The expression of activation antigens, namely CD25, CD69, CD71, and HLA‐DR on T cells from 15 healthy individuals stimulated with different mitogens and specific antigens was evaluated by immunofluorescence assay and flow cytometric analysis and compared with cell proliferation as a function of [3H]thymidine incorporation. CD69 was the earliest expressed antigen on stimulated cells, while HLA‐DR was the latest. Regardless of the stimulus used, lymphocytes expressing CD25 and CD71 were always more numerous than cells expressing CD69 and HLA‐DR. Variations in the proportion of CD4+ and CD8+ T cells expressing each activation marker were observed with different antigenic stimuli. The expression of each activation marker showed overall agreement with the [3H]thymidine incorporation assay in discriminating between positive and negative immune response. However, no correlation was observed between the percentage of CD25‐, CD69‐, CD71‐, and HLA‐DR‐positive T cells and the amount of [3H]thymidine incorporation. Moreover, low doses of mitogens and antigens as well as short time of stimulation were sufficient to induce T cells to express activation antigens but not to proliferate. Our data show that results obtained by flow cytometry and [3H]thymidine incorporation may differ qualitatively, at least under certain conditions; this suggests that the 2 assays are complementary, and when combined, may gives a clearer understanding of events leading to efficient cell‐mediated immune response. Cytometry 27:71–76, 1997. © 1997 Wiley‐Liss, Inc.
Although in decline after successful anti-HIV therapy, B-cell lymphomas are still elevated in HIV-1-seropositive (HIV+) persons, and the mechanisms are obscure. The HIV-1 matrix protein p17 persists in germinal centers long after HIV-1 drug suppression, and some p17 variants (vp17s) activate Akt signaling and promote growth of transformed B cells. Here we show that vp17s derived from four of five non-Hodgkin lymphoma (NHL) tissues from HIV+ subjects display potent B-cell growth-promoting activity. They are characterized by amino acid insertions at position 117-118 (Ala-Ala) or 125-126 (Gly-Asn or Gly-Gln-Ala-Asn-Gln-Asn) among some other mutations throughout the sequence. Identical dominant vp17s are found in both tumor and plasma. Three of seven plasma samples from an independent set of NHL cases manifested multiple Ala insertions at position 117-118, and one with the Ala-Ala profile also promoted B-cell growth and activated Akt signaling. Ultradeep pyrosequencing showed that vp17s with C-terminal insertions are more frequently detected in plasma of HIV+ subjects with than without NHL. Insertion of Ala-Ala at position 117-118 into reference p17 (refp17) was sufficient to confer B-cell growth-promoting activity. In contrast, refp17 bearing the Gly-Asn insertion at position 125-126 did not, suggesting that mutations not restricted to the C terminus can also account for this activity. Biophysical analysis revealed that the Ala-Ala insertion mutant is destabilized compared with refp17, whereas the Gly-Asn form is stabilized. This finding provides an avenue for further exploration of structure function relationships and new treatment strategies in combating HIV-1-related NHL.non-Hodgkin lymphoma | HIV-1 matrix protein p17 | AIDS | p17 variants | B-cell clonogenicity
Vascular diseases supported by aberrant angiogenesis have increased incidence in HIV-1-infected patients. Several data suggest that endothelium dysfunction relies on action of HIV-1 proteins rather than on a direct effect of the virus itself. The HIV-1 matrix protein p17 is known to deregulate the biological activity of different immune cells. Recently, p17 was found to mimic IL-8 chemokine activity by binding to the IL-8 receptor CXCR1. Here we show that p17 binds with high affinity to CXCR2, a CXCR1-related receptor, and promotes the formation of capillary-like structures on human endothelial cells (ECs) by interacting with both CXCR1 and CXCR2 expressed on the EC surface. ERK signaling via Akt was defined as the pathway responsible for p17-induced tube formation. Ex vivo and in vivo experimental models confirmed the provasculogenic activity of p17, which was comparable to that induced by VEGF-A. The hypothesis of a major role for p17 in HIV-1-induced aberrant angiogenesis is enforced by the finding that p17 is detected, as a single protein, in blood vessels of HIV-1-patients and in particular in the nucleus of ECs. Localization of p17 in the nucleus of ECs was evidenced also in in vitro experiments, suggesting the internalization of exogenous p17 in ECs by mechanisms of receptormediated endocytosis. Recognizing p17 interaction with CXCR1 and CXCR2 as the key event in sustaining EC aberrant angiogenesis could help us to identify new treatment strategies in combating AIDSrelated vascular diseases. extracellular viral proteins | virokine | Akt-mediated ERK pathway | vasculogenic assays | surface plasmon resonance A ngiogenesis is a physiological process requiring growth of new blood vessels from preexisting vessels. This process involves coordinated endothelial cell (EC) proliferation, invasion, migration, and tube formation (1). When new vessels are required, proangiogenic factors are produced, whereas restoration of physiological conditions is achieved by producing inhibitors of angiogenesis and vessel stabilization factors. Breakdown of the tight regulated angiogenic balance leads to dysfunctional endothelium, abnormal angiogenesis, and vascular diseases.
