Gene polymorphism and HLA-G expression in patients with childhood-onset systemic lupus erythematosus: A pilot study.

Human leukocyte antigen-G (HLA-G) presents inhibitory functions in immune cells and is located in a chromosomal region associated with systemic lupus erythematosus (SLE) susceptibility. Polymorphisms in 3' untranslated region (3'UTR) of HLA-G gene may influence protein expression. To date, no study analyzing HLA-G polymorphism and expression in childhood-onset systemic lupus erythematosus (cSLE) has been conducted. Therefore, we investigated the influence of HLA-G 3'UTR polymorphisms in 50 cSLE patients and 144 healthy controls. For the expression analysis, the control group included 26 healthy individuals. No significant difference in allele, genotype, and haplotype frequencies was observed between patients and control group. However, both the 14 bp deletion allele (odds ratio [OR] = 2.76, 95% confidence interval [CI] = 1.17-6.52, P = .028) and the 14 bp deletion-deletion genotype (OR = 8.00, 95% CI = 1.57-40.65, P = .006) showed an association with lupus nephritis. After Bonferroni correction, none P-value remained statistically significant. Regarding HLA-G expression, no significant difference was observed between plasma levels of cSLE patients (56.02 U/mL, interquartile range [IQR] = 37.54-75.41) and control group (49.2 U/mL, IQR = 27.84-154.4, P = .952). However, when the patients were stratified according to clinical manifestations, patients with hematological manifestations showed a lower plasma concentration of soluble HLA-G (sHLA-G) (47.08 U/mL, IQR = 34.15-61.56) than patients with no hematological manifestations (65.26 U/mL, IQR = 47.69-102.60, P = .013). These results suggest that HLA-G polymorphism has small effect on cSLE susceptibility and that sHLA-G may be involved in the pathogenesis of the disease.

Cytokine profile in childhood-onset systemic lupus erythematosus: a cross-sectional and longitudinal study.

Childhood-onset systemic lupus erythematosus (cSLE) exhibits an aggressive clinical phenotype and severe complications. This could be due to a pro-inflammatory cytokine milieu. Therefore, we determined plasma levels of Th1 (IL-2, IFN-γ, TNF), Th2 (IL-4), Th17 (IL-17A, IL-6), and Treg (IL-10) cytokines in a cohort of cSLE patients and healthy controls, and we evaluated the association between these cytokines and disease activity. We conducted a cross-sectional study with 51 cSLE patients from two pediatric rheumatology services. Ten cSLE patients participated in a longitudinal follow-up study. Blood samples were collected from the same patient during active and inactive disease. Disease activity was evaluated according to SLE Disease Activity Index 2000 (SLEDAI-2K). Cytokines levels were measured by cytometric bead array technique. cSLE patients had higher IL-6 (P<0.001) and IL-10 (P<0.001) levels than healthy controls. Patients with active disease had higher IL-6 and IL-10 levels than patients with inactive disease (P=0.001 and P=0.014, respectively) and the control group (both P<0.001). IL-6 (P=0.022), IL-10 (P=0.013), and IL-17A (P=0.041) levels were significantly higher during active than inactive disease. Linear regression analysis revealed IL-6 (P=0.002, 95%CI=0.006-0.025) and IL-10 (P=0.01 95%CI=0.021-0.150) as independent factors for increased SLEDAI-2K. IL-6, IL-10, and IL-17A are candidate biomarkers for disease activity in cSLE patients. This is the first longitudinal study to support their pivotal role in the pathogenesis of the disease.

