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Background: Prostate cancer is one of the leading causes of cancer deaths in males and there are currently no effective treatments available for metastatic disease. Although recent clinical trials using dendritic cell (DC) based immunotherapy treatments have demonstrated safety, immunological responses, and some clinical efficacy, better vaccine delivery strategies need to be developed. We have undertaken the first detailed analysis of blood DC (BDC) subsets and their function in prostate cancer patients, with a view to utilizing immunoselected BDC for immunotherapy., Methods: We enumerated the CD11c+CD1c+, CD11c+CD16+, and CD11c-CD123+ BDC subsets in whole blood of prostate cancer patients using a single platform TruCOUNT assay. These subsets were identified and purified using flow cytometry and immunomagnetic selection, and their functional capacity analyzed by costimulatory molecule expression, cytokine secretion, and antigen presenting ability., Results: There were no significant differences in the number or composition of these subsets compared to healthy donors and these cells could be purified with equal efficiency from both groups. The prostate cancer patients BDC had similar levels of key costimulatory molecules and cytokine expression profiles, compared to healthy donors, and these were upregulated to the same extent, in response to exogenous stimuli. BDC from both groups were capable of eliciting allogeneic proliferative responses and inducing autologous CD4+ responses to naïve and recall antigens, and antigen-specific CD8+ responses to influenza matrix protein and prostate specific antigen., Conclusions: These results indicate that an immunoselected CD1c+ BDC preparation could provide a suitable vaccine delivery vehicle for future prostate cancer immunotherapy trials., (Copyright 2005 Wiley-Liss, Inc.)

Langerhans cells (LC) and other antigen-presenting cells are believed to be critical in initiating graft versus host responses that influence the outcome of allogeneic hematopoietic stem cell transplantation. However, their fate in humans is poorly understood. We have sought to define the effect of conditioning regimes and graft versus host disease (GVHD) on the survival of recipient LC and reconstitution of donor cells after transplant. Confocal microscopy of epidermal sheets shows that full intensity transplant (FIT) depletes LC more rapidly than reduced intensity transplant (RIT) at day 0, although the nadir is similar in both at 14-21 d. Recovery occurs rapidly within 40 d in the absence of acute GVHD, but is delayed beyond 100 d when GVHD is active. LC chimerism was determined in sex-mismatched transplants using a two-step Giemsa/fluorescence in situ hybridization assay on isolated cells. Acquisition of donor chimerism at 40 d is more rapid after FIT (97%) than RIT (36.5%), irrespective of blood myeloid engraftment. At 100 d, all transplants achieve at least 90% LC donor chimerism and over half achieve 100%. Complete donor chimerism is associated with prior acute cutaneous GVHD, suggesting a role for allogeneic T cells in promoting LC engraftment.

Lymphoproliferative disorders, including follicular lymphoma (FL), multiple myeloma (MM) and chronic lymphatic leukaemia (CLL), are slowly progressive malignancies which remain incurable despite advances in therapy. Harnessing the immune system to recognise and destroy tumours is a promising new approach to treating these diseases. Dendritic cells (DC) are unique antigen-presenting cells that play a central role in the initiation and direction of immune responses. DC loaded ex vivo with tumour-associated antigens and administered as a vaccine have already shown promise in early clinical trials for a number of lymphoproliferative disorders, but the need for improvement is widely agreed. Recent advances in the understanding of basic DC biology and lessons from early clinical trials have provided exciting new insights into the generation of anti-tumour immune responses and the design of vaccine strategies. In this review we provide an overview of our current understanding of DC biology and their function in patients with lymphoproliferative disorders. We discuss the current status of clinical trials and new approaches to exploit the antigen presenting capacity of DC to design vaccines of the future.

Dendritic cells (DCs) loaded with tumor-associated antigens are a promising treatment to prevent disease relapse in patients with multiple myeloma (MM). Early-phase clinical trials have shown safety, efficacy, and immunologic responses in MM, but a key issue now is the isolation of a functional, clinically relevant DC preparation. The authors have described a unique blood DC (BDC) isolation platform based on positive immunoselection with the CMRF-56 antibody. To validate this as a feasible source of BDCs for immunotherapy, the authors undertook a quantitative and functional analysis of BDCs in MM patients and healthy donors. These data show that MM patients have similar numbers of CD11c+CD16+ and CD11c+CD16- BDCs but about half the number of CD11c-CD123+ BDCs in whole blood compared with healthy donors. BDCs could be isolated by CMRF-56+ immunoselection from all MM patients tested, with similar yields and purity to healthy donors. These BDCs could be activated ex vivo with poly I:C or LPS. Furthermore, CMRF-56+ preparations could induce potent CD4+ and CD8+ T-lymphocyte responses in both MM patients and healthy donors. These data suggest that BDCs with in vitro functional integrity can be isolated from MM patients in sufficient numbers to justify a clinical trial.

Dendritic cells (DC) are specialist antigen presenting cells that play a role in the initiation of innate and adaptive immune response. At the seventh Human Leucocyte Differentiation Antigen workshop, these intriguing cell populations were included as a separate lineage of leucocytes. This paper reports the studies performed in the eighth Human Leucocyte Differentiation Antigen workshop as part of the DC section. Many investigators currently focus on DC that are derived from a number of different leucocyte populations, including those that are differentiated in vitro and cells that are purified ex vivo. The DC section assessed the surface expression of different leucocyte surface molecules on a range of different DC populations. The results summarise the expression of each molecule on dendritic cell populations and differences between different DC preparations. Eleven new CDs were allocated on the basis of monoclonal antibodies and molecular information that identify known cell surface molecules expressed by dendritic cells. This paper gives a brief review of the work that was performed during the HLDA8 and a summary of the CDs represented by submitted mAb.

