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Context: Several curricular initiatives have been developed to improve the acquisition of research competencies by Health Science students. Objectives: To know how students self-perceived of whether their participation in the XIV National Research Congress for Undergraduate Students of Health Sciences had helped them in the acquisition of 36 research-related transferable competencies (TCs) common to Health Science degrees. Methods: A survey design (Cronbach's alpha = 0.924), using a self-administered questionnaire, was conducted among undergraduate students who voluntarily participated in the Congress. Data analysis was performed using SPSS 25 and Statgraphics 19. Statistical significance was considered for P < 0.05. Results: Eighty-one students from 12 Health Science degree programs responded. Key findings are presented in a structured manner, using a Likert-5 scale. Twenty-five of the competencies surveyed obtained an average ≥ 4 highlighting: "Critically evaluate and know how to use sources of clinical and biomedical information to obtain, organize, interpret, and communicate scientific and health information"; "To be able to formulate hypotheses, collect and critically evaluate information for problem solving, following the scientific method", "Critical analysis and research" and "Communicate effectively and clearly, orally and in writing with other professionals". Significance was found in 15 competencies. The development of the competencies "Teamwork", "Critical reasoning" and "Analysis and synthesis abilities" was considered to be of greater "personal utility" by the respondents. Conclusion: Participation in this event contributed to the development of research-related TCs, critical analysis and information management and communication, especially in relation to learning the sources of clinical and biomedical information, to know, following the scientific method, how to formulate hypotheses that allow students to solve problems in their professional activity. The experience was significantly influenced by the respondents' year, the type of participation in the event and the gender of the students. Limitations and suggestions regarding future research are discussed to encourage further exploration of the topic.
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BACKGROUND: Asthma is a heterogeneous disease with several phenotypes, endotypes and severity degrees, in which different T-cell subpopulations are involved. These cells express specific miRNAs (i.e. inflamma-miRs) that can be released to serum in exosomes after activation and be used as biomarkers of underlying inflammation. Thus, we aim to evaluate specific T-cell miRNA signatures in serum exosomes from different subgroups of asthmatic patients. METHODS: Samples from healthy donors (N = 30) and patients (N = 119) with different asthma endotypes (T2high -Atopic/T2high -Non-atopic/T2low ) and severity degrees (mild/MA and moderate-severe/MSA) were used. Demographic, clinical, haematological and biochemical characteristics were collected. Twelve miRNAs previously associated with different Th subsets were preselected and their levels in serum exosome samples were measured using RTqPCR. RESULTS: We detected five miRNAs with high confidence in serum exosomes: miR-16-5p, miR-21-5p, miR-126-3p, miR146a-5p and miR-215-5p. All of them, except miR-16-5p were upregulated in MSA patients compared to MA. A logistic regression model including each of these miRNAs was created to discriminate both conditions, rendering a ROC curve AUC of 0.896 (0.830-0.961). miR-21-5p and miR-126-3p, both involved in Th1/Th2 differentiation, were specifically augmented in T2high -Atopic patients. Of note, all these changes were found in samples collected in autumn. On the contrary, IL-6high patients with MSA, which were more obese, older, with higher neutrophil and basophil counts and TNF levels, displayed a decrease of miR-21-5p, miR-126-3p and miR-146a-5p. CONCLUSION: Immune-related miRNAs, including miR-21-5p, miR-126-3p, miR-146a-5p and miR-215-5p, can be used as clinically relevant non-invasive biomarkers of the phenotype/endotype and severity of asthma.
