Clinical Responses of Oncolytic Coxsackievirus A21 (V937) in Patients With Unresectable Melanoma.

PURPOSE: We evaluated the activity of intratumoral Coxsackievirus A21 (V937) in 57 patients with unresectable stage IIIC or IV melanoma. PATIENTS AND METHODS: In this multicenter, open-label, phase II study, patients received up to a total V937 dose of 3 × 108 TCID50 (50% tissue culture infectious dose) in a maximum 4.0-mL volume by intratumoral injection. Ten sets of V937 injections were administered between days 1 and 127 (NCT01227551). Patients who had stable disease or were responding could continue treatment in an extension study (NCT01636882). Response and progression status were based on contrast-enhanced computed tomography, magnetic resonance imaging, or caliper measurement and were categorized using immune-related Response Evaluation Criteria in Solid Tumors (irRECIST). Other evaluations included monitoring of adverse events and serum levels of V937 and anti-V937 antibody titers. The primary efficacy end point was 6-month progression-free survival (PFS) rate per irRECIST. RESULTS: The primary efficacy end point, 6-month PFS rate per irRECIST, was 38.6% (95% CI, 26.0 to 52.4). Durable response rate (partial or complete response for ≥ 6 months) was 21.1% per irRECIST. Best overall response rate (complete plus partial response) was 38.6% (unconfirmed) and 28.1% (confirmed) per irRECIST. Regression of melanoma was observed in noninjected lesions. Based on Kaplan-Meier estimation, 12-month PFS was 32.9% (95% CI, 19.5 to 46.9) per irRECIST and 12-month overall survival was 75.4% (95% CI, 62.1 to 84.7). No treatment-related grade ≥ 3 adverse events occurred. Viral RNA was detected in serum within 30 minutes of administration. Neutralizing antibody titers increased to > 1:16 in all patients after day 22, without effect on clinical or immunologic response. CONCLUSION: V937 was well tolerated and warrants further investigation for treatment of patients with unresectable melanoma. Studies of combination approaches with V937 and immune checkpoint inhibitors are ongoing.

Myeloid-Derived Suppressor Cells Restrain Natural Killer Cell Activity in Acute Coxsackievirus B3-Induced Myocarditis.

Murine models of coxsackievirus B3 (CVB3)-induced myocarditis well represent the different outcomes of this inflammatory heart disease. Previously, we found that CVB3-infected A.BY/SnJ mice, susceptible for severe acute and chronic myocarditis, have lower natural killer (NK) cell levels than C57BL/6 mice, with mild acute myocarditis. There is evidence that myeloid-derived suppressor cells (MDSC) may inhibit NK cells, influencing the course of myocarditis. To investigate the MDSC/NK interrelationship in acute myocarditis, we used CVB3-infected A.BY/SnJ mice. Compared to non-infected mice, we found increased cell numbers of MDSC in the spleen and heart of CVB3-infected A.BY/SnJ mice. In parallel, S100A8 and S100A9 were increased in the heart, spleen, and especially in splenic MDSC cells compared to non-infected mice. In vitro experiments provided evidence that MDSC disrupt cytotoxic NK cell function upon co-culturing with MDSC. MDSC-specific depletion by an anti-Ly6G antibody led to a significant reduction in the virus load and injury in hearts of infected animals. The decreased cardiac damage in MDSC-depleted mice was associated with fewer Mac3+ macrophages and CD3+ T lymphocytes and a reduced cardiac expression of S100A8, S100A9, IL-1ß, IL-6, and TNF-α. In conclusion, impairment of functional NK cells by MDSC promotes the development of chronic CVB3 myocarditis in A.BY/SnJ mice.

Rac1-dependent endocytosis and Rab5-dependent intracellular trafficking are required by Enterovirus A71 and Coxsackievirus A10 to establish infections.

