Effectiveness of Nitazoxanide and Electrolyzed Oxiding Water in Treating Chagas Disease in a Canine Model.

Chagas disease (CD) is caused by the protozoan Trypanosoma cruzi, and affects seven million people in Latin America. Side effects and the limited efficacy of current treatment have led to new drug research. The objective of this work was to evaluate the effectiveness of nitazoxanide (NTZ) and electrolyzed oxidizing water (EOW) in a canine model of experimental CD. Náhuatl dogs were infected with the T. cruzi H8 strain and NTZ- or EOW-treated orally for 10 days. Seronegativity was shown at 12 months post-infection (mpi) in the NTZ-, EOW-, and benznidazole (BNZ)-treated groups. The NTZ and BNZ groups had high levels of IFN-γ, TNF-α, IL-6, IL-12B, and IL-1ß at 1.5 mpi and low levels of IL-10. Electrocardiographic studies showed alterations from 3 mpi and worsening at 12 mpi; NTZ treatment produced fewer cardiac pathomorphological changes compared to EOW, similar to BNZ treatment. There was no cardiomegaly in any group. In conclusion, although NTZ and EOW did not prevent changes in cardiac conductivity, they were able to avoid the severity of heart damage in the chronic phase of CD. NTZ induced a favorable proinflammatory immune response after infection, being a better option than EOW as a possible treatment for CD after BNZ.

COX-2 expression in mammary invasive micropapillary carcinoma is associated with prognostic factors and acts as a potential therapeutic target in comparative oncology.

Pure human and canine mammary invasive micropapillary carcinoma is a rare malignant epithelial tumor accounting for 0.9 to 2% of all invasive mammary carcinomas and present a high rate of lymphatic invasion and metastasis, with unfavorable prognosis. Surgery and chemotherapy are standard treatments for almost all mammary cancer in both species, as well as hormonal and target therapies available for human patients. However, depending on the patient's clinical staging, satisfactory therapeutic results for invasive micropapillary carcinoma are a challenge due to its high capacity of invasion and metastasis. Cyclooxygenase-2 (COX-2) isoform is an important enzyme stimulated by cytokines, growth factors and oncogenes activation to synthetizes prostaglandins in inflammatory process. COX-2 overexpression is associated with angiogenesis and invasion and contributes to cancer development, disease progression, tumor recurrence and regional lymph node metastasis in human and canine mammary carcinomas. This enzyme can be targeted by non-steroidal anti-inflammatory drugs and its inhibition can reduce tumor growth and metastasis in several cancer types. Given the similarity between both species, the present study aims to elucidate the involvement of COX-2 mRNA and protein expression in canine (cIMPC) and human (hIMPC) pure invasive mammary micropapillary carcinoma, with clinicopathological and survival data. Twenty-nine cases of cIMPC and 17 cases of hIMPC were analyzed regarding histologic type, grade, age, tumor size, lymph node condition, extracapsular extension, inflammatory infiltrate and immunophenotype. When available, information on adjuvant treatment, recurrence, metastasis and overall survival were collected. The present study demonstrated COX-2 protein expression in 65.5% of cIMPC and 92.3% of hIMPC, and an association with more advanced histological grades in bitches and higher Ki67 in women. COX-2 mRNA expression was significantly higher in cIMPC than in hIMPC, and its expression was not associated with COX-2 protein expression in both species. COX-2 mRNA expression was associated with negative-ER hIMPC as well as higher Ki67. cIMPC demonstrated proportional early development, more regional metastasis, and a prevalence of negative estrogen receptor, than hIMPC. This is the first time COX-2 expression is associated with negative prognostic factors in both cIMPC and hIMPC, besides the overexpression of COX-2 protein in such unfavorable histological type, which suggests that COX-2 can act as a potential target in IMPC.

Proteomic Analysis Identifies FNDC1, A1BG, and Antigen Processing Proteins Associated with Tumor Heterogeneity and Malignancy in a Canine Model of Breast Cancer.

