Type 1 diabetes mellitus (T1D) is an organ-specific autoimmune disease characterised by autoimmune destruction of insulin-producing beta cells in the islets of Langerhans. It is a long-term process initiated months or even years prior to the clinical onset. The main role in the pathogenesis is played by T lymphocytes but other cell types are involved as well. The presence of autoantibodies in the circulation is typical even before the disease onset. Nowadays, intensive research is focused on finding individuals at risk and developing an effective prevention. During my postgraduate studies I was involved mainly in the research of T1D prediction and prevention. We investigated the relationship of established autoimmune markers - autoantibodies - and the cellular reactivity to GAD65 and IA2 autoantigens. We discovered that the reaction to autoantigens is very individual and it is influenced by the patient's autoantibody profile. These results could be relevant in planning antigen-specific immunointervention studies and improving their efficacy. We also made an attempt to improve specificity and sensitivity of a beta cell destruction marker (specifically demethylated DNA), which would enable better understanding of the beta cell decline and identification of individuals at risk of T1D development. In...

During treatment of diabetes mellitus by immunointervention or transplantation, it is necessary to monitor the markers of immune destruction or rejection of surviving insulin producing cells. An aim of this thesis is to improve the possibilities of following autoimmunity and to detect the survival of transplanted pancreatic islet in vivo. Partial aims included vitality testing of isolated islets for transplantation by measurement of respiration activity, observing the process of in vitro labeling of isolated islets with superparamagnetic iron oxide (SPIO) contrast agent for subsequent magnetic resonance imaging (MRI) of islets and observing SPIO particles transport after transplantation. We also studied a new dual paramagnetic contrast agent combined with fluorescein intended for identification of the MRI contrast agent in samples for histology. Further, we assessed autoimmune reaction by evaluation of cytokine response to specific stimulation with auto-antigens. We tried to affect beta-cells destruction by polyclonal anti- thymocyte antibodies in a mouse experimental model. A new method of the islet respiration measurement correlated with other methods of islet quality testing and it was suggested as a diagnostic test before clinical transplantation. Results obtained studying the intercellular...

The diploma thesis deals with the attitude of parents and teachers of pupils with chronic diseases in the attenuation of compulsory school attendance at the 1st stage of ordinary primary school. The objective of the work is to identify the problems that parents and teachers of chronically ill students encounter during the educational process. From this basis, the purpose is also to find the basis for the best possible cooperation between parents and teachers. The theoretical idea is represented by individual chronic diseases, namely asthma, cystic fibrosis, pediatric oncological diseases, type 1 diabetes mellitus and epilepsy. In addition, the legislative framework for the education of pupils with these diseases, the methodology of working with pupils with chronic diseases and a search of domestic and foreign research conducted on a similar topic are addressed. The methodological part is devoted to qualitative research, the main goal of which is to find out the attitudes of teachers and parents to the educational process of pupils suffering from the above diseases. Using a questionnaire survey and a semi-structured interview, it is ascertained what are the attitudes of parents and teachers towards the education of pupils with the above-mentioned types of chronic diseases in ordinary primary school....

The physical complications of Type 1 diabetes mellitus (T1DM) have been understood as an accelerated ageing process (Morley, 2008). Do people with T1DM also experience accelerated cognitive and brain ageing? Using findings from research of the normal cognitive and brain ageing process and conceptualized in theories of the functional brain changes in cognitive ageing, a combination of cognitive testing and functional magnetic resonance imaging (fMRI) techniques were used to evaluate evidence of accelerated cognitive and brain ageing in middle-aged adults with T1DM. The first part of this thesis comprises a cognitive study of 94 adults (≥ 45 years of age) with long duration (≥ 10 years) of T1DM. Participants completed cognitive assessment and questionnaires on general mood and feelings about living with diabetes. Findings highlighted the importance of microvascular disease (specifically retinopathy) as an independent predictor of cognitive function. The incidence and predictors of mild cognitive impairment (MCI) were then explored. Results indicate a higher percentage of the group met criteria for MCI than expected based on incidence rates in the general population, providing initial evidence of accelerated cognitive ageing. Psychological factors were explored next. The relationship between the measures of well-being, diabetes health, and cognitive function highlighted the need for attention to patient's psychological well-being in diabetes care. Finally, a subgroup of 30 participants between the ages of 45 and 65 who differed on severity of retinopathy were selected to take part in an fMRI study. Blood oxygen level dependent (BOLD) activity was evaluated while participants were engaged in cognitive tasks and during rest. The findings provided evidence that the pattern of BOLD activation and functional connectivity for those with high severity of retinopathy are similar to patterns found in adults over the age of 65. In line with the theories of cognitive ageing, functional brain changes appear to maintain a level of cognitive function. Evidence of accelerated brain ageing in this primarily middle-aged group, emphasizes the importance of treatments and regimens to prevent or minimize microvascular complications.

