Kumar Visvanathan

Serum hepatitis B surface antigen and hepatitis B e antigen titers: disease phase influences correlation with viral load and intrahepatic hepatitis B virus markers.

Although threshold levels for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) titers have recently been proposed to guide therapy for chronic hepatitis B (CHB), their relationship to circulating hepatitis B virus (HBV) DNA and intrahepatic HBV replicative intermediates, and the significance of emerging viral variants, remains unclear. We therefore tested the hypothesis that HBsAg and HBeAg titers may vary independently of viral replication in vivo. In all, 149 treatment‐naïve CHB patients were recruited (HBeAg‐positive, n = 71; HBeAg‐negative, n = 78). Quantification of HBeAg and HBsAg was performed by enzyme immunoassay. Virological characterization included serum HBV DNA load, HBV genotype, basal core promoter (BCP)/precore (PC) sequence, and, in a subset (n = 44), measurement of intrahepatic covalently closed circular DNA (cccDNA) and total HBV DNA, as well as quantitative immunohistochemical (IHC) staining for HBsAg. In HBeAg‐positive CHB, HBsAg was positively correlated with serum HBV DNA and intrahepatic cccDNA and total HBV DNA (r = 0.69, 0.71, 0.76, P < 0.01). HBeAg correlated with serum HBV DNA (r = 0.60, P < 0.0001), although emerging BCP/PC variants reduced HBeAg titer independent of viral replication. In HBeAg‐negative CHB, HBsAg correlated poorly with serum HBV DNA (r = 0.28, P = 0.01) and did not correlate with intrahepatic cccDNA nor total HBV DNA. Quantitative IHC for hepatocyte HBsAg confirmed a relationship with viral replication only in HBeAg‐positive patients. Conclusion: The correlation between quantitative HBsAg titer and serum and intrahepatic markers of HBV replication differs between patients with HBeAg‐positive and HBeAg‐negative CHB. HBeAg titers may fall independent of viral replication as HBeAg‐defective variants emerge prior to HBeAg seroconversion. These findings provide new insights into viral pathogenesis and have practical implications for the use of quantitative serology as a clinical biomarker. (HEPATOLOGY 2010)

Regulation of Toll‐like receptor‐2 expression in chronic hepatitis B by the precore protein

Toll‐like receptors (TLRs) play a key role in the innate immune response. The aim of this study was to examine the expression of TLR2 and TLR4 in chronic hepatitis B (CHB). The TLR2 and TLR4 expression on hepatocytes and Kupffer cells from fresh liver biopsies was measured from 21 patients with untreated hepatitis B e antigen (HBeAg)‐positive and HBeAg‐negative CHB. Parallel studies were also undertaken on monocytes from their peripheral blood. Expression of TLR2 on hepatocytes, Kupffer cells, and peripheral monocytes was significantly reduced in patients with HBeAg‐positive CHB in comparison with HBeAg‐negative CHB and controls, whereas it was significantly increased in HBeAg‐negative CHB compared with controls. The level of TLR4 expression did not differ significantly between the groups. These results were confirmed in vitro using hepatic cell lines transduced with recombinant HBV baculovirus expressing wild‐type HBV (HBeAg‐positive), precore stop codon (G1896A) mutant HBV (HBeAg‐negative). The functional relevance of these findings was established by the demonstration of significantly reduced cytokine production (TNF‐α) and phospho‐p38 kinase expression in the presence of the HBeAg. In the absence of HBeAg, HBV replication was associated with up‐regulation of the TLR2 pathway leading to increased TNF‐α production. Conclusion: This study demonstrates a potentially important interaction between HBeAg, HBV, and the innate immune response. (HEPATOLOGY 2007;45:102–110.)