Enterovirus A71 (EVA71) and Coxsackievirus A10 (CVA10) are representative types of Enterovirus A. Dependent on the host cell types, the EVA71 entry may utilize clathrin-, caveola-, and endophilin-A2-mediated endocytosis. However, the cell-entry and intracellular trafficking pathways of CVA10, using KREMEN1 as its receptor, are unclear. Here, we tested the relevant mechanisms through RNA interference (RNAi) and chemical inhibitors. We found that endocytosis of EVA71 and CVA10 in rhabdomyosarcoma (RD) cells engaged multiple pathways, and both viruses required Rac1. Interestingly, while CDC42 and Pak1 participated in EVA71 infection, PI3K played a role in CVA10 infection. The functions of Rab proteins in intracellular trafficking of CVA10 and EVA71 were examined by RNAi. Knockdown of Rab5 and Rab21 significantly reduced CVA10 infectivity, while knockdown of Rab5, Rab7 and Rab9 reduced EVA71 infectivity. Confocal microscopy confirmed the colocalization of CVA10 virions with Rab5 or Rab21, and colocalization of EVA71 virions with Rab5 or Rab7. Additionally, we observed that both CVA10 and EVA71 infections were inhibited by endosome acidification inhibitors, bafilomycin-A1 and NH4Cl. Together, our findings comparatively illustrate the entry and intracellular trafficking processes of representative Enterovirus A types and revealed novel enterovirus intervention targets.

Persistent mammalian orthoreovirus, coxsackievirus and adenovirus co-infection in a child with a primary immunodeficiency detected by metagenomic sequencing: a case report.

BACKGROUND: We report a rare case of Mammalian orthoreovirus (MRV) infection in a child with a primary immunodeficiency (PID). Infections with Mammalian orthoreovirus are very rare and probably of zoonotic origin. Only a few cases have been described so far, including one with similar pathogenesis as in our case. CASE PRESENTATION: The patient, age 11, presented with flu-like symptoms and persistent severe diarrhea. Enterovirus has been detected over several months, however, exact typing of a positive cell culture remained inconclusive. Unbiased metagenomic sequencing then detected MRV in stool samples from several time points. The sequencing approach further revealed co-infection with a recombinant Coxsackievirus and Adenovirus. MRV-specific antibodies detected by immunofluorescence proved that the patient seroconverted. CONCLUSION: This case highlights the potential of unbiased metagenomic sequencing in supplementing routine diagnostic methods, especially in situations of chronic infection with multiple viruses as seen here in an immunocompromised host. The origin, transmission routes and implications of MRV infection in humans merit further investigation.

Investigation into the correlations of expressions of Cav-3 and Smad3 with pathogenesis and prognosis of viral myocarditis.

OBJECTIVE: To investigate the correlations of expressions of Caveolae-3 (Cav-3) and sma and mad homologue (Smad3) with the pathogenesis and prognosis of viral myocarditis (VMC). MATERIALS AND METHODS: VMC animal models were prepared and divided into the control group, the virus group and the Shenmai group. We detected the levels of creatine kinase isoenzyme (CK-MB) in the serum that was associated with the myocardial injuries, investigated the pathological features of VMC in BALB/C mice via hematoxylin-eosin (HE) staining, measured the mRNA expressions of Cav-3 and Smad3 via Real-time polymerase chain reaction (RT-PCR) and determined the protein expressions of Cav-3 and Smad3 through Western blotting method. RESULTS: The expressions of CK-MB in the virus group and Shenmai group were significantly higher than those in the control group; in comparison with the virus group, obvious improvement was identified in the pathologic condition of the Shenmai group; also, there was a statistically significant difference in comparison of the pathologic scores of BALB/C mice between the Shenmai group and the virus group. The mRNA expressions of Cav-3 and Smad3 in the virus group and Shenmai group were significantly higher than those in the control group, and the differences had statistical significance; however, higher mRNA expressions were identified in the virus group. Besides, protein expressions of Cav-3 and Smad3 in the virus group and Shenmai group were remarkably higher than those in the control group with statistically significant differences, but those in the virus group were much higher. CONCLUSIONS: Cav-3 and Smad3 may be involved in the occurrence and development of VMC, which provides some theoretical evidence for further research into the pathogenesis of VMC and the development of clinical drugs for treatment of VMC.

Inhibition of 12/15-LO ameliorates CVB3-induced myocarditis by activating Nrf2.

