Moris Cadei

Serum From Patients With Severe Heart Failure Downregulates eNOS and Is Proapoptotic Role of Tumor Necrosis Factor-α

BACKGROUND Cytokine activation and endothelial dysfunction are typical phenomena of congestive heart failure (CHF). We tested the hypothesis that incubating human umbilical vein endothelial cells with serum from patients with CHF will downregulate endothelial constitutive nitric oxide synthase (eNOS) and induce apoptosis. METHODS AND RESULTS We studied 21 patients with severe CHF. Levels of tumor necrosis factor-alpha (TNF-alpha) and several neuroendocrine parameters were assessed. eNOS was measured by Western Blot analysis and apoptosis by optical microscopy and flow cytometry. We observed (1) eNOS downregulation (difference versus healthy subjects at 24 hours [P<0.05] and 48 hours [P<0.001]), (2) nuclear morphological changes typical of apoptosis; and (3) a high apoptotic rate with propidium iodide (increasing from 2.1+/-0.4% to 11.3+/-1.2% at 48 hours; P<0.001 versus healthy subjects) and annexin V. An anti-human TNF-alpha antibody did not completely counteract these effects. A strong correlation existed between eNOS downregulation and apoptosis (r = -0.89; P<0.001). CONCLUSIONS Serum from patients with severe CHF downregulates eNOS expression and increases apoptosis. High levels of TNF-alpha likely play a role, but they cannot be the only factor responsible.

p53 is dispensable for apoptosis but controls neurogenesis of mouse dentate gyrus cells following γ-irradiation

Mammalian cells respond to DNA insults by activating cell-cycle checkpoints. This may result in a temporary cell growth arrest which allows DNA repair before proliferation or induces apoptosis. p53 is one of the main contributors in regulating these activities. To get a better insight on the molecular mechanism underlying these activities we studied the role of p53 in apoptosis and neurogenesis of brain cells from adult p53(+/+) or p53(-/-) mice exposed to gamma-irradiation. Apoptosis and neurogenesis were assessed up to 14 days following the injury. Five-ten hours following gamma-irradiation, cells with TUNEL positive nuclei were identified within the subgranular zone of dentate gyrus (DG) of both p53(+/+) and p53(-/-) mice. At the same time-points, pyknotic and shrinking nuclei were visualized by Hoechst 33258 staining. Furthermore, gamma-irradiation increased the number of proliferating cell nuclear antigen (PCNA) positive cells with a peak at 5-10 h in both animal groups. PCNA immunoreactivity was detected in cells exhibiting condensed nuclei as visualized by Hoechst 33258 staining. Neurogenesis, assessed by mitotic marker p34(cdc2) immunoreactivity, showed a biphasic response to gamma-irradiation both in p53(+/+) and p53(-/-) mice which was characterized by an early inhibition and a delayed stimulation. In p53(-/-) mice, the time required by DG granule cells to recover from the lesion and to stimulate proliferation was significantly shortened in comparison with wild-type mice thus resulting in an accelerated neurogenesis. Our data indicate that following gamma-radiation p53 plays a role in regulating cell-cycle progression rate but it is dispensable for promoting apoptosis of DG granule cells.

Colonic mast cells in controls and slow transit constipation patients.

Aliment Pharmacol Ther 2011; 34: 92–99

Application of alternative fixatives to formalin in diagnostic pathology.

Fixation is a critical step in the preparation of tissues for histopathology. The objective of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved the best fixative for morphology. The morphology obtained with Bouin was comparable to that with formalin. Hollande was the best fixative for histochemistry. Bouin proved equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved possible to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis.

HER-2/neu in breast cancer: a comparative study between histology, immunohistochemistry, and molecular technique (FISH).

