Normal pathogen-specific immune responses mounted by CTLA-4-deficient T cells: a paradigm reconsidered.

CTLA-4 is a critical negative regulator of T cell responses and CTLA-4-deficient (CTLA-4(-/-)) mice die of a lymphproliferative disease. Nevertheless, RAG-2-deficient mice reconstituted with a mixture of CTLA-4(-/-) and normal (CTLA-4(+/+)) bone marrow survive in the absence of any signs of disease, although 50% of their T cells do not express CTLA-4. Using such mixed chimeras, we analyzed the role of CTLA-4 in specific T cell responses to lymphocytic choriomeningitis virus, Leishmania major and mouse mammary tumor virus, which cause acute, chronic and persistent infections, respectively. The populations of antigen-specific CTLA-4(-/-)CD4(+) and CTLA-4(-/-)CD8(+) T cells became activated, expanded and contracted indistinguishably from CTLA-4(+/+)CD4(+) and CTLA-4(+/+)CD8(+) T cells after infection with all three pathogens. Thus, CTLA-4 is not involved in the down-regulation of specific T cell responses and peripheral deletion in a T cell-autonomous fashion.

Oral vitamin B12 for patients suspected of subtle cobalamin deficiency: a multicentre pragmatic randomised controlled trial.

BACKGROUND: Evidence regarding the effectiveness of oral vitamin B12 in patients with serum vitamin B12 levels between 125-200 pM/l is lacking. We compared the effectiveness of one-month oral vitamin B12 supplementation in patients with a subtle vitamin B12 deficiency to that of a placebo. METHODS: This multicentre (13 general practices, two nursing homes, and one primary care center in western Switzerland), parallel, randomised, controlled, closed-label, observer-blind trial included 50 patients with serum vitamin B12 levels between 125-200 pM/l who were randomized to receive either oral vitamin B12 (1000 μg daily, N = 26) or placebo (N = 24) for four weeks. The institution's pharmacist used simple randomisation to generate a table and allocate treatments. The primary outcome was the change in serum methylmalonic acid (MMA) levels after one month of treatment. Secondary outcomes were changes in total homocysteine and serum vitamin B12 levels. Blood samples were centralised for analysis and adherence to treatment was verified by an electronic device (MEMS; Aardex Europe, Switzerland). Trial registration: ISRCTN 22063938. RESULTS: Baseline characteristics and adherence to treatment were similar in both groups. After one month, one patient in the placebo group was lost to follow-up. Data were evaluated by intention-to-treat analysis. One month of vitamin B12 treatment (N = 26) lowered serum MMA levels by 0.13 μmol/l (95%CI 0.06-0.19) more than the change observed in the placebo group (N = 23). The number of patients needed to treat to detect a metabolic response in MMA after one month was 2.6 (95% CI 1.7-6.4). A significant change was observed for the B12 serum level, but not for the homocysteine level, hematocrit, or mean corpuscular volume. After three months without active treatment (at four months), significant differences in MMA levels were no longer detected. CONCLUSIONS: Oral vitamin B12 treatment normalised the metabolic markers of vitamin B12 deficiency. However, a one-month daily treatment with 1000 μg oral vitamin B12 was not sufficient to normalise the deficiency markers for four months, and treatment had no effect on haematological signs of B12 deficiency.

Human epidermal Langerhans cells express the tight junction protein claudin-1 and are present in human genetic claudin-1 deficiency (NISCH syndrome).

Claudin-1 (CLDN1) is a structural tight junction (TJ) protein and is expressed in differentiating keratinocytes and Langerhans cells in the epidermis. Our objective was to identify immunoreactive CLDN1 in human epidermal Langerhans cells and to examine the pattern of epidermal Langerhans cells in genetic human CLDN1 deficiency [neonatal ichthyosis, sclerosing cholangitis (NISCH) syndrome]. Epidermal cells from healthy human skin labelled with CLDN1-specific antibodies were analysed by confocal laser immunofluorescence microscopy and flow cytometry. Skin biopsy sections of two patients with NISCH syndrome were stained with an antibody to CD1a expressed on epidermal Langerhans cells. Epidermal Langerhans cells and a subpopulation of keratinocytes from healthy skin were positive for CLDN1. The gross number and distribution of epidermal Langerhans cells of two patients with molecularly confirmed NISCH syndrome, however, was not grossly altered. Therefore, CLDN1 is unlikely to play a critical role in migration of Langerhans cells (or their precursors) to the epidermis or their positioning within the epidermis. Our findings do not exclude a role of this TJ molecule once Langerhans cells have left the epidermis for draining lymph nodes.

How High Dose(s) of Oral Vitamin D3 Can Correct Insufficiency in a Non Supplemented Rheumatologic Population ?

