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Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disease caused by thymidine phosphorylase (TP) enzyme defect. As gastrointestinal changes do not revert in patients undergone TP replacement therapy, one can postulate that other unexplored mechanisms contribute to MNGIE pathophysiology. Hence, we focused on the local TP angiogenic potential that has never been considered in MNGIE. In this study, we investigated the enteric submucosal microvasculature and the effect of hypoxia on fibrosis and enteric neurons density in jejunal full-thickness biopsies collected from patients with MNGIE. Orcein staining was used to count blood vessels based on their size. Fibrosis was assessed using the Sirius Red and Fast Green method. Hypoxia and neoangiogenesis were determined via hypoxia-inducible-factor-1α (HIF-1α) and vascular endothelial cell growth factor (VEGF) protein expression, respectively. Neuron-specific enolase was used to label enteric neurons. Compared with controls, patients with MNGIE showed a decreased area of vascular tissue, but a twofold increase of submucosal vessels/mm(2) with increased small size and decreased medium and large size vessels. VEGF positive vessels, fibrosis index, and HIF-1α protein expression were increased, whereas there was a diminished thickness of the longitudinal muscle layer with an increased interganglionic distance and reduced number of myenteric neurons. We demonstrated the occurrence of an angiopathy in the GI tract of patients with MNGIE. Neoangiogenetic changes, as detected by the abundance of small size vessels in the jejunal submucosa, along with hypoxia provide a morphological basis to explain neuromuscular alterations, vasculature breakdown, and ischemic abnormalities in MNGIE. NEW & NOTEWORTHY Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is characterized by a genetically driven defect of thymidine phosphorylase, a multitask enzyme playing a role also in angiogenesis. Indeed, major gastrointestinal bleedings are life-threatening complications of MNGIE. Thus, we focused on jejunal submucosal vasculature and showed intestinal microangiopathy as a novel feature occurring in this disease. Notably, vascular changes were associated with neuromuscular abnormalities, which may explain gut dysfunction and help to develop future therapeutic approaches in MNGIE.

Abnormal gut motility is a feature of several mitochondrial encephalomyopathies, and mutations in genes such as TYMP and POLG, have been linked to these rare diseases. The human genome encodes three DNA ligases, of which only one, ligase III (LIG3), has a mitochondrial splice variant and is crucial for mitochondrial health. We investigated the effect of reduced LIG3 activity and resulting mitochondrial dysfunction in seven patients from three independent families, who showed the common occurrence of gut dysmotility and neurological manifestations reminiscent of mitochondrial neurogastrointestinal encephalomyopathy. DNA from these patients was subjected to whole exome sequencing. In all patients, compound heterozygous variants in a new disease gene, LIG3, were identified. All variants were predicted to have a damaging effect on the protein. The LIG3 gene encodes the only mitochondrial DNA (mtDNA) ligase and therefore plays a pivotal role in mtDNA repair and replication. In vitro assays in patient-derived cells showed a decrease in LIG3 protein levels and ligase activity. We demonstrated that the LIG3 gene defects affect mtDNA maintenance, leading to mtDNA depletion without the accumulation of multiple deletions as observed in other mitochondrial disorders. This mitochondrial dysfunction is likely to cause the phenotypes observed in these patients. The most prominent and consistent clinical signs were severe gut dysmotility and neurological abnormalities, including leukoencephalopathy, epilepsy, migraine, stroke-like episodes, and neurogenic bladder. A decrease in the number of myenteric neurons, and increased fibrosis and elastin levels were the most prominent changes in the gut. Cytochrome c oxidase (COX) deficient fibres in skeletal muscle were also observed. Disruption of lig3 in zebrafish reproduced the brain alterations and impaired gut transit in vivo. In conclusion, we identified variants in the LIG3 gene that result in a mitochondrial disease characterized by predominant gut dysmotility, encephalopathy, and neuromuscular abnormalities. Bonora et al. identify a new mitochondrial recessive disorder caused by biallelic variants in the LIG3 gene encoding DNA ligase III, which is responsible for mitochondrial DNA repair. Clinical signs include gut dysmotility and neurological features such as leucoencephalopathy, epilepsy and stroke-like episodes.

