Teaching as a first, second, or third language.

The 2005 National Carl J. Norden/Pfizer Distinguished Teaching Award may bear my name, but clearly it is shared recognition for the institutions, numerous individuals, and varied professional experiences that inspired and influenced a veterinarian who became a teacher whom students seemed to value. My education, practice experience, exposure to some superb educators, and feedback from students have provided a diverse background of knowledge and experiences that have allowed me to adapt to teaching opportunities in courses other than clinical pathology and to develop my own style of instruction, teaching philosophy, and examination strategies and formats. In these times of limited funds and faculty recruitment, teaching programs have been developed to meet our learning objectives and make efficient use of monetary and faculty resources. The description that follows summarizes my thoughts and experiences.

Induction of CYP1A1 and CYP1B1 in liver and lung by benzo(a)pyrene and 7,12-d imethylbenz(a)anthracene do not affect distribution of polycyclic hydrocarbons to target tissue: role of AhR and CYP1B1 in bone marrow cytotoxicity.

Polycyclic aromatic hydrocarbons (PAHs) (50 mg/kg, i.p.) selectively deplete mouse bone marrow (BM) hematopoietic cells through a process that is dependent on CYP1B1. 7,12-dimethylbenz(a)anthracene (DMBA), which forms greater amounts of dihydrodiol-epoxide-DNA adducts in BM, is much more effective in depleting BM cells than benzo(a)pyrene (BP). BM toxicity by BP is restored in congenic mice expressing a weakly responsive aryl hydrocarbon receptor (AhR(d) replaces AhR(b)). BP strongly induces CYP1A1 around the hepatic vein whereas DMBA produces a weaker diffuse response, paralleling differences in CYP1A1 protein. These responses are absent in AhR(d) mice. BP and DMBA broadly and equally induce CYP1A1 in the lung, while CYP1B1 is induced in bronchial blood vessels. In sternum, CYP1B1 is induced in BM and white fat, whereas CYP1A1 is induced only in brown fat. BP and DMBA levels were similar within blood, lung, and BM and did not rise in AhR(d) mice. In liver, selective decrease of BP was consistent with induced metabolism via CYP1A1, which nevertheless does not determine the blood levels and distribution to BM. Effective delivery of BP to BM is indicated by formation of BP-quinone DNA adducts and the effective induction of CYP1B1. The low formation of BP-dihydrodiol-epoxide-DNA adducts suggests effective AhR induction of BM detoxifying reactions that prevents their formation from dihydrodiols. These findings contrast with the substantial hepatic CYP1A1 contribution for PAHs previously seen for intragastric administration where first pass elimination limits the amount of PAHs reaching the BM.

7-12 Dimethylbenz[a]anthracene-induced bone marrow hypocellularity is dependent on signaling through both the TNFR and PKR.

In addition to being carcinogenic, polycyclic aromatic hydrocarbons (PAHs) are known to cause deleterious effects on the immune system, including a marked reduction in bone marrow granulocytes and B lymphocytes. The molecular mechanisms underlying bone marrow hypocellularity are incompletely understood. Hematopoiesis is governed by the production of cytokines and the resultant signaling pathways that they initiate. Our hypothesis was that PAHs may disrupt cytokine production in the bone marrow resulting in the perturbation in bone marrow cellularity observed after PAH administration. TNF-alpha and IFN-gamma are two cytokines that are involved in the regulation of hematopoiesis. Based on observations made in previous research, we sought to determine if the effects of 7-12 dimethylbenz[a]anthracene (DMBA) on the murine bone marrow were mediated through the actions of these molecules. Transgenic mice that were null for either IFN-gamma or TNF-alpha receptors were injected with DMBA and the resulting bone marrow cellularity compared with wild-type mice. We observed that tumor necrosis factor alpha receptor (TNFR) null mice were protected against DMBA-induced bone marrow hypocellularity, while IFN-gamma null mice were not. In addition, we found that dsRNA-dependent protein kinase (PKR) null mice were also protected from DMBA-induced hypocellularity. PKR is an intracellular signaling molecule that has been demonstrated to be activated by TNFR-mediated signaling. Furthermore, we observed upregulation of PKR in the bone marrow after DMBA administration that was dependent on signaling through TNFR. These results point to a role for TNFR-dependent signaling, operating at least in part via PKR activation, as a mechanism for DMBA-induced bone marrow toxicity.

Bone marrow cytotoxicity of benzo[a]pyrene is dependent on CYP1B1 but is diminished by Ah receptor-mediated induction of CYP1A1 in liver.

We have previously used CYP1B1-null mice to demonstrate that dimethylbenz(a)anthracene (DMBA) requires CYP1B1 for bone marrow (BM) toxicity. Benzo(a)pyrene (BP), a much more potent Ah receptor ligand, shows very different responses that nevertheless depend on CYP1B1. Wild-type (AhR(b)) mice treated with DMBA for 48 h exhibit a large loss in BM cellularity and disruption of marrow structure that is not seen for BP treatment. In congenic mice with a low affinity AhR (AhR(d)), DMBA and BP are equally toxic to the BM whereas AhR(d) x CYP1B1-null mice are fully protected. In situ hybridization demonstrates that CYP1B1 mRNA is constitutively expressed in marrow cells and is induced by PAHs according to their AhR affinity (BP>DMBA), including lower levels in AhR(d) mice. Importantly, expression of CYP1A1 mRNA was undetectable in BM. In wild-type mice, BP treatment leads to a fivefold greater induction of hepatic CYP1A1 than that of DMBA treatment. Neither induction occurs in AhR(d) mice. Thus, hepatic metabolism may prevent BP from reaching the BM, where it can be bioactivated by CYP1B1. Flow cytometric analyses of BM cells showed that there were decreases in granulocytes and lymphocytes following DMBA treatment, but not after BP treatment. These data suggest that there is an inverse relationship between liver metabolism and BM toxicity resulting from limitations on the delivery of PAH to CYP1B1 present in BM, where only very low constitutive levels are needed.