O diabetes mellitus tipo 1 (DM-1) e a esclerose múltipla (EM) são doenças auto-imunes órgão-específicas, inflamatórias, mediadas por células T e B auto-reativas e caracterizadas pela destruição seletiva de células b pancreáticas produtoras de insulina e do sistema nervoso central, respectivamente. Acredita-se que a desregulação da expressão de genes reguladores da maquinaria apoptótica possa contribuir para o desenvolvimento da auto-imunidade, visto que algumas dessas moléculas participam nos processos de tolerância central e periférica de linfócitos auto-reativos. O objetivo deste projeto foi analisar a expressão de moléculas reguladoras das vias intrínseca, extrínseca e da Família de proteínas inibidoras da apoptose (IAP) em 33 indivíduos saudáveis, 15 pacientes com DM-1 e 18 com EM submetidos à terapia de imunossupressão em altas doses seguida do transplante autólogo de células-tronco hematopoéticas (IAD/TACTH). As células mononucleares (CMN) foram isoladas do sangue periférico dos controles e de pacientes nos períodos pré-mobilização (pré-mob), pré-condicionamento (pré-cond), D+180, D+360, D+540 e D+720 pós-transplante. As CMN foram utilizadas para extração de RNA, síntese de cDNA, quantificação da expressão por PCR em tempo real dos genes a1, bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bid, bik, bim, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 e cIAP-2 e protéica de Bcl-2, Bcl-xL, Bak, Bim e c-FLIPL por western-blotting. Os resultados de expressão gênica foram representados por unidades relativas de expressão em medianas nas diferentes amostras. Os pacientes com DM-1 apresentaram diminuição da expressão dos genes anti-apoptóticos bcl-2 (mediana: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) e cIAP-1 (1,24; p=0,003) nas CMN dos pacientes no período pré-mob em relação aos indivíduos saudáveis (medianas: bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), enquanto a expressão de cIAP-2 (60,8; p=0,0005) estava aumentada em relação aos controles (23,3). Foi observada redução significativa na expressão dos genes pró-apoptóticos bad (0,002; p<0,0001), bax (0,01; p=0,002) e fasL (1,66; p=0,001) no período pré-mob comparada aos controles sadios (bad: 0,23; bax: 2,79; fasL: 3,56). Os níveis de RNAm de bid (0,10; p=0,001) e bok (0,72; p=0,006) estavam elevados no pré-mob em relação ao grupo controle (bid: 0,004; bok: 0,31). As moléculas bcl-2, bcl-w, bcl-xL, mcl-1, bad, bax, bok, fasL e cIAP-1 atingiram níveis de RNAm similares aos controles após o TACTH. Foi verificado que a expressão de bcl-w, cIAP-1 e noxa estava maior nos pacientes com DM-1 em remissão quando comparados àqueles em recaída. A diminuição da expressão de a1, bcl-2 e bcl-w e o aumento de fas e noxa correlacionaram-se às porcentagens de hemoglobina glicosilada, concentração de auto-anticorpos GAD65, e aos níveis séricos de peptídeo-C após o transplante. Os pacientes com EM mostraram uma expressão reduzida dos genes anti-apoptóticos bcl-w (0,11; p=0,02) e cIAP-1 (1,87; p=0,04) no pré-mob comparada aos valores dos controles (bcl-w: 0,27; cIAP-1: 7,75) e maior expressão dos genes a1 (90,8; p=0,001) e cIAP-2 (58,8; p=0,009) em relação aos controles (a1: 12,7; cIAP-2: 22,3). As moléculas pró-apoptóticas bad (0,007; p=0,01) e bax (0,0007; p=0,004) mostraram menor expressão nas CMN no período pré-mob do que nos controles (bad: 0,27; bax: 1,24). Os genes bid (20,7; p=0,004), bik (0,84; p=0,02) e bok (1,77; p=0,0001) possuíam maior expressão no período pré-mob em relação aos indivíduos sadios (bid: 2,64; bik: 0,33; bok: 0,26). Não foram observadas diferenças significativas na expressão das moléculas da via extrínseca da apoptose nos pacientes com EM (p>0,05) nos períodos avaliados. Os valores de expressão de bcl-w, bak, bax, bik, bok e cIAP-1 atingiram níveis semelhantes aos controles após o transplante. A expressão dos genes bcl-2, cIAP-1, bad e bax estava maior nos pacientes em remissão da EM quando comparados àqueles em progressão neurológica. O aumento da expressão dos genes pró-apoptóticos bax, bak e bimEL correlacionou-se inversamente aos valores de EDSS dos pacientes com EM após o TACTH. Os resultados de expressão protéica foram equivalentes aos de expressão gênica nas duas doenças, com exceção dos dados das proteínas Bcl-2 e Bim. Em conjunto, os resultados demonstraram a desregulação da expressão de várias moléculas anti- e pró-apoptóticas nas CMN dos pacientes com DM-1 e EM. Esses achados sugerem a associação de alterações nos processos de apoptose celular com o surgimento e persistência de células auto-reativas no DM-1 e EM. Os dados indicam que essas alterações, principalmente a diminuição da expressão de moléculas pró-apoptóticas, como bak e bax, possam contribuir para a patogênese do DM-1 e EM. Além disso, a terapia de IAD/TACTH foi capaz de modular a expressão da maioria dos genes anormalmente expressos nas CMN dos pacientes com DM-1 e EM, já que esses atingiram níveis de expressão similares ao grupo controle após o transplante. Esta normalização da expressão de vários genes analisados correlacionou-se com a remissão clínica da doença na maioria dos pacientes / Type 1 diabetes mellitus (T1DM) and multiple sclerosis (MS) are inflammatory, organ-specific autoimmune diseases characterized by selective destruction of insulin-producing pancreatic -cells and central nervous system, respectively, by autoreactive B and T cells. Deregulation of apoptotic machinery is supposed to contribute to self-tolerance breakdown and autoimmune diseases pathogenesis, since apoptotic molecules have an important role in B and T lymphocytes central and peripheral tolerance mechanisms. The aim of this study was to evaluate the expression of pro and anti-apoptotic molecules from intrinsic and extrinsic apoptotic pathways and IAP Family members in 33 healthy individuals, 15 T1DM and 18 MS patients submitted to high-dose immunossupression therapy followed by autologous hematopoietic stem cell transplantation (HDI/AHSCT). Peripheral blood mononuclear cells (PBMC) were isolated from controls and patients at pre-mobilization (pre-mob), pre-conditioning (pre-cond), D+180, D+360, D+540 and D+720 post-transplantation. PBMC were used for RNA extraction, cDNA synthesis, gene quantification of a1, bcl-2, bcl-w, bcl-xL, bad, bak, bax, bid, bik, bimEL, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 and cIAP-2 by Real Time PCR and Bcl-2, Bcl-xL, Bak, BimEL and c-FLIPL proteins detection by western-blotting. Results are expressed as median of relative expression units. Results from T1DM patients indicated that antiapoptotic molecules bcl-2 (median: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) and cIAP-1 (1,24; p=0,003) were downregulated at pre-mob compared with healthy controls (medians bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), while cIAP-2 (60,8; p=0,0005) gene expression was upregulated compared to healthy controls (23,3). We observed a significant decrease in proapoptotic bad (0,002; p<0,0001), bax (0,01; p=0,002) and fasL (1,66; p=0,001) genes expression in patients PBMC at pre-mob period compared to healthy subjects (bad: 0,23; bax: 2,79; fasL: 3,56). mRNA levels of bid (0.10; p=0.001) and bok (0.72; p=0.006) were elevated at pre-mob period when compared to control group (bid: 0.004; bok: 0.31). The bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bok, fasL and cIAP-1 mRNA levels reached controls levels after HDI/AHSCT. We observed that bcl-w, cIAP-1 and noxa gene expression were increased in T1DM patients in remission when compared to relapsed patients. The decreased antiapoptotic gene expression and increased in proapoptotic molecules correlated with decreased glicosilated hemoglobin percentages (Hb A1C) and anti-GAD65 antibodies and increased peptide-C levels. Results from MS patients showed decreased bcl-w (0,11; p=0,02) and cIAP-1 gene expression (1,87; p=0,04) in patients PBMC at pre-mob period compared to healthy controls (bcl-w: 0,27; cIAP-1: 7,75) and increased expression of a1 (90,8; p=0,001) and cIAP-2 (58,8; p=0,009) compared to controls (a1: 12,7; cIAP-2: 22,3). Proapoptotic molecules bad (0.007; p=0.01) and bax (0.0007; p=0.004) showed decreased gene expression at pre-mob compared to control group (bad: 0.27; bax: 1.24). bid (20.7; p=0.004), bik (0.84; p=0.01) and bok genes (1.77; p=0.0001) showed increased expression at pre-mob compared to healthy controls (bid: 2.64; bik: 0.33; bok: 0.26). Significant differences were not observed in the expression of the extrinsic pathway genes in pre-mob and healthy controls samples (p>0.05). bcl-w, bak, bax, bik, bok and cIAP-1 expression values reached healthy control values after transplantation. We observed that bcl-2, cIAP-1, bad and bax gene expression was increased in MS patients in disease remission when compared to patients with neurologic progression. Significant correlation of increased proapoptotic genes expression with decreased EDSS values in MS patients after HDI/AHSCT was observed. Results of protein quantification of apoptotic molecules in PBMC of T1DM and MS patients were similar to the gene expression results of these molecules, except for Bcl-2 and Bim proteins. Taken together, these data indicate a deregulated expression of anti- and proapoptotic genes in T1DM and MS patients PBMC. These data suggest an association of deregulated apoptosis with emergence and maintenance of autoreactive lymphocytes in analyzed patients. Based on these results, we suggest that this altered gene expression profile, mainly the decreased proapoptotic genes expression, as bak and bax, may contribute to T1DM and MS pathogenesis. Furthermore, we showed that the HDI/AHSCT therapy was able to modulate and normalize the expression of most genes abnormally expressed in T1DM and MS patients at pre-transplant period. Many analyzed genes achieved expression levels similar to healthy controls. The normalization of the expression of many evaluated genes correlated to disease remission in the majority of the patients.