BACKGROUND AND PURPOSE: Mitochondrial neurogastrointestinal encephalopathy is a rare disorder due to recessive mutations in the thymidine phosphorylase gene, encoding thymidine phosphorylase protein required for mitochondrial DNA replication. Clinical manifestations include gastrointestinal dysmotility and diffuse asymptomatic leukoencephalopathy. This study aimed to elucidate the mechanisms underlying brain leukoencephalopathy in patients with mitochondrial neurogastrointestinal encephalopathy by correlating multimodal neuroradiologic features to postmortem pathology. MATERIALS AND METHODS: Seven patients underwent brain MR imaging, including single-voxel proton MR spectroscopy and diffusion imaging. Absolute concentrations of metabolites calculated by acquiring unsuppressed water spectra at multiple TEs, along with diffusion metrics based on the tensor model, were compared with those of healthy controls using unpaired t tests in multiple white matters regions. Brain postmortem histologic, immunohistochemical, and molecular analyses were performed in 1 patient. RESULTS: All patients showed bilateral and nearly symmetric cerebral white matter hyperintensities on T2-weighted images, extending to the cerebellar white matter and brain stem in 4. White matter, N -acetylaspartate, creatine, and choline concentrations were significantly reduced compared with those in controls, with a prominent increase in the radial water diffusivity component. At postmortem examination, severe fibrosis of brain vessel smooth muscle was evident, along with mitochondrial DNA replication depletion in brain and vascular smooth-muscle and endothelial cells, without neuronal loss, myelin damage, or gliosis. Prominent periependymal cytochrome C oxidase deficiency was also observed. CONCLUSIONS: Vascular functional and histologic alterations account for leukoencephalopathy in mitochondrial neurogastrointestinal encephalopathy. Thymidine toxicity and mitochondrial DNA replication depletion may induce microangiopathy and blood-brain-barrier dysfunction, leading to increased water content in the white matter. Periependymal cytochrome C oxidase deficiency could explain prominent periventricular impairment.

Gastrointestinal (GI) symptoms can originate from severe dysmotility due to enteric neuropathies. Current methods used to demonstrate enteric neuropathies are based mainly on classic qualitative histopathological/immunohistochemical evaluation. This study was designed to identify an objective morphometric method for paraffin-embedded tissue samples to quantify the interganglionic distance between neighboring myenteric ganglia immunoreactive for neuron-specific enolase, as well as the number of myenteric and submucosal neuronal cell bodies/ganglion in jejunal specimens of patients with severe GI dysmotility. Jejunal full-thickness biopsies were collected from 32 patients (22 females; 16–77 yr) with well-characterized severe dysmotility and 8 controls (4 females; 47–73 yr). A symptom questionnaire was filled before surgery. Mann-Whitney U test, Kruskal-Wallis coupled with Dunn’s posttest and nonparametric linear regression tests were used for analyzing morphometric data and clinical correlations, respectively. Compared with controls, patients with severe dysmotility exhibited a significant increase in myenteric interganglionic distance ( P = 0.0005) along with a decrease in the number of myenteric ( P < 0.00001) and submucosal ( P < 0.0004) neurons. A 50% reduction in the number of submucosal and myenteric neurons correlated with an increased interganglionic distance and severity of dysmotility. Our study proposes a relatively simple tool that can be applied for quantitative evaluation of paraffin sections from patients with severe dysmotility. The finding of an increased interganglionic distance may aid diagnosis and limit the direct quantitative analysis of neurons per ganglion in patients with an interganglionic distance within the control range. NEW & NOTEWORTHY Enteric neuropathies are challenging conditions characterized by a severe impairment of gut physiology, including motility. An accurate, unambiguous assessment of enteric neurons provided by quantitative analysis of routine paraffin sections may help to define neuropathy-related gut dysmotility. We showed that patients with severe gut dysmotility exhibited an increased interganglionic distance associated with a decreased number of myenteric and submucosal neurons, which correlated with symptoms and clinical manifestations of deranged intestinal motility.

