Migrating lymph dendritic cells contain intracellular CD40 that is mobilized to the immunological synapse during interactions with antigen-specific T lymphocytes.

Steady state migrating rat lymph dendritic cells (LDC) are semimature, expressing high levels of surface MHC class II, but low levels of surface costimulatory molecules. In this study, we show that surface CD40 is not detectable, but LDC contain intracellular CD40. Multiple isoforms of CD40 were detected, including the type 1 isoform required for signal transduction. Culture of LDC with syngeneic T cells does not induce redistribution of cytoplasmic CD40. When LDC were cultured with naive allogeneic CD4(+) T lymphocytes, polarization of CD40 to the immune synapse occurred between 3 and 6 h postculture. By 24 h, although large numbers of T cells were engaged with LDC, CD40 could not be detected in LDC or at the synapses. We conclude that migrating LDC contain stores of CD40 that can be mobilized rapidly to the sites of interaction with Ag-specific T cells. The disappearance of CD40 by 24 h may help in the regulation of T cell activation.

Subsets of migrating intestinal dendritic cells.

Dendritic cells (DCs) in the intestine are heterogeneous. Phenotypically different populations of conventional DCs have been identified in the intestinal lamina propria, Peyer's patches, and in the draining mesenteric lymph nodes, to which these DCs constitutively migrate. Markers used to identify these populations include major histocompatibility complex class II, CD11c, CD8 alpha, CD11b, and CD103. Extensive studies in rats, summarized here, which involved collection of migrating DCs by thoracic duct cannulation after mesenteric lymphadenectomy, have clearly demonstrated that the subsets of migrating intestinal lymph DCs have different functional properties. The subsets might play different roles in the induction of oral tolerance and in driving systemic immune responses after vaccination or intestinal stimulation with Toll-like receptor ligands. The use of these surgical techniques allows investigation of the functions of purified subsets of migrating DCs. However, in the rat, these studies are limited by the range of available reagents and are difficult to compare with data from other species in this fast-moving field. Recent refinements have enabled the collection of migrating intestinal DCs from mice; our initial results are described here. We believe that these studies will generate exciting data and have the potential to resolve important questions about the functions of migrating intestinal DC subsets.

Nomenclature of monocytes and dendritic cells in blood.

Monocytes and cells of the dendritic cell lineage circulate in blood and eventually migrate into tissue where they further mature and serve various functions, most notably in immune defense. Over recent years these cells have been characterized in detail with the use of cell surface markers and flow cytometry, and subpopulations have been described. The present document proposes a nomenclature for these cells and defines 3 types of monocytes (classical, intermediate, and nonclassical monocytes) and 3 types of dendritic cells (plasmacytoid and 2 types of myeloid dendritic cells) in human and in mouse blood. This classification has been approved by the Nomenclature Committee of the International Union of Immunological Societies, and we are convinced that it will facilitate communication among experts and in the wider scientific community.

Scrapie pathogenesis: the role of complement C1q in scrapie agent uptake by conventional dendritic cells.

Mice lacking complement components show delayed development of prion disease following peripheral inoculation. The delay could relate to reduced scrapie prion protein (PrP(Sc)) accumulation on follicular dendritic cells (DCs). However conventional DCs (cDCs) play a crucial role in the early pathogenesis of prion diseases and complement deficiency could result in decreased PrP(Sc) uptake by cDCs in the periphery. To explore this possibility, we cultured murine splenic or gut-associated lymph node cDCs with scrapie-infected whole brain homogenate in the presence or absence of complement. Uptake decreased significantly if the serum in the cultures was heat-inactivated. Because heat inactivation primarily denatures C1q, we used serum from C1q(-/-) mice and showed that PrP(Sc) uptake was markedly decreased. PrP(Sc) internalization was saturable and temperature-dependent, suggesting receptor-mediated uptake. Furthermore, uptake characteristics differed from fluid-phase endocytosis. Immunofluorescence showed colocalization of C1q and PrP(Sc), suggesting interaction between these molecules. We evaluated the expression of several complement receptors on cDCs and confirmed that cDCs that take up PrP(Sc) express one of the C1q receptors, calreticulin. Our results show that C1q participates in PrP(Sc) uptake by cDCs, revealing a critical role for cDCs in initial prion capture, an event that takes place before the PrP(Sc) accumulation within the follicular DC network.