O transplante de ilhotas pancreáticas humanas representa uma estratégia promissora para a cura do diabetes mellitus tipo 1 (DM1), mas a aplicação a todos os pacientes diabéticos ainda é impraticável devido à limitada disponibilidade de ilhotas ou células &#946; e à necessidade de utilização de drogas imunossupressoras pelo paciente transplantado. O tratamento com imunossupressores após o transplante de ilhotas pode ser abolido quando se realiza o microencapsulamento das ilhotas pancreáticas. Neste trabalho investigou-se um novo biomaterial, Biodritina® (alginato/sulfato de condroitina) adequado ao microencapsulamento que gelifica na presença de íons de cálcio ou bário. A biocompatibilidade das microcápsulas tem sido avaliada segundo o grau de pureza do alginato utilizado na sua confecção. Amostras de alginato comercial purificado foram analisadas, comprovando-se a presença de impurezas (polifenóis, endotoxinas, proteínas) em níveis elevados, que impedem sua aplicação clínica. Optou-se, portanto pela utilização do alginato comercial ultrapurificado nos experimentos descritos neste trabalho. Das formulações de biomateriais avaliadas, as microcápsulas de bário-Biodritina apresentaram o melhor desempenho em testes de estabilidade físico-química. Estas microcápsulas mantiveram sua morfologia e estabilidade estrutural após permanecerem 30 dias na cavidade peritoneal de camundongos, conforme demonstrado por microscopia eletrônica de varredura (MEV). Análises histológicas mostraram que microcápsulas de bário-Biodritina explantadas, não possuíam adesão celular em sua superfície. Estudos de permeabilidade demonstraram que o tamanho médio dos poros das microcápsulas de bário-Biodritina permite passagem de proteínas de até 70 kDa, enquanto os poros daquelas de cálcio-Biodritina comportam proteínas de até 100 kDa. Experimentos de coResumo | x cultivo de macrófagos peritoneais com ilhotas de rato microencapsuladas demonstraram uma capacidade imunoprotetora maior das microcápsulas de bário-Biodritina em relação às de cálcio- Biodritina, sendo que as primeiras não ativaram os macrófagos. A manutenção da viabilidade e função de ilhotas humanas microencapsuladas com bário-Biodritina foi confirmada através de ensaio funcional in vitro, no qual ilhotas microencapsuladas apresentaram níveis de secreção de insulina idênticos aos de ilhotas nuas. A prova de conceito do biomaterial foi realizada através do implante de ilhotas humanas microencapsuladas em bário-Biodritina em camundongos com DM1 induzido por estreptozotocina. A hiperglicemia desses animais foi corrigida pelo implante por um período superior a 60 dias, durante os quais o teste oral de tolerância à glicose mostrou-se normal, demonstrando completa funcionalidade e eficiência das ilhotas microencapsuladas com bário-Biodritina. Partindo de observações de que animais inoculados com a peçonha do escorpião Tityus serrulatus apresentam nesidioblastose, foi realizado o fracionamento do veneno por HPLC de fase reversa e 24 frações obtidas foram submetidas a ensaios de proliferação celular através da incorporação de 3H-timidina em células de insulinoma de rato RINm5F. Uma dessas frações foi capaz de induzir a proliferação das células RINm5F e quando aplicada a ilhotas humanas isoladas, elevou o índice de secreção de insulina e induziu um aumento da expressão dos mRNAs de insulina e PCNA. Portanto, demonstrou-se que o biomaterial bário-Biodritina possui as características necessárias para microencapsular células/ilhotas com eficiência e que a \"fração ativa\" do veneno do escorpião T. serrulatus induz proliferação de células RINm5F e melhora a secreção de insulina de ilhotas humanas. / Islet transplantation has been proposed as a promising therapeutic strategy for the cure of type 1 diabetes mellitus (DM), however, its application to all diabetic patients is still not possible due to the limited source of islets or &#946; cells and to the need of an immunosuppressive treatment of the recipient to avoid graft rejection. The use of immunosupressors may be abolished when pancreatic islets are microencapsulated prior to transplantation. Here, we investigated the use of a new biomaterial suitable for cell microencapsulation, namely, Biodritin&#174;, composed of alginate and chondroitin sulphate, which is capable of gelation in the presence of barium or calcium ions. Microcapsules biocompatibility has been evaluated according to the purity of the alginate used in its production. Samples of purified commercial alginate were analyzed, but the high levels of contaminants (proteins, endotoxins and polyphenols) detected prevented its use in clinical applications. On the other hand, also commercially available ultrapure alginate fulfills the requirements for this application, therefore, this biomaterial was chosen for our experiments. Among the different biomaterial formulations evaluated, barium-Biodritin microcapsules displayed the best performance in the physico-chemical tests. Scanning electronic microscopy revealed that barium-Biodritin microcapsules maintained their morphology and structural stability after being implanted for 30 days in the peritoneal cavity of mice. No cellular adhesion was detected on the surface of explanted barium-Biodritin microcapsules by histological analysis. Permeability studies determined the medium pore size of barium-Biodritin microcapsules, which allows proteins of up to 70 kDa to pass through the biomaterial, while calcium-Biodritin pores accomodate proteins of up to 100 kDa. Co-culture of peritoneal macrophages with microencapsulated rat islets, revealed a superior immunoprotective capacity of barium-Biodritin microcapsules, which were capable of protecting the islets with no macrophage activation. Microencapsulated and naked human islets presented identical insulin secretion levels upon stimulation with glucose in vitro, confirming that barium-Biodritin microencapsulation maintains the function and viability of human islets. Proof-of-concept experiments in which barium-Biodritin microencapsulated human islets were implanted into chemically-induced diabetic mice, showed that these animals maintained normal blood glucose levels for more than 60 days, during which oral glucose tolerance tests were normal, demonstrating the complete functionality and efficiency of barium-Biodritin microencapsulated human islets. From the observation that animals inoculated with the venom of the scorpion Tityus serrulatus presented nesidioblastosis, we decided to fractionate the venom to isolate the active principle. The venom was fractionated by reversed phase HPLC and 24 fractions were obtained and submitted to cellular proliferation assays, in which rat insulinoma RINm5F cells evaluated for 3H-timidina incorporation. One of these fractions was capable of inducing cell proliferation and was also applied to isolated human islets. Treated islets presented a higher insulin secretion index and an increase in insulin and PCNA mRNA expression. In conclusion, we demonstrated that the barium-Biodritin biomaterial possesses all characteristics required for efficient cell/islet microencapsulation and that the active fraction of Tityus serrulatus venom induces the proliferation of RINm5F cells and improves insulin secretion in human islets.

