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Deficiency of vitamin D is associated with increased risk of several types of cancer including colorectal cancer (CRC). However, factors contributing to low levels of 25-hydroxyvitamin D [25(OH)D] in CRC are not clear. Therefore, in this study serum 25(OH)D levels in 117 CRC patients and 86 controls were analyzed and correlated with the clinicopathological data including morphological subtype (serrated or conventional), quantity of tumor infiltrating immune cells, levels of systemic inflammatory markers, and disease outcome. We found that the patients had lower serum 25(OH)D levels compared to the controls. Interestingly, among the patients mismatch repair deficiency, serrated morphology, and high body mass index associated with lowest serum 25(OH)D levels. In addition, patients operated in summer or autumn had higher serum 25(OH)D levels. Furthermore, serum 25(OH)D levels inversely correlated with several systemic inflammatory markers, e.g. serum C reactive protein, but did not associate with prognosis. Mechanism leading to vitamin D deficiency in these patients are not clear but could be related to the effects of systemic inflammation. Longitudinal studies are warranted to assess vitamin D deficiency as a potential risk factor for serrated colorectal polyps and adenocarcinoma.

Aims: Gremlin1 is a bone morphogenetic protein (BMP) antagonist with a suggested role in colorectal cancer (CRC) progression. We have analysed Gremlin1 protein expression in CRC and assessed its correlation with clinicopathological characteristics, including developmental pathway and prognosis., Methods and Results: Material included a non-selected series of 148 surgically treated CRC cases. The tumour-node-metastasis (TNM) stage, histological grade and inflammatory infiltrate at the invasive margin were assessed, and tumours were classified to serrated or non-serrated types. Immunohistochemistry was conducted to evaluate Gremlin1 expression. Prognosis (60-month follow-up) was analysed by Kaplan-Meier methods and Cox regression analysis. Gremlin1 expression was detected in epithelial cells both in normal mucosa and in carcinomas. Abundant expression in carcinomas associated with low TNM stage (P = 0.044), low histological grade (P = 0.044), serrated histology (P = 0.033 or P = 0.053 depending on the classification cut-off) and intensive inflammatory infiltrate at the invasive margin (P = 0.044), and was a stage independent indicator of extended survival (P = 0.029)., Conclusions: Gremlin1 protein expression in CRC associates with low tumour stage and extended survival independently of tumour stage, suggesting that it represents a relevant prognostic indicator in CRC. High expression in carcinomas with serrated histology suggests a potential role for Gremlin1 in the serrated pathway of CRC., (© 2016 John Wiley & Sons Ltd.)

Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated kinase. In murine intestine, DCLK1 marks tuft cells with characteristic microvilli, features of neuroendocrine cells and also quiescent stem cell-like properties. The occurrence and pathological role of DCLK1-positive cells in human intestinal mucosa is unknown. We analysed DCLK1 expression in healthy duodenal, jejunal and colorectal mucosa samples (n = 35), and in duodenal specimens from patients with coeliac disease (n = 20). The samples were immunohistochemically double-stained with DCLK1, and synaptophysin, chromogranin A and Ki-67. Ultrastructure of DCLK1-expressing duodenal cells was assessed using correlative light and electron microscopy. DCLK1 expression was seen in about 1% of epithelial cells diffusely scattered through the intestinal epithelium. Electron microscopy showed that the duodenal DCLK1-positive cells had short apical microvilli similar to neighbouring enterocytes and cytoplasmic granules on the basal side. DCLK1-positive cells were stained with synaptophysin. The number of DCLK1-positive cells was decreased in villus atrophy in coeliac disease. Our findings indicate that in human intestinal epithelium, DLCK1-positive cells form a subpopulation of non-proliferating neuroendocrine cells with apical brush border similar to that in enterocytes, and their number is decreased in untreated coeliac disease., (© 2016 APMIS. Published by John Wiley & Sons Ltd.)

Toll-like receptor 5 (TLR5) recognizes bacterial flagellin. Increased cytoplasmic and nuclear expression of some TLRs has been previously reported in dysplasia in Barrett's esophagus and various malignant lesions in association with survival and metastasis. We assessed nuclear expression of TLR5 in Barrett's esophagus and esophageal adenocarcinoma (n = 94) by immunohistochemistry and immunofluorescent staining. Nuclear expression was observed in the majority of studied lesions with high variation in the proportion of positive nuclei. Expression in lymph node metastases was significantly higher than in esophageal lesions (p < 0.05), except for intestinal metaplasia and low-grade dysplasia. Furthermore, nuclear expression of TLR5 was associated with the presence of lymph node metastases (p = 0.033). In conclusion, we report nuclear TLR5 expression in Barrett's esophagus and adenocarcinoma. Abundance of positive nuclei in association with lymph node metastases suggests that TLR5 is involved in the pathogenesis and dissemination of esophageal adenocarcinoma through as-yet-uncharacterized mechanisms.

Toll-like receptors (TLRs) are innate immune receptors expressed in all parts of the alimentary tract. However, analyses comparing expression in different segments and data on germ-free animals are lacking. Alimentary tract cancers show increased TLR expression. According to the field effect concept, carcinogenetic factors induce subtle cancer predisposing alterations in the whole organ. We studied TLR1 to TLR9 expression in all segments of the alimentary tract from cancer patients' tumor-adjacent normal mucosa, healthy organ donors, and conventional and germ-free mice by using immunohistochemistry and quantitative PCR. All TLRs were expressed in all segments of the alimentary tract. Expression was most intensive in the small intestine in humans and conventional mice, but germ-free mice showed less expression in the small intestine. TLR expression levels were similar in cancer patients and organ donors. We provide systematic baseline data on the TLR expression in the alimentary tract. Normal epithelium adjacent to tumor seems to have similar TLR expression compared with healthy tissues suggesting absence of any field effect in TLR expression. Accordingly, specimens from cancer patients' normal tumor-adjacent tissue can be used as control tissues in immunohistochemical TLR studies in gastrointestinal cancer., (© 2016 The Authors.)

Background: Juvenile idiopathic arthritis is the most common form of chronic arthritis in children. There is mounting evidence that the microbiota may influence the disease., Main Body: Recent observations in several systemic inflammatory diseases including JIA have indicated that abnormalities in the contents of the microbiota may be factors in disease pathogenesis, while other studies in turn have shown that environmental factors impacting the composition of the microbiota, such as delivery mode and early exposure to antibiotics, affect the risk of chronic inflammatory diseases including JIA. Microbial alterations may predispose to JIA through a variety of mechanisms, including impaired immunologic development, alterations in the balances of pro- versus anti-inflammatory bacteria, and low-grade mucosal inflammation. Additional confirmatory studies of microbiota aberrations and their risk factors are needed, as well as additional mechanistic studies linking these alterations to the disease itself., Conclusions: The microbiota may influence the risk of JIA and other systemic inflammatory conditions through a variety of mechanisms. Additional research is required to improve our understanding of the links between the microbiota and arthritis, and the treatment implications thereof.

Increased inflammatory cell infiltration correlates to improved survival in colorectal cancer (CRC). Development and progression of CRC is associated with alterations in serum cytokine levels but their significance is not well defined. In this study, we investigated the relationships between the serum levels of 13 cytokines and the densities of eight types of tumor infiltrating inflammatory cells and their impact on disease-free survival (DFS), cancer-specific survival (CSS) and overall survival (OS) in a prospectively recruited group of 147 CRC patients. There were strong positive correlations between the serum concentrations of different cytokines, as well as between the different types of tumor infiltrating immune cells, whereas the associations between serum cytokines and tumor infiltrating immune cells were generally weak. High serum IL-12 levels associated with increased densities of peritumoral CD8(+) T cells, intraepithelial CD3(+) T cells and intratumoral neutrophils, while high serum CCL4 levels associated with increased densities of peritumoral CD68(+) cells. In multivariate survival models, increased infiltration of intraepithelial CD3(+) T cells and increased serum CCL4 associated with improved DFS, whereas higher intratumoral CD83(+) dendritic cell density and increased serum interferon gamma levels associated with improved CSS and OS. Also high density of peritumoral CD3(+) T cells associated with improved CSS. In conclusion, serum cytokines and tumor infiltrating immune cells in CRC represent entities with high intragroup correlations but relatively weak intergroup correlations. The results suggest that tumor infiltrating CD3(+) T cells, CD83(+) dendritic cells, serum CCL4 and serum interferon gamma represent relevant markers of disease outcome., (© 2016 UICC.)

