Impact of Repeated Tail Clip and Saphenous Vein Phlebotomy on Rats Used in Toxicology Studies.

Sampling blood for toxicokinetic (TK) evaluation in rodents is typically performed using a satellite group of animals to avoid depleting the blood volume and inducing an additional stressor in the main study animals. This practice does not allow for direct comparison of individual animal toxicity to exposure. These studies evaluated serial collection of twelve, 40-µl blood samples from each rat from either a tail clip or a saphenous vein bleed and its impact on toxicologic parameters over 4- and 14-day periods. The results show the feasibility of successfully collecting TK samples from main study animals, using either of the two techniques. Both procedures were amenable to execution by a single technician using dried blood spot sampling. Any changes observed in the primary markers of erythroid mass between the nonbled control rats and repeat sampled rats were minimal and the range of values often overlapped. This technique would improve the quality of data generated from toxicology studies by allowing a direct comparison of systemic exposure to toxicity while at the same time reducing the number of rats by obviating the need for satellite groups.

Anti-IL5 decreases the number of eosinophils but not the severity of dermatitis in Sharpin-deficient mice.

Sharpin-deficient (Sharpin(cpdm)) mutant mice develop a chronic eosinophilic dermatitis. To determine the efficacy of eosinophil-depletion in chronic inflammation, Sharpin(cpdm) mice were treated with anti-IL5 antibodies. Mice treated with anti-IL5 had a 90% reduction of circulating eosinophils and a 50% decrease in cutaneous eosinophils after 10 days compared with sham-treated littermates. Reducing the number of eosinophils resulted in increased severity of alopecia and erythema and a significant increase in epidermal thickness. Skin homogenates from mice treated with anti-IL5 had decreased mRNA expression of arylsulfatase B (Arsb), diamine oxidase (amiloride-binding protein 1, also called histaminase; Abp1) and Il10, which are mediators that eosinophils may release to quench inflammation. Skin homogenates from mice treated with anti-IL5 also had decreased mRNA expression of Il4, Il5, Ccl11, kit ligand (Kitl) and Tgfa; and increased mRNA expression of Tgfb1, Mmp12 and tenascin C (Tnc). In order to further decrease the accumulation of eosinophils, Sharpin(cpdm) mice were crossed with IL5 null mice. Il5(-/-), Sharpin(cpdm)/Sharpin(cpdm) mice had a 98% reduction of circulating eosinophils and a 95% decrease in cutaneous eosinophils compared with IL5-sufficient Sharpin(cpdm) mice. The severity of the lesions was similar between IL5-sufficient and IL5-deficient mice. Double mutant mice had a significant decrease in Abp1, and a significant increase in Tgfb1, Mmp12 and Tnc mRNA compared with controls. These data indicate that eosinophils are not essential for the development of dermatitis in Sharpin(cpdm) mice and suggest that eosinophils have both pro-inflammatory and anti-inflammatory roles in the skin of these mice.

In Vitro L6 Irritation Assay Predicts Clinical Injection Site Reactions for Small Molecules.

Injection site reactions (ISRs) are commonly encountered in the development of parenteral drugs, and severe ISRs can lead to preclinical and clinical dose limiting toxicities. Tools to assess the risk of clinical ISRs during drug development are not well established. We developed an in vitro ISR screen using L6 rat myotubes to assess compounds for irritation risk. Reference compounds that were either known to induce ISRs or were non-irritating in the clinical setting were used to validate this method. We evaluated three compounds, two with known clinical ISRs (mitoxantrone and doxorubicin) and one without clinical ISR (metoprolol), using a preclinical in vivo rat model and the L6 in vitro model at clinically relevant concentrations, and showed that the L6 assay is a better prognostic indicator for clinical ISR risk. We then utilized this assay during early preclinical development to guide optimization of structure activity relationship (SAR), selection of dose concentrations for pre-clinical in vivo experiments, and prioritization of alternative formulations to minimize ISR risk. Our studies indicate that the L6 assay is a better measure of clinical ISR risk than current in vivo preclinical models, and that it can help guide not only compound selection, but also selection of dose concentration and formulation.

Babesia odocoilei infection in elk.

