Search

Gallbladder cancer (GBC) is one of the deadliest malignancy and treatment options are deplorably limited. Better strategies of prevention are urgently needed but knowledge on risk factors remains scarce. Recent data suggested that arsenic (As) may be involved in GBC carcinogenesis but the question remains debated. To date, there are no data on As measurement in GBC samples. This pilot study aimed to measure As concentrations in tissue samples from patients with GBC compared to non-cancerous gallbladder (NCGB). Included patients underwent cholecystectomy at Hospital Clinico Universidad de Chile, Santiago in Chile, a country with high As exposure, between 2001 and 2020. Tissue samples were preserved in formalin-fixed, paraffin-embedded blocks. Selected samples were retrieved, processed and submitted to inductively coupled plasma mass spectrometry (ICP-MS) to determine As concentrations. A total of 77 patients were included, including 35 GBC and 42 NCGB. The two groups were comparable, except for age (68 vs. 49 years, p 0.001). Measured in 11 GBC and 38 NCGB, total As was detected in 5 GBC (14%) compared to 0 NCGB samples (p 0.001). GBC group also showed higher median values of As compared to NCGB (p 0.001). This pilot study provided a proof-of-concept to measure As concentrations in gallbladder samples and showed higher level of As in GBC samples compared to NCGB, paving the way for future studies aiming to investigate the impact of As on GBC, which may contribute to the prevention of this deadly disease.

Summary Inflammatory bowel diseases (IBDs) are lifelong disorders in which an interaction between genetic and environmental factors is involved. IBDs include two entities: Crohn’s disease (CD) and ulcerative colitis (UC); these can be adequately diagnosed and distinguished with a correct methodological approach based on communicating exhaustive clinical, endoscopic and laboratory information to the pathologist and performing adequate bioptic sampling and precise morphological signs including crypt architecture, distribution of inflammation and granulomas, when present. IBD needs to be distinguished from non-IBD colitis, mostly at its onset. Moreover, IBDs are associated with an increased risk of developing colorectal adenocarcinoma. In daily pathological practice, correct diagnosis of IBD and its subclassification as well as a correct detection of dysplasia is imperative to establish the best therapeutic approach.

Summary Non-IBD colitides (NIBDC) are intestinal diseases clinically and endoscopically overlapping with Inflammatory Bowel Diseases (IBD), sometimes with a similar histological picture. NIBDC include entities such as infectious colitis, ischemic colitis, pseudomembranous colitis, eosinophilic colitis, autoimmune enterocolitis, segmental colitis associated with diverticulosis, drug-induced colitis, radiation-induced colitis, diversion colitis, and microscopic colitis, this last including two entities: collagenous and lymphocytic colitis. The knowledge of the most useful histological features and the main clinical data for each entity is mandatory in daily clinical practice, for correct pathological diagnosis and clinical management.

Simple Summary Therapeutic approaches to increase localization of chemotherapy at the tumor site, reducing systemic toxicity, are under deep investigation. In previous studies, we have shown that microfragmented adipose tissue (MFAT) may act as a natural scaffold able to deliver anti-cancer drugs. We demonstrated that MFAT and its devitalized counterpart (DMFAT) are able to absorb significant amounts of the chemotherapeutic drug Paclitaxel (PTX), with the ability to kill many different human cancer cell lines in vitro and in vivo, preventing tumor relapse when placed in the surgical area of tumor resection. We demonstrated here for the first time that DMFAT loaded with PTX was also very effective in inhibiting the in vivo growth of hepatocellular carcinoma (HCC) in an advanced stage of progression, suggesting it as a new potent and viable drug-delivery system that may be hypothetically translated to treat inoperable primary tumors in humans. Abstract Hepatocellular carcinoma (HCC) is poorly beneficiated by intravenous chemotherapy due to inadequate availability of drugs at the tumor site. We previously demonstrated that human micro-fragmented adipose tissue (MFAT) and its devitalized counterpart (DMFAT) could be effective natural scaffolds to deliver Paclitaxel (PTX) to tumors in both in vitro and in vivo tests, affecting cancer growth relapse. Here we tested the efficacy of DMFAT-PTX in a well-established HCC in nude mice. MFAT-PTX and DMFAT-PTX preparations were tested for anti-cancer activity in 2D and 3D assays using Hep-3B tumor cells. The efficacy of DMFAT-PTX was evaluated after a single-shot subcutaneous injection near a Hep-3B growing tumor by assessing tumor volumes, apoptosis rate, and drug pharmacokinetics in an in vivo model. Potent antiproliferative activity was seen in both in vitro 2D and 3D tests. Mice treated with DMFAT-PTX (10 mg/kg) produced potent Hep-3B growth inhibition with 33% complete tumor regressions. All treated animals experienced tumor ulceration at the site of DMFAT-PTX injection, which healed spontaneously. Lowering the drug concentration (5 mg/kg) prevented the formation of ulcers, maintaining statistically significant efficacy. Histology revealed a higher number of apoptotic cancer cells intratumorally, suggesting prolonged presence of PTX that was confirmed by the pharmacokinetic analysis. DMFAT may be a potent and valid new tool for local chemotherapy of HCC in an advanced stage of progression, also suggesting potential effectiveness in other human primary inoperable cancers.

