Inhibition of experimental proliferative vitreoretinopathy by retroviral vector-mediated transfer of suicide gene. Can proliferative vitreoretinopathy be a target of gene therapy?

PURPOSE: To determine the potential of somatic gene transfer as a treatment for proliferative vitreoretinopathy (PVR), experimental PVR was induced in rabbits by intraocular injection of fibroblasts bearing the herpes simplex virus thymidine kinase (HStk) gene. These transduced cells should be susceptible to cytotoxicity by exposure to ganciclovir (GCV). MATERIALS AND METHODS: Rabbit fibroblasts were transduced with retroviral vectors bearing an HStk gene. Proliferative vitreoretinopathy was induced by injection of 5 x 10(4) normal or HStk gene-transduced fibroblasts (HStk fibroblasts) into rabbit eyes. Ganciclovir (100 micrograms per eye) or saline was injected into the vitreous on days 0 and 4. Experimental animals were divided into three groups: group A received HStk fibroblasts with GCV; group B, normal fibroblasts with GCV; group C, HStk fibroblasts with saline. Proliferative vitreoretinopathy also was induced in several other groups of eyes, some receiving GCV and different proportions of HStk fibroblasts to normal fibroblasts, others receiving only normal fibroblasts and GCV. The eyes were examined by indirect ophthalmoscopy on days 4, 7, 14, and 28, and PVR was classified into six stages (0-5). RESULTS: Proliferative vitreoretinopathy was induced and progressed over time in each group. On day 28, PVR was most severe in animals in group B (average stage, 4.6) and group C (average stage, 4.4). Proliferative vitreoretinopathy was inhibited in group A (average stage, 1.0). The groups that received mixed injection of HStk fibroblasts and normal fibroblasts had intermediate PVR. Results of histologic study showed no apparent toxic or pathologic reaction in the retinochoroidal tissue of group A animals. CONCLUSIONS: Severity of experimental PVR clearly was reduced by transfer of the HStk gene and administration of GCV. This inhibitory effect also was produced by a combination of 10% HStk fibroblasts and 90% normal fibroblasts, indicating a significant bystander effect. These data suggest the potential of somatic gene therapy for the treatment of PVR.     

Myocardial ischemia and infarction postthoracotomy

The best long-term survival for any given lung cancer patient is provided by surgical resection. However, pneumonectomy still has the highest mortality rates, often due to cardiac complications. Risk assessment can be aided by preoperative evaluation of thoracic surgery patients. The role of right heart function, intraoperative management, and postoperative conditions in myocardial ischemia and infraction are analyzed, and the benefits of different kinds of resection are weighed in light of possible cardiac complications.     

Elevated interleukin-12 in progressive multiple sclerosis correlates with disease activity and is normalized by pulse cyclophosphamide therapy

The technique of pulse radiolysis has been used to investigate the possibility of intramolecular charge transfer in the dipeptide histidyltyrosine, following one-electron oxidation of one of its amino acid residues. The radical anion, Br2.- was found to react with the dipeptide at pH 6.0 with a bimolecular rate constant of 2.3+/-0.2 x 10(7) dm3 mol(-1)s(-1) suggesting that it reacts very selectively with the histidine moiety. Spectral observations at, or close to the end of this reaction show only the presence of a tyrosinyl free radical (TyrO.), however, indicating that fast (>10(6) s(-1) intramolecular charge transfer has taken place between histidine radicals (His+.) and tyrosine (TyrOH). This finding was supported by the direct observation of the rate of formation of TyrO. in experiments with the free amino acids, histidine and tyrosine, under conditions where Br2.- reacted selectively with histidine. The bimolecular rate constant for the reaction between His+. and TyrOH was found to be 2.4+/-0.5 x 10(6) dm3 mol(-1)s(-1). Taken together, the results of the study indicate that His+. is a relatively strong oxidising agent where (E (His+./His) > 770 mV at pH 6.0.     

