The Autoimmune Regulator (AIRE), Which Is Defective in Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Patients, Is Expressed in Human Epidermal and Follicular Keratinocytes and Associates With the Intermediate Filament Protein Cytokeratin 17

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, which is caused by mutation of the autoimmune regulator (AIRE) gene, is a highly variable disease characterized by multiple endocrine failure, chronic mucocutaneous candidiasis, and various ectodermal defects. AIRE is a transcriptional regulator classically expressed in medullary thymic epithelial cells, monocytes, macrophages, and dendritic cells. Previous studies have suggested that AIRE can shuttle between the nucleus and cytoplasm of cells, although its cytoplasmic functions are poorly characterized. Through mass spectrometry analysis of proteins co-immunoprecipitating with cytoplasmic AIRE, we identified a novel association of AIRE with the intermediate filament protein cytokeratin 17 (K17) in the THP-1 monocyte cell line. We confirmed AIRE expression in HaCaT epidermal keratinocytes, as well as its interaction with K17. Confocal microscopy of human fetal and adult scalp hair follicles demonstrated a cytoplasmic pattern of AIRE staining that moderately colocalized with K17. The cytoplasmic association of AIRE with the intermediate filament network in human epidermal and follicular keratinocytes may provide a new path to understanding the ectodermal abnormalities associated with the APECED syndrome. (Am J Pathol 2011, 178:983-988; DOI: 10.1016/j.ajpath.2010.12.007)

p38 alpha MAP Kinase Controls IL-17 Synthesis in Vogt-Koyanagi-Harada Syndrome and Experimental Autoimmune Uveitis

PURPOSE. Interleukin (IL)-17, which is responsible for the initial influx of leukocytes into the target tissue, was recently described as the main cytokine involved in autoimmune diseases. Vogt-Koyanagi-Harada (VKH) syndrome is a significant cause of noninfectious blindness in the world. Herein the authors aimed at unraveling the involvement of IL-17 in VKH and in experimental autoimmune uveitis, focusing on the signaling pathways involved in IL-17 synthesis. METHODS. Mice were immunized with 161-180 peptide and pertussis toxin. Draining lymph node cells, harvested 21 days after immunization, were cultured in the presence or absence of p38 alpha mitogen-activated protein kinase (MAPK) inhibitor (SB203580) and assayed for cytokine production and quantification of CD4(+)IL-17(+) cells. Mice received intraocular injections of SB203580, and disease severity was evaluated by histologic examination of the enucleated eyes at day 21. CD4(+) lymphocytes from MSK-1/2-deficient mice, human CD4(+) cells silenced with MSK1 siRNA, or peripheral blood mononuclear cells (PBMCs) from VKH patients were cultured in the presence or absence of p38 alpha MAPK inhibitor and then assayed for IL-17, IFN-gamma, and IL-4 production. RESULTS. The inhibition of p38 alpha MAPK fully blocked the synthesis of IL-17 by PBMCs from VKH patients and lymphocytes from EAU mice. The absence of the msk1/2 gene resulted in failure to produce IL-17 by murine and human lymphocytes. Interestingly, intraocular injections of SB203580 in EAU mice did not suppress development of the disease. CONCLUSIONS. These data show that p38 alpha MAPK-MSK1/2 is involved in the control of IL-17 synthesis by CD4(+) T cells and that inhibition of p38 alpha MAPK in vitro suppresses IL-17 synthesis but that inhibition of this kinase in vivo did not protect from EAU. (Invest Ophthalmol Vis Sci. 2010;51:3567-3574) DOI: 10.1167/iovs.09-4393

