The role of sialoglycans in modulating dendritic cell function and tumour immunity.

Dendritic cells (DCs) are crucial for initiating immune responses against tumours by presenting antigens to T cells. Glycosylation, particularly sialylation, plays a significant role in regulating cell functions, by modulating protein folding and signalling. This review aimed to provide a comprehensive overview of how sialic acids influence key aspects of DC biology, including maturation, migration, antigen presentation, and T cell interactions. Sialic acids influence DC endocytosis, affecting their ability to uptake and present antigens, while guiding their migration to lymph nodes and inflamed tissues. Removing sialic acids enhances DC-mediated antigen presentation to T cells, potentially boosting immune responses. Additionally, sialylated glycans on DCs modulate immune checkpoints, which can impact tumour immunity. Hypersialylation of tumour mucins further promotes immune evasion by interacting with DCs. Understanding the interplay between sialylation and DC functions offers promising avenues for enhancing cancer immunotherapy.

New insights into the immunomodulatory potential of sialic acid on monocyte-derived dendritic cells.

Sialic acids at the cell surface of dendritic cells (DCs) play an important immunomodulatory role, and their manipulation enhances DC maturation, leading to heightened T cell activation. Particularly, at the molecular level, the increased stability of surface MHC-I molecules in monocyte-derived DCs (MoDCs) underpins an improved DC: T cell interaction. In this study, we focused on the impact of sialic acid remodelling by treatment with Clostridium perfringens sialidase on MoDCs' phenotypic and functional characteristics. Our investigation juxtaposes this novel approach with the conventional cytokine-based maturation regimen commonly employed in clinical settings.Notably, C. perfringens sialidase remarkably increased MHC-I levels compared to other sialidases having different specificities, supporting the idea that higher MHC-I is due to the cleavage of specific sialoglycans on cell surface proteins. Sialidase treatment induced rapid elevated surface expression of MHC-I, MHC-II and CD40 within an hour, a response not fully replicated by 48 h cytokine cocktail treatment. These increases were also observable 48 h post sialidase treatment. While CD86 and PD-L1 showed significant increases after 48 h of cytokine maturation, 48 h post sialidase treatment showed a higher increase in CD86 and shorter increase in PD-L1. CCR-7 expression was significantly increased 48 h after sialidase treatment but not significantly affected by cytokine maturation. Both treatments promoted higher secretion of the IL-12 cytokine. However, the cytokine cocktail induced a more pronounced IL-12 production. SNA lectin staining analysis demonstrated that the sialic acid profile is significantly altered by sialidase treatment, but not by the cytokine cocktail, which causes only slight sialic acid upregulation. Notably, the lipid-presenting molecules CD1a, CD1b and CD1c remained unaffected by sialidase treatment in MoDCs, a finding also further supported by experiments performed on C1R cells. Inhibition of endogenous sialidases Neu1 and Neu3 during MoDC differentiation did not affect surface MHC-I expression and cytokine secretion. Yet, sialidase activity in MoDCs was minimal, suggesting that sialidase inhibition does not significantly alter MHC-I-related functions. Our study highlights the unique maturation profile induced by sialic acid manipulation in MoDCs. These findings provide insights into the potential of sialic acid manipulation as a rapid immunomodulatory strategy, offering promising avenues for targeted interventions in inflammatory contexts.

Antitumor Efficacy of Human Monocyte-Derived Dendritic Cells: Comparing Effects of two Monocyte Isolation Methods.

