OBJETIVO: Determinar los predictores no invasivos de la severidad de la fibrosis hepática en pacientes con infección crónica por hepatitis C del Servicio de Gastroenterología del Hospital IV Alberto Sabogal (EsSALUD), durante el período diciembre 2000 – diciembre 2002. MATERIAL Y MÉTODOS: Se incluyeron a 30 pacientes con serología positiva para HCV que fueron sometidos a Laparoscopia más biopsia hepática. Estudio observacional, descriptivo, retrospectivo y de corte transversal. Se usó la base de datos del programa SPSS versión 10.0, y se evaluó la asociación mediante el test de Chi cuadrado (_i2), con una significancia estadística del 5%. RESULTADOS: La edad promedio fue de 56.27 ± 12.55 años, Se demostró asociación entre fibrosis severa y radio TGO/TGP mayor de 1 y esplenomegalia(p=0.018 y 0.001, respectivamente) en forma estadísticamente significativa,mientras que la plaquetopenia menor de 150,000 no mostró asociación con fibrosis hepática severa en el grupo de pacientes estudiados (p=0.136). CONCLUSIONES: El radio TGO/TGP mayor de 1 y la esplenomegalia pueden ser considerados como predictores de fibrosis avanzada en pacientes con infección crónica por hepatitis c. En estos pacientes quizás no sea necesaria una biopsia hepática. / --- OBJETIVE: To Determine the non-invasive predictors from Hepatic Fibrosis severity in patients with Hepatitis C chronic infection at Gastroenterology Service from Alberto Sabogal Hospital (EsSALUD), since december 2000 – december 2002. MATERIAL AND METHODS: 30 patients were included with positive HCV serology who had Laparoscopy and hepatic biopsy. This is an observational, descriptive, retrospective and transversal study. It was used SPSS 10.0 database program, and was evaluated its association with chi-square test (_i2), with 5% statistical significance. RESULTS: The age average was 56.27 ± 12.55 years old. A TGO/TGP ratio of major of 1 and splenomegaly correlated significantly with advance stage of fibrosis (p=0.018 and 0.001 respectively) but there wasn’t correlation between platelet count major of 150,000 and severe fibrosis (p=0.136). CONCLUSIONS: A TGO/TGP ratio of major of 1 and splenomegalia can predict advance stage of fibrosis in patients with chronic hepatitis C infection .In these patients, a liver biopsy may not be necessary. Key words: Hepatitis C chronic infection, Hepatic Fibrosis.

The hepatitis C virus (HCV) currently infects 3% of the world???s population, with chronic infection in 50-80% of exposed individuals. A small subset of individuals who are exposed to HCV do not develop anti-HCV antibodies, persistent viraemia or chronic hepatitis despite generating HCV-specific CD4+ and CD8+ T cells. These individuals are believed to develop an immune response which rapidly clears viraemia prior to the induction of an antibody response. Circumstantial evidence supports the likelihood that some of these individuals may generate these same responses and outcomes on repeated occasions of HCV infection. HCV-specific cellular immune responses in seronegative subjects have been the subject of only limited prior study, in part due to the lack of appropriate recombinant antigens and assay systems. Therefore, this thesis described the development and validation of an interferon-? (IFN-?) ELISPOT assay using overlapping peptides (n=441). Using this assay, HCV-specific cellular immune responses were detected in 5/10 (50%) of chronically infected subjects. Responses were identified more frequently, and were directed against more regions of the HCV genome, than with traditional assay systems. This IFN-? ELISPOT assay, a comparable interleukin (IL)-2 ELISPOT assay, and a multiplex in vitro cytokine production assay were then used to evaluate HCV-specific cellular immune responses in three cohorts of seronegative subjects at high-risk of exposure to HCV ??? babies born to infected mothers, multiply-transfused subjects with thalassaemia, and high risk injecting drug users. Cellular immune responses were evaluated in 23 infants born to HCV-antibody positive women. Responses were not detected in infants born to HCV-PCR negative mothers. IFN-? production was detected in 1/11 infants born to viraemic mothers using the ELISPOT assay, with cytokine production observed in an additional 3/5 infants studied using the in vitro cytokine production assay. HCV-specific cellular immune responses were assessed in a cohort of multiply transfused subjects with thalassaemia using assays for cytotoxic T lymphocyte activity, IFN-? and IL-2 ELISPOT, as well as lymphocyte proliferation and in vitro cytokine production. Responses were detected in 6/13 chronically infected subjects (46%), 4/7 subjects who had cleared infection (71%), and 14/17 seronegative subjects (82%). The seronegative subjects had responses which were broader and higher in magnitude than those with chronic HCV infection, although lower and narrower than in subjects who had cleared prior HCV infection. IFN-? and IL-2 ELISPOT assays, in additional to in vitro cytokine production assays, were performed on 41 injecting drug users (IDUs), with responses detected in 6 (15%). Seronegative IDUs with HCV-specific cellular immune responses had been injecting for a mean of 7.7 years, and reported multiple risk factors for exposure to HCV. The combined data from these three cohorts indicate that the HCV-specific cellular immune responses detected in seronegative subjects were generally broad in specificity. Cytokine production was generally Th1-biased, a pattern which has previously been associated with an increased likelihood of clearance in primary infection. The findings also suggest that responses can be maintained for decades after exposure, and may provide protection against repeated exposures. In summary, cellular immunity against HCV is evident in some seronegative high risk subjects, suggesting that the cellular immune responses may efficiently facilitate viral clearance. Understanding the mechanisms of this immune response pattern will allow better understanding of the host response to HCV and may provide key insights into vaccine design.

