ROCK inhibitor (Y27632) increases apoptosis and disrupts the actin cortical mat in embryonic avian corneal epithelium.

The embryonic chicken corneal epithelium is a unique tissue that has been used as an in vitro epithelial sheet organ culture model for over 30 years (Hay and Revel [1969] Fine structure of the developing Avian cornea. Basel, Switzerland: S. Karger A.G.). This tissue was used to establish that epithelial cells could produce extracellular matrix (ECM) proteins such as collagen and proteoglycans (Dodson and Hay [1971] Exp Cell Res 65:215-220; Meier and Hay [1973] Dev Biol 35:318-331; Linsenmayer et al. [1977] Proc Natl Acad Sci U S A 74:39-43; Hendrix et al. [1982] Invest Ophthalmol Vis Sci 22:359-375). This historic model was also used to establish that ECM proteins could stimulate actin reorganization and increase collagen synthesis (Sugrue and Hay [1981] J Cell Biol 91:45-54; Sugrue and Hay [1982] Dev Biol 92:97-106; Sugrue and Hay [1986] J Cell Biol 102:1907-1916). Our laboratory has used the model to establish the signal transduction pathways involved in ECM-stimulated actin reorganization (Svoboda et al. [1999] Anat Rec 254:348-359; Chu et al. [2000] Invest Ophthalmol Vis Sci 41:3374-3382; Reenstra et al. [2002] Invest Ophthalmol Vis Sci 43:3181-3189). The goal of the current study was to investigate the role of ECM in epithelial cell survival and the role of Rho-associated kinase (p160 ROCK, ROCK-1, ROCK-2, referred to as ROCK), in ECM and lysophosphatidic acid (LPA) -mediated actin reorganization. Whole sheets of avian embryonic corneal epithelium were cultured in the presence of the ROCK inhibitor, Y27632 at 0, 0.03, 0.3, 3, or 10 microM before stimulating the cells with either collagen (COL) or LPA. Apoptosis was assessed by Caspase-3 activity assays and visualized with annexin V binding. The ROCK inhibitor blocked actin cortical mat reformation and disrupted the basal cell lateral membranes in a dose-dependent manner and increased the apoptosis marker annexin V. In addition, an in vitro caspase-3 activity assay was used to determine that caspase-3 activity was higher in epithelia treated with 10 microM Y-27632 than in those isolated without the basal lamina or epithelia stimulated with fibronectin, COL, or LPA. In conclusion, ECM molecules decreased apoptosis markers and inhibiting the ROCK pathway blocked ECM stimulated actin cortical mat reformation and increased apoptosis in embryonic corneal epithelial cells.     

Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response

Congenital nephrogenic diabetes insipidus (NDI) is a rare disease caused most often by mutations in the vasopressin V2 receptor (AVPR2). We studied a family which included a female patient with NDI with symptoms dating from infancy. The patient responded to large doses of desmopressin (dDAVP) which decreased urine volume from 10 to 4 I/day. Neither the parents nor the three sisters were polyuric. The patient was found to be a compound heterozygote for two novel recessive point mutations in the aquaporin-2 (AQP2) gene: L22V in exon 1 and C181W in exon 3. Residue Cys181 in AQP2 is the site for inhibition of water permeation by mercurial compounds and is located near to the NPA motif conserved in all aquaporins. Osmotic water permeability (Pf) in Xenopus oocytes injected with cRNA encoding C181W-AQP2 was not increased over water control, while expression of L22V cRNA increased the Pf to approximately 60% of that for wild-type AQP2. Co-injection of the mutant cRNAs with the wild-type cRNA did not affect the function of the wild-type AQP2. Immunolocalization of AQP2-transfected CHO cells showed that the C181W mutant had an endoplasmic reticulum-like intracellular distribution, whereas L22V and wild-type AQP2 showed endosome and plasma membrane staining. Water permeability assays showed a high Pf in cells expressing wild-type and L22V AQP2. This study indicates that AQP2 mutations can confer partially responsive NDI.      

ATG16L1 and NOD2 polymorphisms enhance phagocytosis in monocytes of Crohn’s disease patients

