Hepatitis C virus (HCV) is a highly pathogenic human virus associated with liver fibrosis, steatosis, and cancer. In infected cells HCV induces oxidative stress. Here, we show that HCV proteins core, ...E1, E2, NS4B, and NS5A activate antioxidant defense Nrf2/ARE pathway via several independent mechanisms. This was demonstrated by the analysis of transient co-expression in Huh7 cells of HCV proteins and luciferase reporters. Expression, controlled by the promoters of stress-response genes or their minimal Nrf2-responsive elements, was studied using luminescence assay, RT-qPCR and/or Western-blot analysis. All five proteins induced Nrf2 activation by protein kinase C in response to accumulation of reactive oxygen species (ROS). In addition, expression of core, E1, E2, NS4B, and NS5A proteins resulted in the activation of Nrf2 in a ROS-independent manner. The effect of core and NS5A was mediated through casein kinase 2 and phosphoinositide-3 kinase, whereas those of NS4B, E1, and E2, were not mediated by either PKC, CK2, PI3K, p38, or ERK. Altogether, on the earliest stage of expression HCV proteins induced a strong up-regulation of the antioxidant defense system. These events may underlie the harmful effects of HCV-induced oxidative stress during acute stage of hepatitis C.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Despite extensive research, there is still no vaccine against the hepatitis C virus (HCV). The aim of this study was to investigate whether MSCs can exhibit adjuvant properties during DNA vaccination ...against hepatitis C. We used the pcNS3-NS5B plasmid encoding five nonstructural HCV proteins and MSCs derived from mice bone marrow. Five groups of DBA mice were immunized with the plasmid and/or MSCs in a different order. Group 1 was injected with the plasmid twice at intervals of 3 weeks; Group 2 with the plasmid, and after 24 h with MSCs; Group 3 with MSCs followed by the plasmid the next day; Group 4 with only MSCs; and Group 5 with saline. When the MSCs were injected prior to DNA immunization, the cell immune response to HCV proteins assessed by the level of IFN-γ synthesis was markedly increased compared to DNA alone. In contrast, MSCs injected after DNA suppressed the immune response. Apparently, the high level of proinflammatory cytokines detected after DNA injection promotes the conversion of MSCs introduced later into the immunosuppressive MSC2. The low level of cytokines in mice before MSC administration promotes the high immunostimulatory activity of MSC1 in response to a DNA vaccine. Thus, when administered before DNA, MSCs are capable of exhibiting promising adjuvant properties.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Hepatitis C virus (HCV) is one of the main triggers of chronic liver disease. Despite tremendous progress in the HCV field, there is still no vaccine against this virus. Potential vaccines can be ...based on its recombinant proteins. To increase the humoral and, especially, cellular immune response to them, more effective adjuvants are needed. Here, we evaluated a panel of compounds as potential adjuvants using the HCV NS5B protein as an immunogen. These compounds included inhibitors of polyamine biosynthesis and urea cycle, the mTOR pathway, antioxidants, and cellular receptors. A pronounced stimulation of cell proliferation and interferon-γ (IFN-γ) secretion in response to concanavalin A was shown for antioxidant
-acetylcysteine (NAC), polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO), and TLR9 agonist CpG ODN 1826 (CpG). Their usage during the immunization of mice with the recombinant NS5B protein significantly increased antibody titers, enhanced lymphocyte proliferation and IFN-γ production. NAC and CpG decreased relative Treg numbers; CpG increased the number of myeloid-derived suppressor cells (MDSCs), whereas neither NAC nor DFMO affected MDSC counts. NAC and DFMO suppressed NO and interleukin 10 (IL-10) production by splenocytes, while DFMO increased the levels of IL-12. This is the first evidence of immunomodulatory activity of NAC and DFMO during prophylactic immunization against infectious diseases.
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Hepatitis C virus (HCV) is one of the major causes of chronic liver disease and leads to cirrhosis and hepatocarcinoma. Despite extensive research, there is still no vaccine against HCV. In order to ...induce an immune response in DBA/2J mice against HCV, we obtained modified mouse mesenchymal stem cells (mMSCs) simultaneously expressing five nonstructural HCV proteins (NS3-NS5B). The innate immune response to mMSCs was higher than to DNA immunization, with plasmid encoding the same proteins, and to naïve unmodified MSCs. mMSCs triggered strong phagocytic activity, enhanced lymphocyte proliferation, and production of type I and II interferons. The adaptive immune response to mMSCs was also more pronounced than in the case of DNA immunization, as exemplified by a fourfold stronger stimulation of lymphocyte proliferation in response to HCV, a 2.6-fold higher rate of biosynthesis, and a 30-fold higher rate of secretion of IFN-γ, as well as by a 40-fold stronger production of IgG2a antibodies to viral proteins. The immunostimulatory effect of mMSCs was associated with pronounced IL-6 secretion and reduction in the population of myeloid derived suppressor cells (MDSCs). Thus, this is the first example that suggests the feasibility of using mMSCs for the development of an effective anti-HCV vaccine.
