Resistance to malignant catarrhal fever in American bison (Bison bison) is associated with MHC class IIa polymorphisms.

The Rhadinovirus ovine herpesvirus-2 (OvHV-2) is the most common causative agent of malignant catarrhal fever (MCF) in clinically susceptible ruminants including cattle and bison. American bison (Bison bison) are highly susceptible to clinical MCF. Nevertheless, approximately 20% of bison on ranches or in feedlots become infected with the virus without developing clinical disease. Defining the genetic basis for differences in susceptibility between bison could facilitate development of improved control strategies for MCF. One genetic region that influences susceptibility to infectious diseases is the major histocompatibility complex (MHC). In this study, a Bison bison (Bibi) DRB3 oligonucleotide microarray was used to type 189 bison from 10 herds where MCF outbreaks had occurred. Binary logistic regression was used to classify DRB3 alleles as resistant (R), susceptible (S) or neutral (N). Animals were reclassified using six DRB3 genotype categories: N/N, N/R, N/S, R/S, R/R and S/S. Analysis of homogeneity across herds showed that there was a herd effect. Consequently, a penalized logistic regression model was run with herd and genotype categories as the explanatory variables. The R/R genotype was associated with resistance to MCF (P = 0.0327), while the S/S genotype was associated with clinical MCF (P = 0.0069). This is the first evidence that MHC class IIa polymorphism is associated with resistance or susceptibility to OvHV-2-induced MCF.

Characterization of promoter function and cell-type-specific expression from viral vectors in the nervous system.

Viral vectors have become important tools to effectively transfer genes into terminally differentiated cells, including neurons. However, the rational for selection of the promoter for use in viral vectors remains poorly understood. Comparison of promoters has been complicated by the use of different viral backgrounds, transgenes, and target tissues. Adenoviral vectors were constructed in the same vector background to directly compare three viral promoters, the human cytomegalovirus (CMV) immediate-early promoter, the Rous sarcoma virus (RSV) long terminal repeat, and the adenoviral E1A promoter, driving expression of the Escherichia coli lacZ gene or the gene for the enhanced green fluorescent protein. The temporal patterns, levels of expression, and cytotoxicity from the vectors were analyzed. In sensory neuronal cultures, the CMV promoter produced the highest levels of expression, the RSV promoter produced lower levels, and the E1A promoter produced limited expression. There was no evidence of cytotoxicity produced by the viral vectors. In vivo analyses following stereotaxic injection of the vector into the rat hippocampus demonstrated differences in the cell-type-specific expression from the CMV promoter versus the RSV promoter. In acutely prepared hippocampal brain slices, marked differences in the cell type specificity of expression from the promoters were confirmed. The CMV promoter produced expression in hilar regions and pyramidal neurons, with minimal expression in the dentate gyrus. The RSV promoter produced expression in dentate gyrus neurons. These results demonstrate that the selection of the promoter is critical for the success of the viral vector to express a transgene in specific cell types.

Percutaneous endovascular repair of infrarenal abdominal aortic aneurysms: a feasibility study.

BACKGROUND: Endovascular grafting has markedly reduced the invasiveness of the treatment of abdominal aortic aneurysms. By using a modification of technique for available closure devices, we have been able to achieve percutaneous repair of aneurysms. This study reviewed our initial experience with this technique. METHODS: Demographics and background data from patients undergoing endovascular repair of abdominal aortic aneurysms were reviewed from prospectively collected registry data. Operative notes and angiographic and computed tomography scan data were retrospectively reviewed to assess the success of the percutaneous approach. RESULTS: Fourteen patients have undergone percutaneous placement of the AneuRx (Medtronic, Sunnyvale, Calif) endovascular graft, with a modification of the technique for the Prostar (Perclose, Redwood City, Calif) device for access site closure. Main graft body introduction with a 22F sheath proved successful in nine of 12 (75%) deployments. Contralateral limb deployment through a 16F sheath was successful in 10 of 14 deployments (71.4%). Reasons for conversion to open groin incisions include inadequate percutaneous hemostasis (six cases), iliofemoral dissection (four cases), device failure (one case), and compromised distal flow (one case). Percutaneous deployment success appears to be improved with larger iliac artery dimensions, decreased calcification, and limited tortuosity, because of the limitation of complications related to delivering a larger diameter sheath. Of the 13 percutaneous endograft insertions that were attempted, six (46.2%) were completely successful. CONCLUSION: Percutaneous deployment of available devices is technically feasible by using modifications of technique with percutaneous closure devices, despite large introducer sizes. Further experience with this technique offers the potential for identifying patients in whom this will prove successful and for even further reducing hospital stay and recovery times for aneurysm repair.