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.

CD123(hi) CD11c(-) dendritic cells (CD123(hi) DC) are a distinct subset of human DC present in bone marrow, blood, lymphoid organs, and peripheral tissues. Pathogen stimulation, cytokine, or CD40 ligation induces CD123(hi) DC maturation, involving a shift from their innate immune to cognate antigen-presenting functions. In this study, we revealed that blood CD123(hi) DC in the presence of cytokine (granulocyte macrophage-colony stimulating factor and interleukin-3) undergo progressive, step-wise maturation through an "early" stage, delineated by expression of the antigen detected by the new monoclonal antibody CMRF58 (CD123(hi)CMRF58(+)CD40(-)CD86(-)CD83(-)) to the "late" stage with costimulatory antigen expression (CD123(hi)CMRF58(+)CD40(+)CD86(+)CD83(+/-)). In this early stage, cytokine-maintained CD123(hi) DC do not display changes in their morphology, no longer produce interferon-alpha (IFN-alpha) in response to bacteria, and develop the capacity to induce proliferation and polarization of allogeneic T cells. CD123(hi)CMRF58(+) DC, phenotypically similar to in vitro cytokine-maintained CD123(hi) DC, were not detected in tonsil but are present in allergen-challenged nasal mucosa of allergic individuals. Thus, CD123(hi) DC in certain tissue environments such as allergen-challenged nasal mucosa share a common CD123(hi)CMRF58(+) phenotype with in vitro cytokine-maintained blood CD123(hi) DC characterized by lack of IFN-alpha production.

CD38 is a cell surface molecule with ADP-ribosyl cyclase activity, which is predominantly expressed on lymphoid and myeloid cells. CD38 has a significant role in B-cell function as some anti-CD38 antibodies can deliver potent growth and differentiation signals, but the ligand that delivers this signal in mice is unknown. We used a chimeric protein of mouse CD38 and human immunogobulin G (IgG) (CD38-Ig) to identify a novel ligand for murine CD38 (CD38L) on networks of follicular dendritic cells (FDCs) as well as dendritic cells (DCs) in the spleen. Flow-cytometry found that all DC subsets expressed cytoplasmic CD38L but only fresh ex vivo CD11c+ CD11b- DCs had cell surface CD38L. Anti-CD38 antibody blocked the binding of CD38-Ig to CD38L, confirming the specificity of detection. CD38-Ig immuno-precipitated ligands of 66 and 130 kDa. Functional studies found that CD38-Ig along with anti-CD40 and anti-major histocompatibility complex (MHC) class II antibody provided maturation signals to DCs in vitro. When CD38-Ig was administered in vivo with antigen, IgG2a responses were significantly reduced, suggesting that B and T cells expressing CD38 may modulate the isotype of antibodies produced through interaction with CD38L on DCs. CD38-Ig also expanded FDC networks when administered in vivo. In conclusion, this study has identified a novel ligand for CD38 which has a role in functional interactions between lymphocytes and DCs or FDCs.

We have studied the mechanisms by which human cytomegalovirus (HCMV) infection of monocyte-derived dendritic cells (moDCs) contribute to immune suppression. Unlike infection of immature moDCs, infection of mature moDCs is not lytic and results in minimally decreased surface major histocompatibility complex (MHC) and costimulatory molecule expression. The presence of a small percentage of CMV-infected mature moDCs, or the transfer of supernatant from infected moDCs depleted of infectious virions, is nevertheless sufficient to cause marked inhibition of immunostimulation by normal uninfected moDCs. Neither viral nor human interleukin 10 (IL-10) nor transforming growth factor-beta-1 (TGF-beta-1) could account for this inhibition. In contrast, we show that infected mature moDCs lose surface CD83 while maintaining intracellular protein expression. Soluble CD83 accumulates in the supernatants of CMV-infected mature moDCs, and CD83 immunodepletion removes the inhibitory effect of these supernatants on normal DC immunostimulation. We have thus discovered a new mechanism by which HCMV infection may establish a nonlytic reservoir in mature moDCs that inhibits DC-mediated T-cell responses.

Therapy for patients with multiple myeloma (MM) is currently unsatisfactory and most patients eventually succumb to relapsed disease. DCs are a subset of leukocytes with the capacity to initiate and control the adaptive immune response against many cancers, including MM. In MM patients, in vivo DC function is often abnormal, however, it appears that it can be restored by in vitro manipulation. This has led to the development of DC immunotherapy for MM patients. We review the background research leading to the recognition of an anti-MM immune response, and discuss abnormalities in DC function, potential tumor-associated Ags, and the results of clinical trials of DC immunotherapy in MM patients.