Background: The disease outcome in colorectal cancer (CRC) can vary in a wide range within the same tumour stage. The aim of this study was to clarify the prognostic value and the determinants of tumour necrosis in CRC., Methods: The areal proportion (%) of tumour tissue showing coagulative necrosis was evaluated in a cohort of 147 CRC patients and correlated with basic clinicopathological characteristics, microvascular density (MVD), cell proliferation rate, KRAS and BRAF mutations, and survival. To validate the prognostic significance of tumour necrosis, an independent cohort of 418 CRC patients was analysed., Results: Tumour necrosis positively correlated with tumour stage (P=8.5E-4)-especially with T class (4.0E-6)-and inversely correlated with serrated histology (P=0.014), but did not significantly associate with cell proliferation rate, MVD, and KRAS or BRAF mutation. Abundant (10% or more) tumour necrosis associated with worse disease-free survival independent of stage and other biological or clinicopathological characteristics in both cohorts, and the adverse effect was directly related to its extent. High CD105 MVD was also a stage independent marker for worse disease-free survival., Conclusions: Tumour necrosis percentage is a relevant histomorphological prognostic indicator in CRC. More studies are needed to disclose the mechanisms of tumour necrosis.

Background: Toll-like receptors (TLRs) recognize microbial and endogenous ligands and have already shown to play a role in esophageal cancer. In this study, we evaluated especially TLRs that sense bacterial cell wall components in Barrett's esophagus, dysplasia and esophageal adenocarcinoma., Methods: TLRs 1, 2, 4 and 6 were stained immunohistochemically and assessed in esophageal specimens from patients with esophageal dysplasia (n = 30) or adenocarcinoma (n = 99). Structures and lesions were evaluated including normal esophagus (n = 88), gastric (n = 67) or intestinal metaplasia (n = 51) without dysplasia, and low-grade (n = 42) or high-grade dysplasia (n = 37), and esophageal adenocarcinoma (n = 99)., Results: We found TLR1, TLR2, TLR4 and TLR6 expression in all lesions. TLR expression increased in Barrett's mucosa and dysplasia. There was profound increase of TLR expression from gastric- to intestinal-type columnar epithelium. In cancers, high nuclear and cytoplasmic staining of TLR4 associated with metastatic disease and poor prognosis., Conclusions: TLR1, TLR2, TLR4 and TLR6 are upregulated during malignant changes of esophageal columnar epithelium. Increased TLR4 expression associates with advanced stage and poor prognosis in esophageal adenocarcinoma., Competing Interests: The authors state no potential conflicts of interest.

Alterations in the intestinal microbial flora have been linked with autoimmune diseases. Our objective was to analyse the composition of the faecal microbiome of children with new-onset juvenile idiopathic arthritis (JIA) compared to healthy controls, and to identify specific gut bacteria associated with JIA. Stool samples from patients were taken at the time of diagnosis of JIA. The microbiome profiles of samples of 30 children with JIA (mean age 6.2 years, 22 girls) were analysed with 16S region-based sequencing profiling and compared to the stool samples of healthy controls (n = 27, mean age 5.4 years, 18 girls). The proportion of bacteria belonging to the phylum Firmicutes was significantly lower in children with JIA [21 % (95 % confident interval [CI]: 17-25 %)] compared to controls [33 % (95 % CI: 26-41 %), p = 0.009]. Bacteria belonging to Bacteroidetes were significantly more abundant in JIA [78 % (95 % CI: 74-82 %)] than in control samples [65 % (95 % CI: 57-73 %), p = 0.008]. Shared operational taxonomic units (OTUs) between the groups revealed that genera Actinobacteria and Fusobacteria were present only in JIA patients and Lentisphaerae only in controls. In summary, faecal flora in JIA is characterised by a low level of Firmicutes and an abundance of Bacteroidetes, resembling the aberration reported in type 1 diabetes. We suggest that alterations in the intestinal microbial flora may challenge the mucosal immune system of genetically susceptible subjects predisposing to local proinflammatory cascades, thus contributing to the development of JIA.

Toll-like receptors (TLRs) are immunological receptors recognizing various microbial and endogenous ligands, such as DNA, RNA, and other microbial and host components thus activating immunological responses. The expression of TLRs in esophageal adenocarcinoma is not well known. The aim of this study was to evaluate expression patterns of those TLRs that sense nucleic acids in Barrett's esophagus with and without dysplasia and in esophageal adenocarcinoma. TLRs 3, 7 and 8 were stained immunohistochemically and evaluated in a cohort of patients with esophageal adenocarcinoma or dysplasia. Specimens with normal esophagus (n = 88), gastric (n = 67) or intestinal metaplasia (n = 51) without dysplasia, and low-grade (n = 42) or high-grade dysplasia (n = 37) and esophageal adenocarcinoma (n = 99) were studied. We used immunofluorescence to confirm the subcellular localization of TLRs. We found abundant expression of TLR3, 7 and 8 in esophageal squamous epithelium, columnar metaplasia, dysplasia and adenocarcinoma. Cytoplasmic expression of TLR3, TLR7 or TLR8 did not associate to clinicopathological parameters or prognosis in esophageal cancer. High nuclear expression of TLR8, confirmed with immunofluorescence, in cancer cells was observed in tumors of high T-stage (p < 0.01) and in tumors with organ metastasis (p < 0.001). High nuclear TLR8 expression was associated with poor prognosis (p < 0.001). The expression of TLR3, TLR7 and TLR8 increased toward dysplasia and adenocarcinoma. We demonstrated nuclear localization of TLR8, which associates with metastasis and poor prognosis. TLR3 and TLR7 do not seem to have prognostic significance in esophageal adenocarcinoma.

Objective: To assess the role of Helicobacter pylori infection and interleukin 6 polymorphism -174 (rs1800795) in dyslipidemia., Design: Case-control study comparing serum lipids between H. pylori positive and negative patients and controlling for IL-6 -174 polymorphism, age, sex and smoking., Setting: 3 hospitals performing outpatient endoscopies in the city of Oulu, Finland., Participants: 199 adult patients with dyspepsia symptoms fulfilling Rome criteria originating from ethnically Finnish population. Patients with an immunosuppressive disorder or malignant disease, treated H. pylori infection, immunosuppressive or anticoagulant medication, previous gastric surgery or ongoing antibiotic treatment were excluded., Primary Outcome Measures: Association of H. pylori infection and serum lipid concentrations in the whole group or in genotype-based subgroups. The associations between peptic ulcer, gastric mucosal inflammation and serum lipid concentrations were assessed as secondary outcomes., Results: The median high-density lipoprotein (HDL) serum concentration was significantly lower in the H. pylori positive group (0.81 mmol/L) than in the negative group (0.95 mmol/L; p<0.001). In the genotype subgroup analyses, a similar association between H. pylori infection and HDL serum levels was seen within the IL-6 -174 CC genotype group (HDL 0.72 vs 1.06 mmol/L, respectively; p<0.001), but no significant associations were seen in the GC or GG genotype groups. Additionally, patients with peptic ulcer demonstrated lower HDL levels (0.75 mmol/L) than H. pylori positive patients without ulcer (0.86 mmol/L; p=0.010)., Conclusions: H. pylori infection associated significantly with low serum levels of HDL in the IL-6 -174 CC genotype patients but not in the other genotypes. This suggests that the association between H. pylori infection and serum HDL could be transmitted through IL-6. We suggest that the role of IL-6 genotype should also be studied in relation to other associations between gastrointestinal microbiome and cardiovascular risk factors., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Fas-mediated induction of apoptosis is a major factor in the selection of lymphocytes and downregulation of immunological processes. In the present study, we have assessed endothelial Fas-ligand (FasL) expression in normal human ileum, appendix, and colon, and compared the expression levels with that in inflammatory bowel disease and in acute appendicitis. In a normal appendix, endothelial FasL levels were constant in almost half of the mucosal vessels; but, in the normal ileum and colon, endothelial FasL was practically restricted to areas in close proximity to lymphatic follicles, and was expressed mainly in the submucosal aspect of the follicles in the vessels with high endothelium. In samples from subjects with either Crohn's disease or ulcerative colitis, the extent of endothelial FasL expression was elevated in the submucosa and associated with an elevated number of lymphoid follicles. In inflammatory bowel disease, ulcers and areas with a high density of mononuclear cells expressing FasL also showed an elevated density of blood vessels with endothelial FasL expression. Although the function of endothelial FasL remains unclear, such a specific expression pattern suggests that endothelial FasL expression has a role in the regulation of lymphocyte access to the peripheral lymphoid tissues, including the intestinal mucosa., (© The Author(s) 2015.)

