D N Hart

Sydney Orthopaedic Research Institute, Sydney, New South Wales, Australia

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Publications (119)465.01 Total impact

  • Molecular Immunology 05/2012; 51(1):28. · 3.00 Impact Factor
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    ABSTRACT: Recombinant monoclonal antibodies currently dominate the protein biologics marketplace. The path from target antigen discovery and screening, to a recombinant therapeutic antibody can be time-consuming and laborious. We describe a set of expression vectors, termed mAbXpress, that enable rapid and sequence-independent insertion of antibody variable regions into human constant region backbones. This method takes advantage of the In Fusion cloning system from Clontech, which allows ligation-free, high-efficiency insertion of the variable region cassette without the addition of extraneous amino acids. These modular vectors simplify the antibody reformatting process during the preliminary evaluation of therapeutic or diagnostic candidates. The resulting constructs can be used directly for transient or amplifiable, stable expression in mammalian cells. The effectiveness of this method was demonstrated by the creation of a functional, fully human anti-human CD83 monoclonal antibody.
    Journal of immunological methods 02/2010; 354(1-2):85-90. · 2.35 Impact Factor
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    ABSTRACT: Dendritic cells (DC) are specialized white blood cells that initiate and direct immune responses. Targeting DC surface proteins to deliver liposomes carrying antigens has demonstrated potential for eliciting antigen-specific immune responses. To evaluate this strategy in preclinical studies, we prepared anti-human DEC-205 immunoliposomes (anti-hDEC-205 iLPSM) and compared their uptake by monocyte-derived DC (MoDC) and blood DC (BDC) with conventional liposomes (cLPSM). Antibody conjugation increased the number of immature MoDC taking up liposomes to 70-80%, regardless of the antibody coupled, whereas less than 20% endocytosed cLPSM. Anti-hDEC-205-IgG specifically increased cell uptake by 15% and the total iLPSM uptake six-fold. The non-specific iLPSM uptake was unlikely to be Fc receptor-mediated as excess immunoglobulins failed to block the uptake. Only a small population (7-24%) of mature MoDC took up cLPSM and control iLPSM. In contrast, approximately 70% of mature MoDC took up anti-hDEC-205 iLPSM, endocytosing 10-fold more iLPSM than the control iLPSM. Anti-hDEC-205 iLPSM uptake by CD1c+ BDC was similar to the immature MoDC, but was five-fold increased compared to the control iLPSM. Confocal microscopy confirmed that the anti-hDEC-205 iLPSM were phagocytosed by DC and available for antigen processing. Thus, DEC-205 is an effective target for delivering liposomes to human DC.
    Vaccine 07/2007; 25(25):4757-66. · 3.49 Impact Factor
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    ABSTRACT: The number and function of human T cells in the periphery are regulated by homeostatic signals received from antigen-presenting cells (APCs) and the common gamma chain (gammac) cytokines interleukin (IL)-7 and IL-15. We found that, in the absence of introduced antigen, blood monocytes or myeloid dendritic cells (MDCs) in the presence of IL-7 and IL-15 (IL-7/IL-15) can regulate CD4(+) T memory (Tm) cell numbers by polyclonal cell proliferation. The dynamics of CD4(+) Tm cell proliferation, in the presence of IL-7/IL-15, was dependent on contact with MDCs and to a lesser extent on contact with monocytes. IL-7/IL-15 either alone or combined with monocytes or MDCs enhanced the proportion of CD4(+) Tm cells with activated and effector phenotype and diminished the helper function of CD4(+) Tm cells. These CD4(+) Tm cells, preconditioned with IL-7/IL-15 alone or with monocytes or MDCs and IL-7/IL-15, reduced T cell-dependent immunoglobulin M (IgM) and IgG responses. This appeared to be a contact-dependent effect involving a reduction in antibody-producing CD27(+) B memory cells, but contact-independent suppression by soluble factors also contributed to the antibody-producing capacity of CD27(+) B memory cells. These results indicate that blood monocytes, MDCs and the cytokines IL-7/IL-15 contribute to homeostasis of CD4(+) Tm cells by regulating their number, activation state and helper/suppressor (regulatory) function. In healthy individuals, this mode of regulating CD4(+) Tm cell homeostasis may provide a basis for the control of autoimmune responses.
