The membrane of methicillin-resistant Staphylococcus aureus (MRSA) contains penicillin-binding proteins (PBPs) in the phospholipidic bilayer, with the protein PBP2a being linked with the resistance ...mechanism. In this work we confirm the role of PBP2a with molecular-level information obtained with Langmuir monolayers as cell membrane models. The MRSA cell membrane was mimicked with a mixed monolayer of dipalmitoyl phosphatidyl glycerol (DPPG) and cardiolipin (CL), also incorporating PBP2a. The surface pressure-area isotherms and the Brewster angle microscopy (BAM) images for these mixed monolayers were significantly affected by the antibiotic meropenem, which is PBP2a inhibitor. The meropenem effects were associated with the presence of PBP2a, as they were absent in the Langmuir monolayers without PBP2a. The relevance of PBP2a was confirmed with results where the antibiotic methicillin, known to be unsuitable to kill MRSA, had the same effects on mixed DPPG/CL and DPPG/CL-PBP2a monolayers since it prevented PBP2a from incorporating in the monolayer. The biological implication of the findings presented here is that a successful antibiotic against MRSA should be able to interact with PBP2a, but in the membrane.
Display omitted
•Langmuir monolayers to represent the MRSA membrane.•Interaction between model membrane and antibiotics against MRSA.•Penicillin-binding proteins determine antibiotic action in model membranes.
Crush injuries in peripheral nerves are frequent and induce long-term disability with motor and sensory deficits. Due to axonal and myelin sheath disruptions, strategies for optimized axonal ...regeneration are needed. Multipotent mesenchymal stromal cells (MSC) are promising because of their anti-inflammatory properties and secretion of neurotrophins. The present study investigated the effect of canine adipose tissue MSC (Ad-MSC) transplantation in an experimental sciatic nerve crush injury. Wistar rats were divided into three groups: sham (n = 8); Crush+PBS (n = 8); Crush+MSC (n = 8). Measurements of sciatic nerve functional index (SFI), muscle mass, and electromyography (EMG) were performed. Canine Ad-MSC showed mesodermal characteristics (CD34-, CD45-, CD44+, CD90+ and CD105+) and multipotentiality due to chondrogenic, adipogenic, and osteogenic differentiation. SFI during weeks 3 and 4 was significantly higher in the Crush+MSC group (p < 0.001). During week 4, the EMG latency in the Crush+MSC groups had better near normality (p < 0.05). The EMG amplitude showed results close to normality during week 4 in the Crush+MSC group (p < 0.04). There were no statistical differences in muscle weight between the groups (p > 0.05), but there was a tendency toward weight gain in the Crush+MSC groups. Better motor functional recovery after crush and perineural canine Ad-MSC transplantation was observed during week 2. This was maintained till week 4. In conclusion, the canine Ad-MSC transplantation showed early pro-regenerative effects between 2–4 weeks in the rat model of sciatic nerve crush injury.
Background Cardiovascular diseases remain leaders as the major causes of mortality in Western society. Restoration of the circulation through construction of bypass surgical treatment is regarded as ...the gold standard treatment of peripheral vascular diseases, and grafts are necessary for this purpose. The great saphenous vein is often not available and synthetic grafts have their limitations. Therefore, new techniques to produce alternative grafts have been developed and, in this sense, tissue engineering is a promising alternative to provide biocompatible grafts. This study objective was to reconstruct the endothelium layer of decellularized vein scaffolds, using mesenchymal stem cells (MSCs) and growth factors obtained from platelets. Methods Fifteen nonpregnant female adult rabbits were used for all experiments. Adipose tissue and vena cava were obtained and subjected to MSCs isolation and tissue decellularization, respectively. MSCs were subjected to differentiation using endothelial inductor growth factor (EIGF) obtained from human platelet lysates. Immunofluorescence, histological and immunohistochemical analyses were employed for the final characterization of the obtained blood vessel substitute. Results The scaffolds were successfully decellularized with sodium dodecyl sulfate. MSCs actively adhered at the scaffolds, and through stimulation with EIGF were differentiated into functional endothelial cells, secreting significantly higher quantities of von Willebrand factor (0.85 μg/mL; P < .05) than cells cultivated under the same conditions, without EIGF (0.085 μg/mL). Cells with evident morphologic characteristics of endothelium were seen at the lumen of the scaffolds. These cells also stained positive for fascin protein, which is highly expressed by differentiated endothelial cells. Conclusions Taken together, the use of decellularized bioscaffold and subcutaneous MSCs seems to be a potential approach to obtain bioengineered blood vessels, in the presence of EIGF supplementation.
