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BACKGROUND: The aim of this double-blind, placebo-controlled study was to investigate the effect of vitamin E supplementation as an addition to a commercial renal diet on survival time of cats with different stages of chronic kidney disease (CKD). In addition, we were interested whether vitamin E supplementation affects selected oxidative stress and clinical parameters. Thirty-four cats with CKD and 38 healthy cats were included in the study. Cats with CKD were classified according to the IRIS Guidelines; seven in IRIS stage 1, 15 in IRIS stage 2, five in IRIS stage 3 and seven in IRIS stage 4. Cats with CKD were treated according to IRIS Guidelines. Cats with CKD were randomly assigned to receive vitamin E (100 IU/cat/day) or placebo (mineral oil) for 24 weeks in addition to standard therapy. Plasma malondialdehyde (MDA) and protein carbonyl (PC) concentrations, DNA damage of peripheral lymphocytes and plasma vitamin E concentrations were measured at baseline and four, eight, 16 and 24 weeks thereafter. Routine laboratory analyses and assessment of clinical signs were performed at each visit. RESULTS: Vitamin E supplementation had no effect on the survival time and did not reduce the severity of clinical signs. Before vitamin E supplementation, no significant differences in vitamin E, MDA and PC concentrations were found between healthy and CKD cats. However, plasma MDA concentration was statistically significantly higher (p = 0.043) in cats with early CKD (IRIS stages 1 and 2) than in cats with advanced CKD (IRIS stages 3 and 4). Additionally, DNA damage was statistically significantly higher in healthy cats (p ≤ 0.001) than in CKD cats. Plasma vitamin E concentrations increased statistically significantly in the vitamin E group compared to the placebo group four (p = 0.013) and eight (p = 0.017) weeks after the start of vitamin E supplementation. During the study and after 24 weeks of vitamin E supplementation, plasma MDA and PC concentrations and DNA damage remained similar to pre-supplementation levels in both the placebo and vitamin E groups. CONCLUSIONS: Vitamin E supplementation as an addition to standard therapy does not prolong survival in feline CKD.
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Enfermedades de los Gatos , Suplementos Dietéticos , Insuficiencia Renal Crónica , Vitamina E , Animales , Gatos , Vitamina E/administración & dosificación , Vitamina E/uso terapéutico , Insuficiencia Renal Crónica/veterinaria , Insuficiencia Renal Crónica/dietoterapia , Insuficiencia Renal Crónica/tratamiento farmacológico , Enfermedades de los Gatos/tratamiento farmacológico , Enfermedades de los Gatos/dietoterapia , Masculino , Femenino , Método Doble Ciego , Estrés Oxidativo/efectos de los fármacos , Malondialdehído/sangre , Daño del ADN/efectos de los fármacos , Alimentación Animal/análisis , Dieta/veterinaria , Carbonilación Proteica/efectos de los fármacosRESUMEN
Chronic kidney disease (CKD) is characterized by chronic inflammation, which mediates the progressive replacement of functional nephrons by fibrotic tissue. Hemogram-derived inflammatory markers are known to serve as markers of pathological conditions; however, their diagnostic value in feline CKD is still unknown. The aim of this retrospective study was to investigate selected hemogram-derived inflammatory markers (neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), platelet-to-lymphocyte ratio (PLR) and the systemic immune-inflammatory index (SII)) in cats at different clinical stages of CKD. Eighty-eight client-owned cats with CKD and thirty-two healthy control cats were included. Cats with CKD were divided into two groups: early CKD (IRIS stage 1 and 2; 62 cats) and progressed CKD (IRIS stage 3 and 4; 26 cats). The values of inflammatory markers were compared between the two CKD groups and the control group. All investigated hemogram-derived inflammatory markers were significantly (p < 0.05) greater in cats with advanced CKD than in those in the other two groups. Additionally, we demonstrated a statistically significant weak to moderate correlation between serum urea, creatinine, selected hematologic and urinary parameters, and the investigated inflammatory markers in cats with CKD. Chronic inflammation can be easily and inexpensively assessed with hemogram-derived markers.
