A Rare Presentation of Pica Disorder in Soap Ingestion Variant: Case Report and Review.

ABSTRACT: Pica indicates the persistent ingestion of inedible substances over a period of at least 1 month, being discordant to the individual's cognitive development level and not directly attributable to cultural or social normative practices. The literature suggests that the prevalence of pica varies significantly according to the social and clinical context. It can co-occur with psychiatric disorders, with its etiology being poorly understood and most likely multifactorial. In this article, we report the case of a patient in her 50s with a clinical setting compatible with soap ingestion variant of pica disorder. In addition to the variety of ingested substances, pica can be associated with heterogeneous behavior, mainly of the obsessive-compulsive spectrum. Despite recent research, this condition is still a target of much speculation. This is a very rare and challenging presentation of a soap ingestion variant of pica with very few similar cases described to date.

Healthy lifestyle and genomic ancestry related to good glycemic control in type 1 diabetes patients from Northeastern Brazil: a hierarchical analysis.

Introduction: This study aimed to investigate the sociodemographic factors, dietary adherence, regular physical activity, and genomic ancestry percentage associated with good glycemic control in Brazilian patients with type 1 diabetes (T1D) using a hierarchical approach. Methods: A cross-sectional study was conducted in 152 T1D patients. Glycated hemoglobin (HbA1C) levels were measured to evaluate the glycemic control status (good, moderate, or poor). Independent factors included sex, age, self-reported skin color, educational level, family income, dietary patterns, and physical activity. The percentage of genomic ancestry (Native American, European, and African) was influenced by a panel of 46 autosomal insertion/deletion ancestry markers. Statistical analyses included receiver operating characteristic curves, and hierarchical logistic regression analysis. Results: The hierarchical analysis, patients who had high dietary adherence showed a positive association with good glycemic control (adjustedOR = 2.56, 95% CI:1.18-5.59, P = 0.016). Thus, age greater than 40 years was associated with good glycemic control compared to the children and adolescents group (adjustedOR = 4.55, 95% CI:1.14-18.1, P = 0.031). Males were associated with good glycemic control (adjustedOR = 2.00, 95% CI:1.01-4.00, P =0.047). Conclusion: The study findings suggest that consistent adherence to dietary regimens is associated with good glycemic control after adjusting for sociodemographic and genomic ancestry factors in an admixed population of T1D patients from Northeast Brazil.

Health Literacy and Complications in People With Type 2 Diabetes: An Exploratory Study.

INTRODUCTION: A person with diabetes is subject to developing micro and macrovascular complications and prevention requires an active role from the person. So, health literacy should have a preponderant role in the health of people with diabetes but this link is yet not fully understood. The objective of this study is to understand the relationship between health literacy and the prevalence of complications in people with diabetes mellitus type 2 (DM2). METHODS: This is a multicentric transversal observational exploratory study. A survey was conducted with two health literacy instruments, the Medical Term Recognition Test (METER) and Newest Vital Sign (NVS), filled out by people with DM2 coming to consultation in primary health centers in three main regions of Portugal.  Results: In this sample (n=141), 50.6% were male, 41 to 88 years old, and 56% earned more than the minimum wage. Using the METER tool, it was found that 57.4% of the diabetic patients had functional literacy. Adequate literacy was found in 24.1% with the NVS tool. Also with the NVS tool it was found that 36.2% of the sample subjects had s high probability of limited literacy. Utilizing the METER tool, a statistically significant decrease in health literacy was observed in individuals with diabetic complications (p=0.001). There was no significant relation between the presence of diabetic complications and present blood pressure values, low-density lipoprotein, and socioeconomic index. CONCLUSION: In this study, we found a significant relation between lower health literacy and the presence of diagnosed DM2 complications (p=0.001).

In- and Out-of-Hospital Deaths by Acute Myocardial Infarction in Brazilian State Capitals. / Óbitos Intra e Extra-Hospitalares por Infarto Agudo do Miocárdio nas Capitais Brasileiras.

