Usutu virus NS4A suppresses the host interferon response by disrupting MAVS signaling.

Usutu virus (USUV) is an emerging flavivirus that can infect birds and mammals. In humans, in severe cases, it may cause neuroinvasive disease. The innate immune system, and in particular the interferon response, functions as the important first line of defense against invading pathogens such as USUV. Many, if not all, viruses have developed mechanisms to suppress and/or evade the interferon response in order to facilitate their replication. The ability of USUV to antagonize the interferon response has so far remained largely unexplored. Using dual-luciferase reporter assays we observed that multiple of the USUV nonstructural (NS) proteins were involved in suppressing IFN-ß production and signaling. In particular NS4A was very effective at suppressing IFN-ß production. We found that NS4A interacted with the mitochondrial antiviral signaling protein (MAVS) and thereby blocked its interaction with melanoma differentiation-associated protein 5 (MDA5), resulting in reduced IFN-ß production. The TM1 domain of NS4A was found to be essential for binding to MAVS. By screening a panel of flavivirus NS4A proteins we found that the interaction of NS4A with MAVS is conserved among flaviviruses. The increased understanding of the role of NS4A in flavivirus immune evasion could aid the development of vaccines and therapeutic strategies.

A synthetic toll-like receptor 7 agonist inhibits porcine reproductive and respiratory syndrome virus replication in piglets.

Toll-like receptor 7 (TLR7) agonists have been shown to exert therapeutic effects against several viruses. However, antiviral potential of TLR7 agonist in inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection has not been assessed in vivo. In our previous study, a synthetic TLR7 agonist, SZU101, was confirmed to inhibit PRRSV infection of porcine alveolar macrophages (PAMs). Here, antiviral effects of SZU101 were evaluated in PRRSV-challenged piglets based on assessments of rectal temperature, viremia, gross and microscopic lung lesions, PRRSV-specific antibodies, PRRSV-specific lymphocyte proliferation and serum IFN-ß level. Our results revealed that SZU101 treatment alleviated PRRSV-induced rectal temperature spikes, pulmonary pathologic changes, and serum viral load. Meanwhile, administration of SZU101 led to increased proliferation of PRRSV-specific lymphocytes and serum IFN-ß levels, but did not enhance PRRSV-specific antibody production. These results demonstrate that SZU101 has potential as a therapeutic treatment for PRRS.

Recombinant MUC1-MBP fusion protein combined with CpG2006 vaccine induces antigen-specific CTL responses through cDC1-mediated cross-priming mainly regulated by type I IFN signaling in mice.

In this study, we explored the initiation and regulation mechanism of antigen-specific CTL responses induced by a novel cancer vaccine containing recombinant human mucin1-maltose-binding protein fusion protein (MUC1-MBP) and CpG2006. First, DC subsets were analyzed by flow cytometry in vivo and in vitro. After vaccination, the proportion and maturation of cDC1s in mouse dLNs were upregulated, and the proportion of cDC2s and pDCs was also increased. In vitro studies on vaccine components showed similar changs, which may mainly depend on the activity of CpG2006. Subsequently, the regulatory effect of type Ⅰ IFN signaling on CTL triggering was confirmed through co-culture of sorted DC subsets and T cells and subsequent CTL activity experiments. CTL killing activity exhibited a 61.9% decrease once type I IFN signaling was blocked. Further analysis showed that blocking IFNAR1 on cDC1s but not on CTLs resulted in significant defects in CTL killing activity. Collectively, M-M combined with CpG2006 vaccine promotes MUC1-specific CTL responses by increasing the cDC1 activity in mice, and this is mainly regulated by type Ⅰ IFN signaling in cDC1s.

Major Vault Protein Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection in CRL2843CD163 Cell Lines and Primary Porcine Alveolar Macrophages.

Porcine reproductive and respiratory syndrome (PRRS), a significant viral infectious disease that commonly occurs among farmed pigs, leads to considerable economic losses to the swine industry worldwide. Major vault protein (MVP) is a host factor that induces type Ⅰ interferon (IFN) production. In this study, we evaluated the effect of MVP on PRRSV infection in CRL2843CD163 cell lines and porcine alveolar macrophages (PAMs). Our results showed that MVP expression was downregulated by PRRSV infection. Adenoviral overexpression of MVP inhibited PRRSV replication, whereas the siRNA knockdown of MVP promoted PRRSV replication. In addition, MVP knockdown has an adverse effect on the inhibitive role of MVP overexpression on PRRSV replication. Moreover, MVP could induce the expression of type Ⅰ IFNs and IFN-stimulated gene 15 (ISG15) in PRRSV-infected PAMs. Based on these results, MVP may be a potential molecular target of drugs for the effective prevention and treatment of PRRSV infection.