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The role of Mycoplasma canis in canine fertility disorders is still poorly understood. As infection is often asymptomatic, there is an increasing need for appropriate diagnostic methods and treatment plans that would allow the reliable detection of M. canis infection and rapid alleviation of infection symptoms in affected dogs. In this study, we included 14 dogs with fertility problems and 16 dogs without fertility disorder signs. We compared clinical examination data and selected laboratory parameters (hematology and biochemistry) between the groups. We performed PCR-based detection of M. canis and 16S rRNA gene-based microbiota profiling of DNA isolated from vaginal and preputial swabs. Dog sera were tested for the presence of M. canis-specific antibodies. Hematological and selected biochemical parameters showed no differences between groups. PCR-based detection of M. canis in the samples was consistent with the results of 16S microbiota profiling. Several other bacterial taxa were also identified that could potentially be involved in different fertility disorders. Serological methods were not accurate enough since high cross-reactivity rates were observed. In the future, more accurate and efficient methods will be needed to determine the role of M. canis and its true role in the pathogenesis of specific fertility disorders in dogs.
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Electrochemotherapy (ECT) in combination with the gene electrotransfer of interleukin 12 (IL-12 GET) has been successfully used in veterinary medicine for the treatment of mast cell tumours (MCT), but the biomarkers that could predict response to this treatment have not yet been investigated. The aim of this study was to determine the plasma nucleosome and serum ferritin concentrations, as well as the lactate dehydrogenase (LDH) activity, in the serum of treated patients before and one and six months after treatment to evaluate their utility as potential biomarkers that could predict response to the combined treatment. The study was conducted in 48 patients with a total of 86 MCTs that we treated with the combined treatment. The blood samples used for analysing the potential predictive biomarkers were taken before treatment and one and six months after treatment, when the response to treatment was also assessed. The Nu. Q® Vet Cancer Test, the Canine Ferritin ELISA Kit, and the RX Daytona+ automated biochemical analyser were used to analyse the blood samples. The results showed that the plasma nucleosome concentration (before treatment (BT): 32.84 ng/mL (median); one month after treatment (1 M AT): 58.89 ng/mL (median); p = 0.010) and serum LDH activity (BT: 59.75 U/L (median); 1 M AT: 107.5 U/L (median); p = 0.012) increased significantly one month after treatment and that the increase correlated significantly with the presence of a more pronounced local reaction (necrosis, swelling, etc.) at that time point for both markers (nucleosome: BT (necrosis): 21.61 ng/mL (median); 1 M AT (necrosis): 69.92 ng/mL (median), p = 0.030; LDH: BT (necrosis): 54.75 U/L (median); 1 M AT (necrosis): 100.3 U/L (median), p = 0.048). Therefore, both the plasma nucleosome concentration and serum LDH activity could serve as early indicators of the effect of the treatment. In this context, the serum ferritin concentration showed no significant predictive potential for treatment response (p > 0.999 for all comparisons). In conclusion, this study provides some new and important observations on the use of predictive biomarkers in veterinary oncology. Furthermore, it emphasises the need for the continued identification and validation of potential predictive biomarkers in dogs with MCT and other malignancies undergoing ECT treatment in combination with IL-12 GET to ultimately improve treatment outcomes.
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The aim of this study was to further describe the oral microbiota of healthy dogs by DNA shotgun sequencing and compare those to dogs with oral tumors. Oral swabs (representative of all niches of the oral cavity) were collected from healthy dogs (n = 24) and from dogs with different oral tumors (n = 7). DNA was extracted from the swabs and shotgun metagenomic sequencing was performed. Only minor differences in microbiota composition were observed between the two groups. At the phylum level, the Bacteroidota, Proteobacteria, Actinobacteriota, Desulfobacterota and Firmicutes were most abundant in both groups. Observed Operational Taxonomic Units-OTUs (species richness) was significantly higher in the healthy patients, but there was no significant difference in the Shannon diversity index between the groups. No significant difference was found in beta diversity between the groups. The core oral microbiota consisted of 67 bacterial species that were identified in all 24 healthy dogs. Our study provides further insight into the composition of the oral microbiota of healthy dogs and in dogs with oral tumors.