Endoscopic and histologic remission (HR) are key therapeutic targets in the management of ulcerative colitis (UC). The aim of this study was to evaluate the reproducibility of the Paddington International virtual ChromoendoScopy ScOre (PICaSSO), a virtual chromoendoscopy score originally validated by use of the iSCAN platform, with the narrow-band imaging (NBI), linked-color imaging (LCI), and blue-laser imaging (BLI) platforms.We evaluated endoscopic activity using the Mayo Endoscopic Score (MES), the Ulcerative Colitis Endoscopic Index of Severity (UCEIS), and PICaSSO in 159 UC patients (78 NBI and 81 BLI/LCI) who underwent colonoscopy in 2 tertiary referral centers. HR was defined by the Robarts Histopathology Index (RHI) and the Nancy Histologic Index (NHI). Receiver operating characteristic curves were plotted to evaluate endoscopic scores for the prediction of HR. Intraclass correlation coefficients (ICC) between endoscopists were evaluated.PICaSSO had an ICC of 0.825 when the NBI and BLI/LCI cohorts were combined, higher than MES and UCEIS. The correlation between PICaSSO and RHI and NHI was 0.83 and 0.79 in the NBI cohort and between 0.63 and 0.65 in LCI/BLI. In the NBI cohort, the accuracy of MES, UCEIS, and PICaSSO was 0.936, 0.897, and 0.808 for HR measured by RHI and 0.897, 0.885, and 0.821 by NHI, respectively. In the BLI/LCI cohort, the accuracy of MES, UCEIS, LCI PICaSSO and BLI PICaSSO was 0.765, 0.778, 0.827, and 0.79 to predict HR with RHI and NHI, respectively.The PICaSSO score can be consistently and accurately reproduced with NBI and LCI/BLI and therefore can be applied to all virtual electronic chromoendoscopy platforms.

Pathologists are often called upon to diagnose colitides that differ from the two main forms of inflammatory bowel disease (IBD). These non-IBD colitides include infectious colitis, microscopic colitis, ischemic colitis, eosinophilic colitis, autoimmune enterocolitis, segmental colitis associated with diverticulosis, drug-induced colitis, radiation colitis and diversion colitis. The diagnosis of these different disease entities relies on the histopathological examination of endoscopic biopsies of the gastrointestinal tract. This paper reviews the main histomorphological characteristics of the various Non-IBD colitides.

Background The aim of the study is to report the experience of the pathologists of the Italian Group for the Study of Inflammatory Bowel Disease (IBD) (group formed by pathologists with various experience) on the morphological assessment of digital slides pertaining to IBD and Non-IBD colitis underlining the necessity to implement this tool in daily routine and its utility to share opinions on difficult cases. Materials and Methods Forty-eight histological slides stained with haematoxylin and eosin obtained from ileo-colorectal endoscopic biopsies were digitized using Menarini D-Sight 2.0 system, uploaded onto a website platform and shared among 40 pathologists participating in the study. Information regarding the site of biopsy was disclosed; clinical data were blinded. Each participant was committed to write a comment on microscopic features purposing diagnostic opinion. One month after the last uploaded case, a form was sent to each participant to evaluate the personal experience on digital slide sharing. Results Sixteen pathologists out of 40 (40%) had consistently accessed to the site,9/40 (22%) commented on all slides, a diagnostic opinion was rendered in 8 slides. Most common critical issues were: A) poor internet connection resulting in ineffective evaluation of the digital slides, B) time-consuming cases raising difficult diagnostic interpretation, C) lack of clinical history. Overall, 24 participants (60%) found the forum valuable for practical training and educational purposes. Conclusions Sharing scanned slides circulating within a dedicated forum is an effective educational tool in both IBDs and Non-IBDs colitis. Although our results demonstrated a substantial compliance of the participants, their limited participation was an objective shortcoming. Hence, further efforts are needed to encourage this potentially rewarding practice among the pathologist community.

Autoimmune gastritis (AIG) is a gastric pathologic condition affecting the mucosa of the fundus and the body and eventually leading to hypo-achlorhydria.We report our clinical and pathological experience with AIG.Data from patients with a diagnosis of AIG seen in the period January 2002-December 2012 were retrieved. Only patients with complete sets of biopsies were analyzed.Data from 138 patients were available for analysis. Pernicious anemia was present in 25% of patients, iron deficiency anemia was found in 29.7% of patients, hypothyroidism in 23% of patients, type 1 diabetes in 7.9% of patients, and vitiligo in 2.8% of patients. Parietal cell antibodies were positive in 65% of patients, and no patient had serology positive for celiac disease. All gastric biopsies showed glandular atrophy associated with enterochromaffin-like (ECL)-cells hyperplasia, features limited to the mucosa of the fundus and body, and focal glandular intestinal metaplasia. Helicobacter pylori was negative in all cases.AIG was strongly associated with anemia; atrophy, intestinal metaplasia and ECL hyperplasia in the gastric fundus and body are hallmarks of this condition.

