Kim M. Newkirk

A Retrospective Study of Eyelid Tumors from 43 Cats

Submissions to the University of Tennessee pathology service from June 1999 to June 2008 were searched for feline cases of tumors involving the eyelids or nictitans. Forty-three tumors were identified. The average age at diagnosis was 10.4 years. Significantly more males than females had eyelid tumors. There were 12 squamous cell carcinomas (SCCs), 11 mast cell tumors (MCTs), 6 hemangiosarcomas (HSAs), 4 adenocarcinomas (ACAs), 3 peripheral nerve sheath tumors (PNSTs), 3 lymphomas, 3 apocrine hidrocystomas (AHCs), and 2 hemangiomas. Cats with MCTs were significantly younger than cats with all other tumor types combined. In contrast, cats with SCCs were significantly older than cats with other tumor types. The HSAs and SCCs were significantly more likely than other tumors to occur in nonpigmented areas. The MCTs, HSAs, AHCs, and hemangiomas did not recur after surgical excision. In contrast, the lymphomas, ACAs, SCCs, and PNSTs frequently recurred and/or resulted in death or euthanasia of the cat. The SCCs were significantly more likely to recur than the MCTs. The average survival time for cats with SCCs was 7.4 months. Although eyelid MCTs have been reported in cats, the prevalence in this study is much higher than previously described.

Detection of papillomavirus in equine periocular and penile squamous cell carcinoma

Squamous cell carcinoma (SCC) is the most common tumor arising in the periocular and penile areas of horses. Both ultraviolet radiation and papillomaviruses have been implicated in the pathogenesis of SCC in various species, including the horse. This retrospective study used polymerase chain reaction (PCR) to detect papillomavirus DNA in archival biopsy samples from equine periocular and penile SCC from 3 different geographic areas (northeast, southeast, and central United States). Forty-two periocular SCCs were tested; none contained papillomavirus DNA. Twenty-two penile SCCs were tested, and papillomavirus DNA was identified in 10 (43%) cases. Sequencing of the PCR products revealed homology with Equus caballus papillomavirus 2 (EcPV-2). No geographic distribution in the detection of papillomavirus was identified. Penile SCCs were significantly more likely to be papillomavirus positive than the periocular SCCs (P < 0.001). The role of papillomavirus in the development of penile SCC requires further investigation. The differing pathogeneses of periocular and penile SCC suggest that the tumors may respond differently to treatment.

Molecular characterization of the L1 gene of papillomaviruses in epithelial lesions of cats and comparative analysis with corresponding gene sequences of human and feline papillomaviruses

OBJECTIVE To characterize the L1 gene of papillomaviruses detected in epithelial lesions of cats and to determine the relationship between those L1 gene nucleotide sequences and known L1 gene sequences of human and feline papillomaviruses. SAMPLE POPULATION 10 tissue samples of epithelial lesions from 8 cats. PROCEDURES DNA was extracted from tissue samples. Primers were designed to amplify the L1 gene of papillomaviruses. Amplicons of DNA were sequenced; nucleotide sequences were compared with known L1 gene nucleotide sequences of papillomaviruses and used for phylogenetic analysis. RESULTS Tissue samples were obtained from lesions (diagnosed as dysplasia [n=1], squamous cell carcinoma in situ [3], or squamous cell carcinoma [6]) of the skin (9) and oral mucosa [1]. Two amplicons had 99% homology with the L1 gene nucleotide sequence of human papillomavirus type 38b subtype FA125. Another amplicon had 84% homology with the L1 gene nucleotide sequence of human papillomavirus type 80 and was considered to be a new type of papillomavirus. Phylogenetic tree analysis revealed that these 3 papillomaviruses were grouped into 2 clades that were not similar to the clades of Felis domesticus papillomavirus type 1 or F domesticus papillomavirus type 2 (FdPV2). The remaining 7 amplicons had 98% to 100% homology with the L1 gene nucleotide sequence of FdPV2. Phylogenetic tree analysis revealed that those 7 papillomaviruses were grouped nto a single clade with FdPV2. CONCLUSIONS AND CLINICAL RELEVANCE Results support the likelihood of transmission of papillomaviruses between humans and cats.

