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Elevated circulating platelet-derived extracellular vesicles (EVs) have been reported in conditions associated with thrombotic risk. The present study aimed to assess the relationship between circulating platelet-derived EV levels, cardiovascular risk stratification and vascular organ damage, as assessed by pulse wave velocity (PWV). A total of 92 patients were included in the present analysis. Platelet EV were evaluated by flow cytometry (CD41+/Annexin v+). The cardiovascular risk was determined using the 2021 ESC guideline stratification and SCORE2 and SCORE-OP. PWV was performed as a surrogate to assess macrovascular damage. Risk stratification revealed significant group differences in EV levels (ANOVA, p = 0.04). Post hoc analysis demonstrated significantly higher levels of EVs in the very high-risk group compared with the young participants (12.53 ± 8.69 vs. 7.51 ± 4.67 EV/µL, p = 0.03). Linear regression models showed SCORE2 and SCORE-OP (p = 0.04) was a predictor of EV levels. EVs showed a significant association with macrovascular organ damage measured by PWV (p = 0.01). PWV progressively increased with more severe cardiovascular risk (p < 0.001) and was also associated with SCORE2 and SCORE-OP (p < 0.001). Within the pooled group of subjects with low to moderate risk and young participants (<40 years), those with EV levels in the highest tertile had a trend towards higher nocturnal blood pressure levels, fasting glucose concentration, lipid levels, homocysteine and PWV. Levels of platelet-derived EVs were highest in those patients with very high CV risk. Within a pooled group of patients with low to moderate risk, an unfavourable cardiometabolic profile was present with higher EV levels.
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Doenças Cardiovasculares , Vesículas Extracelulares , Hipertensão , Anexina A5 , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/etiologia , Glucose , Fatores de Risco de Doenças Cardíacas , Homocisteína , Humanos , Lipídeos , Análise de Onda de Pulso , Fatores de RiscoRESUMO
BACKGROUND: Anaphylaxis is a severe, potentially life-threatening allergic reaction driven primarily by the activation of mast cells. We still fail to understand factors underlying reaction severity. Furthermore, there is currently no reliable diagnostic test to confirm anaphylaxis in the emergency department (ED). OBJECTIVE: This study sought to explore gene expression changes associated with anaphylaxis severity in peripheral blood leucocytes and evaluate biomarker potential. METHODS: Microarray analysis (total RNA) was performed using peripheral blood samples from ED patients with moderate (n = 6) or severe (n = 12) anaphylaxis and sepsis (n = 20) at presentation (T0) and one hour later (T1). Results were compared between groups and healthy controls (n = 10 and n = 11 matched to anaphylaxis and sepsis patients, respectively). Changes in gene expression were determined using R programming language, and pathway analysis applied to explore biological processes and pathways associated with genes. Differentially expressed genes were validated in an independent cohort of anaphylaxis (n = 30) and sepsis (n = 20) patients, and healthy controls (n = 10), using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: Significant up-regulation of small nucleolar RNAs (snoRNAs) was demonstrated in anaphylaxis compared to sepsis patients in the microarray cohort, at T0 and T1. qRT-PCR analysis of the validation cohort showed five genes: SNORD61, SNORD8, SNORD69, SNORD119 and HIST1H1D to be significantly up-regulated (adjusted p < 0.05) in severe anaphylaxis compared to sepsis. Seven genes (SNORD61, SNORD8, SCARNA21, SNORD69, SNORD110, SNORD119 and SNORD59A) were significantly up-regulated (adjusted p < 0.05) in severe anaphylaxis compared to healthy controls. CONCLUSION: This study demonstrates for the first time the unique involvement of snoRNAs in the pathogenesis of anaphylaxis and suggests they are not a general feature of systemic inflammation. Further investigation of snoRNA expression in anaphylaxis could provide insights into disease pathogenesis. CLINICAL RELEVANCE: SnoRNAs are up-regulated during acute anaphylaxis in humans and could potentially be used as biomarkers of severe anaphylaxis.
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Anafilaxia , RNA Nucleolar Pequeno , Anafilaxia/diagnóstico , Anafilaxia/genética , Biomarcadores , Humanos , Mastócitos , Análise em Microsséries , RNA Nucleolar Pequeno/genéticaRESUMO
Tumor growth is associated with a profound alteration in myelopoiesis, leading to recruitment of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). We showed that among factors produced by various experimental tumors, the cytokines GM-CSF, G-CSF, and IL-6 allowed a rapid generation of MDSCs from precursors present in mouse and human bone marrow (BM). BM-MDSCs induced by GM-CSF+IL-6 possessed the highest tolerogenic activity, as revealed by the ability to impair the priming of CD8(+) T cells and allow long term acceptance of pancreatic islet allografts. Cytokines inducing MDSCs acted on a common molecular pathway and the immunoregulatory activity of both tumor-induced and BM-derived MDSCs was entirely dependent on the C/EBPbeta transcription factor. Adoptive transfer of tumor antigen-specific CD8(+) T lymphocytes resulted in therapy of established tumors only in mice lacking C/EBPbeta in the myeloid compartment, suggesting that C/EBPbeta is a critical regulator of the immunosuppressive environment created by growing cancers.
