Immune response to insulin and changes in the gut immune system in children with or at risk for type 1 diabetes

Type 1 diabetes (T1D) is considered to be an autoimmune disease. The cause of T1D is the destruction of insulin-producing β-cells in the pancreatic islets. The autoimmune nature of T1D is characterized by the presence of autoreactive T-cells and autoantibodies against β-cell molecules. Insulin is the only β-cell-specific autoantigen associated with T1D but the insulin autoantibodies (IAAs) are difficult to measure with proper sensitivity. T-cell assays for detection of autoreactive T-cells, such as insulin-specific T-cells, have also proven to be difficult to perform. The genetic risk of T1D is associated with the HLA gene region but the environmental factors also play an important role. The most studied environmental risk factors of T1D are enteroviruses and cow's milk which both affect the immune system through the gut. One hypothesis is that the insulin-specific immune response develops against bovine insulin in cow's milk during early infancy and later spreads to include human insulin. The aims of this study were to determine whether the separation of immunoglobulin (Ig)G from plasma would improve the sensitivity of the IAA assay and how insulin treatment affects the cellular immune response to insulin in newly diagnosed patients. Furthermore, the effect of insulin concentration in mother's breast milk on the development of antibodies to dietary insulin in the child was examined. Small intestinal biopsies were also obtained from children with T1D to characterize any immunological changes associated with T1D in the gut. The isolation of the IgG fraction from the plasma of T1D patients negative for plasma IAA led to detectable IAA levels that exceeded those in the control children. Thus the isolation of IgG may improve the sensitivity of the IAA assay. The effect of insulin treatment on insulin-specific T-cells was studied by culturing peripheral blood mononuclear cells with insulin. The insulin stimulation induced increased expression of regulatory T-cell markers, such as Foxp3, in those patients treated with insulin than in patients examined before initiating insulin treatment. This finding suggests that insulin treatment in patients with T1D stimulates regulatory T-cells in vivo and this may partly explain the difficulties in measuring autoantigen-specific T-cell responses in recently diagnosed patients. The stimulation of regulatory T-cells by insulin treatment may also explain the remission period often seen after initiating insulin treatment. In the third study we showed that insulin concentration in mother's breast milk correlates inversely with the levels of bovine insulin-specific antibodies in those infants who were exposed to cow's milk proteins in their diet, suggesting that human insulin in breast milk induces tolerance to dietary bovine insulin. However, in infants who later developed T1D-associated autoantibodies, the insulin concentration in their mother's breast milk was increased. This finding may indicate that in those children prone to β-cell autoimmunity, breast milk insulin does not promote tolerance to insulin. In the small intestinal biopsies the presence of several immunological markers were quantified with the RT-PCR. From these markers the expression of the interleukin (IL)-18 cytokine was significantly increased in the gut in patients with T1D compared with children with celiac disease or control children. The increased IL-18 expression lends further support for the hypothesis that the gut immune system is involved in the pathogenesis of T1D.

Adipose tissue inflammation, liver fat and insulin resistance in humans

BACKGROUND: Obesity is closely associated with insulin resistance, which is a pathophysiologic condition contributing to the important co-morbidities of obesity, such as the metabolic syndrome and type 2 diabetes mellitus. In obese subjects, adipose tissue is characterized by inflammation (macrophage infiltration, increased expression insulin resistance genes and decreased expression of insulin sensitivity genes). Increased liver fat, without excessive alcohol consumption, is defined as non-alcoholic fatty liver disease (NAFLD) and also associated with obesity and insulin resistance. It is unknown whether and how insulin resistance is associated with altered expression of adipocytokines (adipose tissue-derived signaling molecules), and whether adipose tissue inflammation and NAFLD coexist independent of obesity. Genetic factors could explain variation in liver fat independent of obesity but the heritability of NAFLD is unknown. AIMS: To determine whether acute regulation of adipocytokine expression by insulin in adipose tissue is altered in obesity. To investigate the relationship between adipose tissue inflammation and liver fat content independent of obesity. To assess the heritability of serum alanine aminotransferase (ALT) activity, a surrogate marker of liver fat. METHODS: 55 healthy normal-weight and obese volunteers were recruited. Subcutaneous adipose tissue biopsies were obtained for measurement of gene expression before and during 6 hours of euglycemic hyperinsulinemia. Liver fat content was measured by proton magnetic resonance spectroscopy, and adipose tissue inflammation was assessed by gene expression, immunohistochemistry and lipidomics analysis. Genetic factors contributing to serum ALT activity were determined in 313 twins by statistical heritability modeling. RESULTS: During insulin infusion the expression of insulin sensitivity genes remains unchanged, while the expression of insulin resistance genes increases in obese/insulin-resistant subjects compared to insulin-sensitive subjects. Adipose tissue inflammation is associated with liver fat content independent of obesity. Adipose tissue of subjects with high liver fat content is characterized infiltrated macrophages and increased expression of inflammatory genes, as well as by increased concentrations of ceramides compared to equally obese subjects with normal liver fat. A significant heritability for serum ALT activity was verified. CONCLUSIONS: Effects of insulin infusion on adipose tissue gene expression in obese/insulin-resistant subjects are not only characterized by hyporesponse of insulin sensitivity genes but also by hyperresponse of insulin resistance and inflammatory genes. This suggests that in obesity, the impaired insulin action contributes or self-perpetuates alterations in adipocytokine expression in adipose tissue. Adipose tissue inflammation is increased in subjects with high liver fat compared to equally obese subjects with normal liver fat content. Concentrations of ceramides, the putative mediators of insulin resistance, are increased in adipose tissue in subjects with high liver fat. Genetic factors contribute significantly to variation in serum ALT activity, a surrogate marker of liver fat. These data imply that adipose tissue inflammation and increased liver fat content are closely interrelated, and determine insulin resistance even independent of obesity.