The Hepatitis B e antigen (HBeAg) targets and suppresses activation of the Toll-like receptor signaling pathway

BACKGROUND & AIMS Viruses target innate immune pathways to evade host antiviral responses. Recent studies demonstrate a relationship between hepatitis B disease states and the hosts innate immune response, although the mechanism of immunomodulation is unknown. In humans, the innate immune system recognizes pathogens via pattern recognition receptors such as the Toll-like receptors (TLR), initiating anti-inflammatory responses. TLR expression and pro-inflammatory cytokine production is reduced in hepatitis B e antigen (HBeAg)-positive patients following TLR stimulation. The aim of this study was to investigate interactions between TLR signaling pathways and the mature HBeAg protein localized in the cytosol. METHODS The ability of HBeAg to inhibit TLR signaling and association with TLR adapters was evaluated by immunoprecipitation, immunostaining, and reporter studies. RESULTS Our findings show that HBeAg co-localizes with Toll/IL-1 receptor (TIR)-containing proteins TRAM, Mal, and TLR2 at the sub-cellular level, which was not observed for Hepatitis B core antigen. Co-immunoprecipitation analysis demonstrated HBeAg interacted with TIR proteins Mal and TRAM, while a mutated HBeAg ablated interaction between Mal and MyD88. Importantly, HBeAg also disrupted homotypic TIR:TIR interaction critical for TLR-mediated signaling. Finally, HBeAg suppressed TIR-mediated activation of the inflammatory transcription factors, NF-κB and Interferon-β promoter activity. CONCLUSIONS Our study provides the first molecular mechanism describing HBeAg immunomodulation of innate immune signal transduction pathways via interaction and targeting of TLR-mediated signaling pathways. These finding suggest the mechanism as to how HBeAg evades innate immune responses contributing to the pathogenesis of chronic hepatitis B infection and the establishment of viral persistence.

Epidemiology of sepsis in Victoria, Australia

Objective:To determine the clinical and epidemiologic characteristics of patients with sepsis admitted to hospitals in Victoria, Australia, including the incidence of sepsis and severe sepsis, utilization of intensive care unit (ICU) resources, and hospital mortality. Design:A population-based hospital morbidity database generated from hospital discharge coding. Setting:State of Victoria, Australia (population, 4.5 million), the 4-yr period from July 1, 1999, to June 30, 2003. Patients:A total of 3,122,515 overnight hospitalizations. Interventions:None. Measurements and Main Results:The overall hospital incidence of sepsis was 1.1%, with a mortality of 18.4%. Of septic patients, 23.8% received some care in an ICU. For these patients, hospital mortality was 28.9%. Severe sepsis, defined by sepsis and at least one organ dysfunction, occurred in 39% of sepsis patients and was accompanied by a hospital mortality of 31.1%. Fifty percent of patients with severe sepsis received at least some care in an ICU. Conclusions:Australian state hospital administrative data reveal epidemiologic features of sepsis and severe sepsis that are strikingly similar to those recently reported from comparable populations in North American and Europe. This suggests that lessons learned in this area may be directly applicable internationally.

The oral toll-like receptor-7 agonist GS-9620 in patients with chronic hepatitis B virus infection

BACKGROUND & AIMS GS-9620 is an oral agonist of toll-like receptor 7, a pattern-recognition receptor whose activation results in innate and adaptive immune stimulation. We evaluated the safety, pharmacokinetics, and pharmacodynamics of GS-9620 in patients with chronic hepatitis B. METHODS In two double-blind, phase 1b trials of identical design, 49 treatment-naïve and 51 virologically suppressed patients were randomized 5:1 to receive GS-9620 (at doses of 0.3mg, 1mg, 2mg, 4mg) or placebo as a single dose or as two doses seven days apart. Pharmacodynamic assessment included evaluation of peripheral mRNA expression of interferon-stimulated gene 15 (ISG15), serum interferon gamma-induced protein 10 and serum interferon (IFN)-alpha. RESULTS Overall, 74% of patients were male and 75% were HBeAg negative at baseline. No subject discontinued treatment due to adverse events. Fifty-eight percent experienced ⩾1 adverse event, all of which were mild to moderate in severity. The most common adverse event was headache. No clinically significant changes in HBsAg or HBV DNA levels were observed. Overall, a transient dose-dependent induction of peripheral ISG15 gene expression was observed peaking within 48 hours of dosing followed by return to baseline levels within seven days. Higher GS-9620 dose, HBeAg positive status, and low HBsAg level at baseline were independently associated with greater probability of ISG15 response. Most patients (88%) did not show detectable levels of serum IFN-alpha at any time point. CONCLUSIONS Oral GS-9620 was safe, well tolerated, and associated with induction of peripheral ISG15 production in the absence of significant systemic IFN-alpha levels or related symptoms.

