Stress proteins, or heat shock proteins (hsps) are phylogenetically conserved proteins synthesized during stress and thus providing the cells with a protective mechanism against environmental insult. Stress proteins share sequence homology with a wide range of autoantigens. The study group consisted of 77 consecutive children referred to the outpatient pediatric clinic at the University Hospital of Tampere, Finland, for symptoms and signs suggesting celiac disease. This chapter shows that ingested gluten induces enhanced upregulation of jejunal epithelial hsp 65 in individuals shown to have celiac disease. The staining was for some reasons always uniform throughout the biopsy specimen, but we observed that a blind-tested subjective arbitrary grading worked where the intensity and amount of hsp 65 in individual epithelial cell mitochondrial areas was assessed. The children were suspected to have a gluten-induced enteropathy and therefore biopsied. Thus, epithelial cell expression of the antigen may act as a triggering factor in the ongoing chronic inflammation.

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.

BACKGROUND:The aim of this study was to create a new research strategy to obtain high-quality pancreatic tissues from subjects with preclinical or clinical type 1 diabetes, which would open up new avenues for studying the mechanisms of the β-cell damaging process in humans. RESEARCH DESIGN AND METHODS:A nationwide collaboration network (the PanFin network) was established in Finland to start an on-call screening of diabetes-associated autoantibodies from deceased organ donors and subsequent processing of pancreases from autoantibody-positive donors. This protocol was integrated into the national organ transplantation procedure. RESULTS:Only a few modifications were needed to the normal transplantation practices. One additional blood sample was obtained from donors for autoantibody analyses, the transplantation team was informed about the autoantibody result and the pancreas of autoantibody-positive donors was transported to the core laboratory. Altogether, 307 donors were screened and 22 (7.2%) were positive for at least one autoantibody and 3 tested positive for two or more autoantibodies out of the five tested (islet cell antibodies, insulin autoantibodies and autoantibodies to glutamic acid decarboxylase, islet antigen 2 and zinc transporter 8). The quality of collected pancreatic tissue was superior to that from autopsies and allowed the detection of both RNA and proteins. CONCLUSIONS:The study protocol was proven feasible to be carried out on a nationwide scale. It did not interfere with the normal transplantation activities and provided valuable tissue material for research.

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. (C) 2010 Elsevier B. V. All rights reserved.

BACKGROUND:Peroxiredoxins (Prxs) have recently been suggested to have a role in tumorigenesis.METHODS:We studied the expression of Prx I-VI and their relationship to patient survival in 383 grade II-IV diffuse astrocytic brain tumors.RESULTS:Prx I positivity was found in 68%, Prx II in 84%, Prx III in 90%, Prx IV in 5%, Prx V in 4% and Prx VI in 47% of the tumors. Prx I and Prx II expression decreased significantly with increasing malignancy grade (p < 0.001 and p < 0.001). Patients with Prx I or Prx II positive tumors were significantly younger than the average age of all the patients (p = 0.014 and p = 0.005). A lower proliferation rate was associated with Prx I and Prx VI positive tumors (p = 0.019 and p = 0.033), and a lower apoptotic rate was found within Prx I and Prx II positive tumors (p < 0.001 and p = 0.007). Patients with Prx I and Prx II positive tumors had a significantly better survival rate than their Prx-negative counterparts (p = 0.0052 and p = 0.0002).CONCLUSION:The expression of Prx I and Prx II correlates with astrocytic tumor features, such as grade and patient age and proliferation activity (Prx I), and accordingly with patient survival.

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 coin hi nations 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 12, 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 (Crohn's 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 mu/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 12 was observed in 42% of the [BD patients, this frequency being g 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, 12, 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, 12, 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.

Background Immunoglobulin A (IgA) autoantibodies to tissue transglutaminase (tTG) are commonly used for screening and diagnosing of celiac disease (CD). Seroreactivity for anti- Saccharomyces cerevisiae antibody (ASCA) and bacterial antigens have also been detected in CD patients. The aim of this study was to examine prospectively serologic responses to microbial targets in adult CD patients at the time of diagnosis and during a gluten-free diet (GFD). Further, we wanted to evaluate whether these serologic specificities could provide new tools for the follow-up of CD patients. Methods Data on 55 adult biopsy-proven CD patients were available for follow-up study. Upper gastrointestinal endoscopy was performed on all patients. Sera from patients were tested for antibodies to tTG and ASCA and additionally analyzed with IgA enzyme-linked immunosorbent assays to Pseudomonas fluorescens -associated sequence, I2, and to a Bacteroides caccae TonB-linked outer membrane protein, OmpW. Results At the time of diagnosis, 91% of CD cases were positive for tTG and 49% for ASCA; positive seroreactivity to I2 was found in 86% and to OmpW in 60% of CD patients at the time of diagnosis. The frequency of seropositivity and serum levels of these antibodies decreased during GFD. Moreover, we found that the decline in the serum levels was significant in all of these markers ( p < 0.005). Interestingly, we also found that serum levels of ASCA correlated with the grade of mucosal morphology ( p = 0.021), as the ASCA serum levels declined in accordance with mucosal healing. Conclusions Commensal enteric bacteria seem to play a role in the small intestinal mucosal damage in CD. This was proven by the serological responses to different microbial antigens shown in this study. Serum levels of ASCA, anti-I2, and anti-OmpW antibodies decreased significantly during GFD, indicating that these serologic markers are gluten dependent in CD patients. These specificities could provide new tools in the follow-up of CD patients.

