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Biomedical subjects

D N Hart

Publications and source records attributed to D N Hart.

At least 55 records · Page 3Linked to original sources
PubMed1997

HLA-DR1-restricted bcr-abl (b3a2)-specific CD4+ T lymphocytes respond to dendritic cells pulsed with b3a2 peptide and antigen-presenting cells exposed to b3a2 containing cell lysates.

Chronic myeloid leukemia (CML) is characterized by a specific translocation of the c-abl oncogene on chromosome 9 to the break point cluster region (bcr) on chromosome 22, t(9;22) (q34;q11). This translocation results in the expression of a 210-kD bcr-abl protein fusion gene product. The juxtaposition of the bcr and abl genes produces a novel junctional amino acid sequence, which may be presented by antigen-presenting cells and recognized specifically by human T lymphocytes. We have generated a CD4+ T lymphocyte line (NG-1) which recognizes the peptide epitope (GFKQSSKALQR) in association with HLA-DRbeta1*0101-02. A comparison of antigen-presenting cells showed that CMRF-44+ blood dendritic cell presented a 12mer b3a2 peptide effectively. The b3a2 peptide was able to generate specific primary T-lymphocyte responses in other HLA-DR1 donors. We also show that bcr-abl, b3a2 peptide-specific T-lymphocyte lines proliferate in response to bcr-abl b3a2 containing cell lysates (K562 or CML PBMC derived) but not control (including b2a2 CML PBMC) lysates.

Amino Acid Sequence↗
PubMed1997

Isolation of human blood dendritic cells using the CMRF-44 monoclonal antibody: implications for studies on antigen-presenting cell function and immunotherapy.

Dendritic cells (DC) are potent antigen-presenting cells (APC) with the capacity to stimulate a primary T lymphocyte immune response and are therefore of interest for potential immunotherapeutic applications. Freshly isolated DC or DC precursors may be preferable for studies of antigen uptake and the potential control of APC costimulator activity. In this report, we report that the monoclonal antibody CMRF-44 can be used to detect early DC differentiation. The majority of DC circulating in blood do not express any known DC lineage specific markers, but can be identified by CMRF-44 labeling after a brief period of in vitro culture. The sequential acquisition of DC activation antigens allows the identification of two stages of DC maturation/activation. Cytokines, especially granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)alpha, enhance both phases of this process, whereas CD40-ligand trimer preferentially enhances the final DC maturation to a fully mature, activated phenotype. DC positively selected using CMRF-44 possess potent allostimulatory activity and are efficient at the uptake, processing, and presentation of soluble antigens for both primary and secondary immune responses. CMRF-44+ DC are also more potent than other APC types at restimulation of a chronic myeloid leukemia peptide specific T-cell clone. The use of a purified population of freshly isolated DC may be advantageous in attempts to initiate, maintain, and direct immune responses for immunotherapeutic applications.

Animals↗
PubMed1997

Prolonged remission of longstanding systemic lupus erythematosus after autologous bone marrow transplant for non-Hodgkin's lymphoma.

We describe a patient with longstanding steroid-dependent systemic lupus erythematosus (SLE) in whom clinical and serological remission was achieved following high-dose therapy and autologous bone marrow rescue for high-grade non-Hodgkin's lymphoma. However, 3 years later, autoimmune disease re-presented in the form of immune thrombocytopenia (ITP), which had not previously been a feature of the SLE, necessitating reintroduction of steroid immunosuppression. Relapse of SLE is most likely, although de novo ITP post-BMT is also a possibility. The case suggests that severe long-standing autoimmune disease may be controlled by high-dose therapy and autologous stem cell reconstitution. However, further studies are required to determine the mechanism of re-emergence of autoimmunity and to evaluate optimal regimens and the potential value of such therapy in severe autoimmune diseases.

Anemia, Hemolytic, Autoimmune↗
PubMed1997

Priming to mycobacterial antigen in vivo using antigen-pulsed antigen presenting cells generated in vitro is influenced by the dose and presence of IL-4 in APC cultures.