Background: Toll-like receptor 9 (TLR9) is a cellular receptor, which recognizes bacterial and host-derived DNA. Stimulation of TLR9 induces cellular invasion via matrix metalloproteinase 13 (MMP-13). The aim of this study was to evaluate the role of TLR9 in invasion of oral tongue squamous cell carcinoma (OTSCC)., Methods: The effects of TLR9 ligands on oral squamous cell carcinoma cell lines were studied with invasion and migration assays, as well as in a myoma organotypic model., Results: The TLR9 ligand, CpG-ODN, increased invasion and migration in OTSCC lines. These effects were reduced by TLR9 siRNA or inhibition with TLR9 antibodies. Immunohistochemical analysis of tissues from 195 patients with OTSCC revealed that TLR9 was expressed in 181/195 carcinomas. The expression of TLR9 was higher in the malignant cells than in the normal epithelium. High TLR9 expression was associated with high MMP-13 expression and poor differentiation. High TLR9 expression was also identified as an independent predictor of poor prognosis (HR 1.810, 95% CI [1.053-3.112])., Conclusion: Toll-like receptor 9 mediates OTSCC invasion and migration in vitro and is an independent prognostic factor of OTSCC. Inhibition of TLR9 may be a novel therapeutic opportunity in oral cancer., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Toll-like receptor 4 is a part of the innate immune system and recognizes Helicobacter pylori lipopolysaccharide. The goal of this study was to analyze the role of Toll-like receptor 4 polymorphisms +896 (rs4986790) and +1196 (rs4986791) in the pathogenesis of Helicobacter pylori related gastroduodenal diseases in relation to gastric secretion and inflammation. Toll-like receptor 4 polymorphisms, serum gastrin-17 and pepsinogen I and II concentrations were determined, and gastroscopies with histopathological analyses were performed to 216 dyspeptic patients. As genotype controls, 179 controls and 61 gastric cancer patients were studied. In our study, the Toll-like receptor 4 +896 and +1196 polymorphisms were in total linkage disequilibrium. The homozygous wild types displayed higher gastrin-17 serum concentrations than the mutants (p = 0.001) and this effect was independent of Helicobacter pylori. The homozygous wild types also displayed an increased risk for peptic ulcers (OR: 4.390). Toll-like receptor 4 genotypes did not show any association with Helicobacter pylori positivity or the features of gastric inflammation. Toll-like receptor 4 expression was seen in gastrin and somatostatin expressing cells of antral mucosa by immunohistochemistry. Our results suggest a role for Toll-like receptor 4 in gastric acid regulation and that the Toll-like receptor 4 +896 and +1196 wild type homozygozity increases peptic ulcer risk via gastrin secretion.

Background: Endostatin, a fragment of collagen XVIII, is an endogenous angiogenesis inhibitor with anti-tumour functions. However, elevated circulating endostatin concentrations have been found in several human cancers including colorectal cancer (CRC)., Methods: Serum endostatin levels were measured by enzyme-linked immunoassay from a series of 143 patients with CRC and from 84 controls, and correlated with detailed clinicopathological features of CRC, serum leukocyte differential count and C-reactive protein (CRP) levels., Results: Patients with CRC had higher serum endostatin levels than the controls (P=0.005), and high levels associated with age, tumour invasion through the muscularis propria and poor differentiation, but not with metastases. Endostatin levels showed a positive correlation with the markers of systemic inflammatory response and a negative correlation with the densities of tumour-infiltrating mast cells and dendritic cells. Collagen XVIII was expressed in tumour stroma most strikingly in blood vessels and capillaries, and in the muscle layer of the bowel wall., Conclusions: Elevated endostatin levels in CRC correlate with systemic inflammation and invasion through the muscularis propria. Increased endostatin level may be a result of invasion-related cleavage of collagen XVIII expressed in the bowel wall. The negative correlations between serum endostatin and intratumoural mast cells and immature dendritic cells may reflect angiogenesis inhibition by endostatin.

Background: Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy. Although several biomarkers have been evaluated, histological grade remains the most valuable prognostic marker. Toll-like receptor 9 (TLR9) is an immune receptor recognizing microbial DNA. Its expression associates with prognosis or cancer properties in several cancers. This study examined the role of TLR9 in MEC., Methods: Sixty patients with salivary gland MEC were collected from two Finnish university hospitals, and tumor samples were stained for TLR9. Salivary gland high-grade MEC cell line (UT-MUC-1) was cultured to assess TLR9 and MMP-13 expression. The function of TLR9 was studied in vitro using traditional Matrigel(®) invasion assay and novel human myoma organotypic model., Results: Cancer-specific survival was related with tumor grade (P = 0.01), and there were no deaths in patients with low-grade MEC. TLR9 was expressed in 56 of 60 (93%) tumors. High TLR9 expression indicated better survival in the patient series (P = 0.002) and showed a trend for association with lower disease stage (P = 0.06) and higher differentiation grade (P = 0.068). In multivariate analysis, TLR9 expression was prognostically insignificant due to heavy correlation to disease stage and higher gradus. Treating UT-MUC-1 cells with TLR9 ligand CpG in vitro induced MMP-13 expression and invasion in Matrigel(®) invasion assay, whereas decreased invasion was seen in myoma organotypic model., Conclusion: Functional TLR9 is present in salivary MEC, and high level of expression may indicate good prognosis. However, more studies are needed to evaluate biological consequences of TLR9 interaction in tumor cells., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Severe intestinal mucosal damage and organ failure has been associated in experimental models. Our purpose was to determine whether there is any association between histopathological findings and postoperative mortality among ICU patients undergoing emergency colectomies for various illnesses., Methods: In a retrospective case control study, total colectomy specimens from 50 patients in a mixed ICU were analysed: 18 had sepsis, 11 vascular operations, and 21 Clostridium difficile colitis. Overall thickness, the width of epithelial defects, and presence of cryptal damage were assessed. Extent of necrosis and amount of neutrophils were separately evaluated in the layers of the colonic wall. Clinical features, including sequential organ failure assessment (SOFA) scores and survival, were registered., Results: The histopathological findings for the three clinical entities were similar, except for the abundance of characteristic pseudomembranes in the Clostridium group. Mucosal height (maximum) showed a negative correlation with SOFA score on admission (ρ = -0.296, P = 0.037), and with preoperative blood lactate level (ρ = -0.316; P = 0.027). The nonsurvivors had wider enterocyte defects (60 vs. 40.8, P = 0.002) and more severe crypt damage (61 vs. 27 %; P = 0.024) than the survivors., Conclusions: The histopathological damage involves all layers of the colon wall among ICU patients being largely similar in sepsis, C. difficile infection, and ischemia after vascular operations. Mucosal epithelial damage is associated with clinical severity of the illness and mortality.

