Catharina Hultgren

The hepatitis C virus and immune evasion: non-structural 3/4A transgenic mice are resistant to lethal tumour necrosis factor α mediated liver disease

Background: The hepatitis C virus (HCV) establishes chronic infection by incompletely understood mechanisms. The non-structural (NS) 3/4A protease/helicase has been proposed as a key complex in modulating the infected hepatocyte, although nothing is known about the effects this complex exerts in vivo. Aim: To generate mice with stable and transient hepatocyte expression of the HCV NS3/4A proteins to study its effects in vivo. Methods: NS3/4A expression was determined by western blot and immunohistochemistry. Two independent pathologists determined the liver histology. Hepatic immunity was characterised by quantifying intrahepatic immune cell subsets. Liver damage was induced using carbon tetrachloride (CCl4), lipopolysaccaride (LPS), tumour necrosis factor α (TNFα), and anti-Fas antibody. Results: Expression of NS3/4A was restricted to the cytoplasm of hepatocytes, and did not cause liver cancer or any spontaneous liver pathology. However, the presence of NS3/4A modulated the intrahepatic immunity, as follows: first, the CD4+ T cell and type I/II dendritic cell subsets were reduced in transgenic livers; second, NS3/4A protected hepatocytes from liver damage mediated in vivo by CCl4, LPS, TNFα, but not FAS; and third, both stable and transiently NS3/4A transgenic mice were resistant to lethal doses of liver targeted TNFα, and the resistance could be reverted by treatment with a p38 mitogen activated protein kinase inhibitor (MAPK). Conclusions: Hepatic expression of NS3/4A does not induce spontaneous liver disease. NS3/4A does, however, alter the intrahepatic immune cell subsets and protects hepatocytes against TNFα induced liver damage in vivo. The TNFα resistance can be reverted by treatment with a p38 MAPK inhibitor. This represents a new immune evasion strategy conferred by NS3/4A.

In Vivo Clearance of Hepatitis C Virus Nonstructural 3/4A–Expressing Hepatocytes by DNA Vaccine–Primed Cytotoxic T Lymphocytes

A key question in the development of a therapeutic vaccine against hepatitis C virus (HCV) is whether vaccine-primed T cells enter the liver and eliminate HCV-expressing hepatocytes. In the absence of an infectious small-animal model, we evaluated liver homing of vaccine-primed T cells in mice with transient hepatic transgene expression of the HCV nonstructural 3/4A (NS3/4A) protein. We found that T cells primed by transdermal DNA-based vaccination entered the liver and cleared NS3/4A-expressing hepatocytes in transiently transgenic CD8(+/+) mice but not in CD8(-/-) mice. Hence, peripherally primed NS3/4A-specific CD8(+) T cells home to the liver and clear HCV protein-expressing hepatocytes.

Improving on the Ability of Endogenous Hepatitis B Core Antigen to Prime Cytotoxic T Lymphocytes

Hepatitis B virus core antigen (HBcAg) is thought to be a major target for specific cytotoxic T cells (CTLs) in hepatitis B virus infections. A single dose of hepatitis C virus nonstructural 3/4A DNA (<5 microg) effectively primes functional specific CTLs, independently of CD4(+) T helper cells and by different routes of immunization. In contrast, HBcAg-specific CTL priming was T helper cell dependent and highly sensitive to the dose and route of delivery. Although CTL priming was improved 10-fold by codon optimization and in vivo electroporation, low levels of DNA still failed to prime CTLs effectively. Only high doses (5 microg) of codon-optimized HBcAg delivered by in vivo electroporation primed in vivo lytic and polyfunctional CTLs. The ability of endogenous HBcAg to prime CTLs is surprisingly inefficient and differs from that of nonstructural 3/4A. This has important implications for the design of HBcAg-based therapeutic vaccines in humans.

Improved cell mediated immune responses after successful re-vaccination of non-responders to the hepatitis B virus surface antigen (HBsAg) vaccine using the combined hepatitis A and B vaccine

We successfully re-vaccinated hepatitis B virus (HBV) vaccine non-responders using a double dose of the combined hepatitis A virus (HAV) and HBV vaccine. The hope was to improve priming of hepatitis B surface antigen (HBsAg)-specific cell mediated immune response (CMI) by an increased antigen dose and a theoretical adjuvant-effect from the local presence of a HAV-specific CMI. A few non-responders had a detectable HBsAg-specific CMI before re-vaccination. An in vitro detectable HBsAg-specific CMI was primed equally effective in non-responders (58%) as in first time vaccine recipients (68%). After the third dose a weak, albeit significant, association was observed between the magnitude of HBsAg-specific proliferation and anti-HBs levels. This regimen improves the priming of HBsAg-specific CMIs and antibodies.

Effects by periodontitis on pristane-induced arthritis in rats

BackgroundAn infection-immune association of periodontal disease with rheumatoid arthritis has been suggested. This study aimed to investigate the effect of pre-existing periodontitis on the development and the immune/inflammatory response of pristane-induced arthritis.MethodsWe investigated the effect of periodontitis induced by ligature placement and Porphyromonas gingivalis (P. gingivalis) infection, in combination with Fusobacterium nucleatum to promote its colonization, on the development of pristane-induced arthritis (PIA) in rats (Dark Agouti). Disease progression and severity of periodontitis and arthritis was monitored using clinical assessment, micro-computed tomography (micro-CT)/intraoral radiographs, antibody response, the inflammatory markers such as α-1-acid glycoprotein (α-1-AGP) and c-reactive protein (CRP) as well as cytokine multiplex profiling at different time intervals after induction.ResultsExperimentally induced periodontitis manifested clinically (Pxa0<xa00.05) prior to pristane injection and progressed steadily until the end of experiments (15xa0weeks), as compared to the non-ligated arthritis group. Injection of pristane 8xa0weeks after periodontitis-induction led to severe arthritis in all rats demonstrating that the severity of arthritis was not affected by the pre-existence of periodontitis. Endpoint analysis showed that 89% of the periodontitis-affected animals were positive for antibodies against arginine gingipain B and furthermore, the plasma antibody levels to a citrullinated P. gingivalis peptidylarginine deiminase (PPAD) peptide (denoted CPP3) were significantly (Pxa0<xa00.05) higher in periodontitis rats with PIA. Additionally, there was a trend towards increased pro-inflammatory and anti-inflammatory cytokine levels, and increased α-1-AGP levels in plasma from periodontitis-challenged PIA rats.ConclusionsPre-existence of periodontitis induced antibodies against citrullinated peptide derived from PPAD in rats with PIA. However, there were no differences in the development or severity of PIA between periodontitis challenged and periodontitis free rats.