BACKGROUND:The expression of a neural crest stem cell marker, polysialic acid (polySia), and its main carrier, neural cell adhesion molecule (NCAM), have been detected in some malignant tumors with high metastatic activity and unfavorable prognosis, but the diagnostic and prognostic value of polySia-NCAM in neuroblastoma is unclear.METHODS:A tumor tissue microarray (TMA) of 36 paraffin-embedded neuroblastoma samples was utilized to detect polySia-NCAM expression with a polySia-binding fluorescent fusion protein, and polySia-NCAM expression was compared with clinical stage, age, MYCN amplification status, histology (INPC), and proliferation index (PI).RESULTS:PolySia-NCAM-positive neuroblastoma patients had more often metastases at diagnosis, and polySia-NCAM expression associated with advanced disease (P = 0.047). Most interestingly, absence of polySia-NCAM-expressing tumor cells in TMA samples, however, was a strong unfavorable prognostic factor for overall survival in advanced disease (P = 0.0004), especially when MYCN was not amplified. PolySia-NCAM-expressing bone marrow metastases were easily detected in smears, aspirates and biopsies.CONCLUSION:PolySia-NCAM appears to be a new clinically significant molecular marker in neuroblastoma, hopefully with additional value in neuroblastoma risk stratification.

OBJECTIVES:Expression of anti-Saccharomyces cerevisiae antibodies (ASCA) identifies patients and individuals at risk for Crohn's disease and has also been reported in 40-60% of celiac disease (CD) cases, suggesting a role of host response to enteric microbiota in the development of inflammatory lesions. In this prospective study in patients with suspicion of CD, we evaluate the frequency and association of ASCA to serological responses for other host microbial targets formally associated with Crohn's disease, including the Pseudomonas fluorescens associated sequence I2 and a Bacteroides caccae TonB-linked outer membrane protein, OmpW.METHODS:Small bowel mucosal biopsies were taken from 242 patients with suspicion of CD, their sera were tested for antibodies to tissue transglutaminase (tTG), ASCA, I2, and OmpW. Eighty adult healthy blood donors were used as controls.RESULTS:The diagnosis of CD was confirmed on biopsy in 134 cases. The occurrence of ASCA and I2 positivity was significantly higher in adult CD patients than in patients with non-CD disease. Anti-I2 levels in the sera were significantly higher in adult CD patients than in non-CD disease or the controls and anti-OmpW levels in CD and non-CD patients when compared to controls. Positive seroreactivity to OmpW seemed to increase with age. Of the CD patients, 90% were seropositive for at least one microbial antigen tested.CONCLUSIONS:This study demonstrates a mosaic of disease-related serological responses to microbial antigens in patients with CD. Immune responses to commensal enteric bacteria may play a role in the small intestine mucosal damage in CD.

Carbonic anhydrase XII (CA XII) is a transmembrane enzyme that is associated with neoplastic growth. CA XII has been proposed to be involved in acidification of the extracellular milieu, creating an appropriate microenvironment for rapid tumor growth. Because RNA sequence databases have indicated that two isoforms of CA XII might exist in human tissues, and because alternatively spliced protein forms have been linked to aggressive behavior of cancer cells, we designed a study to evaluate the presence of the two forms of CA XII in diffuse astrocytomas, a tumor type known for its aggressive and often noncurable behavior. Reverse transcription PCR of tumor samples surprisingly revealed that CA XII present in diffuse astrocytomas is mainly encoded by a shorter mRNA variant. We further showed by Western blotting that anti-CA XII antibody recognized both isoforms in the glioblastoma cell lines, and we then evaluated the expression of CA XII in astrocytomas using immunohistochemistry and correlated the results with various clinicopathological and molecular factors. Of 370 diffusely infiltrating astrocytomas, 363 cases (98%) showed immunoreactions for CA XII. Importantly, CA XII expression correlated with poorer patient prognosis in univariate (p = 0.010, log-rank test) and multivariate survival analyses (p = 0.039, Cox analysis). From these results, we conclude that CA XII is commonly expressed in diffuse astrocytomas and that it might be used as a biomarker of poor prognosis. The absence of 11 amino acids in the shorter isoform, which seems to be common in astrocytomas, may affect the normal quaternary structure and biological function of CA XII.

