Biomedical subjects

K Uberla

Publications and source records attributed to K Uberla.

At least 37 records · Page 2Linked to original sources

PubMed2000

Development of a self-inactivating, minimal lentivirus vector based on simian immunodeficiency virus.

In contrast to oncoviruses, lentiviruses do not require target cell division for integration into the host genome. Lentiviral vectors can therefore expand the spectrum of target cells susceptible to retroviral gene transfer. To analyze whether vectors based on simian immunodeficiency viruses (SIVs) could be used for gene transfer, a three-plasmid vector-packaging system was developed, in which Gag-Pol and the vector itself are of SIV origin, while Env is derived either from SIV, amphotropic murine leukemia virus (MuLV), or the G glycoprotein of vesicular stomatitis virus (VSV-G). To increase the safety of the SIV vector system, a self-inactivating SIV vector was constructed. After optimization of the SIV gag-pol expression plasmid, a minimal SIV vector, which contained only SIV sequences present on the multiply spliced nef transcript, could still be produced at titers of 2 x 10(5) infectious units/ml. Growth-arrested cells could be transduced with this vector even if vif, vpr, vpx, and nef had been deleted from the packaging construct and the vector.

Animals↗

PubMed2000

Low level of cross-resistance to amprenavir (141W94) in samples from patients pretreated with other protease inhibitors.

The therapeutic success of an antiretroviral salvage regimen containing protease inhibitors (PI) is limited by PI-resistant viral strains exhibiting various degrees of resistance and cross-resistance. To evaluate the extent of cross-resistance to the new PI amprenavir, 155 samples from 132 human immunodeficiency virus type 1-infected patients were analyzed for viral genotype by direct sequencing of the protease gene. Concomitantly, drug sensitivity to indinavir, saquinavir, ritonavir, nelfinavir, and amprenavir was analyzed by a recombinant virus assay. A total of 111 patients had been pretreated with 1-4 PI, but all were naive to amprenavir. A total of 105 samples (67.7%) were sensitive to amprenavir; 25 samples (16.1%) were intermediately resistant, and another 25 samples were highly resistant (4- to 8-fold- and >8-fold-reduced sensitivity, respectively). The mutations 46I/L, 54L/V, 84V, and 90M showed the strongest association with amprenavir resistance (P < 0. 0001). The scoring system using 84V and/or any two of a number of mutations (10I/R/V/F, 46I/L, 54L/V, and 90M) predicted amprenavir resistance with a sensitivity of 86.0% and a specificity of 81.0% within the analyzed group of samples. Of 62 samples with resistance against 4 PI, 23 (37.1%) were still sensitive to amprenavir. In comparison, only 2 of 23 samples (8.7%) from nelfinavir-naive patients with resistance against indinavir, saquinavir, and ritonavir were still sensitive to nelfinavir. Amprenavir thus appears to be an interesting alternative for PI salvage therapy.

Carbamates↗

PubMed2000

Suppression of acute viremia by short-term postexposure prophylaxis of simian/human immunodeficiency virus SHIV-RT-infected monkeys with a novel reverse transcriptase inhibitor (GW420867) allows for development of potent antiviral immune responses resulting in efficient containment of infection.

A nonnucleoside reverse transcriptase (RT) inhibitor, GW420867, was tested for postexposure prophylaxis (PEP) in rhesus macaques experimentally infected with 100 50% tissue culture infective doses of a chimeric simian/human immunodeficiency virus (SHIV) containing the RT gene of HIV-1 (SHIV-RT). Animals were either mock treated, or treated for 4 weeks starting at 8 or 24 h postinfection (p.i.) with GW420867. While such therapy led to undetectable plasma viremia in three of six monkeys, a transient plasma viremia was noted in the other three treated animals at 2 to 4 weeks following cessation of therapy. Following this transient viremia all drug-treated animals showed low or undetectable levels of plasma viremia up to the last sample examined at 90 weeks p.i. Despite low and/or undetectable viremia, virus-specific cytotoxic T lymphocyte and viral Env-specific proliferative responses were seen in the peripheral blood mononuclear cells of both mock- and drug-treated animals as early as 3 weeks p.i. Such virus-specific cellular responses, however, were better maintained in the drug-treated animals than the mock-treated animals. In contrast to the virus-specific cellular response, the magnitude and kinetics of virus specific humoral responses appeared to correlate with the detection of viremia. These data support the view that a short-term PEP with GW420867 permits the generation and maintenance of long-lasting virus-specific cell-mediated immune responses while markedly reducing viral loads to undetectable levels for a prolonged period of time (90 weeks) and leads to long-term disease protection. This model provides a unique means to define mechanisms and correlates of disease protection.