7,12-Dimethylbenz[a]anthracene-induced bone marrow toxicity is p53-dependent.

Polycyclic aromatic hydrocarbons (PAHs) are known immunotoxins and carcinogens. Our laboratory and others have demonstrated that metabolism of these compounds by CYP1B1 is required for carcinogenicity and immunotoxicity to occur. Previously, our laboratory reported significantly decreased bone marrow cellularity in mice following 7,12-dimethlybenz[a]anthracene (DMBA) administration. In addition, we have observed that DMBA causes apoptosis via activation of both caspase-8 and -9 in pre-B cells co-cultured with bone marrow stromal cells in vitro. In this study, we investigated the importance of the p53 protein in the bone marrow response to DMBA. Through the use of p53 gene knockout mice, we demonstrated that the effect of DMBA on bone marrow cellularity is p53-dependent. In addition, apoptosis of primary cultures of progenitor B cells cultured with bone marrow stromal cells and DMBA is also p53-dependent. The results of this study provide evidence for the importance of p53 in the signaling pathways by which PAHs cause immunotoxicity.

Laboratory evaluation and interpretation of synovial fluid.

Canine and feline joint disease can be a primary disorder limited to joints or a manifestation of multisystemic disease. Collection and analysis of joint fluid provides valuable information for the diagnosis, prognosis, and treatment of diseases that affect the joint space. The cytologic recognition of the cellular components and infectious agents in synovial fluid categorizes the cell response and differentiates inflammatory and noninflammatory joint disorders. This information is supported by the cell counts, protein content, mucin clot test, bacterial culture, and serologic tests for infectious or immune-mediated disease. These results are integrated with the clinical history, physical examination, radiographic findings, and ancillary test results to arrive at a diagnosis and treatment plan.

Serum fructosamine concentration in nondiabetic and diabetic cats.

Differentiating transient hyperglycemia from diabetic hyperglycemia can be difficult in cats since single blood glucose measurements reflect only momentary glucose concentrations, and values may be elevated because of stress-induced hyperglycemia. Glycated protein measurements serve as monitors of longer-term glycemic control in human diabetics. Using an automated nitroblue tetrazolium assay, fructosamine concentration was measured in serum from 24 healthy control cats and 3 groups of hospitalized cats: 32 euglycemic, 19 transiently hyperglycemic, and 12 diabetic cats. Fructosamine concentrations ranged from 2.1 - 3.8 mmol/L in clinically healthy cats; 1.1 - 3.5 mmol/L in euglycemic cats; 2.0 - 4.1 mmol/L in transiently hyperglycemic cats; and 3.4 to >6.0 mmol/L in diabetic cats. Values for with-in-run precision at 2 fructosamine concentrations (2.64 mmol/L and 6.13 mmol/L) were 1.5% and 1.3%, respectively. Between-run coefficient of variation was 3.8% at a fructosamine concentration of 1.85 mmol/L. The mean fructosamine concentration for the diabetic group differed significantly (P=0.0001) from the mean concentrations of the other 3 groups. Poorly regulated or newly diagnosed diabetic cats tended to have the highest fructosamine values, whereas well-regulated or over-regulated diabetic cats had values approaching the reference range. As a single test for differentiating nondiabetic cats from diabetic cats, fructosamine was very sensitive (92%) and specific (96%), with a positive predictive value of 85% and a negative predictive value of 98%. Serum fructosamine concentration shows promise as an inexpensive, adjunct diagnostic tool for differentiating transiently hyperglycemic cats from poorly controlled diabetic cats.

Chylothorax associated with cryptococcal mediastinal granuloma in a cat.

A 10-year-old neutered female cat had chylothorax, precaval syndrome, and a mediastinal granuloma resulting from infection with Cryptococcus neoformans. Diagnosis of a chylous effusion was made by cytologic examination of pleural fluid and by finding higher triglyceride levels in the effusion than in serum (825 vs. 64 mg/dl, respectively). Postmortem examination revealed cryptococcal organisms in the mediastinal granuloma, lungs, cerebral meninges, and connective tissues adjacent to the thyroid gland. Chylous effusion in a cat associated with cryptococcosis has not been reported previously. Cryptococcosis should be included in the differential diagnosis in chylous effusions in cats.

Diagnosis of histoplasmosis in a dog by cytologic examination of CSF.

A mixed inflammatory cell pleocytosis was identified in a cytocentrifuged sample of cerebrospinal fluid (CSF) and diagnosis of histoplasmosis was made on the basis of finding Histoplasma capsulatum organisms within macrophages. This dog had disseminated histoplasmosis with multiple organ involvement including the central nervous system (CNS) and eye.