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Biomedical subjects

H W Doerr

Publications and source records attributed to H W Doerr.

At least 109 records · Page 6Linked to original sources
PubMed1996

Immunomodulatory properties of the metal chelators desferrioxamine and diethylenetriamine penta-acetic acid in vitro.

In this study, we investigated the effects of the intracellular metal chelator desferrioxamine (DFO) and the extracellular metal chelator diethylenetriamine penta-acetic acid (DTPA), which were previously shown to have strong anticytomegalovirus potencies, on their ability to elicit immunomodulatory effects in vitro[fcn,3]. The results showed that nontoxic and in vivo attainable concentrations of both DFO and DTPA inhibited mitogen- and allogen-induced proliferation of peripheral blood lymphocytes. The immunomodulatory effects of DFO/DTPA seem to be due to the impaired expression of interleukin-2 receptor and the reduced secretion of interleukin-2. However, metal chelators were more effective than cyclosporine or tacrolimus (FK506) in our in vitro experiments. Moreover, cytotoxicity mediated by lymphokine-activated killer cells and natural killer cells and the expression of HLA and adhesion molecules on cytokine-stimulated endothelial cells were differentially impaired by DFO/DTPA. These results warrant further study of the immunological effects of metal chelators in vivo.

Adjuvants, Immunologic↗
PubMed1996

Impact of oxidative stress on human cytomegalovirus replication and on cytokine-mediated stimulation of endothelial cells.

Transplantation-related pathogenic factors such as ischemia or allograft-directed inflammation are associated with oxidative changes that might lead to cellular oxidative stress. The aim of this study was to investigate the impact of oxidative stress on: (1) CMV replication in cultured human endothelial cells and (2) the stimulation of endothelial cells by proinfiammatory cytokines. Both pathomechanisms are known to contribute to graft rejection crises in vivo. Oxidative stress was induced in endothelial cell cultures with 10-200 microM buthionine sulfoximine. Western blotting showed a significant increase in the production of CMV-specific immediate early and late proteins in buthionine sulfoximine-treated cultures. Immunocytochemical staining suggested that this effect was caused by increased numbers of CMV antigen expressing cells (66% immediate early; 78%, late). Quantitative polymerase chain reaction for CMV-specific DNA and virus titration revealed that enhanced viral replication levels correlated with increased virion production. As a measure for the endothelial cell activation status, the surface expression of HLA-ABC and HLA-DR and adhesion molecules (ICAM-1, ELAM-1, VCAM-1) was quantified by fluorometric methods. Whereas oxidative stress alone did not modulate any surface molecule expression, the IFN-gamma-mediated expression of HLA-ABC and HLA-DR and the IL-1-mediated expression of ICAM-1, but not of ELAM-1 and VCAM-1 (IL-1 + TNF-alpha), was amplified. Interestingly, the amplification of HLA molecule expression was even higher in CMV-infected endothelial cells. This study provides evidence that oxidative stress contributes to the regulation of CMV replication, virus shedding, and the activation of endothelial cells by proinflammatory cytokines as it is observed in transplant recipients.

Base Sequence↗
PubMed1996

Long-term productive human cytomegalovirus infection of a human neuroblastoma cell line.

Human neuroblastoma cell line UKF-NB-4 persistently infected with human cytomegalovirus (HCMV) strain AD169 was established to study the effects of long-term HCMV infection on virus production and phenotypic characteristics of tumour cells. The cells designated UKF-NB-4AD169 were subcultured (80 subcultures) over a period of more than 2 years after initiation of infection. UKF-NB-4AD169 cells continued to produce infectious virus in successive passages, with a titre ranging from 9 x 10(3) to 1 x 10(5) and from 2 x 10(1) to 2 x 10(2) plaque-forming units per 10(6) cells and 1 ml culture medium, respectively; 10-20% of the cells produced HCMV-specific antigens, while 6-13% produced infectious virus progeny. The number of HCMV-specific DNA copies ranged from 9 x 10(4) to 9 x 10(6) per 10(6) cells. Transmission electron microscopy confirmed the productive nature of HCMV infection. UKF-NB-4AD169 cultures proliferated, with population doubling time ranging from 24.5 to 26.6 hr (19.5 to 20.3 hr for UKF-NB-4) and cell viability from 79% to 85% (91-96% for UKF-NB-4). Significantly lower amounts of tyrosine hydroxylase and decreased activity for dopamine-beta-hydroxylase than in uninfected cells were observed in UKF-NB-4AD169 cells. However, the expression of N-myc oncoprotein was significantly increased in persistently infected cultures. Our results show that long-term productive HCMV infection of UKF-NB-4 cell line is associated with the modulation of phenotypic properties, which may be related to the biological behaviour of neuroblastoma cells.

