Epidermal Langerhans Cells are Dispensable for Humoral and Cell-Mediated Immunity Elicited by Gene Gun Immunization.

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8(+) T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4(+) Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4(+) and CD8(+) T cells, IFN-gamma secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity.

Assessment of cell-mediated immunity in a British population using multiple skin test antigens

An in vivo method of assessing the competence of the cell-mediated immune system (Multitest CMI) was used in 200 healthy volunteers (age range 17-88 years). The profile of reactivity to seven individual antigens was determined. A positive reaction was obtained in 96.5% of the subjects who reacted positively to at least one antigen with 78% reacting to two or more antigens. The number of positive responses and the degree of reactivity was significantly reduced in elderly subjects and in females aged 17-65 years. The Multitest CMI system provides a rapid and convenient method of assessing cell-mediated immunity (CMI) in vivo and could have a wide range of applications in the investigation of immunological, infective and neoplastic conditions.

Immunotoxicity of aflatoxin B1: Impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 mu g pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-alpha, IL-1 beta, IL-6, IFN-gamma) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-gamma and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1. (C) 2008 Elsevier Inc. All rights reserved.

Priming of CD8+ and CD4+ T Cells in Experimental Leishmaniasis Is Initiated by Different Dendritic Cell Subtypes

The biological role of Langerin(+) dendritic cells (DCs) such as Langerhans cells and a subset of dermal DCs (dDCs) in adaptive immunity against cutaneous pathogens remains enigmatic. Thus, we analyzed the impact of Langerin(+) DCs in adaptive T cell-mediated immunity toward Leishmania major parasites in a Lang-DTR mouse model that allows conditional diphtheria toxin (DT)-induced ablation of The biological role of Langerin+ dendritic cells (DCs) such as Langerhans cells and a subset of dermal DCs (dDCs) in adaptive immunity against cutaneous pathogens remains enigmatic. Thus, we analyzed the impact of Langerin+ DCs in adaptive T cell-mediated immunity toward Leishmania major parasites in a Lang-DTR mouse model that allows conditional diphtheria toxin (DT)-induced ablation of Langerin+ DCs in vivo. For the first time, infection experiments with DT-treated Lang-DTR mice revealed that proliferation of L. major-specific CD8+ T cells is significantly reduced during the early phase of the immune response following depletion of Langerin+ DCs. Consequently, the total number of activated CD8+ T cells within the draining lymph node and at the site of infection is diminished. Furthermore, we show that the impaired CD8+ T cell response is due to the absence of Langerin+ dDCs and not Langerhans cells. Nevertheless, the CD4+ T cell response is not altered and the infection is cleared as effectively in DT-treated Lang-DTR mice as in control mice. This clearly demonstrates that Langerin+ DCs are, in general, dispensable for an efficient adaptive immune response against L. major parasites. Thus, we propose a novel concept that, in the experimental model of leishmaniasis, priming of CD4+ T cells is mediated by Langerin− dDCs, whereas Langerin+ dDCs are involved in early priming of CD8+ T cells.

Monochloroacetic acid and 60% salicylic acid as a treatment for simple plantar warts:effectiveness and mode of action

Monochloroacetic acid crystals and 60% salicylic acid ointment was found to be more effective than placebo as a treatment for simple plantar warts in a double blind study on 57 patients. Nineteen (66%) patients in the active treatment group compared with five (18%) patients in the placebo group were cured after 6 weeks (P = 0.002). The active treatment was associated with a significantly higher cure rate 6 months after entry (P = 0.04). Treatments were well tolerated. IgG or IgM antibodies or both to human papilloma virus (HPV) types 1 or 2 or both were detected significantly more frequently in the actively treated group 6 weeks after entry (P = 0.0005). Twelve (50%) patients considered to be cured had no detectable secondary immune response. Our results suggest that cure does not depend primarily on the humoral system but rather on mechanical destruction of wart tissue, or occurs as a result of cell mediated immunity.

