[New infectious diseases caused by viruses. Current knowledge and preliminary information].
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
H W Doerr
Publications and source records attributed to H W Doerr.
Evaluation of the reliability of 6 current anti-HIV-1/HIV-2 enzyme immunoassays.
The sensitivity for early detection of HIV antibodies and specificity of 6 anti-HIV-1/HIV-2 screening enzyme immunoassays (ELISAs) currently on the market were investigated by testing a panel of 249 well-characterized serum samples. The panel included sera from AIDS patients or children with congenital HIV infection, high-risk individuals and patients with conditions unrelated to AIDS. 'Tricky' sera (repeatedly positive results by ELISA and negative or indeterminate results by Western blot; n = 69) were also used in this evaluation along with 6 seroconversion panels. One second-generation assay (Biotest) and two third-generation assays (Abbott and Murex) showed the highest sensitivity for early detection of HIV-1 antibodies in seroconversion panels. A high specificity was achieved with the Cambridge Biotech (100%) and Ortho ELISA (99.4%). A relatively high rate of false-positive results was obtained with the Biotest (n = 10) and the Pasteur assays (n = 8) by testing 'tricky' sera and samples from high-risk individuals and from patients with other acute viral infections. In conclusion, it remains difficult to combine high specificity with an accurate detection of early seroconversion for anti-HIV-1/HIV-2 screening enzyme immunoassays.
Acquired Immunodeficiency Syndrome↗
In vitro inhibition of human cytomegalovirus replication in human foreskin fibroblasts and endothelial cells by ascorbic acid 2-phosphate.
Antiviral activity of L-ascorbic acid-2-phosphate (ASC-2P), a long-acting derivative of L-ascorbic acid, against several human cytomegalovirus (CMV) strains was examined in cultures of human foreskin fibroblasts (HFF) and endothelial cells (EC). ASC-2P at concentrations ranging from 0.2 to 2 mM had no effect on the number of cells expressing 72 kDa CMV immediate early antigen (IEA) while it inhibited expression of 68 kDa late antigen (LA) in infected cultures of both cell types (30% and 55% reduction for EC and HFF, respectively). In HFF cells, virus yield was reduced up to 4-fold, when ASC-2P was added after CMV infection. Antiviral effects were significantly increased in cultures pretreated with ASC-2P. In HFF and EC pretreated for three subcultures (18 days) with 0.2 mM ASC-2P, a significant reduction of cells expressing IEA (75% and 80% reduction in EC and HFF, respectively) and LA (92% and 90% reduction for EC and HFF, respectively) was observed. Pretreatment for three subcultures with ASC-2P inhibited virus yield 50- to 100-fold in EC and 100- to 1000-fold in HFF. The continuous presence of ASC-2P was not required for its antiviral activity. A significantly higher reduction of virus replication with ganciclovir and foscarnet was obtained in ASC-2P pretreated cells than in untreated controls. The results showed that ASC-2P provides L-ascorbic acid with long-lasting antiviral activity against CMV. ASC-2P may be of benefit for the adjunctive treatment of CMV infection.
Antigens, Viral↗
Lymphocytes induce enhanced expression of HLA class I antigens on cytomegalovirus-infected syngeneic human endothelial cells.
Human cytomegalovirus (HCMV) infection has been associated with enhanced expression of HLA antigens on the endothelium and with cellular infiltrates within the graft following human organ transplantation. We investigated the interactions between human cytomegalovirus-infected cultured endothelial cells and cocultured syngeneic as well as allogeneic lymphocytes. Our objective was to find out whether cocultured lymphocytes elicit HCMV-mediated immune responses. In this report we focus on the modified expression of HLA antigens on the surface membrane of human umbilical vein endothelial cells (HUVECs). Endothelial expression of HLA class I and II antigens was measured by means of flow cytometry. Cocultures of HCMV-infected HUVECs with unprimed autologous PBLs led to virus-specific lymphocyte response, resulting in enhanced expression of HLA class I on HUVECs. This effect was only observed when lymphocytes were added to HUVECs during the very early phase after virus inoculation and was due to the stimulation of the CD8+ T-cell subpopulation. The modification of endothelial HLA expression was not observed in transwell cocultures, indicating the importance of cellular contact between endothelial cells and lymphocytes to elicit this effect. We conclude that HCMV-infected endothelial cells may induce virus-specific responses of unprimed syngeneic lymphocytes that lead to upregulated HLA class I expression on the endothelium. This pathway might be of important relevance for graft rejection crises after transplantation.
