Urokinase-type plasminogen activator regulates cranial neural crest cell migration in vitro

Proper migration and differentiation of neural crest (NC) cells are required for normal development of craniofacial structures, heart and great vessels, sensory and autonomic nervous systems, and other organs with vertebrate embryos. Serine-protease inhibitors reduce NC cell migration in vitro, suggesting the extracellular proteases are important mediators of NC cell migration. While plasminogen activator activity levels are high in NC cells relative to other embryonic tissue, its ability to regulate NC cell migration has not been specifically tested in vivo or in vitro through its ability to convert plasminogen to plasmin. Using a transfilter migration assay, NC cell migration was measured in the presence or absence of plasminogen. Our results showed that plasminogen significantly enhanced NC cell migration. This increase could not be attributed to differences in initial NC cell attachment or cytotoxicity and did not require a chemotactic gradient. The plasminogen-enhanced NC cell migration was blocked by aprotinin (a plasmin inhibitor) and was mimicked by the direct addition of plasmin to the NC cells, indicating that the plasminogen effect was mediated through plasmin generation. Furthermore, anticatalytic-uPA antibody blocked the plasminogen-enhanced NC cell migration showing that NC cell-associated uPA activity was required for this effect. Finally, decreasing NC-uPA activity by treating cells with transforming growth factor-Beta, also blocked the plasminogen-dependent increase in cell migration. These data show that in vitro, NC cell migration is regulated by NC-associated uPA activity suggesting that growth factor-regulation of this activity may play a major role in regulating NC cell migratory capacity in vivo.     

Effects of the PIPC model on outcome measures of productivity and costs

The purpose of this experimental research was to determine the effects of the Partners in Care Practice Model (PIPC) on the outcomes of productivity and costs. Over an 18-month study period, no significant differences were found in costs per patient day between the pilot and control units; however, significant differences were found in actual salaries, amount of time spent in documentation, and supply costs. Differences in costs and productivity reflected increased training costs and costs for additional supplies on the pilot unit.     

Initial processing of human proenkephalin in bovine chromaffin cells

The opioid peptide precursor preproenkephalin (PPE) contains seven enkephalin sequences and is synthesized by epinephrine-producing adrenal chromaffin cells and various peripheral and central neurons. After removal of its signal peptide, PPE undergoes processing at dibasic amino acid sites to yield its final opioid products-Met-enkephalin, Leu-enkephalin, and various larger, enkephalin-containing peptides. Processing of PPE was examined in bovine chromaffin cells using a plasmid containing the human PPE (hPPE) cDNA under the control of the cytomegalovirus immediate early enhancer/promoter. Following transfection of this hPPE-containing plasmid into bovine chromaffin cells, several proenkephalin-immunoreactive bands were observed on western blots with monoclonal antibodies that recognize human, but not bovine, proenkephalin sequences. The pattern of hPPE-derived peptides observed was similar to that of bovine PPE processing products. A series of recombinant plasmids containing mutations in the hPPE sequence at putative processing sites was then constructed. Conversion of Lys-Lys and Lys-Arg sequences to Lys-Gln and of Arg-Arg to Arg-Gln altered initial hPPE processing at only three of the putative processing sites. When hPPE cDNA containing mutations at all of these initially processed sites was expressed, one or more alternative processing sites were revealed. These data suggest the importance of structural features in addition to the dibasic sequences that limit the processing of proenkephalin.     

Bioavailability and pharmacokinetic disposition of tacrine in elderly patients with Alzheimer''s disease