    Immunology 04/2007; 120(3):392-403. · 3.74 Impact Factor
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    ABSTRACT: The immune system works through leukocytes interacting with each other, with other cells, with tissue matrices, with infectious agents, and with other antigens. These interactions are mediated by cell-surface glycoproteins and glycolipids. Antibodies against these leukocyte molecules have provided powerful tools for analysis of their structure, function, and distribution. Antibodies have been used widely in hematology, immunology, and pathology, and in research, diagnosis, and therapy. The associated CD nomenclature is commonly used when referring to leukocyte surface molecules and antibodies against them. It provides an essential classification for diagnostic and therapeutic purposes. The most recent (8th) Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA), held in Adelaide, Australia, in December 2004, allocated 95 new CD designations and made radical changes to its aims and future operational strategy in order to maintain its relevance to modern human biology and clinical practice.
    Blood 12/2005; 106(9):3123-6. · 9.78 Impact Factor
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    ABSTRACT: Graft-versus-host disease (GvHD), a life-threatening complication of bone marrow transplantation, is initiated by donor T cells reacting to recipient dendritic cells (DC). GvHD can be controlled by attenuating donor T cells, but few strategies exist to target DC, particularly resident tissue DC, despite recent evidence of their importance. In this report, CMRF-44, a mouse monoclonal IgM reactive to human DC, is tested against human Langerhans cells (LC) in vitro. CMRF-44 antigen is expressed at low level on fresh LC but is up-regulated 40-60-fold during migration. CMRF-44 and complement kill more than 97% of migratory LC in vitro and inhibit allostimulation by LC up to 95%. In comparison, alemtuzumab, which binds CD52, reacts weakly with primary LC and fails to induce significant lysis with complement (less than 5%). These results highlight the potential of new therapeutic antibodies active against tissue DC to control graft-versus-host reactions.
    Transplantation 04/2005; 79(6):722-5. · 3.78 Impact Factor
  • Alison Rice, Derek Hart
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    ABSTRACT: APC-80200 (Mylovenge) has been developed for the treatment of B-cell malignancies and is currently in phase II clinical trials as a therapeutic vaccine for patients with advanced multiple myeloma. This vaccine candidate appears to be of particular benefit in patients who have received high-dose chemotherapy to reduce tumor load following stem cell rescue. APC-80200 is also being tested in patients with amyloidosis.
    Current opinion in molecular therapeutics 11/2002; 4(5):523-7. · 3.42 Impact Factor
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    Blood 06/2002; 99(10):3877-80. · 9.78 Impact Factor
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    The Journal of Immunology 04/2002; 168(5):2083-6. · 5.36 Impact Factor
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    ABSTRACT: This paper reviews the Seventh Human Leucocyte Differentiation Antigen (HLDA7) workshop. Due to the limitations of "blind" antibody screening, which had been evident at the previous meeting in 1996, participants at HLDA7 adopted a more selective approach to the choice of antibodies by identifying new CD specificities. This resulted in the addition of more than 80 new CD specificities. Plans for the eighth and subsequent workshops are also previewed.
    Modern Pathology 02/2002; 15(1):71-6. · 6.36 Impact Factor
  • Gut 01/2002; 99(10):3879-3880. · 13.32 Impact Factor
  • D N Hart
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    ABSTRACT: Dendritic cells (DC) are now recognised as a unique leukocyte type, consisting of two or more subsets. The origins and functional inter-relationships of these cells are the subject of intense basic scientific investigation. They play important roles in initiating and directing immune responses, defending the host from pathogens and maintaining self tolerance. Fundamental studies are defining new molecules and mechanisms associated with DC function. The first methods for counting these rare blood cell populations are already providing interesting new clinical data. Indeed, abnormal DC function may contribute to deficiencies in the immune response against malignancies. Phase I trial data suggests that DC-based cancer vaccination protocols may contribute an important new biological approach to cancer therapy. Manipulation of DC to facilitate allogeneic transplantation and even to manage autoimmune disease are likely developments.
