Targeting ectromelia virus and TNF/NF-κB or STAT3 signaling for effective treatment of viral pneumonia.

Viral causes of pneumonia pose constant threats to global public health, but there are no specific treatments currently available for the condition. Antivirals are ineffective when administered late after the onset of symptoms. Pneumonia is caused by an exaggerated inflammatory cytokine response to infection, but tissue necrosis and damage caused by virus also contribute to lung pathology. We hypothesized that viral pneumonia can be treated effectively if both virus and inflammation are simultaneously targeted. Combined treatment with the antiviral drug cidofovir and etanercept, which targets tumor necrosis factor (TNF), down-regulated nuclear factor kappa B-signaling and effectively reduced morbidity and mortality during respiratory ectromelia virus (ECTV) infection in mice even when treatment was initiated after onset of clinical signs. Treatment with cidofovir alone reduced viral load, but animals died from severe lung pathology. Treatment with etanercept had no effect on viral load but diminished levels of inflammatory cytokines and chemokines including TNF, IL-6, IL-1ß, IL-12p40, TGF-ß, and CCL5 and dampened activation of the STAT3 cytokine-signaling pathway, which transduces signals from multiple cytokines implicated in lung pathology. Consequently, combined treatment with a STAT3 inhibitor and cidofovir was effective in improving clinical disease and lung pathology in ECTV-infected mice. Thus, the simultaneous targeting of virus and a specific inflammatory cytokine or cytokine-signaling pathway is effective in the treatment of pneumonia. This approach might be applicable to pneumonia caused by emerging and re-emerging viruses, like seasonal and pandemic influenza A virus strains and severe acute respiratory syndrome coronavirus 2.

Poxvirus-encoded TNF receptor homolog dampens inflammation and protects from uncontrolled lung pathology during respiratory infection.

Ectromelia virus (ECTV) causes mousepox, a surrogate mouse model for smallpox caused by variola virus in humans. Both orthopoxviruses encode tumor necrosis factor receptor (TNFR) homologs or viral TNFR (vTNFR). These homologs are termed cytokine response modifier (Crm) proteins, containing a TNF-binding domain and a chemokine-binding domain called smallpox virus-encoded chemokine receptor (SECRET) domain. ECTV encodes one vTNFR known as CrmD. Infection of ECTV-resistant C57BL/6 mice with a CrmD deletion mutant virus resulted in uniform mortality due to excessive TNF secretion and dysregulated inflammatory cytokine production. CrmD dampened pathology, leukocyte recruitment, and inflammatory cytokine production in lungs including TNF, IL-6, IL-10, and IFN-γ. Blockade of TNF, IL-6, or IL-10R function with monoclonal antibodies reduced lung pathology and provided 60 to 100% protection from otherwise lethal infection. IFN-γ caused lung pathology only when both the TNF-binding and SECRET domains were absent. Presence of the SECRET domain alone induced significantly higher levels of IL-1ß, IL-6, and IL-10, likely overcoming any protective effects that might have been afforded by anti-IFN-γ treatment. The use of TNF-deficient mice and those that express only membrane-associated but not secreted TNF revealed that CrmD is critically dependent on host TNF for its function. In vitro, recombinant Crm proteins from different orthopoxviruses bound to membrane-associated TNF and dampened inflammatory gene expression through reverse signaling. CrmD does not affect virus replication; however, it provides the host advantage by enabling survival. Host survival would facilitate virus spread, which would also provide an advantage to the virus.

TNF deficiency dysregulates inflammatory cytokine production, leading to lung pathology and death during respiratory poxvirus infection.

