Immune Response and Immunolmodulation in Chronic Hepatiitis B Virus Infection

Despite the presence of an effective vaccine since 1982, chronic hepatitis B virus infection (CHB) still ranks among the highest causes of mortality from infectious diseases worldwide. The studies presented in this thesis were performed to get a better insight into the anti-viral immune response after hepatitis B virus (HBV) infection, and to identify factors in this immune response that contribute to persistent disease. Dendritic cells (DC) play an important role in the induction of anti- viral immune responses. In this thesis we show that two important blood precursor DC, myeloid DC and plasmacytoid DC, are functionally impaired in patients with chronic hepatitis B and this might be an important mechanism by which HBV evades an adequate immune response, leading to viral persistence and disease chronicity. Information about character and grade of the intrahepatic immune response in viral hepatitis is important for evaluation of disease stage and effect of therapy. Complications like haemorrhage provide a limitation to frequently performing standard tissue needle biopsies. The Fine-needle-aspiration-biopsy (FNAB) is an easy and atraumatic alternative and we show that flow cytometry of fine-needle-aspiration- biopsy of the liver allows reliable analysis of lymphocytes obtained from the intrahepatic compartment, in patients with viral hepatitis. Subsequently, the FNAB is used to show that there likely is an important role for intrahepatic HBV-specific CD8+ T-cells in clearing acute HBV infection. Furthermore, we have attempted to boost the impaired T-cell responses in patients with chronic HBV infection, using conventional anti-viral therapy. In in vivo immunization (IVI) of CHB patients, following rapid virus suppression by interferon-lamivudine combination therapy, lamivudine was withdrawn intermittently during continued interferon (IFNα) therapy. Although initially IVI was able to transiently suppress viral replication in two patients with CHB, in a subsequent pilot study the magnitude of the induced T-cell response was insufficient to cause a sustained virological effect in the majority of patients. It is unknown why treatment with IFNα leads to a response in only a minority of patients with chronic HBV. We show that in non-responders and not in responders there was a significant increase in the frequency of regulatory T cells (Treg) and IL-10 secreting cells during treatment with IFNα. Treg depletion resulted in increased proliferation capacity and increased frequencies of HBV-specific INFgamma-producing cells, but did not affect the frequency of IL-10 producing cells measured during the course of the treatment. This study indicates that there may be an important role for Treg in HBV- persistence during and after IFNα therapy

Characterization of hemodialysis membranes by inverse size exclusion chromatography

Inverse size exclusion chromatography (i-SEC) was used to characterize three different cellulosic hollow fiber hemodialysis membranes, i.e. low-flux cuprophan and hemophan and high-flux RC-HP400A. With the i-SEC technique the pore size distribution and porosity of a membrane can be determined and adsorption phenomena can be studied. The membranes showed clear differences in pore size and porosity, the high-flux RC-HP400A membrane has a larger pore size as well as a higher porosity. For all the membranes it was found that the elution curves were best described by a homoporous pore volume distribution. It appeared that the bound or non-freezing water in the membranes was at least partly accessible to solutes. The test molecules creatinine and vitamin B 12 both adsorbed to the cellulosic membranes. The adsorption behavior of creatinine was strongly dependent on the NaCl concentration present. The observations could be explained by assuming that cuprophan and RC-HP400A are negatively charged whereas hemophan is positively charged due to the modification with N,N-diethylaminoethyl ether. The net charge of the hemophan is smaller

