Nicole J. Madrill

First analysis of the secretome of the canine heartworm, Dirofilaria immitis

BackgroundThe characterization of proteins released from filariae is an important step in addressing many of the needs in the diagnosis and treatment of these clinically important parasites, as well as contributing to a clearer understanding of their biology. This report describes findings on the proteins released during in vitro cultivation of adult Dirofilaria immitis , the causative agent of canine and feline heartworm disease. Differences in protein secretion among nematodes in vivo may relate to the ecological niche of each parasite and the pathological changes that they induce.MethodsThe proteins in the secretions of cultured adult worms were run on Tris-Glycine gels, bands separated and peptides from each band analysed by ultra mass spectrometry and compared with a FastA dataset of predicted tryptic peptides derived from a genome sequence of D. immitis.ResultsThis study identified 110 proteins. Of these proteins, 52 were unique to D. immitis . A total of 23 (44%) were recognized as proteins likely to be secreted. Although these proteins were unique, the motifs were conserved compared with proteins secreted by other nematodes.ConclusionThe present data indicate that D. immitis secretes proteins that are unique to this species, when compared with Brugia malayi. The two major functional groups of molecules represented were those representing cellular and of metabolic processes. Unique proteins might be important for maintaining an infection in the host environment, intimately involved in the pathogenesis of disease and may also provide new tools for the diagnosis of heartworm infection.

Effects of the benzimidazole anthelmintic drug flubendazole on rat embryos in vitro

Filarial diseases affect millions of people in poverty-stricken areas. In 2011, an investigation of the potential of flubendazole as a safe, highly efficacious, and field-usable macrofilaricidal drug was begun by Drug for Neglected Diseases initiative. As part of the preclinical development program, whole embryo culture was used to investigate the potential embryotoxicity of flubendazole and its metabolites, reduced and hydrolyzed flubendazole. Albendazole was included as a comparator. Flubendazole and albendazole showed similar potency in affecting rat embryonic development in vitro, inducing retardation of growth and dysmorphogenic effects at concentrations ≥0.5 μg/mL. The head, optic and otic systems, branchial arches and posterior body portion were affected. Diffuse areas of cell death were seen in various embryonic districts. The No Observed Effect Level (NOEL) was 0.25 μg/mL for both drugs. Reduced and hydrolyzed flubendazole were less embryotoxic than the parent compound, with NOELs 4-fold and >40-fold higher than that of flubendazole, respectively.

Comparison of cytogenetics and polymerase chain reaction based detection of the amelogenin gene polymorphism for the diagnosis of freemartinism in cattle

A polymerase chain reaction (PCR) assay which detects a sex-based polymorphism in the bovine amelogenin locus was modified and compared to conventional cytogenetic analysis for diagnosis of freemartinism (XX/XY chimerism) in cattle. The PCR assay is more sensitive than cytogenetic analysis for detection of XY cells, with the limit of detection of the assay falling between 0.2% and 1% XY cells. Seventy-three heifer blood samples submitted for evaluation of freemartinism to the University of Minnesota Diagnostic Laboratory were tested using both cytogenetic and PCR techniques. Poor-quality samples precluded successful lymphocyte culture and recovery of mitotic nuclei for cytogenetic evaluation in 17 cases (23%). Two of these samples (2.7%) also failed to amplify with PCR. There was 100% agreement in the results from the 56 samples that were suitable for testing using both techniques. This PCR-based assay provides an alternative to the more laborious cytogenetic evaluation for diagnosis of freemartinism.

Evaluation of hypoxia in a feline model of head and neck cancer using 64Cu-ATSM positron emission tomography/computed tomography

