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AbstractCytotoxic and antiretroviral activity of cycloSal-d4TMP derivatives were tested in a new AZT-resistant H9 cell subline (H9rAZT250). The results showed, that cycloSal-d4TMP derivatives overcame resistance of HIV-1 to d4T in H9rAZT250cells, which exert decreased thymidine kinase (TK) gene expression.

AbstractA 2,3′-dideoxycytidine (ddC)-resistant T-lymphoid cell line (MOLT-4/8rddC250), in which deoxycytidine kinase (dCK) gene-expression was decreased when compared with parental cells, has been selected. Cytotoxic and antiretroviral activity of ddC and 3TC was significantly lower in MOLT-4/8rddC250than in parental MOLT-4/8 cells. ddC- and 3TC-bis(SATE)phosphotriesters completely overcame cellular resistance mechanisms and showed comparable both cytotoxic and antiretroviral activity in parental and ddC-resistant cells.

Summary Human T cell line H9 was established in a protein-free 1:1 mixture of Ham's F-12 and IMDM. After 230 passages (3 years) in protein-free medium, the cells designated H9-PF were infected with HIV-1. The infectivity titers of HIV-1 in cell culture medium were monitored by determining the median tissue culture infectious doses (TCID50). Additionaly, the production of viral antigen in cells was measured by an immunoenzymatical alkaline phosphatase antialkaline phosphatase (APAAP) method using a monoclonal antibody against HIV-1-p24 antigen. In acutely infected H9-PF and H9 cultures similar TCID50 values and percentage of cells positive for p24 antigen were found. In contrast, both TCID50 values and percentage of cells positive for p24 antigen were by far greater in chronically infected H9-PF than in H9 cultures.

Summary The replication of human cytomegalovirus (HCMV) was investigated in a new human rhabdomyosarcoma cell line (KFR) with morphological and biochemical characteristics of fetal striated muscle precursors (rhabdomyoblasts). KFR cells exhibited the unique property for spontaneous morphological transformation from a poorly-differentiated state into well-differentiated (myotube-like) rhabdomyoblasts. The poorly-differentiated rhabdomyoblasts promoted both complete viral gene expression and the production of infectious virus. In contrast, in well-differentiated rhabdomyoblasts HCMV infection was abortive. The results showed that replication of HCMV in this human rhabdomyosarcoma cell line depended on the state of cellular differentiation.

Human T lymphoid MOLT4/8 cells were grown continuously for more than 2 years in a medium containing 3′-azido-2′,3′-dideoxythymidine (zidovudine; AZT) at a concentration of 250 μM. These cells, designated MOLT-4/8rAZT250, were used to test the cytotoxic and antiviral activity of AZT. Intracellular accumulation of AZT, expression of the multidrug resistance 1 (MDR-1) gene, thymidine kinase (TK) gene and activity of the TK enzyme in cellular extracts were measured. The results showed that both the cytotoxic and antiviral activity of AZT were significantly lower in MOLT4/8rAZT250than in MOLT4/8 cells; concentrations required to inhibit 50% production of the p24 human immunodeficiency virus type 1 (HIV-1) antigen of two laboratory strains were at least 100-fold higher in resistant cells. The MDR-1 gene was not expressed in the resistant cells. TK mRNA expression was significantly lower in the resistant than in the sensitive cells. TK enzymatic activity for deoxythymidine phosphorylation was impaired in MOLT4/8rAZT250cells compared to the sensitive cells. AZT was phosphorylated only in the sensitive cells whereas no phosphorylation of AZT was found in the resistant cells. We tested whether several AZT-monophosphate triesters, which bypass cellular TK, could overcome resistance to the cytotoxic and antiviral activity of AZT. The bis(t-butylSATE) phosphotriester derivative of AZT showed comparable cytotoxic and antiviral activity in sensitive and resistant cells. The results demonstrated that MOLT4/8rAZT250cells exert resistance to the anti-HIV activity of the drug mainly owing to the lack of AZT phosphorylation and that resistance may be bypassed by using AZT-monophosphate SATE prodrugs.

Reduced glutathione (GSH) is present in millimolar concentrations in mammalian cells. It is involved in many cellular functions such as detoxification, amino acid transport, production of coenzymes, and the recycling of vitamins E and C. GSH acts as a redox buffer to preserve the reduced intracellular environment. Decreased glutathione levels have been found in numerous diseases such as cancer, viral infections, and immune dysfunctions. Many antioxidant molecules, such as GSH and N-acetylcysteine (NAC), have been demonstrated to inhibit in vitro and in vivo viral replication through different mechanisms of action. Accumulating evidence suggests that intracellular GSH levels in antigen-presenting cells such as macrophages, influence the Th1/Th2 cytokine response pattern, and more precisely, GSH depletion inhibits Th1-associated cytokine production and/or favours Th2 associated responses. It is known that GSH is not transported to most cells and tissues in a free form. Therefore, a number of different approaches have been developed in the last years to circumvent this problem. This review discusses the capacity of some new molecules with potent pro- GSH effects either to exert significant antiviral activity or to augment GSH intracellular content in macrophages to generate and maintain the appropriate Th1/Th2 balance. The observations reported herein show that pro-GSH molecules represent new therapeutic agents to treat antiviral infections and Th2-mediated diseases such as allergic disorders and AIDS.

