bluuebunny/crossref_metadata_2025 huggingface dataset:
Classifications of Atmospheric Circulation Patterns
Authors: Huth Radan , Beck Christoph , Philipp Andreas , Demuzere Matthias , Ustrnul Zbigniew , Cahynová Monika , Kyselý Jan , Tveito Ole Einar
Published: August 2009

We review recent advances in classifications of circulation patterns as a specific research area within synoptic climatology. The review starts with a general description of goals of classification and the historical development in the field. We put circulation classifications into a broader context within climatology and systematize the varied methodologies and approaches. We characterize three basic groups of classifications: subjective (also called manual), mixed (hybrid), and objective (computer‐assisted, automated). The roles of cluster analysis and principal component analysis in the classification process are clarified. Several recent methodological developments in circulation classifications are identified and briefly described: the introduction of nonlinear methods, objectivization of subjective catalogs, efforts to optimize classifications, the need for intercomparisons of classifications, and the progress toward an optimum, if possible unified, classification method. Among the recent tendencies in the applications of circulation classifications, we mention a more extensive use in climate studies, both of past, present, and future climates, innovative applications in the ensemble forecasting, increasing variety of synoptic–climatological investigations, and steps above from the troposphere. After introducing the international activity within the field of circulation classifications, the COST733 Action, we briefly describe outputs of the inventory of classifications in Europe, which was carried out within the Action. Approaches to the evaluation of classifications and their mutual comparisons are also reviewed. A considerable part of the review is devoted to three examples of applications of circulation classifications: in historical climatology, in analyses of recent climate variations, and in analyses of outputs from global climate models.
Eradication of Helicobactor pylori May Trigger Onset of Type 1 Diabetes
Authors: Tada Ai , Komiya Naoko , Shimada Akira , Katsuki Takeshi , Oikawa Yoichi , Itoh Hiroshi
Published: August 2009

In patients with idiopathic thrombocytopenic purpura (ITP), which is considered an autoimmune disease, eradication of Helicobacter pylori cures a significant number of patients. However, here we report an adult patient who developed type 1 diabetes (T1D), which is also considered an autoimmune disease, after eradication of Helicobacter pylori. Although further investigation is needed to understand the pathophysiology of T1D, we would like to emphasize that clinicians should consider the risk of its development after eradication of Helicobactor pylori.
Prevalence of Organ‐Specific Autoantibodies in Childhood‐ and Adult‐Onset Type 1 Diabetes
Authors: Allen S. , Huber J. , Devendra D.
Published: August 2009

There are no studies that compare the prevalence of organ‐specific autoantibodies (OSAs) between adult (≥ 16 years) and childhood‐onset type 1 diabetes (T1D). We evaluated the prevalence of the following OSAs: thyroid peroxidase, thyroid receptor, parietal cell, intrinsic factor, tissue transglutaminase, adrenal cortex, mitochondrial, smooth muscle, liver kidney microsomal, and ovarian autoantibodies. Three hundred twenty‐seven (327) adults were screened for one or more of these OSAs. The prevalence of all the OSAs studied was similar in both groups. The most prevalent OSA observed was tissue transglutaminase (childhood‐onset disease = 14.3%; adult‐onset disease = 13.6%). This study did not demonstrate a distinct difference in the prevalence of OSAs between adult‐ and childhood‐onset T1D patients.
Intravenous Administration of Proinsulin 1 or 2–Expressing Fiber‐Mutant Recombinant Adenovirus Vector Protects against the Development of Diabetes in NOD Mice
Authors: Yamada Katsumi , Moriyama Hiroaki , Okumachi Yasuyo , Arai Takashi , Kameno Mami , Kishi Minoru , Yasuda Hisafumi , Hara Kenta , Yokono Koichi , Nagata Masao
Published: August 2009

Insulin has been reported as a major autoantigen in both human and murine type 1 diabetes (T1D). Insulin1‐knockout NOD mice with only insulin2 are protected against the development of autoimmune diabetes, suggesting that insulin1 has strong immunogenicity and insulin2 has weak immunogenicity or a possible protective role in the pathogenesis of type 1 diabetes. In this study, we have developed fiber‐mutant adenovirus vectors that express murine proinsulin1 or proinsulin2 (named Ad.Pins1‐RGD/Ad.Pins2‐RGD) and administered those virus vectors to the NOD mouse to evaluate modulation of autoimmune responses. The intravenous administration of either Ad.Pins1‐RGD or Ad.Pins2‐RGD at 3 and 5 weeks of age strongly suppressed the development of overt diabetes, accompanied by a significant reduction of insulin autoantibody (IAA), and suppression of disease was similar between administration of Ad.Pins1‐RGD and that of Ad.Pins2‐RGD. Our study suggests that systemic administration of fiber‐mutant adenovirus vectors, which induce transient expression of proinsulin, may be applicable to a gene therapy inducing tolerance to insulin.
Measurement of CD8+ T Cell Responses in Human Type 1 Diabetes
Authors: Martinuzzi Emanuela , Lemonnier François A. , Boitard Christian , Mallone Roberto
Published: August 2009