Diagnosis of the inflammatory bowel diseases ulcerative colitis (UC) and Crohn's disease (CD) relies mainly on the histopathological examination of endoscopic biopsies of the gastrointestinal tract. To facilitate the accurate diagnosis of these two conditions, this paper addresses key issues on the: (A) gastrointestinal biopsy procedure, (B) histomorphological characteristics of UC and CD, and (C) diagnosis of dysplasia. The 13 statements presented here represent the consensus of two groups of Italian pathologists (IG-IBD and GIPAD).

Background Although antitumor necrosis factor alfa (TNFα) agents are widely used to treat patients with inflammatory bowel diseases (IBD) - both Crohn's disease (CD) and ulcerative colitis (UC) - there is still some uncertainty in the cell type expressing TNFα in human ileo-colonic segments. Aims We investigated the immunohistochemical (IHC) expression of TNFα in the ileo-colonic segments of patients with both active CD and UC, to establish its anatomic and cellular localization in the inflamed sites. Our aim was to identify patients potentially resistant to anti TNFα agents. Patients and methods Ileo-colonic slides of complete histological mapping of patients with CD and UC before any treatment was started were obtained, and serial sections assessed for TNFα expression, together with IHC markers for lymphocytes, macrophages, and plasma cells. Results TNFα was expressed in almost all inflamed segments of IBD patients, albeit with different strength, and was present, in addition to lymphocytes and, to a lesser extent, to macrophages, in plasma cells, where it had a strong positivity, as also demonstrated by colocalization of specific IHC staining. The expression of TNFα was mostly focal in CD patients and more diffuse in UC patients, likely due to the different patterns of inflammation (transmural and mucosal) of the two entities. Conclusions In IBD, TNFα is strongly expressed also in plasma cells, and it is easily evidenced by conventional IHC techniques. It remains to be established whether this observation might be useful in future to establish in routine biopsy samples whether patients may be responsive to treatments toward this cytokine.

Background: The long non-coding RNA (lncRNA) growth arrest&ndash, specific transcript 5 (GAS5) seems to be involved in the regulation of mediators of tissue injury, in particular matrix metalloproteinases (MMPs), implicated in the pathogenesis of inflammatory bowel disease (IBD). We investigated the role of GAS5 in regulating MMP2 and MMP9 expression in pediatric patients with IBD and in vitro. Methods: In total, 25 IBD patients were enrolled: For each patient paired inflamed and non-inflamed biopsies were collected. RNA was extracted and GAS5, MMP2, and MMP9 were quantified by TaqMan assay. The expression of GAS5 and MMPs was also determined in the human monocytic THP1 cells differentiated into macrophages and stimulated with lipopolysaccharide (LPS). The function of GAS5 was assessed by overexpressing the lncRNA and evaluating the MMPs levels. Results: Real-time PCR results demonstrated a downregulation of GAS5 and an upregulation of both MMPs in inflamed tissues. In vitro data confirmed the trend observed in patients for the three genes: The stimulation with LPS promoted a downregulation of GAS5 while an increase of MMPs was observed. Overexpression experiments showed that higher levels of GAS5 lead to a decrease of both enzymes. Conclusion: These results provide new information about the role of GAS5 in IBD: The lncRNA could mediate tissue damage by modulating the expression of MMPs.

Background It is generally thought that gallbladder motility plays a more or less important role in the pathogenesis of gallstones. Some studies have shown that some abnormalities of its intrinsic innervations, but these studies were usually limited to one cell component. Aims We investigated the main cell components of gallbladder intrinsic innervation in patients with and without gallstones. Methods Archival gallbladder specimens from 39 patients, 27 with gallstones (age range 45–69 yrs) and 12 patients without gallstones (age range 39–71 yrs) were obtained. Full thickness sections were obtained from the gallbladder neck and immunohistochemistry was carried out for enteric neurons (neuron-specific enolase and calretinin), enteric glia (S100) and interstitial cells of Cajal (CD117 and CD34); tryptase staining was also done to distinguish the latter from mast cells. Results Apart from calretinin-positive neurons, patients with gallstones featured a significant decrease of neurons, enteric glial cells (EGC) and mast cells compared to those without gallstones; interstitial cells of Cajal were extremely few and only found in two patients, one for each group. Conclusions The intrinsic innervations of the gallbladder is abnormal in gallstone patients, and this may contribute to gallstone formation in these subjects.

Background: In search of novel biomarkers of response to bevacizumab in metastatic colorectal cancer (mCRC), we analyzed the expression and prognostic role of several proteins related to angiogenesis. Methods: A retrospective, multicenter study on 80 surgical samples from mCRC patients treated in first line with bevacizumab plus chemotherapy was accomplished. The following proteins were analyzed by immunohistochemistry: hERG1 potassium channel, β1-integrin, pAKT, NFkB, HIF-1α, HIF-2α, p53, VEGF-A, GLUT-1, and CA-IX. Data were analyzed in conjunction with the clinicopathological characteristics of the patients, KRAS status, response to bevacizumab, and follow-up. Results: (1) All the proteins were expressed in the samples, with statistically significant associations between HIF-1α and gender, HIF-2α and left colon, hERG1 and VEGF-A, β1-integrin and HIF-2α, GLUT-1 and both HIF-1α and HIF-2α, and CA-IX and VEGF-A. (2) At the univariate analysis, positivity for hERG1, VEGF-A, and the active form of HIF-2α (aHIF-2α), and the G3 histological grade showed a positive impact on progression-free survival (PFS). (3) hERG1 and aHIF-2α maintained their positive impact on PFS at the multivariate analysis. (4) hERG1 behaved as a protective factor for PFS independently on KRAS status. Conclusions: hERG1 and aHIF-2α might help to identify patients who would benefit from bevacizumab treatment.

