Encephalomyocarditis Research Articles

Certification of a pharmacopoeial reference standard for potency testing of recombinant interferon α-2b

SCIENTIFIC RELEVANCE. Potency testing of recombinant interferons requires a reference standard. The availability of International Standards (ISs) that are commonly used to assess the quality of recombinant interferons is currently limited. Therefore, the quality of interferons should be assessed using pharmacopoeial reference standards (RSs) certified using ISs (if available).AIM. This study aimed to certify a pharmacopoeial RS for potency testing of recombinant interferon α-2b.MATERIALS AND METHODS. The potency determination involved comparing the inhibition of the virus-induced cytopathic effect observed in cell culture with the candidate RS and the WHO International Standard for Human rDNA-derived interferon α-2b. The study used A-549 and MDBK cell lines with encephalomyocarditis (EMC) and vesicular stomatitis (VSV) viruses. The authors followed the requirements of the State Pharmacopoeia of the Russian Federation (Cell-Culture Bioassays for Interferon Products, OFS.1.7.2.0002.15). The results were recorded using instrumental and visual methods. The calculations used mathematical statistics. To factor in the influence of intermediate precision, the authors applied Student’s t-test.RESULTS. The authors developed a certification programme and procedure and certified the pharmacopoeial RS for recombinant human interferon α-2b (rhIFN α-2b) to identify the corresponding medicinal products (by virus neutralisation) and test their potency (by antiviral activity in cell culture). Upon receipt, the candidate RS was verified for compliance with regulatory specifications. According to the test results, the potency of the candidate pharmacopoeial RS was 4.47×108 IU/mL, and the expanded uncertainty was 8.12×107 IU/mL, with a coverage factor of k=2 and a confidence level of 95%. The pharmacopoeial RS for rhIFN α-2b was considered to have the same shelf-life period as the corresponding medicinal products if stored at a temperature not higher than –40 ºC. When thawed, the pharmacopoeial RS for rhIFN α-2b can be stored at a temperature of 2–8 ºC for up to 1 month.CONCLUSIONS. Upon certification for potency testing, the pharmacopoeial RS for rhIFN α-2b has been included in the Register of Reference Standards of the Russian Pharmacopoeia as Recombinant Human Interferon α-2b (Potency) FSO.3.2.00455. The introduction of this pharmacopoeial RS will help to conduct the identification and potency testing of all Russian interferon α-2b products at an appropriate scientific and methodological level. As a result, Russian pharmaceutical manufacturers and quality control agencies will no longer depend on imported ISs.

Tomatidine inhibits porcine epidemic diarrhea virus replication by targeting 3CL protease

Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in suckling piglets, leading to severe economic losses worldwide. There is an urgent need to find new therapeutic methods to prevent and control PEDV. Not only is there a shortage of commercial anti-PEDV drugs, but available commercial vaccines fail to protect against highly virulent PEDV variants. We screened an FDA-approved library of 911 natural products and found that tomatidine, a steroidal alkaloid extracted from the skin and leaves of tomatoes, demonstrates significant inhibition of PEDV replication in Vero and IPEC-J2 cells in vitro. Molecular docking and molecular dynamics analysis predicted interactions between tomatidine and the active pocket of PEDV 3CL protease, which were confirmed by fluorescence spectroscopy and isothermal titration calorimetry (ITC). The inhibiting effect of tomatidine on 3CL protease was determined using cleavage visualization and FRET assay. Tomatidine-mediated blocking of 3CL protease activity in PEDV-infected cells was examined by western blot detection of the viral polyprotein in PEDV-infected cells. It indicates that tomatidine inhibits PEDV replication mainly by targeting 3CL protease. In addition, tomatidine also has antiviral activity against transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), encephalo myocarditis virus (EMCV) and seneca virus A (SVA) in vitro. These results may be helpful in developing a new prophylactic and therapeutic strategy against PEDV and other swine disease infections.

