-
JAIDS Journal of Acquired Immune Deficiency Syndromes 07/2011; 57(3):e56-8. · 4.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A central, yet unresolved issue in the pathogenesis of HIV disease is the mechanism of antibody perturbation. In this study, HIV-specific memory B-cells were quantified in groups of infected subjects and compared with memory responses to other antigens and antibody titers. HIV-specific memory B-cell responses were vigorous in individuals with CD4(+) T-cell counts >350/microl and weak or undetectable in subjects with CD4(+) T-cell numbers <200/microl. Memory B-cell loss was permanent, because antiretroviral therapy failed to restore HIV-specific memory responses while influenza- and tetanus toxoid-specific memory B-cells remained unaffected or recovered. Antibody titers to Gag strongly correlated with memory B-cell frequencies. In contrast, Env-specific antibodies were maintained in advanced disease despite low or undetectable levels of memory B-cells. These results provide a potential mechanism by which destruction of HIV-specific CD4(+) T-cells affects the humoral immune response against HIV and compromises the ability to maintain an effective antibody response.
Virology 12/2009; 397(1):7-13. · 3.35 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Antibody-mediated elimination of foreign antigens contributes to immune protection from viral infection. We have generated phycoerythrin-conjugated HIV-1 Gag/p24 and influenza virus nucleoprotein tetramers for flow cytometric analysis of blood cell populations involved in antibody-mediated immunity. We show that in the presence of antigen-specific antibodies fluorescent antigen tetramers bound to different blood cell populations including granulocytes, monocytes and lymphocytes. Binding to B-lymphocytes was particularly efficient. The interaction was primarily mediated by complement. Fcgamma receptor-mediated antigen binding by B-cells was significantly less effective. The study shows that fluorescent antigen tetramers are useful to quantify cell populations involved in complement- and Fcgamma-mediated immune responses in viral infections.
Immunobiology 06/2009; 215(3):223-9. · 3.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Protein tagging with a peptide is a commonly used technique to facilitate protein detection and to carry out protein purification. Flexibility with respect to the peptide tag is essential since no single tag suites all purposes. This report describes the usage of two short peptides from the SARS-associated coronavirus nucleocapsid (SARS-N) protein as protein tags. Plasmids for the generation of tagged proteins were generated by ligating synthetic oligonucleotides for the peptide-coding regions downstream of the protein coding sequence. The data show recognition of prokaryotically expressed HIV-1 Gag/p24 fusion protein by Western blot and efficient affinity purification using monoclonal antibodies against the tags. The SARS peptide antibody system described presents an alternative tagging opportunity in the growing field of protein science.
Protein Expression and Purification 06/2008; 61(2):138-41. · 1.59 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A member of the family of coronaviruses has previously been identified as the cause of the severe acute respiratory syndrome (SARS). In this study, several monoclonal antibodies against the nucleocapsid protein have been generated to examine distribution of the nucleocapsid in virus-infected cells and to study antigenic regions of the protein. Confocal microscopic analysis identified nucleocapsids packaged in vesicles in the perinuclear area indicating viral synthesis at the endoplasmic reticulum and Golgi apparatus. The monoclonal antibodies bound to the central and carboxyterminal half of the nucleocapsid protein indicating prominent exposure and immunogenicity of this part of the protein. Antibodies recognised both linear and conformational epitopes. Predictions of antigenicity using mathematical modelling based on hydrophobicity analysis of SARS nucleoprotein could not be confirmed fully. Antibody binding to discontinuous peptides provides evidence that amino acids 274-283 and 373-382 assemble to a structural unit particularly rich in basic amino acids. In addition, amino acids 286-295, 316-325 and 361-367 that represent the epitope recognised by monoclonal antibody 6D11C1 converge indicating a well-structured C-terminal region of the SARS virus nucleocapsid protein and functional relationship of the peptide regions involved. Alternatively, dimerisation of the nucleocapsid protein may result in juxtaposition of the amino acid sequences 316-325 and 361-367 on one nucleoprotein molecule to amino acid 286-295 on the second peptide. The monoclonal antibodies will be available to assess antigenicity and immunological variabilities between different SARS CoV strains.
Virus Research 01/2007; 122(1-2):119-26. · 2.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Infectious pseudovirions based on HIV show the morphology of the parent virus and a genome that is partially expressed in infected cells. The constructs are capable of a single round of infection. In this study, we generated vesicular stomatitis virus (VSV) glycoprotein (G) pseudotyped HIV-1-derived pseudovirions that contain a codonoptimized p17/p24 HIV-1 gag or the green fluorescent protein (GFP) gene as transgene. BALB/c mice were immunized in a DNA prime pseudovirion boost fashion. Immunization induced a Gag-specific antibody response, high titers of neutralizing antibodies directed against the VSV-G protein and a Gag-specific IFN-gamma-secreting cytotoxic T lymphocyte (CTL) response. CTL responses were induced by both structural proteins contained in the pseudovirion preparation and through expression of the transgene. Infection properties similar to those of live attenuated HIV and the immunogenicity observed make infectious pseudovirions valuable tools to further study the mechanism of immune stimulation in models of HIV infection.
