The cost of self-imposed regulatory burden in animal research.

U.S. federal regulations and standards governing the care and use of research animals enacted in the mid- to late 1980s, while having positive effects on the welfare and quality of the animals, have resulted in dramatic increases in overall research costs. In addition to the expenses of housing and caring for animals according to the standards, establishing the requisite internal compliance bureaucracies has markedly driven up costs, in both institutional monetary expenditures and lost research effort. However, many institutions are increasing these costs even further through additional self-imposed regulatory burden, typically characterized by overly complex compliance organizations and unnecessary policies and procedures. We discuss the sources of this self-imposed burden and recommend strategies for avoiding it while preserving an appropriate focus on animal well-being and research success.

Immunologic response enhances atherosclerosis-type 1 helper T cell (Th1)-to-type 2 helper T cell (Th2) shift and calcified atherosclerosis in Bacillus Calmette-Guerin (BCG)-treated apolipoprotein E-knockout (apo E-/-) mice.

Although immunocompetent hosts develop protective type 1 helper T cell (Th1) responses in mycobacterial infections, seroepidemiologic studies show that patients with atherosclerosis commonly express high antibody titers against mycobacterial heat shock protein (HSP) 65 and may develop a nonprotective type 2 helper T cell (Th2) response and advanced disease. These studies were undertaken to define mycobacterial dose requirements and kinetics for development of antibodies to HSP65, the Th1 to Th2 shift of immune response, and calcified atherosclerotic lesion development in the apo E-/- mouse. Fourteen-week apo E-/- female mice were treated intraperitoneally (ip) with heat-killed M. bovis Bacillus Calmette-Guerin (BCG), and 14 days later, cross-sections from the ascending aortas were stained for measurement of lesion size and calcium deposition. At 14 days, 0.01-mg BCG induced Th1 responses against HSP65. In contrast, 1-mg BCG induced splenic PGE2-releasing macrophages with a Th1-to-Th2 shift of responses to HSP65, which was PGE2-dependent. Treatment with 1-mg BCG significantly lowered bone density with increases in marrow osteoclastogenesis and development of calcified lesions in the aorta. At 14 days, 0.01-mg BCG induced Th1-dependent HSP65 responses and did not advance atherosclerosis. In contrast, for 1-mg BCG, a PGE2-dependent Th1-to-Th2 shift of responses to HSP65 and evidence of bone resorption are associated with advanced calcified atherosclerotic lesions.

Cholesteryl ester transfer protein expression prevents diet-induced atherosclerotic lesions in male db/db mice.

OBJECTIVE: Accompanying more atherogenic lipoprotein profiles and an increased incidence of atherosclerosis, plasma cholesteryl ester transfer protein (CETP) is depressed in diabetic obese patients compared with nondiabetic obese counterparts. The depressed levels of CETP in the plasma of diabetic obese individuals may contribute to the development of an atherogenic lipoprotein profile and atherogenesis. We have examined the effect of CETP expression on vascular health in the db/db model of diabetic obesity. METHODS AND RESULTS: Transgenic mice expressing the human CETP minigene were crossed with db/db strain, and 3 groups of offspring (CETP, db, and db/CETP) were placed on an atherogenic diet for 16 weeks. The proximal aorta was then excised and examined for the presence of atherosclerotic plaques. In db mice, 9 of 11 had intimal lesions with a mean area of 26 098+/-7486 microm2. No lesions greater than 1000 microm2 were observed in db/CETP or CETP mice. CETP-expressing mice had lower circulating cholesterol concentrations than db mice. Fractionating plasma lipids by FPLC indicated that the difference in total cholesterol was primarily attributable to differences in VLDL and LDL. CONCLUSIONS: The expression of human CETP in db/db mice prevented the formation of diet-induced lesions, suggesting an antiatherogenic effect of CETP in the context of diabetic obesity.

Aprotinin restores the adhesive capacity of dysfunctional platelets.