Cardiac 12/15-lipoxygenase (12/15-LO) was reported to be markedly up-regulated and involved in the development of heart failure. Nuclear factor E2-related factor 2 (Nrf2) plays anti-inflammatory and anti-oxidation roles in response to oxidative stress. However, the role of 12/15-LO in viral myocarditis (VMC) and its underlying molecular mechanism have not yet been elucidated. Here, we demonstrated that 12/15-LO was up-regulated and Nrf2 was down-regulated in coxsackievirus B3 (CVB3)-infected mice and cardiac myocytes. Baicalein, the specific inhibitor of 12/15-LO, was employed to investigate the role of 12/15-LO and its underlying mechanism in VMC. We found that baicalein treatment alleviated CVB3-induced VMC mouse models, as demonstrated by less inflammatory lesions in the heart tissues and less CK-MB level. Moreover, baicalein treatment attenuated CVB3-induced inflammatory cytokine production and oxidative stress. Mechanistic analysis suggested that baicalein treatment relieved CVB3-induced reduction of Nrf2 and heme oxygenase-1 (HO-1) expressions. Taken together, our study indicated that inhibition of 12/15-LO ameliorates VMC by activating Nrf2, providing a new therapeutic strategy for the therapy of VMC.

2,3,7,8-tetrachlorodibenzo-p-dioxin and the viral infection.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a widespread highly toxic environmental contaminant, suppresses immune response and leads to an increased susceptibility to infectious agents. In particular, several studies have provided evidence that TCDD decreases resistance to numerous viruses. Indeed, in vivo and in vitro investigations showed that the presence of TCDD is able to interfere with the replication of both human and animal viruses, such as influenza A viruses, coxsackie virus B3, immunodeficiency virus type-1 (HIV-1), cytomegalovirus (CMV), herpes simplex II, and bovine herpesvirus 1. Moreover, TCDD could induce an exacerbation of latent infection produced by HIV-1, CMV or Epstein-Barr virus. In this review, we first describe the general effects of TCDD exposure on mammalian cells, then we focus on its influence on the viral infections. Overall, the available data support the concept that TCDD exposure may act as an additional risk factor in promoting of viral diseases.

[Significance of selenium in the pathogenesis and therapy of cardiovascular diseases and those requiring intensive care]. / A szelén szerepe a cardiovascularis és intenzív kezelést igénylo betegségek létrejöttében és kezelésében.

Selenium deficiency results in profound changes in cellular defence mechanisms against oxidative stress, which plays an important role in the development of cardiovascular disease and the associated risk factors. Increased formation and decreased elimination of reactive oxygen radicals contribute to the complicated mechanisms of sepsis and related disorders. Use of selenium in prevention and treatment of the above mentioned conditions is not a new idea, but controversial data were published in relation to both fields recently. The aim of the present review is to summarize the most important results related to this area.

Coxsackievirus B1 peritonitis in a patient treated with continuous ambulatory peritoneal dialysis: a case report and brief review of the literature.

We report a case of viral peritonitis caused by coxsackievirus B1 in a 79-year-old male undergoing continuous ambulatory peritoneal dialysis (CAPD), and review the English language literature. Clinicians should be aware of viral peritonitis in patients on CAPD presenting with a viral syndrome and mononuclear peritoneal dialysis effluent. Currently, viral diagnostic tests are available to confirm the diagnosis and avoid unnecessary treatment with antibiotics.

2012 outbreak of acute haemorrhagic conjunctivitis in Indian Ocean Islands: identification of Coxsackievirus A24 in a returned traveller.

In May 2012, a Coxsackievirus A24 haemorrhagic conjunctivitis was diagnosed in Marseille, France, in a traveller returning from the Comoros Islands. This case allowed identification of the cause of an ongoing outbreak of haemorrhagic conjunctivitis in Indian Ocean Islands, illustrating that returning travellers may serve as sentinels for infectious diseases outbreaks in tropical areas where laboratory investigation is limited.

[Expression of cystathionine-γ-lyase/hydrogen sulfide pathway in CVB3-induced myocarditis in mice].

OBJECTIVE: Previous studies have shown that hydrogen sulfide (H2S) plays key roles in a number of biological processes, including vasorelaxation, inflammation, apoptosis, ischemia/reperfusion and oxidative stress, which are involved in the pathogenesis of myocarditis. This study aimed to examine the expression of cystathionine-γ-lyase(CSE)/H2S pathway in mice with viral myocarditis. METHODS: Six-week-old inbred male mice were randomly assigned to control (n=25) and myocarditis group (n=30). The myocarditis and the control groups were inoculated intraperitoneally with 0.1 mL 10-5.69TCID50/mL CVB3 or vehicle (PBS) alone respectively. Ten mice were sacrificed 4 and 10 days after injection. Blood and heart specimens were harvested for measuring the content of serum H2S and the H2S production rates in cardiac tissues. Heart sections were stained with hematoxylin and eosin. Immunohistochemisty was used to detect the CSE protein expression in the heart. RESULTS: In the myocarditis group, the serum H2S content and H2S production rates in cardiac tissues were significantly higher than those in the control group 4 and 10 days after injection (P<0.05). The expression of CSE protein in the heart in the myocarditis group was also significantly higher than that in the control group (P<0.05). CONCLUSIONS: CSE and its downstream production H2S increase in mice with acute viral myocarditis. The increased expression of CSE/H2S pathway might be involved in the pathogenesis of viral myocarditis.