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Células da Medula Óssea/imunologia , Proteína beta Intensificadora de Ligação a CCAAT/imunologia , Tolerância Imunológica/imunologia , Neoplasias/imunologia , Evasão Tumoral/imunologia , Transferência Adotiva , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Tolerância Imunológica/genética , Interleucina-6/biossíntese , Interleucina-6/imunologia , Camundongos , Neoplasias/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Evasão Tumoral/genéticaRESUMO
OBJECTIVE AND DESIGN: Resistin and neutrophil gelatinase-associated lipocalin (NGAL) are upregulated in circulating leucocytes in sepsis, but the significance of this is uncertain. We evaluated associations between Resistin and NGAL with endothelial cell activation and clinical outcomes in a prospective observational study in the Emergency Department (ED). METHODS: Serum levels of Resistin, NGAL, inflammatory cytokines (IL-6, IL-10) and soluble endothelial adhesion molecules (VCAM-1, ICAM-1) were measured at defined time points up to 24 h. Patterns and relationships between markers were investigated using linear mixed regression models. Predictive values for clinical outcomes for markers at enrollment were assessed by logistic regression and receiver operator characteristic (ROC) curves. RESULTS: 186 participants (89 septic-shock, 69 sepsis, 28 uncomplicated infection) were compared with 29 healthy controls. Median Resistin and NGAL were higher in uncomplicated infection compared to controls, and in septic shock compared to non-shock sepsis. Resistin and NGAL correlated with IL-6 and IL-10, with VCAM-1 and ICAM-1, and with organ failure. Resistin and NGAL were associated with septic shock but had limited predictive utility for mortality. CONCLUSION: Resistin and NGAL correlate with expression of endothelial cell adhesion molecules in sepsis. Further evaluation of the role of Resistin and NGAL in sepsis pathogenesis is warranted.
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Lipocalina-2/sangue , Resistina/sangue , Sepse/sangue , Adulto , Idoso , Biomarcadores/sangue , Feminino , Humanos , Molécula 1 de Adesão Intercelular/sangue , Interleucina-10/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Molécula 1 de Adesão de Célula Vascular/sangueRESUMO
PURPOSE: To investigate the effect of IV fluid resuscitation on endothelial glycocalyx (EG) shedding and activation of the vascular endothelium and inflammation. MATERIALS AND METHODS: A planned biomarker sub-study of the REFRESH trial in which emergency department (ED) patients) with suspected sepsis and hypotension were randomised to a restricted fluid/early vasopressor regimen or IV fluid resuscitation with later vasopressors if required (usual care). Blood samples were collected at randomisation (T0) and at 3 h (T3), 6 h (T6)- and 24 h (T24) for measurement of a range of biomarkers if EG shedding, endothelial cell activation and inflammation. RESULTS: Blood samples were obtained in 95 of 99 enrolled patients (46 usual care, 49 restricted fluid). Differences in the change in biomarker over time between the groups were observed for Hyaluronan (2.2-fold from T3 to T24, p = 0.03), SYN-4 (1.5-fold from T3 to T24, P = 0.01) and IL-6 (2.5-fold from T0 to T3, p = 0.03). No difference over time was observed between groups for the other biomarkers. CONCLUSIONS: A consistent signal across a range of biomarkers of EG shedding or of endothelial activation or inflammation was not demonstrated. This could be explained by pre-existing EG shedding or overlap between the fluid volumes administered in the two groups in this clinical trial. Trial registration Australia New Zealand Clinical Trials Registry ACTRN126160000006448 Registered 12 January 2016.
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Objective: Mechanisms underlying the anaphylactic reaction in humans are not fully understood. Here, we aimed at improving our understanding of anaphylaxis by investigating gene expression changes. Methods: Microarray data set GSE69063 was analysed, describing emergency department (ED) patients with severe anaphylaxis (n = 12), moderate anaphylaxis (n = 6), sepsis (n = 20) and trauma (n = 11). Samples were taken at ED presentation (T0) and 1 h later (T1). Healthy controls were age and sex matched to ED patient groups. Gene expression changes were determined using limma, and pathway analysis applied. Differentially expressed genes were validated in an independent cohort of anaphylaxis patients (n = 31) and matched healthy controls (n = 10), using quantitative reverse transcription-polymerase chain reaction. Results: Platelet aggregation was dysregulated in severe anaphylaxis at T0, but not in moderate anaphylaxis, sepsis or trauma. Dysregulation was not observed in patients who received adrenaline before T0. Seven genes (GATA1 (adjusted P-value = 5.57 × 10-4), TLN1 (adjusted P-value = 9.40 × 10-4), GP1BA (adjusted P-value = 2.15 × 10-2), SELP (adjusted P-value = 2.29 × 10-2), MPL (adjusted P-value = 1.20 × 10-2), F13A1 (adjusted P-value = 1.39 × 10-2) and SPARC (adjusted P-value = 4.06 × 10-2)) were significantly downregulated in severe anaphylaxis patients who did not receive adrenaline before ED arrival, compared with healthy controls. One gene (TLN1 (adjusted P-value = 1.29 × 10-2)) was significantly downregulated in moderate anaphylaxis patients who did not receive adrenaline before ED arrival, compared with healthy controls. Conclusion: Downregulation of genes involved in platelet aggregation and activation is a unique feature of the early anaphylactic reaction not previously reported and may be associated with reaction severity.