Cholesterol, but not cigarette smoke, decreases rabbit carotid artery relaxation.

The purpose of this study was to determine the physiologic effects of cigarette smoke exposure and dietary cholesterol on the availability of nitric oxide in carotid vascular rings. New Zealand white rabbits were placed in an airflow chamber for 3 hr/day over an 8-week period and were exposed to smoke from 600 cigarettes/per day added to the chamber inflow by a robotic smoke generator. New Zealand white rabbits, made hypercholesterolemic, and one group fed a normal diet, were similarly placed in the chamber without exposure to cigarette smoke. In those exposed groups, serum cotinine and cholesterol levels were consistently elevated. After the 8-week period, the carotid arteries were harvested. The vessels were cut into 3-mm rings which were suspended from pressure transducers. The rings were contracted with potassium chloride (KCl) to determine vessel integrity. One ring from each carotid was maximally contracted with 1 x 10(-3) molar norepinephrine (NE) while the experimental ring was contracted to 50% of maximum. Relaxation of the rings was achieved by adding incremental doses of acetylcholine. Our results showed that endothelial dysfunction, as measured by acetylcholine-mediated vasorelaxation, occurs in the rabbit carotid artery when exposed to high dietary cholesterol. Cigarette exposure alone in this particular vessel did not result in significant alteration in acetylcholine-mediated vasorelaxation.

Laparoscopic removal of a right adrenal pheochromocytoma in a pregnant woman.

Pheochromocytomas are a rare cause of hypertension in pregnancy. Laparoscopic adrenalectomy has been used effectively and safely in nonpregnant patients with pheochromocytoma, with the resultant benefits to the patients of less postoperative pain, shorter hospital stay, and quicker return to normal activities than is associated with open techniques. This represents the first report of a laparoscopic adrenalectomy for pheochromocytoma in a pregnant woman. Issues that are unique to laparoscopic surgery in pregnant patients are discussed.

Pentoxifylline prevents endothelial damage due to ischemia and reperfusion injury.

BACKGROUND: Endothelial injury after ischemia and reperfusion is characterized by an increase in permeability, cellular edema, and loss of acetylcholine-mediated vasorelaxation. Three hours of ischemia followed by 2 hr of reperfusion in the New Zealand white rabbit hindlimb has been shown to result in loss of acetylcholine-induced superficial femoral artery vasorelaxation. The purpose of this study was to evaluate the effect of intraarterial pentoxyfylline (PTX) on this endothelial injury. METHODS: New Zealand white rabbits underwent 3 hr of complete hindlimb ischemia followed by 2 hr of reperfusion. Twenty milliliters of either 100 microM PTX or normal saline was infused over 20 min via the circumflex iliac artery at initiation of reperfusion. Superficial femoral artery rings were then evaluated in vitro for endothelial cell-mediated relaxation. Rings were exposed to standardized incremental doses of acetylcholine after norepinephrine-induced contraction and percentage relaxation was measured. Sections of arteries were also sent for hematoxylin and eosin staining. RESULTS: Similar contraction responses following NE stimulation were observed between control and PTX-treated rings. Control rings relaxed a mean of 14.97 +/- 3.64, 23.17 +/- 5.61, and 31.84 +/- 8.43% in response to acetylcholine doses of 6 x 10(-8), 1 x 10(-7), and 1.5 x 10(-7) M, respectively. In contrast, PTX-treated segments relaxed a mean of 47.52 +/- 8.88, 62.32 +/- 6.83, and 76.73 +/- 4.91% to the same doses of acetylcholine. Differences in relaxation between control and PTX-treated vessels were significantly different at each dose (P < 0.05, Student's t test). Histologic examination of the PTX-treated and control arteries revealed an intact endothelium without morphologic differences between the two groups. CONCLUSION: In this model of rabbit hindlimb ischemia, endothelial cell-mediated vasorelaxation was preserved with the administration of intraarterial PTX during reperfusion compared to controls. The different relaxation responses could not be attributed to altered arterial contractility in response to norepinephrine, or explained by histologic changes in the arterial wall. These studies demonstrate a potential modality for therapeutic intervention to reduce reperfusion injury after acute limb ischemia.

Green fluorescent protein expressed in living mosquitoes--without the requirement of transformation.