HER-2/neu is a protooncogene frequently overexpressed in breast cancer. Fluorescence in situ hybridization (FISH) is a technique targeting the gene amplification, while immunohisto-chemistry detects the protein expression. Usually both are applied to paraffin-embedded tissue. The authors studied HER-2 by FISH and immunohistochemistry (HercepTest) in 81 breast carcinomas. The results showed an overall concordance (correlation coefficient 0.64). In all cases with HercepTest score 0 and 1+, nonamplification of the gene was observed. Gene amplification was found in 20% of cases with a 2+ score and in 77.78% of cases with a 3+ score. Data described in literature for 3+ carcinomas showed a 3% to 10% discrepancy between protein expression and gene amplification, while in this study this difference was up to 22.22%. As a consequence, even if it is usually considered important to analyze only 2+ cases by FISH, 3+ scores nonamplified for HER-2/neu may be a new, interesting subset. Furthermore, the authors investigated the two-variables correlation between chromosome 17 copy number, protein over-expression, gene amplification, and presence of metastatic lymph nodes. Interesting results came from the correlation between the HercepTest score and the HER-2/neu gene amplification evaluation, HercepTest and chromosome 17 aneusomy, and gene amplification and lymph nodes status. In conclusion, the FISH technique can be an important and useful diagnostic tool to integrate the results of the HercepTest and to select patients for immunotherapy.

Enteric neuroglial apoptosis in inflammatory bowel diseases.

BACKGROUND Enteric nervous system abnormalities have been described in patients with inflammatory bowel diseases. However, the mechanisms responsible for these abnormalities remain to date largely unknown. AIMS We investigated the potential role of apoptotic phenomena in enteric neurons and enteroglial cells in patients with inflammatory bowel diseases. PATIENTS AND METHODS Full-thickness surgical specimens of 19 patients undergoing surgery for medically refractory disease (9 from the ileum of patients with Crohns disease, 10 from the colon of patients with ulcerative colitis) were assessed for the presence of enteric neurons and enteroglial cells and for their apoptosis by two immunohistochemical methods, one also able to distinguish apoptosis from necrosis. The results were compared with those obtained in control specimens. RESULTS Concerning Crohns disease, the ileal segments displayed a significant increase of apoptotic enteric neurons and enteroglial cells in both the submucous and the myenteric plexus compared to controls. In patients with ulcerative colitis, compared to controls, apoptotic phenomena were significantly reduced in enteric neurons, whereas they were increased in the enteroglial cell population (submucous and myenteric plexus). CONCLUSIONS In patients with inflammatory bowel disease apoptotic phenomena involve both enteric neurons and enteroglial cells, and may play a role in the abnormalities of the enteric nervous system. The importance of these findings in the pathophysiology of these conditions remains to be determined.

An assessment of enteric nervous system and estroprogestinic receptors in obstructed defecation associated with rectal intussusception

Background  The pathophysiological basis of obstructed defecation (OD) is still incompletely understood. In particular, few or no data are available concerning the enteric nervous system (ENS) in this condition. We investigated ENS abnormalities in patients with OD, undergoing surgery, together with the presence of estrogen (α and β) and progesterone receptors, and compare the results with those obtained in controls.

Comparison of three methods to assess enteric neuronal apoptosis in patients with slow transit constipation

Background: The pathophysiological basis of slow transit constipation are scarcely understood. Some recent evidence suggests that increased apoptotic phenomena in the colonic enteric neurons may play a role. However, the best method to assess these phenomena has not been evaluated. Aims. To compare three different methods to detect enteric neuronal apoptosis in these patients. Methods: Serial colonic tissue sections obtained in 10 patients with intractable slow transit constipation were evaluated with immunohistochemical methods aimed at evaluating apoptotic phenomena: the formamide-MAb method, the TUNEL, and the caspase-3. Results: The highest yield of apoptotic neurons was obtained by means of the formamide-MAb method, compared to the other two, whereas the lowest yield was observed with the caspase-3. Conclusions: The formamide-MAb method, which is able to distinguish apoptosis from necrosis and is not influenced by DNA breaks, may prove useful to assess neuronal apoptotic phenomena in the human enteric nervous system. This represents a relevant method to detect enteric neuronal apoptosis.