BACKGROUND: Dendritic cells (DCs), which can be used as anti-cancer vaccines, are generally obtained in vitro from isolated CD14+ monocytes (MoDCs). This generates high cell numbers and allows instructing DCs to guarantee effective antitumor responses. However, the impact of the monocyte isolation step in the antitumor effectiveness of the generated MoDCs is still unknown. Here, we compared the most used immunomagnetic technologies for monocyte isolation: magnetic activated cell sorting (MACS) from Miltenyi Biotec and EasySep from STEM CELL. RESULTS: MACS technology allowed a higher monocyte yield and purity and, by flow cytometry, monocytes displayed higher size and lower granularity. In the resting state, EasySep_MoDCs showed a higher basal expression of HLA-DR, and no significant response to stimulation by LPS and TNF-α. When stimulated with whole tumor cells lysates, both MoDCs expressed similar levels of maturation and co-stimulatory markers. However, when cultured with autologous T cells, MACS_MoDCs induced significantly higher IFN-γ secretion than EasySep_MoDCs, indicating a stronger induction of Th1 cell response profile. Concordantly, T cells induced by MACS_MoDCs also showed a higher release of cytotoxic granules when in contact with tumor cells. CONCLUSIONS: Overall, both the MACS and the EasySep isolation immunomagnetic technologies provide monocytes that differentiate into viable and functional MoDCs. In our experimental settings, resting EasySep_MoDCs showed a higher basal level of maturation but show less responsivity to stimuli. On the other hand, MACS_MoDCs, when stimulated with tumor antigens, showed better ability to stimulate Th1 responses and to induce T cell cytotoxicity against tumor cells. Thus, monocyte isolation techniques crucially affect MoDCs' function and, therefore, should be carefully selected to obtain the desired functionality.

Obesity, acute kidney injury and mortality in patients with sepsis: a cohort analysis.

Although the prognostic effect of obesity has been studied in critically ill patients its impact on outcomes of septic patients and its role as a risk factor for acute kidney injury (AKI) is not consensual. We aimed to analyze the impact of obesity on the occurrence of AKI and on in-hospital mortality in a cohort of critically ill septic patients. This study is retrospective including 456 adult patients with sepsis admitted to the Division of Intensive Medicine of the Centro Hospitalar Lisboa Norte (Lisbon, Portugal) between January 2008 and December 2014. Obesity was defined as a body mass index of 30 kg/m2 or higher. The Kidney Disease Improving Global Outcomes classification was used to diagnose and classify patients developing AKI. AKI occurred in 87.5% of patients (19.5% with stage 1, 22.6% with stage 2 and 45.4% with stage 3). Obese patients developed AKI more frequently than non-obese patients (92.8% versus 85.5%, p = .035; unadjusted OR 2.2 (95% CI: 1.04-4.6), p = .039; adjusted OR 2.31 (95% CI: 1.07-5.02), p = .034). The percentage of obese patients, however, did not differ between AKI stages (stage 1, 25.1%; stage 2, 28.6%; stage 3, 15.4%; p = .145). There was no association between obesity and mortality (p = .739). Of note, when comparing AKI patients with or without obesity in terms of in-hospital mortality there were also no significant differences between those groups (38.4% versus 38.4%, p = .998). Obesity was associated with the occurrence of AKI in critically ill patients with sepsis; however, it was not associated with in-hospital mortality.

Using dendritic cells to evaluate how Burkholderia cenocepacia clonal isolates from a chronically infected cystic fibrosis patient subvert immune functions.

Infection with Burkholderia cepacia complex (Bcc) bacteria is a threat to cystic fibrosis (CF) patients, commonly leading to a fatal pneumonia, the cepacia syndrome. It causes a massive production of pro-inflammatory cytokines and leucocyte recruitment to airway epithelium without resolving infection and contributing to tissue lesion. To dissect how Bcc bacteria subvert the immune response, we developed a co-culture model with human dendritic cells (DCs) and B. cenocepacia clonal variants isolated from a chronically infected CF patient, who died with cepacia syndrome. We demonstrated that the two late variants were sevenfold and 17-fold (respectively) more internalized by DCs than the variant that initiated infection. The late variants showed improved survival within DCs (60.29 and 52.82 CFU/DC) compared to the initial variant (0.38 CFU/DC). All clonal isolates induced high expression of inflammatory cytokines IL-8, IL-6, IL-1ß, IL-12, IL-23, TNF-α and IL-1ß. This pro-inflammatory trait was significantly more pronounced in DCs infected with the late variants than in DCs infected with the variant that initiated patient's infection. All infected DCs failed to upregulate maturation markers, HLA-DR, CD80, CD86 and CD83. Nevertheless, these infected DCs activated approximately twice more T cells than non-infected DCs. Similar T cell activation was observable with respective conditioned media, suggesting a non-antigen-specific activation. Our data indicate that during prolonged infection, B. cenocepacia acquires ability to survive intracellularly, inducing inflammation, while refraining DC's maturation and stimulating non-antigen-specific T cell responses. The co-culture model here developed may be broadly applied to study B. cenocepacia-induced immunomodulation.

