Antibiofilm activity of promethazine against ESBL-producing strains of Escherichia coli in urinary catheters.

The bacterium Escherichia coli is one of the main causes of urinary tract infections. The formation of bacterial biofilms, especially associated with the use of urinary catheters, contributes to the establishment of recurrent infections and the development of resistance to treatment. Strains of E. coli that produce extended-spectrum beta-lactamases (ESBL) have a greater ability to form biofilms. In addition, there is a lack of drugs available in the market with antibiofilm activity. Promethazine (PMZ) is an antihistamine known to have antimicrobial activity against different pathogens, including in the form of biofilms, but there are still few studies of its activity against ESBL E. coli biofilms. The aim of this study was to evaluate the antimicrobial activity of PMZ against ESBL E. coli biofilms, as well as to assess the application of this drug as a biofilm prevention agent in urinary catheters. To this end, the minimum inhibitory concentration and minimum bactericidal concentration of PMZ in ESBL E. coli strains were determined using the broth microdilution assay and tolerance level measurement. The activity of PMZ against the cell viability of the in vitro biofilm formation of ESBL E. coli was analyzed by the MTT colorimetric assay and its ability to prevent biofilm formation when impregnated in a urinary catheter was investigated by counting colony-forming units (CFU) and confirmed by scanning electron microscopy (SEM). PMZ showed bactericidal activity and significantly reduced (p < 0.05) the viability of the biofilm being formed by ESBL E. coli at concentrations of 256 and 512 µg/ml, as well as preventing the formation of biofilm on urinary catheters at concentrations starting at 512 µg/ml by reducing the number of CFUs, as also observed by SEM. Thus, PMZ is a promising candidate to prevent the formation of ESBL E. coli biofilms on abiotic surfaces.

Diet quality and nutritional status in children with human immunodeficiency virus.

Background: Poor diet quality in children and adolescents may contribute to decreased immunity and lead to an increased risk of opportunistic diseases. Aim: To investigate diet quality and its relationship to nutritional status in human immunodeficiency virus (HIV)-infected pediatric patients (HIV-PIHIV). Methods: We conducted a cross-sectional study with 87 patients aged between 6 and 19 years carried out in two University Hospitals. Diet quality was analyzed by an adapted Healthy Eating Index (HEI) and nutritional status. The association between HEI with body mass index-for-age (BMI-for-age) and height-for-age was performed using a linear regression model. Clinical, maternal, anthropometric, and dietary data were collected through a semi-structured questionnaire, based on nutrition service protocols. Results: Diet quality was intermediate (median IAS = 54.8 interquartile range: 47.5 to 65.9 points), due to low consumption of fruits, vegetables, and dairy products and high empty calories and sodium by the PPIHIV. The multivariate regression model indicated that HEI was not significant for explaining BMI-for-age [ß = -0.01; 95% CI = (-0.03; 0.01); p 0.40] nor height-for-age [ß = 0.01; 95% CI = (-0.02; 0.03); p 0.51]. However, it was observed that adolescents showed 1 Z-score [95% CI = (-1.6; -0.44); p 0.001] a reduction in BMI-for-age compared with children, and those black patients showed an increase in BMI-for-age Z-score of 0.57 [95% CI = (0.7; 1.1); p 0.03] compared with non-blacks. Conclusion: The diet quality of the HIV-infected children and adolescents was below desired. No association was found between diet quality and inadequate nutritional status of HIV-PIHIV.

Do combinations of fipronil, eugenol and carvacrol have synergistic effects against Rhipicephalus sanguineus?

