Increase in chromogranin A- and serotonin-positive cells in pouch mucosa of patients with ulcerative colitis undergoing proctocolectomy.

BACKGROUND: Inflammatory bowel disease (IBD) is associated with neuroendocrine cell hyperplasia. AIMS: We investigated neuroendocrine cells in J-pouches of patients with ulcerative colitis undergoing restorative proctocolectomy and ileal pouch-anal anastomosis. METHODS: Sections from pouch biopsies of 17 patients and ileal biopsies of 17 active IBD patients and 16 controls were processed by immunohistochemistry for chromogranin A (CgA) and serotonin. Mucosal tryptophan hydroxylase (TpH)-1 and serotonin-selective reuptake transporter (SERT) transcripts were measured by quantitative RT-PCR. TpH-1 and SERT transcripts were detected in pouch biopsies cultured with infliximab or its isotype control, while interleukin (IL)-6 and IL-8 were measured in biopsy supernatants. RESULTS: A significant increase in CgA-positive cells and serotonin-positive cells was observed in both pouch and IBD ileum compared to control ileum. Significantly raised transcripts of TpH-1, but not SERT, were found in IBD ileum in comparison to control ileum, with no significant difference between pouch and IBD ileum. Infliximab had no influence on ex vivo pouch expression of TpH-1 and SERT, nor on the production of IL-6 and IL-8. CONCLUSION: We here demonstrated neuroendocrine cell hyperplasia in pouch mucosa. Further studies are needed to clarify the pathophysiological implication of this finding.

Four Neuroendocrine Tumor Types and Neuroendocrine Carcinoma of the Duodenum: Analysis of 203 Cases.

BACKGROUND: Several types of neuroendocrine neoplasms (NENs) have been described in the duodenal tract, from low-grade tumors (NETs) to high-grade neuroendocrine carcinomas (NECs). A comprehensive analysis of histology, hormonal profile and prognostic parameters of a sufficiently large duodenal NEN series to cover all main kinds of neoplasms is however lacking. METHODS: We collected a retrospective series of 203 duodenal wall and ampullary region NENs, from six specialized endocrine pathology centers. All were characterized histopathologically and histochemically, and 190 were followed for a median of 9 years. RESULTS: Twenty-seven poorly differentiated NECs, mostly from the ampullary region, were identified and shown to lead to patient demise in a median of 10 months. Among 176 NETs, four subtypes were characterized, including 20 gastrinomas, 37 ampullary-type somatostatin-producing NETs (ASTs), 12 gangliocytic paragangliomas (GPs) and 106 nonfunctioning NETs (nfNETs). ASTs and GPs were mostly localized in the ampullary/periampullary region, while gastrinomas and nfNETs were mainly from the proximal duodenum. ASTs and gastrinomas showed high rates of local infiltration (especially lymphoinvasion and deep duodenal wall/pancreatic tissue invasion) and lymph node metastasis, while nfNETs had significantly lower and more size-dependent local invasive potential. Disease-specific survival differed significantly between NETs and NECs, though not among NET subtypes. NET cases with distant metastases (n = 23) were significantly associated with larger size, higher proliferative grade, lymphovascular invasion, deep invasion and local lymph node metastasis. CONCLUSION: Our careful analysis of a large series of duodenal NENs identified five histologically and prognostically different histotypes of potential clinical relevance.

Chaperone molecules concentrate together with the ubiquitin-proteasome system inside particulate cytoplasmic structures: possible role in metabolism of misfolded proteins.

Ubiquitin-proteasome system (UPS) proteins and proteolytic activity are localized in a recently identified cytoplasmic structure characterized by accumulation of barrel-like particles, which is known as the particulate cytoplasmic structure (PaCS). PaCSs have been detected in neoplastic, preneoplastic, chronically infected, and fetal cells, which produce high amounts of misfolded proteins to be degraded by the UPS. Chaperone molecules are crucial in the early stages of handling misfolded proteins; therefore, we searched for these molecules in PaCSs. Heat shock proteins (Hsp), Hsp90, Hsp70, Hsp40, and Bcl-2-associated athanogene (Bag)3 chaperones, although not Bag6, were selectively concentrated into PaCSs of several cell lines and ex vivo fetal or neoplastic cells. Present findings point to PaCSs as an integrated, active UPS center well equipped for metabolism of misfolded proteins, especially in cells under physiological (fetal development) or pathological (neoplasia or inflammation) stress.