A nefropatia diabética (ND) decorre da hiperglicemia crônica, de fatores de risco como a hipertensão arterial e a dislipidemia e de uma susceptibilidade genética já evidenciada em inúmeros estudos clínicos. Uma das características histológicas da ND é o acúmulo de proteínas de matriz extracelular no mesângio, para o qual contribuem várias vias bioquímicas. O GLUT-1, codificado pelo gene SLC2A1, é o principal transportador de glucose da célula mesangial e sua expressão está aumentada no glomérulo de animais diabéticos, o que constitui uma alça de feedback positivo pela qual a glicose extracelular aumentada estimula ainda mais sua própria captação, piorando a lesão mesangial. O GLUT-2, codificado pelo gene SLC2A2, é expresso nas células tubulares e nos podócitos e sua expressão também está aumentada na ND. A expressão deste transportador de glicose é regulada pelo fator de transcrição HNF-1. Participa, ainda, da lesão renal induzida pela hiperglicemia o fator de crescimento transformante - (TGF-), que exerce vários efeitos deletérios, tais como diminuir a atividade de metaloproteinases de matriz e promover fibrose renal. Esse fator de crescimento determina a ativação transcricional de genes-alvo, mas necessita de outros ativadores e co-ativadores da transcrição, tais como a proteína SMIF, codificada pelo gene DCP1A. Tendo em vista a participação das proteínas mencionadas acima na patogênese da ND, o presente estudo teve o objetivo de avaliar a associação de polimorfismos de um único nucleotídeo (SNPs) nos genes SLC2A1, SLC2A2, HNF1A, TGFB1 e DCP1A com a doença renal em portadores de diabetes mellitus tipo 1 (DM1). Um total de 449 pacientes (56,4% do sexo feminino, idade média de 36,0±11,0 anos) com mais de 10 anos de doença foram incluídos e classificados de acordo com o estágio de ND: (1) Ausência de ND: excreção urinária de albumina (EUA) normal (< 30 mg/24h ou < 20 g/min) e creatinina plasmática < 1,7 mg/dL sem tratamento anti-hipertensivo; (2) ND incipiente: microalbuminúria (EUA de 30 299 mg/24h ou 20 199 g/min) e creatinina plasmática < 1,7 mg/dL sem tratamento anti-hipertensivo e (3) ND Franca: macroalbuminúria (EUA > 300 mg/24h ou > 200 g/min) ou proteinúria ou tratamento para reposição renal. Também foram avaliadas as associações dos SNPs com o ritmo de filtração glomerular estimado (RFGe). Os SNPs foram genotipados pela metodologia de reação em cadeia da polimerase em tempo real, com o uso de sondas fluorescentes. As associações dos SNPs com a ND foram avaliadas por análise de regressão logística e os odds ratios (OR) e respectivos intervalos de confiança (IC) de 95% foram calculados após ajuste para possíveis confundidores, que foram incluídos como co-variáveis no modelo de regressão. Valores de P < 0.05 (bicaudal) foram considerados estatisticamente significantes. As seguintes associações foram observadas: (1) gene SLC2A1: genótipos CT+TT do SNP rs841848 conferiram risco para a ND incipiente na população global (OR 1,88; CI95% 1,06-3,34; P= 0,03) e nos pacientes do sexo masculino (OR 2,67; CI95% 1,13-6,35; P=0,0247) e para a ND franca (OR 2,70; CI95% 1,18-6,31; e P= 0,0197) apenas nos pacientes do sexo masculino; genótipos GA+AA do SNP rs1385129 conferiram risco para a ND franca na população do sexo masculino (OR 3,09; CI95% 1,34-7,25; P=0,0085); genótipos AT + TT do SNP rs3820589, conferiram proteção contra a ND incipiente na população global (OR 0,36; CI95% 0,16-0,78; P=0,0132) e na população do sexo feminino (OR 0,14; CI95% 0,02-0,52; P=0,0122). (2) gene SLC2A2: genótipos GA+GG do SNP rs5396 conferiram proteção contra ND franca nos pacientes do sexo masculino (OR 0,29; CI95% 0,12-0,69; P=0,0052); os genótipos AG+GG do SNP rs6800180 conferiram proteção contra a ND franca nos pacientes do sexo masculino (OR 0,16; CI95% 0,14-0,90; P=0,0324). (3) gene HNF1A: genótipos AC + CC do SNP rs1169288 conferiram risco para ND franca na população global (OR 2,23; CI95% 1,16-4,38; P=0,0175); genótipos CG+GG do SNP rs1169289 conferiram risco para ND franca na população global (OR 3,43; CI95% 1,61-7,73; P=0,002); (4) Gene TGFB1: genótipos CT + TT do SNP 1800468 conferiram risco para ND incipiente na população total (OR 2,99; CI95% 1,26-7,02; P 0,0116) e o alelo polimórfico T do SNP rs1800469 conferiu risco para um menor RFGe (p=0,0271). (5) gene DCP1A: o alelo polimórfico A do SNP rs11925433 também se associou com um menor RFGe (p=0,0075). Em conclusão, SNPs em genes que codificam as proteínas envolvidas na patogênese da ND GLUT-1, GLUT-2, HNF-1, TGF- e SMIF conferem susceptibilidade para essa complicação crônica nos portadores de DM1 avaliados no presente estudo / Diabetic nephropathy (DN) results from chronic hyperglycemia, risk factors such as hypertension and dyslipidemia as well as from genetic susceptibility, already demonstrated in numerous clinical studies. A histological feature of DN is the accumulation of extracellular matrix proteins in the mesangium after activation of multiple biochemical pathways. GLUT-1, encoded by gene SLC2A1, is the major glucose transporter in mesangial cell and its expression is increased in the glomeruli of diabetic animals, comprising a positive feedback loop whereby high extracellular glucose stimulates its own uptake and worsening mesangial