Aspects of a Methodological Approach to Determination of Interferon Alpha Specific Activity

Specific antiviral activity is one of the key indicators characterising pharmaceutical quality and pharmacological efficacy of interferon alpha products (IFN-α). Specific activity is determined using a bioassay measuring antiviral activity in cell culture. The aim of the study was to select the most appropriate conditions for in vitro determination of IFN-α product specific activity. Materials and methods: Vero, MDBK, Hep-2, and A-549 homologous and heterologous cell cultures, as well as vesicular stomatitis Indiana virus (VSV) and murine encephalomyocarditis (EMC) virus at a dose of 100 TCD50 /0.1 mL were used for determination of specific antiviral activity. The international reference standard of recombinant interferon alpha-2b activity (Interferon alpha 2b, human, rDNA, E. coli-derived, 2nd WHO International Standard, 1999, NIBSC Code No. 95/566) and human recombinant interferon alpha 2b in the form of solution (batch No. 040214, Pharmapark LLC, Russia) were used as IFN-α samples. Results: the analysis of the obtained data helped to determine: the combinations of cell lines and the indicator virus most sensitive to IFN-α; the optimal concentration of fetal serum in the medium, and the optimal time parameters; the preferred method of reporting test results. Conclusions: the following test conditions were found to be optimal: the MDBK/VSV and Hep-2/EMC combinations proved to be the most sensitive to IFN-α; the optimal period of interferon and cell culture incubation—24 hours; the optimal concentration of fetal bovine serum in the culture medium used for diluting interferon products—2–5%. The instrumental procedure is preferred for reporting the results of interferon antiviral activity determination, because it is up-to-date, reliable, accurate and time-efficient.

1706-P: A Promoter Variant of TYK2, a Putative Virus-Induced Susceptibility Gene, Is Associated with Impaired Insulin Secretion and Nonobese Type 2 Diabetes

Background: Type 2 diabetes mellitus is typically associated with obesity and insulin resistance. It is well known that viral infection triggers type 1 diabetes, but a potential link with type 2 diabetes has not been fully established. We have reported that a natural mutation in the tyrosine kinase 2 (TYK2) gene, which encodes an interferon receptor signaling molecule, is responsible for encephalomyocarditis (EMC) virus-induced diabetes in mice (Nat Commun 2015). Moreover, a human TYK2 promoter variant (TYK2PV) (NCBI ClinVer ID:440728) is associated with higher risks of not only type 1, but also type 2 diabetes (EBioMedicine 2015). Therefore, we hypothesized that impaired insulin secretion associated with TYK2PV causes type 2 diabetes. Here, we studied the relationships between TYK2PV, and insulin secretion and obesity in patients with type 2 diabetes. Methods: The presence of TYK2PV was determined using direct sequencing, and insulin secretion was evaluated, in type 2 diabetes patients who were not administering either sulfonylureas or insulin. Results: Of the 173 patients assessed, 18 (10.4%) were TYK2PV-positive, and their median BMI was significantly lower than those without the variant (25.3 vs. 23.4 kg/m2, p=0.029). Moreover, the HOMA-β (19.1 vs. 30.0, p=0.027) and fasting serum insulin (3.91 vs. 6.16 μIU/mL, p=0.007) were lower in patients with TYK2PV than in those without. Multivariable analysis showed that TYK2PV was significantly associated with fasting insulin <5μIU/ml (odds ratio 4.14, 95% confidence interval 1.404-12.21, p=0.021). Conclusions: TYK2PV is associated with lower insulin secretion in type 2 diabetes, consistent with it being a virus-induced diabetes susceptibility gene. Therefore, type 2 diabetes patients with TYK2PV should be carefully followed, so that they receive the most appropriate treatment, including insulin. Disclosure H. Mori: None. H. Takahashi: None. K. Mine: None. K. Inoue: None. M. Kojima: None. S. Kuroki: None. Y. Ono: None. S. Inuzuka: None. S. Nagafuchi: None. K. Anzai: None.

Real-Time Temporal Dynamics of Bicistronic Expression Mediated by Internal Ribosome Entry Site and 2A Cleaving Sequence.

Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.

