Effects of antibiotics on morphologic characteristics and migration of canine corneal epithelial cells in tissue culture.

OBJECTIVE: To determine effects of commonly used ophthalmic antibiotics on cellular morphologic characteristics and migration of canine corneal epithelium in cell culture. SAMPLE POPULATION: Corneal epithelial cells harvested from corneas of 12 euthanatized dogs and propagated in cell culture. PROCEDURE: Cells were treated with various antibiotics after a defect was created in the monolayer. Cellular morphologic characteristics and closure of the defect were compared between antibiotic-treated and control cells. RESULTS: Cells treated with ciprofloxacin and cefazolin had the greatest degree of rounding, shrinkage, and detachment from plates. Cells treated with neomycin-polymyxin B-gramicidin and gentamicin sulfate had rounding and shrinkage but with less detachment. Cells treated with tobramycin and chloramphenicol grew similarly to control cells. On the basis of comparisons of defect circumference between control cells and cells exposed to antibiotics, tobramycin affected cellular migration the least. CONCLUSIONS AND CLINICAL RELEVANCE: Effects of ciprofloxacin and cefazolin on morphologic characteristics of canine corneal epithelial cells in vitro should be taken into consideration before using these antibiotics for first-line of treatment for noninfected ulcers. Of the antibiotics tested that have a primarily gram-negative spectrum of coverage, gentamicin inhibited corneal epithelial cell migration and had greater cytopathologic effects than tobramycin did. For antibiotics with a gram-positive coverage, chloramphenicol had no cytopathologic effects on cells in comparison to cefazolin, which caused most of the cells to shrink and detach from the plate. Polymyxin B-neomycin-gramicidin was midrange in its effects on cellular morphologic characteristics and migration.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a nicotine derivative, induces apoptosis of endothelial cells.

Smoking causes endothelial cell (EC) injury; however, neither the components of cigarette smoke nor the mechanisms responsible for this injury are understood. The nitrosated derivative of nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has been implicated in the carcinogenic effects of tobacco; however, the effects of NNK on the cardiovascular system are largely unknown. NNK binds to beta1- and beta2-adrenergic receptors. Because beta-adrenergic receptor activation causes arachidonic acid (AA) release and cellular injury, we postulated that NNK causes EC injury by a mechanism that involves beta-adrenergic-mediated release of AA. NNK stimulated [3H]AA release from ECs, and this effect was mediated by both beta1- and beta2-adrenergic receptors because pretreatment with atenolol or ICI 118,551 inhibited the response. NNK also induced EC apoptosis, as measured by terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling and annexin V staining. NNK-mediated apoptosis was attenuated by pretreatment with atenolol or ICI 118,551. Furthermore, depletion of cellular AA by incubation with eicosapentaenoic acid abolished the apoptotic effect of NNK. These data suggest that NNK causes EC apoptosis by a mechanism that involves beta1- and beta2-adrenergic receptor-mediated release of AA.

Detection and clinical significance of plasma glutathione-S-transferases in dogs with lymphoma.

BACKGROUND: The objective of this study was to determine if glutathione-S-transferases were detectable in the plasma of dogs and to determine if concentrations of the a- and pi-subtypes were related with tumor response to single agent anthracycline (e.g., doxorubicin) chemotherapy in dogs with lymphoma. MATERIALS AND METHODS: Plasma was obtained from 10 healthy, normal dogs and from 11 dogs with lymphoma before treatment, 3 weeks after 1 dose of doxorubicin and every 3 weeks thereafter until relapse (the physical detection of recurrent and enlarged peripheral lymph nodes). Plasma concentration of alpha and pi-GST was determined by use of an ELISA technique with well plates pre-coated with IgG[anti-Canine alpha-GST or anti-Human pi-GST]. RESULTS: Mean plasma alpha-GST concentrations did not significantly decline after 1 dose of doxorubicin chemotherapy; however, mean plasma alpha-GST concentrations were markedly increased (p < 0.05) at the time of relapse (the physical detection of recurrent and enlarged peripheral lymph nodes). CONCLUSIONS: In this study we show that a relationship exists between the plasma alpha-GST concentration and the clinical response of dogs with lymphoma to doxorubicin chemotherapy.