Immature myeloid cells and tolerogenic cytokine profile in lung adenocarcinoma metastatic lymph nodes assessed by endobronchial ultrasound.

In lung cancer, the immune cell compartment of tumor-draining lymph nodes (TDLNs) dictate the response against tumors. This response is predominantly triggered by myeloid antigen-presenting cells (mAPCs) that capture antigens and, if matured, prime anti-tumor-specific T cell populations. However, the clinical role of mAPCs infiltrated in TDLN from lung cancer patients is poorly understood. The purpose of this study was to study mAPCs in TDLN from lung adenocarcinoma patients, in comparison to individuals with non-malignant diseases, using minimally invasive sampling methods. Mediastinal lymph nodes were assessed by endobronchial ultrasound-transbronchial needle aspiration (EBUS-TBNA). mAPCs were characterized by flow cytometry and cytokine expression by quantitative polymerase chain reaction. The association with tumor burden, overall survival, and response to treatment was assessed. TDLN from lung adenocarcinoma patients (n = 24) showed a reduced immune cell compartment, but a higher level of infiltrating mAPCs, when compared with control lymph nodes (n = 17). A decreased expression of co-stimulatory molecules CD80/CD86 by TDLN and blood mAPC was observed. TDLN showed lower levels of TNF-α and IL-12 and increased levels of immunosuppressive cytokines TGF-ß and IL-10. The IL-12 expression was inversely correlated with the percentage of infiltrated tumor cells, while IL-10 was directly correlated. Patients with lower expression of IL-12 in TDLN or lower expression of CD80/86 in blood mAPCs had worse overall survival and response to therapy. mAPCs of lung adenocarcinoma patients express less co-stimulatory molecules, and within TDLN, the cytokine profile is biased towards a tolerance-inducing phenotype. Patients with enhanced immune parameters have better survival and response to treatment. EBUS-TBNA allows the collection of viable specimens from TDLN that may provide further insight on relevant immunological mechanisms.

Inhibition of cellular methyltransferases promotes endothelial cell activation by suppressing glutathione peroxidase 1 protein expression.

S-adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase 1 (GPx-1) and endothelial dysfunction. Here we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNA(Sec) to the Um34 form. The formation of methylated tRNA(Sec) facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNA(Sec) and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase 1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a proinflammatory activation of endothelial cells characterized by up-regulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNA(Sec) hypomethylation, which alters the expression of selenoproteins such as GPx-1 to contribute to a proatherogenic endothelial phenotype.

The phagocytic capacity and immunological potency of human dendritic cells is improved by α2,6-sialic acid deficiency.

Dendritic cells (DCs) play an essential role in immunity against bacteria by phagocytosis and by eliciting adaptive immune responses. Previously, we demonstrated that human monocyte-derived DCs (MDDCs) express a high content of cell surface α2,6-sialylated glycans. However, the relative role of these sialylated structures in phagocytosis of bacteria has not been reported. Here, we show that treatment with a sialidase significantly improved the capacity of both immature and mature MDDCs to phagocytose Escherichia coli. Desialylated MDDCs had a significantly more mature phenotype, with higher expression of MHC molecules and interleukin (IL)-12, tumour necrosis factor-α, IL-6 and IL-10 cytokines, and nuclear factor-κB activation. T lymphocytes primed by desialylated MDDCs expressed more interferon-γ when compared with priming by sialylated MDDCs. Improved phagocytosis required E. coli sialic acids, indicating a mechanism of host-pathogen interaction dependent on sialic acid moieties. The DCs harvested from mice deficient in the ST6Gal.1 sialyltransferase showed improved phagocytosis capacity, demonstrating that the observed sialidase effect was a result of the removal of α2,6-sialic acid. The phagocytosis of different pathogenic E. coli isolates was also enhanced by sialidase, which suggests that modifications on MDDC sialic acids may be considered in the development of MDDC-based antibacterial therapies. Physiologically, our findings shed new light on mechanisms that modulate the function of both immature and mature MDDCs, in the context of host-bacteria interaction. Hence, with particular relevance to DC-based therapies, the engineering of α2,6-sialic acid cell surface is a novel possibility to fine tune DC phagocytosis and immunological potency.

