Soluble TLR2 and 4 concentrations in cerebrospinal fluid in HIV/SIV-related neuropathological conditions.

HIV in the central nervous system (CNS) mainly infects microglial cells which are known to express toll-like receptors (TLRs). This paper aimed to study the role of soluble TLR2 (sTLR2), sTLR4, and other inflammatory markers in cerebrospinal fluid (CSF) in HIV/Simian immunodeficiency virus (SIV)-related neurological sequelae. We determined sTLR2 and sTLR4 levels in CSF and serum/plasma of SIV-infected rhesus macaques with and without neurological sequelae, as well as in HIV-infected patients with and without cognitive impairments and Alzheimer's disease (AD) patients and matched controls. CSF cytokines and chemokines levels were analyzed in macaques as markers of neuroinflammation, while neopterin and S100B CSF concentrations were measured in HIV-infected patients as microglial and astrocyte marker, respectively. We found detectable levels of sTLR2 and sTLR4 in CSF of macaques and humans. Furthermore, CSF sTLR2 and sTLR4 concentrations were higher in SIV-infected macaques with neurological sequelae compared to those without neurological complications (p = 0.0003 and p = 0.0006, respectively). CSF IL-8 and monocyte chemoattractant protein-1 (MCP-1) levels were elevated in macaques with neurological sequelae, and a positive correlation was found between CSF levels of sTLR2/4 and IL-8 and MCP-1. Also in humans, elevated CSF sTLR4 levels were found in HIV-infected patients with cognitive impairments compared to HIV-infected patients with normal cognition (p = 0.019). Unlike CSF S100B levels, neopterin correlated positively with sTLR2 and sTLR4. No difference was found in plasma and CSF sTLR2 and sTLR4 levels between AD patients and control subjects (p = 0.26). In conclusion, CSF sTLR2 and sTLR4 may play a role in HIV/SIV-related neuroinflammation and subsequent neuropathology.

Spontaneous and natural cytotoxicity receptor-mediated cytotoxicity are effector functions of distinct natural killer subsets in hepatitis C virus-infected chimpanzees.

In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV-infected chimpanzees. In HCV-infected chimpanzees increased spontaneous cytotoxicity was observed in CD94(high/dim) CD16(pos) and CD94(low) CD16(pos) subsets. By contrast, increased natural cytotoxicity receptor (NCR)- mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94(dim) CD16(neg) subset. Our findings suggest that spontaneous and NCR-mediated cytotoxicity are effector functions of distinct NK subsets in HCV-infected chimpanzees.

Whole blood stimulation with Toll-like receptor (TLR)-7/8 and TLR-9 agonists induces interleukin-12p40 expression in plasmacytoid dendritic cells in rhesus macaques but not in humans.

Macaques provide important animal models in biomedical research into infectious and chronic inflammatory disease. Therefore, a proper understanding of the similarities and differences in immune function between macaques and humans is needed for adequate interpretation of the data and translation to the human situation. Dendritic cells are important as key regulators of innate and adaptive immune responses. Using a new whole blood assay we investigated functional characteristics of blood plasmacytoid dendritic cells (pDC), myeloid dendritic cells (mDC) and monocytes in rhesus macaques by studying induction of activation markers and cytokine expression upon Toll-like receptor (TLR) stimulation. In a head-to-head comparison we observed that rhesus macaque venous blood contained relatively lower numbers of pDC than human venous blood, while mDC and monocytes were present at similar percentages. In contrast to humans, pDC in rhesus macaques expressed the interleukin (IL)-12p40 subunit in response to TLR-7/8 as well as TLR-9 stimulation. Expression of IL-12p40 was confirmed by using different monoclonal antibodies and by reverse transcription-polymerase chain reaction (RT-PCR). Both in humans and rhesus macaques, TLR-4 stimulation induced IL-12p40 expression in mDC and monocytes, but not in pDC. The data show that, in contrast to humans, pDC in macaques are able to express IL-12p40, which could have consequences for evaluation of human vaccine candidates and viral infection.

