Paul W. Snyder

Overexpression of the EphA2 tyrosine kinase in prostate cancer

Molecules that are highly expressed by human prostate cancers may serve as therapeutically relevant targets or tumor markers. Tyrosine kinases are frequently overexpressed in metastatic tumor cells and this prompted us to screen for tyrosine kinases that are overexpressed in prostate cancer cells.

IMAGING OF SPONTANEOUS CANINE MAMMARY TUMORS USING FLUORESCENT CONTRAST AGENTS

Abstract— We present near‐infrared frequency‐domain photon migration imaging for the lifetime sensitive detection and localization of exogenous fluorescent contrast agents within tissue‐simulating phantoms and actual tissues. We employ intensity‐modulated excitation light that is expanded and delivered to the surface of a tissue or tissue‐simulating phantom. The intensity‐modulated fluorescence generated from within the volume propagates to the surface and is collected using a gain‐modulated image‐intensified charge‐coupled device camera. From the spatial values of modulation amplitude and phase of the detected fluorescent light, micromolar volumes of dieth‐ylthiatricarbocyanine iodide (π= 1.17 ns) and indocyanine green (ICG) (π= 0.58 ns) embedded 1.0 cm deep in a tissue phantom are localized and discriminated on the basis of their lifetime differences. To demonstrate the utility of frequency‐domain fluorescent measurements for imaging disease, we image the fluorescence emitted from the surface of in vivo and ex vivo canine mammary gland tissues containing lesions with preferential uptake of ICG. Pathology confirms the ability to detect spontaneous mammary tumors and regional lymph nodes amidst normal mammary tissue and fat as deep as 1.5 cm from the tissue surface.

Proposal of a 2-Tier Histologic Grading System for Canine Cutaneous Mast Cell Tumors to More Accurately Predict Biological Behavior

Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on histologic grade. However, the use of different grading systems by veterinary pathologists and institutional modifications make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading among veterinary pathologists and the prognostic significance of the Patnaik grading system, 95 cutaneous MCTs from 95 dogs were graded in a blinded study by 28 veterinary pathologists from 16 institutions. Concordance among veterinary pathologists was 75% for the diagnosis of grade 3 MCTs and less than 64% for the diagnosis of grade 1 and 2 MCTs. To improve concordance among pathologists and to provide better prognostic significance, a 2-tier histologic grading system was devised. The diagnosis of high-grade MCTs is based on the presence of any one of the following criteria: at least 7 mitotic figures in 10 high-power fields (hpf); at least 3 multinucleated (3 or more nuclei) cells in 10 hpf; at least 3 bizarre nuclei in 10 hpf; karyomegaly (ie, nuclear diameters of at least 10% of neoplastic cells vary by at least two-fold). Fields with the highest mitotic activity or with the highest degree of anisokaryosis were selected to assess the different parameters. According to the novel grading system, high-grade MCTs were significantly associated with shorter time to metastasis or new tumor development, and with shorter survival time. The median survival time was less than 4 months for high-grade MCTs but more than 2 years for low-grade MCTs.

Mesial Temporal Lobe Epilepsy: Pathogenesis, Induced Rodent Models and Lesions

Mesial temporal lobe epilepsy (MTLE), the most common epilepsy in adults, is generally intractable and is suspected to be the result of recurrent excitation or inhibition circuitry. Recurrent excitation and the development of seizures have been associated with aberrant mossy fiber sprouting in the hippocampus. Of the animal models developed to investigate the pathogenesis of MTLE, post-status epilepticus models have received the greatest acceptance because they are characterized by a latency period, the development of spontaneous motor seizures, and a spectrum of lesions like those of MTLE. Among post-status epilepticus models, induction of systemic kainic acid or pilocarpine-induced epilepsy is less labor-intensive than electrical-stimulation models and these models mirror the clinicopathologic features of MTLE more closely than do kindling, tetanus toxin, hyperthermia, post-traumatic, and perinatal hypoxia/ischemia models. Unfortunately, spontaneous motor seizures do not develop in kindling or adult hyperthermia models and are not a consistent finding in tetanus toxin-induced or perinatal hypoxia/ischemia models. This review presents the mechanistic hypotheses for seizure induction, means of model induction, and associated pathology, especially as compared to MTLE patients. Animal models are valuable tools not only to study the pathogenesis of MTLE, but also to evaluate potential antiepileptogenic drugs.