[cGAS/STING signaling pathways induces the secretion of type â interferon in porcine alveolar macrophages infected with porcine circovirus type 2].

In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.

Bisphenol-A induced antioxidants imbalance and cytokines alteration leading to immune suppression during larval development of Labeo rohita.

Recently, the oxidative stress and immunotoxicity biomarkers have been extensively used in embryotoxicity using fish embryos as promising models especially after exposure to chemical-like environmental estrogens. Bisphenol-A (BPA) is an estrogenic endocrine disruptor and is ubiquitous in the aquatic environment. Larvae of Labeo rohita were exposed to low concentrations of BPA (10, 100, 1000 µg/l) for 21 days. Innate immune system, antioxidants parameters, and developmental alterations were used as biomarkers. Exposure to BPA caused developmental abnormalities including un-inflated swim bladder, delayed yolk sac absorption, spinal curvature, and edema of pericardium. Lipid peroxidation increased and activity of catalase (p < 0.05), superoxide dismutase (p < 0.05), and glutathione peroxidase (p < 0.01) decreased after exposure to BPA. Level of reduced glutathione also decreased (p < 0.05) in BPA-exposed group. Lower expression of tumor necrosis factor-α (p < 0.05) and interferon-γ (p < 0.001) was observed in BPA-exposed groups while expression of interleukin-10 increased (p < 0.05) in larvae exposed to 10 µg/l BPA. Moreover, exposure of BPA caused a concentration-dependent increase in expression of heat shock protein 70 (p < 0.05). The present study showed that the exposure to BPA in early life stages of Labeo rohita caused oxidative stress and suppress NF-κB signaling pathway leading to immunosuppression. The results presented here demonstrate the cross talk between heat shock protein 70 and cytokines expression.

Pharmacokinetics-Based Adjusted Versus Standard Dose of Ribavirin Does Not Improve Virologic Response Rates in Chronic Hepatitis C Genotype 4 Patients: A Randomized Controlled Trial.

Optimal doses of Ribavirin (RBV) for hepatitis C virus (HCV) treatment are not known. To assess the safety and efficacy of PegIFNalfa-2a in combination with an adjusted (ADJ) RBV dose based on early pharmacokinetics versus a fixed standard (STD) dose of RBV in chronic HCV genotype (GT) 4-naive patients in a randomized trial. One hundred eighty-one patients were randomized. The baseline variables were similar in both arms and females were 50.3% of the patients, 76.5% had minimal-moderate fibrosis (F0-2). Sustained virologic response (SVR) was achieved in 99 (54.7%) subjects. SVR was seen in 50/90 (55.6%) of ADJ dose of RBV and 49/91 (53.9%) of STD dose subjects. Prematurely withdrawal or discontinuation of treatment prematurely in the ADJ RBV arm occurred in 11/90 patients (12.2%) compared with 6/91 subjects (6.6%) in the STD arm (P = 0.214). Similarly, virologic relapse was seen in 14/90 (15.6%) patients of the ADJ arm and 12/91 (13.2%) of the STD arm. Anemia grade 3-4 was seen in 36.7% in ADJ versus 17.6% in STD arm (P = 0.003). Occurrence of rapid virologic response and absences of F4 fibrosis predicted SVR in a univariate analysis. However, age, gender, weight, presence of diabetes, baseline alanine aminotransferase, and vitamin D levels were not significantly different in patients achieving SVR. ADJ higher doses of RBV based on its early pharmacokinetics-based RBV do not improve SVR rates in HCV GT4 treated in combination with peg-IFN alpha-2-a versus STD therapy. Patients on ADJ higher doses of RBV experienced higher rates of anemia and require more erythropoietin without increasing SVR.

Optineurin deficiency in mice is associated with increased sensitivity to Salmonella but does not affect proinflammatory NF-κB signaling.

Optineurin (OPTN) is an evolutionary conserved and ubiquitously expressed ubiquitin-binding protein that has been implicated in glaucoma, Paget bone disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases. From in vitro studies, OPTN was shown to suppress TNF-induced NF-κB signaling and virus-induced IRF signaling, and was identified as an autophagy receptor required for the clearance of cytosolic Salmonella upon infection. To assess the in vivo functions of OPTN in inflammation and infection, we generated OPTN-deficient mice. OPTN knockout mice are born with normal Mendelian distribution and develop normally without any signs of spontaneous organ abnormality or inflammation. However, no differences in NF-κB activation could be observed in OPTN knockout mice or fibroblasts derived from these mice upon TNF or LPS treatment. Primary bone marrow-derived macrophages from OPTN-deficient mice had slightly impaired IRF signaling and reduced IFN type I production in response to LPS or poly(I,C). Finally, OPTN-deficient mice were more susceptible to infection with Salmonella, confirming in vivo the importance of OPTN in bacterial clearance.

