Insulin resistance disrupts epithelial repair and niche-progenitor Fgf signaling during chronic liver injury.

Insulin provides important information to tissues about feeding behavior and energy status. Defective insulin signaling is associated with ageing, tissue dysfunction, and impaired wound healing. In the liver, insulin resistance leads to chronic damage and fibrosis, but it is unclear how tissue-repair mechanisms integrate insulin signals to coordinate an appropriate injury response or how they are affected by insulin resistance. In this study, we demonstrate that insulin resistance impairs local cellular crosstalk between the fibrotic stroma and bipotent adult liver progenitor cells (LPCs), whose paracrine interactions promote epithelial repair and tissue remodeling. Using insulin-resistant mice deficient for insulin receptor substrate 2 (Irs2), we highlight dramatic impairment of proregenerative fibroblast growth factor 7 (Fgf7) signaling between stromal niche cells and LPCs during chronic injury. We provide a detailed account of the role played by IRS2 in promoting Fgf7 ligand and receptor (Fgfr2-IIIb) expression by the two cell compartments, and we describe an insulin/IRS2-dependent feed-forward loop capable of sustaining hepatic re-epithelialization by driving FGFR2-IIIb expression. Finally, we shed light on the regulation of IRS2 and FGF7 within the fibrotic stroma and show-using a human coculture system-that IRS2 silencing shifts the equilibrium away from paracrine epithelial repair in favor of fibrogenesis. Hence, we offer a compelling insight into the contribution of insulin resistance to the pathogenesis of chronic liver disease and propose IRS2 as a positive regulator of communication between cell types and the transition between phases of stromal to epithelial repair.

Lack of GDAP1 induces neuronal calcium and mitochondrial defects in a knockout mouse model of charcot-marie-tooth neuropathy.

Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca2+, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria-endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.

Opposite Effects of Mechanical Action of Fluid Flow on Proangiogenic Factor Secretion From Human Adipose-Derived Stem Cells With and Without Oxidative Stress.

Mechanical forces, hypoxia, and oxidative stress contribute to skin renewal, perfusion, and wound healing, but how are they regulating subcutaneous adipose-derived stem cells (ASCs) in the inflammatory microenvironment associated to skin repair and disorders is unknown. In this study, ASCs were isolated from lipoaspirate samples from plastic surgery patients, primary cultured and their differentiation and secretion of a panel of cytokines with pronounced effects on skin repair and angiogenesis were studied under mechanical stimulation by intermittent fluid flow, 1% hypoxia and oxidative stress by glutathione (GSH) depletion with buthionine sulfoximine (BSO) treatment. Mechanical action of fluid flow did not alter mesenchymal phenotype of CD90+ /CD29+ /CD44+ /CD34- /CD106- /CD45- ASCs; however, it remarkably induced ASC secretion of human umbilical vein endothelial cell (HUVEC) migration-stimulating factors. Multiplex Luminex assay further confirmed an increased secretion of VEGF, G-CSF, HGF, Leptin, IL-8, PDGF-BB, Angiopoietin-2, and Follistatin from mechanically-stimulated ASCs via cyclooxygenase-2. Consistent with this mechanism, GSH depletion and hypoxia also increased ASC secretion of VEGF, IL-8, leptin, Angiopoitein-2, and PDGF-BB. However, mechanical action of fluid flow abrogated VEGF and HUVEC migration-stimulating activity from GSH-depleted and hypoxic ASCs. Conversely, GSH depletion and hypoxia abrogated VEGF and HUVEC migration-stimulating activity from mechano-stimulated ASCs. Although mechanical action of fluid flow, hypoxia, and GSH-depletion had independent proangiogenic-stimulating activity on ASCs, mechanical stimulation had opposite effects on proangiogenic factor secretion from ASCs with and without oxidative stress. These data uncover the role of hypoxia and endogenous redox balance during the proangiogenic response of ASCs and other mesenchymal-derived cell types to mechanical action of interstitial fluid flow. J. Cell. Physiol. 232: 2158-2167, 2017. © 2016 Wiley Periodicals, Inc.