injury. GLUT-2, encoded by SLC2A2 gene, is expressed in podocytes and tubular cells and its expression is also increased in DN. The expression of this glucose transporter is regulated by the transcription factor HNF-1. Transforming growth factor - (TGF-) also participates in renal injury induced by hyperglycemia, exerting several deleterious effects, such as to decrease the activity of matrix metalloproteinases and to promote renal fibrosis. This growth factor determines the transcriptional activation of target genes, but needs other activators and co-activators, such as the protein named SMIF, encoded by the gene DCP1A. Given the involvement of the aforementioned proteins in the pathogenesis of DN, the present study aimed to evaluate the association of single nucleotide polymorphisms (SNPs) in the genes SLC2A1, SLC2A2, HNF1A, TGFB1 e DCP1A with renal disease in patients with type 1 diabetes mellitus (T1DM). A total of 449 patients (56.4% female, mean age 36.0±11.0 years) with disease duration > 10 years were included and grouped according to DN stages: (1) absence of DN: normal urinary albumin excretion (UAE) (< 30 mg/24h or < 20 g/min) and plasmatic creatinine < 1.7 mg/dL without antihypertensive treatment; (2) incipient DN: microalbuminuria (UAE 30 299 mg/24h or 20 199 g/min) and plasmatic creatinine < 1.7 mg/dL without antihypertensive treatment and (3) overt DN: macroalbuminúria (UAE > 300 mg/24h or > 200 g/min) or proteinuria or renal replacement therapy. Associations of SNPs with estimated glomerular filtration rate (eGFR) were also evaluated. All SNPs were genotyped by real time polymerase chain reaction using fluorescent-labelled probes. Associations of the SNPs with DN were assessed by logistic regression analyses and odds ratios (OR) were calculated after adjustments for possible confounders included as covariables in the regressive model. P values <0.05 (two-tails) were considered significant. The following associations were observed: (1) SLC2A1: genotypes CT+TT from rs841848 conferred risk to incipient DN in the overall population (OR 1.88; 95%IC 1.06-3.34; P= 0.03) and in the male patients (OR 2.67; CI95% 1.13-6.35; P=0.0247) and to overt DN (OR 2.70; CI95% 1.18-6.31; e P= 0.0197) only in the male patients; genotypes GA+AA from rs1385129 conferred risk to overt DN in the male population (OR 3.09; CI95% 1.34-7.25; P=0.0085); genotypes AT + TT from rs3820589 conferred protection against incipient DN in the overall population (OR 0.36; CI95% 0.16-0.78; P=0.0132) and in the female population (OR 0.14; CI95% 0.02-0.52; P=0.0122). (2) SLC2A2: genotypes GA+GG from rs5396 conferred protection against overt DN in the male patients (OR 0.29; CI95% 0.12-0.69; P=0.0052); genotypes AG+GG from rs6800180 conferred protection against overt DN in the male patients (OR 0.16; CI95% 0.14-0.90; P=0.0324). (3) HNF1A: genotypes AC + CC from rs1169288 conferred risk to overt DN in the overall population (OR 2.23; CI95% 1.16-4.38; P=0.0175); genotypes CG+GG from rs1169289 conferred risk to overt DN in the overall population (OR 3.43; CI95% 1.61-7.73; P=0.002); (4) TGFB1: genotypes CT + TT from 1800468 conferred risk to incipient DN in the overall population (OR 2.99; CI95% 1.26-7.02; P=0.0116) and the polymorphic allele T from SNP rs1800469 conferred risk to a lower eGFR (p=0.0271). (5) DCP1A: the polymorphic allele A from SNP rs11925433 was also associated with a lower eGFR (p=0.0075). In conclusion, SNPs in the genes encoding proteins GLUT-1, GLUT-2, HNF-1, TGF- e SMIF, all involved in the pathogenesis of DN, conferred susceptibility to this chronic complication in the T1DM patients evaluated in the present study

The purpose of this study was to explore the feasibility of using human patient simulation (HPS) to teach Type 1 diabetes (T1DM) management to grandparents of grandchildren with T1DM. Thirty grandparents (11 male, 19 female) of young grandchildren (aged 12 and under) with T1DM were recruited from an urban medical center. Experimental group (n = 14) grandparents received hands-on visual T1DM management education using an HPS intervention, and control group (n = 16) grandparents received similar education using a non-HPS intervention. Post-intervention, researchers interviewed twelve grandparents (50% HPS, 50% non-HPS) who scored highest and lowest on the Hypoglycemia Fear Survey. Using a mixed-method design, researchers integrated study instrument data and post-intervention interview data to describe grandparent’s experience learning T1DM management. Post-intervention, grandparent scores for knowledge, confidence, and fear showed no significant difference by group assignment, however, all grandparent scores showed improvement from Time 1 to Time 2. Grandparents described how taking part in T1DM education heightened their awareness of T1DM risks. GP T1DM knowledge gains aided GPs to make sense of T1DM risks. Newfound T1DM knowledge enhanced GP T1DM management confidence. Improved T1DM knowledge and confidence helped to defuse T1DM management fear. Although study instruments did not measure significant difference between grandparents who received the HPS intervention and those who did not, the consistency of larger HPS-taught grandparent score improvement is suggestive of a benefit for HPS.

