Application of cold atmospheric microwave plasma as an adjunct therapy for wound healing in dogs and cats.

BACKGROUND: Cold atmospheric plasma is a novel innovative approach for wound care, and it is currently underrepresented in veterinary medicine. OBJECTIVES: To investigate the efficacy and safety of using cold atmospheric microwave plasma (CAMP) as an adjunct therapy for wound healing in dogs and cats. METHODS: Wound healing outcomes were retrospectively analyzed using clinical records of client-owned dogs and cats who were first managed through standard wound care alone (pre-CAMP period) and subsequently via CAMP therapy (CAMP period). The degree of wound healing was estimated based on wound size and a modified wound scoring system. RESULTS: Of the 27 acute and chronic wounds included in the analysis, 81.48% showed complete healing after the administration of CAMP as an adjunct therapy to standard care. Most wounds achieved complete healing in < 5 weeks. Compared with the pre-CAMP period, the rate of wound healing significantly increased every week in the CAMP period in terms of in wound size (first week, p < 0.001; second week, p = 0.012; third week, p < 0.001) and wound score (first week, p < 0.001; second week, p < 0.001; third week, p = 0.001). No adverse events were noted except for mild discomfort and transient erythema. CONCLUSIONS: CAMP is a well-tolerated therapeutic option with immense potential to support the treatment of wounds of diverse etiology in small animal practice. Further research is warranted to establish specific criteria for CAMP treatment according to wound characteristics.

Cold atmospheric microwave plasma (CAMP) stimulates dermal papilla cell proliferation by inducing ß-catenin signaling.

Hair loss or alopecia is an unpleasant symptom that exacerbates an individual's self-esteem and requires appropriate treatment. The Wnt/ß-catenin signaling is a central pathway that promotes dermal papilla induction and keratinocyte proliferation during hair follicle renewal. GSK-3ß inactivated by its upstream Akt and ubiquitin-specific protease 47 (USP47) has been shown to inhibit ß-catenin degradation. The cold atmospheric microwave plasma (CAMP) is microwave energy enriched with mixtures of radicals. CAMP has been reported to have antibacterial and antifungal activities with wound healing activity against skin infection; however, the effect of CAMP on hair loss treatment has not been reported. We aimed to investigate the effect of CAMP on promoting hair renewal in vitro and to elucidate the molecular mechanism, targeting ß-catenin signaling and YAP/TAZ, the co-activators in the Hippo pathway, in human dermal papilla cells (hDPCs). We also evaluated plasma effects on the interaction between hDPCs and HaCaT keratinocytes. The hDPCs were treated with plasma-activating media (PAM) or gas-activating media (GAM). The biological outcomes were determined by MTT assay, qRT-PCR, western blot analysis, immunoprecipitation, and immunofluorescence. We found that ß-catenin signaling and YAP/TAZ were significantly increased in PAM-treated hDPCs. PAM treatment also induced ß-catenin translocation and inhibited ß-catenin ubiquitination by activating Akt/GSK-3ß signaling and upregulating USP47 expression. In addition, hDPCs were more aggregated with keratinocytes in PAM-treated cells compared with control. HaCaT cells cultured in a conditioned medium derived from PAM-treated hDPCs exhibited an enhancing effect on activating YAP/TAZ and ß-catenin signaling. These findings suggested that CAMP may be a new therapeutic alternative for alopecic treatment.

In vitro antifungal activity of cold atmospheric microwave plasma and synergistic activity against Malassezia pachydermatis when combined with chlorhexidine gluconate.

BACKGROUND: The antifungal efficacy of cold atmospheric microwave plasma (CAMP) against Malassezia pachydermatis has not been to be evaluated. OBJECTIVE: To examine the antifungal effects of CAMP against M. pachydermatis and its synergistic effects with chlorhexidine gluconate (CHX). METHODS: A M. pachydermatis isolate was collected from a dog with otitis externa and Malassezia dermatitis at the Seoul National University Veterinary Medical Teaching Hospital. The antifungal effect was determined by applying CAMP to a M. pachydermatis isolate that was incubated for 3 days at 37°C. After 1, 2, 3 and 5 min of application, the efficacy of the plasma treatment was determined according to the number of colony forming units (CFUs). A mixture consisting of inoculum and CHX was applied to evaluate the synergistic effect of the plasma treatment in the same way. RESULTS: The application of CAMP showed significant antifungal effects against M. pachydermatis. The antifungal effect of CAMP was enhanced by an increased exposure time and output power. The application of CAMP with 0.02% and 0.2% CHX resulted in lower survival rates against M. pachydermatis when compared with its sole application at 1 or 2 min. CONCLUSIONS: The study findings demonstrate that CAMP has a potential as a new antifungal option for M. pachydermatis and has synergistic antifungal effects with CHX in vitro. Clinical applications for CAMP are necessary to assess the antifungal efficacy for patients.