Cell Differentiation↗
PubMed1996

Hepatitis C plasma viral load is associated with HCV genotype but not with HIV coinfection.

The influence of human immunodeficiency virus (HIV) coinfection and hepatitis C virus (HCV) genotype distribution on HCV viral load and alanine amino transferase (ALT) levels in chronically infected patients remains unclear. In the present study, serum samples from a group of haemophiliac patients were investigated retrospectively. HCV geno- and subtyping was carried out using the Inno line probe assay (Inno LIPA, Innogenetics, Zwijnaarde, Belgium) in 87 patients positive by HCV RT PCR. Of these patients, 31 (35.6%) were HIV coinfected. HCV RNA was quantified with the HCV Monitor kit (Roche, Basel, Switzerland) in 43 patients (22 HIV-negatives, 21 HIV-positives). The most prevalent genotypes were 1 (n = 52) and 3a (n = 16) followed by genotype 2 (n = 9) and 4 (n = 3). Mixed infections were detected in 7 patients. Of genotype 1 positive samples, 24 and 23 were classified as subtype a and b, respectively. Five samples could not be subtyped. Although higher mean values of ALT were observed in genotype 1 infected patients, there was no statistically significant association between HCV genotype or subtype and liver enzymes (P > 0.05). On the other hand, statistically significant higher HCV RNA titres were observed in haemophiliacs infected with HCV genotype 1 in comparison to those infected with other genotypes (P < 0.01). No relationship was found between the presence of HIV coinfection and viral load of HCV RNA. There was no evidence that HCV infection had a more severe outcome in HIV-positive patients who had been infected with HIV and HCV more than ten years ago, even in those with very low CD4+ cell counts. No clear association between high ALT levels and large amounts of viral RNA was observed. In conclusion, a large viral load is associated with HCV genotype 1 infection; HIV coinfection has no clear effect on the intensity of HCV replication. An ongoing prospective study will evaluate the respective role of viral load, genotype, HIV coinfection and ALT level in the response to interferon therapy.

Alanine Transaminase↗
PubMed1996

Evaluation of 11 enzyme immunoassays for the detection of immunoglobulin M antibodies to Epstein-Barr virus.

Laboratory diagnosis of Epstein-Barr virus (EBV) infection is based mainly on serological tests. The analysis of the pattern of class-specific immunoglobulin response against defined viral antigens, i.e. virus capsid antigen (VCA), early antigen (EA) and Epstein-Barr nuclear antigen (EBNA), permits the differentiation between primary, latent and secondary (reactivated) EBV infection. In recent months, numerous test kits for the detection of VCA specific IgM antibody have been introduced on the international market. With a panel of well defined sera, the sensitivity and specificity of eleven different commercially available IgM ELISAs was evaluated. A well established, commercially available, indirect immunofluorescence assay (IFA) served as the reference test. Compared to the IFA, the Biotest and Sigma Diagnostic assays had the highest sensitivity for the detection of IgM antibody to VCA or EA. A variable number of false positive results (n = 0-9) was obtained with the EBV-IgM assays by testing potentially cross-reactive serum samples. Up to 19 serum samples from immunocompromised organ transplant recipients were found positive with the Sigma Diagnostics assay. The results of this study show that there are great differences in quality of current EBV-IgM-ELISA test kits. Depending on the clinical setting, it may be important to use a test kit which detects immunoglobulin M reactivity to EA in order to warrant an optimal sensitivity for the serological diagnosis of EBV reactivation in immunocompromised patients. However, since most immunosuppressed patients have serological reactivations, and in most cases these are asymptomatic, the clinical relevance of the detection of EA-IgM is very low.

Antibodies, Viral↗
PubMed1996

Lack of correlation between different hepatitis C virus screening and confirmatory assays.

