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Biomedical subjects

D N Hart

Publications and source records attributed to D N Hart.

At least 19 recordsLinked to original sources
PubMed2000

Lectin ligands on human dendritic cells and identification of a peanut agglutinin positive subset in blood.

As only a few cell surface markers for dendritic cells (DC) have been identified to date, this study examined the expression of ligands for lectin on different human DC populations. The ability of Concanavalin A (Con A), Wheat Germ Agglutinin (WGA), peanut agglutinin (PNA), and Helix pomatia (HPA) to bind to cell lines and PBMC and DC populations was analyzed by flow cytometry and specificity of binding confirmed using inhibitory and noninhibitory sugars. The cell lines showed non-lineage-restricted binding with Con A and WGA, independent of sialidase treatment. HPA and PNA bound to a restricted number of lines, but showed broad reactivity after sialidase treatment. The peripheral blood mononuclear cells (PBMC) and directly isolated blood DC, activated CD83(+) blood DC, epidermal Langerhans cells (LC), and monocyte-derived DC (Mo-DC) showed strong binding of Con A and WGA, both before and after sialidase treatment. No HPA binding ligands were detected on PBMC populations, including directly isolated blood DC. Following sialidase treatment CD3(+), CD16(+), and a subset of CD19(+) lymphocytes bound HPA. The lectin PNA bound weakly to CD14(+) monocytes and a subpopulation of circulating DC that were HLA-DR(hi)CDw123 Dr(hi)CDw123(dim)/(neg)CD11c(+). The HLA-DR(mod)CDw123(hi)CD11c(neg) subpopulation did not bind PNA. Without sialidase treatment LC expressed both HPA and PNA ligands, but these were either absent on activated CD83(+) blood DC or weakly expressed on Mo-DC. Following sialidase treatment PBMC populations, activated CD83(+) blood DC, and Mo-DC became PNA positive. Thus human DC express several lectin ligands and PNA binding identifies a subset of blood DC. That may reflect discrete changes associated with stages of DC development or functional maturation.

Binding Sites↗
PubMed2000

The role of dendritic cells in the innate immune system.

Dendritic cells (DCs) are bone-marrow-derived leucocytes that are specialised antigen-presenting cells capable of stimulating a primary T-lymphocyte response to specific antigen. In this chapter we discuss the role DCs play in the innate response acting as a critical link with the adaptive response and the influence of the innate response on dendritic cells.

Animals↗
PubMed2000

The CMRF-35H gene structure predicts for an independently expressed member of an ITIM/ITAM pair of molecules localized to human chromosome 17.

The CMRF-35 monoclonal antibody recognizes an epitope found on at least two cell surface molecules, differentially expressed by many leukocytes. These molecules, the CMRF-35H (9) and CMRF-35A (CMRF-35) antigens are both members of the immunoglobulin (Ig) superfamily with a single V-like Ig domain. The function of these molecules is unknown, however the presence of putative immunoreceptor tyrosine-based inhibitory motifs (ITIM) in the cytoplasmic domain of the CMRF-35H molecule suggests that this molecule may play a regulatory role in leukocyte function. The CMRF-35H and CMRF-35A molecules show several similarities to the family of molecules containing ITIM or immunoreceptor tyrosine-based activatory motifs (ITAM) suggesting that CMRF-35H/CMRF-35A may be new members of this family. This would further indicate that, like other ITIM/ITAM containing molecules, CMRF-35H/CMRF-35A will also play an important role in the immune response. To further characterize these molecules, we have isolated genomic clones for the CMRF-35H gene and determined its intron-exon organization. The gene spans approximately 12 kb and consists of seven exons. Furthermore, this gene has been mapped to chromosome 17 and thus is not linked to the known human ITIM containing genes which map to human chromosome 19 or the recently characterized molecule, NKp44, localized to human chromosome 6.

3' Untranslated Regions↗
PubMed2000

Successful high dose therapy for relapsed mediastinal large B cell lymphoma following surgical repair of anterior chest wall defect.