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes.

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

Sialyl Lewisx-dependent binding of human monocyte-derived dendritic cells to selectins.

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewis(x) (sLe(x)) in selectin-dependent mo-DC binding. Our data reveal that sLe(x) is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLe(x)-dependent binding of mo-DC to selectins was further substantiated by using sLe(x) free sugar and anti-sLe(x) antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLe(x) expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.

MHC Class I Stability is Modulated by Cell Surface Sialylation in Human Dendritic Cells.

Maturation of human Dendritic Cells (DCs) is characterized by increased expression of antigen presentation molecules, and overall decreased levels of sialic acid at cell surface. Here, we aimed to identify sialylated proteins at DC surface and comprehend their role and modulation. Mass spectrometry analysis of DC's proteins, pulled down by a sialic acid binding lectin, identified molecules of the major human histocompatibility complex class I (MHC-I), known as human leucocyte antigen (HLA). After desialylation, DCs showed significantly higher reactivity with antibodies specific for properly folded MHC-I-ß2-microglobulin complex and for ß2-microglobulin but showed significant lower reactivity with an antibody specific for free MHC-I heavy chain. Similar results for antibody reactivities were observed for TAP2-deficient lymphoblastoid T2 cells, which express HLA-A*02:01. Using fluorescent peptide specifically fitting the groove of HLA-A*02:01, instead of antibody staining, also showed higher peptide binding on desialylated cells, confirming higher surface expression of MHC-I complex. A decay assay showed that desialylation doubled the half-life of MHC-I molecules at cell surface in both DCs and T2 cells. The biological impact of DC´s desialylation was evaluated in co-cultures with autologous T cells, showing higher number and earlier immunological synapses, and consequent significantly increased production of IFN-γ by T cells. In summary, sialic acid content modulates the expression and stability of complex MHC-I, which may account for the improved DC-T synapses.

Carcinoembryonic antigen is a sialyl Lewis x/a carrier and an E­selectin ligand in non­small cell lung cancer.

The formation of distant metastasis resulting from vascular dissemination is one of the leading causes of mortality in non­small cell lung cancer (NSCLC). This metastatic dissemination initiates with the adhesion of circulating cancer cells to the endothelium. The minimal requirement for the binding of leukocytes to endothelial E­selectins and subsequent transmigration is the epitope of the fucosylated glycan, sialyl Lewis x (sLex), attached to specific cell surface glycoproteins. sLex and its isomer sialyl Lewis a (sLea) have been described in NSCLC, but their functional role in cancer cell adhesion to endothelium is still poorly understood. In this study, it was hypothesised that, similarly to leukocytes, sLe glycans play a role in NSCLC cell adhesion to E­selectins. To assess this, paired tumour and normal lung tissue samples from 18 NSCLC patients were analyzed. Immunoblotting and immunohistochemistry assays demonstrated that tumour tissues exhibited significantly stronger reactivity with anti­sLex/sLea antibody and E­selectin chimera than normal tissues (2.2­ and 1.8­fold higher, respectively), as well as a higher immunoreactive score. High sLex/sLea expression was associated with bone metastasis. The overall α1,3­fucosyltransferase (FUT) activity was increased in tumour tissues, along with the mRNA levels of FUT3, FUT6 and FUT7, whereas FUT4 mRNA expression was decreased. The expression of E­selectin ligands exhibited a weak but significant correlation with the FUT3/FUT4 and FUT7/FUT4 ratios. Additionally, carcinoembryonic antigen (CEA) was identified in only 8 of the 18 tumour tissues; CEA­positive tissues exhibited significantly increased sLex/sLea expression. Tumour tissue areas expressing CEA also expressed sLex/sLea and showed reactivity to E­selectin. Blot rolling assays further demonstrated that CEA immunoprecipitates exhibited sustained adhesive interactions with E­selectin­expressing cells, suggesting CEA acts as a functional protein scaffold for E­selectin ligands in NSCLC. In conclusion, this work provides the first demonstration that sLex/sLea are increased in primary NSCLC due to increased α1,3­FUT activity. sLex/sLea is carried by CEA and confers the ability for NSCLC cells to bind E­selectins, and is potentially associated with bone metastasis. This study contributes to identifying potential future diagnostic/prognostic biomarkers and therapeutic targets for lung cancer.