    Pathology 12/2001; 33(4):479-92. · 2.62 Impact Factor
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    ABSTRACT: Dendritic cells (DC) are the only antigen-presenting cells that can activate naïve T lymphocytes and initiate a primary immune response. They are also thought to have a role in immune tolerance. DC traffic from the blood to peripheral tissue where they become activated. They then present antigen and the costimulating signals necessary to initiate an immune response. In this study, we investigated the number, subsets, and activation pattern of circulating and intestinal DC from patients with clinically mild ulcerative colitis (UC) or Crohn's disease. Patients were recruited, if they were not taking immunosuppressive therapy, and were assessed for clinical severity of their disease using for UC, the Clinical Activity Index, and for Crohn's disease, the Crohn's Disease Activity Index. Blood CD11c+ and CD11c- DC subsets, expression of costimulatory antigens, CD86 and CD40, and the early differentiation/activation antigen, CMRF44, were enumerated by multicolor flow cytometry of lineage negative (lin- = CD3-, CD19-, CD14-, CD16-) HLA-DR+ DC. These data were compared with age-matched healthy and the disease control groups of chronic noninflammatory GI diseases (cGI), acute noninflammatory GI diseases (aGI), and chronic non-GI inflammation (non-GI). In addition, cryostat sections of colonoscopic biopsies from healthy control patients and inflamed versus noninflamed gut mucosa of inflammatory bowel disease (IBD) patients were examined for CD86+ and CD40+ lin- cells. Twenty-one Crohn's disease and 25 UC patients, with mean Crohn's Disease Activity Index of 98 and Clinical Activity Index of 3.1, and 56 healthy controls, five cGI, five aGI, and six non-GI were studied. CD11c+ and CD11c- DC subsets did not differ significantly between Crohn's, UC, and healthy control groups. Expression of CD86 and CD40 on freshly isolated blood DC from Crohn's patients appeared higher (16.6%, 31%) and was significantly higher in UC (26.6%, 46.3%) versus healthy controls (5.5%, 25%) (p = 0.004, p = 0.012) and non-GI controls (10.2%, 22.8%) (p = 0.012, p = 0.008), but not versus cGI or aGI controls. CD86+ and CD40+ DC were also present in inflamed colonic and ileal mucosa from UC and Crohn's patients but not in noninflamed IBD mucosa or normal mucosa. Expression of the CMRF44 antigen was low on freshly isolated DC, but it was upregulated after 24-h culture on DC from all groups, although significantly less so on DC from UC versus Crohn's or healthy controls (p = 0.024). The CMRF44+ antigen was mainly associated with CD11c+ DC, and in UC was inversely related to the Clinical Activity Index (r = -0.69, p = 0.0002). There is upregulation of costimulatory molecules on blood DC even in very mild IBD but surprisingly, there is divergent expression of the differentiation/activation CMRF44 antigen. Upregulation of costimulatory molecules and divergent expression of CMRF44 in blood DC was also apparent in cGI and aGI but not in non-GI or healthy controls, whereas intestinal CD86+ and CD40+ DC were found only in inflamed mucosa from IBD patients. Persistent or distorted activation of blood DC or divergent regulation of costimulatory and activation antigens may have important implications for gut mucosal immunity and inflammation.