BACKGROUND: Acute myocardial infarction (AMI) is the main cause of death in Brazil and the world. Approximately half of these deaths occur outside the hospital. OBJECTIVES: To analyze the distribution, temporal evolution, and sociodemographic characteristics (SDC) of in- and out-of-hospital deaths by AMI in Brazilian state capitals and their relationship with municipal development indicators (MDI). METHODS: This is an ecological study of the number of deaths due to AMI reported annually by the 27 Brazilian state capitals from 2007 to 2016; these were divided into 2 groups: in-hospital (H) and out-of-hospital (OH). We evaluated the temporal evolution of mortality rates in each group and differences in SDC. Negative binomial regression models were used to compare the temporal evolution of the number of deaths in each group with the following variables: residing in the South/Southeast regions (S/SE), municipal human development index (MHDI), Gini coefficient, and expected years of schooling (EYS). We considered p-values<0.05 as statisticallysignificant. RESULTS: The OH mortality rate increased with time for all state capitals. All studied SDC were different between groups (p<0.001). In the OH group, most deaths were of men and patients aged 80 years or older and not married. S/SE increased the incidence of OH deaths (incidence rate ratio [IRR]=2.84; 95% confidence interval [CI]=1.67-4.85), while higher EYS reduced it (IRR=0.86; 95% CI=0.77-0.97). In the H group, higher MHDI reduced the incidence of deaths (IRR=0.44; 95% CI=0.33-0.58), while higher EYS increased it (IRR=1.09; 95% CI=1.03-1.15). CONCLUSIONS: In- and out-of-hospital deaths due to AMI in Brazilian state capitals were influenced by MDI, presented sociodemographic differences and a progressive increase in out-of-hospital occurrences.

FUNDAMENTO: O infarto agudo do miocárdio (IAM) é a principal causa de óbito no Brasil e no mundo. Aproximadamente metade dos óbitos ocorrem fora do ambiente hospitalar. OBJETIVOS: Analisar a distribuição, a evolução temporal e as características sociodemográficas (CSD) dos óbitos intra e extra-hospitalares por IAM nas capitais brasileiras e a sua relação com indicadores municipais de desenvolvimento (IMD). MÉTODOS: Estudo ecológico com contagem anual dos óbitos por IAM nas 27 capitais brasileiras de 2007 a 2016, os quais foram divididos em dois grupos, intra-hospitalar (H) e extra-hospitalar (EH). Avaliou-se a evolução temporal das taxas de mortalidade em cada grupo e as diferenças das CSD. Modelos de regressão binominal negativa compararam temporalmente a contagem de óbitos em cada grupo com as seguintes variáveis: residir nas regiões Sul e Sudeste (S/SE), índice de desenvolvimento humano municipal (IDHM), índice de Gini e expectativa de anos de estudo (EAE). Considerou-se estatisticamente valores significativos de p < 0,05. RESULTADOS: A taxa de mortalidade EH para o conjunto das capitais aumentou ao longo do tempo. Todas as CSD pesquisadas foram difententes entre os grupos (p < 0,001). No grupo EH prevaleceram os óbitos em homens, em pacientes ≥ 80 anos e em solteiros. O S/SE elevou a incidência de óbitos extra-hospitalares (IRR = 2,84; IC 95% = 1,67-4,85), enquanto o maior EAE registrou queda (IRR = 0,86; IC 95% = 0,77-0,97). Para o grupo H, o maior IDHM reduziu a incidência de óbitos (IRR = 0,44; IC 95% = 0,33-0,58), enquanto o maior EAE apresentou crescimento (IRR = 1,09; IC 95% = 1,03-1,15). CONCLUSÃO: Os óbitos intra e extra-hospitalares por IAM nas capitais apresentam diferenças sociodemográficas, incidência influenciada por IMD e progressivo aumento da ocorrência extra-hospitalar.

Óbitos intra e extra-hospitalares por infarto agudo do miocárdio nas capitais brasileiras / In- and out-of-hospital deaths by acute myocardial infarction in brazilian state capitals