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Asma , Exossomos , MicroRNAs , Humanos , Biomarcadores , MicroRNAs/genética , Fenótipo , Asma/diagnóstico , Biomarcadores TumoraisRESUMO
OBJECTIVES: To assess compliance with recommendations to alleviate nipple pain and/or trauma (NPT) and to reduce the rate of breastfeeding abandonment for this reason. INTRODUCTION: As a fundamental priority, health programmes encourage mothers to breastfeed exclusively for the first 6 months of the baby's life and to supplement breast milk with other foods up to the age of 2 years. However, the presence of NPT can reduce or prevent compliance with this recommendation. METHODS: The project was designed and carried out using a framework based on the JBI Practical Application of Clinical Evidence System (JBI-PACES). Six audit criteria were used in preaudits and postaudits to observe any changes in compliance with the recommendations. Between audits, the Getting Research into Practice (GRiP) tool was used to identify stakeholders, barriers and facilitators of the project. RESULTS: Two hundred and sixty-seven breastfeeding women were studied in the baseline phase and 275 during follow-up. Compliance in four criteria improved, and the rates of NPT decreased (pain: from 63.3 to 53.5%; P â=â0.02; trauma: from 37.8 to 24.7%; P â=â0.01). The proportion of women advised by qualified personnel increased from 63 to 88% whereas those who cited pain as the reason for abandoning exclusive breastfeeding decreased from 1.5 to 1.1%. CONCLUSION: This evidence-based implementation project achieved significantly improved compliance rates in most of the evidence-based criteria considered. In consequence, the prevalence of NPT fell significantly. Nevertheless, there was no significant impact on the proportion of mothers abandoning breastfeeding for this reason.
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Aleitamento Materno , Mastodinia , Lactente , Humanos , Feminino , Pré-Escolar , Espanha , Mamilos/lesões , Hospitais UniversitáriosRESUMO
Thoughts about the MIR exam (2020-2021).
Reflexiones sobre el MIR (2020-2021).
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INTRODUCTION: Health care workers experience a tremendous strain while performing their activities, very frequently leading to stress, burnout syndrome, and psychopathological impact. The COVID-19 pandemic may cause physicians to suffer these effects even to a greater extent. Our objective was to describe the frequency of stress, burnout syndrome, anxiety, and depression during the pandemic, and analyze the associations with different independent outcome measures. METHODS: Observational, cross-sectional study conducted 2 months after the lockdown was established in Argentina. Clinical specialists, surgeons, emergency physicians, and those with no direct contact with patients were surveyed using a sociodemographic questionnaire and 3 self-administered inventories: Health Professions Stress Inventory, Maslach Burnout Inventory, and Hospital Anxiety and Depression Scale. RESULTS: The prevalence of stress was 93.7 % (95 % confidence interval [CI]: 90.33-96.2), burnout syndrome 73.5 % (95 % CI: 68.2-78.4), anxiety 44 % (95 % CI: 38.4-49.8), and depression 21.9 % (95 % CI: 17.3-26.9). No association was observed between the frequency and medical specialty. The frequency of burnout syndrome, anxiety, and depression was significantly higher among residents and physicians working in the emergency department. ConcluSions: Residents and emergency physicians working 24-hour shifts showed significantly higher percentages of burnout syndrome, anxiety, and depression compared to staff and head physicians. These findings may be associated with a higher workload and less experience. It is compulsory to take preventive and therapeutic measures to protect those in the pandemic front line.
Introducción. A fin de 2019 se identificó una nueva variedad de coronavirus causante de COVID-19 que alcanzó categoría de pandemia. En Argentina, el área metropolitana de Buenos Aires (AMBA) concentra alrededor del 37 % de la población total y el mayor número de casos diagnosticados. El objetivo de este estudio fue describir las características clínicoepidemiológicas de los pacientes con COVID-19 y describir el impacto en el funcionamiento del Servicio de Pediatría de una institución privada de la zona. Métodos. Diseño retrospectivo, observacional, desarrollado en una institución de la zona oeste del AMBA entre el 12 de marzo y el 31 de agosto de 2020. Se incluyeron todos los menores de 16 años con diagnóstico de COVID-19. Se registraron características demográficas, epidemiológicas, clínicas, indicación de internación/control ambulatorio, número de consultas externas, internación por infecciones virales estacionales, licenciamiento del personal, modificación del número de camas y de las actividades de los profesionales. Resultados. Hubo 5454 consultas ambulatorias pediátricas totales, sospecha de COVID-19 en 753/5 454 (13,8 %), se confirmaron 152/753 (20,2 %). Mediana de edad 82 meses (rango intercuartílico: 20,5-147 m), el 50 % fueron varones. La fiebre fue el síntoma más frecuente. Se internaron 22/152 (14,5 %). Las consultas disminuyeron el 87 %, no hubo internación por infecciones virales estacionales y el 52,9 % (91/172) del personal fue licenciado. Conclusiones. La mayoría de los casos fueron leves y la fiebre fue el principal síntoma. Observamos un notable impacto en el funcionamiento del servicio en cuanto al recurso humano. Destacamos la necesidad de la organización logística del servicio para enfrentar esta contingencia.
