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The increasing prevalence of fluconazole-resistant clinical isolates of Candida spp. strongly hinders the widespread use of the drug. To tackle this problem, great efforts have been made to fully understand the fungal response to fluconazole. In this work, we show that the role of Zap1 in Candida glabrata goes beyond regulating yeast adaptation to zinc deficiency. In line with our previous observation that deletion of ZAP1 makes yeast cells more sensitive to fluconazole, we found that the mutant CgΔzap1 accumulates higher levels of the drug, which correlates well with its lower levels of ergosterol. Surprisingly, Zap1 is a negative regulator of the drug efflux transporter gene CDR1 and of its regulator, PDR1. The apparent paradox of drug accumulation in cells where genes encoding transporters relevant for drug extrusion are being overexpressed led us to postulate that their activity could be impaired. In agreement, Zap1-depleted cells present, in addition to decreased ergosterol levels, an altered composition of membrane phospholipids, which together should impact membrane function and impair the detoxification of fluconazole. Overall, our study brings to light Zap1 as an important hub in Candida glabrata response to fluconazole.
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Candida glabrata , Fluconazol , Proteínas Fúngicas , Antifúngicos/farmacologia , Candida , Candida glabrata/efeitos dos fármacos , Candida glabrata/genética , Farmacorresistência Fúngica , Ergosterol , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
Escherichia coli is an important pathogen responsible for a variety of diseases. We have recently shown that Pic, a serine protease secreted by E. coli, mediates immune evasion by the direct cleavage of complement molecules. The aim of this study was to investigate the action of a Pic-producing bacteria in a murine model of sepsis. Mice were infected with Pic-producing E. coli (F5) or F5∆pic mutant. Animal survival was monitored for five days, and a subset of mice was euthanized after 12 h for sample acquisition. The inoculation of Pic-producing bacteria induced 100% death within 24 h. The colony forming units count in the organs was significantly higher in F5. Hematological analysis showed a decrease of total leukocytes. Nitric oxide and cytokines were detected in serum, as well as on peritoneal lavage of the F5 group in higher levels than those detected in the other groups. In addition, immunophenotyping showed a decrease of activated lymphocytes and macrophages in the F5 group. Therefore, Pic represents an important virulence factor, allowing the survival of the bacterium in the bloodstream and several organs, as well as inducing a high production of proinflammatory mediators by the host, and concomitantly a cellular immunosuppression, leading to sepsis and death.
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Citocinas/metabolismo , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sepse/metabolismo , Serina Endopeptidases/metabolismo , Animais , Citocinas/genética , Escherichia coli/genética , Escherichia coli/patogenicidade , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/genética , Feminino , Inflamação/genética , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/patologia , Camundongos , Sepse/genética , Sepse/microbiologia , Sepse/patologia , Serina Endopeptidases/genéticaRESUMO
Enteroaggregative and uropathogenic Escherichia coli, Shigella flexneri 2a, and the hybrid enteroaggregative/Shiga toxin-producing E. coli strain (O104:H4) are important pathogens responsible for intestinal and urinary tract infections, as well as sepsis and hemolytic uremic syndrome. They have in common the production of a serine protease called Pic. Several biological roles for Pic have been described, including protection of E. coli DH5α from complement-mediated killing. Hereby we showed that Pic significantly reduces complement activation by all 3 pathways. Pic cleaves purified C3/C3b and other proteins from the classic and lectin pathways, such as C4 and C2. Cleavage fragments of C3, C4, and C2 were also observed with HB101(pPic1) culture supernatants, and C3 cleavage sites were mapped by fluorescence resonance energy transfer peptides. Experiments using human serum as a source of complement proteins confirmed Pic proteolytic activity on these proteins. Furthermore, Pic works synergistically with the human complement regulators factor I and factor H, promoting inactivation of C3b. In the presence of both regulators, further degradation of C3 α' chain was observed. Therefore, Pic may contribute to immune evasion of E. coli and S. flexneri, favoring invasiveness and increasing the severity of the disorders caused by these pathogens.
