Successful in vivo and ex vivo transfection of pulmonary artery segments in lung isografts

OBJECTIVE: Gene transfer to lung grafts may be useful in ameliorating ischemia-reperfusion injury and rejection. Efficient gene transfection to the whole organ may prove problematic. Proximal pulmonary artery endothelial transfection might provide beneficial downstream effects on the whole graft. The aim of this study was to determine the feasibility of transfecting proximal pulmonary artery segments in lung isografts. METHODS: Male Fischer rats were divided into six groups. In vivo transfection: In group I (n = 7), a proximal segment of the left pulmonary artery was isolated and injected with saline solution by means of a catheter inserted through the right ventricle. After an exposure period of 20 minutes, clamps were removed and blood flow was restored. In group II (n = 7), the isolated arterial segments were injected with adenovirus carrying the Escherichia coli LacZ gene encoding for beta-galactosidase. Ex vivo transfection: In group III (n = 5), arterial segments were injected ex vivo with saline solution and in group IV (n = 5) with the adenovirus construct. In group V (n = 6), arteries were injected with saline solution and in group VI (n = 11) with liposome chloramphenicol acetyl transferase cDNA. In groups I to IV, animals were killed on postoperative day 3 and transgene expression was assessed by Bluo-Gal staining. In groups V and VI, animals were killed on postoperative day 2 and transgene expression was assessed by chloramphenicol acetyl transferase activity assay. RESULTS: Transgene expression was detected grossly and microscopically in endothelial and smooth muscle cells of pulmonary artery segments from all surviving animals of groups II and IV. In group VI, chloramphenicol acetyl transferase activity was significant in all assessed arterial segments. CONCLUSION: Significant transgene expression is observed in proximal pulmonary artery segments after both in vivo and ex vivo exposure.     

Regulation by gangliosides and sulfatides of phospholipase A2 activity against dipalmitoyl- and dilauroylphosphatidylcholine in small unilamellar bilayer vesicles and mixed monolayers

The modulation by gangliosides GM1 and GD1a, and sulfatide (Sulf) of the activity of porcine pancreatic phospholipase A2 was studied with small unilamellar vesicles of dipalmitoylphosphatidylcholine (L-dpPC) and lipid monolayers of dilauroylphosphatidylcholine (L-dlPC). The presence of Sulf always led to an increase of the maximum rate of the enzymatic reaction, irrespective on whether the vesicles were above, in the range of, or below the bilayer transition temperature. Sulf did not modify the latency period for the reaction that is observed at the bilayer transition temperature. Gangliosides inhibited the maximum rate of enzymatic activity bilayer vesicles in the gel phase but the effect was complex. When the reaction was carried out at a temperature within the range of the bilayer phase transition, the gangliosides inhibited the maximal rate of the reaction in proportion to their content in the bilayer. However, at the same time the latency period observed with vesicles of pure phospholipid at this temperature was shortened in proportion to the mole fraction of gangliosides in the bilayer. At temperatures above the bilayer phase transition, gangliosides stimulated the activity of PLA2. Preincubation of the enzyme with Sulf or gangliosides did not affect the activity against bilayer vesicles of pure substrate. These glycosphingolipids did not modify the rate or extent of desorption of the enzyme from the interface, nor the pre-catalytic steps for the interfacial activation of PLA2, or the enzyme affinity for the phospholipid substrate. Also, the activity of the enzyme was not altered irreversibly by glycosphingolipids. Our results indicate that Sulf and gangliosides modulate the catalytic activity of PLA2 at the interface itself, beyond the initial steps of enzyme adsorption and activation, probably through modifications of the intermolecular organization and surface electrostatics of the phospholipid substrate.     

Sulfated glucuronyl glycolipids and gangliosides in the optic nerve of humans

Pathologically delayed visual evoked potentials may be present in patients with neuropathy associated with IgM M-proteinemia, which is directed against myelin-associated glycoprotein and sulfated glucuronyl glycolipids (SGGLs), but there are no reports of these antigens in the optic nerve. We recently examined human optic nerve and occipital lobe tissues for the occurrence of SGGLs using the technique of immunostaining on thin-layer chromatographic plates and found them in the optic nerve, but not the occipital lobe. SGGLs in the optic nerve may represent target antigens for CNS involvement by the M-protein in patients with neuropathy. We also studied the ganglioside composition of the optic nerve and found it different from that of the brain. Human optic nerve is characterized by an abundance of the b-series gangliosides, including GD1b, GT1b, and GQ1b. GD1a, which is usually a major component of brain gangliosides, is only a minor species of the optic nerve ganglioside fraction.     