Inducible nitric oxide synthase (iNOS) is required in immune response against infections and is involved in granuloma formation in animals; in murine macrophages , iNOS is induced by lipopolysaccharide and interferon-␥.In contrast , the role of iNOS in human immune response against infections is still questioned , and its expression in granulomas is poorly investigated. Using Western blotting and immunohistochemistry , we investigated iNOS expression in human lymph nodes with nonspecific reactions and in tissues containing granulomas caused by mycobacteria , Toxoplasma , Cryptococcus neoformans , Leishmania , Bartonella , noninfectious granulomas (sarcoidosis , foreign body) , and other hystiocitic reactions (Kikuchi's disease , Omenn syndrome). iNOS was undetectable in nonspecific reactive lymphadenitis , foreign-body granulomas , and Omenn syndrome , whereas it was strongly expressed in infectious granulomas , sarcoidosis , and Kikuchi's diseases. Immunohistochemistry demonstrated that iNOS was selectively expressed by the epithelioid and multinucleated giant cells within the granulomas. Use of an antinitrotyrosine antibody , recognizing nitrosilated amino acid residues derived from nitric oxide production , revealed a consistent positivity within the cells expressing iNOS , thus suggesting that iNOS is functionally active. Detection of cytokines by reverse transcriptase-polymerase chain reaction demonstrated that tissues that were positive for iNOS , also expressed the Th1-type cytokine interferon-␥ mRNA , but not the Th2-type cytokine interleukin-4. 1 At least three distinct isoforms of NOS, encoded by separate genes, are expressed in mammalian cells. Two of them are constitutively expressed in brain neurons (NOS1 or nNOS) or in endothelial cells (NOS3 or eNOS).2,3 The third isoform, termed inducible NOS (iNOS or NOS2) is expressed only on stimulation in macrophages and hepatocytes. In vitro, expression of the enzyme is associated with NO production and thereby extracellular release of NO metabolites (eg, nitrites, nitrates, peroxynitrites).4,5 NO release results into tyrosine residues nitrosation, and nitrotyrosine detection has been used as an indicator of iNOS expression and NO function. 6 -9
Purified recombinant HIV-1 p17 matrix protein significantly increased HIV-1 replication in preactivated peripheral blood mononuclear cell cultures obtained from healthy donors. Because HIV-1 infection and replication is related to cell activation and differentiation status, in the present study, we investigated the role played by p17 during the process of T cell stimulation. Using freshly isolated peripheral blood mononuclear cells, we demonstrate that p17 was able to enhance levels of tumor necrosis factor ␣ and IFN-␥ released from cells stimulated by IL-2. IL-4 was found to downregulate IFN-␥ and tumor necrosis factor ␣, and p17 restored the ability of cells to produce both cytokines. The property of p17 to increase production of proinflammatory cytokines could be a mechanism exploited by the virus to create a more suitable environment for HIV-1 infection and replication. Our data show that p17 exerts its biological activity after binding to a specific cellular receptor expressed on activated T lymphocytes. The functional p17 epitope involved in receptor binding was found to be located at the NH 2-terminal region of viral protein. Immunization of BALB͞c mice with a 14-aa synthetic peptide representative of the HIV-1 p17 functional region (SGGELDRWEKIRLR) resulted in the development of p17 neutralizing antibodies capable of blocking the interaction between p17 and its cellular receptor. Our results define a role for p17 in HIV-1 pathogenesis and contribute to our understanding of the molecular mechanism of HIV-1 infection and the development of additional antiviral therapeutic strategies.
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