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Producción Científica, Murine dendritic cells, when pulsed with heat-killed Burkholderia pseudomallei and used to immunise naïve mice, have previously been shown to induce protective immunity in vivo. We have now demonstrated the in vitro priming of naïve human T cells against heat-killed B. pseudomallei, by co-culture with syngeneic B. pseudomallei-pulsed dendritic cells. Additionally, we have enriched the DC fraction such that a study of the differential response induced by pulsed DCs of either myeloid or plasmacytoid lineage in syngeneic human T cells was achievable. Whilst both mDCs and pDCs were activated by pulsing, the mDCs contributed the major response to B. pseudomallei with the expression of the migration marker CCR7 and a significantly greater secretion of the proinflammatory TNFα and IL1β. When these DC factions were combined and used to prime syngeneic T cells, a significant proliferation was observed in the CD4+ fraction. Here, we have achieved human T cell priming in vitro with unadjuvanted B. pseudomallei, the causative organism of melioidosis, for which there is currently no approved vaccine. We propose that the approach we have taken could be used to screen for the human cellular response to candidate vaccines and formulations, in order to enhance the cell-mediated immunity required to protect against this intracellular pathogen and potentially more broadly against other, difficult-to-treat intracellular pathogens. To date, the polysaccharide capsule of B. pseudomallei, fused to a standard carrier protein, e.g., Crm, looks a likely vaccine candidate. Dendritic cells (DCs), providing, as they do, the first line of defence to infection, process and present microbial products to the immune system to direct downstream immune responses. Here, we have sought to use DCs ex vivo to identify immunogenic products from heat-killed B. pseudomallei. Using practical volumes of fresh human donor blood, we show that heat-killed B. pseudomallei activated and stimulated the expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 from both myeloid and plasmacytoid DCs. Furthermore, B. pseudomallei-pulsed DCs cultured with naïve syngeneic T cells ex vivo, induced the activation and proliferation of the CD4+ T-cell population, which was identified by cell surface marker staining using flow cytometry. Thus, both DC subsets are important for driving primary T helper cell responses to B. pseudomallei in healthy individuals and have the potential to be used to identify immunogenic components of B. pseudomallei for future therapies and vaccines., Ministerio de Defensa de Reino Unido - Defence Science and Technology Laboratory (Dstl) - (grant DSTL/AGR/00254/01)

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, an infectious disease endemic to south-east Asia. As B. pseudomallei is antibiotic-resistant, the need for cell-based vaccines and therapies is crucial to managing melioidosis. Dendritic cells (DC) provide the first line of defense to infection and direct downstream immune responses. Using practical volumes of fresh healthy donor blood, we show that heat-killed B. pseudomallei activated and stimulated expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 from both myeloid and plasmacytoid DC. Furthermore, B. pseudomallei-pulsed DC induced activation and proliferation of CD4+ T-cells. Thus, both DC subsets are important for driving primary T helper cell responses to B. pseudomallei in healthy individuals and have the potential to be targeted for future therapies and vaccines.Author SummaryMelioidosis is an infectious disease endemic to south-east Asia and northern Australia caused by the bacterium Burkholderia pseudomallei. Melioidosis presents a significant public health threat because it has no effective vaccine or cure, leading to a high mortality rate of between 10-50%. We highlight the possibility of immune-based strategies targeting Burkholderia pseudomallei to better treat and prevent melioidosis. Specifically, we show that dendritic cells-the sentinel cells of the immune system-respond to B. pseudomallei in healthy individuals and in turn can orchestrate downstream protective immune responses. Thus, dendritic cells may be key players in the development of both vaccines and therapeutics for melioidosis as well as other bacteria-driven diseases.