We describe a man with relapsed large B cell mediastinal lymphoma and associated infected large anterior chest wall defect who required high dose salvage therapy for his underlying disease. An initial mediastinotomy wound, associated with recurrent sepsis, had developed into an abscess, then fistula and eventually a large anterior chest wall defect. Safe use of salvage chemotherapy required reconstructive surgery consisting of a pedicled muscle flap. The subsequent high dose chemotherapy was carried out without complications and 15 months later the patient is alive and well.

Abscess↗
PubMed2000

Cells expressing dendritic cell markers are present in the rheumatoid nodule.

OBJECTIVE: To determine if dendritic antigen-presenting cells (DC) are present in rheumatoid nodules, as has been reported in the synovial lesions of rheumatoid arthritis. METHODS: Nodules (n = 14) were examined with monoclonal antibodies (Mab) recognizing the DC differentiation/activation markers CD83, CMRF44, and CMRF56 and an antibody recognizing the CD1a antigen present on epithelial tissue associated DC. Results. Cells expressing CMRF44 were common in rheumatoid nodules, comprising 22% of nucleated cells versus 13% in synovial membranes (n = 10). Cells positive for CD1a (5%) and CD83 (2%) were less common. A majority (86%) of CMRF44 positive cells were also positive for the macrophage marker CD14. This left a significant minority of putative DC that were single stained with CMRF44. CONCLUSION: Cells bearing DC markers are as frequent in the rheumatoid nodule as in the synovial lesions. A majority are "indeterminate" cells that are CD14 positive but a proportion are single stained putative DC. The lack of lymphoid collections containing DC and T and B lymphocytes in the nodule suggests that local presentation of antigen may not occur in the rheumatoid nodule, as is thought to be the case in synovial membranes containing lymphoid follicles. This difference could potentially be explained by different states of activation, and differentiation of DC within the 2 lesions.

Adult↗
PubMed1999

Monitoring human blood dendritic cell numbers in normal individuals and in stem cell transplantation.

Dendritic cells (DC) originate from a bone marrow (BM) precursor and circulate via the blood to most body tissues where they fulfill a role in antigen surveillance. Little is known about DC numbers in disease, although the reported increase in tissue DC turnover due to inflammatory stimuli suggests that blood DC numbers may be altered in some clinical situations. The lack of a defined method for counting DC has limited patient studies. We therefore developed a method suitable for routine monitoring of blood DC numbers, using the CMRF44 monoclonal antibody (MoAb) and flow cytometry to identify DC. A normal range was determined from samples drawn from 103 healthy adults. The mean percentage of DC present in blood mononuclear cells (MNC) was 0.42%, and the mean absolute DC count was 10 x 10(6) DC/L blood. The normal ranges for DC (mean +/- 1.96 standard deviation [SD]) were 0.15% to 0.70% MNC or 3 to 17 x 10(6) DC/L blood. This method has applications for monitoring attempts to mobilize DC into the blood to facilitate their collection for immunotherapeutic purposes and for counting blood DC in other patients. In preliminary studies, we have found a statistically significant decrease in the blood DC counts in individuals at the time of blood stem cell harvest and in patients with acute illnesses, including allogeneic bone marrow transplant (BMT) recipients with acute graft-versus-host disease (aGVHD).

Adolescent↗
PubMed1999

Synovial fluid transforming growth factor beta inhibits dendritic cell-T lymphocyte interactions in patients with chronic arthritis.

OBJECTIVE: To examine whether rheumatoid synovial fluid (SF) inhibits dendritic cell (DC) expression of the CD80 and CD86 costimulator molecules and contributes to SF T lymphocyte hyporesponsiveness. METHODS: Cell-free rheumatoid SF was tested for its effect on DC-stimulated autologous/allogeneic mixed lymphocyte reactions and for its effect on DC surface antigen expression, as assessed by flow cytometry. Blocking monoclonal antibodies were used to identify the SF cytokines that inhibited DC-T lymphocyte interactions. RESULTS: Low concentrations of SF (2.5%) could inhibit DC-mediated autologous and allogeneic T lymphocyte proliferation. This inhibitory effect could be reversed by neutralizing transforming growth factor beta (TGFbeta) and interleukin-2 (IL-2), but not by IL-12, in the SF. Hyaluronic acid, IL-6, IL-10, and tumor necrosis factor alpha were not associated with SF inhibition. In vitro culture alone and crosslinking with the CD40 ligand up-regulated DC CD80/CD86 expression and costimulator function, and this was not affected by inclusion of SF. In the presence of SF, DC clustered with autologous T lymphocytes showed decreased CD80 and CD86 expression, and variable CD80/CD86 decreases were observed on DC clustered with allogeneic T lymphocytes. CONCLUSIONS: TGFbeta in SF appears to suppress T lymphocyte function, which may affect both signaling to DC and the induction of DC costimulator function.

