Autoantibodies to N-terminally Truncated GAD(65)(96-585) : HLA Associations and Predictive Value for Type 1 Diabetes

Objective To evaluate the role of autoantibodies to N-terminally truncated glutamic acid decarboxylase GAD(65)(96-585) (t-GADA) as a marker for type 1 diabetes (T1D) and to assess the potential human leukocyte antigen (HLA) associations with such autoantibodies. Design In this cross-sectional study combining data from the Finnish Pediatric Diabetes Register, the Type 1 Diabetes Prediction and Prevention study, the DIABIMMUNE study, and the Early Dietary Intervention and Later Signs of Beta-Cell Autoimmunity study, venous blood samples from 760 individuals (53.7% males) were analyzed for t-GADA, autoantibodies to full-length GAD(65) (f-GADA), and islet cell antibodies. Epitope-specific GAD autoantibodies were analyzed from 189 study participants. Results T1D had been diagnosed in 174 (23%) participants. Altogether 631 (83%) individuals tested positive for f-GADA and 451 (59%) for t-GADA at a median age of 9.0 (range 0.2-61.5) years. t-GADA demonstrated higher specificity (46%) and positive predictive value (30%) for T1D than positivity for f-GADA alone (15% and 21%, respectively). Among participants positive for f-GADA, those who tested positive for t-GADA carried more frequently HLA genotypes conferring increased risk for T1D than those who tested negative for t-GADA (77% vs 53%; P < 0.001). Conclusions Autoantibodies to N-terminally truncated GAD improve the screening for T1D compared to f-GADA and may facilitate the selection of participants for clinical trials. HLA class II-mediated antigen presentation of GAD(96-585)-derived or structurally similar peptides might comprise an important pathomechanism in T1D.Peer reviewe

Characteristics of Slow Progression to Type 1 Diabetes in Children With Increased HLA-Conferred Disease Risk

Context: Characterization of slow progression to type 1 diabetes (T1D) may reveal novel means for prevention of T1D. Slow progressors might carry natural immunomodulators that delay beta-cell destruction and mediate preservation of beta-cell function. Objective: To identify demographic, genetic, and immunological characteristics of slow progression from seroconversion to clinical T1D. Design: H LA-susceptible children (n = 7410) were observed from birth for islet cell antibody (ICA), insulin autoantibody (IAA), glutamic acid decarboxylase (GADA), and islet antigen-2 autoantibodies (IA-2A), and for clinical T1D. Disease progression that lasted >= 7.26 years (slowest) quartile from initial seroconversion to diagnosis was considered slow. Autoantibody and genetic characteristics including 45 non-HLA single nucleotide polymorphisms (SNPs) predisposing to T1D were analyzed. Results: By the end of 2015, 1528 children (21 %) had tested autoantibody positive and 247 (16%) had progressed to T1D. The median delay from seroconversion to diagnosis was 8.7 years in slow (n = 62, 25%) and 3.0 years in other progressors. Compared with other progressors, slow progressors were less often multipositive, had lower ICA and IAA titers, and lower frequency of IA-2A at seroconversion. Slow progressors were born more frequently in the fall, whereas other progressors were born more often in the spring. Compared with multipositive nonprogressors, slow progressors were younger, had higher ICA titers, and higher frequency of IAA and multiple autoantibodies at seroconversion. We found no differences in the distributions of non-HLA SNPs between progressors. Conclusions: We observed differences in autoantibody characteristics and the season of birth among progressors, but no characteristics present at seroconversion that were specifically predictive for slow progression.Peer reviewe

Dynamics of Islet Autoantibodies During Prospective Follow-Up From Birth to Age 15 Years

