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Biomedical subjects

D N Hart

Publications and source records attributed to D N Hart.

At least 73 records · Page 4Linked to original sources
PubMed1995

Leukocyte adhesion molecules as a cofactor in AIDS: basic science and pilot study.

It is well known that the AIDS pandemic is a consequence of pandemic HIV infection. However, Koch's postulates are not satisfied for two reasons: 1) AIDS cannot be experimentally produced in animals susceptible to HIV infection and 2) some people have AIDS (idiopathic CD4+ T lymphocytopenia) in the absence of HIV infection. It follows that there is a human immunologic cofactor (HIC) that causes AIDS when certain other conditions are satisfied, and the most common of these other conditions (but not the only one) is HIV infection. Results from microbiology make leukocyte adhesion molecules a good candidate for the HIC. We have tested this hypothesis with a pilot study in which a small number of patients with HIV disease were infused with a monoclonal mouse antibody (MmAb) directed against an LFA-1 adhesion epitope, and then with F(ab) and F(ab)2' fragments that bind to the same epitope but are nonimmunogenic. Both agents reduced peripheral viral burden significantly but fragments were more effective in this respect than the MmAb due to the mitogenic properties of the latter. For the same reason, only the MmAb were highly effective in raising circulating levels of single and double-marked CD4+ T lymphocytes, with a correlated resolution of cutaneous anergy.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Dendritic cell presentation of PPD and 19 kDa protein of Mycobacterium tuberculosis and emergent T helper cell phenotype.

Protection against infection with Mycobacterium tuberculosis is preferentially associated with the development of the T helper 1 subset, IFN-gamma production and a cell-mediated response, rather than with T helper 2 cells, 4 (IL-4) and antibody production. The type of APC interacting with T cells responsive to mycobacterial peptides may influence which of these responses predominates. This investigation focuses on the role of dendritic cells (DC) because they are the most potent APC in both primary and recall immune responses. Our results show that splenic DC-enriched suspensions prepared from C57BL/6 mice and pulsed with either purified protein derivative (PPD) or the immunodominant 19 kDa protein from M. tuberculosis, can activate antigen-primed T cells in vitro, whereas spleen cell suspensions depleted of DC cannot. DC pulsed with PPD or 19 kDa antigen are able to prime naive T cells in vivo. Supernatants collected from cultures containing T cells from mice injected with PPD-pulsed DC and then challenged in vitro with PPD-pulsed DC were found to contain more IL-2 and IFN-gamma than those from control mice which received either DC or PPD alone. No such antigen-specific IFN-gamma response occurred if DC pulsed with 19 kDa were used in place of PPD-pulsed DC. IL-4 was not detected in any of the culture supernatants. We conclude that DC can induce production of cytokines associated with a protective immune response when presenting peptides derived from heterogeneous mycobacterial antigens but not when exposed to the single 19 kDa immunodominant protein.

Animals↗
PubMed1995

Dendritic cells in synovial fluid of chronic inflammatory arthritis lack CD80 surface expression.

Dendritic cells (DC) act as potent primary antigen-presenting cells in many immune responses and therefore may have a role in the initiation and perpetuation of the synovial inflammation in chronic inflammatory arthritis. To examine their function, it is important to isolate fresh DC from arthritic joints without aberrant activation. We have developed a technique using minimal cell manipulation to isolate DC from the synovial fluid of chronic arthritic patients. Using this method, DC were shown to be potent allostimulatory cells, with 63-90% of cells lacking lineage-specific markers (lin-), but positive for MHC class II molecules. Two morphologically distinct populations of these cells were identified in 10 out of 13 DC preparations. Both populations expressed CD40, intercellular adhesion molecule-1 (ICAM-1), ICAM-2, ICAM-3 and leucocyte function associated antigen-3 (LFA-3), but the predominant population, which was larger and more typical of cultured blood DC, had a higher density of these antigens compared with the minor population, which were smaller and morphologically similar to lymphocytes. Two new MoAbs which label activated human blood DC, HB15 (CD83) and CMRF-44, were tested. CD83 labelled very weakly or not at all, whereas CMRF-44 was positive on the larger cells only. Likewise, the costimulator molecule, B7/BB1 (CD80), was not detected on the surface of either synovial lin- cell population, reverse transcriptase polymerase chain reaction (RT-PCR) showed little or no CD80 mRNA, and no binding of the CTLA-4Ig fusion protein was found. These results suggest that synovial DC are not, despite the inflammatory environment, in a fully activated state.

Arthritis↗
PubMed1995

Acute adrenal insufficiency: a new presentation of Castleman's disease.