Introduction: 700 to 1000 UI Vitamin D/day prevent 20% of fall and fracture. Higher dosage could prevent other health problems, such as immune diseases. Adherence to oral daily vitamin D supplementation is low. There is no guideline on how to supplement patients with rheumatic diseases. We evaluated if 1-2 dose(s) of 300'000 UI oral vitamin D3 was enough to reach an optimal level of 25-OH vitamin D in late winter in patients with insufficiency. Methods: During November 2009 (M0) patients attending our Rheumatology Outpatient Clinic had a blood test to measure 25-OH vitamin D. Results were classified as: deficiency <10µg/l, insufficiency 10µg/l to 30µg/l and normal >30µg/l. Patients on daily oral vitamin D3 or who received a single high dose of vitamin D3 in the last 6 months and patients with deficiency or normal results were excluded. Patients included received a single dose of 300'000 IU of oral vitamin D3 and were asked to come back for a blood test for 25-OH vitamin D after 3 (M3) and 6 months (M6). If they were still insufficient at M3, they received a second high dose of 300'000 IU of oral vitamin D3. Results: 292 patients had their level of 25-OH vitamin D determined at M0. 141 patients (70% women) had vitamin D insufficiency (18.5µg/l (10.2-29.1)) and received a prescription for a single dose of 300'000 IU of oral vitamin D3. Men and women were not statistically different in term of age and 25-OH vitamin D level at M0. 124/141 (88%) patients had a blood test at M3. 2/124 (2%) had deficiency (8.1µg/l (7.5-8.7)), 50/124 (40%) normal results (36.7µg/l (30.5-56.5)). 58% (72/124) were insufficient (23.6µg/l (13.8-29.8)) and received a second prescription for 300'000 IU of oral vitamin D3. Of the 50/124 patients who had normal results at M3 and did not receive a second prescription, 36 (72%) had a test at M6. 47% (17/36) had normal results (34.8µg/l (30.3-42.8)), 53% (19/36) were insufficient (25.6µg/l (15.2-29.9)). Out of the 54/72 (75%) patients who received a second prescription, 28/54 (52%) had insufficiency (23.2µg/l (12.8-28.7)) and 26/54 (48%) had normal results (33.8µg/l (30.0-43.7)) at M 6. Discussion: This real life study has shown that one or two oral bolus of 300'000 IU of vitamin D3 in autumn and winter was not enough to completely correct hypovitaminosis D but was a good way of preventing a nadir of 25-OH vitamin D usually observed in spring in a Swiss rheumatologic population.

IRF4 and BATF are critical for CD8(+) T-cell function following infection with LCMV.

CD8(+) T-cell functions are critical for preventing chronic viral infections by eliminating infected cells. For healthy immune responses, beneficial destruction of infected cells must be balanced against immunopathology resulting from collateral damage to tissues. These processes are regulated by factors controlling CD8(+) T-cell function, which are still incompletely understood. Here, we show that the interferon regulatory factor 4 (IRF4) and its cooperating binding partner B-cell-activating transcription factor (BATF) are necessary for sustained CD8(+) T-cell effector function. Although Irf4(-/-) CD8(+) T cells were initially capable of proliferation, IRF4 deficiency resulted in limited CD8(+) T-cell responses after infection with the lymphocytic choriomeningitis virus. Consequently, Irf4(-/-) mice established chronic infections, but were protected from fatal immunopathology. Absence of BATF also resulted in reduced CD8(+) T-cell function, limited immunopathology, and promotion of viral persistence. These data identify the transcription factors IRF4 and BATF as major regulators of antiviral cytotoxic T-cell immunity.

Longevity differs among sexes but is not affected by repeated immune activation in voles (Microtus arvalis)

Investment of resources in immune defences, despite obvious short-term benefits, may be detrimental to long-term maintenance and thus decrease longevity in absence of parasites. In addition, females and males may differ in immune investment and intrinsic longevity because they are subjected to different degrees of sexual competition and extrinsic mortality. In order to test if sex-specific investment in mounting an immune response reduced longevity, we compared the longevity of captive male and female common voles Microtus arvalis regularly challenged with keyhole limpet haemocyanin, an antigen which elicits the production of antibodies, to the longevity of voles injected with the corresponding antigen-free buffer (phosphate-buffered saline). Injections were repeated every 28 days to mimic a chronic infection. The magnitude of immune response did not vary between males and females and did not affect longevity. Overall, females lived longer than males, independently of the immune challenge. Thus, the long-term costs of immunity seem small in voles. The longevity pattern is consistent with the prediction that male-biased predation or parasitism in the wild causes reduced intrinsic lifespan, but this reduction is not mediated by a decrease in male immunity

Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity.