Three-dimensional hydrogels are ideal for tissue engineering applications due to their structural integrity and similarity to native soft tissues; however, they can lack mechanical stability. Our objective was to develop a bioactive and mechanically stable hydrogel for clinical application. Auricular cartilage was decellularised using a combination of hypertonic and hypotonic solutions with and without enzymes to produce acellular tissue. Methacryloyl groups were crosslinked with alginate and PVA main chains via 2-aminoethylmathacrylate and the entire macromonomer further crosslinked with the acellular tissue. The resultant hydrogels were characterised for its physicochemical properties (using NMR), in vitro degradation (via GPC analysis), mechanical stability (compression tests) and in vitro biocompatibility (co-culture with bone marrow-derived mesenchymal stem cells). Following decellularisation, the cartilage tissue showed to be acellular at a significant level (DNA content 25.33 ng/mg vs. 351.46 ng/mg control tissue), with good structural and molecular integrity of the retained extra cellular matrix (s-GAG= 0.19 μg/mg vs. 0.65 μg/mg ±0.001 control tissue). Proteomic analysis showed that collagen subtypes and proteoglycans were retained, and SEM and TEM showed preserved matrix ultra-structure. The hybrid hydrogel was successfully cross-linked with biological and polymer components, and it was stable for 30 days in simulated body fluid (poly dispersal index for alginate with tissue was stable at 1.08 and for PVA with tissue was stable at 1.16). It was also mechanically stable (Young’s modulus of 0.46 ± 0.31 KPa) and biocompatible, as it was able to support the development of a multi-cellular feature with active cellular proliferation in vitro. We have shown that it is possible to successfully combine biological tissue with both a synthetic and natural polymer and create a hybrid bioactive hydrogel for clinical application.

Objective Dendritic cells (DC) mediate intestinal immune tolerance. Despite striking differences between the colon and the ileum both in function and bacterial load, few studies distinguish between properties of immune cells in these compartments. Furthermore, information of gut DC in humans is scarce. We aimed to characterise human colonic versus ileal DC. Design Human DC from paired colonic and ileal samples were characterised by flow cytometry, electron microscopy or used to stimulate T cell responses in a mixed leucocyte reaction. Results A lower proportion of colonic DC produced pro-inflammatory cytokines (tumour necrosis factor-α and interleukin (IL)-1β) compared with their ileal counterparts and exhibited an enhanced ability to generate CD4 + FoxP3 + IL-10 + (regulatory) T cells . There were enhanced proportions of CD103 + Sirpα − DC in the colon, with increased proportions of CD103 + Sirpα + DC in the ileum. A greater proportion of colonic DC subsets analysed expressed the lymph-node-homing marker CCR7, alongside enhanced endocytic capacity, which was most striking in CD103 + Sirpα + DC. Expression of the inhibitory receptor ILT3 was enhanced on colonic DC. Interestingly, endocytic capacity was associated with CD103 + DC, in particular CD103 + Sirpα + DC. However, expression of ILT3 was associated with CD103 − DC. Colonic and ileal DC differentially expressed skin-homing marker CCR4 and small-bowel-homing marker CCR9, respectively, and this corresponded to their ability to imprint these homing markers on T cells. Conclusions The regulatory properties of colonic DC may represent an evolutionary adaptation to the greater bacterial load in the colon. The colon and the ileum should be regarded as separate entities, each comprising DC with distinct roles in mucosal immunity and imprinting.

The pathogenesis of inflammatory bowel disease is incompletely understood but results from a dysregulated intestinal immune response to the luminal microbiota. CD4 T cells mediate tissue injury in the inflammatory bowel disease-associated immune response. Dendritic cells (DC) generate primary T-cell responses and mediate intestinal immune tolerance to prevent overt inflammation in response to the gut microbiota. However, most information regarding function of intestinal DC has come from mouse models, and information in humans is scarce. We show here that intestinal DC subsets are skewed in ulcerative colitis (UC) in humans, with a loss of CD103 lymph-node homing DC; this intestinal DC subset preferentially generates regulatory T cells in mice. We show infiltrates of DC negative for myeloid marker CD11c, with enhanced expression of Toll-like receptors for bacterial recognition. After mixed leukocyte reaction, DC from the inflamed UC colon had an enhanced ability to generate gut-specific CD4 T cells with enhanced production of interleukin-4 but a loss of interferon γ and interleukin-22 production. Conditioning intestinal DC with probiotic strain Lactobacillus casei Shirota in UC partially restored their normal function indicated by reduced Toll-like receptor 2/4 expression and restoration of their ability to imprint homing molecules on T cells and to generate interleukin-22 production by stimulated T cells. This study suggests that T-cell dysfunction in UC is driven by DC. T-cell responses can be manipulated indirectly through effects of bacterial conditioning on gut DC with implications for immunomodulatory effects of the commensal microbiota in vivo. Manipulation of DC to allow generation of DC-specific therapy may be beneficial in inflammatory bowel disease.