Animals↗

PubMed2000

Antiretroviral therapy during primary immunodeficiency virus infection can induce persistent suppression of virus load and protection from heterologous challenge in rhesus macaques.

A limited period of chemotherapy during primary immunodeficiency virus infection might provide a long-term clinical benefit even if treatment is initiated at a time point when virus is already detectable in plasma. To evaluate this strategy, we infected rhesus macaques with the pathogenic simian/human immunodeficiency virus RT-SHIV and treated them with the antiretroviral drug (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) for 8 weeks starting 7 or 14 days postinfection. PMPA treatment suppressed viral replication efficiently in all of the monkeys. After chemotherapy ended, virus replication rebounded and viral RNA in plasma reached levels comparable to that of the controls in four of the six monkeys. However, in the other two animals, virus loads peaked only moderately after withdrawal of the drug and then declined to low or even undetectable levels. These low levels of viremia remained stable for at least 31 weeks after cessation of therapy. At this time point, these two monkeys were challenged with SIV(8980) to evaluate whether the host responses which were able to keep RT-SHIV replication under control were also sufficient to protect against infection with a highly pathogenic heterologous virus. Both monkeys proved to be protected against the heterologous virus. In one of the two animals, low levels of SIV(8980) replication were detected. Thus, by chemotherapy during the acute phase of pathogenic virus replication, we could achieve not only persistent virus load suppression in two out of six monkeys but also protection from subsequent heterologous challenge. By this chemotherapeutic attenuation, the replication kinetics of attenuated viruses could be mimicked and a vaccination effect similar to that induced by live attenuated simian immunodeficiency virus vaccines was achieved.

Adenine↗

PubMed2000

Evidence for recombination of live, attenuated immunodeficiency virus vaccine with challenge virus to a more virulent strain.

Live, attenuated immunodeficiency virus vaccines, such as nef deletion mutants, are the most effective vaccines tested in the simian immunodeficiency virus (SIV) macaque model. In two independent studies designed to determine the breadth of protection induced by live, attenuated SIV vaccines, we noticed that three of the vaccinated macaques developed higher set point viral load levels than unvaccinated control monkeys. Two of these vaccinated monkeys developed AIDS, while the control monkeys infected in parallel remained asymptomatic. Concomitant with an increase in viral load, a recombinant of the vaccine virus and the challenge virus could be detected. Therefore, the emergence of more-virulent recombinants of live, attenuated immunodeficiency viruses and less-aggressive wild-type viruses seems to be an additional risk of live, attenuated immunodeficiency virus vaccines.

Animals↗

PubMed2000

Phenotypic HIV-1 resistance correlates with treatment outcome of nelfinavir salvage therapy.

In order to analyse whether drug sensitivity testing would be beneficial for clinical decision-making in heavily pretreated patients, we retrospectively studied viral genotype and phenotypic drug resistance in 12 HIV-1-infected patients, each of them with a history of failing at least one therapeutic regimen including one or two protease inhibitors (PIs). The salvage therapy included nelfinavir as new PI in all cases. Four patients showed a sustained and five patients a transient viral load decrease. Three patients failed to show a significant decline of plasma HIV-1 RNA. In the baseline samples of these cases, resistance against all components of their combination therapy could be detected, whereas at least one antiretroviral drug was still active in the cases with transient treatment response. All patients with sustained therapy response harboured viruses that were either fully sensitive or resistant to only one of the drugs administered. In our study, phenotypic drug resistance was predictive for the success of antiretroviral salvage regimens.

Amino Acid Sequence↗

PubMed2000

MedIDok--an Intranet tool for XML-based data modelling in medicine.