Cytokine profile in childhood-onset systemic lupus erythematosus: a cross-sectional and longitudinal study

Childhood-onset systemic lupus erythematosus (cSLE) exhibits an aggressive clinical phenotype and severe complications. This could be due to a pro-inflammatory cytokine milieu. Therefore, we determined plasma levels of Th1 (IL-2, IFN-γ, TNF), Th2 (IL-4), Th17 (IL-17A, IL-6), and Treg (IL-10) cytokines in a cohort of cSLE patients and healthy controls, and we evaluated the association between these cytokines and disease activity. We conducted a cross-sectional study with 51 cSLE patients from two pediatric rheumatology services. Ten cSLE patients participated in a longitudinal follow-up study. Blood samples were collected from the same patient during active and inactive disease. Disease activity was evaluated according to SLE Disease Activity Index 2000 (SLEDAI-2K). Cytokines levels were measured by cytometric bead array technique. cSLE patients had higher IL-6 (P<0.001) and IL-10 (P<0.001) levels than healthy controls. Patients with active disease had higher IL-6 and IL-10 levels than patients with inactive disease (P=0.001 and P=0.014, respectively) and the control group (both P<0.001). IL-6 (P=0.022), IL-10 (P=0.013), and IL-17A (P=0.041) levels were significantly higher during active than inactive disease. Linear regression analysis revealed IL-6 (P=0.002, 95%CI=0.006-0.025) and IL-10 (P=0.01 95%CI=0.021-0.150) as independent factors for increased SLEDAI-2K. IL-6, IL-10, and IL-17A are candidate biomarkers for disease activity in cSLE patients. This is the first longitudinal study to support their pivotal role in the pathogenesis of the disease.

Evaluation of multiplex PCR in first episodes of febrile neutropenia as a tool to improve early yeast diagnosis in leukemic/preleukemic patients.

In febrile neutropenic onco-hematological patients, delayed microbiological diagnosis leads to an increase in morbidity and mortality. Identification of the microorganism changes antibiotic therapy in more than half of cases; however, in only 20-30 % of such cases pathogen isolation is achieved. This study evaluates the frequency of fungus infection and its etiology in onco-hematological patients with febrile neutropenia utilizing blood cultures and non-commercial multiplex polymerase chain reaction (MT-PCR) primers. Fifty-three febrile neutropenia episodes in 35 onco-hematological patients were observed, and the results for the first unique 30 episodes are presented. Blood cultures were positive for Candida tropicalis (one case), gram-positive bacteria (two cases), and gram-negative bacteria (four cases), showing a 23.3 % microbiological isolation rate. Multiplex-PCR pan-fungal sequence was positive in 18 cases (60 %), and further sequencing identified fugal pathogens in 11 cases (Candida glabrata and Candida parapsilosis being the most common). MT-PCR pan-fungal sequence amplification was detected in 13 of 16 patients that later received antifungal treatment for clinical reasons only, while positivity was found in 5 out of 14 patients that did not receive antifungal treatment (p = 0.02). These results show that performing in-house non-commercial MT-PCR is feasible and may provide additional information about fungal infection without the need to wait for culture results. Further research is necessary to incorporate this technology into the decision-making process.

Foxp3 expression and nitric oxide production in peripheral blood mononuclear cells of communicants with pulmonary tuberculosis.

The understanding of the mechanisms involved in the immune response is of significant relevance to the control of tuberculosis (TB), especially in individuals living with patients with TB. To characterize the nitric oxide (NO) production and the Foxp3 marker expression in this population, peripheral blood mononuclear cells of intradomiciliary contacts of individuals with pulmonary tuberculosis with (CTb, susceptible) and without (STb, resistant) previous history of active infection were stimulated in vitro with Mycobacterium tuberculosis antigen (TbAg) and with the mitogen Concanavalin A for 24 and 48 h. The groups analysed did not present significant difference in the Foxp3 mRNA expression nor in the NO production. Negative correlation (P = 0.09) between NO and Foxp3 after a 48-h stimulation with TbAg was observed in the STb group. In this group, after a 24-h culture stimulated with TbAg (P = 0.03), this same correlation was observed. In comparison with the cytokines previously studied by our group (Cavalcanti et al., 2009), a positive correlation was observed between IL-10 and Foxp3 after a 48-h culture of cells from communicants susceptible to tuberculosis (STb) stimulated with TbAg (P = 0.04). Evaluating the entire population, a positive correlation was observed between the cytokine TNF-α and the Foxp3 marker in the cultures stimulated for 24 (P = 0.03) and 48 (P = 0.02) hours with TbAg. Therefore, considering the similarity in the exposure and the individual capacity of responding to the contact with M. tuberculosis, the present study contributes to the comprehension of the immune regulation in individuals living with patients with TB.