A nefropatia diabética (ND) decorre da hiperglicemia crônica, de fatores de risco como a hipertensão arterial e a dislipidemia e de uma susceptibilidade genética já evidenciada em inúmeros estudos clínicos. Uma das características histológicas da ND é o acúmulo de proteínas de matriz extracelular no mesângio, para o qual contribuem várias vias bioquímicas. O GLUT-1, codificado pelo gene SLC2A1, é o principal transportador de glucose da célula mesangial e sua expressão está aumentada no glomérulo de animais diabéticos, o que constitui uma alça de feedback positivo pela qual a glicose extracelular aumentada estimula ainda mais sua própria captação, piorando a lesão mesangial. O GLUT-2, codificado pelo gene SLC2A2, é expresso nas células tubulares e nos podócitos e sua expressão também está aumentada na ND. A expressão deste transportador de glicose é regulada pelo fator de transcrição HNF-1. Participa, ainda, da lesão renal induzida pela hiperglicemia o fator de crescimento transformante - (TGF-), que exerce vários efeitos deletérios, tais como diminuir a atividade de metaloproteinases de matriz e promover fibrose renal. Esse fator de crescimento determina a ativação transcricional de genes-alvo, mas necessita de outros ativadores e co-ativadores da transcrição, tais como a proteína SMIF, codificada pelo gene DCP1A. Tendo em vista a participação das proteínas mencionadas acima na patogênese da ND, o presente estudo teve o objetivo de avaliar a associação de polimorfismos de um único nucleotídeo (SNPs) nos genes SLC2A1, SLC2A2, HNF1A, TGFB1 e DCP1A com a doença renal em portadores de diabetes mellitus tipo 1 (DM1). Um total de 449 pacientes (56,4% do sexo feminino, idade média de 36,0±11,0 anos) com mais de 10 anos de doença foram incluídos e classificados de acordo com o estágio de ND: (1) Ausência de ND: excreção urinária de albumina (EUA) normal (< 30 mg/24h ou < 20 g/min) e creatinina plasmática < 1,7 mg/dL sem tratamento anti-hipertensivo; (2) ND incipiente: microalbuminúria (EUA de 30 299 mg/24h ou 20 199 g/min) e creatinina plasmática < 1,7 mg/dL sem tratamento anti-hipertensivo e (3) ND Franca: macroalbuminúria (EUA > 300 mg/24h ou > 200 g/min) ou proteinúria ou tratamento para reposição renal. Também foram avaliadas as associações dos SNPs com o ritmo de filtração glomerular estimado (RFGe). Os SNPs foram genotipados pela metodologia de reação em cadeia da polimerase em tempo real, com o uso de sondas fluorescentes. As associações dos SNPs com a ND foram avaliadas por análise de regressão logística e os odds ratios (OR) e respectivos intervalos de confiança (IC) de 95% foram calculados após ajuste para possíveis confundidores, que foram incluídos como co-variáveis no modelo de regressão. Valores de P < 0.05 (bicaudal) foram considerados estatisticamente significantes. As seguintes associações foram observadas: (1) gene SLC2A1: genótipos CT+TT do SNP rs841848 conferiram risco para a ND incipiente na população global (OR 1,88; CI95% 1,06-3,34; P= 0,03) e nos pacientes do sexo masculino (OR 2,67; CI95% 1,13-6,35; P=0,0247) e para a ND franca (OR 2,70; CI95% 1,18-6,31; e P= 0,0197) apenas nos pacientes do sexo masculino; genótipos GA+AA do SNP rs1385129 conferiram risco para a ND franca na população do sexo masculino (OR 3,09; CI95% 1,34-7,25; P=0,0085); genótipos AT + TT do SNP rs3820589, conferiram proteção contra a ND incipiente na população global (OR 0,36; CI95% 0,16-0,78; P=0,0132) e na população do sexo feminino (OR 0,14; CI95% 0,02-0,52; P=0,0122). (2) gene SLC2A2: genótipos GA+GG do SNP rs5396 conferiram proteção contra ND franca nos pacientes do sexo masculino (OR 0,29; CI95% 0,12-0,69; P=0,0052); os genótipos AG+GG do SNP rs6800180 conferiram proteção contra a ND franca nos pacientes do sexo masculino (OR 0,16; CI95% 0,14-0,90; P=0,0324). (3) gene HNF1A: genótipos AC + CC do SNP rs1169288 conferiram risco para ND franca na população global (OR 2,23; CI95% 1,16-4,38; P=0,0175); genótipos CG+GG do SNP rs1169289 conferiram risco para ND franca na população global (OR 3,43; CI95% 1,61-7,73; P=0,002); (4) Gene TGFB1: genótipos CT + TT do SNP 1800468 conferiram risco para ND incipiente na população total (OR 2,99; CI95% 1,26-7,02; P 0,0116) e o alelo polimórfico T do SNP rs1800469 conferiu risco para um menor RFGe (p=0,0271). (5) gene DCP1A: o alelo polimórfico A do SNP rs11925433 também se associou com um menor RFGe (p=0,0075). Em conclusão, SNPs em genes que codificam as proteínas envolvidas na patogênese da ND GLUT-1, GLUT-2, HNF-1, TGF- e SMIF conferem susceptibilidade para essa complicação crônica nos portadores de DM1 avaliados no presente estudo / Diabetic nephropathy (DN) results from chronic hyperglycemia, risk factors such as hypertension and dyslipidemia as well as from genetic susceptibility, already demonstrated in numerous clinical studies. A histological feature of DN is the accumulation of extracellular matrix proteins in the mesangium after activation of multiple biochemical pathways. GLUT-1, encoded by gene SLC2A1, is the major glucose transporter in mesangial cell and its expression is increased in the glomeruli of diabetic animals, comprising a positive feedback loop whereby high extracellular glucose stimulates its own uptake and worsening mesangial injury. GLUT-2, encoded by SLC2A2 gene, is expressed in podocytes and tubular cells and its expression is also increased in DN. The expression of this glucose transporter is regulated by the transcription factor HNF-1. Transforming growth factor - (TGF-) also participates in renal injury induced by hyperglycemia, exerting several deleterious effects, such as to decrease the activity of matrix metalloproteinases and to promote renal fibrosis. This growth factor determines the transcriptional activation of target genes, but needs other activators and co-activators, such as the protein named SMIF, encoded by the gene DCP1A. Given the involvement of the aforementioned proteins in the pathogenesis of DN, the present study aimed to evaluate the association of single nucleotide polymorphisms (SNPs) in the genes SLC2A1, SLC2A2, HNF1A, TGFB1 e DCP1A with renal disease in patients with type 1 diabetes mellitus (T1DM). A total of 449 patients (56.4% female, mean age 36.0±11.0 years) with disease duration > 10 years were included and grouped according