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The combined treatment of electrochemotherapy (ECT) and interleukin-12 (IL-12) gene electrotransfer (GET) has already been used in clinical studies in dogs to treat various histological types of spontaneous tumors. The results of these studies show that the treatment is safe and effective. However, in these clinical studies, the routes of administration of IL-12 GET were either intratumoral (i.t.) or peritumoral (peri.t.). Therefore, the objective of this clinical trial was to compare the two IL-12 GET routes of administration in combination with ECT and their contribution to the enhanced ECT response. Seventy-seven dogs with spontaneous mast cell tumors (MCTs) were divided into three groups: one treated with a combination of ECT + GET peri. t. (29 dogs), the second with the combination of ECT + GET i.t. (30 dogs), and the third with ECT alone (18 dogs). In addition, immunohistochemical studies of tumor samples before treatment and flow cytometry of peripheral blood mononuclear cells (PBMCs) before and after treatment were performed to determine any immunological aspects of the treatment. The results showed that local tumor control was significantly better in the ECT + GET i.t. group (p < 0.050) than in the ECT + GET peri.t. or ECT groups. In addition, disease-free interval (DFI) and progression-free survival (PFS) were significantly longer in the ECT + GET i.t. group than in the other two groups (p < 0.050). The data on local tumor response, DFI, and PFS were consistent with immunological tests, as we detected an increased percentage of antitumor immune cells in the blood after treatment in the ECT + GET i.t. group, which also indicated the induction of a systemic immune response. In addition, we did not observe any unwanted severe or long-lasting side effects. Finally, due to the more pronounced local response after ECT + GET i.t., we suggest that treatment response assessment should be performed at least two months after treatment, which meets the iRECIST criteria.
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Enfermedades de los Perros , Electroquimioterapia , Trastornos Mieloproliferativos , Neoplasias , Animales , Perros , Enfermedades de los Perros/tratamiento farmacológico , Electroquimioterapia/métodos , Electroquimioterapia/veterinaria , Interleucina-12/genética , Leucocitos Mononucleares , Trastornos Mieloproliferativos/tratamiento farmacológico , Neoplasias/tratamiento farmacológicoRESUMEN
The aim of this study was to evaluate the efficacy of metronomic chemotherapy in the palliative treatment of various malignant oral tumors in dogs. Our focus was to determine the effect of treatment on local disease control and to assess the tolerability and safety of the treatment in dogs with various oral malignancies. Metronomic chemotherapy with cyclophosphamide was used to treat 12 dogs and was combined with non-steroidal anti-inflammatory drugs in 6/12 (50%) of dogs. A clinical benefit was observed in 6/12 (50%) patients 1 month and in 4/12 (33%) 3 months after treatment initiation. The median survival time of the dogs was 155 days (range 21-529 days). At the end of the observation period, the disease had progressed in 10/12 (83.3%) of the patients. Sterile hemorrhagic cystitis was the most commonly reported side effect of treatment, occurring in 4/12 (33.3%) dogs. The results of our study suggest that metronomic chemotherapy with cyclophosphamide can be, in a subset of dogs, beneficial in the palliation of malignant oral tumors.
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Electrochemotherapy (ECT) and/or gene electrotransfer of plasmid DNA encoding interleukin-12 (GET pIL-12) are effective treatments for canine cutaneous, subcutaneous, and maxillofacial tumors. Despite the clinical efficacy of the combined treatments of ECT and GET, data on parameters that might predict the outcome of the treatments are still lacking. This study aimed to investigate whether dynamic contrast-enhanced ultrasound (DCE-US) results of subcutaneous tumors differ between tumors with complete response (CR) and tumors without complete response (non-CR) in dogs treated with ECT and GET pIL-12. Eight dogs with a total of 12 tumor nodules treated with ECT and GET pIL-12 were included. DCE-US examinations were performed in all animals before and immediately after therapy as well as 8 h and 1, 3, and 7 days later. Clinical follow-up examinations were performed 7 and 14 days, 1 and 6 months, and 1 year after treatment. Numerous significant differences in DCE-US parameters were noted between tumors with CR and non-CR tumors; perfusion and perfusion heterogeneity were lower in CR tumors than in non-CR tumors. Therefore, studies with larger numbers of patients are needed to investigate whether DCE-US results can be used to predict treatment outcomes and to make effective decisions about the need for repeated therapy or different treatment combinations in individual patients.
