Desmin as a possible immunohistochemical marker for feline hypertrophic cardiomyopathy

Desmin has been suggested as a possible histopathological marker for hypertrophic cardiomyopathy (HCM) in humans. To test whether a similar pattern of desmin staining applies to HCM in cats, we conducted an immunohistochemical study on myocardial samples from 13 cats (HCM 4, other cardiomyopathies (OCM) 4, and control 5). The pattern of staining for desmin in HCM cats was not the same as that reported in humans, but was weaker than in OCM cats and controls. This suggested that desmin may be a possible histochemical marker for feline HCM, but our data was insufficient to clearly confirm this.     

Characterization of cytoplasmic lysozyme immunoreactivity as a histiocytic marker in normal canine tissues

The immunoreactive lysozyme distribution in normal canine tissues was determined to assess the value of lysozyme as a marker of histiocytic differentiation. Lysozyme was largely confined to monocytes, neutrophils, macrophages, serous cells in mucosal associated exocrine glands, and renal proximal tubular epithelium. Macrophages in the majority of tissues stained intensely for lysozyme, although in lymphoid tissues not all acid phosphatase and nonspecific esterase positive cells contained lysozyme. In particular, dendritic antigen presenting cells, including Langerhans cells, follicular dendritic cells, and interdigitating reticulum cells, lacked immunoreactive lysozyme; hence lysozyme appeared to represent a discriminatory marker with respect to these cells. Also, a small proportion of non-dendritic macrophages appeared to lack lysozyme. It was concluded that the demonstration of immunoreactive lysozyme was a useful adjunct to conventional morphological techniques for the identification of tissue macrophages provided that due caution was exercised in the interpretation of the results of lysozyme staining.     

Expression of inflammation-mediated cluster of genes as a new marker of canine mammary malignancy

Because canine mammary tumours constitute a serious clinical problem and there are no good prognostic markers (only histopathological variables are used), the aim of the presented study was to find new malignancy markers as well as to identify intracellular pathways and biological processes characteristic for canine mammary malignancy. We compared gene expression of the most malignant mammary tumours (poorly differentiated cancers of the 3rd grade of malignancy) with less malignant tumours (well differentiated cancers of the 1st grade of malignancy). The results of our study indicated that in dogs the number of tumour-infiltrating myeloid cells or expression of myeloid-specific antigens by cancer cells is related to the cancer progression and may constitute a new marker of malignancy, however further studies in this field are required.     

Toll-like receptor expression in feline lymphoid tissues

Toll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that activate the innate immune system. While it is clear that TLRs are important in the immune response against pathogens, they may also be exploited by some pathogens. Our objective is to determine whether feline immunodeficiency virus (FIV) infection affects TLR expression or function thereby resulting in innate immune dysfunction. To this end, we cloned partial sequences for feline TLRs 1--3, 5--8, and developed real-time PCR assays to quantify feline TLRs 1--9. TLR expression was quantified in normal cat lymphoid tissues, purified lymphocyte subsets, and FIV-infected cell lines. Different expression patterns of TLRs were found in spleen, mesenteric lymph node, retropharyngeal lymph node, thymus, intestinal intraepithelial lymphocytes, and lamina propria lymphocytes. B lymphocytes, CD4+ T cells, and CD8+ T cells all expressed TLRs 2--5, 7--9; however, the relative levels of expression varied among lymphocyte phenotypes. Infection of cell lines with FIV resulted in altered TLR expression levels that differed depending on cell type. These results demonstrate that tissue distribution of TLRs is associated with the immunological role of a particular tissue, that lymphocytes may also express these 'innate immune' receptors, and that FIV infection can alter TLR expression.     

Telomerase activity in canine osteosarcoma

Appendicular osteosarcoma (OSA) is the most common primary bone tumour in dogs, and the prognosis with standard of care therapy of amputation and adjunctive chemotherapy is generally poor, with median survival times of 1 year. The ability of neoplastic cells to maintain their telomere length, by either telomerase activity or alternate methods, is an important step in tumour development and malignancy. The purpose of this study was to determine the presence of telomerase activity in canine OSA. To evaluate the frequency of alternative lengthening of telomeres in canine OSA, we have used the telomeric repeat amplification protocol in five canine cell lines and in six samples taken from clinical patients at the time of amputation. Our results reveal the presence of telomerase activity in 100% of canine OSA cell lines and 83% of clinical samples evaluated. This is in contrast to human OSA where 25–40% expression levels of telomerase are reported. Importantly, our results not only suggest that canine OSA may serve as a good model for aggressive telomerase‐positive forms of human OSA but also that antitelomerase therapy strategies for treatment of canine OSA may be more successful than in the treatment of majority of human patients with OSA.     