Mosquitoes transmit viruses, protozoa and nematodes that are major causes of morbidity and mortality in humans. Details of arthropod anatomy and development, and the replication and development of pathogens in the arthropod vector, have relied upon examination of dissected or histologically processed material. We constructed a double-subgenomic Sindbis (dsSIN) virus expressing green fluorescent protein to demonstrate the potential of this protein for studying pathogen development in living arthropods. We were able to observe dissemination of virus, and furthermore, it was possible to observe components of the nervous system of mosquito larvae in extraordinary detail and record this on video tape. Although green fluorescent protein has been used as a reporter gene in a number of organisms, expression has relied upon transformation of cells or embryos. Transformation technology has limited applicability, thus we have described an alternative system that, due to the broad host range and viral tropisms of dsSIN viruses, may be useful to scientists in a range of disciplines. Green fluorescent protein may also provide a non-lethal selection method for use in transgenic arthropod research.

Efficiency of transduction by recombinant Sindbis replicon virus varies among cell lines, including mosquito cells and rat sensory neurons.

Recombinant alphaviruses have been used as vehicles for delivery and expression of heterologous genes in mammalian, avian and insect cell lines. We have used a Sindbis replicon virus (Sinreplac) able to express the E. coli lacZ gene to compare the efficiency of transduction in one insect, six mammalian cell lines and cultured rat dorsal neurons which apparently express beta-galactosidase over a 30-day time period. Results show that different cell lines were transduced with varying degrees of efficiency and that this efficiency could be improved in some cell lines by packaging the replicon with a helper derived from a more neurovirulent strain of Sindbis.

Loss of superficial femoral artery relaxation following ischemia-reperfusion.

Acute ischemia followed by reperfusion in skeletal muscle is associated with tissue edema and necrosis. The purpose of this study was to demonstrate superficial femoral artery endothelial injury following complete ischemia with reperfusion. New Zealand white rabbits underwent total devascularization of one hindlimb for 3 hr followed by 0, 1, and 2 hr of reperfusion. Control rabbits underwent a sham operation. Superficial femoral artery rings were then studied for acetylcholine induced relaxation in vitro. The response to acetylcholine was measured as percentage relaxation at three incremental doses (1 x 10(-7) , 3 x 10(-7) and 5 x 10(-7) M). The ischemia-only (26.30 +/- 7.07, 62.63 +/- 8.64, 88.08 +/- 5.25%) and the 1-hr reperfusion group (19.35 +/- 12.99, 39.24 +/- 15.78, 62.01 +/- 14.03%) showed no significant difference (P > or = 0.05, Student's t test) in relaxation as compared to the control group (13.73 +/- 2.11, 47.88 +/- 7.23, 72.44 +/- 9.00%). The 2-hr reperfusion group (6.10 +/- 1.02, 15.33 +/- 2.56, 34.67 +/- 6.31%), however, had a significant loss of relaxation at all three doses of acetylcholine compared to that seen in the control group (P < or = 0.05, Student's t test). In this model of complete ischemia, superficial femoral artery rings lose their ability to relax in response to acetylcholine following 3 hr of ischemia and 2 hr of reperfusion, demonstrating endothelial injury. However, immediately after 3 hr of ischemia or ischemia followed by only 1 hr of reperfusion, superficial femoral artery rings did not lose their ability to relax in response to acetylcholine. This study identifies a window of opportunity for therapeutic intervention after ischemia and prior to endothelial injury from reperfusion.

Potentiation of merocyanine 540-mediated photodynamic therapy by salicylate and related drugs.

Simultaneous exposure to merocyanine 540 (MC540) and light of a suitable wavelength kills leukemia, lymphoma and neuroblastoma cells but is relatively well tolerated by normal pluripotent hematopoietic stem cells. This differential phototoxic effect has been exploited in preclinical models and a phase I clinical trial for the extracorporeal purging of autologous bone marrow grafts. Salicylate is known to potentiate the MC540-mediated photokilling of tumor cells. Assuming that salicylate induces a change in the plasma membrane of tumor cells (but not normal hematopoietic stem cells) that enhances the binding of dye molecules it has been suggested that salicylate may provide a simple and effective means of improving the therapeutic index of MC540-mediated photodynamic therapy. We report here on a direct test of this hypothesis in a murine model of bone marrow transplantation as well as in clonal cultures of normal murine hematopoietic progenitor cells. In both systems, salicylate enhanced the MC540-sensitized photoinactivation of leukemia cells and normal bone marrow cells to a similar extent and thus failed to improve the therapeutic index of MC540 significantly. On the basis of a series of dye-binding studies, we offer an alternative explanation for the potentiating effect of salicylate. Rather than invoking a salicylate-induced change in the plasma membrane of tumor cells, we propose that salicylate displaces dye molecules from serum albumin, thereby enhancing the concentration of free (active) dye available for binding to tumor as well as normal hematopoietic stem cells.