Mulibrey nanism : Clinical characteristics and pathophysiologic features of growth restriction, insulin resistance and tumour development

Background: Mulibrey nanism (MUL; Muscle-liver-brain-eye nanism; OMIM 253250) is an autosomal recessive growth disorder more prevalent in Finland than elsewhere in the world. Clinical characteristics include severe prenatal onset growth restriction, cardiopathy, multiple organ manifestations but no major neurological handicap. MUL is caused by mutations in the TRIM37 gene on chromosome 17q22-23, encoding a peroxisomal protein TRIM37 with ubiquitin E3-ligase activity. Nineteen different mutations have been detected, four of them present in the Finnish patients. Objective: This study aimed to characterize clinical and histopathological features of MUL in the national cohort of Finnish patients. Patients and methods: A total of 92 Finnish patients (age 0.7 to 77 years) participated in the clinical follow-up study. Patients hospital records and growth charts were reviewed. Physical, radiographic and laboratory examinations were performed according to a clinical protocol. Thirty patients (18 females) were treated with recombinant human GH for a median period of 5.7 years. Biopsies and autopsy samples were used for the histopathological and immunohistochemical analyses. Results: MUL patients were born small for gestational age (SGA) with immature craniofacial features after prenatal-onset growth restriction. They experienced a continuous deceleration in both height SDS and weight-for-height (WFH) postnatally. In infancy feeding difficulties and frequent pneumonias were common problems. At the time of diagnosis (median age 2.1 years) characteristic craniofacial, radiological and ocular features were the most constant findings. MUL patients showed a dramatic change in glucose metabolism with increasing age. While the children had low fasting glucose and insulin levels, 90% of the adults were insulin resistant, half had type 2 diabetes and an additional 42% showed impaired glucose tolerance (IGT). Seventy percent fulfilled the National Cholesterol Education Program (NCEP) Adult Treatment Panel III criteria for metabolic syndrome as adults. GH therapy improved pre-pubertal growth but had only minor impact on adult height (+5 cm). Interestingly, treated subjects were slimmer and had less frequent metabolic concerns as young adults. MUL patients displayed histologically a disturbed architecture with ectopic tissues and a high frequency of both benign and malignant tumours present in several internal organs. A total of 232 tumorous lesions were detected in our patient cohort. The majority of the tumours showed strong expression of endothelial cell marker CD34 as well as α-smooth muscle actin (α-SMA). Fifteen of the tumours were malignant and seven of them (five Wilms tumours) occurred in the kidney. Conclusions: MUL patients present a distinct postnatal growth pattern. Short-term response of GH treatment is substantial but the long-term impact remains modest. Although MUL patients form a distinct clinical and diagnostic entity, their clinical findings vary considerably from infancy to adulthood. While failure to thrive dominates early life, MUL adults develop metabolic syndrome and have a tendency for malignancies and vascular lesions in several organs. This speaks for a central role of TRIM37 in regulation of key cellular functions, such as proliferation, migration, angiogenesis and insulin signalling.

