Teemu Honkanen

Type 1 Diabetes Is Associated With Enterovirus Infection in Gut Mucosa

Enterovirus infections have been linked to type 1 diabetes in several studies. Enteroviruses also have tropism to pancreatic islets and can cause β-cell damage in experimental models. Viral persistence has been suspected to be an important pathogenetic factor. This study evaluates whether gut mucosa is a reservoir for enterovirus persistence in type 1 diabetic patients. Small-bowel mucosal biopsy samples from 39 type 1 diabetic patients, 41 control subjects, and 40 celiac disease patients were analyzed for the presence of enterovirus using in situ hybridization (ISH), RT-PCR, and immunohistochemistry. The presence of virus was compared with inflammatory markers such as infiltrating T cells, HLA-DR expression, and transglutaminase 2–targeted IgA deposits. Enterovirus RNA was found in diabetic patients more frequently than in control subjects and was associated with a clear inflammation response in the gut mucosa. Viral RNA was often detected in the absence of viral protein, suggesting defective replication of the virus. Patients remained virus positive in follow-up samples taken after 12 months’ observation. The results suggest that a large proportion of type 1 diabetic patients have prolonged/persistent enterovirus infection associated with an inflammation process in gut mucosa. This finding opens new opportunities for studying the viral etiology of type 1 diabetes.

Fecal Calprotectin Levels and Serological Responses to Microbial Antigens Among Children and Adolescents with Inflammatory Bowel Disease

Background: Noninvasive, sensitive, and specific tools for early identification of chronic inflammatory bowel disease (IBD) are needed for clinical practice. The aim was to identify new noninvasive test combinations for characterization of IBD in children and adolescents by comparing serological responses to microbial antigens and fecal calprotectin, a new promising marker for intestinal inflammation. Methods: Our study included 73 children who underwent endoscopies because of suspicion of IBD. Their sera were tested for antibodies to the Pseudomonas fluorescens‐associated sequence I2, a Bacteroides caccae TonB‐linked outer membrane protein, OmpW, and anti‐Saccharomyces cerevisiae (ASCA). Simultaneously, samples for fecal calprotectin measurements were obtained from 55 subjects. Results: IBD was diagnosed in 60 patients (Crohns disease [CD] in 18 patients, ulcerative colitis [UC] in 36, and indeterminate colitis [IC] in 6). Thirteen children had a non‐IBD disease. Fecal calprotectin levels were elevated (≥100 &mgr;g/g) more frequently in IBD patients (89%, 39/44) compared to non‐IBD cases (9%, 1/11, P < 0.001). ASCA antibodies in sera were detected in 67% (12/18) of patients with CD, in 14% (5/36) of the children with UC, and in 50% (3/6) of patients with IC. Seroreactivity for I2 was observed in 42% of the IBD patients, this frequency being higher than in non‐IBD cases (7.7% seropositive; P = 0.025). Serum anti‐I2 IgA levels (median absorbances) were higher in those with IBD compared to those without gut inflammation (P = 0.039). The combination of the measurements of fecal calprotectin and serological responses to microbial antigens (ASCA, I2, and OmpW) identified 100% of CD patients (sensitivity 100%, specificity 36%, positive predictive value [PPV] 66%, negative predictive value [NPV] 100%) and 89% of UC patients (sensitivity 89%, specificity 36%, PPV 77%, NPV 57%). Conclusions: Increased levels of serological responses to microbial antigens (ASCA, I2, and OmpW) and fecal calprotectin are evident in both CD and UC patients. The combination of these markers provides valuable, noninvasive tools for the diagnosis of IBD.

Elevated serum anti-I2 and anti-OmpW antibody levels in children with IBD

Background Bacteria are implicated as important factors in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to seek evidence of possible bacterial targets of the immune response related to IBD in children. Methods Seventy‐eight children referred to the Department of Paediatrics at Tampere University Hospital on suspicion of IBD were included in the study. Upper and lower gastrointestinal endoscopies with biopsies were performed on all children. Sera from 75 children were tested for antibodies to the Pseudomonas fluorescens‐associated sequence I2, a Bacteroides caccae TonB‐linked outer membrane protein, OmpW, anti‐Saccharomyces cerevisiae, and perinuclear anti‐neutrophil cytoplasmic antibodies. Results The IBD diagnosis was confirmed in 35 children (18 with Crohns disease [CD], 12 with ulcerative colitis [UC], and 5 with indeterminate colitis [IC]); 43 children were found to have no inflammation in the gut. Forty‐three percent (15 of 35) of those with IBD evinced positive seroreactivity to I2 and 46% (16 of 35) to OmpW. In CD, seroreactivity to I2 and OmpW was 50% (9 of 18) and 61% (11 of 18), respectively. Serum anti‐I2 and anti‐OmpW immunoglobulin A levels were significantly elevated in children with CD in comparison with the non‐IBD group (P = 0.007 and P = 0.001, respectively). A combination of OmpW, I2, and anti‐S cerevisiae tests identified 94% of CD patients, and a combination of OmpW, I2, and perinuclear anti‐neutrophil cytoplasmic antibodies detected 83% of UC cases. Conclusions Among children with IBD, strong serological responses to microbial antigens can be found, suggesting that P fluorescens and B caccae antigens have a potential role in the microbiology and immunology of the disease. Furthermore, serologic reactivity to the set of antigens studied here seems to be applicable in the initial differential diagnosis of children with CD and UC.

