected patients that were on the Inochi New Medicine immune booster, as well as, to assess the safety and efficacy of this immune booster in improving the patients’ quality of life.

病原體感染是包括植物的高等生物的主要健康危害之一。為抵禦入侵者，大多數植物會製造防禦蛋白，包括凝集素、蛋白酶抑製劑、抗真菌蛋白、核糖核酸酶和核糖體失活蛋白，並分佈在不同的器官，如葉、根、種子和塊莖。一些植物防禦蛋白被發現能表現出多種生物活性，如抗腫瘤活性、抗細菌活性和抗病毒活性，能抵抗多種植物病原菌和人類病原體。因此，一些植物防禦蛋白可能有潛力用於治療人類疾病，或保護農作物免受感染。 / 我們在研究中從不同的植物來源成功純化出各種防禦蛋白，包括：小芋頭塊莖中的血凝素、日本長芋中的凝集素、東北紅豆中的血凝素和抗真菌多肽、棕色芸豆中的凝集素、抗真菌多肽和胰蛋白酶抑製劑，玉豆一號中的凝集素以及小斑豆中的胰蛋白酶抑製劑。小芋頭血凝素被發現能誘導脾細胞的有絲分裂反應。日本長芋凝集素和東北紅豆血凝素被發現能對一些腫瘤細胞株（如乳腺癌MCF7細胞及鼻咽癌CNE2細胞）發揮抗增殖的作用。棕色芸豆凝集素能誘導脾臟細胞的有絲分裂反應以及抑制腫瘤細胞株（如乳腺癌MCF7細胞、肝癌HepG2及鼻咽癌CNE1和 CNE2細胞）的生長，而棕色芸豆抗真菌蛋白能抑制數種病原真菌物種的生長。研究這些防禦蛋白的生物活性有助找出其潛在應用價值，如藥用前景。 / Infection from pathogens is one of the major health hazards in higher organisms including plants. To defend against harmful invaders, most plants produce a variety of defense proteins including lectins, protease inhibitors, antifungal proteins, ribonucleases and ribosome-inactivating proteins. They may be present in different organs of the plants, such as leaves, roots, seeds and tubers. Some of the plant defense proteins were found to exhibit a variety of biological activities such as anti-tumor activity, anti-bacterial activity and anti-viral activity that act against various plant pathogens and also some human pathogens. Therefore, some plant defense proteins may have potential for therapeutic applications in human diseases, or protecting the crops from infections. / This study involved purification of defense proteins from different plant sources. The proteins that were successfully isolated included a hemagglutinin from small taro tubers, a lectin from Japanese yam tubers, a lectin and an antifungal peptide from northeast red beans, a lectin, an antifungal peptide and a trypsin inhibitor from brown kidney beans, a lectin from French bean cultivar no. 1 and a trypsin inhibitor from mini pinto beans. The small taro hemagglutinin was found to induce mitogenic response in splenocytes. The Japanese yam lectin and northeast red bean hemagglutinin were found to exert anti-proliferative activity toward some tumor cell lines including MCF7 and CNE2 cells. The brown kidney bean lectin induced a mitogenic response from murine splenocytes as well as inhibited the growth of tumor cell lines including MCF7, HepG2, CNE1 and CNE2 cells, while the brown kidney bean antifungal protein inhibited the growth of several pathogenic fungal species including M. arachidicola, S. turcica and B. maydis. Studying the biological activities of these defense proteins helps to find out their potential applications like therapeutic uses. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chan, Yau Sang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves i-xvii). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i-ii / 論文摘要 --- p.iii / Acknowledgements --- p.iv / List of Publications --- p.v / Table of Contents --- p.vi-vii / List of Figures --- p.viii-ix / List of Tables --- p.x / List of Abbreviations --- p.xi / Chapter Chapter 1 --- Introduction on plant defense proteins / Chapter 1.1 --- General introduction to plant defense proteins --- p.1-2 / Chapter 1.2 --- An overview on lectins --- p.3-18 / Chapter 1.2.1 --- History of lectins --- p.3-6 / Chapter 1.2.2 --- Classification of lectins --- p.7-11 / Chapter 1.2.3 --- Biological activities of lectins --- p.12-16 / Chapter 1.2.4 --- Applications of plant lectins --- p.16-18 / Chapter 1.3 --- An overview on defensins --- p.18-25 / Chapter 1.3.1 --- Types of defensins --- p.18-21 / Chapter 1.3.2 --- Mechanism of anti-microbial activity of defensins --- p.22-23 / Chapter 1.3.3 --- Application of defensins --- p.23-25 / Chapter 1.4 --- An overview on trypsin inhibitors --- p.25-38 / Chapter 1.4.1 --- Serpins --- p.26-28 / Chapter 1.4.2 --- Kunitz-type protease inhibitors --- p.29-31 / Chapter 1.4.3 --- Bowman-Birk protease inhibitors --- p.32-34 / Chapter 1.4.4 --- Physiological functions of protease inhibitors --- p.35-38 / Chapter 1.5 --- Aim of study --- p.38-41 / Chapter Chapter 2 --- Isolation and characterization of a hemagglutinin from small taros and a lectin from yam tubers / Chapter 2.1 --- Introduction --- p.42-45 / Chapter 2.2 --- Materials and Methods --- p.46-55 / Chapter 2.3 --- Results --- p.56-78 / Chapter 2.4 --- Discussion --- p.79-84 / Chapter Chapter 3 --- Isolation and characterization of two defense proteins from seeds of Phaseolus vulgaris cv. “northeast red bean“ / Chapter 3.1 --- Introduction --- p.85-86 / Chapter 3.2 --- Materials and Methods --- p.87-93 / Chapter 3.3 --- Results --- p.93-119 / Chapter 3.4 --- Discussion --- p.120-129 / Chapter Chapter 4 --- Isolation and characterization of three defense proteins from seeds of Phaseolus vulgaris cv. “brown kidney bean“ / Chapter 4.1 --- Introduction --- p.130-131 / Chapter 4.2 --- Materials and Methods --- p.131-136 / Chapter 4.3 --- Results --- p.136-175 / Chapter 4.4 --- Discussion --- p.176-189 / Chapter Chapter 5 --- Isolation and characterization of a lectin from French bean cultivar no. 1 beans and a trypsin inhibitor from mini pinto beans / Chapter 5.1 --- Introduction --- p.190-191 / Chapter 5.2 --- Materials and Methods --- p.191-194 / Chapter 5.3 --- Results --- p.195-212 / Chapter 5.4 --- Discussion --- p.213-221 / Chapter Chapter 6 --- General discussion / Chapter 6.1 --- Summary on purification protocols of the defense proteins in the study --- p.222-228 / Chapter 6.2 --- Chemical properties of the defense proteins in the study --- p.228-232 / Chapter 6.3 --- Biological activities of the defense proteins in the study --- p.232-238 / Chapter 6.4 --- Potential application of these defense proteins and future perspectives --- p.238-242 / References --- p.i-xvi

Na infecção crônica pelo vírus da hepatite C (VHC), a persistência do vírus e a resposta à terapia antiviral tem sido associada com a produção de níveis inadequados de diversas citocinas na resposta imunológica e inflamatória. A interleucina 10 (IL10) é uma potente citocina antiinflamatória e parece ter um papel importante na resposta do hospedeiro ao VHC. A produção de IL10 varia de acordo com a composição genética no locus da IL10. Vários sítios polimórficos foram descritos na região promotora do gene da IL10 e um polimorfismo de nucleotídeo único na posição -1082, relativa ao sitio de início de transcrição, é associado com expressão diferencial de IL10. A presença do alelo G nesta região esta associada à alta produção desta citocina. Neste estudo, nós avaliamos a freqüência dos polimorfismos -1082 (G>A), -819 (C>T) e -592 (C>A) da região promotora do gene da IL10 e sua associação com o desfecho à terapia antiviral na infecção crônica pelo VHC. O DNA genômico de 54 pacientes classificados como respondedores e 54 não respondedores à terapia combinada de interferon (convencional ou peguilado) e ribavirina foi genotipado para esses polimorfismos por PCR utilizando iniciadores alelo-específicos, seguidos de eletroforese em gel de agarose a 3%. As distribuições observadas para o polimorfismo -1082 foram: AA 43,3%, GA 40,7% e GG 13,0% entre os pacientes não respondedores; e AA 31,5%, GA 46,3% e GG 22,2% entre os respondedores. Para o polimorfismo -592, as distribuições observadas foram: CC 50,0%, CA 38,7% e AA 11,0% entre os não respondedores a terapia; e CC 42,6%, CA 53,7% e AA 3,7% para os pacientes respondedores. O alelo A do polimorfismo -1082 foi identificado mais freqüentemente entre os pacientes não respondedores do que nos respondedores (P=0,0352). Estes resultados sugerem que a presença deste alelo, que afeta a produção da citocina, possa estar associado a uma desfavorável resposta à terapia antiviral na infecção pelo VHC. / In HCV infections, the persistence of the virus and the response to antiviral therapy has been shown to be associated with the production of inappropriate cytokine levels in inflammatory and immune response. Interleukin-10 (IL10) is a potent anti-inflammatory cytokine and possibly has important role in the host outcome to HCV. IL-10 production varies according to the genetic composition of the IL-10 locus. Several polymorphic sites within the promoter region of the IL-10 gene have been described and a single nucleotide polymorphism at position -1082 (G/A) relative to the transcription start site is associated with differential IL-10 expression. The presence of G allele in this region was involved with high production of the anti-inflammatory cytokine IL-10. In this study, we have evaluated the frequency of the polymorphisms -1082 (G>A), -819 (C>T) and -592 (C>A) of the IL-10 gene promoter and their association with the response to antiviral therapy in chronic hepatitis C infection. Genomic DNA from 54 patients classified as responders and 54 nonresponders to a combination of interferon alpha (conventional and peginterferon) and ribavirin was evaluated by molecular typing by polymerase chain reaction (PCR) with allele-specific primers, followed by electrophoresis on agarose gels (3%). The distribution of the genotypes AA, GA and GG for the polymorphism -1082 in the studied individuals was for nonresponders 46,3%, 40,7% and 13,0%, respectively, and among the responders was 31,5%, 46,3% and 22,2%, respectively. The distribution of the genotypes CC, CA and AA for the polymorphism -592 in the studied individuals was for nonresponders 50,0%, 38,7% and 11,1%, respectively, and among the responders was 42,6%, 53,7% and 3,7%, respectively. The interleukin-10 -1082 A allele was identified more frequently in patients nonresponders than in responders (P=0.0352).These results suggest that presence of the interleukin-10 -1082 A allele, which appears to affect the cytokine production, may be associated with a unfavorable outcome of HCV infection.