CD8-Positive T-Lymphocytes↗
Effects of desferrioxamine on human cytomegalovirus replication and expression of HLA antigens and adhesion molecules in human vascular endothelial cells.
Desferrioxamine (DFO), commonly used in therapy as a chelator of ferric ion in disorders of iron overload, is a potent inhibitor of human cytomegalovirus (HCMV) replication in cultured fibroblast cells. Moreover, DFO has immunomodulatory activity both in vitro and in vivo. We studied DFO effects on HCMV replication in cultured human endothelial cells and on the expression of several cell surface molecules, which mediate interactions of endothelial cells with other cell types in the immune system. The concentrations of DFO required for 50% reduction in the number of endothelial cells expressing HCMV late antigen, ranged for several HCMV strains from 5.2 to 8.8 microM. DFO concentrations ranging from 5 to 40 microM inhibited cellular DNA synthesis in a dose-dependent manner without any significant effects on the cell viability. DFO at 10 microM concentration suppressed expression of intercellular adhesion molecule-1 (ICAM-1) and endothelial leucocyte adhesion molecule-1 (ELAM-1), while it had no significant effect on the expression of vascular cell adhesion molecule-1 (VCAM-1). Expression of HLA class I and class II was not influenced by DFO treatment. The results showed that DFO is both effective in inhibition of HCMV replication and expression of ICAM-1 and ELAM-1 in endothelial cells, a combination that warrants attention to its potential use to prevent HCMV-induced allograft rejection in transplant recipients.
Cell Adhesion Molecules↗
Seroprevalence of HCV, HAV, HBV, HDV, HCMV and HIV in high risk groups/Frankfurt a.M., Germany.
During the period from August 1991 to April 1993, serum samples originating from different risk groups were tested for antibodies against hepatitis C virus (HCV), using a second-generation ELISA. The highest HCV seroprevalence levels were observed in haemophiliacs (87.0%) and intravenous drug abusers (IVDA) (78.9%). The HCV-seropositivity rates of polytransfused and organ transplant recipients were nearly identical (18.4% vs. 16.8%). Significantly lower HCV-seroprevalence rates were determined in chronic haemodialysis patients (8.1%) and homo/bisexuals (10.0%). The lowest HCV seroprevalence levels were found in the groups of female prostitutes (1.4%) and health care staff (0.8%). A strong correlation between HCV seropositivity and the presence of antibodies against HIV-1, HBc and HCMV was observed. There was also an association between HCV seropositivity and HAV. The seroprevalence of HDV and the proportion of HBsAg and HBeAg carriers was not significantly influenced by HCV serostatus. The incidence of HCV infection in renal transplant recipients was 3.9%. In 10 (6.5%) patients, anti-HCV seroconversion was observed in the immediate post-transplantation period followed by a decrease of the index value and finally, disappearance of antibodies at the end of the study. Antibodies passively acquired through intravenous immunoglobulin (IVIG) donation may account for overestimation of HCV seroprevalence in retrospective seroepidemiological surveys as established by our findings.
Adolescent↗
[Selective inhibition of replication of human cytomegalovirus by desferrioxamine in vitro and in vivo (case report)].
UNLABELLED: In AIDS patients cytomegalovirus (HCMV) retinitis is one of the most frequent opportunistic infections. Antiviral therapy aims at preserving vision as long as possible, but more and more HCMV isolates are proving to be resistant to ganciclovir (GCV) and foscarnet (PFA) in vitro. We tested whether desferrioxamine (DFO), an iron chelator with antiherpetic activity, can inhibit clinical virus isolates and laboratory strains. Clinical isolates were obtained from urine samples of AIDS patients with HCMV retinitis. The concentrations of DFO required for 50% and 90% reduction of the production of HCMV in several HCMV strains ranged from 3.1 to 4.9 microns and from 14.2 to 17.3 microns, respectively, Inhibitory effects of DFO on HCMV replication were completely prevented by co-incubation with stoichiometric amounts of FE3+. CASE REPORTS: DFO was administered by daily dose of 1 g i.v. to a patient with AIDS in whom HCMV retinitis continued to progress despite combination therapy with GCV and PFA. The addition of DFO to the combination inhibited progression of the disease. No relapse of HCMV retinitis was seen with 3 months of DFO therapy. The treatment was free of side effects.