Integration of administrative, clinical and scientific documentation in medicine is a challenge because of the complexity and dynamics of the underlying data models. At least 50 items per procedure are necessary for clinically adequate documents. Extensive fine tuning is required to achieve acceptance by physicians, thus rapid prototyping is important. The results of this modelling process should be provided independently of specific implementations for reasons of portability. To address these needs we developed MedIDok (Medical Intranet-based Documentation), a set of intranet tools to generate and maintain specific documentation systems (generated programs, no manual coding) and the associated data models. The data structures are provided in XML format and therefore can be reused with other software architectures. The feasibility of non-redundant Intranet systems for both clinical and scientific information management is assessed covering the following domains at a major university hospital: hip ultrasound, endoscopic cholecystectomy, ESWL (extracorporeal shock wave lithotripsy) and implantable cardiac defibrillators. With the programs generated by MedIDok so far more than 10,000 patient contacts have been documented.

Computer Communication Networks↗

PubMed1999

Rapid, phenotypic HIV-1 drug sensitivity assay for protease and reverse transcriptase inhibitors.

BACKGROUND: Development of drug resistance is one of the major reasons for the failure of antiretroviral therapy of HIV-1 infection. Knowing the drug sensitivity-resistance profile of viruses present in a patient prior to treatment or change in treatment could help to optimize therapy. OBJECTIVE: Development of a rapid standardized phenotypic HIV-1 drug sensitivity assay for protease (PR) and reverse transcriptase (RT) inhibitors. DESIGN: The PR gene (codons 1-99) and the 5' part of the RT gene (codons 1-300) of HIV-1 is amplified from the plasma of infected individuals by RT-PCR and ligated into a proviral clone of HIV-1 containing a deletion of the PR gene and the 5' part of the RT gene. Bacteria are transformed with the ligation product and plasmid DNA is prepared from a library of transformed bacteria. The plasmid DNA is transfected into 293 T cells and recombinant virus is harvested from the supernatant of the transfected cells 2 days after transfection. The sensitivity of the recombinant virus is determined with the help of a sensitive indicator cell line. RESULTS: Recombinant viruses were generated with high efficiency. Determination of the drug sensitivity of the recombinant viruses with an indicator cell line was highly reproducible. The recombinant viruses accurately reflected the sensitivity-resistance profile of the parental viruses. The phenotypic drug sensitivity determined by this assay correlated well with the treatment history of patients. CONCLUSION: This assay system should allow rapid, high-throughput analyses of phenotypic HIV-1 drug sensitivity for PR and RT inhibitors. Due to the efficient generation of recombinant viruses, propagation of the recombinant viruses in cell culture is not required prior to the determination of the sensitivity of the recombinant viruses. The risk of selecting fitter non-resistant viruses due to culture conditions is minimized.

Anti-HIV Agents↗

PubMed1999

Design elements for a computerized patient record.

Computerized medical record systems have to present user- and problem-oriented views of a patient record to health-care professionals. Presentation and manipulation of data must be easily adaptable to current and future demands of medical specialties and specific settings. During the definition, development and evaluation of a prototype of a computerized patient record system, design elements were elaborated to support physicians and other health-care professionals. Our approach shows a high degree of flexibility and adaptability to specific needs, problem orientation and connectivity to other systems, via a hospital information network. The explicit description of the contents of a patient record allows to augment the number of items that can be recorded without modifying the data structure. New views on patient data can be added to the system without interfering with the routine use of the system. Application in several medical specialties proved the feasibility of our prototype.

Medical Records Systems, Computerized↗

PubMed1998

Specificity of helper T-cells generated from macaques infected with attenuated simian immunodeficiency virus.

Deletion of the simian immunodeficiency virus (SIV) nef gene leads to an attenuated virus phenotype in vivo. We have previously shown that these viruses induce a potent cellular immune response in macaques. To extend these studies, we established virus-specific short-term T-cell lines from four rhesus macaques infected with a nef deletion mutant of SIV. These T-cell lines proliferated upon restimulation with whole SIV or SIV gp140 antigen in vitro. The proliferating cells were characterized as CD4+ helper T-cells (TH) and their antigen recognition was MHC class II DR-restricted. After antigenic stimulation, they transcribed mRNA for various TH1- and TH2-like cytokines. Using these SIV-specific cell lines, a variety of helper T-cell epitopes in the SIV Env protein were determined with overlapping peptides. TH epitopes were identified throughout the whole SIV Env including both constant and variable regions. Although the recognition of TH epitopes was heterogeneous among different animals, five more broadly reactive T-cell epitopes were identified. As expected, recognition was associated with the MHC class II DRB background of the animals. This is the first report on helper T-cell epitopes in SIV-infected monkeys. Such studies should be of considerable significance for AIDS/ vaccine research.