Ocular lesions in canine mucopolysaccharidosis I and response to enzyme replacement therapy.

PURPOSE Mucopolysaccharidosis I (MPS I) is an inherited metabolic disorder resulting from deficiency of α-L-iduronidase and lysosomal accumulation of glycosaminoglycans (GAG) in multiple tissues. Accumulation of GAG in corneal stromal cells causes corneal opacity and reduced vision. The purpose of this study was to determine the extent of ocular GAG accumulation and investigate the effectiveness of intravenous enzyme replacement therapy (ERT) on corneal GAG accumulation in dogs. METHODS Ocular tissues were obtained from 58 dogs with mucopolysaccharidosis I and four unaffected controls. Affected dogs received either low-dose ERT, high-dose ERT, or no treatment; some low-dose dogs also received intrathecal treatments. Histologic severity of corneal stromal GAG accumulation was scored. RESULTS Accumulation of GAG was found in corneal stromal cells and scleral fibroblasts but not in corneal epithelium, endothelium, ciliary epithelium, choroid, retina, retinal pigment epithelium, or optic nerve. Corneal GAG accumulation increased in severity with increasing age. Although low-dose ERT did not significantly reduce corneal stromal GAG accumulation in comparison with untreated animals, high-dose ERT did result in significantly less GAG accumulation compared with the untreated dogs (adjusted P = 0.0143) or the low-dose ERT group (adjusted P = 0.0031). Intrathecal treatments did not significantly affect GAG accumulation. Dogs that began ERT shortly after birth also had significantly less (P < 0.0001) GAG accumulation in the corneal stroma than dogs with a later onset of treatment. CONCLUSIONS These data suggest that high-dose, intravenous ERT is effective at preventing and/or clearing corneal stromal GAG accumulation, particularly if initiated early after birth.

Comparison of first-intention healing of carbon dioxide laser, 4.0-MHz radiosurgery, and scalpel incisions in ball pythons (Python regius)

OBJECTIVE To evaluate first-intention healing of CO(2) laser, 4.0-MHz radiowave radiosurgery (RWRS), and scalpel incisions in ball pythons (Python regius). ANIMALS 6 healthy adult ball pythons. PROCEDURES A skin biopsy sample was collected, and 2-cm skin incisions (4/modality) were made in each snake under anesthesia and closed with surgical staples on day 0. Incision sites were grossly evaluated and scored daily. One skin biopsy sample per incision type per snake was obtained on days 2, 7, 14, and 30. Necrotic and fibroplastic tissue was measured in histologic sections; samples were assessed and scored for total inflammation, histologic response (based on the measurement of necrotic and fibroplastic tissues and total inflammation score), and other variables. Frequency distributions of gross and histologic variables associated with wound healing were calculated. RESULTS Gross wound scores were significantly greater (indicating greater separation of wound edges) for laser incisions than for RWRS and scalpel incisions at all evaluated time points. Necrosis was significantly greater in laser and RWRS incisions than in scalpel incision sites on days 2 and 14 and days 2 and 7, respectively; fibroplasia was significantly greater in laser than in scalpel incision sites on day 30. Histologic response scores were significantly lower for scalpel than for other incision modalities on days 2, 14, and 30. CONCLUSIONS AND CLINICAL RELEVANCE In snakes, skin incisions made with a scalpel generally had less necrotic tissue than did CO(2) laser and RWRS incisions. Comparison of the 3 modalities on the basis of histologic response scores indicated that use of a scalpel was preferable, followed by RWRS and then laser.

Magnetic resonance imaging of canine mast cell tumors.

