Biomedical subjects

Hans Wilhelm Doerr

Publications and source records attributed to Hans Wilhelm Doerr.

At least 19 recordsLinked to original sources

PubMed2006

Ribavirin inhibits angiogenesis by tetrahydrobiopterin depletion.

Ribavirin is a broad-spectrum antiviral drug that is used to treat hepatitis C virus (HCV)-infected patients. The virological response after ribavirin treatment appears to be insufficient to fully explain ribavirin-induced beneficial effects. Angiogenesis plays a pathogenic role in HCV-induced liver damage. Here, we investigated the influence of therapeutic ribavirin concentrations on angiogenesis. Ribavirin inhibited endothelial cell tube formation in vitro and vessel formation in the chick chorioallantoic membrane assay in vivo. Ribavirin inhibits inosine monophosphate dehydrogenase, which causes depletion of cellular GTP and in turn reduction of cellular tetrahydrobiopterin levels. The availability of tetrahydrobiopterin limits NO production by endothelial NO synthase. Ribavirin reduced levels of tetrahydrobiopterin (as revealed by HPLC), NO (as revealed by electron spin resonance spectroscopy), and cGMP (as revealed by RIA) in endothelial cells. Addition of tetrahydrobiopterin or NO prevented ribavirin-induced tube formation inhibition. In conclusion, angiogenesis inhibition by ribavirin has not been described before. This inhibition may contribute to ribavirin-induced pharmacological effects including adverse events.

Angiogenesis Inhibitors↗

PubMed2006

Ultrasensitive monitoring of HIV-1 viral load by a low-cost real-time reverse transcription-PCR assay with internal control for the 5' long terminal repeat domain.

BACKGROUND: Current HIV-1 viral-load assays are too expensive for resource-limited settings. In some countries, monitoring of antiretroviral therapy is now more expensive than treatment itself. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. METHODS: We evaluated real-time reverse transcription-PCR with internal control targeting the conserved long terminal repeat (LTR) domain of HIV-1 on reference panels and patient samples from Brazil (n = 1186), South Africa (n = 130), India (n = 44), and Germany (n = 127). RESULTS: The detection limit was 31.9 IU of HIV-1 RNA/mL of plasma (> 95% probability of detection, Probit analysis). The internal control showed inhibition in 3.7% of samples (95% confidence interval, 2.32%-5.9%; n = 454; 40 different runs). Comparative qualitative testing yielded the following: Roche Amplicor vs LTR assay (n = 431 samples), 51.7% vs 65% positives; Amplicor Ultrasensitive vs LTR (n = 133), 81.2% vs 82.7%; BioMerieux NucliSens HIV-1 QT (n = 453), 60.5% vs 65.1%; Bayer Versant 3.0 (n = 433), 57.7% vs 55.4%; total (n = 1450), 59.0% vs 63.8% positives. Intra-/interassay variability at medium and near-negative concentrations was 18%-51%. The quantification range was 50-10,000,000 IU/mL. Viral loads for subtypes A-D, F-J, AE, and AG yielded mean differences of 0.31 log(10) compared with Amplicor in the 10(3)-10(4) IU/mL range. HIV-1 N and O were not detected by Amplicor, but yielded up to 180 180.00 IU/mL in the LTR assay. Viral loads in stored samples from all countries, compared with Amplicor, NucliSens, or Versant, yielded regression line slopes (SD) of 0.9 (0.13) (P < 0.001 for all). CONCLUSIONS: This method offers all features of commercial assays and covers all relevant genotypes. It could allow general monitoring of antiretroviral therapy in resource-limited settings.

Brazil↗

PubMed2006

Flow cytometric analysis of virus-specific T lymphocytes: practicability of detection of HCMV-specific T lymphocytes in whole blood in patients after stem cell transplantation.

The detection and quantification of specific T lymphocytes against human cytomegalovirus (HCMV) has proven an important laboratory marker in the monitoring of patients after stem cell transplantation (SCT). In these patients HCMV infections may cause severe disease and death. However, the determination of HCMV-specific T lymphocytes may be limited by lymphopenia occurring after transplantation. We evaluated a commercial test kit for the reliable determination of HCMV-specific T lymphocyte development in lymphopenic patients after stem cell transplantation. Using a whole blood protocol for the flow cytometric detection of antigen-specific CD4(+) T-helper and CD8(+) cytotoxic T lymphocytes this test kit measures intracellular cytokine production after stimulation with HCMV antigen. The measurement of HCMV-specific T lymphocytes was feasible when at least 3,000 CD4(+) or 1,000 CD8(+) T cells could be counted by flow cytometry. Detection of HCMV-specific T lymphocytes was possible, on average, 67 (SD+/-61) days after transplantation for CD4(+) cells and 27 (SD+/-13) days for CD8(+) cells, thus being still within the critical time for HCMV reactivation. In conclusion, the use of modern test kits permits the measurement of HCMV-specific T lymphocytes in stem cell transplant recipients and may be included in the HCMV monitoring system after SCT.