Dendritic cells (DC) from distinct DC subsets are essential contributors to normal human immune responses. Despite this, reliable assays that enable DC to be counted precisely have been slow to evolve. We have now developed a new single-platform flow cytometric assay based on TruCOUNT beads and the whole blood "Lyse/No-Wash" protocol that allows precise counting of the CD14(-) blood DC subsets: CD11c(+)CD16(-) DC, CD11c(+)CD16(+) DC, CD123(hi) DC, CD1c(+) DC and BDCA-3(+) DC. This assay requires 50 microl of whole blood; does not rely on a hematology blood analyser for the absolute DC counts; allows DC counting in EDTA samples 24 h after collection; and is suitable for cord blood and peripheral blood. The data is highly reproducible with intra-assay and inter-assay coefficients of variation less than 3% and 11%, respectively. This assay does not produce the DC-T lymphocyte conjugates that result in DC counting abnormalities in conventional gradient-density separation procedures. Using the TruCOUNT assay, we established that absolute blood DC counts reduce with age in healthy individuals. In preliminary studies, we found a significantly lower absolute blood CD11c(+)CD16(+) DC count in stage III/IV versus stage I/II breast carcinoma patients and a lower absolute blood CD123(hi) DC count in multiple myeloma patients, compared to age-matched controls. These data indicate that scientific progress in DC counting technology will lead to the global standardization of DC counting and allow clinically meaningful data to be obtained.

This review describes and compares the different DC preparations currently under laboratory and clinical investigation as vehicles for cancer immunotherapy.

Understanding the biology of the DC and its pivotal role in immune response initiation and regulation has enabled new effective anti-cancer therapies to be developed for treatment of a wide range of tumor types. Many studies on DC-based cancer vaccine development have focused on the development of methods that can effectively deliver tumor Ags to DCs. Numerous methods have been developed or evaluated for the delivery of defined and undefined tumor Ags to DCs for processing and presentation to T lymphocytes. This review provides a brief summary of these methods, the techniques that are used in each, how they have -- or could -- be applied clinically, as well as the advantages and disadvantages of each method.

Despite rapid advances in cancer therapeutics, relapsed disease due to failed immunosurveillance remains a major problem in many cancers. Dendritic cells are recognized as key to the induction of immune responses to cancer and intensive study is underway to facilitate their use in cancer immunotherapy. In initial clinical trials, dendritic cell preparations were, with the benefit of hindsight, largely sub-optimal, yet encouraging results have been seen. The challenge now is to expand our knowledge of the interactions between tumors and the immune system, through basic scientific research and coordinated large-scale clinical studies. This review focuses on the anti-tumor immune response, human dendritic cell biology and the results of recent clinical studies of dendritic cell immunotherapy for cancer.

The long-held belief that breast cancer is a weakly immunogenic tumor and a poor candidate for immunotherapy should be reappraised. There is ample evidence for the existence of an immune response, which is, however, attenuated by multiple inhibitory factors. Many tumor-associated antigens (TAA) have been identified in breast cancer, some of which appear to play a critical role in tumorigenesis and may be attractive targets for immunotherapy. There is evidence for DC recruitment and activation within breast cancers, and the presence of intratumoral activated DCs impacts favorably upon survival. Furthermore, there is a striking paucity of activated DCs within the primary draining or sentinel lymph nodes of breast cancers. Tumor infiltrating lymphocytes (TIL) are often documented, however, their function is impaired by inhibitory cytokines, increased regulatory T lymphocyte activity, tumor cell MHC molecule alterations, and aberrant Fas ligand expression, amongst others. DCs are recognized as one of the critical interfaces between a cancer and the immune system, and have emerged as a promising platform for cancer vaccination via ex vivo immunomodulation. Clinical evaluation of DC vaccination in breast cancer is still relatively limited, although evolving. This article details evidence for the immune response in breast cancer and its many failings, and reviews the clinical trials and significant preclinical data which, taken together, validate the concept of DC vaccination in breast cancer.

Classic Hodgkin's lymphoma (HL) tissue contains a small population of morphologically distinct malignant cells called Hodgkin and Reed-Sternberg (HRS) cells, associated with the development of HL. Using 3'-rapid amplification of cDNA ends (RACE) we identified an alternative mRNA for the DEC-205 multilectin receptor in the HRS cell line L428. Sequence analysis revealed that the mRNA encodes a fusion protein between DEC-205 and a novel C-type lectin DCL-1. Although the 7.5-kb DEC-205 and 4.2-kb DCL-1 mRNA were expressed independently in myeloid and B lymphoid cell lines, the DEC-205/DCL-1 fusion mRNA (9.5 kb) predominated in the HRS cell lines (L428, KM-H2, and HDLM-2). The DEC-205 and DCL-1 genes comprising 35 and 6 exons, respectively, are juxtaposed on chromosome band 2q24 and separated by only 5.4 kb. We determined the DCL-1 transcription initiation site within the intervening sequence by 5'-RACE, confirming that DCL-1 is an independent gene. Two DEC-205/DCL-1 fusion mRNA variants may result from cotranscription of DEC-205 and DCL-1, followed by splicing DEC-205 exon 35 or 34-35 along with DCL-1 exon 1. The resulting reading frames encode the DEC-205 ectodomain plus the DCL-1 ectodomain, the transmembrane, and the cytoplasmic domain. Using DCL-1 cytoplasmic domain-specific polyclonal and DEC-205 monoclonal antibodies for immunoprecipitation/Western blot analysis, we showed that the fusion mRNA is translated into a DEC-205/DCL-1 fusion protein, expressed in the HRS cell lines. These results imply an unusual transcriptional control mechanism in HRS cells, which cotranscribe an mRNA containing DEC-205 and DCL-1 prior to generating the intergenically spliced mRNA to produce a DEC-205/DCL-1 fusion protein.