BACKGROUND: Chronic-intestinal-pseudo-obstruction (CIPO) is a rare condition due to severe impairment of gut motility responsible for recurrent sub-occlusive episodes. Although neuro-muscular-glial-ICC abnormalities represent the main pathogenetic mechanism, the pathophysiology of CIPO remains poorly understood. Intestinal epithelial and vascular-endothelial barrier (IEVB) abnormalities can contribute to neuro-epithelial changes by allowing passage of harmful substances. METHODS: To test retrospectively whether IEVB defects occur in patients with CIPO we measured the jejunal protein expression of the major tight junction (TJ) components. CIPO patients were subdivided according to gut neuromuscular histopathology: apparently normal (AN); with inflammation (INF); or with degenerative alterations (DEG). The presence of occludin/claudin oligomers (index of TJ assembly), the amount of occludin, claudin-4 and zonula occludens-1 (ZO-1), as well as the expression of vasoactive intestinal polypeptide (VIP) and glial fibrillary acidic protein (GFAP) immunoreactivities were evaluated on jejunal full thickness biopsies using Western Blot. KEY RESULTS: Oligomers were absent in the 73% of CIPO. Total occludin decreased in CIPO with AN and INF changes. Claudin-4 was upregulated in CIPO with INF and DEG features. ZO-1 and VIP expression decreased selectively in DEG group. GFAP increased in CIPO regardless the histopathological phenotype. CONCLUSIONS & INFERENCES: The absence of oligomers demonstrated in our study suggests that IEBV is altered in CIPO. The mechanism leading to oligomerization is occludin-dependent in AN and INF, whereas is ZO-1-dependent in DEG. Our study provides support to IEVB abnormalities contributing to CIPO clinical and histopathological features.

BACKGROUND: Achalasia is a rare motility disorder characterized by myenteric neuron and interstitial cells of Cajal (ICC) abnormalities leading to deranged/absent peristalsis and lack of relaxation of the lower esophageal sphincter. The mechanisms contributing to neuronal and ICC changes in achalasia are only partially understood. Our goal was to identify novel molecular features occurring in patients with primary achalasia. METHODS: Esophageal full-thickness biopsies from 42 (22 females; age range: 16-82 years) clinically, radiologically, and manometrically characterized patients with primary achalasia were examined and compared to those obtained from 10 subjects (controls) undergoing surgery for uncomplicated esophageal cancer (or upper stomach disorders). Tissue RNA extracted from biopsies of cases and controls was used for library preparation and sequencing. Data analysis was performed with the "edgeR" option of R-Bioconductor. Data were validated by real-time RT-PCR, western blotting and immunohistochemistry. KEY RESULTS: Quantitative transcriptome evaluation and cluster analysis revealed 111 differentially expressed genes, with a P ≤ 10-3 . Nine genes with a P ≤ 10-4 were further validated. CYR61, CTGF, c-KIT, DUSP5, EGR1 were downregulated, whereas AKAP6 and INPP4B were upregulated in patients vs controls. Compared to controls, immunohistochemical analysis revealed a clear increase in INPP4B, whereas c-KIT immunolabeling resulted downregulated. As INPP4B regulates Akt pathway, we used western blot to show that phospho-Akt was significantly reduced in achalasia patients vs controls. CONCLUSIONS & INFERENCES: The identification of altered gene expression, including INPP4B, a regulator of the Akt pathway, highlights novel signaling pathways involved in the neuronal and ICC changes underlying primary achalasia.

Background Although serological tests are useful for identifying celiac disease, it is well established that a minority of celiacs are seronegative. Aim To define the prevalence and features of seronegative compared to seropositive celiac disease, and to establish whether celiac disease is a common cause of seronegative villous atrophy. Methods Starting from 810 celiac disease diagnoses, seronegative patients were retrospectively characterized for clinical, histological and laboratory findings. Results Of the 810 patients, fourteen fulfilled the diagnostic criteria for seronegative celiac disease based on antibody negativity, villous atrophy, HLA-DQ2/-DQ8 positivity and clinical/histological improvement after gluten free diet. Compared to seropositive, seronegative celiac disease showed a significantly higher median age at diagnosis and a higher prevalence of classical phenotype (i.e., malabsorption), autoimmune disorders and severe villous atrophy. The most frequent diagnosis in the 31 cases with seronegative flat mucosa was celiac disease (45%), whereas other diagnoses were Giardiasis (20%), common variable immunodeficiency (16%) and autoimmune enteropathy (10%). Conclusions Although rare seronegative celiac disease can be regarded as the most frequent cause of seronegative villous atrophy being characterized by a high median age at diagnosis; a close association with malabsorption and flat mucosa; and a high prevalence of autoimmune disorders.