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Electroporation is a method of inducing an increase in permeability of the cell membrane through the application of an electric field and can be used as a delivery method for introducing molecules of interest (e.g., chemotherapeutics or plasmid DNA) into cells. Electroporation-based treatments (i.e., electrochemotherapy, gene electrotransfer, and their combinations) have been shown to be safe and effective in veterinary oncology, but they are currently mostly recommended for the treatment of those solid tumors for which clients have declined surgery and/or radiotherapy. Published data show that electroporation-based treatments are also safe, simple, fast and cost-effective treatment alternatives for selected oral and maxillofacial tumors, especially small squamous cell carcinoma and malignant melanoma tumors not involving the bone in dogs. In these patients, a good local response to treatment is expected to result in increased survival time with good quality of life. Despite emerging evidence of the clinical efficacy of electroporation-based treatments for oral and maxillofacial tumors, further investigation is needed to optimize treatment protocols, improve clinical data reporting and better understand the mechanisms of patients' response to the treatment.
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The aim of this study was to evaluate the owners' perception of health-related quality of life (HRQoL) of dogs after treatment with electrochemotherapy (ECT) alone or combined with interleukin-12 gene electrotransfer (IL-12 GET) and/or surgery. The owners of 44 dogs with histologically different tumours were offered the ¼Cancer Treatment Form« at least one month after treatment. The owners assessed their dogs' quality of life (QoL) after treatment as good (mean 7.4) (from 1-very poor to 10-excellent) and the general health compared with the initial diagnosis of cancer as improving (mean 3.9) (from 1-worse to 5-better). The assessment of the current QoL was better within the group of dogs treated with non-invasive treatment (ECT and/or IL-12 GET only), compared with those that received invasive treatment, where, in addition to ECT and/or IL-12 GET, surgery was performed (p < .05). The owners of dogs that achieved an objective response (OR) to the treatment assessed the QoL as significantly better compared with those whose dogs did not respond to the treatment (p < .05). The majority of the owners (86.4%) would opt for the therapy again, regardless of the financial costs. In conclusion, the results of this study demonstrate that the majority of the owners of dogs assessed their dogs' QoL as good and felt that it improved after the treatment, especially in dogs, treated with non-invasive treatment and in those that responded to the treatment. This supports further use of ECT and IL-12 GET as suitable methods for the treatment of selected tumours in veterinary medicine.
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Perros , Electroquimioterapia/veterinaria , Electroporación/veterinaria , Terapia Genética/veterinaria , Interleucina-12/uso terapéutico , Calidad de Vida , Animales , Electroquimioterapia/estadística & datos numéricos , Electroporación/estadística & datos numéricos , Femenino , Terapia Genética/estadística & datos numéricos , Masculino , Estudios ProspectivosRESUMEN
Extracellular vesicles (EVs) are membrane-enclosed fragments shed from all cell types, including tumour cells. EVs contain a wide range of proteins, biolipids and genetic material derived from mother cells and therefore may be potential biomarkers for tumour diagnosis, disease progression and treatment success. We studied the effect of canine mast cell tumours (MCTs) on EV concentrations in blood isolates in association with MCT's histological grade, Ki-67 proliferative index, KIT-staining pattern and number of PLT. The average EV concentration in blood isolates from nine dogs with MCTs was considerably higher than that in blood from eight healthy dogs. But there were no statistically significant differences in EVs concentration in the population of dogs with MCT according to a different histological grade of malignancy (Patnaik, Kiupel), KIT-staining pattern and Ki-67 proliferation index. The results show that these variables statistically do not significantly predicted EV concentrations in blood isolates (P > .05), except the KIT-staining pattern I which added statistically significantly to the prediction (P < .05). The results confirmed the impact of neoplasms on the morphological changes to cell membranes, which result in greater vesiculability and higher EV concentrations.
