Spontaneous colitis occurrence in transgenic mice with altered B7-mediated costimulation.

The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7 interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4(+) T cells. We show that the chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7 double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was more severe in this context, which was associated with the decreased number of Foxp3(+) regulatory T cells in transgenic B7 double knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate T cell function and the development of inflammatory diseases.

Comparison of the protective effect of a commercially available western diamondback rattlesnake toxoid vaccine for dogs against envenomation of mice with western diamondback rattlesnake (Crotalus atrox), northern Pacific rattlesnake (Crotalus oreganus oreganus), and southern Pacific rattlesnake (Crotalus oreganus helleri) venom.

OBJECTIVE: To evaluate effectiveness of a commercially available toxoid manufactured from western diamondback (WD) rattlesnake (Crotalus atrox) venom against envenomation of mice with WD, northern Pacific (NP) rattlesnake (Crotalus oreganus oreganus), and southern Pacific (SP) rattlesnake (Crotalus oreganus helleri) venom. ANIMALS: 90 specific pathogen-free female mice. PROCEDURES: Mice were allocated into 3 cohorts (30 mice/cohort). Mice received SC injections of C atrox toxoid (CAT) vaccine (n = 15/group) or adjuvant (15/group) at day 0 and again at 4 weeks. At 8 weeks, mice were challenge-exposed with 1 of 3 venoms. Survival until 48 hours was evaluated by use of log-rank analysis of survival curves and the z test for proportions. RESULTS: 6 of 15 WD-challenged CAT-vaccinated mice, 3 of 15 NP-challenged CAT-vaccinated mice, and 0 of 15 SP-challenged CAT-vaccinated mice survived until 48 hours. All adjuvant-only vaccinates survived ≤ 21 hours. Mean survival time of CAT vaccinates was longer than that of adjuvant-only vaccinates for all venoms (1,311 vs 368 minutes for WD, 842 vs 284 minutes for NP, and 697 vs 585 minutes for SP). Results of the z test indicated a significantly increased survival rate for vaccinates exposed to WD rattlesnake venom but not for vaccinates exposed to NP or SP rattlesnake venom. Log-rank analysis revealed a significant difference between survival curves of vaccinated versus unvaccinated mice exposed to NP but not WD or SP venom. CONCLUSIONS AND CLINICAL RELEVANCE: CAT vaccination improved survival rate and survival time after challenge exposure with WD rattlesnake venom and may offer limited protection against NP rattlesnake venom but did not provide significant cross-protection against SP rattlesnake venom.

Isoflurane with morphine is a suitable anaesthetic regimen for embryo transfer in the production of transgenic rats.

During our initial attempts to produce transgenic rats, we found that an anaesthetic combination typically used for embryo transfer (intramuscular injection of ketamine [90 mg/kg] with xylazine [10 mg/kg]) yielded extensive variation in both the depth and length of anaesthesia. In the present prospective study, we compared the reproductive outcomes afforded by using either isoflurane (5% for induction, 2% for maintenance, carried in 2 l/min of oxygen) with morphine (5 mg/kg s.c., given immediately after isoflurane induction) or ketamine/xylazine in adult (250-300 g), pseudopregnant Sprague-Dawley rats. Each animal was anaesthetized with either isoflurane/morphine or ketamine/xylazine, after which 30 microinjected eggs were transferred into the left uterine horn. The mean pregnancy rate for isoflurane/morphine (15%) was 50% greater than that achieved with ketamine/xylazine (10%). The mean number of live pups (just over five per litter) was comparable for both regimens. All rats given isoflurane/morphine quickly achieved a surgical depth of anaesthesia and experienced a rapid postoperative recovery (3-5 min). In contrast, 25% of rats injected with ketamine/xylazine did not reach a depth of anaesthesia within 10 min that was sufficient for laparotomy, and all that were anaesthetized successfully required an extended postoperative recovery period (60-90 min). These data show that isoflurane/morphine is well tolerated by microinjected embryos and suggest that its use during embryo transfer may provide a means for both reducing the number of pseudopregnant females used and increasing the speed with which rat transgenic projects are completed.