Mitochondrial neuro-gastro-intestinal encephalomyopathy (MNGIE) is a rare and unavoidably fatal disease due to mutations in thymidine phosphorylase (TP). Clinically it is characterized by gastrointestinal dysfunction, malnutrition/cachexia and neurological manifestations. MNGIE diagnosis remains a challenge mainly because of the complexity and rarity of the disease. Thus, our purposes were to promote a better knowledge of the disease in Emilia-Romagna region (ERR) by creating an accurate and dedicated network; to establish the minimal prevalence of MNGIE in Italy starting from ERR. Blood TP activity level was used as screening test to direct candidates to complete diagnostic work-up. During the study period of 1 year, only 10/71 units of ERR recruited 14 candidates. Their screening did not show TP activity changes. An Italian patient not resident in ERR was actually proved to have MNGIE. At the end of study in Italy there were nine cases of MNGIE; thus, the Italian prevalence of the disease is ~0.15/1,000,000 as a gross estimation. Our study confirms that MNGIE diagnosis is a difficult process which reflects the rarity of the disease and, as a result, a low level of awareness among specialists and physicians. Having available novel therapeutic options (e.g., allogenic hematopoietic stem cell transplantation and, more recently, liver transplantation) and an easy screening test, an early diagnosis should be sought before tissue damage occurs irreversibly.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a fatal, recessive disease caused by mutations in the gene encoding thymidine phosphorylase, leading to reduced enzymatic activity, toxic nucleoside accumulation, and secondary mitochondrial DNA damage. Thymidine phosphorylase replacement has been achieved by allogeneic hematopoietic stem cell transplantation, a procedure hampered by high mortality. Based on high thymidine phosphorylase expression in the liver, a 25-year-old severely affected patient underwent liver transplantation. Serum levels of toxic nucleosides rapidly normalized. At 400 days of follow-up, the patient's clinical conditions are stable. We propose liver transplantation as a new therapy for MNGIE. Ann Neurol 2016;80:448-455.

Background & Aims: Individuals with potential celiac disease have serologic and genetic markers of the disease with little or no damage to the small intestinal mucosa. We performed a prospective study to learn more about disease progression in these people. Methods: We collected data from 77 adults (59 female; median age, 33 years) diagnosed with potential celiac disease (on the basis of serology and HLA type) at Bologna University in Italy from 2004 through 2013. The subjects had normal or slight inflammation of the small intestinal mucosa. Clinical, laboratory, and histologic parameters were evaluated at diagnosis and during a 3-year follow-up period. Results: Sixty-one patients (46 female; median age, 36 years) showed intestinal and extraintestinal symptoms, whereas the remaining 16 (13 female; median age, 21 years) were completely asymptomatic at diagnosis. All subjects tested positive for immunoglobulin A endomysial antibody and tissue transglutaminase antibody, except for 1 patient with immunoglobulin A deficiency; 95% of patients were carriers of HLA-DQ2. Duodenal biopsies from 26% patients had a Marsh score of 0, and 74% had a Marsh score of 1. A higher proportion of symptomatic patients had autoimmune disorders (36%) and antinuclear antibodies (41%) than asymptomatic patients (5% and 12.5%, respectively), and symptomatic patients were of older age at diagnosis (P