Evaluation of Experimental Autoimmune Uveitis in Mice Treated with FTY720

PURPOSE. FTY720 (fingolimod) is an immunomodulatory drug capable of preventing T-cell migration to inflammatory sites by binding to and subsequently downregulating the expression of sphingosine-1 phosphate receptor 1 (S1P(1)) leading in turn to T-cell retention in lymphoid organs. Additional effects of FTY720 by increasing functional activity of regulatory T cells have recently been demonstrated, raising the conversion of conventional T cells into regulatory T cells and affecting the sequestration of regulatory T cells in normal mice. In this study, the action of FTY720 in the ocular autoimmune model in mice was investigated. METHODS. Mice were immunized with 161-180 peptide and pertussis toxin and were treated with 1 mg/kg/d FTY720 by gavage (7-21 days postimmunization [dpi]) or left untreated. Spleen cells, harvested 21 dpi, were cultured and assayed for cytokine production. Draining lymph node, spleen, and eye cells 21 dpi were assayed for quantification of T-cell populations. Disease severity was evaluated by histologic examination of the enucleated eyes at 21 and 49 dpi. In addition, anti-IRBP antibodies were analyzed by ELISA. RESULTS. FTY720 was effective in suppressing the experimental autoimmune uveitis score. Although there was a reduction in the number of eye-infiltrating cells, FTY did not prevent Treg accumulation at this site. FTY720 leads to a significant increase of CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cell percentages in lymph nodes, suggesting that this site could be the source of Treg cells found in the eye. CONCLUSIONS. The data showed that treatment in vivo with FTY720 was able to suppress EAU in mice. These results are indicative of the possible therapeutic use of FTY720 in ocular autoimmune processes. (Invest Ophthalmol Vis Sci. 2010;51:2568-2574) DOI:10.1167/iovs.09-4769

It takes guts for tolerance: The phenomenon of oral tolerance and the regulation of autoimmune response

The intestinal tract is a peculiar environment due to its constant contact with the microbiota agents, food antigens and other molecules. Such exposure requires the establishment of important regulatory mechanisms in order to avoid inflammatory response and self aggression. In this context, the GALT plays a very relevant role due to the presence of several different cellular populations which are the main players in this phenomenon. Moreover, it was described a while ago that the oral ingestion of a given molecule is able to induce systemic tolerance to the same molecule when it is used as an immunogen by parenteral route, known as oral tolerance. This observation led researches to use these mechanisms to induce tolerance against cognate antigens of different autoimmune diseases. In this context, in this review we focused on several tolerance inducing mechanisms which are relevant not only for the maintenance of intestinal tract but also for the suppression of T effector cells, such as Th1, Th2 and the newly described Th17 cells. To name a few, CD103(+) dendritic cells, Tr1 cells derived IL-10 secretion, Foxp3 conversion and CD4(+)LAP(+) regulatory cells induction are among the recently described features of the tolerogenic environment of the intestinal tract. (C) 2009 Elsevier B.V. All rights reserved.

A new model of outbred genetically selected mice which present a strong acute inflammatory response in the absence of complement component C5

Mice selected for a strong (AIRmax) or weak (AIRmin) acute inflammatory response present different susceptibilities to bacterial infections, autoimmune diseases and carcinogenesis. Variations in these phenotypes have been also detected in AIRmax and AIRmin mice rendered homozygous for Slc11a1 resistant (R) and susceptible (S) alleles. Our aim was to investigate if the phenotypic differences observed in these mice was related to the complement system. AIRmax and AIRmin mice and AIRmax and AIRmin groups homozygous for the resistance (R) or susceptibility (S) alleles of the solute carrier family 11a1 member (Slc11a1) gene, formerly designated Nramp-1. While no difference in complement activity was detected in sera from AIRmax and AIRmin strains, all sera from AIRmax Slc11a1 resistant mice (AIRmax(RR)) presented no complement-dependent hemolytic activity. Furthermore, C5 was not found in their sera by immunodiffusion and, polymerase chain reaction and DNA sequencing of its gene demonstrated that AIRmax(RR) mice are homozygous for the C5 deficient (D) mutation previously described in A/J. Therefore, the C5D allele was fixed in homozygosis in AIRmax(RR) line. The AIRmax(RR) line is a new experimental mouse model in which a strong inflammatory response can be triggered in vivo in the absence of C5.