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Esgotamento Profissional , COVID-19 , Pediatria , Esgotamento Profissional/epidemiologia , Controle de Doenças Transmissíveis , Estudos Transversais , Humanos , Pandemias , Instalações Privadas , SARS-CoV-2 , Inquéritos e QuestionáriosRESUMO
The challenges of implementing interventions in healthcare settings have been more apparent during the COVID-19 pandemic. This pre-implementation evaluation used a rapid qualitative approach to explore barriers and facilitators to an intervention in intensive care units in Argentina, aimed to promote the use of personal protection equipment, provide emotional support for professionals, and achieve patient flow goals. Data were collected using semi-structured interviews with health professionals of 15 public hospitals in Argentina. Normalization Process Theory was used to guide content analysis of the data. Participants identified potential barriers such as the incorporation of non-specialist staff, shortage of resources, lack of communication between groups and shifts. Potential facilitators were also identified: regular feedback and communication related to implementation, adequate training for new and non-specialist staff, and incentives (e.g., scholarships). The immediacy of the pandemic demanded rapid qualitative research, sharing actionable findings in real time.
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Introducción. A fin de 2019 se identificó una nueva variedad de coronavirus causante de COVID-19 que alcanzó categoría de pandemia. En Argentina, el área metropolitana de Buenos Aires (AMBA) concentra alrededor del 37 % de la población total y el mayor número de casos diagnosticados. El objetivo de este estudio fue describir las características clínico-epidemiológicas de los pacientes con COVID-19 y describir el impacto en el funcionamiento del Servicio de Pediatría de una institución privada de la zona. Métodos. Diseño retrospectivo, observacional, desarrollado en una institución de la zona oeste del AMBA entre el 12 de marzo y el 31 de agosto de 2020. Se incluyeron todos los menores de 16 años con diagnóstico de COVID-19. Se registraron características demográficas, epidemiológicas, clínicas, indicación de internación/control ambulatorio, número de consultas externas, internación por infecciones virales estacionales, licenciamiento del personal, modificación del número de camas y de las actividades de los profesionales. Resultados. Hubo 5454 consultas ambulatorias pediátricas totales, sospecha de COVID-19 en 753/5 454 (13,8 %), se confirmaron 152/753 (20,2 %). Mediana de edad 82 meses (rango intercuartílico: 20,5-147 m), el 50 % fueron varones. La fiebre fue el síntoma más frecuente. Se internaron 22/152 (14,5 %). Las consultas disminuyeron el 87 %, no hubo internación por infecciones virales estacionales y el 52,9 % (91/172) del personal fue licenciado. Conclusiones. La mayoría de los casos fueron leves y la fiebre fue el principal síntoma. Observamos un notable impacto en el funcionamiento del servicio en cuanto al recurso humano. Destacamos la necesidad de la organización logística del servicio para enfrentar esta contingencia.
Introduction. Towards the end of 2019, a novel coronavirus that causes COVID-19 was identified and became a pandemic. In Argentina, approximately 37 % of the total population lives in the Metropolitan Area of Buenos Aires (AMBA), where most cases have been diagnosed. The objective of this study was to describe the clinical and epidemiological characteristics of COVID-19 patients and the impact on the operations of the Department of Pediatrics of a private facility located in the AMBA. Methods. Retrospective, observational study conducted at a facility in the west of AMBA between March 12th and August 31st, 2020. All patients younger than 16 years diagnosed with COVID-19 were included. Demographic, epidemiological, and clinical characteristics; indication for hospitalization/outpatient follow-up; number of outpatient visits; hospitalization due to seasonal viral infections; staff on leave; changes in bed availability and health care providers' activities were recorded. Results. There were 5454 pediatric outpatient visits, COVID-19 was suspected in 753/5454 (13.8 %) and 152/753 (20.2 %) were confirmed cases. Their median age was 82 months (interquartile range: 20.5-147 months); 50 % were males. Fever was the most common symptom. In total, 22/152 (14.5 %) patients were hospitalized. Outpatients visits decreased by 87 %; there were no hospitalizations due to seasonal viral infections; and 52.9 % (91/172) of staff took a leave. Conclusions. Most cases were mild, and fever was the main symptom. The department operations were considerably affected in terms of human resources. It is worth noting the need for a logistic organization at the Department of Pediatrics to face such contingency.