CD4-Positive T-Lymphocytes↗

PubMed2006

Design and synthesis of dipeptidyl glutaminyl fluoromethyl ketones as potent severe acute respiratory syndrome coronovirus (SARS-CoV) inhibitors.

This paper describes the design and synthesis of dipeptidyl N,N-dimethyl glutaminyl fluoromethyl ketones (fmk) as severe acute respiratory syndrome coronovirus (SARS-CoV) inhibitors. The compounds were tested against SARS-CoV-induced cell death in Vero or CaCo2 cells as a measurement of the inhibiting effects of the compounds on the replication of the virus. Z-Leu-Gln(NMe(2))-fmk (6a) was found to be a potent inhibitor with low toxicity in cells, protecting cells with an EC(50) value of 2.5 microM and exhibiting a selectivity index of >40.

Animals↗

PubMed2006

West Nile virus infection induces interferon signalling in human retinal pigment epithelial cells.

PURPOSE: In addition to neuroinvasive disease, West Nile virus (WNV) infection is frequently associated with self-limiting chorioretinitis and vitritis. However, the mechanisms of ophthalmic WNV infection are rarely investigated, in part because of the lack of reliable in vitro models. The authors therefore established the first model of ocular WNV infection and investigated interaction of WNV with IFN signal-transduction mechanisms. METHODS: Human retinal pigment epithelial (RPE) cells were infected with WNV strain NY385-99 at a multiplicity of infection of 5. Virus replication was evaluated by virus titers at different times after infection. The susceptibility of RPE cells to WNV infection was confirmed by transmission electron microscopy. IFN-beta expression was assessed by quantitative real-time PCR and by measurements of antiviral activity in cell culture supernatants. IFN signaling was evaluated by phosphorylation of transducer and activator of transcription 1 and 2 (STAT1/2) proteins, with immunoblot analysis. RESULTS: RPE cells appeared to be highly sensitive to WNV infection. Maximum viral titers were found 24 hours after infection, followed by a continuous decline during the course of infection. WNV infection of RPE cells was followed by increased IFN-beta expression associated with IFN signaling and subsequent inhibition of WNV replication. CONCLUSIONS: In this study, the first cell culture model of ophthalmic WNV infection was developed and characterized in RPE cells, and the molecular mechanisms of WNV infection were studied. The data suggest that WNV induces a general antiviral state in RPE cells. This general antiviral state correlates with WNV-induced IFN signaling in retinal cells.

Animals↗

PubMed2006

Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants.

BACKGROUND: Experimental animal data show that protection against severe acute respiratory syndrome coronavirus (SARS-CoV) infection with human monoclonal antibodies (mAbs) is feasible. For an effective immune prophylaxis in humans, broad coverage of different strains of SARS-CoV and control of potential neutralization escape variants will be required. Combinations of virus-neutralizing, noncompeting mAbs may have these properties. METHODS AND FINDINGS: Human mAb CR3014 has been shown to completely prevent lung pathology and abolish pharyngeal shedding of SARS-CoV in infected ferrets. We generated in vitro SARS-CoV variants escaping neutralization by CR3014, which all had a single P462L mutation in the glycoprotein spike (S) of the escape virus. In vitro experiments confirmed that binding of CR3014 to a recombinant S fragment (amino acid residues 318-510) harboring this mutation was abolished. We therefore screened an antibody-phage library derived from blood of a convalescent SARS patient for antibodies complementary to CR3014. A novel mAb, CR3022, was identified that neutralized CR3014 escape viruses, did not compete with CR3014 for binding to recombinant S1 fragments, and bound to S1 fragments derived from the civet cat SARS-CoV-like strain SZ3. No escape variants could be generated with CR3022. The mixture of both mAbs showed neutralization of SARS-CoV in a synergistic fashion by recognizing different epitopes on the receptor-binding domain. Dose reduction indices of 4.5 and 20.5 were observed for CR3014 and CR3022, respectively, at 100% neutralization. Because enhancement of SARS-CoV infection by subneutralizing antibody concentrations is of concern, we show here that anti-SARS-CoV antibodies do not convert the abortive infection of primary human macrophages by SARS-CoV into a productive one. CONCLUSIONS: The combination of two noncompeting human mAbs CR3014 and CR3022 potentially controls immune escape and extends the breadth of protection. At the same time, synergy between CR3014 and CR3022 may allow for a lower total antibody dose to be administered for passive immune prophylaxis of SARS-CoV infection.