Resumo Fundamento: O infarto agudo do miocárdio (IAM) é a principal causa de óbito no Brasil e no mundo. Aproximadamente metade dos óbitos ocorrem fora do ambiente hospitalar. Objetivos: Analisar a distribuição, a evolução temporal e as características sociodemográficas (CSD) dos óbitos intra e extra-hospitalares por IAM nas capitais brasileiras e a sua relação com indicadores municipais de desenvolvimento (IMD). Métodos: Estudo ecológico com contagem anual dos óbitos por IAM nas 27 capitais brasileiras de 2007 a 2016, os quais foram divididos em dois grupos, intra-hospitalar (H) e extra-hospitalar (EH). Avaliou-se a evolução temporal das taxas de mortalidade em cada grupo e as diferenças das CSD. Modelos de regressão binominal negativa compararam temporalmente a contagem de óbitos em cada grupo com as seguintes variáveis: residir nas regiões Sul e Sudeste (S/SE), índice de desenvolvimento humano municipal (IDHM), índice de Gini e expectativa de anos de estudo (EAE). Considerou-se estatisticamente valores significativos de p < 0,05. Resultados: A taxa de mortalidade EH para o conjunto das capitais aumentou ao longo do tempo. Todas as CSD pesquisadas foram difententes entre os grupos (p < 0,001). No grupo EH prevaleceram os óbitos em homens, em pacientes ≥ 80 anos e em solteiros. O S/SE elevou a incidência de óbitos extra-hospitalares (IRR = 2,84; IC 95% = 1,67-4,85), enquanto o maior EAE registrou queda (IRR = 0,86; IC 95% = 0,77-0,97). Para o grupo H, o maior IDHM reduziu a incidência de óbitos (IRR = 0,44; IC 95% = 0,33-0,58), enquanto o maior EAE apresentou crescimento (IRR = 1,09; IC 95% = 1,03-1,15). Conclusão: Os óbitos intra e extra-hospitalares por IAM nas capitais apresentam diferenças sociodemográficas, incidência influenciada por IMD e progressivo aumento da ocorrência extra-hospitalar.

Abstract Background: Acute myocardial infarction (AMI) is the main cause of death in Brazil and the world. Approximately half of these deaths occur outside the hospital. Objectives: To analyze the distribution, temporal evolution, and sociodemographic characteristics (SDC) of in- and out-of-hospital deaths by AMI in Brazilian state capitals and their relationship with municipal development indicators (MDI). Methods: This is an ecological study of the number of deaths due to AMI reported annually by the 27 Brazilian state capitals from 2007 to 2016; these were divided into 2 groups: in-hospital (H) and out-of-hospital (OH). We evaluated the temporal evolution of mortality rates in each group and differences in SDC. Negative binomial regression models were used to compare the temporal evolution of the number of deaths in each group with the following variables: residing in the South/Southeast regions (S/SE), municipal human development index (MHDI), Gini coefficient, and expected years of schooling (EYS). We considered p-values<0.05 as statisticallysignificant. Results: The OH mortality rate increased with time for all state capitals. All studied SDC were different between groups (p<0.001). In the OH group, most deaths were of men and patients aged 80 years or older and not married. S/SE increased the incidence of OH deaths (incidence rate ratio [IRR]=2.84; 95% confidence interval [CI]=1.67-4.85), while higher EYS reduced it (IRR=0.86; 95% CI=0.77-0.97). In the H group, higher MHDI reduced the incidence of deaths (IRR=0.44; 95% CI=0.33-0.58), while higher EYS increased it (IRR=1.09; 95% CI=1.03-1.15). Conclusions: In- and out-of-hospital deaths due to AMI in Brazilian state capitals were influenced by MDI, presented sociodemographic differences and a progressive increase in out-of-hospital occurrences.

Birth by cesarean delivery and central adiposity in adolescents from a birth cohort.

The aim of this study was to analyze the association between birth by cesarean section and central adiposity in adolescents in São Luís, Maranhão State, Brazil. This was a cohort study that included 601 participants evaluated at birth and at 18-19 years. At birth we assessed type of delivery, maternal education, family income, maternal marital status, maternal body mass index before pregnancy, prenatal care, maternal smoking habit, gestational age at delivery and intrauterine growth restriction. In the adolescents, we evaluated central adiposity using the dual X-ray energy absorptiometry method. The indicators of central fat used were the trunk-to-total fat mass ratio (T/T), the android-to-gynoid fat mass ratio (A/G), the trunk-to-limb fat mass ratio (T/Lb), and the trunk-to-leg fat mass ratio (T/Lg). A theoretical model for the study of associations was developed using directed acyclic graphs, which allowed selecting the variables that required minimum adjustment for inclusion in the predictive model of exposure to cesarean delivery. The data were analyzed with marginal structural models weighted by the inverse of the probability of selection. A total of 38.6% of the adolescents studied were delivered by cesarean section. There was no significant difference in the central adiposity of adolescents delivered by cesarean section according to the indicators used: T/T ( coefficient = -0.003; 95%CI: -0.013; 0.007), A/G (coefficient = 0.001; 95%CI: -0.015; 0.018); T/Lb (coefficient = -0.016; 95%CI: -0.048; 0.016); T/Lg (coefficient = 0.014; 95%CI: -0.060; 0.030). In conclusion, there was no association between cesarean section delivery and greater central adiposity in the studied adolescents.