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INTRODUCTION: Elevated nocturnal blood pressure (BP) is closely associated with increased risk of cardiovascular (CV) events. Circulating extracellular vesicles (EVs) have been proposed as a potential CV risk biomarker and shown to correlate with BP. The present study aimed to assess whether a reduction in BP is paralleled by respective changes in EVs. METHODS: Fifty-five hypertensive patients (age: 57.7â±â14.1âyears) were included in the study. EVs and BP were assessed at baseline and at 12âweeks follow-up. Interventions to lower BP included advice on life-style modification only or life-style advice combined with additional pharmacotherapy. EVs were evaluated by flow cytometry (CD41+/Annexin V+) and BP by unobserved automated office BP and ambulatory BP monitoring. RESULTS: Nocturnal systolic BP correlated with EV levels at baseline ( P â=â0.01). Multivariable regression models showed that changes in nocturnal systolic BP (adjusted R2 â=â0.23; P â=â0.01) and diastolic BP (adjusted R2 â=â0.18; P â=â0.02) were associated with respective changes in EV levels. Furthermore, intervention-induced improvement of systolic dipping was associated with a reduction in EVs in the univariate analysis (adjusted R2 â=â0.06; P â=â0.03). In contrast, systolic office, 24 h- and daytime-BP did not show significant associations with EVs. Patients whose medication was up-titrated at baseline showed a trend towards lower EV levels at follow-up (absolute change of -1.7â±â1.3âEV/µl; P â=â0.057). CONCLUSIONS: Circulating platelet-derived EVs were positively associated with nocturnal BP and therapy-induced changes over a 12-week treatment period. EVs may provide an integrated measure of BP changes achieved with pharmacotherapy.
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Vesículas Extracelulares , Hipertensão , Adulto , Idoso , Anexina A5 , Pressão Sanguínea/fisiologia , Monitorização Ambulatorial da Pressão Arterial , Ritmo Circadiano/fisiologia , Humanos , Pessoa de Meia-IdadeRESUMO
Endothelial glycocalyx (EG) shedding is associated with septic shock and described following intravenous (IV) fluid administration. To investigate the possible impact of IV fluids on the pathobiology of septic shock we investigated associations between biomarkers of EG shedding and endothelial cell activation, and relationships with IV fluid volume. Serum samples were obtained on admission (T0) and at 24 h (T24) in patients undergoing haemodynamic resuscitation for suspected septic shock in the emergency department. Biomarkers of EG shedding-Syndecan-1 (Syn-1), Syndecan-4 (Syn-4), Hyaluronan, endothelial activation-Endothelin-1 (ET-1), Angiopoeitin-2 (Ang-2), Vascular Endothelial Growth Factor Receptor-1(VEGF-1) and leucocyte activation/inflammation-Resistin, Neutrophil Gelatinase Associated Lipocalin (NGAL) and a marker of cardiac stretch-Pro-Atrial Natriuretic Peptide (Pro-ANP) were compared to the total IV fluid volume administered using Tobit regression. Data on 86 patients (52 male) with a mean age of 60 (SD 18) years were included. The mean fluid volume administered to T24 was 4038 ml (SD 2507 ml). No significant association between fluid volume and Pro-ANP or any of the biomarkers were observed. Syn-1 and Syn-4 were significantly correlated with each other (Spearman Rho 0.43, p < 0.001) but not with Hyaluronan. Syn-1 and Syn-4 both correlated with VEGFR-1 (Rho 0.56 and 0.57 respectively, p < 0.001) whereas Hyaluronan correlated with ET-1 (Rho 0.43, p < 0.001) and Ang-2 (Rho 0.43, p < 0.001). There was no correlation between Pro-ANP and any of the EG biomarkers. Distinct patterns of association between biomarkers of EG shedding and endothelial cell activation were observed among patients undergoing resuscitation for sepsis. No relationship between IV fluid volume and Pro-ANP or any of the other biomarkers was observed.