TRIM5 allelic polymorphism in macaque species/populations of different geographic origins: its impact on SIV vaccine studies.

Tripartite motif 5α (TRIM5α) is a potent antiretroviral immune factor present in the cytoplasm of cells of most tissue types. The rhesus macaque TRIM5 gene has been shown to display polymorphism, with different variants being divided into three groups (TRIM5(TFP), TRIM5(Q), and TRIM5(CypA)), which may have divergent retroviral effects on infection. Along with rhesus macaques, cynomolgus macaques are also used in simian immunodeficiency virus (SIV) infection studies. As a consequence, TRIM5 genotyping of these animals will contribute to interpreting the outcome of such studies. The present communication covers Burmese, Chinese, and a large cohort of Indian-origin rhesus macaques, and describes the first large cohort study on TRIM5 polymorphism in outbred cynomolgus macaques. We demonstrate the presence of the TRIM5(TFP) group in cynomolgus macaques. In addition, we have re-evaluated historical samples of rhesus macaques challenged with SIV(mac251), a virus that has been reported to be partially suppressed by particular rhesus macaque TRIM5 variants.

Adhesion of human B cells to follicular dendritic cells involves both the lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 and very late antigen 4/vascular cell adhesion molecule 1 pathways.

Presentation of antigen in the form of immune complexes to B lymphocytes by follicular dendritic cells (FDC) is considered to be a central step in the generation of memory B cells. During this process, which takes place in the microenvironment of the germinal center, B cells and FDC are in close physical contact. In the present study, we have explored the molecular basis of FDC-B cell interaction by using FDC and B cells derived from human tonsils. We found that FDC express high levels of the adhesion receptors intercellular adhesion molecule 1 (ICAM-1 [CD54]) and vascular cell adhesion molecule 1 (VCAM-1), while the B lymphocytes express lymphocyte function-associated antigen 1 (LFA-1 [CD11a/18]), very late antigen 4 (VLA-4 [CD49d], and CD44. Furthermore, we established that both the LFA-1/ICAM-1 and VLA-4/VCAM-1 adhesion pathways are involved in FDC-B lymphocyte binding, and therefore, these pathways might be essential in affinity selection of B cells and in the formation of B memory cells.

Activated human lymphocytes and aggressive non-Hodgkin's lymphomas express a homologue of the rat metastasis-associated variant of CD44.

A recently described splice variant of CD44 expressed in metastasizing cell lines of rat tumors, has been shown to confer metastatic potential to nonmetastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies raised against a bacterial fusion protein encoded by variant CD44 sequences, we have explored the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and on non-Hodgkin's lymphomas. Normal lymphohematopoietic cells express barely detectable low levels of variant CD44 glycoproteins, whereas T lymphocytes, upon activation by mitogen or antigen, transiently upregulate expression of specific CD44 variant glycoproteins. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that in the rat confers metastatic capability. It is interesting that overexpression of v6 was also found in several aggressive, but not low-grade, non-Hodgkin's lymphomas.

In-beam light absorption measurement of anthracyclines in cells with a flow-through system.

A flow-through cuvette in which cells attach as a monolayer to a quartz plate was developed for measurement of the light absorbance of anthracyclines in cells. Despite the drawback of a short path-length (of the order of the cell diameter), a dynamic flow-through set-up and baseline storage made it possible to measure intracellular absorbance and obtain spectral data for daunomycin and carminomycin. Stopping the flow and allowing the drug to equilibrate between medium and cells led to a 20% decrease of molar light absorption of cellular anthracycline, which permitted measurement of the total cellular concentration. Furthermore, accumulation and efflux kinetics were determined for H35 rat hepatoma cells. On the basis of the reported formation constant of the iron-complex of carminomycin, which is of the order of 10(34), we expected to find this complex within the cells. However, the spectrum of cellular drug did not show absorbance bands characteristic of the complex. A red shift and hypochromism were found in the daunomycin spectrum after intracellular binding, which corresponds with the spectral change observed after intercalation of daunomycin into DNA.

