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Neurônios GABAérgicos , Hormônio Liberador de Gonadotropina , Síndrome do Ovário Policístico , Receptores Androgênicos , Animais , Feminino , Humanos , Camundongos , Neurônios GABAérgicos/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Síndrome do Ovário Policístico/metabolismo , Receptores Androgênicos/metabolismoAssuntos
Integrases , Animais , Camundongos , Alelos , Integrases/genética , Camundongos TransgênicosRESUMO
We employed the whole-cell patch-clamp method and ChAT-Cre mice to study the electrophysiological attributes of cholinergic neurons in the external globus pallidus. Most neurons were inactive, although approximately 20% displayed spontaneous firing, including burst firing. The resting membrane potential, the whole neuron input resistance, the membrane time constant and the total neuron membrane capacitance were also characterized. The current-voltage relationship showed time-independent inward rectification without a "sag". Firing induced by current injections had a brief initial fast adaptation followed by tonic firing with minimal accommodation. Intensity-frequency plots exhibited maximal average firing rates of about 10 Hz. These traits are similar to those of some cholinergic neurons in the basal forebrain. Also, we examined their dopamine sensitivity by acutely blocking dopamine receptors. This action demonstrated that the membrane potential, excitability, and firing pattern of pallidal cholinergic neurons rely on the constitutive activity of dopamine receptors, primarily D2-class receptors. The blockade of these receptors induced a resting membrane potential hyperpolarization, a decrease in firing for the same stimulus, the disappearance of fast adaptation, and the emergence of a depolarization block. This shift in physiological characteristics was evident even when the hyperpolarization was corrected with D.C. current. Neither the currents that generate the action potentials nor those from synaptic inputs were responsible. Instead, our findings suggest, that subthreshold slow ion currents, that require further investigation, are the target of this novel dopaminergic signaling.
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Dopamina , Globo Pálido , Camundongos , Animais , Dopamina/fisiologia , Potenciais de Ação/fisiologia , Neurônios Colinérgicos , Receptores Dopaminérgicos , ColinérgicosRESUMO
Antigen cross-presentation is a vital mechanism of dendritic cells and other antigen presenting cells to orchestrate the priming of cytotoxic responses towards killing of infected or cancer cells. In this process, exogenous antigens are internalized by dendritic cells, processed, loaded onto MHC class I molecules and presented to CD8+ T cells to activate them. Sec22b is an ER-Golgi Intermediate Compartment resident SNARE protein that, in partnership with sintaxin4, coordinates the recruitment of the transporter associated with antigen processing protein and the peptide loading complex to phagosomes, where antigenic peptides that have been proteolyzed in the cytosol are loaded in MHC class I molecules and transported to the cell membrane. The silencing of Sec22b in dendritic cells primary cultures and conditionally in dendritic cells of C57BL/6 mice, critically impairs antigen cross-presentation, but neither affects other antigen presentation routes nor cytokine production and secretion. Mice with Sec22b conditionally silenced in dendritic cells (Sec22b-/-) show deficient priming of CD8+ T lymphocytes, fail to control tumor growth, and are resistant to anti-checkpoint immunotherapy. In this work, we show that Sec22b-/- mice elicit a deficient specific CD8+ T cell response when challenged with sublethal doses of Trypanosoma cruzi trypomastigotes that is associated with increased blood parasitemia and diminished survival.