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Enfermedades de los Perros/sangre , Vesículas Extracelulares , Mastocitoma/veterinaria , Neoplasias Cutáneas/veterinaria , Animales , Estudios de Cohortes , Enfermedades de los Perros/patología , Perros , Femenino , Masculino , Mastocitoma/sangre , Mastocitoma/patología , Neoplasias Cutáneas/sangre , Neoplasias Cutáneas/patologíaRESUMEN
The aim of this study was to evaluate the safety and efficacy of the combination of electrochemotherapy (ECT) with bleomycin and gene electrotransfer (GET) of plasmid encoding canine interleukin 12 (IL-12) for the treatment of canine oral malignant melanoma (OMM). Our focus was to determine the effect of the treatment on achieving local tumor control and stimulation of an antitumor immune response. Nine dogs with histologically confirmed OMM stage I to III were included in a prospective, non-randomized study. The dogs were treated with a combination of cytoreductive surgery, ECT and IL-12 GET, which was repeated up to five times, depending on the clinical response to the treatment, evaluated according to the follow-up protocol (7, 14 and 28â¯days after, the last treatment). One month after treatment, the objective response (OR) rate was 67% (6/9). Median survival time (MST) was 6â¯months and, even though the disease progressed in 8/9 patients at the end of the observation period (2 to 22â¯months), four animals were euthanized due to tumor-unrelated reasons. In addition, we observed a decline in the percentage of regulatory T cells (Treg) in the peripheral blood in the course of the treatment, which could be attributed to a systemic antitumor response to IL-12 GET. The results of this study suggest that a combination of ECT and IL-12 GET may be beneficial for dogs with OMM, especially when other treatment approaches are not acceptable due to their invasiveness or cost.
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Procedimientos Quirúrgicos de Citorreducción , Enfermedades de los Perros , Interleucina-12 , Melanoma , Neoplasias de la Boca , Animales , Perros , Femenino , Terapia Combinada/veterinaria , Procedimientos Quirúrgicos de Citorreducción/veterinaria , Enfermedades de los Perros/terapia , Electroquimioterapia , Interleucina-12/administración & dosificación , Interleucina-12/uso terapéutico , Melanoma/terapia , Melanoma/veterinaria , Neoplasias de la Boca/terapia , Neoplasias de la Boca/veterinaria , Plásmidos , Estudios Prospectivos , Linfocitos T ReguladoresRESUMEN
The electrotransfer of interleukin-12 (IL-12) has been demonstrated as an efficient and safe treatment for tumors in veterinary oncology. However, the plasmids used encode human or feline IL-12 and harbor the gene for antibiotic resistance. Therefore, our aim was to construct plasmids encoding canine IL-12 without the antibiotic resistance genes driven by two different promoters: constitutive and fibroblast-specific. The results obtained in vitro in different cell lines showed that following gene electrotransfer, the newly constructed plasmids had cytotoxicity and expression profiles comparable to plasmids with antibiotic resistance genes. Additionally, in vivo studies showed a statistically significant prolonged tumor growth delay of CMeC-1 tumors compared to control vehicle-treated mice after intratumoral gene electrotransfer. Besides the higher gene expression obtained by plasmids with constitutive promoters, the main difference between both plasmids was in the distribution of the transgene expression. Namely, after gene electrotransfer, plasmids with constitutive promoters showed an increase of serum IL-12, whereas the gene expression of IL-12, encoded by plasmids with fibroblast-specific promoters, was restricted to the tumor. Furthermore, after the gene electrotransfer of plasmids with constitutive promoters, granzyme B-positive cells were detected in the tumor and spleen, indicating a systemic effect of the therapy. Therefore, plasmids with different promoters present valuable tools for focused therapy with local or systemic effects. The results of the present study demonstrated that plasmids encoding canine IL-12 under constitutive and fibroblast-specific promoters without the gene for antibiotic resistance provide feasible tools for controlled gene delivery that could be used for the treatment of client-owned dogs.