Background Nerve injuries are debilitating, leading to long-term motor deficits. Remyelination and axonal growth are supported and enhanced by growth factor and cytokines. Combination of nerve ...guidance conduits (NGCs) with adipose-tissue-derived multipotent mesenchymal stromal cells (AdMSCs) has been performing promising strategy for nerve regeneration. Methods 3D-printed polycaprolactone (PCL)-NGCs were fabricated. Wistar rats subjected to critical sciatic nerve damage (12-mm gap) were divided into sham, autograft, PCL (empty NGC), and PCL + MSCs (NGC multi-functionalized with 10.sup.6 canine AdMSCs embedded in heterologous fibrin biopolymer) groups. In vitro, the cells were characterized and directly stimulated with interferon-gamma to evaluate their neuroregeneration potential. In vivo, the sciatic and tibial functional indices were evaluated for 12 weeks. Gait analysis and nerve conduction velocity were analyzed after 8 and 12 weeks. Morphometric analysis was performed after 8 and 12 weeks following lesion development. Real-time PCR was performed to evaluate the neurotrophic factors BDNF, GDNF, and HGF, and the cytokine and IL-10. Immunohistochemical analysis for the p75.sup.NTR neurotrophic receptor, S100, and neurofilament was performed with the sciatic nerve. Results The inflammatory environment in vitro have increased the expression of neurotrophins BDNF, GDNF, HGF, and IL-10 in canine AdMSCs. Nerve guidance conduits multi-functionalized with canine AdMSCs embedded in HFB improved functional motor and electrophysiological recovery compared with PCL group after 12 weeks. However, the results were not significantly different than those obtained using autografts. These findings were associated with a shift in the regeneration process towards the formation of myelinated fibers. Increased immunostaining of BDNF, GDNF, and growth factor receptor p75.sup.NTR was associated with the upregulation of BDNF, GDNF, and HGF in the spinal cord of the PCL + MSCs group. A trend demonstrating higher reactivity of Schwann cells and axonal branching in the sciatic nerve was observed, and canine AdMSCs were engrafted at 30 days following repair. Conclusions 3D-printed NGCs multi-functionalized with canine AdMSCs embedded in heterologous fibrin biopolymer as cell scaffold exerted neuroregenerative effects. Our multimodal approach supports the trophic microenvironment, resulting in a pro-regenerative state after critical sciatic nerve injury in rats. Keywords: Canine mesenchymal stem cells, Nerve regeneration, Sciatic nerve injury, Cell-based therapy, Tissue engineering, Nerve guidance conduits, 3D printing, Fibrin, Scaffold
Monoclonal antibodies (mAbs) have been a valuable tool to elucidate several biological processes, such as stem cell differentiation and cancer, and contributed to virtually all areas of biomedical ...sciences. Yet, it remains a challenge to obtain mAbs specific to poorly expressed epitopes, or to epitopes that are actually involved in important biological phenomena, such as cell differentiation and metastasis. Drug-induced subtractive immunization, and recently the multiple tolerization subtractive immunization (MTSI) technique, reported by our group, have the potential to level up the field, as they direct the host´s immune response towards these epitopes. However, due to cyclophosphamide (CY) treatment, high mice mortality can be observed, and only a few data are available on how these techniques affect the immune system of mice. Tolerogen and immunogen cells, RWPE-1 and PC-3 cells, respectively, were individually seeded at 2 × 10
cells/cm
, and then adjusted to 2 × 10
cells per mouse before immunization, which was conducted in a subtractive approach (MTSI) with CY. Immunosuppression of mice was recorded
total white blood counting, as well the reactivity of circulating polyclonal antibodies (pAbs). General parameters, including weight, physical appearance, and behavior on mice subjected to three different concentrations of CY were recorded. mAbs were obtained using classical hybridoma techniques, using the spleen of immunized mice. After purification, antibodies were characterized by Western blotting, and Indirect immunofluorescence. In conclusion, all CY dosage were efficient in creating an immunosuppression state, but only the 100 mg/kg body weight was feasible, as the others resulted in extensive mice mortality. pAbs obtained in the peripheral blood of mice showed more reactivity towards tumor cells. MAbs 2-7A50 and 2-5C11 recognized antigens from tumor cells, but not from their non-tumor counterparts, as shown in western blotting and immunofluorescence assays. MTSI technique was successful in generating mAbs that recognize tumor-specific antigens.
This study contributes to the understanding of the mechanisms associated with signs and symptoms of tooth eruption, by investigating the presence of mast cells in pericoronal tissues during the ...intraosseous (Group 1) and submucosal (Group 2) phases of eruption. We compared findings for these two groups with each other and with those for the oral mucosa (Group 3). In each group, 14 specimens were analyzed microscopically after hematoxylin and eosin staining and immunohistochemical analysis of c-Kit and tryptase expression. Results revealed that the number and density of mast cells is different in follicular tissues according to the eruption phase, which may mean that: 1) masticatory trauma of the oral mucosa and dental follicles in the submucosa may explain why reduced enamel epithelium exposes enamel to the cells of the connective tissue; 2) exposure of antigenic enamel proteins might correspond to the release of sequestered antigens, which may lead to the interaction of IgE and a greater number of mast cells in the region; and 3) the consequent degranulation and the local release of mediators, such as histamine, leukotrienes, prostaglandins, proteases, cytokines and growth factors, contribute to the understanding of signs and symptoms associated with tooth eruption.