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Proteínas do Sistema Complemento/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/fisiologia , Evasão da Resposta Imune , Serina Endopeptidases/metabolismo , Fatores de Virulência/metabolismo , Humanos , HidróliseRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Sepsis poses one of the biggest public health problems, necessitating the search for new therapeutic alternatives. For centuries, propolis has been widely used in folk medicine to treat various inflammatory and infectious diseases. Given its extensive use, it has excellent potential as an adjuvant treatment for patients with sepsis. OBJECTIVE: This study evaluated prophylactic treatment with standardized propolis extract (EPP-AF®) and followed the prognosis of sepsis induced by ligation and cecal ligation and puncture (CLP). METHODS: Initially, for survival assessment, Swiss mice were separated into five groups: Sham (false operated), control (PBS), ATB (received antibiotic, 8 mg/kg), P10 (received EPP-AF®, 10 mg/kg), and P100 (received EPP-AF®, 100 mg/kg). The animals received PBS, antibiotic, or EPP-AF® by the subcutaneous route 6 h before the CLP procedure. Animal survival was assessed every 12 h for five days when all of them were euthanized. RESULTS: We show that the treatment with EPP-AF® significantly increased the life expectancy of animals with sepsis compared to the control group. Interestingly, prophylactic treatment with EPP-AF® showed no effect on the number of colony-forming units in the peritoneum, blood, or lung. However, there was a decrease in cellular influx in the peritoneum. This alteration was unrelated to the number of bone marrow cells or the differential counting of peripheral blood cells. The coagulogram remained unchanged, including the number of platelets and prothrombin time-activated partial thromboplastin time. However, the inflammatory infiltrate and bleeding in the lung tissue were lower in the animals that received EPP-AF®. CONCLUSION: Thus, it was possible to conclude that prophylactic treatment with EPP-AF® preserved the lung parenchyma, resulting in an increased lifespan of mice with sepsis. It can be a helpful adjuvant in prophylactic treatment with antibiotics in presurgical conditions.
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Própole , Sepse , Animais , Própole/farmacologia , Sepse/tratamento farmacológico , Sepse/mortalidade , Camundongos , Masculino , Abelhas , Pneumonia/prevenção & controle , Pneumonia/tratamento farmacológico , Modelos Animais de Doenças , Pulmão/efeitos dos fármacos , Pulmão/patologiaRESUMO
Autotransporter (AT) protein-encoding genes of diarrheagenic Escherichia coli (DEC) pathotypes (cah, eatA, ehaABCDJ, espC, espI, espP, pet, pic, sat, and tibA) were detected in typical and atypical enteropathogenic E. coli (EPEC) in frequencies between 0.8% and 39.3%. Although these ATs have been described in particular DEC pathotypes, their presence in EPEC indicates that they should not be considered specific virulence markers.
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Escherichia coli Enteropatogênica/genética , Proteínas de Membrana Transportadoras/genética , Fatores de Virulência/genética , Escherichia coli Enteropatogênica/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Epidemiologia MolecularRESUMO
Environmental factors are the main contributors to type 1 diabetes (T1D) pathogenesis, yet they remain unidentified. Enteroviruses are proposed candidate triggers due to temporal correlations between infection and T1D autoimmunity and to detection of viral proteins in diseased islets. However, such correlations are not universal and may be relatively uncommon. Furthermore, evidence of a cause-effect relationship is lacking, as infection of non-obese diabetic mice with Coxsackie enteroviruses can either trigger or blunt disease. The proposed mechanisms are either non-antigen-specific (i.e. ß-cell destruction and release of sequestered antigens, islet inflammation) or antigen-specific (i.e. epitope mimicry, by which immune responses to enteroviruses may be diverted against homologous ß-cell antigens). The case for the latter mechanisms is even less stringent, as there is little evidence of promiscuous antigen recognition at the single T-cell level. Other infectious agents may thus be implicated. Demonstration of their role will require fulfilling the Koch's postulates, namely isolation of the agent preferentially in T1D patients, including before disease onset; and T1D induction when the agent is inoculated into mice. The same is needed for cross-reactive T cells to support epitope mimicry mechanisms. Generation of alternative (humanized) mouse models that could be challenged with candidate microbes is needed.