Background: Activated dendritic cells (DC) initiate immune responses by presenting antigen, including alloantigen from tissue grafts, to T lymphocytes. The potential to deplete or inactivate differentiated-activated DC during allogeneic transplantation represents a new approach to immunosuppression., Methods: The authors investigated the potential of the monoclonal antibody CMRF-44, which has specificity for a DC-associated differentiation-activation antigen, to induce complement-mediated lysis of activated human DC. Peripheral blood mononuclear cells (PBMC), or purified DC preparations, were cultured overnight to activate endogenous DC, resulting in the expression of CMRF-44 antigen and CD83. These were then treated with CMRF-44 and complement. Depletion of activated DC was monitored by flow cytometry., Results: Eighty-nine percent of activated (CD83+) DC in cultured PBMC were depleted by treatment with CMRF-44 and autologous serum (AS) (complement source; mean percentage of CD83+-CD14--CD19- cells=0.06%; cf 0.50% for heat-inactivated AS controls, P<0.0005, n=7). Ninety-five percent of cultured purified myeloid DC were depleted by this treatment, compared with only 43% of similarly treated lymphoid DC. Overnight culture also increases CMRF-44 antigen on a proportion of B cells and mononuclears, but only 24% of these cells were depleted. This treatment considerably reduced the ability of PBMC to stimulate allogeneic CD4+ CD45RA+ T lymphocytes. Similarly, the T-cell proliferative responses to recall and naive antigens were significantly reduced., Conclusions: CMRF-44 may be a suitable candidate for a new selective immunosuppressive strategy, targeting differentiated-activated but not resting DC. It may have applications in preventing GVHD in allogeneic bone marrow transplantation and facilitate immunoacceptance of solid organ allografts.

Changes in blood dendritic cell (BDC) counts (CD123(hi)BDC and CD11c(+)BDC) and expression of CD62L, CCR7, and CD49d were analyzed in healthy donors, multiple myeloma (MM), and non-Hodgkin lymphoma (NHL) patients, who received granulocyte-colony stimulating factor (G-CSF) containing peripheral blood stem cell (PBSC) mobilization protocols. Low-dose G-CSF in healthy donors (8-10 microg/kg/d subcutaneously) and high-dose G-CSF in patients (30 microg/kg/d) increased CD123(hi)BDC (2- to 22-fold, mean 3.7 x 10(6)/L-17.7 x 10(6)/L and 1.9 x 10(6)/L-12.0 x 10(6)/L) in healthy donors and MM but decreased CD11c(+)BDC (2- to 10-fold, mean 5.7 x 10(6)/L-1.6 x 10(6)/L) in NHL patients, on the day of apheresis, compared with steady state. After apheresis, CD123(hi)BDC counts remained high, whereas low CD11c(+)BDC counts tended to recover in the following 2-5 days. Down-regulation of CD62L and up-regulation of CCR7 on CD123(hi)BDC were found in most healthy donors and MM patients. CD49d expression was unchanged. Thus, PBSC mobilization may change BDC counts by altering molecules necessary for BDC homing from blood into tissues.

Dendritic cells (DC) for cancer immunotherapy protocols are generated most commonly by in vitro differentiation of monocytes with exogenous cytokines (Mo-DC). However, Mo-DC differ in their molecular phenotype and function from blood DC (BDC). Clinical isolation of BDC has been limited to the use of density gradients, which result in low yields of variable purity. We have developed a DC enrichment platform, which uses the CMRF-44 (IgM) or CMRF-56 (IgG) monoclonal antibodies (mAb) to select BDC that express these antigens after a short overnight incubation. After culture of peripheral blood mononuclear cells (PBMC) in autologous/AB serum, biotinylated CMRF-44 was used to select DC in a single step immuno-magnetic bead procedure; this produced populations containing up to 99% CMRF-44(+) cells, including up to 67% CMRF-44(+) CD14(-) CD19(-) DC, from an initial starting population of approximately 0.5%. We observed consistent differences in the purities obtained from individual donors with a mean of 54% CMRF-44(+) cells (range 19-99%). Similar results were obtained using biotinylated CMRF-56 mAb, an antibody identifying a comparable population in cultured PBMC. We recovered an average of 54% and 66% of the available BDC in separations performed with the CMRF-44 and CMRF-56 mAb, respectively. The reproducibility of the procedure and the ability to perform it in a closed sterile system makes it suitable for clinical use. Larger scale preparations starting from apheresis derived PBMC will produce sufficient BDC for immunotherapy protocols. The purified BDC elicited strong allogeneic mixed leukocyte reactions and HLA classes II- and I-restricted antigen-specific primary immune responses.

