Role of matrix metalloproteinases in failure to re-epithelialize after corneal injury.

Delayed re-epithelialization of the cornea after injury usually precedes stromal ulceration. Previous findings using a rat thermal injury model suggested that re-epithelialization is impeded by products of resident corneal cells, which destroy adhesive structures at the basement membrane zone. In this study, we provide additional evidence for this concept. Failure to re-epithelialize was found to correlate with an increase in the amounts of gelatinolytic matrix metalloproteinases present in the rat cornea. One of these gelatinases, gelatinase B, is synthesized by the resident corneal cells, and inhibitions of its synthesis correlated with inhibition of basement membrane dissolution. The matrix metalloproteinases collagenase and stromelysin are also synthesized by resident corneal cells in thermally injured corneas of rabbits, but the timing of bulk enzyme synthesis correlated more closely with deposition of repair tissue in the stroma than with failure to re-epithelialize. Nevertheless, in human corneas with repair defects, gelatinase B and collagenase are synthesized by cells in the basal layer of the epithelium directly adjacent to the basement membrane, suggesting that both could participate in dissolution of this structure. Importantly, treatment of thermally injured corneas with a synthetic inhibitor of matrix metalloproteinases significantly improved basement membrane integrity. These data support the concept that over-expression of matrix metalloproteinases by resident corneal cells impedes re-epithelialization after some types of corneal injury.     

The effect of gamma interferon on IL-1 secretion of in vitro differentiated human macrophages

After being cultured overnight, human monocytes lose their ability to secrete interleukin-1 (IL-1) when stimulated by lipopolysaccharide (LPS). However, when these monocytes were cultured for up to 9 days with various concentrations of interferon-gamma (IFN-gamma), these cells were found to retain their ability to secrete appreciable amounts of IL-1 on LPS stimulation. However, the effect was observed only if the monocytes were exposed to the IFN before LPS stimulation and simultaneous addition of IFN and LPS to macrophages was ineffective. This effect of IFN-gamma was related to the concentration of IFN added to the cultures and was completely neutralized by a monoclonal antibody to IFN-gamma. In addition to inducing IL-1 secretion, IFN-gamma also appeared to increase the overall production of IL-1, since reinduction of IL-1 secretion was not associated with a decrease in intracellular IL-1 content. When these macrophages were initially cultured with IFN-gamma, washed, and further cultured with IFN free medium, these macrophages were found to progressively lose their capacity to secrete IL-1 in response to LPS. Conversely, when monocytes were initially cultured in medium free of IFN, washed, and then further cultured in new medium, but now containing IFN-gamma, these macrophages were found to regain their capacity to secrete IL-1. However, the amount of reinduced IL-1 secretion decreased as the length of the initial culture period without IFN increased, with less than optimal IL-1 secretion occurring if monocytes were allowed to mature for 6 days before IFN-gamma pretreatment. In summary, these studies suggest that IFN-gamma may be important in enhancing IL-1 production and secretion by maturing macrophages and tissue macrophages and consequently may play a role in regulating the accessory cell activity of these cells for a variety of immune responses in vivo.     

Three-dimensional surface reconstructions using a general purpose image processing system

A general purpose two-dimensional (2-D) image processing software system was used to produce high quality three-dimensional (3-D) surface reconstructions from serial sections such as CT scan slices. Depth-encoded 3-D surface images, gradient-shaded 3-D surface images, and weighted sums of these two images were computed. Images that simulate transmission radiographs ("volumetric" views) were created from the same slice data. Hidden surfaces were displayed by reconstructing in 3-D only subvolumes of the original data set. The 2-D image processing functions used were limited to: planar subimage selection and merge, arithmetic and boolean operations, piecewise linear gray scale transform, convolution (1-D), and format conversion (byte-integer-float). Using these methods any user with a general purpose 2-D image processing system can analyze and view multi-slice data as 3-D volume and surface projections.     

