Theiler's virus, a murine picornavirus, causes a persistent infection of macrophage/microglial cells in the central nervous systems of SJL/J mice. Viral replication is restricted in the majority of infected cells, whereas a minority of them contain large amounts of viral RNA and antigens. For the present work, we infected primary cultures of bone marrow monocytes/macrophages from SJL/J mice with Theiler's virus. During the first 10 h postinfection (p.i.), infected monocytes/macrophages were round and covered with filopodia and contained large amounts of viral antigens throughout their cytoplasm. Later on, they were large, flat, and devoid of filopodia and they contained only small amounts of viral antigens distributed in discrete inclusions. These two types of infected cells were very reminiscent of the two types of infected macrophages found in the spinal cords of SJL/J mice. At the peak of virus production, the viral yield per cell was approximately 200 times lower than that for BHK-21 cells. Cell death occurred in the culture during the first 24 h p.i. but not thereafter. No infected cells could be detected after 4 days p.i., and the infection never spread to 100% of the cells. This restriction was unchanged by treating the medium at pH 2 but was abolished by treating it with a neutralizing alpha/beta interferon antiserum, indicating a role for this cytokine in limiting virus expression in monocyte/macrophage cultures. The role of alpha/beta interferon was confirmed by the observation that monocytes/macrophages from IFNA/BR(-/-) mice were fully permissive.

IFN regulatory factor 1 (IRF1) is one of the most important IFN-stimulated genes (ISGs) in cellular antiviral immunity. Although hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide, how ISGs counteract HEV infection is largely unknown. This study was conducted to investigate the effect of IRF1 on HEV replication. Multiple cell lines were used in 2 models that harbor HEV. In different HEV cell culture systems, IRF1 effectively inhibited HEV replication. IRF1 did not trigger IFN production, and chromatin immunoprecipitation sequencing data analysis revealed that IRF1 bound to the promoter region of signal transducers and activators of transcription 1 (STAT1). Functional assay confirmed that IRF1 could drive the transcription of STAT1, resulting in elevation of total and phosphorylated STAT1 proteins and further activating the transcription of a panel of downstream antiviral ISGs. By pharmacological inhibitors and RNAi-mediated gene-silencing approaches, we revealed that antiviral function of IRF1 is dependent on the JAK-STAT cascade. Furthermore, induction of ISGs and the anti-HEV effect of IRF1 overlapped that of IFNα, but was potentiated by ribavirin. We demonstrated that IRF1 effectively inhibits HEV replication through the activation of the JAK-STAT pathway, and the subsequent transcription of antiviral ISGs, but independent of IFN production.-Xu, L., Zhou, X., Wang, W., Wang, Y., Yin, Y., van der Laan, L. J. W., Sprengers, D., Metselaar, H. J., Peppelenbosch, M. P., Pan, Q. IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes.

The possible effect of interferon-alpha (IFNa) on liver fibrosis progression has not been adequately studied in chronic hepatitis B. We evaluated 147 patients with HBeAg-negative chronic hepatitis B who had>> or =2 liver biopsies and had been treated with IFNa (n = 120) or had remained untreated (n = 27). The median interval between the two biopsies was 24 (12-160) months. All biopsies were scored blindly by a single liver histopathologist according to the classification of Ishak et al. (J Hepatol 1995; 22: 696-699). IFNa induced sustained biochemical response in 30, initial response and subsequent relapse in 57 and no response in 33 patients. Fibrosis improved in 17.5% of treated (sustained responders: 40%, relapsers: 9%, nonresponders: 12%) and 4% of untreated patients and worsened in 34% (sustained responders: 7%, relapsers: 40%, nonresponders: 48%) and 70% of cases, respectively (P = 0.002). The annual rate of fibrosis progression was worse in the untreated (0.427 +/- 0.119) than in treated patients (0.067 +/- 0.052, P = 0.001). However, the fibrosis progression rate in the untreated patients was not significantly different than the net fibrosis progression rate (after subtraction of IFNa duration) in nonresponders or relapsers. In multivariate analysis, worse fibrosis progression rate was associated with older age (P = 0.010), worse baseline grading score (P < 0.001), lower baseline fibrosis (P = 0.035) and the type of response to IFNa (P = 0.032). In conclusion, in HBeAg-negative chronic hepatitis B, IFNa significantly reduces the rate of fibrosis progression, but such an effect is mainly observed in patients with sustained biochemical responses. In relapsers and nonresponders, fibrosis benefit equals the treatment period. The strongest factor associated with fibrosis progression is the change in necroinflammatory activity.