Deficiency of thrombospondin-1 reduces Th17 differentiation and attenuates experimental autoimmune encephalomyelitis

Transforming growth factor beta (TGF-beta) plays a role both in the induction of Treg and in the differentiation of the IL-17-secreting T cells (Th17) which drive inflammation in experimental autoimmune encephalomyelitis (EAE). We investigated the role that thrombospondin-1 (TSP-1) dependent activation of TGF-beta played in the generation of an encephalitic Th17 response in EAE. Upon immunization with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)), TSP-1 deficient (TSP-1(null)) mice and MOG(35-55) TCR transgenic mice that lack of TSP-1 (2D2.TSP-1(null)) exhibited an attenuated form of EAE, and secreted lower levels of IL-17. Adoptive transfer of in vitro-activated 2D2.TSP-1(null) T cells induced a milder form of EAE, independent of TSP-1 expression in the recipient mice. Furthermore, in vitro studies demonstrated that anti-CD3/anti-CD28 pre-activated CD4+ T cells transiently upregulated latent TGF-beta in a TSP-1 dependent way, and such activation of latent TGF-beta was required for the differentiation of Th17 cells. These results demonstrate that TSP-1 participates in the differentiation of Th17 cells through its ability to activate latent TGF-beta, and enhances the inflammatory response in EAE. (C) 2009 Elsevier Ltd. All rights reserved.

Autolytic Mycobacterium leprae Hsp65 fragments may act as biological markers for autoimmune diseases

Investigating the proteolytic activity of the recombinant Mycobacterium leprae Heat Shock Protein of 65 kDa (rHsp65), chaperonin 2 (cpn2), we observed that it displays high instability. The fragmentation process starts at the C-terminus followed by progressive degradation of the N-terminus, which leads to a stable fragment comprising the middle region of the molecule. Urea was able to prevent autolysis, probably due to its denaturing action, while EDTA increased degradation levels indicating the need for metal ions. Peptides originated from autolysis were purified and analyzed by mass spectrometry, generating a continuous map. Since the bacteria and mammalian Hsp60 are known to be targets of the immune response and have been implicated in autoimmune diseases and chronic inflammation, the in vivo effect of rHsp65 peptides was evaluated in the spontaneous Systemic Lupus Erythematosus (SLE) model developed by the (NZB/NZW)F(1) mouse hybrids, and their individual anti-rHsp65 IgG2a/IgG1 antibody titer ratio was determined. The results showed orientation toward a T(H)1 responsiveness, and the treatment with the rHsp65 peptides diminished the environmental variance of the survival time of treated animals. These results outline the fact that environmental factors may also act through the modified stability expression of Heat Shock Proteins intervening during autoimmune processes. (C) 2011 Elsevier Ltd. All rights reserved.

Collagenase production and hemolytic activity related to 16S rRNA variability among Parvimonas micra oral isolates

Parvimonas micra are gram positive anaerobic cocci isolated from the oral cavity and frequently related to polymicrobial infections in humans. Despite reports about phenotypic differences, the genotypic variation of P. micra and its role in virulence are still not elucidated. The aim of this study was to determine the genotypic diversity of P. micra isolates obtained from the subgingival biofilm of subjects with different periodontal conditions and to correlate these findings with phenotypic traits. Three reference strains and 35 isolates of P. micro were genotyped by 16S rRNA PCR-RFLP and phenotypic traits such as collagenase production, elastolytic and hemolytic activities were evaluated. 16S rRNA PCR-RFLP showed that P. micra could be grouped into two main clusters: C1 and C2; cluster C1 harbored three genotypes (HG1259-like, HG1467-like and ICBM0583-like) while cluster C2 harbored two genotypes (ATC03270-like and ICBM036). A wide variability in collagenolytic activity intensities was observed among all isolates, while elastolytic activity was detected in only two isolates. There was an association between hemolytic activity in rabbit erythrocytes and cluster C2. There was an association between hemolytic activity in rabbit erythrocytes and cluster C1. Although these data suggest a possible association between P. micra genetic diversity and their pathogenic potential, further investigations are needed to confirm this hypothesis. (C) 2009 Elsevier Ltd. All rights reserved.