Factors associated with ethnical disparity in overall survival for patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is an important cause of cancer-related death worldwide. Ethnical disparity in overall survival has been demonstrated for HCC patients in the United States (U.S.). We aimed to evaluate the contributors to this survival disparity. The SEER database was used to identify HCC patients from 2004 to 2012. Kaplan-Meier curves and Cox proportional hazard models were used to evaluate overall survival by ethnicity and the contributors to ethnical survival disparity. A total of 33 062 patients were included: 15 986 Non-Hispanic Whites, 6535 Hispanic Whites, 4842 African Americans, and 5699 Asians. Compared to Non-Hispanic Whites, African Americans had worse survival (HR, 1.18; 95%CI, 1.14-1.23), while Asians had a better survival (HR, 0.85; 95%CI, 0.82-0.89), and Hispanic Whites had a similar survival (HR, 1.01; 95%CI, 0.97-1.05). Multivariate Cox analysis identified that tumor presentation- and treatment-related factors significantly contributed to the ethnical survival disparity. Especially, tumor size was the most important contributor (HR, 1.11; 95%CI, 1.07-1.16). There is no ethnical survival disparity in patients undergoing liver transplantation and sub-analysis of patients within the Milan criteria for liver transplantation demonstrated no significant survival disparity between African Americans and non-Hispanic Whites in transplantation adjustment analysis (HR, 1.23; 95%CI, 1.11-1.35 in non-adjustment analysis to HR, 1.05; 95%CI, 0.95-1.15 after adjustment). Finally, no important contributor to the superior overall survival in Asians was identified. In conclusion, poor tumor presentation at diagnosis, limited benefit from resection and restricted utilization of liver transplantation are important contributors to poorer survival of African Americans with HCC

Rapid hepatic clearance of full length CCN-2/CTGF: a putative role for LRP1-mediated endocytosis

CCN-2 (connective tissue growth factor; CTGF) is a key factor in fibrosis. Plasma CCN-2 has biomarker potential in numerous fibrotic disorders, but it is unknown which pathophysiological factors determine plasma CCN-2 levels. The proteolytic amino-terminal fragment of CCN-2 is primarily eliminated by the kidney. Here, we investigated elimination and distribution profiles of full length CCN-2 by intravenous administration of recombinant CCN-2 to rodents. After bolus injection in mice, we observed a large initial distribution volume (454 mL/kg) and a fast initial clearance (120 mL/kg/min). Immunosorbent assay and immunostaining showed that CCN-2 distributed mainly to the liver and was taken up by hepatocytes. Steady state clearance in rats, determined by continuous infusion of CCN-2, was fast (45 mL/kg/min). Renal CCN-2 clearance, determined by arterial and renal vein sampling, accounted for only 12 % of total clearance. Co-infusion of CCN-2 with receptor-associated protein (RAP), an antagonist of LDL-receptor family proteins, showed that RAP prolonged CCN-2 half-life and completely prevented CCN-2 internalization by hepatocytes. This suggests that hepatic uptake of CCN-2 is mediated by a RAP-sensitive mechanism most likely involving LRP1, a member of the LDL-receptor family involved in hepatic clearance of various plasma proteins. Surface plasmon resonance binding studies confirmed that CCN-2 is an LRP1 ligand. Co-infusion of CCN-2 with an excess of the heparan sulphate-binding protamine lowered the large initial distribution volume of CCN-2 by 88 % and reduced interstitial staining of CCN-2, suggesting binding of CCN-2 to heparan sulphate proteoglycans (HSPGs). Protamine did not affect clearance rate, indicating that RAP-sensitive clearance of CCN-2 is HSPG independent. In conclusion, unlike its amino-terminal fragment which is cleared by the kidney, fu

Identification of the initial molecular changes in response to circulating angiogenic cells-mediated therapy in critical limb ischemia