In the United States, the estimated number of new cases of renal cell carcinoma (RCC) is approximately 65,000 case with about 15,000 deaths in the year of 2018 (Siegel et al. in CA Cancer J Clin 68(1):7, 2018). RCC as an immunogenic malignancy is supported by many theories and facts which include tumor richness of lymphocytes infiltrate, the occurrence of spontaneous tumor regression, and the proved effect of traditional immunotherapy (Finke et al. in J Immunother 11(1):1-11, 1992), all these factors support the potential therapeutic effect of the novel immunotherapeutic agents in RCC. Historically, complete tumor regression in metastatic RCC is achievable in a minority of patients through traditional immunotherapies such as high-dose interleukin-2 (IL-2) (Fyfe et al. in J Clin Oncol 13(3):688, 1995) and interferon-alfa (IFNa) (Negrier et al. in N Engl J Med 338(18):1272, 1998); however due to the significant rate of toxicities and low efficacy; accordingly the targeted therapy with tyrosine kinase inhibitors (TKIs) and vascular endothelial growth factor-antibodies (VEGF) became the standard and prevalent treatment approach for advanced RCC both in front and subsequent lines of therapy (Escudier et al. in Ann Oncol. 25(Suppl 3):iii49-iii56, 2014). A new avenue of immunotherapy utilizing novel strategy to block immune checkpoints has emerged in a new era for RCC treatment (Ascierto et al. in J Transl Med 12:291, 2014). Results from clinical trials are encouraging in both front-line and second-line settings, in a phase III trial (CheckMate 025) nivolumab compared to everolimus improved overall survival in previously treated metastatic RCC who had progressed on prior treatment with targeting agents (Motzer et al. in N Engl J Med 373:1803, 2015). CheckMate 214, a phase III trial, demonstrated superior overall survival and objective response with combined checkpoint inhibitors compared to sunitinib in Treatment-Naïve Advanced RCC among intermediate- and poor-risk group (Motzer et al. in N Engl J Med. 378(14):1277-1290, 2018). In this review, we discuss the systemic Immunotherapy with checkpoint inhibitors that have been approved or are currently being investigated in RCC, clinical experience with these agents, and its future development.

High-dose interferon alfa-2b (IFNα-2b) is the only approved adjuvant systemic therapy for resected, high risk melanoma in the United States (Fecher and Flaherty, in Natl Compr Cancer Netw 7:295-304, 2009). Recently, two important meta-analyses of randomized trials (Wheatley et al., in J Clin Oncol, 2007; Mocellin et al. in J Natl Cancer Inst, 2010) investigating IFNα-2b versus observation in high risk melanoma patients, showed that adjuvant IFNα-2b has an impact both on relapse-free survival (RFS) and overall survival (OS) independently by dosage, duration and route compared with observation in high risk melanoma patients. Despite of an absolute benefits of 3 % (Wheatley et al., in J Clin Oncol, 2007), this treatment is associated with significant toxicity, which impacts on patient quality of life. A better understanding of the mechanism of action may help to potentiate the clinical efficacy and reduce the toxicity of IFNα-2b/Peg-IFNα-2b. Numerous studies suggest that interferon's mechanism of action in melanoma is primarily immunomodulatory (Table 1) (de La Salmoniere, in Clin Cancer Res 6:4713-4718, 2000; Stuckert, in J Clin Oncol 25:8506, 2007; Gogas et al., in N Engl J Med 354:709-718, 2006; Moschos et al., in J Clin Oncol 24:3164-3171, 2006; Ascierto and Kirkwood, in J Transl Med 6:62, 2008) Recent efforts to elucidate the mechanism of action for interferon have focused upon signal transducers and activators of transcription (STAT) (Simons et al., in J Transl Med 9:52, 2011) signaling and immunoregulatory responses mediated by regulatory T cells (Tregs) (Wang et al., in Clin Cancer Res 13:1523-1531, 2007; Clin Cancer Res 14:8314-8320, 2008). Tregs are a suppressive CD4+ T cell population that is present, along with primed effector T cells, in tumor and tumor-draining lymph nodes (Hiura et al. in J Immunol 175:5058-5066, 2005). Tregs express high levels of surface antigens such as CD25, cytotoxic T lymphocyte associated antigen 4 (CTLA-4), and glucocorticoid-induced tumor necrosis factor receptor (GITR) (Takahashi et al., in J Exp Med 192:303-310, 2000; Shimizu et al., in Nat Immunol 3:135-142, 2002). Moreover, Tregs express a characteristic nuclear transcription regulator, forkhead box P3 (FoxP3) (Hori et al., in Science 299:1057-1061, 2003; Gabriel and Lattime, in Clin Cancer Res 13:785-788, 2007). The presence of Tregs in tumor-draining lymph nodes and tumors provides a potential inhibitory population that may block or balance effector cell function. Thus, depletion of Tregs or blockade of Treg function using targeted antibodies or other strategies might be able to remove Treg suppression and enhance antitumor immunity (Viguier et al., in J Immunol 173:1444-1453, 2004). We conducted an observational study to examine whether the induction phase of the FDA-approved HDI regimen administered iv in patients with stage 3-4 melanoma (20 MU/m(2) intravenously (IV) five times per week for 4 weeks) reduced the number of Treg cells in the peripheral blood.

