Abstract

INTRODUCTION:

Hepatic fibrosis progression in patients with chronic hepatitis C virus infections has been associated with viral and host factors, including genetic polymorphisms. Human platelet antigen polymorphisms are associated with the rapid development of fibrosis in HCV-monoinfected patients. This study aimed to determine whether such an association exists in human immunodeficiency virus-1/hepatitis C virus-coinfected patients.

METHODS:

Genomic deoxyribonucleic acid from 36 human immunodeficiency virus-1/hepatitis C virus-coinfected patients was genotyped to determine the presence of human platelet antigens-1, -3, or -5 polymorphisms. Fibrosis progression was evaluated using the Metavir scoring system, and the patients were assigned to two groups, namely, G1 that comprised patients with F1, portal fibrosis without septa, or F2, few septa (n = 23) and G2 that comprised patients with F3, numerous septa, or F4, cirrhosis (n = 13). Fisher's exact test was utilized to determine possible associations between the human platelet antigen polymorphisms and fibrosis progression.

RESULTS:

There were no deviations from the Hardy-Weinberg equilibrium in the human platelet antigen systems evaluated. Statistically significant differences were not observed between G1 and G2 with respect to the distributions of the allelic and genotypic frequencies of the human platelet antigen systems.

CONCLUSION:

The greater stimulation of hepatic stellate cells by the human immunodeficiency virus and, consequently, the increased expression of transforming growth factor beta can offset the effect of human platelet antigen polymorphism on the progression of fibrosis in patients coinfected with the human immunodeficiency virus-1 and the hepatitis C virus.

Keywords:
Coinfection; Hepatitis C vírus; Human immunodeficiency vírus; Human platelet antigen; Liver fibrosis

INTRODUCTION

The progression of hepatic fibrosis in patients with chronic hepatitis C virus (HCV) infection depends on different viral and host factors. Sex, age at infection, consumption of alcohol, coinfection with the human immunodeficiency virus (HIV) and coinfection with the hepatitis B virus have been associated with liver fibrosis progression ¹1 Poynard T, Ratziu V, Charlotte F, Goodman Z, McHutchison J, Albrecht J. Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis C. J Hepatol 2001; 34:730-739. ²2 Deuffic S, Buffat L, Poynard T, Valleron AJ. Modeling the hepatitis C virus epidemic in France. Hepatol 1999; 29:1596-1601. ³3 Seef LB. Natural history of chronic hepatitis C. Hepatol 2002; 36:35-46. . Similarly, host genetic factors have also been associated with this progression. Besides polymorphisms in the genes coding for human leukocyte antigens, other genetic polymorphisms in the genes coding for interleukin 10, tumor necrosis factor alpha, angiotensinogen, and transforming growth factor (TGF)-β1 have been associated with the progression of fibrosis in HCV-moinfected patients ⁴4 Powell EE, Edwards-Smith CJ, Hay JL, Clouston AD, Crawford DH, Shorthouse C, et al. Host genetic factors influence disease progression in chronic hepatitis C. Hepatol 2000; 31:828-833. .

Similarly, the expression of some integrins, which are present in a variety of cell types, including platelets ⁵5 Takada Y, Simon S. Protein family review: The integrins. Genome Biol 2007; 8:215-219. and liver stellate cells ⁶6 Racine-Samson L, Rockey DC, Bissell DM. The role of α1β1 integrin in wound contraction. A quantitative analysis of liver myofibroblasts in vivo and in primary culture. J Biol Chem 1997; 272:30911-30917. ⁷7 Gao R, Brigstock DR. Connective tissue growth factor (CCN2) induces adhesion of rat activated hepatic stellate cells by binding of its C-terminal domain to integrin alpha(v) beta(3) and heparin sulfate proteoglycan. J Biol Chem2004; 279:8848-8855. , has been associated with the development of fibrosis ⁸8 Nejari M, Couvelard A, Mosnier JF, Moreau A, Feldmann G, Degott C, et al. Integrin up-regulation in chronic liver disease: Relationship with inflammation and fibrosis in chronic hepatitis C. J Pathol 2001; 195:473-481. ⁹9 Popov Y, Patsenker E, Stickel F, Zaks J, Bhaskar KR, Niedobitek G, et al. Integrin αvβ6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies. J Hepatol2008; 48:453-648. . The integrins that are present on platelet membranes express different types of antigens, and, of these, the human platelet antigens (HPA) have received considerable attention ¹⁰10 Schroeder ML, Rayner HL. Red cell, platelet and white cell antigens. : Lee GR, Bithell TC, Foerster J, Athens JW, Lukens JN, editors. Wintrobe´s Clinical Hematology. 9th ed. Philadelphia (PA): Lea and Febiger; 1993. p. 616-646. .

