A novel approach for assessing the quality and effectiveness of IACUC oversight in investigator compliance.

In research facilities that are registered with the U.S. Department of Agriculture (USDA), funded by the Public Health Service, or accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International, the Institutional Animal Care and Use Committee (IACUC) is charged with oversight and evaluation of animal care and use under the terms of the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. Although the committee's oversight of investigator compliance may be evaluated annually during USDA inspections and triennially during AAALAC International site visits, routinely assessing the quality and effectiveness of the IACUC's performance is difficult. To measure the successfulness of IACUC oversight, our committee retained a management consultant to objectively design and conduct a confidential survey that could be used to determine how the IACUC could improve the process of facilitating researcher compliance with federal regulations and accreditation standards. The consultant based the content of the survey on confidential interviews with all IACUC members, the IACUC administrator, and a cross sectional representation of the key animal-user population at the facility. The survey was then distributed to the entire animal-user population. Vice-presidents, directors, principal investigators, and technicians were included in the distribution. With a response rate of 34%, the survey results indicated that the facilitation process warranted refinements. The consultant provided the IACUC with its recommendations, which were based on the discernible trending information indicated in the survey responses. The IACUC developed a specific plan of action to address the consultant's recommendations and intends to re-survey the animal-user population once the action plan has been fully implemented. In summary, the survey is an excellent way to assess the quality and effectiveness of IACUC oversight in investigator compliance by determining the level of researcher satisfaction. The evaluation, review, and follow-up process using a confidential interview and questionnaire technique can enhance the performance and effectiveness of IACUC oversight.

Preclinical toxicology studies of 4-ipomeanol: a novel candidate for clinical evaluation in lung cancer.

4-Ipomeanol (ipomeanol) is being developed as a potential antitumor agent to treat lung cancer. Ipomeanol produced a dose-related toxicity in CD2F1 mice, Fischer 344 rats, and beagle dogs. The LD50 in mice after a single iv dose of ipomeanol was 35 mg/kg in males and 26 mg/kg in females. Minimal cumulative toxicity occurred in mice after seven doses; LD50 was 30 mg/kg/day in males and 21 mg/kg/day in females. In rats, iv doses greater than or equal to 15 mg/kg were lethal. Labored respiration, terminal bronchiolar epithelial necrosis, interstitial inflammation, and alveolar edema were present in rats dosed with ipomeanol at greater than or equal to 9 mg/kg. In addition to pulmonary lesions, splenic and thymic lymphocyte depletion and/or necrosis was present. Ipomeanol had little cumulative toxicity in rats given seven daily doses. In dogs, iv doses greater than 12 mg/kg were lethal. Dogs treated with lethal doses of ipomeanol showed rapid, shallow respiration and pulmonary edema prior to death; diffuse pulmonary congestion or hemorrhage and diffuse renal congestion were present at necropsy. Pulmonary microscopic changes caused by nonlethal doses of ipomeanol included subacute interstitial inflammation and necrosis of respiratory bronchiolar and alveolar duct epithelium. In contrast to rodents, seven daily doses of ipomeanol were cumulatively toxic in dogs. The nonlethal pulmonary effects of ipomeanol were reversible in all three species. Tolerance to lethal doses of ipomeanol occurred in animals of all three species pretreated with multiple nontoxic doses of the drug. The LD50 of ipomeanol in male and female mice increased 2.4- and 4.5-fold, respectively, in tolerant mice. In rats and dogs, previously lethal doses of 48 and 24 mg/kg were nonlethal after tolerance was induced by pretreatment with seven daily doses of ipomeanol.

Effects of ribavirin on red blood cells.

Man and rhesus monkey may develop anemia during treatment with the broad-spectrum antiviral ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide). To assess whether the anemia is due to decreased production of erythrocytes, increased destruction, or a combination of both factors, the transport of ribavirin into erythrocytes and the drug's effect on the osmotic fragility, deformability, and half-life of erythrocytes were evaluated. The rate of uptake of ribavirin by erythrocytes was species and concentration dependent. Monkey cells accumulated the largest concentration of drug followed by human and rat cells. Monkey and human red cells, pretreated in vitro with ribavirin, retained 77 and 45% of the drug, respectively, when reincubated for 2 hr in drug-free medium. Rat red cells retained only 20% of their initial ribavirin content. Neither osmotic fragility nor deformability was altered by exposure of red cells to ribavirin in vitro. The half-life of 1,3-[3H]diisopropyl fluorophosphate (DFP)-labeled erythrocytes was measured in rhesus monkeys treated intramuscularly (im) for 10 days with either 15 or 60 mg/kg of ribavirin. A dose-related decrease in red cell survival was observed from Day 0 to 28. Thereafter, red cell half-lives were comparable to control values. These data indicate that ribavirin at a dose as low as 15 mg/kg decreases the half-life of red cells. This effect was reversible upon discontinuation of the drug. At 60 mg/kg, ribavirin also inhibited the release of red cells from the bone marrow. Termination of treatment was followed by release of red cells from the bone marrow as indicated by a drop in the specific activity of [3H]DFP-labeled red cells and marked reticulocytosis. No inhibition of red cell release from the bone marrow was seen in the low-dose group. These data suggest that ribavirin can decrease red cell survival as well as inhibit red cell release from the bone marrow. Both effects appear fully reversible when treatment is withdrawn.

