Integrative Analysis of the Ethanol Tolerance of Saccharomyces cerevisiae.

Ethanol (EtOH) alters many cellular processes in yeast. An integrated view of different EtOH-tolerant phenotypes and their long noncoding RNAs (lncRNAs) is not yet available. Here, large-scale data integration showed the core EtOH-responsive pathways, lncRNAs, and triggers of higher (HT) and lower (LT) EtOH-tolerant phenotypes. LncRNAs act in a strain-specific manner in the EtOH stress response. Network and omics analyses revealed that cells prepare for stress relief by favoring activation of life-essential systems. Therefore, longevity, peroxisomal, energy, lipid, and RNA/protein metabolisms are the core processes that drive EtOH tolerance. By integrating omics, network analysis, and several other experiments, we showed how the HT and LT phenotypes may arise: (1) the divergence occurs after cell signaling reaches the longevity and peroxisomal pathways, with CTA1 and ROS playing key roles; (2) signals reaching essential ribosomal and RNA pathways via SUI2 enhance the divergence; (3) specific lipid metabolism pathways also act on phenotype-specific profiles; (4) HTs take greater advantage of degradation and membraneless structures to cope with EtOH stress; and (5) our EtOH stress-buffering model suggests that diauxic shift drives EtOH buffering through an energy burst, mainly in HTs. Finally, critical genes, pathways, and the first models including lncRNAs to describe nuances of EtOH tolerance are reported here.

Increased peripheral blood TCD4+ counts and serum SP-D levels in patients with chronic paracoccidioidomycosis, during and after antifungal therapy.

BACKGROUND: The main clinical forms of paracoccidioidomycosis (PCM) are the acute/subacute form (AF) and the chronic form (CF), and they both display considerable clinical variability. The immune responses of PCM patients, during and after treatment, remain neglected, mainly in the case of CF patients, due to the high prevalence of pulmonary sequelae. OBJECTIVE: To evaluate the distribution of whole blood T cell subsets, serum cytokines, and biomarkers of pulmonary fibrosis in PCM patients, according to the clinical form and at different time points, during the antifungal therapy. METHODS: Eighty-seven PCM patients, from an endemic area in Brazil, were categorised into groups, according to the clinical form (AF or CF) and the moment of treatment. The peripheral blood T lymphocyte subsets of these patients were analysed using fluorescence-activated cell sorting. The serum levels of cytokines, basic fibroblast growth factor and surfactant protein-D (SP-D) were also analysed. FINDINGS: In the CF patients, an expansion of the peripheral blood TCD4+ cells was observed during the treatment, and this persisted even after two years of antifungal treatment. In addition, these patients showed high serum levels of SP-D. CONCLUSION: Our findings highlight the immunological changes CF patients undergo, during and after treatment, possibly due to the hypoxia triggered by pulmonary fibrosis and emphysema.

Imbalanced Macrophage and Dendritic Cell Activations in Response to Candida albicans in a Murine Model of Diabetes Mellitus.

Bloodstream infections caused by Candida species are responsible for high morbidity and mortality, and diabetes mellitus (DM) is an important underlying disease in candidemia episodes. Although DM patients show an enhanced proinflammatory profile, they are highly susceptible to mycobacterial and mycotic infections. Attempting to understand this paradox, we investigated if imbalanced macrophage and dendritic cell (DC) activations could be associated to high incidence and/or severity of Candida albicans infection in the hypoinsulinemia-hyperglycemia (HH) milieu. HH alloxan-induced mice were infected with C. albicans and peritoneal aderent phagocytes were co-cultured with or without lipopolyssaccharide or heat-killed C. albicans, and the production of cytotoxic metabolites, cytokines, and chemokines was evaluated. We also evaluated the surface expression of MHC-II and CD86 in splenic DCs. Our findings showed that both uninfected and C. albicans-infected HH mice showed less production of CCL2 and reduced expression of CD86 by peritoneal phagocytes and splenic DCs, respectively.

The Initial Months of Antiretroviral Therapy and Its Influence on AGEs, HMGB1, and sRAGE Levels in Asymptomatic HIV-Infected Individuals.