Crucial role of Reg I from acinar-like cell cluster touching with islets (ATLANTIS) on mitogenesis of beta cells in EMC virus-induced diabetic mice

Recently, we reported the presence of distinct cell clusters named acinar-like cell clusters touching Langerhans islets with thin interstitial surrounding (ATLANTIS) in human pancreas. A morphological study in humans demonstrated that ATLANTIS and islet cell clusters are found together in the microenvironment enclosed by a common basement membrane, and ATLANTIS releases vesicles containing Regenerating gene protein (REG Iα) to islet cell clusters.We examined 1) the presence or absence of ATLANTIS in homozygous Reg I (mouse homologue of human REG Iα) deficient (Reg I−/-) and wild-type mice, and 2) the possible role of ATLANTIS in the regeneration of beta cell clusters after encephalomyocarditis (EMC) virus (D-variant) infection in Reg I−/- and wild-type mice.ATLANTIS was found in both wild-type and Reg I−/- mice. In both groups, mean blood glucose increased transiently to greater than 14.0 mmol/L at 5 days after EMC virus infection and recovered to baseline at 12 days. At 12 days after EMC virus infection, lower BrdU labeling indices were observed in islet beta cells of Reg I−/- mice compared to wild-type mice. Beta cell volume 12 days after EMC virus infection in Reg I−/- mice did not differ from that of wild-type mice.These results suggest that Reg I, which is released from ATLANTIS to islet beta cell clusters, has a crucial role in beta cell regeneration in EMC virus-induced diabetes. The presence of mechanism(s) other than that mediated by Reg I in beta cell restoration after destruction by EMC virus was also suggested.

Prevalence and clinical features of encephalomyocarditis virus infection in children with acute respiratory infection or digestive tract infection

Objective To investigate the possible existence of encephalomyocarditis virus (EMCV) in children with acute respiratory infection or digestive tract infection. Methods Nasopharyngeal aspirates from 577 children with acute respiratory infection and fecal samples from 384 children with digestive tract infection in Tianjin Children′s Hospital were collected from January 2013 to December 2013.Viral nucleic acid was extracted, and EMCV infection was determined by using real-time quantitative PCR.Positive PCR products were sequenced.The sequencing results were aligned with known gene sequences of EMCV sequences in GenBank.Other common respiratory tract or digestive tract viruses were also detected.Direct immunofluorescence was used to detect 7 common respiratory viruses; colloidal gold labeled method was used to detect rotavirus and adenovirus; enzyme linked immunosorbent assay was used to detect avastrovirus; PCR was used to detect human rhinovirus, WU polyomavirus, KI polyomavirus, human bocavirus, norovi-ruses, enteroviruses, Aichi virus and human parechovirus.Real-time PCR was used to detect saffold virus. Results Seven(1.2%) nasopharyngeal aspirates from children with acute respiratory infection were positive for EMCV.These children aged 1-6 months.The infection seasons were in autumn and winter.Eleven(2.9%) fecal samples from children with digestive tract infection were positive for EMCV.Positive specimens were in patients less than 1 year of age and 4-7 years of age.EMCV was identified in other 3 seasons except spring.The clinical diagnosis of the EMCV-po-sitive patients were fever or diarr-hea, which was similar with other respiratory tract or digestive tract viruses.There were 88.9% (16/18 cases) of these EMCV infected children lived in suburbs or countryside.There were a few co-infections with EMCV and other respiratory tract or digestive tract viruses.There was no obvious difference in the clinical symptoms between EMCV-positive patients with or without coinfection. Conclusions Infection of EMCV often occurs in children with acute respiratory infection or digestive tract infection in China.Some of acute respiratory infection or digestive tract infection in pediatric patients was related to EMCV. Key words: Encephalomyocarditis virus; Acute respiratory infection; Digestive tract infection; Child

Activation of type I interferon signaling in the parotid and exorbital lachrymal glands during the acute phase of encephalomyocarditis (EMC) virus infection in mice

