Normalization of deviance in pediatric hospital: perception of health workers.

OBJECTIVE: To know the perception of health workers about the phenomenon of normalization of deviance in a pediatric hospital. METHOD: Exploratory, descriptive, and qualitative study conducted in a public pediatric hospital in northeastern Brazil in 2021. An in-depth interview was applied to 21 health workers, submitted to Thematic Categorical Content Analysis in the MAXQDA® Software. RESULTS: 128 context units emerged from the content analysis. These data were presented in three analytical categories, which address conceptions about normalization of deviance, examples and contributing factors. The omission of the practice of hand hygiene and the correct use of personal protective equipment,and turning off alarms stand out as the main deviance perceived by health workers. As contributing factors, human factors and organizational factors prevailed. CONCLUSION: Workers perceive the normalization of deviance as negligence, recklessness, and violations of good practices, with consequences for patient safety.

Normalization of deviance in pediatric hospital: perception of health workers / Normalización de la desviación en hospital pediátrico: percepción de trabajadores de salud / Normalização do desvio em hospital pediátrico: percepção de trabalhadores de saúde

ABSTRACT Objective: To know the perception of health workers about the phenomenon of normalization of deviance in a pediatric hospital. Method: Exploratory, descriptive, and qualitative study conducted in a public pediatric hospital in northeastern Brazil in 2021. An in-depth interview was applied to 21 health workers, submitted to Thematic Categorical Content Analysis in the MAXQDA® Software. Results: 128 context units emerged from the content analysis. These data were presented in three analytical categories, which address conceptions about normalization of deviance, examples and contributing factors. The omission of the practice of hand hygiene and the correct use of personal protective equipment,and turning off alarms stand out as the main deviance perceived by health workers. As contributing factors, human factors and organizational factors prevailed. Conclusion: Workers perceive the normalization of deviance as negligence, recklessness, and violations of good practices, with consequences for patient safety.

RESUMEN Objetivo: Conocer la percepción de los trabajadores de la salud sobre el fenómeno de la normalización de la desviación en un hospital pediátrico. Método: Estudio exploratorio, descriptivo y cualitativo realizado en un hospital pediátrico público en el noreste de Brasil en 2021. Se aplicó una entrevista en profundidad a 21 trabajadores de la salud, sometidos al Análisis de Contenido Categórico Temático en el Software MAXQDA®. Resultados: 128 unidades de contexto surgieron del análisis de contenido. Estos datos se presentaron en tres categorías analíticas, que abarcan conceptos sobre la normalización de la desviación, ejemplos y factores contribuyentes. La omisión de la práctica de la higiene de manos y el uso correcto de los equipos de protección personal y el apagado de alarmas se destacan como las principales desviaciones percibidas por los trabajadores de la salud. Como factores contribuyentes prevalecieron los factores humanos y los factores organizacionales. Conclusión: Los trabajadores perciben la normalización de la desviación como negligencia, imprudencia y violación de las buenas prácticas, con consecuencias para la seguridad del paciente.

RESUMO Objetivo: Conhecer a percepção de trabalhadores de saúde sobre o fenômeno da normalização do desvio em um hospital pediátrico. Método: Estudo exploratório, descritivo e qualitativo realizado em hospital público pediátrico do nordeste brasileiro em 2021. Aplicou-se entrevista em profundidade a 21 trabalhadores de saúde, submetida à Análise de Conteúdo Categorial Temática no Software MAXQDA®. Resultados: Emergiram 128 unidades de contexto da análise de conteúdo. Esses dados foram apresentados em três categorias analíticas, as quais abordam concepções sobre normalização do desvio, exemplos e fatores contribuintes. Destacam-se a omissão da prática de higienização das mãos e do uso correto dos equipamentos de proteção individual, e o desligamento de alarmes como principais desvios percebidos pelos trabalhadores de saúde. Como fatores contribuintes, preponderaram os fatores humanos e os fatores organizacionais. Conclusão: Os trabalhadores percebem a normalização do desvio como negligência, imprudência e violações de boas práticas, com consequências para a segurança do paciente.