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Sepse , Choque Séptico , Fator Natriurético Atrial/metabolismo , Biomarcadores , Serviço Hospitalar de Emergência , Glicocálix/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Masculino , Pessoa de Meia-Idade , Sepse/metabolismo , Choque Séptico/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Elevated office blood pressure (BP) has previously been associated with increased levels of circulating extracellular vesicles (EVs). The present study aimed to assess the relationship between levels of platelet derived EVs, ambulatory BP parameters, and pulse wave velocity as a marker of macrovascular organ damage. A total of 96 participants were included in the study. Platelet-derived extracellular vesicles (pEVs) were evaluated by flow cytometry (CD41+/Annexin v+). BP evaluation included unobserved automated office BP and ambulatory BP monitoring. Carotid-femoral pulse wave velocity (PWV) was measured as a marker of macrovascular damage. pEVs correlated with nocturnal systolic BP (r = 0.31; p = .003) and nocturnal dipping (r = -0.29; p = .01) in univariable analysis. Multivariable regression models confirmed robustness of the association of EVs and nocturnal blood pressure (p = .02). In contrast, systolic office, 24h- and daytime-BP did not show significant associations with pEVs. No correlations were found with diastolic BP. Circulating pEVs correlated with pulse wave velocity (r = 0.25; p = .02). When comparing different hypertensive phenotypes, higher levels of EVs and PWV were evident in patients with sustained hypertension compared to patients with white coat HTN and healthy persons. Circulating platelet derived EVs were associated with nocturnal BP, dipping, and PWV. Given that average nocturnal BP is the strongest predictor of CV events, platelet derived EVs may serve as an integrative marker of vascular health, a proposition that requires testing in prospective clinical trials.
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Vesículas Extracelulares , Hipertensão , Rigidez Vascular , Biomarcadores , Pressão Sanguínea/fisiologia , Monitorização Ambulatorial da Pressão Arterial , Humanos , Estudos Prospectivos , Análise de Onda de PulsoRESUMO
Dietary nitrate, found predominantly in green leafy vegetables and other vegetables such as radish, celery, and beetroot, has been shown to beneficially modulate inflammatory processes and immune cell function in animals and healthy individuals. The impact of increased nitrate intake on soluble inflammatory mediators in individuals with hypertension is unclear. We assessed whether the daily consumption of dietary nitrate via beetroot juice for 1-week lowered levels of circulating inflammatory markers in men and women with treated hypertension. Twenty-seven male and female participants were recruited to a randomized, placebo-controlled, double-blind crossover trial. The effects of 1-week intake of nitrate-rich beetroot juice versus 1-week intake of nitrate-depleted beetroot juice (placebo) were investigated. Plasma concentrations of circulating soluble adhesion molecules (ICAM-1, VCAM-1, CD62E, CD62P), inflammatory cytokines (IL-1ß, IL-6, IL-10, IL-12p70, TNF-α) and chemokines (IL-8, MCP-1) were measured by multiplex flow cytometric bead array in samples collected on day 7 of each intervention period. Other outcomes included alterations in nitrate metabolism assessed by measuring nitrate and nitrite concentrations in plasma, saliva, and urine. One week of beetroot juice did not alter levels of the soluble adhesion markers or cytokines assessed. A 7-fold increase in salivary nitrite, an 8-fold increase in salivary nitrate, a 3-fold increase in plasma nitrate and nitrite, and a 4-fold increase in urinary nitrate and nitrite compared to the placebo was observed (p < 0.001 for all comparisons). Increasing dietary nitrate consumption over 7 days is not effective in reducing soluble inflammatory mediators in individuals with treated hypertension. This trial was registered at anzctr.org.au as ACTRN 12613000116729.
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Beta vulgaris , Hipertensão , Animais , Nitratos , Nitritos , Citocinas/farmacologia , Sucos de Frutas e Vegetais , Hipertensão/tratamento farmacológico , Antioxidantes/farmacologia , Método Duplo-Cego , Verduras , Estudos Cross-Over , Biomarcadores , Mediadores da Inflamação/farmacologia , Pressão Sanguínea , Suplementos NutricionaisRESUMO
Extracellular vesicles (EVs) represent a promising biomarker in several medical areas. Flow cytometry (FC) is one of the most widely-used methods to characterize EVs, providing quantitative information and determination of EV subtypes. EV evaluation represents a challenge as no standardized methods are available to facilitate assessment across different research centers. This is principally because their size falls below the detection limit of most standard flow cytometers and a thorough optimization process is required to ensure instrument-specific sensitivity. We provide an overview of a standardized method to evaluate large EVs using the Attune™ Nxt Acoustic Focusing Flow Cytometer system (Thermo Fisher Scientific).