Hematological and bone marrow effects of ribavirin in rhesus monkeys.

Ribavirin (Virazole, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), a broad-spectrum antiviral compound, was evaluated for effects on blood and bone marrow of rhesus monkeys when administered by intramuscular injection for 10 days in doses of 30 or 100 mg/kg/day (four monkeys/group). Both groups developed a normochromic, normocytic anemia that was mild in the low-dose group and severe in the high-dose group. A dose-related erythroid hypoplasia occurred during the treatment period. Myeloid precursors were not affected. Differential counts of erythroid precursors showed a significant decrease in late erythroid forms while early erythroid forms were either unchanged or increased. Megakaryocyte numbers were increased in both groups. Qualitative changes in marrow cells included vacuolization of erythroid precursors and of occasional white cell precursors and megakaryocytes, and the appearance of bone marrow histiocytes containing red cells in various stages of disintegration. Thrombocytosis occurred in both treatment groups, with platelet counts returning to control values after drug withdrawal. Platelet function was not affected by treatment. No drug-related changes were seen during the treatment period for total and differential leukocyte counts, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. Reticulocyte counts and mean corpuscular volume increased after treatment then returned to control values. Osmotic fragility of erythrocytes was not changed. These data show that in monkey, ribavirin causes a dose-related decrease in circulating red blood cell mass that is due in part to suppression of late erythroid precursors in bone marrow. These effects are reversible when treatment is discontinued and are not predictive of potentially serious or lasting untoward effects of ribavirin.

Morphological alterations in blood and bone marrow of ribavirin-treated monkeys.

The antiviral drug ribavirin (1, beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) produced significant hematologic effects when administered to rhesus monkeys by intramuscular injection over 10 days in doses of 30 or 100 mg/kg/day. The monkeys developed dose-related progressive anemia and thrombocytosis associated with marrow erythroid hypoplasia and megakaryocyte hyperplasia. In addition, bone marrow examination revealed phagocytosis of erythroid elements by histiocytes; vacuolization of erythroid precursors, and to a lesser extent precursors of other cell types; and occasional erythroid precursors with megaloblastoid appearance. The alterations were transient and disappeared on discontinuation of the drug.

Glucan-induced enhancement of host resistance to selected infectious diseases.

We conducted studies with mice, rats, and monkeys which demonstrated the ability of glucan to induce either nonspecific or specific enhancement of host resistance to infectious diseases. Intravenous pretreatment of mice with glucan significantly enhanced the survival of mice challenged with either Venezuelan equine encephalomyelitis (VEE) virus or Rift Valley fever virus. Pretreatment was beneficial when initiated 3 days before challenge with VEE virus and 7 days before challenge with Rift Valley fever virus. Treatment of mice after VEE challenge did not increase their survival compared with controls. Glucan pretreatment of rats provided increased resistance to both intraperitoneal and low-dose aerosol challenges with virulent Francisella tularensis when the glucan was given intravenously, but not when it was administered intranasally. In contrast, intranasal glucan pretreatment enhanced the survival of mice when they were challenged by aerosol with Pseudomonas pseudomallei, whereas intravenous glucan pretreatment did not increase survival. mice given glucan combined with a marginally immunogenic dose of VEE vaccine were more resistant to homologous virus challenge than were mice given either Freund complete adjuvant plus vaccine or vaccine alone. Similarly, both primary and secondary VEE antibody titers in cynomolgus monkeys given glucan with VEE vaccine were significantly greater than titers in vaccine controls.

In vitro interaction between normal cynolmolgus monkey alveolar macrophages and Legionnaires disease bacteria.

The interaction between normal cynomolgus monkey alveolar macrophages and Legionnaires disease bacteria was studied by transmission electron microscopy. After ingestion of Legionnaires disease bacteria, the organisms replicated within macrophages and destroyed the phagocytic cell.

Cynomolgus monkey model for experimental Q fever infection.

A subhuman primate model was developed for study of the pathogenesis of infection with Coxiella burnetii. Cynomolgus monkeys (Macaca fascicularis) that were exposed to 10(5) mouse median infectious intraperitoneal doses of C. burnetii in a small-particle aerosol developed clinical signs of illness and pathologic changes characteristic of Q fever infection in humans. All monkeys had radiologic evidence of pneumonia by day 9. Antibodies to C. burnetii were detectable by the indirect fluorescent antibody test by day 7. These data indicate that the cynomolgus monkey is a suitable model for study of the pathogenesis of Q fever infection and may prove valuable in the evaluation of C. burnetii vaccines.