Human platelet antigens are polymorphic within the population, and the polymorphism in most of these antigens is caused by the substitution of a single amino acid in the protein, which is the consequence of the substitution of one nucleotide in the deoxyribonucleic acid (DNA) ¹¹11 Lucas GF, Metcalfe P. Platelet and granulocyte polymorphisms. Transfus Med 2000; 10:157-174. ¹²12 Metcalfe P, Watkins NA, Ouwehand WH, Kaplan C, Newman P, Kekomaki R, et al. Nomenclature of human platelet antigens. Vox Sang 2003; 85:240-245. . The polymorphisms of some of the HPA systems have been associated with diseases involving platelets ¹³13 Muller-Eckhardt C, Kiefel V, Santoso S. Review and update of platelet alloantigen systems. Transfus Med Rev 1999; 4:98. and with disorders that do not involve platelet disturbances, including sickle cell anemia ¹⁴14 Castro V, Alberto FL, Costa RN, Lepikson-Neto J, Gualandro SF, Figueiredo MS, et al. Polymorphism of the human platelet antigen-5 system is a risk factor for occlusive vascular complications in patients with sickle cell anemia. Vox Sang2004; 87:118-123. , myocardial infarction ¹⁵15 Rosenberg N, Zivelin A, Chetrit A, Dardik R, Kornbrot N, Freimark D, et al. Effects of platelet membrane glycoprotein polymorphisms on the risk of myocardial infarction in young males. Isr Med Assoc J 2002; 4:411-414. , and venous thrombosis ¹⁶16 Ridker PM, Hennekens CH, Schmitz C, Stampfer MJ, Lindpaintner K PlA1/A2 polymorphism of platelet glycoprotein IIIa and risks of myocardial infarction, stroke and venous thrombosis. Lancet 1997; 349:385-388. . Recently, HPA polymorphism has also been associated with viral infections, including dengue virus infections ¹⁷17 Soundravally R, Hoti SL. Immunopathogenesis of dengue hemorrhagic fever and shock syndrome: Role of TAP and HPA gene polymorphism. Hum Imnunol 2007; 68:973-979. , HCV monoinfections ¹⁸18 Verdichio-Moraes CF, Toralles-Pereira C, Grotto RMT, Silva GF, Pardini MIMC. Allelic frequencies of HPA-1 to -5 human platelet antigens in hepatitis C virus-infected patients. J Med Virol 2009; 81:757-759. , and HIV/HCV coinfection (Grotto et al: in press).

In addition, the findings from a recent study demonstrated an association between the HPA-1a/1b genotype and the rapid development of fibrosis in HCV-monoinfected patients ¹⁹19 Silva GF, Grotto RM, Verdichio-Moraes CF, Corvino SM, Ferrasi AC, Silveira LV, et al. Human Platelet Antigen (HPA) genotype is associated with fibrosis progression in chronic hepatitis C. J Med Virol2012; 84:56-60. . However, whether associations exist between HPA polymorphisms and the progression of fibrosis in HIV/HCV-coinfected patients is unclear.

The aim of this study was to determine possible associations between HPA-1, HPA-3, and HPA-5 polymorphisms and the progression of hepatic fibrosis in individuals coinfected with HIV-1 and HCV.