Type 1 diabetes (T1D) is a T cell–mediated autoimmune disease targeting pancreatic β‐cells. Despite this textbook definition, it is quite striking that neither the diagnosis nor the therapy nor the follow‐up of T1D “belong” to immunologists, but rather to endocrinologists whose only option is to limit the consequences of the disease. Immune therapies would seem better suited to correct the causes of T1D, but critical laboratory tools are missing for early diagnosis, prognostic stratification, and therapeutic follow‐up. The immune markers routinely available are limited to autoantibodies, which have some intrinsic limitations. Because T cells are central pathogenic actors of T1D, the quest for their measurement appeared to offer a path towards new autoimmune markers. Given the strong association between T1D susceptibility and the HLA class II locus, investigators have long been focused on CD4+ T cells. However, data gathered in the NOD mouse and the examination of human insulitis point to a critical role of CD8+ T cells in the pathogenesis of T1D. These observations have revived interest in trying to measure CD8+ T cell responses in human T1D. Achievement of this goal mainly depends on two factors. First, the relevant epitopes need to be identified. Second, appropriate readouts and measurement techniques need to be selected. This review summarizes recent advances on both of these battlefronts, and discusses the potential clinical applications of T cell assays.
Time Course of Islet Loss after Intraportal Transplantation
Authors: Pawelec Krystian , Juszczak Maciej Tadeusz , Kumar Anila , Powis Steve Hugh , Press Martin
Published: August 2009

A high percentage of islets fail to survive intraportal transplantation, but the absolute amount and time course of this loss are uncertain. We have devised a technique to directly quantitate the number of surviving islets using simultaneous selective transplantation of islets and inert beads into the posterior lobes of the liver. Islet:bead ratio did not change significantly within the first 2 hours, but fell progressively thereafter, giving calculated islet survival rates of 89, 43, and 66% at days 1, 7 and 28, respectively. This technique provides a baseline from which to develop strategies to improve the survival of intraportally transplanted islets.
Diverse FOXP3 Expression in Children with Type 1 Diabetes and Celiac Disease
Authors: Kivling Anna , Nilsson Lennart , Fälth‐Magnusson Karin , Söllvander Sofia , Johanson Calle , Faresjö Maria
Published: August 2009

Imbalance between different types of T lymphocytes, such as T helper (Th) and regulatory T cells (Tregs), has been reported to play a part in the pathogenesis behind such autoimmune diseases as type 1 diabetes (T1D) and celiac disease (CD). Defects in Tregs are proposed to at least partly explain the imbalance of Th cells found in children with immunologic diseases. Peripheral blood mononuclear cells from 24 children with T1D and/or CD, and reference children (that is, those without any of these diseases) were stimulated with disease‐associated antigens (insulin, gluten, transglutaminase [tTG]), and phytohemagglutinin (PHA). The mRNA expression of the Treg‐associated marker FOXP3 was analyzed with multiplex real‐time RT‐PCR. Children with T1D showed both a low spontaneous (P < 0.05) and PHA‐induced (P < 0.01) expression of FOXP3 mRNA compared to children with CD. Children with T1D also had a low PHA‐induced FOXP3 mRNA expression compared to the group of children diagnosed with both T1D and CD (P < 0.05). Spontaneous (P < 0.05) and PHA‐induced (P < 0.05) FOXP3 mRNA expression was high in children with CD compared to reference children. In contrast, stimulation with insulin tended to induce high FOXP3 mRNA expression in T1D children compared to reference children (P= 0.057). In conclusion, children with only T1D generally showed a lower FOXP3 mRNA expression than did children with CD, or with T1D in combination with CD, which suggests impaired regulation of the immune system in children with T1D.
Congenic Mapping of the MHC‐Linked Susceptibility to Type 1 Diabetes in the NOD Mouse
Authors: Hiromine Yoshihisa , Fujisawa Tomomi , Noso Shinsuke , Babaya Naru , Kawabata Yumiko , Ikegami Hiroshi
Published: August 2009

Major histocompatibility complex (MHC)‐linked susceptibility to type 1 diabetes consists of multiple components. Previous studies with NOD mice congenic for the MHC from a sister strain, the CTS mouse, mapped Idd16, a second component of MHC‐linked susceptibility to type 1 diabetes, in the region adjacent to, but distinct from Idd1 in the class II region. In this study, three lines of subcongenic strains were established from the original congenic strain. Phenotypic analysis of the strains indicated that MHC‐linked susceptibility to type 1 diabetes consists of at least three components: Idd1, Idd16.2 adjacent to Idd1, and Idd16.1 telomeric to Idd16.2.
The Distribution of Adiponectin Receptors on Human Peripheral Blood Mononuclear Cells
Authors: Pang Terence T.L. , Narendran Parth
Published: August 2009

Adiponectin, an adipocytokine with anti‐inflammatory and insulin‐sensitizing properties, may provide a mechanism by which insulin resistance accelerates autoimmunity in type 1 diabetes (T1D). Its actions are mediated by two receptors, adiponectin receptors 1 (ADIPOR1) and 2 (ADIPOR2). In this study, we measured their distribution on human peripheral mononuclear cells by flow cytometry. ADIPOR1 is present approximately on 1% of T cells, 93% of monocytes, 47% of B cells, and 21% of NK cells (P < 0.01 for difference between subsets). The distribution of ADIPOR2 was found to be similar (r= 0.992, P < 0.01), and staining could be blocked in an antigen‐specific manner. We were also able to confirm our finding at an RNA level by PCR using sequence‐specific primers. Our data are consistent with an immunoregulatory role for adiponectin in T1D.
Closing the Circle between the Bedside and the Bench
Authors: Bortell Rita , Pino Steven C. , Greiner Dale L. , Zipris Danny , Rossini Aldo A.
Published: August 2009

Animal models provide many strategies to unravel the complex interplay of genetic, immunologic, and environmental factors involved in the pathogenesis of type 1A (autoimmune) diabetes. Diabetes can be studied at multiple levels, and new technological advancements provide insights into the functioning of organelle and cellular structures. The role of innate immunity in the response to environmental pathogens has provided possible biochemical and molecular mechanisms which can explain certain clinical events in diabetes. These investigations may uncover new therapies and strategies to prevent type 1A diabetes.
MicroRNA Genes
Authors: Zhou Li , He Hongzhi , Mi Jenny X. , Li Changgui , Lee Byung , Mi Qing‐Sheng
Published: August 2009