Birth by cesarean delivery and central adiposity in adolescents from a birth cohort / Nascimento por parto cesáreo e adiposidade central em adolescentes de uma coorte de nascimentos / Nacimiento por parto con cesárea y adiposidad central en adolescentes de una cohorte de nacimientos

Abstract: The aim of this study was to analyze the association between birth by cesarean section and central adiposity in adolescents in São Luís, Maranhão State, Brazil. This was a cohort study that included 601 participants evaluated at birth and at 18-19 years. At birth we assessed type of delivery, maternal education, family income, maternal marital status, maternal body mass index before pregnancy, prenatal care, maternal smoking habit, gestational age at delivery and intrauterine growth restriction. In the adolescents, we evaluated central adiposity using the dual X-ray energy absorptiometry method. The indicators of central fat used were the trunk-to-total fat mass ratio (T/T), the android-to-gynoid fat mass ratio (A/G), the trunk-to-limb fat mass ratio (T/Lb), and the trunk-to-leg fat mass ratio (T/Lg). A theoretical model for the study of associations was developed using directed acyclic graphs, which allowed selecting the variables that required minimum adjustment for inclusion in the predictive model of exposure to cesarean delivery. The data were analyzed with marginal structural models weighted by the inverse of the probability of selection. A total of 38.6% of the adolescents studied were delivered by cesarean section. There was no significant difference in the central adiposity of adolescents delivered by cesarean section according to the indicators used: T/T ( coefficient = -0.003; 95%CI: -0.013; 0.007), A/G (coefficient = 0.001; 95%CI: -0.015; 0.018); T/Lb (coefficient = -0.016; 95%CI: -0.048; 0.016); T/Lg (coefficient = 0.014; 95%CI: -0.060; 0.030). In conclusion, there was no association between cesarean section delivery and greater central adiposity in the studied adolescents.

Resumo: O estudo teve como objetivo analisar a associação entre nascimento por parto cesáreo e adiposidade central em adolescentes em São Luís, Maranhão, Brasil. O estudo de coorte incluiu 601 participantes avaliados ao nascer e com 18-19 anos de idade. Ao nascer, foram avaliados o tipo de parto, escolaridade materna, renda familiar, estado civil materno, índice de massa corporal pré-gestacional, atendimento pré-natal, tabagismo materno, idade gestacional ao nascer e restrição do crescimento intrauterino. Nos adolescentes, a adiposidade central foi avaliada com o método da absorciometria de raios-X de dupla energia. Como indicadores da adiposidade central, foram usadas a razão gordura tronco/gordura total (T/T), razão gordura androide/gordura ginecoide (A/G), razão gordura tronco/gordura membros (T/Lb) e razão gordura tronco/gordura membros inferiores (T/Lg). Foi desenvolvido um modelo teórico para analisar as associações, usando gráficos acíclicos dirigidos, permitindo a seleção das variáveis que exigiam ajuste mínimo para inclusão no modelo preditivo de exposição ao parto cesáreo. Os dados foram analisados com modelos estruturais marginais, ponderados pelo inverso da probabilidade de seleção. Entre os adolescentes estudados, 38,6% nasceram de parto cesáreo. Não houve diferença significativa na adiposidade central nos adolescentes nascidos de parto cesáreo, de acordo com os indicadores utilizados: T/T (coeficiente = -0,003; IC95%: -0,013; 0,007), A/G (coeficiente = 0,001; IC95%: -0,015; 0,018); T/Lb (coeficiente = -0,016; IC95%: -0,048; 0,016); T/Lg (coeficiente = 0,014; IC95%: -0,060; 0,030), O estudo conclui que não havia associação entre história de parto cesáreo e aumento de adiposidade central nesse grupo de adolescentes.