Decreased expression of IL-2 in central and effector CD4 memory cells during progression to AIDS in rhesus macaques.

OBJECTIVE: HIV-1 infection in humans has been reported to lead to a shift in the cytokine balance, with a relative decrease in T helper 1 type cytokines, especially IL-2. On the basis of the expression of CD45RA, in combination with homing markers CD62L or alpha4beta7, T helper cells can be sub-divided into naive, activated naive, central memory and effector memory cells as well as gut-homing subpopulations. In addition, each subset may have the potential to express distinct cytokines. At present it is unclear whether the changes in cytokine expression observed in HIV-1-infected individuals are secondary to changes within the composition of CD4 T cell subsets or are caused by changes in cytokine expression within each subset. MATERIALS AND METHODS: A new technique was developed to detect cytokine expression in phorbol 12-myristate 13-acetate/ionomycin-activated CD62L and alpha4beta7-expressing CD4 T cell subsets, using the protease inhibitor KD-IX-73-4. RESULTS: In SIV-infected macaques that develop AIDS a marked decrease in IL-2 expression was found within central, effector, or gut-homing memory cell subsets, whereas the expression of IL-2 in naive T cell subsets remained unaffected. This reduced IL-2 expression by memory cells and not a loss of the frequency of CD4 memory cells accounted for the reduced expression of IL-2 by CD4 T cells during SIV infection. CONCLUSION: As defined by the cell surface markers utilized, it appears that progression to AIDS is associated with functional impairment of memory cells, but not changes in lymphocyte circulation patterns.

CD44 antibody stimulates adhesion of peripheral blood T cells to keratinocytes through the leukocyte function-associated antigen-1/intercellular adhesion molecule-1 pathway.

Close contact between T lymphocytes and keratinocytes is an important feature of many inflammatory skin diseases. In vitro studies showed that stimulation of keratinocytes with interferon-gamma or tumor necrosis factor-alpha and of T cells with phorbol esters results in a leukocyte function-associated antigen (LFA)-1/intercellular adhesion molecule (ICAM)-1-mediated adhesion. The present study was performed to investigate the role of the CD44 molecule in keratinocyte/T-cell binding. The CD44 class of lymphocyte adhesion receptors is involved in lymphocyte binding to high endothelial venules and to extracellular matrix compounds and is therefore important in lymphocyte recirculation and homing. Moreover, CD44 can act as a co-stimulating signal in T-cell activation and promotes homotypic adhesion of in vitro cultured CD3-stimulated T cells. Using a cell adhesion assay a sixfold increase in T-cell/keratinocyte adhesion was found after pre-incubating the T cells with anti-CD44. This increased adhesion was found to require an intact cytoskeleton, to be energy and magnesium dependent, and could be completely inhibited by anti-LFA-1 and anti-ICAM-1. Pretreatment of T cells with hyaluronic acid, a ligand for CD44 and an extracellular matrix compound in the dermis and epidermis, did not affect T-cell/keratinocyte adhesion.

Differential cytotoxic T-lymphocyte (CTL) responses in HIV-1 immunised sibling chimpanzees with shared MHC haplotypes.

Cell mediated immune responses to HIV-1 and CTL responses in particular differ dramatically in infected individuals. This may largely be influenced by the immunogenetic differences of different individuals such as those encoded by the MHC. These differences may be difficult to dissect due to the immunosuppressive nature of HIV-1 infection itself. In order to reduce the variables associated with effects of the virus, one recombinant viral antigen was chosen from a particular HIV-1 variant (rgp120 of the clinical isolate HIV-1w6.1D). To minimise differences between outbred hosts, we chose two sibling chimpanzees from which the family pedigree and genetic segregation with respect to polymorphic MHC molecules was known. Immunisation induced strong antigen specific antibody and T-helper immune responses. The magnitude and persistence of the humoral and T-helper immune responses were comparable in both chimpanzees. However, CTL responses were only observed in one sibling. These responses were subsequently mapped to several distinct epitopes. The CTL response to the immunodominant epitope was found to be presented in the context of a MHC molecule which was shared by both siblings. The absence of a CTL response in the other sibling is not yet understood, but could not be attributed to MHC alleles that were not shared by these two chimpanzees. These findings suggest that other polymorphic immunoregulatory mechanisms such as those involved in antigen processing and presentation influence host CTL responses to HIV-1.