Cryoplasty versus angioplasty in the treatment of arterial restenosis in an experimental model of atherosclerosis in rabbits.

Cryoplasty may reduce the incidence of post-angioplasty restenosis in peripheral atherosclerotic arteries. Our study is looking to investigate the mid-term effects (4 weeks) of an FDA-approved cryoplasty catheter (PolarCath(®), Boston Scientific) compared to a conventional angioplasty catheter using a hypercholesterolemic rabbit model of arterial restenosis based on diet plus vessel injury. Thirty-six normolipidemic, 3-month old male New Zealand White rabbits were used. Balloon angioplasty was performed on left external iliac arteries on day 1. Animals were fed with a hypercholesterolemic diet for 60 days. On day 120, three groups of animals were established: conventional PTA (percutaneous transluminal angioplasty) was applied on the PTA group; the CRY group was treated with the PolarCath(®) cryoplasty system and no treatment was given to a control (CTR) group. A broad variety of atheromatous lesions were observed 30 days after treatment, presenting significant differences between groups. Most of the complicated lesions were found in the CRY group, while advanced and early lesions were more often appreciated in the CTR and PTA groups, respectively. The histomorphometric evaluation of the arteries showed significant differences between the CRY group and the other two groups, with the highest percentage of IEM (internal elastic membrane) injury, vascular stenosis and ratio intima/media being registered on animals treated with cryoplasty. Intravascular cryotherapy induces complicated lesions in arterial walls 30 days after treatment in a hypercholesterolemic rabbit model based on diet plus vessel injury. Cryoplasty leads to the production of severe fibrosis and mineralisation and stenosis compared to a conventional angioplasty.

Ultrastructural pathology of anaplastic and grade II ependymomas reveals distinctive ciliary structures--electron microscopy redux.

Ependymoma tumors likely derive from the ependymal cells lining the CNS ventricular system. In grade II ependymomas, tumor cells resemble typical ependymocytes, while anaplastic ependymomas are poorly differentiated. We studied three grade II and one anaplastic ependymoma, focusing on the ciliary structures. To unambiguously characterize the ultrastructure and number of cilia, we performed electron microscopy serial section analysis of individual cells. Differentiated ependymomas contained large basal bodies and up to three cilia, and lacked centrioles. Anaplastic ependymoma cells showed instead two perpendicularly oriented centrioles and lacked cilia or basal bodies. These findings could contribute to understand the mechanisms of ependymoma aggressiveness.

Influence of on-site cytopathology evaluation on the diagnostic accuracy of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of solid pancreatic masses.

OBJECTIVES: The aim of this study was to evaluate the influence of on-site cytopathological evaluation on the diagnostic yield of endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) for the differential diagnosis of solid pancreatic masses in an unselected series of consecutive patients. METHODS: Patients undergoing EUS-guided FNA of solid pancreatic lesions over a 2-year study period were included. Samples were either evaluated on site by a cytopathologist or processed by the endoscopist and sent to the pathology department for evaluation. Diagnostic accuracy for malignancy, number of needle passes, adequate-specimen collection rate, cytological diagnosis, and final diagnosis, and complication rate according to the presence or absence of on-site cytopathologist were evaluated. RESULTS: A total of 182 patients were included. An on-site cytopathologist was available in 95 cases (52.2%). There was no difference between groups in terms of age, sex, location, and size of the lesions. A significantly higher number of needle passes was performed when an on-site cytopathologist was not available (3.5±1.0 vs. 2.0±0.7; P<0.001). The presence of an on-site cytopathologist was associated with a significantly lower number of inadequate samples (1.0 vs. 12.6%, P=0.002), and a significantly higher diagnostic sensitivity (96.2 vs. 78.2%; P=0.002) and overall accuracy (96.8 vs. 86.2%; P=0.013) for malignancy. Three patients developed complications (two acute pancreatitis, one local bleeding), all of them belonging to the group without on-site cytopathology. CONCLUSIONS: On-site cytopathological evaluation improves the diagnostic yield of EUS-guided FNA for the cytological diagnosis of solid pancreatic masses. This is associated with a significantly lower number of inadequate samples and a lower number of needle passes.