Amino Acid Sequence↗

PubMed1998

Env-independent protection induced by live, attenuated simian immunodeficiency virus vaccines.

Live attenuated simian immunodeficiency viruses (SIV), such as nef deletion mutants, are the most effective vaccines tested in the SIV-macaque model so far. To modulate the antiviral immune response induced by live attenuated SIV vaccines, we had previously infected rhesus monkeys with a nef deletion mutant of SIV expressing interleukin 2 (SIV-IL2) (B. R. Gundlach, H. Linhart, U. Dittmer, S. Sopper, S. Reiprich, D. Fuchs, B. Fleckenstein, G. Hunsmann, S. Stahl-Hennig, and K. Uberla, J. Virol. 71:2225-2232, 1997). In the present study, SIV-IL2-infected macaques and macaques infected with the nef deletion mutant SIVDeltaNU were challenged with pathogenic SIV 9 to 11 months postvaccination. In contrast to the results with naive control monkeys, no challenge virus could be isolated from the SIV-IL2- and SIVDeltaNU-infected macaques. However, challenge virus sequences could be detected by nested PCR in some of the vaccinated macaques. To determine the role of immune responses directed against Env of SIV, four vaccinated macaques were rechallenged with an SIV-murine leukemia virus (MLV) hybrid in which the env gene of SIV had been functionally replaced by the env gene of amphotropic MLV. All vaccinated macaques were protected from productive infection with the SIV-MLV hybrid in the absence of measurable neutralizing antibodies, while two naive control monkeys were readily infected. Since the SIV-MLV hybrid uses the MLV Env receptor Pit2 and not CD4 and a coreceptor for virus entry, chemokine inhibition and receptor interference phenomena were not involved in protection. These results indicate that the protective responses induced by live attenuated SIV vaccines can be independent of host immune reactions directed against Env.

Animals↗

PubMed1998

A pathogenic threshold of virus load defined in simian immunodeficiency virus- or simian-human immunodeficiency virus-infected macaques.

To determine if a specific pathogenic threshold of plasma viral RNA could be defined irrespective of virus strain, RNA levels in the plasma of more than 50 infected rhesus macaques (Macaca mulatta) were measured. Animals were inoculated intravenously with either simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) strains of known pathogenic potential (SIV8980, SIVsmm-3, SIVmac32H/J5, SIVmac32H/1XC, reverse transcriptase-SHIV, SHIV89.6p) or with attenuated strains (SHIVW6.1D, SHIVsf13, SHIVhan-2, SIVmacDeltanef, SHIVsf33). In animals inoculated with nonpathogenic strains, shortly after the primary peak of viremia viral RNA levels declined and remained below 10(4) RNA equivalents/ml of plasma between 6 and 12 weeks postinoculation. Animals infected with documented pathogenic strains maintained viral RNA levels higher than 10(5) RNA equivalents/ml of plasma. In animals infected with strains with low virulence, a decline in plasma RNA levels was observed, but with notable individual variation. Our results demonstrate that the disease-causing potential was predicted and determined by a threshold plasma virus load which remained greater than 10(5) RNA equivalents/ml of plasma 6 to 12 weeks after inoculation. A threshold virus load value which remained below 10(4) RNA equivalents/ml of plasma was indicative of a nonpathogenic course of infection.

Animals↗

PubMed1997

Live attenuated SIV vaccines are not effective in a postexposure vaccination model.