PURPOSE: The Baerveldt glaucoma implant is an aqueous shunting device with large surface area that is installed through a single-quadrant conjunctival incision. A rabbit model of the Baerveldt implant was created to obtain serial histology and clinical information over 1 year. METHODS: Modified versions of the Baerveldt implant (110 or 160 mm2) were implanted in 18 normal New Zealand white rabbit eyes. The rabbits were examined periodically and their intraocular pressures (IOPs) recorded. They were killed at monthly intervals to obtain histology of the bleb capsules. RESULTS: Thin capsules were present at 1 month, which consisted of lamellar collagen deposition surrounded by a granulomatous reaction with multinucleate giant cells. Inflammatory cells (probably macrophages) were scattered on the inner bleb surface. The granulomatous reaction resolved after 4 months. Subsequently, capsule thickness and cellularity remained relatively stable, although the collagen stroma became less compact over time. Sixteen rabbit eyes had initial IOP reductions of > or = 3 mm Hg compared with fellow eyes, which persisted up to 4 weeks postoperatively. Seven eyes (39%) exhibited a hypertensive phase (IOP exceeded that of fellow eye by > or = 3 mm Hg) from 2 weeks to 3 months postoperatively. CONCLUSION: The Baerveldt explant is surrounded by a fibrous capsule that matures over time. The bleb histology in the rabbit model is similar to that described with the Molteno implant in primates and humans, except for the eventual development of a fibroblastic inner lining in the rabbit model. This contrasts with primate and human models, in which the inner lining remains an open mesh.     

Serum immunoreactive leptin concentrations in women with polycystic ovary syndrome

Recent data in the mouse demonstrate that leptin, a protein hormone produced by fat cells, is required for fertility. In the absence of leptin the mice become obese, diabetic and infertile. Polycystic ovary syndrome (PCOS), a common cause of infertility in women, is associated with obesity and insulin resistance. Because of the increased frequency of PCOS in obese women we tested the hypothesis that alterations in serum leptin concentrations might be associated with PCOS. Immunoreactive leptin concentrations were measured in 58 women with PCOS and 70 regularly menstruating (control) women. As has previously been shown there was a positive correlation between leptin levels and body mass index (BMI). Although the leptin levels in the majority of women with PCOS fell within the control range, 29% of PCOS women had leptin levels above the 99% prediction interval for their BMI and none had low leptin levels. There were also positive correlations of leptin levels with free testosterone and insulin sensitivity in control women. In women with PCOS, 13% and 9.5% exhibited higher than expected leptin concentrations with respect to free testosterone and insulin sensitivity, respectively. Insulin resistant PCOS women had higher leptin levels than controls. The data demonstrate that a substantial proportion of women with PCOS have leptin levels that are higher than expected for their BMI, free testosterone and insulin sensitivity. These results suggest that abnormalities in leptin signaling to the reproductive system may be involved in certain cases of PCOS.     

Hemodynamic patterns and duration of post-dynamic exercise hypotension in hypertensive humans

It has been shown, that smoking results in a lower yield of surfactant associated phospholipids in bronchoalveolar lavage (BAL). Indirect evidence suggests impaired secretion. In the present study, we investigated the influence of cigarette smoke on surfactant secretion in cultured rat alveolar type II cells. Smoke exposure was achieved by bubbling the smoke of four cigarettes through Dulbecco's modified Eagle's medium (DMEM) which was adjusted to a reference absorption value of 1.36 at 320 nm. Cells were preincubated with various dilutions of cigarette smoke-treated medium for 30 min, and were then exposed to this medium for 2 h. After this time, secretion of 3H-choline-labelled phosphatidylcholine (PC) was measured as a marker of surfactant secretion. A 10 fold dilution of cigarette smoke-treated medium inhibited PC secretion stimulated by a combination of terbutaline, adenosine triphosphate and 12-O-tetradecanoylphorbol-13-acetate by over 50%, but did not alter basal secretion. Exposure to less concentrated cigarette smoke-treated medium resulted in less inhibition. Cellular injury was not observed with the concentrations of cigarette smoke-treated medium used in this study. The gas phase of cigarette smoke was not inhibitory at comparable concentrations. Longer exposure to cigarette smoke-treated medium resulted in increased inhibition of PC secretion. The cigarette smoke ingredients, nicotine and benzo[a]pyrene, failed to inhibit PC secretion. Secretion of type II cells exposed to cigarette smoke-treated medium at lower temperatures was not affected. Addition of antioxidants to medium and cells during the preincubation and secretion period did not alter cigarette smoke-treated medium-induced inhibition of stimulated PC secretion. These results demonstrate a direct inhibitory effect of cigarette smoke constituents on surfactant secretion in type II cells. Inhibition is mediated by compounds contained predominantly in the particulate phase of cigarette smoke. Inactivation of the inhibitory effect by lower temperatures suggests involvement of processes such as enzymatic bioactivation or active transport mechanisms.     

