Deletion of the Met tyrosine kinase in liver progenitor oval cells increases sensitivity to apoptosis in vitro

The hepatocyte growth factor (HGF)/Met signaling system is essential for liver development, homeostasis, and function. In this study, we took advantage of a liver-specific, Met-conditional knockout mouse generated in our laboratory to address the molecular mechanisms of HGF/Met signaling in adult liver progenitor cell (oval cell) biology. For this purpose, we isolated oval cells from 3,5-diethoxycarbonyl-1,4-dihydro-collidine-treated Met(flx/flx) mice and established oval cell-derived cell lines that carried either functional (Met(flx/flx)) or a nonfunctional (Met(-/-)) met gene using virus-mediated Cre-loxP recombination. Oval cells lacking Met tyrosine kinase activity displayed neither Met phosphorylation nor activation of downstream targets and were refractory to HGF stimulation. Although Met(-/-) and Met(flx/flx) cells proliferated at similar rates under 10% serum, Met-deficient cells demonstrated decreased cell viability and were more prone to apoptosis when challenged with either serum starvation or the pro-apoptotic cytokine transforming growth factor-beta. Treatment with HGF reduced transforming growth factor-beta-mediated cell death in Met(flx/flx) but not Met(-/-) cells. Importantly, Met(flx/flx) and Met(-/-) cells both constitutively expressed hgf, and conditioned medium from serum-starved oval cells exhibited anti-apoptotic activity in Met(flx/flx) cells. Furthermore, serum-starved Met(flx/flx) cells showed persistent activation of the Met tyrosine kinase, suggesting HGF/Met autocrine regulation. In conclusion, these data reveal a critical, functional role for Met in oval cell survival through an autocrine mechanism.     

LAMA2 gene analysis in a cohort of 26 congenital muscular dystrophy patients

Congenital muscular dystrophy type 1A (MDC1A) is caused by mutations in the LAMA2 gene encoding laminin-alpha2. We describe the molecular study of 26 patients with clinical presentation, magnetic resonance imaging and/or laminin-alpha2 expression in muscle, compatible with MDC1A. The combination of full genomic sequencing and complementary DNA analysis led to the particularly high mutation detection rate of 96% (50/52 disease alleles). Besides 22 undocumented polymorphisms, 18 different mutations were identified in the course of this work, 14 of which were novel. In particular, we describe the first fully characterized gross deletion in the LAMA2 gene, encompassing exon 56 (c.7750-1713_7899-2153del), detected in 31% of the patients. The only two missense mutations detected were found in heterozygosity with nonsense or truncating mutations in the two patients with the milder clinical presentation and a partial reduction in muscle laminin-alpha2. Our results corroborate the previous few genotype/phenotype correlations in MDC1A and illustrate the importance of screening for gross rearrangements in the LAMA2 gene, which may be underestimated in the literature.     

Abnormal nuclear structures (micronuclei, nucleoplasmic bridges, and nuclear buds) in a pleomorphic giant cell carcinoma of the stomach

A rare case of pleomorphic giant cell carcinoma of the stomach in a 70-year-old man is reported. Characteristic microscopic findings included a general lack of architectural cohesiveness, aggregates of mononucleated or multinucleated giant cells, extensive areas of coagulative necrosis, and numerous mitoses. Immunohistochemically, tumor cells displayed cytoplasmic immunoreactivity for cytokeratin AE1/AE3 as well as overexpression of p53 and Ki-67. Electron microscopy revealed paranuclear tonofilaments bundles in giant cells confirming their epithelial nature. Furthermore, giant cells contained two or more nuclei with heterogeneous size, nucleoplasmic bridges, nuclear buds, and micronuclei. Similar abnormal nuclear structures have been closely related to breakage-fusion-bridge type of mitotic disturbances in tumor cell lines, and have not been previously reported in a human tumor.     

Micropapillary carcinoma of the breast with necrosis-like cell death: a case report

A primary invasive micropapillary carcinoma of the breast in a 46-year-old woman is reported. Histologically, it was composed predominantly of papillary tumor cell clusters without fibrovascular cores, surrounded by a clear space. Tumor cells were positive for cytokeratin (CK) 7, estrogen receptor (ER), and progesterone receptor (PR), but negative for p53, CK 20, CD34, c-Erb-B2, CK5, epidermal growth factor receptor (EGFR), vimentin, and c-kit. MUC1 expression was found at the reversed apical membrane of neoplastic cell clusters. Accordingly, electron microscopy showed the lack of basement membrane and presence of microvilli at the basal surface of the tumor cells. Moreover, ultrastructural examination revealed single tumor cell death characterized by patchy condensations of chromatin throughout the nucleus. These nuclear alterations were associated with the occurrence of empty cytoplasmic vacuoles, conferring a necrosis-like phenotype to this cell death. Alternative programmed cell deaths are reviewed and their morphologic distinction is discussed.     

Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: a comparative study with foot-and-mouth disease virus and vesicular stomatitis virus

The intracellular distribution of swine vesicular disease virus (SVDV) proteins and the induced reorganization of endomembranes in IBRS-2 cells were analyzed. Fluorescence to new SVDV capsids appeared first upon infection, concentrated in perinuclear circular structures and colocalized to dsRNA. As in foot-and-mouth disease virus (FMDV)-infected cells, a vesicular pattern was predominantly found in later stages of SVDV capsid morphogenesis that colocalized with those of non-structural proteins 2C, 2BC and 3A. These results suggest that assembly of capsid proteins is associated to the replication complex. Confocal microscopy showed a decreased fluorescence to ER markers (calreticulin and protein disulfide isomerase), and disorganization of cis-Golgi gp74 and trans-Golgi caveolin-1 markers in SVDV- and FMDV-, but not in vesicular stomatitis virus (VSV)-infected cells. Electron microscopy of SVDV-infected cells at an early stage of infection revealed fragmented ER cisternae with expanded lumen and accumulation of large Golgi vesicles, suggesting alterations of vesicle traffic through Golgi compartments. At this early stage, FMDV induced different patterns of ER fragmentation and Golgi alterations. At later stages of SVDV cytopathology, cells showed a completely vacuolated cytoplasm containing vesicles of different sizes. Cell treatment with brefeldin A, which disrupts the Golgi complex, reduced SVDV (approximately 5 log) and VSV (approximately 4 log) titers, but did not affect FMDV growth. Thus, three viruses, which share target tissues and clinical signs in natural hosts, induce different intracellular effects in cultured cells.     

Biocompatibility of poly(D,L-lactide-co-glycolide) nanoparticles conjugated with alendronate

Nanoparticles made of a conjugate of poly(D,L-lactide-co-glycolide) with alendronate (PLGA-ALE NPs), were prepared by emulsion/solvent evaporation technique. The conjugation yield, determined by MALDI TOF analysis, was 30-35%. PLGA-ALE NPs size, evaluated by photon correlation spectroscopy, was 198.7+/-0.2 nm. Haemocompatibility studies using different concentrations of PLGA-ALE NPs did not show any significant effect on haemolysis, leukocyte number, platelet activation, APTT and complement consumption, in comparison with blood incubated with phosphate buffered saline (PBS). A significant reduction of the prothrombin activity was demonstrated after incubation with 560 microg/ml of PLGA-ALE NPs; a significant increase was observed at the highest dilutions. The viability of human umbilical vein endothelial cells and bone marrow stromal cells (BMSC), evaluated through the neutral red test, was not affected by PLGA-ALE NPs. There were no significant differences in cell-associated alkaline phosphatase between BMSC incubated with PLGA-ALE NP- and PBS-added media. These results demonstrated that PLGA-ALE NPs had an acceptable degree of blood compatibility and were not cytotoxic; therefore, they may be considered suitable for intravenous administration.     

Perturbation of mouse glioma MRS pattern by induced acute hyperglycemia

(1)H MRS is evolving into an invaluable tool for brain tumor classification in humans based on pattern recognition analysis, but there is still room for improvement. Here we propose a new approach: to challenge tumor metabolism in vivo by a defined perturbation, and study the induced changes in MRS pattern. For this we recorded single voxel (1)H MR spectra from mice bearing a stereotactically induced GL261 grade IV brain glioma during a period of induced acute hyperglycemia. A total of 29 C57BL/6 mice were used. Single voxel spectra were acquired at 7 T with point resolved spectroscopy and TE of 12, 30 and 136 ms. Tumors were induced by stereotactic injection of 10(5) GL261cells in 17 mice. Hyperglycemia (up to 338 +/- 36 mg/dL glucose in the blood) was induced by intraperitoneal bolus injection. Maximal increases in glucose resonances of up to 2.4-fold were recorded from tumors in vivo. Our observations are in agreement with extracellular accumulation of glucose, which may suggest that glucose transport and/or metabolism are working close to their maximum capacity in GL261 tumors. The significant and specific MRS pattern changes observed when comparing euglycemia and hyperglycemia may be of use for future pattern-recognition studies of animal and human brain tumors by enhancing MRS-based discrimination between tumor types and grades.     