In vitro antimicrobial activity of cold atmospheric microwave plasma against bacteria causing canine skin and ear infections.

BACKGROUND: Cold atmospheric plasma (CAP) is a new generation medical therapeutic option for bacterial infections. CAP causes physical cell wall rupture and DNA damage, therefore making it highly useful in the treatment of various conditions such as skin infections. HYPOTHESIS/OBJECTIVES: The antimicrobial activity of cold atmospheric microwave plasma (CAMP) against major strains in canine skin infections was tested and the difference in antimicrobial activity between the antibiotic-resistant and antibiotic-susceptible strains of Staphylococcus pseudintermedius was evaluated. METHODS AND MATERIALS: American Type Culture Collection (ATCC) strains (Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli) and clinical isolates identified as methicillin-resistant S. pseudintermedius (n = 27) and methicillin-susceptible S. pseudintermedius (n = 13) were exposed to CAMP for 10 s, 30 s and 60 s. Afterwards, the bacterial survival rate was confirmed. RESULTS: Gram-negative bacteria (P. aeruginosa and E. coli) were more susceptible than Gram-positive bacteria (S. aureus and S. pseudintermedius) for the same duration of CAMP exposure. Only the Gram-negative bacteria were completely killed after 60 s exposure. In S. pseudintermedius isolates, CAMP exposure had similar antibacterial effects regardless of antibiotic resistance. CONCLUSIONS AND CLINICAL IMPORTANCE: CAMP has sufficient antimicrobial activity against major bacterial strains that cause pyoderma and otitis externa in dogs, and may be an alternative therapeutic option for S. pseudintermedius skin infections, for which antibiotics often are ineffective because of antimicrobial resistance in clinical veterinary medicine.

Anticancer Effects of Cold Atmospheric Plasma in Canine Osteosarcoma Cells.

Osteosarcoma is known to be one of the frequently occurring cancers in dogs. Its prognosis is usually very poor, with a high incidence of lung metastasis. Although radiation therapy has become a major therapeutic choice for canine osteosarcoma, the high costs and unexpected side effects prevent some patients from considering this treatment. Cold atmospheric plasma (CAP) is an ionized gas with high energy at low temperatures, and it produces reactive oxygen species that mediate many signaling pathways. Although many researchers have used CAP as an anticancer therapeutic approach in humans, its importance has been neglected in veterinary medicine. In this study, D-17 and DSN canine osteosarcoma cell lines were treated with CAP to observe its anticancer activity. By high-content screening and flow cytometry, CAP-treated cells showed growth arrest and apoptosis induction. Moreover, the osteosarcoma cells exhibited reduced migration and invasion activity when treated with CAP. Overall, CAP exerted an anticancer effect on canine osteosarcoma cell lines. CAP may have the potential to be used as a novel modality for treating cancer in veterinary medicine.

In vitro antibacterial activity of the manuka essential oil from Leptospermum scoparium combined with Tris-EDTA against Gram-negative bacterial isolates from dogs with otitis externa.

BACKGROUND: The increasing prevalence of antimicrobial resistance among bacteria in dogs with otitis externa has led to a need for novel therapeutic agents. HYPOTHESIS/OBJECTIVE: To examine the antibacterial effects of manuka oil combined with ethylenediaminetetraacetic acid-tromethamine (Tris-EDTA) against Gram-negative bacteria isolates from dogs with otitis externa. METHODS AND MATERIALS: A total of 53 clinical isolates including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae ssp. pneumoniae and Proteus mirabilis. Antimicrobial susceptibility was determined using disk diffusion; the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of manuka essential oil, with or without Tris-EDTA, were investigated. RESULTS: A total of 44 isolates were resistant to at least one antibiotic and 19 strains were multidrug-resistant, with resistance to at least one agent in three or more antimicrobial classes. The MICs and MBCs of manuka oil alone were ≥1% (v/v) and ≥2% (v/v), respectively. There was no antimicrobial effect of Tris-EDTA (1.125:0.3 mg/mL) without manuka oil. However, the combination of manuka oil with Tris-EDTA significantly decreased the MICs (ranging from 0.06% to 0.5%, v/v; P < 0.001) and MBCs (ranging from 0.06% to 1%, v/v; P < 0.001). There also was no significant difference between multidrug-resistant and nonresistant bacterial isolates in terms of the antimicrobial activity of manuka oil with Tris-EDTA. CONCLUSIONS AND CLINICAL IMPORTANCE: The study findings suggest that manuka oil, especially when combined with Tris-EDTA, may be a promising alternative therapeutic option for Gram-negative otic pathogens. Clinical studies are needed to assess potential for in vivo ototoxic effects and efficacy.