7-Hydroxymatairesinol (7-HMR) is a plant lignan abundant in various concentrations in plant foods. The objective of this study was to test HMRLignan™, a purified form of 7-HMR, and the correspondingPicea abiesextract (total extractP. abies; TEP) as dietary supplements on a background of a high-fat diet (HFD)-induced metabolic syndrome in mice and in the 3T3-L1 adipogenesis model. Mice, 3 weeks old, were fed a HFD for 60 d. Subgroups were treated with 3 mg/kg body weight 7-HMR (HMRLignan™) or 10 mg/kg body weight TEP by oral administration. 7-HMR and TEP limited the increase in body weight (−11 and −13 %) and fat mass (−11 and −18 %) in the HFD-fed mice. Epididymal adipocytes were 19 and −12 % smaller and the liver was less steatotic (−62 and −65 %). Serum lipids decreased in TEP-treated mice (−11 % cholesterol, −23 % LDL and −15 % TAG) and sugar metabolism was ameliorated by both lignan preparations, as shown by a more than 70 % decrease in insulin secretion and insulin resistance. The expression of several metabolic genes was modulated by the HFD with an effect that was reversed by lignan. In 3T3-L1 cells, the 7-HMR metabolites enterolactone (ENL) and enterodiol (END) showed a 40 % inhibition of cell differentiation accompanied by the inhibited expression of the adipogenic genesPPARγ,C/EBPαandaP2. Furthermore, END and ENL caused a 10 % reduction in TAG uptake in HEPA 1–6 hepatoma cells. In conclusion, 7-HMR and TEP reduce metabolic imbalances typical of the metabolic syndrome and obesity in male mice, whereas their metabolites inhibit adipogenesis and lipid uptakein vitro.

Over the last few years, human microfragmented adipose tissue (MFAT), containing significant levels of mesenchymal stromal cells (MSCs) and obtained from fat lipoaspirate (LP) through a minimal manipulation in a closed system device, has been successfully used in aesthetic medicine as well as in orthopedic and general surgery. Interestingly, in orthopedic diseases, this ready-to-use adipose tissue cell derivative seems to have a prolonged time efficacy even upon a single shot injection into osteoarthritic tissues. Here, we investigated the long-term survival and content of MSCs as well the anti-inflammatory activity of LP and its derived MFAT in vitro, with the aim to better understand a possible in vivo mechanism of action. MFAT and LP specimens from 17 human donors were investigated side by side. During a long-term culture in serum-free medium, we found that the total cell number as well the MSC content in MFAT decreased more slowly if compared to those from LP specimens. The analysis of cytokines and growth factors secreted into the conditioned medium (CM) was similar in MFAT and LP during the first week of culture, but the total amount of cytokines secreted by LP decreased much more rapidly than those produced by MFAT during prolonged culture (up to 28 days). Similarly, the addition of MFAT-CM recovered at early (3-7 days) and late stage (14-28 days) of culture strongly inhibited inflammatory function of U937 monocyte cell line, whereas the anti-inflammatory activity of LP-CM was drastically reduced after only 7 days of culture. We conclude that MFAT is an effective preparation with a long-lasting anti-inflammatory activity probably mediated by a long-term survival of their MSC content that releases a combination of cytokines that affect several mechanisms involved in inflammation processes.

Although glucocorticosteroids (GS) and mesalazine are effective and widely employed to treat moderate-to-severe ulcerative colitis (UC), information regarding the factors responsible for response to such therapy is still scarce. One of these factors is thought to be an increased number of mucosal eosinophils. The aim of our study was to determine whether the presence of hypereosinophilia in colonic mucosa of UC patients might influence the short-term response to l treatment with GS and mesasalazine. Clinical, endoscopic, and pathologic data from patients with a recent diagnosis of moderate UC, who had not undergone treatment, were obtained, and the short-term outcome after 1 month of conventional first-line treatment (mesalazine plus GS) was evaluated. There were 53 patients with a median age of 37 years (95% CI 30–47).Overall, at the end of treatment period 16 (30%) patients responded, whereas a response was not observed in the other 37 (70%) patients. Interestingly, all patients of this latter group had colonic mucosal hypereosinophilia. No significant differences were found between the two groups concerning sex and age at diagnosis, but hypereosinophilia was inversely correlated with the duration of the disease (p = 0.054), and significantly correlated to the localization of UC (p = 0.0023). In addition, The Mayo score was significantly higher in patients with hypereosinophilia (median 8; 95% CI 8–9;) when compared to patients without hypereosinophilia (median 7; 95% CI 7–7, p

