A 69-year-old man presented for endoscopic examination of the upper gastrointestinal tract because of dysphagia for solid food and unintended weight loss. Several months before, he had noticed ...brownish-gray skin lesions in the neck, in the thorax and in both axillae. A dermatological consultant expressed the suspicion of a paraneoplastic disease. Endoscopic examination revealed an adenocarcinoma of the esophagogastric junction as well as multiple small polyps in the middle and the lower thirds of the esophagus. Histological examination showed papilloma-like proliferations without atypia, which were diagnosed as acanthosis nigricans of the esophagus. After completion of the staging investigation regarding the cardiac carcinoma, combination chemotherapy was started because of the presence of liver metastases. Subsequently, partial regression of the carcinoma as well as of the dermal and esophageal lesions was noted. Acanthosis nigricans is a rare paraneoplastic disease of the esophagus. As an indicator lesion, its detection should prompt a search for a malignant tumor in the gastrointestinal tract.
In vitro expansion of PBMC is commonly used prior to a T-cell monitoring to increase low number of peripheral HBV-specific T cells. However, whether in vitro expansion changes functional composition ...of T-cell responses detectable directly ex vivo has not yet been investigated in detail.
We compared quantity and functionality of HBV surface- (HBs) and core- (HBc) specific CD4/CD8 T cells ex vivo and after 10-days in vitro expansion in 41 individuals with different HBV status (acute self-limiting, chronic or resolved infection). T-cell reactivity was analyzed by intracellular cytokine staining (IL-2, IFN-γ, TNF-α) using multiparametric flow cytometry.
The 10-day in vitro expansion significantly increased number of total HBc-specific CD4 and CD8 T cells (median increase factor 12.8 and 7.0, respectively) and HBs-specific CD4 T cells (median increase factor 4.3), but not HBs-specific CD8 T cells. Proliferative capacity of HBV-specific T cells was independent of the detected ex vivo frequency and the donor's HBV status. However proliferative capacity of HBc-specific CD4 T cells negatively correlated with serum HBs antigen (HBsAg) and alanine transferase (ALT) levels in patients with acute self-limiting hepatitis B (r=-0.9 and r=-0.79, respectively). Unexpectedly, in about 20% of the donors a HBs-specific T-cell response was detected ex vivo, which was lost after in vitro expansion (CD4: 8/41; CD8: 9/41). In contrast in vitro expansion increased sensitivity to monitor HBc-specific T-cell responses. Functional composition of ex vivo HBV-specific T-cell responses also differed significantly compared to expanded HBV-specific T cells. Ex vivo, but not any more after expansion detectable HBV-specific T cells were predominantly monofunctional.
In summary our data show that in vitro expansion significantly alters number and functionality of HBV-specific T cells compared to an analysis directly ex vivo. No general increase of assay sensitivity was observed after in vitro expansion. From our results, we conclude that ex vivo monitoring of HBV-specific T cells better reflects the in vivo situation, and should thus be considered for future clinical immune monitoring.
Corresponding author:
Russo, Carolina
E-Mail:
carolina.russo@helmholtz-muenchen.de
Hepatocellular carcinoma (HCC) represents the 3rd most common cause for cancer related death world wide and chronic Hepatitis B (HBV) and Hepatitis C (HCV) infections represent by far the most common ...risk factors driving hepatocarcinogenesis.
Already existing mouse models partially resemble inflammation-driven hepatocarcinogenesis however do not allow discrimination of direct HBV-induced carcinogenic effects from inflammation-induced carcinogenesis.
Here we describe two HBV-transgenic (HBVtg) mouse models as pre-clinical tools to identify molecular and cellular signatures and investigate the involvement of viral proteins in HCC development in the absence of significant cytopathology and inflammation. These two HBVtg -mice express all HBV viral gene products at high levels with or without the HBV-X protein (HBV1.3tg and HBVxfs, respectively). The role of the HBV-X protein in HBV-induced HCC development is still elusive.
These models develop HCC from the age of 20 months with a higher incidence in HBV1.3tg-mice but metastatic disease found exclusively in HBVxfs-mice. Both display all different HCC subtypes as well as a gender disparity, a Stat3 activation, typical chromosomal aberrations and gene expression patterns mimicking different subclasses of HBV-induced human HCC.
These models may thus reveal common mechanisms driving carcinogenesis in chronic HBV infection and help dissect the role of virus proteins in order to unravel new therapeutic approaches in patients facing the burden of HCC.
Marc.Ringelhan@lrz.tum.de
Background and Aims:
Hepatitis B Virus (HBV) remains the most common risk factor for hepatocellular carcinoma (HCC). It has been suggested that, in addition to chronic inflammation, HBV viral ...products directly contribute in HBV-driven hepatocellular carcinogenesis. In patients with low HBV replication, it is proposed that accumulation of HBV surface antigen (HBsAg) in the endoplasmic reticulum (ER) of hepatocytes (referred to as “ground glass hepatocytes”) activates ER stress, directly driving carcinogenesis. In the ER stress response, NF-kB is known to be important for the control of the Unfolded Protein Response (UPR). The role of NF-kB signaling in HCC development has been reported to be on the one hand anti-tumorigenic, due to activation of critical survival signals in hepatocytes. On the other hand, NF-kB has also been identified as a tumor promoter due to its pro-inflammatory function. In this study, we investigate the role of hepatic NF-kB signaling in directly HBV-driven HCC development using mice overexpressing hepatitis B virus surface antigen (HBsAg).
Methods:
HBsAg transgenic mice were crossed with animals expressing a dominant-negative mutant of IKK2 (an upstream kinase in canonical NF-kB signaling) under the control of tetracycline-inducible LAP-promoter to achieve inhibition of hepatocellular canonical NF-kB signaling.
Results:
The incidence of HBsAg-driven hepatocellular carcinogenesis was dramatically increased at the age of 18 months, when canonical NF-kB signaling was inhibited. However, there was no significant change in inflammatory response such as T-lymphocyte or macrophage infiltration. In addition, MyD88 deficiency did not inhibit HBsAg-induced tumor development in mice. Our data suggest that the role of canonical NF-kB signaling in this model does not rely on changes in the inflammatory response, but disrupts UPR control in HBsAg-driven hepatocellular carcinogenesis. Consistently, in HBsAg mice, a critical UPR regulator BiP/GRP78 was not upregulated when canonical NF-kB system was blocked. Furthermore, ER stress-associated cell death factor CHOP was strongly expressed, pointing to a failure of UPR control. In addition, a massive oval cell reaction (ductular reaction) was induced in HBsAg transgenic mice upon inhibition of canonical NF-kB signaling, indicating extensive liver damage and lost capacity of hepatocytes to contribute to compensatory proliferation.
Conclusion:
The role of canonical NF-kB signaling in hepatocellular carcinogenesis depends on the mode of liver damage. In the case of an HBsAg-driven HCC model simulating a direct carcinogenic effect of HBV, NF-kB plays a critical role in controlling the ER stress response. This implies an indispensable role for NF-kB in the control of the UPR and an anti-tumorigenic role in ER stress-related hepatocarcinogenesis.
Corresponding author:
Sunami, Yoshiaki
E-Mail:
yoshiaki.sunami@tum.de
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