Dissecting the order of bacteriophage T4 DNA polymerase holoenzyme assembly

Most biological organisms rely upon a DNA polymerase holoenzyme for processive DNA replication. The bacteriophage T4 DNA polymerase holoenzyme is composed of the polymerase enzyme and a clamp protein (the 45 protein), which functions as a processivity factor by strengthening the interaction between DNA and the holoenzyme. The 45 protein must be loaded onto DNA by a clamp loader ATPase complex (the 44/62 complex). In this paper, the order of events leading to holoenzyme formation is investigated using a combination of rapid-quench and stopped-flow fluorescence spectroscopy kinetic methods. A rapid-quench strand displacement assay in which the order of holoenzyme component addition is varied provided data indicating that the rate-limiting step in holoenzyme assembly is associated with the clamp loading process. Pre-steady-state analysis of the clamp loader ATPase activity demonstrated that the four bound ATP molecules are hydrolyzed stepwise during the clamp loading process in groups of two. Clamp loading was examined with stopped-flow fluorescence spectroscopy from the perspective of the clamp itself, using a site-specific, fluorescently labeled 45 protein. A mechanism for T4 DNA polymerase holoenzyme assembly is proposed in which the 45 protein interacts with the 44/62 complex leading to the hydrolysis of 2 equiv of ATP, and upon contacting DNA, the remaining two ATP molecules bound to the 44/62 complex are hydrolyzed. Once all four ATP molecules are hydrolyzed, the 45 protein is poised on DNA for association with the polymerase to form the holoenzyme.     

Requirement of Src kinase Lyn for induction of DNA synthesis by granulocyte colony-stimulating factor

Treatment of cells with granulocyte colony-stimulating factor (G-CSF) leads to tyrosine phosphorylation of cellular proteins. G-CSF stimulates both the activation of protein tyrosine kinases Lyn, Jak1, and Jak2 and the association of these enzymes with the G-CSF receptor. Wild-type, lyn-deficient, and syk-deficient chicken B lymphocyte cell lines were transfected with the human G-CSF receptor, and stable transfectants were studied. G-CSF-dependent tyrosyl phosphorylation of Jak1 and Jak2 occurred in all three cell lines. Wild-type and syk-deficient transfectants responded to G-CSF in a dose-responsive fashion with increased thymidine incorporation, but none of the clones of lyn-deficient transfectants did. Ectopic expression of Lyn, but not that of c-Src, in the lyn-deficient cells restored their mitogenic responsiveness to G-CSF. Ectopic expression in wild-type cells of the kinase-inactive form of Lyn, but not of the kinase-inactive form of Jak2, inhibited thymidine incorporation in response to G-CSF. These studies show that the absence of Lyn results in the loss of mitogenic signaling in the G-CSF signaling pathway and that activation of Jak1 or Jak2 is not sufficient to cause mitogenesis.     

Ultrasound screening for deep venous thrombosis after total knee arthroplasty. 2-year reassessment

The efficacy of ultrasound compared with ascending venography for the detection of deep venous thrombosis immediately after total knee arthroplasty was assessed after a 2-year interval. One hundred thirty-seven patients were eligible for the study; however, 31 patients received only one of the screening methods and a color Doppler examination was inconclusive in six patients. Therefore, 100 patients had a Doppler examination and a venogram. Overall, the sensitivity of ultrasound was 85%, the specificity 97%, the positive predictive value 85%, the negative predictive value 97%, and the accuracy 95%. The sensitivity in the calf was 83%, in the popliteal vein 86%, and in the femoral vein 100%. Two years ago, the initial assessment of ultrasound for the detection of deep venous thrombosis after surgery in patients who had total joint arthroplasty revealed a 75% sensitivity, 99% specificity, 91% positive predictive value, 97% negative predictive value, and 97% accuracy. The sensitivity in the calf was 83%; the sensitivity in the popliteal vein was 40%; and the sensitivity in the femoral vein was 50%. After 2 years of using this screening test with one technician and one radiologist, an improvement with this noninvasive technique was shown. However, it was found that Doppler imaging is not as sensitive as venography for detecting calf thrombi. Any imaging technique should be validated by each institution to determine the validity of the instrument and the learning curve of the technician administering the examination.