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Humanos , Recém-Nascido , Lactente , Pré-Escolar , Criança , Adolescente , Pediatria , Esgotamento Profissional/epidemiologia , COVID-19 , Controle de Doenças Transmissíveis , Inquéritos e Questionários , Estudos Retrospectivos , Pandemias , Instalações Privadas , SARS-CoV-2RESUMO
Introduction: The worldwide pandemic, coronavirus disease 2019 (COVID-19) is a novel infection with serious clinical manifestations, including death. Our aim is to describe the first non-ICU Spanish deceased series with COVID-19, comparing specifically between unexpected and expected deaths. Methods: In this single-centre study, all deceased inpatients with laboratory-confirmed COVID-19 who had died from March 4 to April 16, 2020 were consecutively included. Demographic, clinical, treatment, and laboratory data, were analyzed and compared between groups. Factors associated with unexpected death were identified by multivariable logistic regression methods. Results: In total, 324 deceased patients were included. Median age was 82 years (IQR 76-87); 55.9% males. The most common cardiovascular risk factors were hypertension (78.4%), hyperlipidemia (57.7%), and diabetes (34.3%). Other common comorbidities were chronic kidney disease (40.1%), chronic pulmonary disease (30.3%), active cancer (13%), and immunosuppression (13%). The Confusion, BUN, Respiratory Rate, Systolic BP and age ≥65 (CURB-65) score at admission was >2 in 40.7% of patients. During hospitalization, 77.8% of patients received antivirals, 43.3% systemic corticosteroids, and 22.2% full anticoagulation. The rate of bacterial co-infection was 5.5%, and 105 (32.4%) patients had an increased level of troponin I. The median time from initiation of therapy to death was 5 days (IQR 3.0-8.0). In 45 patients (13.9%), the death was exclusively attributed to COVID-19, and in 254 patients (78.4%), both COVID-19 and the clinical status before admission contributed to death. Progressive respiratory failure was the most frequent cause of death (92.0%). Twenty-five patients (7.7%) had an unexpected death. Factors independently associated with unexpected death were male sex, chronic kidney disease, insulin-treated diabetes, and functional independence. Conclusions: This case series provides in-depth characterization of hospitalized non-ICU COVID-19 patients who died in Madrid. Male sex, insulin-treated diabetes, chronic kidney disease, and independency for activities of daily living are predictors of unexpected death.
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BACKGROUND: Asthma is heterogeneous disease with different phenotypes, endotypes and severities. Definition of these subgroups requires the identification of biomarkers in biological samples, and serum proteomics is a useful and minimally invasive method for this purpose. Therefore, the aim of this study was to detect serum proteins whose abundance is distinctively associated with different asthma phenotypes (allergic vs nonallergic) or severities. METHODS: For each group of donors (32 healthy controls, 43 allergic rhinitis patients and 192 asthmatics with different phenotypes and severities), we generated two pools of sera that were analysed by a shotgun MS approach based on combinatorial peptide ligand libraries and iTRAQ-LC-MS/MS. RESULTS: MS analyses identified 18 proteins with a differential abundance. Functional/network study of these proteins identified key processes for asthma pathogenesis, such as complement activation, extracellular matrix organization, platelet activation and degranulation, or post-translational protein phosphorylation. Furthermore, our results highlighted an enrichment of the "Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs)" route in allergic asthma and the lectin pathway of complement activation in nonallergic asthma. Thus, several proteins (eg IGFALS, HSPG2, FCN2 or MASP1) displayed a differential abundance between the different groups of donors. Particularly, our results revealed IGFALS as a useful biomarker for moderate-severe allergic asthma. CONCLUSION: Our data suggest a set of serum biomarkers, especially IGFALS, capable of differentiating allergic from nonallergic asthma. These proteins reveal different pathophysiological mechanisms and may be useful in the future for diagnosis, prognosis or targeted therapy purposes.