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Hematopoietic stem cells are maintained in a specialized microenvironment, known as the 'niche', within the bone marrow. Understanding the contribution of cellular and molecular components within the bone marrow niche for the maintenance of hematopoietic stem cells is crucial for the success of therapeutic applications. So far, the roles of crucial mechanisms within the bone marrow niche have been explored in transgenic animals in which genetic modifications are ubiquitously introduced in the whole body. The lack of precise tools to explore genetic alterations exclusively within the bone marrow prevents our determination of whether the observed outcomes result from confounding effects from other organs. Here, we developed a new method - 'whole bone subcutaneous transplantation'- to study the bone marrow niche in transgenic animals precisely. Using immunolabeling of CD45.1 (donor) vs. CD45.2 (recipient) hematopoeitic stem cells, we demonstrated that hematopoeitic stem cells from the host animals colonize the subcutaneously transplanted femurs after transplantation, while the hematopoietic stem cells from the donor disappear. Strikinlgy, the bone marrow niche of these subcutaneously transplanted femurs remain from the donor mice, enabling us to study specifically cells of the bone marrow niche using this model. We also showed that genetic ablation of peri-arteriolar cells specifically in donor femurs reduced the numbers of hematopoietic stem cells in these bones. This supports the use of this strategy as a model, in combination with genetic tools, to evaluate how bone marrow niche specific modifications may impact non-modified hematopoietic stem cells. Thus, this approach can be utilized for genetic manipulation in vivo of specific cell types only within the bone marrow. The combination of whole bone subcutaneous transplantation with rodent transgenic models will facilitate a more precise, complex and comprehensive understanding of existing problems in the study of the hematopoietic stem cell bone marrow niche.
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Medula Óssea , Transplante de Células-Tronco Hematopoéticas , Camundongos , Animais , Células-Tronco Hematopoéticas/metabolismo , Transplante de Medula Óssea , Osso e OssosRESUMO
Background: Antibiotic-resistant gram-negative bloodstream infections (BSI) remain a leading cause morbidity and mortality in pediatric patients with a high impact on the public health system. Data in resource-limited countries, including those in Latin America and the Caribbean region, are scarce. The aim of the study was to identify risk factors for acquiring carbapenem-resistant Enterobacteriaceae (CRE) bacteremia in children and to assess the use of resources. Methods: A retrospective case-control study was conducted to analyze demographic, epidemiological, clinical, microbiological, and outcome data as well as the use of resources between 2014 and 2019. Univariate and logistic regression analysis was performed in order to identify risk factors associated with CRE-BSI. The R software version 4.1.2 was used. Results: A total of 46 cases with CRE-BSI and 92 controls with gram-negative non-CRE-BSI were included. No statistical difference was observed regarding: median age (36 months; IQR, 11.2-117 vs. 48 months, IQR 13-119), male sex (50 vs. 60%), and underlying disease (98 vs. 91%) in cases vs. controls, respectively. The most frequent mechanism of CRE bacteremia were: KPC in 74%, OXA in 15%, and NDM in 6.5%. A total of 54.3% of cases vs. 32.6 % (p = 0.016) of controls were admitted to the pediatric intensive care unit (PICU), and 48 vs. 21% (p = 0.001) required mechanical ventilation. Bacteremia secondary to intra-abdominal infection was observed in 56.5% of cases vs. 35% of controls (p = 0.032). Previous colonization with CRE was detected in 76% of cases vs. 8% of controls. Combination antimicrobial treatment was most frequent in cases vs. control (100 vs. 56.5%). No difference was observed in median length of hospital stay (22 days; IQR, 19-31 in cases vs. 17.5 days; IQR, 10-31 in controls; p = 0.8). Overall case fatality ratio was 13 vs. 5.5%, respectively. The most statistically significant risk factors included previous PICU stay (OR, 4; 95%CI, 2-8), invasive procedures/surgery (OR, 3; 95%CI, 1-7), central venous catheter placement (OR, 6.5; 95%CI, 2-19), urinary catheter placement (OR, 9; 95%CI 4-20), mechanical ventilation (OR, 4; 95%CI, 2-10), liver transplantation (OR, 8; 95%CI, 2-26), meropenem treatment (OR, 8.4; 3.5-22.6) in univariate analysis. The logistic regression model used for multivariate analysis yielded significant differences for previous meropenem treatment (OR, 13; 95%CI, 3-77; p = 0.001), liver transplantation (OR, 13; 95%CI, 2.5-100; p = 0.006), and urinary catheter placement (OR, 9; 95%CI, 1.4-94; p = 0.03). Conclusion: CRE-BSI affects hospitalized children with underlying disease, mainly after liver transplantation, with previous urinary catheter use and receiving broad-spectrum antibiotics, leading to high PICU requirement and mortality. These risk factors will have to be taken into account in our region in order to establish adequate health policies and programs to improve antimicrobial stewardship.
