Nod2 influences microbial resilience and susceptibility to colitis following antibiotic exposure.

Inflammatory bowel disease (IBD) etiology involves genetic susceptibility, environmental triggers, and the gut microbiome. Antibiotic exposure is associated with IBD, both in early life and adulthood. Here, we investigated whether Nod2-deficiency influenced response of the gut microbiota to antibiotics and subsequent colitis susceptibility. Wild-type and Nod2-/- littermate mice were treated with amoxicillin as adults or neonates, and fecal samples were collected for 16S rRNA sequencing. Five weeks after antibiotic exposure, dextran sulfate sodium (DSS) colitis was induced. Antibiotic treatment altered the microbiota of adult WT and Nod2-/- mice, but recovery was delayed in Nod2-/- mice. Neonatal antibiotic treatment significantly changed the microbiota at weaning in WT and Nod2-/- littermates; however, Nod2-/- mice maintained reduced microbial diversity 14 days after cessation of antibiotics. Although treatment of adult mice did not influence susceptibility to colitis, neonatally treated Nod2-/- mice developed a more severe colitis. Moreover, the colitis phenotype was transferable through fecal transplantation into germ-free Nod2-/- recipients, and was associated with changes in intestinal T cells and the cytokine milieu following inflammation. These data demonstrate that neonatal antibiotic exposure has long-lasting influence on the microbiota and mucosal immunity, and may explain how NOD2 contributes to the risk of intestinal inflammation.

Malignancies in a renal transplant population: The St. Michael's Hospital experience.

INTRODUCTION: Previous publications have shown an increased incidence of various malignancies amongst renal transplant populations. The objective of this study was to analyze the rate and types of malignancies occurring in the St. Michael's Hospital renal transplant population and to determine whether our results were comparable to those previously published. METHODS: After approval by the hospital's research ethic board, review of the records and pathology of the 1584 patients in the renal transplant clinic database patients was performed. The reports dated back to the year 1970. RESULTS: Amongst the 1584 renal transplant patients, 106 patients with 132 dysplastic and malignant posttransplant lesions were identified. The highest incidence amid the malignancies was in nonmelanoma skin malignancies squamous cell carcinoma (SCC), basal cell carcinoma, and Kaposi sarcoma, with a total of 32 patients having 54 separate tumors (2.02% of all patients, 43.2% of tumors). Following skin tumors in incidence were genitourinary (28 tumors), gastrointestinal tract (GIT) lesions (8 adenocarcinomas, 14 dysplastic lesions, 1 low grade neuroendocrine tumor/carcinoid), posttransplant lymphoproliferative disorders (PTLDs) (10 cases), gynecologic (6 carcinomas), cervical/anal/vulvar dysplasia and invasive (SCCs) (4), and thyroid (3 papillary tumors). Nine patients had tumors of multiple sites/types. With respect to outcome, 14 patients died of malignancy, with the highest mortality being in the GIT malignancies (six patients). Second in mortality were the PTLD and skin tumor groups. DISCUSSION: Information on the incidence and outcome of various malignancies in renal transplant patients is important in designing guidelines for the follow-up of these patients regarding tumor screening and prevention. The rate of malignancies in our group is comparable to that reported in other centers.

Malignancies arising in allograft kidneys, with a first reported translocation RCC post-transplantation: A case series.

BACKGROUND: The increased risk of malignancy in the post-renal transplant population has been well documented. Renal carcinoma is more common in this population, usually arising in native kidneys. Rarely, tumors arise in the transplanted kidney. Our case series reports four cases of malignancy in allograft kidneys, one of which is a first reported case of translocation RCC in a transplanted kidney. METHODS: The renal transplantation database (1584 patients) at St. Michael's Hospital was reviewed for malignancies arising in allograft kidneys: reports and pathology slides were reviewed. RESULTS: Four cases of malignancies arising in the allograft kidney were identified among our kidney transplant population. One patient developed a high grade urothelial carcinoma in the donor kidney post BK virus infection. The other 3 cases were renal cell carcinomas: one clear cell renal cell carcinoma, one translocation renal cell carcinoma, and one papillary renal cell carcinoma. The translocation renal cell cancer had confirmed TFE3 protein over-expression by immunohistochemistry. Molecular testing of the tumors in all 4 cases identified two separate genetic profiles, favored to represent tumors arising from donor tissues along with infiltrating recipient lymphocytes. DISCUSSION: Previous reports suggested that epithelial malignancies in allograft kidneys are rare. We identified 4 such tumors in 1584 transplant patients. Further, we identified the first reported case of translocation RCC in an allograft kidney. While the rate of malignancy in allograft kidneys is low, screening of the donor kidneys by ultrasound and/or urine cytology may be of use in detecting these lesions.