Acute kidney injury in intensive care unit patients: a comparison between the RIFLE and the Acute Kidney Injury Network classifications.

INTRODUCTION: Whether discernible advantages in terms of sensitivity and specificity exist with Acute Kidney Injury Network (AKIN) criteria versus Risk, Injury, Failure, Loss of Kidney Function, End-stage Kidney Disease (RIFLE) criteria is currently unknown. We evaluated the incidence of acute kidney injury and compared the ability of the maximum RIFLE and of the maximum AKIN within intensive care unit hospitalization in predicting inhospital mortality of critically ill patients. METHODS: Patients admitted to the Department of Intensive Medicine of our hospital between January 2003 and December 2006 were retrospectively evaluated. Chronic kidney disease patients undergoing dialysis or renal transplant patients were excluded from the analysis. RESULTS: In total, 662 patients (mean age, 58.6 +/- 19.2 years; 392 males) were evaluated. AKIN criteria allowed the identification of more patients as having acute kidney injury (50.4% versus 43.8%, P = 0.018) and classified more patients with Stage 1 (risk in RIFLE) (21.1% versus 14.7%, P = 0.003), but no differences were observed for Stage 2 (injury in RIFLE) (10.1% versus 11%, P = 0.655) and for Stage 3 (failure in RIFLE) (19.2% versus 18.1%, P = 0.672). Mortality was significantly higher for acute kidney injury defined by any of the RIFLE criteria (41.3% versus 11%, P < 0.0001; odds ratio = 2.78, 95% confidence interval = 1.74 to 4.45, P < 0.0001) or of the AKIN criteria (39.8% versus 8.5%, P < 0.0001; odds ratio = 3.59, 95% confidence interval = 2.14 to 6.01, P < 0.0001). The area under the receiver operator characteristic curve for inhospital mortality was 0.733 for RIFLE criteria (P < 0.0001) and was 0.750 for AKIN criteria (P < 0.0001). There were no statistical differences in mortality by the acute kidney injury definition/classification criteria (P = 0.72). CONCLUSIONS: Although AKIN criteria could improve the sensitivity of the acute kidney injury diagnosis, it does not seem to improve on the ability of the RIFLE criteria in predicting inhospital mortality of critically ill patients.

Consensus Protocol for the Treatment of Super-Refractory Status Epilepticus.

INTRODUCTION: Super-refractory status epilepticus is defined as status epilepticus that persists or recurs 24 hours after anaesthetic therapy onset or after its withdrawal. It is mostly found in intensive care units and carries high mortality but good long-term prognosis for those who survive. In contrast with the initial phases of status epilepticus, treatment lacks strong scientific evidence and is mostly derived from case reports or small case series. OBJECTIVE: To propose a protocol for the treatment of super-refractory status epilepticus in level III intensive care units, focusing on the treatment strategies to control clinical and/or electroencephalographic epileptic activity. MATERIAL AND METHODS: Narrative review of the literature by PubMed search. Available evidence was discussed in consensus meetings by intensive care and neurology experts' from a level III intensive care unit and one of the Portuguese reference centres for the treatment of refractory epilepsy, respectively. RESULTS: Anaesthetics with the highest level of evidence are propofol, midazolam, thiopental and ketamine. These represent the basis of the treatment of super-refractory status epilepticus and should be used in combination with antiepileptic drugs. The level of evidence for the latter is lower, however, levetiracetam, topiramate, pregabalin, lacosamide, valproic acid, phenytoin and perampanel may be recommended. Alternative therapeutic strategies with very low level of evidence are recommended in cases of total absence of clinical response, such as magnesium sulphate, pyridoxine, ketogenic diet, therapeutic hypothermia and immunosupression. CONCLUSION: We propose a treatment protocol based on a sequential combination of anaesthetics, anti-epileptic drugs and alternative therapies. Strategies to evaluate treatment response and to wean drugs based on clinical results are also proposed.