BackgroundCritical limb ischemia (CLI) constitutes the most aggressive form of peripheral arterial occlusive disease, characterized by the blockade of arteries supplying blood to the lower extremities, significantly diminishing oxygen and nutrient supply. CLI patients usually undergo amputation of fingers, feet, or extremities, with a high risk of mortality due to associated comorbidities.Circulating angiogenic cells (CACs), also known as early endothelial progenitor cells, constitute promising candidates for cell therapy in CLI due to their assigned vascular regenerative properties. Preclinical and clinical assays with CACs have shown promising results. A better understanding of how these cells participate in vascular regeneration would significantly help to potentiate their role in revascularization.Herein, we analyzed the initial molecular mechanisms triggered by human CACs after being administered to a murine model of CLI, in order to understand how these cells promote angiogenesis within the ischemic tissues.MethodsBalb-c nude mice (n:24) were distributed in four different groups: healthy controls (C, n:4), shams (SH, n:4), and ischemic mice (after femoral ligation) that received either 50 mu l physiological serum (SC, n:8) or 5x10(5) human CACs (SE, n:8). Ischemic mice were sacrificed on days 2 and 4 (n:4/group/day), and immunohistochemistry assays and qPCR amplification of Alu-human-specific sequences were carried out for cell detection and vascular density measurements. Additionally, a label-free MS-based quantitative approach was performed to identify protein changes related.ResultsAdministration of CACs induced in the ischemic tissues an increase in the number of blood vessels as well as the diameter size compared to ischemic, non-treated mice, although the number of CACs decreased within time. The initial protein changes taking place in response to ischemia and more importantly, right after administration of CACs to CLI mice, are shown.ConclusionsOur results indicate that CACs migrate to the injured area; moreover, they trigger protein changes correlated with cell migration, cell death, angiogenesis, and arteriogenesis in the host. These changes indicate that CACs promote from the beginning an increase in the number of vessels as well as the development of an appropriate vascular network.Institute of Health Carlos III, ISCIII; Junta de Andaluci

Convergent Transcription of Interferon-stimulated Genes by TNF-α and IFN-α Augments Antiviral Activity against HCV and HEV

IFN-α has been used for decades to treat chronic hepatitis B and C, and as an off-label treatment for

PD-L1, Galectin-9 and CD8+ tumor-infiltrating lymphocytes are associated with survival in hepatocellular carcinoma

Novel systemic treatments for hepatocellular carcinoma (HCC) are strongly needed. Immunotherapy is a promising strategy that can induce specific antitumor immune responses. Understanding the mechanisms of immune resistance by HCC is crucial for development of suitable immunotherapeutics. We used immunohistochemistry on tissue-microarrays to examine the co-expression of the immune inhibiting molecules PD-L1, Galectin-9, HVEM and IDO, as well as tumor CD8+ lymphocyte infiltration in HCC, in two independent cohorts of patients. We found that at least some expression in tumor cells was seen in 97% of cases for HVEM, 83% for PD-L1, 79% for Gal-9 and 66% for IDO. In the discovery cohort (n = 94), we found that lack of, or low, tumor expression of PD-L1 (p < 0.001), Galectin-9 (p < 0.001) and HVEM (p < 0.001), and low CD8+TIL count (p = 0.016), were associated with poor HCC-specific survival. PD-L1, Galectin-9 and CD8+TIL count were predictive of HCC-specific survival independent of baseline clinicopathologic characteristics and the combination of these markers was a powerful predictor of HCC-specific survival (HR 0.29; p <0.001). These results were confirmed in the validation cohort (n = 60). We show that low expression levels of PD-L1 and Gal-9 in combination with low CD8+TIL count predict extremely poor HCC-specific survival and it requires a change in two of these parameters to significantly improve prognosis. In conclusion, intra-tumoral expression of these immune inhibiting molecules was observed in the majority of HCC patients. Low expression of PD-L1 and Galectin-9 and low CD8+TIL count are associated with poor HCC-specific survival. Combining immune biomarkers leads to superior predictors of HCC mortality

Observer variability of absolute and relative thrombus density measurements in patients with acute ischemic stroke

Introduction: Thrombus density may be a predictor for acute ischemic stroke treatment success. However, only limited data on observer variability for thrombus density measurements exist. This study assesses the variability and bias of four common thrombus density measurement methods by expert and non-expert observers. Methods: For 132 consecutive patients with acute ischemic stroke, three experts and two trained observers determined thrombus density by placing three standardized regions of interest (ROIs) in the thrombus and corresponding contralateral arterial segment. Subsequently, absolute and relative thrombus densities were determined using either one or three ROIs. Intraclass correlation coefficient (ICC) was determined, and Bland–Altman analysis was performed to evaluate interobserver and intermethod agreement. Accuracy of the trained observer was evaluated with a reference expert observer using the same statistical analysis. Results: The highest interobserver agreement was obtained for absolute thrombus measurements using three ROIs (ICCs ranging from 0.54 to 0.91). In general, interobserver agreement was lower for relative measurements, and for using one instead of three ROIs. Interobserver agreement of trained non-experts and experts was similar. Accuracy of the trained observer measurements was comparable to the expert interobserver agreement and was better for absolute measurements and with three ROIs. The agreement between the one ROI and three ROI methods was good. Conclusion: Absolute thrombus density measurement has superior interobserver agreement compared to relative density measurement. Interobserver variation is smaller when multiple ROIs are used. Trained non-expert observers can accurately and reproducibly assess absolute thrombus densities using three ROIs