Dendritic cells (DCs) constitute a large family of immune cells with a dendritic morphology and a critical role in all aspects of an immune response and immune regulation, from immunogenicity to tolerance. One of the important characteristic of DCs is maturation, during which DCs undergo significant changes in their phenotypic and functional properties and change from phagocytic cells to highly efficient antigen presenting cells (APCs). Dendritic cells have recently been at the centre of attention as a promising tool in treatment or control of cancer and infectious diseases. Accordingly, DCs have been generated, matured, and loaded with tumor-associated or microbial antigens ex vivo, to be subsequently used as therapeutic tools or vaccine carriers.<p> Hepatitis C virus (HCV) is a hepatotropic virus, which infects the liver in humans and results in a chronic infection in most cases. The persistent infection of the liver eventually results in cirrhosis and/or hepatocellular carcinoma in 15-20 years. Chronic hepatitis C (CHC) has recently become a serious health concern and the leading cause of liver transplantation. The mechanism of persistence of the virus is not clear yet, but as a Th1-type immune response is strongly correlated with elimination of HCV in vivo, it is evident that insufficient cellular immunity is a contributing factor. Non-cytopathic viruses such as HCV may infect immune cells to modify and evade a protective immune response. Dendritic cells, which are the most potent APCs, and uniquely capable of initiating a primary immune response, have been considered as a target for HCV. Inhibition of DC maturation by HCV has been suggested as a potential contributing factor in immune evasion; however, this issue remains controversial as many contradictory results have been reported.<p> To investigate this contention, we initially planned to evaluate the effects of HCV on DCs of CHC patients; however, due to limited access to patients blood, we instead elected to examine the effects of HCV genes products on in vitro generated DCs from healthy volunteers. Specific attention was paid to the generation, maturation, and transfection of DCs in vitro, as variability in procedures might have been responsible for the controversial reports. Viral vectors have generally been used to transfect DCs; however, a vector and HCV genes might have synergistic effects on DC maturation. Thus, our first objective was to develop an efficient non-viral transfection method while retaining high viability of the DCs, as previous efforts in this regard resulted either in low efficiency or in low viability of DCs after transfection. In order to improve the viability of DCs after transfection, we established a new method for fast generation of monocyte-derived DCs (Mo-DCs) in two to three days. By performing a comprehensive study on transfection reagents, electroporation, and nucleofection with DNA or in vitro transcribed (IVT) RNA, we successfully established a new, highly efficient non-viral method for transfection of DCs with long-term viability. This method is based on the use of the X1 program of a nucleofection device with IVT RNA and results in high transfection efficiency of 93%, with 75% viability of DCs 72 h after transfection.<p> Subsequently, we performed a comprehensive study on the effects of different maturation methods on the phenotype, function and gene expression profile of DCs. Three commonly used treatments, TNF-á, LPS and a maturation cocktail (MC) consisting of IL-1â, IL-6, TNF-á, and prostaglandin E2 (PGE2) were compared. Our results showed that there is a significant difference in the level of maturity between these treatments, and MC generated more functionally competent mDCs than TNF-á or LPS. In addition, MC induced Th1-promoting changes in the transcriptional profile of mDCs. This observation was important, as the presence of PGE2 in MC was previously challenged based on the potential induction of Th2-biased immune responses. However, our results suggest retaining PGE2 in the cocktail because of the fact that MC generated highly competent and functional mDCs with a Th1-promoting transcriptional profile. Finally, Mo-DCs were transfected with IVT HCV RNAs, individually or in combination. While HCV genes had no inhibitory effect on DC maturation, transfection of DCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of DCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison to NS3 or GFP RNA treatments, 42 of which are involved in DC maturation. The effects of core on maturation of DCs were further confirmed by a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-ã secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system.<p> The use of a non-viral method of transfection combined with confirmed transcriptional profiles of DCs in this study may make these results conclusive for in vitro generated DCs from healthy volunteers. However, further investigations are required to confirm the effects on DCs from CHC patients.