Interpreting Stress Responses during Routine Toxicity Studies: A Review of the Biology, Impact, and Assessment

Stress often occurs during toxicity studies. The perception of sensory stimuli as stressful primarily results in catecholamine release and activation of the hypothalamic–pituitary–adrenal (HPA) axis to increase serum glucocorticoid concentrations. Downstream effects of these neuroendocrine signals may include decreased total body weights or body weight gain; food consumption and activity; altered organ weights (e.g., thymus, spleen, adrenal); lymphocyte depletion in thymus and spleen; altered circulating leukocyte counts (e.g., increased neutrophils with decreased lymphocytes and eosinophils); and altered reproductive functions. Typically, only some of these findings occur in a given study. Stress responses should be interpreted as secondary (indirect) rather than primary (direct) test article–related findings. Determining whether effects are the result of stress requires a weight-of-evidence approach. The evaluation and interpretation of routinely collected data (standard in-life, clinical pathology, and anatomic pathology endpoints) are appropriate and generally sufficient to assess whether or not changes are secondary to stress. The impact of possible stress-induced effects on data interpretation can partially be mitigated by toxicity study designs that use appropriate control groups (e.g., cohorts treated with vehicle and subjected to the same procedures as those dosed with test article), housing that minimizes isolation and offers environmental enrichment, and experimental procedures that minimize stress and sampling and analytical bias. This article is a comprehensive overview of the biological aspects of the stress response, beginning with a Summary (Section 1) and an Introduction (Section 2) that describes the historical and conventional methods used to characterize acute and chronic stress responses. These sections are followed by reviews of the primary systems and parameters that regulate and/or are influenced by stress, with an emphasis on parameters evaluated in toxicity studies: In-life Procedures (Section 3), Nervous System (Section 4), Endocrine System (Section 5), Reproductive System (Section 6), Clinical Pathology (Section 7), and Immune System (Section 8). The paper concludes (Section 9) with a brief discussion on Minimizing Stress-Related Effects (9.1.), and a final section explaining why Parameters routinely measured are appropriate for assessing the role of stress in toxicology studies (9.2.).

Preparation of 66Ga- and 68Ga-labeled Ga(III)-deferoxamine-folate as potential folate-receptor-targeted PET radiopharmaceuticals

A folate-receptor-targeting radiopharmaceutical, Ga(III)-deferoxamine-folate (Ga-DF-Folate), was radiolabeled with two positron-emitting isotopes of gallium, cyclotron-produced (66)Ga (9.5 hour half-life) and generator-produced (68)Ga (68 minute half-life). The [(66)Ga]Ga-DF-Folate was administered to athymic mice with folate-receptor-positive human KB cell tumor xenografts to demonstrate that microPET mouse tumor imaging is feasible with (66)Ga, despite the relatively high positron energy of this radionuclide. Using the athymic mouse KB tumor xenograft model, dual-isotope autoradiography was also performed following i.v. co-administration of [(18)F]-FDG, a marker of regional metabolic activity, and folate-receptor-targeted [(111)In]In-DTPA-Folate. The autoradiographic images of 1 mm tumor sections demonstrate the gross heterogeneity of the KB cell tumor xenograft, as well as subtle disparity in the regional accumulation of the two radiotracers.

Expression of EphA2 and Ephrin A-1 in Carcinoma of the Urinary Bladder

Purpose: The EphA2 receptor tyrosine kinase is believed to play a role in tumor growth and metastasis. The clinical significance of the expression of EphA2 was observed in breast, prostate, colon, skin, cervical, ovarian, and lung cancers. The purpose of this work was to determine the expression of EphA2 and its ligand, Ephrin A-1, and E-cadherin in carcinoma of the urinary bladder, and determine EphA2 as a new target for therapy in bladder cancer. Experimental Design: EphA2 mRNA and protein expression was investigated by reverse transcription-PCR and Western blot, respectively, in bladder cancer cell lines. In addition, the expression of EphA2, Ephrin A-1, and E-cadherin in tissues from patients with different stages of urinary bladder cancer was determined by immunohistochemistry. Furthermore, the ability of Ephrin A-1 to inhibit growth of bladder cancer cells was also investigated using an adenoviral delivery system. Results: Western blot analysis showed high EphA2 expression in TCCSUP, T24, and UMUC-3 cell lines. In tissues, the staining intensity of EphA2 was less in normal urothelium but increased greatly in advancing stages of urothelial carcinoma (P < 0.05). Similarly, the staining intensity of Ephrin A-1 was low in normal tissues and high in cancerous tissues, but it was similar across the various stages of urothelial carcinoma (Ta-T4). E-cadherin immunoreactivity decreased in urothelial cancer. Association of EphA2 and Ephrin A-1 expression was found to be significant between Ta stage and T1-T2 (P < 0.04) and Ta and T3-T4 stages (P < 0.0001). Adenovirus delivery of Ephrin A-1 inhibited proliferation of TCCSUP cells. Conclusion: EphA2 may serve as a novel target for bladder cancer therapy.