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Bacteriemia , Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Enterobacteriaceae , Sepse , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Argentina/epidemiologia , Bacteriemia/tratamento farmacológico , Bacteriemia/epidemiologia , Bacteriemia/microbiologia , Carbapenêmicos/farmacologia , Carbapenêmicos/uso terapêutico , Estudos de Casos e Controles , Criança , Pré-Escolar , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Hospitais Pediátricos , Humanos , Lactente , Masculino , Meropeném/uso terapêutico , Encaminhamento e Consulta , Estudos Retrospectivos , Sepse/tratamento farmacológicoRESUMO
It is well established that temporal lobe epilepsy (TLE) is often related to oxidative stress and neuroinflammation. Both processes subserve alterations observed in epileptogenesis and ultimately involve distinct classes of cells, including astrocytes, microglia, and specific neural subtypes. For this reason, molecules associated with oxidative stress response and neuroinflammation have been proposed as potential targets for therapeutic strategies. However, these molecules can participate in distinct intracellular pathways depending on the cell type. To illustrate this, we reviewed the potential role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and myeloid differentiation primary response 88 (MyD88) in astrocytes, microglia, and neurons in epileptogenesis. Furthermore, we presented approaches to study genes in different cells, employing single-cell RNA-sequencing (scRNAseq) transcriptomic analyses, transgenic technologies and viral serotypes carrying vectors with specific promoters. We discussed the importance of identifying particular roles of molecules depending on the cell type, endowing more effective therapeutic strategies to treat TLE.
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Background: Healthcare-associated infections by carbapenem-resistant Klebsiella pneumoniae are difficult to control. Virulence and antibiotic resistance genes contribute to infection, but the mechanisms associated with the transition from colonization to infection remain unclear. Objective: We investigated the transition from carriage to infection by K. pneumoniae isolates carrying the K. pneumoniae carbapenemase-encoding gene bla KPC (KpKPC). Methods: KpKPC isolates detected within a 10-year period in a single tertiary-care hospital were characterized by pulsed-field gel electrophoresis (PFGE), multilocus sequencing typing, capsular lipopolysaccharide and polysaccharide typing, antimicrobial susceptibility profiles, and the presence of virulence genes. The gastrointestinal load of carbapenem-resistant Enterobacteriaceae and of bla KPC-carrying bacteria was estimated by relative quantification in rectal swabs. Results were evaluated as contributors to the progression from carriage to infection. Results: No PGFE type; ST-, K-, or O-serotypes; antimicrobial susceptibility profiles; or the presence of virulence markers, such yersiniabactin and colibactin, were associated with carriage or infection, with ST437 and ST11 being the most prevalent clones. Admission to intensive and semi-intensive care units was a risk factor for the development of infections (OR 2.79, 95% CI 1.375 to 5.687, P=0.005), but higher intestinal loads of carbapenem-resistant Enterobacteriaceae or of bla KPC-carrying bacteria were the only factors associated with the transition from colonization to infection in this cohort (OR 8.601, 95% CI 2.44 to 30.352, P<0.001). Conclusion: The presence of resistance and virulence mechanisms were not associated with progression from colonization to infection, while intestinal colonization by carbapenem-resistant Enterobacteriacea and, more specifically, the load of gastrointestinal carriage emerged as an important determinant of infection.
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Enterobacteriáceas Resistentes a Carbapenêmicos , Infecção Hospitalar , Infecções por Klebsiella , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Infecção Hospitalar/microbiologia , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , beta-Lactamases/genéticaRESUMO
Enhancers are regulatory elements of genomes that determine spatio-temporal patterns of gene expression. The human genome contains a vast number of enhancers, which largely outnumber protein-coding genes. Historically, enhancers have been regarded as highly tissue-specific. However, recent evidence has demonstrated that many enhancers are pleiotropic, with activity in multiple developmental contexts. Yet, the extent and impact of pleiotropy remain largely unexplored. In this study we analyzed active enhancers across human organs based on the analysis of both eRNA transcription (FANTOM5 consortium data sets) and chromatin architecture (ENCODE consortium data sets). We show that pleiotropic enhancers are pervasive in the human genome and that most enhancers active in a particular organ are also active in other organs. In addition, our analysis suggests that the proportion of context-specific enhancers of a given organ is explained, at least in part, by the proportion of context-specific genes in that same organ. The notion that such a high proportion of human enhancers can be pleiotropic suggests that small regions of regulatory DNA contain abundant regulatory information and that these regions evolve under important evolutionary constraints.