Background: Dendritic cells (DC) dictate not only the type of T-cell immunity, but also homing patterns of T cells in mice. In humans, we characterized normal human gut DC and tested whether gut-specific homeostatic DC could be generated from blood precursors by factors in the gut microenvironment. Methods: We characterized the phenotype and function of healthy human gut DC compared with blood and skin DC, and studied whether conditioning of blood DC in the presence of colonic biopsy supernatants (Bx-SN) induced gut-like phenotype and functions. Results: Blood DC mostly expressed both gut and skin homing markers, indicating potential to migrate to both major immune surface organs, and induced multi-homing T cells. However, DC within gut or skin did not demonstrate this multi-homing phenotype, were tissue-specific, and induced tissue-specific T cells. Human gut DC were less stimulatory for allogeneic T cells than their dermal and blood counterparts. Human blood DC cultured in vitro lost homing marker expression. Conditioning of human enriched blood DC with colonic Bx-SN from healthy controls induced a gut-homing phenotype and a homeostatic profile. Moreover, Bx-SN-conditioned DC demonstrated a restricted T-cell stimulatory capacity and preferentially induced gut-specific T cells. Retinoic acid and transforming growth factor beta (TGF-β) mediated the acquisition of the gut-homing and homeostatic properties, respectively, induced by colonic Bx-SN on blood enriched DC. Conclusions: Tissue-specific factors manipulate immunity via modulating characteristics of DC and may provide tools to generate tissue-specific immunotherapy. (Inflamm Bowel Dis 2011;)

Off-the-shelf availability of tissue-engineered skin constructs, tailored by different combinations of reagents to produce a highly preserved biological matrix is often the only means to help patients suffering skin damage. This study assessed the effect of five different decellularisation methods on porcine dermal scaffolds with regard to matrix composition, biomechanical strength, and cytotoxicity using an in vitro biocompatibility assay. Results demonstrated that four out of the five tested decellularisation protocols were efficient in producing acellular scaffolds. Nevertheless, decellularisation method using osmotic shock without enzymatic digestion showed to be efficient not only in removing cellular material and debris from dermal scaffolds but was also beneficial in the preservation of extracellular matrix components (glycosaminoglycans and collagen). Histological assessment revealed that the dermal architecture of coarse collagen bundles was preserved. Examinations by scanning electron microscopy and transmission electron microscopy showed that the arrangement and ultrastructure of collagen fibrils in the scaffolds were retained following non-enzymatic method of decellularisation and also after collagen crosslinking using genipin. Moreover, this decellularised scaffold was not only shown to be biologically compatible when co-cultured with bone marrow-derived mesenchymal stem cells and fibroblasts, but also stimulated the cells to release trophic factors essential for tissue regeneration.

Adipose tissue is reported to contain monocyte-like pre-adipocytes, which may mature into macrophages, contributing to local inflammation. Dendritic cells (DC) can be derived from monocytes and initiate and regulate primary immune responses. We hypothesized, therefore, that adipose tissue may provide DC involved in local immune activity. To test this, we studied cells from human omental adipose tissue samples from 17 patients with benign gynecological disease. The hypothesis that adipose tissue DC are involved in inflammatory disease was tested by comparing these cells with those from 18 patients with Crohn's disease, where hypertrophy of adipose tissue suggests involvement in disease. A high proportion of the 1.33 ± 0.12 × 105 CD45-positive cells/mg, obtained from control omenta, expressed CD11c, CD1a, and CD83; costimulatory molecules CD40, CD80, and CD86; and major histocompatibility complex (MHC) Class II but little CD14, CD16, or CD33. Omental cells showing morphological characteristics of DC were also observed. Metrizamide gradient-enriched DC from these populations were potent stimulators of primary proliferation of allogeneic T cells in mixed leukocyte reactions. Increased numbers of CD45+ cells from omentum of Crohn's patients (4.50±1.08×105 CD45+ cells/mg) contained higher percentages of CD11c+ and CD40+ cells (80.8±3.8% vs. 63.4±6, P=0.032; 77.9±4% vs. 58.8±6.5, P=0.029, respectively), but MHC Class II and stimulatory capacity were almost completely lost (P=