Resumen: El objetivo de esta investigación fue estudiar la asociación entre el nacimiento por parto con cesárea y la adiposidad central en adolescentes en São Luís, Maranhão, Brasil. La cohorte de estudio incluyó a 601 participantes evaluados en su nacimiento y con 18-19 años de edad. En el momento del nacimiento se evaluó el tipo de parto, educación de la madre, ingresos familiares, estado civil de la madre, índice de masa corporal de la madre, cuidado prenatal, madre fumadora, edad gestacional en el parto y restricción del crecimiento intrauterino. En los adolescentes, la grasa central se evaluó usando el método de absorciometría con rayos X de energía dual. Los indicadores de grasa central utilizados fueron: el cociente de torso-grasa total (T/T por sus siglas en inglés), el de androide/ginecoide de grasa corporal (A/G), el cociente de masa adiposa torso-extremidades (T/Lb por sus siglas en inglés), y el cociente de masa adiposa torso-pierna (T/Lg por sus siglas en inglés). Se desarrolló un modelo teórico para el estudio de asociaciones, usando grafos acíclicos dirigidos, lo que permitió seleccionar las variables que requerían un mínimo ajuste para su inclusión en el modelo predictivo de exposición al parto por cesárea. Los datos se analizaron con modelos estructurales marginales ponderados por el inverso de la probabilidad de selección. De los adolescentes estudiados, un 38,6% fueron partos por cesárea. No hubo una diferencia significativa en la adiposidad central de los adolescentes que nacieron por cesárea, según los indicadores usados: T/T (coeficiente = -0,003; 95%CI: -0,013; 0,007), A/G (coeficiente = 0,001; 95%CI: -0,015; 0,018); T/Lb (coeficiente = -0,016; 95%CI: -0,048; 0,016); T/Lg (coeficiente = 0,014; 95%CI: -0,060; 0,030). A modo de conclusión, no hubo asociación entre los partos por cesárea y una mayor adiposidad central en los adolescentes estudiados.

Combinatorial detection of autoreactive CD8+ T cells with HLA-A2 multimers: a multi-centre study by the Immunology of Diabetes Society T Cell Workshop.

AIMS/HYPOTHESIS: Validated biomarkers are needed to monitor the effects of immune intervention in individuals with type 1 diabetes. Despite their importance, few options exist for monitoring antigen-specific T cells. Previous reports described a combinatorial approach that enables the simultaneous detection and quantification of multiple islet-specific CD8+ T cell populations. Here, we set out to evaluate the performance of a combinatorial HLA-A2 multimer assay in a multi-centre setting. METHODS: The combinatorial HLA-A2 multimer assay was applied in five participating centres using centralised reagents and blinded replicate samples. In preliminary experiments, samples from healthy donors were analysed using recall antigen multimers. In subsequent experiments, samples from healthy donors and individuals with type 1 diabetes were analysed using beta cell antigen and recall antigen multimers. RESULTS: The combinatorial assay was successfully implemented in each participating centre, with CVs between replicate samples that indicated good reproducibility for viral epitopes (mean %CV = 33.8). For beta cell epitopes, the assay was very effective in a single-centre setting (mean %CV = 18.4), but showed sixfold greater variability across multi-centre replicates (mean %CV = 119). In general, beta cell antigen-specific CD8+ T cells were detected more commonly in individuals with type 1 diabetes than in healthy donors. Furthermore, CD8+ T cells recognising HLA-A2-restricted insulin and glutamate decarboxylase epitopes were found to occur at higher frequencies in individuals with type 1 diabetes than in healthy donors. CONCLUSIONS/INTERPRETATION: Our results suggest that, although combinatorial multimer assays are challenging, they can be implemented in multiple laboratories, providing relevant T cell frequency measurements. Assay reproducibility was notably higher in the single-centre setting, suggesting that biomarker analysis of clinical trial samples would be most successful when assays are performed in a single laboratory. Technical improvements, including further standardisation of cytometry platforms, will likely be necessary to reduce assay variability in the multi-centre setting.

Immunological Balance Is Associated with Clinical Outcome after Autologous Hematopoietic Stem Cell Transplantation in Type 1 Diabetes.

Autologous hematopoietic stem cell transplantation (AHSCT) increases C-peptide levels and induces insulin independence in patients with type 1 diabetes. This study aimed to investigate how clinical outcomes may associate with the immunological status, especially concerning the balance between immunoregulation and autoreactivity. Twenty-one type 1 diabetes patients were monitored after AHSCT and assessed every 6 months for duration of insulin independence, C-peptide levels, frequencies of islet-specific autoreactive CD8+ T cells (CTL), regulatory lymphocyte subsets, thymic function, and T-cell repertoire diversity. In median follow-up of 78 (range 15-106) months, all patients became insulin-independent, resuming insulin after median of 43 (range 6-100) months. Patients were retrospectively divided into short- or prolonged-remission groups, according to duration of insulin independence. For the entire follow-up, CD3+CD4+ T-cell numbers remained lower than baseline in both groups, whereas CD3+CD8+ T-cell levels did not change, resulting in a CD4/CD8 ratio inversion. Memory CTL comprehended most of T cells detected on long-term follow-up of patients after AHSCT. B cells reconstituted to baseline levels at 2-3 months post-AHSCT in both patient groups. In the prolonged-remission-group, baseline islet-specific T-cell autoreactivity persisted after transplantation, but regulatory T cell counts increased. Patients with lower frequencies of autoreactive islet-specific T cells remained insulin-free longer and presented greater C-peptide levels than those with lower frequencies of these cells. Therefore, immune monitoring identified a subgroup of patients with superior clinical outcome of AHSCT. Our study shows that improved immunoregulation may balance autoreactivity endorsing better metabolic outcomes in patients with lower frequencies of islet-specific T cells. Development of new strategies of AHSCT is necessary to increase frequency and function of T and B regulatory cells and decrease efficiently autoreactive islet-specific T and B memory cells in type 1 diabetes patients undergoing transplantation.

