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Biomedical subjects

G Hunsmann

Publications and source records attributed to G Hunsmann.

At least 55 records · Page 3Linked to original sources
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

Protective role of the virus-specific immune response for development of severe neurologic signs in simian immunodeficiency virus-infected macaques.

The pathogenesis of human immunodeficiency virus-associated motor and cognitive disorders is poorly understood. In this context both a protective and a harmful role of the immune system has been discussed. This question was addressed in the present study by correlating the occurrence of neurologic disease in simian immunodeficiency virus (SIV)-infected macaques with disease progression and the humoral and cellular intrathecal antiviral immune response. Overt neurologic signs consisting of ataxia and apathy were observed at a much higher frequency in rapid progressor animals (6 of 12) than in slow progressors (1 of 7). Whereas slow progressors mounted a strong antiviral antibody (Ab) response as evidenced by enzyme-linked immunosorbent and immunospot assays, neither virus-specific Ab titers nor Ab-secreting cells could be found in the cerebrospinal fluid (CSF) or brain parenchyma of rapid progressors. Similarly, increased infiltration of CD8(+) T cells and cytotoxic T lymphocytes specific for viral antigens were detected only in the CSF of slow progressors. The finding that neurologic signs develop frequently in SIV-infected macaques in the absence of an antiviral immune response demonstrates that the immune system does not contribute to the development of motor disorders in these animals. Moreover, the lower incidence of neurologic symptoms in slow progressors with a strong intrathecal immune response suggests a protective role of the virus-specific immunity in immunodeficiency virus-induced central nervous system disease.

AIDS Dementia Complex↗
PubMed1998

Gastrointestinal pathology in rhesus monkeys with experimental SIV infection.

The updated results of current pathomorphological investigations in SIV-infected rhesus monkeys (Macaca mulatta) are summarized. After experimental infection with several SIVmac251 subtypes and various vaccination trails, 147 rhesus monkeys were morphologically examined until now. The pathology of the gastrointestinal tract in SIV-infected animals resembled those of human cases with HIV and AIDS. Alterations were considered to be primary SIV-induced (SIV enteropathy, giant cell disease) or secondary caused by opportunistic agents. Typical secondary gastrointestinal opportunistic infectious agents were parasites (Cryptosporidium sp., Trichuris sp., Trichomonas sp., Spironucleus sp.), viruses (cytomegalovirus, adenovirus) and bacteria (Mycobacterium simiae). Five animals developed malignant lymphomas involving the intestinal tract. The present observations revealed that SIV infection of rhesus monkeys provide an excellent model for studies on the pathogenesis of HIV in man.

Age Factors↗
PubMed1997

Cytokine gene transcription in simian immunodeficiency virus and human immunodeficiency virus-associated non-Hodgkin lymphomas.

Infection of rhesus monkeys with SIV leads to AIDS-like symptoms. Similar to human AIDS patients, some monkeys develop B cell non-Hodgkin lymphoma (NHL). We determined transcription of cytokine genes regulating the activation of B and T cells, which play a role in intratumoral immune surveillance. Therefore, we compared the transcription of the cytokine genes encoding IL-2, IL-4, IL-6, IL-10, IFN-gamma, TNF-alpha, and TGF-beta1, and the Epstein-Barr virus-encoded BCRF 1 gene, in cells from five monkey and two human tumor specimens. The immune-suppressive IL-10 and TGF-beta1 genes were predominantly transcribed in all tumor specimens analyzed. Cytokine gene transcription patterns appeared to be similar in human and animal tumor cells. The transcription patterns corresponded to their histological classification as diffuse large-cell lymphoma according to the REAL classification and as immunoblastic or centroblastic tumors according to the Kiel classification. The determination of cytokine gene transcription pattern in the NHL may improve our understanding of pathogenesis and immune surveillance in this heterogeneous group of tumors. Our data show that SIV-associated NHLs of rhesus monkeys are comparable to human HIV-1-associated EBV-positive non-Hodgkin lymphoma.