APC acting at the early stages of an immune response can shape the nature of that response. Such APC will include dendritic cells (DCs) but may also include populations of B cells such as marginal zone B cells in the spleen. In this study, we analyze APC populations in mouse spleen and compare the phenotype and function of B220+CD11c− populations with those of CD11c+ spleen DC subsets. Low-density B220+ cells had morphology similar to DCs and, like DCs, they could stimulate naive T cells, and expressed high levels of MHC and costimulatory molecules. However, the majority of the B220+ cells appeared to be of B cell lineage as demonstrated by coexpression of CD19 and surface Ig, and by their absence from RAG-2−/− mice. The phenotype of these DC-like B cells was consistent with that of B cells in the marginal zone of the spleen. On bacterial stimulation, they preferentially produced IL-10 in contrast to the DCs, which produced IL-12. Conventional B cells did not produce IL-10. The DC-like B cells could be induced to express low levels of the DC marker CD11c with maturational stimuli. A minority of the B220+CD11c− low-density cells did not express CD19 and surface Ig and may be a DC subset; this population also produced IL-10 on bacterial stimulation. B220+ APC in mouse spleen that stimulate naive T cells and preferentially produce IL-10 may be involved in activating regulatory immune responses.

This is an open access article under the terms of the Creative Commons Attribution License.-- et al., [Scope]: The human/microbiota cross-talk is partially mediated by bacteria-derived peptides like Serine-Threonine peptide (STp), which is resistant to gut proteolysis, is found in the human healthy colon and induces regulatory properties on gut dendritic cells (DCs); here we characterized human gut DC in ulcerative colitis (UC) patients and studied the effect of STp on their properties. [Methods and results]: Human colonic DC from healthy controls and UC patients were isolated, conditioned for 24 h +/- STp and characterized by flow cytometry, immunohistochemistry, and electron microscopy. Expression of immature DC markers DC-SIGN and ILT3, and Toll-like receptors were increased on gut UC-DC. Langerin (involved in phagocytosis), lymph node homing marker CCR7, and activation markers CD40/CD80/CD86 were decreased in UC. Gut DC had restricted stimulatory capacity for T-cells in UC. Conditioning of DC with STp in vitro reduced Toll-like receptor expression, increased CD40 and CD80 expression, and restored their stimulatory capacity. [Conclusion]: Colonic DCs display an abnormal immature phenotype in UC, which was partially restored following STp treatment. Bacteria-derived metabolites, like STp, seem to have a role in gut homeostasis that is missing in UC so they might lead a new era of probiotic products setting the basis for nondrug dietary therapy in inflammatory bowel disease. © 2013 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA Weinheim., The author(s) gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC), this research was funded by the BBSRC Institute Strategic Programme for Gut Health and Food Safety BB/J004529/1. The authors also thank the St Mark’s Hospital Foundation, The Association for International Cancer Research (AICR), Scotland, and the Junta de Castilla y León (GRS175/B/07).

Defective dendritic cell (DC) function caused by abnormal differentiation of these cells is an important mechanism of tumor escape from immune system control. Previously, we have demonstrated that the number and function of DC were dramatically reduced in cancer patients. This effect was closely associated with accumulation of immature cells (ImC) in peripheral blood. In this study, we investigated the nature and functional role of those ImC. Using flow cytometry, electron microscopy, colony formation assays, and cell differentiation in the presence of different cell growth factors, we have determined that the population of ImC is composed of a small percentage (

Human peripheral blood contains two populations of dendritic cells (DC) but their developmental relationship has not been established. Freshly isolated CD11c– DC possessed a lymphoid morphology, lacked myeloid markers but expressed lymphoid markers (CD4+ CD10+) whilst the CD11c+ DC were monocytoid in appearance and expressed myeloid markers. Although both populations were allostimulatory, only the CD11c+ DC were able to take up antigen. Irrespective of the culture conditions the CD11c– cells developed into CD11c– CD13– CD33– CD4+ CD1a– CD83+/– DC. In contrast, cultured CD11c+ cells developed the phenotype CD11c+ CD13+ CD33+/– CD4– CD1a+ CD83+ CD9+. Only the CD11c+ DC expressed macrophage colony-stimulating factor (M-CSF) receptor and gave rise to CD14+, esterase+, phagocytic macrophages when cultured in M-CSF. These data suggest that these two populations of DC represent distinct lineages of antigen-presenting DC.