Standardised reporting protocol for endoscopic resection for Barrett oesophagus associated neoplasia: expert consensus recommendations.

Endoscopic resection (ER) is considered the therapy of choice for intraepithelial neoplasia associated with visible lesions and T1a adenocarcinoma. Pathologists are bound to encounter specimens collected via these techniques more frequently in their practice. A standardised protocol for handling, grossing, and assessing ER specimens should be adopted to ensure that all prognostic information and characteristics influencing treatment are included in reports (see Supplementary Video Abstract, http://links.lww.com/PAT/A22). The entire specimen should be appropriately oriented, processed and assessed. An ER specimen will commonly show intraepithelial neoplasia or invasive carcinoma. There are essential features that should be recorded if invasive carcinoma is found as they dictate further management and follow-up. These features are the margin status, depth of invasion, degree of differentiation and presence or absence of lymphovascular invasion. Important features such as duplication of muscularis mucosae should be recognised to avoid misinterpretation of depth of invasion. Key diagnostic and prognostic elements that are essential for optimal clinical decisions have been included in the reporting format proposed by the Structured Pathology Reporting committee of the Royal College of Pathologists of Australasia (RCPA).

Plasma-derived human antithrombin attenuates ventilator-induced coagulopathy but not inflammation in a Streptococcus pneumoniae pneumonia model in rats.

BACKGROUND: Mechanical ventilation exaggerates pneumonia-associated pulmonary coagulopathy and inflammation. We hypothesized that the administration of plasma-derived human antithrombin (AT), one of the natural inhibitors of coagulation, prevents ventilator-induced pulmonary coagulopathy, inflammation and bacterial outgrowth in a Streptococcus pneumoniae pneumonia model in rats. METHODS: Forty-eight hours after induction of S. pneumoniae pneumonia rats were subjected to mechanical ventilation (tidal volume 12 mL kg(-1), positive end-expiratory pressure 0 cmH(2)O and inspired oxygen fraction 40%). Rats were randomized to systemic treatment with AT (250 IU administered intravenously (i.v.) before the start of mechanical ventilation) or placebo (saline). Non-ventilated, non-infected rats and non-ventilated rats with pneumonia served as controls. The primary endpoints were pulmonary coagulation and inflammation in bronchoalveolar lavage fluid (BALF). RESULTS: Pneumonia was characterized by local activation of coagulation and inhibition of fibrinolysis, resulting in increased levels of fibrin degradation products and fibrin deposition in the lung. Mechanical ventilation exaggerated pulmonary coagulopathy and inflammation. Systemic administration of AT led to supra-normal BALF levels of AT and decreased ventilator-associated activation of coagulation. AT neither affected pulmonary inflammation nor bacterial outgrowth from the lungs or blood. CONCLUSIONS: Plasma-derived human AT attenuates ventilator-induced coagulopathy, but not inflammation and bacterial outgrowth in a S. pneumoniae pneumonia model in rats.

Esophageal papillomatosis complicated by squamous cell carcinoma.

Esophageal papillomatosis is a very rare condition that is believed to have a benign clinical course. Recent reports underscore the potential development of a malignancy in association with squamous papillomatosis of the esophagus. A case of esophageal papillomatosis complicated by the development of esophageal invasive squamous cell carcinoma diagnosed after esophagectomy, despite multiple nondiagnostic endoscopic biopsies, is described. The patient also developed squamous cell carcinoma in the oral cavity and pyloric channel. The finding of extensive esophageal papillomatosis and unremitting dysphagia symptoms should prompt investigations into an underlying associated malignancy.