Proton pump inhibitors prescribing following the introduction of generic drugs

Eur J Clin Invest 2012; 42 (10): 1068-1078 ABSTRACT: Background In many countries, the introduction of generic proton pump inhibitors (PPIs) onto the pharmaceutical market increased the phenomenon of therapeutic substitution in acid-related disorders (ARDs). Aim To investigate the treatment of ARDs in an Italian primary care setting from 2005 to 2008 by verifying: (i) dynamics of PPI prescribing; (ii) predictors of PPI switching; and (iii) healthcare resource consumption costs. Methods This was a retrospective cohort study of 102 general practitioners (GPs) who managed an average of 150?000 inhabitants in Naples. Multilevel logistic regression was used to assess the potential predictors of both PPI switching and termination. Primary care costs were expressed as the cost of ARD management per PPI user year. Results The percentage of PPI users with ARD increased from 5·5% (2005) to 7·0% (2008) (P?相似文献   

ECMO criteria for influenza A (H1N1)-associated ARDS: role of transpulmonary pressure

Purpose

To assess whether partitioning the elastance of the respiratory system (E _RS) between lung (E _L) and chest wall (E _CW) elastance in order to target values of end-inspiratory transpulmonary pressure (PPLAT_L) close to its upper physiological limit (25?cmH₂O) may optimize oxygenation allowing conventional treatment in patients with influenza A (H1N1)-associated ARDS referred for extracorporeal membrane oxygenation (ECMO).

Methods

Prospective data collection of patients with influenza A (H1N1)-associated ARDS referred for ECMO (October 2009?CJanuary 2010). Esophageal pressure was used to (a) partition respiratory mechanics between lung and chest wall, (b) titrate positive end-expiratory pressure (PEEP) to target the upper physiological limit of PPLAT_L (25?cmH₂O).

Results

Fourteen patients were referred for ECMO. In seven patients PPLAT_L was 27.2?±?1.2?cmH₂O; all these patients underwent ECMO. In the other seven patients, PPLAT_L was 16.6?±?2.9?cmH₂O. Raising PEEP (from 17.9?±?1.2 to 22.3?±?1.4?cmH₂O, P?=?0.0001) to approach the upper physiological limit of transpulmonary pressure (PPLAT_L?=?25.3?±?1.7?cm H₂O) improved oxygenation index (from 37.4?±?3.7 to 16.5?±?1.4, P?=?0.0001) allowing patients to be treated with conventional ventilation.

Conclusions

Abnormalities of chest wall mechanics may be present in some patients with influenza A (H1N1)-associated ARDS. These abnormalities may not be inferred from measurements of end-inspiratory plateau pressure of the respiratory system (PPLAT_RS). In these patients, titrating PEEP to PPLAT_RS may overestimate the incidence of hypoxemia refractory to conventional ventilation leading to inappropriate use of ECMO.     

Value of β-d-glucan and Candida albicans germ tube antibody for discriminating between Candida colonization and invasive candidiasis in patients with severe abdominal conditions

Purpose

To assess the value of (1→3)-β-d-glucan (BDG), Candida albicans germ tube antibody (CAGTA), C-reactive protein (CRP), and procalcitonin (PCT) levels for the diagnosis of invasive candidiasis (IC) and for differentiating Candida spp. colonization from infection in ICU patients with severe abdominal conditions (SAC).

Methods

Prospective study of 176 non-neutropenic patients, with SAC at ICU admission, and expected to stay at least 7?days. Surveillance cultures and BDG, CAGTA, CRP, and PCT levels were performed on the third day of ICU stay and twice a week for four consecutive weeks. Patients were grouped into invasive candidiasis (IC), Candida colonization, and neither colonized/nor infected. The classification and regression tree (CART) analysis was used to predict IC in colonized patients. The discriminatory ability of the obtained prediction rule was assessed by the area under the ROC curve (AUC).

Results

The probabilities of IC were 59.3?% for the terminal node of BDG greater than 259?pg/mL and 30.8?% for BDG less than 259?pg/mL and CAGTA positivity, whereas there was a 93.9?% probability in predicting the absence of IC for BDG less than 259?pg/mL and negative CAGTA. Using a cutoff of 30?% for IC probability, the prediction rule showed 90.3?% sensitivity, 54.8?% specificity, 42.4?% positive predictive value, and 93.9?% negative predictive value with an AUC of 0.78 (95?% confidence interval 0.76–0.81). Significant differences in CRP (p?=?0.411) and PCT (p?=?0.179) among the studied groups were not found.

Conclusions

BDG with a positive test for CAGTA accurately differentiated Candida colonization from IC in patients with SAC, whereas CRP and PCT did not.