Species and gender differences in the formation of an active metabolite of a substituted 2,4-thiazolidinedione insulin sensitizer.

1. The metabolism of a substituted 2,4-thiazolidinedione (P1) with dual PPARalpha/gamma activity was evaluated in male and female rats, dogs and monkeys. A para-hydroxylated metabolite (M1) with potent PPARgamma-selective agonist, was a major circulating drug-related component in female rats, dogs and monkeys, but not in male rats (M1-to-P1 exposure ratio of <1, 3-5, 5 and 5-11 in male rat, monkey, female rat, and dog, respectively). 2. M1 (%) formed in vitro (5, 53, 57-65, 67 and 67% in male rat, monkey, female rat, dog, and human liver microsomes, respectively), rank ordered with M1 (%) formed in vivo (24-45, 53-57, 78, 75-85%, for male rat, monkey, female rat and dog, respectively, after oral administration of P1). 3. The plasma clearance of M1 was higher in male rats (32 ml min(-1) kg(-1) compared with 6, 7 and 2 ml min(-1) kg(-1) in female rat, male monkey and male dogs, respectively). 4. The low amounts of M1 observed in male rats, with the appearance of products of the cleavage of the propyl group between the phenyl groups was probably due to the presence of the sex-specific CYP2C11, which cleaves P1 at the propyl bridge. None of the CYPs present in female rats cleaved P1 at this site and M1 was only produced by CYP2C6. In humans, only CYP2C8 and the polymorphic CYP2C19 produced M1.

Hepatotoxicity of an HIV protease inhibitor in dogs and rats.

Oral administration of the HIV protease inhibitor L-689,502 caused cholestasis and hepatocyte injury in rats and dogs. These changes occurred rapidly, with elevations in serum transaminase observed as early as 6 hr after oral dosing in dogs. The acute phase of this hepatotoxic response was characterized in more detail in rats. Following intravenous administration, bile flow was decreased in a dose-dependent manner with greater than 90% decrease in less than 30 min at a dose of 5 mg/kg. The decrease in bile flow was associated with a decrease in erythritol clearance. The decrease in bile flow was not due to disruption of biliary tight junctions. Sucrose clearance was not increased and biliary bile acid concentrations in treated animals were not different from controls. Unlike control animals, bile flow was not stimulated by infusion of the bile acid tauroursodeoxycholic acid in animals treated with L-689,502. These cholestatic effects may be due, in part, to direct hepatocyte injury. Histological examination of perfusion-fixed livers 30 min after L-689,502 administration revealed periportal changes including hepatocyte vacuolation and occasional single cell necrosis. On a subcellular level, the nucleus and mitochondria were intact in less-severely affected cells. However, extensive vacuolation with multilamellar inclusions was pronounced in these cells. In addition, canalicular ectasia was also observed which was consistent with the cholestatic changes that were seen. In summary, L-689,502 is a potent, rapid acting hepatotoxin in dogs and rats. The mechanism by which this agent induces cholestasis is novel compared to other well-characterized cholestatic agents such as alpha-naphtylisothiocyanate and ethinyl estradiol.

Toxicokinetic analysis of losartan during gestation and lactation in the rat.

Previous developmental and reproductive toxicity studies in rats with losartan, a potent AT1-selective angiotensin II (AII) receptor antagonist, correlated maternal treatment during gestation day (GD) 15-20 with irreversible renal abnormalities in the F1 generation (Spence et al., '95a,b). Continued treatment through lactation was also associated with increases in pup mortality and decreases in pup body weights that persisted through weaning. The studies presented here were undertaken to quantify fetal and neonatal exposure to losartan when administered to the dam by oral gavage during early gestation, late gestation, and lactation. Following daily oral dosing of 135 mg/kg/day on GD6-15, fetal drug levels were negligible. However, losartan and its active metabolite, EXP3174 (L-158,641) were readily detectable in fetal plasma on GD 20 (estimated AUC values, 50.70 and 167.70 micrograms/hr/ml, respectively) and maternal milk during lactation (1.61 and 1.67 micrograms/ml, respectively). These studies suggest that the relative increased sensitivity of the fetus as compared to the neonate for losartan-induced renal lesions is related to the degree of exposure which is dependent on the time of administration (early gestation vs. late gestation/lactation) and the route of exposure (transplacental or through the milk). Furthermore, the maximum exposure to losartan and EXP3174 correlates with the ontogeny of the renin angiotensin system on approximately GD 17 and the critical period for losartan-induced renal lesions (GD15-20). The data support the hypothesis that the observed adverse fetal and neonatal effects are pharmacologically mediated, presumably through the lack of AT1 receptor stimulation.