Characterization of tissue autofluorescence in Barrett's esophagus by confocal fluorescence microscopy.

High grade dysplasia and early cancer in Barrett's esophagus can be distinguished in vivo by endoscopic autofluorescence point spectroscopy and imaging from non-dysplastic Barrett's mucosa. We used confocal fluorescence microscopy for ex vivo comparison of autofluorescence in non-dysplastic and dysplastic Barrett's esophagus. Unstained frozen sections were obtained from snap-frozen Barrett's esophagus biopsy samples and scanned with confocal fluorescence microscopy (458 nm excitation; 505-550 nm [green] and > 560 nm [red] emission). Digital micrographs were taken from areas with homogenous and specific histopathology. Visual inspection and statistical analysis were used to evaluate the image datasets. Dysplastic and non-dysplastic Barrett's esophagus epithelia fluoresced mainly in the green spectrum and the main sources of autofluorescence were the cytoplasm and lamina propria. High-grade dysplasia was differentiated from non-dysplastic Barrett's esophagus by microstructural tissue changes. However, there were no specific changes in either the locations or average intensities of intrinsic green and red autofluorescence at the epithelial level that could differentiate between dysplastic and non-dysplastic Barrett's esophagus epithelia, ex vivo. Detectable differences in autofluorescence between BE and dysplasia/cancer in vivo are probably not caused by specific changes in epithelial fluorophores but are likely due to other inherent changes (e.g. mucosal thickening and increased microvascularity) attenuating autofluorescence from the collagen-rich submucosa. Furthermore, confocal fluorescence microscopy provides 'histology-like' imaging of Barrett's tissues and may offer a unique opportunity to exploit microstructural tissue changes occurring during neoplastic transformation for in vivo detection of high-grade dysplasia in Barrett's patients using newly developed confocal fluorescence microendoscopy devices.

Interstitial cells of Cajal in health and disease. Part I: normal ICC structure and function with associated motility disorders.

Ramon y Cajal (1852-1934) is considered to be one of the founders of the field of neuroscience. In 1911, he described interstitial neurons in the gut, noting that they were primitive accessory components that perhaps modify smooth muscle contraction, themselves subject to regulation from principle neurons. The accuracy of his description of their appearance and activities has led to these cells now being called the interstitial cells of Cajal (ICC). Thuneberg and Faussone-Pellegrini were instrumental in bringing these cells to the attention of gastroenterologists and pathologists in the early 1980s. Subsequently, the development of antibodies to c-kit has allowed routine identification of the ICC in pathology specimens. c-Kit is a transmembrane protein kinase which has as ligand stem cell factor and is involved in cell development in a variety of cell lineages. In the gut musculature, ICC and mast cells are the only cells that have prominent c-kit expression. The ICC are now known to play an important role in gut motility and absent or disordered ICC networks have been identified in a variety of motility disorders.

Interstitial cells of Cajal in health and disease. Part II: ICC and gastrointestinal stromal tumours.

Mesenchymal tumours in the gastrointestinal tract have long been problematic in terms of diagnosis, prognosis and therapy, but recent advances in immunohistochemistry and related therapies have allowed more specific diagnosis. In particular, the recognition that both the interstitial cells of Cajal (ICC) and many gastrointestinal stromal tumours (GISTs) are positive for c-kit and CD34 and have other features similar to those of ICC has led to the use of imatinib, a novel small molecule therapy that blocks the CD117/c-kit tyrosine kinase receptor, which shows remarkable efficacy in treatment of malignant and metastatic GISTs as well as other malignancies.

Coeliac disease: an update for pathologists.

Coeliac disease is the manifestation of an immune hypersensitivity reaction towards gluten and related proteins, in genetically predisposed people. Although the precise pathogenesis of this condition remains to be fully elucidated, it is probably multifactorial in origin. The diagnosis of coeliac disease has traditionally depended on intestinal biopsies alone; nowadays, the diagnosis has been expanded to include an array of serological markers. This review is intended to offer pathologists an update of the relevant history and immunopathology pertaining to coeliac disease and also to offer recommendations on the ongoing responsibilities of the pathologist in the diagnosis and reporting of coeliac disease.