Recovery of alveolar macrophages from rhesus and cynomolgus macaques by lung lavage.

A lung lavage technique was developed to recover alveolar macrophages from rhesus and cynomolgus macaques. Sterile saline solution was injected through an endotracheal tube in anesthetized macaques; lung wash fluids containing leukocytes were withdrawn. The lung wash fluids from each animal routinely contained more than 16 x 10(6) leukocytes. The predominant cell type was the alveolar macrophage; lung wash fluids contained more than 53% and 80% alveolar macrophages from rhesus and cynomolgus macaques, respectively. Lung lavage was performed each week for 6 weeks in both species with no ill effects. This technique has many applications in the study of infection and of pulmonary defense mechanisms.

The contribution of renal and extrarenal mechanisms to hypokalemia induced by glucagon.

Other investigators have shown that infusion of glucagon causes the concentration of potassium, [K+], in the arterial plasma to increase rapidly, then to decrease to less than the beginning value. In studies on anesthetized dogs, we found that the magnitude of the initial, rapid rise of [K+] was increased by nephrectomy but not affected by pancreatectomy. The subsequent decline of [K+] and the persistent hypokalemia were not affected significantly by nephrectomy. Plasma [K+] decreased in the nephrectomized-pancreatectomized dogs, as it did in the nephrectomized and the control groups, but the effect was temporary, and [K+] began to increase again, even though the infusion of glucagon continued; after the infusion was ended, plasma [K+] became significantly higher than the beginning value. These data suggest that the hypokalemia caused by infusion of glucagon initally depends on extrarenal factors other than insulin, and, later, depends on insulin.

A rhesus monkey model for the study of Bolivian hemorrhagic fever.

Two groups of rhesus monkeys were inoculated with either 10(5) (group 1) or 10(3) (group 2) plaque-forming units of Machupo virus, the etiologic virus of Bolivian hemorrhagic fever. The monkeys were observed for clinical signs; body temperatures, viremias, hematologic changes, and virus-neutralizing antibody were measured. The onset of clinical signs for groups 1 and 2 occurred on days 4-6 and 7-10, respectively, with fever, anorexia, and depression. These and other signs became more severe, and all of the monkeys died; the respective mean times to death for groups 1 and 2 were 14.3 and 19.5 days. Hematocrit, neutrophil, and lymphocyte values decreased in both groups until a few days before death and then increased slightly. Viremias in the two groups peaked on days 13 and 16, respectively, and persisted until death; the sole exception was one monkey in group 2 that developed neutralizing antibody by day 21. The response of the rhesus monkey to Machupo virus thus provides a useful model for the study of Bolivian hemorrhagic fever.

Pathology of Bolivian hemorrhagic fever in the rhesus monkey.

Gross and microscopic lesions associated with Bolivan hemorrhagic fever virus infection in the rhesus monkey were studied in 10 animals which died following inoculation. Gross lesions included skin rash, lymphadenopathy, splenomegaly, meningeal edema, hydropericardium and enlarged friable livers. Hemorrhagic manifestations of the infection were not consistently observed, but hemorrhages were present in the skin, heart, brain and nares in some monkeys. Histopathologic lesions were fairly consistent. Hepatic necrosis with the presence of acidophilic hyaline bodies, necrotizing enteritis, epithelial necrosis and adrenal cortical necrosis were present in all monkeys. Those monkeys which died after the seventeenth day of infection had nonsupurative meningoencephalitis; lymphoid necrosis was present in 3 monkeys that died after day 18. Other microscopic lesions included myocardial degeneration, lymphoid and reticuloendothelial cell hyperplasia and lymphoid depletion. Most of the histopathologic lesions described in human autopsy material were reproduced; however, the necrosis in the skin and oral mucosa, mucosa of the gastrointestinal tract and the adrenal cortex have not been described in man. Despite these apparent discrepancies the results of this investigation indicate that the rhesus monkey is a good experimental model for the study of Bolivian hemorrhagic fever infection.

Altered immune function in hypercholesterolemic monkeys.

Humoral and cellular immune responses to several antigens were compared in control and hypercholesterolemic groups of monkeys. Chronic hypercholesterolemia, with concomitant hyperphospholipidemia and hypotriglyceridemia, was produced experimentally by feeding monkeys a high-fat, high-cholesterol diet. When studied prior to infection, hypercholesterolemic monkeys exhibited impaired development of precipitating antibodies against ovalbumin, enhanced sensitivity to tuberculin antigen (stimulated apparently by mycobacterial components in complete Freund adjuvant), and an increased rate of clearance of colloidal carbon from blood. During pneumococcal infection the ability of neutrophiles from hypercholesterolemic monkeys to reduce nitroblue tetrazolium dye showed an increase greater than that of control monkeys; both groups exhibited increased but comparable final clearance rates of colloidal carbon, although the increment of increase was smaller in hypercholesterolemic monkeys.