ABSTRACT
In order to identify new potential antiviral drugs, small amounts of extracts or compounds have to be examined for cytotoxicity and antiviral activity in primary screening using a rapid, easy, inexpensive, and highly standardised test system. In this study, high-throughput cytopathic effect (CPE) inhibitory assays were established for coxsackie virus B3 on HeLa Ohio cells, influenza virus A on Madin-Darby canine kidney cells, and herpes simplex virus type 1 (HSV-1) on green monkey kidney cells that meet these requirements. The cytotoxic and the antiviral effects were quantified using a crystal violet uptake assay allowing automated handling of large numbers of candidate agents. To ensure comparable results with plaque reduction assays, the 50 and 90% plaque inhibitory concentrations of guanidine, amantadine, and phosphonoformic acid were used to standardise the anti-coxsackie virus B3, anti-influenza virus A, and anti-HSV-1 tests, respectively. The strong correlation between the antiviral activity determined by CPE-inhibitory assays and plaque reduction assay was further proved for other antivirals. In summary, low amounts of large numbers of compounds may be tested inexpensively and standardised within 24 h (coxsackie virus B3 and influenza virus A) or 48 h (herpes simplex virus type 1) post-infection using CPE inhibitory assays.
Subject(s)
Antiviral Agents/pharmacology , Enterovirus B, Human/drug effects , Herpesvirus 1, Human/drug effects , Influenza A virus/drug effects , Amantadine , Animals , Cell Division , Cell Line , Chlorocebus aethiops , Cytopathogenic Effect, Viral , Dogs , Enterovirus B, Human/pathogenicity , Foscarnet , Guanidine , HeLa Cells , Herpesvirus 1, Human/pathogenicity , Humans , Influenza A virus/pathogenicity , Reproducibility of Results , Staining and Labeling/methods , Time Factors , Viral Plaque AssayABSTRACT
High numbers of inflammatory cells are found in a subgroup of patients with idiopathic dilated cardiomyopathy (IDCM). We hypothesized that the extent of inflammation is linked to myocardial TNF-alpha expression in human IDCM. Fourteen patients who consecutively underwent endomyocardial biopsy (EMB) were stratified into two groups-a group with low and a group with high myocardial inflammatory index (MII)-based on immunohistochemical analysis of cellular infiltration and HLA I and II expression. Myocardial TNF-alpha messenger RNA (mRNA) expression was determined by reverse transcriptase polymerase chain reaction, TNF-alpha protein was localized by immunohistochemistry and TNF-alpha serum levels were measured by EIA. IDCM patients with a high MII (n=6) showed a 1. 9-fold higher TNF-alpha mRNA expression when compared to IDCM patients with low MII (n=8, P=0.020). TNF-alpha protein was detected at perinuclear regions of cardiac myocytes and the endothelium. TNF-alpha serum levels were 3.0 (0.55) pg/ml in patients with high MII compared to 1.35 (0.20) pg/ml in patients with low MII (P=0.017). According to immunolocalization cardiac myocytes and the endothelium seem to be the major source of TNF-alpha production. Whether the elevated systemic level of TNF-alpha found in patients with high MII are elaborated by the myocardium or are produced by other tissues representing a general immune activation is not clear.