Physical and functional interactions between the herpes simplex virus UL15 and UL28 DNA cleavage and packaging proteins

Herpes simplex virus (HSV) DNA is cleaved from concatemers and packaged into capsids in infected cell nuclei. This process requires seven viral proteins, including UL15 and UL28. UL15 expressed alone displays a nuclear localization, while UL28 remains cytoplasmic. Coexpression with UL15 enables UL28 to enter nuclei, suggesting an interaction between the two proteins. Additionally, UL28 copurified with UL15 from HSV-infected cells after ion-exchange and DNA affinity chromatography, and the complex sedimented as a 1:1 heterodimer upon sucrose gradient centrifugation. These findings are evidence of a physical interaction of UL15 and UL28 and a functional role for UL15 in directing UL28 to the nucleus.     

Interleukin-1 mediates a rapid inflammatory response after injection of adenoviral vectors into the brain

Adenovirus-mediated gene transfer into the brain is associated with significant inflammation and activation of anti-vector and anti-transgene immune responses that curtail the gene delivery of adenoviruses and therapeutic efficacy. Elucidating the molecular mediators of inflammatory and immune responses to adenoviruses injected into the brain should allow us to inhibit their inflammatory actions, thereby reducing vector clearance and enhance adenoviral-mediated gene transfer into the CNS. Cytokines are primary mediators of the immune response and are released during inflammation. Here we report for the first time that injection of replication-deficient adenovirus vectors into the cerebral ventricles of rats causes a rapid increase in body temperature. This fever response precedes any vector-encoded transgene expression and occurs with vectors encoding no transgene, as well as with vectors encoding a therapeutic transgene i.e., HSV1-thymidine kinase. No fever is detected after infection of the striatum, an important brain target in studies on neurodegeneration. After infection of the brain ventricles, CSF levels of immunoreactive tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta increase significantly (up to 300-fold). In the hypothalamus, the locus of thermoregulation in the brain, only IL-1beta and IL-6 are significantly elevated. A neutralizing TNF-alpha antibody has no effect on adenovirus-induced fever. However, pretreatment with either the IL-1 receptor antagonist or the cyclooxygenase inhibitor flurbiprofen completely abolishes adenovirus-induced fever, suggesting that IL-1 and prostaglandins are direct mediators of this response. These results are the first to demonstrate that IL-1, but not TNF-alpha, is the main mediator of a very early inflammatory response to adenovirus in the brain.     

Progression detection of stage I nonseminomatous testis cancer on surveillance: implications for the followup protocol

PURPOSE: To optimize followup in patients with stage I nonseminomatous testis cancer on surveillance we evaluated the contribution of each followup modality to the detection of progression as well as morbidity and mortality outcomes. MATERIALS AND METHODS: After orchiectomy 170 patients with clinical stage I nonseminoma were prospectively placed on a surveillance protocol. History, physical examination, serum tumor markers, abdominal and pelvic computerized tomography (CT), and chest x-ray were used for followup. The number of failures, methods and timing of progression detection, treatments required, mortality rate and subsequent contralateral primary tumors were recorded. RESULTS: The 170 surveillance patients were followed a median of 6.3 years. Within 2 years (median 6.9 months) postoperatively 48 patients (28.2%) had disease progression. History, physical examination, markers, CT and chest radiography provided the initial evidence of progression in 18 (37.5%), 34 (70.8%), 34 (70.8%), and 4 (8.3%) patients, respectively. Each modality was the only indicator of failure in 2 (4.2%), 4 (8.3%), 10 (20.8%) and 0 cases, respectively. Of the 170 patients 122 (71.8%) required no additional treatment beyond orchiectomy, 26 (15.3%) received 1 and 22 (12.9%) underwent more than 1 therapeutic modality. Only 1 patient (0.6%) died of disease. Contralateral tumors developed in 5 cases (2.9%) therapeutic a mean of 8.1 years after orchiectomy. CONCLUSIONS: In stage I nonseminoma patients, surveillance history, physical examination, tumor markers and abdominopelvic CT are necessary components of the followup protocol. Removal of routine chest x-ray from the protocol would not have changed progression detection. The initial surveillance visit must occur by 2 months postoperatively. Patients should be followed beyond 5 years and likely for life in addition to regular patient self-examination.