Excessive tumor necrosis factor (TNF) is known to cause significant pathology. Paradoxically, deficiency in TNF (TNF-/-) also caused substantial pathology during respiratory ectromelia virus (ECTV) infection, a surrogate model for smallpox. TNF-/- mice succumbed to fulminant disease whereas wild-type mice, and those engineered to express only transmembrane TNF (mTNF), fully recovered. TNF deficiency did not affect viral load or leukocyte recruitment but caused severe lung pathology and excessive production of the cytokines interleukin (IL)-6, IL-10, transforming growth factor beta (TGF-ß), and interferon gamma (IFN-γ). Short-term blockade of these cytokines significantly reduced lung pathology in TNF-/- mice concomitant with induction of protein inhibitor of activated STAT3 (PIAS3) and/or suppressor of cytokine signaling 3 (SOCS3), factors that inhibit STAT3 activation. Consequently, inhibition of STAT3 activation with an inhibitor reduced lung pathology. Long-term neutralization of IL-6 or TGF-ß protected TNF-/- mice from an otherwise lethal infection. Thus, mTNF alone is necessary and sufficient to regulate lung inflammation but it has no direct antiviral activity against ECTV. The data indicate that targeting specific cytokines or cytokine-signaling pathways to reduce or ameliorate lung inflammation during respiratory viral infections is possible but that the timing and duration of the interventive measure are critical.

Therapeutic Targeting of Inflammation and Virus Simultaneously Ameliorates Influenza Pneumonia and Protects from Morbidity and Mortality.

Influenza pneumonia is a severe complication caused by inflammation of the lungs following infection with seasonal and pandemic strains of influenza A virus (IAV), that can result in lung pathology, respiratory failure, and death. There is currently no treatment for severe disease and pneumonia caused by IAV. Antivirals are available but are only effective if treatment is initiated within 48 h of onset of symptoms. Influenza complications and mortality are often associated with high viral load and an excessive lung inflammatory cytokine response. Therefore, we simultaneously targeted the virus and inflammation. We used the antiviral oseltamivir and the anti-inflammatory drug etanercept to dampen TNF signaling after the onset of clinical signs to treat pneumonia in a mouse model of respiratory IAV infection. The combined treatment down-regulated the inflammatory cytokines TNF, IL-1ß, IL-6, and IL-12p40, and the chemokines CCL2, CCL5, and CXCL10. Consequently, combined treatment with oseltamivir and a signal transducer and activator of transcription 3 (STAT3) inhibitor effectively reduced clinical disease and lung pathology. Combined treatment using etanercept or STAT3 inhibitor and oseltamivir dampened an overlapping set of cytokines. Thus, combined therapy targeting a specific cytokine or cytokine signaling pathway and an antiviral drug provide an effective treatment strategy for ameliorating IAV pneumonia. This approach might apply to treating pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Targeting tumour necrosis factor to ameliorate viral pneumonia.

Pneumonia is a serious complication associated with inflammation of the lungs due to infection with viral pathogens. Seasonal and pandemic influenza viruses, variola virus (agent of smallpox) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; agent of COVID-19) are some leading examples. Viral pneumonia is triggered by excessive inflammation associated with dysregulated cytokine production, termed 'cytokine storm'. Several cytokines have been implicated but tumour necrosis factor (TNF) plays a critical role in driving lung inflammation, severe lung pathology and death. Despite this, the exact role TNF plays in the aetiology and pathogenesis of virus infection-induced respiratory complications is not well understood. In this review, we discuss the pathological and immunomodulatory roles of TNF in contributing to immunopathology and resolution of lung inflammation, respectively, in mouse models of influenza- and smallpox (mousepox)-induced pneumonia. We review studies that have investigated dampening of inflammation on the outcome of severe influenza and orthopoxvirus infections. Most studies on the influenza model have evaluated the efficacy of treatment with anti-inflammatory drugs, including anti-TNF agents, in animal models on the day of viral infection. We question the merits of those studies as they are not transferable to the clinic given that individuals generally present at a hospital only after the onset of disease symptoms and not on the day of infection. We propose that research should be directed at determining whether dampening lung inflammation after the onset of disease symptoms will reduce morbidity and mortality. Such a treatment strategy will be more relevant clinically.

Propagation and Purification of Ectromelia Virus.