The development of the typical comorbidities of aging which currently affects people living with HIV/AIDS (PLWHA) can be partially ascribed to the persistent immune activation and chronic inflammation characterizing these individuals. The aim of this study was to analyze the effect exerted by combined antiretroviral therapy (cART) administration on plasma levels of HMGB1 (high mobility group box protein-1), AGEs (advanced glycation end products), their soluble receptor sRAGE, cytokines, C-reactive protein (CRP), and some metabolic markers in asymptomatic PLWHA. Analyses were performed longitudinally in 30 PLWHA, before and about 6-12 months after cART initiation. We observed that lower levels of AGEs in post-cART group were accompanied by an increase of CRP and triglyceride levels already in the early months of therapy. Because of the current ever-earlier recommendations to start cART and its prolonged use, these and other markers should be investigated in order to monitor and postpone the appearance of non-AIDS comorbidities in PLWHA.

Phenotypic and functional evaluations of peripheral blood monocytes from chronic-form paracoccidioidomycosis patients before and after treatment.

BACKGROUND: Paracoccidioidomycosis (PCM) is systemic mycosis caused by the thermal dimorphic fungus of genus Paracoccidioides, leading to either acute/subacute (AF) or chronic (CF) clinical forms. Numerous CF patients after treatment exhibit sequels, such as pulmonary and adrenal fibrosis. Monocytes are cells that are involved in the inflammatory response during active infection as well as in the fibrogenesis. These cells comprise a heterogeneous population with distinct phenotypic and functional activities. The scope of this study was to identify changes regarding functional and phenotypical aspects in monocytes comparing CF PCM patients on antifungal treatment versus non-treated patients (PMC-p). METHODS: Twenty-three CF PCM composed of 11 non-treated patients (NTG) and 12 patients in apparent cure (ACG) were studied. Sixteen healthy individuals were used as control group (CG). Monocyte subsets were determined by immunophenotyping based on CD14 and CD16 expression. Cellular function was measured in vitro with and without stimulation with lipopolysaccharide (LPS) and P. brasiliensis exoantigen (PbAg) for 24 hours. Independent samples were compared using unpaired t tests, dependent samples were analyzed by paired t-test. Groups of more than two independent samples were analyzed using an ANOVA, with Tukey's post-test. Significance was set up at p <0.05. RESULTS: Our results showed high counts of peripheral blood CD14+CD16+ and CD14+CD16++ monocytes in untreated PCM-p accompanied by intense production of pro-inflammatory cytokines (IL-1ß and TNF-α) and profibrotic growth factors (TGF-ß1 and bFGF) by monocytes challenged with P. brasiliensis antigens. After the introduction of antifungal therapy, the counts of CD14+CD16+ cells returned to baseline while CD14+CD16++ counts remained high. Interestingly, counts of CD14+CD16++ monocytes remained elevated even 52 ± 7 months after successful antifungal treatment. Furthermore, the ACG-patients showed preserved pro-inflammatory activity in the presence of specific antigen stimuli and high spontaneous production of TNF-α by monocytes. CONCLUSIONS: Infection with Paracoccidioides leads to initiation of a specific proinflammatory response by monocytes of PCM-p during active disease and in the apparent cure. A profibrotic profile by monocytes was observed only at admission. Furthermore, PCM-p with apparent cure showed high spontaneous production of TNF-α and high counts of CD14+CD16++ monocytes, probably induced by hypoxia duo to fibrotic sequelae.

Propolis anti-inflammatory effects on MAGE-1 and retinoic acid-treated dendritic cells and on Th1 and T regulatory cells.

Background: Propolis exhibits huge potential in the pharmaceutical industry. In the present study, its effects were investigated on dendritic cells (DCs) stimulated with a tumor antigen (MAGE-1) and retinoic acid (RA) and on T lymphocytes to observe a possible differential activation of T lymphocytes, driving preferentially to Th1 or Treg cells. Methods: Cell viability, lymphocyte proliferation, gene expression (T-bet and FoxP3), and cytokine production by DCs (TNF-α, IL-10, IL-6 and IL-1ß) and lymphocytes (IFN-γ and TGF-ß) were analyzed. Results: MAGE-1 and RA alone or in combination with propolis inhibited TNF-α production and induced a higher lymphoproliferation compared to control, while MAGE-1 + propolis induced IL-6 production. Propolis in combination with RA induced FoxP3 expression. MAGE-1 induced IFN-γ production while propolis inhibited it, returning to basal levels. RA inhibited TGF-ß production, what was counteracted by propolis. Conclusion: Propolis affected immunological parameters inhibiting pro-inflammatory cytokines and favoring the regulatory profile, opening perspectives for the control of inflammatory conditions.

