Hepatic enrichment and activation of myeloid dendritic cells during chronic hepatitis C virus infection.

UNLABELLED: Chronic hepatitis C virus (HCV) infection is a serious disease that can result in numerous long-term complications leading to liver failure or death. Approximately 80% of people fail to clear their infection, largely as the result of weak, narrowly targeting or waning antiviral T-cell responses. Although professional antigen presenting cells (APCs) like dendritic cells (DCs) might serve as targets for modulation of T-cell immunity, the particular role of DCs in immunity to HCV is not known. Moreover the identity, phenotype, and functional characteristics of such populations in the liver, the site of HCV replication, have proven difficult to elucidate. Using a multicolor flow-based approach, we identified six distinct populations of professional APCs among liver interstitial leukocytes isolated from uninfected and HCV-infected patients. Although a generalized enrichment of DCs in the liver compared to blood was observed for all patients, HCV infection was characterized by a significant increase in the frequency of intrahepatic myeloid DCs (both CD1c+ and CD141+). Phenotypic analyses of liver plasmacytoid (pDC) and myeloid DCs (mDC) further revealed the HCV-induced expression of maturation molecules CD80, CD83, CD40, and programmed death ligand-1. Importantly, pDC and mDCs from HCV-infected liver were capable of secreting effector cytokines, interferon-alpha and interleukin-12, respectively, in response to Toll-like receptor stimulation in vitro. CONCLUSION: Chronic HCV infection facilitates the "customized" recruitment of liver DC subsets with established functional roles in antigen presentation. These DCs are characterized by a mature, activated phenotype and are functionally responsive to antigenic stimulation in vitro. Such findings highlight an important paradox surrounding liver DC recruitment during HCV infection, where despite their activation these cells do not provide adequate protection from the virus.

Transient CD86 expression on hepatitis C virus-specific CD8+ T cells in acute infection is linked to sufficient IL-2 signaling.

Costimulatory signals via B7/CD28 family molecules (signal 2) are critical for effective adaptive CD8(+) T cell immune responses. In addition to costimulatory signals, B7/CD28 family coinhibitory receptor/ligands that modulate immune responses have been identified. In acute hepatitis C virus (HCV) infection, programmed death receptor 1, an inhibitory receptor in the CD28 family, is highly expressed on virus-specific CD8(+) T cells, yet vigorous immune responses often develop. We hypothesized that other costimulatory signals present during the acute phase of HCV infection would be important to counter this negative signaling. In this study, we found that CD86 was highly expressed on HCV-specific CD8(+) T cells early in acute HCV infection and was lost on transition to chronic HCV infection; the expression of CD86 was different from other activation markers, because expression was delayed after in vitro TCR stimulation and required sufficient IL-2 signaling; and HCV-specific CD8(+) T cells in the liver of patients with chronic HCV infection were highly activated (CD69, CD38, and HLA-DR expression), but only a minority expressed CD86 or showed evidence of recent IL-2 signaling (low basal phosphorylated STAT5), despite persistent viremia. Our study identified B7 ligand expression on HCV-specific CD8(+) T cells as a distinct marker of effective T cell stimulation with IL-2 signaling in acute HCV infection. Expression of costimulatory molecules, such as CD86, early in HCV infection may be essential in overcoming inhibitory signals from the high level of programmed death receptor 1 expression also seen at this phase of infection.

Screening high risk individuals for hepatitis B: physician knowledge, attitudes, and beliefs.

BACKGROUND: Although the overall incidence of hepatitis B virus (HBV) has declined since the introduction of universal vaccine guidelines, the incidence remains elevated in high risk groups. Recent guidelines from the Centers for Disease Control (CDC) have underscored the importance of vaccination against HBV in high risk individuals. However, the incidence of HBV in this group remains elevated, suggesting underuse of vaccinations by healthcare providers. AIM: The purpose of this study was to measure practice patterns of HBV vaccination, and identify predictors of vaccination underuse. METHODS: We created a survey with four vignettes describing patients at high risk for contracting HBV, followed by questions regarding knowledge, attitudes, and beliefs (KAB) of HBV screening and vaccination. A random sample of 1,000 physicians, including internists, family medicine, OB/GYN, gastroenterologists, and experts in HBV epidemiology were surveyed. Regression analysis on composite guideline adherence scores identified KAB profiles that predict scores. RESULTS: On average, responders endorsed 71% of the CDC HBV vaccination guidelines. There were three predictors of diminished screening proclivity: (1) younger provider age (P = 0.028), (2) lower awareness that adult HBV is contracted primarily through heterosexual sex (P = 0.023), and (3) being a provider other than a gastroenterologist (P = 0.009). CONCLUSIONS: Respondents endorsed most-but not all-CDC supported HBV screening practices. Lower adherence was predicted by specific and modifiable KAB profiles, and by younger age. Future efforts to improve adherence should target trainees, emphasize the importance of obtaining sexual histories in high risk patients, and inform that HBV is predominantly a heterosexually transmitted infection.