Particle-rich cytoplasmic structure (PaCS): identification, natural history, role in cell biology and pathology.

Cytoplasmic structures showing a selective concentration of both polyubiquitinated proteins and proteasome have been described in various epithelial, hematopoietic, mesenchymal and neural cells in vitro or in fetal tissues, as well as in chronically-infected, mutated preneoplastic and neoplastic tissues. These cytoplasmic structures differ from other ubiquitin-reactive cytoplasmic bodies, like sequestosomes, aggresome-like-induced structures in dendritic cells (DALIS)/non-dendritic cells (ALIS) and aggresomes in showing distinctive ultrastructural organization (particle-rich cytoplasmic structure or PaCS), a cytochemical pattern and a functional profile. Their formation can be induced in vitro in dendritic or natural killer cells by trophic factors and interleukin treatment. They originate in close connection with ribosomes, while, as a result of their growth, the cytoskeleton and other surrounding organelles are usually dislocated outside their core. Interestingly, these particulate cytoplasmic structures are often found to fill cytoplasmic blebs forming proteasome- and polyubiquitinated protein-discharging vesicles, called ectosomes, which are found to detach from the cell and freely float in the extracellular space. To clearly point out the importance of the polyubiquitinated proteins and proteasome containing cytoplasmic structures, their role in cell biology and pathology has been carefully analyzed.

Increase in neuroendocrine cells in the duodenal mucosa of patients with refractory celiac disease.

OBJECTIVES: Several immune-mediated gastrointestinal disorders, including celiac disease (CD), are associated with neuroendocrine cell hyperplasia. However, neuroendocrine cells have never been explored in refractory CD (RCD). METHODS: Serial duodenal sections from 17 patients with RCD (6 type 1 and 11 type 2), 16 uncomplicated CD patients before and after gluten-free diet, 14 patients with potential CD, 27 patients with non-CD villous atrophy, i.e., common variable immunodeficiency (n=12), Whipple's disease (n=10) and giardiasis (n=5), and 16 healthy subjects were processed for the immunohistochemical detection of chromogranin A (CgA), serotonin, and somatostatin. Mucosal tryptophan hydroxylase (TpH)-1 and serotonin-selective reuptake transporter (SERT) transcripts were measured by quantitative reverse transcription-PCR. Serum CgA and 24-h urine 5-hydroxyindoleacetic acid (5-HIAA) were assessed. Biopsies from treated CD patients were cultured with serotonin or peptic tryptic digest of gliadin (PT-gliadin), and interferon (IFN)-γ was detected by ELISA in culture supernatants. RESULTS: Epithelial cells positive for CgA and serotonin, but not somatostatin, were significantly increased in RCD. Raised mucosal transcripts of TpH-1, but not SERT, were found in RCD. On biopsies from treated CD patients, serotonin upregulated IFN-γ production at levels comparable to those induced by PT-gliadin. Serum CgA, but not urine 5-HIAA, was increased in RCD. No significant difference was found between RCD type 1 and type 2 in terms of neuroendocrine cells, mucosal TpH-1 transcripts, and serum CgA. CONCLUSIONS: Serotonin-producing neuroendocrine cells are increased in RCD mucosa. IFN-γ upregulation induced by serotonin suggests that this monoamine may have a role in sustaining the local inflammatory response in CD.

Role of the advanced glycation end products receptor in Crohn's disease inflammation.