Evaluation of feline serum amyloid A (SAA) as an inflammatory marker

The concentration of feline serum amyloid A (fSAA) was determined by a direct enzyme-linked immunosorbent assay (ELISA) by using fSAA specific monoclonal antibodies, to evaluate the fSAA as an inflammatory marker in cats. The mean concentration +/- standard deviation of fSAA was found to be 0.60 +/- 1.06 microg/m l and 33.65 +/- 67.59 microg/ml in serum samples from normal cats (n=45) and cats (n=312) with various diseases and disorders, respectively. A significant difference (p<0.001) was found between the two groups. It was also found that the concentration of fSAA begins to increase rapidly at approximately 3-6 hr after spay, and increases up to significantly high levels in some disorders, like injury, renal failure, infectious diseases, etc.     

Evaluation of formalin-fixed paraffin-embedded tissues from feline vaccine site-associated sarcomas for feline foamy virus DNA

OBJECTIVE: To evaluate a group of vaccine site-associated sarcomas (VSS) for the presence of feline foamy virus (FeFV) DNA, using polymerase chain reaction (PCR) methods. SAMPLE POPULATION: 50 formalin-fixed paraffin-embedded (FFPE) tissue blocks from VSS of cats. PROCEDURE: DNA was extracted from FFPE sections of each tumor, and regions of the gag and pol genes of FeFV were amplified by use of PCR methods, using 1 primer set for each region. Sensitivity of the method was compared between fresh and FFPE cells, using mouse kidney tissue that was injected with FeFV-infected cultured cells and using agarose-cell pellets. Results-Feline foamy virus DNA was not detected in VSS tissues. Sensitivity of the method was 10 times greater in fresh versus FFPE mouse tissues. Sensitivity of the method in fresh FeFV-infected cultured cells versus FFPE agarose-cell pellets was equal when fixation was 24 or 48 hours and 10 times greater when fixation was 72 hours or 1 week. CONCLUSIONS AND CLINICAL RELEVANCE: A PCR-based method can be successfully applied to FFPE tissues for FeFV DNA detection. Results suggest there is no direct FeFV involvement in the pathogenesis of VSS in cats.     

Telomerase enzyme activity as a diagnostic tool to distinguish effusions of malignant and benign origin

Telomerase enzyme activity is high in populations of cells that are dividing, and is low or undetectable in quiescent cell populations. Activation of telomerase in tissues that normally lack the capacity for self-renewal is strongly correlated with neoplasia. Telomerase activity can be detected in samples containing very small numbers of cells and studies of human patients suggest that measurement of telomerase activity may be useful for the evaluation of samples that can be obtained in a minimally invasive manner. This study compares the presence or absence of telomerase activity with cytologic evaluation of body cavity effusions, to determine if neoplasia is the underlying cause for the effusion in dogs and cats. Detection of telomerase in effusions was no more sensitive than cytologic evaluation for the identification of underlying neoplasia, and was less specific (telomerase assay: sensitivity = 50%, specificity = 83%; cytology: sensitivity = 50%, specificity = 100%). We conclude that although the telomerase assay may constitute a useful adjunctive test for the diagnosis of neoplasia in some dogs and cats with body cavity effusions, the results of this assay are not sufficiently reliable to be used as a sole diagnostic test.     

Paradigms for pharmacologic use as a treatment component in feline behavioral medicine

Veterinary behavioral medicine remains an under-supported, under-appreciated, and under-taught specialty within veterinary medicine. Neuropsychopharmacology is the aspect that has provided the field with the most scientific legitimacy, but is also one of the most hotly debated. Paradigms for use of pharmacologic intervention include firstly ruling out any underlying medical cause. If a behavioral diagnosis can be made, treatment with psychotropic medication may be considered, although their use is most effective as part of an integrated treatment program that includes behavior modification. Used without an understanding of the mechanism of action, pharmacologic intervention may only blunt or mask behavior without altering processes or environments that produced the behavior. This paper reviews specific drugs, mechanism of action of those drugs, and relevant uses are reviewed for cats. Future advances in treatment in veterinary behavioral medicine will be pharmacological and neurophysiological. As the field of veterinary behavioral medicine expands, its paradigm will enlarge to include routine combination therapy and the implementation of neuropharmacological intervention as a diagnostic tool.     