AIM:To investigate if the presence of relevant genetic polymorphisms has effect on the effectual clearance of bacteria by monocytes and granulocytes in patients with Crohn’s disease(CD).METHODS:In this study,we assessed the differential responses in phagocytosis by measuring the phagocytic activity and the percentage of active phagocytic monocytes and granulocytes in inflammatory bowel disease patients as well as healthy controls.As both autophagy related like 1(ATG16L1)and immunityrelated guanosine triphosphatase gene are autophagy genes associated with CD and more recently nucleo-tide-binding ligomerization domain-containing protein2(NOD2)has been identified as a potent inducer of autophagy we genotyped the patients for these variants and correlated this to the phagocytic reaction.The genotyping was done with restriction fragment length polymorphisms analysis and the phagocytosis was determined with the pHrodo?Escherichia coli Bioparticles Phagocytosis kit for flowcytometry.RESULTS:In this study,we demonstrate that analysis of the monocyte and granulocyte populations of patients with CD and ulcerative colitis showed a comparable phagocytic activity(ratio of mean fluorescence intensity)between the patient groups and the healthy controls.CD patients show a significantly higher phagocytic capacity(ratio mean percentage of phagocytic cells)compared to healthy controls(51.91%±2.85%vs 37.67%±7.06%,P=0.05).The extend of disease was not of influence.However,variants of ATG16L1(WT:2.03±0.19 vs homozygoot variant:4.38±0.37,P<0.009)as well as NOD2(C-ins)(heterozygous variant:42.08±2.94 vs homozygous variant:75.58±4.34(P=0.05)are associated with the phagocytic activity in patients with CD.CONCLUSION:Monocytes of CD patients show enhanced phagocytosis associated with the presence of ATG16L1 and NOD2 variants.This could be part of the pathophysiological mechanism resulting in the disease.     

Evaluation of Efficacy of Microwave Irradiation in Disinfecting Dental Gypsum Casts: An Ex Vivo Study

The aim of this study is to assess the efficacy of microwave irradiation in disinfecting gypsum casts and also to compare its efficacy with validated method of chemical disinfection. The present study is an ex vivo study conducted on a sample of five irreversible hydrocolloid impressions in vitro and on ten patients gypsum casts in vivo following standard impression techniques to check the efficacy of microwave oven irradiation and compare its efficacy with standard chemical method of disinfection. Results were analysed using Mann–Whitney test and Wilcoxon signed rank test. Untreated gypsum casts showed cfu/ml counts with a median log value of 6, while microwave-irradiated ones had median cfu/ml counts of 0. Casts poured from chemically disinfected impressions demonstrated cfu/ml counts with a median log value of 5. Microwave irradiation was found to be effective in disinfecting gypsum casts when compared to chemical disinfectant in disinfecting dental impressions.     

Acoustic radiation force impulse (ARFI) elastography in acute liver failure: necrosis mimics cirrhosis

Acoustic radiation force Impulse (ARFI) technology correlates shear-wave velocity with fibrosis. It can differentiate between advanced fibrosis and normal tissue in chronic liver disease. However, specificity is impaired by cholestasis, inflammation or oedema in acute hepatitis. In patients with acute liver failure (ALF) necessitating liver transplantation ARFI has not been evaluated yet. We investigated 3 patients with ALF and compared their ARFI results to those of healthy controls (n = 33) and cases with liver cirrhosis (n = 21). In the 3 ALF patients shear-wave velocities were 3.0, 2.5, and 2.7 m/s, respectively. These results were significantly increased compared to those of healthy controls (median: 1.13 m/s; p < 0.001) and similar to those of cirrhotic individuals (median: 2.93 m/s). Two individuals underwent liver transplantation. Explants showed massive necrosis, but no signs of chronic liver disease. Patient 3 recovered spontaneously and showed decreasing ARFI results during follow-up. In conclusion, hepatic necrosis can mimic liver cirrhosis at ARFI evaluation in ALF patients and this impairs the specificity of ARFI.     

The clinical psychological diagnostic system (KPD‐38): Sensitivity to change and validity of a self‐report instrument for outcome monitoring and quality assurance

Background: Monitoring psychotherapeutic progress requires valid and economic measures to detect change of clinical relevance. We addressed validity and sensitivity to change of the ‘Klinisch Psychologisches Diagnosesystem 38’ (KPD‐38, Clinical Psychological Diagnosis System), an instrument for outcome monitoring and quality assurance. Methods: Data from an inpatient sample (n = 1.377) were used to investigate the concurrent validity, the sensitivity to change, and the criterion validity of the KPD‐38 in comparison to the Outcome Questionnaire (OQ‐45) and the Brief Symptom Inventory (BSI). Results: Sensitivity‐specificity analyses showed similar sensitivity and higher specificity for the KPD‐38 scales compared with the BSI and OQ total scores. Change rates on the KPD‐38 distress scale (DIS) were found to be lower than on the BSI (KPD‐38 DIS: 36.6%, GSI: 67.7%) and the OQ‐45 (KPD‐38 DIS: 44.3%, OQ tot: 57.1%) total scores. Concordance was low with the BSI ([κ = 0.24] and moderate with the OQ‐45 [κ = 0.45]). Discussion: Implications for applications in the field of outcome assessment and the challenge of further validation are discussed. The relation between sensitivity to change and criterion validity is highlighted. Copyright © 2010 John Wiley & Sons, Ltd. Key Practitioner Messages: ? The KPD‐38 is a self‐report questionnaire for outcome monitoring and quality assurance, which is free of charge for research purposes. ? Both OQ‐45 and BSI show higher sensitivity to change than the KPD‐38, overall concordance of change is moderate. ? Aspects of validity should be considered in the evaluation of sensitivity to change.