Glycyrrhizin (GL) was shown to inhibit SARS-coronavirus (SARS-CoV) replication in vitro. Here the anti-SARS-CoV activity of 15 GL derivatives was tested. The introduction of 2-acetamido-β-d-glucopyranosylamine into the glycoside chain of GL resulted in 10-fold increased anti-SARS-CoV activity compared to GL. Amides of GL and conjugates of GL with two amino acid residues and a free 30-COOH function presented up to 70-fold increased activity against SARS-CoV but also increased cytotoxicity resulting in decreased selectivity index.

90K is a secreted serum glycoprotein with immune stimulatory activity. In this study, 90K plasma levels were determined by an enzyme-linked immunosorbent assay in 18 HIV-1-infected children and 10 uninfected control children. 90K levels in HIV-1-infected children (median, 12.5 μg/ml) were higher than in HIV-1 uninfected control group (6.3 μg/ml; P< 0.05). 90K levels of HIV-1-infected children classified as stage B and C (median, 15.0 μg/ml and 22.7 μg/ml, respectively) were higher compared to children with stage A disease (median, 7.0 μg/ml; P< 0.05). A positive correlation (r= 0.5; P< 0.05) was found between 90K levels and HIV-1 RNA levels in 137 plasma samples of 18 HIV-1-infected children collected during a period of 1 year. No correlation was found between 90K levels and CD4 cell counts. These results suggest that 90K plasma levels may represent a novel marker of disease progression in HIV-1-infected children.

For the development of effective prophylaxis (hyperimmune globulins) and improvement of serological testing for human cytomegalovirus (HCMV) infection in immunocompromised patients it is essential to characterize the viral encoded proteins and the humoral immune response in terms of neutralizing antibodies and immunglobulin class and IgG subclass reactivity to “early” and “late” HCMV proteins. The major neutralizing epitopes have been identified and screening of donor sera for neutralizing antibody by either conventional neutralization assays or enzyme-linked immunosorbent assay using recombinant antigens may help to improve the efficacy of hyperimmune globulin prophylaxis. The humoral response to individual HCMV proteins has been thoroughly investigated in immunocompromised patients. Antibodies against HCMV induced “early” antigens are not exclusively associated with active infection but may indicate an elevated risk for cytomegalic inclusion disease in immunocompromised patients. With a sensitive western blot technique. IgM and IgA antibodies against HCMV “late” proteins can be detected in sera from healthy seropositive individuals. Serum samples from subjects suffering from cytomegalic inclusion disease show significantly larger broader immune responses compared with healthy HCMV antibody carriers. Promising results using recombinant antigens corresponding to immunodominant epitopes for the detection of HCMV specific antibodies have been published.

A crude hydroalcoholic extract from HAMAMELIS VIRGINIANA bark was subjected to ultrafiltration (UF) with a cut-off limit of 3 kDa to obtain a higher and a lower molecular weight fraction. Characterisation of the fractions was attempted with TLC, HPLC, acidic hydrolysis, and chromatography over Sephadex LH-20. The UF-concentrate was shown to consist mainly of oligomeric to polymeric proanthocyanidins (PA). This fraction was found to exhibit significant antiviral activity against HERPES SIMPLEX virus type 1 (HSV-1). In addition, the UV-concentrate displayed radical scavenging properties, inhibited ?-glucosidase as well as human leukocyte elastase (HLE), and exhibited strong antiphlogistic effects in the croton oil ear edema test in the mouse. With the exception of the antioxidant potential and the inhibition of HLE-action the lower molecular fraction possessed weaker activities and contained mainly hamamelitannin, catechin, and further, unidentified constituents.

AbstractThe bis(S-pivaloyl-2-thioethyl) phosphotriesters of Ara-C and Ara-A were synthesized as potential bioreversible mononucleotide prodrugs. Some N- and O-acylated derivatives were also prepared with the aim to modify the lipophilicity of the title pronucleotides. Compounds were tested for their antitumor/antiviral activity against a variety of tumor cells and viruses.

Rapid Chelex extraction combined with an automated hybridization assay for the detection of PCR-amplified human cytomegalovirus DNA from cerebrospinal fluid was established. Quantitation of DNA was performed with a plasmid being used as an external standard. The detection limit was 10 copies per microliter. Quantitative detection of human cytomegalovirus DNA could be achieved over a range from 10 to 10(4) copies per microliter.