Yard vegetation is associated with gut microbiota composition.

Gut microbes play an essential role in the development and functioning of the human immune system. A disturbed gut microbiota composition is often associated with a number of health disorders including immune-mediated diseases. Differences in host characteristics such as ethnicity, living habit and diet have been used to explain differences in the gut microbiota composition in inter-continental comparison studies. As our previous studies imply that daily skin contact with organic gardening materials modify gut microflora, here we investigated the association between living environment and gut microbiota in a homogenous western population along an urban-rural gradient. We obtained stool samples from 48 native elderly Finns in province Häme in August and November 2015 and identified the bacterial phylotypes using 16S rRNA Illumina MiSeq sequencing. We assumed that yard vegetation and land cover classes surrounding homes explain the stool bacterial community in generalized linear mixed models. Diverse yard vegetation was associated with a reduced abundance of Clostridium sensu stricto and an increased abundance of Faecalibacterium and Prevotellaceae. The abundance of Bacteroides was positively and strongly associated with the built environment. Exclusion of animal owners did not alter the main associations. These results suggest that diverse vegetation around homes is associated with health-related changes in gut microbiota composition. Manipulation of the garden diversity, possibly jointly with urban planning, is a promising candidate for future intervention studies that aim to maintain gut homeostasis.

Association of Picornavirus Infections With Acute Otitis Media in a Prospective Birth Cohort Study.

BACKGROUND: Human rhinoviruses (HRVs), human enteroviruses (HEVs) and human parechoviruses (HPeVs) have been linked to acute otitis media (AOM). We evaluated this association in a prospective birth cohort setting. METHODS: A total of 324 healthy infants were followed up from birth to age 3 years. Nasal swab samples were collected at age 3, 6, 12, 18, 24, and 36 months and screened for HRV and HEV using real-time reverse-transcription quantitative polymerase chain reaction. Stool samples were collected monthly and analyzed for HRV, HEV, and HPeV. AOM episodes diagnosed by physicians were reported by parents in a diary. The association of viruses with AOM was analyzed using generalized estimation equations, and their relative contributions using population-attributable risk percentages. RESULTS: A clear association was found between AOM episodes and simultaneous detection of HEV (adjusted odds ratio for the detection of virus in stools, 2.04; 95% confidence interval, 1.06-3.91) and HRV (1.54; 1.04-2.30). HPeV showed a similar, yet nonsignificant trend (adjusted odds ratio, 1.44; 95% confidence interval, .81-2.56). HRV and HEV showed higher population-attributable risk percentages (25% and 20%) than HPeV (11%). CONCLUSIONS: HEVs and HRVs may contribute to the development of AOM in a relatively large proportion of cases.

Rhinoviruses in infancy and risk of immunoglobulin E sensitization.

Previous data about the role of viruses in the development of allergic immunoglobulin E (IgE) sensitization are contradictory. The aim of this study was to determine the possible associations between exposure to different viruses (rhinovirus, enterovirus, norovirus, and parechovirus) during the first year of life and IgE sensitization. Viruses were analyzed from stool samples collected monthly from infants participating in a prospective birth cohort study. From that study, 244 IgE sensitized case children and 244 nonsensitized control children were identified based on their allergen-specific IgE antibody levels at the age of 6, 18, and 36 months. Stool samples (n = 4576) from the case and control children were screened for the presence of rhinovirus, enterovirus, norovirus, and parechovirus RNA by reverse transcription quantitative polymerase chain reaction. The study showed that rhinovirus was the most prevalent virus detected, present in 921 (20%) samples. None of the viruses were associated with IgE sensitization in the full cohort but after stratifying by sex, the number of rhinovirus positive samples was inversely associated with IgE sensitization in boys (odds ratio [OR]: 0.81; 95% confidence interval [CI]: 0.69-0.94; P = 0.006). There was also a temporal relation between rhinoviruses and IgE sensitization, as rhinovirus exposure during the first 6 months of life was associated with a reduced risk of subsequent IgE sensitization in boys (OR: 0.76; 95% CI: 0.6-0.94; P = 0.016). In conclusion, early exposure to rhinoviruses was inversely associated with IgE sensitization but this protective association was restricted to boys.