The hepatitis C virus (HCV) causes chronic liver disease leading to fibrosis, cirrhosis, and hepatocellular carcinoma. HCV infection triggers various types of cell death which contribute to hepatitis ...C pathogenesis. However, much is still unknown about the impact of viral proteins on them. Here we present the results of simultaneous immunocytochemical analysis of markers of apoptosis, autophagy, and necrosis in Huh7.5 cells expressing individual HCV proteins or their combinations, or harboring the virus replicon. Stable replication of the full-length HCV genome or transient expression of its core, Е1/Е2, NS3 and NS5B led to the death of 20-47% cells, 72 h posttransfection, whereas the expression of the NS4A/B, NS5A or NS3-NS5B polyprotein did not affect cell viability. HCV proteins caused different impacts on the activation of caspases-3, -8 and -9 and on DNA fragmentation. The structural core and E1/E2 proteins promoted apoptosis, whereas non-structural NS4A/B, NS5A, NS5B suppressed apoptosis by blocking various members of the caspase cascade. The majority of HCV proteins also enhanced autophagy, while NS5A also induced necrosis. As a result, the death of Huh7.5 cells expressing the HCV core was induced via apoptosis, the cells expressing NS3 and NS5B via autophagy-associated death, and the cells expressing E1/E2 glycoproteins or harboring HCV the replicon via both apoptosis and autophagy.
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Objectives:
Diagnostic between multisystem inflammatory syndrome associated with COVID-19 in children (MIS-C) and Kawasaki disease (KD) can make difficulties due to many similarities. Our study aimed ...to create a Kawasaki/MIS-C differentiation score (KMDscore) allowing discrimination of MIS-C and KD.
Study design:
The retrospective multicenter cohort study included clinical, laboratory, and instrumental information about MIS-C (
n
= 72) and KD (
n
= 147). The variables allowed to discriminate both conditions used to construct and validate the diagnostic score called the KMDscore.
Results:
Patients with MIS-C were older, had earlier admission to the hospital, had a shorter time before fever resolution, two times frequently had signs of GI and CNS involvement observed, and had more impressive thrombocytopenia, higher level of CRP, ferritin, ALT, AST, LDH, creatinine, triglycerides, troponin, and D-dimer compared to KD patients. Respiratory signs in MIS-C were presented with pleuritis, acute respiratory distress syndrome, oxygen dependency, lung infiltration, and ground-glass opacities in CT. The heart involvement with fast progression of myocarditis provided the severity of MIS-C and ICU admission due to 12 times higher arterial hypotension or shock and required cardiotonic. No differences in the frequency of CA lesions were seen in the majority of cases. Five criteria, CRP >11 mg/dl (18 points), D-dimer >607 ng/ml (27 points), age >5 years (30 points), thrombocytopenia (25 points), and GI involvement (28 points), were included in the KMDscore. The summa >55 points allowed to discriminate MIS-C from KD with a sensitivity of 87.5% and specificity of 89.1%.
Conclusion:
The KMDscore can be used to differentiate the diagnostic of MIS-C from KD.
JAK-inhibitors are small molecules blocking the JAK-STAT pathway that have proven effective in the treatment of different immune-mediated diseases in adults and juvenile idiopathic arthritis (JIA).
...To evaluate the safety and efficacy of tofacitinib in children with different rheumatic diseases.
We extracted information from 24 children with the following diagnosis: JIA (
= 15), undifferentiated systemic autoinflammatory diseases (SAIDs) (
= 7), and juvenile dermatomyositis (JDM) (
= 2) who have been treated with tofacitinib for a period of longer than 6 months. The treatment outcomes were classified according to the opinion of the attending physicians as having a complete response (CR), i.e., the absence of disease activity, or a partial response (PR)-a significant improvement of symptoms and disease activity, or no response (NR)-no changes in disease activity.
CR was achieved in 10/24 patients; 7/15 among JIA patients, 1/2 among JDM patients, 4/7 among SAID patients, and PR in 5/15 of JIA, 1/2 of JDM, and 3/7 of SAID patients. Three non-responders with JIA discontinued tofacitinib. Corticosteroids were successfully tapered off in 11/14 patients and discontinued in 2/14 patients. Four patients had side effects not requiring treatment discontinuation: liver enzyme elevation (
= 2), hypercholesterolemia (
= 1), lymphadenitis (
= 1).