Introdução: O estado de mal epiléptico super-refractário define-se como um estado de mal epiléptico que persiste ou recorre 24 horas após o início da terapêutica anestésica ou após a sua suspensão. Encontra-se fundamentalmente em unidades de cuidados intensivos e está associado a uma elevada mortalidade apesar de ter um bom prognóstico a longo prazo nos doentes que sobrevivem. Ao contrário das fases iniciais do estado de mal epiléptico, o tratamento não é baseado numa forte evidência científica e deriva principalmente de relatos ou pequenas séries de casos. Objectivo: Propor um protocolo de tratamento do estado de mal epiléptico super-refractário em unidades de cuidados intensivos de nível III, focando-se nas estratégias de tratamento para controlar a actividade epiléptica clínica e/ou electroencefalográfica. Material e Métodos: Revisão narrativa da literatura no PubMed, seguida de discussão em reuniões de consenso de peritos de medicina intensiva e neurologia de uma unidade de cuidados intensivos de nível III e de um dos centros de referência para o tratamento da epilepsia refractária em Portugal, respectivamente. Resultados: Os fármacos anestésicos com maior nível de evidência são o propofol, midazolam, tiopental e ketamina. Estes representam a base do tratamento do estado de mal super-refractário e devem ser utilizados em combinação com fármacos antiepilépticos. O nível de evidência para estes últimos é menor, contudo, podem ser recomendados o levetiracetam, topiramato, pregabalina, lacosamida, perampanel, ácido valpróico, fenitoína e perampanel. São recomendadas estratégias terapêuticas alternativas com muito baixo nível de evidência, em casos de ausência total de resposta clínica, tais como o sulfato de magnésio, piridoxina, dieta cetogénica, hipotermia terapêutica e imunossupressão. Conclusão: Propomos um protocolo de tratamento baseado numa combinação sequencial de fármacos anestésicos, antiepilépticos e terapêuticas alternativas. São também propostas estratégias de avaliação da eficácia da terapêutica e de desmame farmacológico progressivo de acordo com a resposta clínica obtida.

A Stewardship Program to Optimize the Use of Inhaled Nitric Oxide in Pediatric Critical Care.

PURPOSE: Inhaled nitric oxide (iNO) is a pulmonary vasodilator that is approved for use in term and near-term neonates with hypoxic respiratory failure associated with evidence of pulmonary hypertension. However, it is commonly used in infants and children to treat a variety of other cardiopulmonary diseases associated with pulmonary hypertension and hypoxic respiratory failure. In critically ill children, iNO therapy may be continued for a prolonged period, and this increases the risk for adverse consequences including toxicity and unnecessary costs. We implemented an iNO Stewardship Program with the aim of improving adherence to guidelines and reducing unnecessary iNO utilization. METHODS: Between April 1, 2011, and March 31, 2015, a before and after cohort study was conducted at The Hospital for Sick Children. Prospective iNO usage and outcome variables in the poststewardship period were examined. RESULTS: Patient characteristics and outcomes were similar before and after stewardship implementation. The number of iNO therapy courses were also similar in the before and after period. Inhaled nitric oxide utilization in the pediatric intensive care unit and the cardiac critical care unit decreased from 15 765 hours in the prestewardship period (April 2011 to March 2013) to 10 342 hours in the poststewardship period (April 2013 to March 2015), with significant improvement in adherence to the iNO guideline and a small decrease in expenditure (3%). CONCLUSION: Implementation of the iNO Stewardship was successful at reducing overall iNO utilization. This quality improvement initiative helped us optimize practice and subsequently expand the methodology to inform the clinical indication for iNO.

Halilectin-3, a Lectin from the Marine Sponge Haliclona caerulea, Induces Apoptosis and Autophagy in Human Breast Cancer MCF7 Cells Through Caspase-9 Pathway and LC3-II Protein Expression.