AIDS-Related Opportunistic Infections↗
[Polio vaccination today: critical remarks].
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Age Factors↗
Induction of myogenic differentiation in a human rhabdomyosarcoma cell line by phenylacetate.
Sodium phenylacetate (NaPA) at concentrations ranging from 2 to 10 mM promoted myogenic differentiation of the human alveolar rhabdomyosarcoma cell line KFR. These concentrations inhibited DNA synthesis of the cells in a dose-dependent manner without significant effect on cell viability. The morphological differentiation of small mononuclear elements to terminal, elongated multinuclear structures resembling myotubes was accompanied by the expression of skeletal muscle myosin. The proportion of differentiated myosin-positive cells which was around 0.8-1.7% in control cultures 12 days after seeding was increased by NaPA treatment up to 47%. In the cytoplasm of differentiated cells, features of sarcomerogenesis were observed. These results suggest that NaPA is an effective inducer of rhabdomyosarcoma cell differentiation at concentrations that have been achieved in humans with no significant adverse effects.
Cell Differentiation↗
Low correlation of human cytomegalovirus DNA amplification by polymerase chain reaction with cytomegalovirus disease in organ transplant recipients.
Seventy-five organ transplant recipients underwent prolonged virological and serological follow-up for early detection of human cytomegalovirus (HCMV) infection after transplantation. HCMV DNA detection by nested polymerase chain reaction (PCR) and HCMV early structural antigen (pp65) detection were carried out in 576 peripheral blood leucocyte (PBL) samples. Furthermore, 563 blood specimens were investigated by a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulins G, M, and A against HCMV structural antigens. In eight of nine symptomatic organ transplant recipients, HCMV DNA was detected in one or more consecutive blood samples. HCMV DNA PCR was also positive in one or more samples from eight patients who never developed HCMV-related symptoms. HCMV pp65 antigen was detected almost exclusively in PBL samples from organ transplant recipients suffering from HCMV disease. However, antigenaemia was not detected in four PCR positive patients presenting clinical signs attributable to HCMV infection. Two of the initially HCMV DNA positive samples were not confirmed by retesting and hybridisation. The results of the present study demonstrate that despite the high specificity of nested PCR, HCMV DNA may be detected in the absence of clinical symptoms attributable to HCMV infection. In asymptomatic reactivation, limited replication of viral DNA may be responsible for positive results of PCR without any clinical relevance. In this context, pp65-antigen detection from PBL seems to have a better prognostic value, but is not always detected when clinical symptoms are present.
Antigens, Viral↗
Aphidicolin induces myogenic differentiation in the human rhabdomyosarcoma cell line KFR.
The effects of aphidicolin, a specific inhibitor of DNA polymerase alpha, on cell growth, DNA synthesis and myogenic differentiation in the human alveolar rhabdomyosarcoma cell line KFR were studied. The treatment with aphidicolin at 5 x 10(-6) M concentration, which completely inhibited DNA synthesis and cell growth, induced morphological differentiation of small mononuclear cells to elongated, multinucleated (myotube-like) structures. The morphological differentiation was accompanied by the expression of skeletal muscle myosin; about 30% myosin-positive cells were observed after 14 days of treatment, compared to 2.3% in untreated cultures. The results showed that aphidicolin induces differentiation of human rhabdomyosarcoma cells and that multinucleated myotube-like elements may develop simply by cell fusion without cell division and DNA synthesis.
Aphidicolin↗
Replication of human cytomegalovirus in a rhabdomyosarcoma cell line depends on the state of differentiation of the cells.
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.
Antigens, Viral↗
Comparison of two hybridization assays for the rapid detection of PCR amplified HSV genome sequences from cerebrospinal fluid.