Alternative splicing and differential expression of the islet autoantigen IGRP between pancreas and thymus contributes to immunogenicity of pancreatic islets but not diabetogenicity in humans.

AIMS/HYPOTHESIS: Thymic expression of self-antigens during T-lymphocyte development is believed to be crucial for preventing autoimmunity. It has been suggested that G6PC2, the gene encoding islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), is differentially spliced between pancreatic beta cells and the thymus. This may contribute to incomplete elimination of IGRP-specific T lymphocytes in the thymus, predisposing individuals to type 1 diabetes. We tested whether specific splice variation in islets vs thymus correlates with loss of tolerance to IGRP in type 1 diabetes. METHODS: Expression of G6PC2 splice variants was compared among thymus, purified medullary thymic epithelial cells and pancreatic islets by RT-PCR. Differential immunogenicity of IGRP splice variants was tested in patients and healthy individuals for autoantibodies and specific cytotoxic T lymphocytes using radiobinding assays and HLA class I multimers, respectively. RESULTS: Previously reported G6PC2 splice variants, including full-length G6PC2, were confirmed, albeit that they occurred in both pancreas and thymus, rather than islets alone. Yet, their expression levels were profoundly greater in islets than in thymus. Moreover, three novel G6PC2 variants were discovered that occur in islets only, leading to protein truncations, frame shifts and neo-sequences prone to immunogenicity. However, autoantibodies to novel or known IGRP splice variants did not differ between patients and healthy individuals, and similar frequencies of IGRP-specific cytotoxic T lymphocytes could be detected in both patients with type 1 diabetes and healthy individuals. CONCLUSIONS/INTERPRETATION: We propose that post-transcriptional variation of tissue-specific self-proteins may affect negative thymic selection, although this need not necessarily lead to disease.

Plasmid-encoded proinsulin preserves C-peptide while specifically reducing proinsulin-specific CD8⁺ T cells in type 1 diabetes.

In type 1 diabetes (T1D), there is an intense inflammatory response that destroys the ß cells in the pancreatic islets of Langerhans, the site where insulin is produced and released. A therapy for T1D that targets the specific autoimmune response in this disease while leaving the remainder of the immune system intact, has long been sought. Proinsulin is a major target of the adaptive immune response in T1D. We hypothesized that an engineered DNA plasmid encoding proinsulin (BHT-3021) would preserve ß cell function in T1D patients through reduction of insulin-specific CD8⁺ T cells. We studied 80 subjects over 18 years of age who were diagnosed with T1D within the past 5 years. Subjects were randomized 2:1 to receive intramuscular injections of BHT-3021 or BHT-placebo, weekly for 12 weeks, and then monitored for safety and immune responses in a blinded fashion. Four dose levels of BHT-3021 were evaluated: 0.3, 1.0, 3.0, and 6.0 mg. C-peptide was used both as an exploratory efficacy measure and as a safety measure. Islet-specific CD8⁺ T cell frequencies were assessed with multimers of monomeric human leukocyte antigen class I molecules loaded with peptides from pancreatic and unrelated antigens. No serious adverse events related to BHT-3021 were observed. C-peptide levels improved relative to placebo at all doses, at 1 mg at the 15-week time point (+19.5% BHT-3021 versus -8.8% BHT-placebo, P < 0.026). Proinsulin-reactive CD8⁺ T cells, but not T cells against unrelated islet or foreign molecules, declined in the BHT-3021 arm (P < 0.006). No significant changes were noted in interferon-γ, interleukin-4 (IL-4), or IL-10 production in CD4 T cells. Thus, we demonstrate that a plasmid encoding proinsulin reduces the frequency of CD8⁺ T cells reactive to proinsulin while preserving C-peptide over the course of dosing.

Genetically engineered human islets protected from CD8-mediated autoimmune destruction in vivo.