BackgroundHuman and feline head and neck squamous cell carcinoma (HNSCC) share histology, certain molecular features, as well as locally aggressive and highly recurrent clinical behavior. In human HNSCC, the presence of significant hypoxia within these tumors is considered an important factor in the development of a more aggressive phenotype and poor response to therapy. We hypothesized that feline head and neck tumors, particularly HNSCC, would exhibit hypoxia and that 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) positron emission tomography/computed tomography (PET/CT) would permit detection of intratumoral hypoxia.Methods12 cats with measureable head and neck tumors were given 64Cu-ATSM and iodinated contrast for PET/CT scan. The presence or absence of hypoxia was also assessed using an intratumoral fluorescent life-time probe to quantitate pO2 and pimonidazole immunohistochemical staining in biopsy specimens. In two cats, intratumoral O2 and 64Cu-ATSM uptake was measured before and after treatment with anti-angiogenic agents to determine the effect of these agents on hypoxia.ResultsEleven of twelve feline tumors demonstrated significant 64Cu-ATSM uptake, regardless of malignant or benign etiology. The presence (and absence) of hypoxia was confirmed using the fluorescent O2 detection probe in nine tumors, and using pimonidazole staining in three tumors. Squamous cell carcinomas (HNSCC) demonstrated the highest degree of hypoxia, with Tmax/M ratios ranging from 4.3 to 21.8. Additional non-neoplastic tissues exhibited 64Cu-ATSM uptake suggestive of hypoxia including reactive draining lymph nodes, non-malignant thyroid pathology, a tooth root abscess, and otitis media. In two cats with HNSCC that received anti-vascular agents, the pattern of 64Cu-ATSM uptake was altered after treatment, demonstrating the potential of the feline model to study the modulation of tumor oxygenation.ConclusionFeline HNSCC serves as a clinically relevant model for the investigation of intratumoral hypoxia including its measurement, modulation and targeting.

Microsatellite instability in canine mammary gland tumors.

BACKGROUND Genomic instability is a hallmark of cancer and may be required for the accumulation of cancer-causing mutations within cells. One form of genomic instability occurs in tandem nucleotide repeats and is known as microsatellite instability (MSI). HYPOTHESIS We hypothesized that MSI can be observed in canine mammary gland tumors (MGT) and represents a potential carcinogenic mechanism in dogs. ANIMALS Thirty-five dogs with MGTs and 9 dogs with other tumors were recruited from the University of Minnesota Veterinary Medical Center and referring veterinary clinics. METHODS A panel of 21 canine microsatellite (MS) markers was amplified by polymerase chain reaction (PCR) from deoxyribonucleic acid obtained from blood and from fresh or formalin-fixed, paraffin-embedded tumor tissues. PCR products were evaluated by using capillary electrophoresis, and the chromatograms were analyzed by using genotyping software. MS genotypes obtained from fresh and formalin-fixed tumor tissues were compared, as were MS genotypes from normal tissue and tumor tissue. RESULTS Genotypes obtained from formalin-fixed and fresh tissues were identical for all MS in 9 tumors evaluated, suggesting excellent concordance between the 2 sample types. For the 35 canine mammary tumors evaluated, 13 (37%) had stable genotypes; 22 (63%) exhibited aberrations in 1 or 2 MS; and 4 tumors (11%) demonstrated high-level instability, with aberrations in 29 to 61% of MS. CONCLUSIONS AND CLINICAL IMPORTANCE Although some low-level MSI often is observed, high-level MSI is an infrequent finding in canine mammary tumors. Further evaluations are required to better characterize this phenomenon and to determine its relevance to canine carcinogenesis.

Evaluation of Microsatellite Instability in Urine for the Diagnosis of Transitional Cell Carcinoma of the Lower Urinary Tract in Dogs

BACKGROUND The accumulation of frame-shift mutations in microsatellites (MS), termed microsatellite instability (MSI), is associated with certain tumors. MSI and its detection in urine samples has been used to aid in the detection of human bladder cancer. HYPOTHESIS Evaluation of MSI in urine is a useful assay test for diagnosis of transitional cell carcinoma (TCC) in dogs and is more specific than the commercially available, veterinary bladder tumor analyte (V-BTA) test. ANIMALS Seventy-three dogs: healthy controls (n=21), proteinuric (n=12), lower urinary tract disease excluding TCC (n=17), and TCC (n=23). METHODS Prospective observational study. Urine samples collected from each animal were evaluated for MSI and using the V-BTA. For MSI detection, 22 MS sequences were polymerase chain reaction amplified from urine and blood, subjected to capillary electrophoresis, and the MS genotypes were compared. Aberration in ≥15% of MS was considered indicative of MSI. RESULTS MSI was detected in 11 of 23 (48%) urine samples from dogs with TCC. MSI was also detected in 12 of 50 (24%) of the control animals, including 29, 16, and 24% of healthy, proteinuric, and lower urinary disease dogs, respectively. In this population, sensitivity and specificity of MSI analysis was 48 and 76%, respectively, compared with 83 and 64%, respectively, for the V-BTA test. CONCLUSIONS MS analysis as performed in this study is not useful in the diagnosis of TCC.

A histochemical study of the Nras/let-60 activity in filarial nematodes.