Context: We set out to characterize the dynamics of islet autoantibodies over the first 15 years of life in children carrying genetic susceptibility to type 1 diabetes (T1D). We also assessed systematically the role of zinc transporter 8 autoantibodies (ZnT8A) in this context. Design: HLA-predisposed children (N = 1006, 53.0% boys) recruited from the general population during 1994 to 1997 were observed from birth over a median time of 14.9 years (range, 1.9-15.5 years) for ZnT8A, islet cell (ICA), insulin (IAA), glutamate decarboxylase (GADA), and islet antigen-2 (IA-2A) antibodies, and for T1D. Results: By age 15.5 years, 35 (3.5%) children had progressed to T1D. Islet autoimmunity developed in 275 (27.3%) children at a median age of 7.4 years (range, 0.3-15.1 years). The ICA seroconversion rate increased toward puberty, but the biochemically defined autoantibodies peaked at a young age. Before age 2 years, ZnT8A and IAA appeared commonly as the first autoantibody, but in the preschool years IA-2A- and especially GADA-initiated autoimmunity increased. Thereafter, GADA-positive seroconversions continued to appear steadily until ages 10 to 15 years. Inverse IAA seroconversions occurred frequently (49.3% turned negative) and marked a prolonged delay from seroconversion to diagnosis compared to persistent IAA (8.2 vs 3.4 years; P = .01). Conclusions: In HLA-predisposed children, the primary autoantibody is characteristic of age and might reflect the events driving the disease process toward clinical T1D. Autoantibody persistence affects the risk of T1D. These findings provide a framework for identifying disease subpopulations and for personalizing the efforts to predict and prevent T1D.Peer reviewe

Estrogenic regulation of skeletal muscle proteome : a study of premenopausal women and postmenopausal MZ cotwins discordant for hormonal therapy

Female middle age is characterized by a decline in skeletal muscle mass and performance, predisposing women to sarcopenia, functional limitations, and metabolic dysfunction as they age. Menopausal loss of ovarian function leading to low circulating level of 17-estradiol has been suggested as a contributing factor to aging-related muscle deterioration. However, the underlying molecular mechanisms remain largely unknown and thus far androgens have been considered as a major anabolic hormone for skeletal muscle. We utilized muscle samples from 24 pre- and postmenopausal women to establish proteome-wide profiles, associated with the difference in age (30-34 years old vs. 54-62 years old), menopausal status (premenopausal vs. postmenopausal), and use of hormone replacement therapy (HRT; user vs. nonuser). None of the premenopausal women used hormonal medication while the postmenopausal women were monozygotic (MZ) cotwin pairs of whom the other sister was current HRT user or the other had never used HRT. Label-free proteomic analyses resulted in the quantification of 797 muscle proteins of which 145 proteins were for the first time associated with female aging using proteomics. Furthermore, we identified 17-estradiol as a potential upstream regulator of the observed differences in muscle energy pathways. These findings pinpoint the underlying molecular mechanisms of the metabolic dysfunction accruing upon menopause, thus having implications for understanding the complex functional interactions between female reproductive hormones and health.Peer reviewe

Autoantibodies to N-Terminally Truncated GAD65(96-585): HLA Associations and Predictive Value for Type 1 Diabetes

Objective To evaluate the role of autoantibodies to N-terminally truncated glutamic acid decarboxylase GAD65(96-585) (t-GADA) as a marker for type 1 diabetes (T1D) and to assess the potential HLA-associations with such autoantibodies.DesignIn this cross-sectional study combining data from the Finnish Pediatric Diabetes Register, the Type 1 Diabetes Prediction and Prevention (DIPP) Study, the DIABIMMUNE Study, and the Early Dietary Intervention and Later Signs of Beta-Cell Autoimmunity (EDIA) Study, venous blood samples from 760 individuals (53.7% males) were analyzed for t-GADA, autoantibodies to full-length GAD65 (f-GADA), and islet cell antibodies. Epitope-specific GAD autoantibodies were analyzed from 189 study participants.ResultsT1D had been diagnosed in 174 (23%) participants. Altogether 631 (83%) individuals tested positive for f-GADA and 451 (59%) for t-GADA at a median age of 9.0 years (range 0.2-61.5). t-GADA demonstrated higher specificity (46%) and positive predictive value (30%) for T1D than positivity for f-GADA alone (15% and 21%, respectively). Among participants positive for f-GADA, those who tested positive for t-GADA carried more frequently HLA genotypes conferring increased risk for T1D than those who tested negative for t-GADA (77 vs. 53%; PConclusionsAutoantibodies to N-terminally truncated GAD improve the screening for T1D compared to f-GADA and may facilitate the selection of participants for clinical trials. HLA class II-mediated antigen presentation of GAD(96-585)-derived or structurally similar peptides might comprise an important pathomechanism in T1D.</p

Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake

Naturally competent bacteria acquire DNA from their surroundings to survive in nutrient-poor environments and incorporate DNA into their genomes as new genes for improved survival. The secretin HofQ from the oral pathogen Aggregatibacter actinomycetemcomitans has been associated with DNA uptake. Cytokine sequestering is a potential virulence mechanism in various bacteria and may modulate both host defense and bacterial physiology. The objective of this study was to elucidate a possible connection between natural competence and cytokine uptake in A. actinomycetemcomitans. The extramembranous domain of HofQ (emHofQ) was shown to interact with various cytokines, of which IL-8 exhibited the strongest interaction. The dissociation constant between emHofQ and IL-8 was 43 nM in static settings and 2.4 μM in dynamic settings. The moderate binding affinity is consistent with the hypothesis that emHofQ recognizes cytokines before transporting them into the cells. The interaction site was identified via crosslinking and mutational analysis. By structural comparison, relateda type I KH domain with a similar interaction site was detected in the Neisseria meningitidis secretin PilQ, which has been shown to participate in IL-8 uptake. Deletion of hofQ from the A. actinomycetemcomitans genome decreased the overall biofilm formation of this organism, abolished the response to cytokines, i.e., decreased eDNA levels in the presence of cytokines, and increased the susceptibility of the biofilm to tested β-lactams. Moreover, we showed that recombinant IL-8 interacted with DNA. These results can be used in further studies on the specific role of cytokine uptake in bacterial virulence without interfering with natural-competence-related DNA uptake

Characterization of the micro-environment of the testis that shapes the phenotype and function of testicular macrophages