The granulocyte colony-stimulating factor (G-CSF) and Flt-3 receptor agonist progenipoietin-1 (ProGP-1) has potent effects on dendritic cell (DC) expansion and may be an alternative to G-CSF for the mobilization of stem cells for allogeneic stem cell transplantation (SCT). We studied the ability of stem cell grafts mobilized with this agent to induce graft-versus-host disease (GVHD) to minor and major histocompatibility antigens in the well-described B6 --> B6D2F1 SCT model. ProGP-1, G-CSF, or control diluent was administered to donor B6 mice. ProGP-1 expanded all cell lineages in the spleen, and unseparated splenocytes from these animals produced large amounts of interleukin 10 (IL-10) and transforming growth factor beta (TGFbeta) whereas the expression of T-cell adhesion molecules was diminished. Transplantation survival was 0%, 50%, and 90% in recipients of control-, G-CSF-, and ProGP-1-treated allogeneic donor splenocytes, respectively (P <.0001). Donor pretreatment with ProGP-1 allowed a 4-fold escalation in T-cell dose over that possible with G-CSF. Donor CD4 T cells from allogeneic SCT recipients of ProGP-1 splenocytes demonstrated an anergic response to host antigen, and cytokine production (interferon gamma [IFNgamma], IL-4, and IL-10) was also reduced while CD8 T-cell cytotoxicity to host antigens remained intact. Neither CD11c(hi) DCs nor CD11c(dim)/B220(hi) DCs from ProGP-1-treated animals conferred protection from GVHD when added to control spleen. Conversely, when equal numbers of purified T cells from control-, G-CSF-, or ProGP-1-treated allogeneic donors were added to allogeneic T-cell-depleted control spleen, survival at day 60 was 0%, 15%, and 90%, respectively (P <.0001). The improved survival in recipients of ProGP-1 T cells was associated with reductions in systemic tumor necrosis factor alpha generation and GVHD of the gastrointestinal tract. We conclude that donor pretreatment with ProGP-1 is superior to G-CSF for the prevention of GVHD after allogeneic SCT, primarily due to incremental affects on T-cell phenotype and function.

Dendritic cells (DCs) are key antigen-presenting cells for stimulating immune responses and they are now being investigated in clinical settings. Although defined as lineage-negative (Lin(-)) HLA-DR(+) cells, significant heterogeneity in these preparations is apparent, particularly in regard to the inclusion or exclusion of CD14(+), CD16(+), and CD2(+) cells. This study used flow cytometry and a panel of monoclonal antibodies (mAbs), including reagents from the 7th Leukocyte Differentiation Antigen Workshop, to define the cellular composition of 2 standardized peripheral blood mononuclear cell (PBMCs)-derived Lin(-) HLA-DR(+) preparations. Lin(-) cells were prepared from PBMCs by depletion with CD3, CD14, CD19, CD11b, and either CD16 or CD56 mAbs. Analysis of the CD16-replete preparations divided the Lin(-) HLA-DR(+) population into 5 nonoverlapping subsets (mean +/- 1 SD): CD123 (mean = 18.3% +/- 9.7%), CD1b/c (18.6% +/- 7.6%), CD16 (49.6% +/- 8.5%), BDCA-3 (2.7% +/- 1.4%), and CD34 (5.0% +/- 2.4%). The 5 subsets had distinct phenotypes when compared with each other, monocytes, and monocyte-derived DCs (MoDCs). The CD85 family, C-type lectins, costimulatory molecules, and differentiation/activation molecules were also expressed differentially on the 5 Lin(-) HLA-DR(+) subsets, monocytes, and MoDCs. The poor viability of CD123(+) DCs in vitro was confirmed, but the CD16(+) CD11c(+) DC subset also survived poorly. Finally, the individual subsets used as stimulators in allogeneic mixed leukocyte reactions were ranked by their allostimulatory capacity as CD1b/c > CD16 > BDCA-3 > CD123 > CD34. These data provide an opportunity to standardize the DC populations used for future molecular, functional and possibly even therapeutic studies.

T cells are present in the inflammatory infiltrates of periodontal disease lesions and require antigen presentation by antigen-presenting cells (APCs). While it is still not known whether Th1 or Th2 cells predominate in these lesions, it has been reported that different APCs may induce activation of different T-cell subsets. An immunoperoxidase technique was used to investigate the presence of CD1a+, CMRF-44+, CMRF-58+ and CD83+ dendritic cells, CD14+ macrophages or dendritic cell precursors and CD19+ B cells in gingival biopsies from 21 healthy or gingivitis and 25 periodontitis subjects. The samples were divided into three groups according to the size of infiltrate (group 1, small infiltrates; group 2, medium infiltrates; group 3, extensive infiltrates). The presence of numerous CD1a+ Langerhans cells was noted in the epithelium with no differences between the healthy/gingivitis and periodontitis groups. The percentage of CD83+ dendritic cells in the infiltrates was higher than the percentage of CD1a+, CMRF-44+ or CMRF-58+ dendritic cells. Endothelial cells positive for CD83 were found predominantly in areas adjacent to infiltrating cells, CD83+ dendritic cells being noted in the region of CD83+ endothelium. The percentage of CD14+ cells in the inflammatory infiltrates was similar to that of CD83+ dendritic cells. B cells were the predominant APC in group 2 and 3 tissues. The percentage of B cells in group 3 periodontitis lesions was increased in comparison with group 1 periodontitis tissues and also in comparison with group 3 healthy/gingivitis sections. Functional studies are required to determine the roles of different APC subpopulations in periodontal disease.