Rhesus macaque and chimpanzee DC-SIGN act as HIV/SIV gp120 trans-receptors, similar to human DC-SIGN.

Dendritic cells (DC) have been implicated in the pathogenesis of both human and simian immunodeficiency viruses (HIV and SIV, respectively). The DC-specific HIV-1 trans-receptor DC-SIGN is thought to be essential for viral dissemination by DC. Abundant expression in lymphoid tissues also implies a function for DC-SIGN in chronic HIV-1 infections, in facilitating persistent infection of T cells. We have therefore isolated the rhesus macaque and chimpanzee homologues of DC-SIGN to investigate their function in a primate model. Both rhesus macaque and chimpanzee DC-SIGN are highly similar to the human homologue. Three monoclonal antibodies against human DC-SIGN, AZN-D1, -D2 and -D3, cross-react with rhesus macaque DC-SIGN, whereas AZN-D2 does not cross-react with chimpanzee DC-SIGN. The primate homologues are abundantly expressed in lymphoid tissues such as lymph nodes, as well as in mucosal tissues involved in sexual transmission of HIV-1, and are functionally similar to human DC-SIGN. They have a high affinity for the immunological ligands of DC-SIGN: ICAM-2 and -3. Moreover, both homologues bind the HIV-1 envelope glycoprotein gp120 and therefore can act as a HIV-1 trans-receptor in the same way as human DC-SIGN. These data demonstrate that primate models are suitable to further dissect the role of DC-SIGN in the transmission and pathogenesis of infection with immunodeficiency viruses.

HIV-1 vaccine-induced immune responses which correlate with protection from SHIV infection: compiled preclinical efficacy data from trials with ten different HIV-1 vaccine candidates.

The specific immune mechanisms necessary and/or sufficient to elicit HIV-vaccine protection remain undefined. Utilising the SHIV rhesus macaque model the immunogenicity as well as the efficacy of ten different HIV-1 vaccine candidates was evaluated. Comparison of the immune responses induced, with the ability of the vaccine to protect from SHIV infection provided a means to determine which type of immune responses were necessary for protection. Vaccine candidates included VLPs, DNA, subunit protein with novel adjuvant formulations, ISCOMs and pox-virus vectors. Protection from SHIV infection was achieved in approximately half of the animals which received a primary intravenous cell-free challenge. The presence of CTL in the absence of other effector responses did not correlate with protection from this route and type of challenge. Virus neutralising antibodies (Nab) appeared to be necessary but alone were insufficient for protection. If Ag-specific IFN-gamma and/or IL-4 as well as lymphoproliferative (LP) responses were found with the lack of a detectable IL-2 response, then protection was not observed. Immunity correlated with the magnitude of Nab responses, beta-chemokines and as well as balanced, qualitative T-helper responses.

Changes in dendritic cell subsets in the lymph nodes of rhesus macaques after application of glucocorticoids.

Corticosteroids are used therapeutically as potent immunosuppressive and antiinflammatory drugs for a broad spectrum of diseases. Although corticosteroids are known to inhibit the production of many cytokines in activated T cells, there is also evidence for increases in IL-4 and in some cases IFNgamma production. These conflicting results may be caused by contrary effects of corticosteroids on different cell types involved in immune regulation, for instance antigen presenting cells (APC) versus T cells. In the present study we simultaneously investigated the effect of local as well as systemic application of glucocorticoids (GCC) on the phenotype of APC in the skin as well as the lymph nodes in a model primate species, the rhesus macaque. Using a range of APC markers, including CD68, HAM56, HLA-DR, CD1a, p55, RFD-1, and costimulatory molecules CD40, CD80, and CD86 we document the close phenotypic resemblance of rhesus and human APC. We noted that topical GCC treatment specifically lead to a marked decrease in the number of CD1a expressing cells in the draining lymph nodes. However, the number of CD1a positive cells in peripheral lymph nodes was not affected by systemic GCC treatment. Importantly, by performing double staining of CD1a with RFD-1 we observed a shift in the expression pattern of these dendritic cell markers in the lymph nodes, with an increase in the number of RFD-1 single positive cells relative to CD1a single positive and CD1a/RFD-1 double positive cells. These findings suggest that GCC treatment results in the presence of phenotypically more mature APC.