The +3187A/G HLA-G polymorphic site is associated with polar forms and reactive reaction in leprosy.

Considering that variability in immune response genes has been associated with susceptibility to leprosy and with disease severity, leprosy presents clinicopathological variants that are highly associated with the immune response, HLA-G has a well-recognized role in the modulation of the immune response, and polymorphisms at the 3' untranslated region (UTR) of the HLA-G gene may influence HLA-G production, we studied the polymorphic sites at the 3' UTR of the HLA-G gene in leprosy and their association with disease severity. We evaluated by sequencing analysis the allele, genotype, and haplotype frequencies of the 3' UTR HLA-G polymorphic sites (14-bpINDEL/+3003C-T/+3010C-G/+3027A-C/+3035C-T/+3142C-G/+3187A-G/+3196C-G) in 146 individuals presenting reactive leprosy from a highly endemic area, and associated with bacillary load and the type of reactive leprosy. A total of 128 healthy subjects were also studied. Allele, genotype, and haplotype frequencies for the 3' UTR HLA-G polymorphisms in leprosy patients did not differ from those observed in healthy donors. The +3187A allele was responsible for protection against the development of multibacillary leprosy in a dominant model (AA + AG)/GG, OR = 0.11, P = 0.018), and the +3187A allele and +3187A-A genotype were overrepresented in type II reactive leprosy reaction. The effect of genetic factors on leprosy susceptibility may be hidden by environmental components in highly endemic areas. The HLA-G + 3187A polymorphic site, which is related to unstable mRNA production, was associated with the development of polar forms of leprosy and reactive leprosy reaction.

Haplotype frequencies based on eight polymorphic sites at the 3' untranslated region of the HLA-G gene in individuals from two different geographical regions of Brazil.

The Brazilian population represents an admixture of native Amerindians, Portuguese settlers and Africans who were brought as slaves during the colonization period that began in the 16th century and was followed by waves of immigrations of Europeans and Asians in the 20th century. The contribution of these different ethnic groups to the constitution of Brazilian populations from different geographic regions is variable and, in addition to environmental factors, might act by determining different allele profiles among Brazilian populations from different regions. We studied polymorphic sites at the 3' untranslated region of the HLA-G gene in individuals from a Northeastern Brazilian region and compared them to our previously published data about a Southeastern Brazilian region, located at a distance of 2589 km. Our results showed that most polymorphic sites present a similar distribution in both populations, except for the lower frequency of the +3003C allele in the Northeastern population compared to the Southeastern population. Although differences in genotypic distribution were only significant for the +3003 locus (P = 0.0201), the diversity of haplotypes was distinct for each population. These results are important for case-control studies on the association of human leucocyte antigen-G polymorphism with disease and also in terms of the genetic structure of two distinct Brazilian populations.

Caracterização genotípica dos isolados de Escherichia coli provenientes de frangos de corte / Genotypically characterization of Escherichia coli isolates from poultry

Caracterizaram-se genotipicamente os isolados de Escherichia coli oriundos de fígado de frangos provenientes de dois matadouros avícolas. Foram coletadas 62 amostras de fígados de frangos, sendo 30 macroscopicamente inalterados e 32 com alteração macroscópica e que originaram no descarte da carcaça. Isolaram-se 30 cepas de Escherichia coli pelo método clássico, sendo 21 isoladas de fígados inalterados e nove provenientes de carcaças rejeitadas. Utilizou-se a reação em cadeia de polimerase para verificação de genes de virulência de E. coli, incluindo o gene de resistência sérica (iss) para identificação de E. coli patogênica para aves, o gene para Shiga cytotoxin 1 e 2 (stx) para identificação de E. coli enteroemorrágica, o gene bfpA para identificação de E. coli enteropatogênica e os genes para toxinas LT-I (elt) e ST-I (stI) para identificação de E. coli enterotoxigênica. Identificou-se iss em 83,3 por cento (25/30) dos isolados, sendo 76,2 por cento (16/21) provenientes de fígados de animais hígidos, e detectou-se stx em 13,3 por cento (4/30). Os genes stx e iss foram identificados em três fígados, caracterizando infecção mista. Os genes não foram observados em um isolado de E. coli pelo método clássico. Faz-se necessária a utilização de tecnologias para identificação e prevenção de Escherichia coli nos aviários e matadouros avícolas.