Extent and persistence of streptozotocin-induced DNA damage and cell proliferation in rat kidney as determined by in vivo alkaline elution and BrdUrd labeling assays.

The extent of DNA damage and cellular proliferation induced in rat kidneys by single doses of the diabetogenic alkylating agent streptozotocin (STZ) and the time course of repair of that damage were evaluated using an in vivo alkaline elution assay for DNA strand breaks and a bromodeoxyuridine (BrdUrd) labeling assay for cell replication. Male Sprague-Dawley rats were given iv injections of 0.25 to 60 mg/kg STZ and kidneys were harvested 3 hr later for alkaline elution. A dose of 2.5 mg/kg STZ was the lowest dose to induce detectable DNA strand breaks and extensive damage was produced by the commonly used diabetogenic dose of 60 mg/kg. To characterize the repair of the drug-induced DNA damage, kidneys were harvested from a 60 mg/kg group of animals 3 hr to 27 days after dosing. BrdUrd-labeled kidney sections were also evaluated to assess any cellular proliferative response associated with STZ administration. Significant DNA damage was detected up to 14 days after dosing with return to near background levels by 20 days. Similarly, treatment with 60 mg/kg STZ was associated with increases in BrdUrd labeling indices 4 and 9 days after treatment with resolution by 27 days. These results indicate that the cellular and molecular repair responses to a single diabetogenic dose of STZ are prolonged, requiring up to 3 weeks to complete. Thus, to avoid potential additive or synergistic effects on STZ-induced nephrotoxicity and/or genotoxicity, a delay in the start of experimental therapies in this model (other than insulin) should be considered.

Development and submission of a nonclinical (pharmacology/toxicology) CANDA.

The experience with the submission of a nonclinical (pharmacology and toxicology) computer-assisted New Drug Application (CANDA) is reviewed. This system consisted of a stand-alone personal computer running several commercial programs in Microsoft Windows to access both text and data. WordPerfect was used as the word processor that contained all the documents and data tables (in read-only format) that were submitted in hard copy, and Andyne GQL was used as a tool to query the data in an Oracle relational database. Microsoft Excel was provided as a spreadsheet for graphics and analysis of data. Documents appeared virtually identical to those in the hard copy NDA submission. Searching the text was facilitated by the use of buttons on the screen, which allowed the NDA to be searched for a particular term. Data could be located either in WordPerfect documents, or in an Oracle database (using Andyne GQL) by querying the data. The data queries could be performed ad hoc, in which the reviewer selected all the parameters for a search, or with predefined query buttons, which retrieved data for principal treatment-related changes. This type of system also could serve as a useful model for both in-house nonclinical review and the submission of INDs and IND amendments.

Defining the susceptible period of developmental toxicity for the AT1-selective angiotensin II receptor antagonist losartan in rats.