Live attenuated simian immunodeficiency virus (SIV) vaccines, like nef deletion mutants, have been the most effective vaccines tested in the SIV/macaque model so far. The efficacy of live attenuated SIV vaccines in therapeutic vaccination and postexposure prophylaxis has not been determined. Inoculation of macaques with a pathogenic challenge virus and an attenuated SIV vaccine at the same time mimics postexposure vaccination, whereby vaccination with the attenuated virus is performed as rapidly as possible after exposure to pathogenic SIV. In the study presented here, four rhesus macaques were coinfected with pathogenic SIV and a nearly 3000-fold excess of a nef deletion mutant of SIV. Four macaques received pathogenic SIV and an approximately 200-fold excess of a nef deletion mutant expressing interleukin 2 (IL-2). The IL-2-expressing SIV had been previously constructed to enhance the immunogenicity of live attenuated SIV vaccines. All coinfected macaques had a high viral load, and some of them developed AIDS-like symptoms and pathological alterations rapidly. In the presence of pathogenic SIV, both live attenuated SIV vaccines did not protect from disease in this postexposure vaccination model.

Animals↗

PubMed1997

SIV/HIV-1 hybrid virus expressing the reverse transcriptase gene of HIV-1 remains sensitive to HIV-1-specific reverse transcriptase inhibitors after passage in rhesus macaques.

We have previously described an animal model for the therapy of human immunodeficiency virus type 1 (HIV-1) infection with HIV-1-specific reverse transcriptase (RT) inhibitors based on a simian immunodeficiency virus (SIV), in which the RT gene of SIV was replaced by the RT gene of HIV-1. In vitro, replication of the hybrid virus, RT-SHIV, was delayed compared with parental SIV. RT-SHIV could induce AIDS-like symptoms and pathologic alterations in rhesus macaques. Characterization of re-isolates recovered from RT-SHIV-infected macaques one-half year after infection revealed that the re-isolates replicated with kinetics similar to those of SIV. Inefficient processing of the Gag-Pol precursor of RT-SHIV may be one reason for the retarded growth of RT-SHIV, because the protease cleavage site between the protease gene and the RT gene was frequently mutated in the RT-SHIV re-isolates. Adaptation of RT-SHIV to the growth in macaques did not result in a relevant loss of sensitivity to nonnucleoside RT inhibitors (NNRTIs). However, because a minor sub-population of the RT-SHIV re-isolates contained a mutation conferring low-level resistance to ddI and ddC, the RT-SHIV/macaque model may underestimate the efficacy of these drugs. Nevertheless, this report further supports the suitability, reliability, and usefulness of the RT-SHIV/macaque model to investigate the antiviral properties of most RT inhibitors in an in vivo setting.

Animals↗

PubMed1997

Maternal smoking during pregnancy and lactation increases the risk for atopic eczema in the offspring.

BACKGROUND: Maternal smoking during pregnancy has been shown to lead to immunologic changes in the offspring. However, little is known about the influence of this exposure on atopic manifestations. OBJECTIVE: Our purpose was to investigate the influence of air pollutants on manifestations of atopy in preschool children. METHODS: Unselected cohorts of a total of 678 5- to 6-year-old preschool children (350 boys, 328 girls) were investigated in areas with different degrees of air pollution in Bavaria. Data on the history of atopic diseases and other relevant factors were obtained by questionnaire. A skin-prick test was performed with common aeroallergens. Manifestation of atopy was defined as personal history of atopic disease or positive prick test to either grass pollen, house dust mite, or cat and analyzed multivariately. RESULTS: Of all children, 38.9% exhibited at least one manifestation of atopy. Atopic eczema was reported in 7.9% to 15.5%, hayfever in 4.1% to 25.6%, and asthma in 3.0% to 8.1%. Of the mothers, 12.6% smoked during pregnancy or lactation or both. Analysis of the manifestation of atopy including sex, location, nitrogen oxide and sulfur dioxide exposure and maternal smoking as covariates revealed an influence of the maternal smoking during pregnancy/lactation. Of children whose mothers had smoked during pregnancy/lactation, 52.2% exhibited manifestations of atopy in contrast to 35.7% of children of nonsmoking mothers (p < 0.044). A history of atopic eczema was the only component of the variable "manifestation of atopy" that was significantly associated with maternal smoking during pregnancy and lactation. A causal interpretation of this finding, however, was not supported by a follow-up study. CONCLUSION: Maternal smoking during pregnancy or lactation or both might play a role in the development of atopic eczema and should be avoided.

Allergens↗

PubMed1997

Construction, replication, and immunogenic properties of a simian immunodeficiency virus expressing interleukin-2.