to DN stages: (1) absence of DN: normal urinary albumin excretion (UAE) (< 30 mg/24h or < 20 g/min) and plasmatic creatinine < 1.7 mg/dL without antihypertensive treatment; (2) incipient DN: microalbuminuria (UAE 30 299 mg/24h or 20 199 g/min) and plasmatic creatinine < 1.7 mg/dL without antihypertensive treatment and (3) overt DN: macroalbuminúria (UAE > 300 mg/24h or > 200 g/min) or proteinuria or renal replacement therapy. Associations of SNPs with estimated glomerular filtration rate (eGFR) were also evaluated. All SNPs were genotyped by real time polymerase chain reaction using fluorescent-labelled probes. Associations of the SNPs with DN were assessed by logistic regression analyses and odds ratios (OR) were calculated after adjustments for possible confounders included as covariables in the regressive model. P values <0.05 (two-tails) were considered significant. The following associations were observed: (1) SLC2A1: genotypes CT+TT from rs841848 conferred risk to incipient DN in the overall population (OR 1.88; 95%IC 1.06-3.34; P= 0.03) and in the male patients (OR 2.67; CI95% 1.13-6.35; P=0.0247) and to overt DN (OR 2.70; CI95% 1.18-6.31; e P= 0.0197) only in the male patients; genotypes GA+AA from rs1385129 conferred risk to overt DN in the male population (OR 3.09; CI95% 1.34-7.25; P=0.0085); genotypes AT + TT from rs3820589 conferred protection against incipient DN in the overall population (OR 0.36; CI95% 0.16-0.78; P=0.0132) and in the female population (OR 0.14; CI95% 0.02-0.52; P=0.0122). (2) SLC2A2: genotypes GA+GG from rs5396 conferred protection against overt DN in the male patients (OR 0.29; CI95% 0.12-0.69; P=0.0052); genotypes AG+GG from rs6800180 conferred protection against overt DN in the male patients (OR 0.16; CI95% 0.14-0.90; P=0.0324). (3) HNF1A: genotypes AC + CC from rs1169288 conferred risk to overt DN in the overall population (OR 2.23; CI95% 1.16-4.38; P=0.0175); genotypes CG+GG from rs1169289 conferred risk to overt DN in the overall population (OR 3.43; CI95% 1.61-7.73; P=0.002); (4) TGFB1: genotypes CT + TT from 1800468 conferred risk to incipient DN in the overall population (OR 2.99; CI95% 1.26-7.02; P=0.0116) and the polymorphic allele T from SNP rs1800469 conferred risk to a lower eGFR (p=0.0271). (5) DCP1A: the polymorphic allele A from SNP rs11925433 was also associated with a lower eGFR (p=0.0075). In conclusion, SNPs in the genes encoding proteins GLUT-1, GLUT-2, HNF-1, TGF- e SMIF, all involved in the pathogenesis of DN, conferred susceptibility to this chronic complication in the T1DM patients evaluated in the present study

O diabetes mellitus tipo 1 (DM-1) e a esclerose múltipla (EM) são doenças auto-imunes órgão-específicas, inflamatórias, mediadas por células T e B auto-reativas e caracterizadas pela destruição seletiva de células b pancreáticas produtoras de insulina e do sistema nervoso central, respectivamente. Acredita-se que a desregulação da expressão de genes reguladores da maquinaria apoptótica possa contribuir para o desenvolvimento da auto-imunidade, visto que algumas dessas moléculas participam nos processos de tolerância central e periférica de linfócitos auto-reativos. O objetivo deste projeto foi analisar a expressão de moléculas reguladoras das vias intrínseca, extrínseca e da Família de proteínas inibidoras da apoptose (IAP) em 33 indivíduos saudáveis, 15 pacientes com DM-1 e 18 com EM submetidos à terapia de imunossupressão em altas doses seguida do transplante autólogo de células-tronco hematopoéticas (IAD/TACTH). As células mononucleares (CMN) foram isoladas do sangue periférico dos controles e de pacientes nos períodos pré-mobilização (pré-mob), pré-condicionamento (pré-cond), D+180, D+360, D+540 e D+720 pós-transplante. As CMN foram utilizadas para extração de RNA, síntese de cDNA, quantificação da expressão por PCR em tempo real dos genes a1, bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bid, bik, bim, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 e cIAP-2 e protéica de Bcl-2, Bcl-xL, Bak, Bim e c-FLIPL por western-blotting. Os resultados de expressão gênica foram representados por unidades relativas de expressão em medianas nas diferentes amostras. Os pacientes com DM-1 apresentaram diminuição da expressão dos genes anti-apoptóticos bcl-2 (mediana: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) e cIAP-1 (1,24; p=0,003) nas CMN dos pacientes no período pré-mob em relação aos indivíduos saudáveis (medianas: bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), enquanto a expressão de cIAP-2 (60,8; p=0,0005) estava aumentada em relação aos controles (23,3). Foi observada redução significativa na expressão dos genes pró-apoptóticos bad (0,002; p<0,0001), bax (0,01; p=0,002) e fasL (1,66; p=0,001) no período pré-mob comparada aos controles sadios (bad: 0,23; bax: 2,79; fasL: 3,56). Os níveis de RNAm de bid (0,10; p=0,001) e bok (0,72; p=0,006) estavam elevados no pré-mob em relação ao grupo controle (bid: 0,004; bok: 0,31). As moléculas bcl-2, bcl-w, bcl-xL, mcl-1, bad, bax, bok, fasL e cIAP-1 atingiram níveis de RNAm similares aos controles após o TACTH. Foi verificado que a expressão de bcl-w, cIAP-1 e noxa estava maior nos pacientes com DM-1 em remissão quando comparados àqueles em recaída. A diminuição da expressão de a1, bcl-2 e bcl-w e o aumento de fas e noxa correlacionaram-se às porcentagens de hemoglobina glicosilada, concentração de auto-anticorpos GAD65, e aos níveis séricos de peptídeo-C após o transplante. Os pacientes com EM mostraram uma expressão reduzida dos genes anti-apoptóticos bcl-w (0,11; p=0,02) e cIAP-1 (1,87; p=0,04) no pré-mob comparada aos valores dos controles (bcl-w: 0,27; cIAP-1: 7,75) e maior expressão dos genes a1 (90,8; p=0,001) e cIAP-2 (58,8; p=0,009) em relação aos controles (a1: 12,7; cIAP-2: 22,3). As moléculas pró-apoptóticas bad (0,007; p=0,01) e bax (0,0007; p=0,004) mostraram menor expressão nas CMN no período pré-mob do que nos controles (bad: 0,27; bax: 1,24). Os genes bid (20,7; p=0,004), bik (0,84; p=0,02) e bok (1,77; p=0,0001) possuíam maior expressão no período pré-mob em relação aos indivíduos sadios (bid: 2,64; bik: 0,33; bok: 0,26). Não foram observadas diferenças significativas na expressão das moléculas da via extrínseca da apoptose nos pacientes com EM (p>0,05) nos