Human type 1 diabetes (T1D) is a chronic autoimmune disorder with complex genetic inheritance. To date, more than 19 insulin‐dependent diabetes mellitus (IDDM) susceptibility loci have been mapped to specific chromosome regions in the human. MicroRNAs (miRNAs) are a recently discovered class of evolutionarily conserved small noncoding RNAs that negatively regulate the expression of protein‐coding genes without affecting mRNA levels. There are a growing number of reports that miRNAs link to the regulation of different biological pathways associated with human diseases. However, the potential role of miRNAs in human T1D is still unknown. To investigate the possible involvements of miRNAs in human T1D on a genome‐wide basis, we have mapped 530 miRNAs and compared their locations to the current IDDM loci. We found that at least 27 miRNAs are located in 9 human IDDM loci. More interestingly, some of them potentially target autoimmune‐ and β‐cell‐related genes. Our data represent a genome‐wide search for a potential correlation between the genomic position of miRNAs and specific IDDM loci, indicating that miRNAs may be susceptibilty candidates or biomarkers for human T1D.
Humoral Immune Response to Islet Autoantigens in Japanese Patients with Type 1 Diabetes
Authors: Kawasaki Eiji , Kuriya Genpei , Satoh Tsuyoshi , Fujishima Keiichiro , Moriuchi Akie , Fukushima Keiko , Ozaki Masako , Abiru Norio , Yamasaki Hironori , Eguchi Katsumi
Published: August 2009

In this study, we evaluated autoantibodies to IA‐2 (IA‐2As), glutamic acid decarboxylase 65 (GADAs), and islet cell antibodies (ICAs) in 233 patients with type 1 diabetes (M:F = 90:143, mean duration 4.0 ± 6.7 yr) as a cross‐sectional study. Of 233 patients with type 1 diabetes, IA‐2A was detected in 58% of patients with duration within 2 weeks, 61% of patients with duration <1 yr, 41% of patients with diabetes for 1–3 yr, 29% for 4–9 yr, and 21% for ≥10 yr. These prevalences were similar to those of ICA, while the prevalence of GADA was not influenced by duration of diabetes with positivity of 63–74%. Thus, as the duration of diabetes became longer, the frequency of GADA+/IA‐2A− patients increased and the frequency of GADA+/IA‐2A+ patients decreased. However, the frequency of GADA−/IA‐2A+ patients was not influenced by duration of diabetes. The prevalence of IA‐2A was significantly higher in abrupt‐onset group (68%, n= 79) compared to the slowly progressive group (23%, n= 22) in new‐onset patients (P= 0.0001). However, there was no difference in the IA‐2A frequency between these two groups (abrupt‐onset 26%, n= 53 vs. slowly progressive 24%, n= 21) in patients with long‐standing disease, suggesting that IA‐2A positivity might persist in patients with slowly progressive type 1 diabetes. These results emphasize the heterogeneity of humoral autoimmunity to protein tyrosine phosphatase–like molecules, but not to GAD, in patients with type 1 diabetes.
Identification of a Major Humoral Epitope in Slc30A8 (ZnT8)
Authors: Wenzlau Janet M. , Moua Ong , Liu Yu , Eisenbarth George S. , Hutton John C. , Davidson Howard W.
Published: August 2009

The human zinc transporter Slc30A8 (ZnT8) is a major target of humoral autoimmunity in human type 1A diabetes. However, despite extensive conservation, the majority of human autoimmune sera fail to recognize the murine ortholog. Moreover, Slc30A8 appears not to be a significant target of humoral autoimmunity in the NOD mouse. We therefore “humanized” the murine protein by site‐directed mutagenesis. Only conversion of Q324 to arginine (equivalent to R325 in the human protein) partially restored reactivity to a pool of sera selected for high titers to the human probe. Additionally, the reciprocal mutation (human R325 to Q) abolished reactivity for 38/103 (36.9%) of ZnT8+ sera. We conclude that the C‐terminal domain of human ZnT8 contains at least two discrete epitopes, one of which is critically dependent upon the arginine residue at position 325.
TLR Agonists Abrogate Co‐stimulation Blockade‐Induced Mixed Chimerism and Transplantation Tolerance
Authors: Miller David M. , Thornley Thomas , Pearson Todd , Yamazaki Masahiro , Brehm Michael A. , Rossini Aldo A. , Greiner Dale L.
Published: August 2009

We investigated the mechanisms by which Toll‐like receptor (TLR) agonists affect the induction of mixed chimerism and skin allograft survival in mice treated with co‐stimulation blockade (CB). We report that TLR agonists prevent the generation of mixed chimerism by breaking tolerance in the alloreactive CD4+ and CD8+ T cell compartments, and that type I interferon (IFN) is important in this process. Understanding how environmental perturbations affect CB‐induced transplantation tolerance may lead to more effective regimens that can be used as an approach for the treatment of type I diabetes, for which the transplantation of pancreatic islets is a promising therapy.
Carboxypeptidase‐H Autoantibodies Differentiate a More Latent Subset of Autoimmune Diabetes from Phenotypic Type 2 Diabetes among Chinese Adults
Authors: Yang L. , Zhou Z.G. , Tan S.Z. , Huang G. , Jin P. , Yan X. , Li X. , Peng H. , Hagopian W.
Published: August 2009