Long-term care pharmacy services: a new dimension of pharmacy practice

Pharmacists providing care to patients must have a commitment to quality patient care, have well-developed operations systems, possess refined clinical skills, and be able to effect excellent communication with the clinical and administrative staff. These attributes of pharmacy practice should exist in both the acute care and long-term care settings. Pharmacy practice in the long-term care setting, a unique position due to the everchanging definition of long-term care, may best be referred to as an alternative site for the application of contemporary pharmacy distribution and clinical systems for patient care over an extended period.     

Protein covalent binding of maxipost through a cytochrome P450-mediated ortho-quinone methide intermediate in rats.

(3S)-(+)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one) (MaxiPost, BMS-204352) is a potent and specific opener for maxi-K channels and has potential to prevent and treat ischemic stroke. Following single intravenous doses of [14C]BMS-204352 to rats, only 10 to 12% of radioactivity was extractable from plasma with organic solvents. The unextractable radioactivity remained associated with the proteins (mostly albumin) after SDS-polyacrylamide gel electrophoresis or dialysis. Following acid hydrolysis in 6 M HCl for 24 h at 110 degrees C from plasma proteins collected from nine rats dosed with [14C]BMS-204352, one major radioactive product was isolated and identified as a lysine-adduct of des-fluoro des-O-methyl BMS-204352 by liquid chromatography/mass spectrometry and NMR analyses as well as by comparison with the synthetic analog, lysine-adduct of des-fluoro BMS-204352 (BMS-349821). The covalent binding of BMS-204352 results from the displacement of the ring-fluorine atom of des-O-methyl BMS-204352 with the epsilon-amino group of a lysine residue. Microsomal incubations of [14C]BMS-204352 resulted in low levels of covalent binding of radioactivity to proteins. This in vitro covalent binding required cytochrome P450-reductase cofactor NADPH and was attenuated by glutathione. P4503A inhibitors ketoconazole and troleadomycin selectively prevented the covalent binding in vitro. Based on these observations, a two-step bioactivation process for the protein covalent binding of BMS-204352 was postulated: 1) P4503A-mediated O-demethylation leading to spontaneous release of HF and the formation of an ortho-quinone methide reactive metabolite and 2) nucleophilic addition of the epsilon-amino group of protein lysine residue(s) in protein to form des-fluoro des-O-methyl BMS-204352 lysine adduct.     

Computer simulated modeling of healthy and diseased right ventricular and pulmonary circulation

We have previously developed a simulated cardiovascular physiology model for in-silico testing and validation of novel closed-loop controllers. To date, a detailed model of the right heart and pulmonary circulation was not needed, as previous controllers were not intended for use in patients with cardiac or pulmonary pathology. With new development of controllers for vasopressors, and looking forward, for combined vasopressor-fluid controllers, modeling of right-sided and pulmonary pathology is now relevant to further in-silico validation, so we aimed to expand our existing simulation platform to include these elements. Our hypothesis was that the completed platform could be tuned and stabilized such that the distributions of a randomized sample of simulated patients’ baseline characteristics would be similar to reported population values. Our secondary outcomes were to further test the system in representing acute right heart failure and pulmonary artery hypertension. After development and tuning of the right-sided circulation, the model was validated against clinical data from multiple previously published articles. The model was considered ‘tuned’ when 100% of generated randomized patients converged to stability (steady, physiologically-plausible compartmental volumes, flows, and pressures) and ‘valid’ when the means for the model data in each health condition were contained within the standard deviations for the published data for the condition. A fully described right heart and pulmonary circulation model including non-linear pressure/volume relationships and pressure dependent flows was created over a 6-month span. The model was successfully tuned such that 100% of simulated patients converged into a steady state within 30 s. Simulation results in the healthy state for central venous volume (3350?±?132 ml) pulmonary blood volume (405?±?39 ml), pulmonary artery pressures (systolic 20.8?±?4.1 mmHg and diastolic 9.4?±?1.8 mmHg), left atrial pressure (4.6?±?0.8 mmHg), PVR (1.0?±?0.2 wood units), and CI (3.8?±?0.5 l/min/m²) all met criteria for acceptance of the model, though the standard deviations of LAP and CI were somewhat narrower than published comparators. The simulation results for right ventricular infarction also fell within the published ranges: pulmonary blood volume (727?±?102 ml), pulmonary arterial pressures (30?±?4 mmHg systolic, 12?±?2 mmHg diastolic), left atrial pressure (13?±?2 mmHg), PVR (1.6?±?0.3 wood units), and CI (2.0?±?0.4 l/min/m²) all fell within one standard deviation of the reported population values and vice-versa. In the pulmonary hypertension model, pulmonary blood volume of 615?±?90 ml, pulmonary arterial pressures of 80?±?14 mmHg systolic, 36?±?7 mmHg diastolic, and the left atrial pressure of 11?±?2 mmHg all met criteria for acceptance. For CI, the simulated value of 2.8?±?0.4 l/min/m² once again had a narrower spread than most of the published data, but fell inside of the SD of all published data, and the PVR value of 7.5?±?1.6 wood units fell in the middle of the four published studies. The right-ventricular and pulmonary circulation simulation appears to be a reasonable approximation of the right-sided circulation for healthy physiology as well as the pathologic conditions tested.     