New insights into the underlying biological processes of breast cancer are needed for the development of improved markers and treatments. The complex nature of mammary cancer in dogs makes it a great model to study cancer biology since they present a high degree of tumor heterogeneity. In search of disease-state biomarkers candidates, we applied proteomic mass spectrometry imaging in order to simultaneously detect histopathological and molecular alterations whilst preserving morphological integrity, comparing peptide expression between intratumor populations in distinct levels of differentiation. Peptides assigned to FNDC1, A1BG, and double-matching keratins 18 and 19 presented a higher intensity in poorly differentiated regions. In contrast, we observed a lower intensity of peptides matching calnexin, PDIA3, and HSPA5 in poorly differentiated cells, which enriched for protein folding in the endoplasmic reticulum and antigen processing, assembly, and loading of class I MHC. Over-representation of collagen metabolism, coagulation cascade, extracellular matrix components, cadherin-binding and cell adhesion pathways also distinguished cell populations. Finally, an independent validation showed FNDC1, A1BG, PDIA3, HSPA5, and calnexin as significant prognostic markers for human breast cancer patients. Thus, through a spatially correlated characterization of spontaneous carcinomas, we described key proteins which can be further validated as potential prognostic biomarkers.

Echocardiographic Findings in Canine Model of Chagas Disease Immunized with DNA Trypanosoma cruzi Genes.

Chagas disease (ChD) is considered an emerging disease in the USA and Europe. Trypanosoma cruzi genes encoding a trans-sialidase protein and an amastigote-specific glycoprotein were tested as vaccines in canine model. The aim for this study was determining the prophylactic effect of these genes in experimentally infected dogs by echocardiography evaluation to compare with our findings obtained by other techniques published previously. Low fractional-shortening values of non-vaccinated dogs suggested an impairment in general cardiac function. Low left ventricular ejection fraction values found in infected dogs suggested myocardial injury regardless of whether they were vaccinated. Low left ventricular diastolic/systolic diameters suggested that progressive heart damage or heart dilation could be prevented by DNA vaccination. Systolic peak time was higher in non-vaccinated groups, increasing vulnerability to malignant arrhythmias and sudden death. High left ventricular volume suggested a decrease in wall thickness that might lead to increased size of the heart cavity, except in the pBCSP plasmid-vaccinated dogs. There was an echocardiographic evidence of left ventricular dilation and reduction in systolic function in experimental chagasic dogs. Echocardiography allowed a more complete follow-up of the pathological process in the living patient than with other techniques like electrocardiography, anatomopathology, and histopathology, being the method of choice for characterizing the clinical stages of ChD.

Detection of allopurinol and oxypurinol in canine urine by HPLC/MS-MS: Focus on veterinary clinical pharmacokinetics.

Allopurinol is the most commonly used drug for the treatment of hyperuricemia in people, and in view of the risks of fatal hypersensitivity in patients with renal dysfunction, doses based on the glomerular filtration rate are proposed. In veterinary medicine, allopurinol is used in the treatment of canine leishmaniasis (CanL) caused by Leishmania infantum owing to the drug action of inhibiting the parasite's RNA synthesis. However, renal dysfunction frequently ensues from disease progression in dogs. The purpose of the present study was to standardize and validate a sensitive high-performance liquid chromatography-mass spectrometric (HPLC-MS/MS) method to determine the concentration of allopurinol and its active metabolite oxypurinol in canine urine for clinical pharmacokinetic investigation. Urine samples of eleven (11) dogs with naturally occurring CanL and in the maintenance phase of the treatment with alopurinol were used. For the chromatographic analysis of urine, the mobile phase consisted of a solution of 0.1 % formic acid (88 %) in 10 mM ammonium acetate. Separation of allopurinol and oxypurinol occurred in a flow of 0.8 mL/min on a C8 reverse phase column 5 µm, and acyclovir was the internal standard. The HPLC-MS/MS method was validated by reaching the limits of detection and quantification, reproducibility and linearity. The lower limit of quantification achieved by the method was 10 µg/mL for both allopurinol and oxypurinol. Calibration curves were prepared in blank urine added with allopurinol at concentrations of 10-1000 µg/mL, and oxypurinol at 10-200 µg/mL. Coefficients of variation of less than 15 % between intracurrent and intercurrent accuracy values were observed for both allopurinol and oxypurinol. Urine test samples remained stable after being subjected to freeze-thaw cycles and remaining at room temperature for 4 h. The method proved to be adequate to quantify allopurinol and oxypurinol in urine samples from dogs under treatment.