Amino Acid Substitution↗

PubMed2006

Anti-cancer effects of bortezomib against chemoresistant neuroblastoma cell lines in vitro and in vivo.

The proteasome inhibitor bortezomib (Velcade) was recently approved for the treatment of therapy-refractive multiple myeloma and is under investigation for numerous other types of cancer. A phase I clinical trial in paediatric patients resulted in tolerable toxicity. Since the emergence of chemoresistance represents one of the major drawbacks in cancer therapy, we investigated the influence of bortezomib on multi-drug resistant human neuroblastoma cell lines characterised by P-glycoprotein expression and p53 mutation. Nanomolar concentrations of bortezomib inhibited the cell cycle and induced apoptosis in chemosensitive as well as in chemoresistant cell lines. In vivo growth of chemosensitive and chemoresistant neuroblastoma cell lines was inhibited to a similar extent. In addition, bortezomib inhibited vessel formation in neuroblastoma xenografts. These findings and the favourable toxicity profile of bortezomib in children make it reasonable to further pursue additional development of the drug for the treatment of neuroblastoma and other paediatric solid tumours.

Animals↗

PubMed2005

[How dangerous is avian flu for mankind?].

BACKGROUND: Avian influenza, an infectious disease of birds, is caused by type A strain of the influenza virus. The disease, which was first identified in Italy more than 100 years ago, occurs worldwide. Avian influenza viruses are mainly distributed by migratory birds. Various animals like birds, pigs, horses, sea mammals and, finally, humans are susceptible to influenza A viruses. The high possibility of genomic changes like gene shift and drift are caused by the segmented RNA genome. EPIDEMIOLOGY: Since December 2003 a total of eleven countries (Vietnam, Thailand, Indonesia, Cambodia, China, Laos, Japan, Malaysia, South Korea, Russia, Kazakhstan) have reported outbreaks of highly pathogenic avian influenza A (H5N1) virus affecting poultry. Mongolia reported detection of H5N1 virus in migratory birds in August 2005, and actually also Romania and Turkey. During the avian flu outbreak approximately 150 million birds have died or been culled. The virus also jumped to humans; a total of 117 H5N1 cases in humans have been reported to the WHO, in four countries (Vietnam, Thailand, Indonesia, and Cambodia), 60 patients of them died. That avian influenza could also be a threat to humans has been known since 1997. OUTLOOK: Because of the natural virus reservoir like wild and/or domesticated ducks and others, actually there is rarely no chance to eradicate influenza. Furthermore, the virus could mutate and jump to humans with the threat of a global influenza pandemic. However, this is a statistically rare event.

Adult↗

PubMed2005

NK sensitivity of neuroblastoma cells determined by a highly sensitive coupled luminescent method.

The measurement of natural killer (NK) cells toxicity against tumor or virus-infected cells especially in cases with small blood samples requires highly sensitive methods. Here, a coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase release from injured target cells was used to evaluate the cytotoxicity of interleukin-2 activated NK cells against neuroblastoma cell lines. In contrast to most other methods, CLM does not require the pretreatment of target cells with labeling substances which could be toxic or radioactive. The effective killing of tumor cells was achieved by low effector/target ratios ranging from 0.5:1 to 4:1. CLM provides highly sensitive, safe, and fast procedure for measurement of NK cell activity with small blood samples such as those obtained from pediatric patients.

Cell Line, Tumor↗

PubMed2005

Cytomegalovirus-specific CD4 T-cell and glycoprotein B specific antibody response in recipients of allogenic stem cell transplantation.

BACKGROUND: The human cytomegalovirus (HCMV) causes severe complications in immunosuppressed patients, resulting in increased morbidity and mortality. The immunological components important for the control of HCMV are still not completely understood. OBJECTIVE AND STUDY DESIGN: To evaluate the importance of cellular and humoral immunity in stem cell transplant (SCT) recipients, we analysed levels of HCMV specific IFN-gamma producing CD4+ cells and glycoprotein B (gB) specific antibodies in HCMV positive SCT patients with and without reactivation episodes after SCT. RESULTS AND CONCLUSION: Patients without HCMV reactivation episodes showed a slow but steady increase in both parameters after SCT, indicating that initial high levels of gB specific antibodies or HCMV specific CD4+ IFN-gamma+ cells are not necessary to prevent reactivation of HCMV. In contrast, patients with reactivation episodes showed a steep, significant increase in HCMV specific CD4+ IFN-gamma+ counts just prior to HCMV reactivation, followed by a decline after the reactivation period. Patients who underwent only a single reactivation generated significant higher amounts of CD4+ IFN-gamma+ cells, than did patients with further reactivation episodes. The course of gB specific antibodies for reactivating patients was different, with significantly higher average values in the patients with HCMV reactivation. This indicates that patients with a HCMV reactivation exhibit a stronger humoral dominated immune response.