Kinetics of Plasma Cytokines During Two Different Modalities of Extracorporeal Blood Purification in the Critically Ill Covid 19 Patients: A Cohort Study.

Background: In the absence of direct therapy for COVID-19, extracorporeal blood treatment (EBT) could represent an option for cytokine removal. Objective: This study aimed to describe and compare cytokine removal during intermittent haemodialysis (IHD) and continuous renal replacement therapy (CRRT) in COVID-19 patients with Acute Kidney Injury (AKI). Methods: It was a cohort study that studied patients with COVID-19-related AKI according to KDIGO criteria and admitted at Intensive Care Unit (ICU). Blood samples were collected at the start and end of both IHD using high flux (HF) membranes (10 patients) and continuous venovenous haemodiafiltration (CVVHDF:10 patients) in two sessions for measuring 13 different plasma interleukins and calculating the cytokine removal rate. Results: There was no difference between the two groups regarding mechanical ventilation, vasoactive drug, age or prognostic scores. Patients treated by CRRT presented higher levels of IL-2 and IL-8 than patients treated by IHD at dialysis start. Cytokine removal ranged from 9% to 78%. Patients treated by CRRT presented higher cytokine removal for IL-2, IL-6 IL-8, IP-10 and TNF. The removal rates of IL-4, IL-10, IL-17A, IFN, MCP-1 and TGF-B1 were similar in two groups. After one session of CVVHDF (24 h), IL-2 and IL-1ß levels did not vary significantly, whereas IL-4, IL-6, IL-8, IL-10, IL-17A, TNF, IFN, IP-10, MCP-1, IL-12p70 and TGF-B1 decreased by 33.8-76%, and this decrease was maintained over the next 24 h. In IHD groups, IL-2, IL-6, TNF, IP-10 and IL-1ß levels did not decrease significantly whereas IL-4, IL-8, IL-10, IL-17A, IFN, MCP-1, IL-12p70 and TGF-B1 decreased by 21.8-72%; however, cytokine levels returned to their initial values after 24 h. Conclusion: Cytokine removal is lower in IHD using HF membranes than in CVVHDF, and in IHD the removal is transient and selective, which can be associated with mortality during cytokines storm-related COVID-19.

Cirrhosis regression after SVR with indirect methods of fibrosis analysis: How far is it real?

Hepatitis C virus has infected over 71 million people worldwide, and it is the main cause of cirrhosis in the western world. Currently, the treatment involves direct-acting antiviral agents (DAAs) and its main goal is to achieve sustained virologic response (SVR). The aim of this study was to evaluate the impact of SVR using DAAs in the improvement of liver fibrosis using scores evaluation by indirect method, liver function, and inflammation indirect biomarkers. Patients with cirrhosis with SVR after treatment (n = 104) were evaluated using liver function scores, indirect fibrosis methods, alpha-fetoprotein, and ferritin at t-base and t-SVR. Statistically significant positive results in all parameters were observed: 54 patients were classified as 5 in the CP score in t-base, and 77 in t-SVR; a significant decrease was observed in MELD score, alpha-fetoprotein, ferritin, APRI, FIB-4 and liver stiffness in liver elastography. We did not observe difference in the liver function scores between regressors and non-regressors of liver stiffness, as well as in indirect inflammation biomarkers. The measurements of fibrosis using the indirect methods have significantly decreased in patients with cirrhosis treated who achieved SVR associated with decreased indirect inflammation biomarkers and improved liver function scores.

Production of Cytotoxic Antibodies After Intra-Articular Injection of Allogeneic Synovial Membrane Mesenchymal Stem Cells With and Without LPS Administration.