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Vesículas Extracelulares/química , Citometria de Fluxo/normas , Vesículas Extracelulares/imunologia , Humanos , Padrões de ReferênciaRESUMO
BACKGROUND: Carbon monoxide (CO) interferes with inflammatory and apoptotic processes associated with ischemia-reperfusion injury and graft rejection. Here, the in vitro effects of carbon monoxide releasing molecule-3 (CORM-3), a novel water-soluble carbonyl CO carrier, have been investigated on porcine aortic endothelial cells (PAEC) and primate peripheral blood mononuclear cells (PBMC). Furthermore, the pharmacodynamics and pharmacotolerance of CORM-3 after administration of single and multiple doses in the primate have been assessed in view of its potential application in pig-to-primate xenotransplantation models. METHODS: For in vitro studies, PAEC and primate PBMC were exposed for 24, 48 and 72 h to CORM-3 (20 to 1000 microm) and viability was measured using an MTS assay. PAEC and primate PBMC proliferation after exposure to CORM-3 was assessed by CFSE labelling. Proliferation of primate PBMC against irradiated pig lymphocytes was also assessed. Tumor necrosis factor alpha (TNF-alpha) production and Caspase-3 and -7 activity in Concanavalin A (conA)-stimulated primate PBMC were measured following treatment with CORM-3. In vivo, CORM-3 was administered i.v. to cynomolgus monkeys at 4 mg/kg, as single or multiple doses for up to 30 days. The effect of CORM-3 was evaluated by the assessment of production of TNF-alpha and interleukin 1beta following PBMC stimulation with LPS by species-specific ELISA. Complete hematologic and biochemical analyses were routinely performed in treated primates. RESULTS: At concentrations <500 microm, CORM-3 did not alter the viability of PAEC or primate PBMC cultures in vitro, nor did it induce significant levels of apoptosis or necrosis. Interestingly, at concentrations of 300 and 500 microm, significant PAEC proliferation was observed, whilst concentrations > or =50 microm inhibited conA-activated primate lymphocyte proliferation (IC(50) of 345.8 +/- 51.9 microm) and the primate xenogeneic response against pig PBMC. Such responses were demonstrated to be CO-dependent. In addition, CORM-3 significantly inhibited caspase-3 and -7 activity at concentrations between 200 and 500 microm and caused a significant reduction in TNF-alpha production (IC(50) 332.8 +/- 33.9 microm). In vivo, following the administration of multiple doses, TNF-alpha production was significantly reduced in comparison to pre-treatment responses, with decreased levels maintained throughout the study. Moreover, a slight and transient increase in transaminases and bilirubin was observed in animals exposed to multiple doses of CORM-3. CONCLUSIONS: These studies suggest that CORM-3 has anti-inflammatory and immunomodulatory properties in primates that may result in clinical benefit to allo- and xenografted organs.
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Monóxido de Carbono/metabolismo , Células Endoteliais/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Transplante Heterólogo , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Concanavalina A/farmacologia , Células Endoteliais/citologia , Humanos , Interleucina-1beta/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Macaca fascicularis , Mitógenos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sus scrofa , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE AND DESIGN: Hypertonic saline administered during fluid resuscitation may mitigate endothelial glycocalyx (EG) shedding and inflammation. The objective of this pilot randomised controlled trial was to measure the effect of hypertonic saline, compared to isotonic saline, on biomarkers of EG shedding and inflammation in emergency department patients with suspected sepsis. METHODS: Patients received either 5â¯mL/kg of 3% saline (hypertonic group, nâ¯=â¯34) or 10â¯mL/kg of 0.9% saline (isotonic group, nâ¯=â¯31). Change in serum biomarker concentrations of syndecan-1, hyaluronan, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, interleukin-6, -8, -10, interferon-γ, neutrophil gelatinase-associated lipocalin and resistin were compared from baseline (T0) to after fluid (T1), 3â¯h (T3) and 12-24â¯h (T24) later, as was serum osmolality, using linear mixed effects models. RESULTS: The hypertonic group had significantly increased mean serum osmolality compared to the isotonic group at T1 (Pâ¯<â¯.001) and T3 (Pâ¯=â¯.004). Minor differences were found in some biomarker outcomes, including a decreased fold-change in syndecan-1 at T1 (Pâ¯=â¯.012) and in interleukin-10 at T24 (Pâ¯=â¯.006) in the isotonic group, compared to the hypertonic group. CONCLUSIONS: Although a single bolus of hypertonic saline increased serum osmolality, it did not reduce biomarkers of EG shedding or inflammation, compared to patients that received isotonic saline. TRIAL REGISTRATION: ANZCTR.org.au, ACTRN12611001021965, Registered on 23rd September 2011.