Tissue-specific macrophages are important for the activation of innate immune responses and general organ homeostasis. Testicular macrophages (TM) reside in the testicular interstitial space and comprise the largest leukocyte population in the testis and are assumed to play a role in maintaining testicular immune privilege. Numerous studies have indicated that the interstitial fluid (IF) surrounding the TM has immunosuppressive properties, which may influence the TM phenotype. However, the identity of the immunosuppressive molecules present in the IF is poorly characterized. In this thesis it is shown that in the rat, IF shifts the M1 phenotype of granulocyte macrophage-colony stimulating factor induced bone marrow derived macrophages towards the M2 phenotype. M2 macrophages polarized by IF mimic the properties of TM such as increased expression of CD163, high secretion of IL-10 and low secretion of TNF-alpha. In addition, IF-polarized macrophages display immunoregulatory functions by inducing the expansion of immunosuppressive regulatory T cells. This thesis provides evidence that PGE2, PGI2, testosterone and corticosterone are important immunoregulatory molecules in the IF, playing a relevant role in determining the phenotype of TM. Except corticosterone, all of these factors are able to inhibit the NF-kB signaling pathway to suppress the production of pro-inflammatory cytokines and thus maintain an immunosuppressive microenvironment of the testis. Corticosterone was found to be the principal immunosuppressive molecule in the IF. Its receptor, the glucocorticoid receptor, was found to be present in TM immunohistochemically. In addition, TM locally produce small amounts of corticosterone, which suppress the expression of inflammatory genes and render TM refractory to inflammatory stimuli. Taken together, these results suggest that testicular corticosterone shapes the immunosuppressive function and phenotype of TM. This steroid hormone may therefore play also an important role in the establishment and maintenance of the immune privilege of the testis.Gewebsspezifische Makrophagen haben eine wichtige Funktion bei der Aktivierung angeborener Immunantworten und der Organhomeostase. Testikuläre Makrophagen (TM) befinden sich im Interstitium des Hodens und stellen die größte Leukozytenpopulation in der männlichen Gonade dar. Es wird angenommen, dass sie eine wichtige Funktion in der Aufrechterhaltung des Immunprivilegs des Hodens ausüben. Studien haben gezeigt, dass die interstitielle Flüssigkeit (IF), wleche die TM umgibt, immunsuppressive Eigenschaften aufweist, die den Phänotyp der TM beeinflussen könnten. Allerdings konnten immunsuppressive Moleküle in der IF bislang kaum charakterisiert werden. In der vorliegenden Arbeit wird für die Ratte als Modell gezeigt, dass die IF den durch Granulozyten- Makrophagen-Kolonie-stimulierenden Faktor (GM-CSF) induzierten M1 Phänotyp von Makrophagen, die aus dem Knochenmark isoliert wurden, in Richtung des M2 Phänotyps verschieben kann. IF-polarisierte M2-Makrophagen zeigen damit charakteristische Eigenschaften von TM, wie z. Bsp. erhöhte Expression von CD163, hohe Level von sezerniertem IL-10 bei geringer TNF-alpha Sekretion. Darüber hinaus zeigen IF-polarisierte Makrophagen immunoregulatorische Funktionen, indem sie die Expansion von immunsuppressiven regulatorischen T-Zellen induzieren. In dieser Studie werden erstmals auch Ergebnisse vorgestellt, die zeigen, dass PGE2, PGI2, Testosteron und Corticosteron wichtige immunregulatorische Moleküle in der IF darstellen und eine wesentliche Rolle bei der Bestimmung des TM-Phänotyps spielen. Mit Ausnahme von Corticosteron sind die genannten Faktoren in der Lage, den NF-kB-Signalweg zu hemmen, und damit die Produktion von entzündungshemmenden Zytokinen zu unterdrücken. Bei Corticosteron war der NFkB Signalweg bei der Immunsuppression nicht blockiert. Corticosteron konnte als wichtigster immunsuppressiver Faktor in der IF identifiziert werden. Dessen Rezeptor, der Glucocorticoidrezeptor, konnte in TM mittels Immunhistochemie gefunden werden. TM produzieren lokal moderate Mengen an Corticosteron, die die Expression inflammatorischer Gene unterdrücken und TM unempfindlich gegenüber entzündlichen Stimuli machen können. Zusammengenommen zeigen diese Ergebnisse, dass testikuläres Corticosteron maßgeblich für die immunsuppressive Funktion und den spezifischen Phänotyp der TM verantwortlich ist. Damit könnte das Steroidhormon auch eine wichtige Rolle bei der Etablierung und Aufrechterhaltung des Immunprivilegs im Hoden spielen

Power training and postmenopausal hormone therapy affect transcriptional control of specific co-regulated gene clusters in skeletal muscle

At the moment, there is no clear molecular explanation for the steeper decline in muscle performance after menopause or the mechanisms of counteractive treatments. The goal of this genome-wide study was to identify the genes and gene clusters through which power training (PT) comprising jumping activities or estrogen containing hormone replacement therapy (HRT) may affect skeletal muscle properties after menopause. We used musculus vastus lateralis samples from early stage postmenopausal (50–57 years old) women participating in a yearlong randomized double-blind placebo-controlled trial with PT and HRT interventions. Using microarray platform with over 24,000 probes, we identified 665 differentially expressed genes. The hierarchical clustering method was used to assort the genes. Additionally, enrichment analysis of gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was carried out to clarify whether assorted gene clusters are enriched with particular functional categories. The analysis revealed transcriptional regulation of 49 GO/KEGG categories. PT upregulated transcription in “response to contraction”—category revealing novel candidate genes for contraction-related regulation of muscle function while HRT upregulated gene expression related to functionality of mitochondria. Moreover, several functional categories tightly related to muscle energy metabolism, development, and function were affected regardless of the treatment. Our results emphasize that during the early stages of the postmenopause, muscle properties are under transcriptional modulation, which both PT and HRT partially counteract leading to preservation of muscle power and potentially reducing the risk for aging-related muscle weakness. More specifically, PT and HRT may function through improving energy metabolism, response to contraction as well as by preserving functionality of the mitochondria