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Activating mutations of the leucine-rich repeat kinase 2 ( LRRK2 ) gene are associated with Parkinson disease (PD), prompting development of LRRK2 inhibitors as potential treatment for PD. However, kidney safety concerns have surfaced from LRRK2 knockout (KO) mice and rats and from repeat-dose studies in rodents administered LRRK2 inhibitors. To support drug development of this therapeutic target, we conducted a study of 26 weeks' duration in 2-month-old wild-type and LRRK2 KO Long-Evans Hooded rats to systematically examine the performance of urinary safety biomarkers and to characterize the nature of the morphological changes in the kidneys by light microscopy and by ultrastructural evaluation. Our data reveal the time course of early-onset albuminuria at 3 and 4 months in LRRK2 KO female and male rats, respectively. The increases in urine albumin were not accompanied by concurrent increases in serum creatinine, blood urea nitrogen, or renal safety biomarkers such as kidney injury molecule 1 or clusterin, although morphological alterations in both glomerular and tubular structure were identified by light and transmission electron microscopy at 8 months of age. Diet optimization with controlled food intake attenuated the progression of albuminuria and associated renal changes.

PI3Kδ catalytic activity is required for immune cell activation, and has been implicated in inflammatory diseases as well as hematological malignancies in which the AKT pathway is overactive. A purine PI3Kδ inhibitor bearing a benzimidazolone-piperidine motif was found to be poorly tolerated in dog, which was attributed to diffuse vascular injury. Several strategies were implemented to mitigate this finding, including reconstruction of the benzimidazolone-piperidine selectivity motif. Structure-based design led to the identification of O- and N-linked heterocycloalkyls, with pyrrolidines being particularly ligand efficient and kinome selective, and having an improved safety pharmacology profile. A representative was advanced into a dog tolerability study where it was found to be well tolerated, with no histopathological evidence of vascular injury.

The effects of the cathepsin K inhibitor odanacatib (ODN) on fracture healing were monitored for ~6 and 15 weeks post-fracture in two separate studies using the unilateral transverse mid-ulnar osteotomy model in skeletally mature female rabbits. Rabbits were pre-treated for 3-4 weeks with vehicle (Veh), ODN (2 mg/kg, po, daily), or alendronate (ALN) (0.3 mg/kg, sc, twice-weekly) prior to osteotomy. In Study 1, the animals were maintained on the same respective treatment for ~6 weeks. In Study 2, the animals were also continued on the same therapy or switched from Veh to ODN or ODN to Veh for 15 weeks. No treatment-related impairment of fracture union was seen by qualitative histological assessments in the first study. Cartilage retention was detected in the calluses of ALN-treated rabbits at week-6, while calluses in the ODN and Veh groups contained bony tissue with significantly less residual cartilage. ODN treatment also markedly increased the number of cathepsin K-(+) osteoclasts in the callus, indicating enhanced callus remodeling. From the second study, ex vivo DXA and pQCT confirmed that ODN treatment pre- and post-osteotomy increased callus bone mineral content and bone mineral density (BMD) versus Veh (p < 0.001) and discontinuation of ODN post-surgery returned callus BMD to Veh. Peak load of ODN- or ALN-treated calluses were comparable to Veh. ODN increased callus yield load (20%, p = 0.056) and stiffness (26%, p < 0.05) versus Veh. These studies demonstrated that ODN increased mineralized callus during the early phase of fracture repair without impairing callus formation or biomechanical integrity at the fracture site., (© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Epithelioid hemangiosarcoma is a rare malignant endothelial neoplasia with a unique, predominantly epithelioid morphology. A 4-y-old rhesus monkey from our laboratory had multiple neoplastic nodules in a digit, limb skin, hindlimb muscle, and visceral organs including lung, heart, and brain. The nodules were composed of pleomorphic, polygonal, epithelioid, neoplastic cells that were arranged in sheets, nests, and cords and supported by variably dense fibrovascular connective tissue. The morphologic features of this tumor were predominantly epithelioid. However, some regions contained cystic spaces, clefts, and channel-like structures, all of which were lined with morphologically distinct neoplastic endothelial cells. These neoplastic cells, with or without epithelioid morphology, were positive immunohistochemically for CD31, factor VIII-related antigen, and vimentin. The presence of multiple metastatic nodules, high mitotic rate, and extensive Ki67-positive staining were consistent with malignancy. This report is the first description of epithelioid hemangiosarcoma in a rhesus monkey.

The blood pressure (BP)-lowering effects of mineralocorticoid receptor (MR) antagonists in salt-sensitive rat models of hypertension are well understood. However, studies in salt-independent models have yielded mixed results, and therefore, we measured the hemodynamic effects of MR blockade in spontaneously hypertensive rats. We treated spontaneously hypertensive rats for 8 weeks with 30-300 mg.kg.d eplerenone or 20 mg.kg.d losartan and monitored BP using radiotelemetry and performed histopathological analyses of the hearts. Eplerenone, in contrast to losartan, caused only a small reduction in systolic BP at the highest dose tested. Both reduced left ventricular wall thickness, although eplerenone was less effective than losartan. Only losartan decreased heart weight. We observed foci of cardiomyopathy characterized by combinations of infiltrating monocytes, necrotic myocytes, and interstitial fibrosis in hearts of control animals. The number of foci seemed to be decreased in hearts of losartan- and eplerenone-treated animals. In a second study, using quantitative histomorphometry, the number of foci was significantly reduced by 20 mg.kg.d losartan (by 68%) or by 300 mg.kg.d eplerenone (by 50%). Our data support the hypothesis that a direct BP-independent effect on the progression of cardiomyopathy in the heart may be one basis for the cardiac protection afforded by MR antagonism.