Antibodies, Monoclonal↗
PubMed1999

Human dendritic cells express a 95 kDa activation/differentiation antigen defined by CMRF-56.

Despite the unique functions of dendritic cells (DC), only two cell surface antigens (CMRF-44 and CD83) with relatively restricted expression on human DC have been described to date. We describe a third mAb, CMRF-56, which recognizes another DC early activation/differentiation antigen with limited expression on other haemopoietic cell populations. Circulating blood leukocytes did not express the CMRF-56 antigen and, following either in vitro culture or activation of PBMC populations, CMRF-56 antigen expression was detected only on DC and a subpopulation of CD19+ lymphocytes. Circulating blood DC were CMRF-56 but induced expression within 6 h of in vitro culture. This, together with the finding that tonsil and synovial fluid DC upregulate the antigen following short-term in vitro culture, confirmed that CMRF-56 recognizes an early activation antigen on DC. Isolated Langerhan's cells, dermal DC, migratory dermal DC and monocyte derived DC (GM-CSF/IL-4/TNFalpha) also express the CMIRF-56 antigen. Antigen modulation studies demonstrated that the amount of cell surface bound CMRF-56 and CMRF-44 (but not CD83) mAb was dramatically reduced by short-term incubation at 37 degrees C. This effect was not due to internalization and the reduction in CMRF-56 binding was a reversible, temperature-dependent process. In contrast, the decrease in CMRF-44 binding was irreversible, suggesting that following ligation the CMRF-44 antigen undergoes an irreversible conformational change or shedding at 37 degrees C. Western blotting confirmed that CMRF-56 recognizes a previously undescribed 95 kDa activation antigen whose cellular distribution and expression kinetics overlaps with, but is clearly distinguishable from, that of the CD83 and CMRF-44 antigens. CMRF-56 therefore provides a useful additional marker for studies on human DC.

Antibodies, Monoclonal↗
PubMed1999

Dendritic cells in chronic myelomonocytic leukaemia.

Blood dendritic cells (DC) differentiate in vitro via two separate pathways: either directly from blood DC precursors (DCp) or from CD14+ monocytes. In chronic myelomonocytic leukaemia (CMML) abnormal bone marrow precursors contribute to blood monocyte development but DC development has not been studied previously. Monocytes comprised 60% of blood MNC in 15 CMML patients studied, compared with 20% in 16 age-matched controls. The increase in blood monocytes was accompanied by a reciprocal decrease in mean blood DC percentage (from 0.42% of MNC in normal individuals to 0.16% of MNC in CMML patients). Absolute blood DC numbers showed a minimal (non-significant) reduction from 9.8 x 10(6)/l in normal individuals to 7.5 x 10(6)/l in CMML patients. The CD14(low) WCD16+ monocyte subpopulation was not found in CMML patients. After culture in GM-CSF/IL-4, CMML CD14+ monocytes acquired the phenotype of immature monocyte derived DC (Mo-DC) with similar yields to normal blood Mo-DC generation. Addition of TNF-alpha or LPS induced both normal and CMML Mo-DC to express prominent dendritic processes, the CMRF44+ and CD83+ antigens and high levels of HLA-DR, CD80 and CD86. Treatment either with TNF-alpha or LPS increased the allostimulatory activity of normal Mo-DC, but had little effect on the allostimulatory activity of CMML Mo-DC, perhaps reflecting the underlying neoplastic changes in monocyte precursors. We conclude that the blood DC numbers are relatively unaffected in CMML, suggesting discrete regulation of monocyte and DC production.