Enterovirus infection during pregnancy is inversely associated with atopic disease in the offspring.

BACKGROUND: Prenatal environment has been shown to influence child's risk of atopic diseases. Laboratory-confirmed data about the role of maternal infections during pregnancy is scarce. OBJECTIVE: The aim of this study was to determine the associations between serologically confirmed maternal infections during pregnancy and atopic disease in the offspring. METHODS: This was a nested case-control study within a prospective birth cohort study. Altogether 202 atopic case children and 333 matched non-atopic control children were included. Atopic outcome was defined as having an atopic disease and IgE sensitization by the age of 5 years. We analysed serologically acute enterovirus (EV), influenza virus A (IAV) and Mycoplasma pneumoniae (M. pneumoniae) infections during pregnancy, and mother's seropositivity against human cytomegalovirus (CMV) and Helicobacter pylori. RESULTS: Maternal EV infection during pregnancy was inversely associated with atopic outcome in the offspring (odds ratio 0.43; 95% confidence interval: 0.23-0.80, P = 0.008). Acute IAV or M. pneumoniae infections or seropositivity against CMV or Helicobacter pylori were not associated with the atopic outcome. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that maternal EV infections during pregnancy are inversely associated with atopic disease in the offspring. Our finding provides further support to the previous studies suggesting an important role of the in utero environment in the development of atopic diseases.

Molecular epidemiology of enteroviruses in young children at increased risk of type 1 diabetes.

BACKGROUND: Young children are susceptible to enterovirus (EV) infections, which cause significant morbidity in this age group. However, the current knowledge regarding the epidemiology of EVs and the circulating virus strains is mostly based on viruses detected in children with severe diseases leading to contact with the health care system, while the vast reservoir of EVs that circulate in the general population is less characterized. METHODOLOGY: The present study investigates the types and the prevalence of EVs circulating in the young children of the background population in Georgia, Colorado, and Washington State in the USA, and Germany, Sweden, and Finland in Europe. A total of 4018 stool samples, collected monthly from 300 healthy and non-hospitalized children at the age of 3-18 months in 2005-2009, were analyzed for the presence of EVs using RT-PCR, followed by sequencing of the VP1-2A region of the viral genome to type the EV(s) present. All of the children carried type HLA-DQ2 or -DQ8 alleles associated with type 1 diabetes. PRINCIPAL FINDINGS: Altogether 201 children (67%) were found to be EV positive. The prevalence was much lower in Finnish children (26%) than in the children of the other counties combined (75%). Infections increased by age and showed a nadir during the winter months. Children who carried both the HLA-DQ2 and -DQ8 alleles had less infections than children who were homozygous for these alleles. Coxsackieviruses type A were the most frequently detected viruses in all geographical regions. Coxsackievirus type A4, Echovirus type 18, and Echovirus type 25 were shed for longer time periods than the other EV types. CONCLUSIONS: Compared to prevalence data from symptomatic patients requiring medical attention, this study provides a better view of EVs circulating in young children in the USA and in Europe. The observations may prove useful for the selection of strategies for designing EV vaccines in the future. The study also confirms our previous serological findings suggesting that EV infections are relatively rare in Finland.

Detection of enteroviruses in stools precedes islet autoimmunity by several months: possible evidence for slowly operating mechanisms in virus-induced autoimmunity.

AIMS/HYPOTHESIS: This case-control study was nested in a prospective birth cohort to evaluate whether the presence of enteroviruses in stools was associated with the appearance of islet autoimmunity in the Type 1 Diabetes Prediction and Prevention study in Finland. METHODS: Altogether, 1673 longitudinal stool samples from 129 case children who turned positive for multiple islet autoantibodies and 3108 stool samples from 282 matched control children were screened for the presence of enterovirus RNA using RT-PCR. Viral genotype was detected by sequencing. RESULTS: Case children had more enterovirus infections than control children (0.8 vs 0.6 infections per child). Time-dependent analysis indicated that this excess of infections occurred more than 1 year before the first detection of islet autoantibodies (6.3 vs 2.1 infections per 10 follow-up years). No such difference was seen in infections occurring less than 1 year before islet autoantibody seroconversion or after seroconversion. The most frequent enterovirus types included coxsackievirus A4 (28% of genotyped viruses), coxsackievirus A2 (14%) and coxsackievirus A16 (11%). CONCLUSIONS/INTERPRETATION: The results suggest that enterovirus infections diagnosed by detecting viral RNA in stools are associated with the development of islet autoimmunity with a time lag of several months.