Numerous 2nd and 3rd generation screening and confirmatory assays for the detection of anti-HCV antibodies have been introduced on the international market. The aim of the present study was to compare the performance of five different commercially available screening assays and four 'confirmatory' assays in a panel of serum samples that had tested positive or borderline with a 2nd generation EIA (Abbott HCV EIA 2nd generation). Considerable discrepancies were observed between the different screening assays and confirmatory tests. The antigens from the putative 'core' region of HCV were recognized most frequently by the confirmatory assays. By considering the reactivity to either NS5 (RIBA III and Inno-LIA) or E2/NS1 antigens (Inno-LIA Ab III) no sample could be identified as anti-HCV positive that would otherwise have been regarded as borderline or negative according to its banding pattern with core, NS3 and NS4 proteins. All 24 HCV-RT-PCR positive samples were anti-HCV reactive by the screening EIAs but only 18 and 21 samples were confirmed anti-HCV positive with the RIBA II and III, respectively. A clear association was observed between HCV-RNAemia in serum samples and index values (O.D. sample/O.D. cut-off) of the screening EIAs as well as with the number of reactive proteins in the confirmatory assays. In conclusion, the results of current screening and confirmatory assays are highly divergent. The additional diagnostic significance of the relatively expensive and labour-intensive immunoblots appears to be very limited. For the serological diagnosis of HCV infection and for blood donor screening, confirmatory assays should only be used if there is a borderline result by HCV EIA. The determination of infectivity by qualitative PCR and the follow-up of patients undergoing IFN therapy by HCV-RNA quantification appears to be much more useful.

Hepacivirus↗
PubMed1996

In vitro inhibition of human cytomegalovirus replication by calcium trinatrium diethylenetriaminepentaacetic acid.

Desferrioxamine (DFO) has been shown to inhibit human cytomegalovirus (CMV) replication in vitro. In the present study, we compared antiviral effects of DFO in human foreskin fibroblast (HFF) cells against several CMV strains with those of other chelators that interact with iron and other ions from different pools. DFO, a hydrophilic chelator, that may chelate both intracellular and extracellular ions inhibited production of CMV late antigen at 50% effective concentrations (EC50S) ranging from 6.2 to 8.9 microM. EC50S for calcium trinatrium diethylenetriaminepentaacetic acid (CaDTPA) ranged from 6.1 to 9.9 microM. EC50S for 2,2'-bipyridine (BPD), a hydrophobic chelator, which diffuses into cell membranes ranged from 65 to 72 microM. Concentrations which inhibited BrdU incorporation into cellular DNA by 50% (IC50S) ranged from 8.2 to 12.0 microM (DFO), from 65 to 89 microM (BPD), and from 139 to 249 microM (CaDTPA). CaDTPA was the only chelator which completely inhibited production of infectious virus in HFF and vascular endothelial cells at concentrations which had no significant effects on cellular DNA synthesis and growth. Addition of stoichiometric amounts of Fe3+ in the culture medium of HFF cells completely eliminated antiviral effects of DFO while antiviral effects of CaDTPA and BPD were only moderately affected. Fe2+ and Cu2+ were stronger inhibitors of CaDTPA than Fe3+; however, Mn2+ and Zn2+ completely suppressed antiviral effects of CaDTPA. The results show that CaDTPA is a novel nontoxic inhibitor of CMV replication. The antiviral activity of CaDTPA is suppressed by metal ions with a decreasing potency order of Mn2+/Zn2+ > Fe2+ > Cu2+ > Fe3+.

2,2'-Dipyridyl↗
PubMed1996

Antiviral and antiphlogistic activities of Hamamelis virginiana bark.

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 alpha-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.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Antitumor activity of sodium valproate in cultures of human neuroblastoma cells.

Valproic acid (VPA) is a simple branched-chain fatty acid that has anticonvulsant activity and is widely used in the treatment of epilepsy. VPA was found to effect growth and differentiation of human neuroblastoma (NB) cells in vitro at concentrations that have been achieved in humans with no significant adverse effects. Treatment of UKF-NB-2 and UKF-NB-3 NB cell lines with VPA at concentrations ranging from 0.5 to 2 mM resulted in neuronal morphological differentiation characterized by extension of cellular processes without significant effects on cell viability. Ultrastructural features of VPA-treated cells were consistent with the neuronal type of differentiation. VPA treatment of NB cells was associated with decreased expression of N-myc oncoprotein and increased expression of neural cell adhesion molecule in their membrane. Treatment of NB cells with 0.5 mM VPA increased their sensitivity to lymphokine-activated killer lysis. The results indicate that VPA, at non-toxic pharmacological concentrations, arrests the growth, induces differentiation and increases immunogenicity of NB cells through non-toxic mechanisms.