Echocardiographic Measurements in a Preclinical Model of Chronic Chagasic Cardiomyopathy in Dogs: Validation and Reproducibility.

Background: The failure to translate preclinical results to the clinical setting is the rule, not the exception. One reason that is frequently overlooked is whether the animal model reproduces distinctive features of human disease. Another is the reproducibility of the method used to measure treatment effects in preclinical studies. Left ventricular (LV) function improvement is the most common endpoint in preclinical cardiovascular disease studies, while echocardiography is the most frequently used method to evaluate LV function. In this work, we conducted a robust echocardiographic evaluation of LV size and function in dogs chronically infected by Trypanosoma cruzi. Methods and Results: Echocardiography was performed blindly by two distinct observers in mongrel dogs before and between 6 and 9 months post infection. Parameters analyzed included end-systolic volume (ESV), end-diastolic volume (EDV), ejection fraction (EF), and fractional shortening (FS). We observed a significant LVEF and FS reduction in infected animals compared to controls, with no significant variation in volumes. However, the effect of chronic infection in systolic function was quite variable, with EF ranging from 17 to 66%. Using the cut-off value of EF ≤ 40%, established for dilated cardiomyopathy (DCM) in dogs, only 28% of the infected dogs were affected by the chronic infection. Conclusions: The canine model of CCC mimics human disease, reproducing the percentage of individuals that develop heart failure during the chronic infection. It is thus mandatory to establish inclusion criteria in the experimental design of canine preclinical studies to account for the variable effect that chronic infection has on systolic function.

Morphological and ultrastructural evaluation of the golden retriever muscular dystrophy trachea, lungs, and diaphragm muscle.

Duchenne muscular dystrophy (DMD) is a genetic disease, characterized by atrophy and muscle weakness. The respiratory failure is a common cause of early death in patients with DMD. Golden retriever muscular dystrophy (GRMD) is a canine model which has been extensively used for many advances in therapeutics applications. As the patients with DMD, the GRMD frequently died from cardiac and respiratory failure. Observing the respiratory failure in DMD is one of the major causes of mortality we aimed to describe the morphological and ultrastructural data of trachea, lungs (conductive and respiratory portion of the system), and diaphragm muscle using histological and ultrastructural analysis. The diaphragm muscle showed discontinuous fibers architecture, with different diameter; a robust perimysium inflammatory infiltrate and some muscle cells displayed central nuclei. GRMD trachea and lungs presented collagen fibers and in addition, the GRMD lungs showed higher of levels collagen fibers that could limit the alveolar ducts and alveoli distension. Therefore, the most features observed were the collagen areas and fibrosis. We suggested in this study that the collagen remodeling in the trachea, lungs, and diaphragm muscle may increase fibrosis and affect the trachea, lungs, and diaphragm muscle function that can be a major cause of respiratory failure that occur in patients with DMD.

Effect of acute endotoxemia on analog estimates of mean systemic pressure.