BackgroundControl and elimination of filarial pathogens is a central focus of major global health efforts directed at parasitic diseases of developing countries. Accomplishment of these goals would be markedly enhanced by the enhanced destruction of the adult stage of filariae. The identification of new, more quantitative biomarkers that correlate with mortality or chemotherapeutic damage to adult filariae, would greatly facilitate, for example, the development of new macrofilaricides.MethodsAn immunocytochemical approach using an antibody against human Nras was used to identify and detect changes in the nematode homolog let-60 that is associated with cell growth and maintenance. Single Onchocerca volvulus nodules were removed from each of 13 patients treated with ivermectin (as part of a community-wide mass drug administration programme), and from each of 13 untreated individuals; these 26 nodules were stained with the anti-Nras antibody. The localization and degree of positivity of Nras/let-60 staining were assessed subjectively and compared between the two groups; the positivity of staining was also quantified, using image analysis, in a subgroup of these nodules. In addition, the specific morphological association between Nras/let-60 and the Wolbachia endosymbiont present in these parasites was also observed in 4 additional filarial species using an anti-Wolbachia surface protein (WSP) antibody under light and confocal microscopy.ResultsNras/let-60 is present in many structures within the adult female worms. A statistically significant decrease in the general staining intensity of Nras/let-60 was observed in adult female O. volvulus treated with ivermectin when compared with parasites from untreated patients. Nras/let-60 staining was frequently observed to be co-localized with WSP in O.volvulus, Brugia malayi, Litomosoides sigmodontis and Dirofilaria immitis. Nras/let60 is also present in Onchocerca ochengi.ConclusionNras/let-60, as detected by immunocytochemical staining, is decreased in ivermectin-treated adult female O. volvulus relative to untreated control specimens, suggesting a suppressive effect of ivermectin on the overall biochemical activity of these parasites. Co-localization of Nras/let-60 and WSP suggests the possibility that the endosymbiont utilizes this nematode protein as part of a mutualistic relationship. Nras/let60 appears to be a useful biomarker for assessing the health of filariae.

Abstract 5258: Characterization of canine lower urinary tract carcinoma cell lines with variable DNA mismatch repair proficiency derived from a single tumor

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Deficiencies in the DNA mismatch repair system (MMR) have been well described in a variety of neoplasms in humans; however, naturally occurring animal models of MMR deficiency associated carcinogenesis are lacking. Recently, our lab has shown that deficiencies in the MMR are common in canine lower urinary tract carcinomas through the identification of microsatellite instability and decreased expression of MSH2. In order to further investigate MMR in the dog, cell lines were derived from canine lower urinary tract carcinomas. Briefly, samples of a prostatic urothelial carcinoma from an 8-year-old neutered male Beagle were minced into 0.5-1mm3 pieces and incubated in culture plates with 1:1 DMEM/F12 supplemented with 10% fetal bovine serum, penicillin, streptomycin, and l-glutamine. Resulting colonies of epithelioid cells were harvested with 0.05% trypsin and subcultured. Two distinct cell populations emerged following repeated subculture, which were subsequently isolated from one another through selective trypinization. Thus, two distinct cell lines, TYLER1 and TYLER2, were established. While these cell lines were derived from the same tumor, they differ greatly in terms of morphology and expression of DNA mismatch repair genes. Cells of TYLER1 are polygonal with regular dimensions, and grow in fairly discrete aggregates. Cells of TYLER2 are spindle-shaped to stellate, and grow in haphazardly arranged interlacing patterns. Both cell lines express cytokeratins confirming their epithelial origin; however, the specific cytokeratins expressed by each line differs and both cell lines strongly express vimentin. In contrast to the primary tumor, both cell lines express prostatic acid phosphatase (PAP), a marker of prostatic differentiation, and neither cell line expresses uroplakin III, an urothelial marker. This is likely a reflection of plasticity of cells of the lower urinary tract in terms of their ability to differentiate. Comparing microsatellite sequences obtained from a blood sample from the dog of origin to those in the cell lines, there is variability in the length of select microsatellites suggesting functional MMR deficiency. The gene and protein expression of MSH2 and MSH6 are significantly lower in TYLER2 than TYLER1 as determined by Western blot and RT PCR, respectively. In preliminary studies using XTT assays, the MMR deficient TYLER2 cell line is more sensitive to gemcitabine and carboplatin than TYLER1, although both are more sensitive to these agents than other canine urothelial carcinoma cell lines that strongly express MSH2 and MSH6. Overall, given the differences in apparent MMR expression and differences observed in morphology and response to treatment, these cell lines will undoubtedly prove valuable in future studies of MMR in dogs and the effects MMR deficiency on cancer treatment response and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5258. doi:1538-7445.AM2012-5258