CD11c(high )dendritic cells are essential for activation of CD4+ T cells and generation of specific antibodies following mucosal immunization.

To generate vaccines that protect mucosal surfaces, a better understanding of the cells required in vivo for activation of the adaptive immune response following mucosal immunization is required. CD11c(high) conventional dendritic cells (cDCs) have been shown to be necessary for activation of naive CD8(+) T cells in vivo, but the role of cDCs in CD4(+) T cell activation is still unclear, especially at mucosal surfaces. The activation of naive Ag-specific CD4(+) T cells and the generation of Abs following mucosal administration of Ag with or without the potent mucosal adjuvant cholera toxin were therefore analyzed in mice depleted of CD11c(high) cDCs. Our results show that cDCs are absolutely required for activation of CD4(+) T cells after oral and nasal immunization. Ag-specific IgG titers in serum, as well as Ag-specific intestinal IgA, were completely abrogated after feeding mice OVA and cholera toxin. However, giving a very high dose of Ag, 30-fold more than required to detect T cell proliferation, to cDC-ablated mice resulted in proliferation of Ag-specific CD4(+) T cells. This proliferation was not inhibited by additional depletion of plasmacytoid DCs or in cDC-depleted mice whose B cells were MHC-II deficient. This study therefore demonstrates that cDCs are required for successful mucosal immunization, unless a very high dose of Ag is administered.

Murine cecal patch M cells transport infectious prions in vivo.

We show that following oral inoculation, prions bind to ileal Peyer patch and cecal patch microfold cells (M cells) in vivo. Furthermore, we show evidence that the cecum acts a biological sump holding large concentrations of prions for relatively long periods, thus increasing the exposure time of cecal patch M cells. Our results show a critical initial step in the translocation of prions from the intestinal lumen of mammals in vivo, which is a precursor to infection.

A framework to explore micronutrient deficiency in maternal and child health in Malawi, Southern Africa.

BACKGROUND: Global food insecurity is associated with micronutrient deficiencies and it has been suggested that 4.5 billion people world-wide are affected by deficiencies in iron, vitamin A and iodine. Zinc has also been identified to be of increasing concern. The most vulnerable are young children and women of childbearing age. A pilot study has been carried out in Southern Malawi, to attempt to link the geochemical and agricultural basis of micronutrient supply through spatial variability to maternal health and associated cultural and social aspects of nutrition. The aim is to establish the opportunity for concerted action to deliver step change improvements in the nutrition of developing countries. RESULTS: Field work undertaken in August 2007 and July/August 2008 involved the collection of blood, soil and crop samples, and questionnaires from ~100 pregnant women. Complex permissions and authorisation protocols were identified and found to be as much part of the cultural and social context of the work as the complexity of the interdisciplinary project. These issues are catalogued and discussed. A preliminary spatial evaluation is presented linking soil quality and food production to nutritional health. It also considers behavioural and cultural attitudes of women and children in two regions of southern Malawi, (the Shire Valley and Shire Highlands plateau). Differences in agricultural practice and widely varying soil quality (e.g. pH organic matter, C/N and metal content) were observed for both regions and full chemical analysis of soil and food is underway. Early assessment of blood data suggests major differences in health and nutritional status between the two regions. Differences in food availability and type and observations of life style are being evaluated through questionnaire analysis. CONCLUSION: The particular emphasis of the study is on the interdisciplinary opportunities and the barriers to progress in development support in subsistence communities. Engaging at the community level and the balance of expectations from both study subjects and research team highlight the merit of careful and detailed planning and project delivery.

Micronutrient deficiencies in maternity and child health: a review of environmental and social context and implications for Malawi.

It is well documented that micronutrient malnutrition is of increasing concern in the developing world, resulting in poor health and high rates of mortality and morbidity. During pregnancy, deficiency of iron and zinc can produce cognitive and growth impairment of the foetus, which may continue into infancy. Iron and zinc are essential micronutrients for both plant growth and human nutrition. Despite significant work in the areas of soil fertility, crop biofortification and dietary interventions, the problems of micronutrient deficiencies persist in Africa. There is a need to examine why communities have not embraced intervention strategies which may offer health benefits. Bottom-up, interdisciplinary approaches are required to effectively study the relationships between local communities and their environment, and to assess the impact their behaviour has on the cycling of micronutrients within the soil-plant-human system. From a detailed consideration of diverse influencing factors, a methodological model is suggested for studying the barriers to improving micronutrient uptake within rural communities. It combines environmental understanding with health and social factors, emphasising the need for and potential benefits of understanding and coherence in true interdisciplinary working.

