Maturity-related Variability of the Thymus in Cynomolgus Monkeys (Macaca fascicularis).

Terminal body weights (TBWs), thymus weight parameters, and thymus morphology were retrospectively evaluated in 453 cynomolgus monkeys assigned to control groups on nonclinical toxicity studies. Morphology of bone, ovary, and testis/epididymis were used to determine maturity status of individual animals. There was no correlation between TBW and thymus weight (absolute and/or relative to TBW or brain weight). Thymus weight parameters and grades of decreased lymphocytes in the thymus were highly variable in immature animals compared to mature animals. There was also high (up to 11-fold) variability of thymus weight parameters within a given control group on the same study (generally 3 or 4 animals per sex). Several parameters evaluated had more pronounced age-related changes in males when compared to females. Our results demonstrate the inherent variability of thymus weight parameters and morphologic observations for cynomolgus monkeys on toxicology studies. Changes in thymus parameters in cynomolgus monkeys are unreliable indicators of immunomodulation or immunotoxicity in the absence of other relevant findings. Therefore, the thymus parameters commonly evaluated in preclinical safety assessments should not be the primary data set used to determine the presence of a direct test article-related effect on the immune system.

Spontaneous complex polysaccharide inclusions in the skeletal muscle of purpose-bred beagle dogs.

Amylase-resistant, periodic acid-Schiff (PAS)-positive inclusions were identified in the skeletal muscle of four of twenty-four purpose-bred beagle dogs from a routine toxicology study. Affected myofibers contained amorphous material filling up to 20% of the sarcoplasm that stained lightly basophilic with hematoxylin and eosin and was strongly PAS-positive with amylase resistance. Transmission electron micrographic examination of the inclusions revealed granular, non-membrane-bound, electron-dense material, consistent with polysaccharide. Although skeletal muscle inclusions with similar features have been reported in dogs in conjunction with systemic metabolic disorders and less often in muscle adjacent to nonmyogenic sarcomas, all four of these dogs lacked clinical or pathological findings diagnostic of a concurrent systemic metabolic or localized skeletal muscle disorder. Furthermore, these skeletal muscle inclusions were present in both vehicle- and test article-treated dogs and were considered an incidental finding that may occur spontaneously in clinically normal beagle dogs; as such, their presence in drug-treated animals should be interpreted with caution.

Increased connective tissue extracellular matrix in the op/op model of osteopetrosis.

Mice that are homozygous for the recessive osteopetrosis spontaneous mutation (op/op) develop severe osteopetrosis due to a defect in the production of macrophage colony-stimulating factor (M-CSF) and a deficiency in monocyte-derived osteoclasts. Our study describes a novel soft tissue finding in an osteopetrosis (B6C3Fe a/a-Csf1(op)/J) mouse model. Tissues were obtained from B6C3Fe a/a-Csf1(op)/J mice and age-matched wild-type mice, processed for hematoxylin and eosin sections, and comprehensive light microscopic tissue evaluation was performed. Mutant mice had characteristic traits of op/op deficiency including missing incisors and domed skulls. Histologically, the bone marrow cavity was effaced by interweaving thick bony trabeculae consistent with osteopetrosis. An increase in a finely granular, basophilic interstitial extracellular matrix (ECM) was observed in the subcutaneous connective tissue of the op/op mice when compared with controls. Histochemically, the ECM was negative with periodic acid Schiff and stained dark blue with alcian blue at a pH of 2.5, indicating that it is composed primarily of nonsulfated glycosaminoglycans (GAGs). This work suggests an increased ECM that is composed mainly of GAGs located in the subcutaneous tissue in op/op mice. This increase in ECM may be related to altered matrix production or turnover because of changes in M-CSF production.

Chronic lymphocytic thyroiditis in a cynomolgus macaque (Macaca fascicularis).

Chronic lymphocytic thyroiditis characterized by multifocal follicular lymphoid cell infiltrates with germinal centers, thyroid acinar atrophy and pituitary cell hyperplasia/hypertrophy of the adenohypophysis was detected in a vehicle control, 4-year-old female Cynomolgus macaque in a routine toxicology study. Lymphoid cells of germinal centers were positive for the B-cell marker CD20 by immunohistochemistry (IHC), while remaining lymphocytes were positive for the T-cell marker CD3. Hypertrophied/hyperplastic pituitary cells were positive for thyroid stimulating hormone (TSH) by IHC, consistent with an adaptive response due to removal of hormonal negative feedback from the diseased thyroid gland. Features of this case are similar to chronic lymphocytic thyroiditis in humans, an autoimmune disorder also known as Hashimoto's disease. Chronic lymphocytic thyroiditis with compensatory pituitary changes may occur spontaneously in young, clinically normal cynomolgus macaques and its presence in drug treated animals should be interpreted with caution.

