Factors That Increase Risk of Celiac Disease Autoimmunity After a Gastrointestinal Infection in Early Life.

BACKGROUND & AIMS: Little is known about the pathogenic mechanisms of gluten immunogenicity in patients with celiac disease. We studied temporal associations between infections and the development of celiac disease autoimmunity, and examined effects of HLA alleles, rotavirus vaccination status, and infant feeding. METHODS: We monitored 6327 children in the United States and Europe carrying HLA risk genotypes for celiac disease from 1 to 4 years of age for presence of tissue transglutaminase autoantibodies (the definition of celiac disease autoimmunity), until March 31, 2015. Parental reports of gastrointestinal and respiratory infections were collected every third month from birth. We analyzed time-varying relationships among reported infections, rotavirus vaccination status, time to first introduction of gluten, breastfeeding, and risk of celiac disease autoimmunity using proportional hazard models. RESULTS: We identified 13,881 gastrointestinal infectious episodes (GIE) and 79,816 respiratory infectious episodes. During the follow-up period, 732 of 6327 (11.6%) children developed celiac disease autoimmunity. A GIE increased the risk of celiac disease autoimmunity within the following 3 months by 33% (hazard ratio [HR], 1.33; 95% confidence interval [CI], 1.11-1.59). This risk increased 2-fold among children born in winter and introduced to gluten before age 6 months (HR, 2.08; 95% CI, 1.46-2.98), and increased 10-fold among children without HLA-DQ2 alleles and breastfed for fewer than 4 months (HR, 9.76; 95% CI, 3.87-24.8). Risk of celiac disease autoimmunity was reduced in children vaccinated against rotavirus and introduced to gluten before age 6 months (HR, 0.57; 95% CI, 0.36-0.88). CONCLUSIONS: Gastrointestinal infections increase the risk of celiac disease autoimmunity in children with genetic susceptibility to this autoimmune disorder. The risk is modified by HLA genotype, infant gluten consumption, breastfeeding, and rotavirus vaccination, indicating complex interactions among infections, genetic factors, and diet in the etiology of celiac disease in early childhood.

Steroid-like signalling by interferons: making sense of specific gene activation by cytokines.

Many cytokines, hormones and growth factors use the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) pathway for cell signalling and specific gene activation. In the classical model, ligand is said to interact solely with the receptor extracellular domain, which triggers JAK activation of STATs at the receptor cytoplasmic domain. Activated STATs are then said to carry out nuclear events of specific gene activation. Given the limited number of STATs (seven) and the activation of the same STATs by cytokines with different functions, the mechanism of the specificity of their signalling is not obvious. Focusing on IFNγ (interferon γ), we have shown that ligand, receptor and activated JAKs are involved in nuclear events that are associated with specific gene activation, where the receptor subunit IFNGR1 (IFNγ receptor 1) functions as a transcription/co-transcription factor and the JAKs are involved in key epigenetic events. RTKs (receptor tyrosine kinases) such as EGFR [EGF (epidermal growth factor) receptor] and FGFR [FGF (fibroblast growth factor) receptor] also undergo nuclear translocation in association with their respective ligands. EGFR and FGFR, like IFNGR1, have been shown to function as transcription/co-transcription factors. The RTKs also regulate other kinases that have epigenetic effects. Our IFNγ model, as well as the RTKs EGFR and FGFR, have similarities to that of steroid receptor signalling. These systems consist of ligand-receptor-co-activator complexes at the genes that they activate. The co-activators consist of transcription factors and kinases, of which the latter play an important role in the associated epigenetics. It is our view that signalling by cytokines such as IFNγ is but a variation of specific gene activation by steroid hormones.

TWO APPLICATIONS OF PERMUTATION TESTS IN BIOSTASTICS.

We show two examples of how we answer biological questions by converting them into statistical hypothesis testing problems. We consider gene abundance data, and apply permutation tests. Though these tests are simple, they allow us to test biologically relevant hypotheses. Here we present the analysis of data rising from two studies on Type 1 Diabetes. In the first study [3] are interested in comparing the gut bacterial biodiversity in children at risk and not at risk of developing diabetes. In the second study, [4] compare the gut bacterial biodiversity of children in six different sites in USA and Europe. The statistical analyses presented here are parts of the "statistical methods" in two papers mentioned above. Here we offer a detailed explanation of the "Statistical Methods" addressed to readers with a statistics background.