Previous developmental and reproductive toxicity studies conducted in rats with Losartan, a potent AT1 subtype selective angiotensin II receptor antagonist, noted treatment-related effects on the pups of dams treated beyond the second trimester through lactation, as demonstrated by increases in pre- and postweaning pup deaths and decreased pup body weights [Spence et al. (1995) Teratology 51:000-000]. The studies presented here were designed to define the critical period for the induction of neonatal toxicity and to examine the effects of Losartan on kidney development when the drug is administered to the dam beyond the second trimester through lactation. In a developmental toxicity study with postweaning evaluation, pregnant rats were administered 5, 20, and 100 mg Losartan/kg/day on gestation days 6 through 15 (GD 6-15). There were no adverse effects on the F1 generation as assessed by mortality, clinical signs, weight gain, external examinations, developmental signs, behavioral tests, and gross or microscopic examination of the kidney. In a fostering/cross-fostering study, pregnant rats were administered 100 mg Losartan/kg/day on GD 15 through lactation day 20 (LD 20). Following delivery, pups from dams treated with Losartan were fostered to control dams, pups from control dams were fostered to Losartan-treated dams, and pups were also fostered to dams within the same group. Maternal exposure to Losartan during lactation increased the incidence of pup deaths on postnatal days 1-3 (PND 1-3), caused decreased pup weights on PND 7, and decreased performance in the auditory startle test in females and increased performance on the second swim maze test in males, relative to controls. Maternal exposure to Losartan during gestation was associated with decreased pup weight on PND 21 and effects observed on male performance in the swim maze test. Treatment during gestation was also associated with decreased pup cardiac weight as well as drug-induced histopathological changes of the kidneys from F1 pups, including medial hypertrophy of intracortical arterioles and dilatation of the renal pelvis. While the cardiac and renal vascular effects disappeared with time, significant renal lesions were still evident by PND 90. In a late-gestation/lactation study with renal evaluation, pregnant rats were administered 0.5, 1.0, 5.0, 20, and 100 mg Losartan/kg/day on GD 15-LD 20. Maternal toxicity was evident as decreased body weight gain in the 100 mg Losartan/kg/day group.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the reproductive and developmental toxicity of the AT1-selective angiotensin II receptor antagonist losartan in rats.

Losartan, an AT1-selective angiotensin II receptor antagonist, was evaluated in female rats for effects on fertility, reproduction, and perinatal and postnatal development. In a range-finding study, pregnant rats were treated orally from gestation days 6-17 (GD 6-17) with doses of 25, 75, 150, 225, and 300 mg Losartan/kg/day. There were treatment-related decreases in maternal body weight gain, slight treatment-related decreases in hemoglobin concentration, and slight treatment-related increases in serum urea nitrogen in the 225 and 300 mg/kg/day groups. In a fertility study, female rats were treated for 15 days prior to mating, during mating, and GD 0-19 with doses of 25, 100, and 300 mg Losartan/kg/day. The initial dose of 300 mg/kg/day was lowered to 200 mg/kg/day at the start of mating due to excessive body weight loss during the premating treatment interval. There were no treatment-related effects on reproductive performance, mating, or fertility indices in the F0 generation. There was no evidence of treatment-related or dose-related fetal malformations. However, decreased F1 pup body weights were observed in all drug-treated groups. In the 100 and 300/200 mg/kg/day groups there were treatment-related increases in F1 pup mortality and alterations in the pattern of postweaning body weight gains. There was also a delay in developmental signs in the 100 and 300/200 mg/kg/day groups, which were likely secondary to the decreased weight of the pups in these groups. In a developmental toxicity study, pregnant rats were administered 50, 100, and 200 mg Losartan/kg/day on GD 6-17. There was no evidence of developmental toxicity in any dose group. Maternal toxicity was evident in the 200 mg/kg/day group as a treatment-related decrease in body weight gain during gestation. In a late-gestation/lactation study, pregnant rats were administered 10, 25, and 100 mg Losartan/kg/day on GD 15 through lactation day 20 (LD 20). There were treatment-related decreases in maternal body weight gain during gestation and lactation in the 100 mg/kg/day group. Decreased pup weights were noted in all dose groups, and pre- and postweaning pup deaths were observed in the high dose group which were comparable to those observed in the female fertility study. The lack of fetal body weight effects at 100 mg Losartan/kg/day in the developmental toxicity study, with treatment ending on GD 17, indicates that adverse effects observed in the F1 generation in the fertility and late-gestation/lactation studies were due to exposure during late gestation and/or lactation.(ABSTRACT TRUNCATED AT 400 WORDS)

The morphology of juxtaglomerular cell hyperplasia and hypertrophy in normotensive rats and monkeys given an angiotensin II receptor antagonist.