The present study was carried out to clarify the mechanisms of EMC virus-induced sialodacryoadenitis in mice during the acute phase infection focusing on the activation of type I interferon (IFN) signaling in the parotid and exorbital lachrymal glands. In the parotid gland, a few apoptotic acinar cells were detected at 2days post inoculation (DPI). The ratio of apoptotic acinar cells increased at 3 and 4DPI. On the other hand, in the exorbital lachrymal gland, apoptosis of acinar cells and infiltration of inflammatory cells mainly composed of mononuclear cells started at 3DPI, and prominent acinar cell damage developed at 4DPI. Viral RNA was detected at 3 and 4DPI in both glands and the expression level was higher in the exorbital lachrymal gland than in the parotid gland. The up-regulation of IFN-stimulated genes (ISGs), such as Irf7, Pkr and Oas, was quickly induced at 2DPI in the parotid gland, and this probably contributed to suppress viral replication and to eliminate affected cells by apoptosis. In the exorbital lachrymal gland, the expression levels of ISGs mRNAs were not elevated at 2DPI, suggesting no induction of an effective anti-viral response such as apoptosis at this time point. In the exorbital lachrymal gland, the mRNA expression of IFN beta and IFN alpha (type I IFNs) was weak- to strong-positive at 1DPI, and became negative at 2DPI. The weak- to strong-positive expression of IFNs at 1DPI is likely related to the abrupt viral replication and pathological changes in the exorbital lachrymal gland through activating the negative feedback regulation that depressed the IFN signaling cascade at 2DPI. In conclusion, the present study showed the changes in factors involved in the activation of type I IFN signaling cascade in the parotid and exorbital lachrymal glands and their differences between the two glands during the acute phase of EMC virus infection in mice.

Antibody-Interferon-Alpha Fusion Protein Therapy for the Treatment of B-Cell Non-Hodgkin Lymphoma: Enhanced ADCC, Inhibition of Proliferation, and In Vivo Eradication of CD20+ Human Lymphomas

Background: Interferon-alpha (IFNα) is a pleiotrophic cytokine with direct anti-tumor and immunostimulatory effects. Currently IFNα is approved for the treatment of multiple hematologic malignancies, including non-Hodgkin lymphoma (NHL). However, its clinical utility has been hindered by dose-limiting toxicitiy due to systemic activation of the interferon receptor. To overcome this limitation, we engineered anti-tumor antibody-IFNα fusion proteins to selectively increase delivery of IFN to the tumor site and reduce systemic toxicity. We previously reported that IGN002, an anti-CD20-IFNα fusion protein, exhibits enhanced complement-dependent cytotoxicity (CDC) compared to rituximab, and inhibits proliferation and induces apoptosis of human B-cell NHL (Yamada et al, ASCO 2013). We now extend these previous findings and show that IGN002 possesses enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) effector function and superior in vivo anti-tumor activity against B-cell NHL, compared to rituximab.Methods: IGN002 was evaluated against a panel of human Burkitt and diffuse large B-cell lymphoma (DLBCL) cell lines. Proliferation was measured by [3 H]-thymidine incorporation, STAT1 activation by flow cytometry, ADCC by lactate dehydrogenase release using human PBMC effectors, and IFN bioactivity by encephalomyocarditis (EMC) viral protection assay. NHL xenografts were grown in SCID mice.Results: IGN002 more potently inhibited the growth of NHL cell lines expressing CD20 than rituximab or unfused IFNα. Intrinsic IFNα activity of IGN002 was reduced in viral protection and anti-proliferation assays using cells lacking CD20 expression. STAT1 activation by IGN002 was enhanced on cells expressing the target antigen, whereas a control antibody-IFNα fusion protein showed reduced STAT activation activity compared to unfused IFNα. Together, these results indicate that fusion of IFNα to the antibody results in reduced IFN effects on cells not bearing the tumor antigen target. IGN002 exhibited enhanced ADCC activity compared to rituximab against Daudi, Ramos, and Raji NHL cells in long-term (overnight incubation) assays, demonstrating both higher potency and higher maximal cytotoxicity. This result is possibly due to activation of the effector cell populations by the fused IFNα moiety, as IFN is known to activate both NK cells and monocytes. The in vivo anti-tumor efficacy of IGN002 was compared to rituximab and a control antibody-IFNα fusion protein against 10-day established Raji NHL xenografts. IGN002 was superior to both rituximab and the control fusion protein, achieving a longer median survival and higher long-term survival rate (p = 0.0015 and < 0.0001 vs. rituximab and control fusion protein, respectively). The in vivo anti-tumor efficacy of IGN002 was also compared to rituximab at three equimolar dose levels (5 mg/kg, 1 mg/kg, and 0.2 mg/kg antibody) against 10-day established Daudi NHL xenografts. IGN002 showed superior efficacy compared to rituximab at all doses (p < 0.001), achieving tumor eradication (100% long-term survival) in all mice treated at all three dose levels, whereas rituximab only delayed tumor progression.Conclusions: IGN002 demonstrated more robust direct anti-proliferative and antibody effector functions than rituximab against human NHL cells in vitro, and also showed the ability to eradicate established NHL xenografts in vivo. Against cells expressing the CD20 target antigen, IGN002 exhibited greater anti-proliferative potency than unfused IFNα. In contrast, the anti-proliferative and anti-viral potency of IGN002 was reduced against cells lacking CD20, compared to unfused IFNα. These findings support the hypothesis that tumor antigen-targeted IFN therapeutics may possess a broader therapeutic index than unfused IFNα, inhibiting tumor growth by multiple mechanisms while reducing systemic toxicity. These results support the further development of IGN002 for the treatment of B-cell NHL, and a first-in-human phase I clinical study will begin later this year in the United States. DisclosuresTimmerman:Janssen: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Valor Biotherapeutics: Research Funding. Steward:ImmunGene, Inc.: Employment. Minning:Valor Biotherapeutics, LLC: Consultancy. Sachdev:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Gresser:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Khare:Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Morrison:ImmunGene, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Encephalomyocarditis virus infection in a splenectomised calf