Abstract 3293: Activity and safety of an antiangiogenic peptide in spontaneous head and neck squamous cell carcinoma in cats

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Anginex is a rationally designed, synthetic peptide that inhibits proliferation of human and mouse endothelial cells and demonstrates anti-tumor activity in murine tumor models. The anti-tumor and anti-angiogenic activity of Anginex is attributable, at least in part, to its interaction with galectin-1, a carbohydrate binding protein. As an intermediate step in the clinical translation of Anginex and related anti-vascular agents, we are investigating the effects of Anginex in spontaneous feline head and neck squamous cell carcinoma (HNSCC). Feline HNSCC shares morphology, clinical behavior, and biologic features such as intratumoral hypoxia and expression of EGFR, COX-2, and mutant p53 with its human counterpart. Our objectives were to investigate the activity of Anginex in feline cells and provide evidence of its safety in a phase I trial in cats with HNSCC. To establish Anginex activity in vitro, we evaluated the effect of 24 hour exposure to Anginex doses ranging from 2.5 – 25 uM on proliferation of both feline aortic endothelial cells (FAECs) and feline HNSCC cells (SCCF1) using flow cytometry and labeling with propidium iodide and bromodeoxyuridine (BrdU). With increasing Anginex dose, we observed accumulation of FAECs in G1 and decreased uptake of BrdU. We observed no effect in SCCF1 cells. To investigate safety, cats were treated with Anginex in cohorts of at least three animals at doses of 1, 3, and 9 mg/kg daily for 5 days. Clinical, laboratory and histologic features were evaluated before and after treatment. There were no serious adverse events during treatment. Continuous variables including body weight and laboratory variables were compared before and after treatment using a Wilcoxon signed rank test. No statistically significant changes occurred in these parameters. These data indicate that Aniginex is active against feline endothelial cells specifically and appears to be safe in cats. We are currently investigating the effects of Anginex on tumor physiology and in combination with radiation therapy for feline HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3293. doi:10.1158/1538-7445.AM2011-3293

Abstract 439: Evaluation of hypoxia in a feline model of head and neck squamous cell carcinoma using 64Cu-ATSM positron emission tomography

Tumor hypoxia substantially impacts the prognosis of cancer patients by impairing the response to therapy and by promoting the acquisition of a malignant cellular phenotype. A variety human tumors exhibit hypoxia including head and neck squamous cell carcinoma (HNSCC) where it is considered a barrier to successful management of these patients. In the feline, HNSCC is a common and aggressive malignancy that shares morphologic, clinical and molecular features with human HNSCC. Therefore, feline HNSCC may serve as an informative model. To date, there are no data concerning the occurrence of hypoxia in feline HNSCC. We hypothesized that like its human counter part, feline HNSCC would demonstrate regions of intratumoral hypoxia and that hypoxia would be a feature of malignant rather than benign oral tumors. To test this hypothesis, cats with spontaneously occurring oral tumors (N=7) were administered 2 mCi 64 Cu- diacetyl-bis(N4-methylthiosemicarbazone) (ATSM) IV 20 minutes prior to PET-CT scan (Discovery ST, GE Healthcare). Histologically, 4 cats had invasive squamous cell carcinoma, 1 had osteosarcoma and 2 had inflammatory lesions (1 polyp and 1 granuloma). All four squamous cell carcinomas and both inflammatory lesions demonstrated strong diffuse uptake of 64 Cu within the tumor. Two cats with metastases to regional lymph nodes also demonstrated significant 64 Cu uptake. Tumor hypoxia was confirmed by fluorescent lifetime probe measurements (Oxylab pO2), Oxford Optronix) in 4 cats and pimonidazole (Hypoxyporbe TM -1, Hypoxyprobe Inc) uptake in biopsy samples in 4 cats. These data indicate that HNSCC in the cat exhibits significant, diffuse hypoxia. Hypoxia was also significant in inflammatory lesions and thus was not a marker of malignancy, but could indicate that inflammatory disease may provide a selective microenvironement to promote tumorigenesis in the cat. The feline model may provide an important tool for studying clinical methods to modulate hypoxia and to study the role of hypoxia on oral carcinogenesis. We are currently evaluating the effect of vascular modulating agents on feline HNSCC and intratumoral hypoxia as a means to optimize this therapy for combination with irradiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 439.