BACKGROUND: An ideal strategy for cancer treatment is the specific induction of tumor cell death, sparing normal cells. Marine sponges are rich biological reservoirs of biomolecules, especially lectins, which have attracted considerable attention due to potential biological effect on human cells. Lectins are proteins that bind specific carbohydrate signatures and some gained further interest for their capacity to bind tumor associated carbohydrates antigens and induce tumor cell apoptosis. OBJECTIVE: This study aimed to evaluate the antitumor potential of H3, a lectin, recently reported from marine sponge Haliclona caerulea on the human breast cancer cell line MCF7. RESULTS: H3 reduced MCF7 cell viability with an IC50 of 100 µg/ml, without a significant effect on normal cells. At 24 h, H3 induced a significant arrest in the G1 cell cycle phase. Consistently, almost 50% of the cells were in early apoptosis and showed remarkable increased expression of caspase-9 (CASP 9). H3 impaired dramatically the adhesiveness of MCF7 cells in culture. Assays conducted with Lysotracker Red probe showed increased organelle acidity, suggesting autophagic cell death, which was further supported by increased expression of microtubuleassociated protein light chain 3 (LC3) and observable conversion of LC3-I in LC3-II by western blot. CONCLUSION: The apoptotic effect of H3 may be related to a balance between apoptotic and autophagic cell death, mediated by increased expression of CASP 9 and LC3-II. To the best of our knowledge this is the first report about a sponge lectin triggering both apoptosis and autophagy in MCF7 cell.

Gla-rich protein function as an anti-inflammatory agent in monocytes/macrophages: Implications for calcification-related chronic inflammatory diseases.

Calcification-related chronic inflammatory diseases are multifactorial pathological processes, involving a complex interplay between inflammation and calcification events in a positive feed-back loop driving disease progression. Gla-rich protein (GRP) is a vitamin K dependent protein (VKDP) shown to function as a calcification inhibitor in cardiovascular and articular tissues, and proposed as an anti-inflammatory agent in chondrocytes and synoviocytes, acting as a new crosstalk factor between these two interconnected events in osteoarthritis. However, a possible function of GRP in the immune system has never been studied. Here we focused our investigation in the involvement of GRP in the cell inflammatory response mechanisms, using a combination of freshly isolated human leucocytes and undifferentiated/differentiated THP-1 cell line. Our results demonstrate that VKDPs such as GRP and matrix gla protein (MGP) are synthesized and γ-carboxylated in the majority of human immune system cells either involved in innate or adaptive immune responses. Stimulation of THP-1 monocytes/macrophages with LPS or hydroxyapatite (HA) up-regulated GRP expression, and treatments with GRP or GRP-coated basic calcium phosphate crystals resulted in the down-regulation of mediators of inflammation and inflammatory cytokines, independently of the protein γ-carboxylation status. Moreover, overexpression of GRP in THP-1 cells rescued the inflammation induced by LPS and HA, by down-regulation of the proinflammatory cytokines TNFα, IL-1ß and NFkB. Interestingly, GRP was detected at protein and mRNA levels in extracellular vesicles released by macrophages, which may act as vehicles for extracellular trafficking and release. Our data indicate GRP as an endogenous mediator of inflammatory responses acting as an anti-inflammatory agent in monocytes/macrophages. We propose that in a context of chronic inflammation and calcification-related pathologies, GRP might act as a novel molecular mediator linking inflammation and calcification events, with potential therapeutic application.

Acute kidney injury in patients with severe sepsis or septic shock: a comparison between the 'Risk, Injury, Failure, Loss of kidney function, End-stage kidney disease' (RIFLE), Acute Kidney Injury Network (AKIN) and Kidney Disease: Improving Global Outcomes (KDIGO) classifications.

PURPOSE: Using the Risk, Injury, Failure, Loss of kidney function, End-stage kidney disease (RIFLE), Acute Kidney Injury Network (AKIN) and Kidney Disease: Improving Global Outcomes (KDIGO) systems, the incidence of acute kidney injury (AKI) and their ability to predict in-hospital mortality in severe sepsis or septic shock was compared. MATERIALS AND METHODS: We performed a retrospective analysis of 457 critically ill patients with severe sepsis or septic shock hospitalized between January 2008 and December 2014. Multivariate logistic regression was employed to evaluate the association between the RIFLE, AKIN and KDIGO systems with in-hospital mortality. Model fit was assessed by the goodness-of-fit test and discrimination by the area under the receiver operating characteristic (AUROC) curve. Statistical significance was defined as P < 0.05. RESULTS: RIFLE (84.2%) and KDIGO (87.5%) identified more patients with AKI than AKIN (72.8%) (P < 0.001). AKI defined by AKIN and KDIGO was associated with in-hospital mortality {AKIN: adjusted odds ratio [OR] 2.3[95% confidence interval (CI) 1.3-4], P = 0.006; KDIGO: adjusted OR 2.7[95% CI 1.2-6.2], P = 0.021} while AKI defined by RIFLE was not [adjusted OR 2.0 (95% CI 1-4), P = 0.063]. The AUROC curve for in-hospital mortality was similar between the three classifications (RIFLE 0.652, P < 0.001; AKIN 0.686, P < 0.001; KDIGO 0.658, P < 0.001). CONCLUSIONS: RIFLE and KDIGO diagnosed more patients with AKI than AKIN, but the prediction ability for in-hospital mortality was similar between the three systems.