We tested the tissue reactions and mechanical strength of a novel biodegradable craniomaxillofacial plating system, Inion CPS™, in the course of degradation. Plates and screws composed of l-lactide, d-lactide and trimethylene carbonate were implanted to the mandible and dorsal subcutis of 12 sheep. The animals were sacrificed at 6–156 weeks. Histological evaluation was done using paraffin and methylmetacrylate techniques. Degradative and mechanical properties during the follow-up were measured both of in vivo and in vitro implants. In light microscopy, the in vivo implant material began to fragment at 52 weeks and could not be detected at 104 weeks. No significant foreign body reactions were seen in the mandibles. The dorsal subcutis disclosed mild reactions, which were, however, not of clinical significance. The implants in vitro maintained their entire mass for 26 weeks and lost 63–80% of the mass by week 104. The inherent viscosity of the implants in vitro and in vivo diminished uniformly. The screws retained their shear strength for 12–16 weeks. The plates maintained their tensile strength for at least 6 weeks. The maximum capacity of the plates in 3-point bending tests diminished gradually by 87% in 26 weeks. In conclusion, the plates and screws examined maintain adequate strength for the healing period of a bone fracture or osteotomy, producing no harmful foreign body reactions.

Carbonic anhydrase isozyme II (CA II) is a cytosolic enzyme that is highly expressed in most organs, including the brain, where it is mainly located in the oligodendrocytes. Recent studies have shown that its expression is induced in the endothelium of neovessels in melanoma and esophageal, renal, and lung cancer. Immunological studies further indicate that CA II represents a major target antigen stimulating an autoantibody response in melanoma patients. These results prompted us to investigate endothelial CA II expression in two types of brain cancer: oligodendrogliomas and astrocytomas. A series of 255 astrocytoma and 71 oligodendroglial tumor specimens was immunostained for CA II. The staining results were correlated with a number of different clinicopathological factors and survival data. CA II showed weak or no expression in low-grade tumors, while grade 3 mixed oligoastrocytoma and glioblastoma multiforme were the most positively stained tumor types. Survival analysis indicated that endothelial CA II staining is significantly associated with a poor prognosis in patients with astrocytomas. About 17% of patients with CA II-negative tumors (weak or no endothelial signal) were still alive at the end of the follow-up period of five years. The presence of CA II in the tumor endothelium suggests that it may play an important functional role in tumor metabolism. From a clinical perspective, the results also open new avenues for selecting tumor types for dendritic cell therapy trials.

The study was aimed to test the mechanical strength, structural stability, and tissue reactions of optically amorphous and crystalline polyetheretherketone (PEEK) plates during a 3-year follow-up in vivo and in vitro. The injection-moulded PEEK plates were implanted to the dorsal subcutis of 12 sheep, which were sacrificed at 6-156 weeks. Thereafter, the plates were subjected to tensile tests, and levels of crystallinity were assessed by differential scanning calorimetry (DSC). Histological evaluation was carried out using the paraffin technique. In vitro properties were examined with the tensile test and DSC at 0-156 weeks. Tissue reactions were mild and fairly similar for the amorphous and crystalline plates at corresponding points in time. The mechanical characteristics of the plates remained stable over the entire follow-up. The tensile yield load and elongation at the yield load of the crystalline plates were roughly double ( approximately 500 vs. 270 N and 2.4 vs. 1.4 mm, respectively) in comparison to the amorphous plates. The elongation at break load of the crystalline plates was smaller than that of the amorphous ones (6 vs. 10). The level of crystallinity in both the optically amorphous ( approximately 15%) and crystalline (32-34%) plates remained invariable during the follow-up. The in vitro and in vivo data coincided remarkably well. In conclusion, both optically amorphous and crystalline PEEK plates are suitable for the fixation of fractures and osteotomies.

Mechanisms of prostate cancer progression during hormonal therapy and the pathobiologic consequences of androgen receptor (AR) gene amplification are inadequately known. To further investigate the hypothesis that AR gene amplification is associated with increased cell proliferation, we analyzed 123 paraffin-embedded prostate cancer specimens from men who experienced tumor relapse during androgen withdrawal therapy. We used fluorescence in situ hybridization to quantify AR gene copy number and Ki-67 immunohistochemistry to determine cell proliferation. One third of the tumors showed AR gene amplification. Among tumors with AR amplification, the mean cell proliferation rate was 19.8 (SD, 12.3; 95% confidence interval [CI], 15.4-24.1), whereas it was 13.0 (SD, 15.9; 95% CI, 9.1-16.8) in tumors without amplification (P = .032). In the best fitting logistic regression model, only proliferation remained significant (P = .040). When the median Ki-67 labeling index (6.7%) of all tumors was used as a cutoff point, the tumors with AR amplification were more frequently highly proliferating than tumors with no amplification (P = .010; odds ratio, 3.4; 95% CI, 1.4-8.3). Our results imply that progression of prostate cancer during androgen withdrawal therapy is associated with AR gene amplification and increased cell proliferation rate in one third of tumors. We suggest that AR gene amplification is an important molecular mechanism underlying the increase in proliferation rate of a substantial fraction of recurrent prostate carcinomas. However, efforts should be targeted to develop prostate cancer cell lines to study causal relationships between AR gene amplification and various biologic variables.