    The American Journal of Gastroenterology 11/2001; 96(10):2946-56. · 9.21 Impact Factor
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    ABSTRACT: Background: Dendritic cells (DC) are believed to be one of the first cell types infected during HIV transmission. Recently a single C-type lectin receptor (CLR), DC-SIGN, has been reported to be the predominant receptor on monocyte derived DC (MDDC) rather than CD4. The role of other CLRs in HIV binding and HIV binding by CLRs on other types of DC in vivo is largely unknown. Objectives and study design: Review HIV binding to DC populations, both in vitro and in vivo, in light of the immense interest of a recently re-identified CLR called DC-SIGN. Results and conclusions: From recent work, it is clear that immature MDDC have a complex pattern of HIV gp120 binding. In contrast to other cell types gp120 has the potential to bind to several receptors on DC including CD4 and several types of C type lectin receptor, not just exclusively DC-SIGN. Given the diverse types of DC in vivo future work will need to focus on defining the receptors for HIV binding to these different cell types. Mucosal transmission of HIV in vivo targets immature sessile DCs, including Langerhans cells which lack DC-SIGN. The role of CLRs and DC-SIGN in such transmission remains to be defined. (C) 2001 Elsevier Science B.V. All rights reserved.
    Journal of Clinical Virology 11/2001; · 3.47 Impact Factor
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    ABSTRACT: Dendritic cells (DCs) are important targets for human immunodeficiency virus (HIV) because of their roles during transmission and also maintenance of immune competence. Furthermore, DCs are a key cell in the development of HIV vaccines. In both these settings the mechanism of binding of the HIV envelope protein gp120 to DCs is of importance. Recently a single C-type lectin receptor (CLR), DC-SIGN, has been reported to be the predominant receptor on monocyte-derived DCs (MDDCs) rather than CD4. In this study a novel biotinylated gp120 assay was used to determine whether CLR or CD4 were predominant receptors on MDDCs and ex vivo blood DCs. CLR bound more than 80% of gp120 on MDDCs, with residual binding attributable to CD4, reconfirming that CLRs were the major receptors for gp120 on MDDCs. However, in contrast to recent reports, gp120 binding to at least 3 CLRs was observed: DC-SIGN, mannose receptor, and unidentified trypsin resistant CLR(s). In marked contrast, freshly isolated and cultured CD11c(+ve) and CD11c(-ve) blood DCs only bound gp120 via CD4. In view of these marked differences between MDDCs and blood DCs, HIV capture by DCs and transfer mechanisms to T cells as well as potential antigenic processing pathways will need to be determined for each DC phenotype.
    Blood 11/2001; 98(8):2482-8. · 9.78 Impact Factor
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    Stem Cells 10/2001; 19(6):556 - 562. · 7.70 Impact Factor
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    Journal of Leukocyte Biology 08/2001; · 4.30 Impact Factor
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    ABSTRACT: CD83 is an inducible glycoprotein expressed predominantly by dendritic cells (DC) and B lymphocytes. Expression of membrane CD83 (mCD83) is widely used as a marker of differentiated/activated DC but its function and ligand(s) are presently unknown. We report the existence of a soluble form of CD83 (sCD83). Using both a sCD83-specific ELISA and Western blotting, we could demonstrate the release of sCD83 by mCD83(+) B cell and Hodgkin's disease-derived cell lines, but not mCD83(-) cells. Inhibition of de novo protein synthesis did not affect the release of sCD83 during short-term (2 h) culture of cell lines although mCD83 expression was significantly reduced, suggesting sCD83 is generated by the release of mCD83. Isolated tonsillar B lymphocytes and monocyte-derived DC, which are mCD83(low), released only low levels of sCD83 during culture. However, the differentiation/activation of these populations both up-regulated mCD83 and increased sCD83 release significantly. Analysis of sera from normal donors demonstrated the presence of low levels (121 +/- 3.6 pg/ml) of circulating sCD83. Further studies utilizing purified sCD83 and the analysis of sCD83 levels in disease may provide clues to the function and ligand(s) of CD83.