Na infecção crônica pelo vírus da hepatite C (VHC), a persistência do vírus e a resposta à terapia antiviral tem sido associada com a produção de níveis inadequados de diversas citocinas na resposta imunológica e inflamatória. A interleucina 10 (IL10) é uma potente citocina antiinflamatória e parece ter um papel importante na resposta do hospedeiro ao VHC. A produção de IL10 varia de acordo com a composição genética no locus da IL10. Vários sítios polimórficos foram descritos na região promotora do gene da IL10 e um polimorfismo de nucleotídeo único na posição -1082, relativa ao sitio de início de transcrição, é associado com expressão diferencial de IL10. A presença do alelo G nesta região esta associada à alta produção desta citocina. Neste estudo, nós avaliamos a freqüência dos polimorfismos -1082 (G>A), -819 (C>T) e -592 (C>A) da região promotora do gene da IL10 e sua associação com o desfecho à terapia antiviral na infecção crônica pelo VHC. O DNA genômico de 54 pacientes classificados como respondedores e 54 não respondedores à terapia combinada de interferon (convencional ou peguilado) e ribavirina foi genotipado para esses polimorfismos por PCR utilizando iniciadores alelo-específicos, seguidos de eletroforese em gel de agarose a 3%. As distribuições observadas para o polimorfismo -1082 foram: AA 43,3%, GA 40,7% e GG 13,0% entre os pacientes não respondedores; e AA 31,5%, GA 46,3% e GG 22,2% entre os respondedores. Para o polimorfismo -592, as distribuições observadas foram: CC 50,0%, CA 38,7% e AA 11,0% entre os não respondedores a terapia; e CC 42,6%, CA 53,7% e AA 3,7% para os pacientes respondedores. O alelo A do polimorfismo -1082 foi identificado mais freqüentemente entre os pacientes não respondedores do que nos respondedores (P=0,0352). Estes resultados sugerem que a presença deste alelo, que afeta a produção da citocina, possa estar associado a uma desfavorável resposta à terapia antiviral na infecção pelo VHC. / In HCV infections, the persistence of the virus and the response to antiviral therapy has been shown to be associated with the production of inappropriate cytokine levels in inflammatory and immune response. Interleukin-10 (IL10) is a potent anti-inflammatory cytokine and possibly has important role in the host outcome to HCV. IL-10 production varies according to the genetic composition of the IL-10 locus. Several polymorphic sites within the promoter region of the IL-10 gene have been described and a single nucleotide polymorphism at position -1082 (G/A) relative to the transcription start site is associated with differential IL-10 expression. The presence of G allele in this region was involved with high production of the anti-inflammatory cytokine IL-10. In this study, we have evaluated the frequency of the polymorphisms -1082 (G>A), -819 (C>T) and -592 (C>A) of the IL-10 gene promoter and their association with the response to antiviral therapy in chronic hepatitis C infection. Genomic DNA from 54 patients classified as responders and 54 nonresponders to a combination of interferon alpha (conventional and peginterferon) and ribavirin was evaluated by molecular typing by polymerase chain reaction (PCR) with allele-specific primers, followed by electrophoresis on agarose gels (3%). The distribution of the genotypes AA, GA and GG for the polymorphism -1082 in the studied individuals was for nonresponders 46,3%, 40,7% and 13,0%, respectively, and among the responders was 31,5%, 46,3% and 22,2%, respectively. The distribution of the genotypes CC, CA and AA for the polymorphism -592 in the studied individuals was for nonresponders 50,0%, 38,7% and 11,1%, respectively, and among the responders was 42,6%, 53,7% and 3,7%, respectively. The interleukin-10 -1082 A allele was identified more frequently in patients nonresponders than in responders (P=0.0352).These results suggest that presence of the interleukin-10 -1082 A allele, which appears to affect the cytokine production, may be associated with a unfavorable outcome of HCV infection.