Encephalomyocarditis (EMC) caused by EMC virus (EMCV) was diagnosed in a 5-month-old splenectomised calf, which died suddenly on an experimental farm that had a high infestation of rodents. At postmortem examination, the lungs were dark purple and diffusely congested. On histological examination, the calf had severe necrotising myocarditis. EMCV was isolated from the heart. The polyprotein gene of the EMCV isolate was amplified by PCR and had 85-91% identity with published EMCV sequences, including 89% identity with isolates from Queensland. On phylogenetic analysis, the polyprotein gene had highest sequence identity with South Korean EMCV strain, CBNU. This is the first report of naturally occurring EMC in cattle in Australia and the first report of naturally occurring bovine EMC from which EMCV has been isolated.

The significance of tyrosine kinase 2 gene in encephalomyocarditis-D virus-induced diabetes as studied in Tyk2 gene knockout mice (P6334)

Abstract Type 1 diabetes mellitus results form progressive loss of pancreatic β cells. The virus, as one of environmental factors, has been long considered to play a role in the development of type 1 diabetes. The D variant of encephalomyocarditis (EMC-D) virus induced susceptible strain of mice to become type 1 diabetes. We have already reported that innate immunity may play an important role in protection against EMC-D virus-induced diabetes (Arch Virol, 2008). In the present study, we analyzed the role of Tyk2 gene in the pathogenic mechanism of EMC-D virus-induced diabetes by using Tyk2 gene knockout (KO) mice. Tyk2 gene KO C57BL/6J mice, mouse strain resistant to EMC-D virus-induced diabetes, developed diabetes. In order to examine whether Tyk2 gene expression was important in parenchymal or immune cells to protect against EMC-D virus-induced diabetes, we developed splenic chimera mice. Tyk2 gene expression was important in parenchymal cells, but not in spleen cells, to resist against EMC-D virus-induced diabetes. Consistently, mice specifically expressed Tyk2 gene only in pancreatic β cells prevented EMC-D virus-induced diabetes. These observations suggest that Tyk2 gene expressed in pancreatic β cells plays a significant role in preventing EMC-D virus-induced diabetes in mice.