Allogeneic mesenchymal stem cells (MSC) are widely used in clinical routine due to the shorter expansion time and reliability of its quality. However, some recipients can produce alloantibodies that recognize MSCs and activate the immune system, resulting in cell death. Although antibody production was already described after MSC injection, no previous studies described the immune response after intra-articular MSC injection in acute synovitis. This study aimed to evaluate the influence of inflammation on immune response after single and repeated intra-articular injections of synovial membrane MSC (SMMSC). Horses were divided in three groups: control group (AUTO) received autologous synovial membrane MSCs; whereas group two (ALLO) received allogeneic SMMSCs and group three (ALLO LPS) was submitted to acute experimental synovitis 8 h before SMMSCs injection. The procedure was repeated for all groups for 28 days. Physical and lameness evaluations and synovial fluid analysis were performed. Sera from all animals were obtained before and every 7 days after each injection up to 4 weeks, to perform microcytotoxicity assays incubating donor SMMSCs with recipients' sera. The first injection caused a mild and transient synovitis in all groups, becoming more evident and longer in ALLO and ALLO LPS groups after the second injection. Microcytotoxicity assays revealed significant antibody production as soon as 7 days after SMMSC injection in ALLO and ALLO LPS groups, and cytotoxicity scores of both groups showed no differences at any time point, being equally different from AUTO group. Although inflammation is capable of inducing MHC expression in MSCs, which enhances immune recognition, cytotoxicity scores were equally high in ALLO and ALLO LPS groups, making it difficult to determine the potentiation effect of inflammation on antibody production. Our findings suggest that inflammation does not display a pivotal role in immune recognition on first allogeneic MSC injection. In a translational way, since specific antibodies were produced against MSCs, patients that need more than one MSC injection may benefit from a first allogeneic injection followed by subsequent autologous injections.

Genotoxicity, cytotoxicity and gene expression in patients undergoing elective surgery under isoflurane anaesthesia.

There are numerous studies reporting on the effects of inhalation anaesthesia in cells of exposed individuals but not much is known about the ability of isoflurane (ISF) to induce oxidative DNA damage. However, surgery is often associated with a temporary perioperative immunological alteration, and some volatile anaesthetics seem to contribute to a transient lymphocytopenia after surgery. We conducted a study to evaluate a possible genotoxic effect, including oxidative DNA damage, and apoptosis in peripheral lymphocytes of 20 patients American Society of Anaesthesiologists physical status I undergoing minor elective surgery lasting at least 120 min, under anaesthesia with ISF. We also investigated the expression of several genes in blood cells. Blood samples were collected at three time points: before anaesthesia (T(1)), 2 h after the beginning of anaesthesia (T(2)) and on the first post-operative day (T(3)). General DNA damage and oxidised bases (Fpg and endo III-sites) in blood lymphocytes were evaluated using the comet assay. Lymphocytes were phenotyped and apoptosis was evaluated by flow cytometry. In addition, expressions of hOGG1 and XRCC1, genes involved in DNA repair, and BCL2, a gene related to apoptosis, were assessed by quantitative real-time polymerase chain reaction. Results showed no statistically significant difference in the level of DNA damage and oxidised bases among the three sampling times. Anaesthesia with ISF did not increase the percentage of cells in early or late apoptosis in cytotoxic or helper T lymphocytes. Lower hOGG1 and BCL2 expressions were detected at T(3) in comparison to the other two previous time points, and there was significantly lower expression of XRCC1 at T(3) in relation to T(2). In conclusion, the exposure to ISF did not result in genotoxicity and cytotoxicity in lymphocytes and in toxicogenomic effect in leukocytes, although DNA repair and apoptosis-related genes were down-regulated on the first post-operative day.

Inflammation response and liver stiffness: predictive model of regression of hepatic stiffness after sustained virological response in cirrhotics patients with chronic hepatitis C.

Cirrhotic patients with chronic hepatitis C should be monitored for the evaluation of liver function and screening of hepatocellular carcinoma even after sustained virological response (SVR). The stage of inflammatory resolution and regression of fibrosis is likely to happen, once treatment and viral clearance are achieved. However, liver examinations by elastography show that 30-40% of patients do not exhibit a reduction of liver stiffness. This work was a cohort study in cirrhotic patients whose purpose was to identify immunological factors involved in the regression of liver stiffness in chronic hepatitis C and characterize possible serum biomarkers with prognostic value. The sample universe consisted of 31 cirrhotic patients who underwent leukocyte immunophenotyping, quantification of cytokines/chemokines and metalloproteinase inhibitors in the pretreatment (M1) and in the evaluation of SVR (M2). After exclusion criteria application, 16 patients included were once more evaluated in M3 (like M1) and classified into regressors (R) or non-regressors (NR), decrease or not ≥ 25% stiffness, respectively. The results from ROC curve, machine learning (ML) and linear discriminant analysis showed that TCD4 + lymphocytes (absolute) are the most important biomarkers for the prediction of the regression (AUC = 0.90). NR patients presented levels less than R of liver stiffness since baseline, whereas NK cells were increased in NR. Therefore, it was concluded that there is a difference in the profile of circulating immune cells in R and NR, thus allowing the development of a predictive model of regression of liver stiffness after SVR. These findings should be validated in greater numbers of patients.