Adult↗

PubMed2005

Development of antiviral therapy for severe acute respiratory syndrome.

A new disease, the severe acute respiratory distress syndrome (SARS), caused by the SARS coronavirus (SARS-CoV), emerged at the beginning of 2003 and rapidly spread throughout the world. Although the disease had disappeared in June 2003 its re-emergence cannot be excluded. The development of vaccines against SARS-CoV may take years. Therefore, the availability of effective antiviral drugs against SARS-CoV may be crucial for the control of future SARS outbreaks. In this review, experimental and clinical data about potential anti-SARS drugs is summarised and discussed. Animal model studies will be needed to help to determine which interventions warrant controlled clinical testing.

Animals↗

PubMed2005

The human eye (retina): a site of persistent HCMV infection?

BACKGROUND: Human cytomegalovirus (HCMV) retinitis frequently occurs in severely naturally and iatrogenically immunocompromised patients. It has been shown that the immune-privileged retina is a major site of HCMV infection in AIDS patients. It is conceivable either that during the immunosuppression HCMV infection reactivates in various other organs viremically affecting the retina or that HCMV persisting in the retina may locally reactivate and result in HCMV retinitis. METHODS: As there is still controversy about the sites of HCMV latency and persistence we investigated 75 eyes of HIV-seronegative patients undergoing enucleation due to a variety of malignant and non-viral benign ophthalmic disorders for the retinal presence of HCMV antigen and DNA. RESULTS: None of the analyzed patients had symptoms of HCMV retinitis. Immunohistologic staining as well as Taq Man DNA PCR analysis showed all samples to be free of HCMV. CONCLUSIONS: Our data suggest that the human eye is rather unlikely to be a site of productive or latent HCMV persistence.

Adult↗

PubMed2005

Impact of persistent cytomegalovirus infection on human neuroblastoma cell gene expression.

In a model of human neuroblastoma (NB) cell lines persistently infected with human cytomegalovirus (HCMV) we previously showed that persistent HCMV infection is associated with an increased malignant phenotype, enhanced drug resistance, and invasive properties. To gain insights into the mechanisms of increased malignancy we analyzed the global changes in cellular gene expression induced by persistent HCMV infection of human neuroblastoma cells by use of high-density oligonucleotide microarrays (HG-U133A, Affymetrix) and RT-PCR. Comparing the gene expression of different NB cell lines with persistently infected cell sub-lines revealed 11 host cell genes regulated in a similar manner throughout all infected samples. Nine of these 11 genes may contribute to the previously observed changes in malignant phenotype of persistently HCMV infected NB cells by influencing invasive growth, apoptosis, angiogenesis, and proliferation. Thus, this work provides the basis for further functional studies.

Cell Line↗

PubMed2005

Role of tumor cell immune escape mechanisms in cytomegalovirus-mediated oncomodulation.

It has been known for a long time that cytomegalovirus (CMV) has evolved mechanisms that allow the escape from the host immune surveillance. In the past, many efforts have been done to elucidate the molecular mechanisms underlying this virus-mediated immune escape and thus virus persistence. However, it is unknown, whether CMV may also impair immune responses directed against tumor cells. This might have severe consequences on tumor progression and may explain the growing evidence for CMV-mediated oncomodulation. This review summarizes recent work on CMV-mediated immune escape mechanisms of tumor cells and oncomodulation and proposes novel aspects that may be important for understanding the CMV-associated tumor progression.

Animals↗

PubMed2005

Comparative evaluation of the Cobas Amplicor HIV-1 Monitor Ultrasensitive Test, the new Cobas AmpliPrep/Cobas Amplicor HIV-1 Monitor Ultrasensitive Test and the Versant HIV RNA 3.0 assays for quantitation of HIV-1 RNA in plasma samples.