L-694,492 (DUP 532), an angiotensin II (AII) receptor antagonist, was given orally at 125 mg/kg/day to rats and monkeys for up to 6 mo to assess the effects of the compound on juxtaglomerular (JG) cells. In rats, mild JG cell hypertrophy/hyperplasia occurred and was associated with a 12-fold increase in the bromodeoxyuridine-labeling index of JG cells and a 10-fold increase in renal renin content. Ultrastructurally, intermediate cells with characteristics of both smooth muscle cells and granulated renin-producing cells as well as hypertrophied renin-synthesizing cells were seen in the afferent arterioles. In monkeys, marked hypertrophy and hyperplasia were seen with an 80% increase in JG cell numbers, mitotic activity, and a greatly increased renin content compared to controls. Three mo after drug withdrawal, an increased number of cells remained, which showed features of smooth muscle cells with essentially no renin. These results show that AII receptor antagonism stimulates increased renal renin production by hypertrophy of existing granulated cells, metaplasia of smooth muscle cells to renin-synthesizing cells, and cell proliferation. When treatment was discontinued, the renin-producing cells redeveloped the features of smooth muscle, but, as we have shown with enalapril (angiotensin-converting enzyme inhibitor), the increase in their number persists for at least 3 mo.

The morphology of juxtaglomerular cell hyperplasia and hypertrophy in normotensive rats and monkeys given an angiotensin II receptor antagonist.

L-694,492 (DUP 532), an angiotensin II (AII) receptor antagonist, was given orally at 125 mg/kh/day to rats and monkeys for up to 6 mo to assess the effects of the compound on juxtaglomerular (JG) cells. In rats, mild JG cell hypertrophy/hyperplasia occurred and was associated with a 12-fold increase in the bromodeoxyuridine-labeling index of JG cells and a 10-fold increase in renal renin content. Ultrastructurally, intermediate cells with characteristics of both smooth muscle cells and granulated renin-producing cells as well as hypertrophied renin-synthesizing cells were seen in the afferent arterioles. In monkeys, marked hypertrophy and hyperplasia were seen with an 80% increase in JG cell numbers, mitotic activity, and a greatly increased renin content compared to controls. Three mo after drug withdrawal, an increased number of cells remained, which showed features of smooth muscle cells with essentially no renin. These results show that AII receptor antagonism stimulates increased renal renin production by hypertrophy of existing granulated cells, metaplasia of smooth muscle cells to renin-synthesizing cells, and cell proliferation. When treatment was discontinued, the renin-producing cells redeveloped the features of smooth muscle cells, but, as we have shown with enalapril (augioteusin-converting enzyme inhibitor), the increase in their number persists for at least 3 mo.

Juxtaglomerular cell hypertrophy and hyperplasia induced in rhesus monkeys by angiotensin II receptor antagonists.

BACKGROUND: Juxtaglomerular (JG) cell hypertrophy and hyperplasia were investigated in rhesus monkeys given angiotensin II (AII) AT1 receptor antagonists L-158,338 and DUP 753 (MK-0954, losartan). EXPERIMENTAL DESIGN: In 2 initial studies, L-158,338 was given orally at 10, 30, and 90 mg/kg/day for 3 or 14 weeks. To investigate the observed JG hypertrophy and hyperplasia, in a third 5-week experiment L-158,338 was given alone at 90 mg/kg/day, or with physiologic saline supplementation at 25 ml/kg/day, or coadministered with the angiotensin converting enzyme inhibitor enalapril at 10 mg/kg/day. Physiologic saline was given to attempt to suppress renin release through volume expansion and/or sodium retention. Enalapril was given to lower plasma AII levels and observe whether JG cell hypertrophy and hyperplasia were increased or decreased. For comparison, DUP 753 was given at 90 and 300 mg/kg/day. Plasma renin activity and AII concentration were measured in this study. RESULTS: Dose- and time-dependent increases in JG cell hypertrophy and hyperplasia were seen in the 2 initial experiment. In the third experiment, plasma renin activity and AII concentration were increased 3-fold and 6-fold over pretest values by L-158,338 at 90 mg/kg/day for 5 weeks. Saline supplementation had no effect on these parameters but diminished the group mean severity grade for JG hypertrophy and hyperplasia from 1.5 to 1.0. Enalapril coadministration had no effect on plasma renin activity, whereas it blunted the plasma AII increase caused by L-158,338 and increased the group mean grade to 2.5. DUP 753 at 300 mg/kg/day produced similar increases in plasma renin activity and AII concentration but only resulted in grade 1 JG cell hypertrophy and hyperplasia. CONCLUSIONS: L-158,338-induced JG cell hypertrophy and hyperplasia is an exaggerated pharmacologic response that can be modulated by saline supplementation and angiotensin converting enzyme inhibition. These results suggest that decreased renal perfusion or altered sodium homeostasis and plasma AII concentration are important variables that contribute to AT1 receptor blockade to induce JG cell hypertrophy and hyperplasia.