AIM: To investigate the level of mucosal expression and the involvement of the receptor for the advanced glycation end products (RAGE) in delayed apoptosis and tumor necrosis factor (TNF)-α production in Crohn's disease (CD). METHODS: Surgical and endoscopic specimens from both inflamed and non-inflamed areas of the ileum and/or colon were collected from 20 and 14 adult CD patients, respectively, and used for the assessment of RAGE expression by means of immunohistochemistry and western blotting analysis. Normal tissues from 21 control subjects were used for comparison. The same polyclonal anti-human RAGE antibody (R and D System) was used in all experimental conditions. RAGE staining was quantized by a score including both the amount of positive cells and intensity of immunoreactivity; cellular pattern was also described. The effects of RAGE blocking on apoptotic rate and TNF-α production were investigated on immune cells freshly isolated from CD mucosa and incubated both with and without the muramyl dipeptide used as antigenic stimulus. Statistical analysis was performed via the test for trend, with regression models to account for intra-patient correlations. A 2-sided P < 0.05 was considered significant. RESULTS: In inflamed areas, RAGE expression in both the epithelial and lamina propria compartments was higher than control tissues (P = 0.001 and 0.021, respectively), and a cluster of positive cells were usually found in proximity of ulcerative lesions. Similar results were obtained in the lamina propria compartment of non-inflamed areas (P = 0.025). The pattern of staining was membranous and granular cytosolic at the epithelial level, while in the lamina propria it was diffuse cytosolic. When evaluating the amount of protein expression by immunoblotting, a significant increase of both surface area and band intensity (P < 0.0001 for both) was observed in CD inflamed areas compared to control tissue, while in non-inflamed areas a significant increase was found only for band intensity (P < 0.005). Moreover, a significantly lower expression in non-inflamed areas in comparison with inflamed areas was found for both surface area and band intensity (P < 0.0006 for both). Finally, RAGE blocking largely affects both the apoptotic rate of mucosal cells (towards an increase in both non-inflamed and inflamed areas of P < 0.001 and < 0.0001, respectively) and TNF-α secretion (towards a decrease in both non-inflamed and inflamed areas of P < 0.05 and < 0.01, respectively), mainly in the presence of antigenic stimulation. CONCLUSION: RAGE is up-regulated in CD, especially in inflamed areas, and it appears to play a role in the mechanisms involved in chronic inflammation.

Histologic changes in type A chronic atrophic gastritis indicating increased risk of neuroendocrine tumor development: the predictive role of dysplastic and severely hyperplastic enterochromaffin-like cell lesions.

The role of putative preneoplastic enterochromaffin-like cell lesions, either hyperplastic or dysplastic, in the genesis of type 1 enterochromaffin-like cell neuroendocrine tumors associated with type A chronic atrophic gastritis, their actual neoplastic risk, and their precise histogenetic mechanism deserve further clarification by specific histopathologic studies coupled with patient follow-up. A total of 100 patients with severe type A chronic atrophic gastritis, enterochromaffin-like cell hyperplasia, and antral G-cell hyperplasia were endoscopically and histologically followed up for a median of 90.1 months (total of 9118 person-months). Preneoplastic enterochromaffin-like cell lesions and newly developed neuroendocrine tumors were investigated histologically and histochemically, in parallel with enterochromaffin-like cell lesions found in nontumor mucosa of another 32 well-characterized and previously reported type 1 neuroendocrine tumors. Both neuroendocrine and nonneuroendocrine mucosa changes were analyzed and statistically evaluated. During follow-up, 7 of 100 patients developed neuroendocrine tumors: 5 were in a group of 20 cases with previous enterochromaffin-like cell dysplasia and 2 were among 80 cases showing only enterochromaffin-like cell hyperplasia throughout the study (hazard ratio, 20.7; P < .001). The severity of enterochromaffin-like cell hyperplasia at first biopsy, with special reference to linear hyperplasia with 6 chains or more per linear millimeter, also increased the risk of neuroendocrine tumor development during follow-up (hazard ratio, 13.0; P < .001). Enterochromaffin-like cell microinvasive dysplastic lesions arising at the epithelial renewal zone level, in connection with immature proliferating mucous-neck cells, were found to be linked to early intramucosal neuroendocrine tumor histogenesis. Both enterochromaffin-like cell dysplasia and severe hyperplasia indicate increased risk of neuroendocrine tumor development in type A chronic atrophic gastritis with hypergastrinemia/G-cell hyperplasia.

The contribution of cell phenotype to the behavior of gastric cancer.