The isolates of Escherichia coli from chicken livers from two slaughterhouses were genotypically characterized in 62 samples. Thirty samples were macroscopically unchanged and 32 demonstrated alterations that led to the disposal of carcass for sanitary inspection. Thirty Escherichia coli strains from 21 unchanged and 9 from carcasses that were rejected were isolated through the classical method. Polymerase Chain Reaction was performed to verify E. coli virulence of the following genes: serum resistance (iss), to identify avian pathogenic E. coli; Shiga cytotoxin 1 and 2 (stx), to identify enterohaemorrhagic E. col; bfpA, to identify enteropathogenic E. coli; LT-I (elt) and ST-I (stI) toxins to identify enterotoxigenic E. coli. Iss gene was identified in 83.3 percent (25/30), being 76.2 percent (16/21) from E. coli isolated strains from healthy animals. stx gene was identified in 13.3 percent (4/30) of E. coli isolates, and in three of these samples was identified as stx and iss, featuring a mixed infection. The genes were not identified in one E. coli isolated from the classic method. Thus, it is necessary to use advanced technologies to identify and prevent Escherichia coli contamination in poultry farms and slaughterhouses.

Relatedness and HLA-DRB1 typing may discriminate the magnitude of the genetic susceptibility to tuberculosis using a household contact model.

BACKGROUND: Tuberculosis clusters in families may be due to increased household exposure, shared genetic factors, or both. Household contact studies are useful to control exposure because socioeconomic and environmental conditions are similar to all subjects, allowing the evaluation of the contribution of relatedness to disease development. METHODS: In this study, the familial aggregation of tuberculosis using relatedness and a specific inherited marker (HLA-DRB1) was evaluated. Fifty families, which had at least two cases of tuberculosis diagnosed within the past 5 years, were selected from a cohort of tuberculosis carried out in Recife, Brazil. The first case diagnosed was considered to be a primary case. The secondary attack rate of tuberculosis in household contacts was estimated according to the degree of relatedness. The relative risk of having tuberculosis based on the degree of relatedness household and the population attributable fraction to relatedness were also estimated. HLA-DRB1 typing and attributable etiologic/preventive fractions were calculated among sick and healthy household contacts. RESULTS: Compared to unrelated contacts, the relative risk for tuberculosis adjusted for age was 1.38 (95% CI 0.86 to 2.21). Relatedness contributed 23% to the development of tuberculosis at the population levels. The HLA-DRB1*04 allele group (OR=2.44; p=0.0324; etiologic fraction=0.15) was overrepresented and the DRB1*15 allele group (OR=0.48; p=0.0488; protective fraction=0.19) was underrepresented among household contacts exhibiting tuberculosis. The presence of DRB1 shared alleles between primary cases and their contacts was a risk factor for tuberculosis (p=0.0281). CONCLUSION: This household contact model together with the utilisation of two genetic variables permitted the evaluation of genetic factors contributing towards tuberculosis development.

Evaluation of memory immune response to mycobacterium extract among household contact of tuberculosis cases.

The human immune response to tuberculosis (TB) is especially mediated by T CD4(+)lymphocytes. However, more studies are needed in order to understand the exact role of each cytokine in the mechanisms for cures. In this article, our aim was to analyze the production of TNF-alpha, IL-10, and IFN-gamma in peripheral blood mononuclear cells (PBMCs) among the household contacts of common primary TB cases, with or without histories of active TB infection, who were negative to parasitological and HIV tests. In order to characterize the cytokine production, PBMCs from these groups were stimulated with whole-protein extract of M. tuberculosis (WPE) antigen (rAgTb) for 24 and 48 hr. The culture supernatants were collected and IFN-gamma, TNF-alpha, and IL-10 were assayed using capture ELISA. There were no statistical differences between primary TB cases and their household contacts with or without previous histories of lung TB. Our results suggest that T memory cells, T regulatory cells, and the Th1/Th2 dichotomy may be responsible for the results described in this article. Further studies are currently underway.