This study aimed to investigate whether carboxypeptidase‐H antibody (CPH‐Ab) can help identify latent autoimmune diabetes in adults (LADA). Phenotypic type 2 diabetic (T2D) patients (n= 1296) were studied for CPH Abs and autoantibodies to glutamic acid decarboxylase (GAD‐Abs). CPH‐Ab+ T2D patients also underwent testing for insulinoma protein tyrosine phosphatase (IA‐2A). Clinical features were compared among CPH‐Ab+, GAD‐Ab+, and Ab− T2D patients. Some of the antibody‐positive patients were followed up for 3 years to assess beta cell function. The prevalence of CPH‐Abs in T2D patients was 4.8%, significantly higher than that in controls. Double positivity was rare between CPH‐Abs and GAD‐Abs or IA‐2A. Compared to patients with Ab− T2D, those with CPH‐Ab+ T2D had lower BMI, lower fasting C‐peptide (FCP) levels, and more frequent ketosis, while not as much as did those with GAD‐Ab+ T2D. The mild beta cell dysfunction in patients with CPH‐Ab+ T2D was associated with their longer duration of diabetes. No marked change of C‐peptide in the CPH‐Ab+ group was found during follow‐up. These findings demonstrated that CPH‐Abs may allow discrimination of a more latent subset of adult‐onset autoimmune diabetes (LADA) whose features are intermediate between those with classic GAD‐Ab+ LADA and patients with Ab− T2D.
The Role of Toll‐Like Receptors 3 and 9 in the Development of Autoimmune Diabetes in NOD Mice
Authors: Wong F. Susan , Hu Changyun , Zhang Li , Du Wei , Alexopoulou Lena , Flavell Richard A. , Wen Li
Published: August 2009

Innate immunity is mediated, at least in part, through a number of receptors known as Toll‐like receptors (TLRs), which are activated by different microbial stimuli. Adaptive immunity, including autoimmunity, follows the innate response in a more specific manner. To investigate the roles of TLR3 and TLR9 in the development of type 1 diabetes, we generated NOD mice that are deficient in TLR3 and 9, respectively. There was no obvious difference in the incidence of spontaneous diabetes between TLR3‐deficient mice and TLR3 heterozygous mice. However, TLR9‐deficient mice were markedly protected from the disease compared to TLR9 heterozygous mice. Our results suggest that different TLRs play a varying role in autoimmune diseases.
Role of Increased ROS Dissipation in Prevention of T1D
Authors: Chen Jing , Gusdon Aaron M. , Thayer Terri C. , Mathews Clayton E.
Published: August 2009

Protection of pancreatic β cells is an approach to prevent autoimmune type 1 diabetes (T1D) and to protect transplanted islets. Reactive oxygen species (ROS) are important mediators of β cell death during the development of T1D. We have examined the role of elevated ROS dissipation in the prevention of T1D using the ALR mouse strain. The selection of ALR, for resistance against alloxan‐induced free radical–mediated diabetes, led to a strain of mice with an elevated systemic as well as pancreatic ROS dissipation. Independent genetic mapping studies have identified ALR‐derived diabetes protective loci. Conplastic and congenic mouse as well as cell line studies have confirmed the genetic mapping and demonstrated that the elevated ROS dissipation protects ALR β cells from autoimmune destruction. Our data support the hypothesis that elevated ROS dissipation protects β cells against autoimmune destruction and prevents T1D development.
Immunoregulatory Pathways Controlling Progression of Autoimmunity in NOD Mice
Authors: You Sylvaine , Alyanakian Marie‐Alexandra , Segovia Berta , Damotte Diane , Bluestone Jeffrey , Bach Jean‐François , Chatenoud Lucienne
Published: August 2009

The activation, expansion, and survival of regulatory T cells (Tregs) as well as the expression of their suppressive capacities result from distinct signaling pathways involving various membrane receptors and cytokines. Multiple studies have shown that thymus‐derived naturally occurring Tregs constitutively express the forkhead/winged helix transcription factor FoxP3 in addition to high levels of CD25, the negative co‐stimulatory molecule CTLA‐4, and the glucocorticoid‐induced TNF receptor‐related protein GITR. At variance, adaptive or induced Tregs acquire these phenotypic markers as they differentiate in the periphery, following adequate stimulation in the appropriate environment, together with their capacity to produce immunomodulatory cytokines (mainly, IL‐4, IL‐10 and TGF‐β) and to display regulatory capacities. However, none of these molecules but FoxP3 are restricted to Tregs since they may also be expressed and upregulated on activated effector T cells. This explains why different hypotheses were proposed to interpret interesting reports showing that in vivo abrogation of CTLA‐4 signaling using neutralizing CTLA‐4 antibodies triggers different autoimmune or immune‐mediated manifestations. Thus, an effect on pathogenic T cell effectors and/or Tregs has been proposed. Here we present and discuss recent results we obtained in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes, arguing for a key role of CTLA‐4 in the functional activity of Tregs. Moreover, data are presented that simultaneous blockade of CTLA4 and TGF‐β further impairs immunoregulatory circuits that control disease progression.
The Sentinel Role of CD8 T Cells in Regulating CD4 T Cell Responses to Proinsulin in β‐Islet Cell Autoimmunity
Authors: Batarelo Vedran , Durinovic‐Belló Ivana
Published: August 2009

Circulating CD4 T cells specific for peptide epitopes of proinsulin and other autoantigens are markers of autoimmune β cell destruction in type 1 diabetes, while the role of CD8 T cells is still largely unknown. Here we show that CD8 T cells of a diabetic patient—after rechallange with proinsulin peptides—secrete IFNγ and granzyme B, markers of their effector capacity. On the other hand, CD8 T cells of the same patient in a “cross‐talk” with proinsulin‐specific CD4 T cells suppress their proliferation. If confirmed in larger numbers of subjects with β‐islet cell autoimmunity, these results may help us to understand the role of CD8 cells in disease progression and extend our knowledge of disease pathogenesis.
Autologous Hematopoietic Stem Cell Transplantation for Type 1 Diabetes
Authors: Voltarelli Júlio C. , Couri Carlos E.B. , Stracieri Ana B.P.L. , Oliveira Maria C. , Moraes Daniela A. , Pieroni Fabiano , Barros George M.N. , Madeira Maria Isabel A. , Malmegrim Kelen C.R. , Foss‐Freitas Maria C. , Simões Belinda P. , Foss Milton C. , Squiers Elizabeth , Burt Richard K.
Published: August 2009

