Toll-Like Receptor 4 Modulates Small Intestine Neuromuscular Function through Nitrergic and Purinergic Pathways

Objective: Toll-like receptors (TLRs) play a pivotal role in the homeostatic microflora-host crosstalk. TLR4-mediated modulation of both motility and enteric neuronal survival has been reported mainly for colon with limited information on the role of TLR4 in tuning structural and functional integrity of enteric nervous system (ENS) and in controlling small bowel motility. Methods: Male TLR4 knockout (TLR4-/-, 9 \ub1 1 weeks old) and sex- and age-matched wild-type (WT) C57BL/6J mice were used for the experiments. Alterations in ENS morphology and neurochemical code were assessed by immunohistochemistry whereas neuromuscular function was evaluated by isometric mechanical activity of ileal preparations following receptor and non-receptor-mediated stimuli and by gastrointestinal transit. Results: The absence of TLR4 induced gliosis and reduced the total number of neurons, mainly nNOS+ neurons, in ileal myenteric plexus. Furthermore, a lower cholinergic excitatory response with an increased inhibitory neurotransmission was found together with a delayed gastrointestinal transit. These changes were dependent on increased ileal non-adrenergic non-cholinergic (NANC) relaxations mediated by a complex neuronal-glia signaling constituted by P2X7 and P2Y1 receptors, and NO produced by nNOS and iNOS. Conclusion: We provide novel evidence that TLR4 signaling is involved in the fine-tuning of P2 receptors controlling ileal contractility, ENS cell distribution, and inhibitory NANC neurotransmission via the combined action of NO and adenosine-5\u2032-triphosphate (ATP). For the first time, this study implicates TLR4 at regulating the crosstalk between glia and neurons in small intestine and helps to define its role in gastrointestinal motor abnormalities during dysbiosis

Prognostic Impact of miR-224 and RAS Mutations in Medullary Thyroid Carcinoma

Little is known about the function of microRNA-224 (miR-224) in medullary thyroid cancer (MTC). This study investigated the role of miR-224 expression in MTC and correlated it with mutation status in sporadic MTCs. A consecutive series of 134 MTCs were considered. Patients had a sporadic form in 80% of cases (107/134). In this group, REarranged during transfection (RET) and rat sarcoma (RAS) mutation status were assessed by direct sequencing in the tumor tissues. Quantitative real-time polymerase chain reaction was used to quantify mature hsa-miR-224 in tumor tissue. RAS (10/107 cases, 9%) and RET (39/107 cases, 36%) mutations were mutually exclusive in sporadic cases. miR-224 expression was significantly downregulated in patients with the following: high calcitonin levels at diagnosis (p=0.03, r=−0.3); advanced stage (p=0.001); persistent disease (p=0.001); progressive disease (p=0.002); and disease-related death (p=0.0001). We found a significant positive correlation between miR-224 expression and somatic RAS mutations (p=0.007). Patients whose MTCs had a low miR-224 expression tended to have a shorter overall survival (log-rank test p=0.005). On multivariate analysis, miR-224 represented an independent prognostic marker. Our data indicate that miR-224 is upregulated in RAS-mutated MTCs and in patients with a better prognosis and could represent an independent prognostic marker in MTC patients

Frequency and significance of Ras, Tert promoter, and Braf mutations in cytologically indeterminate thyroid nodules: A monocentric case series at a tertiary-level Endocrinology unit

PurposeThe management of thyroid nodules of indeterminate cytology is controversial. Our study aimed to establish the frequency and significance of H-,K-,N-RAS, TERT promoter, and BRAF gene mutations in thyroid nodes of indeterminate cytology and to assess their potential usefulness in clinical practice.MethodsH-,K-,N-RAS, TERT promoter and BRAF gene mutations were examined in a series of 199 consecutive nodes of indeterminate cytology referred for surgical excision.Results69/199 (35%) were malignant on histopathological review. RAS mutations were detected in 36/199 (18%), and 19/36 cases (53%) were malignant on histological diagnosis. TERT promoter mutations were detected in 7/199 (4%) nodules, which were all malignant lesions. BRAF mutations were detected in 15/199 (8%), and a BRAF K601E mutation was identified in 2 follicular adenomas and 1 noninvasive follicular thyroid neoplasm with papillary-like nuclear features. Altogether, this panel was able to identify 48% of the malignant lesions, achieving a specificity, positive predictive value, and negative predictive value for malignancy of 85, 62, and 75%, respectively.ConclusionThe residual malignancy risk in mutation-negative nodes is 25%. These nodes still need to be resected, but mutation analysis could help to orient the appropriate surgical strategy