Islet transplantation is a promising therapy for type 1 diabetes, but graft function and survival are compromised by recurrent islet autoimmunity. Immunoprotection of islets will be required to improve clinical outcome. We engineered human ß cells to express herpesvirus-encoded immune-evasion proteins, "immunevasins." The capacity of immunevasins to protect ß cells from autoreactive T-cell killing was evaluated in vitro and in vivo in humanized mice. Lentiviral vectors were used for efficient genetic modification of primary human ß cells without impairing their function. Using a novel ß-cell-specific reporter gene assay, we show that autoreactive cytotoxic CD8(+) T-cell clones isolated from patients with recent onset diabetes selectively destroyed human ß cells, and that coexpression of the human cytomegalovirus-encoded US2 protein and serine proteinase inhibitor 9 offers highly efficient protection in vitro. Moreover, coimplantation of these genetically modified pseudoislets with ß-cell-specific cytotoxic T cells into immunodeficient mice achieves preserved human insulin production and C-peptide secretion. Collectively, our data provide proof of concept that human ß cells can be efficiently genetically modified to provide protection from killing mediated by autoreactive T cells and retain their function in vitro and in vivo.

Efeito de diferentes técnicas de desinfecção na precisão de moldes de alginato avaliada em modelos de gesso / Effect of different disinfection techniques on the accuracy of alginate impressions evaluated on stone casts

Objetivo: Avaliar o efeito de diferentes técnicas de desinfecção de moldes de alginato (imersão e pulverização) sobre a precisão em modelos de gessos. Métodos: A partir de um troquel mestre em formato de tronco de cone e moldeiras de PVC, foram gerados moldes de alginato (Jeltrate Dustless), e então distribuídos em cinco grupos (n = 7) de acordo com o método de desinfecção a ser realizado (controle ? sem desinfecção; HipPul pulverizado com hipoclorito de sódio; HipIm ? imersão em hipoclorito de sódio; CloPul ? pulverizado com clorexedina; CloIm ? imersão em clorexedina). Após a desinfecção, os moldes foram vazados em gesso pedra tipo III (Herodent), segundo recomendações do fabricante, e então medidos com um paquímetro digital e submetidos à análise estatística (Anova e Tukey e Krsukal-Wallise Dunn). Resultados: Todos os grupos apresentaram um determinado grau de distorção mesmo que não significativa. Entretanto, o método de imersão com hipoclorito de sódio a 1%, apresentou alteração dimensional significativamente maior quando comparado ao grupo de controle. Conclusão: A imersão em hipoclorito de sódio 1% se mostrou o método menos indicado para desinfecção dos moldes de alginato.

Islet-specific CTL cloned from a type 1 diabetes patient cause beta-cell destruction after engraftment into HLA-A2 transgenic NOD/scid/IL2RG null mice.

Despite increasing evidence that autoreactive CD8 T-cells are involved in both the initiation of type 1 diabetes (T1D) and the destruction of beta-cells, direct evidence for their destructive role in-vivo is lacking. To address a destructive role for autoreactive CD8 T-cells in human disease, we assessed the pathogenicity of a CD8 T-cell clone derived from a T1D donor and specific for an HLA-A2-restricted epitope of islet-specific glucose-6-phosphatase catalytic-subunit related protein (IGRP). HLA-A2/IGRP tetramer staining revealed a higher frequency of IGRP-specific CD8 T-cells in the peripheral blood of recent onset human individuals than of healthy donors. IGRP(265-273)-specific CD8 T-cells that were cloned from the peripheral blood of a recent onset T1D individual were shown to secrete IFNγ and Granzyme B after antigen-specific activation and lyse HLA-A2-expressing murine islets in-vitro. Lytic capacity was also demonstrated in-vivo by specific killing of peptide-pulsed target cells. Using the HLA-A2 NOD-scid IL2rγ(null) mouse model, HLA-A2-restricted IGRP-specific CD8 T-cells induced a destructive insulitis. Together, this is the first evidence that human HLA-restricted autoreactive CD8 T-cells target HLA-expressing beta-cells in-vivo, demonstrating the translational value of humanized mice to study mechanisms of disease and therapeutic intervention strategies.

Discovery of low-affinity preproinsulin epitopes and detection of autoreactive CD8 T-cells using combinatorial MHC multimers.