In this review, we present (1) the scientific basis for the use of high‐dose immunosuppression followed by autologous peripheral blood hematopoietic stem cell transplantation for newly diagnosed type 1 diabetes (T1D); (2) an update of the clinical and laboratory outcome of 20 patients transplanted at the University Hospital of the Ribeirão Preto Medical School, University of São Paulo, Brazil, and followed up to January/2008, including 4 relapses among 19 patients without previous ketoacidosis; (3) a commentary on criticisms to our article that appeared in four articles from the scientific literature; and (4) a discussion of the prospectives for cellular therapy for T1D.
Insulitis in Human Type 1 Diabetes
Authors: Hanafusa Toshiaki , Imagawa Akihisa
Published: August 2009

Pancreatic tissues were analyzed immunohistologically in patients with autoimmune and fulminant type 1 diabetes (T1D) and control subjects. Both β and alpha cells were decreased in fulminant T1D, but only β cells were significantly decreased in autoimmune T1D. Insulitis was seen in both subtypes of T1D, but it remained longer in autoimmune than in fulminant T1D. Lymphocytic infiltration to the exocrine pancreatic tissue was observed only in fulminant T1D, whereas immunologically abnormal findings, such as increased expression of MHC class I molecule and Fas antigen in islet cells and Fas‐ligand expression in infiltrating lymphocytes, were detected only in autoimmune T1D. From these findings, together with clinical features, it could be concluded that in autoimmune T1D, β cells are assumed to be destroyed through a long‐standing autoimmune process, whereas in fulminant T1D, β cells seem to be destroyed very rapidly, probably by a destructive process triggered by viral infection.
Type 1 Diabetes TrialNet–An International Collaborative Clinical Trials Network
Authors: Skyler Jay S. , Greenbaum Carla J. , Lachin John M. , Leschek Ellen , Rafkin‐Mervis Lisa , Savage Peter , Spain Lisa ,   the Type 1 Diabetes TrialNet Study Group
Published: August 2009

Type 1 Diabetes TrialNet is an international consortium of clinical research centers aimed at the prevention or delay of type 1 diabetes (T1D). The fundamental goal of TrialNet is to counter the T1D disease process by immune modulation and/or enhancement of β cell proliferation and regeneration. To achieve this goal, TrialNet researchers are working to better understand the natural history of the disease, to identify persons at risk, and to clinically evaluate novel therapies that balance potential risks and benefits. The particular focus is on studies of preventive measures. In addition, TrialNet evaluates therapies in individuals with newly diagnosed T1D with preserved β cell function to help determine the risk/benefit profile and gain an initial assessment of potential efficacy in preservation of β cell function, so that promising agents can be studied in prevention trials. In addition, TrialNet evaluates methodologies that enhance the conduct of its clinical trials, which includes tests of outcome assessment methodology, the evaluation of surrogate markers, and mechanistic studies laying the foundation for future clinical trials.
Variations in Incidence of Type 1 Diabetes in Different Municipalities of Stockholm
Authors: Gopinath Sreevalsam , Örtqvist Eva , Norgren Svante , Green Anders , Sanjeevi Carani B.
Published: August 2009

This article reports a test of the hypothesis that municipalities within the County of Stockholm have varying incidence rates of type 1 diabetes (T1D), suggesting a strong etiologic environmental component to the disease. The study group included T1D patients in the age group from birth to 18 years who were diagnosed each year from 20 municipalities in Stockholm County during the 1990–2003. Specific incidence rates by age, sex, and socioeconomic characteristics (income level, proportion of taxpayers, proportion of foreigners, population density and green cover) were estimated annually together with age standardization. χ2 analyses were used for the statistical assessment of variability in incidence. During the study period, 733 newly diagnosed T1D patients aged 0–18 years were recorded from the 20 municipalities under study. The overall age‐standardized incidence in these 20 municipalities was 24.38 (22.65–26.21) per 100,000, with 45.35 (32.08–62.29) as highest and 13.41 (9.53–18.35) as lowest estimated incidence. For all socioeconomic variables statistically significant heterogeneity was demonstrated in the standardized incidence rate. High green index was positively associated with the incidence of T1D, as was low population density. For the three remaining socioeconomic variables no clear patterns of associations with incidence of T1D were seen. This study demonstrates a considerable and statistically significant variation between the lowest and highest values in the incidence and prevalence rates for T1D in municipalities of Stockholm County. Such variation seems unlikely to be explained by genetic differences since the population is homogeneous. Our study provides support for the hypothesis that environmental factors have a major influence on the pathogenesis of T1D.
The Environmental Determinants of Diabetes in the Young (TEDDY) Study
Authors:   TEDDY Study Group
Published: August 2009