Multiple pituitary hormone deficiencies in a patient with spinocerebellar ataxia: magnetic resonance imaging and hormonal studies

Degenerative spinocerebellar ataxia has a rare association with hypogonadotropic hypogonadism. In this report we present the results of the detailed endocrine evaluation and magnetic resonance imaging in one such patient. A 20-year-old male with progressive cerebellar ataxia, hypogonadism, and short stature was investigated. Basal testing revealed hypogonadotropic hypogonadism (LH < 5 mU/L, FSH < 5 mU/L, testosterone 2.5 nM/L). There was no rise in LH after stimulation with LHRH, peak LH level being < 5 mU/L. Insulin hypoglycemia testing was consistent with GH deficiency, with peak GH being 3.2 mU/L. On TRH stimulation, there was no significant rise in prolactin, though the TSH response was normal. Magnetic resonance imaging revealed cerebellar atrophy. The anterior pituitary was atrophic, with a height of 1.4 mm. The posterior pituitary and the pituitary stalk were normal in size and position. This patient with degenerative spinocerebellar ataxia had multiple pituitary hormone deficiencies. The results of our endocrine evaluation and MR imaging lead us to believe that these deficits may result from a lesion at the level of the pituitary gland.     

Three-point correlation functions in uniformly and randomly driven diffusive systems

We have previously shown that human antibody (Ab) directed against the capsular polysaccharide of the important bacterial pathogen, Haemophilus influenzae type b (Hib) is encoded by a small group of VH3 gene family members. The majority of anti-Hib PS Ab use members of the smaller VH3b subfamily. To examine directly the available human VH3 repertoire, we have used PCR to amplify and clone candidate germ-line VH3b H chain V region genes from two unrelated subjects from whom anti-Hib polysaccharide mAb had been previously obtained. A single functional VH3b germ-line gene was obtained from one subject. This gene is identical throughout the coding region to the previously identified gene 9.1. Twelve distinct VH3b germ-line sequences, 87.6-99.8% homologous to one another, were obtained from the second subject. One of these genes, LSG1.1, is also identical to the 9.1 germ-line gene, and a second, LSG6.1, is identical to a previously reported cDNA, M85. These germ-line VH3b genes are 82.7-94.1% homologous to rearranged anti-Hib PS VH3b segments obtained from these subjects. Our findings further demonstrate that considerable polymorphism of VH segments exists in the human population. Despite the presence of very highly homologous VH elements in the germ line, particular genes are highly conserved within the outbred human population.     

Dye leakage of four root end filling materials: effects of blood contamination

OBJECTIVES: A measurement of cell DNA content would be highly useful in determining the malignant nature of thyroid tumours in cases without distinctive features such as metastases, capsule invasion or emboli. Abnormal cell ploidy can be recognized with flow cytometry, but it is not known whether such results have diagnostic value. We therefore compared--in a double blind prospective study--the results of flow cytometry and pathologic diagnosis in fresh tumoural and non-tumoural thyroid cells. METHODS: Fifty unselected cold thyroid nodules were obtained from 50 consecutive patients (40 women, 10 men; age 18-80 years; mean 46) who underwent surgery within a 6 month period. Surrounding non-tumoural tissue was also obtained in 46 of them. Cell ploidy and the percentage of cells in each cell phase was determined with flow cytometry for both tumoural and nontumoural tissues. Two pathologists, unaware of the flow cytometric results, independently established the histologic diagnosis according to the WHO classification. RESULTS: The pathologic diagnosis was carcinoma in 7 cases (papillary carcinoma 6, vesicular carcinoma 1) and benign adenomas in 43 (29 macrovesicular, 11 microvesicular, 3 oncocytal). All the non-tumoural tissue samples were diploid. All 7 carcinomas were diploid and 10 of the 43 benign adenomas were aneuploid (4 near-diploid, 3 hyperploid, 1 near-tetraploid, 2 multiploid). The mean proliferation index was increased in 5 diploid tumours. CONCLUSION: These findings confirm that cell ploidy measured by flow cytometry is of no diagnostic value in the thyroid gland. It was also revealed that aneuploidy in adenomas may be related to tissue rearrangements of undetermined prognostic significance.