The relative resistance of HIV type 1-infected chimpanzees to AIDS correlates with the maintenance of follicular architecture and the absence of infiltration by CD8+ cytotoxic T lymphocytes.

Lymphoid tissues are the focus of critical events in HIV pathogenesis. Persistent and high levels of virus production, extensive trapping of virus particles in germinal centers, and progressive degenerative changes in lymph node architecture are characteristics of progressive HIV-1 infection. Infiltrates of granzyme B- and TIA-expressing CD8+ "cytotoxic" T lymphocytes (CTLs) precede involution of germinal centers in humans who develop AIDS. Similar to humans, HIV-1 infection in chimpanzees is active and persistent. However, in contrast to humans, they remain relatively resistant to AIDS. Lymph node biopsies from chimpanzees infected with HIV-1 or a related chimpanzee lentivirus were studied for the level and pattern of virus expression, changes in lymphoid architecture, CD8+ T cell infiltrates and the presence or absence of CTL markers. In stark contrast to HIV-1-infected humans, lymph nodes from infected chimpanzees had little virus deposition in germinal centers and a paucity of virus-expressing cells. Although some of the lymph nodes examined from infected animals had moderate follicular hyperplasia with infiltrating CD8+ T cells, none had evidence of follicular fragmentation. Most importantly, in marked contrast to infected humans, CD8+ T cells infiltrating the germinal center were negative for the CTL marker granzyme B. This evidence suggests that the infiltration of CD8+ CTLs into the germinal centers of lymph nodes may be a key determinant in AIDS pathogenesis.

Expression of granzyme B by cytotoxic T lymphocytes in the lymph nodes of HIV-infected patients.

During HIV infection the architecture of secondary lymphoid tissues is severely disrupted. In particular the germinal centers, which play a key role in the orchestration of the secondary immune response, undergo gross phenotypic alterations, leading to a complete destruction of the germinal center microenvironment. The precise mechanisms responsible for the lymphoid tissue destruction in HIV infection are still unknown. However, the large influx of CD8+ T lymphocytes suggests an important role for T cell-mediated cytotoxicity. To establish whether the infiltrating CD8+ T lymphocytes are killing competent, we investigated the expression of granzyme B, which is known to be present in the cytotoxic granules of NK cells and "activated" CTLs with cytolytic potential. We observed a 20-fold increase in the percentage of granzyme B-expressing CD8+ T cells in both the germinal center and the interfollicular areas in HIV patients relative to HIV-negative controls. This increase was present in patients with early-stage disease (i.e., absolute CD4+ T cell count > 500/microliters) as well as in patients with intermediate and late-stage disease. Thus, from relatively early stages of HIV infection onward large numbers of killing competent T lymphocytes are present in the lymphoid tissues, a finding that supports the notion that CTL act as mediators of destruction of immune function during HIV infection.

CD27-CD70 interaction: unravelling its implication in normal and neoplastic B-cell growth.

Members of the Tumour Necrosis Factor-Receptor (TNFR) family play an essential role in the control of lymphoid cell growth and differentiation. The ligand of one of its lymphoid-specific members, CD27, was recently characterized as CD70, a type II transmembrane molecule with homology to TNF that is expressed on activated T and B cells. Ligation of CD27 by its natural ligand generates a potent costimulatory signal for cytokine production and proliferation of activated T cells. In contrast to normal B cells, where CD27 expression is confined to germinal centre cells and to a small subset of circulating B lymphocytes, CD27 expression is found on a large array of distinct B-cell neoplasia. Here, we review recent data on the expression and function of TNFR family members on normal and malignant lymphocytes and propose a role for CD27-CD70 interaction in B-cell development.