A tight link exists between dietary factors and irritable bowel syndrome (IBS), one of the most common functional syndromes, characterized by abdominal pain/discomfort, bloating and alternating bowel habits. Amongst the variety of foods potentially evoking “food sensitivity”, gluten and other wheat proteins including amylase trypsin inhibitors represent the culprits that recently have drawn the attention of the scientific community. Therefore, a newly emerging condition termed non-celiac gluten sensitivity (NCGS) or non-celiac wheat sensitivity (NCWS) is now well established in the clinical practice. Notably, patients with NCGS/NCWS have symptoms that mimic those present in IBS. The mechanisms by which gluten or other wheat proteins trigger symptoms are poorly understood and the lack of specific biomarkers hampers diagnosis of this condition. The present review aimed at providing an update to physicians and scientists regarding the following main topics: the experimental and clinical evidence on the role of gluten/wheat in IBS; how to diagnose patients with functional symptoms attributable to gluten/wheat sensitivity; the importance of double-blind placebo controlled cross-over trials as confirmatory assays of gluten/wheat sensitivity; and finally, dietary measures for gluten/wheat sensitive patients. The analysis of current evidence proposes that gluten/wheat sensitivity can indeed represent a subset of the broad spectrum of patients with a clinical presentation of IBS.

In Western countries the dietary guidance emphasizes the need to decrease the intake of saturated fatty acids and to replace them with polyunsaturated fatty acids (PUFA), particularly long chain n-3 PUFA (LC-PUFA). The production of poultry meat having a lower fat content and healthier fatty acid (FA) profile is a hot topic for the poultry industry, and the possibility to identify genotypes able to produce meat with a higher LC-PUFA content deserves attention. The aims of the present study were to evidence in chicken (i) a genotype-related different expression of the desaturating enzymes delta-6 (Δ6, EC 1.14.99.25), delta-5 (Δ5, EC 1.14.19.) and delta-9 (Δ9, EC 1.14.19.1); (ii) the impact of the hypothesized different expression on the meat FA composition; (iii) the distribution of desaturase products in the different lipid classes. Slow (SG), medium (MG) and fast (FG) growing chickens fed the same diet were evaluated either for the relative expression of FADS1, FADS2 and SCD1 genes in liver (by q-PCR), or for the FA composition of breast meat. MG and particularly SG birds showed a greater expression of FADS2 and FADS1 genes, a higher Δ6 and Δ5 activity (estimated using desaturase indices), and consequently a higher LC-PUFA content in the breast meat than FG birds. The relationship between genotype and desaturating ability was demonstrated, with a significant impact on the PUFA content of breast meat. Due to the high consumption rate of avian meat, the identification of the best genotypes for meat production could represent an important goal not only for the food industry, but also for the improvement of human nutrition.

Hypoxia/reoxygenation is one of the causes of the increased expression of inducible NO synthase in cardiomyocytes. In a recent study we demonstrated that a single, high dose of green tea extract (GT) supplemented to the medium of cultured cardiomyocytes just before hypoxia/reoxygenation is able to prevent the increased expression of inducible NO synthase, therefore reducing NO overproduction. In the present study we investigated the mechanism by which GT reduces NO production. Since a molecular mechanism for polyphenol activity has been postulated, and PPAR activation is related to the transcription of the inducible NO synthase gene, we evaluated the activation of PPAR by GT. A moderate GT concentration, supplemented to the cardiomyocyte medium since the initial seeding, selectively activated the PPAR-β/δ isoform. Furthermore, we observed a reduction in NO production and an increase in total antioxidant activity, indicating that GT components may act on both reactive oxygen species, via an antioxidant mechanism, and NO overproduction. PPAR-β/δ activation could represent the key event in the reduction of NO production by GT. Although PPAR activation by GT was lower than activation by fenofibrate, it is very interesting to note that it was selective for the β/δ isoform, at least in neonatal cardiomyocytes.

Magnetic Resonance Spectroscopy visible mobile lipids are considered important markers in the diagnosis of human cancer and are thought to be closely involved in various aspects of tumour transformation, such as cell proliferation, necrosis, apoptosis, hypoxia and drug resistance. A method allowing the straightforward identification of the lipid classes contributing to the mobile lipids in human malignant tissues is highly advisable. Ex vivo High Resolution Magic Angle Spinning Magnetic Resonance Spectroscopy was done directly on human cerebral, renal and colorectal malignant tissue specimens. A diffusion edited sequence, based on stimulated echo and bipolar gradient pulses, was used to characterize molecules with low diffusion rates, arising from mobile lipid components. Cholesterol, triglycerides and phosphatidylcholine are simultaneously detected and all contribute to the mobile lipid resonances present in malignant glioma and clear cell renal carcinoma tissue specimens spectra. On the contrary, papillary cell renal carcinoma spectrum is predominated by phosphatidylcholine resonances and that of colorectal adenocarcinoma is characterized by signals arising from triglycerides. Ex vivo diffusion edited High Resolution Magic Angle Spinning Magnetic Resonance Spectroscopy, done on intact tissue, is a powerful analytical tool to obtain a simple and immediate identification of mobile lipid components. This can offer a significant contribution to better understanding their involvement in cancer tissues. Furthermore, ex vivo high resolution spectroscopic measurements allow to improve the interpretation of in vivo Magnetic Resonance spectra, increasing its clinical potentiality.