Adult↗
PubMed1999

Expression of the RelB transcription factor correlates with the activation of human dendritic cells.

The RelB gene product is a member of the nuclear factor (NF)-kappaB family of transcription factors. It has been identified recently within mouse antigen-presenting cells and human monocyte-derived dendritic cells (DC). Disruption of the mouse RelB gene is accompanied, amongst other phenotypes, by abnormalities in the antigen-presenting cell lineages. In order to define RelB expression during human DC differentiation, we have analysed RelB mRNA by reverse transcriptase-polymerase chain reaction and RelB protein by intracellular staining in CD34+ precursors and different types of DC preparations. RelB mRNA was not detected in CD34+ precursor populations. Fresh blood DC (lineage-human leucocyte antigen-DR+ (lin-HLA-DR+)) lacked RelB mRNA and cytoplasmic RelB protein but a period of in vitro culture induced RelB expression in blood DC. Purified Langerhans' cells (LC) (CD1a+ HLA-DR+) failed to express RelB mRNA. Immunocytochemical staining identified RelB protein in human skin epithelium. RelB protein was expressed in a very few CD1a+, CD83+ or CMRF-44+ dermal DC but was not present in CD1a+ LC. Tonsil DC (lin-HLA-DR+ CMRF-44+) were positive for RelB mRNA and RelB protein. Intestinal DC (HLA-DR+) also lacked immunoreactive RelB protein. The majority of interdigitating CD83+, CMRF-44+, CMRF-56+ or p55+ DC located in paracortical T-lymphocyte areas of lymph node and tonsil contained RelB protein. The expression of RelB mRNA and RelB protein correlates with the activated phase of blood DC and the postmigration cell (activated) stage of tissue DC development.

Antigens, CD34↗
PubMed1999

Induction of dendritic cell costimulator molecule expression is suppressed by T cells in the absence of antigen-specific signalling: role of cluster formation, CD40 and HLA-class II for dendritic cell activation.

Full activation of T lymphocytes by dendritic cells (DC) during antigen presentation is known to require the interaction of several inducible receptor-ligand pairs. We have postulated that the reciprocal activation of DC by T lymphocytes is also important. Potential signalling molecules that might increase the stimulatory capacity of DC during antigen presentation to T lymphocytes were tested using an in vitro model. Fresh human blood DC were cocultured with CD4+ and CD8+ allogeneic or with autologous T lymphocytes plus Staphylococcus superantigen A (SEA). Surprisingly, costimulator expression on DC cocultured with T lymphocytes was reduced in comparison to DC cultured alone. However, the minority (10-30%) of DC clustering with T lymphocytes showed antigen-specific up-regulation of the CD40, CD80 and CD86 costimulator molecules, whereas the non-clustered DC (70-90%) had less up-regulation than control DC cultured alone and did not respond to antigen-specific triggering. Monoclonal antibodies (mAb) to CD40 ligand (CD40L) and human leucocyte antigen (HLA)-DR, but not lymphocyte function-associated antigen-1 (LFA-1), LFA-3 or HLA-class I, significantly inhibited the T-lymphocyte induction of DC costimulator expression. Since HLA-class II, but not HLA-class I mAb, inhibited allogeneic T-lymphocyte-mediated activation of DC, CD4 T lymphocytes appear to be the main subset activating DC in the mixed lymphocyte reaction. Cross-linking of CD40, but not HLA-class II, up-regulated DC or B-cell costimulator expression. Although direct class II signalling does not appear to play a role in DC activation, antigen-specific T-cell recognition contributes via other mechanisms to regulate DC activation.

Antibodies, Monoclonal↗
PubMed1999

Dendritic cell immunotherapy for cancer: application to low-grade lymphoma and multiple myeloma.

