Subcutaneous Implants of a Cholesterol-Triglyceride-Buprenorphine Suspension in Rats.

A Target Animal Safety protocol was used to examine adverse events in male and female Fischer F344/NTac rats treated with increasing doses of a subcutaneous implant of a lipid suspension of buprenorphine. A single injection of 0.65 mg/kg afforded clinically significant blood levels of drug for 3 days. Chemistry, hematology, coagulation, and urinalysis values with 2- to 10-fold excess doses of the drug-lipid suspension were within normal limits. Histopathology findings were unremarkable. The skin and underlying tissue surrounding the drug injection were unremarkable. Approximately 25% of a cohort of rats given the excess doses of 1.3, 3.9, and 6.5 mg/kg displayed nausea-related behavior consisting of intermittent and limited excess grooming and self-gnawing. These results confirm the safety of cholesterol-triglyceride carrier systems for subcutaneous drug delivery of buprenorphine in laboratory animals and further demonstrate the utility of lipid-based carriers as scaffolds for subcutaneous, long-acting drug therapy.

Pathobiology of aging mice and GEM: background strains and experimental design.

The use of induced and spontaneous mutant mice and genetically engineered mice (and combinations thereof) to study cancers and other aging phenotypes to advance improved functional human life spans will involve studies of aging mice. Genetic background contributes to pathology phenotypes and to causes of death as well as to longevity. Increased recognition of expected phenotypes, experimental variables that influence phenotypes and research outcomes, and experimental design options and rationales can maximize the utility of genetically engineered mice (GEM) models to translational research on aging. This review aims to provide resources to enhance the design and practice of chronic and longevity studies involving GEM. C57BL6, 129, and FVB/N strains are emphasized because of their widespread use in the generation of knockout, transgenic, and conditional mutant GEM. Resources are included also for pathology of other inbred strain families, including A, AKR, BALB/c, C3H, C57L, C58, CBA, DBA, GR, NOD.scid, SAMP, and SJL/J, and non-inbred mice, including 4WC, AB6F1, Ames dwarf, B6, 129, B6C3F1, BALB/c,129, Het3, nude, SENCAR, and several Swiss stocks. Experimental strategies for long-term cross-sectional and longitudinal studies to assess causes of or contributors to death, disease burden, spectrum of pathology phenotypes, longevity, and functional healthy life spans (health spans) are compared and discussed.

Immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice.

Inbred laboratory mouse strains are highly divergent in their immune response patterns as a result of genetic mutations and polymorphisms. The generation of genetically engineered mice (GEM) has, in the past, used embryonic stem (ES) cells for gene targeting from various 129 substrains followed by backcrossing into more fecund mouse strains. Although common inbred mice are considered "immune competent," many have variations in their immune system-some of which have been described-that may affect the phenotype. Recognition of these immune variations among commonly used inbred mouse strains is essential for the accurate interpretation of expected phenotypes or those that may arise unexpectedly. In GEM developed to study specific components of the immune system, accurate evaluation of immune responses must take into consideration not only the gene of interest but also how the background strain and microbial milieu contribute to the manifestation of findings in these mice. This article discusses points to consider regarding immunological differences between the common inbred laboratory mouse strains, particularly in their use as background strains in GEM.

Of mice and microflora: considerations for genetically engineered mice.

The phenotype of genetically engineered mice is a combination of both genetic and environmental factors that include the microflora of the mouse. The impact a particular microbe has on a mouse reflects the host-microbe interaction within the context of the mouse genotype and environment. Although often considered a confounding variable, many host-microbe interactions have resulted in the generation of novel model systems and characterization of new microbial agents. Microbes associated with overt disease in mice have been the historical focus of the laboratory animal medical and pathology community and literature. The advent of genetic engineering and the complex of mouse models have revealed previously unknown or disregarded agents that now oblige the attention of the biomedical research community. The purpose of this article is to describe and illustrate how phenotypes can be affected by microflora by focusing on the infectious diseases present in genetically engineered mouse (GEM) colonies of our collective institutions and by reviewing important agents that are rarely seen in most research facilities today. The goal is to introduce the concept of the role of microflora on phenotypes and in translational research using GEM models.

Spontaneous unilateral brainstem infarction in Swiss mice.

