From Inflammation to Cutaneous Repair: Topical Application of Lupeol Improves Skin Wound Healing in Rats by Modulating the Cytokine Levels, NF-κB, Ki-67, Growth Factor Expression, and Distribution of Collagen Fibers.

Skin wound healing is a highly complex event that involves different mediators at the cellular and molecular level. Lupeol has been reported to possess different biological activities, such as anti-inflammatory, antioxidant, antidiabetic, and in vitro wound healing properties, which motivated us to proceed with in vivo studies. We aimed to investigate the wound healing effect of lupeol-based cream for 3, 7, and 14 days. Wound excisions were induced on the thoraco-lumbar region of rats and topically treated immediately after injury induction. Macroscopic, histopathological, and immunohistochemical analyses were performed. Cytokine levels were measured by ELISA and gene expression was evaluated by real-time RT-qPCR. Our results showed a strong wound-healing effect of lupeol-based cream after 7 and 14 days. Lupeol treatment caused a reduction in proinflammatory cytokines (TNF-a, IL-1ß, and IL-6) and gene and protein NF-κB expression, and positively altered IL-10 levels, showing anti-inflammatory effects in the three treatment periods. Lupeol treatment showed involvement in the proliferative phase by stimulating the formation of new blood vessels, increasing the immunostaining of Ki-67 and gene expression, and immunolabeling of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), and increasing gene expression of transforming growth factor beta-1 (TGF-ß1) after seven days of treatment. Lupeol was also involved in the tissue regeneration phase by increasing the synthesis of collagen fibers noted in the three treatment periods analyzed. Our findings suggest that lupeol may serve as a novel therapeutic option to treat cutaneous wounds by regulating mechanisms involved in the inflammatory, proliferative, and tissue-remodeling phases.

Evaluation of heart rate variability and behavior of electrocardiographic parameters in dogs affected by chronic Monocytic Ehrlichiosis.

Canine Monocytic Ehrlichiosis (CME) is a systemic disease prevalent in the entire world caused by the obligate intracellular bacteria Ehrlichia canis. The occurrence of myocarditis with a high prevalence of arrhythmias in dogs affected by this disease in the cytopenic phase has already been proven. This study aims to evaluate the concentrations of CK MB, cTnI and NT-proBNP in dogs affected by Ehrlichia canis in the chronic phase since the intense stimulation of the immune system can lead to myocarditis; to evaluate if the condition can lead to arrhythmic events and, if so, define their frequency and classification through conventional and ambulatory electrocardiogram tests (Holter method) for a period of 24 hours; to analyze heart rate variability in the time domain and whether the condition can lead to autonomic imbalance; and to determine the survival rate of affected dogs, identifying possible risk factors for mortality at this stage of the disease. For this purposes, we evaluated clinical, hematological and biochemical data, as well as the concentrations of cardiac biomarkers Creatine Kinase-MB (CK MB), Cardiac Troponin I (cTnI) and N-terminal pro-peptide natriuretic type B (NT-proBNP). We also analyzed conventional and ambulatory electrocardiography (24-hour Holter) and heart rate variability (HRV) in 20 dogs afflicted by cytopenic CME in the chronic phase of the disease (G1) and compared the results with a control group comprised of ten healthy dogs (G2). G1 was monitored during the treatment for 28 days, during which eight (8) of the 20 infected dogs died (40%). Anorexia, vomiting, fatigue, hypoalbuminemia, heart murmurs and increased concentrations of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were common clinical signs. The mean concentrations of cTnI and CKMB were significant (0.24 ng / mL ± 0.5, 229 ± 205 IU / mL) in comparison to the control group (0.042 ± 0.07 ng / mL, 126 ± 46.12 IU / mL). No significant differences were observed between NT-proBNP concentrations in G1 (135.46 ± 29.7) and G2 (138.28 ± 19.77). Nine of the twenty dogs (45%) presented a high frequency of arrhythmias during 24-hour recording, ranging from first and second-degree atrioventricular block, ventricular and supraventricular ectopic events and sinus tachycardia. No sinus pause was observed. One dog had 120 episodes of unsustained ventricular tachycardia and two episodes of sustained ventricular tachycardia. The short-term and long-term HRV data, represented by SDNN (ms), SDANN (ms) and pnn50 (%) were also significant lower (83 ± 65, 56.05 ± 37.3 and 14.56 ± 20, respectively) in comparison to the healthy animals (268 ± 74.6, 168.3 ± 39.14 and 55.87 ± 12.8, respectively). These results suggest that cytopenic CME is characterized by an arrhythmogenic component and intense stimulation of the sympathetic autonomic nervous system in the heart, reflecting an imbalance in the activity of the ANS.