Antigen presenting cells (APC) similar to immature dendritic cells can be generated in vitro from bone marrow precursors. The authors have compared the yield, the phenotype and the function of murine bone marrow cells cultured for 7 or 11 days in either granulocyte macrophage colony stimulating factor alone (GM BMAPC) or in combination with interleukin-4 (GM/IL-4 BMAPC). The results showed that GM/IL-4 BMAPC expressed the highest levels of MHC Class 2 molecules, CD86/B7-2 and CD80/B7-1 co-stimulatory molecules and the lowest levels of F4/80 macrophage marker. However, when these APC were pulsed with BCG culture filtrate antigen or PPD they were not correspondingly more effective at stimulating activated T lymphocytes in vitro or priming naive T lymphocytes in vivo. Also, in contrast to GM BMAPC, high backgrounds recorded following injections of GM/IL-4 BMAPC without antigen were not consistently reduced by lowering the dose and irradiating the cells prior to administration. The authors conclude that the degree of maturity of BMAPC varies with culture conditions and that this may be an important consideration where BMAPC are to be used in vivo in immunotherapeutic regimens.

Animals↗
PubMed1997

Hodgkin's cells express CD83, a dendritic cell lineage associated antigen.

Hodgkin's cells (HC) are considered to be the malignant cells of Hodgkin's disease (HD), but despite extensive studies, no conclusive evidence has emerged regarding their non-malignant counterpart and the ontogeny of these cells remains controversial. The analysis of a possible dendritic cell (DC) origin of HC has been hampered to date by the lack of a DC lineage specific marker. The expression of the two DC-associated antigens CD83 and CMRF-44, the B lymphocyte restricted molecule CD79, and the costimulator molecule CD86, was examined in lymph nodes from 23 HD patients using immunohistological techniques. The majority of HC expressed the CD83 (22/23) and CD86 antigens (20/23), whereas expression of the CMRF-44 antigen was variable (10/23) and usually only a subpopulation of HC stained. In contrast, the CD79 antigen was absent from most HC (17/23). The presence of the CD83 antigen on HC in the absence of the CD79 antigen supports a possible DC lineage origin for some HC. Regardless of its role in lineage assignment, CD83 may become a useful immunohistological marker for HD as the CD83 antigen was present on most HC.

Antibodies, Monoclonal↗
PubMed1996

Human T lymphocytes and hematopoietic cell lines express CD24-associated carbohydrate epitopes in the absence of CD24 mRNA or protein.

The CD24 surface antigen is a small glycophosphatidylinositol (GPI)-anchored glycoprotein found on human granulocytes and most B lymphocytes. Many CD24 monoclonal antibodies (MoAbs) have been described that identify several epitopes, with the majority of them related to carbohydrate structures associated with the CD24 molecule. Considerable variation has been observed in the apparent tissue distribution of the CD24 antigen depending on the MoAb used, and hence the CD24 epitope studied. In this study, CD24 expression by human cell lines and normal hematopoietic call populations was assessed using a panel of carbohydrate and protein core-specific CD24 MoAbs and reverse transcriptase polymerase chain reaction (RT-PCR) analysis. A number of CD24 carbohydrate epitope-reactive MoAbs bound to both T lymphocytes and several hematopoietic cell lines, despite the absence of concomitant CD24 mRNA or detectable surface CD24 core protein in the same cells. This additional CD24 MoAb reactivity on T lymphocytes was, in common with that observed on granulocytes (CD24 protein+), specifically inhibited by the presence of both sialyllactose and mucin. Similarly, the binding of carbohydrate epitops-reactive CD24 MoAb was reduced on both T lymphocytes and granulocytes by pretreatment with phospholipase C, pronase, or neuraminidase. Together, the data indicate that a number of CD24-associated carbohydrate epitopes have a broader tissue distribution than the CD24 protein and are expressed on additional GPI-linked molecule(s). These findings have immediate implications for both leukemia phenotyping and attempts to examine CD24 function with CD24 MoAb.