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Elementos Facilitadores Genéticos , Genoma Humano , Evolução Biológica , Cromatina/genética , HumanosRESUMO
BACKGROUND: The generation of animals expressing reporter proteins (e.g., GFP, mCherry or tdTomato) under the control of genes of interest has become a valuable tool in neuroscience. However, the histological reuse of brain sections of these genetically modified animals in unplanned experiments is often infeasible since the constitutive expression of fluorescent reporter proteins interferes with further fluorescent staining procedures. Thus, expensive or time-demanding experiments frequently need to be repeated using additional experimental animals. NEW METHOD: To improve the reuse of tissues of reporter animals for fluorescent staining procedures, we developed fast, inexpensive and simple methods that induce denaturation of constitutively expressed fluorescent proteins in free-floating brain slices. These procedures consist of incubation of brain sections either in a 1% sodium hydroxide alkaline solution (pH 13.0) for one hour at room temperature or at 95 °C for 10-30 min. RESULTS: The strong fluorescence of tdTomato, mCherry and eGFP was completely eliminated after incubation of brain sections of different reporter mice in a pH 13.0 solution for one hour. hrGFP was resistant to denaturation in an alkaline solution, but incubation of brain sections at 95 °C for 10 min eliminated the fluorescence of hrGFP, as well as of tdTomato, mCherry and eGFP. The denaturing procedures did not prevent the reuse of brain tissues in free-floating immunofluorescence staining using multiple antibodies. Furthermore, the quality of the labeling remained unaffected. Although pretreatment in pH 13.0 solution maintained good tissue integrity, as a side effect, brain sections exhibited increased autofluorescence. However, a rinse in 0.25% Sudan Black B solution was efficient in eliminating the autofluorescence without impairing the immunofluorescence staining or DAPI counterstaining. CONCLUSIONS: The present study provides simple procedures capable of inducing denaturation of fluorescent proteins in free-floating brain slices.
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Anticorpos , Encéfalo , Animais , Encéfalo/metabolismo , Corantes/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Coloração e RotulagemRESUMO
OBJECTIVES: This study analysed the impact of antimicrobial stewardship team (AST) evaluation on time to susceptible in vitro therapy and mortality of patients with carbapenem-resistant Enterobacterales (CRE) bacteraemia. METHODS: We performed a retrospective cohort study (February 2018 to July 2020) to evaluate the impact of AST evaluation, along with other clinical and microbiological variables, on time to appropriate antibiotics, 14-day mortality and in-hospital mortality in patients aged >18 years with CRE bacteraemia. A Cox regression model was used for multivariate analysis. RESULTS: A total of 142 patients were included. The proportion of patients who received appropriate antibiotics in the first 5 days after bacteraemia was 82/92 (89.1%) versus 29/50 (58.0%) evaluated and not evaluated by the AST, respectively (P < 0.01). AST evaluation reduced the median time to appropriate therapy (49.8 h vs. 71.1 h; P = 0.01). AST intervention was independently associated with earlier prescription of appropriate therapy (P = 0.02) when controlled for septic shock (P < 0.01) and CRE isolation in the previous 90 days (P = 0.04). Regarding mortality, 51 patients (35.9%) died within 14 days (25.8% vs. 44.7% with and without AST intervention, respectively; P = 0.02) and 82 patients (57.7%) in hospital (52.2% vs. 68.0% evaluated and not evaluated by the AST, respectively; P = 0.08). AST intervention was independently protective for 14-day mortality (P = 0.03) when controlled for septic shock status (P < 0.01). CONCLUSION: AST guidance improves the quality of antibiotic prescriptions and clinical outcomes in patients with CRE bacteraemia.
