RESUMO
BACKGROUND: Bipolar electrocautery systems used during neurosurgical procedures have been shown to induce thermal injury to surrounding tissue. The goal of this study was to compare the thermal injury induced by two different systems commonly used in neurosurgical procedures (Silverglide by Stryker Corporation and SpetzlerMalis by Codman Neuro), with that of a newly introduced device (TRIOwand by NICO Corporation). METHODS: A farm swine underwent craniectomy and durotomy with subsequent exposure of cortical brain tissue. Electrocoagulation for the duration of 3 s was conducted with three different bipolar systems under comparable power settings. The maximal depth of thermal injury and mean area of injury in Hematoxylin and Eosin stained slides were quantified using Image J. The tissues were evaluated for vacuolization and ischemic damage. One-way ANOVA followed by post hoc Tukey test was utilized for statistical analysis. Alpha level was set at 0.05. RESULTS: TRIOwand lesions showed less depth of injury when compared to both Spetzler-Malis (P < 0.001) and Silverglide lesions (P = 0.048). Silverglide lesions showed significantly less depth of injury when compared to SpetzlerMalis lesions (P < 0.001). The injury area induced by the TRIOwand was significantly less than that of Spetzler-Malis (P < 0.001) and Silverglide systems (P < 0.001). Ischemic changes and vacuolization were seen in all three groups. CONCLUSION: Thermal damage is induced to varying extents by all bipolar systems. In this porcine model and under the conditions tested, bipolar cauterization with the TRIOwand resulted in less depth and decreased mean area of injury. Further studies are needed to characterize the injury caused by different bipolar systems with other settings and under surgical conditions in humans.
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BACKGROUND: The reported incidence of postoperative ileus (POI) after spine surgery depends on the surgical approach and definition used. It is therefore possible that the overall incidence is substantially higher than previously thought. POI has consequences for both the patient and hospital resources, and can significantly increase health care costs. METHODS: We retrospectively reviewed all patients aged 18 years or older who underwent elective complex spine surgery at our tertiary referral institution from 2011 through 2017. Preoperative comorbidities, operating time and approach, estimated blood loss, postoperative complications, and length of stay (LOS) were analyzed for patients meeting the inclusion criteria. RESULTS: Of 174 patients included in the study, 32 patients (18.4%) developed POI, leading to a significant increase in their median LOS (9 vs. 7 days; P = 0.020). Total estimated blood loss (1649.5 ± 1266.2 vs. 1124.6 ± 936.3 mL; P = 0.009) and total surgical time (501.6 ± 170.5 vs. 388.4 ± 159.8 minutes; P < 0.001) were significantly higher in the POI cohort. The use of nonselective µ-opioid receptor antagonists in 66% of patients with POI did not significantly impact the median LOS (9 vs. 8 days; P = 0.477) compared with patients with POI who did not receive this intervention. The incidence of postoperative adverse events other than ileus was similar between the 2 patient groups. CONCLUSIONS: Despite use of early interventions, the median LOS remains significantly longer in patients who develop POI after complex spine surgery. Knowledge of the associated predictive risk factors could potentially assist with the development of rigorous, evidence-based preventative strategies.