Dendritic cells (DCs) constitute a large family of immune cells with a dendritic morphology and a critical role in all aspects of an immune response and immune regulation, from immunogenicity to tolerance. One of the important characteristic of DCs is maturation, during which DCs undergo significant changes in their phenotypic and functional properties and change from phagocytic cells to highly efficient antigen presenting cells (APCs). Dendritic cells have recently been at the centre of attention as a promising tool in treatment or control of cancer and infectious diseases. Accordingly, DCs have been generated, matured, and loaded with tumor-associated or microbial antigens ex vivo, to be subsequently used as therapeutic tools or vaccine carriers.<p> Hepatitis C virus (HCV) is a hepatotropic virus, which infects the liver in humans and results in a chronic infection in most cases. The persistent infection of the liver eventually results in cirrhosis and/or hepatocellular carcinoma in 15-20 years. Chronic hepatitis C (CHC) has recently become a serious health concern and the leading cause of liver transplantation. The mechanism of persistence of the virus is not clear yet, but as a Th1-type immune response is strongly correlated with elimination of HCV in vivo, it is evident that insufficient cellular immunity is a contributing factor. Non-cytopathic viruses such as HCV may infect immune cells to modify and evade a protective immune response. Dendritic cells, which are the most potent APCs, and uniquely capable of initiating a primary immune response, have been considered as a target for HCV. Inhibition of DC maturation by HCV has been suggested as a potential contributing factor in immune evasion; however, this issue remains controversial as many contradictory results have been reported.<p> To investigate this contention, we initially planned to evaluate the effects of HCV on DCs of CHC patients; however, due to limited access to patients blood, we instead elected to examine the effects of HCV genes products on in vitro generated DCs from healthy volunteers. Specific attention was paid to the generation, maturation, and transfection of DCs in vitro, as variability in procedures might have been responsible for the controversial reports. Viral vectors have generally been used to transfect DCs; however, a vector and HCV genes might have synergistic effects on DC maturation. Thus, our first objective was to develop an efficient non-viral transfection method while retaining high viability of the DCs, as previous efforts in this regard resulted either in low efficiency or in low viability of DCs after transfection. In order to improve the viability of DCs after transfection, we established a new method for fast generation of monocyte-derived DCs (Mo-DCs) in two to three days. By performing a comprehensive study on transfection reagents, electroporation, and nucleofection with DNA or in vitro transcribed (IVT) RNA, we successfully established a new, highly efficient non-viral method for transfection of DCs with long-term viability. This method is based on the use of the X1 program of a nucleofection device with IVT RNA and results in high transfection efficiency of 93%, with 75% viability of DCs 72 h after transfection.<p> Subsequently, we performed a comprehensive study on the effects of different maturation methods on the phenotype, function and gene expression profile of DCs. Three commonly used treatments, TNF-á, LPS and a maturation cocktail (MC) consisting of IL-1â, IL-6, TNF-á, and prostaglandin E2 (PGE2) were compared. Our results showed that there is a significant difference in the level of maturity between these treatments, and MC generated more functionally competent mDCs than TNF-á or LPS. In addition, MC induced Th1-promoting changes in the transcriptional profile of mDCs. This observation was important, as the presence of PGE2 in MC was previously challenged based on the potential induction of Th2-biased immune responses. However, our results suggest retaining PGE2 in the cocktail because of the fact that MC generated highly competent and functional mDCs with a Th1-promoting transcriptional profile. Finally, Mo-DCs were transfected with IVT HCV RNAs, individually or in combination. While HCV genes had no inhibitory effect on DC maturation, transfection of DCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of DCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison to NS3 or GFP RNA treatments, 42 of which are involved in DC maturation. The effects of core on maturation of DCs were further confirmed by a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-ã secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system.<p> The use of a non-viral method of transfection combined with confirmed transcriptional profiles of DCs in this study may make these results conclusive for in vitro generated DCs from healthy volunteers. However, further investigations are required to confirm the effects on DCs from CHC patients.