Helicobacter pylori and Iron-deficiency Anemia in Adolescents in Brazil

Aim: The aim of the study was to evaluate the association between Helicobacter pylori infection and iron deficiency (ID) in adolescents attending a public school. Patients and Methods: From March to June 2001, a cross-sectional study was conducted among adolescents (10-16 years) enrolled in a single public school in Sao Paulo, Brazil. Of 400 eligible students, 195 agreed to participate, but 1 was excluded due to sickle cell disease. A blood sample was collected from each subject to measure hemoglobin and ferritin. H pylori status was investigated with the 13 C-urea breath test. All of the subjects with either anemia or ID were given iron therapy. Results: H pylori prevalence was 40.7% (79/194), being higher in male subjects (45/90 vs 34/104, P = 0.014). There was no relation between infection and nutritional status. Abnormally low serum ferritin was observed in 12 subjects, half of whom were positive for H pylori (odds ratio [OR] 1.49, 95% confidence interval [CI] 0.38-5.81). The median serum ferritin was 33.6 ng/mL (interquartile range 23.9-50.9) in infected subjects and 35.1 ng/mL (interquartile range 23.7-53.9) in uninfected subjects. Anemia was detected in 2% (4/194) of the students, half of whom were infected (OR 1.47, 95% CI 0.1-20.6). The mean hemoglobin value in infected subjects was 13.83 g/dL +/- 1.02 versus 14 g/dL +/- 1.06 in uninfected subjects. Conclusions: The study was not able to find a relation between H pylori infection and ID or anemia.

Does hepatocellular carcinoma in non-alcoholic steatohepatitis exist in cirrhotic and non-cirrhotic patients?

Non-alcoholic steatohepatitis (NASH) has been associated with hepatocellular carcinoma (HCC) often arising in histologically advanced disease when steatohepatitis is not active (cryptogenic cirrhosis). Our objective was to characterize patients with HCC and active, histologically defined steatohepatitis. Among 394 patients with HCC detected by ultrasound imaging over 8 years and staged by the Barcelona Clinic Liver Cancer (BCLC) criteria, we identified 7 cases (1.7%) with HCC occurring in the setting of active biopsy-proven NASH. All were negative for other liver diseases such as hepatitis C, hepatitis B, autoimmune hepatitis, Wilson disease, and hemochromatosis. The patients (4 males and 3 females, age 63 ± 13 years) were either overweight (4) or obese (3); 57% were diabetic and 28.5% had dyslipidemia. Cirrhosis was present in 6 of 7 patients, but 1 patient had well-differentiated HCC in the setting of NASH without cirrhosis (fibrosis stage 1) based on repeated liver biopsies, the absence of portal hypertension by clinical and radiographic evaluations and by direct surgical inspection. Among the cirrhotic patients, 71.4% were clinically staged as Child A and 14.2% as Child B. Tumor size ranged from 1.0 to 5.2 cm and 5 of 7 patients were classified as early stage; 46% of all nodules were hyper-echoic and 57% were <3 cm. HCC was well differentiated in 1/6 and moderately differentiated in 5/6. Alpha-fetoprotein was <100 ng/mL in all patients. HCC in patients with active steatohepatitis is often multifocal, may precede clinically advanced disease and occurs without diagnostic levels of alpha-fetoprotein. Importantly, HCC may occur in NASH in the absence of cirrhosis. More aggressive screening of NASH patients may be warranted.

Antibody responses elicited in mice immunized with Bacillus subtilis vaccine strains expressing Stx2B subunit of enterohaemorragic Escherichia coli O157:H7

No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) producedbyenterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.

New insights into Vogt-Koyanagi-Harada disease

Vogt-Koyanagi-Harada disease (VKH), a well-established multiorgan disorder affecting pigmented structures, is an autoimmune disorder of melanocyte proteins in genetically susceptible individuals. Several clinical and experimental data point to the importance of the effector role of CD4+ T cells and Th1 cytokines, the relevance of searching a target protein in the melanocyte, and the relevance of the HLA-DRB1*0405 in the pathogenesis of the disease. Vogt-Koyanagi-Harada disease has a benign course when early diagnosed and adequatey treated. Full-blown recurrences are rare after the acute stage of Vogt-Koyanagi-Harada disease is over. On the other hand, clinical findings, such as progressive tissue depigmentation (including sunset glow fundus) and uveitis recurrence, indicate that ocular inflammation may persist after the acute phase. Additionally, indocyanine green angiography findings suggest the presence of choroidal inflammation in eyes without clinically detectable inflammation. The aim of this paper is to review the latest research results on Vogt-Koyanagi-Harada disease pathogenesis and chronic/convalescent stages, which may help to better understand this potentially blinding disease and to improve its treatment.