Human bocavirus infection diagnosed serologically among children admitted to hospital with community-acquired pneumonia in a tropical region.

Human bocavirus (HBoV) is a human virus associated with respiratory disease in children. Limited information is available on acute infection with HBoV among children admitted to hospital with community-acquired pneumonia in tropical regions and the current diagnosis is inadequate. The aims were to diagnose and describe acute HBoV infections among children hospitalized for community-acquired pneumonia. In Salvador, Brazil, 277 children with community-acquired pneumonia were prospectively enrolled. Paired serum samples were tested by IgG, IgM, and IgG-avidity enzyme immunoassays (EIAs) using recombinant HBoV VP2. HBoV DNA was detected in nasopharyngeal aspirates and serum by a quantitative polymerase-chain reaction (PCR). HBoV DNA was detected in nasopharyngeal aspirates of 62/268 (23%) children and 156/273 (57%) were seropositive. Acute primary HBoV infection was reliably diagnosed (bearing at least two acute markers: Positive IgM, a fourfold increase/conversion of IgG, low IgG avidity or viremia) in 21 (8%) of 273 patients, 90% of 20 had HBoV DNA in nasopharyngeal aspirates, 83% with a high DNA load. The median age of infection with HBoV was 16 months, range 5-36. Community-acquired pneumonia was confirmed radiographically in 85% of 20 patients with acute HBoV infection diagnosed serologically. HBoV DNA was found in nasopharyngeal aspirates of 42/246(17%) children without an acute primary HBoV infection and available nasopharyngeal aspirate. Four children with HBoV secondary immune responses were detected, lacking both IgM and viremia. HBoV infection was diagnosed accurately in children aged 5-36 months with community-acquired pneumonia confirmed radiographically. PCR of nasopharyngeal aspirates is not a reliable marker of acute HBoV infection.

Occurrence of human bocaviruses and parvovirus 4 in solid tissues.

Human bocaviruses 1-4 (HBoV1-4) and parvovirus 4 (PARV4) are recently discovered human parvoviruses. HBoV1 is associated with respiratory infections of young children, while HBoV2-4 are enteric viruses. The clinical manifestations of PARV4 remain unknown. The objective of this study was to determine whether the DNAs of HBoV1-4 and PARV4 persist in human tissues long after primary infection. Biopsies of tonsillar tissue, skin, and synovia were examined for HBoV1-4 DNA and PARV4 DNA by PCR. Serum samples from the tissue donors were assayed for HBoV1 and PARV4 IgG and IgM antibodies. To obtain species-specific seroprevalences for HBoV1 and for HBoV2/3 combined, the sera were analyzed after virus-like particle (VLP) competition. While HBoV1 DNA was detected exclusively in the tonsillar tissues of 16/438 individuals (3.7%), all of them ≤8 years of age. HBoV2-4 and PARV4 DNAs were absent from all tissue types. HBoV1 IgG seroprevalence was 94.9%. No subject had HBoV1 or PARV4 IgM, nor did they have PARV4 IgG. The results indicate that HBoV1 DNA occurred in a small proportion of tonsils of young children after recent primary HBoV1 infection, but did not persist long in the other tissue types studied, unlike parvovirus B19 DNA. The results obtained by the PARV4 assays are in line with previous results on PARV4 epidemiology.

Clinical assessment and improved diagnosis of bocavirus-induced wheezing in children, Finland.

Human bocavirus (HBoV) is a widespread respiratory virus. To improve diagnostic methods, we conducted immunoglobulin (Ig) G and IgM enzyme immunoassays with recombinant virus-like particles of HBoV as antigen. Acute-phase and follow-up serum samples from 258 wheezing children and single serum samples from 115 healthy adults in Finland were examined. Our assays had a sensitivity of 97% and a specificity of 99.5%. Of adults, 96% had immunity; none had an acute infection. Of 48 children with serologically diagnosed acute HBoV infections, 45 were viremic and 35 had virus in nasopharyngeal aspirates (NPAs). Of 39 HBoV NPA PCR-positive children co-infected with another virus, 64% had a serologically verified HBoV infection. HBoV caused illness of longer duration than rhinovirus and of equal severity to that of respiratory syncytial virus. Among children with bronchiolitis, >25% had acute HBoV infections. Accurate HBoV diagnosis requires serologic analysis or PCR of serum; PCR of NPAs alone is insufficient.

Association Between Early-Life Antibiotic Use and the Risk of Islet or Celiac Disease Autoimmunity.