Mouse superkiller-2-like helicase DDX60 is dispensable for type I IFN induction and immunity to multiple viruses.

IFN-α/ß allow cells to fight virus infection by inducing the expression of many genes that encode effectors of antiviral defense. One of these, the Ski2-like DExH-box helicase DDX60, was recently implicated in resistance of human cells to hepatitis C virus, as well as in induction of IFN-α/ß by retinoic acid inducible gene 1-like receptors (RLRs) that detect the presence of RNA viruses in a cell-intrinsic manner. Here, we sought to investigate the role of DDX60 in IFN-α/ß induction and in resistance to virus infection. Analysis of fibroblasts and myeloid cells from Ddx60-deficient mice revealed no impairment in IFN-α/ß production in response to RLR agonists, RNA viruses, or other stimuli. Moreover, overexpression of DDX60 did not potentiate IFN induction and DDX60 did not interact with RLRs or capture RLR agonists from virally infected cells. We also failed to identify any impairment in Ddx60-deficient murine cells or mice in resistance to infection with influenza A virus, encephalomyocarditis virus, Sindbis virus, vaccinia virus, or herpes simplex virus-1. These results put in question the reported role of DDX60 as a broad-acting positive regulator of RLR responses and hint at the possibility that it may function as a restriction factor highly specific for a particular virus or class of viruses.

STS-1 promotes IFN-α induced autophagy by activating the JAK1-STAT1 signaling pathway in B cells.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the overexpression of IFN-α. IFN-α induces autophagy via the JAK1-STAT1 signaling pathway, contributing to the pathogenesis of SLE. Recent studies reported that B cells from patients with SLE and NZB/W F1 mice had enhanced autophagy activity; however, the mechanism still remains unknown. Here, we show that the protein tyrosine phosphatase STS-1 (suppressor of T-cell receptor signaling 1) was significantly overexpressed in B cells from patients with SLE and MRL/lpr mice. Notably, STS-1 promoted IFN-α-induced autophagy in B cells by enhancing the JAK1-STAT1 signaling activation. STS-1 inhibited the phosphorylation of the E3 ubiquitin protein ligase c-cbl, and subsequently promoted IFN-α-induced phosphorylation of tyrosine kinase 2, leading to JAK1-STAT1 signaling activation. Furthermore, STAT1 and JAK1 inhibitors blocked the IFN-α-induced autophagy promoted by STS-1, indicating that STS-1 promotes IFN-α-induced autophagy via the JAK1-STAT1 signaling. Our results demonstrate the importance of STS-1 in regulating IFN-α-induced autophagy in B cells, and this could be used as a therapeutic approach to treat SLE.

Policitemia Vera. Experiencias en el diagnóstico y tratamiento en el Instituto de Hematología e Inmunología / Polycythemia vera: Experiences in diagnosis and treatment in the Institute of Hematology and Immunology

La policitemia Vera se encuentra entre las neoplasias mieloides según la clasificación más reciente de la Organización Mundial de la Salud (OMS). Sus criterios diagnósticos han tenido variaciones en los últimos años y en este trabajo se realiza un análisis de estos criterios, así como de la respuesta a los tratamientos empleados en 349 pacientes atendidos en el Instituto de Hematología e Inmunología en los últimos 40 años. Se sugiere, dada su factibilidad y eficacia, continuar utilizando por el momento la clasificación OMS del 2001, y de acuerdo con la experiencia acumulada, se propone el tratamiento con medicamentos de primera y segunda líneas según la edad y las características clínicas de cada enfermo(AU)

The Polycythemia vera is located among the myeloid neoplasms according to the more recent classification of the HWO. Its diagnostic criteria have underwent variations in past years and in present paper authors made an analysis of such criteria, as well as of the response to treatments applied in 349 patients seen in the Institute of Hematology and Immunology during the past 40 years. Due to feasibility and effectiveness, for the moment, is has been suggested to carry on with the use of the classification of WHO of 2001, and according to the experience gained, it is proposed the treatment with first and second line drugs by age and the clinical features of each patient(AU)