Propolis from southeastern Brazil produced by Apis mellifera affects innate immunity by modulating cell marker expression, cytokine production and intracellular pathways in human monocytes.

OBJECTIVES: Propolis is a bee-made product used for centuries due to its diverse biological properties, including its immunomodulatory action. This work aimed at investigating whether propolis may affect monocyte functions challenged with retinoic acid (RA), B subunit of Escherichia coli heat-labile enterotoxin (EtxB), human melanoma-associated antigen-1 (MAGE-1) and lipopolysaccharide (LPS). METHODS: Monocytes from healthy donors were treated with the stimuli separately or in the presence of propolis. Cell viability was evaluated by MTT assay, cell marker expression was assessed by flow cytometry, cytokine production by ELISA, gene expression by RT-qPCR. KEY FINDINGS: Propolis alone maintained TLR-2, TLR-4, HLA-DR, CD40 and CD80 expression in the monocytes; however, its combination with either MAGE-1 or LPS decreased CD40 expression triggered by the stimuli. Propolis maintained RA action on cell marker expression. Propolis inhibited TNF-α (with either EtxB or MAGE-1) and IL-6 (with either RA or MAGE-1), and increased IL-10 (with MAGE-1) production. Propolis downmodulated LC3 expression induced by LPS. It also induced a lower NF-kB expression than control cells and its combination with RA induced a higher expression than the stimulus alone. CONCLUSIONS: Propolis potentially affected innate immunity by downmodulating the monocytes pro-inflammatory activity.

Systemic Cytokine Profiles of CD4+ T Lymphocytes Correlate with Clinical Features and Functional Status in Stable COPD.

Aims: To evaluate the expressions of intracellular cytokines in CD4+ T lymphocytes and to investigate the correlation between biomarker expressions and clinical and functional characteristics of stable COPD patients. Patients and Methods: Peripheral blood was collected from 36 COPD patients, and the expression of cytokines (IL-8, IL-13, IL-17, IL-6, IL-2, IL-10, and TNF-α) in T lymphocytes CD4 + was investigated. In addition, lung function, dyspnea symptoms, quality of life, vital signs, body composition, level of physical activity, peripheral muscle strength, and functional capacity were assessed. Results: Individuals with greater bronchial obstruction present a higher proportion of CD4 + IL-2 + lymphocytes compared to individuals with less severe bronchial obstruction. We found a positive correlation between the expression of the cytokines IL-13, IL-17, IL-6, IL-2, IL-10, and TNF-α in CD4+ T lymphocytes. In addition, we found a positive correlation between CD4+ IL-10+ T lymphocytes and lower limb muscle strength and a negative correlation between CD4+ IL-8+ T lymphocytes and peripheral oxygen saturation and steps per day. Conclusion: Systemic CD4+IL-2+, IL-8+, and IL-10+ T lymphocytes presented a correlation with clinical characteristics and functional status in stable COPD.

Highly effective fibrin biopolymer scaffold for stem cells upgrading bone regeneration.

Fibrin scaffold fits as a provisional platform promoting cell migration and proliferation, angiogenesis, connective tissue formation and growth factors stimulation. We evaluated a unique heterologous fibrin biopolymer as scaffold to mesenchymal stem cells (MSCs) to treat a critical-size bone defect. Femurs of 27 rats were treated with fibrin biopolymer (FBP); FBP + MSCs; and FBP + MSC differentiated in bone lineage (MSC-D). Bone repair was evaluated 03, 21 and 42 days later by radiographic, histological and scanning electron microscopy (SEM) imaging. The FBP + MSC-D association was the most effective treatment, since newly formed Bone was more abundant and early matured in just 21 days. We concluded that FBP is an excellent scaffold for MSCs and also use of differentiated cells should be encouraged in regenerative therapy researches. The FBP ability to maintain viable MSCs at Bone defect site has modified inflammatory environment and accelerating their regeneration.