The etiology of type 1 diabetes (T1D) remains unknown, but a growing body of evidence points to infectious agents and/or components of early childhood diet. The National Institutes of Health has established the TEDDY Study consortium of six clinical centers in the United States and Europe and a data coordinating center to identify environmental factors predisposing to, or protective against, islet autoimmunity and T1D. From 2004–2009, TEDDY will screen more than 360,000 newborns from both the general population and families already affected by T1D to identify an estimated 17,804 children with high‐risk HLA‐DR,DQ genotypes. Of those, 7,801 (788 first‐degree relatives and 7,013 newborns with no family history of T1D) will be enrolled in prospective follow‐up beginning before the age of 4.5 months. As of May 2008, TEDDY has screened more than 250,000 newborns and enrolled nearly 5,000 infants—approximately 70% of the final cohort. Participants are seen every 3 months up to 4 years of age, with subsequent visits every 6 months until the subject is 15 years of age. Blood samples are collected at each visit for detection of candidate infectious agents and nutritional biomarkers; monthly stool samples are collected for infectious agents. These samples are saved in a central repository. Primary endpoints include (1) appearance of one or more islet autoantibodies (to insulin, GAD65 or IA‐2) confirmed at two consecutive visits; (2) development of T1D. By age 15, an estimated 800 children will develop islet autoimmunity and 400 will progress to T1D; 67 and 27 children have already reached these endpoints.
Toll‐Like Receptors and Diabetes
Authors: Wong F. Susan , Wen Li
Published: August 2009

Toll‐like receptors (TLRs) recognize molecular patterns relating to a variety of microbial infections. Stimulation through TLRs leads to activation of antigen‐presenting cells, production of inflammatory cytokines creating inflammation, and production of type 1 interferons (IFNs) that include IFN‐α and ‐β, and exerts direct effects on regulatory cells. These effects can direct the immune response, dealing with the immediate problems of infection and activating more specific responses of the adaptive immune system. However, it has recently been recognized that these receptors may recognize endogenous ligands that include DNA, RNA, and proteins that arise from cellular stress. This may have an effect on autoimmune responses in a number of ways, both activating and inhibitory. The means by which infection or endogenous stimuli through TLRs may influence autoimmunity will be discussed.
Normalization of Circulating Nucleic Acid Results
Authors: Holford N.C. , Ramoutar A. , Butt A.N. , Swaminathan R.
Published: August 2009

Nucleic acids, both DNA and mRNA, have been detected in the circulation and have been demonstrated to be useful in such areas as fetal medicine, oncology, and transplantation. When mRNA is measured in circulating blood, the results are expressed in relation to a reference gene product in order to correct for any differences in extraction, volume of starting material or other differences. Many authors use β‐actin mRNA and express results as a ratio of target mRNA to β‐actin mRNA. We have used a similar approach when studying diabetic retinopathy. Recently, we planned to investigate the expression of thyroid dependent gene expression in acutely ill patients. As a control study, we examined the expression of thyroid hormone–dependent gene expression in subjects with hyperthyroidism and found that the expression of β‐actin mRNA was affected by thyroid hormone status. Blood samples were taken into PAX gene™ tubes from 31 healthy subjects (mean age, 43 ± 16 yrs) and 7 patients with hyperthyroidism (mean age, 43 ± 5 yrs). Diagnosis of hyperthyroidism was confirmed by clinical findings and biochemical results. After extraction of mRNA, cDNA was synthesized using reverse transcription. Quantification of Na/K‐ATPase, T3 receptor, and β‐actin cDNA was carried out by RT‐PCR. Median β‐actin levels were significantly higher in hyperthyroid subjects compared to healthy subjects (18.2 versus 2.30; P < 0.00042). When mRNA for the T3 receptor was expressed in relation to β‐actin, there was a significantly higher __________________ in hyperthyroidism (0.0168 versus 0.218, P < 0.05). However, this was significantly lower when expressed in relation to total RNA (12.2 versus 2.24, P < 0.00015).We conclude that normalizing results to β‐actin may not be appropriate in all circumstances.
Hypertension and Circulating mRNA for 11β‐Hydroxysteroid Dehydrogenase Type II
Authors: Agalou S. , Butt A. N. , Chowienczyk Phil , Swaminathan R.
Published: August 2009

Circulating DNA and mRNA for 11β‐hydoxysteroid dehydrogenase (HSD) type II were measured in patients with hypertension and in healthy subjects. DNA and RNA levels in hypertensive patients and controls were quantified using real‐time RT–PCR. Messenger RNA for 11β‐HSD type II was significantly lower in the hypertensive patients (median: 0.18) (P= 0.032) than in healthy subjects (median: 0.42). Plasma DNA was also significantly lower (P= 0.016) in hypertension. It is suggested that measurement of mRNA for 11β‐HSD type II in hypertension may identify subjects who may be salt‐sensitive.
Circulating 11β‐Hydroxysteroid Dehydrogenase Type 1 mRNA and Cardiovascular Risk Factors
Authors: Al Bakir Maise , Butt Asif N. , Swaminathan R.
Published: August 2009

11β‐Hydroxysteroid dehydrogenase type 1 (11β‐HSD1) has been implicated in the pathogenesis of central obesity and metabolic syndrome. It has been shown that tissue‐specific mRNA is present in blood, raising the possibility that quantifiable levels of 11β‐HSD1 mRNA may also be present in circulation. The aim of this study was to determine whether a relationship exists between circulating 11β‐HSD1 mRNA levels and various cardiovascular risk factors. Whole blood was collected using PAXgene Blood RNA tubes from 73 subjects. Total RNA was extracted, reverse‐transcribed to cDNA, and 11β‐HSD1 mRNA was measured by quantitative real time PCR. The median BMI was 24 (range = 17–36) and the concentration of 11β‐HSD1 mRNA tended to decrease with increasing BMI (r=−0.206; P= 0.08). Multiple linear regression analysis identified BMI as an independent predictor of mRNA levels (β=−0.412, P= 0.002). Other factors included in the analysis are age (β= 0.952, P < 0.001), smoking (β=−0.228, P= 0.027), alcohol intake (β= 0.479, P < 0.001), and duration of menopause (β=−1.602, P < 0.001). The results suggest a net decrease in systemic expression of 11β‐HSD1 with increased smoking, BMI, and duration of menopause. Increased systemic expression is seen with increasing age and alcohol intake.
Retina‐Specific mRNA in the Assessment of Diabetic Retinopathy
Authors: Shalchi Z. , Sandhu H.S. , Butt A.N. , Smith S. , Powrie J. , Swaminathan R.
Published: August 2009