Ectromelia virus (ECTV) is an orthopoxvirus that causes mousepox in mice. Members of the genus orthopoxvirus are closely related and include variola (the causative agent of smallpox in humans), monkeypox, and vaccinia. Common features of variola virus and ECTV further include a restricted host range and similar disease progression in their respective hosts. Mousepox makes an excellent small animal model for smallpox to investigate pathogenesis, vaccine and antiviral agent testing, host-virus interactions, and immune and inflammatory responses. The availability of a wide variety of inbred, congenic, and gene-knockout mice allows detailed analyses of the host response. ECTV mutant viruses lacking one or more genes encoding immunomodulatory proteins are being used in numerous studies in conjunction with wild-type or gene-knockout mice to study the functions of these genes in host-virus interactions. The methods used for propagation of ECTV in cell culture, purification, and quantification of infectious particles through viral plaque assay are described. © 2018 by John Wiley & Sons, Inc.

Diagnostic Accuracy of GeneXpert MTB/RIF Assay in Comparison to Conventional Drug Susceptibility Testing Method for the Diagnosis of Multidrug-Resistant Tuberculosis.

Xpert MTB/RIF assay is regarded as a great achievement of modern medicine for the rapid diagnosis of multidrug-resistant tuberculosis (MDR-TB). The main purpose of this study was to determine the performance of Xpert MTB/RIF assay compared to conventional drug susceptibility testing (DST) method for the diagnosis of MDR-TB. A comparative cross sectional study was carried out at German-Nepal Tuberculosis Project, Kathmandu, Nepal, from April 2014 to September 2014. A total of 88 culture positive clinical samples (83 pulmonary and 5 extra-pulmonary) received during the study period were analyzed for detection of multidrug-resistant tuberculosis by both GeneXpert MTB/RIF assay and conventional DST method. McNemar chi square test was used to compare the performance of Xpert with that of DST method. A p-value of less than 0.05 was considered as statistically significant. Of total 88 culture positive samples, one was reported as invalid while 2 were found to contain nontuberculous Mycobacteria (NTM). Among remaining 85 Mycobacterium tuberculosis culture positive samples, 69 were found to be MDR-TB positive by both methods. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of GeneXpert MTB/RIF assay were found to be 98.6%, 100%, 100% and 93.8% respectively. Statistically, there was no significant difference between the diagnostic performance of Xpert and conventional DST method for detection of MDR-TB. GeneXpert MTB/RIF assay was found to be highly sensitive, specific and comparable to gold standard conventional DST method for the diagnosis of MDR-TB.

Cardiovascular Risk Factors in Subclinical Hypothyroidism: A Case Control Study in Nepalese Population.

Objectives. To assess cardiovascular risk factors in Nepalese population with subclinical hypothyroidism as compared to age and sex matched controls. Materials and Methods. A case control study was conducted among 200 subjects (100 subclinical hypothyroid and 100 euthyroid) at B.P. Koirala Institute of Health Sciences, Dharan, Nepal. Demographic and anthropometric variables including systolic and diastolic blood pressure (BP) were taken. Blood samples were assayed for serum free triiodothyronine (fT3), free thyroxine (fT4), thyroid stimulating hormone (TSH), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and high sensitivity C reactive protein (hs-CRP). Results. Subclinical hypothyroid patients had significantly higher diastolic BP, total cholesterol, LDL cholesterol, and hs-CRP than controls. The odds ratio of having hypercholesterolemia (>200 mg/dL), low HDL cholesterol (<40 mg/dL), undesirable LDL-cholesterol (>100 mg/dL), high hs-CRP (>1 mg/L), and high diastolic BP (>80 mmHg) and being overweight (BMI ≥ 23 Kg/m(2)) in subclinical hypothyroidism was 2.29 (95% CI; 1.2-4.38, p = 0.011), 1.73 (95% CI; 0.82-3.62, p = 0.141), 3.04 (95% CI; 1.66-5.56, p < 0.001), 2.02 (95% CI; 1.12-3.64, p = 0.018), 3.35 (95% CI; 1.72-6.55, p < 0.001), and 0.9 (95% CI; 0.48-1.67, p = 0.753), respectively, as compared to controls. Conclusion. Subclinical hypothyroid patients are associated with higher risk for cardiovascular disease than euthyroid subjects.