Assuntos
Íleus/etiologia , Procedimentos Ortopédicos/efeitos adversos , Complicações Pós-Operatórias/etiologia , Curvaturas da Coluna Vertebral/cirurgia , Adulto , Idoso , Feminino , Humanos , Íleus/epidemiologia , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/prevenção & controle , Estudos Retrospectivos , Fatores de RiscoRESUMO
BACKGROUND: Sinonasal epithelial cells participate in host defense by initiating innate immune mechanisms against potential pathogens. Antimicrobial innate mechanisms have been shown to involve Th1-like inflammatory responses. Although epithelial cells can also be induced by Th2 cytokines to express proeosinophilic mediators, no environmental agents have been identified that promote this effect. METHODS: Human sinonasal epithelial cells from patients with chronic rhinosinusitis with nasal polyps (CRSwNPs) and controls were harvested and grown in primary culture. Cell cultures were exposed to a range of concentrations of chitin for 24 hours, and mRNA for acidic mammalian chitinase (AMCase), eotaxin-3, and thymic stromal-derived lymphopoietin (TSLP) were assessed. Other cultures were exposed to interleukin 4 (IL- 4) alone and in combination with dust-mite antigen (DMA) for 36 hours. Extracted mRNA and cell culture supernatant were analyzed for expression of AMCase and eotaxin-3. RESULTS: Chitin induced a dose-dependent expression of AMCase and eotaxin-3 mRNA but not TSLP. Patients with recalcitrant CRSwNPs showed lower baseline expression of AMCase when compared with treatment-responsive CRSwNP and less induction of AMCase expression by chitin. DMA did not directly induce expression of AMCase or eotaxin-3. Expression of eotaxin-3 was stimulated by IL-4 and further enhanced with the addition of DMA. Levels of AMCase were not significantly affected by either IL-4 or DMA exposure. In some cases, the combination of IL-4 and DMA was able to induce AMCase expression in cell cultures not producing AMCase at baseline. CONCLUSION: The abundant biopolymer chitin appears to be recognized by a yet uncharacterized receptor on sinonasal epithelial cells. Chitin stimulates production of AMCase and eotaxin-3, two pro-Th2 effector proteins. This finding suggests the existence of a novel innate immune pathway for local defense against chitin-containing organisms in the sinonasal tract. Dysregulation of this function could precipitate or exacerbate Th2 inflammation, potentially acting as an underlying factor in recalcitrant CRSwNP.
Assuntos
Quimiocinas CC/genética , Quitina/farmacologia , Quitinases/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mucosa Nasal/metabolismo , RNA/genética , Reanimação Cardiopulmonar , Células Cultivadas , Quimiocina CCL26 , Quimiocinas CC/biossíntese , Quimiocinas CC/efeitos dos fármacos , Quitinases/biossíntese , Quitinases/efeitos dos fármacos , Eosinófilos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Sinusite Etmoidal/genética , Sinusite Etmoidal/imunologia , Sinusite Etmoidal/patologia , Regulação da Expressão Gênica/genética , Humanos , Imunidade Celular/efeitos dos fármacos , Mucosa Nasal/patologia , Rinite/genética , Rinite/imunologia , Rinite/patologia , Células Th2/efeitos dos fármacos , Células Th2/imunologiaRESUMO
Questions surround the mechanism of mutually exclusive expression by which Plasmodium falciparum mediates activation and silencing of var genes. These encode PfEMP1 proteins, which function as cytoadherent and immunomodulatory molecules at the surface of parasitised erythrocytes. Current evidence suggests that promoter silencing by var introns might play a key role in var gene regulation. To evaluate the impact of cis-acting regulatory regions on var silencing, we generated P. falciparum lines in which luciferase was placed under the control of an UpsA var promoter. By utilising the Bxb1 integrase system, these reporter cassettes were targeted to a genomic region that was not in apposition to var subtelomeric domains. This eliminated possible effects from surrounding telomeric elements and removed the variability inherent in episomal systems. Studies with highly synchronised parasites revealed that the UpsA element possessed minimal activity in comparison with a heterologous (hrp3) promoter. This may result from the integrated UpsA promoter being largely silenced by the neighbouring cg6 promoter. Our analyses also revealed that the DownsA 3' untranslated region further decreased the luciferase activity from both cassettes, whereas the var A intron repressed the UpsA promoter specifically. By applying multivariate analysis over the entire cell cycle, we confirmed the significance of these cis-elements and found the parasite stage to be the major factor regulating UpsA-promoter activity. Additionally, we observed that the UpsA promoter was capable of nucleating reversible silencing that spread to a downstream promoter. We believe these studies are the first to analyse promoter activity of Group A var genes, which have been implicated in severe malaria, and support the model that var introns can further suppress var expression. These data also suggest an important suppressive role for the DownsA terminator. Our findings imply the existence of multiple levels of var gene regulation in addition to intrinsic promoter-dependent silencing.