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Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos/imunologia , Infecções/imunologia , Mimetismo Molecular/fisiologia , Animais , Autoimunidade/imunologia , Meio Ambiente , Humanos , Infecções/complicações , CamundongosRESUMO
The relationship between efficiency, quality, and access in healthcare is far from being well defined. In particular, there is no consensus on whether there is a trade-off between hospital performance and its social dimensions, such as the care appropriateness, safety, and access to proper health care. This study proposes a new approach based on the Network Data Envelopment Analysis (NDEA) to evaluate the existence of potential trade-offs between efficiency, quality, and access. The aim is to contribute for the heated debate around this topic with a novel approach. The suggested methodology combines a NDEA model with the weak disposability of outputs to handle with undesirable outputs related to the poor quality of care or the lack of access to appropriate and safe care. This combination results in a more realistic approach that has not yet been used to investigate this topic. We utilised data of the Portuguese National Health Service from 2016 to 2019, with four models and nineteen variables selected to quantify the efficiency, quality, and access to public hospital care in Portugal. A baseline efficiency score was calculated and compared with the performance scores obtained under two hypothetical scenarios to quantify the impact of each quality/access-related dimension on efficiency. The first scenario considers that each variable, individually, is at its best situation (for example, absence of septicaemia cases), and the second one, at its worst (e.g., all seen inpatients had a septicaemia case). The findings suggest that there might exist meaningful trade-offs between efficiency, quality, and access. Most variables exhibited a considerable and negative impact on the overall hospital efficiency. That is, we may expect a trade-off between efficiency and quality/access.
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AIMS/HYPOTHESIS: Anti-zinc transporter (ZnT)8 autoantibodies are commonly detected in type 1 diabetic patients. We hypothesised that ZnT8 is also recognised by CD8(+) T cells and aimed to identify HLA-A2 (A*02:01)-restricted epitope targets. METHODS: Candidate epitopes were selected by ZnT8 plasmid DNA immunisation of HLA-A2/DQ8 transgenic mice and tested for T cell recognition in peripheral blood mononuclear cells of type 1 diabetic, type 2 diabetic and healthy participants by IFN-γ enzyme-linked immunospot. RESULTS: White HLA-A2(+) adults (83%) and children (60%) with type 1 diabetes displayed ZnT8-reactive CD8(+) T cells that recognised a single ZnT8(186-194) (VAANIVLTV) epitope. This ZnT8(186-194)-reactive fraction accounted for 50% to 53% of total ZnT8-specific CD8(+) T cells. Another sequence, ZnT8(153-161) (VVTGVLVYL), was recognised in 20% and 25% of type 1 diabetic adults and children, respectively. Both epitopes were type 1 diabetes-specific, being marginally recognised by type 2 diabetic and healthy participants (7-12% for ZnT8(186-194), 0% for ZnT8(153-161)). CONCLUSIONS/INTERPRETATION: ZnT8-reactive CD8(+) T cells are predominantly directed against the ZnT8(186-194) epitope and are detected in a majority of type 1 diabetic patients. The exceptional immunodominance of ZnT8(186-194) may point to common environmental triggers precipitating beta cell autoimmunity.
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Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte de Cátions/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/imunologia , Adolescente , Adulto , Animais , Autoanticorpos/genética , Linfócitos T CD4-Positivos/imunologia , Proteínas de Transporte de Cátions/genética , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/genética , Mapeamento de Epitopos , Epitopos de Linfócito T/genética , Feminino , Antígeno HLA-A2/genética , Humanos , Lactente , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Transportador 8 de ZincoRESUMO
Several natural products have been investigated for their bactericidal potential, among these, cinnamaldehyde. In this study, we aimed to evaluate the activity of cinnamaldehyde in the treatment of animals with sepsis induced by extraintestinal pathogenic E. coli. Initially, the E. coli F5 was incubated with cinnamaldehyde to evaluate the minimum inhibitory and minimum bactericidal concentration. Animal survival was monitored for five days, and a subset of mice were euthanized after 10 h to evaluate histological, hematological, and immunological parameters, as well as the presence of bacteria in the organs. On the one hand, inoculation of bacterium caused the death of 100% of the animals within 24 h after infection. On the other hand, cinnamaldehyde (60 mg/kg) was able to keep 40% of mice alive after infection. The treatment significantly reduced the levels of cytokines in serum and peritoneum and increased the production of cells in both bone marrow and spleen, as well as lymphocytes at the infection site. Cinnamaldehyde was able to reduce tissue damage by decreasing the deleterious effects for the organism and contributed to the control of the sepsis and survival of animals; therefore, it is a promising candidate for the development of new drugs.