Comprehensive NGS Panel Validation for the Identification of Actionable Alterations in Adult Solid Tumors.

The increasing identification of driver oncogenic alterations and progress of targeted therapies addresses the need of comprehensive alternatives to standard molecular methods. The translation into clinical practice of next-generation sequencing (NGS) panels is actually challenged by the compliance of high quality standards for clinical accreditation. Herein, we present the analytical and clinical feasibility study of a hybridization capture-based NGS panel (Action OncoKitDx) for the analysis of somatic mutations, copy number variants (CNVs), fusions, pharmacogenetic SNPs and Microsatellite Instability (MSI) determination in formalin-fixed paraffin-embedded (FFPE) tumor samples. A total of 64 samples were submitted to extensive analytical validation for the identification of previously known variants. An additional set of 166 tumor and patient-matched normal samples were sequenced to assess the clinical utility of the assay across different tumor types. The panel demonstrated good specificity, sensitivity, reproducibility, and repeatability for the identification of all biomarkers analyzed and the 5% limit of detection set was validated. Among the clinical cohorts, the assay revealed pathogenic genomic alterations in 97% of patient cases, and in 82.7%, at least one clinically relevant variant was detected. The validation of accuracy and robustness of this assay supports the Action OncoKitDx's utility in adult solid tumors.

Human adipose-derived mesenchymal stem cells accelerate decellularized neobladder regeneration.

Decellularized natural bladder matrices (neobladders) represent an exciting means to regenerate the bladder following bladder cancer-associated cystectomy. In this study, we compare the evolution of decellularized matrices with recellularized matrices by seeding it with human adipose-derived mesenchymal stem cells (ADSC) after implantation following partial cystectomy in rats. We discovered significant anatomical differences since 10 days after neobladder implantation with the ADSC-containing matrices promoting a significant recovery of mature p63- and cytokeratin 7-positive urothelium. We also discovered significantly induced expression of the vimentin mesoderm marker in the submucosal layer in ADSC-seeded matrices. Interestingly, we found a higher expression of smooth muscle actin in transversal and longitudinal smooth muscle layers with ADSC-seeded matrices. Furthermore, ADSC also showed increased vascularization and nerve innervation of the neobladder as determined by the distribution of CD31 and S100ß reactivity, respectively. We believe that ADSC and their paracrine-acting pro-regenerative secretome within decellularized matrices represent an efficient bladder substitution strategy; however, we require a fuller understanding of the mechanisms involved before clinical studies can begin.

Clinicopathological and immnuohistochemical findings in a series of folliculosebaceous cystic hamartoma.

BACKGROUND: Folliculo-sebaceous cystic hamartoma (FSCH) is an uncommon skin condition presenting as a slow-growing papulo-nodular lesion, in or around the nose. Most cases are not clinically suspected and only histopathological examination allows the diagnosis. Pathological features include a dermal-located infundibulo-cystic structure with sebaceous glands radiating around, a stromal component encircling the epithelial structures, with clefts between the lesional epithelial and stromal parts, as well as between this and the adjacent dermis. RESULTS: We report eight patients with the diagnosis of FSCH (5 females and 3 males), with ages ranging from 35 to 77 years. Most cases (5 out of 8) were located in or around the nose and sizes were comprised between 0.6 and 1.2 cm. Lesions had grown for long periods of time, up to ten years in one case. Immunohistochemistry showed staining for p63 in the epithelial component of all lesions, while CD10 was only present in some sebocytes. CD34 and Factor XIIIa positive cells were present in the lesional stroma. Staining for androgen and alpha-estrogen receptors was also usually noticed. CONCLUSIONS: FCSH is a hamartomatous skin lesion, clinically indistinct but with well-defined histopathological features. Immunohistochemistry shows a profile very close to normal sebaceous glands.