Mast cell tumors (MCT) are the most common cutaneous tumors in dogs. Our purpose was to describe the magnetic resonance (MR) imaging characteristics of cutaneous MCT and to identify imaging characteristics that allow differentiation of metastatic from normal lymph nodes. Eight dogs with a total of nineMCT were imaged as were their presumed draining and associated contralateral lymph nodes. The signal intensity of tumors and lymph nodes was compared to adjacent musculature. On T2-W images, 7/9 MCT were hyperintense to muscle and 2/9 were isointense. On T1-W images, 8/9 MCT were isointense and 1/9 were mildly hypointense. All tumors were strongly contrast enhancing; 5/9 were homogeneous and 4/9 heterogeneous in their enhancement patterns. Six lymph node pairs were included in the evaluation (five sentinel lymph nodes with metastases, one without, and six contralateral lymph nodes). Metastatic lymph nodes were significantly larger than their contralateral lymph nodes (P = 0.039). All lymph nodes were isointense on T1-W images and hyperintense on T2-W images. 5/5 metastatic and 2/7 normal lymph nodes were heterogeneously T2-hyperintense. All lymph nodes were moderately to strongly contrast enhancing. 4/5 metastatic and 2/7 normal lymph nodes had heterogeneous enhancement patterns. While heterogeneity was more common in metastatic than in normal lymph nodes, this difference was not significant (P = 0.058 for T2-W images; P = 0.234 for postcontrast images). MR imaging may be useful in the presurgical evaluation and clinical staging of cutaneous MCT.

Evaluation of the role of the cyclooxygenase signaling pathway during inflammation in skin and muscle tissues of ball pythons (Python regius)

OBJECTIVE To determine degrees of production of cyclooxygenase (COX)-1 and -2 and other mediators of inflammation in noninflamed and inflamed skin and muscle tissues in ball pythons (Python regius). ANIMALS 6 healthy adult male ball pythons. PROCEDURES Biopsy specimens of noninflamed skin and muscle tissue were collected from anesthetized snakes on day 0. A 2-cm skin and muscle incision was then made 5 cm distal to the biopsy sites with a CO2 laser to induce inflammation. On day 7, biopsy specimens of skin and muscle tissues were collected from the incision sites. Inflamed and noninflamed tissue specimens were evaluated for production of COX-1, COX-2, phosphorylated protein kinase B (AKT), total AKT, nuclear factor κ-light-chain-enhancer of activated B cells, phosphorylated extracellular receptor kinases (ERKs) 1 and 2, and total ERK proteins by western blot analysis. Histologic evaluation was performed on H&E-stained tissue sections. RESULTS All biopsy specimens of inflamed skin and muscle tissues had higher histologic inflammation scores than did specimens of noninflamed tissue. Inflamed skin specimens had significantly greater production of COX-1 and phosphorylated ERK than did noninflamed skin specimens. Inflamed muscle specimens had significantly greater production of phosphorylated ERK and phosphorylated AKT, significantly lower production of COX-1, and no difference in production of COX-2, compared with production in noninflamed muscle specimens. CONCLUSIONS AND CLINICAL RELEVANCE Production of COX-1, but not COX-2, was significantly greater in inflamed versus noninflamed skin specimens from ball pythons. Additional research into the reptilian COX signaling pathway is warranted.

Evaluation of delivery agents used for introduction of small interfering RNAs into feline corneal cells.