períodos avaliados. Os valores de expressão de bcl-w, bak, bax, bik, bok e cIAP-1 atingiram níveis semelhantes aos controles após o transplante. A expressão dos genes bcl-2, cIAP-1, bad e bax estava maior nos pacientes em remissão da EM quando comparados àqueles em progressão neurológica. O aumento da expressão dos genes pró-apoptóticos bax, bak e bimEL correlacionou-se inversamente aos valores de EDSS dos pacientes com EM após o TACTH. Os resultados de expressão protéica foram equivalentes aos de expressão gênica nas duas doenças, com exceção dos dados das proteínas Bcl-2 e Bim. Em conjunto, os resultados demonstraram a desregulação da expressão de várias moléculas anti- e pró-apoptóticas nas CMN dos pacientes com DM-1 e EM. Esses achados sugerem a associação de alterações nos processos de apoptose celular com o surgimento e persistência de células auto-reativas no DM-1 e EM. Os dados indicam que essas alterações, principalmente a diminuição da expressão de moléculas pró-apoptóticas, como bak e bax, possam contribuir para a patogênese do DM-1 e EM. Além disso, a terapia de IAD/TACTH foi capaz de modular a expressão da maioria dos genes anormalmente expressos nas CMN dos pacientes com DM-1 e EM, já que esses atingiram níveis de expressão similares ao grupo controle após o transplante. Esta normalização da expressão de vários genes analisados correlacionou-se com a remissão clínica da doença na maioria dos pacientes / Type 1 diabetes mellitus (T1DM) and multiple sclerosis (MS) are inflammatory, organ-specific autoimmune diseases characterized by selective destruction of insulin-producing pancreatic -cells and central nervous system, respectively, by autoreactive B and T cells. Deregulation of apoptotic machinery is supposed to contribute to self-tolerance breakdown and autoimmune diseases pathogenesis, since apoptotic molecules have an important role in B and T lymphocytes central and peripheral tolerance mechanisms. The aim of this study was to evaluate the expression of pro and anti-apoptotic molecules from intrinsic and extrinsic apoptotic pathways and IAP Family members in 33 healthy individuals, 15 T1DM and 18 MS patients submitted to high-dose immunossupression therapy followed by autologous hematopoietic stem cell transplantation (HDI/AHSCT). Peripheral blood mononuclear cells (PBMC) were isolated from controls and patients at pre-mobilization (pre-mob), pre-conditioning (pre-cond), D+180, D+360, D+540 and D+720 post-transplantation. PBMC were used for RNA extraction, cDNA synthesis, gene quantification of a1, bcl-2, bcl-w, bcl-xL, bad, bak, bax, bid, bik, bimEL, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 and cIAP-2 by Real Time PCR and Bcl-2, Bcl-xL, Bak, BimEL and c-FLIPL proteins detection by western-blotting. Results are expressed as median of relative expression units. Results from T1DM patients indicated that antiapoptotic molecules bcl-2 (median: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) and cIAP-1 (1,24; p=0,003) were downregulated at pre-mob compared with healthy controls (medians bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), while cIAP-2 (60,8; p=0,0005) gene expression was upregulated compared to healthy controls (23,3). We observed a significant decrease in proapoptotic bad (0,002; p<0,0001), bax (0,01; p=0,002) and fasL (1,66; p=0,001) genes expression in patients PBMC at pre-mob period compared to healthy subjects (bad: 0,23; bax: 2,79; fasL: 3,56). mRNA levels of bid (0.10; p=0.001) and bok (0.72; p=0.006) were elevated at pre-mob period when compared to control group (bid: 0.004; bok: 0.31). The bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bok, fasL and cIAP-1 mRNA levels reached controls levels after HDI/AHSCT. We observed that bcl-w, cIAP-1 and noxa gene expression were increased in T1DM patients in remission when compared to relapsed patients. The decreased antiapoptotic gene expression and increased in proapoptotic molecules correlated with decreased glicosilated hemoglobin percentages (Hb A1C) and anti-GAD65 antibodies and increased peptide-C levels. Results from MS patients showed decreased bcl-w (0,11; p=0,02) and cIAP-1 gene expression (1,87; p=0,04) in patients PBMC at pre-mob period compared to healthy controls (bcl-w: 0,27; cIAP-1: 7,75) and increased expression of a1 (90,8; p=0,001) and cIAP-2 (58,8; p=0,009) compared to controls (a1: 12,7; cIAP-2: 22,3). Proapoptotic molecules bad (0.007; p=0.01) and bax (0.0007; p=0.004) showed decreased gene expression at pre-mob compared to control group (bad: 0.27; bax: 1.24). bid (20.7; p=0.004), bik (0.84; p=0.01) and bok genes (1.77; p=0.0001) showed increased expression at pre-mob compared to healthy controls (bid: 2.64; bik: 0.33; bok: 0.26). Significant differences were not observed in the expression of the extrinsic pathway genes in pre-mob and healthy controls samples (p>0.05). bcl-w, bak, bax, bik, bok and cIAP-1 expression values reached healthy control values after transplantation. We observed that bcl-2, cIAP-1, bad and bax gene expression was increased in MS patients in disease remission when compared to patients with neurologic progression. Significant correlation of increased proapoptotic genes expression with decreased EDSS values in MS patients after HDI/AHSCT was observed. Results of protein quantification of apoptotic molecules in PBMC of T1DM and MS patients were similar to the gene expression results of these molecules, except for Bcl-2 and Bim proteins. Taken together, these data indicate a deregulated expression of anti- and proapoptotic genes in T1DM and MS patients PBMC. These data suggest an association of deregulated apoptosis with emergence and maintenance of autoreactive lymphocytes in analyzed patients. Based on these results, we suggest that this altered gene expression profile, mainly the decreased proapoptotic genes expression, as bak and bax, may contribute to T1DM and MS pathogenesis. Furthermore, we showed that the HDI/AHSCT therapy was able to modulate and normalize the expression of most genes abnormally expressed in T1DM and MS patients at pre-transplant period. Many analyzed genes achieved expression levels similar to healthy controls. The normalization of the expression of many evaluated genes correlated to disease remission in the majority of the patients.