Pathologic Features of Naturally Occurring Juvenile Polyarteritis in Beagle Dogs

Eighteen young Beagle dogs (eight males and 10 females), ages 6-40 months, with canine juvenile polyarteritis syndrome (CJPS), a naturally occurring vasculitis and perivasculitis of unknown etiology, were necropsied, and their tissues were examined by histopathologic and histochemical methods. The condition is characterized by recurring episodes of an acute onset of fever (>40 C) and neck pain that persist for 3-7 days. The major histopathologic alterations were a systemic vasculitis and perivasculitis. During the febrile, painful period of CJPS, the vascular lesions ranged from a histiocytic-lymphocytic periarterial infiltration to transmural arterial inflammation with concomitant fibrinoid necrosis and vascular thrombosis. Massive periarterial accumulations of inflammatory cells were common and often extended into adjacent tissues. The small- to medium- sized muscular arteries of the heart, cranial mediastinum, and cervical spinal meninges were consistently involved. Vasculitis occasionally occurred in other organ systems. The vascular lesions in dogs examined during clinically normal periods consisted of intimai and medial fibrosis, ruptured elastic laminae, and mild perivasculitis; these lesions were probably related to previous episodes of vasculitis. Eight dogs that had experienced repeated acute episodes also developed splenic, hepatic, and renal amyloidosis. The clinical signs, laboratory abnormalities, and the vascular lesions suggest that the condition may be immune-system mediated. CJPS may serve as a naturally occurring animal model of human immune-system-mediated vasculitides such as polyarteritis nodosa, infantile polyarteritis, and Kawasaki disease.

Comparison of pain and postoperative stress in dogs undergoing natural orifice transluminal endoscopic surgery, laparoscopic, and open oophorectomy

BACKGROUND Few studies are available to compare the potential benefits of natural orifice transluminal endoscopic surgery (NOTES) approaches to traditional surgery. OBJECTIVE To compare complications, surgical stress, and postoperative pain. DESIGN Prospective study in dogs. SETTING Research laboratory. SUBJECTS Thirty dogs. INTERVENTIONS Oophorectomy procedures were performed via NOTES and laparoscopic and traditional open surgery. MAIN OUTCOME MEASUREMENTS Operative time, pain scores, systemic stress parameters (cortisol, glucose), surgical stress markers (interleukin 6, C-reactive protein), 3-day observation. RESULTS Median operative times were 76, 44, and 35 minutes for the NOTES, laparoscopic, and open procedures, respectively, with the NOTES procedure being significantly longer than the other 2 procedures. All ovaries were completely excised, and all the animals survived without complications. The NOTES animals had greater increases in serum cortisol concentrations at 2 hours but no statistically significant differences in glucose concentrations compared with the other groups. Serum interleukin 6 and C-reactive protein concentrations were significantly increased at specific times compared with baseline in the NOTES group, but not in the open or laparoscopic surgery groups. Based on the cumulative pain score and nociceptive thresholds, the animals in the NOTES group demonstrated less evidence of pain. LIMITATIONS Small sample size, limited follow-up. CONCLUSIONS Although the NOTES oophorectomy procedures took approximately twice as long and there may be more evidence of tissue damage as judged by increases in serum cortisol and interleukin 6 concentrations, the dogs in the NOTES group had lower pain scores, especially when compared with animals undergoing open surgery.

Cyclooxygenase inhibitors in urinary bladder cancer: in vitro and in vivo effects

More than 14,000 people die from invasive transitional cell carcinoma (TCC) of the urinary bladder yearly in the United States. Cyclooxygenase (COX)-inhibiting drugs are emerging as potential antitumor agents in TCC. The optimal in vitro or in vivo systems to investigate COX inhibitor antitumor effects have not been defined. The purpose of this study was to determine COX-1 and COX-2 expression and antitumor effects of COX inhibitors in human TCC cell lines (HT1376, RT4, and UMUC3 cells) and xenografts derived from those cell lines. COX-2 expression (Western blot, immunocytochemistry) was high in HT1376, modest in RT4, and absent in UMUC3 cells in vitro. Similarly, COX-2 expression was noted in RT4 but not UMUC3 xenografts. COX-2 expression in HT1376 xenografts was slightly lower than that observed in vitro. None of four COX inhibitors evaluated (celecoxib, piroxicam, valeryl salicylate, and NS398) reduced TCC growth in standard in vitro proliferation assays at concentrations that could be safely achieved in vivo (≤5 μmol/L). Higher celecoxib concentrations (≥50 μmol/L) inhibited proliferation and induced apoptosis in all three cell lines. Celecoxib or piroxicam treatment in athymic mice significantly delayed progression of HT1376 xenografts, which express COX-2, but not UMUC3 xenografts that lack COX-2 expression. In conclusion, standard in vitro assays were not useful in predicting COX inhibitor antitumor effects observed in vivo. Athymic mice bearing TCC xenografts provide a useful in vivo system for COX inhibitor studies. Results of this study provide justification for further evaluation of COX inhibitors as antitumor agents against TCC. [Mol Cancer Ther 2006;5(2):329–36]