Toll-like receptors and their role in gastrointestinal disease.

The innate immune response to invading pathogens is centred upon a family of non‐clonal, germline‐encoded pattern recognition receptors (PRRs), the Toll‐like receptors (TLRs). These provide specificity for a vast range of microbial pathogens, and offer an immediate anti‐microbial response system. Thirteen mammalian TLRs have been described; 10 are expressed in humans, each responsible for the recognition of distinct, invariant microbial structures originating from bacteria, viruses, fungi and protozoa. The two most thoroughly studied are TLR4 and TLR2, the PRRs for Gram‐negative and Gram‐positive bacterial products, respectively. TLR4 is also the major receptor recognising endogenous ligands released from damaged or dying cells. Activation of a TLR by its relevant ligand rapidly ignites a complex intracellular signaling cascade that ultimately results in upregulation of inflammatory genes and production of proinflammatory cytokines, interferons and recruitment of myeloid cells. It also stimulates expression, upon antigen presenting cells, of co‐stimulatory molecules required to induce an adaptive immune response. Whilst a robust TLR response is critical for survival and defence against invading pathogens, inappropriate signaling in response to alterations in the local microflora environment can be detrimental. Such ‘unhelpful TLR responses’ could form the basis for a large number of gastrointestinal and liver disorders, including inflammatory bowel disease, viral hepatitis, autoimmune liver diseases and hepatic fibrosis. As our understanding of TLRs expands, the pathogenesis of a number of gastrointestinal disorders will be further elucidated, and this offers potential for specific therapies aimed directly at TLR signaling.

Regulation of Toll-like receptor (TLR)2 and TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens

Rapid overproduction of proinflammatory cytokines are characteristic of sepsis. CD14dimCD16+ monocytes are thought to be major producers of cytokine and have been shown to be elevated in septic patients. Toll‐like receptors (TLR) are pattern recognition receptors important in mediating the innate immune response and their activation can lead to production of cytokines. Using whole blood culture and flow cytometry we have investigated TLR2 and TLR4 regulation after stimulation with sepsis‐relevant antigens [lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB) and peptidoglycan (PGN)]. The percentage of CD14dimCD16+ monocyte population expanded at 20 h post‐stimulation, after a rise in tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 at 2 h. A strong positive correlation between the percentage of CD14dimCD16+ monocytes and secreted TNF‐α was demonstrated (r = 0·72). Furthermore, we were able to induce expansion of the CD14dimCD16+ population to approximately 35% of all monocytes with the addition of recombinant TNF‐α to the whole blood culture. TLR4 was found to be expressed 2·5 times higher on CD14dimCD16+ compared to CD14+ CD16– monocytes, while TLR2 expression was similar in both subpopulations. The CD14dimCD16+ and CD14+ CD16– monocyte populations were different in their response to various antigens. LPS down‐regulated TLR4 by 4·9 times in CD16+ monocytes compared to only 2·3 times in CD16– monocytes at 2 h. LPS was able to up‐regulate TLR2 by 6·2 times after 2 h, with no difference between the subpopulations. LPS further up‐regulated TLR2 by 18·4 times after 20 h only in the CD14+ CD16– population. PGN and SEB induced no significant changes in TLR2 or TLR4 expression. We hypothesize that following exposure to bacterial antigens, subsequent TNF‐α drives a differentiation of monocytes into a CD14dimCD16+ subpopulation.