METHODS

Patients

Samples of ethylenediaminetetraacetic acid-anticoagulated peripheral venous blood were collected from 36 HIV-1/HCV-coinfected patients who were seen at the Specialized Outpatient Service "Domingos Alves Meira" and at the Department of Internal Medicine, Gastroenterology Division, Botucatu School of Medicine, São Paulo State University, UNESP, Botucatu, SP, Brazil. The study's inclusion criteria were the presence of an HIV-1/HCV coinfection that was determined using the reverse transcription-polymerase chain reaction (RT-PCR) a liver biopsy performed before antiviral treatment began, the absence of other hepatic diseases, and the provision of signed informed consent.

The levels of fibrosis were determined from liver biopsies, which were obtained using Menghini or Tru-Cut needles, and the fragments were analyzed when at least eight portal spaces were present. The tissues underwent hematoxylin and eosin, Masson trichrome, and reticulin staining. The biopsies were analyzed by a pathologist who used the Metavir scoring system ²⁰20 Poynard T, Bedossa P, Opolon P. For the OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Natural history of liver fibrosis progression in patients with chronic hepatitis C. Lancet1997; 349:825-832. to evaluate fibrosis progression. The patients were assigned to Group 1 (G1) that comprised HIV-1/HCV-coinfected patients with lower stages of fibrosis (F1, portal fibrosis without septa or F2, few septa) or Group 2 (G2) that comprised HIV-1/HCV-coinfected patients with higher degrees of fibrosis (F3, numerous septa or F4, cirrhosis). In the study, patients who were infected with the HCV genotypes 1, 2, or 3, and the HIV subtypes B, F, or BF recombinant were included. Information was obtained from the databank regarding each patient's sex, age, and time of infection, which was defined as the time that had elapsed between the presumed date of infection and the biopsy date; this information was available for 27 of the 36 patients studied. Information was also obtained about the degree of alcohol abuse, which was defined as more than 40g per day for women and over 80g per day for men; this information was available for 29 of the 36 patients studied.

Deoxyribonucleic acid extraction and human platelet antigen genotyping

Genomic DNA was isolated from whole blood using a commercial AxyPrep Blood Genomic DNA Miniprep Kit (Axygen Scientific, Inc., Union City, CA, USA). HPA-1 and HPA-3 were genotyped using PCR sequence-specific primers according to Kluter et al. ²¹21 Kluter H, Fehlau K, Panzer S, Kirchner H, Bein G. Rapid typing for human platelet antigen systems -1, -2, -3 and -5 by PCR amplification with sequence-specific primers. Vox Sang1996; 71:121-125. , and the CRP gene was used as the internal positive control in all reactions. HPA-5 was genotyped using PCR-restriction fragment length polymorphism analysis according to Kalb et al. ²²22 Kalb R, Santoso S, Unkelbach K, Kiefel V, Mueller-Eckhardt C. Localization of the Br polymorphism on a 144bp exon of the GPIa gene and its application for platelet DNA typing. Thromb Haemost 1994; 71:651-654. . A negative control was used in all of the PCR assays.

Statistical analysis

The Hardy-Weinberg equilibrium test was carried out to evaluate the distributions of the allelic frequencies of HPA-1, HPA-3, and HPA-5 according to the degrees of fibrosis. Fisher's exact test was used to investigate possible associations between the HPA alleles, genotypes, and the degrees of fibrosis in HIV-1/HCV-coinfected patients. The level of significance for all of the statistical tests was set at 0.05.

Ethical considerations

The study was approved by the Research Ethics Committee of Botucatu Medical School, UNESP (3750-2010). A written informed consent was obtained from patients participating in the study.

RESULTS

Table 1 shows the demographic and virological characteristics of the patients who participated in the study.

There were no deviations from the Hardy-Weinberg equilibrium in the HPA systems evaluated. No statistically significant differences were found between the patient groups in relation to the distributions of the allelic and genotypic frequencies of the HPA systems ( Table 2 ).

DISCUSSION

The progression of hepatic fibrosis in patients with HCV infections has been associated with factors that include sex, alcohol consumption, the age at infection ¹1 Poynard T, Ratziu V, Charlotte F, Goodman Z, McHutchison J, Albrecht J. Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis C. J Hepatol 2001; 34:730-739. ²2 Deuffic S, Buffat L, Poynard T, Valleron AJ. Modeling the hepatitis C virus epidemic in France. Hepatol 1999; 29:1596-1601. ³3 Seef LB. Natural history of chronic hepatitis C. Hepatol 2002; 36:35-46. , and genetic factors ⁴4 Powell EE, Edwards-Smith CJ, Hay JL, Clouston AD, Crawford DH, Shorthouse C, et al. Host genetic factors influence disease progression in chronic hepatitis C. Hepatol 2000; 31:828-833. .