Assuntos
Inativação Gênica/fisiologia , Malária/parasitologia , Plasmodium falciparum/genética , Regiões Promotoras Genéticas/genética , Proteínas de Protozoários/genética , Regiões 3' não Traduzidas/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , Íntrons/genética , Análise Multivariada , Plasmodium falciparum/metabolismo , Reação em Cadeia da PolimeraseRESUMO
Quinine (QN) continues to be an important treatment option for severe malaria, however resistance to this drug has emerged in field isolates of the etiologic agent Plasmodium falciparum. Quantitative trait loci investigations of QN resistance have mapped three loci of this complex trait. Two coincide with pfcrt and pfmdr1, involved in resistance to chloroquine (CQ) and other quinoline-based antimalarials. A third locus on chromosome 13 contains the sodium-proton exchanger (pfnhe) gene. Previous studies have associated pfnhe polymorphisms with reduced QN sensitivity in culture-adapted field isolates. Here, we provide direct evidence supporting the hypothesis that pfnhe contributes to QN resistance. Using allelic exchange, we reduced pfnhe expression by introducing a truncated 3' untranslated region (UTR) from pfcrt into the endogenous pfnhe 3'UTR. Transfections were performed with 1BB5 and 3BA6 (both CQ- and QN-resistant) as well as GC03 (CQ- and QN-sensitive), all progenies of the HB3xDd2 genetic cross. RNA and protein analyses of the ensuing recombinant clones demonstrated a approximately 50% decrease in pfnhe expression levels. A statistically significant 30% decrease in QN IC(50) values was associated with these decreased expression levels in 1BB5 and 3BA6 but not in GC03. CQ, mefloquine and lumefantrine IC(50) values were unaltered. Cytosolic pH values were similar in all parental lines and recombinant clones. Our observations support a role for pfnhe in QN resistance in a strain-dependent manner, which might be contingent on pre-existing resistance to CQ and/or QN. These data bolster observations that QN resistance is a complex trait requiring the contribution of multiple transporter proteins.
Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Quinina/farmacologia , Trocadores de Sódio-Hidrogênio/genética , Animais , Citosol/química , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Concentração de Íons de Hidrogênio , Plasmodium falciparum/metabolismo , Proteínas Recombinantes/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Especificidade da Espécie , Fatores de TempoRESUMO
The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.
Assuntos
Fígado/parasitologia , Plasmodium berghei/patogenicidade , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/metabolismo , Animais , Antimaláricos/farmacologia , Deleção de Genes , Malária/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Parasitemia , Plasmodium berghei/enzimologia , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Triclosan/farmacologiaRESUMO
Azithromycin (AZ), a broad-spectrum antibacterial macrolide that inhibits protein synthesis, also manifests reasonable efficacy as an antimalarial. Its mode of action against malarial parasites, however, has remained undefined. Our in vitro investigations with the human malarial parasite Plasmodium falciparum document a remarkable increase in AZ potency when exposure is prolonged from one to two generations of intraerythrocytic growth, with AZ producing 50% inhibition of parasite growth at concentrations in the mid to low nanomolar range. In our culture-adapted lines, AZ displayed no synergy with chloroquine (CQ), amodiaquine, or artesunate. AZ activity was also unaffected by mutations in the pfcrt (P. falciparum chloroquine resistance transporter) or pfmdr1 (P. falciparum multidrug resistance-1) drug resistance loci, as determined using transgenic lines. We have selected mutant, AZ-resistant 7G8 and Dd2 parasite lines. In the AZ-resistant 7G8 line, the bacterial-like apicoplast large subunit ribosomal RNA harbored a U438C mutation in domain I. Both AZ-resistant lines revealed a G76V mutation in a conserved region of the apicoplast-encoded P. falciparum ribosomal protein L4 (PfRpl4). This protein is predicted to associate with the nuclear genome-encoded P. falciparum ribosomal protein L22 (PfRpl22) and the large subunit rRNA to form the 50 S ribosome polypeptide exit tunnel that can be occupied by AZ. The PfRpl22 sequence remained unchanged. Molecular modeling of mutant PfRpl4 with AZ suggests an altered orientation of the L75 side chain that could preclude AZ binding. These data imply that AZ acts on the apicoplast bacterial-like translation machinery and identify Pfrpl4 as a potential marker of resistance.