BACKGROUND: Several histochemical studies suggest a role of tumor cell phenotype and related differentiation markers in the prognostic assessment of gastric cancer. Unfortunately, most studies have dealt with single or a few markers and have paid limited attention to their interplay with tumor histological types, which are potentially informative of prognosis. METHODS: In this study, 292 invasive (T1b to T4) gastric cancers with prolonged follow-up and carefully analyzed histotype, inclusive of histotype-based grade, were investigated histochemically with a panel of 14 phenotypic markers known to be expressed in normal gut tissues and gastric cancer. RESULTS: Three of seven intestinal type markers investigated showed a trend for improved prognosis, one of which, CDX2, was stage independent. Three among gastric and pancreatobiliary duct markers (MUC1, MUC6, and pepsinogen II), predicted more severe prognosis stage independently, as did a combination of eight potentially informative (p < 0.1 at univariable Cox analysis) markers. Cancers with predominantly intestinal phenotype had significantly better prognosis than those with predominantly gastric, mixed, or poorly defined phenotypes; among the latter, those with high lymphocyte response, with favorable outcome, were separated from anaplastic cancers, with ominous prognosis. At multivariable analysis, CDX2 and the eight marker combination proved to be stage- and grade-independent predictors. CONCLUSIONS: When individually considered, and with the exception of CDX2, the biomarkers investigated gave an appreciable, although moderate, contribution to the prognostic evaluation of gastric cancer. Combined analysis of all potentially informative markers gave more important information, highly additive to both stage and histotype-based grade.

Mesenchymal stromal cell infusions as rescue therapy for corticosteroid-refractory adult autoimmune enteropathy.

Adult autoimmune enteropathy (AIE) is a rare cause of malabsorption syndrome unresponsive to dietary restriction. Its diagnostic hallmarks are small-bowel villous atrophy and antienterocyte autoantibodies. Therapy is based mainly on nutritional support and immunosuppression. We treated a 61-year-old woman with corticosteroid-refractory AIE and life-threatening malabsorption syndrome with systemic infusions of autologous, bone marrow-derived, mesenchymal stromal cells (MSCs) as rescue therapy. The MSCs were expanded ex vivo following a previously used Good Manufacturing Practice procedure, and 2 intravenous infusions of 1.8 × 10(6) MSCs/kg body weight were administered 2 weeks apart. Analysis of circulating and mucosal regulatory T-and B-cell numbers, and of serum and secretory immunoglobulin levels, was performed before and after treatment. The MSC infusions were safe and effective, leading to disappearance of disease hallmarks and recovery from the life-threatening condition. Increases in mucosal regulatory T-cell numbers and secretory immunoglobulin levels were also observed. The benefit, however, was transient, and a further MSC infusion resulted in the same short efficacy. This case encourages the use of MSCs to treat patients with life-threatening, corticosteroid-refractory AIE and suggests that MSC infusion can attenuate, albeit transiently, the autoimmune attack.

Proteasome particle-rich structures are widely present in human epithelial neoplasms: correlative light, confocal and electron microscopy study.

A novel cytoplasmic structure has been recently characterized by confocal and electron microscopy in H. pylori-infected human gastric epithelium, as an accumulation of barrel-like proteasome reactive particles colocalized with polyubiquitinated proteins, H. pylori toxins and the NOD1 receptor. This proteasome particle-rich cytoplasmic structure (PaCS), a sort of focal proteasome hyperplasia, was also detected in dysplastic cells and was found to be enriched in SHP2 and ERK proteins, known to play a role in H. pylori-mediated gastric carcinogenesis. However, no information is available on its occurrence in neoplastic growths. In this study, surgical specimens of gastric cancer and various other human epithelial neoplasms have been investigated for PaCSs by light, confocal and electron microscopy including correlative confocal and electron microscopy (CCEM). PaCSs were detected in gastric cohesive, pulmonary large cell and bronchioloalveolar, thyroid papillary, parotid gland, hepatocellular, ovarian serous papillary, uterine cervix and colon adenocarcinomas, as well as in pancreatic serous microcystic adenoma. H. pylori bodies, their virulence factors (VacA, CagA, urease, and outer membrane proteins) and the NOD1 bacterial proteoglycan receptor were selectively concentrated inside gastric cancer PaCSs, but not in PaCSs from other neoplasms which did, however, retain proteasome and polyubiquitinated proteins reactivity. No evidence of actual microbial infection was obtained in most PaCS-positive neoplasms, except for H. pylori in gastric cancer and capsulated bacteria in a colon cancer case. Particle lysis and loss of proteasome distinctive immunoreactivities were seen in some tumour cell PaCSs, possibly ending in sequestosomes or autophagic bodies. It is concluded that PaCSs are widely represented in human neoplasms and that both non-infectious and infectious factors activating the ubiquitin-proteasome system are likely to be involved in their origin. PaCS detection might help clarify the role of the ubiquitin-proteasome system in carcinogenesis.