[Laryngeal adenocarcinoma: case report]. / Adenocarcinoma de laringe: presentación de un caso.

Laryngeal adenocarcinomas are extremely unusual (only 0.35 %-0.5 % of all laryngeal malignancies). It usually behaves as a highly malignant tumour, with tendency for local lymph node and distant organ metastases. We report a case of a 64-year-old man with an adenocarcinoma of the larynx with cervical lymph node involvement at the moment of the diagnosis. The patient was treated with surgery (total laryngectomy and cervical lymph node dissection) and post-operative radiotherapy. Seven months later, the patient had a local recurrence.

Prognostic Significance of Cyclins A2, B1, D1, and E1 and CCND1 Numerical Aberrations in Oral Squamous Cell Carcinomas.

We analysed the expression of cyclins A2, B1, D1, and E1 by immunohistochemistry and numerical aberrations in CCND1 gene by fluorescence in situ hybridization technique in 67 primary oral squamous cell carcinomas (OSCC). Cyclin A2 expression was observed in 54 (83.1%) tumours, cyclin D1 in 58 (89.2%), cyclin B1 in 39 (60%), and cyclin E in 21 (32.8%). CCND1 region analysis revealed 26 (43.3%) tumours with the presence of numerical aberrations which were correlated with cyclin D1 high expression (Rho = 0.48; p < 0.001). Twenty-nine (45.3%) tumours were classified as high proliferative tumours assessed by Ki-67 protein expression and correlated with tumours with high expression of cyclin A2 (Rho = 0.30; p = 0.016) and cyclin B1 (Rho = 0.37; p = 0.003). In multivariate analysis for an overall five-year survival (OS), we found an adverse independent prognostic value for cyclin A2 high expression (p = 0.031) and for advanced tumour stage (p < 0.001). Our results confirm that several cyclins are commonly expressed in OSCC. CCND1 gene is abnormal in more than one-third of the cases and is frequently associated with cyclin D1 high expression. Moreover, cyclin A2 high expression is an independent indicator of worse OS suggesting that this protein may serve as a reliable biological marker to identify high-risk subgroups with poor prognosis.

Imbalance of immunological synapse-kinapse states reflects tumor escape to immunity in glioblastoma.

Since the proper activation of T cells requires the physical interaction with target cells through the formation of immunological synapses (IS), an alteration at this level could be a reason why tumors escape the immune response. As part of their life cycle, it is thought that T cells alternate between a static phase, the IS, and a dynamic phase, the immunological kinapse (IK), depending on high or low antigen sensing. Our investigation performed in tissue samples of human glioma shows that T cells are able to establish synapsing interactions not only with glioma tumorigenic cells, but also with stromal myeloid cells. Particularly, the IS displaying a T cell receptor-rich (TCR-rich) central supramolecular activation cluster (cSMAC) is preferentially established with stromal cells, as opposed to malignant cells. Conversely, T cells in the malignant areas showed distinct morphometric parameters compared with nonneoplastic tissue - the former characterized by an elongated shape, well-suited to kinaptic dynamics. Importantly, high-resolution 3-dimensional analyses demonstrated the existence of bona-fide IK preferentially arranged in malignant areas of the tumor. This imbalance of IS/IK states between these 2 microenvironments reveals the low antigenic sensing of T cells when patrolling tumorigenic cells and reflects the immunoevasive environment of the tumor.

Human Suprapatellar Fat Pad-Derived Mesenchymal Stem Cells Induce Chondrogenesis and Cartilage Repair in a Model of Severe Osteoarthritis.

