Acute changes of coagulation and fibrinolysis parameters after experimental thromboembolic stroke and thrombolytic therapy

Thrombolysis is the only effective pharmaceutical therapy in acute ischemic stroke in humans but has a high risk of intracerebral hemorrhage. We aimed to establish an animal model to study changes of coagulation and fibrinolytic parameters during thromboembolic ischemic stroke and thrombolysis with recombinant tissue plasminogen activator (rt-PA). We used a thromboembolic stroke model in the rat. Animals were treated with rt-PA thrombolysis (n = 10) and compared with untreated (n = 10), sham operated (n = 10) and control animals (n = 20). Coagulation parameters (APTT, PT, TT, fibrinogen, AT III, TAT) and fibrinolytic parameters (t-PA antigen concentration, t-PA activity, PAI-1 concentration, PAI activity, plasminogen, antiplasmin) were measured at two time points (2.5 and 5 h after stroke induction) with a battery of commercially available test kits. We observed an (1) initiation of coagulation and inhibition of fibrinolysis by the operation procedure itself, (2) simultaneous activation of fibrinolysis and its inhibitors after stroke induction and (3) potent initiation of fibrinolysis and consumption of fibrinolysis inhibitors after rt-PA therapy of stroke. We established a model system to monitor coagulation and fibrinolysis during thrombolytic therapy of stroke in the rat. This model may be used to study the influence of these parameters on hemorrhagic stroke transformation and outcome in experimental stroke in future.     

Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines

Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4(+) helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-gamma enzyme-linked immunospot assay (ELISPOT) assays and HLA-peptide tetramer staining. Single-cell cloning resulted in both CD4(+) and CD8(+) T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8(+) and CD4(+) T cell epitopes.     

Migrating anterior mesoderm cells and intercalating trunk mesoderm cells have distinct responses to Rho and Rac during Xenopus gastrulation.

Rho GTPases have been shown recently to be important for cell polarity and motility of the trunk mesoderm during gastrulation in Xenopus embryos. This work demonstrated that Rho and Rac have both distinct and overlapping roles in regulating cell shape, and the dynamic properties, polarity, and type of protrusive activity of these cells. Overexpression of activated or inhibitory versions of these GTPases also disrupts development of the head in Xenopus embryos. In this study, we have undertaken a detailed analysis of Rho and Rac function in migrating anterior mesendoderm cells. Scanning electron micrographs of these cells in situ revealed that their normal shingle arrangement is disrupted and both the cells and their lamellipodia are disoriented. Anterior mesendoderm explants plated on their natural blastocoel roof matrix, however, still migrated towards the animal pole, although the tendency to move in this direction is reduced compared to controls. Analysis of a number of parameters in time-lapse recordings of dissociated cells indicated that Rho and Rac also have both distinct and overlapping roles in the motility of the prospective head mesoderm; however, their effects differ to those previously seen in the trunk mesoderm. Both GTPases appear to modulate cell polarization, migration, and protrusive activity. Rho alone, however, regulates the retraction of the lagging edge of the cell. We propose that within the gastrulating Xenopus embryo, two types of mesoderm cells that undergo different motilities have distinct responses to Rho GTPases.     

Expression of mRNAs related to connective tissue metabolism in rat hepatic stellate cells and myofibroblasts

Hepatic stellate cells (HSC) and liver myofibroblasts (MFB) are two cell populations most likely responsible for the synthesis of most connective tissue components in fibrotic liver. They differ in their origin and location, and possibly in patterns of gene expression. Normal and carbon tetrachloride-cirrhotic livers from rats were used to isolate HSC. Liver was perfused with pronase and collagenase solutions, followed by centrifugation of the cell suspension on a density gradient. HSC were quiescent 2 days after plating on plastic but they became activated after another 5 days in culture. When the culture was passaged 5 times, its character changed profoundly as HSC were replaced by MFB. Microarray analysis was used to determine gene expression in quiescent HSC, activated HSC and MFB. The expression of 49 genes coding for connective tissue proteins, proteoglycans, metalloproteinases and their inhibitors, growth factors and cellular markers was determined. The pattern of gene expression changed during HSC activation and there were distinct differences between HSC and MFB. Little difference between normal cells and cells isolated from cirrhotic liver was found.     