We report the case of a 35-year-old patient who presented with acute adrenal insufficiency, then developed fever, hypoalbuminuria, anasarca, thrombocytopaenia and anaemia. Lymphadenopathy appeared later with microscopic features typical of Castleman's disease. Clinical remission followed treatment with intravenous immunoglobulin. Circulating interleukin-6 levels were elevated initially but were normal after immunoglobulin therapy. We surmise that high circulating levels of interleukin-6 (and ACTH) may have induced haemorrhagic necrosis of the adrenal glands and accounted for the constitutional symptoms.

Acute Disease↗
PubMed1995

Antigen-pulsed, interleukin-4-treated B cells activate primed T cells in vitro but not naive T cells in vivo.

The ability of B cells to act as effective antigen-presenting cells is a source of debate which centres on the degree of activation of either B cells or T cells. We have investigated whether B cells treated with interleukin 4 (IL-4) can express the two signals required to activate T cells: MHC Class 2/antigenic peptide complexes(signal 1) and the costimulatory molecules B7-1 and B7-2 (signal 2). We have also determined whether these cells could activate antigen-experienced T cells in vitro and whether they could prime naive T cells in vivo. We found that B cells expressed abundant MHC Class 2 molecules and moderate levels of B7-2 after 24 h culture in IL-4 with or without purified protein derivative (PPD) but B7-1 was not detectable. PPD-pulsed, IL-4 treated B cells induced antigen-experienced T cells to proliferate in vitro but these cells failed to prime naive T cells in vivo when injected into mice. We conclude that signals, in addition to those induced with IL-4, are required for B cells to initiate an immune response to antigen.

Animals↗
PubMed1995

The phenotype of freshly isolated and cultured human bone marrow allostimulatory cells: possible heterogeneity in bone marrow dendritic cell populations.

Putative dendritic cells (DC) and their precursors have been obtained from human bone marrow but their origin and relationship to other myeloid cells remains obscure. A minor bone marrow mononuclear cell (BMMC) population, which contains the most potent allostimulatory cells and lacks mature cell lineage markers (CD3, CD11b, CD14, CD15, CD16, CD19, CD57 and glycophorin A; lineage-negative) was enriched by immunoselection. These preparations, which contain cells with similar characteristics to freshly isolated human blood DC, were further subdivided by serial fluorescent-activated cell sorting (FACS). Potent allostimulatory cells were detected in the CD34, CD33 and CD4 positive and negative subpopulations. Cells with putative DC morphology were present in both the CD33 and CD4 positive and negative fractions. No significant CD13 or Thy-1 staining was seen in the lineage-negative population. In vitro culture of lineage-negative BMMC for 7 days in conditioned medium resulted in a up to fivefold expansion of cells and generated many lineage-positive progeny. This lineage-positive population was as allostimulatory as the negative progeny. Likewise, the CD14-positive and the CD14-negative cell progeny were equally allostimulatory. In contrast, the freshly isolated lineage-positive BMMC (containing CD14-positive monocytes) remained poor stimulators of the mixed lymphocyte reaction (MLR), even after culture in the presence of cytokines. These data suggest that there are at least two phenotypically diverse forms of potent allostimulatory cells in the lineage-negative fraction of human BM, at least some of which express the early haemopoietic precursor antigens CD34 or CD33. Some of these precursors generate CD14-positive allostimulatory cells upon in vitro culture, suggesting an intimate link between DC ontogeny and myeloid differentiation.

Antigens, CD↗
PubMed1995

Monocyte-macrophage antigen expression on chondrocytes.

OBJECTIVE: To characterize chondrocytes in normal and arthritic joints and compare their phenotype to that of synovial macrophages present in rheumatoid joints. METHODS: Using an immunoperoxidase staining technique, we examined the presence and distribution of a number of leukocyte activation and differentiation antigens on samples of cartilage obtained from resected joints of normal controls and subjects with rheumatoid arthritis or osteoarthritis. RESULTS: Chondrocytes in each group were CD14+, CD68+, Thy-1+, CD11a+, CD18+, MAX.3-, and MAX.24-. Staining was variable for MAX.1 and CD45. HLA-DR and CD71 were expressed only on cells located in the superficial layer of rheumatoid cartilage. We found lower levels of expression of CD14 on chondrocytes in arthritic joints, whereas CD58 was expressed at higher levels. Surface expression of CD14 was confirmed on normal chondrocytes using flow cytometry and further supported by the detection of CD14 mRNA by polymerase chain reaction. CONCLUSION: Our findings demonstrate that chondrocytes express several antigens that are also found on monocytes and macrophages.

Arthritis, Rheumatoid↗
PubMed1995

Role of tumour necrosis factor-alpha in dendritic cell-mediated primary mixed leucocyte reactions.