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Gestão de Antimicrobianos , Bacteriemia , Farmacorresistência Bacteriana , Gammaproteobacteria , Choque Séptico , Antibacterianos/uso terapêutico , Bacteriemia/microbiologia , Carbapenêmicos/uso terapêutico , Estudos de Coortes , Humanos , Prescrições , Estudos Retrospectivos , Choque Séptico/tratamento farmacológicoRESUMO
BACKGROUND: 6S RNA is a regulator of cellular transcription that tunes the metabolism of cells. This small non-coding RNA is found in nearly all bacteria and among the most abundant transcripts. Lactic acid bacteria (LAB) constitute a group of microorganisms with strong biotechnological relevance, often exploited as starter cultures for industrial products through fermentation. Some strains are used as probiotics while others represent potential pathogens. Occasional reports of 6S RNA within this group already indicate striking metabolic implications. A conceivable idea is that LAB with 6S RNA defects may metabolize nutrients faster, as inferred from studies of Echerichia coli. This may accelerate fermentation processes with the potential to reduce production costs. Similarly, elevated levels of secondary metabolites might be produced. Evidence for this possibility comes from preliminary findings regarding the production of surfactin in Bacillus subtilis, which has functions similar to those of bacteriocins. The prerequisite for its potential biotechnological utility is a general characterization of 6S RNA in LAB. RESULTS: We provide a genomic annotation of 6S RNA throughout the Lactobacillales order. It laid the foundation for a bioinformatic characterization of common 6S RNA features. This covers secondary structures, synteny, phylogeny, and product RNA start sites. The canonical 6S RNA structure is formed by a central bulge flanked by helical arms and a template site for product RNA synthesis. 6S RNA exhibits strong syntenic conservation. It is usually flanked by the replication-associated recombination protein A and the universal stress protein A. A catabolite responsive element was identified in over a third of all 6S RNA genes. It is known to modulate gene expression based on the available carbon sources. The presence of antisense transcripts could not be verified as a general trait of LAB 6S RNAs. CONCLUSIONS: Despite a large number of species and the heterogeneity of LAB, the stress regulator 6S RNA is well-conserved both from a structural as well as a syntenic perspective. This is the first approach to describe 6S RNAs and short 6S RNA-derived transcripts beyond a single species, spanning a large taxonomic group covering multiple families. It yields universal insights into this regulator and complements the findings derived from other bacterial model organisms.
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Regulação Bacteriana da Expressão Gênica , Lactobacillales/genética , RNA Bacteriano/genética , RNA não Traduzido/genética , Bacillus subtilis/genética , Sequência Conservada/genética , Humanos , Sintenia/genéticaRESUMO
Infections by carbapenem-resistant Klebsiella pneumoniae (CRKp) are an increasing global threat with limited therapeutic options. Our objective was to evaluate clinical and microbiological outcomes of patients treated with amikacin for CRKp infections. We did a retrospective cohort of patients > 18 years old, with CRKp infections treated with amikacin in two tertiary care hospitals in Porto Alegre, Brazil. The impact of clinical factors, antibiotic treatment, and amikacin minimum inhibitory concentration (MIC) on patients' 30-day mortality was assessed. Microbiological clearance and nephrotoxicity (assessed by RIFLE score) were evaluated as secondary outcomes. A Cox regression analysis was done for mortality. We included 84 patients for analysis. Twenty-nine (34.5%) patients died in 30 days. Amikacin MIC values ranged from 0.125 to 8 µg/mL and did not influence on mortality, regardless of the prescribed dose of this antibiotic (P = 0.24). Bacterial clearance occurred in 17 (58.6%) of 29 patients who collected subsequent cultures. Two (16.6%) of the 12 persistently positive cultures changed the amikacin susceptibility profile from susceptible to intermediate. Twenty-nine (37.2%) patients developed acute kidney injury (AKI): risk 13, injury 11, and failure 5. Risk factors for AKI were higher baseline eGFR (P < 0.01) and combination therapy with colistin (P = 0.02). Comparing patients who received combination with colistin vs polymyxin B, AKI occurred in 60.0% vs 20.6%, respectively, P < 0.01. Fifteen of the 16 (16.6%) patients who developed renal injury or failure were receiving colistin. In conclusion, amikacin was an effective treatment for CRKp infections. Within susceptible range, amikacin MIC values did not influence on clinical outcomes. Combination therapy of amikacin and colistin was highly nephrotoxic and should be used with caution.