Cell Adhesion Molecules↗
PubMed1996

New PCR assay for rapid and quantitative detection of human cytomegalovirus in cerebrospinal fluid.

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.

AIDS-Related Opportunistic Infections↗
PubMed1996

Quantitative detection of human immunodeficiency virus (HIV) antigen by the Enzymun-Test: comparison with alternative assays and nucleic acid sequence-based amplification of HIV type 1 RNA.

A new modular automated enzyme immunoassay (EIA) (Enzymun-Test HIV Ag: Boehringer Mannheim) for quantitative human immunodeficiency virus (HIV) antigen detection was evaluated by testing a panel of 1,506 serum samples, including seroconversions, dilution series, follow-up samples from patients under antiretroviral therapy, single serum specimens from HIV-seropositive individuals in different stages of infection, potentially cross-reactive samples, and sera from HIV-negative hospitalized patients. The Abbott HIV type 1 (HIV-1) antigen monoclonal antibody assay served as the reference assay, and nucleic acid sequence-based amplification (Organon Teknika) for quantitative amplification of HIV-1 RNA was used for follow-up of patients under antiretroviral chemotherapy. The Boehringer Mannheim and Abbott EIAs showed concordant results for the early detection of HIV antigen in all the seroconversion panels. The follow-up samples from 29 HIV-infected individuals under antiretroviral therapy gave divergent results between both antigen tests. For the detection of HIV antigen in single serum samples from HIV-infected patients in different stages of HIV infection, a higher number of positive samples was detected with the Abbott HIV-1 antigen monoclonal antibody assay in samples from patients in stages II and III of HIV infection. The Enzymun-Test detected three or more positive samples than did the Abbott assay among the samples of patients with AIDS. The concordance on a sample-to-sample basis between the Boehringer Mannheim and Abbott EIAs was 98.6%. The sensitivity of the Enzymun-Test in comparison to the reference assay was 97.2%; the specificity was 98.8%. Although no close correlation could be found between the amount of viral RNA in serum detected by nucleic acid sequence-based amplification and the concentration of HIV antigen, a high HIV-1 RNA copy number was mostly associated with high levels of HIV antigen. In conclusion, the Enzymun-Test permits accurate HIV antigen detection and offers, in contrast to previous assays, the possibility of completely automated detection.

AIDS Serodiagnosis↗
PubMed1996

Modulatory effects of human cytomegalovirus infection on malignant properties of cancer cells.

Although there is no definitive evidence of the association of human cytomegalovirus (HCMV) infection with human cancers, the oncogenic potential of HCMV has been well established by in vitro studies demonstrating the ability of UV-irradiated or infectious virus to transform a variety of cells. After prolonged passaging the transformed cell type was maintained while HCMV DNA sequences were no more detectable. Three morphological transforming regions (mtr) of HCMV have been identified. The effects of HCMV on cellular functions which may be associated with the malignant phenotype include the expression of oncogenes and transcriptional activation of growth factors and interleukin synthesis. In infected cells, HCMV induces cytoskeletal alterations and changes in expression of cell surface receptors for extracellular matrix proteins which could result in increased motility and dissemination of cancer cells. Several human neuroblastoma cell lines undergo maturation in different neural crest derived cell types upon treatment with oncogenic potential agents, i.e. retinoic acid. The persistent HCMV infection of neuroblastoma cells (> 1 year) is accompanied by the increased expression of oncoproteins (i.e. N-myc) and decreased expression of tyrosine hydroxylase and dopamine-beta-hydroxylase. The activation of the cellular metabolism is due to HCMV binding to cellular receptors (prior to virus gene expression) and to the activity of HCMV immediate early (IE) gene products. IE proteins act directly as transcriptional activators or their activity is mediated by a variety of cellular transcription factors. HCMV infection may result in activation of promoters of cellular genes coding for cytokines, replication enzymes, proto-oncogenes and viral promoters. Recently it has been demonstrated that HCMV IE proteins block apoptosis probably by suppressing the ability of the antioncogene p53 to activate a reporter gene. The interactions of HCMV with tumor suppressor proteins such as p53 or retinoblastoma (pRb) susceptibility protein are reminiscent of those mediated by the oncoproteins of DNA tumor viruses. The acquisition of a fully malignant phenotype by normal cells is thought to require several mutations in a number of cellular genes. In this connection, HCMV may play the role of a nonobligate either direct or indirect cofactor for tumor genesis, e.g. by blocking apoptosis, which may be an essential requirement for tumor progression. Due to the stimulation of growth factors and/or inhibition of antioncogenes by its gene products, HCMV may modulate the malignant potential for tumor cells.