OBJETIVO: Determinar los predictores no invasivos de la severidad de la fibrosis hepática en pacientes con infección crónica por hepatitis C del Servicio de Gastroenterología del Hospital IV Alberto Sabogal (EsSALUD), durante el período diciembre 2000 – diciembre 2002. MATERIAL Y MÉTODOS: Se incluyeron a 30 pacientes con serología positiva para HCV que fueron sometidos a Laparoscopia más biopsia hepática. Estudio observacional, descriptivo, retrospectivo y de corte transversal. Se usó la base de datos del programa SPSS versión 10.0, y se evaluó la asociación mediante el test de Chi cuadrado (_i2), con una significancia estadística del 5%. RESULTADOS: La edad promedio fue de 56.27 ± 12.55 años, Se demostró asociación entre fibrosis severa y radio TGO/TGP mayor de 1 y esplenomegalia(p=0.018 y 0.001, respectivamente) en forma estadísticamente significativa,mientras que la plaquetopenia menor de 150,000 no mostró asociación con fibrosis hepática severa en el grupo de pacientes estudiados (p=0.136). CONCLUSIONES: El radio TGO/TGP mayor de 1 y la esplenomegalia pueden ser considerados como predictores de fibrosis avanzada en pacientes con infección crónica por hepatitis c. En estos pacientes quizás no sea necesaria una biopsia hepática. / OBJETIVE: To Determine the non-invasive predictors from Hepatic Fibrosis severity in patients with Hepatitis C chronic infection at Gastroenterology Service from Alberto Sabogal Hospital (EsSALUD), since december 2000 – december 2002. MATERIAL AND METHODS: 30 patients were included with positive HCV serology who had Laparoscopy and hepatic biopsy. This is an observational, descriptive, retrospective and transversal study. It was used SPSS 10.0 database program, and was evaluated its association with chi-square test (_i2), with 5% statistical significance. RESULTS: The age average was 56.27 ± 12.55 years old. A TGO/TGP ratio of major of 1 and splenomegaly correlated significantly with advance stage of fibrosis (p=0.018 and 0.001 respectively) but there wasn’t correlation between platelet count major of 150,000 and severe fibrosis (p=0.136). CONCLUSIONS: A TGO/TGP ratio of major of 1 and splenomegalia can predict advance stage of fibrosis in patients with chronic hepatitis C infection .In these patients, a liver biopsy may not be necessary.

Liver transplantation was first performed in 1963 (1) as an experimental treatment for end stage liver disease. Three patients were transplanted, all of whom died within 3 weeks. Since then it has become an established therapy resulting in improved quality of life (2), with 675 transplants from cadaveric donors taking place in the UK in 2001 and 706 in 2002 (3). This level of activity compares with 10 years ago when 502 liver transplants were performed in 1992. Figures released for survival up to the year 2000 show that early (1 year) survival has improved to 88% for patients transplanted from 1998 – 1999, with 3 year survival for the period 1996 – 1997 being 73% and 5 year survival for the period 1994 – 1995 being 64% (3). This improvement is probably due to a combination of factors such as improved surgical and anaesthetic technique, changes in medical management after transplantation, the improved recognition of other harmful factors like hypertension, choice of immune suppression and better prediction of patients in whom liver transplantation is not likely to be appropriate such as those with cholangiocarcinoma or multiple large hepatocellular carcinomas. [Taken from Introduction]