Preliminary study on the identification methods for encephalomyocarditis virus

Objective To explore and develop methods for encephalomyocarditis virus (EMCV) identification.Methods According to the genetic sequence VR-129B of EMCV recorded in the GenBank,five gene fragments were selected to design primer sequence pairs.RNA was extracted to run RT-PCR,and then the products of amplification were identified by agarose gel electrophoresis.The results of DNA sequences were compared with the sequences in GenBank of the same EMCV strains.Antiserum was prepared based on the EMCV cultured in RK cells for establishing indirect immunofluorescence assay (IIFA) and neutralization test method,and verification for precision and specificity of the two methods were carried out after it.Antiserum that was prepared with GST-VP1 and GST-VP2 expressed in E.coli was reacted with the purified EMCV in Western blot test.Results By sequencing and comparing,the similarity of DNA fragments between the obtained and the GenBank recorded was reached 98％ to 100％.The antiserum of No.20100901 batch that was chosen as the first antibody at a dilution of 1 ∶ 160 to develop IIFA brought about a better specificity.The neutralization titers of 20100901 batch antiserum was 1 ∶ 30 211 measured by fixing virus and diluting serum method,which showed good specificity and precision.The results of the Western blot test showed two clear bands above and under 33×103 respectively,which matched the theoretical value.Conclusion The RT-PCR,indirect immunofluorescence,neutralization test and Western blot method for EMCV strains identification were established initially. Key words: Encephalomyocarditis virus; Indirect immunofluorescence assay; Neutralization test

Lessons learned from experimental myocarditis

We have developed murine models of viral myocarditis induced by encephalomyocarditis (EMC) virus in which severe myocarditis, congestive heart failure and dilated cardiomyopathy occur in high incidence. From these models, we have learned the natural history and pathogenesis and assessed not only new diagnostic methods but also therapeutic and preventive interventions. Autoantibodies against cardiac troponin I appeared in spontaneously developing autoimmune myocarditis in PD-1 deficient mice, who lack the T-cell receptor costimulatory molecule PD-1. The passive transfer of this antibody induced myocardial dysfunction. Later, this autoantibody was found in patients with myocarditis. Mast cell deficiency had beneficial effects in the viral myocarditis model, and anti-allergic agents prevented viral myocarditis. Angiotensin-converting enzyme inhibitors, angiotensin II receptor blocker and an aldosterone receptor antagonist improved viral myocarditis, suggesting that the renin-angiotension-aldosterone system may play an important role in the pathogenesis of viral myocarditis. Differential modulation of cytokine production was seen with various calcium channel blockers, and some calcium channel blocker improved viral myocarditis. Viral infection could lead to increased synthesis of immunoglobulin light chains (FLC). Serum levels of FLC were increased in myocarditis, and exogenously given FLC inhibited viral replication and improved myocarditis. We suggest that a strategy of drug development specifically addressing inflammation in myocarditis may provide increased benefit in terms of target organ damage.

Effects of Free Immunoglobulin Light Chains on Viral Myocarditis

In recent work, we have demonstrated a crucial role of mast cells in the development of viral myocarditis. Viral infection could lead to increased synthesis of free immunoglobulin light chains (FLC) and our earlier work showed that FLC can trigger mast cell activation. We studied the possible involvement of FLC in the pathogenesis of viral myocarditis, and therapeutic effects of FLC using an animal model of viral myocarditis. DBA/2 mice were inoculated intraperitoneally with encephalomyocarditis (EMC) virus. Serum levels and concentrations in the heart of kappa FLC on day 14 in mice inoculated with EMC virus were significantly increased compared with controls. Myocardial viral concentration was significantly inhibited, the area of myocardial lesions was smaller in mice treated with kappa or lambda FLC, and survival of mice given FLC significantly improved. In contrast, an FLC antagonist deteriorated myocarditis. kappa and lambda FLC chains inhibited EMC viral replication in human amnion cells in vitro. lambda FLC significantly increased the gene expression of interleukin-10 in the heart which was previously shown to improve viral myocarditis when given exogenously. FLC also tended to increase the gene expressions of interferon-alpha and -gamma in the heart mice. FLC have antiviral and antiinflammatory effects and improved viral myocarditis in mice. FLC may be promising agents for the treatment of viral myocarditis.