Two-center collaborative evaluation of the performance of the BD Phoenix automated microbiology system for identification and antimicrobial susceptibility testing of Enterococcus spp. and Staphylococcus spp

The performance of the BD Phoenix Automated Microbiology System (BD Diagnostic Systems, Sparks, Md.) was assessed for identification (ID) and antimicrobial susceptibility testing (AST) for the majority of clinically encountered bacterial isolates in a European collaborative two-center trial. A total of 469 bacterial isolates of the genera Staphylococcus (275 isolates), Enterococcus (179 isolates), and Streptococcus (15 isolates, for ID only) were investigated; of these, 367 were single patient isolates, and 102 were challenge strains tested at one center. Sixty-four antimicrobial drugs were tested, including the following drug classes: aminoglycosides, beta-lactam antibiotics, beta-lactam-beta-lactamase inhibitors, carbapenems, cephems, folate antagonists, quinolones, glycopeptides, macrolides-lincosamides-streptogramin B (MLS), and others. Phoenix ID results were compared to those of the laboratories' routine ID systems (API 32 Staph, API 32 Strep, and VITEK 2 [bioMérieux, Marcy l'Etoile, France]); Phoenix AST results were compared to those of frozen standard broth microdilution (SBM) panels according to NCCLS guidelines (NCCLS document M 100-S 9, approved standard M 7-A 4). Discrepant results were repeated in duplicate. Concordant IDs of 97.1, 98.9, and 100% were observed for staphylococci, enterococci, and streptococci, respectively. For AST results the overall essential agreement was 93.3%; the category agreement was 97.3%; and the very major error rate, major error rate, and minor error rate were 1.2, 1.9, and 1.3%, respectively. In conclusion, the Phoenix ID results showed high agreement with results of the systems to which they were being compared; the AST performance was highly equivalent to that of the SBM reference method.     

p62 Is a Common Component of Cytoplasmic Inclusions in Protein Aggregation Diseases

Exposure of cells to stress, particularly oxidative stress, leads to misfolding of proteins and, if they are not refolded or degraded, to cytoplasmic protein aggregates. Protein aggregates are characteristic features of a variety of chronic toxic and degenerative diseases, such as Mallory bodies (MBs) in hepatocytes in alcoholic and non-alcoholic steatohepatitis, neurofibrillary tangles in neurons in Alzheimer's, and Lewy bodies in Parkinson's disease. Using 2D gel electrophoresis and mass spectrometry, we identified p62 as a novel MB component. p62 and cytokeratins (CKs) are major MB constituents; HSP 70, HSP 25, and ubiquitinated CKs are also present. These proteins characterize MBs as a prototype of disease-associated cytoplasmic inclusions generated by stress-induced protein misfolding. As revealed by transfection of tissue culture cells overexpressed p62 did not induce aggregation of regular CK filaments but selectively bound to misfolded and ubiquitinated CKs. The general role of p62 in the cellular response to misfolded proteins was substantiated by detection of p62 in other cytoplasmic inclusions, such as neurofibrillary tangles, Lewy bodies, Rosenthal fibers, intracytoplasmic hyaline bodies in hepatocellular carcinoma, and alpha1-antitrypsin aggregates. The presence of p62 along with other stress proteins and ubiquitin in cytoplasmic inclusions indicates deposition as aggregates as a third line of defense against misfolded proteins in addition to refolding and degradation.     