Fibrosis develops mainly as a consequence of the activity of the liver stellate cells ²³23 Gabele E, Brenner DA, Rippe RA. Liver fibrosis: signals leading to the amplification of the fibrogenic hepatic stellate cell. Front Biosci 2003; 8:69-77. ²⁴24 Friedman SL. Liver fibrosis - from bench to bedside. J Hepatol2003; 38:38-53. . The activation of the liver stellate cells has been associated with regulation by the integrin family of proteins ⁸8 Nejari M, Couvelard A, Mosnier JF, Moreau A, Feldmann G, Degott C, et al. Integrin up-regulation in chronic liver disease: Relationship with inflammation and fibrosis in chronic hepatitis C. J Pathol 2001; 195:473-481. ⁹9 Popov Y, Patsenker E, Stickel F, Zaks J, Bhaskar KR, Niedobitek G, et al. Integrin αvβ6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies. J Hepatol2008; 48:453-648. , and some of these are expressed on hepatic stellate cells ⁶6 Racine-Samson L, Rockey DC, Bissell DM. The role of α1β1 integrin in wound contraction. A quantitative analysis of liver myofibroblasts in vivo and in primary culture. J Biol Chem 1997; 272:30911-30917. ⁷7 Gao R, Brigstock DR. Connective tissue growth factor (CCN2) induces adhesion of rat activated hepatic stellate cells by binding of its C-terminal domain to integrin alpha(v) beta(3) and heparin sulfate proteoglycan. J Biol Chem2004; 279:8848-8855. .

In 2012, Silva and coworkers ¹⁹19 Silva GF, Grotto RM, Verdichio-Moraes CF, Corvino SM, Ferrasi AC, Silveira LV, et al. Human Platelet Antigen (HPA) genotype is associated with fibrosis progression in chronic hepatitis C. J Med Virol2012; 84:56-60. found an association between the genotype HPA-1a/1b and the more rapid progression of fibrosis in HCV-monoinfected patients, and the patients reached F3/F4 earlier. However, this was not observed among the HIV-1/HCV-coinfected patients in the present study, which suggests that the effect of HPA polymorphism on the progression of hepatic fibrosis that has been observed in HCV-monoinfected patients is lost in the presence of HIV.

Unlike the HCV, the HIV does not replicate in human hepatocytes. However, the chemokine coreceptors CCR5 and CXCR4, which allow the HIV to enter its target cells, are expressed on hepatocytes and stellate cells. The interactions between the HIV and these cells via CCR5 and CXCR4 induce cell signaling ²⁵25 Lin W, Weinberg EM, Tai AW, Peng LF, Brockman MA, Kim KA, et al. HIV increases HCV replication in a TGF-β1-dependent manner. Gastroenterol 2008; 134:803-811. ²⁶26 Bruno R, Galastri S, Sacchi P, Cima S, Caligiuri A, DeFranco R, et al. gp120 modulates the biology of human hepatic stellate cells: a link between HIV infection and liver fibrogenesis. Gut 2010; 59:612-622. ²⁷27 Tuyama AC, Hong F, Saiman Y, Wang C, Ozkok D, Mosoian A, et al. Human immunodeficiency virus (HIV)-1 infects human hepatic stellate cells and promotes collagen I and monocyte chemoattractant protein-1 expression: implications for the pathogenesis of HIV/hepatitis C virus-induced liver fibrosis. Hepatol 2010; 52:612-622. , which subsequently leads to the greater expression of pro-fibrogenic factors, such as TGF-β1, which are involved in the activation of the stellate cells. Thus, the presence of HIV-1 and the activation of signaling pathways via non-integrin receptors may lead to the greater stimulation of the stellate cells as a consequence of the increased expression of TGF-β, which would offset the effect of the HPA polymorphism on the progression of hepatic fibrosis.

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