Proteasome activator REGgamma enhances coxsackieviral infection by facilitating p53 degradation.

Coxsackievirus B3 (CVB3) is a small RNA virus associated with diseases such as myocarditis, meningitis, and pancreatitis. We have previously demonstrated that proteasome inhibition reduces CVB3 replication and attenuates virus-induced myocarditis. However, the underlying mechanisms by which the ubiquitin/proteasome system regulates CVB replication remain unclear. In this study, we investigated the role of REGγ, a member of the 11S proteasome activator, in CVB3 replication. We showed that overexpression of REGγ promoted CVB3 replication but that knockdown of REGγ led to reduced CVB3 replication. We further demonstrated that REGγ-mediated p53 proteolysis contributes, as least in part, to the proviral function of REGγ. Although total protein levels of REGγ remained unaltered after CVB3 infection, virus infection induced a redistribution of REGγ from the nucleus to the cytoplasm, rendering an opportunity for a direct interaction of REGγ with viral proteins and/or host proteins (e.g., p53), which controls viral growth and thereby enhances viral infectivity. Further analyses suggested a potential modification of REGγ by SUMO following CVB3 infection, which was verified by both in vitro and in vivo sumoylation assays. Sumoylation of REGγ may play a role in its nuclear export during CVB3 infection. Taken together, our results present the first evidence that the host REGγ pathway is utilized and modified during CVB3 infection to promote efficient viral replication.

Care of the acutely ill pediatric heart transplant recipient.

Young children may present to the emergency department after undergoing orthotopic heart transplantation. Emergency department care of pediatric heart transplant recipients with an acute illness is not clearly defined. To assist the emergency physician with treating these children, 2 cases are detailed to demonstrate the variation in outcome of young children presenting to the emergency department with acute illness months after their transplant. The cases typify the varied outcomes possible after emergency department presentation. After the cases, a review of the medical conditions common in transplant recipients precedes a description of management suggestions.

Proteome alterations in human host cells infected with coxsackievirus B3.

Coxsackievirus B3 (CVB3) is a common factor in human myocarditis. The interplay between host factors and virus components is crucial for the fate of the infected cells. Despite that, host protein responses, which characterize CVB3-induced diseases, have not yet been determined in detail. To investigate the nature of modified protein patterns in infected human cells compared with uninfected cells, two-dimensional gel electrophoresis in combination with matrix-assisted laser desorption/ionization-mass spectrometry were used. The regulated proteins, e.g. nucleophosmin (nucleolar protein B23), lamin, the RNA-binding protein UNR and the p38 mitogen-activated protein kinase, were sorted according to their functional groups and interpreted in the context of the myocarditis process.

Host nutritional status: the neglected virulence factor.

The emergence of new infectious diseases and old diseases with new pathogenic properties is a burgeoning worldwide problem. Severe acute respiratory syndrome (SARS) and acquired immune deficiency syndrome (AIDS) are just two of the most widely reported recent emerging infectious diseases. What are the factors that contribute to the rapid evolution of viral species? Various hypotheses have been proposed, all involving opportunities for virus spread (for example, agricultural practices, climate changes, rainforest clearing or air travel). However, the nutritional status of the host, until recently, has not been considered a contributing factor to the emergence of infectious disease. In this review, we show that host nutritional status can influence not only the host response to the pathogen, but can also influence the genetic make-up of the viral genome. This latter finding markedly changes our concept of host-pathogen interactions and creates a new paradigm for the study of such phenomena.

Coxsackievirus B4 infection of human fetal thymus cells.

