Antimicrobial Activity of Plasma Rich in Platelets (PRP) on the Ocular Microbiota of Healthy Horses from Minas Gerais: In Vitro Study.

In equine ophthalmology, ulcerative keratitis is among the most common conditions and, in general, arises as a consequence of some trauma suffered. Secondarily, subsequent contamination by pathogenic or resident bacteria of the horse's ocular microbiota may have undesirable consequences. Under physiological conditions, the normal microbiota coexists with the immune status of the host, serving as a barrier, ensuring the health of the ocular surface, and inhibiting the proliferation of pathogens. However, in the imbalance of immune barriers, the normal microbiota can become pathogenic and lead to infection, acting as an opportunistic agent. The present study aims to demonstrate the antimicrobial effect of platelet-rich plasma (PRP), its time of action, and its correlation with the concentration of its same components in vitro on Staphylococcus sciuri, a bacterium with high prevalence in the normal ocular microbiota of horses in the municipality of Minas Gerais. For the preparation of the PRP, eight adult Quarter Horse (QH) horses were used. The individual PRP was prepared by the double centrifugation protocol, and then, the PRPs were added to a pool, followed by testing their interaction in culture with Brain Heart Infusion (BHI) broth at different dilutions against five strains collected from different animals. After 3, 6, 12, and 18 hours, the colony formation units (CFU) count on a 5% horse blood agar plate was evaluated for each time point. Our study showed that Staphylococcus sciuri, the resident microorganism of the ocular conjunctival microbiota of horses, is more susceptible when compared to the standard strain "American Type Culture Collection" (ATCC-29213) Staphylococcus aureus, a pathogenic microorganism, which was used for the validation of our study. The antibacterial effect shown in this study was bacteriostatic for up to 6 hours. The most concentrated PRP dilutions, 1 : 1 and 1 : 2, were also most effective, suggesting that the antibacterial effect is volume dependent.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Flow rates of double-lumen over-the-wire catheters for high-volume fluid resuscitation in large animal species.

OBJECTIVES: To evaluate the maximum in vitro flow rate of 6 types of polyurethane over-the-wire double lumen catheters using both ports, for high volume fluid resuscitation in large animal species. SETTINGS: University teaching hospital. DESIGN: Prospective in vitro experimental study. INTERVENTIONS: The flow rate of both ports of 6 polyurethane double lumen over-the-wire catheters (11 and 13-Fr, 15 and 20 cm long, elliptical and tapered tip designs) and 2 types of infusion (with or without pressure bags) were tested on a factorial scheme (6 × 2) in triplicate, using commercial isotonic crystalloid (0.9% NaCl) and synthetic colloid (6% Hydroxyethyl starch, 130/0.4). MEASUREMENTS AND MAIN RESULTS: Flow rates were influenced by catheter diameter, length, tip design and presence or absence of pressure bags (P < 0.05). Mean flow rates during non-pressurized 0.9% NaCl infusion ranged from 584 mL/min (35 L/h; 11-Fr x 15 cm x tapered tip catheter) to 905 mL/min (54 L/h; 13-Fr x 15 cm x elliptical tip catheter). Mean flow rates during non-pressurized synthetic colloid infusion varied from 404 mL/min (24 L/h; 11-Fr x 15 cm x tapered tip catheter) to 724 mL/min (43 L/h; 13-Fr x 15 cm x elliptical tip catheter). Mean flow rates during pressurized infusion were 1.72 and 2.02 times greater than those obtained by gravity alone for 0.9% NaCl and synthetic colloid, respectively (P < 0.05). CONCLUSIONS: Highest in vitro flow rates were achieved when larger diameter, shorter and elliptical tip catheters were used during 0.9% NaCl infusion. Catheter diameter, tip design but not length influenced the flow rate during synthetic colloid infusion. The use of pressure bags significantly increased the flow rate of all catheters, for both solutions.

miR-210 and miR-152 as Biomarkers by Liquid Biopsy in Invasive Ductal Carcinoma.