A subset of colorectal cancers (CRCs) exhibits so-called Crohn's like lymphoid reaction (CLR), an inflammatory reaction pattern that consists of numerous transmural lymphoid aggregates. However, the composition of these aggregates, their biological mechanisms and their prognostic significance are not well-defined. We analyzed two CRC cohorts (418 and 149 patients) and determined clinicopathological features including survival. A new method for evaluating CLR based on counting the areal density of the lymphoid follicles (CLR density) was adopted. Immune cell densities at intratumoral and peritumoral regions, as well as the composition of the lymphoid follicles, were studied by immunohistochemistry. We found that CLR comprised of lymphoid aggregates with no evidence of granuloma formation. High CLR density associated with lower tumor stage, lack of preoperative radiotherapy or chemoradiotherapy and deficient mismatch repair enzyme expression. CLR density had positive correlations with peritumoral and intratumoral densities of CD83(+) mature dendritic cells and T cells. High CLR density associated with better survival and had prognostic value that was independent of stage, Klintrup-Mäkinen score for peritumoral inflammation and the numbers of tumor infiltrating T cells. CLR density evaluation had excellent intraobserver and interobserver agreement. In conclusion, the results suggest that CLR contributes to the adaptive antitumor immunity. Quantitative evaluation of CLR density is a relevant prognostic indicator in CRC., (© 2013 UICC.)

Toll-like receptor 5 (TLR5) is an immune receptor, which recognizes bacterial flagellin. Increased expression has been reported in various premalignant and malignant lesions indicating a role in carcinogenesis. We assessed the expression of TLR5 in normal esophageal squamous epithelium, Barrett's esophagus with and without dysplasia, and in esophageal adenocarcinoma. Specimens with normal esophagus (n=93), gastric (n=75) or intestinal metaplasia (n=53) without dysplasia, and low-grade (n=56) or high-grade dysplasia (n=33) and esophageal adenocarcinoma (n=94) were studied. TLR5 immunohistochemical stainings were analyzed for the proportion of positive cells and the intensity of expression. In normal squamous epithelium, only the basal third showed TLR5 expression. In esophageal gastric or intestinal metaplasia, expression was present in majority of the cells but significantly weaker (p<0.001) than in dysplastic epithelium. In dysplasia, expression extended to the apical cytoplasm, contrasting basolateral expression in non-dysplastic columnar epithelium. Receiver operating characteristic curve analysis showed that moderate to high expression intensity of TLR5 indicates low-grade dysplasia with 86 % sensitivity and 83 % specificity. Carcinomas showed increased expression in comparison with non-dysplastic columnar epithelium, but there was no association with prognosis. Our results indicate that the esophageal columnar dysplasia is associated with clear increase of TLR5 expression and dissolution of regular polarized expression. TLR5 staining provides a possible biomarker for the recognition of low-grade dysplasia. In addition, the findings suggest a role for abnormal expression of TLR5 in the pathogenesis of esophageal adenocarcinoma and suggest importance of altered microbiome in the pathogenesis of complications of Barrett's esophagus.

The aim of this study was to assess the significance of the interleukin 6 gene polymorphism -174 in gastric cancer risk. The interleukin 6 -174 G/C (rs1800795) gene polymorphisms was analyzed in gastric cancer, peptic ulcer, and nonulcer dyspepsia patients and in healthy control subjects and the data were correlated with the histopathological features of the patients' biopsies. The interleukin 6 -174 GG and GC genotypes have been previously associated with high interleukin 6 serum levels. We discovered that the interleukin 6 -174 GG and GC genotypes are associated with an increased risk of the diffuse histologic subtype of gastric carcinomas (OR: 6.809, P = 0.034), but absent in the intestinal type carcinomas (OR: 1.109, P = 0.908). No significant associations with peptic ulcer, gastric atrophy, or intestinal metaplasia were seen. Our results demonstrate that the interleukin 6 -174 GG and GC genotypes increase the risk of the diffuse type gastric carcinoma, but not the intestinal type gastric carcinoma or its precursor conditions, including atrophy or intestinal metaplasia. Thus, interleukin 6 seems to be an important carcinogenetic factor in the diffuse type gastric adenocarcinoma and its carcinogenetic effect could be noninflammatory., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: Higher-grade inflammatory infiltrate is a promising marker for better prognosis in colorectal cancer (CRC). However, the knowledge on the interrelationships between different inflammatory cells and classifications is fragmentary., Methods: We analysed the densities of eight types of inflammatory cells in a prospectively recruited group of 117 CRC patients and determined their interrelationships and contributions to Klintrup-Mäkinen (K-M) score of overall peritumoural inflammation. We characterised the inflammatory infiltrate in relation to stage and recurrences in 24-month follow-up., Results: There were high positive correlations between the inflammatory cell densities, with the exception of mast cells and CD1a+ immature dendritic cells. High K-M score associated with high peri- and intratumoural densities of CD3+, CD8+, CD68+, CD83+, and FoxP3+ cells and neutrophils. Advanced stage associated with low K-M score, as well as low CD3+, CD8+, CD83+, and FoxP3+ cell counts, of which low K-M score, low CD3(+) T-cell count, and low FoxP3+ T-cell count were linked to higher recurrence rate., Conclusion: The density of CRC inflammatory infiltrate declines as stage advances. Especially, low K-M score and low T-cell counts predict higher recurrence rate. The high positive correlations between the individual inflammatory markers support the value of overall inflammatory reaction scoring.

The objective of this study was to assess the expression of protease inhibitor 9, a granzyme B inhibitor, in human small intestine, and to evaluate its cytoprotective role in the celiac disease of children. Twelve subjects with untreated celiac disease and thirteen healthy controls were examined by endoscopy. The expression of protease inhibitor 9 was analyzed immunohistochemically from duodenal biopsies and compared to granzyme B expression, apoptosis rate, number of intraepithelial lymphocytes and villus and crypt height data from the biopsies. We discovered that protease inhibitor 9 is expressed in the cytoplasm of the duodenal epithelial cells in the majority of cases. The enterocyte expression of protease inhibitor 9 was lower in celiac disease patients than in controls. Protease inhibitor 9 expression also showed a negative correlation with the number of apoptotic cells, overall density of granzyme B expressing intraepithelial lymphocytes, the height of the crypts and the severity of villous atrophy in duodenum. Therefore, we conclude that the protease inhibitor 9 is constantly expressed in the enterocytes of normal duodenum and the expression is decreased in celiac disease. These findings suggest that protease inhibitor 9 has a role in duodenal homeostasis and in the protection of enterocytes from misdirected granzyme B. Indeed, observed associations of lowered protease inhibitor 9 expression together with increased granzyme B expression, apoptosis rate and severity of villous atrophy suggest that impaired balance between granzyme B mediated cytotoxicity and its inhibition by protease inhibitor 9 forms an important factor in the pathogenesis of villous atrophy in celiac disease.

Toll-like receptor 9 (TLR9) recognizes both bacterial and self-DNA and it is abundantly expressed in the gastrointestinal tract. In this study, we investigated the influences of both bacterial DNA and specific short DNA sequences on TLR9-mediated gastrointestinal cancer cell invasion. We assessed the effect of various DNA ligands on cellular invasion and on TLR9 and matrix metalloproteinase expression of three gastrointestinal cancer cell lines. DNA-ligands described in this study include CpG-ODN M362, 9-mer (hairpin), human telomeric sequence h-Tel22 G-quadruplex, and bacterial DNAs from Escherichia coli and Helicobacter pylori. All of the DNAs studied were demonstrated to induce invasion in the studied cells. The DNA-induced invasion was inhibited with a broad-spectrum MMP inhibitor and partly also with chloroquine suggesting that it could be mediated via MMP activation, endosomal signaling, and TLR9. Interestingly, H. pylori DNA was shown to induce a more pronounced invasion in a gastric cancer cell line than in the other cell lines. Our results suggest that bacterial DNA as well as deoxynucleotides having stable secondary structures (i.e. hairpins or G-quadruplex structures) may serve as endogenous, invasion-inducing TLR9-ligands and promote local progression and metastasis of cancers in the alimentary tract., (© 2012 APMIS Published by John Wiley & Sons Ltd.)

Toll-like receptor 5 (TLR5) is an pattern recognition receptor expressed by several cells of the immune system that recognizes bacterial flagellin. We studied the influence of TLR5 in oral tongue squamous carcinoma and found that its expression levels predict patient survival and recurrence. Considering the role of microbiome in oral cancer, TLR5 may represent an important link between bacteria and oral oncogenesis.