In chronic hepatitis C virus (HCV) infections, the current standard of care (combination therapy with pegylated alpha interferon (PEG-IFNalpha) and ribavirin) is only effective in around 50% of cases. The aim of the present study was to analyze the relationship between the HCV load and the PEG-IFN concentration during the first week of treatment. Fifteen treatment-naive patients with chronic hepatitis C infection (genotypes 1, 2, 3, and 4) underwent PEG-IFNα-2b/ribavirin combination therapy. Blood samples were collected before the first injection (T(0)) and then at different time points until the next injection a week later. The PEG-IFN concentration and the HCV load were assayed. The serum interferon concentration peaked 2 days after the first injection (mean value for the study population; T(max) = 40.9 hr; C(max) = 490 pg/ml) and a trough in viral load was seen at day 3. The PEG-IFNalpha-2b concentration decreased from day 2 to day 7, enabling a viral rebound in all patients. The change in viral load between day 0 and day 3 differed significantly according to whether the patients were responders at week 12 (Deltalog d(0)/d(3) = 2.729 +/- 1.419 log(10) IU/ml) or not (Deltalog d(0)/d(3) = 1.102 +/- 0.472 log(10) IU/ml). Our results emphasize the potential clinical importance of achieving viral decay immediately after initiation of interferon-ribavirin combination therapy. J. Med. Virol. 82:1640-1646, 2010. 2010 Wiley-Liss, Inc.

BACKGROUND

To elucidate relationship between amino acid sequence of non-structural protein 5A (NS5A) and outcome of HCV (1 b) patients after interferon (IFNa) therapy.

METHODS

Sera of 24 patients were collected before, during and after IFNa therapy. Pretreatment RNA levels and the sequences of HCV NS5A interferon sensitivity determining region (ISDR) were determined. NS5A full-length sequences of 5 HCV isolates from 3 patients with different response types were also analyzed. Phylogenetic tree analysis and protein secondary structure prediction were undertaken.

RESULTS

Pretreatment RNA levels of sustained response group were significantly lower than that of non-response group and relapse group (4.50X104 copies/ml versus 1.82X107 copies/ml, P < 0.01).ISDR sequences of NS5A from pretreatment sera were compared with HCV-J strain (prototype). Thirteen of 24 isolates were wild type,11 of 24 were intermediate type and none of them was mutant type. 3 of 6 sustained responders were infected with wild-type isolates, the rest with intermediate type isolates. Phylogenetic tree based on NS5A full-length sequences classified 5 isolates with 3 different response types into 3 groups. Non-response isolates belonged to the same group as HCV-J. Secondary structure prediction of 5 isolates revealed significant differences existing in 2 255- 2 289. This region was partly overlapped with PKR-binding domain.