Two male North American elk from a commercial herd were evaluated because of a sudden onset of lethargy, anorexia, and voiding of red urine. These 2 elk were kept in the same pen as 4 other male elk that had died during the preceding 2 months. Laboratory analyses revealed anemia and intraerythrocytic parasites, later confirmed as Babesia odocoilei (a protozoal hemoparasite of cervids). Of the 240 elk remaining in the herd, 59 were screened for B odocoilei by microscopic evaluation of blood smears, protozoal culture of blood, and immunofluorescent antibody testing of serum. Of those 59 elk, 34 (58%) were infected with B odocoilei. Babesia odocoilei infection in elk can be fatal and should be considered in cases of sudden death or acute hemolytic anemia. Familiarity with the disease in elk is essential for practitioners because of the increasing popularity of commercial elk farming.

Expression of chitinase-like proteins in the skin of chronic proliferative dermatitis (cpdm/cpdm) mice.

Mammalian chitinase-like proteins belong to a family of proteins structurally related to chitinases but devoid of enzymatic activity. They have a postulated role in remodeling of extracellular matrix and defense mechanisms against chitin-containing pathogens. The expression of these proteins is increased in parasitic infections and allergic airway disease, but their expression in dermatitis has not been examined. The mRNA expression of two chitinase 3-like (Chi3L) proteins, Chi3L3 (Ym1) and Chi3L4 (Ym2), was determined in the skin of normal mice, chronic proliferative dermatitis (cpdm/cpdm) mutant mice and mice with experimentally induced contact hypersensitivity reaction. The localization of Chi3L3 and Chi3L4 proteins in cells was determined by fluorescence microscopy of double-labeled frozen sections of skin, and confirmed in vitro by stimulation of macrophages and mast cells with cytokines. Quantitative RT-PCR demonstrated a 976-fold increase of Chi3l4 mRNA expression and a 24-fold increase of Chi3l3 mRNA expression in the skin of cpdm/cpdm mice. Their expression was also increased in the ears of mice with 2,4-dinitrofluorobenzene-induced contact hypersensitivity, but the increase was greater for Chi3l3 mRNA (51-fold) than Chi3l4 mRNA (32-fold). Western blot analysis with an antibody against Chi3L3 and Chi3L4 confirmed the increased amount of these proteins in the skin of cpdm/cpdm mice. Two-color immunofluorescence identified macrophages, dendritic cells and mast cells as cellular sources of Chi3L3 and Chi3L4 proteins. Eosinophils and neutrophils did not contain detectable concentrations of these proteins. Treatment of macrophages and mast cells in vitro with interleukin-4 induced expression of Chi3l3 and Chi3l4 mRNA.

Increased expression of chemokines in the skin of chronic proliferative dermatitis mutant mice.

Chemokines direct the migration of leukocytes to sites of inflammation and are potential targets for anti-inflammatory therapy. Chronic proliferative dermatitis (cpdm/cpdm) mutant mice develop a persistent eosinophilic dermatitis associated with increased T(H)2 cytokines in the skin. Expression patterns of chemokines in the skin of cpdm/cpdm mice were evaluated to define the mechanisms driving cutaneous infiltration by leukocytes. RNA isolated from the skin of mutant and littermate control mice revealed a significant increase in Ccl1 (TCA-3), Ccl2 (MCP-1), Ccl11 (eotaxin), Ccl17 (TARC), Cxcl10 (IP-10), and the chemokine receptor Ccr3. The concentration of CCL11 protein was increased two- to threefold in the skin of cpdm/cpdm mice by enzyme-linked immunosorbent assay. In vitro culture of primary dermal fibroblasts from cpdm/cpdm and control mice with tumor necrosis factor, IL-4, and IL-13 stimulation did not reveal differences in their ability to secrete CCL11, suggesting that the increased chemokine expression observed in the skin of cpdm/cpdm mice is most likely caused by the increased T(H)2 cytokines in the dermis of this mouse model. Treatment of cpdm/cpdm mice with CCL11-neutralizing polyclonal antibodies did not affect the number of eosinophils in the skin or the severity of the dermatitis. Neutralizing multiple chemokines or chemokine receptors may be necessary to decrease eosinophil accumulation. The cpdm/cpdm mutant mouse is a potentially useful model to determine the role of various chemokines in eosinophil accumulation in chronic inflammation.

Dexamethasone-induced exophthalmos in a group of Holstein calves.

Six Holstein calves were evaluated for progressive exophthalmos. The affected calves were receiving daily injections of dexamethasone (30 microg/kg subcutaneously twice daily) as part of a metabolic study. The control calves did not exhibit exophthalmos. The ocular examinations were normal except for the exophthalmos, which ranged from mild to marked in severity. Upon postmortem examination, marked deposition of retrobulbar adipose tissue was noted in the affected calves. Dexamethasone administration appears to increase deposition of retrobulbar adipose tissue resulting in a progressive exophthalmos.