Serum alpha 1-acid glycoprotein concentrations before and after relapse in dogs with lymphoma treated with doxorubicin.

OBJECTIVE: To determine whether serum alpha 1-acid glycoprotein (AGP) concentration was a useful marker of relapse in dogs with lymphoma that were in clinical remission following treatment with doxorubicin. DESIGN: Cohort study. ANIMALS: 12 dogs with lymphoma and 10 healthy dogs. PROCEDURE: Serum AGP concentration was measured in the healthy dogs and in the dogs with lymphoma before treatment, 3 weeks after the first dose of doxorubicin was administered, and every 3 weeks thereafter until relapse (i.e., recurrence of clinically detectable disease such as palpable enlargement of peripheral lymph nodes). Serum AGP concentrations were determined by use of a radial immunodiffusion kit. RESULTS: Mean serum AGP concentration in healthy dogs was significantly less than concentration in dogs with lymphoma prior to treatment. Mean serum AGP concentrations after the first and each subsequent dose of doxorubicin were not significantly different from concentration in healthy dogs. However, mean serum AGP concentrations 3 weeks prior to and at the time of relapse were significantly higher than concentration measured after the first dose of doxorubicin, and were not significantly different from concentration measured before treatment. CLINICAL IMPLICATIONS: Results suggest that measuring serum AGP concentration may be a useful method of predicting relapse before recurrence of clinically detectable disease in dogs with lymphoma undergoing treatment with doxorubicin.

Inhibitors of intracellular chloride regulation induce cisplatin resistance in canine osteosarcoma cells.

The objective of this study was to examine the role of ion transport mechanisms in clinical anticancer drug resistance. Reduction in intracellular accumulation of cisplatin is believed to be an early change in cisplatin-resistant cells, and may be dependent on the concentration of intracellular chloride (Cl-) ions and intracellular pH. The primary aim of this study was to describe the modifying effects of NHMA (5-N,N hexamethylene; amiloride), a Na+/H+ antiport inhibitor, and/or SITS (4-acetamido-4';isothiocyanostilbene-2,2'-disulfonic acid), a HCO3-/Cl- transport inhibitor, in bicarbonate-containing or bicarbonate-free media on cisplatin (cis-diamminedichloroplatinum(II); CDDP) toxicity between known cisplatin-sensitive (COS31) and cisplatin-resistant (COS31/rCDDP) canine osteosarcoma cells. This study has shown that cell survival can be influenced by the inhibition of the Na(+)-dependent HCO3-/Cl- exchanger using SITS. The addition of SITS increases the intracellular Cl- concentration in canine osteosarcoma cells cultured in a bicarbonate-containing media. In a bicarbonate-free media, the addition of SITS results in a decrease in the cytotoxic action of cisplatin.

An in vivo/in vitro experimental model system for the study of human osteosarcoma: canine osteosarcoma cells (COS31) which retain osteoblastic and metastatic properties in nude mice.

BACKGROUND: In this report we describe the establishment, characterization, and research utility of a cell line derived from a dog having a spontaneously occurring osteosarcoma. MATERIALS AND METHODS: Tumor samples were collected from a dog with a naturally occurring osteosarcoma and processed for light microscopy, electron microscopy, immunocytochemistry, immunohistochemistry, karyology, and cell culture. Established cells from passage 31 (COS31; canine Qsteosarcoma cells from passage 31) were inoculated subcutaneously between the scapula and in the right abdominal side of athymic nude mice and evaluated similarly. RESULTS: COS31 cells derived in cell culture and in nude mice had morphological and biochemical properties comparable in all respects to the original canine tumor specimen. CONCLUSIONS: The ability of COS31 cells to produce tumors in nude mice (i.e. a small animal model) typical of canine osteosarcoma (i.e. a large animal model) with a similar pathological and biological behavior (e.g. alkaline phosphatase and osteocalcin positive immunostaining, osteoid production, rapid growth, and wide spread metastases) demonstrates the potential utility of COS31 cells as a in vitro and in vivo model system in the development of new strategies in the treatment of human osteosarcoma.

In vitro reversal of glutathione-S-transferase-mediated resistance in canine osteosarcoma (COS31) cells.

