scAAV-mediated gene transfer of interleukin-1-receptor antagonist to synovium and articular cartilage in large mammalian joints.

With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for adeno-associated virus (AAV)-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1-receptor antagonist (hIL-1Ra) or green fluorescent protein was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 10(11) vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA.

Effect of tumor necrosis factor antibody on synovial fluid cytokine activities in equine antebrachiocarpal joints injected with endotoxin.

Six horses received intra-articular injections of a mixture of 1 micrograms of endotoxin/5 mg of equine tumor necrosis factor (eqTNF) monoclonal antibody in 1 antebrachiocarpal joint and an equal volume (2 ml) of 1 micrograms of endotoxin/5 mg of control antibody in the opposite joint. Synovial fluid sample collection (1 ml) was accomplished by use of an indwelling, intra-articular catheter at postinjection hours (PIH) 0, 1, 1.5, 2, 5, and 8, and by arthrocentesis at PIH 24. Joint fluid samples were analyzed for nucleated cell count, protein concentration, and TNF, interleukin 6 (IL-6), IL-1, and IL-1-inhibitory activities. To monitor local inflammation, each carpus was graded semiquantitatively for swelling prior to each sample collection. Tumor necrosis factor, IL-1, or IL-1-inhibitory activity was not detected in any synovial fluid sample collected before endotoxin/antibody was administered. However, low IL-6 activity (< 100 U/ml) was found in 2 of 12 preinjection samples. In joints injected with endotoxin/control antibody mixture, maximal mean +/- SEM activities for TNF (1,019 +/- 310 U/ml), IL-1 (173 +/- 102 U/ml), and IL-6 (10.8 +/- 3.1 x 10(4) U/ml) were observed at PIH 2, 5, and 8, respectively. Tumor necrosis factor and IL-1 activities returned to baseline values by PIH 8 and 24, respectively; however, IL-6 activity remained high. Interleukin 1 inhibitory activity (27.4 +/- 2.25 IU/ml) was detected in all PIH-24 samples from control joints, but was not detected at any other time in control joints (limit of detection, 20 IU/ml). Tumor necrosis factor activity was not detected in any synovial fluid sample from joints treated with endotoxin/eqTNF antibody. In contrast, endotoxin IL-1 inhibitory activity (PIH 24) was higher in eqTNF antibody-treated joints (41.0 +/- 7.7 IU/ml) than in control joints, but the difference was not significant. Mean WBC count and protein concentration in control and treated joints were maximal at PIH 8. The curves for mean values of WBC count and total protein concentration were not significantly different in treated versus control joints. Swelling in each treated joint was either less than or the same as that in the opposite control joint at even, time in the initial 8 PIH. There was significant (P = 0.043) difference between treated and control joints at PIH 5 and 8. These results describe a profile of synovial fluid TNF, IL-1, IL-6 bioactivities, and IL-1-inhibitory activity during the initial 24 hours of synovitis induced by intra-articular administration of endotoxin in horses. Our eqTNF monoclonal antibody was effective in neutralizing TNF activity in synovial fluid when administered intra-articularly with endotoxin in horses. The induction of IL-1, IL-1 inhibitory activity IL-6, WBC, and total protein concentration responses are largely independent of TNF activity in synovial fluid of horses receiving endotoxin intra-articularly.

Septic metritis secondary to torsion of a pedunculated uterine fibroleiomyoma in a filly.

A 2-year-old Arabian filly was referred for evaluation of a serosanguineous vaginal discharge. Palpation per rectum revealed a large, fluid-filled uterus and a uterine mass. The filly developed septic metritis and secondary laminitis as a result of torsion and necrosis of a pedunculated uterine mass. Ovariohysterectomy was performed. The entire cervix was removed with the uterus. Gross examination of the excised uterus revealed 3 intraluminal masses. Histologic evaluation identified the tumors as fibroleiomyoma. The filly recovered completely, and there was no recurrence of the tumor.

Abdominal adhesions after small intestinal surgery in the horse.

One hundred thirteen of 172 horses (66%) undergoing exploratory celiotomy for a small intestinal lesion survived 4 or more days after surgery. Intra-abdominal adhesions causing clinical problems requiring additional surgery or euthanasia were documented in 25 horses (22.1%). Problems developed in significantly more males than females. The most common initial small bowel lesion was ileal impaction (12 horses); 21 horses underwent small intestinal resection or bypass. However, there was no significant difference in the incidence of intra-abdominal adhesions between horses that underwent intestinal resection or bypass and those that did not. Only 4 of the 25 horses (16%) with problems associated with postoperative adhesions survived. The mean interval between surgical procedures or between the initial procedure and euthanasia for all horses was 84 days (range, 7-512 days; median, 25 days). However, 70% of the subsequent celiotomies were performed within 60 days of the previous surgery. The mean interval between celiotomies was 221 days (range, 9-512 days) for the survivors and 61 days (range, 7-358 days) for the nonsurvivors. These results indicated that most of the problems related to postoperative intra-abdominal adhesions occurred within 2 months of the initial small intestinal surgery. Furthermore, the earlier development of postoperative adhesions was associated with a poorer prognosis for survival.

Effect of reduced food intake on blood levels of a hypertensive factor in spontaneously hypertensive rats.

We have previously demonstrated the existence of an active component(s) of extracts prepared from rat erythrocytes that stimulates the in vitro uptake of calcium in aortic rings and induces a sustained elevation in the blood pressure of normotensive rats. The apparent concentration of the calcium stimulatory component and the responsiveness of aortic rings to stimulation of calcium uptake by the compound is exaggerated in adult spontaneously hypertensive rats (SHR) compared with normotensive controls. Although no effect was observed on tissue responsiveness to the compound, a decrease in the concentration of the compound was observed in the blood of adult SHR subjected to dietary manipulation designed to blunt the elevation of blood pressure. These data suggest that blood concentration of the active compound may be influenced by external stimuli such as dietary manipulation.

Particle distribution around a copper beam stop for 72 MeV protons.

Results are presented of an experiment to study neutron yields at different production angles from 72 MeV protons stopping in copper. Activation detectors were used to provide data on neutron energies and flux densities. The results of a spectrum unfolding analysis are presented and compared with a Monte Carlo calculation. Some dosimetric data are also included.