Cetirizine a histamine H1 receptor antagonist improves viral myocarditis

BackgroundWe showed that mast cells played a critical role in the progression of heart failure induced by pressure overload and viral myocarditis in mice. In this study, we investigated the effect of cetirizine, a selective H1 receptor antagonist, on experimental viral myocarditis induced by encephalomyocarditis (EMC) virus.MethodsFour-week-old inbred male DBA/2 mice were inoculated intraperitoneally with 10 plaque-forming units (pfu) of the EMC virus. Cetirizine was administered orally at a dose of 1 or 10 mg/kg per day for the survival study, and 1 mg/kg for the histologic and gene expression studies, beginning on the day of viral inoculation.ResultsCetirizine improved survival dose dependently. Heart weight to body weight ratio was significantly decreased in mice treated with cetirizine. The area of myocardial necrosis was significantly smaller in the hearts of mice treated with cetirizine compared with controls. Gene expressions of tumor necrosis factor, interleukin 6, and metalloproteinase 2 were significantly suppressed in the hearts of mice treated with cetirizine.ConclusionThese results suggest that cetirizine exerts its beneficial effects on viral myocarditis by suppressing expression of pro-inflammatory cytokines, genes related to cardiac remodeling in the hearts of mice.

Oncolytic Effects of the E Variant of Encephalomyocarditis Virus to Human Neuroblastoma in in Vitro and in Vivo

The oncolytic effects of encephalomyocarditis (EMC) virus on of human neuroblastoma (NB) were studied in vitro employing NB cell cultures and in vivo employing tumors grown in nude mice and nude rats. In short-term studies, human NB cell (HTB10) cultures were exposed to the virus 104 Tissue Culture Infectious Doses (TCIDs). The TCIDs were used to titer the maximum effect of EMC virus on L-929 cells. In vitro studies showed 100% Cytopathic Effect (CPE) at 48 hours. The CPE was used to observe pathologic effects of the cells. The in vivo studies showed necrosis and degeneration in the tumors grown in nude mice at 7 days following treatment with the virus. The nude mice exhibited a high mortality rate after exposure to the virus. The long-term studies were performed on tumors grown in nude rats, since they were more tolerant to the viral treatment. On the 58th day after the viral treatment of tumor bearing rats, 8 out of the 12 tumors had disappeared, and the remaining 4 tumors were grossly necrotic. The average size of the infected tumors was 2.13 cm2 as compared with 30.5 cm2 in the control. Microscopically the treated tumors showed severe necrosis and microcystic degeneration. The observed gross decrease in treated tumor size together with the presented light and electron microscopic studies, support the potential value of viral treatment of neuroblastoma.

Coinfecting Viruses and Mycoplasmas Can Appear To Cooperate

Very different kinds of pathogen—in this case, one a virus, the other a mycoplasma—can act as if cooperating when infecting cultured cells, with one augmenting the potency of the other, according to Peter Lidsky and Vadim I. Agol of the Russian Academy of Medical Sciences, Moscow, Russia, and their collaborators at the Academy and the nearby M. V. Lomonosov Moscow State University, as well as at Radboud University Nijmegen Medical Centre in Nijmegen, the Netherlands. Thus a DNAase from a simultaneously infecting mycoplasma enhances the cell-killing activity of the encephalomyocarditis (EMC) virus, they report in the October Journal of Virology (83:9940–9951).

Inactivation of bacterial spores and viruses in biological material using supercritical carbon dioxide with sterilant.