In a previous study we demonstrated the presence and diagnostic usefulness of circulating rhodopsin mRNA in the assessment of diabetic retinopathy (DR). In the present study we investigated three further retina‐specific markers in blood to determine their suitability as markers of DR. The markers were RPE65, retinoschisin, and melanopsin. Whole blood was collected from diabetic patients and healthy controls into PAXgene Blood RNA tubes and RNA was extracted using the PAXgene Blood RNA System. Quantitative real‐time PCR was used to quantify mRNA for RPE65, retinoschisin, and melanopsin. β‐actin mRNA was used for normalization. RPE65, retinoschisin, and β‐actin mRNA were detected in 100% of subjects; melanopsin was not detected in either controls or diabetic patients. Circulating RPE65 mRNA concentration was 63% higher in diabetic patients than in healthy individuals (P= 0.019), whereas retinoschisin showed no change between the two groups. Compared with healthy controls, circulating RPE65 mRNA concentration was higher in diabetics with no retinopathy (30%; P= NS), background DR (93%; P= 0.01), preproliferative DR (20%; P= NS), and proliferative DR (107%; P= 0.004). Compared with diabetics with no retinopathy, levels of RPE65 mRNA were also significantly higher (60%) in the presence of proliferative DR (P= 0.029). In contrast, levels of retinoschisin mRNA were lower in background DR (34%; P= 0.033), preproliferative DR (43%; P= 0.026), and proliferative DR (47%; P= 0.038) compared to that in diabetics without retinopathy. We conclude that not all retina‐specific mRNA species are detectable in circulation (e.g., melanopsin). This may be related to differences in expression levels for the individual markers. Both RPE65 and retinoschisin were detectable and demonstrated contrasting trends in diabetics with and without retinopathy. In combination with rhodopsin, RPE65, and retinoschisin, mRNA may offer a useful tool in developing a blood test for DR.
Circulating DNA in the Blood of Gastric Cancer Patients
Authors: Kolesnikova Elena V. , Tamkovich Svetlana N. , Bryzgunova Olga E. , Shelestyuk Petr I. , Permyakova Valentina I. , Vlassov Valentin V. , Tuzikov Aleksandr S. , Laktionov Pavel P. , Rykova Elena Y.
Published: August 2009

The concentration of cell‐free DNA and promoter methylation status of the MGMT, p15, and hMLH1 genes were analyzed by a fluorescence‐based assay and methylation‐specific PCR (MSP) in the blood of gastric cancer patients (n= 20) and healthy subjects (n= 22). Gastric cancer patients were characterized by an increased concentration of circulating DNA in the plasma; the amount of cell‐surface‐bound DNA was not decreased compared with controls and amounted to 80 ± 15% of the total circulating DNA. MSP analysis of three genes in the cell‐surface‐bound DNA permits the detection of gastric cancer patients with a sensitivity of 75% and a specificity of 54%. Thus, the cell‐surface‐bound DNA is a convenient source of DNA for MSP analysis of cancer‐specific markers. The data on the presence of methylated DNA in plasma combined with the analysis of other cancer‐related changes in DNA can significantly contribute to cancer diagnostics.
Effect of Adjuvant Chemotherapy on Integrity of Free Serum DNA in Patients with Breast Cancer
Authors: Deligezer Ugur , Eralp Yesim , Akisik Elif Z. , Akisik Ebru E. , Saip Pinar , Topuz Erkan , Dalay Nejat
Published: August 2009

It is not known how chemotherapy‐induced cell death influences the size distribution of circulating free DNA (cf‐DNA) in serum or plasma of cancer patients. In the present study, we investigated the integrity of cf‐DNA during adjuvant systemic therapy in patients (n= 41) with invasive breast cancer. Sera taken at the beginning and the end of the adjuvant chemotherapy were comparatively analyzed for the integrity of cf‐DNA. The assay was based on quantification of shorter and longer fragments representing apoptotic or non‐apoptotic DNA from abundant genomic ALU repeats by quantitative real‐time PCR. The ratio of longer to shorter fragments showed the integrity of free serum DNA. During chemotherapy, in half of the patients (51.2%), total DNA levels increased, but decreased in the other half. The distribution of the DNA integrity in the whole patient group after the systemic therapy (median 0.31) did not significantly differ from that at the beginning (median 0.29, P= 0.39). However, in the subgroups, the variation of the DNA integrity was related to the course of the total DNA level. In the subgroup with an increasing DNA level, the median DNA integrity was elevated from 0.27 to 0.39 (P= 0.005), whereas in the group with a decrease it declined from 0.34 to 0.28 (P= 0.044). Our results show that longer fragments released from non‐apoptotic cells are the main contributors to increasing DNA levels during adjuvant systemic therapy. This information might be helpful in evaluating the response of patients to adjuvant systemic therapy.
Quantification of LINE1 in Circulating DNA as a Molecular Biomarker of Breast Cancer
Authors: Sunami Eiji , Vu Anh‐Thu , Nguyen Sandy L. , Giuliano Armando E. , Hoon Dave S. B.
Published: August 2009