In vitro efficacy of the essential oil from Leptospermum scoparium (manuka) on antimicrobial susceptibility and biofilm formation in Staphylococcus pseudintermedius isolates from dogs.

BACKGROUND: Staphylococcus pseudintermedius is a common pathogen of skin and ear infections in dogs. The widespread and rapid emergence of meticillin-resistant S. pseudintermedius (MRSP) has created therapeutic challenges in veterinary medicine and the need for alternative treatments. HYPOTHESIS/OBJECTIVES: We aimed to evaluate the in vitro antimicrobial activity of the essential oil manuka (Leptospermum scoparium) against S. pseudintermedius. METHODS: This study was performed using S. pseudintermedius strains isolated from dogs with skin and ear infections collected throughout Korea between 2009 and 2011. The in vitro antimicrobial activity of manuka oil against 39 MRSP and 11 meticillin-susceptible S. pseudintermedius (MSSP) strains was analysed by measuring minimal inhibitory concentrations (MICs) using the agar dilution method and biofilm inhibition activity as assessed by the colorimetric microtitre plate assay. RESULTS: Our results indicated that manuka oil had excellent activity against all bacterial isolates. The MICs for MRSP and MSSP to manuka oil were in the range of 2(-9) to 2(-6) and 2(-9) to 2(-7) % (v/v), respectively. Manuka oil was a potent inhibitor of S. pseudintermedius biofilm formation, and the majority of bacteria decreased by >50%. No significant differences were observed in the MICs or biofilm formation between the MRSP and MSSP strains. CONCLUSIONS AND CLINICAL IMPORTANCE: These results suggest that manuka oil has the potential to be a useful therapeutic option for treating superficial infections caused by MRSP and MSSP; further clinical investigations are required.

Characterization and zoonotic potential of uropathogenic Escherichia coli isolated from dogs.

The aim of this study was to investigate the characteristics of canine uropathogenic Escherichia coli (UPEC) and the interaction between canine UPEC and human bladder epithelial cells. Ten E. coli isolates collected from dogs with cystitis were analyzed for antimicrobial resistance patterns, the presence of virulence factors, and biofilm formation. The ability of these isolates to induce cytotoxicity, invade human bladder epithelial cells, and stimulate an immune response was also determined. We observed a high rate of antimicrobial resistance among canine UPEC isolates. All virulence genes tested (including adhesins, iron acquisition, and protectin), except toxin genes, were detected among the canine UPEC isolates. We found that all isolates showed varying degrees of biofilm formation (mean, 0.26; range, 0.07 to 0.82), using a microtiter plate assay to evaluate biofilm formation by the isolates. Cytotoxicity to human bladder epithelial cells by the canine UPEC isolates increased in a time-dependent manner, with a 56.9% and 36.1% reduction in cell viability compared with the control at 6 and 9 h of incubation, respectively. We found that most canine UPEC isolates were able to invade human bladder epithelial cells. The interaction between these isolates and human bladder epithelial cells strongly induced the production of proinflammatory cytokines such as IL-6 and IL-8. We demonstrated that canine UPEC isolates can interact with human bladder epithelial cells, although the detailed mechanisms remain unknown. The results suggest that canine UPEC isolates, rather than dogspecific pathogens, have zoonotic potential.

C-reactive protein as an indicator of inflammatory responses to experimentally induced cystitis in dogs.

The aim of this study was to demonstrate and assess C-reactive protein (CRP) changes in dogs with induced bacterial cystitis with or without antibiotics. We also evaluated availability of CRP levels to serve as an indicator for monitoring or diagnosing bacterial cystitis. Serial CRP concentrations in dogs with induced bacterial cystitis were higher than those of controls (p < 0.001). CRP concentrations peaked on day 7 and gradually decreased thereafter. In the treatment group, CRP concentrations decreased after medication compared to the untreated group (p = 0.032). CRP levels had a linear correlation with urine white blood cell counts among all groups (r = 0.837, p < 0.001, n = 140). Compared to the negative urine culture group, dogs with positive urine culture results had higher CRP concentrations (median 43.8 mg/L vs. 5.9 mg/L; p < 0.001). Area under the receiver operating characteristic curve was 0.955; when cut-off value was 12.2 mg/L, CRP measurements were found to have a sensitivity of 92.3% and specificity of 86.4%. This result indicates that rapid increases of CRP occurred after inducing bacterial cystitis and CRP may be a useful indicator for monitoring or diagnosing canine bacterial cystitis together with sediment urinalysis and urine bacterial culture.