The use of the 5-alpha reductase inhibitors (5-ARIs) finasteride and dutasteride for prostate cancer prevention is still under debate. The FDA recently concluded that the increased prevalence of ...high-grade tumors among 5-ARI-treated patients must not be neglected, and they decided to disallow the use of 5-ARIs for prostate cancer prevention. This study was conducted to verify the effects of finasteride on prostate cell migration and invasion and the related enzymes/proteins in normal human and tumoral prostatic cell lines.
RWPE-1, LNCaP, PC3 and DU145 cells were cultivated to 60% confluence and exposed for different periods to either 10 µM or 50 µM finasteride that was diluted in culture medium. The conditioned media were collected and concentrated, and MMP2 and MMP9 activities and TIMP-1 and TIMP-2 protein expression were determined. Cell viability, migration and invasion were analyzed, and the remaining cell extracts were submitted to androgen receptor (AR) detection by western blotting techniques. Experiments were carried out in triplicate.
Cell viability was not significantly affected by finasteride exposure. Finasteride significantly downregulated MMP2 and MMP9 activities in RWPE-1 and PC3 cells and MMP2 in DU145 cells. TIMP-2 expression in RWPE-1 cells was upregulated after exposure. The cell invasion of all four tested cell lines was inhibited by exposure to 50 µM of finasteride, and migration inhibition only occurred for RWPE-1 and LNCaP cells. AR was expressed by LNCaP, RWPE-1 and PC3 cells.
Although the debate on the higher incidence of high-grade prostate cancer among 5-ARI-treated patients remains, our findings indicate that finasteride may attenuate tumor aggressiveness and invasion, which could vary depending on the androgen responsiveness of a patient's prostate cells.
To evaluate the influence of mesenchymal stem cells from adipose tissue in the end-to-side neurorrhaphy, focusing in the nerve regeneration and the muscle reinnervation in acute trauma.
140 animals ...were randomly divided in seven groups: control, denervated, end-to-side neurorrhaphy between distal stump of common peroneal nerve and tibial nerve (ESN), ESN wrapped in fascia, ESN wrapped in fascia and platelet gel, ESN wrapped in platelet gel, ESN wrapped in fascia and platelet gel within stem cells (without culture) removed from the adipose tissue. Mass measurements of the animal and of cranial tibial muscles, electromyography, walking track analysis tests and histological examinations of the nerves and muscles after 180 days was performed.
In the groups where the ESN was performed, the results were always better when compared to the denervated group, showing reinnervation in all ESN groups. The most sensitive methods were walking track and histological analysis. Only the group with stem cells showed values similar to the control group, as well as the functional indices of peroneal nerve and the number of nerve fibers in the peroneal nerve.
Stem cells were effective in ESN according with the functional index of the peroneal nerve, evaluated by walking track analysis and the number of nerve fibers in the peroneal nerve.
•S. aureus photoinactivation pattern was different in media with blood derivatives.•Plasma appears to be the component responsible for hindering bacteria inactivation.•Photodynamic inactivation ...showed lower erythrocytes hemolysis when in whole blood.•Plasma is fundamental to the hemolysis process occur in a smaller percentage.•S. aureus inactivation in whole blood resulted in better outcomes.
Blood can be the target of microbial cells in the human body. Erythrocytes, platelets, and plasma concentrates in blood bags used in hemotherapy for blood transfusion are contamination targets, which can trigger serious diseases in blood. These infections can cause septicemia that can lead to death if not recognized rapidly and treated adequately. The aim of this study was to evaluate the photodynamic inactivation in the in vitro decontamination of Staphylococcus aureus in whole blood, erythrocytes and platelet-rich plasma.
Photodynamic inactivation using light doses of 10, 15 and 30 J/cm2 at 630 nm and an hematoporphyrin-derivative photosensitizer (Photogem®) solutions at 25 and 50 μg/mL were evaluated. Toxicity of treatment was determined by hemolysis and cell viability assays. Results: The S. aureus reduction in phosphate buffered saline (PBS), whole blood, erythrocytes and platelet-rich plasma at 15 J/cm2 and 50 μg/mL were 7.2, 1.0, 1.3 and 0.4 log CFU/mL, respectively. Quantitative and qualitative analyses were performed in whole blood samples, and Photogem® showed a low risk of hemolysis (10.7%) in whole blood. However, 100% of erythrocytes suffered hemolysis in the absence of plasma. The cell viability assay showed 13.9% of apoptosis in erythrocytes, but normal platelet viability.
S. aureus inactivation of whole blood samples using 50 μg/mL Photogem® and 15 J/cm2 resulted in better outcomes, providing promising indications for treatment of bacterial contamination of blood, and in this work, alternative possibilities to apply the technique for blood decontamination are discussed.
PDF