The concept of remission for patients with inflammatory bowel diseases has recently evolved, and should also include histological healing of the mucosa, difficult to evaluate since there is no agreement on pathological scores and those available are quite complex to use in the daily routine. We evaluated the possible usefulness of a simplified pathological score to assess histological healing of the mucosa in inflammatory bowel diseases patients compared with four commonly proposed pathological scores. Slides from 24 patients (12 Crohn’s disease, 12 ulcerative colitis, age range 24–62 years), pre- and post-treatment with biological agents and displaying endoscopic remission were assessed by two pathologists. Pre- and post-treatment results and the time employed to calculate the various scores were obtained. All scores were useful to document highly significant post-treatment decreases of histological activity. However, the simplified score needed significant less time to be calculated for each slide, had high inter-rater agreement, and avoided subjectivity from the pathologists. The simplified score is easy to calculate and seems apt to document histological healing of the mucosa, in a manner similar to the more complex scores. It remains to be established whether this score could simplify the daily routinary practice in this context.

Background Epithelial ovarian cancer (EOC) is a spectrum of different diseases, which makes their treatment a challenge. Forkhead box M1 (FOXM1) is an oncogene aberrantly expressed in many solid cancers including serous EOC, but its role in non-serous EOCs remains undefined. We examined FOXM1 expression and its correlation to prognosis across the three major EOC subtypes, and its role in tumorigenesis and chemo-resistance in vitro. Methods Gene signatures were generated by microarray for 14 clear-cell and 26 endometrioid EOCs, and 15 normal endometrium snap-frozen biopsies. Validation of FOXM1 expression was performed by RT–qPCR and immunohistochemistry in the same samples and additionally in 50 high-grade serous EOCs and in their most adequate normal controls (10 luminal fallopian tube and 20 ovarian surface epithelial brushings). Correlations of FOXM1 expression to clinic-pathological parameters and patients’ prognosis were evaluated by Kaplan-Meier and Cox proportional-hazards analyses. OVCAR-3 and two novel deeply characterized EOC cell lines (EOC-CC1 and OSPC2, with clear-cell and serous subtype, respectively) were employed for in vitro studies. Effects of FOXM1 inhibition by transient siRNA transfection were evaluated on cell-proliferation, cell-cycle, colony formation, invasion, and response to conventional first- and second-line anticancer agents, and to the PARP-inhibitor olaparib. Gene signatures of FOXM1-silenced cell lines were generated by microarray and confirmed by RT-qPCR. Results A significant FOXM1 mRNA up-regulation was found in EOCs compared to normal controls. FOXM1 protein overexpression significantly correlated to serous histology (p = 0.001) and advanced FIGO stage (p = 0.004). Multivariate analyses confirmed FOXM1 protein overexpression as an independent indicator of worse disease specific survival in non-serous EOCs, and of shorter time to progression in platinum-resistant cases. FOXM1 downregulation in EOC cell lines inhibited cell growth and clonogenicity, and promoted the cytotoxic effects of platinum compounds, doxorubicin hydrochloride and olaparib. Upon FOXM1 knock-down in EOC-CC1 and OSPC2 cells, microarray and RT-qPCR analyses revealed the deregulation of several common and other unique subtype-specific FOXM1 putative targets involved in cell cycle, metastasis, DNA repair and drug response. Conclusions FOXM1 is up-regulated in all three major EOCs subtypes, and is a prognostic biomarker and a potential combinatorial therapeutic target in platinum resistant disease, irrespective of tumor histology. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0536-y) contains supplementary material, which is available to authorized users.

The association between gluten related disorders and psychiatric diseases has been firmly demonstrated. Non-celiac gluten sensitivity (NCGS) is a syndrome diagnosed in patients responsive to gluten-free diet after ruling out celiac disease and wheat allergy. The pathogenesis of neuro-psychiatric disorders in NCGS is unclear. An association between gluten and schizophrenia was described for the first time in 1950 by Bender et al. In the 1950's, Dicke noted that gluten-free diet improved mood in celiac patients. In 1970, Goldberg et al., in a study of 80 celiac patients, found that 34% of them showed minor affective disorders. Bipolar disorder patients show an increase of blood anti gliadin deamidated antibodies (IgG). The effect of diet and nutrition on autistic spectrum disorders has been investigated in the last two decades, particularly focusing on the symptoms of hyperactivity and attention. Toxoplasma gondii and other neurotropic pathogens as Influenzavirus and Coronavirus may be associated with mood disorders, probably secondary to an increased intestinal permeability. Abnormalities of host-microbiota interactions or of gut-microbiota composition have been associated with central nervous system disorders, such as autism, anxiety, depression and the integrity of intestinal microbiota may be considered a potential therapeutic goal to treat these conditions.