    International Immunology 08/2001; 13(7):959-67. · 3.18 Impact Factor
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    ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells that have the unique ability to initiate a primary immune response. The effect of physiologic stress on circulating blood DCs has thus far not been studied. In this study, we applied a recently developed method of counting blood DCs to test the hypothesis that significant stress to the body such as surgery and exercise might induce measurable changes in the DC numbers, subsets, phenotype, and function. Twenty-six patients scheduled for elective laparoscopic cholecystectomy, 4 for elective hysterectomy, 56 controls, and 5 volunteers who underwent a stress exercise test were enrolled in the study. Absolute DC counts increased acutely (71.7% +/- 11% [SEM], P =.0001) in response to the stress of surgery and dropped below preoperative levels (-25% +/- 14% [SEM], P =.05) on days 2-3. The perioperative DC subset balance remained constant. Interestingly, DC counts changed independently of monocyte counts. Exercise also induced a rise in DC counts but coincidentally with monocyte counts. Surprisingly, no phenotypic or functional activation of DCs was seen in either stress situations in vivo. DCs are rapidly mobilized into the circulation in response to surgical and exercise stress, which may serve to prepare the host's immune defenses against trauma. The independent regulation of the DC and monocyte counts reinforces the distinction between these 2 cell populations.
    Blood 08/2001; 98(1):140-5. · 9.78 Impact Factor
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    ABSTRACT: Heterogeneous expression of several antigens on the three currently defined tonsil dendritic cell (DC) subsets encouraged us to re-examine tonsil DCs using a new method that minimized DC differentiation and activation during their preparation. Three-color flow cytometry and dual-color immunohistology was used in conjunction with an extensive panel of antibodies to relevant DC-related antigens to analyze lin(-) HLA-DR(+) tonsil DCs. Here we identify, quantify, and locate five tonsil DC subsets based on their relative expression of the HLA-DR, CD11c, CD13, and CD123 antigens. In situ localization identified four of these DC subsets as distinct interdigitating DC populations. These included three new interdigitating DC subsets defined as HLA-DR(hi) CD11c(+) DCs, HLA-DR(mod) CD11c(+) CD13(+) DCs, and HLA-DR(mod) CD11c(-) CD123(-) DCs, as well as the plasmacytoid DCs (HLA-DR(mod) CD11c(-) CD123(+)). These subsets differed in their expression of DC-associated differentiation/activation antigens and co-stimulator molecules including CD83, CMRF-44, CMRF-56, 2-7, CD86, and 4-1BB ligand. The fifth HLA-DR(mod) CD11c(+) DC subset was identified as germinal center DCs, but contrary to previous reports they are redefined as lacking the CD13 antigen. The definition and extensive phenotypic analysis of these five DC subsets in human tonsil extends our understanding of the complexity of DC biology.
    American Journal Of Pathology 08/2001; 159(1):285-95. · 4.60 Impact Factor

Publication Stats

3k Citations
465.01 Total Impact Points

Institutions

  • 2012
    • Sydney Orthopaedic Research Institute
      Sydney, New South Wales, Australia
  • 2005
    • Newcastle University
      Newcastle-on-Tyne, England, United Kingdom
  • 2002
    • University of Oxford
      • Nuffield Division of Clinical Laboratory Sciences
      Oxford, ENG, United Kingdom
  • 2001–2002
    • Oxford University Hospitals NHS Trust
      • Department of Haematology
      Oxford, ENG, United Kingdom
    • University of Queensland
      Brisbane, Queensland, Australia
  • 1999–2001
    • Mater Hospital
      Brisbane, Queensland, Australia
  • 1985–2001
    • Canterbury District Health Board
      • • Department of Haematology
      • • South Island Bone Marrow Transplant Unit (BMTU)
      Christchurch, Canterbury, New Zealand
  • 2000
    • University of Otago
      • Department of Medicine (Dunedin)
      Dunedin, Otago, New Zealand
  • 1999–2000
    • Mater Medical Research Institute
      South Brisbane, Queensland, Australia