Measurement of plasma angiotensin II: purification by cation-exchange chromatography.

Measurement of plasma angiotensin II (AII) by radioimmunoassay (RIA) usually requires prior purification of the plasma to remove substances that cross-react in the RIA, most notably angiotensin III (AIII). Purification of AII is generally accomplished by solid-phase extraction (SPE) followed by reverse-phase HPLC, with tedious evaporation and resuspension steps in between, and requires collection of many HPLC fractions per sample for RIA. In this report, we describe a rapid two-step SPE procedure for the purification of plasma AII, including an improved protease inhibitor cocktail for preventing the formation or degradation of AII in vitro. Plasma is first extracted on an S-Sepharose cation-exchange column, in which AII is separated from AIII by virtue of their difference in net charge, and then extracted on a C8 SPE column, without need for intermediate sample handling. The two-step SPE method is fast, results in only a single fraction for RIA per sample, and yields consistently high recoveries (77-86%) of AII, reducing the volume of plasma needed from 2 to 0.5 ml. Rat plasma was used in the present study, but the complete conservation of angiotensin peptide sequences (except angiotensinogen) in mammals suggests that this method will be applicable for other species including humans. In summary, the two-step SPE method offers the speed and simplicity of solid phase extraction while achieving a purity in AII (i.e., free of AIII) previously only obtained by laborious procedures involving HPLC.

Studies of early hepatocellular proliferation and peroxisomal proliferation in Sprague-Dawley rats treated with tumorigenic doses of clofibrate.

Clofibrate, a peroxisome proliferator, is hepatocarcinogenic in rats in a dose-dependent fashion. While there is a relationship between peroxisome proliferation and rodent liver carcinogenesis, recent evidence also suggests an association between the tumorigenicity of peroxisome proliferators and sustained cell proliferation. To investigate the role of early cell proliferation in clofibrate-induced carcinogenesis and the predictive potential of this endpoint, in a 3-month study, rats were fed clofibrate doses equivalent to those used in the chronic bioassay, and cell proliferation was determined after 1 week and 3 months, using a 1-week continuous bromodeoxyuridine (BrdU)-labeling technique. Adult Sprague-Dawley rats were fed clofibrate at 1500, 4500, or 9000 ppm. Six rats/sex/group were killed after 1 or 13 weeks of treatment. Osmotic minipumps containing BrdU were implanted into rats 7 days prior to necropsy to determine the cumulative 7-day hepatocyte labeling index immunohistochemically. A dose-related increase in hepatocyte labeling index was seen after 1 week of treatment. However, at 13 weeks, sustained increases in hepatocyte proliferation were not seen; but a dose-related decrease in the hepatocyte labeling index was observed. Liver stereology at 13 weeks demonstrated a dose-related increase in liver weight and volume, but a decrease in hepatocyte nuclei per unit volume, a minimal increase or no change in the total number of hepatocyte nuclei per liver, and an absolute decline in the total number of BrdU-labeled hepatocyte nuclei per liver. These data suggest that in rats, clofibrate may influence hepatocarcinogenicity by decreases in normal hepatocyte proliferation over time and this effect may influence the pathogenesis of tumors at time points beyond 13 weeks of treatment.

Spontaneous ophthalmic lesions in young Sprague-Dawley rats.