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Escherichia coli is responsible for cases of diarrhea around the world, and some studies have shown the benefits of cinnamaldehyde in the treatment of bacterial disease. Therefore, the objective of this study was to evaluate the effects of cinnamaldehyde in mice colonized by pathogenic E. coli, as well as to provide more insights into its antimicrobial action mechanism. After determination of minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations, the interference of cinnamaldehyde in macromolecular pathways (synthesis of DNA, RNA, protein, and cell wall) was measured by incorporation of radioisotopes. The anti-adhesive properties of cinnamaldehyde towards E. coli 042 were evaluated using human epithelial type 2 (HEp-2) cells. Intestinal colonization was tested on mice, and the effect of cinnamaldehyde on Tenebrio molitor larvae. Cinnamaldehyde showed MIC and MBC values of 780 µg/mL and 1560 µg/mL, respectively; reduced the adhesion of E. coli 042 on HEp-2 cells; and affected all the synthetic pathways evaluated, suggesting that compost impairs the membrane/cell wall structure leading bacteria to total collapse. No effect on the expression of genes related to the SOS pathway (sulA and dinB1) was observed. The compound did not interfere with cell viability and was not toxic against T. molitor larvae. In addition, cinnamaldehyde-treated mice exhibited lower levels of colonization by E. coli 042 than the untreated group. Therefore, the results show that cinnamaldehyde is effective in treating the pathogenic E. coli strain 042 and confirm it as a promising lead molecule for the development of antimicrobial agents.
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Acroleína/análogos & derivados , Escherichia coli/efeitos dos fármacos , Acroleína/farmacologia , Animais , Antibacterianos/farmacologia , Produtos Biológicos/farmacologia , Humanos , Intestinos/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Tenebrio/microbiologiaRESUMO
AIMS/HYPOTHESIS: Displaying immunomodulatory capacities, mesenchymal stem cells (MSCs) are considered as beneficial agents for autoimmune diseases. The aim of this study was to examine the ability of MSCs to prevent autoimmune diabetes in the NOD mouse model. METHODS: Prevention of spontaneous insulitis or of diabetes was evaluated after a single i.v. injection of MSCs in 4-week-old female NOD mice, or following the co-injection of MSCs and diabetogenic T cells in irradiated male NOD recipients, respectively. The frequency of CD4(+)FOXP3(+) cells and Foxp3 mRNA levels in the spleen of male NOD recipients were also quantified. In vivo cell homing was assessed by monitoring 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled T cells or MSCs. In vitro, cell proliferation and cytokine production were assessed by adding graded doses of irradiated MSCs to insulin B9-23 peptide-specific T cell lines in the presence of irradiated splenocytes pulsed with the peptide. RESULTS: MSCs reduced the capacity of diabetogenic T cells to infiltrate pancreatic islets and to transfer diabetes. This protective effect was not associated with the modification of diabetogenic T cell homing, but correlated with a preferential migration of MSCs to pancreatic lymph nodes. While injection of diabetogenic T cells resulted in a decrease in levels of FOXP3(+) regulatory T cells, this decrease was inhibited by MSC co-transfer. Moreover, MSCs were able to suppress both allogeneic and insulin-specific proliferative responses in vitro. This suppressive effect was associated with the induction of IL10-secreting FOXP3(+) T cells. CONCLUSIONS/INTERPRETATION: MSCs prevent autoimmune beta cell destruction and subsequent diabetes by inducing regulatory T cells. MSCs may thus offer a novel cell-based approach for the prevention of autoimmune diabetes and for islet cell transplantation.