The tick Rhipicephalus sanguineus is one of the main ectoparasites that affects dogs, causing direct and indirect damage to parasitized animals. Currently, infestation control is mainly carried out by using synthetic acaricidal drugs. However, a decrease in efficacy and an increase in resistance to the main therapeutic protocols against tick infestations have been increasingly reported and confirmed, a factor that has driven research into the potential acaricide activity of natural compounds, including in association with synthetic molecules. The aim of this work was to evaluate whether the combinations of fipronil (FIP) and eugenol (EUG), FIP and carvacrol (CAR), and EUG and CAR would have synergistic effects against immature and unfed adult stages of R. sanguineus through in vitro bioassays. Bioassays were carried out using the larval packet test (FAO 2004) adapted for nymphs and adults. The synergistic activity was explored by combining each solution, based on the estimated LC50, in a 1:1 ratio (FIP: EUG, FIP: CAR and EUG: CAR). CompuSyn software was used to evaluate the various pairwise combinations of FIP, EUG and CAR, checking if there was synergism or antagonism between them. FIP and EUG and FIP and CAR showed combination index (CIn) values above 1.45, indicating antagonism. The synergistic activity between EUG and CAR was verified against all unfed phases of R. sanguineus, since the CIn was below 0.70, a value that indicates synergism. The combination of fipronil with either eugenol or carvacrol presented antagonistic effects against R. sanguineus larvae. On the other hand, carvacrol and eugenol had excellent pharmacological synergism against all tick stages with mortality values in the range of 80 to 100%, including the adult stage, which is less susceptible than immature stages.

[Spatial-temporal distribution of visceral leishmaniasis in Brazil from 2007 to 2020Distribución espaciotemporal de la leishmaniasis visceral en Brasil en el período 2007-2020]. / Distribuição espaço-temporal da leishmaniose visceral no Brasil no período de 2007 a 2020.

Objective: To analyze the dynamics of the spatial and temporal distribution of visceral leishmaniasis in Brazil from 2007 to 2020. Method: This ecological study focused on the spatial pattern of visceral leishmaniasis cases in Brazil from 2007 to 2020 using data from the Information System for Notifiable Diseases (SINAN). The variables analyzed were the incidence rate of visceral leishmaniasis and the visceral leishmaniasis composite indicator (VLCI), from which triennial thematic maps were constructed. The global Moran index was calculated to assess the existence of spatial autocorrelation, and the local Moran index was used to identify areas with higher and lower risk of visceral leishmaniasis in Brazil. Results: During the study period, there were 48 705 cases of visceral leishmaniasis, with an incidence rate of 25.53 cases per 100 000 population. There was spatial autocorrelation in all triennial blocks, with municipalities in the North and Northeast regions having an incidence equal to or greater than 50 cases per 100 000 population. Regarding the VLCI there was an increase in the number of municipalities classified as low risk for transmission, and a growing presence of state capitals with a classification of very high risk. Conclusion: The incidence of visceral leishmaniasis varied over the trienniums. The border region between the states of Tocantins, Maranhão, and Pará, along with the state of Ceará, stood out in the spatial distribution of the disease incidence and risk stratification by VLCI. These areas should be a priority for surveillance and control efforts for the disease.

Objetivo: Analizar la dinámica de la distribución espaciotemporal de la leishmaniasis visceral en Brasil en el período 2007-2020. Métodos: En este estudio ecológico del patrón de distribución espacial de los casos de leishmaniasis visceral ocurridos en Brasil en el período 2007-2020 se utilizaron datos del sistema de información de enfermedades de declaración obligatoria (SINAN). Las variables analizadas fueron el coeficiente de incidencia de leishmaniasis visceral y el índice compuesto de leishmaniasis visceral, a partir de los cuales se construyeron mapas temáticos trienales. Se calcularon el índice general de Moran para verificar la existencia de autocorrelación espacial, y el índice local de Moran para delimitar las zonas de mayor y menor riesgo de leishmaniasis visceral en Brasil. Resultados: En el período estudiado, hubo 48 705 casos de leishmaniasis visceral, con una incidencia de 25,53 casos por 100 000 habitantes. En todos los bloques trienales hubo una autocorrelación espacial, en la cual varios municipios de las regiones norte y nordeste registraron una incidencia de 50 casos por 100 000 habitantes o una tasa superior. En relación con el índice compuesto de leishmaniasis visceral, se observó un aumento del número de municipios considerados de riesgo bajo de transmisión y una tendencia ascendente del número de ciudades capitales con clasificación de riesgo muy alto. Conclusión: La incidencia de leishmaniasis visceral varió a lo largo de los períodos trienales. La región fronteriza entre Tocantins, Maranhão y Pará, junto con el estado de Ceará, se destacó en términos de la distribución espacial de la incidencia de la enfermedad y la estratificación del riesgo según el índice compuesto de leishmaniasis visceral. Es preciso dar prioridad a estos lugares en lo referente a las medidas y los servicios de vigilancia y control de esta enfermedad.