The post-operative coagulopathy associated with cardiopulmonary bypass (CPB) is known to be predominantly related to platelet dysfunction. The use of the serine protease inhibitor aprotinin dramatically reduces CPB associated hemorrhage and is thought to act primarily through the inhibition of plasmin without directly influencing platelets. Our data indicate that there is a direct effect of aprotinin on platelet adhesion, which has not been previously reported. We found that when aprotinin was added to blood samples with poorly adhesive platelets, platelet adhesion significantly increased as measured by the percent coverage of denuded arterial segments in the Baumgartner perfusion chamber. In preliminary experiments using expired platelet concentrates or fresh whole blood, the addition of aprotinin induced a positive increase of 22+/-7.5 and 14+/-6.2 percentage point in platelet adhesion, respectively. A simulated CPB model that recirculated a unit of anticoagulated whole blood for 2 h was used (n=14) to induce a platelet adhesion defect similar to that seen in clinical CPB. At initiation of recirculation, platelet adhesion was 55+/-9.5% but dropped to 13+6.5% coverage after 2 h simulated CPB. The addition of aprotinin to the post-recirculation samples induced a significant restoration of platelet adhesion back to 38+/-11% coverage. When epsilon amino-caproic acid with soybean trypsin inhibitor was added to post recirculation samples, there was no similar effect on adhesion scores. To compare these findings with surgical CPB, we collected one blood sample at the beginning and two at the end of CPB from each of seven open-heart patients. Aprotinin was added to one of each of the post-CPB samples. Platelet adhesion at the onset of surgical CPB was only 39+/-11% in this patient group but dropped to 7+/-7% by the end. Similar to the model, the addition of aprotinin post-CPB restored adhesion to 29+/-11%. These results suggest some action of aprotinin other than its antiplasmin effect, and that platelet adhesion in general can be promoted by aprotinin.

Comparative healing of surgical incisions created by a standard "bovie," the Utah Medical Epitome Electrode, and a Bard-Parker cold scalpel blade in a porcine model: a pilot study.

A comparative study of skin incision healing using a standard "bovie" and a new design electroscalpel, Utah Medical Products Epitome Electrode (Midvale, UT), was conducted in a porcine model. Wounds were evaluated objectively at 14 and 28 days after surgery using wound bursting strength measurements and histologic wound scoring. Each electrosurgical device was compared with wound healing of cold scalpel incisions as the gold standard using the same criteria. Statistical differences of healing between the bovie and the Epitome indicating preferential healing for the Epitome wounds were demonstrated for bursting strength at 14 days (p = 0.002). Comparisons of the measured "zone of coagulation necrosis" produced by the electroscalpels demonstrated significantly decreased thermal tissue damage favoring the Epitome (p = 0.0003). Greater differences in wound healing favoring the cold scalpel occurred in comparisons of bovie with cold scalpel than Epitome with cold scalpel, and overall results demonstrated healing for the Epitome wounds closely approximated that for cold scalpel. The authors conclude that this new generation electroscalpel provides measurable improvements in incisional wound healing compared to established electrosurgical technology.

Induction of cytolytic anti-Gal antibodies in alpha-1,3-galactosyltransferase gene knockout mice by oral inoculation with Escherichia coli O86:B7 bacteria.

Naturally occurring antibodies against [Gal alpha-1,3-Gal] structures (anti-Gal antibodies) are the primary effectors of human hyperacute rejection (HAR) of nonhuman tissue. Unlike most mammals, humans lack a functional alpha-1,3-galactosyltransferase (GalT) gene and produce abundant anti-Gal antibodies, putatively in response to GalT(+) enteric bacteria. GalT knockout (KO) mice have been generated as a small animal model of HAR but inconsistently express anti-Gal antibodies. We hypothesized that enteric exposure of GalT KO mice to live GalT(+) bacteria would produce cytolytic anti-Gal antibodies. Naive mice lacking anti-Gal antibodies were orally immunized with 10(10) live GalT(+) Escherichia coli O86:B7 bacteria and assayed for anti-Gal antibody titer, isotype, and cytolytic activity. Fecal samples were tested for E. coli O86:B7 prior to and after inoculation. In two separate experiments, 77 to 100% (n = 31) of mice developed serum anti-Gal immunoglobulin G (IgG; titer, 1:5 to 1:80) and/or anti-Gal IgM antibodies (titer, 1:5 to 1:1,280) 14 days postinoculation. Induced anti-Gal antibodies caused complement-mediated cytolysis of GalT(+) target cells, with extensive cytolysis observed consistently at serum IgM titers of >/=1:320. Absorption with synthetic [Gal alpha-1,3-Gal] inhibited both antibody binding and cytolysis. E. coli O86:B7 was recovered from stool samples from 83 to 94% of inoculated mice but not from naive mice, thus confirming enteric exposure. These findings demonstrate that oral inoculation with E. coli O86:B7 is a novel and effective method to induce cytolytic anti-Gal antibodies in GalT KO mice and support the premise that enteric exposure to GalT(+) bacteria induces anti-Gal antibodies in humans. These studies also suggest a role for GalT KO mice in elucidating anti-Gal responses in microbial immunity.