Adipose-derived stem cells and adipose-derived stem cell-conditioned medium modulate in situ imbalance between collagen I- and collagen V-mediated IL-17 immune response recovering bleomycin pulmonary fibrosis.

The immunogenic collagen V (Col V) and the proinflammatory cytokine interleukin (IL)-17 have been implicated in the pathogenesis of multiple autoimmune diseases. Col V is also up-regulated during adipogenesis and can stimulate adipocyte differentiation in vitro. Conditioned medium (CM) generated from adipose-derived mesenchymal stem cells (MSCs) reduces bleomycin (BLM)-induced lung injury in rats, suggesting a crucial role in situ of immunomodulatory factors secreted by MSCs in these beneficial effects. In the present work, we investigated this hypothesis, analyzing levels of plasma inflammatory mediators and inflammatory and fibrotic mediators in the lung tissue of BLM-injured rats after treatment with MSCs and CM. Pulmonary fibrosis was intratracheally induced by BLM. After 10 days, BLM animals were further randomized into subgroups receiving saline, MSCs, or CM intravenously. On days 14 and 21, the animals were euthanized, and the lungs were examined through protein expression of nitric oxide synthase (NOS), IL-17, transforming growth factor-ß (TGF-ß), vascular endothelial growth factor, endothelin-1, and the immunogenic Col V through histological quantitative evaluation and plasma levels of fibrinogen, Von Willebrand factor, and platelet-derived growth factor (PDGF). Rats that had been injected with MSCs and CM showed a significant increase in weight and significant improvements at 14 and 21 days after intravenous injection at both time points of analysis of plasma fibrinogen, PDGF, and Von Willebrand factor and NOS-2 expression, supporting an early anti-inflammatory action, thus reducing TGF-ß and collagen I fibers. In contrast, intravenous injection of CM was able to significantly increase the deposition of Col V fibers and IL-17 on both day 14 and day 21 as compared with the amount observed in rats from the BLM group and MSC groups. In conclusion, this study reinforces previous observations on the therapeutic properties of MSCs and CM and is the first report to demonstrate the association of its actions with immunomodulatory biomarkers on lung tissue. We concluded that adipose-derived stem cells and adipose-derived stem cells-CM modulate an in situ imbalance between collagen I- and Col V-mediated IL-17 immune response, emerging as a promising therapeutic option for recovering from BLM pulmonary fibrosis.

Enhanced expression of NLRP3 inflammasome components by monocytes of patients with pulmonary paracoccidioidomycosis is associated with smoking and intracellular hypoxemia.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thermally dimorphic fungi of the genus Paracoccidioides that affects predominantly 30-60-year-old male rural workers. The main clinical forms of the disease are acute/subacute, chronic (CF); almost all CF patients develop pulmonary fibrosis, and they also exhibit emphysema due to smoke. An important cytokine in this context, IL-1ß, different from the others, is produced by an intracellular multimolecular complex called inflammasome that is activated by pathogens and/or host signs of damage. Inflammasome has been recognized for its contribution to chronic inflammatory diseases, from that, we hypothesized that this activation could be involved in paracoccidioidomycosis, contributing to chronic inflammation. While inflammasome activation has been demonstrated in experimental models of Paracoccidioides brasiliensis infection, no information is available in patients, leading us to investigate the participation of NLRP3-inflammasome machinery in CF/PCM patients from a Brazilian endemic area. Our findings showed increased priming in mRNA levels of NLRP3 inflammasome genes by monocytes of PCM patients in vitro than healthy controls. Similar intracellular protein expression of NLRP3, CASP-1, ASC, and IL-1ß were also observed in freshly isolated monocytes of PCM patients and smoker controls. Increased expression of NLRP3 and ASC was observed in monocytes from PCM patients under hypoxia in comparison with smoker controls. For the first time, we showed that primed monocytes of CF-PCM patients were associated with enhanced expression of components of NLRP3-inflammasome due to smoke. Also, hypoxemia boosted this machinery. These findings reinforce the systemic low-grade inflammation activation observed in PCM during and after treatment.

Photodynamic inactivation for in vitro decontamination of Staphylococcus aureus in whole blood.

BACKGROUND: 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. METHODS: 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. CONCLUSION: 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.

Evaluation of alginate hydrogel encapsulated mesenchymal stem cell migration in horses.

