Type 1 DM Is Associated With Enterovirus in Gut Mucosa
Small intestinal biopsy samples were first screened for the presence of enterovirus genome using ISH method. Seventy-four percent of the type 1 diabetic patients compared with 29% of the control subjects were found to be virus positive (P < 0.001) (Table 2 and Fig. 1). A higher frequency of clearly positive hybridization signals was seen in diabetic patients especially (51 vs. 5%; P < 0.001) (Table 2). Enterovirus RNA was mainly located in the epithelial cells of villi and crypts; however, occasional staining in lamina propria was also observed. A follow-up sample was available from three diabetic patients who were enterovirus positive in the initial sample (taken 1 year after the initial sample). All of these remained enterovirus positive. The detection of enteroviruses was not associated with age, sex, HLA DQ2 and/or DQ8 alleles, or duration of diabetes or celiac disease (data not shown).
(Enlarge Image)
Figure 1.
Detection of enterovirus RNA by ISH in intestinal biopsies of type 1 diabetic patients including weak-positive (A), moderate-positive (B), and strong-positive (C) cases. D: Immunohistochemical staining for enterovirus VP1 protein. Arrows point to cells positive for enterovirus RNA (A) and VP1 protein (D). (A high-quality digital representation of this figure is available in the online issue.)
Next, the presence of viral RNA was confirmed using RT-PCR. A freshly frozen biopsy sample was available from 26 of the diabetic patients, 34 celiac disease patients, and 31 control subjects, and RT-PCR found the virus in 19, 15, and 10% of them, respectively. The virus was detected more frequently in ISH-positive than in ISH-negative subjects (12 of 50 vs. 1 of 41; P < 0.01).
In the next phase, we analyzed whether the presence of viral RNA was associated with the production of viral VP1 protein (see Supplementary Table 5>). The majority of subjects who were positive for virus RNA in ISH were negative for VP1 protein in immunohistochemistry. Overall, VP1 protein was found in 22, 20, and 22% of altogether 30 diabetic patients, 37 celiac disease patients, and 41 control subjects, respectively (Fig. 1). VP1 protein was mainly observed in the epithelial cells of the crypts. The proportion of VP1-negative subjects among all RNA-positive subjects tended to be higher among diabetic and celiac disease patients than in control subjects (78 and 86 vs. 66%).
Finally, the presence of viral RNA (ISH) was correlated with inflammation markers in the intestinal mucosa. A total of 23 type 1 diabetic patients, 40 celiac disease patients, and 41 control subjects was analyzed for the presence of CD3, αβ, and γδ intraepithelial lymphocytes and HLA-DR cells. Viral RNA was associated with the infiltration of αβ, γδ, and HLA-DR cells (Table 3). The presence of inflammation markers was associated with enterovirus RNA positivity in the whole study cohort, and the trend was also seen in diabetic patients in γδ IELs (46 vs. 17%, respectively) and HLA-DR expression (46 vs. 0%), but because of the low number of diabetic patients positive for inflammation markers, the results did not reach statistical significance (data not shown). The mean γδ-to-CD3 cell ratio was also higher in virus-positive than in virus-negative subjects (0.24 vs. 0.17; P = 0.043). Besides T-cell–mediated inflammation, antibody-mediated inflammation (IgA deposits) was more frequent in virus-positive than in virus-negative subjects (54 vs. 31%; P = 0.023) (Table 3). As expected, the inflammatory markers were most frequent in celiac disease patients in general, but diabetic patients who did not have celiac disease also had a tendency of elevated γδ IELs, HLA-DR expression, and IgA deposits compared with control subjects (Table 4 and Supplementary Figs. 2 and 3). They also had a higher mean γδ-to-CD3 cell ratio (0.18 vs. 0.09; P = 0.028). However, the inflammation was clearly much weaker in the diabetes group than in the celiac disease group.
Results
Small intestinal biopsy samples were first screened for the presence of enterovirus genome using ISH method. Seventy-four percent of the type 1 diabetic patients compared with 29% of the control subjects were found to be virus positive (P < 0.001) (Table 2 and Fig. 1). A higher frequency of clearly positive hybridization signals was seen in diabetic patients especially (51 vs. 5%; P < 0.001) (Table 2). Enterovirus RNA was mainly located in the epithelial cells of villi and crypts; however, occasional staining in lamina propria was also observed. A follow-up sample was available from three diabetic patients who were enterovirus positive in the initial sample (taken 1 year after the initial sample). All of these remained enterovirus positive. The detection of enteroviruses was not associated with age, sex, HLA DQ2 and/or DQ8 alleles, or duration of diabetes or celiac disease (data not shown).
(Enlarge Image)
Figure 1.
Detection of enterovirus RNA by ISH in intestinal biopsies of type 1 diabetic patients including weak-positive (A), moderate-positive (B), and strong-positive (C) cases. D: Immunohistochemical staining for enterovirus VP1 protein. Arrows point to cells positive for enterovirus RNA (A) and VP1 protein (D). (A high-quality digital representation of this figure is available in the online issue.)
Next, the presence of viral RNA was confirmed using RT-PCR. A freshly frozen biopsy sample was available from 26 of the diabetic patients, 34 celiac disease patients, and 31 control subjects, and RT-PCR found the virus in 19, 15, and 10% of them, respectively. The virus was detected more frequently in ISH-positive than in ISH-negative subjects (12 of 50 vs. 1 of 41; P < 0.01).
In the next phase, we analyzed whether the presence of viral RNA was associated with the production of viral VP1 protein (see Supplementary Table 5>). The majority of subjects who were positive for virus RNA in ISH were negative for VP1 protein in immunohistochemistry. Overall, VP1 protein was found in 22, 20, and 22% of altogether 30 diabetic patients, 37 celiac disease patients, and 41 control subjects, respectively (Fig. 1). VP1 protein was mainly observed in the epithelial cells of the crypts. The proportion of VP1-negative subjects among all RNA-positive subjects tended to be higher among diabetic and celiac disease patients than in control subjects (78 and 86 vs. 66%).
Finally, the presence of viral RNA (ISH) was correlated with inflammation markers in the intestinal mucosa. A total of 23 type 1 diabetic patients, 40 celiac disease patients, and 41 control subjects was analyzed for the presence of CD3, αβ, and γδ intraepithelial lymphocytes and HLA-DR cells. Viral RNA was associated with the infiltration of αβ, γδ, and HLA-DR cells (Table 3). The presence of inflammation markers was associated with enterovirus RNA positivity in the whole study cohort, and the trend was also seen in diabetic patients in γδ IELs (46 vs. 17%, respectively) and HLA-DR expression (46 vs. 0%), but because of the low number of diabetic patients positive for inflammation markers, the results did not reach statistical significance (data not shown). The mean γδ-to-CD3 cell ratio was also higher in virus-positive than in virus-negative subjects (0.24 vs. 0.17; P = 0.043). Besides T-cell–mediated inflammation, antibody-mediated inflammation (IgA deposits) was more frequent in virus-positive than in virus-negative subjects (54 vs. 31%; P = 0.023) (Table 3). As expected, the inflammatory markers were most frequent in celiac disease patients in general, but diabetic patients who did not have celiac disease also had a tendency of elevated γδ IELs, HLA-DR expression, and IgA deposits compared with control subjects (Table 4 and Supplementary Figs. 2 and 3). They also had a higher mean γδ-to-CD3 cell ratio (0.18 vs. 0.09; P = 0.028). However, the inflammation was clearly much weaker in the diabetes group than in the celiac disease group.
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