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Amicacina , Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Klebsiella , Klebsiella pneumoniae , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Amicacina/efeitos adversos , Amicacina/farmacologia , Amicacina/uso terapêutico , Amicacina/toxicidade , Antibacterianos/efeitos adversos , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Carbapenêmicos/farmacologia , Colistina/efeitos adversos , Feminino , Humanos , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
INTRODUCTION: Carbapenemases are ß-lactamases able to hydrolyze a wide range of ß-lactam antibiotics, including carbapenems. Carbapenemase production in Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter spp., with and without the co-expression of other ß-lactamases is a serious public health threat. Carbapenemases belong to three main classes according to the Ambler classification: class A, class B, and class D. AREAS COVERED: Carbapenemase-bearing pathogens are endemic in Latin America. In this review, we update the status of carbapenemases in Latin America and the Caribbean. EXPERT OPINION: Understanding the current epidemiology of carbapenemases in Latin America and the Caribbean is of critical importance to improve infection control policies limiting the dissemination of multi-drug-resistant pathogens and in implementing appropriate antimicrobial therapy.
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Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/enzimologia , Infecções por Bactérias Gram-Negativas/epidemiologia , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/classificação , Região do Caribe/epidemiologia , Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , América Latina/epidemiologia , beta-Lactamases/classificaçãoRESUMO
The genetic manipulation of Trypanosoma cruzi continues to be a challenge, mainly due to the lack of available and efficient molecular tools. The CRE-lox recombination system is a site-specific recombinase technology, widely used method of achieving conditional targeted deletions, inversions, insertions, gene activation, translocation, and other modifications in chromosomal or episomal DNA. In the present study, the CRE-lox system was adapted to expand the current genetic toolbox for this hard-to-manipulate parasite. For this, evaluations of whether direct protein delivery of CRE recombinase through electroporation could improve CRE-mediated recombination in T. cruzi were performed. CRE recombinase was fused to the C-terminus of T. cruzi histone H2B, which carries the nuclear localization signal and is expressed in the prokaryotic system. The fusion protein was affinity purified and directly introduced into epimastigotes and tissue culture-derived trypomastigotes. This enabled the control of gene expression as demonstrated by turning on a tandem dimer fluorescent protein reporter gene that had been previously transfected into parasites, achieving CRE-mediated recombination in up to 85% of parasites. This system was further tested for its ability to turn off gene expression, remove selectable markers integrated into the genome, and conditionally knock down the nitroreductase gene, which is involved in drug resistance. Additionally, CREditing also enabled the control of gene expression in tissue culture trypomastigotes, which are more difficult to transfect than epimastigotes. The considerable advances in genomic manipulation of T. cruzi shown in this study can be used by others to aid in the greater understanding of this parasite through gain- or loss-of-function approaches.
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Genes Reporter , Engenharia Genética , Trypanosoma cruzi , Doença de Chagas , Eletroporação , Histonas , Humanos , Integrases/genética , Plasmídeos , Trypanosoma cruzi/genéticaRESUMO
The exponential increase in the numbers of isolates of Carbapenem-Resistant Enterobacteriaceae (CRE) creates the need for using novel therapeutic approaches to save the lives of patients. Fosfomycin has long been considered a rational option for the treatment of CRE to be used as part of a combined therapy scheme. However, the assessment of fosfomycin susceptibility in the laboratory presents a great challenge due to the discrepancies found between different methodologies. Thus, our goal was to evaluate fosfomycin susceptibility in a group of 150 Enterobacteriaceae bacterial isolates using agar dilution as the gold standard technique to compare the results with those obtained by disk diffusion. We found a fosfomycin susceptibility of 79.3% in general terms. By comparing both methodologies, we reported a categorical agreement of 96% without Very Major Errors (VMEs) or Major Errors (MEs) and 4% of minor Errors (mEs). Our results suggest that fosfomycin could provide a rational alternative treatment for those patients that are infected by a Multidrug-Resistant (MDR) microorganism that is currently untreatable and that the disk diffusion and classical agar dilution techniques are adequate to assess the resistance profile of CRE to fosfomycin.