Importance: Evidence is lacking regarding the consequences of antibiotic use in early life and the risk of certain autoimmune diseases. Objective: To test the association between early-life antibiotic use and islet or celiac disease (CD) autoimmunity in genetically at-risk children prospectively followed up for type 1 diabetes (T1D) or CD. Design, Setting, and Participants: HLA-genotyped newborns from Finland, Germany, Sweden, and the United States were enrolled in the prospective birth cohort of The Environmental Determinants of Diabetes in the Young (TEDDY) study between November 20, 2004, and July 8, 2010. The dates of analysis were November 20, 2004, to August 31, 2014. Individuals from the general population and those having a first-degree relative with T1D were enrolled if they had 1 of 9 HLA genotypes associated with a risk for T1D. Exposures: Parental reports of the most common antibiotics (cephalosporins, penicillins, and macrolides) used between age 3 months and age 4 years were recorded prospectively. Main Outcomes and Measures: Islet autoimmunity and CD autoimmunity were defined as being positive for islet or tissue transglutaminase autoantibodies at 2 consecutive clinic visits at least 3 months apart. Hazard ratios and 95% CIs calculated from Cox proportional hazards regression models were used to assess the relationship between antibiotic use in early life before seroconversion and the development of autoimmunity. Results: Participants were 8495 children (49.0% female) and 6558 children (48.7% female) enrolled in the TEDDY study who were tested for islet and tissue transglutaminase autoantibodies, respectively. Exposure to and frequency of use of any antibiotic assessed in this study in early life or before seroconversion did not influence the risk of developing islet autoimmunity or CD autoimmunity. Cumulative use of any antibiotic during the first 4 years of life was not associated with the appearance of any autoantibody (hazard ratio [HR], 0.98; 95% CI, 0.95-1.01), multiple islet autoantibodies (HR, 0.99; 95% CI, 0.95-1.03), or the transglutaminase autoantibody (HR, 1.00; 95% CI, 0.98-1.02). Conclusions and Relevance: The use of the most prescribed antibiotics during the first 4 years of life, regardless of geographic region, was not associated with the development of autoimmunity for T1D or CD. These results suggest that a risk of islet or tissue transglutaminase autoimmunity need not influence the recommendations for clinical use of antibiotics in young children at risk for T1D or CD.

Early childhood gut microbiomes show strong geographic differences among subjects at high risk for type 1 diabetes.

OBJECTIVE: Gut microbiome dysbiosis is associated with numerous diseases, including type 1 diabetes. This pilot study determines how geographical location affects the microbiome of infants at high risk for type 1 diabetes in a population of homogenous HLA class II genotypes. RESEARCH DESIGN AND METHODS: High-throughput 16S rRNA sequencing was performed on stool samples collected from 90 high-risk, nonautoimmune infants participating in The Environmental Determinants of Diabetes in the Young (TEDDY) study in the U.S., Germany, Sweden, and Finland. RESULTS: Study site-specific patterns of gut colonization share characteristics across continents. Finland and Colorado have a significantly lower bacterial diversity, while Sweden and Washington state are dominated by Bifidobacterium in early life. Bacterial community diversity over time is significantly different by geographical location. CONCLUSIONS: The microbiome of high-risk infants is associated with geographical location. Future studies aiming to identify the microbiome disease phenotype need to carefully consider the geographical origin of subjects.

The methylome of the gut microbiome: disparate Dam methylation patterns in intestinal Bacteroides dorei.

Despite the large interest in the human microbiome in recent years, there are no reports of bacterial DNA methylation in the microbiome. Here metagenomic sequencing using the Pacific Biosciences platform allowed for rapid identification of bacterial GATC methylation status of a bacterial species in human stool samples. For this work, two stool samples were chosen that were dominated by a single species, Bacteroides dorei. Based on 16S rRNA analysis, this species represented over 45% of the bacteria present in these two samples. The B. dorei genome sequence from these samples was determined and the GATC methylation sites mapped. The Bacteroides dorei genome from one subject lacked any GATC methylation and lacked the DNA adenine methyltransferase genes. In contrast, B. dorei from another subject contained 20,551 methylated GATC sites. Of the 4970 open reading frames identified in the GATC methylated B. dorei genome, 3184 genes were methylated as well as 1735 GATC methylations in intergenic regions. These results suggest that DNA methylation patterns are important to consider in multi-omic analyses of microbiome samples seeking to discover the diversity of bacterial functions and may differ between disease states.