Spontaneous vestibular syndrome in mice, characterized clinically by head tilt, circling or rolling, can be caused by otitis media, arteritis or central nervous system lesions. Postmortem examination of eleven non-inbred Swiss mice submitted for necropsy due to acute onset of vestibular signs revealed lesions consistent with brainstem infarction. The lesions were characterized by unilateral, well-demarcated areas of necrosis, malacia, and gliosis, with variable amounts of hemorrhage, in the lateral aspect of the medulla and caudal pons. The affected area included the medial, lateral and superior vestibular nuclei, the facial nucleus and the spinal trigeminal nucleus. While vestibular disease secondary to otitis media, periarteritis, and central nervous system neoplasia has been reported in many mouse strains, these unilateral brainstem infarctions were only seen in Swiss mice. These lesions share features with Wallenberg's Lateral Medullary Syndrome, the most common type of brainstem infarct in humans.

The differential roles of LFA-1 and Mac-1 in host defense against systemic infection with Streptococcus pneumoniae.

Mice deficient in CD18, which lack all four CD11 integrins, have leukocytosis and increased susceptibility to bacterial infection. To determine the effect of deficiencies in LFA-1 (CD11a/CD18) or Mac-1 (CD11b/CD18) on host defense against systemic bacterial infection, knockout mice were inoculated i.p. with Streptococcus pneumoniae. Increased mortality occurred in both LFA-1(-/-) (15 of 17 vs 13 of 35 in wild type (WT), p < 0.01) and Mac-1(-/-) (17 of 34 vs 6 of 25, p < 0.01) mice. All deaths in LFA-1(-/-) mice occurred after 72 h, whereas most deaths in Mac-1(-/-) mice occurred within 24-48 h. At 24 h, 21 of 27 Mac-1(-/-) mice were bacteremic, vs 15 of 25 WT (p = 0.05); no difference was observed between LFA-1(-/-) and WT. Increased bacteria were recovered from Mac-1(-/-) spleens at 2 h (p = 0.03) and 6 h (p = 0.002) and from livers (p = 0.001) by 6 h. No difference was observed at 2 h in LFA-1(-/-) mice, but by 6 h increased bacteria were recovered from spleens (p = 0.008) and livers (p = 0.04). Baseline and peak leukocyte counts were similar between Mac-1(-/-) and WT, but elevated in LFA-1(-/-). At 8 h, peritoneal neutrophils were increased in Mac-1(-/-), but not significantly different in LFA-1(-/-). Histopathologically, at 24 h Mac-1(-/-) animals had bacteremia and lymphoid depletion, consistent with sepsis. LFA-1(-/-) mice had increased incidence of otitis media and meningitis/encephalitis vs WT at 72 and 96 h. Both Mac-1 and LFA-1 play important but distinct roles in host defense to S. pneumoniae.

Evaluating mutant mice: anatomic pathology.

As the human and mouse genome projects approach their goals, initiatives in functional genomics are advancing. When the nucleotide sequences are available, identification of gene functions will assume even greater importance. Determination of gene products and their proximal biochemical functions provide a part of the picture, but determination of their functions in the context of the whole organism is the ultimate goal. The manipulated mouse genome has become accepted as a model for understanding the genetic basis of human conditions and diseases. Consequently, biomedical research institutions have seen significant increases in the use of mice since the early 1980s, and these increases are largely attributable to the use of genetically modified mice. The role of comparative pathology in research on mutant mouse models of disease is increasing in response to these trends. Evaluation and phenotypic characterization of mutant mice, via clinical and anatomic pathology techniques, will be an important component of functional genomics initiatives.

The material basis for reduced mechanical properties in oim mice bones.

Osteogenesis imperfecta (OI), a heritable disease caused by molecular defects in type I collagen, is characterized by skeletal deformities and brittle bones. The heterozygous and homozygous oim mice (oim/+ and oim/oim) exhibit mild and severe OI phenotypes, respectively, serving as controlled animal models of this disease. In the current study, bone geometry, mechanics, and material properties of 1-year-old mice were evaluated to determine factors that influence the severity of phenotype in OI. The oim/oim mice exhibited significantly smaller body size, femur length, and moment of area compared with oim/+ and wild-type (+/+) controls. The oim/oim femur mechanical properties of failure torque and stiffness were 40% and 30%, respectively, of the +/+ values, and 53% and 36% of the oim/+ values. Collagen content was reduced by 20% in the oim/oim compared with +/+ bone and tended to be intermediate to these values for the oim/+. Mineral content was not significantly different between the oim/oim and +/+ bones. However, the oim/oim ash content was significantly reduced compared with that of the oim/+. Mineral carbonate content was reduced by 23% in the oim/oim bone compared with controls. Mineral crystallinity was reduced in the oim/oim and oim/+ bone compared with controls. Overall, for the majority of parameters examined (geometrical, mechanical, and material), the oim/+ values were intermediate to those of the oim/oim and +/+, a finding that parallels the phenotypes of the mice. This provides evidence that specific material properties, such as mineral crystallinity and collagen content, are indicative and possibly predictive of bone fragility in this mouse model, and by analogy in human OI.