Animals↗
PubMed1996

Immunological findings in HIV-Leishmania coinfection.

It is supposed that Leishmania infection increases human immunodeficiency virus (HIV) replication in seropositive individuals. Two groups of 9 HIV-infected intravenous drug users each, one group with HIV-Leishmania coinfection (as determined by bone marrow microscopy, culture and an immunofluorescent assay, the other with HIV infection alone, but no evidence of Leishmania coinfection were matched for sex, age, time since first diagnosis of HIV infection, number of AIDS-defining diseases, proportion of patients treated with AZT and months of treatment, CD4/CD8 ratio, beta 2-microglobulin level and HIV p24 antigen positivity rate. IL-4, -6, -10 and -12 and IFN-gamma levels were determined by commercial enzyme immunoassays. The HIV-1 RNA copy number was quantified with the nucleic-acid-sequence-based amplification method (NASBA). The differences between the two groups were highly significant for all markers determined except for IL-12 and IFN-gamma. We found a higher viral load in the patients with HIV-Leishmania coinfection compared to the patients with HIV infection alone (p < 0.009). In 6 HIV-positive individuals without Leishmania coinfection, the HIV-1 RNA copy number was below the detection limit of NASBA (i. e. < 400 copies/100 microliters). Plasma levels of IL-4, -6, and -10 were significantly elevated in the coinfected group (p < 0.0001; p < 0.02, and p < 0.005). The results of our study show that the viral load is increased in patients with HIV-Leishmania coinfection in comparison to the controls. This might be partly due to Th2 immune activation, as demonstrated by higher plasma levels of IL-4, -6 and -10 in HIV-Leishmania-coinfected patients than in HIV-infected individuals.

AIDS-Related Opportunistic Infections↗
PubMed1996

How accurate is serologic testing of plasma pools for hepatitis B virus surface antigen, anti-human immunodeficiency virus 1 and 2, and anti-hepatitis C virus?

OBJECTIVE: The European pharmacopeia prescribes that, during the manufacture of blood derivates, the first homogeneous pool of plasma (for example, after removal of cryoprecipitate) must be tested for hepatitis B virus surface antigen (HBsAg), for hepatitis C virus (HCV) antibodies and for human immunodeficiency virus (HIV) antibodies using test methods of suitable sensitivity and specificity, in order to reduce the residual risk of infection originating from blood products. The present study was performed to verify if commercially available immunoassays, which are licensed for the screening of single serum and plasma samples, are suitable for the determination of HBsAg, anti-HCV, and anti-HIV in plasma pools. DESIGN: Plasma tools originating from different countries were spiked with HBsAg- and anti-HCV-positive standards (National Institut für Biological Standards, Hertfordshire, UK) and anti-HIV-positive serum. For the determination of HBsAg with and without immune complex dissociation (ICD), anti-HCV and anti-HIV antibodies the Abbott (Delkenheim, Germany) IMx system was used. RESULTS: In contrast to the testing for anti-HIV and anti-HCV, the detection of HBsAg (0.125 IU/ml detection limit) was influenced by the presence of anti-HBs in plasma pools. Statistically significant differences could be observed between anti-HBs-positive plasma pools and anti-HBs-negative serum samples spiked with HBsAg. The kinetics of HBsAg-anti-HBs complex formation showed that in a plasma pool with a high anti-HBs (329.4 IU/ml) concentration HBsAg was not detectable after 5 h of incubation. After ICD, HBsAg was still detectable in the pool with high anti-HBs content. Anti-HIV antibodies could be detected up to a dilution of 1/480,000, anti-HCV up to a dilution of 1/80, both in a spiked negative serum. Overall, the divergences between spiked plasma and serum were relatively low for anti-HCV and anti-HIV detection. CONCLUSION: The Abbott IMx assay permits a highly sensitive detection of HBsAg (after ICD or immediately after thawing of anti-HBs-positive plasma pools), anti-HCV, and anti-HIV in plasma pools. However, in case of a contamination with donations originating from individuals with acute HBV, HIV, or HCV infection or with a poor humoral immune response, serologic testing for HBsAg, anti-HIV, and anti-HCV may fail to detect potentially infectious plasma pools.

AIDS Serodiagnosis↗