Dendritic cells (DCs) initiate and direct immune responses. Recent studies have defined different DC populations, therefore we undertook this study comparing 2 types of myeloid DCs: blood CD11c(+) DCs and in vitro monocyte-derived DCs (Mo-DCs), which are both candidates as cellular adjuvants for cancer immunotherapy. Blood CD11c(+) DCs were prepared by cell sorting from peripheral blood mononuclear cells cultured overnight in RPMI 1640 medium supplemented with autologous or pooled AB serum. Mo-DCs were prepared in the same medium using granulocyte macrophage-colony-stimulating factor (GM-CSF)/interleukin 4 (IL-4) and differentiated/activated with lipopolysaccharide or monocyte-conditioned medium (ActMo-DCs). Morphologically, differences between the DC preparations were noted both at a light and and electron microscopic level. Blood CD11c(+) DCs expressed similar levels of HLA-DR, CD40, CD86, and CD83 as Mo-DCs. CD209 was present on Mo-DCs but not on blood CD11c(+) DCs. Blood CD11c(+) DCs generated a lower proliferative mixed leukocyte response (MLR) than Mo-DCs. Blood CD11c(+) DCs loaded with 0.1 microg/mL tetanus toxoid (TT)-generated greater T lymphocyte proliferative responses than did Mo-DCs or ActMo-DCs, but when loaded with higher TT concentrations no difference in T lymphocyte proliferative response was observed. Keyhole limpet hemocyanin (KLH)-loaded blood CD11c(+) DCs generated greater T lymphocyte proliferative responses than Mo-DCs or ActMo-DCs. Allogeneic MLR- or KLH-specific responses induced by blood CD11c(+) DCs generated more Th1 effectors than the responses induced by Mo-DCs or ActMo-DCs. These data establish several differences in the properties of blood CD11c(+) DCs, Mo-DCs, and ActMo-DCs, which suggest that blood DCs merit further consideration as DC preparations for clinical programs are evolved.

The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

The fundamental role of dendritic cells (DC) in initiating and directing the primary immune response is well established. Furthermore, it is now accepted that DC may be useful in new vaccination strategies for preventing certain malignant and infectious diseases. As blood DC (BDC) physiology differs from that of the DC homologues generated in vitro from monocyte precursors, it is becoming more relevant to consider BDC for therapeutic interventions. Until recently, protocols for the isolation of BDC were laborious and inefficient; therefore, their use for investigative cancer immunotherapy is not widespread. In this study, we carefully documented BDC counts, yields and subsets during apheresis (Cobe Spectra), the initial and essential procedure in creating a BDC isolation platform for cancer immunotherapy. We established that an automated software package (Version 6.0 AutoPBPC) provides an operator-independent reliable source of mononuclear cells (MNC) for BDC preparation. Further, we observed that BDC might be recovered in high yields, often greater than 100% relative to the number of circulating BDC predicted by blood volume. An average of 66 million (range, 17-179) BDC per 10-l procedure were obtained, largely satisfying the needs for immunization. Higher yields were possible on total processed blood volumes of 15 l. BDC were not activated by the isolation procedure and, more importantly, both BDC subsets (CD11c(+)CD123(low) and CD11c(-)CD123(high)) were equally represented. Finally, we established that the apheresis product could be used for antibody-based BDC immunoselection and demonstrated that fully functional BDC can be obtained by this procedure.

Purpose: Prostate specific antigen (PSA) is found in high concentration in prostate tissue and in semen, in which its physiological function appears to be liquefaction. In prostate cancer the peripheral PSA concentration is elevated, which may be used as a disease marker. Systemic and local immune defects have been demonstrated in prostate cancer and we postulated a role for PSA in this immunosuppression. We explored the effects of PSA on human T-lymphocyte proliferation in vitro., Materials and Methods: PSA was purified from normal seminal plasma using a modified chromatographic technique. The effect of PSA or control protein on lymphocyte responses to mitogens, tetanus toxoid and alloantigens was tested. The inhibitory effect observed was further explored by varying the time of PSA addition, denaturing PSA and including interleukin-2 and anti-PSA antibodies., Results: PSA suppressed in vitro phytohemagglutinin and alloantigen stimulated lymphocyte proliferation in a dose dependent manner. This effect was reversed by adding anti-PSA antibodies but not by interleukin-2., Conclusions: These in vitro PSA effects suggest another T-lymphocyte mediated immunosuppressive mechanism. In vivo high levels of PSA may compromise natural immune responses to cancer and current attempts at immunotherapy for prostate cancer.

Differentiation of CD34(+) haematopoietic stem cells into functional dendritic cells (DC) was investigated using the mAb CMRF-44 and other mAb against DC-associated markers. GM-CSF mobilized peripheral blood stem cells were obtained from healthy donors by leukapheresis. CD34(+) cells were purified using CD34(+)-positive selection,and subsequent immunomagnetic depletion of CD14 and CD2 cells. CD34(+) cells were cultured in medium supplemented with one or more of GM-CSF,TNF-alpha, IL-4 or IL-6. CMRF-44 Ag expression was monitored by flow cytometry, and DC function by allogeneic MLR and tetanus toxoid(TT) presentation assays. CD34(+) cells quickly acquired the CMRF-44 Ag when cultured in the presence of TNF-alpha. By day 3, more than 50% of the cells were double-positive for CD34 and CMRF-44. CD34 expression was gradually lost, so that by day 9, the majority of the cells were CD34(-)/CMRF-44(+).GM-CSF and TNF-alpha also induced CD40 expression, and up-regulation of CD54 and MHC class II on CD34(+) cells; their expression was correlated to the CMRF-44 Ag. Day 3 CD34(+)/CMRF-44(+) cells,but not CD34(+)/CMRF-44(-) cells, become potent APC when cultured further with GM-CSF plus TNF-alpha. These CMRF-44(+) cells were potent inducers of Th1-type immune response in the primary allogeneic MLR and present TT to autologous CD4(+) T cells. TNF-alpha alone is sufficient to induce CMRF-44 expression on CD34(+) cells, but in combination with GM-CSF expands the CMRF-44(+) population. CMRF-44 expression correlates with DC function and may be a useful early marker for commitment of CD34(+) cells to the DC differentiation pathway.