BACKGROUND: In this report we describe the establishment, characterization, and research utility of a cell line derived from a dog having a spontaneously occurring osteosarcoma (COS) made resistant to the cytotoxic effects of cisplatin chemotherapy. MATERIALS AND METHODS: Established cells from passage 31 (COS31) were exposed to increasing sublethal concentrations of cisplatin in vitro. RESULTS: A 2.2-fold increase in glutathione-S-transferase (GST) activity was found to be induced in this cell line (COS31/rCDDP) compared to parent cells. Furthermore, these cells were 7.8-fold more resistant to the cytotoxic effects of higher concentrations of cisplatin compared to parent cells. Ethacrynic acid was found to inhibit GST enzymatic activity and increase cisplatin cytotoxicity in resistant COS31 (COS31/rCDDP) cells. CONCLUSIONS: Inhibiting the function of GST with ethacrynic acid pretreatment in humans and dogs with osteosarcoma may result in more tumor cells than normal cells killed in vivo by cisplatin, thus significantly prolonging lifespan without increasing host toxicity.

Effect of hypoxia on the frequency of bleomycin-induced micronuclei.

Normal G(0) pig lymphocytes were maintained in a hypoxic or aerobic environment for one hour and then concurrently exposed to bleomycin at varying concentrations for a second hour. The cytochalasin-block micronucleus assay was used to determine cytotoxicity. Exposure to transient hypoxia significantly decreased the frequency of bleomycin-induced micronuclei per cell as compared to that observed in oxygenated cells. Furthermore, the frequency of micronuclei per hypoxic cell did not correspond to any increase in bleomycin dose as observed in oxygenated cells. These data demonstrate the significance of transient hypoxia in the resistance of G(0) cells to the cytotoxic effects of bleomycin and may explain the observed selectivity of tumor tissue response to bleomycin.

Tripartite differentiation in a carcinoma of the duodenum.

BACKGROUND: Carcinomas containing three distinctly different cell lines have been encountered in the colon and rectum, but a tripartite malignancy in the small intestine has not been reported previously. METHODS: A duodenal carcinoma was studied by light and electron microscopic examination and immunohistochemistry. RESULTS: The duodenal carcinoma was found to have tripartite glandular, squamous, and neuroendocrine differentiation. Histologically, an adenocarcinoma, which originated in a villous adenoma, was continuous with squamous cell carcinoma and small cell carcinoma components. Tumor cells of the squamous cell carcinoma component had conspicuous intercellular bridges but did not form keratin pearls. Immunohistochemical analysis showed strong expression of carcinoembryonic antigen (CEA) by the adenocarcinomatous component. The squamous cell carcinoma component demonstrated focal weak CEA and neuron specific enolase (NSE) reactivity. Ultrastructurally, tumor cells of this component had frequent desmosomes and free tonofilaments. The small cell carcinoma had clusters of dense core granules in tumor cell cytoplasmic processes, which are indicative of neuroendocrine differentiation. This neuroendocrine component was immunoreactive for somatostatin and NSE. CONCLUSIONS: This case of tripartite duodenal carcinoma supports the theory of an origin from an intestinal pluripotential stem cell capable of differentiating into multiple cell types.

Quantification of phthalocyanine concentration in rat tissue using laser-induced fluorescence spectroscopy.

Quantification of photosensitizer concentration in tissue should improve planning and outcome of photodynamic therapy. Laser-induced fluorescence (LIF) can be used to measure in vivo fluorescence of photosensitizers in tissue. This study was designed to determine if in vivo fluorescence intensity of chloroaluminum phthalocyanine tetrasulfonate correlates with its concentration in different rat tissues. Following LIF measurements, the animals were humanely euthanized and the concentration of phthalocyanine in different tissues was determined using chemical extraction technique. The correlation of phthalocyanine fluorescence intensity and its concentration was determined for each tissue using Pearson product-moment correlation analysis. A strong correlation between in vivo phthalocyanine fluorescence intensity and its concentration was found for spleen, kidney, liver, and chemically induced mammary adenocarcinoma. Low correlation was found for thigh skin and planum of nose. No correlation was found for thigh muscle and tongue.

Effect of multidrug-resistant P-glycoprotein gene expression on chloroaluminum tetrasulfonate phthalocyanine concentration.