Background: μ opioid receptors (μORs) are expressed by neurons and inflammatory cells, and mediate immune response. We tested whether activation of peripheral μORs ameliorates the acute and delayed phase of colitis. Methods: C57BL/6J mice were treated with 3% dextran sodium sulfate (DSS) in water, 5 days with or without the peripherally acting μOR agonist, [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin (DAMGO) or with DAMGO+μOR antagonist at day 2-5, then euthanized. Other mice received DSS followed by water for 4 weeks, or DSS with DAMGO starting at day 2 of DSS for 2 or 3 weeks followed by water, then euthanized at 4 weeks. Disease activity index (DAI), histological damage, and myeloperoxidase assay (MPO), as index of neutrophil infiltration, were evaluated. Cytokines and μOR mRNAs were measured with RT-PCR, and nuclear factor-kB (NF-kB), the antiapoptotic factor Bcl-xL, and caspase 3 and 7 with Western blot. Key Results: DSS induced acute colitis with elevated DAI, tissue damage, apoptosis and increased MPO, cytokines, μOR mRNA, and NF-kB. DAMGO significantly reduced DAI, inflammatory indexes, cytokines, caspases, and NF-kB, and upregulated Bcl-xL, effects prevented by μOR antagonist. In DSS mice plus 4 weeks of water, DAI, NF-kB, and μOR were normal, whereas MPO, histological damage, and cytokines were still elevated; DAMGO did not reduce inflammation, and did not upregulate Bcl-xL. Conclusions & Inferences: μOR activation ameliorated the acute but not the delayed phase of DSS colitis by reducing cytokines, likely through activation of the antiapoptotic factor, Bcl-xL, and suppression of NF-kB, a potentiator of inflammation. μ opioid receptors (μORs) mediate a variety of biological processes, including gastrointestinal functions and immune response. This study showed that activation of peripheral μOR with DAMGO, a selective μOR agonist, reduces the inflammatory response and disease activity index in acute experimental colitis through a mechanism involving the activation of the antiapoptotic factor, Bcl-xL, a member of the Bcl-2 family of apoptosis regulator proteins, and the suppression of the transcription nuclear factor-kB (shown here), which in turns reduces the release of pro-inflammatory cytokines and immune cells infiltration. By contrast, the delayed phase of colitis, which is characterized by lower level of inflammation, does not appear to benefit for additional treatment with peripherally acting μOR agonists as indicated by the lack of effect of DAMGO on the expression of Bcl-xL and NF-kB.

It is becoming increasingly apparent that responsiveness to dietary fat composition is heterogeneous and dependent on the genetic make-up of the individual. The aim of this study was to evidence a genotype-related differential effect of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) on the modulation of hepatic genes involved in cholesterol metabolism. Fourteen spontaneously hypertensive (SH) rats, which present a naturally occurring variation in the gene encoding for sterol responsive element binding protein 1 (SREBP-1), contributing to their inherited variation in lipid metabolism, and 14 Wistar-Kyoto (WK) rats were fed a control diet or an n-3 LC-PUFA enriched diet for 90 days. Plasma lipid profile, total lipid fatty acid composition in plasma and liver, and the expression of SREBP-1 and 2, 3-hydroxy-3-methyl-glutaryl-CoA reductase, low-density lipoprotein receptor, and acyl-CoA:cholesterol acyltransferase 2 encoding genes and proteins were determined. The positive effect of the enriched diet on the serum lipid profile, particularly on total cholesterol and triglyceride level, was clearly evidenced in both WK and SH rats, but n-3 LC-PUFA acted through a different modulation of gene and protein expression that appeared related to the genetic background. Our study evidences a different transcriptional effect of specific nutrients related to genetic variants.