Policitemia Vera. Experiencias en el diagnóstico y tratamiento en el Instituto de Hematología e Inmunología / Polycythemia vera: Experiences in diagnosis and treatment in the Institute of Hematology and Immunology

La policitemia Vera se encuentra entre las neoplasias mieloides según la clasificación más reciente de la Organización Mundial de la Salud (OMS). Sus criterios diagnósticos han tenido variaciones en los últimos años y en este trabajo se realiza un análisis de estos criterios, así como de la respuesta a los tratamientos empleados en 349 pacientes atendidos en el Instituto de Hematología e Inmunología en los últimos 40 años. Se sugiere, dada su factibilidad y eficacia, continuar utilizando por el momento la clasificación OMS del 2001, y de acuerdo con la experiencia acumulada, se propone el tratamiento con medicamentos de primera y segunda líneas según la edad y las características clínicas de cada enfermo

The Polycythemia vera is located among the myeloid neoplasms according to the more recent classification of the HWO. Its diagnostic criteria have underwent variations in past years and in present paper authors made an analysis of such criteria, as well as of the response to treatments applied in 349 patients seen in the Institute of Hematology and Immunology during the past 40 years. Due to feasibility and effectiveness, for the moment, is has been suggested to carry on with the use of the classification of WHO of 2001, and according to the experience gained, it is proposed the treatment with first and second line drugs by age and the clinical features of each patient

Autoimmune hemolytic anemia in HCV/HIV coinfected patients during treatment with pegylated alpha-2a interferon plus ribavirin

Two cases of autoimmune hemolytic anemia that occurred during the treatment of chronic hepatitis C with pegylated alpha-2a interferon and ribavirin, in HIV coinfected patients, are presented and described. The late occurrence (after six months of therapy) of this severe hemolytic anemia leads to the recommendation that hemoglobin levels should be monitored throughout the treatment period, even among patients who presented stable hemoglobin levels in the preceding months.

São apresentados e discutidos dois casos de anemia hemolítica auto-imune que ocorreram durante o tratamento da hepatite crônica pelo vírus C, com interferon peguilado alfa 2a e ribavirina, em pacientes co-infectados pelo HIV. A ocorrência de anemia hemolítica grave em etapa tardia, após o sexto mês da terapêutica, recomenda que o controle dos níveis de hemoglobina deva ser feito durante todo o período do tratamento , mesmo nos pacientes que apresentam níveis estáveis de hemoglobina nos meses precedentes.

The Degree of TGF-beta1 and alpha-SMA Expression in Liver Tissues with its Prognostic Value for the Therapeutic Efficacy of Interferon in Chronic Hepatitis B / 대한간학회지

BACKGROUND/AIMS: The aim of this study was to evaluate the effect of alpha-interferon (IFN) on liver histology as well as on activation of hepatic stellate cell ( HSC) and trans for ming growth fact or beta-1 (TGF beta-1) expression. We had also investigated the clinical usefulness of TGFbeta-1 and alpha-smooth muscle actin (alpha-SMA) expression in liver tissue for predicting a response to alpha-IFN therapy in chronic hepat it is B. METHODS: We studied the expression of TGFbeta-1 and alpha-SMA in liver biopsys pecimens from 51 chronic hepatitis B pat ients. Using immunohistochemical staining and a semiquant it ative scoring met hod, we also evaluated TGF-beta1 and alpha-SMA expression in liver stellate cells before and after alpha-IFN therapy in liver tissue from rebiopsys pecimen of the 12 chronic hepatitis B pat ients. Recombinant IFN alpha-2b (Intron A) in doses of 6 MU/ d was given to patients intramus cularly three times per week for 6 months (total doses , 432 MU). The patients were divided into two groups according to serum alanine aminotransferase levels as well as HBV- DNA and HBeAg s eroconversion stat e. Histological grading and staging scores were according to modified Histological Activity Index (HAI) grading systems of Ishak (1995). RESULTS: The index of portal inflammation and total scores of HAI grading significantly decreased in biopsies after alpha-IFN treatment, but the scores of fibrosis staging showed no significant change in biopsies after IFN treatment. A significant decrease in alpha-SMA expression, especially in periportal area, was found, but the change of TGFbeta-1 expression was not significant. The immunoreactivity of alpha-SMA was significantly lower in responders than in non-responders, whereas the diffference of immunoreactivity of TGF-beta1 between these two groups was not found. CONCLUSIONS: These findings suggest that alpha-IFN therapy may reduce the necroinflammatory activity in liver tissues of chronic B viral hepatitis and that the degree of alpha-SMA expression before treatment may be employed as a pottent predicting indicator for the therapeutic efficacy of IFN-alpha.