Rapid diagnosis of herpes simplex encephalitis (HSE) can only be achieved by the polymerase chain reaction (PCR). In order to carry out PCR under routine conditions, it is of great importance to establish an easy DNA extraction protocol and especially a rapid and sensitive DNA detection method. In the present study, two different solid phase hybridization assays (Gen-Eti-K-DNA Enzyme Immunoassay (DEIA), Sorin Biomedica, Italy and Enzymun-Test DNA detection, Boehringer Mannheim, Germany) were compared for detection of PCR amplified HSV DNA polymerase genome region, using standard primers, from cerebrospinal fluid (CSF) samples. 122 CSF samples obtained from patients suffering from encephalitis and hospitalized at the University Clinics of Frankfurt and Graz during the period January 1992 to July 1993 were tested. To ascertain the sensitivity of the hybridization assays, dilution series of a plasmid, encoding the amplified region of the polymerase gene, were investigated. The detection limit of the DEIA assay was one copy of the plasmid/microliter, and the lowest amount of DNA which could be detected by the Enzymun assay as well as Southern blot was 10 copies/microliter. 15 CSF samples obtained from patients with HSE were found positive by the three assays. Concordant results were also obtained with CSF samples from non-HSE patients. The results of this study show that new hybridization systems guarantee a fast and high-sensitive detection of amplified HSV DNA. HSV PCR in CSF can be carried out routinely by the combined use of rapid hybridization and a simple extraction procedure.
Base Sequence↗
In vitro inhibition of human cytomegalovirus replication by desferrioxamine.
Desferrioxamine (DFO) is commonly used in therapy as a chelator of ferric ion in disorders of iron overload. We found that DFO inhibits human cytomegalovirus (HCMV) replication in infected cultures of human foreskin fibroblasts (HFF) at concentrations that have been achieved in humans with no significant adverse effects. The concentrations of DFO required for 50 and 90% reduction in the production of a HCMV-late antigen ranged for several HCMV strains from 3.1 to 4.9 microM and from 14.2 to 17.3 microM, respectively. DFO concentration of 60 microM had no significant effect on the viability of HFF cells. Inhibitory effects of DFO on HCMV replication were completely prevented by co-incubation with stoichiometric amounts of Fe3+.
Cell Division↗
Serological diagnosis of cytomegalovirus infection: comparison of 8 enzyme immunoassays for the detection of HCMV-specific IgM antibody.
BACKGROUND: Recently, numerous commercial ELISA kits for the detection of HCMV-specific IgM antibodies have been developed and are available on the international market. OBJECTIVE: The aim of the present study was to compare eight different ELISAs for the early serological diagnosis of HCMV infection. STUDY DESIGN: A panel of paired sera obtained from patients suffering from primary or secondary infection and potential cross-reactive serum samples positive for IgM antibodies against other herpesviruses, antinuclear antibody and rheumatoid factor were investigated with 5 indirect ELISAs (1-5) and 3 mu-capture assays (ELISAs 6-8). RESULTS: The highest sensitivity for IgM detection was achieved by a mu-capture assay (ELISA 8) and a traditional indirect ELISA (4). Their respective sensitivities were 78.0 and 76.0%. Overall, mu-capture immunoassays (ELISAs 6-8) detected HCMV infection earlier than indirect ELISAs (1-5) and showed a higher sensitivity for antibody detection in dilution series of IgM positive serum samples. Whereas ELISAs 1, 2, 5 and 6 showed a specificity of 100%, the alternative assays were affected by a variable number of false positive results obtained by testing potential cross-reactive serum samples. Their specificities ranged from 72.0% (ELISA 8) to 96.0% (ELISA 3). CONCLUSION: The results of the present study show that recent mu-capture and indirect ELISAs permit a relatively sensitive and reliable diagnosis of HCMV infection. Cross-reactive antigens and interference from antinuclear antibody may limit the specificity of commercial kits.
Journal Article↗
Typing and strain differentiation of clinical herpes simplex virus type 1 and 2 isolates by polymerase chain reaction and subsequent restriction fragment length polymorphism analysis.