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Asma , Proteômica , Asma/diagnóstico , Biomarcadores , Cromatografia Líquida , Humanos , Espectrometria de Massas em TandemRESUMO
CD26 displays variable levels between effector (TH17 â« TH1 > TH2 > Treg) and naïve/memory (memory > naïve) CD4+ T lymphocytes. Besides, IL-6/IL-6R is associated with TH17-differentiation and asthma severity. Allergic/atopic asthma (AA) is dominated by TH2 responses, while TH17 immunity might either modulate the TH2-dependent inflammation in AA or be an important mechanism boosting non-allergic asthma (NAA). Therefore, in this work we have compared the expression of CD26 and CD126 (IL-6Rα) in lymphocytes from different groups of donors: allergic (AA) and non-allergic (NAA) asthma, rhinitis, and healthy subjects. For this purpose, flow cytometry, haematological/biochemical, and in vitro proliferation assays were performed. Our results show a strong CD26-CD126 correlation and an over-representation of CD26- subsets with a highly-differentiated effector phenotype in AA (CD4+CD26-/low T cells) and NAA (CD4-CD26- γδ-T cells). In addition, we found that circulating levels of CD26 (sCD26) were reduced in both AA and NAA, while loss of CD126 expression on different leukocytes correlated with higher disease severity. Finally, selective inhibition of CD26-mRNA translation led to enhanced T cell proliferation in vitro. These findings support that CD26 down-modulation could play a role in facilitating the expansion of highly-differentiated effector T cell subsets in asthma.
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Asma/imunologia , Dipeptidil Peptidase 4/metabolismo , Receptores de Interleucina-6/metabolismo , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th2/imunologia , Adolescente , Adulto , Idoso , Asma/patologia , Estudos de Casos e Controles , Dipeptidil Peptidase 4/sangue , Dipeptidil Peptidase 4/genética , Feminino , Citometria de Fluxo , Humanos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Interleucina-6/genética , Índice de Gravidade de Doença , Adulto JovemRESUMO
Asthma is a heterogeneous and chronic inflammatory family of disorders of the airways with increasing prevalence that results in recurrent and reversible bronchial obstruction and expiratory airflow limitation. These diseases arise from the interaction between environmental and genetic factors, which collaborate to cause increased susceptibility and severity. Many asthma susceptibility genes are linked to the immune system or encode enzymes like metalloproteases (e.g., ADAM-33) or serine proteases. The S9 family of serine proteases (prolyl oligopeptidases) is capable to process peptide bonds adjacent to proline, a kind of cleavage-resistant peptide bonds present in many growth factors, chemokines or cytokines that are important for asthma. Curiously, two serine proteases within the S9 family encoded by genes located on chromosome 2 appear to have a role in asthma: CD26/dipeptidyl peptidase 4 (DPP4) and DPP10. The aim of this review is to summarize the current knowledge about CD26 and to provide a structured overview of the numerous functions and implications that this versatile enzyme could have in this disease, especially after the detection of some secondary effects (e.g., viral nasopharyngitis) in type II diabetes mellitus patients (a subset with a certain risk of developing obesity-related asthma) upon CD26 inhibitory therapy.
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Asma/etiologia , Asma/metabolismo , Dipeptidil Peptidase 4/metabolismo , Animais , Asma/diagnóstico , Asma/terapia , Biomarcadores , Líquidos Corporais/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/genética , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Família Multigênica , Fenótipo , Prolil Oligopeptidases , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Índice de Gravidade de Doença , Relação Estrutura-Atividade , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
A major obstacle hampering the therapeutic application of regulatory T (Treg) cells is the lack of suitable extracellular markers, which complicates their identification/isolation. Treg cells are normally isolated via CD25 (IL-2Rα) targeting, but this protein is also expressed by activated CD4(+) effector T (Teff) lymphocytes. Other extracellular (positive or negative) Treg selection markers (e.g., HLA-DR, CD127) are also nonspecific. CD26 is an extracellular peptidase whose high expression has been traditionally used as an indicator of immune activation and effector functions in T cells. Now, we provide flow cytometry data showing high levels of CD26 within CD4(+)CD25(-) or CD4(+)FoxP3(-/low) effector T (Teff) lymphocytes, but negative or low levels (CD26(-/low)) in Treg cells selected according to the CD4(+)CD25(high) or the CD4(+)FoxP3(high) phenotype. Unlike the negative marker CD127 (IL-7Rα), which is down modulated in CD4(+) Teff lymphocytes after TCR triggering, most of these cells upregulate CD26 and take a CD4(+)CD25(+/high) CD26(+) phenotype upon activation. In contrast, there is only a slight upregulation within Treg cells (CD4(+)CD25(high) CD26(-/low)). Thus, differences in CD26 levels between Treg and Teff subsets are stable, and assessment of this marker, in combination with others like CD25, FoxP3, or CD127, may be useful during the quantitative evaluation or the isolation of Treg cells in samples containing activated Teff lymphocytes (e.g., from patients with autoimmune/inflammatory diseases).
