Type 1 Diabetes is Associated With Enterovirus Infection in Gut Mucosa

Maarit Oikarinen; Sisko Tauriainen; Sami Oikarinen; Teemu Honkanen; Pekka Collin; Immo Rantala; Markku Mäki; Katri Kaukinen; Heikki Hyöty


Diabetes. 2012;61(3):687-691. 

In This Article

Research Design and Methods

Study Series

The study series consisted of 120 individuals altogether who underwent gastroscopy because of various gastrointestinal complaints. Small-bowel mucosal biopsies were taken from distal duodenum for morphological analyses and for basic research. Samples were taken during the period 1995–2000 at the Department of Gastroenterology, Tampere University Hospital. Formalin-fixed samples were available from all 120 study subjects and frozen samples from 86 subjects.

The study subjects comprised 39 type 1 diabetic patients, 40 patients with celiac disease who did not have type 1 diabetes, and 41 control subjects, including subjects who had neither of these diseases but nonspecific gastrointestinal symptoms (25 dyspepsia, 10 irritable bowel syndrome, 3 aphthous stomatitis, 1 ulcerative colitis, 1 collagenous colitis, and 1 eventually healthy). Furthermore, one control subject had psoriasis and one IgA nephropathy. All control subjects were nondiabetic and showed no signs of celiac disease (Table 1).

The diagnosis of type 1 diabetes was based on World Health Organization criteria,[19] and all the patients were on insulin treatment. Sixteen also had celiac disease, 10 thyroidal autoimmune disorder, 1 IgA nephropathy, 1 Addison disease, 1 alopecia, and 1 rheumatoid arthritis. The duration of diabetes varied from 0 to 38 years (median 20 years; exact information available from only 23 patients).

Celiac disease was diagnosed in study subjects by a positive endomysial antibody result and by small-bowel mucosal villous atrophy and crypt hyperplasia. Celiac disease had recently been diagnosed in all the patients, and all were on a normal gluten-containing diet at the time of the biopsy. One celiac disease patient had IgA nephropathy, and one had hyperthyroidism.

The study protocol was approved by the ethical committee of the Tampere University Hospital. All subjects gave informed consent.

In situ Hybridization

Primary screening of enterovirus was carried out on formalin-fixed and paraffin-embedded biopsy samples (5-μm sections) using an in situ hybridization (ISH) assay as previously described.[4,18,20] This is based on a single enterovirus-specific probe targeting a highly conserved, group-common sequence in the 5′-noncoding region of the enteroviral genome. This assay covers most if not all known enterovirus types. All analyses were performed blind to the case-control status of the subjects. Positive hybridization signal was semiquantified by counting positive cells per microscopic field into the following categories: negative (no positive cells), weak positive (an average of 1–10 positive cells per microscopic field using 400-fold magnification), moderate positive (10–100 positive cells per field), and strong positive (>100 cells per field).

Immunohistochemical Staining

Formalin-fixed paraffin-embedded biopsy samples (5-μm sections) were stained with antienterovirus VP1 antibody (clone 5-D8/1, 1:300; DakoCytomation, Glostrup, Denmark) using Ventana BencMark LT (Ventana Medical Systems) and the ultraView Universal detection system as previously described.[20] Known virus-positive tissue and cell culture samples were used to confirm the staining reliability of all separate staining batches. CD3+ intraepithelial lymphocytes (IELs) were stained with monoclonal antibody Leu-4 (Becton Dickinson, San Jose, CA), αβ+ IELs with monoclonal βF1 antibody (Endogen, Woburn, MA), and γδ+ IELs with T-cell receptor γ antibody (Endogen). Positive IELs were counted with a ×100 objective throughout the surface epithelium, and IEL reference values were set at 37 cells/mm for CD3+, 25 for αβ+, and 4.3 for γδ+ IELs as previously described.[21] Mucosal HLA-DR expression was detected with monoclonal HLA-DR antibody (Becton Dickinson) and graded as previously described.[22] IgA deposits were analyzed from unfixed frozen sections using double staining with fluorescein isothiocyanate–labeled rabbit antibody against human IgA (Dako, Glostrup, Denmark) and monoclonal mouse antibody against transglutaminase 2 (CUB7402; NeoMarkers, Fremont, CA) followed by rhodamine-conjugated anti-mouse IgG antibodies (Dako). It was earlier shown that the mucosal IgA deposits are specifically targeted against transglutaminase 2.[23,24] All evaluations were carried out blind to disease history or laboratory findings.

Enterovirus RT-PCR

For RT-PCR, unfixed biopsy samples were stored frozen in optimal cutting temperature medium at −70°C. The biopsy samples were removed from the optimal cutting temperature medium and homogenized using a SilentCrusher S homogenizer (Heidolph, Schwabach, Germany). RNA was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed using two independent methods: a previously described method amplifying a sequence common to all known enterovirus serotypes[25] and a real-time RT-PCR using the same primers and probes. A sample was considered enterovirus positive if it gave a positive signal in at least one of these assays.

Statistical Analysis

Statistical analyses were performed using SPSS 18.0 for Windows. Frequency comparison was performed with the Pearson χ2 and Fisher exact tests, and continuous variables were analyzed by independent sample t test.


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