Metacyclic Leishmania promastigotes (PM), transmitted by sand-fly bite, are likely to interact initially with cells of the dendritic cell (DC) lineage(s) in the epidermis or dermis. Epidermal Langerhans cells internalize L. major amastigotes (AM) and transport them to draining lymph nodes (Moll, H., Fuchs, H., Blank, C. and Rollinghoff, M., Eur. J. Immunol. 1993. 23: 1595) but little is known about the interaction of DC with PM. The present study demonstrates that DC are able to internalize PM and that the fate of the parasites within DC differs from that within macrophages (Mphi). DC took up small numbers of PM which did not differentiate into AM but appeared to be degraded; Mphi internalized large numbers of PM into parasitophorous vacuoles where they differentiated into AM. In response to direct stimulation with PM, DC from both C3H ("resistant" to L. major infection) and BALB/c ("susceptible") up-regulated production of IL-12 p40. In contrast, IL-12 production by Mphi was not detected. DC exposed to either metacyclic PM or PM culture supernatants were also able to stimulate proliferative responses in lymph node T cells from naive mice. These data indicate that DC have the capacity to promote protective Th1 immune responses in Leishmania infection and suggest that DC exposed to PM may be useful in immunotherapy and vaccination.

Infection of dendritic cells (DC) by human immunodeficiency virus (HIV) has been disputed. Employing a fluorescence-activated cell sorter, DC, identified by the absence of membrane markers for T, B, natural killer (NK) and monocytic cells and by high levels of MHC class II DR antigen, were shown to express low levels of CD4. Immunomagnetic beads were used to separate blood low density cells, which are enriched for DC, into CD4-positive and -negative populations. Examination of these cells by electron microscopy showed an increase in the percentage of cells with DC morphology in the CD4-positive fraction and a reduction in the CD4-negative fraction. Electron microscopy of semi-purified DC preparations infected in vitro for 5 days with HIV-1 revealed morphologically distinct veiled DC with mature virions on the cell surface and virus budding through the cell membrane. Further evidence for the growth of HIV in DC was provided by experiments in which DC were extensively depleted of contaminating lymphocytes and monocytes prior to infection. Estimation of provirus load by a nested PCR indicated that after 5 days an infection level of one provirus copy per five cells could be achieved. After 7 days the provirus copy number could exceed the cellular genome copy number, suggesting that some cells had more than one provirus. Infectious virus could not be demonstrated in these cultures after 24 h but was detected after 5 or 7 days. Infection of DC in the presence of antibodies against CD4 was inhibited and suggests infection occurs via a CD4-dependent pathway. These results confirm that DC are susceptible to HIV infection in vitro. The immunological consequences of DC infection in vivo may be significant in the pathogenesis of AIDS.

Summary Previous studies from this laboratory indicated infection of dendritic cells (DC) from peripheral blood of individuals infected with HIV1. Here, further evidence for the infection of peripheral blood DC with HIV1 is presented. Low-density cells (LDC) were prepared from blood mononuclear cells of HIV-infected individuals at different clinical stages of disease. These cells are enriched (10–40 %) for MHC class-II-bearing DC, while most of the remaining cells are monocytes, and 2–10 % are lymphocytes. A quantitative polymerase chain technique (PCR) was used to estimate the HIV provirus load in LDC and lymphocytes of patients in different disease categories. HIV provirus was detected in every LDC preparation, and for many individuals, particularly CDC stage IV patients, the load was higher in the LDC than in the lymphocyte fraction. These findings suggested that patient DC are infected with HIV. In order to provide confirmatory evidence for this conclusion, PCR was performed on DC that were highly purified from LDC by panning to remove contaminating T, B, natural killer and monocytic cells. High levels of HIV provirus were found in these purified DC. These findings suggest that DC provide a reservoir of HIV and that the consequences of such infection may be relevant to the development of disease.