Protease-activated receptor 2 enhances innate and inflammatory mechanisms induced by lipopolysaccharide in macrophages from C57BL/6 mice.

OBJECTIVE: This study was conducted to investigate the effects of the synthetic PAR2 agonist peptide (PAR2-AP) SLIGRL-NH2 on LPS-induced inflammatory mechanisms in peritoneal macrophages. METHODS: Peritoneal macrophages obtained from C57BL/6 mice were incubated with PAR2-AP and/or LPS, and the phagocytosis of zymosan fluorescein isothiocyanate (FITC) particles; nitric oxide (NO), reactive oxygen species (ROS), and cytokine production; and inducible NO synthase (iNOS) expression in macrophages co-cultured with PAR-2-AP/LPS were evaluated. RESULTS: Co-incubation of macrophages with PAR2AP (30 µM)/LPS (100 ng/mL) enhanced LPS-induced phagocytosis; production of NO, ROS, and the pro-inflammatory cytokines interleukin (IL)-1ß, tumour necrosis factor (TNF)-α, IL-6, and C-C motif chemokine ligand (CCL)2; and iNOS expression and impaired the release of the anti-inflammatory cytokine IL-10 after 4 h of co-stimulation. In addition, PAR2AP increased the LPS-induced translocation of the p65 subunit of the pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) and reduced the expression of inhibitor of NF-κB. CONCLUSION: This study provides evidence of a role for PAR2 in macrophage response triggered by LPS enhancing the phagocytic activity and NO, ROS, and cytokine production, resulting in the initial and adequate macrophage response required for their innate response mechanisms.

Rapid scale-up of COVID-19 training for frontline health workers in 11 African countries.

BACKGROUND: The global spread of the SARS-CoV-2 virus highlights both the importance of frontline healthcare workers (HCW) in pandemic response and their heightened vulnerability during infectious disease outbreaks. Adequate preparation, including the development of human resources for health (HRH) is essential to an effective response. ICAP at Columbia University (ICAP) partnered with Resolve to Save Lives and MOHs to design an emergency training initiative for frontline HCW in 11 African countries, using a competency-based backward-design approach and tailoring training delivery and health facility selection based on country context, location and known COVID-19 community transmission. METHODS: Pre- and post-test assessments were conducted on participants completing the COVID-19 training. Parametric and non-parametric methods were used to examine average individual-level changes from pre- to post-test, and compare performance between countries, cadres, sex and facility types. A post-evaluation online training survey using Qualtrics was distributed to assess participants' satisfaction and explore training relevance and impact on their ability to address COVID-19 in their facilities and communities. RESULTS: A total of 8797 HCW at 945 health facilities were trained between June 2020 and October 2020. Training duration ranged from 1 to 8 days (median: 3 days) and consisted of in person, virtual or self guided training. Of the 8105 (92%) HCW working at health facilities, the majority (62%) worked at secondary level facilities as these were the HF targeted for COVID-19 patients. Paired pre- and post-test results were available for 2370 (25%) trainees, and 1768 (18%) participants completed the post-evaluation training survey. On average, participants increased their pre- to post-test scores by 15 percentage points (95% CI 0.14, 0.15). While confidence in their ability to manage COVID-19 was high following the training, respondents reported that lack of access to testing kits (55%) and PPE (50%), limited space in the facility to isolate patients (45%), and understaffing (39%) were major barriers. CONCLUSION: Ongoing investment in health systems and focused attention to health workforce capacity building is critical to outbreak response. Successful implementation of an emergency response training such as this short-term IPC training initiative in response to the COVID-19 pandemic, requires speed, rigor and flexibility of its design and delivery while building on pre-existing systems, resources, and partnerships.

Determination of Fipronil and Fipronil-Sulfone in Surface Waters of the Guandu River Basin by High-Performance Liquid Chromatography with Mass Spectrometry.