Protective immunity of single and multi-antigen DNA vaccines against schistosomiasis.

We evaluated the usefulness of the combination of three plasmids encoding tegumental (pECL and pSM14) and muscular (pIRV5) antigens of the Schistosoma mansoni on improving the protective immunity over the use of a single antigen as DNA vaccines. Female BALB/c mice were inoculated twice with 25 micro g DNA plasmid within two weeks interval. The challenge was performed with 80 cercarias of a regional isolate of S. mansoni (SLM) one week after the last immunization. Six weeks after challenge, all mice were perfused for worm load determination. The following groups were analyzed: saline; empty vector; monovalent formulations of pECL; pSM14 and pIRV5 and also double combinations of pECL/pIRV5 and pIRV5/pSM14 and a triple combination of pECL/pIRV5/pSM14. The protection was expressed as a percentage of worm loads in each group compared with the saline group. The results obtained were 41% (p < 0.05); 52% (p < 0.05); 51% (p < 0.05); 48% (p < 0.05); 55% (p < 0.05); 45% (p < 0.05); 65% (p < 0.05) for each group respectively.

Protective immunity of single and multi-antigen DNA vaccines against schistosomiasis

We evaluated the usefulness of the combination of three plasmids encoding tegumental (pECL and pSM14) and muscular (pIRV5) antigens of the Schistosoma mansoni on improving the protective immunity over the use of a single antigen as DNA vaccines. Female BALB/c mice were inoculated twice with 25 æg DNA plasmid within two weeks interval. The challenge was performed with 80 cercarias of a regional isolate of S. mansoni (SLM) one week after the last immunization. Six weeks after challenge, all mice were perfused for worm load determination. The following groups were analyzed: saline; empty vector; monovalent formulations of pECL; pSM14 and pIRV5 and also double combinations of pECL/pIRV5 and pIRV5/pSM14 and a triple combination of pECL/pIRV5/pSM14. The protection was expressed as a percentage of worm loads in each group compared with the saline group. The results obtained were 41 percent (p < 0.05); 52 percent (p < 0.05); 51 percent (p < 0.05); 48 percent (p < 0.05); 55 percent (p < 0.05); 45 percent (p < 0.05); 65 percent (p < 0.05) for each group respectively

Characterization of Sm13, a tegumental antigen of Schistosoma mansoni.

Sm13, a 13-kDa Schistosoma mansoni tegumental antigen, is one of the principal polypeptides recognized by antibodies from mice protectively vaccinated with adult-worm tegumental membranes. To obtain the complete gene encoding Sm13 we subcloned and sequenced a cDNA and a fragment of a genomic clone. The collated sequence contains 1,088 nucleotides and represents the full-length open reading frame of the gene, encoding a protein of 104 amino acids with a calculated molecular mass of 11,923 Da, compatible with the native protein identified in the tegumental membranes. The sequence derived from genomic DNA contains a 45-nucleotide intron. The analysis of the predicted protein suggests the presence of both N- and C-terminal hydrophobic membrane-spanning segments, and the coding region contains no homology in the currently available data bases. Additionally, the coding region is preceded by putative CCAAT and TATA boxes that may be involved in the control of expression. Western-blot analysis and indirect immunofluorescence resulted in the identification of a 13-kDa protein (Sm13) in the tegument of adult worms. The present study reveals that Sm13 behaves as an integral membrane protein upon partitioning in Triton X-1 14 and that it is present in worms of 3 weeks or older but not in schistosomula or miracidia. Moreover, it is also specifically recognized by sera from some schistosomiasis patients in enzyme-linked immunosorbent assay and Western-blot analysis, suggesting that it is immunogenic in human schistosomiasis.