BACKGROUND: There are several commercially available assays for the quantitation of HIV RNA. A new automated specimen preparation system, the Cobas AmpliPrep, was developed to automate this last part of the PCR. OBJECTIVES AND STUDY DESIGN: We compared the results obtained by the Roche Cobas Amplicor HIV-1 Monitor Ultrasensitive Test (MCA, manual sample preparation) with those by the Versant HIV-1 RNA 3.0 assay (bDNA). Secondly we compared the MCA with the new Cobas AmpliPrep/Cobas Amplicor HIV Monitor Ultrasensitive Test (CAP/CA, automated specimen preparation) by investigating clinical patient samples and a panel of HIV-1 non-B subtypes. Furthermore, we assessed the assay throughput and workflow (especially hands-on time) for all three assays. RESULTS: Seventy-two percent of the 140 investigated patient samples gave concordant results in the bDNA and MCA assays. The MCA values were regularly higher than the bDNA values. One sample was detected only by the MCA within the linear range of quantification. In contrast, 38 samples with results <50 copies/ml in the MCA showed in the bDNA results between 51 and 1644 copies/ml (mean value 74 copies/ml); 21 of these specimens were shown to have detectable HIV RNA < 50 copies/ml in the MCA assay. The overall agreement between the MCA and the CAP/CA was 94.3% (551/584). The quantification results showed significant correlation, although the CAP/CA generated values slightly lower than those generated by the manual procedure. We found that the CAP/CA produced comparable results with the MCA test in a panel of HIV-1 non-B subtypes. CONCLUSIONS: All three assays showed comparable results. The bDNA provides a high sample throughput without the need of full automation. The new CAP/CA provides reliable test results with no HIV-subtype specific influence and releases time for other works in the laboratory; thus it is suitable for routine diagnostic PCR.

Automation↗

PubMed2005

HBV reactivation after kidney transplantation.

Recent studies suggest that reappearance of hepatitis B surface antigen (HBsAg) and loss of anti-HBs antibodies may be common events in bone marrow recipients and patients with chemotherapy. In this study, we reviewed the virologic laboratory records from kidney recipients. Out of 1512 patients, 228 had been diagnosed with resolved HBV infection (anti-HBc positive, HBsAg negative) but normal liver enzyme levels prior to kidney transplantation. Reappearance of HBsAg after kidney transplantation was observed in two (0.9%) of those patients, which may be attributed to reactivation of a latent infection or to a new HBV infection. In both of the patients, HBV infection may have been reactivated although immunosuppression was just on a low level over the whole period. We conclude that natural immunity to HBV may not protect against reactivation in patients with suppression of the immune system. Periodic follow-up of HBV serology for early diagnosis of reactivation is highly recommended in transplant recipients.

Aged↗

PubMed2005

Molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus.

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.

Amino Acid Sequence↗

PubMed2005

Increased replication of human cytomegalovirus in retinal pigment epithelial cells by valproic acid depends on histone deacetylase inhibition.

PURPOSE: Human cytomegalovirus (HCMV) replication depends on different cellular pathways, including histone acetylation and extracellular-signal regulated kinases 1 and 2 (Erk 1/2). In the present study, the influence of therapeutic valproic acid (VPA) concentrations was investigated on HCMV replication in retinal pigment epithelial (RPE) cells. METHODS: HCMV antigen expression and replication were detected by immunostaining, real-time RT-PCR, and determination of virus titers. Histone acetylation and Erk 1/2 phosphorylation were detected by Western blot. RESULTS: Pretreatment with VPA < or =1 mM enhanced HCMV antigen expression and replication by up to ninefold. In addition to histone deacetylase (HDAC) inhibition, VPA stimulated Erk 1/2 phosphorylation in RPE cells. Investigation of six VPA derivatives revealed that S-2-pentyl-4-pentynoic acid was the only derivative that induced histone hyperacetylation, indicating HDAC inhibition, in the observed concentrations < or =1 mM and that increased HCMV antigen expression. Other derivatives did not enhance HCMV replication in the tested concentrations, although some were found to induce Erk 1/2 phosphorylation. The mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 inhibited VPA-induced Erk 1/2 phosphorylation but did not affect VPA-induced increased HCMV replication. In addition, the structurally nonrelated HDACI trichostatin A enhanced HCMV replication but did not affect Erk 1/2 phosphorylation in RPE cells. CONCLUSIONS: The data demonstrate that VPA stimulates HCMV replication by HDAC inhibition independent of Erk 1/2 phosphorylation in therapeutic concentrations in RPE cells. Therefore, patients at risk of HCMV retinitis who are treated with VPA or other HDAC inhibitors should be carefully monitored.

Acetylation↗