Background: Toll-like receptor 5 (TLR5) is an immune receptor recognising bacterial flagellin. Activation of TLR5 results in cancer invasion and cytokine release. As certain bacteria have been linked to oral cancer, we wanted to study TLR5 expression in oral tongue squamous cell carcinoma (OTSCC)., Methods: Samples from 119 patients with OTSCC were obtained, including 101 samples of adjacent normal lingual mucosa. The TLR5 histoscore (0-300) was assessed semiquantitatively by immunohistochemistry in a blinded manner., Results: Toll-like receptor 5 was expressed in 84 normal epithelia and 118 cancer samples. Expression of TLR5 was increased in cancer when compared with normal lingual epithelium (median histoscore 15 vs 135). In cancer, higher TLR5 was associated with age of >70 years at the time of diagnosis, female gender and disease recurrence. No association between TLR5 expression and tumour grade, stage or treatment was found. In multivariate analysis, TLR5 was an independent predictor of cancer mortality (hazard ratio (HR) 3.587, 95% confidence interval (CI) (1.632-7.882)) and disease recurrence (HR 4.455, 95% CI (2.168-9.158))., Conclusion: Toll-like receptor 5 has a previously undescribed role in the pathophysiology of OTSCC and might represent a link between bacteria and cancer. It could be a useful marker for predicting recurrence or survival of OTSCC patients.

We aimed to study intestinal immune activation status in juvenile idiopathic arthritis (JIA) by assessing intestinal human leucocyte antigen (HLA) class II expression and the mRNA expression levels of the pro- and anti-inflammatory mediators and pattern recognition receptors. HLA-D-related (HLA-DR) expression was assessed using immunohistochemical staining of frozen sections in 11 children with JIA and 17 controls. The gene expression levels of the anti- and proinflammatory cytokines, lymphocyte recognition receptors and pattern recognition receptors were studied with reverse transcription-polymerase chain reaction (RT-PCR) in 14 children with JIA and 12 controls. All subjects had various gastrointestinal (GI) symptoms indicating endoscopic examinations, but eventually were not diagnosed with GI disease. In JIA patients, the expression of HLA-DR was increased in the crypt epithelial cells and in the epithelial basement membrane of the ileum when compared with the controls. Positive HLA-DR staining in the ileal mucosa was associated with the presence of high clinical disease activity of JIA and low mRNA expression of anti-inflammatory mediators, such as forkhead box protein P3 (FoxP3), glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR) and transforming growth factor (TGF)-beta. Low ileal expression of interleukin (IL)-10, TGF-β, FoxP3, Toll-like receptor 2 (TLR-2) and TLR-4 transcripts correlated significantly with a high clinical disease activity in the JIA patients. The increased HLA-DR expression suggests enhanced intestinal antigen presentation in JIA. A correlation between clinical disease activity and low gene expression of tolerogenic mediators in the ileum supports the hypothesis that a link exists between the gut immune system and JIA., (© 2012 British Society for Immunology.)

Background: Inflammation contributes to the pathogenesis of colorectal cancer (CRC), and cytokine levels are altered during colorectal carcinogenesis., Methods: The serum levels of 13 cytokines and their relation to clinical and pathological parameters, and systemic inflammatory response (mGPS, CRP and neutrophil-lymphocyte ratio), were analysed from a prospective series of 148 CRC patients and 86 healthy age- and sex-matched controls., Results: CRC patients had higher serum platelet-derived growth factor, interleukin (IL)-6, IL-7, and IL-8 levels and lower monocyte chemotactic protein-1 (MCP-1) levels than the controls. A logistic regression model for discriminating the patients from the controls - including the five most predictive cytokines (high IL-8, high IL-6, low MCP-1, low IL-1ra, and low IP-10) - yielded an area under curve value of 0.890 in receiver operating characteristics analysis. Serum cytokines showed distinct correlation with other markers of systemic inflammatory response, and advanced CRCs were associated with higher levels of IL-8, IL-1ra, and IL-6. A metastasised disease was accompanied by an orientation towards Th2 cytokine milieu., Conclusion: CRC is associated with extensive alterations in serum cytokine environment, highlighting the importance of studying relative cytokine level alterations. Serum cytokine profile shows promise in separating CRC patients from healthy controls but its clinical value is yet to be confirmed.

Background: The histopathologic characteristics of the antral erosions, and a comparison with samples systematically collected from the background antral mucosa, have not been studied previously. Similarly, unknown is the association of these features with suspected etiological factors and chronicity of erosion., Material and Methods: We studied 117 patients with gastric erosions in the absence of peptic ulcer disease. With 28 patients available for a follow-up 19 years later, sites of erosions and background mucosa were biopsied and histopathology of both independently assessed at both visits. Helicobacter pylori status was examined from the biopsies taken in the initial and follow-up gastroscopies. Only subjects originally displaying antral erosions were included. The presence of Herpes simplex virus (HSV) antibodies was analyzed and use of nonsteroidal anti-inflammatory drug (NSAID) was inquired., Results:   Initially, the inflammation was more active in the region of erosions than elsewhere in antral mucosa. More active inflammation in the erosion was associated with HSV seropositivity, Helicobacter pylori infection, and the recent use of NSAIDs. In the follow-up visit, antral erosions were present in 38% (3/8) of Helicobacter pylori negatives and in 35% (7/20) of positives (p = ns). The Helicobacter pylori positive subjects with chronic or recurrent erosions had initially higher scores of neutrophils compared to subjects with nonrecurrent or nonchronic erosions (2.7 ± 0.5 vs 1.2 ± 1.0; p = .002)., Conclusions: Focally enhanced inflammation is characteristic for gastric erosions. This focal inflammation was associated with HSV seropositivity or NSAID use suggesting that such inflammation may be important in the pathogenesis of gastric antral erosions. Highly active inflammation in the erosions associates with their chronicity., (© 2011 Blackwell Publishing Ltd.)

Aims: Toll-like receptor 9 (TLR-9) is a cellular DNA receptor that has been linked previously to invasion in various cancers. The aim of this study was to investigate TLR-9 expression and its possible association with prognosis in oesophageal adenocarcinoma., Methods and Results: Immunohistochemical TLR-9 expression was graded in clinical specimens (n = 76) of oesophageal adenocarcinoma. The TLR-9 immunostaining intensity was compared with tumour grade, stage and indicators of proliferation, apoptosis and tumour vascular supply. High TLR-9 expression correlated with advanced tumour stage, tumour unresectability, poor differentiation and high proliferation. Strong immunoreactivity of TLR-9 also indicated poor overall survival., Conclusions: High TLR-9 expression is associated with poor differentiation, a high proliferation rate and disseminated disease. Accordingly, increased TLR-9 expression may contribute to the growth and metastatic properties of oesophageal adenocarcinoma., (2011 Blackwell Publishing Limited.)

Objectives: Microscopic colitis (MC) is a chronic inflammatory disease with unknown pathogenesis. Very little is known about polymorphisms in the cytokine genes in MC. We have investigated the occurrence of well-characterized polymorphisms of interleukins (IL-6, IL-1β, IL-1 receptor antagonist, IL-10) and CD14 in MC. We also determined the serum IL-6 levels., Methods: We genotyped 81 patients with MC and 178 controls for polymorphisms of IL-6-174, IL-1β-511, IL-1β-3953, IL-1 receptor antagonist, IL-10-1082 and CD14-159. Serum concentration of IL-6 was measured in 72 patients., Results: Genotype GG of IL-6-174 was more prevalent in MC compared with the controls (P=0.030; odds ratio: 1.941; confidence interval: 1.078-3.495), and the frequency of allele G of IL-6-174 was higher in MC (0.55 vs. 0.47; P=0.036; odds ratio: 1.514; confidence interval: 1.041-2.203). However, after correction for multiple comparisons, the difference became nonsignificant. IL-6 genotype and the serum IL-6 concentration showed no association. The concentration of IL-6 was higher in patients with collagenous colitis than in those with lymphocytic colitis (median 1.73 vs. 1.34 pg/ml, P=0.011). No association between polymorphisms of other cytokine genes and MC was seen., Conclusion: The IL-6-174 gene polymorphism has a possible association with MC, as the IL-6 GG genotype was more frequent in patients with the disease. As this genotype may be linked with an enhanced IL-6 production, we speculate that this polymorphism can influence the pathogenesis of MC by evoking a proinflammatory bias in the mucosal cytokines. The enhanced concentration of IL-6 in collagenous colitis compared with lymphocytic colitis supports a difference in the pathogenetic mechanisms between the two subgroups of MC.