TRIO amplification and abundant mRNA expression is associated with invasive tumor growth and rapid tumor cell proliferation in urinary bladder cancer

Studies by comparative genome hybridization have suggested that 5p amplification is related to tumor progression in urinary bladder cancer. In this study seven genes (TAS2R, ADCY2, DNAH5, CTNND2, TRIO, ANKH, and MYO10) located to 5p15.31-5p15.1 were analyzed by fluorescence in situ hybridization using a tissue microarray containing samples from tumors and cell lines with known 5p amplification by comparative genome hybridization. Amplification frequency was highest for TRIO, which maps to 5p15.2 and encodes a protein with a putative role in cell-cycle regulation. To further investigate the role of TRIO amplification in bladder cancer, a tissue microarray containing samples from 2317 bladder tumors was used for fluorescence in situ hybridization analysis. TRIO amplification was strongly associated with invasive tumor phenotype, high tumor grade, and rapid tumor cell proliferation (Ki67 LI) (P < 0.0001 each). Only 7 of 456 pTaG1/G2 tumors (1.5%) but 62 of 485 pT1-4 carcinomas (12.8%) had TRIO amplification. TRIO amplification was not associated with poor prognosis. Using a frozen bladder tumor tissue microarray RNA in situ hybridization confirmed that TRIO is up-regulated in amplified tumors. It is concluded that TRIO up-regulation through amplification has a potential role in bladder cancer progression.     

A Novel Macroinitiator for the Synthesis of Triblock Copolymers via Atom Transfer Radical Polymerization: Polystyrene‐block‐poly(bisphenol A carbonate)‐block‐polystyrene and Poly(methyl methacrylate)‐block‐poly(bisphenol A carbonate)‐block‐poly(methyl methacrylate)

Summary: Bis(hydroxy)telechelic bisphenol A polycarbonate (PC) was prepared via melt polycondensation of bisphenol A (BPA) and diphenyl carbonate (DPC) using lanthanum(III ) acetylacetonate as a catalyst for transesterification. Subsequently, the polycarbonate was converted to a bifunctional macroinitiator for atom transfer radical polymerization (ATRP) with the reagent, α‐chlorophenylacetyl chloride. The macroinitiator was used for the polymerization of styrene (S) and methyl methacrylate (MMA) to give PS‐block‐PC‐block‐PS and PMMA‐block‐PC‐block‐PMMA triblock copolymers. These block copolymers were characterized by NMR and GPC. When styrene and methyl methacrylate were used in large excess, significant shifts toward high molecular weights were observed with quantitative consumption of the macroinitiator. Several ligands were studied in combination with CuCl as the ATRP catalyst. Kinetic studies reveal the controlled nature of the polymerization reaction for all the ligands used.

Formation of a bifunctional ATRP macroinitiator by esterification of bis(hydroxy)telechelic PC with α‐chlorophenylacetyl chloride.     

Comparison of the Automated Mycobacteria Growth Indicator Tube System (BACTEC 960/MGIT) with Löwenstein-Jensen medium for recovery of mycobacteria from clinical specimens

We examined whether the BACTEC/Mycobacteria Growth Indicator Tube (MGIT) system alone could supplant the use of a supplemental L?wenstein-Jensen (LJ) slant for routine recovery of Mycobacterium species from clinical specimens. A total of 6,062 specimens were included in the study. Of these, 273 specimens were positive for 278 mycobacterial isolates while 15 specimens were smear positive but culture negative using both media. Further analysis showed that 143 (51.4%) of the 278 total isolates were recovered from both the MGIT and LJ media. An additional 106 isolates (38.1%) were recovered from the MGIT only, while 29 (10.4%) isolates grew only on the LJ slant. The overall sensitivities of the MGIT and LJ media were 86.5% and 59.7%, respectively, for the recovery of mycobacteria from clinical materials. This study shows that although the MGIT system demonstrates better sensitivity for the recovery of mycobacteria from clinical specimens, both media types are necessary to maximize the sensitivity of detection.