The effect of multidrug-resistant P-glycoprotein gene expression (MDR1) in 3T3 cells on cellular concentrations and cytotoxicity induced by the photodynamic agent chloroaluminum tetrasulfonate phthalocyanine (AlSPc) was evaluated. 3T3 cells transfected with a retroviral vector expressing human MDR1 cDNA were resistant to colchicine. Resistant cells incubated with daunomycin accumulated only 40-50% of the quantity of daunomycin accumulated in control cells. Resistant cells incubated with daunomycin in the presence of verapamil had intracellular daunomycin concentrations approximately equal to control cells without verapamil. When these MDR1 3T3 cells were incubated with AlSPc, cellular concentrations of AlSPc did not differ between cells resistant to colchicine and those that were not. Similarly, there was little difference in cytotoxicity demonstrated by 51Cromium release in the two cell lines exposed to AlSPc and light (675 nm; 6 J/cm2). This study suggests photodynamic therapy using AlSPc may be a useful treatment modality for tumors in which the MDR1 P-glycoprotein confers resistance to cancer chemotherapeutics.

Comparative pharmacokinetics of the photosensitizer tin-etiopurpurin in dogs and rats.

Photodynamic therapy is a promising new treatment for local eradication of cancer. Little work has been done to define the pharmacokinetics of photodynamic drugs or the variability in drug disposition that may occur between different species and pathophysiological states of tissues. Pharmacokinetic studies of tin-etiopurpurin (SnET2), a lipophilic photosensitizer, were conducted on six Beagle dogs and six Sprague-Dawley rats. Blood was collected up to 24 h following drug administration for measurement of tin-etiopurpurin concentration. Dogs and rats were euthanatized 24 h post-administration and tissues were collected for drug analyses. The plasma drug concentrations were best described by a 2-compartment model (Ct = Ae-alpha t + Be-beta t). Median distribution and elimination half-lives were 0.24 and 0.34 h and 10.21 and 5.25 h for dogs and rats, respectively. The apparent volumes of distribution were 4.26 +/- 1.75 L/kg for dogs and 1.84 +/- 0.36 L/kg for rats. Systemic clearance was 7.56 +/- 2.45 ml/kg/min and 6.63 +/- 0.91 ml/kg/min for dogs and rats, respectively. Drug was detected in all tissues analyzed 24 h after drug administration. Drug was detected only sporadically in skin and muscle and was generally below the limit of detection of the assay. Where comparisons could be made, concentrations of SnET2 were significantly greater in all tissues except jejunum of rats compared to dogs 24 h after drug administration.

Neutrophil function in dogs with congenital ciliary dyskinesia.

In vitro neutrophil function was assessed in two English Springer Spaniel dogs, two Bichon Frise dogs, and one Chow Chow dog with congenital ciliary dyskinesia; three clinically normal English Springer Spaniel dogs that were presumed heterozygous for congenital ciliary dyskinesia; and five control dogs. Chemotaxis and random migration in affected and heterozygous dogs were found to be comparable to those of control dogs. Increased (P less than or equal to 0.05) neutrophil adhesion, antibody dependent cell-mediated cytotoxicity, iodination of proteins, and oxygen radical production in neutrophils from affected dogs were probably the result of chronic bacterial infection in vivo. Bacterial ingestion by neutrophils from the three heterozygous English Springer Spaniel dogs was significantly increased compared to control dogs but was not different from affected English Springer Spaniel dogs, suggesting a breed-related phenomenon. Significant decreases in neutrophil function were not seen in any of the dogs with congenital ciliary dyskinesia, indicating that a defective microtubular system is not shared by respiratory cilia and neutrophils and that defective neutrophil function does not contribute to respiratory infection.

The histologic spectrum of pigmented spindle cell nevus: a review of 120 cases with emphasis on atypical variants.