Animals↗
PubMed1997

Comparison of in vitro and in vivo infectivity of different clade B HIV-1 envelope chimeric simian/human immunodeficiency viruses in Macaca mulatta.

The use of HIV-1 env/SIVmac chimeric viruses expressing divergent HIV-1 envelopes of clinical isolates, facilitates homologous and heterologous evaluation of various recombinant HIV-1 envelope vaccine candidates in lower primates. In this study we compare the in vitro and in vivo infectivity, via intravenous (IV) and intravaginal (IVAG) routes of infection, of stocks of chimeric viruses expressing env from four different clade B HIV-1 isolates. The TCID50/ml was 7.1 x 10(4), 1.0 x 10(4), 6.3 x 10(4), and 1.2 x 10(3) for SHIVsf13, SHIVHan2, SHIVNM-3rn, and SHIVW6.1D, respectively, with a MID50/ml upon IV inoculation of 3.2 x 10(3), 3.2 x 10(4), 3.2 x 10(4), and 3.2 x 10(3), respectively. The same SHIVsf13 stock was infectious after IVAG administration, requiring a 300-fold higher virus dose. Plasma antigenemia and cell-associated viremia were generally highest at weeks 2 or 4 after infection and decreased to subdetectable levels after 8-12 weeks. All infected animals tested developed anti-HIV-1 gp120 antibodies. Inoculated virus dose showed no (linear) quantitative correlation with cellular virus load, duration of viremia, plasma antigenemia, and anti-gp120 antibody titers. No significant changes in peripheral blood CD4 cell levels were observed and none of the animals has shown evidence of disease progression to date (i.e., 13 months postinfection). Four in vivo passages of cell-associated SHIVW6.1D did not result in increased virulence. Vaccine development studies in macaques monkeys have become feasible with the use of various clade B HIV-1 env SHIV chimeras.

Animals↗
PubMed1997

Detection of Epstein-Barr virus small RNAs EBER1 and EBER2 in lymphomas of SIV-infected rhesus monkeys by in situ hybridization.

SIV infection of macaques is a well-established animal model for studying the pathogenesis of HIV infection in humans. During the course of SIV infection, up to 40% of cynomolgus macaques (Macaca fascicularis) develop SIV-associated non-Hodgkin's lymphomas. In the present study, we characterized malignant lymphomas of SIV(mac) 251/32H-infected rhesus macaques (Macaca mulatto) of our cohort in terms of clinical outcome, histopathology and EBV association. Histopathologic changes of lymphoid malignancies were classified according to the Kiel classification. For detection of the EBV-encoded small RNAs EBER1 and EBER2, a method of non-isotopic in situ hybridization was established. The presence of EBNA-2 antigens was assessed by immunohistochemistry. Seven of 43 rhesus macaques developed highly malignant B-cell lymphomas of the centroblastic, immunoblastic and Burkitt subtypes within 18-29 months post-experimental SIV infection. In situ hybridization revealed the presence of small EBER1 and -2 RNAs in 6 of 7 disease cases. EBNA-2 antigens could be demonstrated in only 4 of 7 tissue specimens. As expected, the Burkitt-type of lymphoma was negative for EBNA-2 antigen staining. In accordance with findings on SIV-associated lymphomas of cynomolgus macaques, infection with an EBV-related herpesvirus could be demonstrated in almost 90% of lymphomas in SIV-infected rhesus macaques. In contrast, the presence of EBV in lymphomas had been documented previously in only 30-40% of HIV-infected patients. Further studies should thus define the precise role of herpesvirus infection for lymphomagenesis in SIV- and HIV-induced immunodeficiency.

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

Generation of herpes virus saimiri-transformed T-cell lines from macaques is restricted by reactivation of simian spuma viruses.