Apoptotic tubular cell death during acute renal allograft rejection.

Tubular cells are important targets during acute renal allograft rejection and induction of apoptosis might be a mechanism of tubular cell destruction. Susceptibility to induction of apoptosis is regulated by the homologous Bcl-2 and Bax proteins. Expression of Bcl-2 and Bax is regulated by p53, which down-regulates expression of Bcl-2, while simultaneously up-regulating expression of Bax. We studied apoptotic tubular cell death in 10 renal allograft biopsies from transplant recipients with acute rejection by in situ end-labelling and the DNA-binding fluorochrome propidium iodide. Tubular expression of p53, Bcl-2 and Bax was studies by immunohistochemistry. Five renal allograft biopsies from transplant recipients with uncomplicated clinical course and histologically normal renal tissue present in nephrectomy specimens from 4 patients with renal adenocarcinoma served as control specimens. Apoptotic cells and apoptotic bodies were detected in tubular epithelia and tubular lumina in 9 out of 10 acute rejection biopsies. In control renal tissue, apoptotic cells were detected in 1 biopsy only. Compared to control renal tissue, acute renal allograft rejection was, furthermore, associated with a shift in the ratio of Bcl-2 to Bax in favour of Bax in tubular epithelia and increased expression of p53 in tubular nuclei. These observations demonstrate that apoptosis contributes in part to tubular cell destruction during acute renal allograft rejection. In accordance, the shift in the ratio of Bcl-2 to Bax in favour of Bax indicates increased susceptibility of tubular epithelia to induction of apoptosis. The expression of p53 in tubular nuclei during acute renal allograft rejection indicates the presence of damaged DNA, which can be important in initiation of part of the observed apoptosis. These findings elucidate part of the mechanisms controlling apoptotic tubular cell death during acute renal allograft rejection.

Comparison of intranasal with targeted lymph node immunization using PR8-Flu ISCOM adjuvanted HIV antigens in macaques.

The rapidly spreading HIV epidemic requires a vaccine that elicits potent mucosal immunity to halt or slow transmission. Induction of these responses will depend on the use of appropriate adjuvants and targeting of the mucosal immune system. Previously, immune stimulating complexes (ISCOM) have shown great potency as adjuvant in the induction of mucosal responses in mice and systemic responses in non-human primates. In this study, HIV formulated in PR8-Flu ISCOM adjuvant was applied to immunize rhesus macaques against HIV; targeting the mucosa either via intranasal (IN) application or via targeted lymph node immunization (TLNI). While, strong systemic, HIV specific, cytokine, lymphoproliferative, and antibody responses were induced via the TLNI route, the IN application generated only low responses. Furthermore, all four animals immunized via TLNI developed vaginal IgA antibodies against gp120. In conclusion, in contrast to what has been demonstrated in mice, the IN application of PR8-Flu ISCOM did not induce strong immune responses in rhesus macaques unlike those immunized by the TLNI route.

Cellular interactions in the germinal center: role of adhesion receptors and significance for the pathogenesis of AIDS and malignant lymphoma.

The germinal center forms a specialized microenvironment that is thought to play a key role in the induction of antibody synthesis, affinity maturation of B cells, isotype switching, and memory B-cell formation. Moreover, the germinal center may also be involved in the maintenance of T-cell memory. In this paper we focus on the role of adhesion receptors in cellular interactions in the germinal center, and discuss evidence indicating that these molecules play an important role in regulating B-cell activation and differentiation. Furthermore, we discuss two important diseases involving the germinal center, i.e., HIV infection and malignant lymphoma. In HIV infection, destruction of the FDC network may explain the selective loss of memory cells observed in otherwise asymptomatic patients and is likely to represent a major pathway leading to AIDS. In follicular lymphoma, escape from physiological apoptosis in the germinal center by overexpression of Bcl-2 appears be a major pathogenetic pathway.