Soluble CD38 significantly prolongs the lifespan of memory B-cell responses.

The development and maintenance of memory B cells (MBC) is dependent on germinal centres (GC) with follicular dendritic cell (FDC) networks. We have previously shown that FDC networks within GC of the spleen express a novel ligand for CD38 and that the administration of soluble CD38 induces an expansion of these cellular structures. We therefore used adoptive transfer studies to investigate whether the expansion of FDC networks with soluble CD38 affected the generation and maintenance of antigen-specific MBC. These studies found that the administration of soluble CD38 significantly extended the period after which MBC could be activated and that the frequencies of these cells also were increased. In conclusion, soluble CD38 appears to significantly extend the lifespan of antibody memory by increasing the numbers of MBC.

Evaluation of human fetal bone implants in SCID mice as a model of prostate cancer bone metastasis.

The metastasis of prostate cancer cells to the bone marrow constitutes the major source of morbidity and mortality in prostate cancer. Studying this process has been hampered by the lack of preclinical models to evaluate novel therapeutics and to study the biology of the disease. One proposed model utilizes human fetal bone implants to serve as the target for prostate cancer cells injected via the tail vein. We employed this model to test the ability of zoledronic acid to prophylax and to treat bone metastases. To improve the rate of bone metastasis, we used two bone implants instead of one to evaluate the cell lines PC3 and PC3M, a more metastatic subline. For this purpose we generated the novel cell line PC3EGFPLuc, which can be used for luminescence and/or fluorescence imaging in vivo. We did not observe bone implant metastases in 52 mice, with 90 bone implants following tail vein injection of 1x10(6) PC3 or PC3M cells. Soft tissue lesions in the buttocks and hind limbs as well as cellular growth in the hindlimbs were observed via bioluminescence imaging. This evidence together with literature findings suggests that this model produces artifactual 'bone metastasis' lesions.

Thalidomide.

Despite its history as a human teratogen, thalidomide is emerging as a treatment for cancer and inflammatory diseases. Although the evolution of its clinical application could not have been predicted from the tragedy associated with its misuse in the past, its history serves as a lesson in drug development that underscores the need to understand the molecular pharmacology of a compound's activity, including associated toxicities. Here, we summarise the applications for thalidomide with an emphasis on clinical trials published over the past 10 years, and consider our knowledge of the molecular pharmacology of the drug in the context of clinical trial data, attempting to provide a mechanism-guided understanding of its activity.

Antitumor effects of thalidomide analogs in human prostate cancer xenografts implanted in immunodeficient mice.

PURPOSE: Thalidomide has demonstrated clinical activity in various malignancies including androgen-independent prostate cancer. The development of novel thalidomide analogs with better activity/toxicity profiles is an ongoing research effort. Our laboratory previously reported the in vitro antiangiogenic activity of the N-substituted thalidomide analog CPS11 and the tetrafluorinated analogs CPS45 and CPS49. The current study evaluated the therapeutic potential of these analogs in the treatment of prostate cancer in vivo. EXPERIMENTAL DESIGN: Severely combined immunodeficient mice bearing s.c. human prostate cancer (PC3 or 22Rv1) xenografts were treated with the analogs at their maximum tolerated doses. Tumors were then excised and processed for ELISA and CD31 immunostaining to determine the levels of various angiogenic factors and microvessel density (MVD), respectively. RESULTS: CPS11, CPS45, and CPS49 induced prominent and modest growth inhibition in PC3 and 22Rv1 tumors, respectively. Thalidomide had no effect on tumor growth in either xenograft. Vascular endothelial growth factor and basic fibroblast growth factor levels were not significantly altered by any of the thalidomide analogs or thalidomide in both PC3 and 22Rv1 tumors. CPS45, CPS49, and thalidomide significantly reduced PC3 tumor platelet-derived growth factor (PDGF)-AA levels by 58-82% (P < 0.05). Interestingly, treatment with the analogs and thalidomide resulted in differential down-regulation (>/=1.5-fold) of genes encoding PDGF and PDGF receptor isoforms as determined by DNA microarray analysis. Intratumoral MVD of 22Rv1 xenografts was significantly decreased by CPS45 and CPS49. CPS49 also reduced MVD in PC3 xenografts. CONCLUSIONS: Thalidomide analogs CPS11 and 49 are promising anti-cancer agents. PDGF signaling pathway may be a potential target for these thalidomide analogs. Detailed microarray and functional analyses are under way with the aim of elucidating the molecular mechanism(s) of action of these thalidomide analogs.