Dynamic estimates of mean systemic pressure based on a Guytonian analog model (Pmsa) appear accurate under baseline conditions but may not remain so during septic shock because blood volume distribution and resistances between arterial and venous beds may change. Thus, we examined the effect of acute endotoxemia on the ability of Pmsa, estimated from steady-state cardiac output, right atrial pressure, and mean arterial pressure, to reflect our previously validated instantaneous venous return measure of mean systemic pressure (Pmsi), derived from beat-to-beat measures of right ventricular stroke volume and right atrial pressure during positive pressure ventilation. We studied 6 splenectomized pentobarbital-anesthetized close chested dogs. Right ventricular stroke volume was measured by a pulmonary arterial electromagnetic flow probe. Instantaneous venous return measure of mean systemic pressure and Pmsa were calculated during volume loading and removal (±100-mL bolus increments×5) both before (control) and 30 minutes after endotoxin infusion (endo). Cardiac output increased (2628±905 vs 3560±539 mL/min; P<.05) and mean arterial pressure decreased (107±16 vs 56±12 mm Hg; P<.01) during endo. Changes in Pmsi and Pmsa correlated during both control and endo (r2=0.7) with minimal bias by Bland-Altman analysis (mean difference±95% confidence interval, 0.47±5.04 mm Hg). We conclude that changes in Pmsa accurately tracts Pmsi under both control and endo.

Estudo da biodistribuição de células tronco de polpa de dente decíduo humana (CTPDDh) após o transplante intra-uterino no modelo canino (Canis lupus familiares) / Biodistribution of human immature dental pulp stem cells following in utero transplantation in canine model (Canis lupus familiaris)

O transplante intrauterino de células-tronco (TIUCT) é um método de tratamento de doenças genéticas, congênitas, hematológicas e imunológicas em um feto durante a gestação. Em pesquisa básica este modelo permite o estudo da dinâmica de migração, enxertia e estado funcional de diferentes tipos de células-tronco (CT). Estas células podem ser transplantadas em diferentes momentos do período gestacional, que pode ser dividido em três momentos do desenvolvimento fetal, sendo estes, diferentes funcionalmente. A escolha deste momento para o transplante influenciará tanto no comportamento celular quanto no resultado. Para o TIUCT são utilizadas as CT mesenquimais derivadas da medula óssea ou fetais ou hematopoiéticas. Para esta pesquisa utilizamos células-tronco derivadas da polpa dentária imatura humana (CTIPDh) as quais apresentam potencial pluripotente e propriedades imunomodulatórias. Nosso principal objetivo foi avaliar a capacidade migratória, bem como de proliferação e endereçamento (homing) das CTIPDh durante o terceiro período gestacional do desenvolvimento fetal no modelo canino. Todos os procedimentos experimentais foram elaborados sob protocolo anestésico apropriado e aprovados pelo comitê de ética da FMVZ da USP. Foram transplantadas via intraperitoneal (IP) 1x106 CTIPDh GFP+ em cada feto, durante procedimento cirúrgico de laparotomia exploratória com ultrassonografia guiada intraoperatóriamente em quatro fetos com idade gestacional aproximada de 45 dias, e outros dois fetos os quais não receberam o transplante, utilizados como controle. Avaliamos os fetos pré e pós-transplante através do ultrasson. Após sete dias, realizamos a ovário-salpingo-histerectomia (OSH) para a colheita dos fetos. Em seguida coletamos seus órgãos e tecidos os quais foram fixados em paraformoldeído a 4% e criopreservados a temperatura de -80oC. Analisamos a biodistribuição das CTIPDh dentro dos órgãos e tecidos em criocortes de 5µm sob microscopia Confocal.

Intra-uterine stem cells transplantation (IUSCT) is a method for the treatment of genetic, congenital, hematological, and immunological diseases. In basic research it provides a model for studying the dynamics of migration, graft and functional status of different types of stem cells. The cells can be transplanted in different moments of gestational period, which can be divided into quarters that are not functionally equivalent. The choice of the cells and quarter where the stem cells will be applied can influence cells behavior and results of transplantation. Fetal and adult hematopoietic or bone marrow derived mesenchymal stem cells (MSCs) were mainly used for IUSCT. We previously obtained human immature dental pulp stem cell (IDPSCs), which showed pluripotent potential and immune-compatible properties. The goal of our study was to evaluate migration capacity, proliferation and homing of IDPSCs after IUSCT during the third fetal period in dogs.