Fipronil (FIP) is a broad-spectrum insecticide used in the industrial, residential and agricultural sectors, which presents environmental and human health risks. Studies report its presence in aquatic environments in several countries, but in Brazil reports are scarce. The aim of this work was to evaluate the presence of fipronil and fipronil sulfone in surface waters through a validated analytical method by LC-MS according to INMETRO and USEPA in eight sampling sites in two seasons (summer and autumn, 2020) in Guandu River basin, in the state of Rio de Janeiro. FIP was quantified in the concentration range of 0.132-2.44 µg/L, while FIP-S was detected in most samples. This is the first study on the occurrence of FIP and FIP-S in the Guandu River basin, presenting values high enough to justify the need for monitoring studies in a region of great importance for the city of Rio de Janeiro.

Mimosa tenuiflora's antimicrobial activity on bacteria and fungi from medical importance: an integrative review.

The misuse of antimicrobials has led to the appearance of several multi-resistant pathogens and, as a result, it has been necessary to search for new compounds that are effective against these microorganisms. Faced with this scenario, one of the forms that has been arousing the interest of scholars is the obtaining of antimicrobial compounds from medicinal plants, since, through them, effective compounds are usually obtained against pathogens and with low toxicity to humans and animals. In Brazilian Caatinga, there are several medicinal plants with antimicrobial properties, and one of them is Mimosa tenuiflora (Willd.) Poir., also known as Jurema Preta, popularly used to treat comorbidities that affect mainly the skin. In view of this, to investigate its antimicrobial potential, a bibliographical survey of experimental articles, published in the last 10 years, which analyzed the antimicrobial activity of M. tenuiflora in bacteria and fungi of importance, was made. Thus, it was possible to observe that the extracts obtained from M. tenuiflora showed in vitro antimicrobial activity. In addition, the compounds flavonoids and tannins are likely to be the main responsible for Mimosa tenuiflora's antimicrobial activity. Thus, M. tenuiflora presents a high study potential in microbial growth control.

Effects of ketamine in methicillin-resistant Staphylococcus aureus and in silico interaction with sortase A.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main human pathogens and is responsible for many diseases, ranging from skin infections to more invasive infections. These infections are dangerous and expensive to treat because these strains are resistant to a large number of conventional antibiotics. Thus, the antibacterial effect of ketamine against MRSA strains, its mechanism of action, and in silico interaction with sortase A were evaluated. The antibacterial effect of ketamine was assessed using the broth microdilution method. Subsequently, the mechanism of action was assessed using flow cytometry and molecular docking assays with sortase A. Our results showed that ketamine has a significant antibacterial activity against MRSA strains in the range of 2.49-3.73 mM. Their mechanism of action involves alterations in membrane integrity and DNA damage, reducing cell viability, and inducing apoptosis. In addition, ketamine had an affinity for S. aureus sortase A. These results indicate that this compound can be used as an alternative to develop new strategies to combat infections caused by MRSA.

Effect of Homeopathic Preparations on Lettuce, Parasitized or Not by Meloidogyne enterolobii.

BACKGROUND: There are relatively few scientific works on the use of homeopathy to manage plant pathogens, particularly nematodes. A handful of studies focused on Meloidogyne spp. parasitizing vegetables have brought contradictory results on nematode control and enhancement of plant tolerance to parasitism. OBJECTIVE: Our goal was to assess the effect of Cina-a well-known anti-nematode ingredient-on Meloidogyne enterolobii parasitizing lettuce. METHODS: Cina was applied daily on nematode-inoculated plants, from the seedling stage until harvest. We tested an evenly spaced range of Hahnemannian concentrations (c), which were applied though irrigation with a constant dose of the ingredient. Several absolute and relative controls were employed to allow the assessment of the effect of Cina on nematode reproduction and lettuce growth. RESULTS: Cina affected growth of non-parasitized plants, both positively and negatively; this effect was modulated by the c applied and the thermal stress suffered by the plants in one of the assays. The effect of Cina on the growth of nematode-parasitized plants was neutral or negative. Cina reduced nematode reproduction by 25-36%. CONCLUSION: Based on the moderate negative effect of Cina on M. enterolobii reproduction, it seems this ingredient may be useful as a complementary strategy for Meloidogyne control. But Cina did not enhance the tolerance of lettuce to Meloidogyne spp.