AIDS Research and Human Retroviruses 12/2006; 22(11):1162-6. · 2.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: CD95 (Fas/Apo-1) triggers apoptotic cell death via a caspase-dependent pathway. Inhibition of caspase activation blocks proapoptotic signaling and thus, prevents execution of apoptosis. Besides induction of apoptotic cell death, CD95 has been reported to trigger necrotic cell death in susceptible cells. In this study, we investigated the interplay between apoptotic and necrotic cell death signaling in T cells. Using the agonistic CD95 antibody, 7C11, we found that caspase inhibition mediated by the pancaspase inhibitor, zVAD-fmk, prevented CD95-triggered cell death in Jurkat T cells but not in A3.01 T cells, although typical hallmarks of apoptosis, such as DNA fragmentation or caspase activation were blocked. Moreover, the caspase-independent cell death in A3.01 cells exhibited typical signs of necrosis as detected by a rapid loss of cell membrane integrity and could be prevented by treatment with the radical scavenger butylated hydroxyanisole (BHA). Similar to CD95-induced cell death, apoptosis triggered by the DNA topoisomerase inhibitors, camptothecin or etoposide was shifted to necrosis when capsase activation was inhibited. In contrast to this, ZVAD was fully protective when apoptosis was triggered by the serpase inhibitor, Nalpha-tosyl-phenyl-chloromethyl ketone (TPCK). TPCK was not protective when administered to anti-CD95/ZVAD-treated A3.01 cells, indicating that TPCK does not possess anti-necrotic activity but fails to activate the necrotic death pathway. Our findings show (a) that caspase inhibition does not always protect apoptotic T cells from dying but merely activates a caspase-independent mode of cell death that results in necrosis and (b) that the caspase-inhibitor-induced shift from apoptotic to necrotic cell death is dependent on the cell type and the proapoptotic stimulus.
Journal of Cellular Biochemistry 05/2006; 97(6):1350-61. · 2.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Synthetic caspase inhibitors are known to block death receptor-induced apoptosis. We have reported previously that in addition to apoptosis inhibition, caspase inhibitors induce a switch from proapoptotic to proinflammatory signaling in T cells. This switch sensitizes TNF-R1 to trigger TNF-alpha-induced reactivation of HIV in latently infected T cells. Here we ponder the prospects of caspase inhibitors as a supplement in immune activation therapies (IAT) to achieve eradication of HIV from the latent virus reservoirs in HIV-infected patients.
Annals of the New York Academy of Sciences 01/2004; 1010:209-12. · 3.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: NFAT factors control HIV-1 transcription. We show here that, in addition to binding to two NF-kappaB/NFAT sites within the U3 HIV LTR, NFATc1 and NFATc2 bind to an NFAT site within the LTR's U5 region. Mutations in this site which abolish NFAT binding reduce the ability of NFATs to transactivate LTR-mediated transcription. Mutations in all three NFAT sites strongly interfered with LTR induction, but affected moderately the stimulatory effect of Tat.
Immunobiology 02/2003; 208(4):361-5. · 3.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: CD95 is a major apoptosis receptor that induces caspase activation and programmed cell death in susceptible cells. CD95-induced apoptosis can be blocked by peptidic caspase inhibitors such as benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or Ile-Glu-Thr-Asp-fluoromethyl ketone. Here we show that stimulation of CD95 in the presence of these inhibitors induces necrosis and expression of various proinflammatory cytokines in primary T lymphocytes, such as TNF-alpha, IFN-gamma and granulocyte/macrophage colony-stimulating factor. In the absence of caspase inhibition CD95 stimulation did not result in cytokine expression, indicating that this proinflammatory signaling pathway is suppressed by active caspases. Further analysis with A3.01 T cells revealed that the proinflammatory signaling activity of CD95 was mediated by MEK/ERK, p38 and NF-kappaB signaling pathways. These findings point to a pivotal role of caspases not only as mediators of apoptosis but also as enzymes that prevent proinflammatory signaling during CD95-induced apoptosis. Moreover, our findings may be useful for the development of novel pharmacological strategies.
European Journal of Immunology 10/2002; 32(9):2471-80. · 5.10 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Stimulation of tumor necrosis factor receptor 1 (TNF-R1) triggers both caspase-dependent and caspase-independent signaling activities. The caspase-dependent signaling pathway induces apoptotic cell death in susceptible cells, whereas the caspase-independent signaling cascade leads to activation of nuclear factor kappa B and induces antiapoptotic signaling activities. Stimulation of nuclear factor kappa B via TNF-R1 is known to activate human immunodeficiency virus (HIV) replication in infected cells. Here we show that the broad range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD) activates HIV replication in the chronically infected T-cell line ACH-2. Virus activation was caused by a sensitization of TNF-R1 toward endogenously produced tumor necrosis factor alpha (TNF-alpha). Neutralizing anti-TNF-alpha antibodies completely abolished the virus-inducing activity of ZVAD. Treatment of cells with TNF-alpha in the presence of ZVAD caused increased expression of TNF-alpha and induced enhanced virus replication. Activation of CD95, another member of the TNF receptor family, similarly triggered HIV replication, which was further enhanced in the presence of ZVAD. Our data show that caspase inhibitors sensitize both CD95 and TNF-R1 to mediate activation of HIV in latently infected cells. Activation of HIV replication in latent virus reservoirs is currently discussed as a therapeutic strategy to achieve eradication of HIV in patients treated with antiretroviral therapy. Our results point to a novel role for caspase inhibitors as activators of virus replication in vivo.
Journal of Biological Chemistry 06/2002; 277(18):15459-64. · 4.77 Impact Factor
-
Advances in Virus Research 02/2002; 58:157-202. · 3.97 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Infection with the human immunodeficiency virus (HIV) causes gradual depletion of CD4+ T helper lymphocytes and destruction of the lymphoid tissue, which ultimately leads to a fatal defect of the cellular immune system. Paramount to the understanding of the pathogenesis of HIV infection is to elucidate the mechanism which underlies the loss of T helper cells. Various ideas have been proposed in order to explain this issue. Several hypotheses have focused on the role of the envelope glycoprotein in this process. This review summarizes the data obtained and concepts proposed regarding the involvement of the HIV glycoprotein in the pathology of CD4+ T cell depletion.
Pathology & Oncology Research 02/1997; 3(1):62-67. · 1.37 Impact Factor