Animals↗
PubMed1996

High dose therapy and autologous bone marrow versus blood cell rescue. South Island Bone Marrow Transplant Unit.

AIMS: To compare haematological recovery and supportive care requirements for patients receiving high dose therapy (HDT) supported by rescue with either autologous cytokine mobilised blood cells (BC) or autologous bone marrow (BM). To identify stem cell harvest criteria predictive of rapid haematological recovery after rescue with BC. METHODS: Single arm, open study in four groups of 69 consecutive patients undergoing HDT in a single institution from 1986-95. Group 1 contained patients with solid tumours or myeloma rescued with BC alone (n = 14); group 2 patients with solid tumours rescued with BM (n = 31); group 3 patients with acute leukaemia rescued with BM (n = 21); and group 4 patients with solid tumours rescued with both BC and BM (n = 3). RESULTS: Haemopoietic recovery was most rapid for group 1 where, in comparison with the BM group transplanted for similar disease (group 2), highly significant reductions were observed for median days to 1) neutrophil s >/= 0.5 x 10(9)L (12 v 22; p 0.0001); 2) neutrophils >/= 1.0 x 10(9)/L (14 v 27 ; p = 0.0001); platelets >/= 20 x 10(9) /L (11 v 20; p = 0.0005); and 4) platelets >/= 50 x 10(9)/L (15 v 27; p = 0.001). Similar significant reductions for the BC group over group 2 were also observed for median, (1) inpatient days (22 v 30; p = 0.0001); (2) red cell transfusions (2 v 5; p = 0.01) ; (3) platelet transfusions (2 v 6; p = 0.0001); (4) days of fever (2 v 8; p = 0.001); and (5) days on IV antibiotics (8 v 14; p = 0.02). Group 3 patients yielded data either similar to or less advantageous to that in group 2 and group 4 patients yielded data intermediate between groups 1 and 2. Data from BC harvests suggested that yields of CD34+ cells of > 2.0 x 10(6)/kg and/or of colony forming units-granulocyte-macrophage (CFU-GM) of > 6.0 x 10(4)/kg were predictive of rapid haemopoietic recovery. CONCLUSION: For haemopoietic rescue following single HDT for solid tumours BC has considerable advantages over BM.

Adolescent↗
PubMed1996

Expression and function of CD80 and CD86 costimulator molecules on synovial dendritic cells in chronic arthritis.

OBJECTIVE: To examine CD86 expression on dendritic cells isolated from the synovial fluid (SFDC) of patients with chronic arthritis, and to determine the importance of both CD80 and CD86 molecules in SFDC-T lymphocyte interactions. METHODS: CD86 messenger RNA (mRNA) and surface expression were analyzed in SFDC using reverse transcriptase-polymerase chain reaction and flow cytometry, respectively. The costimulator activity of the SFDC CD80 and CD86 molecules was determined by allogeneic mixed lymphocyte reaction (MLR). CD80 and CD86 induction on SFDC during in vitro culture was also examined. RESULTS: Fresh SFDC either lacked or showed very weak surface expression of CD86 molecules (as shown previously for CD80), yet contained CD86 mRNA. CD80 antibodies minimally inhibited an allogeneic MLR, whereas CD86 antibodies and CTLA-4 Ig showed significant inhibition. Both CD80 and CD86 molecules were inconsistently induced on SFDC following culture in either media, interferon-gamma, or granulocyte-macrophage colony-stimulating factor. CONCLUSION: SFDC may be defective antigen-presenting cells in vivo. The ability of CD80 and CD86 molecules to be induced and become functional on SFDC in vitro implies the presence of a negative regulatory compound(s) in the synovial environment.

Abatacept↗
PubMed1996

Human dendritic cells activate T lymphocytes via a CD40: CD40 ligand-dependent pathway.