Development and maturation of norovirus antibodies in childhood.

The burden of norovirus (NoV) gastroenteritis is substantial in young children. Maternal antibodies are thought to protect a child from NoV infection in early infancy but subsequent development of NoV-specific protective immunity in children is still largely unexplored. We have determined NoV-specific antibody seroconversion to GII.4 virus-like particles as an indicator of NoV infection in two children prospectively followed from birth to eight years of age. Blocking activity and affinity maturation of maternal and serum IgG antibodies were evaluated. Our results show that multiple infections occur in children up to eight years of age. The titer, blocking activity and avidity of maternal antibodies determined susceptibility of an infant to NoV infection. NoV GII.4-specific antibodies with high blocking potential and avidity were developed at two to three years of age and were retained throughout the follow-up. Subsequent NoV infections may have contributed to the duration of protective NoV-specific immune responses that lasted for several years. This study adds to current understanding of the duration of passive protection by maternal antibodies and the duration and quality of acquired immunity following primary and subsequent NoV infections in infants and young children, who are the main target group for NoV vaccine development.

Age-associated DNA methylation changes in immune genes, histone modifiers and chromatin remodeling factors within 5 years after birth in human blood leukocytes.

BACKGROUND: Age-related changes in DNA methylation occurring in blood leukocytes during early childhood may reflect epigenetic maturation. We hypothesized that some of these changes involve gene networks of critical relevance in leukocyte biology and conducted a prospective study to elucidate the dynamics of DNA methylation. Serial blood samples were collected at 3, 6, 12, 24, 36, 48 and 60 months after birth in ten healthy girls born in Finland and participating in the Type 1 Diabetes Prediction and Prevention Study. DNA methylation was measured using the HumanMethylation450 BeadChip. RESULTS: After filtering for the presence of polymorphisms and cell-lineage-specific signatures, 794 CpG sites showed significant DNA methylation differences as a function of age in all children (41.6% age-methylated and 58.4% age-demethylated, Bonferroni-corrected P value <0.01). Age-methylated CpGs were more frequently located in gene bodies and within +5 to +50 kilobases (kb) of transcription start sites (TSS) and enriched in developmental, neuronal and plasma membrane genes. Age-demethylated CpGs were associated to promoters and DNAse-I hypersensitivity sites, located within -5 to +5 kb of the nearest TSS and enriched in genes related to immunity, antigen presentation, the polycomb-group protein complex and cytoplasm. CONCLUSIONS: This study reveals that susceptibility loci for complex inflammatory diseases (for example, IRF5, NOD2, and PTGER4) and genes encoding histone modifiers and chromatin remodeling factors (for example, HDAC4, KDM2A, KDM2B, JARID2, ARID3A, and SMARCD3) undergo DNA methylation changes in leukocytes during early childhood. These results open new perspectives to understand leukocyte maturation and provide a catalogue of CpG sites that may need to be corrected for age effects when performing DNA methylation studies in children.

Aberrant gut microbiota composition at the onset of type 1 diabetes in young children.

AIMS/HYPOTHESIS: Recent studies indicate that an aberrant gut microbiota is associated with the development of type 1 diabetes, yet little is known about the microbiota in children who have diabetes at an early age. To this end, the microbiota of children aged 1-5 years with new-onset type 1 diabetes was compared with the microbiota of age-matched healthy controls. METHODS: A deep global analysis of the gut microbiota composition was established by phylogenetic microarray analysis using a Human Intestinal Tract Chip (HITChip). RESULTS: Principal component analyses highlighted the importance of age when comparing age-matched pairs. In pairs younger than 2.9 years, the combined abundance of the class Bacilli (notably streptococci) and the phylum Bacteroidetes was higher in diabetic children, whereas the combined abundance of members of Clostridium clusters IV and XIVa was higher in the healthy controls. Controls older than 2.9 years were characterised by a higher fraction of butyrate-producing species within Clostridium clusters IV and XIVa than was seen in the corresponding diabetic children or in children from the younger age groups, while the diabetic children older than 2.9 years could be differentiated by having an increased microbial diversity. CONCLUSIONS/INTERPRETATION: The results from both age groups suggest that non-diabetic children have a more balanced microbiota in which butyrate-producing species appear to hold a pivotal position.