Effect of Nosodes on Lettuce, Parasitized or Not by Meloidogyne enterolobii.

BACKGROUND: Nosodes are homeopathic preparations (HPs) obtained from tissues or substances associated with the targeted disease or from culture of the pathogenic agent. Nosodes are thought to modulate host resistance, easing symptoms or promoting cure. A few studies have been published about control of plant-parasitic nematodes with HPs, but none with nosodes. Conceptually, nosodes prepared from nematode infective stages might interact with the plant's pathogen-recognition system and initiate or modulate plant resistance to nematodes. OBJECTIVE: Our goal was to investigate whether nosodes prepared from second-stage juveniles (J2) of Meloidogyne enterolobii can affect the moderate resistance already existing in the lettuce cultivar 'Elisa'. METHODS: Nosodes at the Hahnemannian concentrations (cH) 6, 18, 30 and 42 were applied on lettuce plants through irrigation, with a constant daily dosage. The nosode treatment started at the seedling stage, before nematode inoculation with 3,000 eggs + J2 per plant. A series of absolute and relative controls, and 10 replicates per treatment, were employed. At harvest, variables related to plant growth and nematode reproduction were assessed. RESULTS: The nosode at 6, 18, and 30cH reduced (p <0.05) the nematode reproduction factor and root density. The nosode effect was cH-dependent since nematode reproduction was favored by treatment with 42cH. The nosode also affected (p <0.05) lettuce roots, which presented higher or lower fresh weight and volume depending on the cH applied and the condition-parasitized or not. CONCLUSION: Nosodes obtained from Meloidogyne J2 may affect plant parasitism by nematodes, possibly by interfering with plant resistance. The nature-positive or negative-and intensity of the nosode effect depends on the cH applied to the plants. Further studies are necessary to identify which cH values are more effective in reducing nematode reproduction without causing negative side effects on plant growth.

Chitosan gels for buccal delivery of Schinus molle L. essential oil in dogs: characterization and antimicrobial activity in vitro.

Periodontal disease is considered the main oral cavity disorder in dogs. Essential oils have the potential for use in the prevention and treatment of oral diseases. The antimicrobial activity of Schinus molle L. essential oil (SMEO) has already been reported. Chitosan, a natural product with antimicrobial activity and good biocompatibility has potential in biodental applications. In this study, we evaluated the in vitro antimicrobial activity of SMEO against bacteria associated with periodontal disease in dogs, developed and evaluated the physicochemical properties of a novel chitosan-based buccal delivery system containing SMEO. SMEO showed antimicrobial activity against Gram positive and Gram negative bacteria associated with canine periodontitis, with MIC values of 750 µg.mL-1 for Staphylococcus spp. and Streptococcus spp, 1000 µg.mL-1 for Corynebacterium spp. and 1250 µg.mL-1 for Pseudomonas spp. All formulations evaluated presented adequate physicochemical properties, good stability, and pH values close to buccal pH (5.0-7.0). Chitosan gel loaded with SMEO showed potential as a SMEO delivery system, having the ideal physicochemical and rheological properties (pseudoplastic and apparent viscosities) required for application on buccal tissue. Thus, we can conclude that formulation has the potential to be used for buccal mucosa delivery in the prevention and treatment of periodontal disease in dogs.

Serotonin decreases the production of Th1/Th17 cytokines and elevates the frequency of regulatory CD4+ T-cell subsets in multiple sclerosis patients.