Granular cell tumours of the gastrointestinal tract: expression of nestin and clinicopathological evaluation of 11 patients.

AIMS: Granular cell tumours (GCTs) in the gastrointestinal (GI) tract are rare, with few series reported in the literature. Nestin is a recently identified intermediate filament protein that is expressed in neuroectodermal stem cells and skeletal muscle progenitor cells and has been shown to be expressed in gastrointestinal stromal tumours (GISTs) and GI schwannomas. Herein, we describe the clinicopathological and immunohistochemical features of 11 GI GCTs, introducing nestin as an additional marker that identifies these tumours. METHODS AND RESULTS: The archives of the departments of pathology at London Health Sciences Centre (London, Ontario) and St Michael's Hospital (Toronto, Ontario) were searched for GCTs occurring in the GI tract, yielding 11 cases. Histological features were assessed and immunohistochemistry was performed with S100 protein, nestin, glial fibrillary acidic protein (GFAP), CD34, desmin, CD117, and inhibin-alpha. Charts were reviewed for clinical information. Ages at diagnosis ranged from 31 to 73 years; there were six males and four females. All GCTs were solitary, six in the oesophagus, three in the caecum, one in the rectum and one perianal. Most lesions were discovered incidentally. The size of the GCTs ranged from 4 mm to 30 mm. All were submucosal, typically firm, with a white-yellow appearance. Histologically, the GCTs showed moderate cellularity, predominantly solid growth with areas of nesting. While lesional cells were mainly plump and polygonal, areas of spindling were present in several tumours, more frequently in the colorectum. Margins were circumscribed. Nuclei were round to oval, with even chromatin and small nucleoli. Mitoses were rare to absent and necrosis was absent in all cases. Staining with periodic acid-Schiff, with diastase predigestion, showed globular and diffuse positivity within the cytoplasm. Moderate to strong expression of S100 protein and nestin was observed in 11 of 11 and seven of seven tumours, respectively. GFAP, CD34, desmin, CD117 and inhibin-alpha were negative. While patients were variably managed with resection or observation, all remain clinically well, without disease progression. CONCLUSIONS: Although rare, GI GCTs have characteristic clinicopathological features. Nestin may be a useful immunohistochemical marker for identifying these tumours; the presence of this persistent stem cell cytoskeletal filament within GI GCTs suggests that these lesions may arise from a multipotential stem cell in the GI tract.

Increased epithelial uptake of protein antigens in the ileum of Crohn's disease mediated by tumour necrosis factor alpha.

BACKGROUND AND AIMS: The exact nature of the epithelial barrier defect in Crohn's disease remains to be elucidated. Previously we showed increased permeability to proteins in ileal Crohn's disease. Our aims were to study if this barrier defect (a) involves endocytotic uptake of antigens and (b) is related to low grade inflammation not detectable by histology. METHODS: Macroscopically normal segments of distal ileum of Crohn's disease patients (n = 10) were subgrouped into non-inflamed (histologically unaffected) and slightly inflamed tissues and studied in Ussing chambers, with normal ileal specimens from colon cancer patients (n = 9) as controls. Endocytotic uptake into enterocytes of the protein antigen horseradish peroxidase was assessed by measuring the area of horseradish peroxidase containing endosomes in electron photomicrographs. Mucosal tumour necrosis factor alpha (TNF-alpha) mRNA was quantified using real time polymerase chain reaction. For comparison, the effects of low doses of TNF-alpha on endosomal uptake of horseradish peroxidase were studied in cultured T84 cells grown on filter supports. RESULTS: The area of horseradish peroxidase containing endosomes was increased (p<0.001) in enterocytes of non-inflamed ileum of Crohn's disease (2.8 (0.7) mum(2)/300 mum(2)) compared with control ileum (0.6 (0.06)). In non-inflamed mucosa, a significant association between endosomal uptake and mucosal expression of TNF-alpha mRNA (p = 0.03) was found. Low concentrations of TNF-alpha (0.25-1.0 ng/ml) enhanced the endosomal uptake of horseradish peroxidase in polarised T84 cells, without affecting transepithelial electrical resistance. CONCLUSIONS: Our findings suggest increased endosomal uptake of antigens in ileal Crohn's disease that may be mediated by TNF-alpha. These data highlight the transcellular route of antigen uptake in barrier dysfunction and implicate the interaction between epithelial cells and the innate immune system in the development of mucosal inflammation.