Cartilage degeneration is associated with degenerative bone and joint processes in severe osteoarthritis (OA). Spontaneous cartilage regeneration is extremely limited. Often the treatment consists of a partial or complete joint implant. Adipose-derived stem cell (ASC) transplantation has been shown to restore degenerated cartilage; however, regenerative differences of ASC would depend on the source of adipose tissue. The infra- and suprapatellar fat pads surrounding the knee offer a potential autologous source of ASC for patients after complete joint substitution. When infrapatellar- and suprapatellar-derived stromal vascular fractions (SVF) were compared, a significantly higher CD105 (+) population was found in the suprapatellar fat. In addition, the suprapatellar SVF exhibited increased numbers of colony formation units and a higher population doubling in culture compared to the infrapatellar fraction. Both the suprapatellar- and infrapatellar-derived ASC were differentiated in vitro into mature adipocytes, osteocytes, and chondrocytes. However, the suprapatellar-derived ASC showed higher osteogenic and chondrogenic efficiency. Suprapatellar-derived ASC transplantation in a severe OA mouse model significantly diminished the OA-associated knee inflammation and cartilage degenerative grade, significantly increasing the production of glycosaminoglycan and inducing endogenous chondrogenesis in comparison with the control group. Overall, suprapatellar-derived ASC offer a potential autologous regenerative treatment for patients with multiple degenerative OA.

Immunohistochemical expression of HIF-1alpha in response to early myocardial ischemia.

This study aims to evaluate the effects of ischemia on the myocardial fibers and the expression of the transcriptional factor for angiogenesis hypoxia-inducible factor-1 alpha (HIF-1alpha) in human heart specimens. We have prospectively analyzed the HIF-1alpha expression in human ischemic hearts with the ABC-inmunohistochemistry technique and amplification by biotinylated tyramide. The relationship between the expression of HIF-1alpha and the temporal evolution of ischemia has also been evaluated. As pathomorphological diagnosis of early myocardial ischemia has many problems in human autopsy material with less than 4 to 6 h after clinical onset, we suggest that HIF-1alpha is helpful in the early acute myocardial infarction diagnosis, so it stains necrotic areas within the first 2 h. The amplification procedure provides a higher intensity of the final staining without losing specificity. It is concluded that in normal cardiac fibers, basal expression of HIF-1alpha is not appreciable, but it steadily increases after ischemia. With regard to the practical applicability in forensic field, our observations suggest that positive immunohistochemical expression of HIF-1alpha on heart samples may be used as a reliable indicator of myocardial damage in cases without cardiac lesion evidence, using conventional microscopy. This method is especially useful and may provide definitive proof of myocardial ischemia in unexpected deaths without previous symptoms, or in forensic cases with a short period of clinical manifestations. In addition, it may have been involved in possible future cardiovascular therapies.

Controversial origin of Pseudomyxoma peritonei.

Pseudomyxoma peritonei describes the accumulation of mucinous material in the abdominal cavity. The main diagnostic problem appears when the primary site of origin could be appendix or ovary. In this paper describe clinicopathological features and biological markers that support appendiceal origin.

Family history and breast cancer hormone receptor status in a Spanish cohort.

BACKGROUND: Breast cancer is a heterogenous disease that impacts racial/ethnic groups differently. Differences in genetic composition, lifestyles, reproductive factors, or environmental exposures may contribute to the differential presentation of breast cancer among Hispanic women. MATERIALS AND METHODS: A population-based study was conducted in the city of Santiago de Compostela, Spain. A total of 645 women diagnosed with operable invasive breast cancer between 1992 and 2005 participated in the study. Data on demographics, breast cancer risk factors, and clinico-pathological characteristics of the tumors were collected. Hormone receptor negative tumors were compared with hormone receptor postive tumors on their clinico-pathological characteristics as well as risk factor profiles. RESULTS: Among the 645 breast cancer patients, 78% were estrogen receptor-positive (ER+) or progesterone receptor-positive (PR+), and 22% were ER-&PR-. Women with a family history of breast cancer were more likely to have ER-&PR- tumors than women without a family history (Odds ratio, 1.43; 95% confidence interval, 0.91-2.26). This association was limited to cancers diagnosed before age 50 (Odds ratio, 2.79; 95% confidence interval, 1.34-5.81). CONCLUSIONS: An increased proportion of ER-&PR- breast cancer was observed among younger Spanish women with a family history of the disease.