Central nervous system relapses in 3 dogs with B-cell lymphoma.

This report describes cases of central nervous system (CNS) relapse that occurred during chemotherapy in 3 dogs with lymphoma. Diagnosis was made by a combination of clinical signs and cytology of cerebrospinal fluid. The suspected risk factors, clinical features common to the 3 dogs, and treatment options are discussed.

Elevated serum levels of S100 calcium binding protein A8 (S100A8) reflect disease severity in canine atopic dermatitis.

A monoclonal antibody to canine S100 calcium binding protein A8 (S100A8) was developed to determine the association between S100A8 and the disease severity of canine atopic dermatitis. Serum S100A8 concentrations were studied in dogs with canine atopic dermatitis (n=213) and healthy dogs (n=213). Statistical correlations between these indices and atopic dermatitis activity were established, and dermatitis severity was assessed according to the CADESI score. Serum S100A8 concentrations were measured with an enzyme-linked immunosorbent assay (ELISA). S100A8 serum levels were significantly higher in canine atopic dermatitis patients than in healthy dogs. A strong positive correlation was identified between S100A8 levels and canine atopic dermatitis patients. Our findings suggested that S100A8 is actively involved in the pathogenesis and clinical picture of canine atopic dermatitis.

Effect of recombinant lactobacillus expressing canine GM-CSF on immune function in dogs.

Many Lactobacillus strains have been promoted as good probiotics for the prevention and treatment of diseases. We engineered recombinant Lactobacillus casei, producing biologically active canine granulocyte macrophage colony stimulating factor (cGM-CSF), and investigated its possibility as a good probiotic agent for dogs. Expression of the cGM-CSF protein in the recombinant Lactobacillus was confirmed by SDS-PAGE and Western blotting methods. For the in vivo study, 18 Beagle puppies of 7 weeks of age were divided into three groups; the control group was fed only on a regular diet and the two treatment groups were fed on a diet supplemented with either 1 x 10(9) colony forming units (CFU)/day of L. casei or L. casei expressing cGM-CSF protein for 7 weeks. Body weight was measured, and fecal and blood samples were collected from the dogs during the experiment for the measurement of hematology, fecal immunoglobulin (Ig)A and IgG, circulating IgA and IgG, and canine corona virus (CCV)-specific IgG. There were no differences in body weights among the groups, but monocyte counts in hematology and serum IgA were higher in the group receiving L. casei expressing cGMCSF than in the other two groups. After the administration of CCV vaccine, CCV-specific IgG in serum increased more in the group supplemented with L. casei expressing cGM-CSF than the other two groups. This study shows that a dietary L. casei expressing cGM-CSF enhances specific immune functions at both the mucosal and systemic levels in puppies.

Effects of GM-CSF gene transfer using silica-nanoparticles as a vehicle on white blood cell production in dogs.

OBJECTIVE: We sought to test two concepts: that nanoparticles can be used for in vivo gene delivery and that canine granulocyte-macrophage colony-stimulating factor (GM-CSF)/nanoparticles can have possibility to be used to treat transient (acute) canine leukopenia. MATERIALS AND METHODS: We have generated a novel fluorescent-silica nanoparticle binding of canine GM-CSF gene; canine GM-CSF gene was inserted between the cytomegalovirus promoter and poly-adenylation sequences of simian virus 40, and the gene construct was ligated to fluorescent silica nanoparticles functionalized with tertiary amine. RESULTS: When the GM-CSF/nanoparticles were injected into normal dogs, the GM-CSF was expressed in peripheral blood mononuclear cells for at least 9 days and there were significant increases in white blood cell counts, as confirmed by complete blood count, differential count, and flow cytometry. Significant increases in expression of major histocompatibility complex class II on granulocytes and in serum GM-CSF were also observed. Readministration of the nanoparticles was also effective and expression in various tissues was confirmed by reverse transcriptase polymerase chain reaction. CONCLUSIONS: These GM-CSF/nanoparticles may be useful for correction of acute leukopenia, such as chemotherapy-induced myelosuppression without developing neutralizing antibodies.