Increased methylglyoxal (MG) concentrations and formation of advanced glycation end-products (AGEs) are major pathways of glycaemic damage in diabetes, leading to vascular and neuronal complications. Diabetes patients also suffer increased susceptibility to many common infections, the underlying causes of which remain elusive. We hypothesized that immune glycation damage may account for this increased susceptibility. We previously showed that the reaction mixture (RM) for MG glycation of peptide blocks up regulation of CD83 in myeloid cells and inhibits primary stimulation of T cells. Here, we continue to investigate immune glycation damage, assessing surface and intracellular cytokine protein expression by flow cytometry, T-cell proliferation using a carboxyfluorescein succinimidyl ester assay, and mRNA levels by RT-PCR. We show that the immunomodulatory component of this RM was MG itself, with MG alone causing equivalent block of CD83 and loss of primary stimulation. Block of CD83 expression could be reversed by MG scavenger N-acetyl cysteine. Further, MG within RM inhibited stimulated production of interleukin (IL)-10 protein from myeloid cells plus interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha from T cells. Loss of IL-10 and IFN-gamma was confirmed by RT-PCR analysis of mRNA, while TNF-alpha message was raised. Loss of TNF-alpha protein was also shown by ELISA of culture supernatants. In addition, MG reduced major histocompatibility complex (MHC) class I expression on the surface of myeloid cells and increased their propensity to apoptose. We conclude that MG is a potent suppressor of myeloid and T-cell immune function and may be a major player in diabetes-associated susceptibility to infection.

The large intestine is a major site of infection and disease, yet little is known about how immunity is initiated within this site and the role of dendritic cells (DCs) in this process. We used the well-established model of Trichuris muris infection to investigate the innate response of colonic DCs in mice that are inherently resistant or susceptible to infection. One day postinfection, there was a significant increase in the number of immature colonic DCs in resistant but not susceptible mice. This increase was sustained at day 7 postinfection in resistant mice when the majority of the DCs were mature. There was no increase in DC numbers in susceptible mice until day 13 postinfection. In resistant mice, most colonic DCs were located in or adjacent to the epithelium postinfection. There were also marked differences in the expression of colonic epithelial chemokines in resistant mice and susceptible mice. Resistant mice had significantly increased levels of epithelium-derived CCL2, CCL3, CCL5, and CCL20 compared with susceptible mice. Furthermore, administering neutralizing CCL5 and CCL20 Abs to resistant mice prevented DC recruitment. This study provides clear evidence of differences in the kinetics of DC responses in hosts inherently resistant and susceptible to infection. DC responses in the colon correlate with resistance to infection. Differences in the production of DC chemotactic chemokines by colonic epithelial cells in response to infection in resistant vs susceptible mice may explain the different kinetics of the DC response.

AIM: Recent studies demonstrating the direct involvement of dendritic cells (DC) in the activation of pathogenic T cells in animal models of inflammatory bowel disease identify DC as important antigen presenting cells in the colon. However, very little is known about the properties of colonic DC. METHODS: Using immunohistochemistry, electron microscopy and flow cytometry we have characterized and compared colonic DC in the colon of healthy animals and interleukin-2-deficient (IL2-/-) mice that develop colitis. RESULTS: In the healthy colon, DC resided within the lamina propria and in close association with the basement membrane of colonic villi. Type 1 myeloid (CD11c+, CD11b+, B220-, CD8α-) DC made up the largest (40-45%) population and all DC expressed low levels of CD80, CD86, and CD40, and had high endocytic activity consistent with an immature phenotype. In colitic IL2-/- mice, colonic DC numbers increased four- to five-fold and were localized within the epithelial layer and within aggregates of T and B cells. They were also many more DC in mesenteric lymph nodes (MLN). The majority (>85%) of DC in the colon and MLN of IL2-/- mice were type 1 myeloid, and expressed high levels of MHC class II, CD80, CD86, CD40, DEC 205, and CCR5 molecules and were of low endocytic activity consistent with mature DC. CONCLUSION: These findings demonstrate striking changes in the number, distribution and phenotype of DC in the inflamed colon. Their intimate association with lymphocytes in the colon and draining lymph nodes suggest that they may contribute directly to the ongoing inflammation in the colon.