Background: We have assessed gastroduodenal, endoscopical and histopathological findings in a series of patients with microscopic colitis (MC)., Methods: We studied 75 patients with MC, 27 with collagenous colitis (CC) and 48 with lymphocytic colitis (LC), and 60 controls. Data of endoscopical findings were collected and biopsies were assessed., Results: Helicobacter pylori infection rate was 15% in MC and 28% in the controls (p = 0.088). Age at diagnosis of MC was higher in H. pylori positive than negative patients (63.4 ± 9.6 vs. 54.4 ± 13.1 years; p = 0.034). Gastric endoscopic erosions were more prevalent in CC than in LC (25.9% vs. 6.2%; p = 0.030) and associated with thick body glands and antral predominance of gastritis in H. pylori positive patients. Rates of focal gastritis (5.6% vs. 6.9%) and lymphocytic gastritis (5.6% vs. 10%) were similar in MC and controls. LC was associated with gastric epithelial lymphocytosis and lymphocytic gastritis. Fifteen patients (20%) had celiac disease., Conclusions: Unlike LC, CC is associated with endoscopic erosions, likely related with the high acid secretion capacity as indicated by the ample body glands and antral predominance of gastritis in H. pylori associated cases of CC. The presence of some divergent gastroduodenal features in LC and CC, and in comparison with those reported in inflammatory bowel disease (IBD), supports the concept that these two conditions differ not only from IBD but also from each other. The findings also suggest the presence of pathogenetic links between colorectal and gastroduodenal abnormalities.

Aims: To define the occurrence of KRAS and BRAF mutations, microsatellite instability (MSI), and MGMT and hMLH1 methylation and expression in colorectal serrated adenocarcinoma., Methods and Results: KRAS codon 12/13 and 59/61 and BRAF V600E mutations, MSI, and MGMT and hMLH1 methylation and expression in 42 serrated adenocarcinomas and 17 serrated adenomas were compared with those in 59 non-serrated colorectal carcinomas (CRCs) and nine adenomas. KRAS and BRAF mutations were observed in 45% and 33% of serrated adenocarcinomas and in 27% and 0% of non-serrated CRCs (P < 0.001). The KRAS c12G→A transition was the predominant type of mutation in serrated adenocarcinomas. Forty-two per cent of BRAF-mutated serrated adenocarcinomas showed high-level MSI (MSI-H) (P = 0.075), 100% showed hMLH1 methylation (P = 0.001) and 90.9% showed MGMT methylation (P = 0.019). Fifty-six per cent of serrated adenocarcinomas with microsatellite stability/low-level microsatellite instability harboured KRAS mutations. In non-serrated cancers, KRAS mutations were not associated with MSI status., Conclusions: A high combined mutation rate (79-82%) of KRAS and BRAF in serrated adenomas and adenocarcinomas indicates that mitogen-activated protein kinase activation is a crucial part of the serrated pathway. BRAF mutations are specific for serrated adenocarcinoma and identify a subset of serrated adenocarcinomas with gene methylation and a tendency for MSI-H. A high frequency of KRAS mutations in serrated adenocarcinomas suggests that a significant proportion of KRAS-mutated CRCs originate from serrated precursors, thus challenging the traditional model of Vogelstein., (© 2011 Blackwell Publishing Limited.)

Colorectal serrated adenocarcinoma forms about 15-20% of colorectal carcinomas. We have previously shown that downregulation of PTCH1 is distinctive for this type of colorectal cancer. In several other tumor types, somatic inactivating PTCH1 mutations have been shown to lead to aberrant Hedgehog signaling, but in colorectal cancer the role of PTCH1 mutations has not been thoroughly studied. Here, we have analyzed the mutation status of PTCH1 in a series of 33 colorectal serrated adenocarcinomas by sequencing all 23 coding exons. We detected 11 previously known SNPs and eight new alterations. The latter included five synonymous changes and two previously unknown missense variations, somatic M319V, and germline V1231A. V1231A was also present in population controls and likely represents polymorphism. The somatic M319V variant does not appear to be an attractive candidate for a disease-associated mutation because in silico analyses did not support the pathogenic nature of the change. A somatic, intronic 1-bp deletion was detected in a short poly(T) stretch in two microsatellite unstable tumors. None of the three changes had predicted effect on splicing when analyzed in silico. Our results did not reveal any clearly deleterious inactivating PTCH1 mutations in our collection of colorectal serrated adenocarcinomas. This suggests that other mechanisms are involved in the observed downregulation of the PTCH1 gene. These might include, e.g., constantly active MAPK signaling by KRAS or BRAF mutations or silencing of PTCH1 by hypermethylation, and further studies are needed to reveal these mechanisms.

Background: Stimulation of Toll-like receptor 9 (TLR9) has been linked to invasion in various cancer cells in vitro. We investigated TLR9 expression in normal, dysplastic and malignant esophageal squamous epithelium., Methods: TLR9 expression was analyzed by immunohistochemistry in 46 cases of esophageal squamous cell carcinoma, including 12 cases with adjacent squamous dysplasia and 24 cases with normal esophageal epithelium. TLR9 expression was compared with tumor grade, stage, proliferation, apoptosis and vascular density., Results: In normal esophageal squamous epithelium, TLR9 staining intensity decreased linearly from the basal layers to the superficial layers (p < 0.001). Strong TLR9 expression was detected across full thickness of high-grade dysplasia, the intensity clearly differing from the normal squamous epithelium and squamous cell carcinoma (p < 0.001). All squamous cell carcinomas exhibited TLR9 expression that was positively associated with a high grade (p < 0.05), the presence of lymph node metastases (p < 0.05) and previously undetected distant metastases (p < 0.05)., Conclusions: Expression of TLR9 in the basal parts of normal esophageal epithelium suggests a role related to cell proliferation and differentation. TLR9 upregulation detected in dysplastic epithelium and in disseminated carcinomas indicates that this protein may serve as a novel marker for esophageal squamous dysplasia and carcinoma with metastatic potential., (Copyright © 2011 S. Karger AG, Basel.)

We have optimized an immunohistochemical double-staining method combining immunohistochemical lymphocyte lineage marker detection and apoptosis detection with terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling. The method was used to trace Fas-mediated apoptosis in human reactive lymph nodes according to cell lineage and anatomical location. In addition to Fas, we also studied the expression of Fas ligand (FasL), CD3, CD20, CD19, CD23, and CD68 of apoptotic cells. The presence of simultaneous Fas and FasL positivity indicated involvement of activation-induced death in the induction of paracortical apoptosis. FasL expression in the high endothelial venules might be an inductor of apoptosis of Fas-positive lymphoid cells. In addition to B-lymphocyte apoptosis in the germinal centers, there was often a high apoptosis rate of CD23-expressing follicular dendritic cells. In summary, our double-staining method provides valuable new information about the occurrence and mechanisms of apoptosis of different immune cell types in the lymph node compartments. Among other things, we present support for the importance of Fas/FasL-mediated apoptosis in lymph node homeostasis.

