Neutralizing Ljungan virus antibodies in children with newly diagnosed type 1 diabetes.

Ljungan virus (LV), a Parechovirus of the Picornavirus family, first isolated from a bank vole at the Ljungan river in Sweden, has been implicated in the risk for autoimmune type 1 diabetes. An assay for neutralizing Ljungan virus antibodies (NLVA) was developed using the original 87-012 LV isolate. The goal was to determine NLVA titres in incident 0-18 years old newly diagnosed type 1 diabetes patients (n=67) and school children controls (n=292) from Jämtland county in Sweden. NLVA were found in 41 of 67 (61 %) patients compared to 127 of 292 (44 %) controls (P=0.009). In the type 1 diabetes patients, NLVA titres were associated with autoantibodies to glutamic acid decarboxylase (GADA) (P=0.023), but not to autoantibodies against insulin (IAA) or islet antigen-2 (IA-2A). The NLVA assay should prove useful for further investigations to determine levels of LV antibodies in patients and future studies to determine a possible role of LV in autoimmune type 1 diabetes.

First trimester enterovirus IgM and beta cell autoantibodies in mothers to children affected by type 1 diabetes autoimmunity before 7 years of age.

BACKGROUND: Autoimmune (type 1) diabetes (T1D) is a frequent chronic disease in children and adolescents globally. Gestational enterovirus (EV) infections have been associated with an increased risk for T1D in the offspring. We test the hypothesis that EV infections during the first trimester were associated with beta cell autoantibodies in mothers of children who developed islet autoantibodies before 7 years of age. MATERIALS AND METHODS: Local registries were used to identify mothers to children born 2000-2007 who developed either beta cell autoantibodies or T1D during follow up. Serum samples from the first trimester were located in the Biobank. A total of 448 index mothers were identified and compared to 891 matched control mothers. EV-IgM was determined in a capture enzyme immunoassay. Beta cell autoantibodies were analyzed in standard radio binding assays. RESULTS: The frequency of EV-IgM in index mothers was 20% (89/448), which did not differ from the control mothers 20% (175/891) (p = 0.922). Index mothers had multiple beta cell autoantibodies more often than control mothers (p = 0.037). Beta cell autoantibodies were increased during the November-April winter months in index compared to control mothers (p = 0.022). The observed difference was possibly explained by the months of February-April (p = 0.014). Concomitant EV-IgM and beta cell autoantibodies tended to be more common among index compared to control mothers (p = 0.039). CONCLUSION: EV-IgM during the first trimester may be associated with beta cell autoantibodies in mothers to children who developed either beta cell autoantibodies or T1D before 7 years of age.

Genome-wide analysis of DNA methylation in subjects with type 1 diabetes identifies epigenetic modifications associated with proliferative diabetic retinopathy.

BACKGROUND: Epigenetic variation has been linked to several human diseases. Proliferative diabetic retinopathy (PDR) is a major cause of vision loss in subjects with diabetes. However, studies examining the association between PDR and the genome-wide DNA methylation pattern are lacking. Our aim was to identify epigenetic modifications that associate with and predict PDR in subjects with type 1 diabetes (T1D). METHODS: DNA methylation was analyzed genome-wide in 485,577 sites in blood from cases with PDR (n = 28), controls (n = 30), and in a prospective cohort (n = 7). False discovery rate analysis was used to correct the data for multiple testing. Study participants with T1D diagnosed before 30 years of age and insulin treatment within 1 year from diagnosis were selected based on 1) subjects classified as having PDR (cases) and 2) subjects with T1D who had had diabetes for at least 10 years when blood DNA was sampled and classified as having no/mild diabetic retinopathy also after an 8.7-year follow-up (controls). DNA methylation was also analyzed in a prospective cohort including seven subjects with T1D who had no/mild diabetic retinopathy when blood samples were taken, but who developed PDR within 6.3 years (converters). The retinopathy level was classified by fundus photography. RESULTS: We identified differential DNA methylation of 349 CpG sites representing 233 unique genes including TNF, CHI3L1 (also known as YKL-40), CHN2, GIPR, GLRA1, GPX1, AHRR, and BCOR in cases with PDR compared with controls. The majority of these sites (79 %) showed decreased DNA methylation in cases with PDR. The Natural Killer cell-mediated cytotoxicity pathway was found to be significantly (P = 0.006) enriched among differentially methylated genes in cases with PDR. We also identified differential DNA methylation of 28 CpG sites representing 17 genes (e.g. AHRR, GIPR, GLRA1, and BCOR) with P <0.05 in the prospective cohort, which is more than expected by chance (P = 0.0096). CONCLUSIONS: Subjects with T1D and PDR exhibit altered DNA methylation patterns in blood. Some of these epigenetic changes may predict the development of PDR, suggesting that DNA methylation may be used as a prospective marker of PDR.