Summary On maturation, dendritic cells (DCs) become highly active cells equipped for antigen uptake, migration and clustering and activation of T cells. We therefore asked whether DCs acquire fat and glycogen stores as they mature. DCs were generated from mouse bone marrow stem cells by culturing with granulocyte–macrophage colony-stimulating factor (GM-CSF) for 7–8 days. Stimulation of the DCs with lipopolysaccharide (LPS) for the last 24 hr of culture, or exposure to 1–15 ng/ml of interleukin (IL)-4 during development, resulted in production of DCs not only with an increased ability to stimulate T cells but also with an increasingly lacy appearance on transmission electron microscopy, with multiple unstained areas in the cytoplasm. This changed morphology was associated with the presence of increasing amounts of fat and glycogen, identified by Sudan Black and periodic acid leukofushin/Schiff (PAS) staining, respectively. Lacy DCs up-regulated type 1 and type 2 scavenger receptors, providing possible mechanisms contributing to these changes. Lacy DCs were found occasionally amongst freshly isolated splenic and lymph node DCs. DCs can be isolated from human adipose tissue, and we tested whether lacy DCs acquiring fat and glycogen were present in mouse omentum. CD45 + cells migrating from the omentum expressed specific DC markers CD11c and 33D1, costimulatory molecules and major histocompatibility complex (MHC) class II, and most showed darkly staining fat inclusions. Thus, during development, DCs can acquire large amounts of fat and glycogen, accumulation of which is promoted by antigen exposure and modulated by the cytokine milieu and location, and which may act as a link between energy stores and immune function.

Dendritic cells are potent activators of the immune system and have a key role in linking innate and adaptive immune responses. In the current study we have used ex vivo pulsed bone marrow dendritic cells (BMDC) in a novel adoptive transfer strategy to protect against challenge with Bacillus anthracis, in a murine model. Pre-pulsing murine BMDC with either recombinant Protective Antigen (PA) or CpG significantly upregulated expression of the activation markers CD40, CD80, CD86 and MHC-II. Passive transfusion of mice with pulsed BMDC, concurrently with active immunisation with rPA in alum, significantly enhanced (p

Dendritic cells (DC) in the colon may regulate intestinal immunity but remain poorly characterized. In this study a CD11c+HLA-DR+lin− (CD3−CD14−CD16−CD19−CD34−) population has been identified by flow cytometry in cells obtained by rapid collagenase digestion of human colonic and rectal biopsies. These day 0 (d0) CD11c+HLA-DR+lin− cells comprised ∼0.6% of the mononuclear cells obtained from the lamina propria, were endocytically active, and had the phenotype of immature DC; they were CD40+ and expressed low levels of CD83 and CD86, but little or no CD80 or CD25. Similar d0 DC populations were isolated from the colonic mucosa of healthy controls and from both inflamed and noninflamed tissue from patients with Crohn’s disease. The lamina propria also contained a population of cells capable of migrating out of biopsies during an overnight culture and differentiating into mature DC with lower levels of endocytic activity and high cell surface expression of CD40, CD80, CD86, CD83, and CD25. This mature DC population was a potent stimulator of an allogeneic mixed leukocyte (MLR). Overnight culture of cells isolated by enzymatic digestion on d0 yielded DC with a phenotype intermediate between that of the d0 cells and that of the cells migrating out overnight. Overnight culture of colonic cells in which DC and HLA-DR+lin+ cells were differentially labeled with FITC-dextran suggested that some of the maturing DC might differentiate from HLA-DR+lin+ progenitors. This study presents the first analysis of the phenotype, maturational status, and migratory activity of human gut DC.

Nucleotide sequences of HIV-1 from plasma virus RNA and proviral DNA extracted from blood dendritic cells (DCs) and from T cells were analyzed to determine whether blood DCs may harbor a restricted population of virus variants. The sequence of the V3 loop and 51 bases from the 3' flanking region were determined in four patients not receiving antiviral therapy. There was no evidence of a unique or more restricted population of variants in DCs for any of the four patients studied. However, for one patient there was evidence of differences between plasma virus and virus in the T cell population, with virus in the plasma showing a closer relationship to DC-derived sequences.

Blood dendritic cells (DC) express CD4 and are susceptible to HIV infection. By electron microscopy two morphologically distinct types of DC were identified in peripheral blood. Only one of these two types was susceptible to infection with a lymphotropic strain of HIV-1. By FACS two populations could be defined based on the expression of CD11c. The morphology of cultured FACS-purified CD11c negative DC was similar to that DC population shown to be susceptible to infection with the lymphotropic strain of HIV. Furthermore after several hours in culture CXCR4, the co-receptor for lymphotropic strains of HIV-1, was expressed at a significantly higher level on the CD11c negative DC than on the CD11c positive cells. This study suggests that there are subpopulations of DC that show differences in susceptibility to infection with some strains of HIV-1.

