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.

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.

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.

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.

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.

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.

Isolation of rat intestinal lymph DC.

Dendritic cells (DCs) migrate constitutively from the intestine via the lymph to the mesenteric lymph nodes. These migrating intestinal lymph DCs (ilDCs) carry antigens acquired in the intestine and play important roles in both the initiation of immune responses and the maintenance of oral tolerance. The ilDC population is made up of at least three functionally different DC subsets. Like many DC populations, ilDCs are exquisitely sensitive to their environment, changing their phenotype and maturing in response to the procedures associated with their extraction from solid tissues. We have developed and refined a method for collecting and purifying these DC subsets from rats, without inducing them to mature. This method involves two separate surgical procedures, separated by at least 6 weeks. Initially, mesenteric lymph nodes are removed. After the animals have fully recovered we cannulate the thoracic duct and collect the ilDCs on ice, minutes after they have left the lymph vessel. The DCs are then enriched using magnetic beads and purified by flow cytometric sorting. We describe this method here, including our recent refinements to limit the use of the restraining "Bollman" cage.

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.

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.

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.

Exploring the diversity of marine-derived fungal polyketide synthases.

Using an approach based on polymerase chain reaction (PCR), we examined the diversity of polyketide synthase (PKS) genes present in 160 marine fungal isolates, representing 142 species. We obtained ketosynthase (KS) domain PCR products from 99 fungal isolates, representing Dothideomycetes, Sordariomycetes, Eurotiomycetes, and incertae sedis. Sequence similarity searches and phylogenetic analysis of 29 marine partial-KS-encoding sequences revealed domains predicted to encode reducing, nonreducing, and 6-methylsalicylic acid PKSs. Bioinformatic analysis of an alignment of the KS sequences from marine-derived fungi revealed no unique motifs in this region. However, several specificity-determining positions were apparent between fungal 6-methylsalicylic acid PKSs as compared with either reducing or nonreducing PKSs. Evaluation of these positions in the context of a modelled three-dimensional protein structure highlighted their potential use as PKS classification markers. Evaluating primer-binding sites was necessary to obtain KS domain fragments from putative PKSs while maintaining a level of sequence information adequate to properly classify and characterize them.

Regulation of intestinal immunity: effects of the oral adjuvant Escherichia coli heat-labile enterotoxin on migrating dendritic cells.

Escherichia coli heat-labile enterotoxin (Etx) is an oral adjuvant in mice. We show that this is also true for rats. To understand this adjuvant activity we examined lymph dendritic cells (DC) migrating from the intestine to mesenteric lymph nodes (MLN) in animals fed Etx. These DC can prime antigen-specific antibody responses. We show that in rats the small intestine contains 7-24 million DC and 8 x 10(5 )of these migrate to MLN each day. Surprisingly, Etx does not stimulate increased migration of lymph DC. However, oral Etx affects the activation, antigen transport and localization of migratory DC. Specifically, expression of CD25 increases on the CD172a(high) subset of lymph DC. Oral Etx also increases the number of CD172a(high) lymph DC containing co-administered ovalbumin. CD172a(high) lymph DC treated with Etx in vitro, or purified from the lymph of animals fed Etx, stimulate stronger proliferative responses from primed T cells. Etx also directs more of the CD172a(high) lymph DC into the central region of the MLN T cell areas. This change in DC localization is associated with an increase in the expression of CCR7. These data help advance our understanding of the role of DC in initiating mucosal immune responses in vivo.

Eosinophilic bowel disease controlled by the BB rat-derived lymphopenia/Gimap5 gene.

BACKGROUND & AIMS: Many models of autoimmunity are associated with lymphopenia. Most involve a T-helper cell (Th)1-type disease, including the diabetic BioBreeding (BB) rat. To investigate the roles of identified susceptibility loci in disease pathogenesis, we bred PVG-RT1(u), lymphopenia (lyp)/lyp rats, congenic for the iddm1 (RT1(u)) and iddm2 (lyp, Gimap5(-/-)) diabetes susceptibility loci on the PVG background. Surprisingly, these rats developed a spontaneous, progressive, inflammatory bowel disease. To understand the disease pathogenesis, we undertook investigations at the genetic, histologic, and cellular levels. METHODS: Genetically lymphopenic rats and congenic wild-type partners were compared for gross pathologic, histologic, and immunologic parameters, the latter including cytokines and autoantibodies. RESULTS: Genetic analysis demonstrated that homozygosity at the lyp locus was required for disease. All rats developed disease, and the median age at humane killing was approximately 36 weeks. This panintestinal disease showed a conspicuous eosinophilic infiltrate in the submucosa and muscle layers, but the villi were unaffected. Diseased rats showed splenomegaly and massive enlargement of the mesenteric lymph nodes. This pathology resembles human eosinophilic gastroenteritis, and several further features indicate a Th2 basis. The rats developed high serum IgE and made IgG autoantibodies that detected a nonleukocytic cell present in the intestinal wall of all rats (including germ free). CONCLUSIONS: The T-lymphopenic state associated with GIMAP5 deficiency renders rats generally susceptible to T-cell-mediated autoimmunity, but the immunoregulatory bias (Th1/Th2) of any disease depends on other genetic (or environmental) factors. In the present model, we suggest that defective peripheral tolerance to an intestine-specific autoantigen leads to uncontrolled inflammation of the intestinal wall.

