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.     

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.     

Investigations of a novel ferrocene-containing lipid by cyclic voltammetry and cryogenic transmission electron microscopy

A novel chiral redox-active ferrocene compound (FcVI) with amphiphilic properties has been synthesized. Cryogenic Transmission Electron Microscopy (cryo-TEM) has been used to estimate the shape and size of the FcVI aggregates in solution. Uni- and multi-lamellar vesicles (between 40 and 300 nm in diameter) were observed in water. Large particles (of more than 1 μm in diameter) with a hexagonal fine structure were found in 50 mM aqueous Na₂SO₄ solution. Sonication transformed the latter into ‘rosette’-like structures. Cyclic voltammetry has been employed to investigate the electrochemical properties of FcVI. The amphiphile adsorbed on graphite electrodes and a reversible electrochemical behaviour, characteristic of ferrocene, was observed with redox potentials between 330 and 350 mV.     

Acro-pectoro-renal field defect with contralateral ureteropelvic junction obstruction

Ureteropelvic junction obstruction was noted in a newborn male infant with acro-pectoro-renal field defect. To our knowledge, this association has not previously been reported. Ultrasonography of the urinary tract should be performed on all children with aplasia of the pectoralis major muscle.     

Holographic interferometry forIn Vitro investigation of bioprosthetic valves

Dysfunction of heart valve prostheses—mechanical as well as biological—is a common problem in cardiac surgery. The reasons for the valve failures are still not well understood. Biological valves especially have an unsatisfactory durability; degeneration and calcification very often lead to the failure of the valves. In our opinion, hidden defects present in the valve material prior to implantation of the valves is a plausible explanation for the dysfunction. Hitherto there has been no technique to detect these defects without destructing the specimen. Holographic interferometry proved to be applicable forin vitro evaluation of mechanical heart valve prostheses. In the present paper we describe application of this method to biological valves. Nine porcine bioprostheses and four fresh porcine aortic valves were investigated by means of holographic interferometry. In eight of nine bioprostheses, the results showed irregularities of the leaflet structure which depend on anomalies of the connective tissue of the leaflets of the valves. To make sure that these findings are not due to normal variations of the morphology, the investigations were carried out with fresh and unfixated porcine aortic valves. In the latter, no such anomalies of the structure were detected. The results obtained confirm the above hypothesis on the origin of the later valve dysfunction. Thus, holographic interferometry tests of bioprostheses prior to their implantation prevent the use of potentially dysfunctional valves.Presented at the 35th World Congress, International College of Angiology, Copenhagen, Denmark, July 1993