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Dipeptidil Peptidase 4/imunologia , Epitopos de Linfócito T/imunologia , Imunofenotipagem/métodos , Linfócitos T Reguladores/metabolismo , Biomarcadores/metabolismo , Dipeptidil Peptidase 4/genética , Epitopos de Linfócito T/genética , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologiaRESUMO
OBJECTIVE: To determine the epidemiological features, course, and outcomes of critically ill pediatric patients with Influenza A (H1N1) virus. DESIGN: Prospective cohort of children in pediatric intensive care units (PICUs) due to Influenza A (H1N1) virus infection. SETTING: Seventeen medical-surgical PICUs in tertiary care hospital in Argentina. PATIENTS: All consecutive patients admitted to the PICUs with influenza A (H1N1) viral infection from 15 June to 31 July 2009. MEASUREMENTS AND MAIN RESULTS: Of 437 patients with acute lower respiratory infection in PICUs, 147 (34%) were diagnosed with influenza A (H1N1) related to critical illness. The median age of these patients was 10 months (IQR 3-59). Invasive mechanical ventilation was used in 117 (84%) on admission. The rate of acute respiratory distress syndrome (ARDS) was 80% (118 of 147 patients). Initial non-invasive ventilation failed in 19 of 22 attempts (86%). Mortality at 28 days was 39% (n = 57). Chronic complex conditions (CCCs), acute renal dysfunction (ARD) and ratio PaO(2)/FiO(2) at day 3 on MV were independently associated with a higher risk of mortality. The odds ratio (OR) for CCCs was 3.06, (CI 95% 1.36-6.84); OR for ARD, 3.38, (CI 95% 1.45-10.33); OR for PaO(2)/FiO(2), 4 (CI 95% 1.57-9.59). The administration of oseltamivir within 24 h after admission had a protective effect: OR 0.2 (CI 95% 0.07-0.54). CONCLUSIONS: In children with ARDS, H1N1 as an etiologic agent confers high mortality, and the presence of CCCs in such patients increases the risk of death.
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Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana , Unidades de Terapia Intensiva Neonatal , Argentina/epidemiologia , Estudos de Coortes , Estado Terminal/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Influenza Humana/diagnóstico , Influenza Humana/tratamento farmacológico , Influenza Humana/epidemiologia , Influenza Humana/fisiopatologia , Masculino , Estudos Prospectivos , Análise de Sobrevida , Resultado do TratamentoRESUMO
Serum is a typical sample for non-invasive studies in clinical research. Its proteome characterization is challenging, since requires extensive protein depletion. Methods used nowadays for removal of high-abundance proteins are expensive or show quite often a low loading capacity, which has strong repercussions on the number of samples and replicates per analysis. In order to deplete immunoglobulins (Igs) and albumin (HSA) from 1 mL serum samples, we have developed a protocol based on a combination of thiophilic chromatography, not previously used in clinical proteomics, and a HSA-specific resin. Ig/HSA-depleted samples, immunoglobulinome and albuminone were analyzed by 2-DE. Thiophilic chromatography, coupled with HSA-depletion, allows a good 2-DE resolution as well as the visualization of new spots. Moreover, it yields enough protein to evaluate technical variability and facilitate subsequent protein identification. To validate the protocol, we carried out a preliminary comparative study between triplicate Igs/HSA-depleted serum samples from healthy control individuals and recently diagnosed/untreated rheumatoid arthritis (RA) patients. RA patients showed several acute phase proteins, as well as additional serum proteins, differentially and significantly regulated. Therefore, thiophilic chromatography can be used as an efficient and economical method in 2-DE to deplete immunoglobulins from large human serum samples before a more extensive fractioning.