Automated entire thrombus density measurements for robust and comprehensive thrombus characterization in patients with acute ischemic stroke

Background and Purpose: In acute ischemic stroke (AIS) management, CT-based thrombus density has been associated with treatment success. However, currently used thrombus measurements are prone to inter-observer variability and oversimplify the heterogeneous thrombus composition. Our aim was first to introduce an automated method to assess the entire thrombus density and then to compare the measured entire thrombus density with respect to current standard manual measurements. Materials and Method: In 135 AIS patients, the density distribution of the entire thrombus was determined. Density distributions were described usingmedians, interquartile ranges (IQR), kurtosis, and skewedness. Differences between themedian of entire thrombusmeasurements and commonly applied manualmeasurements using 3 regions of interest were determined using linear regression. Results: Density distributions varied considerably with medians ranging from 20.0 to 62.8 HU and IQRs ranging from 9.3 to 55.8 HU. The average median of the thrombus density distributions (43.5 ± 10.2 HU) was lower than the manual assessment (49.6 ± 8.0 HU) (p<0.05). The difference between manual measurements and median density of entire thrombus decreased with increasing density (r = 0.64; p<0.05), revealing relatively higher manual measurements for low density thrombi such that manual density measurement tend overestimates the real thrombus density. Conclusions: Automatic measurements of the full thrombus expose a wide variety of thrombi density distribution, which is not grasped with currently used manual measurement. Furthermore, d

Tumour antigen expression in hepatocellular carcinoma in a low-endemic western area

Background: Identification of tumour antigens is crucial for the development of vaccination strategies against hepatocellular carcinoma (HCC). Most studies come from eastern-Asia, where hepatitis-B is the main cause of HCC. However, tumour antigen expression is poorly studied in low-endemic, western areas where the aetiology of HCC differs. Methods: We constructed tissue microarrays from resected HCC tissue of 133 patients. Expression of a comprehensive panel of cancer-testis (MAGE-A1, MAGE-A3/4, MAGE-A10, MAGE-C1, MAGE-C2, NY-ESO-1, SSX-2, sperm protein 17), onco-fetal (AFP, Glypican-3) and overexpressed tumour antigens (Annexin-A2, Wilms tumor-1, Survivin, Midkine, MUC-1) was determined by immunohistochemistry. Results: A higher prevalence of MAGE antigens was observed in patients with hepatitis-B. Patients with expression of more tumour antigens in general had better HCC-specific survival (P=0.022). The four tumour antigens with high expression in HCC and no, or weak, expression in surrounding tumour-free-liver tissue, were Annexin-A2, GPC-3, MAGE-C1 and MAGE-C2, expressed in 90, 39, 17 and 20% of HCCs, respectively. Ninety-five percent of HCCs expressed at least one of these four tumour antigens. Interestingly, GPC-3 was associated with SALL-4 expression (P=0.001), an oncofetal transcription factor highly expressed in embryonal stem cells. SALL-4 and GPC-3 expression levels were correlated with vascular invasion, poor differentiation and higher AFP levels before surgery. Moreover, patients who co-expressed higher levels of both GPC-3 and SALL-4 had worse HCC-specific survival (P=0.018). Conclusions: We describe a panel of four tumour antigens with excellent coverage and good tumour specificity in a western area, low-endemic for hepatitis-B. The association between GPC-3 and SALL-4 is a novel finding and suggests that GPC-3 targeting may specifically attack the tumour stem-cell compartment