Genome-wide association study of urinary albumin excretion rate in patients with type 1 diabetes

Aims/hypothesis: An abnormal urinary albumin excretion rate (AER) is often the first clinically detectable manifestation of diabetic nephropathy. Our aim was to estimate the heritability and to detect genetic variation associated with elevated AER in patients with type 1 diabetes. 
 Methods: The discovery phase genome-wide association study (GWAS) included 1,925 patients with type 1 diabetes and with data on 24 h AER. AER was analysed as a continuous trait and the analysis was stratified by the use of antihypertensive medication. Signals with a p value <10^{−4 were followed up in 3,750 additional patients with type 1 diabetes from seven studies.} 
 Results: The narrow-sense heritability, captured with our genotyping platform, was estimated to explain 27.3% of the total AER variability, and 37.6% after adjustment for covariates. In the discovery stage, five single nucleotide polymorphisms in the GLRA3 gene were strongly associated with albuminuria (p < 5 × 10^{−8). In the replication group, a nominally significant association (p = 0.035) was observed between albuminuria and rs1564939 in GLRA3, but this was in the opposite direction. Sequencing of the surrounding genetic region in 48 Finnish and 48 UK individuals supported the possibility that population-specific rare variants contribute to the synthetic association observed at the common variants in GLRA3. The strongest replication (p = 0.026) was obtained for rs2410601 between the PSD3 and SH2D4A genes. Pathway analysis highlighted natural killer cell mediated immunity processes.} 
 Conclusions/interpretation: This study suggests novel pathways and molecular mechanisms for the pathogenesis of albuminuria in type 1 diabetes.

Aflatoxin Exposure and Associated Human Health Effects, a Review of Epidemiological Studies

flatoxins are fungal toxins that possess acute life threatening toxicity, carcinogenic properties and other potential chronic adverse effects. Dietary exposure to aflatoxins is considered a major public health concern, especially for subsistence farming communities in sub-Saharan Africa and South Asia, where dietary staple food crops such as groundnuts and maize are often highly contaminated with aflatoxin due to hot and humid climates and poor storage, together with low awareness of risk and lack of enforcement of regulatory limits. Biomarkers have been developed and applied in many epidemiological studies assessing aflatoxin exposure and the associated health effects in these high-risk population groups. This review discusses the recent epidemiological evidence for aflatoxin exposure, co-exposure with other mycotoxins and associated health effects in order to provide evidence on risk assessment, and highlight areas where further research is necessary. Aflatoxin exposure can occur at any stage of life and is a major risk factor for hepatocellular carcinoma, especially when hepatitis B infection is present. Recent evidence suggests that aflatoxin may be an underlying determinant of stunted child growth, and may lower cell-mediated immunity, thereby increasing disease susceptibility. However, a causal relationship between aflatoxin exposure and these latter adverse health outcomes has not been established, and the biological mechanisms for these have not been elucidated, prompting further research. Furthermore, there is a dearth of information regarding the health effects of co-exposure to aflatoxin with other mycotoxins. Recent developments of biomarkers provide opportunities for important future research in this area.

Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus

The interaction between microorganisms and host defense mechanisms is a decisive factor for the survival of marine bivalves. They rely on cell-mediated and humoral reactions to overcome the pathogens that naturally occur in the marine environment. In order to understand host defense reactions in animals inhabiting extreme environments we investigated some of the components from the immune system of the deep sea hydrothermal vent mussel Bathymodiolus azoricus. Cellular constituents in the hemolymph and extrapallial fluid were examined and led to the identification of three types of hemocytes revealing the granulocytes as the most abundant type of cell. To further characterize hemocyte types, the presence of cell surface carbohydrate epitopes was demonstrated with fluorescent WGA lectin, which was mostly ascribed to the granulocytes. Cellular reactions were then investigated by means of phagocytosis and by the activation of putative MAPKs using the microbial compounds zymosan, glucan, peptidoglycan and lipopolysaccharide. Two bacterial agents, Bacillus subtilis and Vibrio parahaemolyticus, were also used to stimulate hemocytes. The results showed that granulocytes were the main phagocytic cells in both hemolymph and extrapallial fluid of B. azoricus. Western blotting analyses using commercially available antibodies against ERK, p38 and JNK, suggested that these putative kinases are involved in signal transduction pathways during experimental stimulation of B. azoricus hemocytes. The fluorescent Ca²⁺ indicator Fura-2 AM was also insightful in demonstrating hemocyte stimulation in the presence of laminarin or live V. parahaemolyticus. Finally, the expression of the antibacterial gene mytilin was analyzed in gill tissues by means of RT-PCR and whole-mount in situ hybridization. Mytilin transcripts were localized in hemocytes underlying gill epithelium. Moreover, mytilin was induced by exposure of live animals to V. parahaemolyticus. These findings support the premise of a conserved innate immune system in B. azoricus. Such system is comparable to other Bivalves and involves the participation of cellular and humoral components. © 2008 Elsevier Inc. All rights reserved.

Loss of the LAT adaptor converts antigen-responsive T cells into pathogenic effectors that function independently of the T cell receptor.