Herpes virus saimiri (HVS) transforms human T-cells in vitro to stable growth. These T-cell lines retain their immunological characteristics of the parent cells and do not release infectious virus. Recently, lymphocytes of Old World monkeys were efficiently transformed by HVS. In parallel to these studies we initiated transformation experiments by infecting peripheral blood cell cultures of 45 monkeys, 35 rhesus and 10 cynomolgus macaques. In only three cases, we obtained transformed T-cell lines. The transformed T-cells were largely double-positive for CD4 and CD8. They responded with increased proliferation to mitogenic or IL-2 stimulation and transcribed mRNA for IL-2, IL-4, and IL-10. However, most initiated T-cell cultures from macaques developed giant cells. The cytopathic agent was identified as simian foamy virus (SFV) as confirmed by PCR, immunofluorescence, and coculture experiments. Treatment of the T-cell cultures with AZT- and SFV-specific sera did only shortly prolong the life-span of the cultures. Therefore, the reactivation of SFV caused remarkable difficulties in the establishment of macaque T-cell lines by HVS. This seems to be a general problem since most animals from several breeding colonies are SFV-positive.

Animals↗
PubMed1997

Detection of JC virus by anti-VP1 immunohistochemistry in brains with progressive multifocal leukoencephalopathy.

We have assessed the diagnostic efficacy of a novel polyclonal rabbit antiserum directed to the recombinant major capsid protein VP1 of JC virus (JCV). Immunohistochemistry for VP1 was compared to non-radioactive JCV DNA in situ hybridization (ISH) in ten cases of progressive multifocal leukoencephalopathy (PML). Tissue sections from postmortem brains were studied from PML patients suffering from immunodeficient conditions of various causes: immunodeficiency syndrome (AIDS, n = 7), severe combined immune deficiency due to adenosine deaminase deficiency (n = 1), sarcoidosis (n = 1) and leukemia (n = 1). VP1 immunohistochemistry demonstrated the presence of JCV in lesional oligodendrocytes of all PML patients, whereas ISH was able to detect JCV in nine out of ten cases. We conclude that VP1 immunohistochemistry is a specific, sensitive and rapid method for confirming the diagnosis of PML.

Adult↗
PubMed1997

Mutations in the SIV env and the M13 lacZa gene generated in vitro by reverse transcriptases and DNA polymerases.

To investigate the accuracy of retroviral in vitro DNA replication we have examined with two fidelity assays the reverse transcriptases (RTs) from SIVagm, HIV-1, MoMLV as well for comparison the Klenow fragment from E. coli and DNA polymerase a from calf-thymus. These forward mutation assays measured the loss of bacteriophage M13 lacZa gene function by mutations. In the EnvlacZa assay frameshift mutations occurring during polymerisation of a 176 b long simian immunodeficiency virus (SIV) envelope (env) sequence were phenotypically detected by blue/white-plaque screening. To measure in addition substitutions, a 116 b long M13 lacZa gene DNA template was used as the mutational target (LacZa assay). With the SIVagm env gene DNA template, we observed similar levels of frameshift fidelity for all three RTs. Nevertheless, the SIVagm RT was slightly more accurate than the other RTs and nearly all frameshifts were observed at two homopolymeric runs of its homologous template. Measuring also substitution errors at the lacZa template the mutation frequency of the SIVagm RT increased 2.5 fold and that of the HIV-1 RT was enhanced by a factor of 3.

Animals↗
PubMed1997

Detection of CSF-specific oligoclonal antibodies to recombinant JC virus VP1 in patients with progressive multifocal leukoencephalopathy.

The intrathecal synthesis of antibodies against recombinant VP1, the major structural protein of JC virus (JCV), was studied in 18 patients with progressive multifocal leukoencephalopathy (PML) and in 31 patients with various neurological disorders. Two methods were used, the calculation of an antibody specific index (ASI) on one hand and an antigen-driven immunoblotting for the detection of oligoclonal antibodies on the other. Most PML patients displayed an elevated (> 1.5) ASI (78%) and anti-VP1 oligoclonal antibodies restricted to the cerebrospinal fluid (55%). Only two other patients (one case each of multiple sclerosis and of neuroborreliosis) also showed an intrathecal synthesis of anti-VP1 oligoclonal antibodies, likely as a result of a 'polyspecific' reaction within the central nervous system.

Antibodies, Viral↗
PubMed1997

Efficient production of JC virus in SVG cells and the use of purified viral antigens for analysis of specific humoral and cellular immune response.