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Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Transplante de Células-Tronco Mesenquimais , Estado Pré-Diabético/terapia , Linfócitos T Reguladores/citologia , Animais , Movimento Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Células Secretoras de Insulina/imunologia , Interleucina-10/metabolismo , Teste de Cultura Mista de Linfócitos , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Estado Pré-Diabético/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
BACKGROUND: The congenital muscular torticollis is characterized by a persistent lateral flexion of the head to the affected side and cervical rotation to the opposite side due to unilateral shortening of the sternocleidomastoid muscle. The majority of the cases resolve with conservative management, with parents/caregivers education and physical therapy.The aim of this study was to assess demographic and clinical characteristics, treatment options, and outcome, amongst infants referred to pediatric rehabilitation consultation due to congenital muscular torticollis. METHODS: Retrospective cohort study of infants diagnosed with congenital muscular torticollis between January 2012 and December 2014. Obstetric and perinatal data, clinical presentation, comorbidities, treatment, and outcome were abstracted from clinical records. RESULTS: One hundred six infants were included. There was no sex predominance and mean age at first pediatric rehabilitation consultation was 11.6 (10.4) weeks. Most women were primiparous (76.4%), dystocic labor predominated (73.6%), and pelvic fetal presentation occurred in 20.8%. At examination, 49.1% of the infants had abnormalities, beyond the tilt cervical, mainly range of motion restrictions and palpable nodule in sternocleidomastoid muscle. Among the 87 children who performed the cervical ultrasound, 29 (27.4%) had anomalies. Associated clinical conditions such as hip dysplasia were identified. The majority (71.7%) were submitted to conservative treatment, 30.2% in the Pediatric Rehabilitation Department. Most infants (97.2%) showed a complete resolution of the torticollis. CONCLUSIONS: Congenital muscular torticollis is the most common cause of torticollis in the infants. Early diagnosis, parent/caregivers education, and conservative treatment are crucial to achieving good results.
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BACKGROUND: Physical activity (PA) is recommended as adjuvant therapy to control blood pressure (BP). The effectiveness of simple recommendations is not clear. We aimed to assess the agreement between self-report of adherence to PA in clinical routine and International Physical Activity Questionnaire (IPAQ) interview and its association with BP control. METHODS: A cross-sectional study was conducted with hypertensive outpatients. Adherence to recommendation to PA was assessed by the physician and IPAQ interview. A cutoff of 150 minutes/week was used to classify active or nonactive patients. High sitting time was considered >4 hours/day. A total of 127 individuals (SBP 144.9±24.4 mmHg/DBP 82.0±12.8 mmHg) were included. RESULTS: A total of 69 subjects (54.3%) reported to be active to their physician, whereas 81 (63.8%) were classified as active by IPAQ (6.3% active in leisure time PA). Kappa test was 0.22 (95% CI, 0.06-0.37). The rate of BP control was 45.7%. There was no association with the reported PA assessed by both methods nor with sitting time. Our results demonstrated poor agreement between self-report adherence and IPAQ interview, and neither evaluation was associated with BP control. CONCLUSION: Our findings underpin evidences that a simple PA recommendation has low association with BP control in clinical settings.
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Background: The spinocerebellar ataxias (SCAs) are a group of autosomal dominant degenerative diseases characterized by cerebellar ataxia. Classified according to gene discovery, specific features of the SCAs - clinical, laboratorial, and neuroradiological (NR) - can facilitate establishing the diagnosis. The purpose of this study was to review the particular NR abnormalities in the main SCAs. Methods: We conducted a literature search on this topic. Results: The main NR characteristics of brain imaging (magnetic resonance imaging or computerized tomography) in SCAs were: (1) pure cerebellar atrophy; (2) cerebellar atrophy with other findings (e.g., pontine, olivopontocerebellar, spinal, cortical, or subcortical atrophy; "hot cross bun sign", and demyelinating lesions); (3) selective cerebellar atrophy; (4) no cerebellar atrophy. Discussion: The main NR abnormalities in the commonest SCAs, are not pathognomonic of any specific genotype, but can be helpful in limiting the diagnostic options. We are progressing to a better understanding of the SCAs, not only genetically, but also pathologically; NR is helpful in the challenge of diagnosing the specific genotype of SCA.