Programa de de prevençäo do câncer bucal no município de Goiânia / Program of prevention of the oral cancer in the municipal district of Goiânia

No ano de 1996, foi desenvolvido no município de Goiânia um programa de prevençäo contra o câncer bucal cujas informaçöes eram transmitidas, à populaçäo em geral, através dos meios de comunicaçäo, palestras, painés o auto-exame bucal. Os pacientes eram examinados, conscientizados e, quando necessário, encaminhados para as clínicas da Faculdade de Odontologia - UFG e do Hospital Araújo Jorge. Os resultados mostraram que os pacientes ainda säo pouco informados sobre as consequências das neoplasias e que a eficácia e o sucesso de um programa de prevençäo contra o câncer bucal necessita, fundamentalmente, da ajuda dos programas de saúde bucal desenvolvido no Estado de Goiás

Identification of the oim mutation by dye terminator chemistry combined with automated direct DNA sequencing.

The homozygous oim/oim mouse, a model of moderate-to-severe human osteogenesis imperfecta, contains a G-nucleotide deletion in the Cola-2 gene (the murine pro alpha(I) collagen gene) that results in accumulation of alpha1(I) homotrimer collagen. Although these mice have a distinctive phenotype that includes multiple fractures and deformities, genotyping is necessary to distinguish them from their wildtype (+/+) and heterozygote (oim/+) littermates. In this study, the dye primer and dye terminator chemistry methods, in combination with automated direct DNA sequencing, were compared for accuracy and ease in genotyping. A total of 82 mice from 14 litters were bred and genotyped; this resulted in 18 +/+, 35 oim/+, and 29 oim/oim mice. The dye primer and dye terminator chemistry methods worked equally well for identification of the deletion mutation and thus the genotype of all of the mice. However, the dye terminator method was found to be superior on the basis of the reduced amount of sample handling and reduced quantity of reagent required.

Amyloidosis, hemochromatosis, and atherosclerosis in a roseate flamingo (Phoenicopterus ruber).

An aged male roseate flamingo, in a private collection in the British Virgin Islands, was found acutely "down." After four days of supportive therapy, the flamingo succumbed. At necropsy gross lesions included emaciation; collapsed and thickened, yellow abdominal air sac; dark red liver, partially covered by friable yellow material; and a raised, intimal plaque in the aorta near the iliac trifurcation. Histologic examination revealed severe, diffuse, pyogranulomatous air sacculitis with associated locally extensive pleuroperitonitis/perihepatitis. Pansystemic, predominantly periarteriolar distribution of amyloid deposition was evident, as was massive intrahepatocellular accumulation of iron pigment (hemachromatosis/hemosiderosis). A locally extensive, nonobstructive, fibroatheromatous plaque was present in the distal aorta. Amyloidosis, hemochromatosis/hemosiderosis, and atherosclerosis have been recognized in Phoenicopteriformes and other marine or aquatic birds. Their pathogenesis and pathogenicity remain a matter of debate.

Wasting disease associated with cutaneous and renal nematodes, in commercially obtained Xenopus laevis.

Xenopus laevis, the South African clawed frog or toad, is a member of the family Pipidae. Now in high demand for research purposes, they are available commercially. Its reported lifespan is up to fifteen years. Investigators and caretakers are frustrated when commercially obtained, young frogs (four years or younger), not subjected to any studies, "waste" and die. The wasting syndrome is characterized by anorexia, color change, and "flaky skin." Often the first sign of this syndrome is the presence of large fragments of "flakes" of desquamated epithelium in the water. At necropsy, these frogs are thin and have rough skin instead of smooth slimy skin. Histologic examination reveals tortuous intraepithelial cavities or tunnels that contain nematodes, and associated mild to moderate granulomatous inflammation. Nematodes are also found in the kidneys of some of these frogs, usually in Bowman's space, wrapped around the glomerulus. The cutaneous capillarid nematode is identified as Pseudocapillaroides xenopi. Successful treatment with ivermectin and thiabendizole has been reported. The renal glomerular nematode has not been identified.

Fucoidan is a non-anticoagulant inhibitor of intimal hyperplasia.

We previously reported that heparin inhibits the proliferation of fibroblasts and vascular smooth muscle cells (SMC), in part, by binding to and increasing the antiproliferative activity of transforming growth factor-beta 1 (TGF-beta 1). We now report that certain other polyanions which are structurally distinct from heparin, such as fucoidan and polyinosinic acid, are more avid ligands for TGF-beta 1 and more potent antiproliferative agents than heparin. Fucoidan possessed more potent antiproliferative activity than heparin against rat and bovine aortic SMC in vitro, though possessing much lower anticoagulant activity than heparin. Furthermore, fucoidan suppressed in vivo intimal hyperplasia when continuously infused into rats subjected to balloon-catheter injury. Unlike heparin, which also suppressed intimal hyperplasia, fucoidan did not cause systemic anticoagulation. Thus, fucoidan may be useful as a non-anticoagulant inhibitor of post-angioplasty intimal hyperplasia.