Palliative intravenous cisplatin treatment for concurrent peritoneal and pleural mesothelioma in a dog.

A 10-year-old Maltese dog was presented with abdominal distention and dyspnea. Cytological examination of pleural and peritoneal effusion was suggestive of malignant effusion of glandular origin. Numerous, multifocal, tan to white nodules were disseminated throughout the surface of the abdominal organs and peritoneum at biopsy. Histologically, the tumors were revealed to be an epithelial type of mesothelioma. Neoplastic cells co-expressed cytokeratin and vimentin. Intravenous administration of cisplatin was chosen as the treatment. During treatment, the dog's overall body condition improved and the clinical signs were relieved without significant side effects. The survival time from diagnosis to sudden death by unknown cause was 153 days.

Antimicrobial effects of silver nanoparticles.

The antimicrobial effects of silver (Ag) ion or salts are well known, but the effects of Ag nanoparticles on microorganisms and antimicrobial mechanism have not been revealed clearly. Stable Ag nanoparticles were prepared and their shape and size distribution characterized by particle characterizer and transmission electron microscopic study. The antimicrobial activity of Ag nanoparticles was investigated against yeast, Escherichia coli, and Staphylococcus aureus. In these tests, Muller Hinton agar plates were used and Ag nanoparticles of various concentrations were supplemented in liquid systems. As results, yeast and E. coli were inhibited at the low concentration of Ag nanoparticles, whereas the growth-inhibitory effects on S. aureus were mild. The free-radical generation effect of Ag nanoparticles on microbial growth inhibition was investigated by electron spin resonance spectroscopy. These results suggest that Ag nanoparticles can be used as effective growth inhibitors in various microorganisms, making them applicable to diverse medical devices and antimicrobial control systems.

Mediastinal lymphoma in a young Turkish Angora cat.

An 8-month old intact male Turkish Angora cat was referred to the veterinary Medical Teaching Hospital (VMTH), Seoul National University, for an evaluation of anorexia and severe dyspnea. The thoracic radiographs revealed significant pleural effusion. A cytology evaluation of the pleural fluid strongly suggested a lymphoma containing variable sized lymphocytes with frequent mitotic figures and prominent nucleoli. The feline leukemia virus and feline immunodeficiency virus tests were negative. The cat was euthanized at his owner's request and a necropsy was performed. A mass was detected on the mediastinum and lung lobes. A histopathology evaluation confirmed the mass to be a lymphoma. Immunohistochemistry revealed the mass to be CD3 positive. In conclusion, the cat was diagnosed as a T-cell mediastinal lymphoma.

Epitheliotropic cutaneous lymphoma (mycosis fungoides) in a dog.

A seven-year-old castrated male Yorkshire terrier dog was presented for a recurrent skin disease. Erythematous skin during the first visit progressed from multiple plaques to patch lesions and exudative erosion in the oral mucosa membrane. Biopsy samples were taken from erythematous skin and were diagnosed with epitheliotropic T cell cutaneous lymphoma by histopathology and immunochemical stain. In serum chemistry, the dog had a hypercalcemia (15.7 mg/dl) and mild increased alkaline phosphatase (417 U/l). Immunohistochemistry was performed to detect parathyroid hormone-related peptide (PTH-rP) in epitheliotropic cutaneous lymphoma tissues but the neoplastic cells were not labeled with anti-PTH-rP antibodies. The patient was treated with prednisolone and isotretinoin. However, the dog died unexpectedly.

Molecular cloning of the cDNA of canine homeodomain-interacting protein kinase 2.

The research of p53 is being conducted to find the mechanisms of tumorigenesis and to treat various cancers. Homeodomain-interacting protein kinase2 (HIPK2) is an important factor to regulate p53 and to increase the stability of p53. Activation of HIPK2 leads to the selective phosphorylation of p53, resulting in growth arrest and the enhancement of apoptosis. In this study, the canine HIPK2 cDNA fragments were obtained, and their overlapping regions were aligned to give a total sequence of 3489 bp. The canine HIPK2 cDNA (GenBank accession number; AY800385) shares 93% and 90% sequence identity with those of human and mouse HIPK2, respectively. The canine HIPK2 cDNA contains an open reading frame encoding 1163 amino acid residues and the predicted amino acid sequence has 98% and 96% identity with those of human and mouse, respectively. The deduced amino acid sequence of canine HIPK2 has also all domains' sites compared with human and mouse HIPK2. Therefore, these structural similarities suggested that the canine HIPK2 shares the basic biological functions that HIPK2 exhibit in other species.