Assuntos
Antimaláricos/farmacologia , Azitromicina/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Amodiaquina/farmacologia , Animais , DNA de Protozoário/metabolismo , Resistência a Medicamentos , Eritrócitos/parasitologia , Humanos , Malária Falciparum/parasitologia , Modelos Estruturais , Testes de Sensibilidade Parasitária , Proteínas de Protozoários/metabolismoRESUMO
The x-ray crystal structures of five triclosan analogs, in addition to that of the isoniazid-NAD adduct, are described in relation to their integral role in the design of potent inhibitors of the malarial enzyme Plasmodium falciparum enoyl acyl carrier protein reductase (PfENR). Many of the novel 5-substituted analogs exhibit low micromolar potency against in vitro cultures of drug-resistant and drug-sensitive strains of the P. falciparum parasite and inhibit purified PfENR enzyme with IC50 values of <200 nM. This study has significantly expanded the knowledge base with regard to the structure-activity relationship of triclosan while affording gains against cultured parasites and purified PfENR enzyme. In contrast to a recent report in the literature, these results demonstrate the ability to improve the in vitro potency of triclosan significantly by replacing the suboptimal 5-chloro group with larger hydrophobic moieties. The biological and x-ray crystallographic data thus demonstrate the flexibility of the active site and point to future rounds of optimization to improve compound potency against purified enzyme and intracellular Plasmodium parasites.
Assuntos
Antimaláricos/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Triclosan/química , Animais , Antimaláricos/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Resistência a Medicamentos/efeitos dos fármacos , Modelos Moleculares , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Estrutura Terciária de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Triclosan/análogos & derivados , Triclosan/metabolismoRESUMO
Here we report an efficient, site-specific system of genetic integration into Plasmodium falciparum malaria parasite chromosomes. This is mediated by mycobacteriophage Bxb1 integrase, which catalyzes recombination between an incoming attP and a chromosomal attB site. We developed P. falciparum lines with the attB site integrated into the glutaredoxin-like cg6 gene. Transfection of these attB(+) lines with a dual-plasmid system, expressing a transgene on an attP-containing plasmid together with a drug resistance gene and the integrase on a separate plasmid, produced recombinant parasites within 2 to 4 weeks that were genetically uniform for single-copy plasmid integration. Integrase-mediated recombination resulted in proper targeting of parasite proteins to intra-erythrocytic compartments, including the apicoplast, a plastid-like organelle. Recombinant attB x attP parasites were genetically stable in the absence of drug and were phenotypically homogeneous. This system can be exploited for rapid genetic integration and complementation analyses at any stage of the P. falciparum life cycle, and it illustrates the utility of Bxb1-based integrative recombination for genetic studies of intracellular eukaryotic organisms.
Assuntos
Cromossomos/genética , Engenharia Genética/métodos , Integrases/genética , Mutagênese Sítio-Dirigida/métodos , Micobacteriófagos/genética , Plasmodium falciparum/genética , Recombinação Genética/genética , Proteínas Repressoras/genética , Proteínas Virais/genética , Animais , Sequência de Bases , Dados de Sequência Molecular , Transgenes/genéticaRESUMO
A male gametocyte defect in the Plasmodium falciparum Dd2 parasite was previously discovered through the observation that all progeny clones in a Dd2 x HB3 genetic cross were the result of fertilization events between Dd2 female and HB3 male gametes. A determinant linked to the defect in Dd2 was subsequently mapped to an 800-kb segment on chromosome 12. Here, we report further mapping of the determinant to an 82-kb region and the identification of a candidate gene, P. falciparum male development gene 1 (pfmdv-1), that is expressed at a lower level in Dd2 compared with the wild-type normal male gametocyte-producing ancestor W2. Pfmdv-1 protein is sexual-stage specific and is located on the gametocyte plasma membrane, parasitophorous vacuole membrane, and the membranes of cleft-like structures within the erythrocyte. Disruption of pfmdv-1 results in a dramatic reduction in mature gametocytes, especially functional male gametocytes, with the majority of sexually committed parasites developmentally arrested at stage I. The pfmdv-1-knockout parasites show disturbed membrane structures, particularly multimembrane vesicles/tubes that likely derive from deformed cleft-like structures. Mosquito infectivity of the knockout parasites was also greatly reduced but not completely lost. The results suggest that pfmdv-1 plays a key role in gametocyte membrane formation and integrity.