High-level detection of gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer using FISH: a new approach for retrospective studies

A novel application of fluorescence in situ hybridization (FISH) to isolated nuclei is described. The method detects gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer with greater sensitivity and specificity than existing FISH methods. In this study, the method is applied to signal detection of the HER-2/neu (c-erbB-2) gene, whose amplification is one of the most common genetic alterations associated with human breast cancer. Nuclei were extracted and isolated from formalin fixed, paraffin embedded tissue of 43 different carcinomas (breast, ovary, endometrium, gastrointestinal stromal tumor and malignant mesothelioma). FISH was performed both on sections and extracted nuclei of each tissue using chromosome enumeration probes (CEP) for the centromeric regions of chromosomes 8 and 17, and a locus specific identifier (LSI) for the HER-2/neu oncogene. Differences between ploidy calculated in sections and extracted nuclei were seen in 3 breast carcinomas and 1 gastrointestinal stromal tumor (GIST). Furthermore, 1 breast cancer, previously considered to be borderline for HER-2/neu gene amplification turned out to be clearly amplified. Nuclei extraction and isolation bypass all the problems related to signal interpretation in tissue sections, and the adoption of this new technique, which improves the signal quality in several neoplastic samples, is suggested.

Duodenal histological features in suspected non-celiac gluten sensitivity: new insights into a still undefined condition

Since the late seventies, when gluten-related disorders were first described, up to nowadays, non-celiac gluten sensitivity or non-celiac wheat sensitivity (NCGS/NCWS) is still a not well-defined clinical entity. Epidemiological data are inconsistent providing a wide range of prevalence from 0.5 to 13% in Western countries [1]. The clinical spectrum of this condition is characterized by the presence, in different association, of gastrointestinal (especially bloating and abdominal pain/discomfort with or without alteration in bowel movement) and extra-intestinal symptoms (fatigue, headache, joint pain, foggy mind, and so forth) with a wide overlap of its presentation with irritable bowel syndrome (IBS), other functional gastrointestinal disorders, and other organic etiologies such as bacterial or parasitic infections, autoimmune disorders, and inflammatory bowel diseases (IBD). In June 2015, Salerno Experts’ criteria were published with the proposal of a double (or single)-blind, placebo-controlled, (DBPC), crossover gluten challenge as the gold standard to NCGS diagnosis [2]. According to a recent systematic review, data from 10 DBPC gluten challenge trials were published, revealing still heterogeneity in the methodology of the challenge and highlighting the role of the nocebo effect among these types of studies [1]. In another review article [3], the authors discuss the limitations and the interpretative issues in DBPC gluten challenge studies, suggesting multiple testing to improve accuracy of the results. Given the limitation of DBPC diagnosis criteria, active research in finding systemic biological markers and histological features is ongoing. In a study by Uhde and colleagues, the authors investigated the levels of multiple biomarkers in patients with diagnosis of NCWS according to the criteria of the Salerno consensus [2]: among others IgA, IgM and IgG antibodies to native gliadin (AGA), antibodies to bacterial antigens, lipopolysaccharide-binding protein (LBP), intestinal fatty acid-binding protein (FABP2) were measured before and after 6 months of gluten-free diet. The results of this study suggest the presence of a gluten-induced gut epithelial cell damage with a compromised intestinal permeability [4]. The histologic characteristics of NCGS/NCWS are still under investigations. In a recent editorials, Talley and colleagues reported the presence of lymphocytic duodenosis and increased duodenal eosinophils as the histological hallmark in some cases of NCGS, but underlined the overlap of these findings in patients with functional gastrointestinal disorders [5]. In order to investigate histological findings of people with suspicion of NCGS, we retrospectively evaluated duodenal biopsies of a cohort of patients undergoing clinical diagnostic algorithm for NCGS as proposed by the Salerno consensus.