Eight hundred eight Sprague-Dawley rats were examined for ophthalmic abnormalities during a pretest period in various preclinical safety assessment studies. Persistent pupillary membrane, corneal crystal, healed minor trauma, synechia, coloboma of the iris, lens luxation, cataract, persistent hyperplastic primary vitreous, vitreous hemorrhage, coloboma of the optic disc or choroid, remnant of hyaloid arterial system, retinal hemorrhage, retinal detachment, retinal folding and choroidal defect were observed. The incidences of corneal crystal, synechia, and nuclear cataract in this survey were higher than those reported previously. On the other hand, retinal folding in this survey was less common than that reported previously. These results suggest that background data of eye problems in albino rats should be accumulated in each own laboratory colony. In addition, since spontaneous eye problems are common in young albino rats, elimination of rats with ophthalmic abnormalities from study groups by an ophthalmic examination during a pretest period would facilitate to evaluate toxicity potential of test compounds in safety assessment studies.

HMG-CoA reductase inhibitor-induced myopathy in the rat: cyclosporine A interaction and mechanism studies.

Recent clinical evidence indicates a potential for skeletal muscle toxicity after therapy with HMG-CoA reductase inhibitors (HMGRIs) in man. Although the incidence of drug-induced skeletal muscle toxicity is very low (0.1-0.2%) with monotherapy, it may increase following concomitant drug therapy with the immunosuppressant, cyclosporine A (CsA), and possibly with certain other hypolipidemic agents. In the Sprague-Dawley rat, very high, pharmacologically comparable dosages (150-1200 mg/kg/day) of structurally similar HMGRIs (lovastatin, simvastatin, pravastatin and L-647, 318) produced dose-related increases in the incidence and severity of skeletal muscle degeneration. Physical signs included inappetence, decreased activity, loss of body weight, localized alopecia and mortality. To evaluate the interaction between HMGRIs and CsA, a rat model of CsA-induced cholestasis was developed. In this 2-week model, the skeletal muscle toxicity of the HMGRIs was clearly potentiated by CsA (10 mg/kg/day). Doses of HMGRIs which did not produce skeletal muscle toxicity when given alone caused between 75 and 100% incidence of myopathy (very slight to marked skeletal muscle degeneration) when CsA was coadministered. Typical light microscopic changes included myofiber necrosis with interstitial edema and inflammatory infiltration in areas of acute injury. Histochemical characterization of the muscle lesion indicated that type 2B fibers (primarily glycolytic white fibers) were most sensitive to this toxicity but that, with prolonged administration, all fiber types were ultimately affected. Results of pharmacokinetic studies in rats treated with various HMGRIs +/- CsA indicated that coadministration of CsA alters the disposition of these compounds, resulting in increased systemic exposure (e.g., increased area under the plasma drug concentration vs. time curve-AUC) and consequent (up to 13-fold) increases in skeletal muscle drug levels. Evaluation of the potential interaction between the HMGRI, lovastatin and CsA at the level of hepatic microsomal metabolism indicated that CsA did not inhibit the metabolism of lovastatin in isolated microsomes from female rats. In light of the above findings, it appears that HMGRI-induced myopathy is a class effect in the rat, which is potentiated by CsA as the result of altered clearance and resultant increased tissue exposure. Cholestasis associated with CsA and HMGRIs may form the basis for decreased elimination and the resultant elevated systemic exposure. Furthermore, this toxicity is muscle fiber-selective and may be associated with impaired skeletal muscle energy metabolism.

Investigations of the potential for five beta-lactam antibiotics to elicit type II hypersensitivity reactions in rats and monkeys.