Toll-like receptor expression in chronic hepatitis C: Correlation with pro-inflammatory cytokine levels and liver injury

Abstract.Background/AimsToll-like receptors (TLR’s) are critical receptors that promote innate immune responses to pathogen-associated molecular patterns. Activation of TLR’s leads to production of pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α. This study investigates whether peripheral blood monocyte expression of TLR’s is disturbed in patients with chronic hepatitis C and whether levels of expression of these molecules are significantly correlated with hepatitis C virus (HCV) genotype, viral load, hepatic necroinflammatory activity, histological stage and circulating TNF-α concentrations.MethodsIn 18 non-cirrhotic patients with biopsy-proven, virologically-confirmed chronic hepatitis C and 32 controls, we measured expression of TLR2 and TLR4 on peripheral blood monocytes. HCV genotype, viral load, serum alanine aminotransferase (ALT) levels, histological stage of disease and circulating TNF-α and endotoxin levels were also determined.ResultsPeripheral blood monocyte expression of TLR2 and TLR4 were significantly increased in patients with chronic hepatitis C compared to controls, irrespective of HCV genotype or histological stage of disease. Circulating levels of TNF-α were also significantly increased in patients with chronic hepatitis C. In both the overall study cohort and patients with chronic hepatitis C, monocyte expression of TLR2, but not of TLR4, correlated significantly with serum TNF-α levels. In patients with chronic hepatitis C, monocyte expression of TLR2, but not of TLR4, also correlated significantly with serum ALT levels. Expression of TLR’s was not significantly correlated with viral load.ConclusionsUp-regulation of peripheral blood monocyte expression of TLR2 and TLR4 occurs in patients with chronic hepatitis C. Increased monocyte expression of TLR2, but not of TLR4, correlates significantly with both increased circulating TNF-α levels and hepatic necroinflammatory activity in this disorder.

Inhibition of Bacterial Superantigens by Peptides and Antibodies

ABSTRACT The pyrogenic exotoxins of group A streptococci and staphylococcal enterotoxins are a family of structurally related superantigens with similar biological activity. Two distinct areas have been identified which have a highly conserved amino acid homology in all of the toxin families. A number of peptides were constructed from these regions, some of which were concatenated and polymerized to enhance their immunogenicity in animals. Antibodies prepared against these polymerized peptides were used to serologically identify the majority of the superantigen toxins, block the biological activities of the superantigens, and protect an experimental animal model against shock. In addition certain peptides were able per se to block up to 90% of the proliferative responses induced by the toxins. The peptide also proved protective in a septic shock model in mice. Binding experiments indicate that the peptide binds tightly to the major histocompatibility complex class II molecule, thus preventing binding and hence activation of the superantigen. The selective and rapid binding of the peptide to the major histocompatibility complex class II molecule may lead to a novel therapeutic modality in treatment of superantigen-mediated diseases.

Immunopathogenesis of hepatic flare in HIV/hepatitis b virus (Hbv)-coinfected individuals after the initiation of Hbv-active antiretroviral therapy

BACKGROUND The pathogenesis of and risk factors for hepatic flare (HF) after the initiation of hepatitis B virus (HBV)-active antiretroviral therapy (ART) in HIV/HBV-coinfected individuals is not well understood. METHODS We studied HF in ART-naive HIV/HBV-coinfected individuals in Thailand (n = 36) who were beginning HBV-active ART as part of a prospective clinical trial. HF was defined as an alanine aminotransferase (ALT) level>5 times the upper limit of normal or >200 IU/L higher than that at baseline. Immune mediators (interleukin [IL]-18, IL-2, IL-6, IL-8, IL-10, soluble CD26 [sCD26], sCD30, sCD8, CXCL-10, CCL-2, tumor necrosis factor-alpha, interferon [IFN]-gamma, and IFN-alpha) and activated NK cells were quantified. RESULTS HBV DNA and ALT levels at baseline were higher in patients with HF (n=8) than in patients without HF (n=28) (P=.01). After the initiation of ART, CXCL-10 levels remained elevated in patients with HF but decreased in patients without HF (P<.01). sCD30 levels increased and were significantly higher at week 8 in patients with HF (P<.05). There was a positive correlation between levels of ALT and levels of CXCL-10, sCD30, CCL-2, and IL-18 at week 8 (the time of peak ALT level) but not at other time points. CONCLUSION Elevated HBV DNA and ALT levels before the initiation of HBV-active ART are risk factors for HF. The pathogenesis of HF after the initiation of HBV-active ART is probably consistent with immune restoration disease.