Developing a noninvasive test for detecting and monitoring breast cancer progression would help in providing better procedures for the treatment of breast cancer. Increases in the absolute quantity of circulating DNA and DNA integrity have been previously reported in breast cancer patients. LINE1 is one of the most abundant sequences in the human genome, with about 520,000 copies per genome. To assess the combination of circulating DNA quantity and DNA integrity, we developed a long LINE1 (about 300‐bp amplicon size) quantitative method. A quantitative real‐time PCR (qPCR) technique was used to detect long LINE1. Breast cancer patients' sera was assessed preoperatively before primary tumor surgery. LINE1 could be detected in high levels of breast cancer patients' sera and in limited levels in normal females. We demonstrated that long LINE1 quantification of circulating DNA was useful for detecting early‐stage breast cancer, and that copy number correlated with tumor size. This preliminary study demonstrates the potential clinical utility of LINE1 copy numbers in breast cancer patients.
DNA Integrity in Plasma and Serum of Patients with Malignant and Benign Diseases
Authors: Holdenrieder Stefan , Burges Alexander , Reich Oliver , Spelsberg Fritz W. , Stieber Petra
Published: August 2009

This study aimed to test the diagnostic utility of the DNA integrity index for detection of cancer. Matched serum and plasma samples of 42 patients with various forms of cancer and 17 patients with corresponding benign diseases were analyzed. DNA was isolated from 1 mL serum and plasma using the MagNA Pure LC system. Cell‐free DNA was quantified by real‐time PCR using the reference gene of the LightCycler t(14;18) kit. Subsequently, the DNA integrity index was calculated as the ratio in relative abundance of 347‐bp versus 137‐bp PCR products. We found that DNA concentrations were not different in plasma (median: 4.5 ng/mL) and serum (67.1 ng/mL) of patients with benign diseases when compared with values in plasma (5.1 ng/mL) and serum (65.4 ng/mL) of cancer patients. Similarly, the DNA integrity index in plasma (0.38), and serum of patients with benign diseases (0.29) was comparable to values in plasma (0.33) and serum (0.37) of cancer patients. Diagnostic sensitivity of DNA (AUC 0.53) and DNA integrity (AUC 0.45) was poor in plasma, and was increased only slightly by the combination of both (AUC 0.57). In serum, sensitivity of DNA (AUC 0.52) and DNA integrity (AUC 0.64) was higher and was further improved by the combination of both (AUC 0.72) reaching a sensitivity of 30% at 100% specificity. In conclusion, we could not confirm a high diagnostic utility of the DNA integrity index. However, a combination with other markers such as DNA may enhance sensitivity for cancer detection.
Deoxyribonuclease Activity and Circulating DNA Concentration in Blood Plasma of Patients with Prostate Tumors
Authors: Cherepanova Anna V. , Tamkovich Svetlana N. , Bryzgunova Olga E. , Vlassov Valentin V. , Laktionov Pavel P.
Published: August 2009

The DNase activity and circulating DNA (cirDNA) concentration in blood plasma of healthy donors, patients with chronic prostatitis, and patients with prostate tumors were analyzed. The concentration of the cirDNA from plasma was determined by PicoGreen fluorescent assay. DNase activity in blood was measured using the immunoassay based on the cleavage of a hapten‐labeled 974‐bp DNA substrate. The mean cirDNA concentration in the plasma of healthy donors was low (21 ± 4 ng/mL total blood) and was accompanied by high DNase activity (0.17 ± 0.04 U/mL blood). The mean cirDNA concentration was 90 ng/mL blood (10–234 ng/mL) in the patients with nonmalignant prostate tumors and 115 ng/mL blood (13–339 ng/mL) in those with prostate cancer. The mean DNase activity in blood plasma of the patients with prostate tumors was 0.06 U/mL blood (0–0.12 U/mL). The results obtained demonstrate that increased concentrations of cirDNA in blood of the patients with prostate tumors is accompanied by a decreased DNase activity, confirming our previous data that a low DNase activity in blood plasma of cancer patients is one reason for a high cirDNA concentration.
Phenotype and mRNA Expression of Syncytiotrophoblast Microparticles Isolated from Human Placenta
Authors: Kumpel Belinda , King May‐Jean , Sooranna Suren , Jackson Dave , Eastlake Jane , Cheng Rong , Johnson Mark
Published: August 2009

Fetal and neonatal alloimmune thromboctyopenia due to maternal human platelet antigen (HPA)‐1a antibodies affects primigravidas. Immunization must occur early in pregnancy before fetal platelets enter maternal blood via fetomaternal hemorrhage. The HPA‐1a antigen is located on platelet glycoprotein (GP)IIIa (CD61, β3 integrin), which is also present on the placental syncytiotrophoblast (ST) and in direct contact with maternal blood. Since ST debris is shed into maternal blood during pregnancy, this material might be immunogenic in vivo. For experimental purposes, we prepared and characterized ST microparticles (STMPs) in vitro from term placentas. Phenotype analysis by flow cytometry and Western blotting showed that STMP expressed more placental alkaline phosphatase (PLAP) than GPIIIa. Quantitative real‐time PCR demonstrated expression of human placental lactogen (HPL), human chorionic gonadotrophin (HCG), and GPIIIa by STMP, in the order HPL > HCG > GPIIIa. PLAP, HPL, and HCG are trophoblast‐specific proteins. These STMPs may be a useful model for studying the natural ST debris in plasma of pregnant women.
Isolation and Sequencing of Short Cell‐Surface‐Bound DNA
Authors: Mal'shakova Vera S. , Pyshnyi Dmitry V. , Bondar Alexander A. , Vlassov Valentin V. , Laktionov Pavel P.
Published: August 2009

To isolate the DNA fragments bound to cell‐surface proteins with high affinity, peptide–DNA complexes were obtained by mild treatment of human endothelial cells with trypsin. After binding of nucleoprotein complexes to BrCN‐Sepharose and extensive washing, 7–11 nt DNA fragments were eluted with acidic buffer, labeled with 32P, and isolated by PAGE. The fragments were ligated to double‐stranded oligonucleotide adapters, amplified by PCR, and cloned in pBluescript II KS (+) vector. Sequences of 12 clones containing the insert of cell‐surface‐bound 7–11‐mer DNA fragments were obtained.