A constitutive active MAPK/ERK pathway due to BRAFV600E positively regulates AHR pathway in PTC

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor mediating the toxicity and tumor-promoting properties of dioxin. AHR has been reported to be overexpressed and constitutively active in a variety of solid tumors, but few data are currently available concerning its role in thyroid cancer. In this study we quantitatively explored a series of 51 paired-normal and papillary thyroid carcinoma (PTC) tissues for AHR-related genes. We identified an increased AHR expression/activity in PTC, independently from its nuclear dimerization partner and repressor but strictly related to a constitutive active MAPK/ERK pathway. The AHR up-regulation followed by an increased expression of AHR target genes was confirmed by a meta-analysis of published microarray data, suggesting a ligand-independent active AHR pathway in PTC. In-vitro studies using a PTC-derived cell line (BCPAP) and HEK293 cells showed that BRAF(V600E) may directly modulate AHR localization, induce AHR expression and activity in an exogenous ligand-independent manner. The AHR pathway might represent a potential novel therapeutic target for PTC in the clinical practice

PROGNOSTIC SIGNIFICANCE OF TERT PROMOTER AND BRAF MUTATIONS IN TIR-4 AND TIR-5 THYROID CYTOLOGY

Objective: Follicular-derived thyroid cancers generally have a good prognosis, but in a minority of cases, they have an aggressive behavior and develop distant metastases, with an increase in the associated mortality. None of the prognostic markers currently available prior to surgery can identify such cases. Methods: TERT promoter and BRAF gene mutations were examined in a series of 436 consecutive TIR-4 and TIR-5 nodes referred for surgery. Follow-up (median: 59 months, range: 7-293 months) was available for 384/423 patients with malignant nodes. Results: TERT promoter and BRAF mutations were detected in 20/436 (4.6%) and 257/434 thyroid nodules (59.2%), respectively. At the end of the follow-up, 318/384 patients (82.8%) had an excellent outcome, 48/384 (12.5%) had indeterminate response or biochemical persistence, 18/384 (4.7%) had a structural persistence or died from thyroid cancer. TERT promoter mutations correlated with older age (P < 0.0001), larger tumor size (P = 0.0002), oxyntic and aggressive PTC variants (P = 0.01), higher tumor stages (P < 0.0001), distant metastases (<0.0001) and disease outcome (P < 0.0001). At multivariate analysis, TERT promoter mutation was not an independent predictor of disease outcome. TERT promoter mutation- (OR: 40.58; 95% CI: 3.06-539.04), and N1b lymph node metastases (OR: 40.16, 95% CI: 3.48-463.04) were independent predictors of distant metastases. BRAF mutation did not predict the outcome, and it correlated with a lower incidence of distant metastases (P = 0.0201). Conclusions: TERT promoter mutation proved an independent predictor of distant metastases, giving clinicians the chance to identify many of the patients who warranted more aggressive initial treatment and closer follow-up

MiR-21 up-regulation in ampullary adenocarcinoma and its pre-invasive lesions

Poor information is available on the molecular landscape characterizing the carcinogenetic process leading to ampullary carcinoma. MiR-21 is one of the most frequently up-regulated miRNAs in pancreatic adenocarcinoma, a tumor sharing similar molecular features with ampullary adenocarcinomas (AVCs), above all with the pancreatic-biliary type. We profiled, by in situ hybridization (ISH), miR-21 expression in a series of 26 AVCs, 50 ampullary dysplastic lesions (35 low-grade [LG-IEN] and 15 high-grade [HG-IEN]) and 10 normal duodenal mucosa samples. The same series was investigated by immunohistochemistry for β-catenin, p53 and HER2 expression. HER2 gene amplification was evaluated by chromogenic in situ hybridization. To validate miR-21 ISH results we performed miR-21 qRT-PCR analysis in a series of 10 AVCs and their matched normal samples. All the normal control samples showed a negative or faint miR-21 expression, whereas a significant miR-21 up-regulation was observed during the carcinogenetic cascade (p &lt; 0.001), with 21/26 (80.8%) of cancer samples showing a miR-21 overexpression. In comparison to control samples, a significant overexpression was found in samples of LG-IEN (p = .0003), HG-IEN (p = .0001), and AVCs (p &lt; 0.0001). No significant difference in miR-21 overexpression was observed between LG-IEN, HG-IEN and AVCs. By qRT-PCR analysis, AVCs showed a 1.7-fold increase over the controls (p = .003). P53 was frequently dysregulated in both dysplastic and carcinoma samples (44 out of 76; 57.9%). A 20% (10/50) of dysplastic lesions and 11% (3/26) of carcinomas were characterized by a nuclear localization of β-catenin. Only 2 AVCs (7.7%; both intestinal-type) showed a HER2 overexpression (both 2+), which corresponded to a HER2 gene amplification at CISH analysis. This is the first study demonstrating a miRNA dysregulation in the whole spectrum of ampullary carcinogenesis. MiR-21 overexpression is an early molecular event during ampullary carcinogenesis and its levels increase with the neoplastic progression