Controlled delivery of high vs low humidity vs mist therapy for croup in emergency departments: a randomized controlled trial.

CONTEXT: Children with croup are often treated with humidity even though this is not scientifically based, consumes time, and can be harmful. Although humidity using the traditional blow-by technique is similar to room air and no water droplets reach the nasopharynx, particles sized for laryngeal deposition (5-10 microm) could be beneficial. OBJECTIVE: To determine whether a significant difference in the clinical Westley croup score exists in children with moderate to severe croup who were admitted to the emergency department and who received either 100% humidity or 40% humidity via nebulizer or blow-by humidity. DESIGN AND SETTING: A randomized, single-blind, controlled trial conducted between 2001 and 2004 in a tertiary care pediatric emergency department. PARTICIPANTS: A convenience sample of 140 previously healthy children 3 months to 10 years of age with Westley croup score of more than 1 or 2 or higher (scoring system range, 0-17); 21 families refused participation. INTERVENTION: Thirty-minute administration of humidity using traditional blow-by technique (commonly used placebo, n = 48), controlled delivery of 40% humidity (optimally delivered placebo, n = 46), or 100% humidity (n = 46) with water particles of mass median diameter 6.21 microm. MAIN OUTCOME MEASURE: A priori defined change in the Westley croup score from baseline to 30 and 60 minutes in the 3 groups. RESULTS: Groups were comparable before treatment. At 30 minutes the difference in the improvement in the croup score between the blow-by and low-humidity groups was 0.03 (95% confidence interval [CI], -0.72 to 0.66), between low- and high-humidity groups, 0.16 (95% CI, -0.86 to 0.53), and between blow-by and high-humidity groups, 0.19 (95% CI, -0.87 to 0.49). Results were similar at 60 minutes. Differences between groups in pulse and respiratory rates and oxygen saturation changes were insignificant, as were proportions of excellent responders; proportions with croup score of 0 at study conclusion; and proportions receiving dexamethasone, epinephrine, or requiring additional medical care or hospitalization. CONCLUSIONS: One hundred percent humidity with particles specifically sized to deposit in the larynx failed to result in greater improvement than 40% humidity or humidity by blow-by technique. This study does not support the use of humidity for moderate croup for patients treated in the emergency department. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00230841.

Impact of Stewardship on Inhaled Nitric Oxide Utilization in a Neonatal ICU.

OBJECTIVES: Inhaled nitric oxide (iNO) remains the "gold standard" therapy for hypoxemic respiratory failure in newborns. Despite good quality evidence to guide iNO use in this population, we observed considerable practice variation, particularly in timing and rate of weaning. To promote evidence-based practice, we launched an iNO stewardship program in April 2013. Our objective was to determine whether iNO stewardship led to changes in iNO utilization and weaning. METHODS: We conducted a quality improvement project in an outborn quaternary NICU, targeting improved iNO guideline compliance. We compared patterns of iNO utilization between 2 cohorts: prestewardship (April 2011-March 2013; retrospective data collection) and poststewardship (April 2013-March 2015; prospective data collection). RESULTS: Eighty-seven neonates received 88 courses of iNO in the 2 years prestewardship, and 64 neonates received 64 courses of iNO in the 2 years poststewardship. There were no significant differences (P > .05) in patient demographics, in the proportion of patients receiving iNO "off-label," in proportion initiated at the referring hospital, or in outcomes (death or extracorporeal membrane oxygenation). There were significant (P < .05) reductions in median total hours on iNO per patient (47 vs 20; P < .001), in iNO hours per patient from maximum dose to initial wean (28 vs 9; P < .01), and in hours from initial wean to discontinuation (14 vs 8; P < .05). CONCLUSIONS: The introduction of iNO stewardship was associated with improved adherence to evidence-based guidelines and an overall reduction in total and per-patient iNO use.