In the present study, different combinations of primers were investigated for PCR amplification and typing of clinical herpes simplex virus type 1 (HSV-1) (n = 22) and herpes simplex virus type 2 (HSV-2) (n = 12) isolates. Intratypic strain differentiation was performed by restriction fragment length polymorphism (RFLP) analysis of different PCR amplified HSV genome regions. Enzymatic amplification of HSV DNA from all the clinical isolates could be achieved using primer combinations DNAP5/DNAp3-1 (HSV-1), DNAP5/DNAP3-2 (HSV-2). With the primer pair corresponding to the thymidine kinase (TK) genome region, amplification of all the HSV-1 isolates was only possible by nested PCR. With primers HSV UP/DOWN, 10 of 12 HSV-2 isolates could be detected. HSV typing by type-specific primers DNAP5/DNAP3-1 (HSV-1), DNAP5/DNAP3-2 (HSV-2) or restriction enzyme analysis (Ava II) of amplified DNA with HSV-specific UP/DOWN primers showed results which concorded with serotyping by monoclonal antibodies. RFLP analysis of the PCR products using selected restriction enzymes showed sufficient diversity of profiles among strains to differentiate all the HSV-1 isolates and to distinguish four groups of HSV-2 isolates. Amplification of HSV DNA from clinical isolates with subsequent typing and strain differentiation represents a valuable alternative to conventional methods (virus isolation, serotyping and restriction fragment analysis of the entire genomic DNA) and may be suitable for the study of HSV transmission and pathogenesis, especially in labour-intensive clinical samples with low levels of virus replication (i.e. cerebrospinal fluid, vitreous fluid and corneal transplants).
Base Sequence↗
Quantitative detection of neutralizing antibodies against polioviruses and non-polio enteroviruses (NPEV) using an automated microneutralization assay: a seroepidemiologic survey.
A seroepidemiological survey for the assessment of immunity against polioviruses and non-polio enteroviruses (NPEV) seroprevalence was performed using a recently established automated microneutralization assay. The seroprevalences of neutralizing antibodies against Coxsackie viruses B1-B6, and A9, echoviruses 6, 9, 11, and 30, and polioviruses 1, 2, and 3 were investigated in serum samples collected over a period of 30 months (January 1990 to June 1992). Overall, NPEV seroprevalence ranged from 8.3% (Coxsackie virus B6) to 69.8% (echovirus 30). Age-related increases of seroprevalence were only observed for Coxsackie viruses B2 and A9. Relatively low antibody titres were found, the average antibody titre being the highest for Coxsackie virus B2 (1/55.4). The overall poliovirus seropositivity rate reached 80%. The highest seroprevalence was observed in the higher age groups (> or = 50 years). A relatively high proportion (7%) of individuals did not show neutralizing antibodies against all the three poliovirus serotypes. The results of our study have shown that, using an automated microneutralization assay, it is possible to perform seroepidemiological surveys in large patient populations. Until now, no comparable study concerning the NPEV seroprevalence has been performed.
Adolescent↗
Failure of antiretroviral therapy: role of viral and cellular factors.
Effective therapy of human immunodeficiency virus (HIV) infection is mainly based on inhibition of reverse transcriptase by nucleoside analogues such as zidovudine (azidothymidine; AZT), didanosine, and zalcitabine. A major problem associated with long-term AZT therapy is the waning efficacy ('clinical resistance') over time. Clinical isolates of HIV-1 with reduced susceptibility to AZT can be recovered from HIV-infected individuals under prolonged treatment. However, the clinical importance of AZT resistance is uncertain. Other factors such as increased virus burden, increased virulence, and AZT toxicity could contribute, singly or in combination, to the loss of therapeutic benefit. Recent observations based on experimental models and clinical trials suggest that cellular mechanisms ('cellular resistance') may account for clinical resistance to antiviral agents. In vitro experiments demonstrated that in analogy to antitumoral therapy, the acquisition of multidrug resistance, i.e., resistance of cells to multiple, structurally unrelated chemotherapeutic agents, may play a role in the failure of long-term antiretroviral therapy. The 'cellular resistance' may contribute directly to the failure of antiviral therapy by the generation of subtherapeutic levels of antiviral compounds and/or their active forms. Indirectly, such subtherapeutic concentrations of active substances which permit limited replication of virus may represent a selective pressure for emergence and development of a resistant virus population. Hence it is of great importance to investigate the role of cellular factors in 'clinical resistance' to AZT and other anti-HIV agents. More detailed knowledge of cellular interactions and antiviral agents could help to improve or develop new strategies for antiviral therapy regimens.
Antiviral Agents↗