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Objectives: The Dominican Republic lacks reliable information on antimicrobial resistance (AMR), which would allow physicians to prescribe the best treatment for common infectious diseases. This study aimed to define the antimicrobial resistance profiles of the more common pathogens from pediatric services, where data is even more important due to the vulnerability of the population. Methods: We collected data from patients admitted in the pediatric unit of three third level hospitals in the city of Santiago de los Caballeros, Dominican Republic, showing positive bacterial cultures, during a period of two years. Results: Half of the Gram negative pathogens exhibited third generation cephalosporins (3GC) resistance, 17% were resistant to carbapenems. Serratia marcescens presented an exceptionally high proportion of resistance to 3GC (95.9%). Staphylococcus aureus showed elevated resistance to methicillin (58.4%) and even to clindamycin (35.8%). Conclusion: There are elevated levels of antimicrobial resistance among the Enterobacteriaceae family and the Staphylococcus genus isolated from the pediatric population. Necessary measures should be taken to tackle AMR in the country.
Objetivos: La República Dominicana carece de información confiable sobre las resistencias antimicrobianas en el país, lo que permitiría al personal médico prescribir los mejores tratamientos para infecciones comunes. El objetivo de este estudio es definir los perfiles de resistencia antimicrobiana de los patógenos más comunes en servicios pediátricos, donde esta información es esencial, debido a la vulnerabilidad de la población. Métodos: Se tomaron los datos de reportes microbiológicos con cultivo bacteriano positivo procedentes de pacientes admitidos en la unidad pediátrica de tres hospitales de tercer nivel en la ciudad de Santiago de los Caballeros, República Dominicana, durante un periodo de dos años. Resultados: La mitad de los patógenos Gram negativos mostraron resistencia a cefalosporinas de tercera generación (3GC), 17% eran resistentes a carbapenémicos. Serratia marcescens presentó una resistencia excepcionalmente elevada a 3GC (95.9%). Staphylococcus aureus mostró alta resistencia a meticilina (58.4%) e incluso a clindamicina (35.8%). Conclusión: Existen elevados niveles de resistencia antimicrobiana entre las enterobacterias y los estafilococos en la población pediátrica dominicana. Es necesario tomar medidas para abordar este problema en el país.
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Humanos , Masculino , Feminino , Criança , Farmacorresistência Bacteriana , Pediatria , Atenção Terciária à Saúde , Clindamicina , Carbapenêmicos , República Dominicana , MeticilinaRESUMO
The hippocampus is an extended structure displaying heterogeneous anatomical cell layers along its dorsoventral axis. It is known that dorsal and ventral regions show different integrity when it comes to functionality, innervation, gene expression, and pyramidal cell properties. Still, whether hippocampal interneurons exhibit different properties along the dorsoventral axis is not known. Here, we report electrophysiological properties of dorsal and ventral oriens lacunosum moleculare (OLM) cells from coronal sections of the Chrna2-cre mouse line. We found dorsal OLM cells to exhibit a significantly more depolarized resting membrane potential compared to ventral OLM cells, while action potential properties were similar between the two groups. We found ventral OLM cells to show a higher initial firing frequency in response to depolarizing current injections but also to exhibit a higher spike-frequency adaptation than dorsal OLM cells. Additionally, dorsal OLM cells displayed large membrane sags in response to negative current injections correlating with our results showing that dorsal OLM cells have more hyperpolarization-activated current (Ih ) compared to ventral OLM cells. Immunohistochemical examination indicates the h-current to correspond to hyperpolarization-activated cyclic nucleotide-gated subunit 2 (HCN2) channels. Computational studies suggest that Ih in OLM cells is essential for theta oscillations in hippocampal circuits, and here we found dorsal OLM cells to present a higher membrane resonance frequency than ventral OLM cells. Thus, our results highlight regional differences in membrane properties between dorsal and ventral OLM cells allowing this interneuron to differently participate in the generation of hippocampal theta rhythms depending on spatial location along the dorsoventral axis of the hippocampus.