The histopathologic features of 120 cases of pigmented spindled nevus (PSCN) from the years 1973 through 1988 were reviewed from a consultative practice heavily weighted with difficult nevomelanocytic lesions. The patients' mean age was 25.2 years, and females outnumbered males (68 versus 52). Extremity lesions made up 69.6% of the total, with the thigh the most common site. The lesions were categorized into one of four variants of PSCN, based on the presence or absence of various architectural and cytologic parameters and involvement of the reticular dermis. Thirteen cases (10.8%) were designated typical PSCN, and were characterized by fascicles of uniform pigmented spindle cells without cellular atypia and limited to the epidermis or papillary dermis. Ninety-five cases (79.2%) were classified as atypical PSCN (PSCN with architectural and/or cytologic atypia). Some of the latter also demonstrated substantial numbers of epithelioid cells, thus exhibiting some overlap with Spitz nevus. Eight cases showed striking features of dysplastic nevus. Ten cases had fascicles of pigmented spindle cells involving the reticular dermis ("plexiform" PSCN). Two cases were designated as combined PSCN because of the presence of banal nevus cells in addition to the spindle cell component. Clinical follow-up of a small group of patients has not suggested, to date, any aggressive behavior. Knowledge of PSCN and its atypical variants is important for discrimination from malignant melanoma.

Angiotensin converting enzyme system in the normal canine eye: pharmacological and physiological aspects.

The function of the renin-angiotensin-aldosterone system (RAAS) in the mammalian eye remains unclear, although alterations in the concentrations of various pathway components can influence intraocular pressure and the electroretinogram. Angiotensin Converting Enzyme (ACE) has been localized to ocular tissues and fluids. Aqueous humor and serum values of ACE are increased in sarcoid uveitis patients. We used the dog to simultaneously examine the effects of a topically administered ACE inhibitor on the intraocular pressure (IOP), on components of the renin-angiotensin pathway in the serum and aqueous humor, and to monitor any systemic effects of the ACE inhibitor. The novel ACE inhibitor, SCH 33861 (Schering Corporation), decreased IOP in amounts similar to timolol when applied topically to the canine eye. Serum ACE values significantly decreased in SCH 33861 treated dogs, while aqueous ACE values were only slightly decreased. A decrease in heart rate, and systolic and diastolic blood pressure in these dogs during the treatment period indicated probable systemic absorption. Normal values of aqueous humor and serum angiotensin-I were established for the dog. Plasma renin activity and angiotensin-I values were not significantly changed for any of the treatment groups. Topical application of SCH 33861 to the canine eye is a useful model to further evaluate the role of the renin-angiotensin system in the eye.

Comparative results with five cannabinoid immunoassay systems at the screening threshold of 100 micrograms/L.

Five commercially available immunoassay techniques were compared for the detection of cannabinoid metabolites in urine at a positive cutoff of 100 micrograms/L. Numerical values, available for four of these techniques, were used to evaluate inter-assay equivalence at this threshold. The total number of positive results differed for each assay, with some individual samples exhibiting wide variations in numerical values. As a group, the radioimmunoassay systems gave a greater proportion of positive specimens than did enzyme-linked or fluorescence-polarization-based techniques. The single threshold of detection suggested by federal guidelines does not provide for equivalent screening of cannabinoids with these immunoassay techniques.

Lowered susceptibility of K-562 cells treated with gamma interferon in serum-free medium to natural killer cell-mediated cytolysis.

K-562 cells grown in serum-free medium were treated with gamma interferon (IFN-gamma) and they became significantly less susceptible to natural killer (NK) cell-mediated cytolysis. To examine if this loss in susceptibility was related to induced differentiation events, the presence of various antigens was determined after induction. There was a coincident expression of class I HLA common antigen, although it is not clear if this is a direct causal relationship. The level of the constitutively expressed myelomonocytic antigen, reactive with anti-Leu-M1, was not affected by IFN-gamma induction and three normally nonexpressed monocytic antigens, defined by monoclonal antibodies, remained unexpressed. IFN-gamma did induce an enhanced expression of IL-2 receptors on K-562 cells after 2 days of treatment but, thereafter, the expression appeared to be suppressed. Electron microscopy of IFN-gamma-treated cells revealed the development of increased surface blebbing and electron-dense cytoplasmic inclusions. These ultramicroscopic changes could not be correlated with definitive differentiation events. We suggest that IFN-gamma treatment of K-562 cells induces class I HLA expression and morphological changes that may be important to differentiation events that render the cells less susceptible ot NK-mediated cytolysis.