A new in vitro system for the production of the human polyomavirus JC virus (JCV) was established to circumvent the need for virus growth in primary human fetal glial cells (PHFG). The permanent cell line SVG, transformed by an origin-defective mutant of Simian Virus 40 (SV40) was used to grow JCV. JCV-specific RNA could be detected at day 5 and viral antigen at day 6 post infection (p.i.). Virus production peaked at day 16. Virus could be purified by differential centrifugation. The purified fraction consisted mainly of mature particles but contained also pentamers of the major structural virus protein 1 (VP1). The VP1-pentamers could be purified to near homogeneity. The purified virus particles stimulated a specific T-cell proliferation of peripheral blood monocytes (PBMCs) of a patient with progressive multifocal leukoencephalopathy (PML) and of two healthy individuals. In addition, JCV-particles and VP1-pentamers reacted specifically in an ELISA with a series of five PML-patient sera and four sera of individuals not affected by PML. These results demonstrate that purified whole virus particles are suitable for the analysis of specific cellular and humoral immune responses to JCV.

Animals↗
PubMed1997

Association of simian immunodeficiency virus Nef with cellular serine/threonine kinases is dispensable for the development of AIDS in rhesus macaques.

The nef gene of simian immunodeficiency virus (SIV) is essential for high viral load and induction of AIDS in rhesus monkeys. A mutant form of the SIVmac239 Nef, which contains changes in a putative SH3-binding domain (amino acids 104 and 107 have been changed from PxxP to AxxA), does not associate with cellular serine/threonine kinases, but is fully active in CD4 downregulation and associates with the cellular tyrosine kinase Src. Infection of two rhesus macaques with SIVmac239 containing the mutant AxxA-Nef caused AIDS and rapid death in both animals. No reversions were observed in the majority of nef sequences analyzed from different time points during infection and from lymphatic tissues at the time of death. Our findings indicate that the putative SH3-ligand domain in SIVmac Nef and the association with cellular serine/threonine kinases are not important for efficient replication and pathogenicity of SIVmac in rhesus macaques.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Analysis of the systemic and intrathecal humoral immune response in progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a subacute viral infection of oligodendrocytes by JC virus occurring almost exclusively in immunocompromised patients. By use of partially purified recombinant VP1 as antigen, the IgG response was analyzed by a quantitative ELISA of paired cerebrospinal fluid (CSF) and serum samples. An intrathecal immune response to VP1, defined as an antibody-specificity index of CSF to serum antibody titers > or =1.5, was found in 76% of PML patients (47/62) but in only 3.2% of controls (5/155) (P < .001). Intra-blood-brain barrier synthesis of VP1-specific IgG antibodies is 76% sensitive and 96.8% specific for the diagnosis of PML. Furthermore, the excellent correlation (r = .985) between the plasma cell count in brain tissue and the humoral intrathecal immune response to VP1 in PML patients suggests a role for B cells in this disorder.

Adolescent↗
PubMed1997

Impaired mitogen-driven proliferation and cytokine transcription of lymphocytes from macaques early after simian immunodeficiency virus (SIV) infection.

Altered cytokine transcription might play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection in humans. The infection of rhesus macaques with simian immunodeficiency virus (SIV) provides a relevant animal model for HIV infection. Therefore, we evaluated the cyokine transcription of phytohemagglutinin (PHA)-stimulated lymphocytes in the early phase after infection of four rhesus macaques with pathogenic SIV-mac239. To determine transcription of interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-6, and IL-10 we established a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). After inoculation with SIV, all monkeys became productively infected and developed an acquired immunodeficiency syndrome (AIDS) like disease. Infection was associated with a proliferation dysfunction of monkey lymphocytes in response to PHA. In addition, a decreasing overall cytokine transcription could be observed during the course of SIV infection. These findings demonstrate that an impairment of the lymphocyte function is associated with a reduced cytokine transcription in the early phase of an immunodeficiency virus infection. The observed differences of cytokine expression might contribute to the impaired immune response of SIV-infected monkeys and HIV-infected humans.

Animals↗