Tumour necrosis factor (TNF alpha) is a major inflammatory cytokine with potentiating effects on specific immune responses, including graft-versus-host disease. This study examined the contribution of TNF alpha to dendritic cell (DC)-mediated primary allogeneic T lymphocyte responses. Purified blood DC were shown to produce minimal amounts of TNF alpha mRNA but no significant TNF biological activity or secreted TNF alpha as measured by ELISA. Amplification of DC mRNA by PCR using oligonucleotide primers to CD120a (TNFRI, p55) and CD120b (TNFRII, p75) and probing with specific internal oligonucleotides, suggested that DC express the CD120b but little if any CD120a. These results were confirmed using monoclonal antibodies to the TNF receptors. Polyclonal antiserum specific for TNF alpha blocked the blood DC-stimulated allogeneic mixed leucocyte reaction (MLR). The addition of TNF alpha to suboptimal MLRs (limited DC stimulators), increased the proliferation of responding T lymphocytes. Having confirmed that T lymphocytes produce TNF alpha and express CD120b after stimulation, we sought to clarify whether the contributing effect of TNF alpha to the allogeneic MLR resulted from a TNF alpha-mediated signal stimulating DC activity, or as a result of autocrine stimulation of T lymphocytes. Pre-incubation of DC with TNF alpha did not increase DC stimulatory capacity and late addition of anti-TNF serum (up to 72 h) still had a significant inhibitory effect on the MLR. We conclude that TNF alpha is probably not involved in the initial DC-T lymphocyte interaction, but acts as an autocrine growth factor for DC induced T lymphocyte proliferation.

Antigens, CD↗
PubMed1994

Characterization of a novel leucocyte activation antigen recognized by the antibody CMRF-37.

We describe a novel activation antigen recognized by the monoclonal antibody CMRF-37, which is absent or only weakly expressed on resting cells but is rapidly induced on T cells, B cells and monocytes following stimulation. Its kinetics of expression (12-24 h after the addition of the inductive signal) indicate that it is an early activation antigen. The cellular distribution and expression kinetics of the CMRF-37 antigen differ from other known activation antigens and as such should be a useful marker of human leucocyte activation.

Animals↗
PubMed1994

Co-expression of the CD45RA and CD45RO antigens on T lymphocytes in chronic arthritis.

The site of T lymphocyte activation in chronic arthritis is unknown. Peripheral blood (PB) lymphocytes from chronic arthritis patients are in a 'naive' or non-activated state, as defined by expression of the CD45RA antigen and lack of HLA class II expression. In contrast, most synovial fluid (SF) T lymphocytes express a 'memory' or activated phenotype, as defined by the CD45RO antigen and high HLA class II expression. Following stimulation, naive cells lose CD45RA and gain CD45RO expression to become memory cells with a transitional stage of dual CD45RA, CD45RO antigen expression. To localize where this change in phenotype occurs we used dual colour immunofluorescence labelling to compare the percentage of dual CD45RA, CD45RO-positive T lymphocytes in PB and SF from chronic arthritic patients and from normal PB, assuming this population would be increased at the primary site of T lymphocyte activation. Expression of the intermediate and late activation marker, HLA-DR, was also analysed using dual colour immunofluorescence labelling. The percentage of dual positive T lymphocytes was similar between arthritic PB, SF, and normal PB, as was the density of both CD45RA and CD45RO antigens. Thus, CD45 isoform expression did not indicate where T lymphocytes were activated. However, we identified a previously unreported population of CD45RA+ CD45RO+ HLA-DR- T lymphocytes in arthritic and normal PB. In SF, this population was absent, but a substantial number of dual CD45RA, CD45RO-positive HLA-DR+ T lymphocytes were identified. This population would not be predicted by the current model of T lymphocyte activation. Division of T lymphocytes into functional groups on the basis of CD45 isoform expression is likely to be more complicated than previously thought. Based on our findings we propose an alternative model of T lymphocyte differentiation.

Arthritis↗
PubMed1994

Characterization of CMRF-44, a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood, including dendritic cells.