The purpose of this study was to investigate SCID-bg mice engrafted with bovine haematolymphoid tissues (SCID-bo) as a model for studying bovine Mannheimia haemolytica serotype 1- induced pneumonia, in which leucotoxin (LKT) plays a major role. In experiment A, SCID-bo and SCID-bg mice were inoculated intratracheally with either (1) phosphate-buffered saline (PBS), (2) M. haemolytica wild-type strain 89010807N ("LKT(+)WT"), (3) a M. haemolytica leucotoxin-deficient mutant of strain 89010807N ("LKT(-)mutant"), or (4) the M. haemolytica wild-type Oklahoma strain. Mice were killed for examination at intervals between 20 and 44h after inoculation. Lung lesions consisted of thickened alveolar septa and neutrophil and macrophage infiltrates in the bronchioles and alveoli. Lung lesion scores in the SCID-bo mice inoculated with LKT(+)WT or LKT(-) mutant were significantly (P<0.05) greater than those of the PBS control group, but the two bacterial strains produced results that did not differ significantly. M. haemolytica was isolated from lung, liver and spleen after inoculation but less frequently as time progressed. In experiment B, SCID-bg mice were inoculated intratracheally with live LKT(+)WT or formalin-killed LKT(+)WT and killed 24, 48 or 96 h later. Lung lesions were histologically similar to those observed in experiment A; however, there were no significant differences in the lung lesion scores between groups. It was concluded that the lesions seen in this study were probably not due to LKT, and that the SCID-bo mouse does not provide a good rodent model for bovine pneumonia.

PER.C6, a cell line derived from human embryonic retinal cells transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes, is used to produce E1-deleted Ad5 vectors such as the MRKAd5 HIV-1 gag vaccine. While whole, live PER.C6 cells are capable of growing as tumours when transplanted subcutaneously into immunodeficient nude mice at a high dosage, the process for vaccine production includes filtration steps and other methods which effectively preclude contamination by intact viable substrate cells. However, because of the neoplastic nature of this cell line, we carried out a series of investigations to assess the tumorigenic risk posed by residuals from the cell substrate in a vaccine. To address concerns about transmission of oncogenic DNA, we demonstrated that purified PER.C6 cellular DNA does not induce tumours in newborn hamsters or nude mice. To address concerns about other potential residuals, including hypothetical adventitious tumour viruses, we demonstrated that a PER.C6 cell lysate and a MRKAd5 HIV-1 gag vaccine produced on PER.C6 cells do not induce tumours in newborn hamsters or newborn rats. These results, in conjunction with the wide panel of viral safety tests performed on these cells, support the safety of the PER.C6 as a cell substrate for vaccine production.

Over a 21-month period, three Beagle dogs and one mixed-breed dog at our facility developed fatal pneumonia. The four dogs, all purpose bred, came from three vendors and had received the standard canine vaccines prior to shipment. In each instance, the affected dog had been shipped to our facility within the past 10 days. Three cases presented as a peracute clinical syndrome, and all had gross and microscopic findings consistent with hemorrhagic pneumonia. Escherichia coli was isolated from the lungs of all four dogs. Results of testing of lung tissue for canine parainfluenza virus and canine adenovirus were negative. Escherichia coli was also isolated from blood of three of the four dogs. Serotyping of the E. coli isolates indicated that two were serotype 06 and two were 04. Isolates from all four dogs were positive for the virulence factors alpha hemolysin and cytotoxic necrotizing factor 1 and for the adhesin factor class-III papG allele. These traits place the isolates in the class of extraintestinal pathogenic E. coli, which is being increasingly implicated as a cause of extraintestinal infections in animals and humans and may represent a zoonotic risk to humans working with research dogs.

Pasteurella multocida A:3 is a major cause of bovine pneumonia. A major antigenic heat-modifiable 28kDa outer membrane protein (Omp28) was previously identified. The purpose of this study was to purify and characterize Omp28 immunologically and structurally. Omp28 was extracted from N-lauroylsarcosine-insoluble protein preparations by a combination of detergent fractionation with Zwittergent 3-14 and chromatography. Partial N-terminal amino acid sequence confirmed Omp28 as a member of the OmpA-porin family. However, porin activity could not be demonstrated in a lipid-bilayer assay. Heat modifiability of purified Omp28 was demonstrated, and Omp28 was found in outer membrane fraction of P. multocida. Surface exposure of Omp28 was demonstrated by partial protease digestion of intact bacteria, by binding of anti-Omp28 polyclonal ascites fluid to the bacterial surface, and by partial inhibition of anti-outer membrane antiserum binding by previous incubation of the bacteria with anti-Omp28 serum. CD-1 mice vaccinated with purified Omp28 developed a significant antibody titer (P<0.05) compared to the control treatment group but were not protected from a homologous intraperitoneal bacterial challenge. By contrast, treatment groups vaccinated with P. multocida outer membrane, formalin-killed P. multocida or a commercial vaccine were significantly protected from challenge. In vitro complement-mediated killing of P. multocida was observed in post-vaccination sera of outer membrane, formalin-killed P. multocida, and commercial vaccine-treatment groups, but not with sera from the Omp28-treatment group. In conclusion, although Omp28 is surface exposed and antigenic, it may not be a desirable immunogen for stimulating immunity to P. multocida.