Hypophysectomy as a treatment for canine and feline Cushing's disease.

The microsurgical technique of transsphenoidal hypophysectomy performed with the dogs and cats positioned in sternal recumbency enables the treatment of Cushing's disease, independent of skull type, in a safe and effective manner. In dogs, the short-term survival rate after hypophysectomy is comparable to that after treatment with o,p'-DDD, whereas the recurrence rate in this period is lower. When the surgeon has gone through a learning curve, the results of the 1- to 3-year follow-up interval may be better than those after adrenocorticolysis with o,p'-DDD. CT enables assessment of localization and size of the pituitary before surgery. In general, dogs with Cushing's disease and normal-sized pituitaries or moderately enlarged pituitaries (up to 12 mm in diameter) are suitable candidates for transsphenoidal surgery. In dogs with larger pituitary tumors and tumor extension rostrally or caudally over the dorsum sellae, transsphenoidal debulking surgery may be only a palliative treatment. The main complications are postoperative hypernatremia, keratoconjunctivitis sicca, diabetes insipidus, and secondary hypothyroidism. In cats, special attention should be paid to closure of the soft palate. The neurosurgeon must be familiar with these complications so as to recognize them as early as possible and to treat them immediately and effectively. It is concluded that microsurgical transsphenoidal hypophysectomy in dogs and cats with Cushing's disease is an effective method of treatment.     

Gemcitabine as a radiosensitizer for nonresectable feline oral squamous cell carcinoma

Eight cats with locally advanced, oral squamous cell carcinoma (SCC) were treated with a combination of gemcitabine and palliative radiotherapy. Low-dose gemcitabine was administered twice weekly (25 mg/m2) in conjunction with megavoltage radiation in 6 Gray (Gy) fractions for a total dose of 36 Gy. Responses included two complete and four partial responses, and two cats had no response to therapy. Median duration of remission was 42.5 days (range, 11 to 85 days). Median survival time was 111.5 days (range, 11 to 234 days). This data suggests that a combination of low-dose gemcitabine and palliative radiation therapy may be tolerable for cats with oral SCC and may cause a therapeutic benefit.     

Evaluation of tizanidine as a marker of canine CYP1A2 activity

The dog CYP1A2 enzyme is likely an important contributor to the metabolism of veterinary drugs. Dog CYP1A2 is expressed in liver, plus it is inducible and polymorphic, creating the potential for intersubject differences in pharmacokinetics. Hence, the ability to probe dog CYP1A2 activity and inhibition is relevant toward veterinary drug development and drug–drug interaction assessment. Previous studies have relied on human probes with questionable specificity for CYP1A2, so it was hypothesized that recombinant CYP1A2 could be used to find a specific CYP1A2 substrate. Intrinsic clearance experiments demonstrated that tizanidine was a substrate of CYP1A2. Profiling of tizanidine metabolites generated by CYP1A2 identified the imidazole metabolite that was detectable in dog plasma. The imidazole metabolite was subsequently used to evaluate tizanidine as a CYP1A2 probe. Co‐administration of the CYP1A inhibitor enrofloxacin with tizanidine significantly decreased (30%; n = 3) the formation of the imidazole metabolite vs. control experiments. As enrofloxacin is a weak inhibitor, further studies are required to confirm the sensitivity of tizanidine as an in vivo probe. However, tizanidine may be a more selective CYP1A2 probe than phenacetin when conducting in vitro studies due to the presence of other phenacetin‐metabolizing enzymes in dog liver microsomes.     

Expression of podoplanin in various types of feline tumor tissues

Podoplanin is expressed in various human tumors where it promotes tumor progression, epithelial-mesenchymal transition, and distant metastasis. Podoplanin is also expressed in cancer-associated fibroblasts and induces tumor malignancy. The objective of this study was to evaluate podoplanin expression in various types of feline tumor tissues. Immunohistochemical analysis revealed that podoplanin was expressed in cells of 13/15 (87%) squamous cell carcinomas and 5/19 (26%) fibrosarcomas. Moreover, cancer-associated fibroblasts expressed podoplanin in most tumor types, including 18/21 (86%) mammary adenocarcinoma tissues. Our findings demonstrate that various types of feline tumor tissues expressed podoplanin, indicating the importance of the comparative aspects of podoplanin expression, which may be used as a novel research model for podoplanin biology.     