The CD40:CD40 ligand (CD40L) interaction provides T lymphocyte-mediated help for B lymphocyte and monocyte function but has also been shown to serve as a co-stimulus for T lymphocyte activation. In this report, we studied the regulation of CD40 expression and its functional relevance for the human dendritic cell (DC) stimulation of T lymphocytes. Only a small subpopulation of directly isolated blood DC expressed CD40. However, CD40 was rapidly up-regulated by culture, and its expression was further enhanced by interleukin (IL)-1 alpha, IL-1 beta, IL-3, tumor necrosis factor-alpha and granulocyte/macrophage-colony-stimulating factor. Expression of CD40L on DC was not detected. The proliferation of T lymphocytes in an allogeneic mixed leukocyte reaction, stimulated by blood DC or epidermal Langerhans cells, was significantly reduced in the presence of the CD40 immunoglobulin (CD40Ig) fusion protein or CD40L monoclonal antibodies. Cross-linking of CD40 on directly isolated DC with mouse CD40L trimer (mCD40LT) markedly augmented CD80 and CD86 up-regulation. Nevertheless, the same cross-linking mCD40LT inhibited DC stimulated T lymphocyte proliferation. When CD40Ig was added simultaneously with CTLA-4Ig, only minimal and variable additional inhibition of DC-stimulated allogeneic T lymphocyte proliferation and IL-2 secretion was observed, compared to each fusion protein alone. These results suggest that both CD80/CD86-dependent and -independent components of DC-T lymphocyte CD40:CD40L co-stimulation exist and further emphasize that the majority of blood DC have to differentiate or be activated to express co-stimulatory molecules.

Abatacept↗
PubMed1996

Identification of a novel dendritic cell surface antigen defined by carbohydrate specific CD24 antibody cross-reactivity.

Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC however, cross-reactive sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation differentiation in vitro.

Antibodies, Monoclonal↗
PubMed1996

Donor leukocyte infusions in the treatment of chronic myeloid leukemia in relapse post bone marrow transplantation.

A 25-yr-old Caucasian man presented in 1988 with Philadelphia chromosome (Ph) negative, bcr-abl rearranged, chronic phase chronic myeloid leukemia (CML). He was treated with human leukocyte antigen matched sibling allogeneic bone marrow transplantation but relapsed 5 yrs later. At this time he was given donor leukocyte infusions from the original bone marrow donor, seeking an immune anti-leukemic effect. This treatment induced graft versus host disease and severe bone marrow aplasia, requiring immunosuppression and repeat donor marrow infusion (without prior conditioning). Graft versus host disease was controlled and full donor hematopoiesis was restored, resulting in complete eradication of the leukemic clone at a molecular level. The patient remains in complete clinical and molecular remission and off all immunosuppression 24 mths later. This emphasizes a potentially powerful graft versus leukemia effect in CML.

Adult↗
PubMed1995

Isolation of human blood dendritic cells by discontinuous Nycodenz gradient centrifugation.

The most potent antigen presenting cell present in peripheral blood, lymphoid and non-lymphoid tissue is the dendritic cell (DC). The study of human DC has been restricted by their low frequency in the tissues and the lack of a truly DC specific surface marker to assist in identification and isolation. Standard techniques for the isolation of blood DC generally employ a period of in vitro culture followed by flotation on dense albumin gradients, or more recently, discontinuous gradients of metrizamide. Dense albumin gradients are time consuming to prepare, giving low and variable yields of DC. Metrizamide is more convenient, although exposure of monocytes to metrizamide can decrease the expression of CD14 and alter the accessory cell properties of antigen presenting cells. Here we demonstrate that Nycodenz gradient centrifugation of 16 h cultured, T lymphocyte depleted, peripheral blood mononuclear cells (PBMC) reliably yields a population of low density cells that is highly enriched for DC. Most B and residual T lymphocytes are depleted and NK cell numbers are reduced two-fold from the interface cell population. The high density pellet fraction exhibits very little allostimulatory activity, indicating that few DC pass into the pellet. The low density fraction contains a significant population (20 +/- 5 (SD)%, n = 8) of cells which fail to stain for the lineage markers CD3, CD11b, CD14, CD16, CD19 and CD57. Nycodenz exhibits low toxicity, does not alter the allostimulatory activity of antigen presenting cells, and is therefore ideal for the isolation of cultured DC.