The confirmation that most cancers express one or more molecular changes, which may act as tumour-associated antigens (TAA), combined with the knowledge that T lymphocytes recognize even single amino acid differences in MHC presented peptides has stimulated renewed clinical interest in immunotherapeutic strategies. Dendritic cells (DC) are now recognized as specialist antigen-presenting cells, which initiate, direct and regulate immune responses. Recent data suggest that DC are not recruited into, or activated by, cancers and that other abnormalities in DC function are associated with malignancy, including multiple myeloma. This provides a rationale for designing immunotherapeutic strategies, which exploit DC as nature's adjuvant either in vivo or in vitro. Low-grade lymphoma and multiple myeloma are slowly progressive malignancies, which generally express a unique immunoglobulin idiotype as a potential TAA. Data from animal models and clinical studies suggest that DC-based immunotherapy strategies, applied when the patient has minimal residual disease, may improve the long-term prognosis in these diseases.

Animals↗
PubMed1999

CD1a dendritic cells predominate in transitional cell carcinoma of bladder and kidney but are minimally activated.

PURPOSE: In this study, we employed similar techniques to detail dendritic cell subsets within bladder transitional cell carcinoma and kidney transitional cell carcinoma. MATERIALS AND METHODS: To identify both the CD1a+ and CD1a- antigen-expressing dendritic cell populations we employed a double labeling technique to identify non-lineage-expressing leukocytes similar to that employed to isolate blood dendritic cells. RESULTS: Dendritic cells were found in significant numbers within both bladder and kidney derived transitional cell carcinoma. Almost all the dendritic cells among the tumor cells belonged to the CD1a+ subset of epithelial dendritic cells. Similar numbers of dendritic cells were observed in the lamina propria adjacent to the tumor. These dendritic cells belonged predominantly to the CD1a- subset. These differences appear to reflect the different dendritic cell phenotypes reported for the epidermis and dermis. CONCLUSIONS: The number of dendritic cells increased as the grade of the tumor increased, reflecting an overall higher leukocyte density in higher grade tumors. However, a possible trend for less dendritic cell activation in higher grade cancers was noted, raising the intriguing possibility that this might be a relevant prognostic factor, to be confirmed in a larger study.

Antigens, CD1↗
PubMed1999

Presentation of tumor antigens.

Dendritic cells, with their extraordinary capacity for initiating primary and secondary T-lymphocyte responses, may be pivotal in the development of immunotherapeutic strategies for multiple myeloma. Although host lymphocytes are able to recognize tumor-associated antigens (TAAs), many tumors are able to avoid dendritic cell-mediated immune surveillance. One reason may be that the tumor environment inhibits the maturation and activation of dendritic cells. A recently developed strategy to use dendritic cells in immunotherapy involves removing them from the tumor, pulsing them in vitro with antigen, and reinfusing them into the patient to generate responding T cells in vivo. Methods for reliably obtaining dendritic cells for therapeutic use are currently being investigated. Among other efforts to induce T-cell-mediated immunity against cancer, the presentation of tumor antigens by the tumor cells themselves is being investigated. Issues to be resolved include defects of antigen presentation by tumor cells and whether all cells present the same set of peptides. Moreover, as long as all the tumor antigens have not been identified, the tumor cell itself remains the primary source of unknown antigens and, therefore, is a worthwhile subject for study. Phase I trials of immunotherapy using adenovirus-infected autologous plasma cells have recently been undertaken. The adenoviral vectors carry genes with therapeutic potential, including interleukin-2 (IL-2), interleukin-12, and B7-1. Initial results showed that the vector can be readily detected in tumor cells at 13 days postinjection, and IL-2 expression was evident at 7 days. The chief side effect reported was inflammation.

Antigen Presentation↗
PubMed1998

Optimisation of the conditions for generating human DC initiated antigen specific T lymphocyte lines in vitro.