The hepatitis C virus (HCV) currently infects 3% of the world???s population, with chronic infection in 50-80% of exposed individuals. A small subset of individuals who are exposed to HCV do not develop anti-HCV antibodies, persistent viraemia or chronic hepatitis despite generating HCV-specific CD4+ and CD8+ T cells. These individuals are believed to develop an immune response which rapidly clears viraemia prior to the induction of an antibody response. Circumstantial evidence supports the likelihood that some of these individuals may generate these same responses and outcomes on repeated occasions of HCV infection. HCV-specific cellular immune responses in seronegative subjects have been the subject of only limited prior study, in part due to the lack of appropriate recombinant antigens and assay systems. Therefore, this thesis described the development and validation of an interferon-? (IFN-?) ELISPOT assay using overlapping peptides (n=441). Using this assay, HCV-specific cellular immune responses were detected in 5/10 (50%) of chronically infected subjects. Responses were identified more frequently, and were directed against more regions of the HCV genome, than with traditional assay systems. This IFN-? ELISPOT assay, a comparable interleukin (IL)-2 ELISPOT assay, and a multiplex in vitro cytokine production assay were then used to evaluate HCV-specific cellular immune responses in three cohorts of seronegative subjects at high-risk of exposure to HCV ??? babies born to infected mothers, multiply-transfused subjects with thalassaemia, and high risk injecting drug users. Cellular immune responses were evaluated in 23 infants born to HCV-antibody positive women. Responses were not detected in infants born to HCV-PCR negative mothers. IFN-? production was detected in 1/11 infants born to viraemic mothers using the ELISPOT assay, with cytokine production observed in an additional 3/5 infants studied using the in vitro cytokine production assay. HCV-specific cellular immune responses were assessed in a cohort of multiply transfused subjects with thalassaemia using assays for cytotoxic T lymphocyte activity, IFN-? and IL-2 ELISPOT, as well as lymphocyte proliferation and in vitro cytokine production. Responses were detected in 6/13 chronically infected subjects (46%), 4/7 subjects who had cleared infection (71%), and 14/17 seronegative subjects (82%). The seronegative subjects had responses which were broader and higher in magnitude than those with chronic HCV infection, although lower and narrower than in subjects who had cleared prior HCV infection. IFN-? and IL-2 ELISPOT assays, in additional to in vitro cytokine production assays, were performed on 41 injecting drug users (IDUs), with responses detected in 6 (15%). Seronegative IDUs with HCV-specific cellular immune responses had been injecting for a mean of 7.7 years, and reported multiple risk factors for exposure to HCV. The combined data from these three cohorts indicate that the HCV-specific cellular immune responses detected in seronegative subjects were generally broad in specificity. Cytokine production was generally Th1-biased, a pattern which has previously been associated with an increased likelihood of clearance in primary infection. The findings also suggest that responses can be maintained for decades after exposure, and may provide protection against repeated exposures. In summary, cellular immunity against HCV is evident in some seronegative high risk subjects, suggesting that the cellular immune responses may efficiently facilitate viral clearance. Understanding the mechanisms of this immune response pattern will allow better understanding of the host response to HCV and may provide key insights into vaccine design.

Infection with hepatitis C virus (HCV) causes significant morbidity and mortality. An understanding of the factors associated with both acquisition and clearance of HCV infection is critical to prevention strategies including vaccine development. Although research in the prison environment is logistically challenging, inmates are a premier risk group. Accordingly, a prospective cohort study of prisoners with monthly sampling for HCV viraemia was undertaken to assess the incidence of, and risk factors for, infection; and to assess the natural history of infection when detected by viraemia. The incidence of infection was 8 per 100 person years, with the incidence of "high risk" and "possible" HCV transmission risk events being 61 and 210 per 100 person years respectively. The first case of HCV infection in prison with tattooing as the probable route of acquisition was reported. A novel phenotype of HCV infection with HCV viraemia and subsequent clearance without the development of symptoms, biochemical hepatitis or seroconversion on HCV specific enzyme immunoassay (EIA), despite more than one year of follow-up, was reported. HCV-specific cell mediated immune responses were detected in the subjects analysed. These subjects also had indeterminate HCV serological responses directed against non-structural proteins detected on a recombinant immunob10t assay (RIBA) that were stable over time and typically predated HCV viraemia. The prevalence of such responses ranged from 29-79% in other relevant cohorts, including injecting drug users (IDUs) and multiply-transfused patients with thalassaemia. The antibody response against the non-structural protein, NS5 was the most reproducible. This reactivity was blocked in 57% of subjects when sera were pre-incubated with recombinant HCV proteins, suggesting HCV-specificity. A case-control study was undertaken to examine whether such responses predicted protection from "classical" HCV infection with EIA seroconversion. Cases that developed HCV viraemia and EIA seroconversion were more likely to have these responses at baseline (when aviraemic) than controls, demonstrating that they do not protect against acute infection. However, the rate of viral clearance in subjects with indeterminate RIBA responses that subsequently developed acute infection and were followed for viral clearance was high (88%), suggesting that such subjects have immune responses that are associated with viral clearance.

Thesis (Ph.D.)--Australian National University, 2004.

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor in Philosophy in Experimental Surgery, Department of Surgery. Title from pdf file main screen (viewed on October 13, 2009). Includes bibliographical references.