Transforming growth factor-beta activity is potentiated by heparin via dissociation of the transforming growth factor-beta/alpha 2-macroglobulin inactive complex.

The control of smooth muscle cell (SMC) proliferation is determined by the combined actions of mitogens, such as platelet-derived growth factor, and the opposing action of growth inhibitory agents, such as heparin and transforming growth factor-beta (TGF-beta). The present studies identify an interaction between heparin and TGF-beta in which heparin potentiates the biological action of TGF-beta. Using a neutralizing antibody to TGF-beta, we observed that the short term antiproliferative effect of heparin depended upon the presence of biologically active TGF-beta. This effect was observed in rat and bovine aortic SMC and in CCL64 cells, but not in human saphenous vein SMC. Binding studies demonstrated that the addition of heparin (100 micrograms/ml) to medium containing 10% plasma-derived serum resulted in a 45% increase in the specific binding of 125I-TGF-beta to cells. Likewise, heparin induced a twofold increase in the growth inhibitory action of TGF-beta at concentrations of TGF-beta near its apparent dissociation constant. Using 125I-labeled TGF-beta, we demonstrated that TGF-beta complexes with the plasma component alpha 2-macroglobulin, but not with fibronectin. Heparin increases the electrophoretic mobility of TGF-beta apparently by freeing TGF-beta from its complex with alpha 2-macroglobulin. Dextran sulfate, another highly charged antiproliferative molecule, but not chondroitin sulfate or dermatan sulfate, similarly modified TGF-beta's mobility. Relatively high, antiproliferative concentrations of heparin (1-100 micrograms/ml) were required to dissociate the TGF-beta/alpha 2-macroglobulin complex. Thus, it appears that the antiproliferative effect of heparin may be partially attributed to its ability to potentiate the biological activity of TGF-beta by dissociating it from alpha 2-macroglobulin, which normally renders it inactive. We suggest that heparin-like agents may be important regulators of TGF-beta's biological activity.

Mycoplasma pulmonis infection augments natural killer cell activity in mice.

The goal of this study was to determine if experimental Mycoplasma pulmonis infection augmented splenic natural killer (NK) cell activity in mice. A 4 hour 51Cr-release in vitro assay using YAC-1 tumor target cells was employed to measure splenic NK cell activity in C57BL/6J mice infected intraperitoneally with M. pulmonis and in uninfected controls. Transient augmentation of the NK cells was observed, peaking at day 3 postinoculation (PI) and gradually returning to normal levels by day 10 PI. Selective depletion studies showed that the cells responsible for killing target cells were NK cells. They were nonadherent to nylon wool, not susceptible to Thy-1.2 antibody and susceptible to asialo GM1 ganglioside antibody. Inadvertent augmentation of the NK cell system due to M. pulmonis infection may complicate the interpretation of research data, especially in immunology and cancer studies.

Dimethyl sulfoxide (DMSO): a review.

Dimethyl sulfoxide (DMSO) is a very simple compound that has stimulated much controversy in the scientific and popular literature. Fig. 1 It is an aprotic solvent. Therapeutic and toxic agents that are not soluble in water are often soluble in DMSO. DMSO has a very strong affinity for water; on exposure to air, pure DMSO is rapidly diluted. DMSO's physiologic and pharmacologic properties and effects are incompletely understood. Properties that are considered to be particularly important to its therapeutic and toxic effects include: its own rapid penetration and enhanced penetration of other substances across biologic membranes; free radical scavenging; effects on coagulation; anticholinesterase activity; and DMSO-induced histamine release by mast cells. DMSO's systemic toxicity is considered to be low. Combinations of DMSO with other toxic agents probably constitute its greatest toxic potential. The scientific literature is reviewed with particular attention to mechanisms underlying DMSO's reported therapeutic and toxic effects. Currently approved, veterinary applications of DMSO are limited. DMSO's potential value in specific, approved and unapproved veterinary applications is discussed.

Synthesis of type 2 adenovirus DNA in the presence of cycloheximide.

Adenovirus type 2 DNA synthesis, either in permissive human cells or nonpermissive monkey cells, becomes independent of protein synthesis after the appearance of progeny viral DNA. In the presence of cycloheximide, semiconservative replication and initiation of progeny molecules can occur.