Anti-angiogenic activity of human endostatin is HIF-1-independent in vitro and sensitive to timing of treatment in a human saphenous vein assay.

Endostatin is a 20-kDa endogenous angiogenesis inhibitor that has recently been shown to inhibit the expression of vascular endothelial growth factor (VEGF), an angiogenic growth factor that is up-regulated by hypoxia via the HIF-1 transcription factor complex. To determine if the anti-angiogenic activity of endostatin involves a modulation of the HIF-1/VEGF pathway in cancer cells, experiments were conducted to establish what effect endostatin has on HIF-1 activity, HIF-1alpha protein production, and cellular localization in prostate cancer cells and endothelial cells. Endothelial cell tube formation was inhibited by endostatin purchased from Calbiochem (San Diego, CA) but not endostatin obtained from EntreMed (Rockville, MD). Subsequent experiments using Calbiochem endostatin showed that it did not alter HIF-1alpha protein production or cellular localization in any of the cell lines tested, nor did it alter HIF-1 transactivational activity in hypoxia. Whether or not this is also true in vivo remains to be determined. Nevertheless, these data suggest that the anti-angiogenic activity of endostatin is independent of the HIF-1/VEGF pathway. Immunocytochemical staining results do not indicate a decreased production of VEGF in Calbiochem endostatin-treated LNCaP or human umbilical vein endothelial cells (HUVEC). Treatment of rat aortic cross sections with human endostatin from Calbiochem resulted in a dose-dependent inhibition of microvessel outgrowth. Importantly, inhibition of vessel outgrowth by Calbiochem endostatin in a human saphenous vein angiogenesis assay required early treatment. In view of this in vitro data, we suggest that clinical trials involving endostatin treatment of late-stage disease may not adequately represent the efficacy of this drug in early-stage cancer.

Current status of thalidomide and its role in the treatment of metastatic prostate cancer.

Following the discovery of its anti-angiogenic properties and despite its tragic history, thalidomide has re-surfaced in the field of oncology. Concurrent with its evaluation in various clinical trials for cancer, thalidomide's mechanism of action is sought and new analogues with improved efficacy and pharmacological profile are emerging. This review is a critical evaluation of thalidomide metabolism, molecular targets, anti-angiogenic activity and clinical efficacy with an emphasis on metastatic prostate cancer.

Small molecule-mediated anti-cancer therapy via hypoxia-inducible factor-1 blockade.

Despite reservations regarding potential toxicities, small molecule-mediated blockade of the hypoxia-inducible factor-1 transcription factor has emerged as a viable anti-cancer strategy in vivo. Recent experiments by Welsh et al. revealed unprecedented anti-tumor responses of various aggressive solid tumors to the HIF-1-inhibitory small molecule drug PX-478. Compared with other anti-cancer drugs, PX-478 had markedly improved regression, growth delay and log10 cell kill profiles, particularly against large tumors that are normally refractory to small molecule drug therapy. Importantly, pharmacokinetic and toxicity profiles were within acceptable limits, providing rationale for the clinical development of HIF-1 inhibitors in general. Though the mechanism of action for PX-478 is not completely understood, inhibition of glycolysis rather than angiogenesis appeared to be the primary mode of anti-cancer activity.

Genotyping and functional analysis of the D104N variant of human endostatin.

Endostatin is an endogenous inhibitor of angiogenesis derived from the extracellular matrix protein collagen XVIII. It has been reported that a variation at the 104 position (D104N) of human endostatin is associated with an increased risk of prostate cancer, potentially indicating that this protein variant is less active as an anti-angiogenic agent. Herein we reported the results of genotyping 389 patients with androgen independent prostate cancer (AIPC) and 352 normal control individuals for D104N endostatin. There was no significant association between the frequency of 104N endostatin and the incidence of AIPC in either Caucasian or African American patients compared to controls (15% Caucasian AIPC versus 13.7% in Caucasian controls, p=0.79; 7.4% African American AIPC versus 5.6% in African American controls, p=0.64). Actuarial analysis revealed no statistically significant association between incidence of the DN heterozygous genotype and survival (p=0.62 by logrank test). To study the functional significance of the D104N conversion, we have expressed and purified insoluble recombinant human 104D and 104N endostatin and compared their respective activities in human umbilical vein endothelial cell (HUVEC) tube formation assays. The 104N variant of human endostatin inhibited HUVEC tube formation at least as well as the wild-type form. We concluded that the D104N variation in human endostatin is neither clinically relevant nor suitable as a pharmacogenomic endpoint to assess the risk for developing AIPC.