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Farmacorresistencia Microbiana , Terapia Genética/métodos , Interleucina-12 , Melanoma , Plásmidos , Animales , Línea Celular Tumoral , Perros , Femenino , Técnicas de Transferencia de Gen , Humanos , Interleucina-12/biosíntesis , Interleucina-12/genética , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Melanoma/terapia , Ratones , Ratones Desnudos , Plásmidos/genética , Plásmidos/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Mast cell tumour, sebaceous gland adenoma, and less common squamous papilloma are skin tumours in ferrets (Mustela putorius furo), and early excisional surgery is usually the treatment of choice. The aim of our study was to investigate the effectiveness of electrochemotherapy (ECT), a new, minimally invasive non-surgical method, as first treatment option of different types of ferret skin tumours located on surgically difficult sites. MATERIALS AND METHODS: A 5-year-old castrated male ferret with two cutaneous masses, presenting 4 months apart and a 7-year-old spayed female ferret with two cutaneous masses, that appeared simultaneously on two locations are presented. In the first patient, both masses were diagnosed as mast cell tumours, and in the second patient, squamous papilloma and sebaceous adenoma were diagnosed. One session of ECT with bleomycin injected intratumourally was applied in all tumours. RESULTS: Complete response (CR) of all tumours was obtained, without recurrence during observation period of 15 months after ECT for first tumour and 11 months after ECT of the tumour located on the right hock in first patient, and 8 months after treatment for the second patient. CONCLUSIONS: In present study, ECT with bleomycin proved to be safe and effective against different cutaneous tumours in ferrets. Due of good results, low cost and relatively easy procedure, ECT could be the treatment of choice instead of surgery for the selected skin tumours in ferrets.
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BACKGROUND: The study was aimed to characterize tumor response after combined treatment employing electrochemotherapy with IL-12 gene electrotransfer in dogs with spontaneous mast cell tumors (MCT). MATERIALS AND METHODS: Eleven dogs with eleven MCTs were included in the study. Histological changes were investigated in biopsy specimens collected before the treatment (T0), and 4 (T1) and 8 weeks (T2) later. Cellular infiltrates were characterized immunohistochemically by using anti CD3, CD20, Foxp3 (Treg), CD68 and anti MHC-class II antibodies. Proliferation and anti-apoptotic activity of neoplastic cells were assessed using anti Ki-67 and Bcl-2 antibodies. Angiogenetic processes were investigated immunohistochemically by using anti Factor VIII and anti CD31 antibodies and micro vessel density quantification. RESULTS: Histopathological examination of samples at T0 confirmed the diagnosis and the presence of scanty infiltrates consisted mainly of T-lymphocytes and macrophages. At T1 and T2 neoplastic cells were drastically reduced in 7/11 cases, small clusters of neoplastic cells were detected in 3/11 cases and 1/11 cases neoplastic cells were still evident. Proliferation activity of neoplastic cells was significantly reduced at T1 and T2 and expression of anti-apoptotic protein at T1. Microvessel density was drastically reduced in all samples after treatment. The number of T-lymphocytes increased at T1, although not significant, while Treg were significant higher at T1 and macrophages at T2. CONCLUSIONS: The combined electrochemotherapy and IL-12 gene electrotransfer effectively induced a cellular response against neoplastic cells characterized mainly by the recruitment of T-lymphocytes and macrophages and a fibrotic proliferation with reduction of microvessels.