CONCLUSIONS

Low HCV RNA levels in serum are associated with favorable outcome of IFNa therapy. ISDR sequence alone could not predict outcome of IFN treatment. Combination of determination of HCV RNA levels in serum with sequence analysis of PKR-binding domain may be helpful in predicting the efficacy of IFN therapy.

The genetic alterations responsible for the development of cutaneous lymphoma are largely unknown. Chromosome region 9p21 contains a gene locus encoding an inhibitor of cyclin-dependent kinase 4, and heterozygous deletions of this tumor suppressor gene (p16) have been shown in a variety of malignant tumors. We studied 11 randomly selected cutaneous CD30-positive large cell lymphomas. Several areas containing 20-50 CD30-positive lymphocytes were microdissected in each case and subjected to single-step DNA extraction. Loss of heterozygosity analysis was performed using polymorphic markers at 9p21 (IFNA, D9S171, D9S169) and 17p13 (TP53). Samples from normal cells apart from CD30-positive lymphocytes, e.g., CD30-negative lymphohistiocytic infiltrates and normal epidermal layer, were also obtained in all cases from the same slide for comparison with the tumor samples. Expression of CD30 and T-lineage antigens (CD3, CD45Ro) was confirmed in all cases. Immunohistochemical staining for p16 and p53 was performed using the monoclonal antibodies sc-1661 and DO-7, respectively. Of the 11 informative cases, seven (64%) exhibited loss of heterozygosity at least for one marker at 9p21 (p16), whereas no allelic deletions were found for the polymorphic marker at 17p13 (p53). On immunohistochemistry loss of the p16 protein was detected in two of 11 cases. Nuclear staining for p53 protein was found in four of 11 cases. Here, we provide the first evidence of the involvement of the tumor suppressor gene p16 in primary cutaneous large cell lymphoma. Whether p16 deletion in these lymphomas is associated with disease progression and whether this method could serve as an early marker to detect lymphomas at an early stage needs to be addressed in future studies. J Invest Dermatol 115:1104-1107 2000

Previously published studies revealed that variation in expression of the DNA-binding protein ARID3a in B lymphocytes from patients with systemic lupus erythematosus (SLE) correlated with levels of disease activity ("Disease activity in systemic lupus erythematosus correlates with expression of the transcription factor AT-rich-interactive domain 3A" (J.M. Ward, K. Rose, C. Montgomery, I. Adrianto, J.A. James, J.T. Merrill et al., 2014) [1]). The data presented here compare DNA methylation patterns from SLE peripheral blood mononuclear cells obtained from samples with high numbers of ARID3a expressing B cells (ARID3a(H)) versus SLE samples with normal numbers of ARID3a(+) B cells (ARID3a(N)). The methylation data is available at the gene expression omnibus (GEO) repository, "Gene Expression Omnibus: NCBI gene expression and hybridization array data repository" (R. Edgar, M. Domrachev, A.E. Lash, 2002) [2]. Isolated B cells from SLE ARID3a(H) and ARID3a(N) B samples were also evaluated via qRT-PCR for Type I interferon (IFN) signature and pathway gene expression levels by qRT-PCR. Similarly, healthy control B cells and B cells stimulated to express ARID3a with the TLR agonist, CpG, were also compared via qRT-PCR. Primers designed to detect 6 IFNa subtype mRNAs were tested in 4 IFNa, Epstein-Barr Virus-transformed B cell lines ("Reduced interferon-alpha production by Epstein-Barr virus transformed B-lymphoblastoid cell lines and lectin-stimulated lymphocytes in congenital dyserythropoietic anemia type I" (S.H. Wickramasinghe, R. Hasan, J. Smythe, 1997) [3]). The data in this article support the publication, "Human effector B lymphocytes express ARID3a and secrete interferon alpha" (J.M. Ward, M.L. Ratliff, M.G. Dozmorov, G. Wiley, J.M. Guthridge, P.M. Gaffney, J.A. James, C.F. Webb, 2016) [4].