Phenotype and function of rat dendritic cell subsets.

Dendritic cells (DC) comprise phenotypically-distinct subsets that sub-serve distinct functions in immune induction. Understanding the biology of DC subsets in vivo is crucial for the understanding of immune regulation and its perturbations in disease. This review focuses on the phenotype and functions of rat DC subsets and compares these with subsets identified in other species. Our research has concentrated on DC migrating in lymph. DC migrate constitutively from peripheral tissues to draining nodes, probably to induce/maintain tolerance to self- or harmless foreign antigens. After removal of mesenteric lymph nodes (MLN) in the rat, healing of afferent and efferent lymphatics permits migrating intestinal DC (iLDC) to be collected from the thoracic duct. We have shown that iLDC consist of least two subsets that differ in phenotype, in situ distribution and function. CD4+/SIRPalpha+ iLDC are highly immunostimulatory, but are excluded from T cell areas of MLN. In contrast, CD4-/SIRPalpha- iLDC are less potent stimulators of T cells, but carry material from apoptotic enterocytes to T cell areas of MLN. Similar subsets exist in both lymph nodes and spleen. It has been shown that phenotypically-similar subsets migrate in skin-draining lymph in cattle and sheep. We and others have shown that splenic CD4-/SIRPalpha- DC can phagocytose allogeneic cells in vitro, are poor stimulators of CD8+ T cells, and can lyse NK-sensitive target cells. Although some of our data suggest that rat CD4-/SIRPalpha- DC may equate to murine CD8+ DC, there is at present insufficient evidence to be confident of this.

Uptake of antigens from the intestine by dendritic cells.

The intestinal immune system responds to ingested antigens in a variety of ways, ranging from tolerance to full immunity. How T cells are instructed to make these differential responses is still unclear. Dendritic cells (DCs) sample enteric antigens in the lamina propria and Peyer's patches, and transport them within the patch or to mesenteric nodes where they are presented to lymphocytes. It is probable that DCs also transmit information that influences the outcome of T cell activation, but the nature of this information and the factors in the intestine that regulate DC behavior and properties are far from clear. We have developed a model in the rat that permits analysis of DCs actually in the process of migration from the intestine to mesenteric nodes. In this paper we will review those aspects of our research that relate to antigen uptake and discuss these in the context of other experimental systems.

Identification and phenotypic characterization of γδ T cells in rat lymph.

γδ T cells represent an unconventional subset of T lymphocytes that are abundant in epithelial tissues and serve as an early immune defense against microbes. We have, for the first time, identified γδ T cells in steady-state thoracic duct lymph (TDL) from rats. The lymph contains γδ T cells expressing CD8 but not CD4, CD25, MHC-II or CD103. The percentage of TDL γδ T cells in rats does not change when the mesenteric lymph nodes (MLN) are surgically removed. Our data suggest that a proportion of γδ T cells migrate from the intestine into rat TDL, under steady-state conditions.

Retrovirus-transformed erythroleukemia cells induce central nervous system failure in a new syngeneic mouse model of meningeal leukemia.

Lack of suitable mouse models for central nervous system (CNS)-associated leukemias has hindered mechanism-guided development of therapeutics. By transplanting retrovirus-transformed mouse erythroleukemia cells into syngeneic mice, we developed a new animal model of meningeal leukemia associated with rapid paralysis. Necropsy revealed massive proliferation of the leukemic cells in the bone marrow (BM) followed by pathological angiogenesis and invasion of the leukemic cells into the meninges of the CNS. Further analysis demonstrated that the erythroleukemia cells secreted high levels of VEGF and preferentially adhered in vitro to fibronectin. This unique animal model for meningeal leukemia should facilitate studies of engraftment and proliferation of leukemic cells in the BM and their invasion of the CNS as well as pre-clinical evaluation of experimental therapeutics for CNS-associated leukemias.