Despite compromised T cell antigen receptor (TCR) signaling, mice in which tyrosine 136 of the adaptor linker for activation of T cells (LAT) was constitutively mutated (Lat(Y136F) mice) accumulate CD4(+) T cells that trigger autoimmunity and inflammation. Here we show that equipping postthymic CD4(+) T cells with LATY136F molecules or rendering them deficient in LAT molecules triggers a lymphoproliferative disorder dependent on prior TCR engagement. Therefore, such disorders required neither faulty thymic T cell maturation nor LATY136F molecules. Unexpectedly, in CD4(+) T cells recently deprived of LAT, the proximal triggering module of the TCR induced a spectrum of protein tyrosine phosphorylation that largely overlapped the one observed in the presence of LAT. The fact that such LAT-independent signals result in lymphoproliferative disorders with excessive cytokine production demonstrates that LAT constitutes a key negative regulator of the triggering module and of the LAT-independent branches of the TCR signaling cassette.

A Paradoxical Effect of Systemic IL-23 in EAE – Limitation of Autoimmune Inflammatory Demyelination

The heterodimeric cytokine IL-23 plays a non-redundant function in the development of cell-mediated, organspecific autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE). To further characterize the mechanisms of action of IL-23 in autoimmune inflammation, we administered IL-23 systemically at different time points during both relapsing and chronic EAE. Surprisingly, we found suppression of disease in all treatment protocols. We observed a reduction in the number of activated macrophages and microglia in the CNS, while T cell infiltration was not significantly affected. Disease suppression correlated with reduced expansion of myelin-reactive T cells, loss of T-bet expression, loss of lymphoid structures, and increased production of IL-6 and IL-4. Here we describe an unexpected function of exogenous IL-23 in limiting the scope and extent of organ-specific autoimmunity.

Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts

Bystander responses underlie some of the current efforts to develop gene therapy approaches for cancer treatment. Similarly, they may have a role in strategies to treat tumours with targeted radioisotopes. In this study we show direct evidence for the production of a radiation-induced bystander response in primary human fibroblasts, We utilize a novel approach of using a charged-particle microbeam, which allows individual cells within a population to be selected and targeted with counted charged particles. Individual primary human fibroblasts within a population of 600-800 cells were targeted with between 1 and 15 helium ions (effectively, alpha -particles). The charged particles were delivered through the centre of the nucleus with an accuracy of +/- 2 mum and a detection and counting efficiency of greater than 99%. When scored 3 days later, even though only a single cell had been targeted, typically an additional 80-100 damaged cells were observed in the surviving population of about 5000 cells. The yield of damaged cells was independent of the number of charged particles delivered to the targeted cell, Similar results of a 2-3-fold increase in the background level of damage present in the population were observed whether 1 or 4 cells were targeted within the dish. Also, when 200 cells within one quadrant of the dish were exposed to radiation, there was a 2-3-fold increase in the damage level in an unexposed quadrant of the dish, This effect was independent of the presence of serum in the culture medium and was only observed when a cell was targeted, but not when only the medium was exposed, confirming that a cell-mediated response is involved. (C) 2001 Cancer Research Campaign.

Langerhans cells protect from allergic contact dermatitis in mice by tolerizing CD8+ T cells and activating Foxp3+ regulatory T cells

Allergic contact dermatitis is the most frequent occupational disease in industrialized countries. It is caused by CD8(+) T cell-mediated contact hypersensitivity (CHS) reactions triggered at the site of contact by a variety of chemicals, also known as weak haptens, present in fragrances, dyes, metals, preservatives, and drugs. Despite the myriad of potentially allergenic substances that can penetrate the skin, sensitization is relatively rare and immune tolerance to the substance is often induced by as yet poorly understood mechanisms. Here we show, using the innocuous chemical 2,4-dinitrothiocyanobenzene (DNTB), that cutaneous immune tolerance in mice critically depends on epidermal Langerhans cells (LCs), which capture DNTB and migrate to lymph nodes for direct presentation to CD8(+) T cells. Depletion and adoptive transfer experiments revealed that LCs conferred protection from development of CHS by a mechanism involving both anergy and deletion of allergen-specific CD8(+) T cells and activation of a population of T cells identified as ICOS(+)CD4(+)Foxp3(+) Tregs. Our findings highlight the critical role of LCs in tolerance induction in mice to the prototype innocuous hapten DNTB and suggest that strategies targeting LCs might be valuable for prevention of cutaneous allergy.