Preterm delivery and selected biomarkers : phosphorylated insulin-like growth factor-binding protein-1 and matrix metalloproteinase-8 – in cervical fluid

Premature delivery is a major cause of neonatal morbidity and mortality. The incidence of premature deliveries has increased around the world. In Finland 5.3%, or about 3,000 children per year are born prematurely, before 37 weeks of gestation. The corresponding figure in the United States is about 13%. The morbidity and mortality are highest among infants delivered before 32 weeks of gestation - about 600 children each year in Finland. Approximately 70% of premature deliveries are unexplained. Preterm delivery can be caused by an asympto-matic infection between uterus and the fetal membranes, such can begin already in early pregnancy. It is difficult to predict preterm delivery, and many patients are therefore unnecessarily admitted to hospital for observation and exposed to medical treatments. On the other hand, the high risk women should be identified early for the best treatment of the mother and preterm infant. --- In the prospective study conducted at the Department of Obstetric and Gynecology, Helsinki University Central Hospital two biochemical inflammation related markers were measured in the lower genital tract fluids of asymp-tomatic women in early and mid pregnancy in an order to see whether these markers could identify women with an increased risk of preterm delivery. These biomarkers were phosphorylated insulin-like growth factor binding protein-1 (phIGFBP-1) and matrix metalloproteinase-8 (MMP-8). The study involved 5180 asymptomatic pregnant women, examined during the first and second ultrasound screening visits. The study samples were taken from the vagina and cervicix. In addition, 246 symptomatic women were studied (pregnancy weeks 22 – 34). The study showed that increased phIGFBP-1 concentration in cervical canal fluid in early pregnancy increased the risk for preterm delivery. The risk for very premature birth (before 32 weeks of gestation) was nearly four-fold. Low MMP-8 concentration in mid pregnancy increased the risk of subsequent premature preterm rupture of fetal membranes (PPROM). Significantly high MMP-8 concentrations in the cervical fluid increased the risk for prema-ture delivery initiated by preterm labour with intact membranes. Among women with preterm contractions the shortened cervical length measured by ultrasound and elevated cervical fluid phIGFBP-1 both predicted premature delivery. In summary, because of the relatively low sensitivity of cervical fluid phIGFBP-1 this biomarker is not suitable for routine screening, but provides an additional tool in assessing the risk of preterm delivery. Cervical fluid MMP-8 is not useful in early or mid pregnancy in predicting premature delivery because of its dual role. Further studies on the role of MMP-8 are therefore needed. Our study confirms that phIGFBP-1 testing is useful in predicting pre-term delivery.

Testicular function in adolescent boys with Klinefelter syndrome

Klinefelter syndrome (KS) is the most frequent karyotype disorder of male reproductive function. Since its original clinical description in 1942 and the identification of its chromosomal basis 47,XXY in 1959, the typical KS phenotype has become well recognized, but the mechanisms behind the testicular degeneration process have remained unrevealed. This prospective study was undertaken to increase knowledge about testicular function in adolescent KS boys. It comprised a longitudinal follow-up of growth, pubertal development, and serum reproductive hormone levels in 14 prepubertal and pubertal KS boys. Each boy had a testicular biopsy that was analyzed with histomorphometric and immunohistochemical methods. The KS boys had sufficient testosterone levels to allow normal onset and progression of puberty. Their serum testosterone levels remained within the low-normal range throughout puberty, but from midpuberty onwards, findings like a leveling-off in testosterone and insulin-like factor 3 (INSL3) concentrations, high gonadotropin levels, and exaggerated responses to gonadotropin-releasing hormone stimulation suggest diminished testosterone secretion. We also showed that the Leydig cell differentiation marker INSL3 may serve as a novel marker for onset and normal progression of puberty in boys. In the KS boys the number of germ cells was already markedly lower at the onset of puberty. The pubertal activation of the pituitary-testicular axis accelerated germ cell depletion, and germ cell differentiation was at least partly blocked at the spermatogonium or early primary spermatocyte stages. The presence of germ cells correlated with serum reproductive hormone levels. The immature Sertoli cells were incapable of transforming to the adult type, and during puberty the degeneration of Sertoli cells increased markedly. The older KS boys displayed an evident Leydig cell hyperplasia, as well as fibrosis and hyalinization of the interstitium and peritubular connective tissue. Altered immunoexpression of the androgen receptor (AR) suggested that in KS boys during puberty a relative androgen deficiency develops at testicular level. The impact of genetic features of the supernumerary X chromosome on the KS phenotype was also studied. The present study suggests that parental origin of the supernumerary X chromosome and the length of the CAG repeat of the AR gene influence pubertal development and testicular degeneration. The current study characterized by several means the testicular degeneration process in the testes of adolescent KS boys and confirmed that this process accelerates at the onset of puberty. Although serum reproductive hormone levels indicated no hypogonadism during early puberty, the histological analyses showed an already markedly reduced fertility potential in prepubertal KS boys. Genetic features of the X chromosome affect the KS phenotype.