JAK-inhibitors are effective new therapies for the treatment of multiple immune-mediated diseases. Our experience has shown the best results in patients with JIA and JIA-associated alopecia, and type I interferonopathies. More data from randomized controlled clinical trials are needed to use JAK-inhibitors safely in pediatric rheumatic diseases.
Hepatitis C virus (HCV) is one of the basic culprits behind chronic liver disease, which may result in cirrhosis and hepatocarcinoma. In spite of the extensive research conducted, a vaccine against ...HCV has not been yet created. We have obtained human mesenchymal stem cells (hMSCs) and used them for expressing the HCV NS5A protein as a model vaccination platform. Sixteen hMSC lines of a different origin were transfected with the pcNS5A-GFP plasmid to obtain genetically modified MSCs (mMSCs). The highest efficiency was obtained by the transfection of dental pulp MSCs. C57BL/6 mice were immunized intravenously with mMSCs, and the immune response was compared with the response to the pcNS5A-GFP plasmid, which was injected intramuscularly. It was shown that the antigen-specific lymphocyte proliferation and the number of IFN-γ-synthesizing cells were two to three times higher after the mMSC immunization compared to the DNA immunization. In addition, mMSCs induced more CD4+ memory T cells and an increase in the CD4+/CD8+ ratio. The results suggest that the immunostimulatory effect of mMSCs is associated with the switch of MSCs to the pro-inflammatory phenotype and a decrease in the proportion of myeloid derived suppressor cells. Thus, the possibility of using human mMSCs for the creation of a vaccine against HCV has been shown for the first time.
Acute and chronic liver diseases are a major global public health problem; nevertheless, the etiology of 12–30% of cases remains obscure. The purpose of this research was to study the incidence of ...human herpesviruses (HHVs) cytomegalovirus, Epstein–Barr virus (EBV), and HHV-6 in patients with hepatitis and to examine the effect of HHV on the disease severity. We studied the clinical materials of 259 patients with hepatitis treated in Infectious Clinic n.1 (Moscow) and the archived materials of 118 patients with hepatitis C. HHV DNA was detected in the whole blood in 13.5% of patients with hepatitis B or C and in 10.1% of patients with hepatitis of unspecified etiology. EBV demonstrated the highest incidence (58.1%). Cirrhosis was diagnosed in 50% of patients with HHV and in 15.6% of patients without HHV. In patients with hepatitis C, the frequency of HHV was higher in liver biopsy (38.7%) compared to blood. The clinical and virological indicators of hepatitis were considerably higher in patients with coinfection. Conclusion: HHV detected in patients with viral hepatitis has been associated with a significant effect on the severity of the disease, and we suggest monitoring HHV DNA in patients with severe hepatitis and/or poor response to antiviral drugs.
Introduction. Hepatitis C is a liver disease with high chronicity, the cause of cirrhosis and hepatocarcinoma. The main obstacle to controlling hepatitis C is the lack of vaccines.
The aim of the ...work was to compare the immunogenic activity of nonstructural recombinant proteins NS3, NS4 and NS5B of hepatitis C virus (HCV) as components of a subunit candidate vaccine and to analyze the adjuvant properties of two available commercial drugs, polymuramil and pyrogenalum.
Materials and methods. BALB/c, DBA/2J and C57BL/6 mice were immunized with nonstructural proteins without adjuvants or with polymuramyl (NOD1 and NOD2 agonist) and pyrogenalum (TLR-4 agonist). The activity of antibodies was determined in ELISA, the cellular response by antigen-specific lymphocyte proliferation and by production of IFN- in vitro.
Results. Recombinant proteins showed different immunogenicity. NS4 induced antibodies more efficiently than NS3 and NS5B. Significant differences were found in the immune response of three inbred lines mice: the level of IFN- in BALB/c and DBA/2J mice induced by NS5B protein was 30 times higher than in C57Bl/6 mice. In contrast, the induction of antibodies in BALB/c mice was lower than in C57Bl/6 and DBA/2J. Polymuramil did not increase the humoral response to NS5B and enhanced the cellular response only in C57BL/6 mice. The combined use of polymuramil with pyrogenalum significantly increased both the humoral and cellular response of mice to all recombinant HCV proteins.
Conclusion. Different immunogenic properties and different functions of recombinant non-structural HCV proteins indicate the feasibility of their combined inclusion in subunit vaccines. It was established for the first time that immunization with HCV proteins with a complex adjuvant (polymuramyl + pyrogenalum) has a synergistic effect, significantly exceeding the effect of each of them separately.
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