Naive T lymphocytes specific for a given primary antigen occur in low frequencies and require the relevant antigen to be presented by specialist antigen presenting cells (APC), i.e., dendritic cells (DC). For these reasons, the in vitro induction of primary T lymphocyte responses remains a significant technical challenge. We have attempted to improve current strategies for generating in vitro responses by optimising (i) isolation and concomitant activation of DC from peripheral blood, (ii) uptake, processing and presentation of antigen by DC and (iii) antigen driven T lymphocyte proliferation. We established that RPMI 1640 media supplemented with 10% autologous serum resulted in the best yield of CMRF-44+, CD14-, CD19- DC after enrichment over a Nycodenz gradient. Optimal presentation of whole protein and peptide antigen was achieved by addition after the purification of the APC, i.e., at the start of the T lymphocyte proliferation assay. RPMI 1640 supplemented with 10% autologous serum or plasma supported the best antigen driven specific T lymphocyte responses. Using these optimised conditions, we compared the efficacy of PBMC and purified blood DC for priming T lymphocyte responses to the chronic myeloid leukaemia (CML) specific bcr-abl (b3a2) peptide. Peptide specific T lymphocyte responses were generated with both purified DC and whole PBMC, suggesting that T lymphocyte precursor frequency was the limiting factor in these experiments. These results will aid in the generation of human T lymphocyte lines to primary antigens, for in vitro and therapeutic applications.

Animals↗
PubMed1998

Type I IFNs enhance the terminal differentiation of dendritic cells.

This study identifies type I IFNs as activating cytokines in a serum-free system in which human dendritic cells (DC) were generated from CD34+ progenitor cells. After 14 days of culture in GM-CSF, TNF-alpha, and IL-4, CD34+ progenitors gave rise to a population of large, immature DC expressing CD1a and CD11b but lacking CD14, CD80, CD83, CD86, and CMRF44. During the next 2 wk, this population spontaneously matured into nonadherent, CD1a(low/-), CD11b(low/-), CD14-, CD80+, CD83+, CD86+, CMRF44+ DC with high allostimulatory activity in the MLR. To examine which factors influenced this maturation, 25 different cytokines or factors were added to the immature DC culture. Only type I IFNs (alpha or beta) accelerated this maturation in a dose-dependent manner, so that after only 3 days the majority of large cells acquired the morphology, phenotype, and function characteristics of mature DC. Furthermore, supernatants from cultures containing spontaneously maturing DC revealed low levels of endogenous IFN production. Because of the similarity of the activation of DC in our culture system with the phenotypic and functional changes observed during Langerhans cells activation and migration in vivo, we investigated the effect of IFN-alpha on human Langerhans cell migration. IFN-alpha also activated the migration of human split skin-derived DC, demonstrating that this effect was not limited to DC derived in vitro from hemopoietic progenitor cells. DC activation by type I IFNs represents a novel mechanism of immunomodulation by these cytokines, which could be important during antiviral responses and autoimmune reactions.

Adjuvants, Immunologic↗
PubMed1998

cDNA cloning of human DEC-205, a putative antigen-uptake receptor on dendritic cells.

Dendritic cells (DC) are specialist antigen presenting cells which capture antigens in the periphery, migrate centrally, and present the processed antigens in the context of major histocompatibility complex and appropriate co-stimulatory molecules to T lymphocytes for the initiation of an immune response. DEC-205 has been identified as a putative antigen-uptake receptor, which is expressed abundantly on mouse DC. The recently cloned mouse DEC-205 cDNA predicts a molecular structure which has a marked similarity to the macrophage mannose receptor. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and cDNA library screening, we obtained the full coding region of human DEC-205 cDNA from the Hodgkin's disease-derived L428 cell line. The predicted protein structure is a type I transmembrane protein of 1722 amino acids consisting of a signal peptide, cysteine-rich domain, fibronectin type II domain, ten carbohydrate recognition-like domains, transmembrane domain, and a cytoplasmic tail. Human DEC-205 is 77% identical to the mouse protein with completely conserved cysteines. The DEC-205 gene (LY75) was mapped to chromosome band 2q24 by somatic cell hybrid panel analysis and fluorescent in situ hybridization. Northern blot analysis detected 7.8 and 9.5 kilobase DEC-205 transcripts in myeloid, B lymphoid, and Hodgkin's disease-derived cell lines. RT-PCR analysis indicated that immature blood DC contain a barely detectable amount of DEC-205 transcripts but these were markedly increased upon differentiation/activation.

Amino Acid Sequence↗