OBJECTIVE To evaluate agents used for delivery of small interfering RNAs (siRNAs) into feline corneal cells, toxicity of the delivery agents, and functionality of anti-feline herpesvirus 1 (FHV-1)-specific siRNA combinations. SAMPLE Feline primary corneal cells and 19 six-month-old colony-bred cats. PROCEDURES siRNA delivery into corneal cells via various delivery agents was evaluated via flow cytometric detection of labeled siRNAs. Cellular toxicity was evaluated with a proliferation assay. Functionality was tested via quantitative reverse transcriptase PCR assay, plaque assay, and flow cytometry. In vivo safety was evaluated with an ocular scoring method following topical application of delivery agents containing siRNAs into eyes. Corneal biopsy specimens were used to assess safety and uptake of siRNAs into corneal cells. RESULTS Use of 3 delivery agents resulted in > 95% transfection of primary corneal cells. Use of a peptide for ocular delivery yielded approximately 82% transfection of cells in vitro. In cultured corneal cells, use of the siRNA combinations resulted in approximately 76% to 89% reduction in FHV-1-specific mRNA, 63% to 67% reduction of FHV-1-specific proteins in treated cells, and 97% to 98% reduction in FHV-1 replication. The agents were nonirritating in eyes, caused no substantial clinical ocular signs, and were nontoxic. Histologically, corneal epithelium and stroma were normal in treated cats. However, none of the agents were effective in delivering siRNAs into the corneal cells in vivo. CONCLUSIONS AND CLINICAL RELEVANCE The tested anti-FHV-1-specific siRNAs could potentially be used as a treatment for FHV-1 if a successful means of in vivo delivery can be achieved.

Hepatic copper and iron accumulation and histologic findings in 104 feline liver biopsies

In contrast to dogs, the role of copper and iron accumulation in feline hepatic disease remains poorly characterized. Therefore, the objective of the current study was to compare the amount and distribution of copper and iron accumulation for different disease processes in feline liver biopsies. Liver biopsies (from 104 privately owned cats) were categorized by primary histopathologic lesion. Copper (by rubeanic acid) and iron (by Prussian blue) accumulation were graded by amounts (0–3) and location (centrilobular, midzonal, periportal, random). The Kruskal–Wallis test and Pearson chi-square test were used to assess differences in metal grade and location, respectively, between diagnostic categories. Histologic diagnoses were normal (n = 12), congenital (n = 6), neoplastic (n = 16), infectious and/or inflammatory (n = 39), and other (n = 31). Hepatocellular iron staining was negative in 18 samples; remaining samples had grade 1 (n = 38), 2 (n = 40), and 3 (n = 8) accumulation. Ninety-two samples were negative for copper; remaining samples had grade 1 (n = 5), 2 (n = 6), and 3 (n = 1) accumulation. No significant differences were found in the amount of iron or copper accumulation between the different diagnostic categories. Diagnostic category and the location of copper or iron accumulation were not associated. Hepatic iron accumulation was common and not associated with histologic diagnosis. Hepatocellular copper accumulation was more common in cats than previously reported, had a similar pattern of distribution to fibrotic changes, and was not present in histologically normal liver biopsies.

Effect of laser treatment on first-intention incisional wound healing in ball pythons (Python regius).

OBJECTIVE To evaluate effects of laser treatment on incisional wound healing in ball pythons (Python regius). ANIMALS 6 healthy adult ball pythons. PROCEDURES Snakes were sedated, a skin biopsy specimen was collected for histologic examination, and eight 2-cm skin incisions were made in each snake; each incision was closed with staples (day 0). Gross evaluation of all incision sites was performed daily for 30 days, and a wound score was assigned. Four incisions of each snake were treated (5 J/cm(2) and a wavelength of 980 nm on a continuous wave sequence) by use of a class 4 laser once daily for 7 consecutive days; the other 4 incisions were not treated. Two excisional skin biopsy specimens (1 control and 1 treatment) were collected from each snake on days 2, 7, 14, and 30 and evaluated microscopically. Scores were assigned for total inflammation, degree of fibrosis, and collagen maturity. Generalized linear models were used to investigate the effect of treatment on each variable. RESULTS Wound scores for laser-treated incisions were significantly better than scores for control incisions on day 2 but not at other time points. There were no significant differences in necrosis, fibroplasia, inflammation, granuloma formation, or bacterial contamination between control and treatment groups. Collagen maturity was significantly better for the laser-treated incisions on day 14. CONCLUSIONS AND CLINICAL RELEVANCE Laser treatment resulted in a significant increase in collagen maturity at day 14 but did not otherwise significantly improve healing of skin incisions.