Anatomical localization of cartilage degradation markers in a surgically induced rat osteoarthritis model.

Osteoarthritis (OA) is a degenerative disease characterized by an irreversible loss of articular cartilage. Although surgically induced animal OA models are commonly used in drug efficacy assessment, degradation of type II collagen, an important component of articular cartilage is not routinely evaluated. Here, the medial meniscectomy surgical model (MMT) in Lewis rats was evaluated for proteoglycan loss with toluidine blue staining and collagen degradation with immunohistochemical staining for a collagen cleavage C-neoepitope, using a novel anti-type II collagen neoepitope antigen (TIINE) antibody. Femorotibial joints were collected for histology at 0 (no surgery), 3, 7, 14, 21, 28, 35, and 42 days postsurgery. Following MMT surgery, the medial tibial articular cartilage had proteoglycan matrix loss by day 3 that reached subchondral bone by days 28-42. Femoral cartilage damage occurred by day 14. TIINE staining was present at basal levels in growth plates and articular cartilage of all joints while all MMT-treated animals had increased intensity and area of staining in erosions that colocalized with proteoglycan loss. The MMT model produces a progressive pattern of cartilage damage resembling human OA lesions, making it useful, when evaluated with cartilage biomarkers, for assessing changes in cartilage degradation.

T-cell-dependent antibody response: assay development in cynomolgus monkeys.

The current study was designed to develop and test a T-cell dependent antibody response to keyhole limpet hemocyanin (KLH) in cynomolgus monkeys. In an optimization experiment, monkeys (3/sex) were given a single intramuscular injection of KLH at 10 mg/animal to evaluate the kinetics of the antibody response. Serum samples were collected pretest, and on Days 4, 6, 8, 11, 15 and 22 for measurement of anti-KLH IgM and IgG endpoint titers. In a subsequent experiment, female monkeys (3/group) were treated once daily by gavage with the immunosuppressive agent cyclosporine (Neoral) at 0, 10 and 50 mg/kg for 21 days, and the effects of drug treatment on anti-KLH IgM and IgG responses were determined. The effects of cyclosporine on hematology, biochemistry, bone marrow, organ weights, gross and histopathology, and peripheral lymphocyte subsets also were evaluated. Robust anti-KLH IgM and IgG responses were seen in monkeys given a single intramuscular injection of KLH at 10 mg/animal, with peak antibody responses at approximately 10-14 days post-immunization for anti-KLH IgM, and 14-21 days for anti-KLH IgG. Decreases in anti-KLH IgG endpoint titers were seen in 1 monkey given cyclosporine at 10 mg/kg, and 1 monkey dosed at 50 mg/kg. Relative to vehicle control animals, mild lymphoid depletion was evident in lymph nodes and tonsil of monkeys with suppressed anti-KLH IgG titers. Collectively, these findings in individual animals provided evidence of cyclosporine-induced immunosuppression. Cyclosporine at 10 and 50 mg/kg did not alter anti-KLH IgM production, hematology, biochemistry, bone marrow, organ weights, or peripheral lymphocyte subsets. Lastly, the results of this study demonstrated that KLH immunization at 10 mg/animal did not alter the standard toxicity endpoints evaluated in control animals.

In vivo micro computed tomography of subchondral bone in the rat after intra-articular administration of monosodium iodoacetate.