Impaired hepatitis C virus (HCV)-specific effector CD8+ T cells undergo massive apoptosis in the peripheral blood during acute HCV infection and in the liver during the chronic phase of infection.

A majority of patients infected with hepatitis C virus (HCV) do not sustain an effective T-cell response, and viremia persists. The mechanism leading to failure of the HCV-specific CD8(+) T-cell response in patients developing chronic infection is unclear. We investigated apoptosis susceptibility of HCV-specific CD8(+) T cells during the acute and chronic stages of infection. Although HCV-specific CD8(+) T cells in the blood during the acute phase of infection and in the liver during the chronic phase were highly activated and expressed an effector phenotype, the majority was undergoing apoptosis. In contrast, peripheral blood HCV-specific CD8(+) T cells during the chronic phase expressed a resting memory phenotype. Apoptosis susceptibility of HCV-specific CD8(+) T cells was associated with very high levels of programmed death-1 (PD-1) and low CD127 expression and with significant functional T-cell deficits. Further evaluation of the "death phase" of HCV-specific CD8(+) T cells during acute HCV infection showed that the majority of cells were dying by a process of cytokine withdrawal, mediated by activated caspase 9. Contraction during the acute phase occurred rapidly via this process despite the persistence of the virus. Remarkably, in the chronic phase of HCV infection, at the site of infection in the liver, a substantial frequency of caspase 9-mediated T-cell death was also present. This study highlights the importance of cytokine deprivation-mediated apoptosis with consequent down-modulation of the immune response to HCV during acute and chronic infections.

Detection of latent tuberculosis among HIV-infected patients after initiation of highly active antiretroviral therapy.

This cross-sectional study of 110 individuals examined skin testing for latent tuberculosis infection (LTBI) after the initiation of highly active antiretroviral therapy. Skin test reactivity to one or more of four antigens was found in 98 out of 110 subjects (89%), and was maximal in those whose CD4 cell counts recovered to >= 100 cells/mm3. Skin testing is reliable for the identification or exclusion of LTBI once the CD4 cell count recovers to >= 100 cells/mm3.

Tracking dendritic cells in vivo: insights into DC biology and function.

Dendritic cells (DCs) play a vital role in the generation of immune responses, participating both in innate immunity as well as in the initiation of adaptive immunity. However, study of this rare cell population in vivo has been hampered by their low frequency as well as by inadequate means to track antigen-bearing DCs. Our laboratory has developed a novel strategy to genetically tag these DCs in the skin, and to monitor their migration from the periphery to the draining lymph nodes. These studies have provided new insights into the frequency of DC migration, the longevity of DCs in the lymphoid organs, as well as the ability of these DCs to function as antigen-presenting cells. Furthermore, the potential applications of this technique include the ability to evaluate DC function after silencing of specific genes.

Hepatocellular carcinoma in an adult with repaired tetralogy of fallot.

Liver fibrosis is a growing concern among adults with congenital heart disease, particularly for those who have undergone a Fontan operation. Liver fibrosis leads to cirrhosis, a precursor of hepatocellular carcinoma. A few cases of hepatocellular carcinoma in patients with prior palliative surgery for congenital heart disease have been identified in the literature. The current case reports the first known case of hepatocellular carcinoma in a 45-year-old male with repaired tetralogy of Fallot.

B lymphocytes participate in cross-presentation of antigen following gene gun vaccination.