Virus antibody survey in different European populations indicates risk association between coxsackievirus B1 and type 1 diabetes.

Enteroviruses (EVs) have been connected to type 1 diabetes in various studies. The current study evaluates the association between specific EV subtypes and type 1 diabetes by measuring type-specific antibodies against the group B coxsackieviruses (CVBs), which have been linked to diabetes in previous surveys. Altogether, 249 children with newly diagnosed type 1 diabetes and 249 control children matched according to sampling time, sex, age, and country were recruited in Finland, Sweden, England, France, and Greece between 2001 and 2005 (mean age 9 years; 55% male). Antibodies against CVB1 were more frequent among diabetic children than among control children (odds ratio 1.7 [95% CI 1.0-2.9]), whereas other CVB types did not differ between the groups. CVB1-associated risk was not related to HLA genotype, age, or sex. Finnish children had a lower frequency of CVB antibodies than children in other countries. The results support previous studies that suggested an association between CVBs and type 1 diabetes, highlighting the possible role of CVB1 as a diabetogenic virus type.

Coxsackievirus B1 is associated with induction of ß-cell autoimmunity that portends type 1 diabetes.

The rapidly increasing incidence of type 1 diabetes implies that environmental factors are involved in the pathogenesis. Enteroviruses are among the suspected environmental triggers of the disease, and the interest in exploring the possibilities to develop vaccines against these viruses has increased. Our objective was to identify enterovirus serotypes that could be involved in the initiation of the disease process by screening neutralizing antibodies against 41 different enterovirus types in a unique longitudinal sample series from a large prospective birth-cohort study. The study participants comprised 183 case children testing persistently positive for at least two diabetes-predictive autoantibodies and 366 autoantibody-negative matched control children. Coxsackievirus B1 was associated with an increased risk of ß-cell autoimmunity. This risk was strongest when infection occurred a few months before autoantibodies appeared and was attenuated by the presence of maternal antibodies against the virus. Two other coxsackieviruses, B3 and B6, were associated with a reduced risk, with an interaction pattern, suggesting immunological cross-protection against coxsackievirus B1. These results support previous observations suggesting that the group B coxsackieviruses are associated with the risk of type 1 diabetes. The clustering of the risk and protective viruses to this narrow phylogenetic lineage supports the biological plausibility of this phenomenon.

Human rhinoviruses including group C are common in stool samples of young Finnish children.

BACKGROUND: Human rhinoviruses (HRVs) are common causes of viral respiratory infections. They have been widely studied in respiratory samples in hospital patient series but only a few studies have been performed to assess their occurrence in other sample types and their circulation in healthy children background population. OBJECTIVES: To analyze the frequency of HRVs in the background population in Finland by screening HRV RNA from stool samples longitudinally collected in a cohort of young children. STUDY DESIGN: Altogether 4184 stool samples were collected regularly from a cohort of children who were observed from birth. Samples were screened for the presence of RNA of HRVs using RT-PCR. HRV specific sequences were identified by sequencing the VP1 or VP4/VP2 coding region. Virus isolation was performed using four different cell lines and the result was confirmed by real time PCR. RESULTS: A total of 9% of the stool samples were positive for HRV RNA. Sequence analysis indicated that the most prevalent species was HRV-A, and the most prevalent serotype was HRV61. HRV-B and HRV-C species were also detected. One of the six tested rhinovirus positive samples retained its infectivity and was able to grow in RD and GMK cells. CONCLUSIONS: Our study shows that HRVs are frequently detected in the stool samples from the population of young children. We also show that HRV-C, which can cause severe illnesses in children, is commonly circulating in young children in Finland.