Basal plasmacytosis is an early-onset and highly predictive feature of inflammatory bowel disease (IBD), but may have several restrictions in routine histology. Considering evidences about cooperation between eosinophils and plasma cells in IBD pathogenesis, we investigated immunostain of these two cells as a marker of disease. 343 samplings from 83 patients (52 IBD, 31 non-IBD colitis) were evaluated. The sections were stained with monoclonal antibodies against plasma cells (CD138 and MUM1), and eosinophils (CD193). Eosinophilia-associated basal plasmacytosis (EBP) was related with the histologic diagnosis of IBD (90.3% IBD and 35.4% non-IBD colitides, p < 0.005, sensitivity 90.4%). A strong relation was detected between the occurrence of EBP and (i) the achieving of a complete endoscopic mapping; (ii) the presence of other characteristic lesions of IBD in single segmental sampling, although EBP was evident in more than 40% of samples without other IBD-related lesions. EBP is a sensitive histologic feature of IBD, especially at the first endoscopic sampling, even in the absence of the other characteristic histologic lesions, and may help in formulating a more precise diagnosis in this setting.

OBJECTIVES Dilated intercellular spaces (DIS) in the esophageal epithelium can be induced by acid and reduced by proton pump inhibitors (PPI), and are thus considered a marker of gastroesophageal reflux disease (GERD). Over the years, however, DIS have also been reported in esophagitis unrelated to GERD. Because DIS have never been formally measured in eosinophilic esophagitis (EoE), we aimed at detecting and measuring DIS in EoE before and after nutritional or pharmacological therapy. METHODS In 22 children with EoE, DIS were measured by morphometry and transmission electron microscopy (TEM), before and after treatment with topical steroids (n = 16) and/or exclusion diet (n = 13). A total of 30 children undergoing upper gastrointestinal endoscopy with biopsy for nonesophageal disorders acted as controls. RESULTS In controls, the mean (± standard deviation [SD]) number of esophageal eosinophils was 0.91 (± 0.47) and the mean DIS values were 0.62 (± 0.08) μm at morphometry and 0.33 (± 0.24) μm at TEM. In patients with EoE, the mean (± SD) number of esophageal eosinophils decreased from 31.8 (± 6.96) to 6.64 (± 5.01) (P < 0.0001), and the mean DIS values decreased from 2.26 (± 0.21) to 1.23 (± 0.20) μm at morphometry (P < 0.0001), and from 2.24 (± 0.28) to 0.98 (± 0.19) μm at TEM (P < 0.0001), respectively, before and after treatment. CONCLUSIONS DIS are a prominent morphological feature of EoE, in which they can be significantly reduced by an appropriate non-PPI therapy.

Background Glioblastoma (GBM) represents the most aggressive malignant brain tumor in adults, with a risible median life expectancy despite gold standard treatment. Novel drug-delivery methods have been explored. Here we evaluated the possibility to use mononuclear cells (MCs) belonging to the monocytic-dendritic lineage as drug-carrier. Methods MCs were obtained from 10 patients harboring a GBM, and from healthy volunteers, considered as controls. GBM tissue was also obtained from patients. MCs were cultured and the adherent population on fibronectin (FN-MCs), after immunocytochemistry and flow cytometry characterization, was loaded with Paclitaxel (FN-MCs-PTX). Antiproliferative and migration activity of FN-MCs-PTX was evaluated in two-dimensional (2D) and three-dimensional (3D) co-culture assays with red fluorescent U87 Malignant Glioma cells and primary GBM cells. Antiangiogenic properties of FN-MCs-PTX were tested on cultures with endothelial cells. Results Phenotypical characterization showed a high expression of monocytic-dendritic markers in GBM cells and FN-MCs. FN-MCs demonstrated to effectively uptake PTX and to strongly inhibit GBM growth in vitro ( P 0.01). Moreover, tumor-induced migration of MCs, although partially affected by the PTX cargo, remained statistically significant when compared with unprimed cells and this was confirmed in a 3D Matrigel model ( P 0.01) and in a Trans-well assay ( P 0.01). FN-MCs-PTX also disclosed considerable antiangiogenic properties. Discussion Our results suggest that the fibronectin-adherent population of MCs isolated from peripheral blood can be an effective tool to inhibit GBM growth. Given the relative facility to obtain such cells and the short time needed for their culture and drug loading this approach may have potential as an adjuvant therapy for GBM.

The study of proto-oncogene Her-2/neu using the fluorescence in situ hybridization (FISH) technique in routinely paraffin-embedded formalin-fixed tissue has become commonplace over the past decade and mandatory among invasive breast cancer expressing a score 2+ by immunohistochemical analysis of c-erbB2 protein. The patient's eligibility for treatment with the biological drug trastuzumab/herceptin is based on the evidence of a Her-2/neu proto-oncogene amplification (ratio Her-2/neu/CEP-172.2). However, although the exclusion is declared in the absence of Her-2/neu gene amplification (ratio Her-2/neu/CEP-171.8) according to the American Society of Clinical Oncology/College of American Pathologists recommendations, there are borderline cases (1.8ratio Her-2/neu/CEP-172.2) that need to be investigated (eg, ductal carcinoma in situ with microinvasion, metastatic breast cancer). In such cases with Her-2/neu genetic heterogeneity it is difficult to count the nuclear signals in the areas of invasive tumor using fluorescence. The availability of a Fluorescence Immunophenotyping and Interphase Cytogenetics as a Tool for Investigation of Neoplasms technique, based on the simultaneous evaluation of immunostaining with anticytokeratins (CKAE1/AE3 and CK19), together with FISH for Her-2/neu gene status [it is therefore useful and of current applicability in breast cancer blocks (formalin-fixed and paraffin-embedded)], permits a more easy identification of even single neoplastic cells by immunofluorescence and then a better evaluation of Her-2/neu status gene by the FISH technique, as shown in our study.