Regulatory T cells (Tregs), particularly the CD4(+)CD25(+)Foxp3(+) Tregs, down regulate immunity and promote tumor cell growth by directly suppressing CD8(+) and CD4(+) T cells. Alternatively they can promote tumor growth by generating interleukin-10 (IL-10) and transforming growth factor β (TGFβ) in situ, which help tumor cells to evade the immune system.

In vivo tumor models were prepared via subcutaneous injection with a suspension of B16 melanoma cells into the left upper flank of C57BL/6 J mice. The mice were randomized into five groups: radiotherapy (RT), chemotherapy (CT), radiochemotherapy (RCT), Inteferon α (INFα) groups, and a control group. Flow cytometry was used to determine the Tregs levels in the spleen and peripheral blood, and immunohistochemistry was performed to determine the expression levels of TGFβ and IL-10 in the tumor microenvironment.

Tumor weight was significantly reduced in the CT or RCT groups (40.91 % and 41.83 %, respectively), while the reduction in tumor weight was relatively lower for the RT and IFNα groups (15.10 % and 13.15 %, respectively). The flow cytometry results showed that the ratios of CD4(+)CD25(+)Foxp3(+) Tregs to lymphocytes and CD4(+) cells in the spleen and in peripheral blood were significantly decreased after treatment with IFNα (P < 0.05). Expression of TGFβ and IL-10 in the tumor microenvironment in the CT and RT groups was higher compared with the control group (P < 0.01), while the expression of TGFβ and IL-10 in the INFα group was not significantly different (P>> 0.05).

The results show that INFα-2b inhibits cancer cell immune evasion by decreasing the levels of CD4(+)CD25(+)Foxp3(+) Tregs and suppressing the expression of TGFβ and IL-10 in the tumor microenvironment.

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that causes immunosuppression and neoplastic diseases in poultry. Cytokine signal-transduction inhibitor molecule 3 (SOCS3) is an important negative regulator of the JAK2/STAT3 signaling pathway and plays certain roles in ALV-J infection. It is of significance to confirm the roles of SOCS3 in ALV-J infection and study how this gene affects ALV-J infection. In this study, we assessed the expression of the SOCS3 gene in vivo and in vitro, and investigated the roles of SOCS3 in ALV-J infection using overexpressed or interfered assays with the SOCS3 in DF-1 cells. The results showed that the SOCS3 expression of ALV-J infected chickens was different from uninfected chickens in the spleen, thymus and cecal tonsil. Further, SOCS3 is mainly expressed in the nucleus as determined by immunofluorescence assay. Overexpression of SOCS3 in DF-1 cells promoted the replication of ALV-J virus, and the expression of interferons (IFNα and INFβ), inflammatory factors (IL-6 and TNFα) along with interferon-stimulating genes (CH25H, MX1, OASL, and ZAP). Conversely, interference of SOCS3 showed the opposite results. We also observed that SOCS3 promoted ALV-J virus replication by inhibiting JAK2/STAT3 phosphorylation. In conclusion, SOCS3 promotes ALV-J replication via inhibiting the phosphorylation of the JAK2/STAT3 signaling pathway. These results would advance further understanding of the persistent infection and the viral immune evasion of the ALV-J virus.

Keywords: ALV-J; JAK2/STAT3; SOCS3; chicken; immune.

Type I interferons (IFNa/b) form a family of related cytokines that include INFa, b, e/s, j, x (human) and limitin (mouse). These cytokines exert a potent antiviral activity, control cell proliferation and modulate the immune response. They are used in the fight against viral infections, tumors, and multiple sclerosis. Expression of IFNs is typically induced by viral infections. Cells express cytoplasmic helicases as well as endosomial toll-like receptors acting as sensors to detect endogenous and exogenous viral infections, respectively. Signal transduction from these sensors induces the transcription of IFN genes. IFNs are secreted and bind to a cell surface receptor expressed by most cells of the organism. Upon receptor binding, IFNs induce the transcription of hundreds of genes whose products exert antiviral, antiproliferative and immunomodulatory functions. Antiviral activity of IFNs is so potent that most (if not all) viruses developed strategies to antagonize the IFN response.

Keywords: Tolllike receptor; antiviral defence; innate immunity; type I interferon; virus.