Human enterovirus 71 strains in the background population and in hospital patients in Finland.

BACKGROUND: Human enterovirus 71 (HEV71) is a common cause of severe outbreaks of hand-foot- and mouth disease, aseptic meningitis and encephalitis in Asian populations but has not caused such epidemics in all populations. OBJECTIVES: To analyze the frequency of HEV71 in the background childhood population in Finland by screening in stool and serum samples and by measuring neutralizing antibodies against HEV71 in serum and to compare the genetic relationship of virus strains detected in asymptomatic children and those causing severe illness in Finland to the strains found in other countries. STUDY DESIGN: 4185 stool samples and 5686 serum samples were collected and clinical symptoms recorded from children who were observed from birth. Additional stool samples were available from four children hospitalized due to EV71 infection. Samples were screened for the presence of RNA of human enteroviruses using RT-PCR and HEV71 amplicons were identified by sequencing. Phylogenetic analyses were carried out to study genetic relationships between different virus strains. Neutralizing antibodies against HEV71 were screened from 522 children. RESULTS: A total of 0.3% of stool samples and two serum samples from healthy children were positive for HEV71 genome. 1.6% of the children had neutralizing antibodies against HEV71. Most infections were asymptomatic or mild in contrast to the clear symptoms in the children hospitalized due to HEV71. All viruses were C strains. CONCLUSIONS: HEV71 is circulating in Finland but it is rare. No clear difference was seen between strains circulating in the Finnish background population and those found in hospitalized patients or those causing severe outbreaks worldwide.

Seroconversion to islet autoantibodies after enterovirus infection in early pregnancy.

Gestational enterovirus (EV) infections have been associated with an increased risk for type 1 diabetes in the offspring. We therefore analyzed non-diabetic mothers for EV exposure in early pregnancy in relation to type 1 diabetes HLA-DQ risk genotypes and seroconversion to islet autoantibodies during pregnancy. Non-diabetic mothers who had islet autoantibodies (n=365) against glutamic acid decarboxylase (GADA), islet antigen-2 autoantibodies (IA-2A), or insulin autoantibodies (IAA), in early pregnancy and at delivery were compared to islet autoantibody-negative mothers (n=1457) matched for age and sampling date. Mothers were genotyped for HLA-DQ and analyzed for both EV-RNA and EV-IgM. EV-IgM, but not EV-RNA, was detected during early pregnancy in 12% of islet autoantibody-positive mothers compared to 11% of the controls. In early pregnancy, mothers with HLA-DQ 2/2 or 2/X genotypes showed an increased risk for islet autoantibodies at delivery (OR 1.85; p=0.001). After adjusting for parity, maternal age, year of birth, and season of early pregnancy, early pregnancy EV-IgM combined with DQ2/2 or 2/X increased the risk for islet autoantibodies (OR 3.10; 95% CI 1; p=0.008). EV-IgM in early pregnancy increased the risk for islet autoantibodies at delivery in non-diabetic mothers with HLA-DQ 2/2 or 2/X type 1 diabetes risk genotypes.

Viral interference induced by live attenuated virus vaccine (OPV) can prevent otitis media.

BACKGROUND: The goal of this study was to evaluate whether a live attenuated poliovirus vaccine (OPV) has clinically relevant interfering effect with non-polio infections causing otitis media in young children. METHODS: Open trial in which the intervention group (64 children) received OPV at the age of 2, 3, 6 and 12 months. The control group (250 children) received IPV (inactivated polio vaccine) at the age of 6 and 12 months. Clinical symptoms were recorded by a questionnaire at the age of 3, 6, 12, 18 and 24 months. RESULTS: Otitis media episodes were less frequent in the OPV than in the control group. A significant difference was seen at the age of 6-18 months (IRR=0.76 [95% CI 0.59-0.94], P=0.011) and was particularly clear among children, who attended daycare (IRR 0.37 [95% CI 0.19-0.71], P=0.003). CONCLUSIONS: OPV provides some protection against otitis media. This effect may be mediated by viral interference with non-polio viruses.