Разработка и управление проектом строительства жилого дома в г. Краснодар

Текст работы публикуется с изъятиями

F-actin dynamics regulates mammalian organ growth and cell fate maintenance.

BACKGROUND & AIMS: In vitro, several data indicate that cell function can be regulated by the mechanical properties of cells and of the microenvironment. Cells measure these features by developing forces via their actomyosin cytoskeleton, and respond accordingly by transducing forces into biochemical signals that instruct cell behavior. Among these, the transcriptional coactivators YAP/TAZ recently emerged as key factors mediating multiple responses to actomyosin contractility. However, whether mechanical cues regulate adult liver tissue homeostasis, and whether this occurs through YAP/TAZ, remains largely unaddressed. METHODS & RESULTS: Here we show that the F-actin capping protein CAPZ is a critical negative regulator of actomyosin contractility and mechanotransduction. Capzb inactivation alters stress fiber and focal adhesion dynamics leading to enhanced myosin activity, increased cellular traction forces, and increased liver stiffness. In vitro, this rescues YAP from inhibition by a small geometry; in vivo, inactivation of Capzb in the adult mouse liver induces YAP activation in parallel to the Hippo pathway, causing extensive hepatocyte proliferation and leading to striking organ overgrowth. Moreover, Capzb is required for the maintenance of the differentiated hepatocyte state, for metabolic zonation, and for gluconeogenesis. In keeping with changes in tissue mechanics, inhibition of the contractility regulator ROCK, or deletion of the Yap1 mechanotransducer, reverse the phenotypes emerging in Capzb-null livers. CONCLUSIONS: These results indicate a previously unrecognized role for CAPZ in tuning the mechanical properties of cells and tissues, which is required in hepatocytes for the maintenance of the differentiated hepatocyte state and to regulate organ size. More in general, it indicates for the first time a physiological role of mechanotransduction in maintaining tissue homeostasis in mammals. LAY SUMMARY: The mechanical properties of cells and tissues (i.e. whether they are soft or stiff) are thought to be important regulators of cell behavior. A recent advancement in our understanding of these phenomena has been the identification of YAP and TAZ as key factors mediating the biological responses of cells to mechanical signals in vitro. However, whether the mechanical properties of cells and/or the mechanical regulation of YAP/TAZ are relevant for mammalian tissue physiology remains unknown. Here we challenge this issue by genetic inactivation of CAPZ, a protein that regulates the cytoskeleton, i.e. the cells' scaffold by which they sense mechanical cues. We found that inactivation of CAPZ alters cells' and liver tissue's mechanical properties, leading to YAP hyperactivation. In turn, this profoundly alters liver physiology, causing organ overgrowth, defects in liver cell differentiation and metabolism. These results reveal a previously uncharacterized role for mechanical signals for the maintenance of adult liver homeostasis.This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) Investigator Grant 15307, WCR (Worldwide Cancer Research) Grant 15-1192, CARIPARO Eccellenza Program 2017 and University of Padua BIRD Grant to SD, AIRC ‘Hard ROCK Café’ Fellowship to GS, Marie Sklodowska-Curie Individual Fellowship (796547) to AG, AIRC Special Program Molecular Clinical Oncology ‘5 per mille’ 10016 to SB, UK Medical Research Council and Sackler Foundation Doctoral Training Grant RG70550 to ACL, UK Medical Research Council Career Development Award G1100312/1 and an Isaac Newton Trust Research Grant 17.24(p) to KF

Veno-occlusive disease nurse management: Development of a dynamic monitoring tool by the GITMO nursing group