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Potenciais de Ação/fisiologia , Hipocampo/fisiologia , Interneurônios/fisiologia , Potenciais da Membrana/fisiologia , Receptores Nicotínicos/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos TransgênicosRESUMO
In this article, we report a case series of patients with infections caused by Enterobacteriales coresistant to carbapenems and polymyxins who were treated with ceftazidime/avibactam (CAZ-AVI) salvage therapy on a compassionate-use protocol. We enrolled 29 adult patients in 3 centers that had an infection due to a resistant microorganism and for whom the treatments available were considered ineffective, treated them with CAZ-AVI, and assessed clinical and microbiological cure at the end of treatment and all-cause mortality at 14 days and 30 days. The antimicrobial susceptibility profile was determined using broth microdilution, and total genomic DNA was sequenced. Twelve (41.4%) patients had bacteremia, and 48.3% (14/29) of the infections were treated with combination therapy. All strains were producers of KPC-2 and were susceptible to CAZ-AVI (MIC90, 1 µg/ml). Clinical success was high (24/29 [82.7%; 95% confidence interval, 64.2 to 94.2%]), even for the bacteremic cases (75%). The 14-day and 30-day mortality rates were 9/29 (31%) and 15/29 (51.7%), respectively. The 14-day mortality rate for pneumonia was the same as that for bloodstream infections (33.3%) and although not significant, we found that patients with renal impairment that received adjusted doses of CAZ-AVI had high mortality (4/9 [44%]; P = 0.22). We concluded that CAZ-AVI is an option for the treatment of severe infections due to difficult-to-treat drug-resistant Enterobacteriales.
Assuntos
Antibacterianos/uso terapêutico , Compostos Azabicíclicos/uso terapêutico , Bacteriemia/tratamento farmacológico , Ceftazidima/uso terapêutico , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Infecções por Enterobacteriaceae/tratamento farmacológico , Pneumonia Bacteriana/tratamento farmacológico , Terapia de Salvação/métodos , Adulto , Bacteriemia/microbiologia , Bacteriemia/mortalidade , Bacteriemia/patologia , Carbapenêmicos/uso terapêutico , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/crescimento & desenvolvimento , Enterobacteriaceae/patogenicidade , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/mortalidade , Infecções por Enterobacteriaceae/patologia , Feminino , Expressão Gênica , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/mortalidade , Pneumonia Bacteriana/patologia , Polimixinas/uso terapêutico , Estudos Prospectivos , Análise de Sobrevida , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Many functions of glial cells depend on the formation of selective glial networks mediated by gap junctions formed by members of the connexin family. Olfactory ensheathing cells (OECs) are specialized glia associated with olfactory sensory neuron axons. Like other glia, they form selective networks, however, the connexins that support OEC connectivity in vivo have not been identified. We used an in vivo mouse model to selectively delete candidate connexin genes with temporal control from OECs and address the physiological consequences. Using this model, we effectively abolished the expression of connexin 43 (Cx43) in OECs in both juvenile and adult mice. Cx43-deleted OECs exhibited features consistent with the loss of gap junctions including reduced membrane conductance, largely reduced sensitivity to the gap junction blocker meclofenamic acid and loss of dye coupling. This indicates that Cx43, a typically astrocytic connexin, is the main connexin forming functional channels in OECs. Despite these changes in functional properties, the deletion of Cx43 deletion did not alter the density of OECs. The strategy used here may prove useful to delete other candidate genes to better understand the functional roles of OECs in vivo.