Osteoarthritis (OA) is a degenerative disease that is characterized by joint discomfort, loss of articular cartilage, and changes to the subchondral bone. Studies to elucidate the pathophysiology of OA have been hampered by the lack of a rapid, reproducible animal model that mimics the structural changes associated with the disease. A single intra-articular injection of mono-iodoacetate (MIA), an inhibitor of glycolysis, into the femorotibial joint of rodents promotes loss of articular cartilage similar to that noted in human OA. The purpose of the present study was to determine whether in vivo three-dimensional micro computed tomography (microCT) was of use for detecting progressive changes over time to the subchondral bone (femorotibial joint) of Wistar rats treated with a single intra-articular injection of MIA. MIA-treated right knee joints and left contralateral control knee joints were imaged in vivo at 0, 1, 7, 14, 28, and 56 days postinjection by using microCT. Analysis of 50- and 100- micro m resolution images demonstrated that changes to the subchondral bone, as determined by visual and bone mineral density analysis, are apparent by day 14 post-MIA. By day 28, there were marked changes to lateral aspect of the medial tibial plateaus of the subchondral bone in MIA-treated joints. These changes were progressive through day 56. It was concluded that intra-articular injection of MIA induces progressive changes to subchondral bone that can be assessed using in vivo microCT imaging. In light of these data, in vivo microCT imaging represents a valuable tool for investigating bone remolding and has the potential to be used for routine, high-throughput analysis and screening of investigation therapeutics.

Mono-iodoacetate-induced histologic changes in subchondral bone and articular cartilage of rat femorotibial joints: an animal model of osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease characterized by joint pain and a progressive loss of articular cartilage. Studies to elucidate the pathophysiology of OA have been hampered by the lack of a rapid, reproducible animal model that mimics both the histopathology and symptoms associated with the disease. Injection of mono-iodoacetate (MIA), an inhibitor of glycolysis, into the femorotibial joint of rodents promotes loss of articular cartilage similar to that noted in human OA. Here, we describe the histopathology in the subchondral bone and cartilage of rat (Wistar) knee joints treated with a single intra articular injection of MIA (1 mg) and sacrificed at 1, 3, 5, 7, 14, 28, and 56 days postinjection. Histologically, the early time points (days 1-7) were characterized by areas of chondrocyte degeneration/necrosis sometimes involving the entire thickness of the articular cartilage in the tibial plateaus and femoral condyles. Changes to the subchondral bone, as evidenced by increased numbers of osteoclasts and osteoblasts, were noted at by day 7. By 28 days, there was focal fragmentation and collapse of bony trabeculae with fibrosis and increased osteoclastic activity. By 56 days there were large areas of bone remodeling evidenced by osteoclastic bone resorption and newly formed trabeculae with loss of marrow hematopoietic cells. Subchondral cysts and subchondral sclerosis were present in some rats. In conclusion, intra-articular injection of MIA induces loss of articular cartilage with progression of subchondral bone lesions that mimic those of OA. This model offers a rapid and minimally invasive method to reproduce OA-like lesions in a rodent species.

Inhibition of nuclear protein phosphatase activity in mouse hepatocytes by the cyanobacterial toxin microcystin-LR.

Microcystin-LR (MCLR) is a cyanobacterial hepatotoxin and protein phosphatase inhibitor that contaminates water reservoirs worldwide. MCLR localizes to the cytosol of hepatocytes, however, immunohistochemical studies indicate that it accumulates in the nucleus. MCLR toxicosis is associated with decreased hepatic protein phosphatase activity, but effects in nuclear protein phosphatase activity have not been investigated. Balb/c mice were given lethal (100 microg/kg) or sublethal (12, 23 and 45 microg/kg) i.p. doses of MCLR and hepatic nuclear extracts were analyzed for protein phosphatase 1 and 2A activity. There was profound inhibition of nuclear protein phosphatase activity within 50 min of lethal dosing, however an inhibition was not detected with sublethal doses. MCLR immunohistochemistry revealed widespread lobular staining in the lethal group and centrilobular staining in the sublethal groups. At the cellular level there was nuclear and cytoplasmic staining of equal intensity. As an indicator of nuclear protein phosphatase activity, the phosphorylation of p53, a nuclear phosphoprotein and known substrate for protein phosphatases 1 and 2A, was evaluated. Balb/c mice were treated with sublethal doses of MCLR or saline vehicle after induction of hepatic p53 by the DNA damaging agent diethylnitrosamine (DEN). P53 was immunoprecipitated and probed with phosphoserine specific antibodies by Western blotting. There was greater phosphoserine reactivity of p53 protein in animals treated with MCLR relative to saline treated controls, consistent with increased phosphorylation of serine sites. It is concluded that an interaction of this toxin with nuclear protein phosphatases occurs within 50 min of lethal dosing, which leads to a profound inhibition of enzymatic activity. Even sublethal doses of MCLR that do not result in significant inhibition of activity in bulk nuclei, result in detectable changes in phosphorylation of p53.