Photodamaging effects of merocyanine 540 on neutrophils and HL-60 cells.

Merocyanine 540 (MC540) is a photosensitizing dye that has been used in several preclinical models and in a phase I clinical trial for the extracorporeal purging of tumor cells from autologous bone marrow grafts. The mechanism of the cytotoxic activity of MC540 is not yet fully understood, and the subcellular targets of MC540-mediated photodynamic damage remain to be identified. The human neutrophil provides an attractive model with which to study the effects of photoactivated MC540 on several well-defined cellular functions. As we report in this paper, simultaneous exposure of neutrophils to MC540 and light inhibited phagocytosis, random migration, chemotaxis, hydrogen peroxide production, and oxygen consumption. By contrast, the ability of neutrophils to kill engulfed bacteria and to produce superoxide radical was not compromised. Intracellular ATP levels and the activities of the cytosolic enzymes superoxide dismutase, catalase, and myeloperoxidase were only slightly reduced. Even in HL-60 leukemia cells, which bind more dye and are more readily killed by MC540-mediated photodynamic therapy than neutrophils, superoxide dismutase, catalase, and myeloperoxidase activities remained at normal or near-normal levels. These results are compatible with the view that plasma membrane components are primary targets of MC540-mediated photodynamic damage.

Limited cell-cycle dependence of the merocyanine 540-sensitized photoinactivation of L1210 leukemia cells.

L1210 leukemia cells were synchronized by a double thymidine block technique and then characterized with regard to their susceptibility to merocyanine 540 (MC540)-sensitized photoinactivation. Cells harvested 5 (G2/M phase) h after release from the second thymidine block were most susceptible to MC540-sensitized photoinactivation followed, in order of decreasing sensitivity, by cells harvested 2 (S phase) h and by cells harvested 7 (G1 phase) h after release from the second block. The expression of dye-binding sites changed very little during the cell cycle.

Interactions of merocyanine 540 with human brain tumor cells.

Merocyanine 540 (MC 540), a photosensitizing dye, has been used in preclinical studies and in a phase I clinical trial for the purging of leukemia, lymphoma, and neuroblastoma cells from bone marrow grafts. We evaluated MC 540 as an agent for the inactivation of brain tumor cell lines of medulloblastoma or glioma origin. The U373 glioma and 74SA medulloblastoma demonstrated significantly reduced survival as determined by in vitro clonogenic assay compared to normal glial cells when exposed to MC 540 and light. U87 glioma and Daoy medulloblastoma, however, were less sensitive than normal glial cells to MC 540 photoinactivation. In vivo injection of MC 540 into mice with malignant brain tumors disclosed greater dye incorporation into the malignant tissue compared with normal control mice brains or normal tissue surrounding the brain tumor. Increased uptake of MC 540 was observed in mice injected with either photosensitive (U373 and 74SA) or photoresistant (Daoy) cell lines. These data suggest that MC 540 may be an effective agent against certain brain tumors and that dye uptake in vivo does not reflect photosensitivity.

Evaluation of merocyanine 540-sensitized photoirradiation as a method for purging malarially infected red cells from blood.

The photosensitizing dye merocyanine 540 (MC 540) was evaluated as a means for purging malarially infected red cells from murine blood using the rodent malarial pathogens, Plasmodium yoelii and Plasmodium berghei, as models of human malaria. Malarially infected red cells bound more MC 540 and were more sensitive to MC 540-sensitized photoirradiation than were noninfected erythroid cells. Extracorporeal exposure of infected red cells to the dye and white light prevented the transmission of the disease in a transfusion model. P. berghei-infected red cells were more resistant to the antimalarial activity of MC 540 than were P. yoelii-infected cells, presumably because P. berghei preferentially infects reticulocytes whereas P. yoelii infects mature red cells. The possibility of using photoirradiation sensitized by MC 540 or related dyes to purge malarially infected donor blood is discussed.

Flexible fiberoptic sigmoidoscopy--the Monroe Clinic experience. A prospective study of 5000 examinations.

Analysis of 5000 consecutive flexible fiberoptic sigmoidoscopies form the basis of this report. It is concluded that this method of examination of the distal large bowel is not only safe and comfortable for the patient but is a more appropriate examination than the rigid proctosigmoidoscopy because of the significant increase in pathologic material found. This examination has proven practical and acceptable in a multispecialty clinic setting and has completely replaced rigid proctosigmoidoscopy. Flexible sigmoidoscopy is now the standard "routine" examination of the rectum and distal colon. The rationale for this conclusion is presented in this timely report.