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Glicocálix/química , Solução Salina Hipertônica/farmacologia , Solução Salina/farmacologia , Sepse/terapia , Adulto , Biomarcadores/sangue , Serviço Hospitalar de Emergência , Feminino , Hidratação , Humanos , Inflamação , Masculino , Pessoa de Meia-Idade , Concentração Osmolar , Projetos Piloto , Sepse/sangue , Resultado do TratamentoRESUMO
BACKGROUND: We have previously identified the upregulation of the innate immune response, neutrophil activation, and apoptosis during anaphylaxis using a microarray approach. This study aimed to validate the differential gene expression and investigate protein concentrations of "hub genes" and upstream regulators during anaphylaxis. METHODS: Samples were collected from patients with anaphylaxis on their arrival at the emergency department, and after 1 and 3 h. mRNA levels of 11 genes (interleukin-6 [IL-6], IL-10, oncostatin M [OSM], S100A8, S100A9, matrix metalloproteinase 9 [MMP9], FASL, toll-like receptor 4 [TLR4], MYD88, triggering receptor expressed on myeloid cells 1 [TREM1], and cluster of differentiation 64 [CD64]) were measured in peripheral blood leucocytes using qPCR. Serum protein concentrations were measured by ELISA or cytometric bead array for 6 of these candidates. RESULTS: Of 69 anaphylaxis patients enrolled, 36 (52%) had severe reactions, and 38 (55%) were female. Increases in both mRNA and protein of IL-10, S100A9, MMP9, and TREM1 were observed. OSM, S100A8, TLR4, and CD64 were upregulated and IL-6 protein concentrations were increased during anaphylaxis. Both FASL and soluble Fas ligand decreased during anaphylaxis. CONCLUSION: These results provide evidence for the involvement of innate immune pathways and myeloid cells during human anaphylaxis, validating previous microarray findings. Elevated S100A8, S100A9, TLR4, and TREM1 expression, and increased S100A9 and soluble TREM1 protein concentrations strongly suggest that neutrophils are activated during acute anaphylaxis.
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Anafilaxia/imunologia , Imunidade Inata , Células Mieloides/imunologia , Ativação de Neutrófilo , Neutrófilos/imunologia , Adulto , Anafilaxia/sangue , Anafilaxia/genética , Citocinas/genética , Citocinas/metabolismo , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Proteínas Inflamatórias de Macrófagos/genética , Proteínas Inflamatórias de Macrófagos/metabolismo , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Adulto JovemRESUMO
OBJECTIVE: To investigate the association between synthetic colloids and biomarkers of acute kidney injury (AKI) in dogs with hemorrhagic shock. DESIGN: Experimental interventional study. SETTING: University. ANIMALS: Twenty-four healthy ex-racing Greyhounds. INTERVENTIONS: Anesthetized Greyhounds subjected to hemorrhage for 60 min were resuscitated with 20 mL/kg of fresh whole blood (FWB), 6% hydroxyethyl starch (HES) 130/0.4, 4% succinylated gelatin (GELO), or 80 mL/kg of isotonic crystalloid (CRYST) over 20 min (n = 6 per treatment). Concentrations of biomarkers of AKI were measured at baseline, end of hemorrhage, and at 40 (T60), 100 (T120), and 160 (T180) min after fluid bolus. Biomarkers included neutrophil gelatinase-associated lipocalin in urine and serum (uNGAL; sNGAL), and urine cystatin C (uCYSC), kidney injury molecule-1 (uKIM), clusterin (uCLUST), osteopontin, gamma-glutamyl transferase, monocyte chemoattractant protein-1 (uMCP), interleukin-6, interleukin-8, protein (uPROT), hyaluronan, and F2 -isoprostanes. Renal histology was scored for tubular injury and microvesiculation. Biomarker fold-change from baseline was compared between groups using mixed effects models (Bonferroni-Holm corrected P<0.05). Frequencies of histology scores were compared by Fisher's exact test. MEASUREMENTS AND MAIN RESULTS: In dogs treated with GELO, uNGAL fold-change was markedly greater compared with all other groups at T60, T120, and T180 (all P<0.001), and uCYSC was greater at T60 compared with CRYST (P<0.001), and at T120 and T180 compared with all other groups (all P<0.001). Smaller, albeit significant, between-group differences in uKIM, uCLUST, uMCP, and urine protein concentration were observed across the FWB, GELO, and HES groups, compared with CRYST. The GELO group more frequently had marked tubular microvesiculation than the other groups (P = 0.015) although tubular injury scores were comparable. CONCLUSION: In dogs with hemorrhagic shock, GELO was associated with greater magnitude increases in urine biomarkers of AKI and more frequent marked tubular microvesiculation, compared with FWB, CRYST, and HES.