Immunologic reactions are occasionally elicited in patients by various beta-lactam antibiotics (e.g., penicillins and cephalosporins). A relatively rare reaction (type II hypersensitivity) may involve antibody-mediated destruction of erythrocytes, leukocytes, and/or platelets. During the safety evaluation of several modified beta-lactam compounds (carbapenems), hemolytic anemia and/or neutropenia were observed in rhesus monkeys, and anemia, neutropenia, and thrombocytopenia in rats, after approximately 2 weeks of intravenous administration. Antiglobulin tests and other clinicopathologic findings indicated an immune basis for the cytopenias. A review of summaries of the preclinical data for numerous marketed beta-lactam antibiotics revealed that various cytopenias of unknown etiology were commonly seen in animals given high doses of these compounds. To determine whether these hematologic abnormalities were related to those produced by the above carbapenems, we investigated the potential of five widely used beta-lactam antibiotics (penicillin G, cephalothin, cefazolin, cefoperazone, and cefamandole) to elicit immune-mediated cytopenias in rhesus monkeys and Sprague-Dawley rats when given intravenously. After approximately 1 month of administration of these compounds at a dose level of 500 mg/kg/day, slight anemia occurred in several drug-treated monkeys; however, direct and indirect antiglobulin tests were negative for all animals, indicating that the anemias were not immune-mediated. In rats, no drug-induced hematologic changes were observed after 1 month of intravenous administration of 500 and 1000 mg/kg/day of each of the beta-lactams. In addition, direct antiglobulin tests were negative in rats. Therefore, it appears that the ability of certain carbapenem antibiotics to produce a high incidence of type II hypersensitivity reactions in animals is not typical of beta-lactam compounds in general.

Kinetics of viral replication and local and systemic immune responses in experimental rotavirus infection.

Rotavirus-seronegative mice were orally inoculated with murine rotavirus in order to study the kinetics of rotavirus replication and the relationship of viral replication to immunity and disease and to assess the effects of local and systemic antibodies on viral clearance and disease resolution.

Virus-specific immunity in neonatal and adult mouse rotavirus infection.

Mouse rotavirus (epizootic diarrhea of infant mice) was used as a model to study the role of virus-specific immunity in infection and diarrheal disease. The distribution of viral antigen in intestinal tissues was determined by immunofluorescent staining with anti-simian rotavirus (SA-11) serum. The location and proportion of antigen-positive cells appeared to vary as a function of time postinfection and age of the animal at the time of infection. In animals infected at 1 and 7 days of age, antigen-positive cells (5 to 25%) were first detected (1 day postinfection) in the proximal segment of the small intestine, and infection progressed to the middle and distal segments. At 10 days postinfection, virus-infected cells were no longer observed in the proximal segment. In animals infected at 21 days of age (disease-free), a significantly lower proportion of cells were antigen positive (2 to 5%), and they were restricted to the middle and distal segments of the small intestine. Infection, defined according to the presence of virus and viral antigens in intestinal tissues and by seroconversion in the immunoglobulin M (IgM) isotype as determined by enzyme-linked immunosorbent assay with SA-11 antigen, was observed for all age groups (neonatal to adult), even in the presence of virus-specific serum or intestinal immunoglobulins. On the other hand, diarrheal disease was not detected in neonatal mice (1 to 3 days old) positive for passively acquired virus-specific intestinal IgG. The presence of virus-specific IgA in the intestinal tract at the time of infection did not protect from subsequent diarrheal disease. Virus-specific, cell-mediated immunity, determined by a delayed-type hypersensitivity response, did not develop in neonatal mice infected at 5 and 12 days of age. Reinfection of adult mice was associated with suppression of virus-specific delayed-type hypersensitivity and a significant decrease in the titers of the virus-specific serum IgG and IgA.

Intraductal mammary carcinoma and benign ovarian teratoma in a rhesus monkey.

A benign ovarian teratoma and an intraductal mammary carcinoma were found in an adult rhesus monkey that had been used in reproductive studies and received human luteinizing hormone, follicle-stimulating hormone, and human chorionic gonadotropin.

Hemorrhagic streptococcal pneumonia in newly procured research dogs.

An acute necrotizing hemorrhagic pneumonia syndrome was recognized among 14 newly arrived research dogs. Typically, there were acute deaths without clinical signs. Necropsy revealed diffuse hemorrhagic pneumonia, and Lancefield group C Streptococcus zooepidemicus was isolated consistently from the lungs. In many cases, septic thrombi were seen in the small vessels of the kidneys, lymph nodes, spleen, brain, and adrenal glands. The syndrome was reproduced by intratracheal inoculation of the isolant into a susceptible dog.