Cell Separation↗
PubMed1995

Activation of human peripheral blood dendritic cells induces the CD86 co-stimulatory molecule.

Maximal T lymphocyte responses require presentation of antigen by major histocompatibility complex molecules and delivery of one or more co-stimulatory signals. Interaction of the CD28 molecule on T lymphocytes with its ligands on antigen-presenting cells (APC) initiates a critical co-stimulatory pathway inducing T lymphocyte proliferation and cytokine secretion. Dendritic cells (DC) are potent APC for a primary T lymphocyte response and potential CD28/CTLA-4 ligands on DC are, therefore, of particular functional relevance. In these experiments, the expression and function of the CD28/CTLA-4 ligands B7.1 (CD80) and B7.2 (CD86) were examined on human blood DC. Resting DC populations directly isolated by immunodepletion of lineage marker-positive cells lacked cell membrane expression of CD80 and expressed little or no CD86, although CD86, but not CD80 mRNA was detected by reverse transcription-polymerase chain reaction analysis. In contrast, low-density DC isolated after culture in vitro strongly expressed CD86 surface protein, but expressed limited or no CD80, although mRNA for both molecules were detected. Short-term culture of directly isolated DC up-regulated both CD80 and CD86 expression. Analysis of the kinetics of CD28/CTLA-4 ligand induction showed that surface CD86 was present within 8 h, whereas CD80 antigen was first detected after 24 h of culture. The functional importance of CD28/CTLA-4 ligand up-regulation on DC during T lymphocyte interactions was demonstrated by the ability of both CTLA-4Ig and CD86 monoclonal antibodies (mAb), but not CD80 mAb, to block an allogeneic mixed lymphocyte reaction stimulated by DC populations initially negative for CD80 and CD86. These results demonstrate that CD86 is both the earliest and functionally the predominant co-stimulatory CD28/CTLA-4 ligand on DC.

Abatacept↗
PubMed1995

Intercellular adhesion molecule-3 is the predominant co-stimulatory ligand for leukocyte function antigen-1 on human blood dendritic cells.

Dendritic cells (DC) are potent stimulators of primary T lymphocyte responses to foreign antigen. The initial DC-T lymphocyte interaction involves the binding of the adhesion molecule leukocyte function antigen-1 (LFA-1; CD11a/CD18) on the T lymphocyte to an intercellular adhesion molecule (ICAM) on the DC. Although blood and tonsil DC express ICAM-1 (CD54) and ICAM-2 (CD102) on their surface, anti-ICAM-1 and anti-ICAM-2 monoclonal antibodies (mAb) have little inhibitory activity on the DC-stimulated mixed leukocyte reaction (MLR). We therefore examined the expression of the more recently identified LFA-1 ligand, ICAM-3 (CD50), in comparison to ICAM-1 and ICAM-2 on blood DC and sought a functional role for ICAM-3 in DC-mediated T lymphocyte responses. Resting blood DC expressed significantly more ICAM-3 than ICAM-1 or ICAM-2 as assessed by flow cytometry. Treatment of resting DC with interferon-gamma led to increased expression of ICAM-1; however, ICAM-2 and ICAM-3 levels remained relatively constant. Solid-phase recombinant chimeric molecules ICAM-1-, ICAM-2- and ICAM-3-Fc were able to co-stimulate CD4+ T lymphocyte proliferation in conjunction with suboptimal solid-phase CD3 mAb 64.1. However, the anti-ICAM-3 mAb CAL 3.10 inhibited a DC-stimulated MLR to a greater extent than anti-ICAM-1 or anti-ICAM-2 reagents and appeared to act by blocking the DC ICAM-3- T lymphocyte LFA-1 interaction. As ICAM-3 is the predominant LFA-1 ligand on resting blood DC, we postulate that DC may utilize ICAM-3 for initial DC- T lymphocyte interactions, and that ICAM-1, which is up-regulated upon DC activation, and/or ICAM-2, may contribute to DC migration or later phases of the T lymphocyte activation process.

Antigens, CD↗