Conventional and Holter Electrocardiographic Assessment of Dogs Infected Naturally With Acute Canine Monocytic Ehrlichiosis.

Canine Monocytic Ehrlichiosis (CME) is a disease of worldwide distribution caused by the bacteria Ehrlichia canis, appearing primarily in hot climates due to the massive prevalence of its vector, the tick Rhipicephalus sanguineus. Previous studies have shown that dogs afflicted by CME in the chronic phase can develop infectious myocarditis, arrhythmias, and alterations in heart rate variability (HRV), but there are few studies correlating cardiac diseases with the acute phase of CME. This study aims at assessing electric cardiac alterations and HRV in the time and frequency domains during the acute phase of CME. This study assessed 22 animals divided into 2 distinct groups: the control group, comprised by 10 healthy dogs, and the sick group, comprised of 12 dogs infected naturally with ehrlichiosis which presented clinical and hematological signs compatible with the acute phase of the disease. The animals underwent conventional and Holter electrocardiographic evaluations, systolic blood pressure measurement, complete blood count and biochemical assays (urea, creatinine, alanine aminotransferase (ALT), alkaline phosphatase (AP), and gamma glutamyl transferase (GGT)). The sick group presented higher activity in the sympathetic nervous system than in the parasympathetic nervous system, manifest as a significant increase in mean heart rate and a reduction in the HRV indexes for the time and frequency domains. The frequency-domain HRV indexes presented sympathetic prevalence during the sleep and vigilance states. Sinus tachycardia was the predominant heart rhythm in 58.33% of the animals. The mean systolic blood pressure diverged between the groups and no significant arrhythmias were observed during monitoring. The serum concentrations of urea, creatinine, AP, ALT, and GGT were within the established reference values for the species. We observed no indication that there was enough time during the acute phase for the disease to evolve in a way that resulted in arrhythmias, as is common in the chronic phase, but we observed that animals in the acute phase already present reduced HRV indexes.

Avaliação do perfil liquórico de caninos (Canis lupus familiaris) naturalmente infectados com o vírus da cinomose antes e após tratamento com ribavirina (Ribaviron C®)

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The relationship between the degree of thrombocytopenia and infection with Ehrlichia canis in an endemic area.

Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis. In order to evaluate platelet counts as a screening test for E. canis in an endemic area, 217 whole blood samples from dogs were divided into three groups: 71 non-thrombocytopenic samples (group A, platelet counts greater than 200 000/mL) and 146 thrombocytopenic samples (less than 200 000/mL). The thrombocytopenic group was further divided into 62 with platelet counts between 100 000-200 000/mL (Group B) and 84 samples with less than 100 000 platelets/mL (Group C). All samples were examined for the presence of a segment of the Ehrlichia canis 16S rRNA gene using a nested polymerase chain reaction. Sixty-seven of the 217 samples (30.9%) were positive for the presence of the E. canis 16S rRNA gene; 53 (63.1%) of the group C samples and 13 (21%) of group B. Only one (1.4%) of the non-thrombocytopenic samples (Group A) was positive. These data support the concept that platelet counts may be a good screening test for canine monocytic ehrlichiosis, and that the magnitude of thrombocytopenia may increase the reliability of diagnosis.