Strain variation of immature female rats in response to various superovulatory hormone preparations and routes of administration.

The purpose of this study was to examine the response of rats of different genetic backgrounds to various superovulatory hormonal treatments. Immature Sprague Dawley (SD), FBNF1, and F344 female rats (30 to 35 days of age) were used for this study as representatives of outbred, hybrid, and inbred strains respectively. Animals from each strain were allocated into four groups of hormone treatments as follows: 1) 30 IU pregnant mare serum gonadotrophin (PMSG) intraperitoneally (i.p.) followed 52 h later with 25 IU human chorionic gonadotrophin (HCG) i.p.; 2) 15 IU PMSG i.p. followed 52 h later with 7.5 IU HCG i.p.; 3) 1.0 IU follicle stimulating hormone (FSH) daily via Alzet mini-pumps for 60 h; and 4) 1.0 IU FSH daily via Alzet mini-pumps for 54 h followed by 10 mg luteinizing hormone (LH). The efficacies of the hormone treatments were evaluated using the following criteria: % mated, % ovulated, total oocytes per female, and % fertilized. The % mated of SD rats treated with PMSG(30)+HCG(25) was significantly higher (P < 0.05) than that of animals treated with PMSG(15)+HCG(7.5); in addition, the total oocytes per female was significantly higher (P < 0.05) for SD animals receiving PMSG(30)+HCG(25) than all other treatments. The % ovulated of SD rats was significantly lower (P < 0.05) in response to FSH alone as compared to all other treatments. The % ovulated for FBNF1 rats was significantly greater (P < 0.05) in response to both PMSG+HCG treatments as compared to FSH and FSH+LH. The % ovulated and % fertilized were significantly lower (P < 0.05) in F344 rats treated with FSH alone as compared to all other treatments. F344 rats produced significantly (P < 0.05) more oocytes per female in response to both PMSG+HCG treatments as compared to FSH and FHS+LH. The % ovulated of SD and F344 rats were significantly higher (P < 0.05) than that of FBNF1 rats in response to FSH and FSH+LH. SD rats produced a significantly greater (P < 0.05) number of oocytes per female than did FBNF1 rats in response to PMSG(30)+HCG(25) and a significantly greater (P < 0.05) number of oocytes per female as compared to those of FBNF1 and F344 rats in response to FSH and FSH+LH. The % fertilized of SD and FBNF1 rats were significantly higher (P < 0.05) than that of F344 rats in response to PMSG(15)+HCG(7.5). Our study demonstrates that treatment with PMSG+HCG is an effective method of eliciting superovulatory responses according to most criteria examined. We have also shown that outbred (SD) rats generally produced more oocytes per female in response to hormonal stimulation than did inbred and hybrid rats. Our results indicate that different strains of rats have various degrees of hormone sensitivity and response to different superovulation protocols.

Core body temperature as adjunct to endpoint determination in murine median lethal dose testing of rattlesnake venom.

Median lethal dose (LD50) testing in mice is the 'gold standard' for evaluating the lethality of snake venoms and the effectiveness of interventions. As part of a study to determine the murine LD50 of the venom of 3 species of rattlesnake, temperature data were collected in an attempt to more precisely define humane endpoints. We used an 'up-and-down' methodology of estimating the LD50 that involved serial intraperitoneal injection of predetermined concentrations of venom. By using a rectal thermistor probe, body temperature was taken once before administration and at various times after venom exposure. All but one mouse showed a marked, immediate, dose-dependent drop in temperature of approximately 2 to 6°C at 15 to 45 min after administration. The lowest temperature sustained by any surviving mouse was 33.2°C. Surviving mice generally returned to near-baseline temperatures within 2 h after venom administration, whereas mice that did not survive continued to show a gradual decline in temperature until death or euthanasia. Logistic regression modeling controlling for the effects of baseline core body temperature and venom type showed that core body temperature was a significant predictor of survival. Linear regression of the interaction of time and survival was used to estimate temperatures predictive of death at the earliest time point and demonstrated that venom type had a significant influence on temperature values. Overall, our data suggest that core body temperature is a useful adjunct to monitoring for endpoints in LD50 studies and may be a valuable predictor of survival in venom studies.