Veno-occlusive disease (VOD) is a complication arising from the toxicity of conditioning regimens that have a significant impact on the survival of patients who undergo stem cell transplantation. There are several known risk factors for developing VOD and their assessment before the start of conditioning regimens could improve the quality of care. Equally important are early identification of signs and symptoms ascribable to VOD, rapid diagnosis, and timely adjustment of support therapy and treatment. Nurses have a fundamental role at the stages of assessment and monitoring for signs and symptoms; therefore, they should have documented skills and training. The literature defines nurses' areas of competence in managing VOD, but in the actual clinical practice, this is not so clear. Moreover, there is an intrinsic difficulty in managing VOD due to its rapid and often dramatic evolution, together with a lack of care tools to guide nurses. Through a complex evidence-based process, the Gruppo Italiano per il Trapianto di Midollo Osseo (GITMO), cellule staminali emopoietiche e terapia cellulare nursing board has developed an operational flowchart and a dynamic monitoring tool applicable to haematopoietic stem cell transplantation patients, whether they develop this complication or not

F-actin dynamics regulates mammalian organ growth and cell fate maintenance.

BACKGROUND & AIMS: In vitro, several data indicate that cell function can be regulated by the mechanical properties of cells and of the microenvironment. Cells measure these features by developing forces via their actomyosin cytoskeleton, and respond accordingly by transducing forces into biochemical signals that instruct cell behavior. Among these, the transcriptional coactivators YAP/TAZ recently emerged as key factors mediating multiple responses to actomyosin contractility. However, whether mechanical cues regulate adult liver tissue homeostasis, and whether this occurs through YAP/TAZ, remains largely unaddressed. METHODS & RESULTS: Here we show that the F-actin capping protein CAPZ is a critical negative regulator of actomyosin contractility and mechanotransduction. Capzb inactivation alters stress fiber and focal adhesion dynamics leading to enhanced myosin activity, increased cellular traction forces, and increased liver stiffness. In vitro, this rescues YAP from inhibition by a small geometry; in vivo, inactivation of Capzb in the adult mouse liver induces YAP activation in parallel to the Hippo pathway, causing extensive hepatocyte proliferation and leading to striking organ overgrowth. Moreover, Capzb is required for the maintenance of the differentiated hepatocyte state, for metabolic zonation, and for gluconeogenesis. In keeping with changes in tissue mechanics, inhibition of the contractility regulator ROCK, or deletion of the Yap1 mechanotransducer, reverse the phenotypes emerging in Capzb-null livers. CONCLUSIONS: These results indicate a previously unrecognized role for CAPZ in tuning the mechanical properties of cells and tissues, which is required in hepatocytes for the maintenance of the differentiated hepatocyte state and to regulate organ size. More in general, it indicates for the first time a physiological role of mechanotransduction in maintaining tissue homeostasis in mammals. LAY SUMMARY: The mechanical properties of cells and tissues (i.e. whether they are soft or stiff) are thought to be important regulators of cell behavior. A recent advancement in our understanding of these phenomena has been the identification of YAP and TAZ as key factors mediating the biological responses of cells to mechanical signals in vitro. However, whether the mechanical properties of cells and/or the mechanical regulation of YAP/TAZ are relevant for mammalian tissue physiology remains unknown. Here we challenge this issue by genetic inactivation of CAPZ, a protein that regulates the cytoskeleton, i.e. the cells' scaffold by which they sense mechanical cues. We found that inactivation of CAPZ alters cells' and liver tissue's mechanical properties, leading to YAP hyperactivation. In turn, this profoundly alters liver physiology, causing organ overgrowth, defects in liver cell differentiation and metabolism. These results reveal a previously uncharacterized role for mechanical signals for the maintenance of adult liver homeostasis.This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) Investigator Grant 15307, WCR (Worldwide Cancer Research) Grant 15-1192, CARIPARO Eccellenza Program 2017 and University of Padua BIRD Grant to SD, AIRC ‘Hard ROCK Café’ Fellowship to GS, Marie Sklodowska-Curie Individual Fellowship (796547) to AG, AIRC Special Program Molecular Clinical Oncology ‘5 per mille’ 10016 to SB, UK Medical Research Council and Sackler Foundation Doctoral Training Grant RG70550 to ACL, UK Medical Research Council Career Development Award G1100312/1 and an Isaac Newton Trust Research Grant 17.24(p) to KF