Recommendations for folate intake in women: implications for public health strategies

Folate deficiency has been associated with anemia and other adverse outcomes in pregnancy such as neural tube defects. The current recommendations for prevention of such outcomes are difficult to achieve through diet only, and folic acid supplementation and food fortification are feasible public health strategies. However, it is necessary to determine the usual diet and supplement use among women of reproductive age, including an accurate assessment of other dietary micronutrients. In addition to the beneficial effects observed in randomized clinical trials, health risks to the population have also been widely evaluated and discussed in the scientific community: for a minority to benefit from fortification programs, many are exposed to high folic acid intake levels.

Análise de diferentes medidas antropométricas na identificação de síndrome metabólica, com ou sem alteração do metabolismo glicídico

OBJETIVO: Este estudo comparou parâmetros antropométricos e de resistência à insulina de indivíduos sem e com síndrome metabólica (SM), subestratificados pela presença de anormalidades glicêmicas. SUJEITOS E MÉTODOS: Foram incluídos 454 indivíduos (66% mulheres, 54% brancos), sendo 155 alocados para o grupo 1 (sem SM, sem anormalidade glicêmica), 32 para o grupo 2 (sem SM, com anormalidade glicêmica), 104 no grupo 3 (com SM, sem anormalidade glicêmica) e 163 no grupo 4 (com SM e anormalidade glicêmica). Os grupos foram comparados por ANOVA. RESULTADOS: Os grupos com SM (3 e 4) apresentaram os piores perfis antropométrico e lipídico; no grupo 2, apesar de glicemias significantemente mais elevadas, as médias das variáveis antropométricas e lipídicas não diferiram do grupo 1. Os maiores valores médios de HOMA-IR foram encontrados nos grupos com SM, enquanto o grupo 2 apresentou o menor HOMA-β. A trigliceridemia foi a variável metabólica com coeficientes de correlação mais elevados com a antropometria. Porém, as correlações mais fortes foram da circunferência da cintura (r = 0,503) e da razão cintura-altura (r = 0,513) com o HOMA-IR (p < 0,01). CONCLUSÃO: Nossos achados revelam que, em amostra da população brasileira, qualquer das medidas antropométricas identifica indivíduos com SM, mas não parece capaz de diferenciar aqueles com distúrbio glicêmico. Reforçamos a relação mais forte das medidas de adiposidade central com resistência à insulina, sugerindo utilidade da razão cintura-altura. É possível que componente autoimune contribua para o comprometimento do metabolismo glicídico dos indivíduos do grupo 2.

Frequência à creche e estado nutricional de pré-escolares: uma revisão sistemática

OBJETIVO: Descrever e comparar estudos longitudinais que permitam inferir sobre a influência da creche no estado nutricional de crianças pré-escolares. FONTES DE DADOS: Revisão sistemática de trabalhos científicos publicados entre janeiro de 1990 e dezembro de 2008. Buscaram-se os estudos nas seguintes bases de dados: Lilacs, Scielo e PubMed. Realizou-se também pesquisa manual dos artigos referenciados. A busca ocorreu no período de março de 2008 a junho de 2009, e os descritores utilizados foram: "creche", "estado nutricional", "antropometria", "consumo alimentar", "anemia" e "alimentação escolar". SÍNTESE DOS DADOS: Na primeira etapa do estudo, obtiveram-se 78 artigos, mas somente sete puderam ser incluídos. Os outros 71 não apresentaram dados para contribuir com o objetivo específico deste estudo. Entre os artigos pesquisados na literatura, existem poucos que permitem inferir sobre a influência que a creche pode ter em relação ao estado nutricional de pré-escolares. Contudo, estudos longitudinais têm mostrado a relação causal entre a presença frequente da criança na creche e a melhoria do estado nutricional. CONCLUSÕES: Existe uma relação positiva entre a frequência da criança na creche e a melhoria do estado nutricional.