Excessive levels of proinflammatory cytokines in the CNS are associated with reduced serotonin (5-HT) synthesis, a neurotransmitter with diverse immune effects. In this study, we evaluated the ability of exogenous 5-HT to modulate the T-cell behavior of patients with MS, a demyelinating autoimmune disease mediated by Th1 and Th17 cytokines. Here, 5-HT attenuated, in vitro, T-cell proliferation and Th1 and Th17 cytokines production in cell cultures from MS patients. Additionally, 5-HT reduced IFN-γ and IL-17 release by CD8+ T cells. By contrast, 5-HT increased IL-10 production by CD4+ T cells from MS patients. A more accurate analysis of these IL-10-secreting CD4+ T cells revealed that 5-HT favors the expansion of FoxP3+ CD39+ regulatory T cells (Tregs) and type 1 regulatory T cells. Notably, this neurotransmitter also elevated the frequency of Treg17 cells, a novel regulatory T-cell subset. The effect of 5-HT in upregulating CD39+ Treg and Treg17 cells was inversely correlated with the number of active brain lesions. Finally, in addition to directly reducing cytokine production by purified Th1 and Th17 cells, 5-HT enhanced in vitro Treg function. In summary, our data suggest that serotonin may play a protective role in the pathogenesis of MS.

High abdominal adiposity and low phase angle in overweight renal transplant recipients.

Obesity is associated with increased risk of cardiovascular disease (CVD). Body mass index (BMI) is the most used parameter for obesity screening. However, the evaluation of CVD risk in overweight individuals should include the assessment of body fat distribution and body composition. Renal transplant recipients (RTR) have a high CVD risk and frequently present weight gain and loss of lean mass. The aim of this study was to evaluate body fat distribution and body composition in overweight RTR. This cross-sectional study was conducted with 86 RTR and 86 hypertensive individuals (comparison group, CG) presenting BMI 25-35 Kg/m2 and 45-70 years. Anthropometric evaluation included BMI, waist circumference, waist-to-height ratio, and a body shape index. Body composition was evaluated with bioelectrical impedance analysis (BIA). Glomerular filtration rate was estimated (eGFR) by CKD-EPI equation. RTR group (RTRG) and CG presented similar age and BMI. RTRG when compared to CG presented lower percentage of women and eGFR; higher central adiposity; and lower values of reactance, intracellular water, body cell mass and phase angle, more consistently observed in women. This study suggests that overweight RTR present higher abdominal adiposity and impairment in BIA parameters that are sensitive indicators of impaired membrane integrity, water distribution, and body cell mass.

Phenotypic, molecular and genomic characterization of Actinobaculum suis isolated from swine in Brazil.

Urinary tract infections (UTI) are considered one of the most important diseases of sows due to its close relationship with reproductive problems such as reduced litter size, increase in the rate of return to estrous, vulvar discharge, abortion, mastitis and anestrus. Actinobaculum suis is one of the main agents involved in porcine urinary tract infection and is responsible for the most severe and fatal cases in sows. In the present report, 23 A. suis strains isolated from a sow and boars in Brazil were identified by PCR and further characterized by broth microdilution, molecular typing by pulsed-field gel electrophoresis (PFGE), single-enzyme amplified fragment length polymorphism (SE-AFLP), and whole-genome sequencing. All strains were sensitive to ceftiofur, linezolid, nitrofurantoin, quinupristin-dalfopristin and vancomycin. Ciprofloxacin, daptomycin, lincomycin, erythromycin and tylosin resistance was observed in 100% of tested strains. Tetracycline and tigecycline also presented high resistance rates (87% and 30.4%, respectively). PFGE with eight different restriction enzymes and three programs did not enable strain characterization; however, all strains were typed by SE-AFLP that clustered strains according to their origin, thus proving an effective tool for A. suis genotyping. Whole-genome sequencing and comparative analysis enabled species differentiation from closely related genus. This is the first report of genomic characterization of A. suis.

Fipronil Tablets: Development and Pharmacokinetic Profile in Beagle Dogs.

Increased human-pet interactions have led to concerns related to the prevention and treatment of ectoparasite infestations. Fipronil (FIP) is a widely used ectoparasiticide in veterinary medicine available for topical administration; however, its use may cause damage to the owners and the environment. The aim of the study was to develop immediate-release tablets of FIP, as well as to determine its pharmacokinetic properties after oral administration in beagle dogs. The prepared FIP tablets were evaluated for pre-compression (angle of repose, speed flow, and Carr's index) and post-compression (weight variation, friability, thickness, hardness, disintegration time, and dissolution rate) parameters. Orally administered FIP at a dose of 2 mg/kg was rapidly absorbed with Cmáx of 3.13 ± 1.39 µg/mL at 1.83 ± 0.40 h post treatment (P.T.) and metabolized with 1.27 ± 1.04 µg/mL at 2.33 ± 0.82 h P.T. for fipronil sulfone (SULF) (the primary metabolite). The elimination of FIP and SULF occurred slowly and had maintained quantifiable plasma levels in the blood for up to 28 days P.T. The goal of the study is aligned with the concept of One Health, which aims to collaboratively achieve the best health for people, animals, and the environment. Therefore, the use of FIP tablets for the control of ectoparasites in dogs may be a safer alternative for owners and the environment.