Dendritic cells (DC) are required to initiate primary cellular immune responses. Human immunodeficiency virus type 1 (HIV-1) infection of DC may be central to transmission and persistence of virus and in the pathogenesis of AIDS. In symptomatic HIV-1-infected patients the proportion of DC in the mononuclear cell population was reduced. Provirus load in the T cells was 3-100 times higher than in DC and there was no correlation between the levels of infection in the two cell types. Phylogenetic analysis of amino acids in the V3 loop and flanking regions indicated intermingling of sequences and thus provides the first evidence for transfer of virus between DC and T cells in vivo. In one of three patients analysed there were significant differences in amino acid residues in the V3 region. This may reflect reduced interactions between DC and T cells in infected individuals and for the existence of variants with a stronger tropism for DC, which could play a role in transmission by initiating infection in mucosal DC.

Summary In this pilot study, the numbers of dendritic cells (DC) in peripheral blood of AIDS patients and the level of infection with HIV1 were determined before and after AZT treatment. Mononuclear cells were cultured overnight and DC were identified by their lack of labelling with antibodies specific for T, B and natural killer (NK) cells and monocytes and by their high level of staining with antibodies for MHC class II molecules. Although the numbers of DC identified by this method were lower than those identified morphologically in earlier studies (Macatonia et al. , 1990), the numbers in three untreated AIDS patients were below the range seen in normals. There was also a marked rise in DC number in patients given AZT therapy. In two patients, there was a significant provirus load in the DCs which was decreased two to three weeks after the commencement of AZT therapy. The studies suggest that DC numbers and their infection levels may be markers of disease in HIV infection.

The question of whether blood dendritic cells (DC) can be infected with HIV is controversial (1,2,3). In further studies aimed at resolving this problem DC were shown to express CD4 and cells with the distinctive morphology of veiled DC were found to support productive virus growth. In vitro infection was also demonstrated by PCR on highly purified preparations of DC. Finally, PCR was used to show that highly purified DC from HIV-infected individuals contained HIV provirus DNA.

Apoptosis is a universal form of “programmed” cell death that appears to have a key role in the development and regulation of the immune system1. Induction of the apoptotic pathway is a complex event, but in most instances requires an initial stimulus. Such a stimulus can include the removal of key growth factors, stimulation of immature T cells or inappropriate activation signals. Both activated and immature T lymphocytes appear to undergo apoptosis when stimulated through the T cell receptor, indicating that a combination of the stimulatory events and the state of maturation of the T cells is important in determining a cell’s outcome.

INTRODUCTION: The immunopathology of ulcerative colitis (UC) relates to an inappropriate mucosal immune response to constituents of the intestinal microbiota in genetically susceptible individuals. HLA-DR+ lin-/dim (lin = anti-CD3,14,16,19,34) cells extracted from human intestinal tissue contain CD11c+ myeloid dendritic cells (DC) that contribute to the immunoregulatory events that normally limit inflammatory responses to commensal bacteria. Also present within the HLA-DR+ lin-/dim population are hitherto poorly characterized CD11ccells. We hypothesized that this population in the gut may also contribute to intestinal inflammation in active UC AIMS & METHODS: HLA-DR+ lin-/dim cells were identified in freshly isolated lamina propria mononuclear cells by multicolour flow cytometry, from patients with UC (n=48) and controls (n=22). The proportion and number of CD11c+ and CD11ccells within this population were determined. Surface expression of the activation/maturation markers CD40, CD86, Toll-like receptors (TLR), TLR-2, TLR-4, and the natural killer cell marker, CD56 was assessed on each cell population. Morphology was assessed by electron microscopy and T cell stimulatory capacity measured in allogenic mixed leucocyte reaction (MLR). RESULTS: Lamina propria colonic HLA-DR+ lin-/dim cells, of the CD11c-, but not the CD11c+ subset, were significantly increased in inflamed tissue of patients with UC compared with control tissue (UC 447±94 versus Control 104±17 per mg; p