Studies on purified blood dendritic cells (DCs) are hampered by poor viability in tissue culture. We, therefore, attempted to study some of the interactions/relationships between DCs and other blood cells by culturing unseparated peripheral blood mononuclear cell (PBMC) preparations in vitro. Flow cytometric techniques were used to undertake a phenotypic and functional analysis of DCs within the cultured PBMC population. We discovered that both the CD11c(+) and CD11c(-) CD123(hi) DC subsets maintained their viability throughout the 3-day culture period, without the addition of exogenous cytokines. This viability was accompanied by progressive up-regulation of the surface costimulatory (CD40, CD80, CD86) and activation (CMRF-44, CMRF-56, CD83) molecules. The survival and apparent production of DCs in PBMC culture (without exogenous cytokines) and that of sorted DCs (with cytokines) were evaluated and compared by using TruCOUNT analysis. Absolute DC counts increased (for CD123(hi) and CD11c(+) subsets) after overnight culture of PBMCs. Single-cell lineage depletion experiments demonstrated the rapid and spontaneous emergence of "new" in vitro generated DCs from CD14(+)/CD16(+) PBMC radioresistant precursors, additional to the preexisting ex vivo DC population. Unlike monocyte-derived DCs, blood DCs increased dextran uptake with culture and activation. Finally, DCs obtained after culture of PBMCs for 3 days were as effective as freshly isolated DCs in stimulating an allogeneic mixed leukocyte reaction.

Vaccines are one of the most cost effective methods of improving public health thereby increasing the quality of life. Prophylactic and therapeutic treatment by vaccines can prevent infectious diseases and some cancers and could also be used in the treatment of autoimmune disorders. An appreciation of this potential has resulted in a burgeoning literature which not only describes the scientific efforts being made into designing new and improved vaccines but also drives the efforts being made by public health organizations world-wide in delivering vaccines to the community. At the forefront of technologies being applied to the design of vaccines is the use of synthetic peptides; the chemical technologies used to assemble peptides have made great strides over the last decade and assembly of hi-fidelity peptides which can be of high molecular weight, multimeric or even branched is now almost routine. Together with the advances in peptide technology our understanding of the molecular events that are necessary to induce immune responses has also made great strides. The central role that peptides play in immune recognition is now recognised and rules are emerging that are being applied to the construction of peptide-based vaccines that, in the right context, can induce humoral (antibody) and cellular (cytotoxic and helper T cell) immune responses. Synthetic peptides are exquisitely placed to answer questions about immune recognition and along the way to provide us with new and improved vaccines.

Dendritic cells (DC) are the professional antigen-presenting cells that initiate immune responses. While DC take up antigen, migrate to lymph nodes and present processed antigen to T lymphocytes, little is known of the intracellular biochemical pathways controlling these events. Using the differential display technique, employing the activated blood DC-like cell line L428, we isolated a cDNA induced during DC differentiation likely to have a regulatory function. This cDNA encoded a putative 530-amino-acid (aa) protein consisting of a unique hydrophilic domain (106 aa) and a domain (424 aa) similar to the methylation pathway enzyme S-adenosylhomocysteine hydrolase (AHCY). Therefore, this molecule was termed DC-expressed AHCY-like molecule (DCAL). DCAL mRNA was expressed moderately in fresh blood DC, but was not detectable in other peripheral blood mononuclear cells. DCAL mRNA increased markedly during activation of blood and skin DC (Langerhans cells), but was diminished in terminally differentiated tonsil DC. Cultured monocytes expressed little DCAL mRNA, but levels increased markedly when differentiated into DC by cytokines GM-CSF and IL-4. The DCAL gene [Chromosome (Chr) 1] and another previously identified DCAL-like molecule KIAA0828 (Chr 7) differed from the AHCY gene (Chr 20) in gene organization. Thus, DCAL may have a role in controlling critical events in DC differentiation and belong to a novel family of AHCY-like molecules.

Low CD1a-positive putative dendritic cell numbers in human breast cancer has recently been described and may explain the apparent 'poor immunogenicity' previously reported in breast cancer. Little attention has been given to dendritic cell activation within the tumour microenvironment, which is another reason why the in-situ immune response may be severely deficient. We have therefore examined CD1a expression as a marker for dendritic cells, together with CMRF-44 and -56 as markers of dendritic cell activation status, in 40 human breast cancers. The results demonstrate few or no CD1a-positive putative dendritic cells and minimal or no expression of the dendritic cell activation markers. Both dendritic cell number and dendritic cell activation appear substantially deficient in human breast cancers, regardless of tumour histological grade.

Dendritic cells (DCs) initiate and direct the immune response. Their inability to detect danger signals from transformed cells and to generate an effective immunological response may allow cells with a malignant phenotype to evolve into cancers. This defect can be corrected for many cancer types and the immune response boosted to eliminate malignant cells by means of DC-based vaccines/therapies. Rapid advances in our understanding of basic DC physiology and improved methods for DC isolation have made clinical application of DC therapy practical, and encouraging phase I/II results are emerging.

