In Vivo Molecular Imaging Characterizes Pulmonary Gene Expression During Experimental Lung Transplantation

Experimental gene therapy is a promising strategy to prevent ischemia-reperfusion (I/R) injury and allograft rejection after lung transplantation, and methods will eventually be needed to characterize pulmonary transgene expression in vivo in humans. Therefore, we studied positron emission tomography (PET) as a means of performing in vivo molecular imaging in rodent models of lung transplantation. Rats were transfected endotracheally with adenovirus encoding a fusion gene of a mutant Herpes simplex virus-1 thymidine kinase and the green fluorescent protein gene (the former serving as an imaging reporter gene). Twenty-four hours after transfection, lungs were transplanted in groups representing normal transplantation, I/R injury and acute allograft rejection. Imaging was obtained either 24 h after transplantation to study reperfusion injury or 4 days after transplantation to study graft rejection. After imaging, lungs were excised and analyzed for thymidine kinase activity. Imaging detected transgene expression in transplanted lungs even in the presence of acute rejection or I/R injury. The PET imaging signal correlated with in vitro lung tissue assays of thymidine kinase activity (r(2) = 0.534). Thus, noninvasive molecular imaging with PET is a feasible, sensitive and quantitative method for characterizing pulmonary transgene expression in experimental lung transplantation.     

Molecular genetics and transfusion management in a child with Bernard Soulier syndrome.

We present a case of Bernard Soulier syndrome in a 9-year-old boy caused by a novel genetic mutation. This child was shown to be homozygous for a single nucleotide deletion (c.1077delG) in the GP1BA gene not previously reported. Clinically, the boy has become refractory to platelet transfusions with both allo-antibodies and iso-antibodies and a massive transfusion requirement for ongoing haemorrhage. We describe the critical role that the blood product transfusion continues to play in the management of Bernard Soulier syndrome and discuss therapeutic options in these patients.     

Improved Performance of Hip DXA Using a Novel Region of Interest in the Upper Part of the Femoral Neck: In Vitro Study Using Bone Strength as a Standard of Reference

We tested the hypothesis that bone mineral density (BMD) and bone mineral content (BMC) in proximal human femur specimens in the upper neck region of interest (ROI) and femoral neck axis length (FNAL) provide a significantly better prediction of femoral bone strength than standard ROIs in vitro. BMD and BMC were measured in 110 proximal femur specimens using a standard dual-energy X-ray absorptiometry (DXA) scanner. The analysis included a new ROI in the upper neck as well as the standard ROIs. FNAL was obtained from the scan images. The specimens' failure-load was measured in a mechanical loading device, simulating a fall on the greater trochanter. For the standard ROIs, correlations between failure-load and BMD ranged from R2 = 0.64 (shaft ROI) to R2 = 0.70, p < 0.001 (femoral neck). Prediction of strength by BMD did not significantly differ from those of BMC (R2 ranging from 0.65 to 0.75, p < 0.001). In the upper neck ROI, for both BMD and BMC correlations with failure-load were higher (R2 = 0.76 and 0.81, respectively; p < 0.001). A lower, yet still significant, correlation was found between FNAL and bone strength (R2 = 0.23, p < 0.001). Normalization of failure-load with respect to FNAL did not significantly increase the correlations with densitometric measures. This study provides in vitro evidence indicating that among the ROIs of the proximal femur the newly defined upper neck ROI provides the best prediction of bone strength. Only a weak association was observed between failure load and FNAL.     

Anticholinergic sensitivity in patients with dementia of the Alzheimer type and age-matched controls. A dose-response study

We compared the cognitive and behavioral responses to three intravenous doses of scopolamine (0.1, 0.25, and 0.5 mg) and placebo of ten patients with dementia of the Alzheimer type (DAT) and ten age- and sex-matched elderly control subjects. The patients with DAT showed significant behavioral and cognitive but not physiologic changes at a lower scopolamine dose (0.25 mg) than did the normal elderly controls. Cognitive tests of new learning and semantic knowledge revealed significant impairments at the 0.25-mg scopolamine dose in the patients with DAT, while the responses of the control population were essentially unchanged. Behaviorally, mild euphoria, motor incoordination, and hostility occurred in the patients with DAT but not the controls at the 0.25-mg dose. These differences were unrelated to peripheral physiologic changes produced by the different scopolamine doses. These results indicate that central nervous system functions such as cognition and certain elements of behavior are more sensitive to temporary cholinergic blockade in patients with DAT than in normal age-matched controls. We review implications concerning the status of central cholinergic function in patients with DAT in light of neuropathologically demonstrated cholinergic system lesions in DAT.     

In-vitro/in-vivo correlation of pulsatile drug release from press-coated tablet formulations: a pharmacoscintigraphic study in the beagle dog.