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Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Neuroimagem , Ataxias Espinocerebelares/diagnóstico por imagem , Ataxias Espinocerebelares/patologia , HumanosRESUMO
Staphylococcus aureus is a notorious human pathogen associated with serious nosocomial and community-acquired infections, such as pneumonia, meningitis, endocarditis, toxic shock syndrome, and sepsis, among others. The objective of this study was to investigate the molecular profile, antimicrobial resistance, and clonal diversity of S. aureus isolated from the bloodstream. The determination of the minimum inhibitory concentration (MIC) of the antimicrobial was performed by an automated method. The presence of several virulence and resistance genes was evaluated by PCR. In addition, multilocus sequence typing (MLST) was used to analyze the clonal diversity of S. aureus. A high resistance to oxacillin (78%), clindamycin (78%), erythromycin (70%), ciprofloxacin (61%), and gentamicin (52%) was observed among the isolates. In most of them, the following virulence genes were detected: hlb (83%), ebpS (61%), icaA (57%), fnbpA (17%), and clfA (13%). Only one isolate carried the pvl gene. MLST analysis identified five new sequence types (STs): 5429, 5430, 5431, 5432, and 5433, as well as another seven-ST5, ST97, ST398, ST101, ST30, ST461, and ST2779-among the remaining strains. These seven STs and the four new STs are clustered in four clonal complexes: CC1, CC2, CC7, and CC17. Phylogenetic analysis showed the genetic relationship of the five new ST strains with another 18 strains. Altogether, these analyses indicate the horizontal transfer acquisition of virulence factor genes and multidrug resistance.
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Per-operative management of patients with cardiovascular pathology, has been regarded, since ever, as a challenge for anesthesiologists and vascular surgeons. Vascular disease, often diffuse and asymptomatic, has a high prevalence in the community and is associated to an annual mortality rate of 12%. This requires that anesthetic technique should be concentrated in the preservation of myocardial, renal, pulmonary and cerebral functions, during the hemodynamic alterations related to surgery, in order to lower the per-operative complications and to promote faster and safer post-operative recovery. In this paper we describe the experience of the Anesthesiology Department of S. Joatilde;o Hospital, Porto, particulary with the superficial and deep cervical plexus block, in carotid endarterectomy surgery.
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Anestesia Local , Procedimentos Cirúrgicos Vasculares , Idoso , Feminino , Humanos , MasculinoRESUMO
Leptospires are highly motile spirochetes equipped with strategies for efficient invasion and dissemination within the host. Our group previously demonstrated that pathogenic leptospires secrete proteases capable of cleaving and inactivating key molecules of the complement system, allowing these bacteria to circumvent host's innate immune defense mechanisms. Given the successful dissemination of leptospires during infection, we wondered if such proteases would target a broader range of host molecules. In the present study, the proteolytic activity of secreted leptospiral proteases against a panel of extracellular matrix (ECM) and plasma proteins was assessed. The culture supernatant of the virulent L. interrogans serovar Kennewicki strain Fromm (LPF) degraded human fibrinogen, plasma fibronectin, gelatin, and the proteoglycans decorin, biglycan, and lumican. Interestingly, human plasminogen was not cleaved by proteases present in the supernatants. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. Moreover, production of proteases might be an important virulence determinant since culture-attenuated or saprophytic Leptospira did not display proteolytic activity against ECM or plasma components. Exoproteomic analysis allowed the identification of three metalloproteases that could be involved in the degradation of host components. The ability to cleave conjunctive tissue molecules and coagulation cascade proteins may certainly contribute to invasion and tissue destruction observed upon infection with Leptospira.