Dendritic cells (DC) initiate tumor specific immune responses in animal studies and initial human trials suggest that certain tumor-antigen loaded DC preparations generate clinical responses. DC may be obtained from blood or generated in vitro from precursor cells. In vitro generation of DC from precursor cells, under the influence of cytokines, has been favoured to date as a source because of the greater numbers of DC produced. However, the different cytokine combinations and serum or plasma component(s) used, differentiate precursor cells into DC with different physiological properties and ultimate immunogenicity. Thus, the quality of in vitro cytokine derived DC may have a profound influence on clinical outcomes. The administration of certain growth factors, which increase the number of circulating blood DC, may provide an alternative source of DC for use in clinical trials. Although clinical trials in prostate cancer, melanoma and metastatic renal carcinoma patients are encouraging, some data suggest certain DC preparations and administration protocols are sub optimal, even potentially tumor enhancing. As basic scientific studies establish how to provide DC with stable phenotype, resistance to tumour inhibitory factors and high migratory capacity, the technology for producing cytokine derived DC in vitro using Good Manufacturing Practise (GMP) conditions needs to be developed. Future DC vaccination protocols will require careful control of the DC used for tumor-antigen loading and repetitive long term DC vaccination may be necessary to maintain effective anti-tumor immune responses.

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.

Objective: 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., Methods: 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., Results: 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)., Conclusions: 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.

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.

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.

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.

The immunoregulatory signaling (IRS) family includes several molecules, which play major roles in the regulation of the immune response. The CMRF-35A and CMRF-35H molecules are two new members of the IRS family of molecules, that are found on a wide variety of haemopoietic lineages. The extracellular functional interactions of these molecules is presently unknown, although CMRF-35H can initiate an inhibitory signal and is internalized when cross-linked. In this paper, we described the gene structure for the CMRF-35A gene and its localization to human chromosome 17. The gene consists of four exons spanning approximately 4.5 kb. Exon 1 encodes the 5' untranslated region and leader sequence, exon 2 encodes the immunoglobulin (Ig)-like domain, exon 3 encodes the membrane proximal region and exon 4 encodes the transmembrane region, the cytoplasmic tail and the 3' untranslated region. A region in the 5' flanking sequence of the CMRF-35A gene, that promoted expression of a reporter gene was identified. The genes for the CMRF-35A and CMRF-35H molecules are closely linked on chromosome 17. Similarity between the Ig-like exons and the preceding intron of the two genes suggests exon duplication was involved in their evolution. We also identified a further member of the CMRF-35 family, the CMRF-35J pseudogene. This gene appears to have arisen by gene duplication of the CMRF-35A gene. These three loci - the CMRF-35A, CMRF-35J and CMRF-35H genes-form a new complex of IRS genes on chromosome 17.

Dendritic cells (DC) are potent antigen-presenting cells and understanding their mechanisms of antigen uptake is important for loading DC with antigen for immunotherapy. The multilectin receptors, DEC-205 and macrophage mannose receptor (MMR), are potential antigen-uptake receptors; therefore, we examined their expression and FITC-dextran uptake by various human DC preparations. The RT-PCR analysis detected low levels of DEC-205 mRNA in immature blood DC, Langerhans cells (LC) and immature monocyte-derived DC (Mo-DC). Its mRNA expression increased markedly upon activation, indicating that DEC-205 is an activation-associated molecule. In Mo-DC, the expression of cell-surface DEC-205 increased markedly during maturation. In blood DC, however, the cell-surface expression of DEC-205 did not change during activation, suggesting the presence of a large intracellular pool of DEC-205 or post-transcriptional regulation. Immature Mo-DC expressed abundant MMR, but its expression diminished upon maturation. Blood DC and LC did not express detectable levels of the MMR. FITC-dextran uptake by both immature and activated blood DC was 30- to 70-fold less than that of LC, immature Mo-DC and macrophages. In contrast to immature Mo-DC, the FITC-dextran uptake by LC was not inhibited effectively by mannose, an inhibitor for MMR-mediated FITC-dextran uptake. Thus, unlike Mo-DC, blood DC and LC do not use the MMR for carbohydrate-conjugated antigen uptake and alternative receptors may yet be defined on these DC. Therefore, DEC-205 may have a different specificity as an antigen uptake receptor or contribute to an alternative DC function.

Cardiac failure is a known complication of haemopoietic stem cell transplantation (HSCT) and is often difficult to diagnose as patients may have multiple medical problems. Since brain natriuretic peptide (BNP) is largely a hormone of cardiac ventricular origin and is released early in the course of ventricular dysfunction, we have examined the value of serial plasma BNP levels for detecting cardiac failure in patients undergoing cytotoxic conditioning for HSCT. Fifteen patients undergoing HSCT were evaluated (10 undergoing autologous HSCT; five undergoing allogeneic HSCT). BNP was measured by radioimmunoassay prior to therapy and weekly for 5 weeks. Seven patients had a significant rise in BNP level (above a previously established threshold of 43 pmol/l associated with cardiac failure), occurring 1-4 weeks post commencement of conditioning. In three of these patients, cardiac failure was subsequently diagnosed clinically 3, 9 and 23 days after a BNP level of 43 pmol/l had been detected. These three patients had the highest peak BNP levels for the group and in each case elevation in BNP level occurred for a period exceeding 1 week. Although numbers were relatively small, a BNP >43 pmol/l was significantly associated with the inclusion of high-dose cyclophosphamide in the preparative regimen (P = 0.02). BNP levels showed no relationship to febrile episodes. In conclusion, these results show that plasma BNP may be used as a marker for early detection of cardiac dysfunction in patients undergoing HSCT, particularly if levels are increased for periods exceeding 1 week. Measurement of BNP during HSCT may be helpful in patients at risk of cardiac failure, in complex clinical situations and in monitoring the cardiotoxicity of preparative regimens.

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.

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.

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., (Copyright 2000 Academic Press.)

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.

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.

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.

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.