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) involved in the initiation of primary T-lymphocyte responses. However, despite their importance, no DC-specific surface marker has been identified in humans and many aspects of their ontogeny and the mechanisms underlying their potent functional activity remain unknown. In this report we describe a novel monoclonal antibody (mAb), CMRF-44, which recognizes an early activation antigen expressed by human DC and acts as a marker of allostimulatory activity within preparations of peripheral blood mononuclear cells (PBMC). The CMRF-44 antigen was expressed strongly on DC isolated from blood and tonsil by standard techniques, but was not detectable on Langerhans' cells within skin or on DC isolated directly from blood using a cell-sorting method which involves minimal DC manipulation/activation. Normal resting peripheral blood leucocytes did not label with CMRF-44, although weak staining of a small subpopulation (15%) of blood B lymphocytes was identified by double labelling. However, following overnight culture at 37 degrees, moderate staining of a subpopulation of PBMC was detected. Confirmation that CMRF-44 recognized an early marker of activated DC and hence allostimulatory activity was obtained by sorting cultured cell preparations on the basis of CMRF-44 reactivity. A marked enrichment of allostimulatory activity was observed in the CMRF-44-positive cellular population, whereas the CMRF-44-negative cells showed only minimal stimulatory activity. Activation studies established that the CMRF-44 antigen was an early activation marker, expressed constitutively on the majority of tonsil B lymphocytes, which can be induced on peripheral blood B lymphocytes and subpopulations of monocytes. Expression of the CMRF-44 antigen on cell lines was similarly restricted, CMRF-44 antigen being detected only on Hodgkin's disease-derived and B-lymphoid lines. The cellular distribution, expression kinetics and biochemical characteristics of the CMRF-44 antigen identify it as a new early marker of activated allostimulatory (DC) populations.

Animals↗
PubMed1993

Identification of potent mixed leukocyte reaction-stimulatory cells in human bone marrow. Putative differentiation stage of human blood dendritic cells.

Dendritic cells (DC) have been isolated from blood, lymphoid tissue, and other tissues, as potential members of a hemopoietic lineage of specialist APC for naive T lymphocyte activation. To define human bone marrow (BM) DC we have attempted to identify allostimulatory cells with DC-like characteristics among human BM mononuclear cells (BMMC) by FACS cell sorting and immunophenotyping, monitoring the APC function of different cell lineages in the human primary MLR. We show that fresh human BM stimulates allogeneic T lymphocytes with an activity equal to or greater than that of peripheral blood. As with DC from other tissue sources, the most potent stimulatory activity was found in the low density BMMC, and these cells, like peripheral blood, stimulated a maximal allogeneic MLR response at days 5 to 6. FACS purification of the allostimulatory population in fresh human BMMC was undertaken by using a wide range of mAb directed against lineage-associated molecules of mature and immature lymphoid, erythroid, and myeloid cells. The most potent constitutive BMMC stimulatory activity was located in the CD3-, CD11b-, CD14-, CD15-, CD16-, CD19-, CD57-, and glycophorin A- population. A mixture of antibodies to these Ag was used to isolate a "mix-negative" BMMC population, which contained the most highly potent MLR-stimulatory cells. Further cytologic and immunophenotypic analysis of this population revealed an enriched population of HLA-DP+, HLA-DQ+, HLA-DR+, and CD45+ cells, with morphologic similarities to the human tonsil and blood DC. These cells were CD4- and CD1a- and were weakly CD33+ (but CD15-), suggesting a possible early myeloid origin distinct from both the committed granulocytic and monocytic lineages. In addition, they lacked both CD10 and CD20, making a lymphoid origin unlikely. Further identification of these putative DC precursors will allow analysis of the early phases of DC hemopoiesis, whereas the characterization of the MLR-stimulatory cells in human BM will be of major importance in the understanding of BM transplant failure and graft-vs-host disease.

Animals↗
PubMed1993

Intercellular adhesion molecule-2 (ICAM-2) expression on human dendritic cells.

Dendritic cells (DC) form a distinct hemopoietic lineage of specialist antigen-presenting cells (APC) with unique abilities to stimulate a primary T lymphocyte response in vitro. In vivo data indicate that the interstitial DC in allografted tissue likewise stimulate an allogeneic response. DC express a range of adhesion molecules which are relevant to their interaction with T lymphocytes including ICAM-1, a ligand for T lymphocyte LFA-1. The T lymphocyte LFA-1 interaction with its DC ligand(s) plays a major role in DC-mediated activation of allogeneic T lymphocytes. This study details the expression of ICAM-1 and ICAM-2, a ligand for LFA-1, on cells of the DC lineage and examines their contribution to the DC-stimulated MLR response. Sensitive immunofluorescence and immunoperoxidase studies using newly available anti-ICAM-2 reagents failed to detect ICAM-2 or ICAM-1 on skin Langerhans cells, liver, and kidney interstitial DC. Isolated blood and tonsil DC were ICAM-1 and weakly ICAM-2 positive. Functional studies with CBR-IC2/2, an antibody known to block ICAM-2 binding to LFA-1, did not have a significant inhibitory effect on the DC-stimulated allogeneic MLR. Likewise we were again unable to show a major role for ICAM-1 in these DC-T cell interactions. These results suggest that other DC ligands for LFA-1, perhaps ICAM-3, may be present on DC and act as the major functional ligand for T cell activation.

Antigens, CD↗