Characterization of the beta-ketoadipate pathway in Sinorhizobium meliloti.

Aromatic compounds represent an important source of energy for soil-dwelling organisms. The beta-ketoadipate pathway is a key metabolic pathway involved in the catabolism of the aromatic compounds protocatechuate and catechol, and here we show through enzymatic analysis and mutant analysis that genes required for growth and catabolism of protocatechuate in the soil-dwelling bacterium Sinorhizobium meliloti are organized on the pSymB megaplasmid in two transcriptional units designated pcaDCHGB and pcaIJF. The pcaD promoter was mapped by primer extension, and expression from this promoter is demonstrated to be regulated by the LysR-type protein PcaQ. Beta-ketoadipate succinyl-coenzyme A (CoA) transferase activity in S. meliloti was shown to be encoded by SMb20587 and SMb20588, and these genes have been renamed pcaI and pcaJ, respectively. These genes are organized in an operon with a putative beta-ketoadipyl-CoA thiolase gene (pcaF), and expression of the pcaIJF operon is shown to be regulated by an IclR-type transcriptional regulator, SMb20586, which we have named pcaR. We show that pcaR transcription is negatively autoregulated and that PcaR is a positive regulator of pcaIJF expression and is required for growth of S. meliloti on protocatechuate as the carbon source. The characterization of the protocatechuate catabolic pathway in S. meliloti offers an opportunity for comparison with related species, including Agrobacterium tumefaciens. Differences observed between S. meliloti and A. tumefaciens pcaIJ offer the first evidence of pca genes that may have been acquired after speciation in these closely related species.

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.

Collection of lymph-borne dendritic cells in the rat.

Dendritic cells (DCs) are crucial in immune induction. Not only do they collect antigens in peripheral tissues, and transport and process them for presentation to lymphocytes in draining lymph nodes, but they also regulate the immune response by modulating T-cell differentiation. Intestinal and hepatic DCs migrating in lymph can be collected from rats under near-physiological conditions. Initially, the mesenteric or celiac lymph nodes are removed from young rats (30 min). The afferent and efferent lymph vessels subsequently heal, permitting DCs to enter the thoracic duct. After at least 6 wk, the duct is cannulated (40 min). Lymph can be collected for up to 48 h. DCs can subsequently be identified, enriched and sorted to high degrees of purity. This two-stage technique generates large numbers of immunologically relevant DCs under near-physiological conditions. Lymph collection requires 2-3 h per animal over 6 wk.

Network of dendritic cells within the muscular layer of the mouse intestine.

Dendritic cells (DCs) are located at body surfaces such as the skin, respiratory and genital tracts, and intestine. To further analyze intestinal DCs, we adapted an epidermal sheet separation technique and obtained two intestinal layers, facing the lumen and serosa. Unexpectedly, immunolabeling of the layer toward the serosa revealed a regular, dense, planar network of cells with prominent dendritic morphology within the external muscular layer and with increasing frequency along the length of the intestine. Direct examination of the serosal-disposed layers showed a significant fraction of the DCs to express DEC-205/CD205, CD11c, Langerin/CD207, Fcgamma receptor/CD16/32, CD14, and low levels of activation markers, CD25, CD80, CD86, and CD95. By more sensitive FACS analyses, cells from this layer contained two CD11c(+) populations of CD45(+) CD205(+), CD19(-) leukocytes, MHC II(+) and MHC II(-). When ovalbumin conjugated to an anti-DEC-205 antibody was injected into mice, the conjugate targeted to these DCs, which upon isolation were able to stimulate ovalbumin-specific, CD4(+) and CD8(+) T cell antigen receptor-transgenic T cells. In vivo, these DCs responded to two microbial stimuli, systemic LPS and oral live bacteria, by up-regulating CD80, CD86, DEC-205, and Langerin within 12 h. This network of DCs thus represents a previously unrecognized antigen-presenting cell system in the intestine.

Association of a common variant of the CASP8 gene with reduced risk of breast cancer.

Apoptosis, or programmed cell death, is perturbed in many cancers. We tested the hypothesis that coding polymorphisms of the death receptor 4 (DR4), caspase-8 (CASP8), and caspase-10 (CASP10) genes might act as low-penetrance breast cancer genes. Single-nucleotide polymorphisms (SNPs) of these genes were genotyped in a series of 999 breast cancer case patients and 996 control subjects from Sheffield, U.K., and in a second, independent U.K. population of 2192 case patients and 2262 control subjects from East Anglia. In the Sheffield study, the rare H allele of CASP8, D302H, was associated with a reduced risk of breast cancer in a dose-dependent manner (P(trend) = .007). Furthermore, the CASP8 D302H association, but not that of the other CASP8 SNPs examined (T21914C, G50121A, and G50358A), was replicated in the East Anglian study. The combined adjusted odds ratios for breast cancer were 0.83 (95% confidence interval [CI] = 0.74 to 0.94) for the DH heterozygote and 0.58 (95% CI = 0.39 to 0.88) for the HH homozygote (P(trend) = .0002, adjusted for study). The reproducible, dose-dependent association of CASP8 D302H with breast cancer indicates the potential importance of inherited variation in the apoptosis pathway in breast cancer susceptibility.