To study the effect of interleukin-2 (IL-2) on simian immunodeficiency virus (SIV) replication, pathogenesis, and immunogenicity, we replaced the nef gene of SIVmac239 by the IL-2 coding region. The virus, designated SIV-IL2, stably expressed high levels of IL-2 in cell culture. In comparison to SIVmac239, SIV-IL2 replicated more efficiently in peripheral blood mononuclear cells in the absence of exogenously added IL-2. To determine whether this growth advantage would be of relevance in vivo, four juvenile rhesus monkeys were infected with SIV-IL2 and four monkeys were infected with a nef deletion mutant of SIV (SIVdeltaNU). After a peak in the cell-associated viral load 2 weeks postinfection, the viruses could barely be isolated 3 to 7 months postinfection. Mean capsid antigen levels were higher in the SIV-IL2 group than in the nef deletion group 2 weeks postinfection. Viruses reisolated from the SIV-IL2-infected animals expressed high levels of IL-2 during the acute phase of infection. Deletions in the IL-2 coding region of SIV-IL2 were observed in two of the SIV-IL2-infected macaques 3 months postinfection. Urinary neopterin levels, a marker for unspecific immune stimulation, were higher in the SIV-IL2-infected macaques than in SIVdeltaNU-infected animals during the acute phase of infection. The SIV-specific T-cell-proliferative response and antibody titers were similar in both groups. Cytotoxic T cells directed against viral antigens were detected in all SIV-IL2-infected macaques and in two of the SIVdeltaNU-infected animals. Expression of IL-2 did not seem to alter the attenuated phenotype of nef deletion mutants fundamentally, although there might have been a slight increase in virus replication and immune stimulation during the acute phase of infection. Deletion of the viral IL-2 gene 3 months postinfection could be a consequence of a selective disadvantage due to local coexpression of viral antigen and IL-2 in the presence of an antiviral immune response.

Animals↗

PubMed1997

Replication-competent chimeric lenti-oncovirus with expanded host cell tropism.

Baboon bone marrow was grafted into human immunodeficiency virus type 1-infected patients in the course of recent trials for AIDS treatment. Since the baboon genome harbors multiple copies of an endogenous oncovirus, chimeric lenti-oncoviruses could emerge in the xenotransplant recipient. To analyze the potential replication competence of hybrid viruses between different genera of retroviruses, we replaced most of the env gene of simian immunodeficiency virus with the env gene of an amphotropic murine leukemia virus. The hybrid virus could be propagated in human T-cell lines, in peripheral blood mononuclear cells of rhesus macaques, and in CD4- B-cell lines. Because of the expanded cell tropism, the hybrid virus might have a selective advantage in comparison to parental viruses. Therefore, emerging chimeric viruses may be considered a serious risk of xenotransplantation. A note of caution is also suggested for the use of pseudotyped lentiviral vectors for human gene therapy.

Animals↗

PubMed1997

Simian immunodeficiency virus variants with differential T-cell and macrophage tropism use CCR5 and an unidentified cofactor expressed in CEMx174 cells for efficient entry.

The recent identification of coreceptors that mediate efficient entry of human immunodeficiency virus type 1 (HIV-1) suggests new therapeutic and preventive strategies. We analyzed simian immunodeficiency virus (SIV) entry cofactors to investigate whether the macaque SIV model can be used as an experimental model to evaluate these strategies. Similar to primary HIV-1 isolates, a well-characterized molecular clone, SIVmac239, which replicates poorly but efficiently enters into rhesus alveolar macrophages and an envelope variant, SIVmac239/316Env, with an approximately 1,000-fold-higher replicative capacity in macrophages used the beta-chemokine receptor CCR5 for efficient entry. The transmembrane portion of 316Env allowed low-level entry into cells expressing CCR1, CCR2B, and CCR3. A single amino acid substitution in the V3 loop of SIVmac239/316Env, 321P-->S, impaired the ability to enter into the T-B hybrid cell line CEMx174 but had relatively little effect on entry into primary cells and HOS.CD4 cells expressing CCR5. Although CEMx174 cells do not express CCR5, most SIVmac variants entered this hybrid cell line efficiently but did not enter the parental T-cell line CEM. It seems likely that CEMx174 cells express an as-yet-unidentified, perhaps B-cell-derived cofactor which allows efficient entry of SIVmac.

Animals↗