Objective: Intestinal gamma/delta- intraepithelial lymphocytes (IEL) have increased in children with juvenile idiopathic arthritis (JIA). To further characterise intestinal immune activation in these children, we have quantitated cytotoxic lymphocytes in intestinal mucosa., Methods: We studied 23 children with JIA suffering from gastrointestinal symptoms with gastroduodenoscopy and colonoscopy. The control children (n=20) had GI-symptoms but eventually shown not to have any significant gastrointestinal disease. Granzyme A (GrA) and Granzyme B (GrB) expressing lymphocytes in the epithelium and lamina propria were counted in immunostained sections of ileal and duodenal biopsies., Results: The number of GrB expressing IELs was increased in duodenal mucosa in patients with JIA. In the ileum the number of both GrB and GrA positive IELs was similarly increased. No significant differences in the counts of the lamina propria GrA or GrB expressing cells were observed. Granzyme expression was not associated with the duration or with the severity of the disease, or with medication., Conclusions: These observations suggest that lymphocyte cytotoxicity is abnormally increased in the intestinal mucosa in JIA. Since a similar pattern of activation has been seen in food allergy and celiac disease, we speculate that some luminal, possibly a nutritional factor may be involved in JIA as well. Further studies are needed to see whether cytotoxic activation plays any role in the pathogenesis of JIA.

Objectives: To examine the prevalence of immune activation in gastrointestinal (GI) mucosa in children with juvenile idiopathic arthritis (JIA) or connective tissue disease (CTD)., Study Design: We studied 27 children (15 girls, mean age 9.8+/-4.8 years) with JIA/CTD and GI symptoms, including nine with oligoarthritis, nine with polyarthritis, two with systemic arthritis, three with enthesitis-related arthritis, and four with various CTDs. The control group consists of 54 children (31 girls, mean age 11.3+/-6.3 years) with GI symptoms but shown to have no significant GI or rheumatoid disorder. The subjects were examined by gastroduodenoscopy (22 patients, 50 controls) and colonoscopy (23 patients, 16 controls). Intraepithelial CD3+, alpha/beta+, and gamma/delta+ lymphocytes were counted from duodenal and ileal biopsies., Results: Five patients with JIA/CTD (19%) had ulcerative colitis. Lymphoid nodular hyperplasia (LNH) was more common in the patients [74% (20/27)] than in the controls [16% (8/50), p = 0.001], as well in the duodenal bulb [29% (7/24) vs. 10% (5/50)], terminal ileum [74% (14/19) vs. 38% (5/13)], and the colon [50% (11/22) vs. 14% (2/14)]. In the duodenum, CD3, alpha/beta+, and gamma/delta+ lymphocytes counts were higher in JIA/CTD (p<0.05). In the ileum, gamma/delta+ cell numbers had increased in JIA/CTD (p<0.05). Either LNH, increased gamma/delta+ count, or both were more common in JIA/CTD [89% (24/27)] than in the controls [13% (7/54), p<0.0001]., Conclusions: The majority of children suffering from JIA or CTD with GI symptoms show abnormalities consistent with activation of the intestinal immune system. The aetiology of this reaction remains unknown, but similar features are seen in delayed-type food allergy.

Serrated colorectal carcinomas (CRCs) are morphologically different from conventional CRCs and have been proposed to follow a distinct pathway of CRC formation. Despite studies of single molecular events in this tumor type, the diagnosis of serrated CRC relies on morphology and the putative unique biological character of these tumors has not been established. Here we show that the gene expression profiling of 37 CRCs separated serrated and conventional CRCs into two distinct branches in unsupervised hierarchical clustering (P-value 7.8 x 10(-7)), and revealed 201 differentially expressed genes representing potential biomarkers for serrated CRC. Immunohistochemistry was utilized to verify the key findings in the 37 CRCs examined by expression profiling, and a separate validation set of 37 serrated and 86 conventional CRCs was examined to evaluate the candidate biomarkers in an extended sample material. Ephrin receptor B2, hypoxia-inducible factor 1-alpha and patched appeared as proteins important for genesis of serrated CRC. This study establishes serrated CRCs as a biologically distinct subclass of CRC and represents a step forward in the molecular classification of these cancers. The study also provides a platform to understand the molecular basis of serrated CRC and in long term may contribute to the development of specific treatment options for this tumor type.

Objectives: The aim of the study was to investigate the characteristics of intestinal immune activation (ie, a chemokine receptor and cytokine expression profile) in delayed-type cow's milk allergy (CMA) appearing in the form of gastrointestinal symptoms., Patients and Methods: In all biopsy samples taken from the duodenum and/or the terminal ileum, 30 were studied for the expression of interferon-gamma, transforming growth factor-beta, chemokine receptor (CCR)-4, CCR-5, IL-2, IL-6, IL-10, IL-12p35, IL-12p40 and IL-18 specific mRNA by real-time quantitative reverse transcriptase-polymerase chain reaction in 26 children ages 3 to 15 years: 10 with untreated delayed-type CMA, 6 with celiac disease (CD) and 10 controls., Results: The children with delayed-type CMA showed lower IL-2 and IL-18 mRNA expression in the duodenum (both P = 0.055) and higher CCR-4 and IL-6 mRNA expression in the terminal ileum (P = 0.055, P = 0.016) compared with the controls. The children with CD exhibited slightly higher expression of interferon-gamma and CCR-4 mRNA (P = 0.054, P = 0.053) and lower expression of IL-18 mRNA (P = 0.004) in the duodenal samples compared with the controls. The mRNA expression levels of regulatory cytokines, transforming growth factor-beta and IL-10 remained similar in all 3 groups., Conclusions: The children with delayed-type gastrointestinal CMA showed a unique pattern of local intestinal hypersensitivity with Th2 response-related characteristics, a profile differing clearly from the children with CD.

The precursors for colorectal cancer include polypoid (conventional), flat and serrated adenomas. Polypoid growth in polypoid adenomas and serrated adenomas is associated with K-ras mutations. The regulation of polypoid or nonpolypoid growth is not well known, but could be related to trophic stimuli, such as thyroid hormones. Hence, we investigated the expression pattern of thyroid hormone receptor TRbeta1 in colorectal mucosa and in colorectal tumours and its relationship to tumour growth type. One hundred fourteen colorectal carcinoma specimens were evaluated for TRbeta1. Normal mucosa, adjacent adenomatous component (N = 46) and lymph node metastases (N = 28) were analysed when present, and the results were confirmed by Western blot analysis in selected cases. Nuclear TRbeta1 was almost always present in normal epithelium (96%), but less frequent in adenomas (83%) and in cancer (68%; p < 0.001 and p < 0.001, respectively). TRbeta1 was associated with polypoid growth, presence of K-ras mutations and also with a higher WHO histological grade and advanced Dukes' stage. Cytoplasmic expression of TRbeta1 was observed in nonneoplastic and neoplastic epithelium. In Western blot analysis, a 58 kDa band corresponding to TRbeta1 was expressed in normal mucosa and in colorectal cancer specimens with positive immunohistochemistry. Association of TRbeta1 expression with growth pattern and the presence of K-ras mutations suggest that abnormalities in thyroid hormone signalling involving TRbeta1 play a role in the development of some types of colorectal adenocarcinomas.

Our purpose was to evaluate the long-term clinical significance of gastric erosions. A series of 117 patients with gastric erosions without peptic ulcer disease, and matched controls were studied in 1974-1979. All available subjects were reinvestigated 17 years later, including detailed clinical history and laboratory analysis. At follow-up, erosions were still more prevalent (39%; 20/50) in the erosion group than in the controls (11; 7/66). In Helicobacter pylori-positive participants, peptic ulcer or a scar was more common in the erosion group (17%; 9/52) than in controls (5%; 3/66). Overall malignancy rate was higher in controls (15%; 17/117) than in erosion group (5%; 6/117; P = .025), but no other differences were seen between the groups or related with current erosion. We conclude that a significant proportion of gastric erosions are chronic or recurrent but mostly without serious complications. However, H. pylori-positive patients with erosions have significant risk to develop a peptic ulcer.

The three-dimensional structure, digestibility, and immunological properties of bovine beta-lactoglobulin (beta-lg) are modified by heat treatments used in processing of liquid milk products. Because it is not known if such treatments also modify the intestinal transport properties of beta-lg, the transport of native and heat-denatured bovine beta-lg was investigated in experimental cell models using Caco-2 cells and M cells. Transport of beta-lg labeled with a fluorescent marker was followed with fluorometric measurements, electrophoretic analyses, and fluorescence microscopy. The data show that both cell types transported native beta-lg more efficiently than they did heat-denatured beta-lg. In addition, M cells transported native beta-lg more than Caco-2 cells. Transport of native and heat-denatured beta-lg was transcellular. The electrophoretic data also suggest that heat-denatured beta-lg may have degraded more than native beta-lg during the transport.