Osteoarthritis is an incapacitating disease characterized by pain and a progressive decrease in joint mobility. The implantation of mesenchymal stem cells (MSCs) has shown promising results for its treatment. The challenge remains to keep the cells longer at the site of action, increasing their therapeutic potential. The aim of this study was to evaluate the effectiveness of the Qtracker® 655 nanocrystal marking on allogeneic synovial membrane (SM) MSCs, encapsulated in alginate hydrogel, evaluating the migration of these cells. The 10 radiocarpal joints were submitted to arthroscopic surgery (D0), divided into two groups. The chondral defect was treated according to the group: GA free-labelled MSCSM and GB labelled MSCSM microcapsules. Seven days after lesion induction and implantation of labelled cells, biopsies of the lesion site were performed in two animals, and fragments of SM and joint capsule also collected, which were frozen and later processed for fluorescence microscopy. The synovial fluid of the three animals was analyzed by flow cytometry three times - 3, 7 and 21 days after application. The cellular marking with the nanocrystals allowed the visualization of the cells in cartilage, synovial membrane, synovial fluid and articular capsule, but with a predilection for the synovial membrane and the lesion site was scarce. The labelled MSCSM in microcapsules were scarce in the synovial fluid and could be related to the small quantity of MSCs leaving the pores of the microcapsules, also favorable results, as the cells release paracrine effects acting for a long period until the cellular differentiation.

Fibrin biopolymer as scaffold candidate to treat bone defects in rats.

BACKGROUND: Bone tissue repair remains a challenge in tissue engineering. Currently, new materials are being applied and often integrated with live cells and biological scaffolds. The fibrin biopolymer (FBP) proposed in this study has hemostatic, sealant, adhesive, scaffolding and drug-delivery properties. The regenerative potential of an association of FBP, biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSCs) was evaluated in defects of rat femurs. METHODS: Adult male Wistar rats were submitted to a 5-mm defect in the femur. This was filled with the following materials and/or associations: BPC; FBP and BCP; FBP and MSCs; and BCP, FBP and MSCs. Bone defect without filling was defined as the control group. Thirty and sixty days after the procedure, animals were euthanatized and subjected to computed tomography, scanning electron microscopy and qualitative and quantitative histological analysis. RESULTS: It was shown that FBP is a suitable scaffold for bone defects due to the formation of a stable clot that facilitates the handling and optimizes the surgical procedures, allowing also cell adhesion and proliferation. The association between the materials was biocompatible. Progressive deposition of bone matrix was higher in the group treated with FBP and MSCs. Differentiation of mesenchymal stem cells into osteogenic lineage was not necessary to stimulate bone formation. CONCLUSIONS: FBP proved to be an excellent scaffold candidate for bone repair therapies due to application ease and biocompatibility with synthetic calcium-based materials. The satisfactory results obtained by the association of FBP with MSCs may provide a more effective and less costly new approach for bone tissue engineering.

Influence of the inflammatory response on treatment of hepatitis C with triple therapy.

INTRODUCTION: Chronic hepatitis C is a leading cause of liver disease. Infection triggers an immediate immune response in the host that is mediated by humoral/cellular mechanisms. T cells respond to infection via secretion of cytokines, which inhibit or stimulate one another, leading to cytokine imbalance and ultimately affecting treatment. Studies using interferon (IFN) and ribavirin (RBV) showed that TCD8+ cells and cytokine levels are associated with sustainable virological response (SVR). However, studies that investigated the effects of triple therapy (TT) are limited. METHODS: The study included hepatitis C virus (HCV)+ RNA, naives, genotype 1, ≥18 years, and advanced fibrosis (F≥3) patients. Samples were collected at baseline and after 12 weeks (W12) of TT. Six cytokines were analyzed by flow cytometry. RESULTS: Of 31 patients, four were excluded (two deaths, one interrupted TT, and one F2 patient). Of the 27 remaining patients, 21 (78%) were cirrhotic. SVR was achieved in 63% of the patients. The patients had a mean age of 55.11 ± 10.03 years. Analyses at baseline showed that the chemokine CCL5/Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) (p=0.04) and interleukin (IL)-6 (p=0.02), which was associated with SVR. RANTES (p=0.04) and IL-8 (p=0.01) levels were associated with SVR at W12. CONCLUSIONS: Similar to patterns observed during double therapy, IL-6, IL-8, and RANTES levels were associated with SVR in TT, indicating the potential role of interferon in immune response to hepatitis C virus.