The infection of human fetal thymus organ cultures (FTOC) with coxsackievirus B4 E2 (CVB4 E2) was investigated. Both positive- and negative-strand viral RNA were detected by real-time quantitative reverse transcription-PCR (RT-PCR) in CVB4 E2-infected FTOC, which supported high yields of virus production (approximately 10(6) 50% tissue culture infective doses/ml), and in flow-sorted thymocyte populations for 7 days after inoculation. Cortical CD4+ CD8+ thymocytes were found to be the principal targets of infection. Inoculation of human FTOC with CVB4 E2 led to a marked and progressive depletion of immature thymocytes (CD4+ CD8+ cells) with no enhancement of Annexin V-positive cells. CVB4 E2 replication caused significant major histocompatibility complex (MHC) class I upregulation on these cells. MHC class I upregulation was correlated with positive- and negative-strand RNA quantitative detection and the release of infectious particles. In addition, chloroquine treatment of FTOC and single-thymocyte suspensions suggested that MHC class I upregulation on thymocytes was the result of direct infection rather than caused by production of soluble factors such as alpha interferon. Thus, CVB4 E2 can infect human fetal thymocytes, which subsequently results in quantitative and qualitative abnormalities of these cells.

Animal models for autoimmune myocarditis and autoimmune thyroiditis.

This chapter describes four murine models of autoimmune diseases: two related to autoimmune myocarditis and two related to autoimmune thyroiditis. The first model, Coxsackie virus B3 (CB3)-induced myocarditis, results in the development of acute myocarditis in susceptible as well as resistant mouse strains, whereas chronic myocarditis develops only in genetically susceptible mice. CB3-induced myocarditis closely resembles the course of human myocarditis, which is believed to be initiated by viral infection. Mouse cardiac myosin heavy chain has been identified as the major antigen associated with the late chronic phase of viral myocarditis. The second model is cardiac myosin-induced experimental autoimmune myocarditis (EAM) and, in a modification, cardiac alpha-myosin heavy chain peptide-induced myocarditis. In the EAM model, cardiac myosin or the relevant peptide in Freund's complete adjuvant (FCA) is injected subcutaneously into mice. The immune response, the histological changes, and the genetic susceptibility seen in EAM are similar to those of CB3-induced myocarditis. The third model is experimental autoimmune thyroiditis (EAT). EAT can be induced in genetically susceptible strains of mice by immunization with mouse thyroglobulin in FCA or lipopolysaccharide. Mice susceptible to EAT have the H-2A(k), H-2A(s), or H-2A(q) alleles. We describe here a standard technique for the induction of EAT; it was developed in our laboratory and is widely used as a model for studying Hashimoto's thyroiditis. The fourth model presented in this chapter is that of spontaneous autoimmune thyroiditis in NOD.H2h4 mice. These mice express the H-2A(k) allele on an NOD genetic background and develop spontaneous thyroiditis, which is exacerbated with dietary iodine.

Heparan sulfates and coxsackievirus-adenovirus receptor: each one mediates coxsackievirus B3 PD infection.

Amino acid exchanges in the virus capsid protein VP1 allow the coxsackievirus B3 variant PD (CVB3 PD) to replicate in decay accelerating factor (DAF)-negative and coxsackievirus-adenovirus receptor (CAR)-negative cells. This suggests that molecules other than DAF and CAR are involved in attachment of this CVB3 variant to cell surfaces. The observation that productive infection associated with cytopathic effect occurred in Chinese hamster ovary (CHO-K1) cells, whereas heparinase-treated CHO-K1 cells, glucosaminoglycan-negative pgsA-745, heparan sulfate (HS)-negative pgsD-677, and pgsE-606 cells with significantly reduced N-sulfate expression resist CVB3 PD infection, indicates a critical role of highly sulfated HS. 2-O-sulfate-lacking pgsF-17 cells represented the cell line with minimum HS modifications susceptible for CVB3 PD. Inhibition of virus replication in CHO-K1 cells by polycationic compounds, pentosan polysulfate, lung heparin, and several intestinal but not kidney HS supported the hypothesis that CVB3 PD uses specific modified HS for entry. In addition, recombinant human hepatocyte growth factor blocked CVB3 PD infection. However, CAR also mediates CVB3 PD infection, because this CVB3 variant replicates in HS-lacking but CAR-bearing Raji cells, infection could be prevented by pretreatment of cells with CAR antibody, and HS-negative pgsD-677 cells transfected with CAR became susceptible for CVB3 PD. These results demonstrate that the amino acid substitutions in the viral capsid protein VP1 enable CVB3 PD to use specific modified HS as an entry receptor in addition to CAR.