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Proteínas Sanguíneas/química , Eletroforese em Gel Bidimensional/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Compostos de Sulfidrila/química , Artrite Reumatoide/diagnóstico , Proteínas Sanguíneas/análise , Cromatografia em Gel , Humanos , Imunoglobulinas/isolamento & purificação , Albumina Sérica/isolamento & purificação , Tripsina/metabolismoRESUMO
According to some authors, membrane compartmentalization is a key regulator of CD45 function. Indeed, it has been described that CD45 repositioning from raft microdomains to phospholipid-rich plasma membrane areas leads to the activation of extracellular signal-regulated kinases (ERKs). We have previously shown that interleukin-12 (IL-12) increases the expression of CD26, promoting the interaction of CD26 with CD45R0 (a CD45 isoform) and removing CD45R0 from lipid rafts. Thus, this IL-12-dependent removal of CD45RO from rafts could, hypothetically, fulfill functions like the activation of the ERK1/2 pathway. IL-12 is an important interleukin for T cells. Upon interaction with its receptor (interleukin-12 receptor; IL-12R), this cytokine triggers a signalling cascade, where the classical Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway and other additional routes participate. Due to the promitogenic effect of IL-12 and the influence of this cytokine on CD45RO compartmentalization, ERK kinases were likely candidates to be downstream of IL-12R. However, several research groups have rejected a role for these kinases. Now, results in this paper show that the IL-12R binding, similar to the stimulation via T cell receptor (TCR), promotes the activation of the Raf/MEK-1/ERK1/2 pathway. In addition, the IL-12R-associated Janus kinase JAK2, but not TYK2, seems upstream of this important pathway for the proliferation of human T cells. However, even though c-Myc is slightly up-regulated by IL-12 and partially mediates the proliferative effect of IL-12, this transcription factor was not found downstream of ERK1/2.
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Interleucina-12/metabolismo , Janus Quinase 2/metabolismo , Subunidades Proteicas/metabolismo , Receptores de Interleucina-12/metabolismo , Linfócitos T/metabolismo , Proliferação de Células , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Interleucina-12/imunologia , Janus Quinase 2/imunologia , Antígenos Comuns de Leucócito , Ativação Linfocitária/efeitos dos fármacos , Fito-Hemaglutininas/imunologia , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/imunologia , Transdução de Sinais/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Tirfostinas/farmacologiaRESUMO
IL-12 is a cytokine that stimulates the expression of CD26, a T cell- and raft-associated ectopeptidase. IL-12 also enhances the interaction between CD26 and CD45RO, which removes the phosphatase CD45RO from raft microdomains. Since Janus kinases are known CD45 substrates, our hypothesis was that this relocation of CD45RO in nonraft areas of the membrane could be important to switch off the signaling via cytokine receptors, e.g., the IL-12 receptor (IL-12R). Accordingly, both IL-12R and CD45RO should be equally positioned in the cell membrane upon IL-12R ligation. However, there were no data available on the membrane distribution of IL-12R on human T cells. Working with phytohemagglutinin (PHA) lymphoblasts, we tried to fill that gap. The high-affinity IL-12R is made of two chains: IL-12Rbeta1 and IL-12Rbeta2. Using flow cytometry, Western blot and confocal microscopy, we obtained data suggesting that IL-12Rbeta1 is mainly associated to phospholipid-rich membrane areas, a location even enhanced upon IL-12 incubation of PHA blasts. Instead, IL-12Rbeta2 is found more segregated into membrane rafts, which could explain why two IL-12-triggered events, T-cell proliferation and ERK1/2 activation, are both methyl-beta-cyclodextrin-sensitive events. Ligation of IL-12R with IL-12 seems to induce a partial enrichment of IL-12Rbeta2 in phospholipid-rich areas, where according to our data IL-12Rbeta1 is already present. Therefore, although new data will be required, the present results support the initial hypothesis.