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Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane permeabilization by means of "electroporation". To achieve the "electroporation" of the cells, electric pulses are generated by a generator and delivered to the target tissue by the use of electrodes. Electroporation is a physical method which is used to introduce molecules, like cytostatic drugs, into the cells that could not pass the cell membrane on their own. In electrochemotherapy, currently, cisplatin and bleomycin are clinically used. Electrochemotherapy antitumor effectiveness is high, for example up to 100% complete response of canine mast cell tumors smaller than 2 cm3 was achieved. Additionally, electrochemotherapy can be used for the treatment of inoperable tumors. One of the important characteristics of electrochemotherapy is that it can be effective as a one-time treatment only. However, in the case of failure or partial tumor response it can be repeated several times with equal or improved effectiveness. Electrochemotherapy is already a standard treatment for cutaneous and subcutaneous tumors of various histologies in human and veterinary oncology. Furthermore, several clinical studies exploiting electrochemotherapy for deep-seated tumors are on-going.
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Electroquimioterapia , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Bleomicina/uso terapéutico , Gatos , Cisplatino/uso terapéutico , Perros , Electroporación , Humanos , Neoplasias/veterinariaRESUMEN
A gene electrotransfer (GET) of interleukin 12 (IL-12) had already given good results when treating tumors in human and veterinary clinical trials. So far, plasmids used in veterinary clinical studies encoded a human or a feline IL-12 and an ampicillin resistance gene, which is not recommended by the regulatory agencies to be used in clinical trials. Therefore, the aim of the current study was to construct the plasmid encoding a canine IL-12 with kanamycin antibiotic resistance gene that could be used in veterinary clinical oncology. The validation of the newly constructed plasmid was carried out on canine malignant melanoma cells, which have not been used in GET studies so far, and on human malignant melanoma cells. Canine and human malignant melanoma cell lines were transfected with plasmid encoding enhanced green fluorescence protein at different pulse parameter conditions to determine the transfection efficiency and cell survival. The IL-12 expression of the most suitable conditions for GET of the plasmid encoding canine IL-12 was determined at mRNA level by the qRT-PCR and at protein level with the ELISpot assay. The obtained results showed that the newly constructed plasmid encoding canine IL-12 had similar or even higher expression capacity than the plasmid encoding human IL-12. Therefore, it represents a promising therapeutic plasmid for further IL-12 gene therapy in clinical studies for spontaneous canine tumors. Additionally, it also meets the main regulatory agencies' (FDA and EMA) criteria.
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Electroporación/métodos , Terapia Genética/métodos , Interleucina-12/administración & dosificación , Melanoma/terapia , Plásmidos/administración & dosificación , Transfección/métodos , Animales , Gatos , Supervivencia Celular , Perros , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Interleucina-12/metabolismo , Melanoma/genética , Melanoma/patología , Células Tumorales CultivadasRESUMEN
Mycoplasma (M.) cynos is a proven pathogen of dogs causing respiratory infections including pneumonia. We examined 19 M. cynos strains isolated from different organs of dogs in Austria, Denmark and Israel. All strains agglutinated mammalian and chicken erythrocytes. Using erythrocytes of chickens or dogs as specific ligands we isolated an approximately 65 kDa protein from cell-free supernatants of 3 M. cynos strains, which showed an apparent capacity for haemagglutination. The N-terminal sequence of a 25 kDa fragment of this protein was identified as NNEMTPKVTVEAKSMELLLSVEK. The identical amino acid sequence is encoded by the gene MCYN_0308 in the genome of M. cynos C142. This gene belongs to a family of some 20 genes which encode putative lipoproteins with proline-rich regions (PRR) in the first third of their molecules. We termed the 65 kDa haemagglutinin HapA and sequenced hapA gene homologues of 16 M. cynos strains. Analyses of hapA gene homologues revealed similar but not identical sequences, some having insertions and/or deletions in the PRR. We produced a recombinant HapA protein (rHapA) and also mouse monoclonal antibodies (mAbs) recognizing HapA. However, enzyme immunoassays using native M. cynos colonies and mAbs 5G2 or 3B7 showed variable expression of HapA in all M. cynos strains. This was further confirmed by Western blot analyses which showed different HapA quantities and also size-variation of HapA among strains. Analyses of cDNA of the expressed hapA genes showed that besides the hapA gene cultures of M. cynos (strains 105, 2002, 2297) can also express other forms of hap genes. In addition, in cloned cultures of strain 2297 altered HapA epitopes for mAbs 5G2 and 3B7 with distinct hapA gene mutations that resulted in altered HapA amino acid sequence were found. Most of the dogs examined had serum antibodies to rHapA. In conclusion, we characterized the M. cynos haemagglutinin HapA protein and encoding gene hapA, a factor involved in cytadherence to host cells and therefore important for M. cynos infection, and showed that expression of HapA is varied in M. cynos by two distinct mechanisms; differential gene expression and nucleic acid substitution within hapA homologues.