Diverticular disease of the colon is frequent in clinical practice, and a large number of patients each year undergo surgical procedures worldwide for their symptoms. Thus, there is a need for better knowledge of the basic pathophysiologic mechanisms of this disease entity.Because patients with colonic diverticular disease have been shown to display abnormalities of the enteric nervous system, we assessed the frequency of myenteric plexitis (i.e. the infiltration of myenteric ganglions by inflammatory cells) in patients undergoing surgery for this condition.We analyzed archival resection samples from the proximal resection margins of 165 patients undergoing left hemicolectomy (60 emergency and 105 elective surgeries) for colonic diverticulitis, by histology and immunochemistry.Overall, plexitis was present in almost 40% of patients. It was subdivided into an eosinophilic (48%) and a lymphocytic (52%) subtype. Plexitis was more frequent in younger patients; and it was more frequent in those undergoing emergency surgery (50%), compared to elective (28%) surgery (p = 0.007). All the severe cases of plexitis displayed the lymphocytic subtype.In conclusion, myenteric plexitis is frequent in patients with colonic diverticular disease needing surgery, and it might be implicated in the pathogenesis of the disease.

Objective. - Arginase is a nitric oxide synthase-alternative pathway for l-arginine breakdown leading to biosynthesis of urea and l-ornithine. Arginase pathway is inducible by inflammatory molecules-such as cytokines and bacterial endotoxin-in macrophages and smooth muscle cells. The presence of an arginase pathway in human endothelial cells and its possible modulation by inflammation are unknown. Methods. - We have: (i) characterised arginase pathway in terms of activity, isoform type and gene expression in a primary human umbilical vein endothelial cells (HUVEC) line; (ii) evaluated arginase functional role in cell proliferation with the aid of l-norvaline, an arginase inhibitor and (iii) determined the effects of tumour necrosis factor-alpha and endotoxin on arginase pathway. Results. - HUVEC showed a baseline arginase activity and expression of both arginase isoforms (arginase I and II (A-I and A-II, respectively)) which resulted in l-norvaline-inhibitable cellular polyamine synthesis. The baseline arginase activity is important for HUVEC proliferation as cell cycle analysis and nuclear factor Ki-67 immunostaining revealed. Following incubation with inflammatory molecules, arginase activity increased but HUVEC cell cycling decreased. Conclusions. - A-I and A-II are constitutively expressed in HUVEC where they take part to the regulation of cell cycling. Although arginase activity is positively modulated by inflammatory molecules, it is insufficient to counteract the overall cell cycling inhibiting effects of inflammation.

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.

Purpose and background The focus was directed to the study of two of the most lignan-rich food sources: sesame and flaxseeds. Recent epidemiological and experimental evidences suggesting that these foods may improve metabolic functions underlying metabolic syndrome (MetS). Methods To characterize the effect of these oilseeds on metabolic functions, we conducted an experimental study aimed at preventing adiposity and metabolic imbalance in a mouse model of high-fat diet (HFD)-induced MetS. Statistical analysis was performed by two-way analysis of variance test followed by post hoc Bonferroni analysis. Results We studied the effect of the oilseeds sesame and flaxseed on metabolic parameters in mice on a HFD. When the HFD was integrated with 20 % of sesame or flaxseed flours, the mice showed a decrease in body fat, already at day 15, from time 0. The size of the adipocytes was smaller in epididymal fat, liver steatosis was inhibited, and insulin sensitivity was higher in mice on the supplemented diets. The supplemented diets also resulted in a significant increase in the serum levels of the lignan metabolites enterodiol and enterolactone compared with the controls. The expression of genes associated with the inflammatory response, glucose metabolism, adipose metabolism and nuclear receptor were altered by the oilseed-supplemented diets. Some of the most abundant lignans in these oilseeds were studied in 3T3-L1 preadipocyte cells and were effective in inhibiting adipocyte differentiation at the minimal dose of 1 nM. Conclusions The consumption of sesame and flaxseed may be beneficial to decrease metabolic parameters that are generally altered in MetS.

Flow cytometric DNA analysis is an important prognostic tool in breast cancer. We evaluated the possibility of performing DNA analysis on cell suspensions obtained by scraping the cut surface of breast tumors; 31 breast tumor nodules, including six benign and 25 malignant lesions, were studied. From each case, cell suspensions acquired by mechanical mincing of a fresh frozen tissue fragment and by two different scrapings (central and peripheral) from the cut surface of the tumor were analyzed via flow cytometry. In all cases, comparison of the DNA histograms for three samples showed no significant differences in the appearance of debris or in the value of coefficient of variation of the G0-G1 peak. All benign nodules showed a normal DNA stemline in all specimens. In 23 of 25 cases of breast carcinoma, the ploidy of the three preparations was similar, with a concordance in 12/14 (85, 71%) cases in DNA nondiploid tumors. Linear regression analysis showed a good correlation in DNA index between either scraping sample and the tissue fragment (r = .955 and r = .905). The results indicate that the scraping technique provides excellent cell suspensions and DNA histograms comparable to those obtained from mechanical mincing of tissue fragments. The technique minimizes preparation time and avoids consuming much tissue and, thus, is the method of choice when very small cancers have to be analyzed.