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Injúria Renal Aguda/veterinária , Biomarcadores/urina , Doenças do Cão/tratamento farmacológico , Lipocalina-2/urina , Choque Hemorrágico/veterinária , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/urina , Animais , Cuidados Críticos , Modelos Animais de Doenças , Cães , Feminino , Gelatina/administração & dosagem , Gelatina/efeitos adversos , Derivados de Hidroxietil Amido/administração & dosagem , Derivados de Hidroxietil Amido/efeitos adversos , Masculino , Substitutos do Plasma/administração & dosagem , Substitutos do Plasma/efeitos adversos , Choque Hemorrágico/tratamento farmacológico , Succinatos/administração & dosagem , Succinatos/efeitos adversosRESUMO
Apoptotic phenomena observed in vitro following isolation and following transplantation contribute significantly to islet graft loss. Strategies to reduce apoptosis of islet tissue prior to and posttransplantation may improve graft survival and function and reduce the amount of tissue necessary to achieve insulin independence. The expression of cytoprotective proteins is one such strategy that may prolong islet survival. In this light, heme-oxygenase 1 (HO-1) upregulation has been studied in both allo- and xenotransplantation models. In this study, the effect of HO-1 on apoptosis in neonatal porcine islet-like cell clusters (NPICC) was assessed. In in vitro assessments of NPICC apoptosis, NPICC showed a high sensitivity to apoptotic stimulation using a combination of TNF-alpha and cycloheximide. Stimulation with TNF-alpha alone was sufficient to induce reproducible apoptotic responses as demonstrated by caspase-3,-7 activation and subdiploid DNA analysis. Dose-dependent, high-level HO-1 protein expression was achieved following culture of NPICC in medium containing either cobalt protoporphyrin (CoPP) or cobalt mesoporphyrin (CoMP). CoPP treatment resulted in the reduction of caspase-3,-7 enzyme activity following TNF-alpha stimulation. However, such an effect was not associated with a reduction in the levels of cell death. Indeed, the inhibition of caspase enzyme activity resulted in decreased PARP-1 cleavage, which may lead to heightened levels of necrosis in treated NPICC cultures, possibly explaining the observed commitment of NPICC to the death pathway.
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Caspase 3/metabolismo , Caspase 7/metabolismo , Morte Celular/fisiologia , Ilhotas Pancreáticas/enzimologia , Protoporfirinas/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Animais Recém-Nascidos , Morte Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Cicloeximida/farmacologia , Inibidores de Cisteína Proteinase/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Heme Oxigenase-1/metabolismo , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Oligopeptídeos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Protoporfirinas/metabolismo , Sus scrofa , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
PURPOSE OF REVIEW: Porcine islets are a major focus of current research in nonhuman primate xenotransplantation models. Major advances have been obtained recently and these are briefly described. RECENT FINDINGS: Reports by three independent centres have described 6-month porcine islet xenograft survival in nonhuman primates. Two of these have obtained such results by interfering with the CD40/CD154 co-stimulatory pathway. While these results are groundbreaking, the immunosuppressive regimens used are not viewed as clinically applicable and will need to be modified before islet xenotransplantation can be considered for clinical trials. In contrast, preliminary results by Gianello and colleagues have demonstrated 6-month survival of diabetic nonhuman primates transplanted subcutaneously with encapsulated porcine islets, in the complete absence of immunosuppression. The confirmation and full assessment of these results are eagerly awaited. Importantly, to date, no evidence of xenozoonoses has been observed following porcine islet xenotransplantation to nonhuman primates. SUMMARY: Of the possible organ candidates for xenotransplantation, porcine islets are the closest to a possible clinical application. Prior to the initiation of ethical and safe clinical trials, however, further efforts to generate additional efficacy and safety data in the nonhuman primate model will be indispensable.
Assuntos
Transplante das Ilhotas Pancreáticas , Modelos Animais , Primatas , Suínos , Transplante Heterólogo , Animais , Engenharia Genética , Terapia de Imunossupressão , Transplante das Ilhotas Pancreáticas/métodosRESUMO
PURPOSE: Endocan, a component of the endothelial glycocalyx (EG), has been linked with respiratory failure in sepsis. This study explored the temporal patterns of three EG biomarkers, including endocan, and their relationships with inflammation and respiratory failure. MATERIALS AND METHODS: Plasma endocan, syndecan-1, and hyaluronan concentrations were measured in Emergency Department (ED) patients with sepsis due to pneumonia (nâ¯=â¯44) on ED arrival (T0), 1â¯h (T1), 3â¯h (T3) and 12-24â¯h (T24) later, with change over time tested using mixed regression models. Biomarker associations with inflammatory cytokine concentrations and with respiratory failure on days 1, 2 or 3, need for mechanical ventilation and 30-day mortality were also tested. RESULTS: Endocan concentration significantly decreased over time (T0-T24, Pâ¯=â¯0.003) whereas both syndecan-1 (T0-T3, Pâ¯=â¯0.010; T0-T24, Pâ¯<â¯0.001) and hyaluronan (T0-T1, Pâ¯=â¯0.010; T0-T3, Pâ¯<â¯0.001; T0-T24, Pâ¯=â¯0.003) significantly increased over time. Increased syndecan-1 was significantly correlated with neutrophil activation biomarkers and significantly increased the odds of respiratory failure (OR 1.18, 95% CI 1.05-1.33, Pâ¯=â¯0.004), need for mechanical ventilation (OR 1.24, 95% CI 1.04-1.48, Pâ¯=â¯0.014) and 30-day mortality (OR 1.29, 95% CI 1.07-1.55, Pâ¯=â¯0.008). CONCLUSION: Syndecan-1, but not endocan, was associated with neutrophil activation and was the best EG biomarker predictor of adverse clinical outcomes.