Bacteroides dorei dominates gut microbiome prior to autoimmunity in Finnish children at high risk for type 1 diabetes.

The incidence of the autoimmune disease, type 1 diabetes (T1D), has increased dramatically over the last half century in many developed countries and is particularly high in Finland and other Nordic countries. Along with genetic predisposition, environmental factors are thought to play a critical role in this increase. As with other autoimmune diseases, the gut microbiome is thought to play a potential role in controlling progression to T1D in children with high genetic risk, but we know little about how the gut microbiome develops in children with high genetic risk for T1D. In this study, the early development of the gut microbiomes of 76 children at high genetic risk for T1D was determined using high-throughput 16S rRNA gene sequencing. Stool samples from children born in the same hospital in Turku, Finland were collected at monthly intervals beginning at 4-6 months after birth until 2.2 years of age. Of those 76 children, 29 seroconverted to T1D-related autoimmunity (cases) including 22 who later developed T1D, the remaining 47 subjects remained healthy (controls). While several significant compositional differences in low abundant species prior to seroconversion were found, one highly abundant group composed of two closely related species, Bacteroides dorei and Bacteroides vulgatus, was significantly higher in cases compared to controls prior to seroconversion. Metagenomic sequencing of samples high in the abundance of the B. dorei/vulgatus group before seroconversion, as well as longer 16S rRNA sequencing identified this group as Bacteroides dorei. The abundance of B. dorei peaked at 7.6 months in cases, over 8 months prior to the appearance of the first islet autoantibody, suggesting that early changes in the microbiome may be useful for predicting T1D autoimmunity in genetically susceptible infants. The cause of increased B. dorei abundance in cases is not known but its timing appears to coincide with the introduction of solid food.

Type I IFN receptor controls activated TYK2 in the nucleus: implications for EAE therapy.

Recent studies have suggested that activated wild-type and mutant Janus kinase JAK2 play a role in the epigenetics of histone modification, where it phosphorylates histone H3 on tyrosine 41(H3pY41). We showed that type I IFN signaling involves activated TYK2 in the nucleus. ChIP-PCR demonstrated the presence of receptor subunits IFNAR1 and IFNAR2 along with TYK2, STAT1, and H3pY41 specifically at the promoter of the OAS1 gene in IFN treated cells. A complex of IFNAR1, TYK2, and STAT1α was also shown in the nucleus by immunoprecipitation. IFN treatment was required for TYK2 activation in the nucleus. The presence of IFNAR1, IFNAR2, and activated STAT1 and STAT2, as well as the type I IFN in the nucleus of treated cells was confirmed by the combination of Western blotting and confocal microscopy. Trimethylated histone H3 lysine 9 underwent demethylation and subsequent acetylation specifically in the region of the OAS1 promoter. Resultant N-terminal truncated IFN mimetics functioned intracellularly as antivirals as well as therapeutics against experimental allergic encephalomyelitis without the undesirable side effects that limit the therapeutic efficacy of IFNß in treatment of multiple sclerosis. The findings indicate that IFN signaling is complex like that of steroid signaling.

Human bocavirus and rhino-enteroviruses in childhood otitis media with effusion.

BACKGROUND: Viral respiratory infections play an important role in the pathogenesis of otitis media with effusion (OME) in children. The most common human rhinoviruses (HRVs) have been detected in middle ear effusions (MEE), but there is only limited data available about the closely related human enteroviruses (HEVs). The newly discovered human bocavirus (HBoV) has not, however, been identified in MEE of OME children. OBJECTIVES: The aim of our study was to determine the presence of HBoV and HRV/HEV and the rate of coinfection in a set of MEE samples collected from OME children. STUDY DESIGN: Seventy-five MEE samples from 54 children with no acute respiratory symptoms were studied with reverse transcription polymerase chain reaction (RT-PCR) for detection of HRV/HEV and quantitative PCR for detection of HBoV. RESULTS: Twenty-six (35%) of 75 MEE samples were positive for viral nucleic acid, 22 (29%) for HEV, 10 (13%) for HRV and 2 (3%) for HBoV. There was no statistically significant difference between mucoid and serous effusions in the rate of virus detection. Forty-three percent of bilateral cases showed a contra-lateral difference in viral finding. CONCLUSIONS: Our results suggest that these common respiratory viruses can be associated with OME in children. Whether these viruses are causative etiologic factors of MEE persistence or merely remnants of previous infections is not known.