Besides being cholesterol-lowering agents, phytosterols (PS) can inhibit the growth and development of tumours. The anti-neoplastic activity is accounted for by PS incorporation into cell membranes, resulting in the interference of membrane functionality. The similarity between the PS cholesterol-lowering and anti-neoplastic effective doses deserves attention on the possible adverse effects even in non-neoplastic cells. To date, few studies have addressed the clarification of this important issue. In the present study, we supplemented primary, non-neoplastic neonatal rat cardiomyocytes with two different PS concentrations (3 or 6 μg/ml), both within the range of human plasma concentration. Cardiac cells were chosen as an experimental model since the heart has been reported as the target organ for subchronic toxicity of PS. Following supplementation, a dose-dependent incorporation of PS and a decrease in cholesterol content were clearly evidenced. PS did not induce apoptosis but caused a reduction in metabolic activity (measured as 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion) and a slowing down of cell growth. The lower MTT conversion and the similar lactate dehydrogenase release could suggest that PS more efficiently target mitochondria than plasma membrane integrity. The replacement of cholesterol by PS could also have caused the observed slowing down of cell growth and the reduction in metabolic activity, which could rely on the PS increase, cholesterol decrease, or both. The present study is the first report on the effect of PS in cardiac cells, and although it is difficult to translate the obtained results to the health of heart tissue, it raises concerns about the safety of long-term exposure to physiologically relevant PS concentrations.

Background: Cronkhite-Canada Syndrome (CCS) is an uncommon gastrointestinal polyposis syndrome that exists mostly in Japan characterized by dermatologic manifestations associated with chronic diarrhea, malnutrition, and protein wasting that can be fatal in as high as 50% if untreated or if treatment is delayed or inadequate. Due to its rarity, its subjects cannot be studied in clinical trials. Here, we report patient characteristics derived from a national database in an effort to identify prognostic factors and to improve treatment outcomes. Method: Retrospective analysis of 150 patients with CCS from a national database including 80 institutions specializing in gastroenterology. Clinical features including endoscopic findings, treatment, prognosis, and response to medical therapy were included. This study was supported by Intractable Disease, Health and Labor Sciences research groups for the Japanese Ministry of Health, Labor and Welfare. Results: Patient's age was 68.1±9.7 and treatment duration was 6.0±4.6years. CCS-related polyposis endoscopically confirmed to be throughout the gastrointestinal tract. Of the 140 patients who received steroids, 5 failed to respond, 2 underwent colectomy due to massive bleeding, 2 received cyclosporine, and 1 received biologics. Improvement of diarrhea followed by recovery of hypoalbuminemia was observed usually within 3 months after the start of medical therapy, with weight gain and improvement of dermatologic manifestations occurring at 5-6.5 months, and improvement of endoscopic appearance by 7.7 months. In 17%, no improvement of polyposis occurred despite clinical response. CCS-related gastric and colonic polyps progressed to advanced gastric and colon cancer in 7 of 107 and 15 of 120, respectively, suggesting the importance of endoscopic follow-up. Among those who discontinued steroid therapy, 7.4% developed advanced cancer, compared with 2.7% in those who maintained clinical remission with steroids. No patient who received nutritional support alone experienced clinical remission. An induction dose of steroid of >0.5mg/kg, followed, in some cases, with steroidmaintenance appeared beneficial for preventing the risk of recurrence of CCS-related polyps, reducing cancer risk, underscoring the importance of maintaining endoscopic remission. As for prognosis, 108 patients were alive and 22 had expired. The causes of death were colon cancer (1), gastric cancer (2), other malignancies (5), and other diseases, but none died of malnutrition. Conclusion: Contrary to earlier reports, the prognosis of CCS has greatly improved through improved medical treatment, although the course of CCS is relentlessly progressive with high cancer risk. A sufficiently high induction steroid dose, long-term steroidmaintenance, and aggressive nutritional support appear to improve the natural history of CCS.