Detection of species-specific helicobacter ribosomal DNA in intestinal biopsy samples from a population-based cohort of patients with ulcerative colitis.

The inflammatory bowel diseases are considered an abnormal host immune response to an environmental stimulus. Evidence suggests a role for intestinal bacteria in initiating and/or providing an ongoing stimulus for inflammation in inflammatory bowel disease. Helicobacter pylori is the major cause of active chronic gastritis and peptic ulcers in humans and has been linked to gastric carcinoma and lymphoma. Studies in various animal models, particularly mice, have identified enterohepatic Helicobacter species that are capable of causing hepatitis and enterocolitis. We hypothesize that Helicobacter species may have a role in maintaining inflammation in humans with inflammatory bowel disease. In order to investigate this, biopsy specimens were obtained from patients with and without inflammatory bowel disease. DNA was extracted from the tissues and subjected to PCR with primers designed to detect the ribosomal DNA of members of the Helicobacter species. DNA from six biopsy samples from 60 inflammatory bowel disease patients tested positive. This included 5 of 33 ulcerative colitis patients that were positive compared to 0 of 29 age-matched controls (P < 0.04). Sequencing of the bands produced by PCR amplification revealed >or=99% homology with H. pylori. These results indicate that a member of the Helicobacter species may be involved in some cases of ulcerative colitis.

Loss of CD117 (c-kit)- and CD34-positive ICC and associated CD34-positive fibroblasts defines a subpopulation of chronic intestinal pseudo-obstruction.

Chronic idiopathic intestinal pseudo-obstruction is a syndrome in which symptoms of intestinal obstruction are present in the absence of mechanical obstruction. Lack of normal pacemaker activity, usually generated by the interstitial cells of Cajal (ICC), could account for the apparent obstruction. ICC are normally located around and between the myenteric plexus ganglia and within muscle and also in the deep muscular plexus of the small bowel and the submuscular plexus of the large intestine, just within the circular muscle. ICC can be demonstrated immunohistochemically with CD117 (c-kit) as well as with CD34, although this is less specific. CD34 also stains a population of fibroblasts that are intimately associated with ICC. To determine whether there is a relative deficiency of ICC and CD34-positive fibroblasts in patients with chronic idiopathic intestinal pseudo-obstruction, tissue from 30 patients of large intestine and eight patients with small intestine pseudo-obstruction was obtained. Controls (large intestinal specimens from 12 patients, small intestinal specimens from six patients) were chosen from resections for Crohn's disease and colorectal neoplasia, both with and without dilatation. Examination of pseudo-obstruction cases identified 10 patients (nine large intestinal and one small intestinal) in which both CD117 and CD34 were absent or severely reduced in all three of the examined areas. In contrast, the control cases, including those with preobstructive dilatation, showed relatively constant ICC staining. These results suggest that there is a proportion of pseudo-obstruction cases in which the ICC are markedly reduced. These results also demonstrate that, in these cases, loss of the kit immunoreactivity is correlated with the loss of CD34 staining: this indicates that both the ICC and the CD34-positive fibroblasts associated with the ICC are absent. These findings will allow surgical pathologists to identify this subpopulation of patients with CIIP using tissue obtained by laparoscopic biopsy of the muscularis propria or surgical resection.

Utilization of cytokeratins 7 and 20 does not differentiate between Barrett's esophagus and gastric cardiac intestinal metaplasia.

