Nonproliferative and Proliferative Lesions of the Rat and Mouse Skeletal Tissues (Bones, Joints, and Teeth).

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is an initiative of the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the skeletal tissues and teeth of laboratory rats and mice, with color photomicrographs illustrating examples of many common lesions. The standardized nomenclature presented in this document is also available on the internet (http://www.goreni.org/). Sources of material were databases from government, academic and industrial laboratories throughout the world.

Toxicity of hydroxyurea in rats and dogs.

The toxicity of hydroxyurea, a treatment for specific neoplasms, sickle-cell disease, polycythemia, and thrombocytosis that kills cells in mitosis, was assessed in repeat-dose, oral gavage studies in rats and dogs and a cardiovascular study in telemetered dogs. Hydroxyurea produced hematopoietic, lymphoid, cardiovascular, and gastrointestinal toxicity with steep dose response curves. In rats dosed for 10 days, 50 mg/kg/day was tolerated; 500 mg/kg/day produced decreased body weight gain; decreased circulating leukocytes, erythrocytes, and platelets; decreased cellularity of thymus, lymph nodes, and bone marrow; and epithelial degeneration and/or dysplasia of the stomach and small intestine; 1,500 mg/kg/day resulted in deaths on day 5. In dogs, a single dose at ≥ 250 mg/kg caused prostration leading to unscheduled euthanasia. Dogs administered 50 mg/kg/day for 1 month had decreased circulating leukocytes, erythrocytes, and platelets; increased bone marrow cellularity with decreased maturing granulocytes; increased creatinine kinase activity; and increased iron pigment in bone marrow and hepatic sinusoidal cells. In telemetered dogs, doses ≥ 15 mg/kg decreased systolic blood pressure (BP); 50 mg/kg increased diastolic BP, heart rate, and change in blood pressure over time (+dP/dt), and decreased QT and PR intervals and maximum left ventricular systolic and end diastolic pressures with measures returning to control levels within 24 hr.

IL-22 induces an acute-phase response.

IL-22 is made by a unique set of innate and adaptive immune cells, including the recently identified noncytolytic NK, lymphoid tissue-inducer, Th17, and Th22 cells. The direct effects of IL-22 are restricted to nonhematopoietic cells, its receptor expressed on the surface of only epithelial cells and some fibroblasts in various organs, including parenchymal tissue of the gut, lung, skin, and liver. Despite this cellular restriction on IL-22 activity, we demonstrate that IL-22 induces effects on systemic biochemical, cellular, and physiological parameters. By utilizing adenoviral-mediated delivery of IL-22 and systemic administration of IL-22 protein, we observed that IL-22 modulates factors involved in coagulation, including fibrinogen levels and platelet numbers, and cellular constituents of blood, such as neutrophil and RBC counts. Furthermore, we observed that IL-22 induces thymic atrophy, body weight loss, and renal proximal tubule metabolic activity. These cellular and physiological parameters are indicative of a systemic inflammatory state. We observed that IL-22 induces biochemical changes in the liver including induction of fibrinogen, CXCL1, and serum amyloid A that likely contribute to the reported cellular and physiological effects of IL-22. Based on these findings, we propose that downstream of its expression and impact in local tissue inflammation, circulating IL-22 can further induce changes in systemic physiology that is indicative of an acute-phase response.

Selective functional inhibition of JAK-3 is sufficient for efficacy in collagen-induced arthritis in mice.

OBJECTIVE: All gamma-chain cytokines signal through JAK-3 and JAK-1 acting in tandem. We undertook this study to determine whether the JAK-3 selective inhibitor WYE-151650 would be sufficient to disrupt cytokine signaling and to ameliorate autoimmune disease pathology without inhibiting other pathways mediated by JAK-1, JAK-2, and Tyk-2. METHODS: JAK-3 kinase selective compounds were characterized by kinase assay and JAK-3-dependent (interleukin-2 [IL-2]) and -independent (IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF]) cell-based assays measuring proliferation or STAT phosphorylation. In vivo, off-target signaling was measured by IL-22- and erythropoietin (EPO)-mediated models, while on-target signaling was measured by IL-2-mediated signaling. Efficacy of JAK-3 inhibitors was determined using delayed-type hypersensitivity (DTH) and collagen-induced arthritis (CIA) models in mice. RESULTS: In vitro, WYE-151650 potently suppressed IL-2-induced STAT-5 phosphorylation and cell proliferation, while exhibiting 10-29-fold less activity against JAK-3-independent IL-6- or GM-CSF-induced STAT phosphorylation. Ex vivo, WYE-151650 suppressed IL-2-induced STAT phosphorylation, but not IL-6-induced STAT phosphorylation, as measured in whole blood. In vivo, WYE-151650 inhibited JAK-3-mediated IL-2-induced interferon-gamma production and decreased the natural killer cell population in mice, while not affecting IL-22-induced serum amyloid A production or EPO-induced reticulocytosis. WYE-151650 was efficacious in mouse DTH and CIA models. CONCLUSION: In vitro, ex vivo, and in vivo assays demonstrate that WYE-151650 is efficacious in mouse CIA despite JAK-3 selectivity. These data question the need to broadly inhibit JAK-1-, JAK-2-, or Tyk-2-dependent cytokine pathways for efficacy.

Development of an early biomarker for the ovarian liability of selective estrogen receptor modulators in rats.