TMC114/ritonavir substitution for protease inhibitor(s) in a non-suppressive antiretroviral regimen: a 14-day proof-of-principle trial

OBJECTIVE: To evaluate antiviral activity, tolerability, and safety of the protease inhibitor (PI) TMC114 boosted with low-dose ritonavir (RTV). DESIGN: A randomized, open-label, controlled, phase IIA clinical trial in 15 sites in Europe with 50 HIV-1-infected patients who had taken multiple PIs. METHODS: At entry, PIs in non-suppressive regimens were replaced with TMC114/RTV (300/100 or 600/100 mg twice daily, or 900/100 mg once daily) or left unchanged for 14 days. The time-averaged difference (DAVG) in HIV-1 RNA from baseline, change in HIV-1 RNA from baseline, proportions achieving plasma HIV-1 RNA < 400 copies/ml and > or = 0.5 and > or = 1.0 log10 copies/ml reductions in HIV-1 RNA, and safety were assessed. RESULTS: DAVG responses in all TMC114/RTV groups (range, -0.56 to -0.81 log10 copies/ml) were significantly greater (P < 0.001) than in the controls (-0.03 log10 copies/ml). Median change at day 14 was -1.38 and +0.02 log10 copies/ml for all TMC114/RTV groups and the control group, respectively. A reduction of > or = 0.5 and > or = 1.0 log10 copies/ml was attained by 97% and 76% of patients, respectively, in all TMC114/RTV groups and by 25% and 17%, respectively, in the control group. HIV-1 RNA < 400 copies/ml at any time during treatment was achieved by 40% in the TMC114/RTV groups and 8% in the control group. Most common reported adverse events were gastrointestinal and central nervous system disorders (mild to moderate severity). No dose relationship was observed. Biochemical, haematological and electrocardiographic parameters showed no significant changes. CONCLUSIONS: TMC114/RTV demonstrated a potent antiretroviral effect over 14 days in multiple-PI-experienced patients and was generally well tolerated.     

Understanding macrographia in children with autism spectrum disorders

It has been consistently reported that children with autism spectrum disorders (ASD) show considerable handwriting difficulties, specifically relating to accurate and consistent letter formation, and maintaining appropriate letter size. The aim of this study was to investigate the underlying factors that contribute to these difficulties, specifically relating to motor control. We examined the integrity of fundamental handwriting movements and contributions of neuromotor noise in 26 children with ASD aged 8–13 years (IQ > 75), and 17 typically developing controls. Children wrote a series of four cursive letter l's using a graphics tablet and stylus. Children with ASD had significantly larger stroke height and width, more variable movement trajectory, and higher movement velocities. The absolute level of neuromotor noise in the velocity profiles, as measured by power spectral density analysis, was significantly higher in children with ASD; relatively higher neuromotor noise was found in bands >3 Hz. Our findings suggest that significant instability of fundamental handwriting movements, in combination with atypical biomechanical strategies, contribute to larger and less consistent handwriting in children with ASD.