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Proteínas de Bactérias/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/microbiologia , Leptospira interrogans/enzimologia , Leptospirose/metabolismo , Leptospirose/microbiologia , Peptídeo Hidrolases/metabolismo , Proteínas de Bactérias/genética , Proteínas Sanguíneas/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Interações Hospedeiro-Patógeno , Humanos , Leptospira interrogans/genética , Leptospirose/sangue , Peptídeo Hidrolases/genética , ProteóliseRESUMO
Chronic use of statins may have anti-inflammatory action, promoting immunomodulation and survival in patients with sepsis. This study aimed to analyze the effects of pretreatment with simvastatin in lethal sepsis induced by cecal ligation and puncture (CLP). Male Swiss mice received prophylactic treatment with simvastatin or pyrogen-free water orally in a single daily dose for 30 days. After this period, the CLP was performed. Naïve and Sham groups were performed as non-infected controls. Animal survival was monitored for 60 h after the CLP. Half of mice were euthanized after 12 h to analyze colony-forming units (CFUs); hematological parameters; production of IL-10, IL-12, IL-6, TNF-α, IFN-γ, and MCP-1; cell counts on peritoneum, bronchoalveolar lavage (BAL), bone marrow, spleen, and mesenteric lymph node; immunephenotyping of T cells and antigen presenting cells and production of hydrogen peroxide (H2O2). Simvastatin induced an increase in survival and a decrease in the CFU count on peritoneum and on BAL cells number, especially lymphocytes. There was an increase in the platelets and lymphocytes number in the Simvastatin group when compared to the CLP group. Simvastatin induced a greater activation and proliferation of CD4+ T cells, as well as an increase in IL-6 and MCP-1 production, in chemotaxis to the peritoneum and in H2O2 secretion at this site. These data suggest that simvastatin has an impact on the survival of animals, as well as immunomodulatory effects in sepsis induced by CLP in mice.
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Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Sepse , Sinvastatina/farmacologia , Animais , Linfócitos T CD4-Positivos/patologia , Modelos Animais de Doenças , Peróxido de Hidrogênio/imunologia , Masculino , Camundongos , Sepse/imunologia , Sepse/patologia , Sepse/prevenção & controleRESUMO
Complement is a crucial arm of the innate immune response against invading bacterial pathogens, and one of its main functions is to recognize and destroy target cells. Similar to other pathogens, Escherichia coli has evolved mechanisms to overcome complement activation. It is well known that capsular polysaccharide may confer resistance to complement-mediated killing and phagocytosis, being one of the strategies adopted by this bacterium to survive in serum. In addition, proteases produced by E. coli have been shown to downregulate the complement system. Pic, an autotransporter secreted by different pathogens in the Enterobacteriaceae family, is able to cleave C2, C3/C3b, and C4/C4b and works synergistically with human Factor I and Factor H (FH), thereby promoting inactivation of C3b. Extracellular serine protease P, a serine protease of enterohemorrhagic E. coli (EHEC), downregulates complement activation by cleaving C3/C3b and C5. StcE, a metalloprotease secreted by EHEC, inhibits the classical complement-mediated cell lysis by potentiating the action of C1 inhibitor, and the periplasmic protease Prc contributes to E. coli complement evasion by interfering with the classical pathway activation and by preventing membrane attack complex deposition. Finally, it has been described that E. coli proteins interact with negative complement regulators to modulate complement activation. The functional consequences resulting from the interaction of outer membrane protein A, new lipoprotein I, outer membrane protein W, and Stx2 with proteins of the FH family and C4b-binding protein (C4BP) are discussed in detail. In brief, in this review, we focused on the different mechanisms used by pathogenic E. coli to circumvent complement attack, allowing these bacteria to promote a successful infection.
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Enteroaggregative Escherichia coli (EAEC) is an agent of acute and persistent diarrhea worldwide, categorized in typical or atypical subgroups. Some EAEC virulence factors are members of the serine protease autotransporters of Enterobacteriaceae (SPATE). The presence of SPATE-encoding genes of different E. coli pathotypes was searched in a large collection of EAEC strains, and a possible association between SPATEs and E. coli phylogroups was investigated. Among 108 typical and 85 atypical EAEC, pic was the most prevalent gene, detected in 47.1% of the strains, followed by sat (24.3%), espI (21.2%), pet (19.2%), sepA (13.5%), sigA (4.1%), eatA (4.1%), vat (1.0%), espP and tsh, detected in one strain (0.5%) each; while epeA and espC were not detected. Phylogenetic analysis demonstrated that 39.9% of the strains belonged to group A, 23.3% to B1, 10.9% to B2, 7.8% to D, 8.8% to E and 1.5% to F. The majority of the SPATE genes were distributed in typical and atypical strains without association with any phylogroup. In addition, pic and pet were strongly associated with typical EAEC and sepA was detected in close association with atypical EAEC. Our data indicate that SPATEs may represent important virulence traits in both subgroups of EAEC.