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Enfermedades de los Perros/microbiología , Regulación Bacteriana de la Expresión Génica , Hemaglutininas/genética , Mycoplasma/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Proteínas Bacterianas/genética , Secuencia de Bases , Pollos , ADN Complementario/genética , Perros , Epítopos , Eritrocitos/inmunología , Lipoproteínas/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Mycoplasma/citología , Mycoplasma/inmunología , Mycoplasma/aislamiento & purificación , Proteínas Recombinantes , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
Alterations in serum protein profile, presence of circulating immune complexes (CIC) and proteinuria were investigated in a large group of dogs naturally infected with the Anaplasma phagocytophilum bacterium. Our aim was to evaluate the presence of hypergammaglobulinaemia, CIC and proteinuria as a possible result of an immune-mediated disease following infection by or exposure to A. phagocytophilum. Dogs were divided into three groups - IFA positive (188 dogs with confirmed exposure to A. phagocytophilum), PCR positive (31 dogs with confirmed infection), and control (IFA and PCR negative) (19 dogs). Serum and urine protein patterns were determined by electrophoresis and CIC concentrations by absorbance nephelometry. No significant differences in hypergammaglobulinaemia were observed between the different groups, as shown by the presence of acute phase proteins α2 and ß1-2 globulins. CIC concentrations in the IFA and PCR positive groups were, on average, higher than in controls by 151.3µg/ml, though the differences were not significant. The proportion of dogs with proteinuria did not differ significantly between groups. Our results confirm the assumption that anaplasmosis in dogs is most probably a disease with an acute course, with a good prognosis under the right treatment.
Asunto(s)
Anaplasma phagocytophilum/inmunología , Anaplasmosis/inmunología , Anticuerpos Antibacterianos/sangre , Complejo Antígeno-Anticuerpo/sangre , Enfermedades de los Perros/inmunología , Proteinuria/veterinaria , Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/aislamiento & purificación , Anaplasmosis/complicaciones , Anaplasmosis/microbiología , Animales , Enfermedades de los Perros/microbiología , Perros , Técnica del Anticuerpo Fluorescente Directa/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Proteinuria/complicaciones , Proteinuria/inmunología , Proteinuria/microbiología , alfa-Macroglobulinas/inmunologíaRESUMEN
The aim of this study was to evaluate the efficacy and safety of electrochemotherapy (ECT) with bleomycin for treatment of squamous cell carcinoma (SCC) in cats. Between March 2008 and October 2011, 11 cats with 17 superficial SCC nodules in different clinical stages (ranging from Tis to T4), located on nasal planum (6/11), pinnae (3/11) and both locations (2/11), were included in a prospective non-randomised study. Sixteen of 17 SCC nodules were treated with ECT (15/16 with single session and in one case with two sessions); one nodule was surgically removed. Altogether, complete response (CR) was achieved for 81.8% (9/11) cats and 87.5% (14/16) nodules, lasting from 2 months up to longer than 3 years. Only 2/9 cats in which CR was initially observed, had recurrence 2 and 8 months after the ECT procedure. In the remaining two cats with highly infiltrative spread into adjacent tissues, progression of the disease was observed, despite ECT, and both were euthanased 4 and 5 months after the procedure. ECT in cats was well tolerated and no evident local or systemic side effects were observed. The results of this study suggest that ECT is a highly effective and safe method of local tumour control of feline cutaneous SCCs. It should be considered as an alternative treatment option, especially when other treatment approaches are not acceptable by the owners, owing to their invasiveness, mutilation or high cost.