Long segment Barrett's esophagus (LSBE) is a recognized risk factor for the development of esophageal dysplasia and carcinoma. However, the risk of dysplasia arising within intestinal metaplasia below a normal-appearing Z-line (i.e., in native cardiac mucosa) is unknown. Regular endoscopic surveillance is required in patients with LSBE and is frequently performed in short segment BE (SSBE), but the need for surveillance in cardiac intestinal metaplasia (CIM) is unknown. Unfortunately IM arising in SSBE and immediately below a normal Z-line can be indistinguishable histologically on H&E stains. Previous reports suggest that the appearance of superficial CK20 immunohistochemical staining accompanied by intermediate and deep CK7 positivity is characteristic of BE, whereas CIM specimens show superficial and deep CK20 positivity and weak to absent CK7 staining. We hypothesized that CK7/20 immunostaining of metaplastic biopsies from the esophagus and stomach would allow complete differentiation of these two entities when correlated with the endoscopic appearance. We undertook an evaluation of gastric and esophageal specimens to determine whether these characteristics were valid. Cases of both BE (long and short segment) and CIM, as well as cases of gastric cardiac biopsies lacking IM, were evaluated for CK7 and CK20 and correlated with the endoscopic appearance. We observed that, although the "Barrett's" pattern of CK7/20 was maintained for many cases of BE, the sensitivity and specificity were only moderate (65% and 56%, respectively). The pattern of staining for the CIM was variable, i.e., some cases showed a CK7/20 Barrett's pattern despite a normal appearance at endoscopy. The differences between this and previous studies may be due to inaccurate visualization of SSBE on endoscopy, the development of very early SSBE cases, inter-observer variability, fixation differences, or antibody differences. Whatever the cause of the differences, if results between laboratories are not comparable, CK7/20 immunostaining cannot be used to differentiate reliably between IM present in biopsy specimens taken from above versus below the Z-line. However, further studies should be performed to determine whether the presence or absence of a Barrett's pattern of CK7/20 immunostaining could predict progression to dysplasia or carcinoma.

Telomerase activity as a prognostic factor in neuroblastomas.

Neuroblastoma is the most common extracranial solid tumor of early childhood. This tumor demonstrates significant heterogeneity with respect to pathologic, genetic, and clinical features. The outcome varies from spontaneous regression or maturation to rapid progression, despite aggressive therapy. Prognostic factors have been found that identify those tumors which have a high probability of aggressive behavior; these factors include unfavorable histology, MYCN copy number, deletions of the short arm of chromosome 1, DNA content, and TRK-A (high-affinity receptor protein for nerve growth factor) expression. Recent studies have suggested that high levels of telomerase activity also correlate with poor clinical outcome. We investigated this relationship in 40 patients with untreated neuroblastoma, using a PCR-ELISA assay for telomerase activity. In these patients, 23 tumors had no or minimal telomerase activity whereas 15 had high levels of activity. In two tumors, telomerase activity was not assessable. There was significant correlation between the telomerase activity and MYCN copy number, 1p deletions, and TRK-A expression, as well as patient age, clinical stage, and outcome. The histological classification of the tumors was not significantly different between the two groups, being predominantly unfavorable by the Shimada classification. In addition, for 17 patients tumor tissue was assessed for telomerase activity post-chemotherapy. In those cases where the tumor was negative for telomerase activity before and after chemotherapy, the patients uniformly did well. In cases where the tumor was positive before and negative or weakly positive after treatment, two of the seven patients did well clinically. However, in cases that were positive after chemotherapy, all had recurrence or died. In conclusion, telomerase activity appears to be a prognostic factor for neuroblastomas. In addition, assessment of tumors post-chemotherapy may be a further indicator of clinical outcome.

Malignant melanoma in a patient with oculocutaneous albinism.

BACKGROUND: Sun-induced malignancies (basal cell and squamous cell carcinomas) are common in oculocutaneous albinism, however, the incidence of malignant melanoma is a topic of controversy. OBJECTIVE: We have reviewed the literature and report a case of a woman with oculocutaneous albinism with an amelanotic melanoma of the anterior chest wall. RESULTS: There are only 26 previously reported cases (both case reports and African albino population studies) in 25 patients in the literature. A 27th case with immunohistochemical and ultrastructural evaluation is presented. CONCLUSIONS: It appears that melanoma, a malignancy for which sun exposure and light colouration are felt to be major risk factors, has a low incidence among a population that is both hypopigmented and often exposed to high levels of ultraviolet light. This low incidence is poorly understood and frequently disputed.