Although endocytosed proteins are commonly presented via the class II MHC pathway to stimulate CD4(+) T cells, professional APCs can also cross-present Ags, whereby these exogenous peptides can be complexed with class I MHC for cross-priming of CD8(+) T cells. Whereas the ability of dendritic cells (DCs) to cross-present Ags is well documented, it is not known whether other APCs may also play a role, or what is the relative contribution of cross-priming to the induction of acquired immunity after DNA immunization. In this study, we compared immune responses generated after gene gun vaccination of mice with DNA vaccine plasmids driven by the conventional CMV promoter, the DC-specific CD11c promoter, or the keratinocyte-specific K14 promoter. The CD11c promoter achieved equivalent expression in CD11c(+) DCs in draining lymph nodes over time, as did a conventional CMV-driven plasmid. However, immunization with DC-restricted DNA vaccines failed to generate protective humoral or cellular immunity to model Ags influenza hemagglutinin and OVA, despite the ability of CD11c(+) cells isolated from lymph nodes to stimulate proliferation of Ag-specific T cells directly ex vivo. In contrast, keratinocyte-restricted vaccines elicited comparable T and B cell activity as conventional CMV promoter-driven vaccines, indicating that cross-priming plays a major role in the generation of immune responses after gene gun immunization. Furthermore, parallel studies in B cell-deficient mu-MT mice demonstrated that B lymphocytes, in addition to DCs, mediate cross-priming of Ag-specific T cells. Collectively, these data indicate that broad expression of the immunogen is required for optimal induction of protective acquired immunity.

The hybrid cytomegalovirus enhancer/chicken beta-actin promoter along with woodchuck hepatitis virus posttranscriptional regulatory element enhances the protective efficacy of DNA vaccines.

DNA vaccines represent a novel and powerful alternative to conventional vaccine approaches. They are extremely stable and can be produced en masse at low cost; more importantly, DNA vaccines against emerging pathogens or bioterrorism threats can be quickly constructed based solely upon the pathogen's genetic code. The main drawback of DNA vaccines is that they often induce lower immune responses than traditional vaccines, particularly in nonrodent species. Thus, improving the efficacy of DNA vaccines is a critical issue in vaccine development. In this study we have enhanced the efficacy of DNA vaccines by adopting strategies that increase gene expression. We generated influenza-hemagglutinin (HA)-encoding DNA vaccines that contain the hybrid CMV enhancer/chicken beta-actin (CAG) promoter and/or the mRNA-stabilizing post-transcriptional regulatory element from the woodchuck hepatitis virus (WPRE). Mice were immunized with these DNA vaccines, and the influenza-HA-specific cellular and humoral immune responses were compared with a conventional, HA-encoding DNA vaccine whose gene expression was driven by the CMV immediate-early promoter (pCMV-HA). CAG promoter-driven DNA vaccines elicited significantly higher humoral and cellular immune responses compared with the pCMV-HA vaccine. DNA vaccines consisting of both CAG and WPRE elements (pCAG-HA-WPRE) induced the highest level of protective immunity, such that immunization with 10-fold lower DNA doses prevented death in 100% of the mice upon lethal viral challenge, whereas all mice immunized with the conventional pCMV-HA vaccine succumbed to influenza infection.

bcl-xL is critical for dendritic cell survival in vivo.

Dendritic cells (DC) are important regulators of immune function, transporting Ags from the periphery to draining lymph nodes (dLN) where they prime Ag-specific T lymphocytes. The magnitude of the immune response generated depends upon the longevity of the Ag-bearing DC in lymphoid tissues. We hypothesized that the control of DC survival is regulated by the antiapoptotic factor bcl-x(L). Gene gun immunization of dual-expression DNA vaccines into a bcl-x(fl/fl) mouse resulted in the delivery of Ag, as well as selective deletion of the bcl-x gene in directly transfected, skin-residing DC. bcl-x-deficient DC failed to mount effective immune responses, and this corresponded to their rapid disappearance from the dLN due to apoptosis. We confirmed these results using RNA interference to specifically silence the antiapoptotic bcl-x(L) isoform in targeted skin-residing DC of C57BL/6 mice. In addition, delivery of bcl-x(L) in trans complemented the bcl-x deficiency in DC of bcl-x(fl/fl) mice, resulting in the maintenance of normal levels of Ag-bearing DC in the dLN. Taken together, our work demonstrates that the bcl-x(L) isoform is critical for survival of skin-derived, Ag-bearing DC in vivo.