Myc activity is emerging as a key element in acquisition and maintenance of stem cell properties. We have previously shown that c-Myc deficiency results in accumulation of defective hematopoietic stem cells (HSCs) due to niche-dependent differentiation defects. Here we report that immature HSCs coexpress c-myc and N-myc mRNA at similar levels. Although conditional deletion of N-myc in the bone marrow does not affect hematopoiesis, combined deficiency of c-Myc and N-Myc (dKO) results in pancytopenia and rapid lethality. Interestingly, proliferation of HSCs depends on both myc genes during homeostasis, but is c-Myc/N-Myc independent during bone marrow repair after injury. Strikingly, while most dKO hematopoietic cells undergo apoptosis, only self-renewing HSCs accumulate the cytotoxic molecule Granzyme B, normally employed by the innate immune system, thereby revealing an unexpected mechanism of stem cell apoptosis. Collectively, Myc activity (c-Myc and N-Myc) controls crucial aspects of HSC function including proliferation, differentiation, and survival.

Programming of marginal zone B-cell fate by basic Kruppel-like factor (BKLF/KLF3).

Splenic marginal zone (MZ) B cells are a lineage distinct from follicular and peritoneal B1 B cells. They are located next to the marginal sinus where blood is released. Here they pick up antigens and shuttle the load onto follicular dendritic cells inside the follicle. On activation, MZ B cells rapidly differentiate into plasmablasts secreting antibodies, thereby mediating humoral immune responses against blood-borne type 2 T-independent antigens. As Krüppel-like factors are implicated in cell differentiation/function in various tissues, we studied the function of basic Krüppel-like factor (BKLF/KLF3) in B cells. Whereas B-cell development in the bone marrow of KLF3-transgenic mice was unaffected, MZ B-cell numbers in spleen were increased considerably. As revealed in chimeric mice, this occurred cell autonomously, increasing both MZ and peritoneal B1 B-cell subsets. Comparing KLF3-transgenic and nontransgenic follicular B cells by RNA-microarray revealed that KLF3 regulates a subset of genes that was similarly up-regulated/down-regulated on normal MZ B-cell differentiation. Indeed, KLF3 expression overcame the lack of MZ B cells caused by different genetic alterations, such as CD19-deficiency or blockade of B-cell activating factor-receptor signaling, indicating that KLF3 may complement alternative nuclear factor-κB signaling. Thus, KLF3 is a driving force toward MZ B-cell maturation.

Parasitism, host immune function, and sexual selection.

Parasite-mediated sexual selection may arise as a consequence of 1) females avoiding mates with directly transmitted parasites, 2) females choosing less-parasitized males that provide parental care of superior quality, or 3) females choosing males with few parasites in order to obtain genes for parasite resistance in their offspring. Studies of specific host-parasite systems and comparative analyses have revealed both supportive and conflicting evidence for these hypotheses. A meta-analysis of the available evidence revealed a negative relationship between parasite load and the expression of male secondary sexual characters. Experimental studies yielded more strongly negative relationships than observations did, and the relationships were more strongly negative for ectoparasites than for endoparasites. There was no significant difference in the magnitude of the negative effect for species with and without male parental care, or between behavioral and morphological secondary sexual characters. There was a significant difference between studies based on host immune function and those based on parasite loads, with stronger effects for measures of immune function, suggesting that the many negative results from previous analyses of parasite-mediated sexual selection may be explained because relatively benign parasites were studied. The multivariate analyses demonstrating strong effect sizes of immune function in relation to the expression of secondary sexual characters, and for species with male parental care as compared to those without, suggest that parasite resistance may be a general determinant of parasite-mediated sexual selection.

Members of the PHO1 gene family show limited functional redundancy in phosphate transfer to the shoot, and are regulated by phosphate deficiency via distinct pathways.

The PHO1 family comprises 11 members in Arabidopsis thaliana. In order to decipher the role of these genes in inorganic phosphate (Pi) transport and homeostasis, complementation of the pho1 mutant, deficient in loading Pi to the root xylem, was determined by the expression of the PHO1 homologous genes under the control of the PHO1 promoter. Only PHO1 and the homologue PHO1;H1 could complement pho1. The PHO1;H1 promoter was active in the vascular cylinder of roots and shoots. Expression of PHO1;H1 was very low in Pi-sufficient plants, but was strongly induced under Pi-deficient conditions. T-DNA knock-out mutants of PHO1;H1 neither showed growth defects nor alteration in Pi transport dynamics, or Pi content, compared with wild type. However, the double mutant pho1/pho1;h1 showed a strong reduction in growth and in the capacity to transfer Pi from the root to the shoot compared with pho1. Grafting experiments revealed that phenotypes associated with the pho1 and pho1/pho1;h1 mutants were linked to the lack of gene expression in the root. The increased expression of PHO1;H1 under Pi deficiency was largely controlled by the transcription factor PHR1 and was suppressed by the phosphate analogue phosphite, whereas the increase of PHO1 expression was independent of PHR1 and was not influenced by phosphite. Together, these data reveal that although transfer of Pi to the root xylem vessel is primarily mediated by PHO1, the homologue PHO1;H1 also contributes to Pi loading to the xylem, and that the two corresponding genes are regulated by Pi deficiency by distinct signal transduction pathways.