Autoreactive cytotoxic CD8 T-cells (CTLs) play a key pathogenic role in the destruction of insulin-producing beta-cells resulting in type 1 diabetes. However, knowledge regarding their targets is limited, restricting the ability to monitor the course of the disease and immune interventions. In a multi-step discovery process to identify novel CTL epitopes in human preproinsulin (PPI), PPI was digested with purified human proteasomes, and resulting COOH-fragments aligned with algorithm-predicted HLA-binding peptides to yield nine potential HLA-A1, -A2, -A3 or -B7-restricted candidates. An UV-exchange method allowed the generation of a repertoire of multimers including low-affinity HLA-binding peptides. These were labeled with quantum dot-fluorochromes and encoded in a combinatorial fashion, allowing parallel and sensitive detection of specific, low-avidity T-cells. Significantly increased frequencies of T-cells against four novel PPI epitopes (PPI(4-13)/B7, PPI(29-38)/A2, PPI(76-84)/A3 and PPI(79-88)/A3) were detected in stored blood of patients with recent onset diabetes but not in controls. Changes in frequencies of circulating CD8 T-cells against these novel epitopes were detected in blood of islet graft recipients at different time points after transplantation, which correlated with clinical outcome. In conclusion, our novel strategy involving a sensitive multiplex detection technology and requiring minimal volumes of stored blood represents a major improvement in the direct ex-vivo characterization and enumeration of immune cells in the pathogenesis of type 1 diabetes.

Silencing the expression of Ras family GTPase homologues decreases inflammation and joint destruction in experimental arthritis.

Changes in the expression and activation status of Ras proteins are thought to contribute to the pathological phenotype of stromal fibroblast-like synoviocytes (FLS) in rheumatoid arthritis, a prototypical immune-mediated inflammatory disease. Broad inhibition of Ras and related proteins has shown protective effects in animal models of arthritis, but each of the Ras family homologues (ie, H-, K-, and N-Ras) makes distinct contributions to cellular activation. We examined the expression of each Ras protein in synovial tissue and FLS obtained from patients with rheumatoid arthritis and other forms of inflammatory arthritis. Each Ras protein was expressed in synovial tissue and cultured FLS. Each homolog was also activated following FLS stimulation with tumor necrosis factor-α or interleukin (IL)-1ß. Constitutively active mutants of each Ras protein enhanced IL-1ß-induced FLS matrix metalloproteinase-3 production, while only active H-Ras enhanced IL-8 production. Gene silencing demonstrated that each Ras protein contributed to IL-1ß-dependent IL-6 production, while H-Ras and N-Ras supported IL-1ß-dependent matrix metalloproteinase-3 and IL-8 production, respectively. The overlap in contributions of Ras homologues to FLS activation suggests that broad targeting of Ras GTPases in vivo suppresses global inflammation and joint destruction in arthritis. Consistent with this, simultaneous silencing of H-Ras, K-Ras, and N-Ras expression significantly reduces inflammation and joint destruction in murine collagen-induced arthritis, while specific targeting of N-Ras alone is less effective in providing clinical benefits.

Sustained T cell Rap1 signaling is protective in the collagen-induced arthritis model of rheumatoid arthritis.

OBJECTIVE: Defective activation of T cell receptor-proximal signaling proteins, such as the small GTPase Rap1, is thought to contribute to the pathologic behavior of rheumatoid arthritis (RA) synovial T cells. This study was undertaken to determine whether maintaining Rap1 signaling in murine T cells modifies disease onset or severity in collagen-induced arthritis (CIA). METHODS: CIA experiments were conducted using wild-type and RapV12-transgenic mice, which express an active mutant of Rap1 in the T cell compartment. Mice were assessed using macroscopic, microscopic, and radiologic measures, and serum levels of anticollagen antibodies were measured by enzyme-linked immunosorbent assay. Phenotypic and functional characterization of wild-type and RapV12-transgenic T cells under homeostatic conditions and during disease onset was performed by flow cytometry. RESULTS: Disease incidence and severity, synovial infiltration, joint destruction, and anticollagen antibody production were significantly reduced in RapV12-transgenic mice. Although the numbers and percentages of CD3+, CD4+, and CD8+ (naive, effector, and memory) T cells, Treg cells, and Th17 cells were equivalent in wild-type and RapV12-transgenic mice, a significant decrease in the percentage of tumor necrosis factor α-secreting CD8+ T cells was observed in RapV12-transgenic mice during CIA. RapV12-transgenic T cells also inefficiently expressed inducible costimulator and CD40L costimulatory proteins involved in B cell immunoglobulin class switching. CONCLUSION: Our findings indicate that maintenance of T cell Rap1 signaling in murine T cells reduces disease incidence and severity in CIA, which are associated with specific defects in T cell effector function. Therefore, the restoration of Rap1 function in RA synovial T cells may have therapeutic benefit in RA.