Mast cells are involved in visceral hypersensitivity and motor activity of the gastrointestinal tract. However, there is almost no information concerning mast cells in constipated patients. The purpose of this study was to investigate mast cells distribution in all colonic layers in controls and severely constipated patients with obstructed defecation. Full-thickness specimens from colons of patients undergoing surgery for obstructed defecation due to refractoriness to other therapeutic interventions (n = 11), compared to controls, were obtained and the number of mast cells (evaluated by specific monoclonal antibodies) were counted in the whole viscus and in the various colonic segments (cecum, ascending, transverse, descending, and sigmoid). Compared to controls, constipated patients had significantly higher numbers of mast cells, both as an overall number and in single colonic segments. This increase was especially evident in the mucosa and submucosa. Mast cells were homogeneously represented in the various segment of the large bowel, in both controls and patients. Degranulated mast cells were found to be close to enteric glial cells and glial filaments. Colonic mast cells are increased in obstructed defecation patients. This might represent a vicariating mechanism to the impaired colonic propulsive activity of these patients.

Gut mast cells represent an important cell population involved in intestinal homeostasis and inflammatory processes. However, their possible role has not to date been investigated in colonic diverticular disease.This study aims to evaluate colonic mast cells in patients undergoing surgery for diverticular disease.Surgical resection samples from 27 patients undergoing surgery for diverticular disease (12 emergency procedures for severe disease and 15 elective procedures) were evaluated. The number of mast cells was assessed in the various layers by means of a specific antibody (tryptase) and compared with those evaluated in ten controls. In patients with mast cells degranulation, double immunohistochemistry, also assessing nerve fibres, was carried out. In addition, the presence of myenteric plexitis was sought.Compared with controls, the number of mast cells in diverticular patients was significantly increased, both as an overall figure and in the various layers of the large bowel. In patients in whom mast cells degranulation was present, these were always closed to nerve fibres. No differences were found between the two subgroups of patients with respect to the number and distribution of mast cells; however, all patients undergoing emergency surgery (but none of those undergoing elective procedures) had myenteric plexitis, represented by lymphocytic infiltration in 67 % and eosinophilic infiltration in 33 % of cases.Patients with diverticular disease display an increase of mast cells in the large bowel. The presence of myenteric plexitis in those with complicated, severe disease, suggest that this could represent a histopathologic marker of more aggressive disease.

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.Full-thickness rectal samples were obtained from 17 patients undergoing stapled transanal rectal resection for OD associated with rectal intussusception. Samples were analyzed by immunohistochemistry for enteric neurons, enteric glial cells, interstitial cells of Cajal (ICC), and for estrogen and progesterone receptors. Data were compared with those obtained in 10 controls.No differences between patients and controls were found for enteric neurons, whereas (compared with controls) OD patients displayed a significant decrease of enteric glial cells in both the submucous (P = 0.0006) and the myenteric (P0.0001) plexus. ICC were significantly increased in patients in the submucosal surface (P0.0001) and the myenteric area (P0.0001). Concerning estroprogestinic receptors, both were present on ICC in patients and controls. Estrogen receptors α and progesterone receptors were absent on enteric neurons and enteric glial cells in patients and controls, whereas estrogen receptors β were present in all controls and in 69% of patients' enteric neurons (P = 0.18) and in 12% of patients' glial cells (P = 0.0001).Patients with OD associated to rectal intussusception display abnormalities of the ENS and of estrogen receptors β.

Cisplatin (cisPt) use in chemotherapy is limited by the occurrence of a severe nephrotoxicity. Both autophagy and apoptosis seem to contribute in kidney response to cisPt, however their cross-talk is still controversial, since the role played by autophagy (cytoprotective or harmful) and the cellular site driving their switch, are still unclear. Here, we used a multidisciplinary approach to study the correlation between autophagy and apoptosis in renal NRK-52E cells exposed to cisPt. We showed two “sensitivity-thresholds” to cisPt, stating whether apoptosis or autophagy would develop: 10 μM dose of cisPt activated autophagy that preserved cell homeostasis; however 3-methyladenine co-administration affected cell viability and induced apoptosis. In contrast, 50 μM cisPt determined cell death by apoptosis, whereas the pre-conditioning with taurine contributed to cell rescue, delaying apoptosis and maintaining autophagy. Hence, autophagy protects NRK-52E cells from cisPt injury. By studying the expression of ER specific hallmarks, such as GRP78, GRP94 and GADD153/CHOP, we found a possible pivotal role of ER signaling modulation in the crosstalk between autophagy and apoptosis induced by cisPt. To the best of our knowledge, this is the first demonstration that taurine enhances autophagic protection against apoptosis by reducing ER stress, thus making it possible to develop new strategies to reduce severe cisPt-induced side-effects such as nephrotoxicity.

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.

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.

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 Crohn's 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 Crohn's 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.

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.

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.