A validation of 10 feline reference genes for gene expression measurements in snap-frozen tissues

For a proper determination of relative mRNA expression levels with real-time quantitative PCR (Q-PCR) internal standards, such as the expression of reference genes, are of utmost importance. For cats, in contrast to dogs, no validation of reference genes has been published. Our goal was to evaluate frequently used reference genes for the analysis of relative mRNA levels from feline tissues in a SYBR Green-based Q-PCR protocol. First, primers were optimized on mRNA-derived cDNA from liver and kidney tissues of randomly chosen (healthy and diseased) cats. Then, the expression variation and stability of each reference gene within a specific tissue was determined. Dental roots and crowns, heart (left ventricle), renal, liver, lung, and mammary gland tissues from 3 to 11 cats of different breeds, sexes, ages, and disease status were included in this study. Averaging relative stabilities over these six tissues revealed the usefulness of each tested gene as reference gene. In order to compensate for the expression variation of a reference gene within a specific tissue, as much as six reference genes (e.g. RPL17, RPL30, RPS7, YWHAZ, and HPRT) were required to obtain highly reliable data in cat tissues. The optimal set of reference genes depended on the tissue analyzed and should, ideally, be selected and evaluated at the start of each experimental condition. A comparison with a similar evaluation in dogs revealed three issues: (i) most ribosomal genes are suitable in both species; (ii) good non-ribosomal reference genes differ; (iii) more feline than canine reference genes are required for proper analysis.     

p63: a novel myoepithelial cell marker in canine mammary tissues

Several immunohistochemical markers have been used to demonstrate the presence of myoepithelial cells in order to determine their role in the histogenesis of mammary tumors. p63, a recently characterized p53 homologue, is consistently expressed in myoepithelial cells of the human breast; however, no assessment of its immunoreactivity has been reported so far in canine mammary tissues. We investigated p63 immunohistochemical expression, as a novel myoepithelial cell nuclear marker, in 81 samples of normal (n = 2), hyperplastic (n = 11), and neoplastic (n = 68) canine mammary tissues. Myoepithelial phenotype was confirmed by using complementary monoclonal antibodies: alpha-smooth muscle actin, cytokeratin 14, cytokeratin AE1/AE3, and vimentin. p63 expression was observed in 91.4% (74/81) of the samples evaluated. Normal mammary glands, mammary hyperplasias, and benign tumors showed 100% immunoreactivity, with p63 expression restricted to myoepithelial cell nuclei. In general, benign mixed tumors showed a basal cell compartment immunoreactive to p63, with a gradual decrease of its expression during myoepithelial transformation. p63 expression was found in 72% of malignant tumors, allowing myoepithelial or basal cell identification in spindle-cell carcinomas (2/2), tubulopapillary carcinomas (8/9), solid carcinomas (7/10), and carcinosarcomas (1/3). The osteosarcoma analyzed was p63 negative. In our series, stromal components were consistently nonreactive to p63. In conclusion, the present study reveals p63 as a sensitive and highly specific marker of myoepithelial cells in canine mammary tissues, and the authors suggest p63 as an additional marker for defining myoepithelial histogenesis.     

Porphyrins are not present in feline ocular tissues or corneal sequestra

Objective To determine if feline lacrimal glands, glands of the third eyelid, corneas, and corneal sequestra contain porphyrins, which could be responsible for the brown/amber discoloration of corneal sequestra and tears in affected cats. Procedures Samples of grossly normal cornea, lacrimal gland, gland of the third eyelid, and sequestra obtained via keratectomy were collected. Porphyrin concentrations of the homogenate were determined by spectrofluorometry with protoporphyrin IX and coproporphyrin III dihydrochloride used as standards. A hamster harderian gland was used as a positive control. Results Normal tissues were harvested from one eye each of 14 nonclient owned, adult, mixed‐breed, short‐hair cats euthanized for reasons not associated with this study. Eighteen sequestra were acquired from cats undergoing unilateral lamellar keratectomies. Breeds of the affected cats included eight Himalayan, five domestic shorthair, and one each of four other breeds. Only the positive control and standards contained levels of porphyrins above background. All feline samples examined were histologically normal with no evidence of porphyrins. Conclusions Porphyrins are absent in normal feline lacrimal glands, corneas, and corneal sequestra. Porphyrins do not appear to be the cause of the brown/amber color of feline corneal sequestra.