Background: Allergic reactions to cow's milk are common in small children. One of the main protein allergens found in cow's milk is beta-lactoglobulin (beta-Lg). Reindeer and bovine milk both contain related beta-Lg proteins, but the allergenicity of reindeer beta-Lg has not previously been studied. The purpose of this study was to analyze the immunological cross-reactivity of IgE antibodies from children with cow's milk allergy to reindeer and bovine beta-Lg., Methods: Sera from 17 children and a serum pool of 4 patients with elevated cow's milk-specific IgE were investigated. Beta-Lg from bovine and reindeer milk was isolated in native form and an enzyme-linked immunosorbent inhibition assay was developed. Bovine beta-Lg was used as a capturing antigen and the inhibiting effects of reindeer and bovine beta-Lg on the IgE binding were measured., Results: Cross-reactivity patterns of bovine milk beta-Lg specific IgE to reindeer beta-Lg varied among patients. In general, reindeer beta-Lg showed significantly lower inhibition (mean 43%) of IgE binding to the capturing antigen than did bovine beta-Lg (mean 89%). In some patients, even high concentrations of reindeer beta-Lg only partly eliminated the IgE binding to bovine beta-Lg., Conclusions: The partial cross-reactivity of human anti-bovine IgE with reindeer beta-Lg suggests that it lacks important bovine epitopes and those that are recognized are only weakly bound.

Objective: Pathogenetic mechanisms of cow's milk protein-sensitive enteropathy (CMSE) are poorly defined, but elevated serum granzyme levels and an increase in duodenal intraepithelial lymphocytes (IELs) expressing TIA-1 suggest the involvement of abnormal lymphocyte cytotoxicity. To evaluate cytotoxicity in CMSE we analysed the expression of cytotoxic granule components in duodenal IELs. For comparison, we studied subjects with coeliac disease (CD), in which lymphocyte cytotoxicity is pathogenically important., Material and Methods: Fifty-four children were examined by endoscopy for gastrointestinal complaints. Twenty-one subjects had a final diagnosis of CMSE, 15 children had untreated CD and 18 controls showed no definite gastrointestinal disease. Mucosal samples furnished from the bulb and descending duodenum were stained for CD3, perforin, granzymes A and B and TIA-1., Results: In both CMSE and CD, increase of mid-duodenal TIA-1, perforin and granzyme A expressing IELs was seen, the counts in CD being much higher, and increased expression was also seen in the bulb. Granzyme B expression was increased only in CD. In CMSE, no evidence of villous atrophy was seen., Conclusions: Increase in duodenal IELs expressing cytotoxic granules is a characteristic feature in CMSE, although to a lesser degree than in CD. Cytotoxicity is suggested to be involved in the pathogenesis of intestinal dysfunction in CMSE, but based on the absence of villous abnormalities may not be mainly targeted to enterocytes. The mechanisms leading to the accumulation of these cells in CMSE need further investigation.

Aims: To illustrate the histopathological features of acute acalculous cholecystitis (AAC) of critically ill patients and to compare them with those of acute calculous cholecystitis (ACC) and normal gallbladders., Methods and Results: We studied 34 gallbladders with AAC and compared them with 28 cases of ACC and 14 normal gallbladders. Histological features were systematically evaluated. Typical features in AAC were bile infiltration, leucocyte margination of blood vessels and lymphatic dilation. Bile infiltration in the gallbladder wall was more common and extended wider and deeper into the muscle layer in AAC compared with ACC. Epithelial degeneration and defects and widespread occurrence of inflammatory cells were typical features in ACC. Necrosis in the muscle layer was also more common and extended wider and deeper in ACC. There were no differences in the occurrence of capillary thromboses, lymphatic follicles or Rokitansky-Aschoff sinuses between the AAC and ACC samples., Conclusions: There are characteristic differences in histopathology between AAC and ACC, although due to overlap, none appeared to be specific as such for either condition. These results suggest that AAC is largely a manifestation of systemic critical illness, whereas ACC is a local disease of the gallbladder.

Colorectal serrated adenocarcinoma originates from serrated adenoma, but definite histological criteria have not yet been established. It presents with frequent DNA microsatellite instability (MSI), but the frequency of low-level (MSI-L) and high-level MSI (MSI-H) and the expression of mismatch-repair (MMR) enzymes in serrated adenocarcinoma are not known. To address these questions, morphological criteria for serrated cancers were established, their validity was tested, and MSI analysis was performed with NIH consensus markers and MMR enzyme immunohistochemistry for hMLH1, hMSH2, and hMSH6 in 35 serrated and 75 non-serrated colorectal carcinomas. Serrated carcinomas frequently showed a serrated, mucinous or trabecular growth pattern; abundant eosinophilic cytoplasm; chromatin condensation; preserved polarity; and the absence of necrosis. With these features, it was possible to distinguish them from non-serrated cancers, with the mean kappa score for five observers being 0.509. MSI analysis was successful in 31 serrated and 73 non-serrated carcinomas. 54.8% of serrated carcinomas were microsatellite-stable (MSS), 29.0% presented with MSI-L, and 16.1% presented with MSI-H, whereas 78.1% of non-serrated carcinomas were MSS, 13.7% were MSI-L, and 8.2% were MSI-H. MSI-L was more common in serrated cancers (p=0.035) and it was associated with patchy immunohistochemical staining (33.3%) of MLH1. MSI-H did not differ between serrated and non-serrated cancers (p=0.14). These results suggest that the biological background of serrated carcinomas differs from sporadic non-serrated colorectal cancer, but is not directly related to MSI., (Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Objective: Gastric erosions are mainly associated with Helicobacter pylori infection and non-steroidal anti-inflammatory drugs (NSAIDs), but there has been no information available on the long-term evolution of gastritis in subjects with erosions., Material and Methods: A series of 117 patients with gastric erosions without peptic ulcer disease and matched controls without erosions or ulcers were studied. Available subjects underwent endoscopy and biopsy 17 years later. Parietal cell antibodies were analysed at the first visit., Results: Fifty-two patients and 67 controls were available for follow-up. Since H. pylori was a major determinant of gastritis, only subjects with unchanged H. pylori status were included in the evaluation of gastritis progression. At the follow-up visit, gastric erosions were present in 38% (16/42) of the patients and 11% (5/46) of the controls (p=0.005). In H. pylori-negative subjects, no evolution of histological changes was seen. In H. pylori-positive subjects, body gastritis was initially less active in the erosion group. With time, antral gastritis worsened only in the erosion group. Parietal cell antibodies were more common in the control group (23%; erosion patients 0%; p=0.01), which also showed worsening of gastritis (p=0.003) and aggravation of atrophy (p=0.002) in the body mucosa., Conclusions: Gastritis in H. pylori-positive subjects with gastric erosions shows evolution of antral predominance, body predominance including development of atrophic changes being rare. Accordingly, patients with erosions share the characteristics of gastritis of the duodenal ulcer phenotype. These findings support the importance of H. pylori and acid in the pathogenesis of gastric erosions in H. pylori-positive patients.

Aim of the Study: Previous work has indicated that quantification of inflammatory cell reaction is of prognostic value in colorectal cancer. We evaluated the prognostic significance of inflammatory cell reaction patterns in colorectal cancer and developed a grading method which could be used in the routine assessment of tumours., Methods: The intensity of overall inflammatory cell reaction, numbers of neutrophilic and eosinophilic granulocytes, lymphoid cells and macrophages in both the central region and the invasive margin were estimated in 386 colorectal cancer patients. Prognostic significance was analysed by uni- and multivariate analysis., Results: Our method for classification of inflammatory reaction was reliable. High-grade inflammation at the invasive margin in Dukes' stage A and B cancers (pT1-2N0 and pT3N0, respectively) was associated with better 5-year-survival (87.6%) than low-grade inflammation (47.0%)., Conclusions: Inflammatory cell response at the invasive border is a relevant prognostic indicator and could be easily incorporated into the routine evaluation of histopathological specimens.