Evaluation of immunohistochemistry and in situ hybridization methods for the detection of enteroviruses using infected cell culture samples

BACKGROUND Enterovirus infections are frequent in all age groups. In addition to acute infections, they have been connected to chronic diseases such as cardiomyopathies and type 1 diabetes. Based on this there is an increasing need for the reliable detection of enteroviruses in different kinds of tissue samples. OBJECTIVES The aim of this study was to set up a test panel which can detect a wide range of different enteroviruses in paraffin-embedded samples and fresh frozen samples using immunohistochemical and in situ hybridization methods. STUDY DESIGN A panel of nine enterovirus antibodies was optimized for the detection of different enterovirus types in both paraffin-embedded and frozen cell culture samples. In addition, an oligonucleotide probe detecting all human enteroviruses was evaluated for ISH in formalin-fixed paraffin-embedded cell culture samples. RESULTS Most antibodies worked well in both sample types. Some antibodies detected only one of the tested serotypes, whereas others detected several serotypes. ISH was able to detect all tested enterovirus types. CONCLUSIONS This test panel makes it possible to detect a wide range of different enterovirus types in both formalin-fixed paraffin-embedded and frozen samples. The same methods can also be applied for tissue sections, but may need further optimization for each tissue type.

Myocardial infarction induces early increased remote ADAM8 expression of rat hearts after cardiac arrest.

Abstract Background. A disintegrin and metalloproteinase-8 (ADAM8) is a potential surrogate of inflammation which has recently been associated with myocardial infarction. We evaluated in a rat cardiac transplantation model whether ischemia-reperfusion injury alone (IRI) or with early regional myocardial infarction (MI) would suffice to induce inflammatory myocardial remodeling and ADAM8 expression. Material and methods. Isogenic heterotopic cardiac transplantation after cardiac arrest was performed to 48 Fischer 344 rats to induce ischemia-reperfusion injury (IRI), of which 27 rats also underwent ligation of the left anterior coronary artery (LAD) of the heart to yield MI. Histology was performed at 0.5, 24 and 48 h after transplantation. ADAM8 was evaluated by qRT-PCR after graft harvesting. Results. After 0.5 and 48 h respectively, edematous intramyocardial artery nuclei and periadventitial inflammation were more prominent in MI after transplantation, as compared with IRI alone and Controls (57.0 vs 40.0 and 5.0; 1.9 vs 1.1 and 0.9, point score units, p < 0.05, respectively). The expression of ADAM-8 was increased in MI as compared with Controls (1.9 vs 1.0, 1.9 fold increase) at 48 h. In grafts with MI, ADAM8 was localized using immunohistochemistry to the vicinity of the area corresponding to the developing infarction as well as in intramyocardial arteries remote to the infarction area. Conclusions. Remote histopathological changes of ischemic cardiac grafts are associated with increased expression of ADAM8 thus emphasizing a global myocardial impact of MI.

Indoleamine 2,3-dioxygenase expression in patients with allergic rhinitis: a case-control study

BackgroundIndoleamine 2,3-dioxygenase (IDO) is a tryptophan catalyzing enzyme. It has been suggested that it has a role in lower airway allergic inflammations, but its role in allergic rhinitis has not been investigated.ObjectiveOur aim was to evaluate the expression of IDO in the nasal mucosa of allergic rhinitis patients allergic to birch pollen during peak exposure to birch pollen allergen and compare it to non-atopic patients.MethodsIDO expression was immunohistochemically evaluated from nasal specimens obtained in- and off-season from otherwise healthy non-smoking volunteers both allergic to birch pollen (having mild or moderate allergic rhinoconjunctivitis) and non-allergic controls. Results: The IDO expression levels were low in healthy controls and remained low also in patients allergic to birch pollen. There were no differences in the expression of IDO in- and off-season in either healthy or allergic subjects.ConclusionsThere is a controversy in the role of IDO in upper and lower airways during allergic airway disease. It seems that IDO is associated to allergic inflammations of the lower airways, but does not have a local role in the nasal cavity at least in mild or moderate forms of allergic rhinitis.

Calcium Carbonate versus Sevelamer Hydrochloride as Phosphate Binders after Long-Term Disease Progression in 5/6 Nephrectomized Rats

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The effect of fundoplication on proliferative and anti‐apoptotic activity of esophageal mucosa in gastroesophageal reflux disease: 4‐year follow‐up study

OBJECTIVE:  The capacity of fundoplication to prevent esophageal adenocarcinoma is controversial. Development of cancer is associated with proliferation and anti‐apoptosis, for which little data exist as to their response to fundoplication. Therefore, we wanted to clarify the effect of fundoplication on the magnitude of Ki‐67 and B‐cell lymphoma 2 (Bcl‐2) during 48 months of follow up.