The purpose of this study was to validate supercritical carbon dioxide (SC-CO(2)) as a terminal sterilization method for biological materials, specifically acellular dermal matrix. In this study, bacterial spores, Bacillus atrophaeus, were inoculated onto porcine acellular dermal matrix to serve as a "worst case" challenge device. The inactivation of the spores by SC-CO(2) with peracetic acid (PAA) sterilant was analyzed as a function of exposure times ranging from 1 to 30 min. A linear inactivation profile for the Bacillus atrophaeus spores was observed, and a SC-CO(2) exposure time of 27 min was determined to achieve a sterility assurance level of 10(-6). The inactivation of viruses was also studied using Encephalomyocarditis (EMC) viruses. After 15 min of exposure to SC-CO(2) with PAA sterilant, more than a 6 log(10) reduction was observed for EMC viruses. Biochemical and biomechanical evaluations showed that the SC-CO(2) treatment with PAA sterilant did not cause significant changes in porcine acellular matrix's susceptibility to collagenase digestion, tensile or tear strength, indicating limited alteration of the tissue structure following SC-CO(2) sterilization.

Anti‐inflammatory effects of eplerenone on viral myocarditis

Inflammation contributes to increased cardiovascular morbidity and mortality associated with activation of the renin-angiotensin-aldosterone system. The aim of this study was to investigate whether eplerenone, a selective aldosterone receptor antagonist, has anti-inflammatory effects on viral myocarditis. Four-week-old inbred male DBA/2 mice were inoculated intraperitoneally with 10 plaque-forming units (pfu) of the encephalomyocarditis (EMC) virus. Mice were fed with standard chow (control) or with chow containing 2.5 mg/kg of eplerenone, starting either on day 0 (inoculation) or day 7. Survival at 28 days was significantly higher in the mice which started eplerenone treatment on day 0 (35 vs. 15% in controls, each n = 40, P < 0.05). The area of myocardial fibrosis on day 28 was significantly smaller in the eplerenone-treated mice than in controls (19.8 +/- 2.6%, n = 14, vs. 33.4 +/- 5.4%, n = 6, mean +/- SEM, P < 0.05). Gene expression of mouse mast cell proteases-4 and -5, matrix metalloproteinase-9, and type I procollagen on day 6 after EMC virus inoculation was significantly decreased in the hearts of eplerenone-treated mice. These results suggest that eplerenone has anti-inflammatory effects, and exerts its beneficial effects on viral myocarditis by suppression of genes related to mast cells and cardiac remodelling in the hearts of mice.

The significance of T cells, B cells, antibodies and macrophages against encephalomyocarditis (EMC)-D virus-induced diabetes in mice

In order to clarify the significance of protective mechanisms against encephalomyocarditis (EMC) virus-induced diabetes in mice, we studied the relative importance of T cells, B cells, antibodies and macrophages in the prevention of virus-induced diabetes. Neither T cell-deficient athymic nude mice nor B cell-deficient microMT/microMT mice showed an enhanced clinical course of EMC-D virus-induced diabetes, indicating that neither T cells nor B cells played a major role in the protection against EMC-D-virus-induced diabetes. Transfer of a large amount of antiserum to EMC-D-virus-infected mice protected the development of diabetes only when transferred within 36 h of infection, the timing of which was earlier than that for the production of natural neutralizing antibodied. Since pretreatment of mice with the macrophage-activating immunopotentiator Corynebacterium parvum (CP) completely prevented the development of diabetes, we studied the clinical outcome of EMC-D-virus-infected mice pretreated with CP. Mice treated with CP showed reduced proliferation of EMC-D virus in the affected organs, including the pancreas, while the levels of development of neutralizing antibody and serum interferon were not enhanced compared with the controls. Finally, we studied the macrophages derived from mice pretreated with CP and found that they inhibited the growth of EMC-D virus in vitro more than those derived from non-treated and thioglycolate-treated mice. Taken together, it can be suggested that neither T cells nor B cells, which have to do with adaptive immunity, play a significant role in the pathogenesis of EMC-D-virus-induced diabetes, while innate immunity, which is dependent on activated macrophages, contributes to in vivo resistance against EMC-D-virus-induced diabetes.