Detecting circulating microRNAs (miRNAs; miRs) by means of liquid biopsy is an important tool for the early diagnosis and prognosis of breast cancer (BC). We aimed to identify and validate miR-210 and miR-152 as non-invasive circulating biomarkers, for the diagnosis and staging of BC patients, confirming their involvement in tumor angiogenesis. METHODS: RT-qPCR was performed and MiRNA expression analysis was obtained from plasma and fragments of BC and benign breast condition (BBC) women patients, plus healthy subjects. Additionally, the immunohistochemistry technique was carried out to analyze the expression of target proteins. RESULTS: Tumor fragments showed increased expression of oncomiR-210 and decreased expression of miR-152 tumoral suppressor. Both miRNAs were increased in plasma samples from BC patients. The receiver operating characteristic (ROC) curve analysis revealed that only the expression of oncomiR-210 in tissue samples and only the expression of the miR-152 suppressor in plasma have the appropriate sensitivity and specificity for use as differential biomarkers between early/intermediate and advanced stages of BC patients. In addition, there was an increase in the expression of hypoxia-inducible factor 1-alpha (HIF-1α), insulin-like growth factor 1 receptor (IGF-1R), and vascular endothelial growth factor (VEGF) in BC patients. On the contrary, a decrease in Von Hippel-Lindau (VHL) protein expression was observed. CONCLUSIONS: This study showed that increased levels of miR-210 and decreased levels of miR152, in addition to the expressions of their target proteins, could indicate, respectively, the oncogenic and tumor suppressive role of these miRNAs in fragments. Both miRNAs are potential diagnostic biomarkers for BC by liquid biopsy. In addition, miR-152 proved to be a promising biomarker for disease staging.

Signalling pathways involved in p47phox -dependent reactive oxygen species in platelets of endotoxemic rats.

Thrombocytopenia during sepsis is associated with a less favourable clinical outcome. Overproduction of reactive oxygen species (ROS) by different cell types contributes to sepsis. Platelets generate ROS, but the upstream pathways of NADPH oxidase activation are not completely understood. Here, we designed experiments in washed platelets from lipopolysaccharide (LPS)-treated rats to investigate the p47phox activation and ROS generation, and its modulation by c-Src family kinase (c-Src), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC) and protein kinase G (PKG). Rats were injected intraperitoneally with LPS (1 mg/kg), and at 48 hours thereafter, arterial blood was collected and washed platelets were obtained. Washed platelets were pre-incubated with different inhibitors and subsequently activated or not with ADP. Flow cytometry, Western blotting and ELISA were performed. We found that LPS significantly increased the p47phox phosphorylation and ROS generation compared with the control group (P < 0.05). The enhanced ROS production in the LPS group was unaffected by the non-selective SFKs inhibitor PP2, the PI3K inhibitor wortmannin or the Akt inhibitor PPI-1. The cyclic GMP levels were 115% higher in activated platelets of LPS compared with the saline group (P < 0.05). Moreover, in the LPS group, the sGC inhibitor ODQ, the PKG inhibitor Rp-8-Br and the PKC inhibitor GF109203X abrogated the increased p47phox phosphorylation and reduced the ROS levels. In conclusion, selective inhibitors of cGMP-PKG and PKC-p47phox pathways that regulate ROS generation by LPS in platelets may help control the redox balance in sepsis improving the survival of patients.

Melatonin restrains angiogenic factors in triple-negative breast cancer by targeting miR-152-3p: In vivo and in vitro studies.

AIMS: Breast cancer represents the second most prevalent tumor-related cause of death among women. Although studies have already been published regarding the association between breast tumors and miRNAs, this field remains unclear. MicroRNAs (miRNAs) are defined as non-coding RNA molecules, and are known to be involved in cell pathways through the regulation of gene expression. Melatonin can regulate miRNAs and genes related with angiogenesis. This hormone is produced naturally by the pineal gland and presents several antitumor effects. The aim of this study was to understand the action of melatonin in the regulation of miRNA-152-3p in vivo and in vitro. MAIN METHODS: In order to standardize the melatonin treatment in the MDA-MB-468 cells, we carried out the cell viability assay at different concentrations. PCR Array plates were used to identify the differentiated expression of miRNAs after the treatment with melatonin. The relative quantification of the target gene expression (IGF-IR, HIF-1α and VEGF) was performed by real-time PCR. For the tumor development, MDA-MB-468 cells were implanted in female BALB/c mice, and treated or not treated with melatonin. Moreover, the quantification of the target genes protein expression was performed by immunocytochemistry and immunohistochemistry. KEY FINDINGS: Relative quantification shows that the melatonin treatment increases the gene expression of miR-152-3p and the target genes, and decreased protein levels of the genes both in vitro and in vivo. SIGNIFICANCE: Our results confirm the action of melatonin on the miR-152-3p regulation known to be involved in the progression of breast cancer.