Different interleukin-17-secreting Toll-like receptor+ T-cell subsets are associated with disease activity in multiple sclerosis.

Signalling through Toll-like receptors (TLRs) may play a role in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS). In the present study, the expression of TLR-2, -4 and -9 was significantly higher on CD4+ and CD8+ T-cells from MS patients compared to healthy individuals. Following in-vitro activation, the proportion of interleukin (IL)-17+ and IL-6+ CD4+ and CD8+ T-cells was higher in the patients. In addition, the proportion of IFN-γ-secreting TLR+ CD8+ T-cells was increased in MS patients. Among different IL-17+ T-cell phenotypes, the proportion of IL-17+ TLR+ CD4+ and CD8+ T-cells producing IFN-γ or IL-6 were positively associated with the number of active brain lesions and neurological disabilities. Interestingly, activation of purified CD4+ and CD8+ T-cells with ligands for TLR-2 (Pam3Csk4), TLR-4 [lipopolysaccharide (LPS)] and TLR-9 [oligodeoxynucleotide (ODN)] directly induced cytokine production in MS patients. Among the pathogen-associated molecular patterns (PAMPs), Pam3Csk4 was more potent than other TLR ligands in inducing the production of all proinflammatory cytokines. Furthermore, IL-6, IFN-γ, IL-17 and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels produced by Pam3Csk4-activated CD4+ cells were directly associated with disease activity. A similar correlation was observed with regard to IL-17 levels released by Pam3Csk4-stimulated CD8+ T-cells and clinical parameters. In conclusion, our data suggest that the expansion of different T helper type 17 (Th17) phenotypes expressing TLR-2, -4 and -9 is associated with MS disease activity, and reveals a preferential ability of TLR-2 ligand in directly inducing the production of cytokines related to brains lesions and neurological disabilities.

Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis.

BACKGROUND: Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE: The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS: B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS: TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS: Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.

The influence of dietary and supplemental calcium on postprandial effects of a high-fat meal on lipaemia, glycaemia, C-reactive protein and adiponectin in obese women.

Non-fasting hypertriacylglycerolaemia is a risk factor for CVD and the amount of fat in a meal seems to be the main factor influencing postprandial lipaemia. Although several studies suggest that Ca can increase faecal fat excretion, it is not known whether Ca can decrease postprandial TAG. This study aimed to evaluate the influence of dietary Ca (DC) and supplemental Ca (SC) on lipaemia, glucose metabolism, C-reactive protein (CRP) and adiponectin during postprandial period in obese women challenged with a high-fat meal. In this cross-over controlled trial, sixteen obese women aged 20-50 years were randomly assigned to receive three test meals (approximately 2900 kJ; 48 % fat): high DC (547 mg DC), high SC (HSCM; 500 mg SC-calcium carbonate) and low Ca (42 mg DC). Blood samples were collected in the fasting period and at minutes 120 and 240 after meals to evaluate total cholesterol and fractions, TAG, glucose, insulin, high-sensitivity CRP and adiponectin. Serum levels of TAG and insulin increased significantly after all test meals. Only after HSCM total cholesterol did not present a significant increase and LDL-cholesterol had a significant decrease. Postprandial glucose, HDL-cholesterol, CRP and adiponectin did not present significant changes after the three test meals. The comparative analysis of the effects of the three test meals on serum lipids, glucose, insulin, CRP and adiponectin revealed no significant meal-by-time interaction. These results suggest that in obese women challenged with a high-fat meal DC and SC do not interfere with postprandial lipaemia, glucose metabolism, CRP and adiponectin.