No excess risk for colorectal cancer among subjects seropositive for the JC polyomavirus.

The human polyomaviruses JC virus (JCV) and BK virus (BKV) are oncogenic in experimental systems and commonly infect humans. JCV DNA has been reported to be present in human colon mucosa and in colorectal cancers. To investigate whether the risk for colorectal cancer is associated with JCV or BKV infection, we performed a case-control study nested in the Janus biobank, a cohort of 330,000 healthy Norwegian subjects. A 30-year prospective follow-up using registry linkages identified 386 men with colorectal cancer who had baseline serum samples taken >3 months before diagnosis. Control subjects were matched for sex, age and date of blood sampling and county of residence. Seropositivity for JCV or BKV had high (97-100%) sensitivity for detection of viral DNA-positive subjects and discriminated the different polyomaviruses. Seropositivity was mostly stable over time in serial samples. The relative risk for colorectal cancer among JCV seropositive subjects was 0.9 (95% CI: 0.7-1.3) and the BKV-associated relative risk was 1.1 (95% CI: 0.8-1.5). Determining seropositivity using alternative cutoffs also found no evidence of excess risk. In summary, this prospective study found no association between JCV or BKV infections and excess risk for colorectal cancer.

No detection of SV40 DNA in mesothelioma tissues from a high incidence area in Sweden.

Simian virus 40 (SV40), a polyoma virus of the rhesus macaque was discovered in 1960 as a contaminant of human polio vaccines produced in monkey cells. A number of studies have reported the detection of SV40 nucleotide sequences in human tumors, mainly mesotheliomas, but the reports have not been consistent. The presence of SV40 in 26 consecutive cases of malignant mesothelioma of biphasic type was investigated using a SV40 quantitative real time polymerase chain reaction (PCR) with a sensitivity of 10 copies of viral DNA per sample. All the samples were also tested for amplifiability using a real-time PCR for the beta-globin gene. Eighteen tumors were amplifiable, but none contained SV40 DNA. The results do not support an association between mesothelioma and SV40.

Serological diagnosis of human polyomavirus infection.

Measurement of antibody titres to the human polyomaviruses BK and JC has for many years had to rely on Hemagglutination inhibition. In recent years, viral serology based on virus-like particles (VLPs) in enzyme immunoassays (EIAs) has become widely used for a variety of viruses. We sought to establish a modern method for serological diagnosis of BK and JC viruses, by using purified VLPs containing the VP1 major capsid proteins. Antibody titres in assays based on VLPs of BKV (strain SB) showed no correlation to the titres in similar JCV assays. BKV (SB) seropositivity increases rapidly with increasing age of the children and reaches a 98 % seroprevalence at 7-9 years of age, whereas JCV seroprevalences increase more slowly with increasing age reaching 51 % positivity among children 9-11 years of age. The antibody levels are almost identical in serial samples taken up to 5 years apart, suggesting that both BKV and JCV VLP seropositivitities are usually stable over time and can be used to measure cumulative exposure to these viruses. Serology using SV40 VLPs showed strong cross-reactivity with human polyomaviruses, in particular with BKV strain AS, and establishing a specific VLP-based serology assay for SV40 required blocking with several hyperimmune sera to the human polyomaviruses. SV40-specific seropositivity also increased with increasing age of children, reaching 14% seroprevalence among children 7-9 years of age, but had limited stability over time in serial samples.

Prevalence and stability of human serum antibodies to simian virus 40 VP1 virus-like particles.

Possible human infection with simian virus 40 (SV40) has been of great concern ever since SV40 was discovered in polio vaccines. Human populations are SV40-seropositive, but because of serological cross-reactivity between SV40 and the human polyomaviruses BK virus (BKV) and JC virus (JCV), it is debatable whether these antibodies are specific. An SV40-specific serological assay was established, based on purified virus-like particles (VLPs), where the SV40 VLPs were blocked with hyperimmune sera to BKV and JCV. Competition with SV40 hyperimmune sera was used as a confirmatory test. Among 288 Swedish children of between 1 and 13 years of age, 7.6 % had SV40-specific antibodies. SV40 seroprevalence reached a peak of 14 % at 7-9 years of age. Among 100 control patients with benign tumours, 9 % were SV40-seropositive. However, SV40 DNA was not detectable in corresponding buffy-coat samples. In serial samples taken up to 5 years apart from 141 Finnish women participating in the population-based serological screening for congenital infections, only two of 141 women were SV40-seropositive in both samples. Six women seroconverted and eight women had a loss of antibodies over time. None of the SV40-seropositive samples contained detectable SV40 DNA. In conclusion, there is a low prevalence of SV40-specific antibodies in the Nordic population. The SV40 antibodies appear to have a low stability over time and their origin is not clear.