Selective estrogen receptor modulators (SERMs) have the potential to treat estrogen sensitive diseases such as uterine leiomyoma and endometriosis, which are prevalent in reproductive age women. However, SERMs also increase the risk of developing ovarian cysts in this population, a phenomenon that is not seen in postmenopausal women. It is believed that current SERMs partially block estradiol's ability to downregulate gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus thereby interfering with estradiol's negative feedback, leading to increased ovarian stimulation by gonadotropins, and cyst formation. It has been postulated that a SERM with poor brain exposure would have less negative effect on the HPO axis, therefore reducing the risk of developing ovarian cysts. In order to test this hypothesis, we identified an early marker of SERM-dependent ovarian effects: upregulation of Cyp17a1 mRNA. SERMs known to cause ovarian cysts upregulate Cyp17a1 after only 4 days of dosing and suppression of the HPO axis prevented this regulation, indicating that ovarian expression of Cyp17a1 was secondary to SERM's effect on the brain. We then characterized three SERMs with similar binding affinity and antagonist effects on the uterus for their relative brain/plasma exposure and ovarian effects. We found that the degree of brain exposure correlated very well with Cyp17a1 expression.

1-(2-Hydroxy-2-methyl-3-phenoxypropanoyl)indoline-4-carbonitrile derivatives as potent and tissue selective androgen receptor modulators.

We present a novel series of selective androgen receptor modulators (SARMs) which shows excellent biological activity and physical properties. 1-(2-Hydroxy-2-methyl-3-phenoxypropanoyl)-indoline-4-carbonitriles showed potent binding to the androgen receptor (AR) and activated AR-mediated transcription in vitro. Representative compounds demonstrated diminished activity in promoting the intramolecular interaction between the AR carboxyl (C) and amino (N) termini. This N/C-termini interaction is a biomarker assay for the undesired androgenic responses in vivo. In orchidectomized rats, daily administration of a lead compound from this series showed anabolic activity by increasing levator ani muscle weight. Importantly, minimal androgenic effects (increased tissue weights) were observed in the prostate and seminal vesicles, along with minimal repression of circulating luteinizing hormone (LH) levels and no change in the lipid and triglyceride levels. This lead compound completed a two week rat toxicology study, and was well tolerated at doses up to 100 mg/kg/day, the highest dose tested, for 14 consecutive days.

Double-knockout of ADAMTS-4 and ADAMTS-5 in mice results in physiologically normal animals and prevents the progression of osteoarthritis.

OBJECTIVE: To phenotypically characterize ADAMTS-4- and ADAMTS-5-double-knockout mice, and to determine the effect of deletion of ADAMTS-4 and ADAMTS-5 on the progression of osteoarthritis (OA) in mice. METHODS: Mice lacking the catalytic domain of ADAMTS-4 and ADAMTS-5 were crossed to generate ADAMTS-4/5-double-knockout animals. Twelve-week-old and 1-year-old male and female ADAMTS-4/5-double-knockout mice were compared with age- and sex-matched wild-type (WT) mice by evaluating terminal body weights, organ weights, clinical pathology parameters, PIXImus mouse densitometry findings, and macroscopic and microscopic observations. ADAMTS-4/5-double-knockout mice were challenged by surgical induction of joint instability to determine the importance of these genes in the progression of OA. Articular and nonarticular cartilage explants from WT and ADAMTS-4/5-double-knockout mice were treated with interleukin-1 (IL-1) plus retinoic acid ex vivo, to examine proteoglycan degradation. RESULTS: There were no genotype-related phenotype differences between ADAMTS-4/5-double-knockout and WT mice through 1 year of age, with the exception that female ADAMTS-4/5-double-knockout mice had a lower mean terminal body weight at the 12-week time point. Eight weeks after surgical induction of joint instability, OA was significantly less severe in ADAMTS-4/5-double-knockout mice compared with WT mice. Following stimulation of cartilage explants with IL-1 plus retinoic acid, aggrecanase-mediated degradation in ADAMTS-4/5-double-knockout mice was ablated, to a level comparable with that in ADAMTS-5-knockout mice. CONCLUSION: Dual deletion of ADAMTS-4 and ADAMTS-5 generated mice that were phenotypically indistinguishable from WT mice. Deletion of ADAMTS-4/5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of murine OA. These effects in the ADAMTS-4/5-double-knockout mice were comparable with those observed with deletion of ADAMTS-5 alone.

A role for endothelial selectins in allergic and nonallergic inflammatory disease.

BACKGROUND: Several studies indicate that selectin-mediated leukocyte migration may depend on the types of initiating inflammatory stimuli or on the vascular beds involved in the inflammatory response. Thus, targeting selectin interactions to treat inflammation may have variable effects depending on the site and origin of the inflammatory response. OBJECTIVE: To address whether selectin-mediated leukocyte recruitment is stimulus or tissue dependent. METHODS: We examined pulmonary and cutaneous allergic inflammatory responses and silica-induced nonallergic lung inflammation and fibrosis in wild-type and P- and E-selectin-deficient (P/E-/-) double knockout mice. Allergen-sensitized wild-type and P/E-/- double knockout mice were challenged either intradermally or via the airways to induce allergic responses in the skin or lung, respectively. Other animals were subjected to intranasal silica administration to induce a nonallergic lung inflammatory/fibrotic response. RESULTS: The P/E-/- mice exhibited significantly reduced allergic inflammation in the skin and lung. Allergic late-phase ear swelling and allergic lung airway hyperresponsiveness were also significantly attenuated in the P/E-/- mice compared with identically treated wild-type animals. In contrast, pulmonary inflammation and fibrosis induced by intranasal administration of silica particles resulted in a more severe phenotype in the P/E-/- mice. CONCLUSIONS: Selectin interactions drive allergic inflammation in the lung and skin. Silica-induced pulmonary inflammation and fibrosis, however, was more pronounced in the absence of selectin interactions, suggesting that selectin-mediated leukocyte migration may depend on the types of initiating inflammatory stimuli.