The aim of the current study was to investigate the in-vitro and in-vivo performance of a press-coated tablet (PCT) intended for time delayed drug release, consisting of a rapidly disintegrating theophylline core tablet, press-coated with barrier granules containing glyceryl behenate (GB) and low-substituted hydroxypropylcellulose (L-HPC). The PCTs showed pulsatile release with a lag time dependent upon the GB and L-HPC composition of the barrier layer. In-vivo gamma-scintigraphic studies were carried out for PCTs containing GB:L-HPC at 65:35 w/w and 75:25 w/w in the barrier layer in four beagle dogs, in either the fed or fasted state. The in-vivo lag time in both the fed and fasted states did not differ significantly (p>0.05) from the in-vitro lag time. Additionally, no significant difference (p<0.05) between in-vivo fed and fasted disintegration times was observed, demonstrating that in-vivo performance of the PCT was not influenced by the presence or absence of food in the gastrointestinal tract. A distinct lag time was obtained prior to the appearance of drug in plasma and correlated (R2=0.98) with disintegration time observed from scintigraphic images. However, following disintegration, no difference in pharmacokinetic parameters (AUC(0-6 dis), K(el), Cmax) was observed. The current study highlighted the potential use of these formulations for chronopharmaceutical drug delivery.     

Viral cysteine proteinases

Summary Dozens of novel cysteine proteinases have been identified in positive single-stranded RNA viruses and, for the first time, in large double-stranded DNA viruses. The majority of these proteins are distantly related to papain or chymotrypsin and may be direct descendants of primordial proteolytic enzymes. Virus genome synthesis and expression, virion formation, virion entry into the host cell, as well as cellular architecture and functioning can be under the control of viral cysteine proteinases during infection. RNA virus proteinases mediate their liberation from giant multidomain precursors in which they tend to occupy conserved positions. These proteinases possess a narrow substrate specificity, can cleave in cis and in trans, and may also have additional, nonproteolytic functions. The mechanisms of catalysis, substrate recognition and RNA binding were highlighted by the recent analysis of the three-dimensional structure of the chymotrypsin-like cysteine proteinases of two RNA viruses.     

A Comparison of Oral and Rectal Absorption of L-Dopa Esters in Rats and Mice

Short-chain alkyl esters of L-dopa were administered to rats and mice via oral and rectal routes. Plasma L-dopa esters and L-dopa were determined in the systemic and portal circulation by HPLC. A comparison of isopropyl, butyl, and 4-hydroxybutyl esters of L-dopa demonstrated significantly higher levels of the esters in both systemic and portal blood samples following rectal administration than following oral administration. In most cases, oral administration resulted in nondetectable (<0.01 µg/ml) levels of the esters in plasma. Correspondingly, the plasma levels of L-dopa itself were consistently higher following rectal administration. At very high oral doses (500 mg L-dopa equivalents/kg body weight), systemic plasma levels of the butyl ester could be detected (1.25 µg/ml at 10 min), which might indicate saturation of the esterase activity of the small intestine. These studies indicate that the systemic availability of L-dopa from short-chain alkyl esters of L-dopa may be best optimized by rectal administration, which avoids the relatively high esterase activity characteristic of the small intestine.     

Serum and Cerebrospinal Fluid Pharmacokinetics of Intravenous and Oral Lamivudine in Human Immunodeficiency Virus-Infected Children

We studied the pharmacokinetics of intravenously and orally administered lamivudine at six dose levels ranging from 0.5 to 10 mg/kg of body weight in 52 children with human immunodeficiency virus infection. A two-compartment model with first-order elimination from the central compartment was simultaneously fitted to the serum drug concentration-time data obtained after intravenous and oral administration. The maximal concentration at the end of the 1-h intravenous infusion and the area under the concentration-time curve after oral and intravenous administration increased proportionally with the dose. The mean clearance of lamivudine (± standard deviation) in the children was 0.53 ± 0.19 liter/kg/h (229 ± 77 ml/min/m² of body surface area), and the mean half-lives at the distribution and elimination phases were 0.23 ± 0.18 and 2.2 ± 2.1 h, respectively. Clearance was age dependent when normalized to body weight but age independent when normalized to body surface area. Lamivudine was rapidly absorbed after oral administration, and 66% ± 25% of the oral dose was absorbed. Serum lamivudine concentrations were maintained above 1 μM for ≥8 h of 24 h on the twice daily oral dosing schedule with doses of ≥2 mg/kg. The cerebrospinal fluid drug concentration measured 2 to 4 h after the dose was 12% (range, 0 to 46%) of the simultaneously measured serum drug concentration. A limited-sampling strategy was developed to estimate the area under the concentration-time curve for concentrations in serum at 2 and 6 h.