Assuntos
Glicocálix/metabolismo , Proteínas de Neoplasias/sangue , Proteoglicanas/sangue , Insuficiência Respiratória/sangue , Sepse/sangue , Sindecana-1/sangue , Idoso , Biomarcadores/sangue , Citocinas/sangue , Feminino , Humanos , Ácido Hialurônico/sangue , Inflamação , Masculino , Pessoa de Meia-Idade , Pneumonia/sangue , Respiração com Pressão Positiva , Respiração Artificial , Sepse/mortalidade , Fatores de TempoRESUMO
OBJECTIVE: Clinical diagnosis of anaphylaxis is principally based on symptoms and signs. However, particularly for patients with atypical symptoms, laboratory confirmation of anaphylaxis would be useful. This study investigated the utility of mast cell tryptase, an available clinical biomarker, for differentiating anaphylaxis from other causes of critical illness, which can also involve mast cell activation. METHODS: Tryptase was measured (ImmunoCAP) in serum from patients with anaphylaxis and non-anaphylactic critical illness (controls) at ED arrival, and after 1-2, 3-4 and 12-24 h. Differences in both peak and delta (difference between highest and lowest) tryptase concentrations between groups were investigated using linear regression models, and diagnostic ability was analysed using Receiver Operating Characteristic curve analysis. RESULTS: Peak tryptase was fourfold (95% CI: 2.9, 5.5) higher in anaphylaxis patients (n = 67) than controls (n = 120) (P < 0.001). Delta-tryptase was 5.1-fold (95% CI: 2.9, 8.9) higher in anaphylaxis than controls (P < 0.001). Optimal test characteristics (sensitivity: 72% [95% CI: 59, 82] and specificity: 72% [95%CI: 63, 80]) were observed when peak tryptase concentrations were >11.4 ng/mL and/or delta-tryptase ≥2.0 ng/mL. For hypotensive patients, peak tryptase >11.4 ng/mL had improved test characteristics (sensitivity: 85% [95% CI: 65, 96] and specificity: 92% [95% CI: 85, 97]); the use of delta-tryptase reduced test specificity. CONCLUSION: While peak and delta tryptase concentrations were higher in anaphylaxis than other forms of critical illness, the test lacks sufficient sensitivity and specificity. Therefore, mast cell tryptase values alone cannot be used to establish the diagnosis of anaphylaxis in the ED. In particular, tryptase has limited utility for differentiating anaphylactic from non-anaphylactic shock.
Assuntos
Anafilaxia/diagnóstico , Mastócitos/microbiologia , Triptases/análise , Adulto , Anafilaxia/sangue , Serviço Hospitalar de Emergência/organização & administração , Feminino , Humanos , Hipóxia/sangue , Hipóxia/diagnóstico , Modelos Lineares , Masculino , Mastócitos/classificação , Pessoa de Meia-Idade , Curva ROC , Sensibilidade e Especificidade , Choque/sangue , Choque/diagnóstico , Triptases/sangueRESUMO
We previously reported on a 26-year-old patient who presented early during a large and eventually fatal cerebral infarct. Microarray analysis of blood samples from this patient demonstrated initially up-regulated and subsequently down-regulated Granzyme B (GzmB) expression, along with progressive up-regulation of genes for S100 calcium binding protein A12 (S100A12) and matrix metalloproteinase 9 (MMP-9). To confirm these findings, we investigated these parameters in patients with suspected stroke presenting within 6h of symptom onset to a single centre. Blood samples were taken at enrolment, then 1h, 3h and 24h post-enrolment for the examination of cellular, protein and genetic changes. Patients with subsequently confirmed ischaemic (n=18) or haemorrhagic stroke (n=11) showed increased intracellular concentrations of GzmB in all cell populations investigated (CD8+, CD8- and Natural Killer [NK] cells). Infarct patients, however, demonstrated significantly reduced GzmB gene expression and increased circulating MMP-9 and S100A12 levels in contrast to transient ischaemic attack (TIA) patients or healthy controls. Furthermore, a pronounced neutrophilia was noted in the infarct and haemorrhage groups, while TIA patients (n=9) reflected healthy controls (n=10). These findings suggest a spectrum of immune response during stroke. TIA showed few immunological changes in comparison to infarct and haemorrhage, which demonstrated inhibition of GzmB production and a rise in neutrophil numbers and neutrophil-associated mediators. This implies a greater role of the innate immune system. These markers may provide novel targets for inhibition and reduction of secondary injury.