Purpose: Carbonic anhydrase IX (CA IX) is a hypoxia-inducible enzyme, which is associated with neoplastic growth. Ectopic CA IX expression has been observed in several tumors, whose normal counterparts do not express this enzyme. Normal human brain tissue shows only slight or no expression of CA IX.Experimental Design: We describe CA IX expression in human diffusely infiltrating astrocytomas. The association of CA IX is evaluated with clinicopathologic and molecular factors including cell proliferation and apoptosis as well as the expression of p53 and epidermal growth factor receptor.Results: CA IX immunopositivity was observed in 284 cases of 362 (78%) tumors. The positive areas were often located in close proximity to necrotic regions (P < 0.001). The CA IX immunoreactivity showed strong association with tumor malignancy grades (P < 0.0001). CA IX showed no association with p53 expression nor did it correlate with epidermal growth factor receptor-amplification, apoptosis, or cell proliferation. CA IX intensity had significant prognostic value in univariate (P= 0.0011, log-rank test) and multivariate survival analysis (P = 0.038, Cox analysis).Conclusions: CA IX expression is common in diffusely infiltrating high-grade astrocytomas. Our results suggest that CA IX is a useful biomarker for predicting poor prognosis of astrocytic tumors. It may also be a promising target molecule for the improvement of therapeutic interventions in astrocytomas.

1699 Amplification of the 17q23 chromosome region is frequently found in primary breast tumours and has been associated with more advanced disease. The PPM1D gene is located within the epicentre of the amplified region at 17q23 and based on its function represents an attractive putative amplification target gene. PPM1D encodes a serine/threonine protein phosphatase that mediates a negative feedback regulation of the p53 tumour suppressor signalling pathway and thus prevents prolonged activation of p53. PPM1D amplification might therefore represent another mechanism to inactivate p53 in cancer. Here we determined copy numbers and expression levels of PPM1D gene in breast cancer cell lines and primary breast tumours. The PPM1D copy numbers were determined in 27 breast cancer cell lines using interphase FISH and expression levels were determined by Northern blot –method. PPM1D was amplified (3- to 12-fold) in six cell lines (22%). Increased PPM1D expression levels (compared to normal human mammary epithelial cells) were observed in 5 of the 6 cell lines with PPM1D gene amplification. To determine the PPM1D gene copy number levels in primary breast tumours, a tissue microarray containing 147 primary tumour samples was constructed. FISH on the tissue microarray showed PPM1D amplification in 9.8% of tumours, with lobular breast carcinomas showing amplification in 34.5% of the cases. A set of 40 frozen tumour tissue samples, including both amplified and non-amplified cases, was chosen for expression analysis and PPM1D mRNA expression levels will be determined using quantitative real-time PCR (LightCycler). This analysis will allow us to evaluate the possible correlation between PPM1D amplification and overexpression in primary breast tumors. In conclusion, our preliminary results show that the PPM1D gene amplification in breast cancer leads to overexpression of PPM1D.

Androgen action is mediated through androgen receptor (AR), which appears to undergo structural and functional alterations during prostate cancer (CaP) progression. AR mutations have been infrequently reported in CaP before hormonal therapy, but in untreated, advanced tumors AR mutations are suggested to be more common. To investigate the frequency of AR mutations in aggressive CaP before hormonal therapy, we have analyzed AR coding region for aberrations in 21 paraffin-embedded prostate carcinoma samples (14 primary tumors, 7 metastases) of poor histologic differentiation. Single-stranded conformational polymorphism and sequencing analyses revealed AR missense mutations in 29% (4/14) of the primary tumors and in one (14%) metastasis. Mutations resided in the transactivation domain and in the hinge region. One of the hinge region mutants, Ser646Phe, that was identified in a patient with short endocrine therapy response, exhibited a markedly increased transcriptional activity on single androgen response element-containing promoters. In conclusion, AR mutations are frequent in high-grade CaP before initiation of hormonal therapy, and these mutations may play a role in poor therapy response and emergence of hormone-refractory CaP in some cases.