A Brazilian Inter-Hospital Candidemia Outbreak Caused by Fluconazole-Resistant Candida parapsilosis in the COVID-19 Era.

Horizontal transmission of fluconazole-resistant Candida parapsilosis (FRCP) through healthcare workers' hands has contributed to the occurrence of candidemia outbreaks worldwide. Since the first COVID-19 case in Brazil was detected in early 2020, hospitals have reinforced hand hygiene and disinfection practices to minimize SARS-CoV-2 contamination. However, a Brazilian cardiology center, which shares ICU patients with a cancer center under a FRCP outbreak since 2019, reported an increased FRCP candidemia incidence in May 2020. Therefore, the purpose of this study was to investigate an inter-hospital candidemia outbreak caused by FRCP isolates during the first year of the COVID-19 pandemic in Brazil. C. parapsilosis bloodstream isolates obtained from the cancer (n = 35) and cardiology (n = 30) centers in 2020 were submitted to microsatellite genotyping and fluconazole susceptibility testing. The ERG11 gene of all isolates from the cardiology center was sequenced and compared to the corresponding sequences of the FRCP genotype responsible for the cancer center outbreak in 2019. Unprecedentedly, most of the FRCP isolates from the cardiology center presented the same genetic profile and Erg11-Y132F mutation detected in the strain that has been causing the persistent outbreak in the cancer center, highlighting the uninterrupted horizontal transmission of clonal isolates in our hospitals during the COVID-19 pandemic.

Environmental Clonal Spread of Azole-Resistant Candida parapsilosis with Erg11-Y132F Mutation Causing a Large Candidemia Outbreak in a Brazilian Cancer Referral Center.

Clonal outbreaks due to azole-resistant Candida parapsilosis (ARCP) isolates have been reported in numerous studies, but the environmental niche of such isolates has yet to be defined. Herein, we aimed to identify the environmental niche of ARCP isolates causing unremitting clonal outbreaks in an adult ICU from a Brazilian cancer referral center. C. parapsilosis sensu stricto isolates recovered from blood cultures, pericatheter skins, healthcare workers (HCW), and nosocomial surfaces were genotyped by multilocus microsatellite typing (MLMT). Antifungal susceptibility testing was performed by the EUCAST (European Committee for Antimicrobial Susceptibility Testing) broth microdilution reference method and ERG11 was sequenced to determine the azole resistance mechanism. Approximately 68% of isolates were fluconazole-resistant (76/112), including pericatheter skins (3/3, 100%), blood cultures (63/70, 90%), nosocomial surfaces (6/11, 54.5%), and HCW's hands (4/28, 14.2%). MLMT revealed five clusters: the major cluster contained 88.2% of ARCP isolates (67/76) collected from blood (57/70), bed (2/2), pericatheter skin (2/3), from carts (3/7), and HCW's hands (3/27). ARCP isolates were associated with a higher 30 day crude mortality rate (63.8%) than non-ARCP ones (20%, p = 0.008), and resisted two environmental decontamination attempts using quaternary ammonium. This study for the first time identified ARCP isolates harboring the Erg11-Y132F mutation from nosocomial surfaces and HCW's hands, which were genetically identical to ARCP blood isolates. Therefore, it is likely that persisting clonal outbreak due to ARCP isolates was fueled by environmental sources. The resistance of Y132F ARCP isolates to disinfectants, and their potential association with a high mortality rate, warrant vigilant source control using effective environmental decontamination.

Medo de Covid-19 e fatores associados ao possível bruxismo do sono e/ou em vigília entre universitários da região sudeste do Brasil durante a pandemia Covid-19 / Fear of Covid-19 and factors associated with sleep and/or wakefulness bruxism among university students in southeastern Brazil during the Covid-19 pandemic

Introdução: O possível bruxismo é um comportamento autorrelatado, caracterizado pelo ranger e/ou apertar dos dentes, estando associado a fatores psicológicos. A pandemia da COVID-19 tem afetado o emocional das pessoas, podendo ser associada a este comportamento. Objetivo: Analisar a associação entre o nível de medo da COVID-19, assim como outros fatores associados à prevalência de possível bruxismo entre universitários. Materiais e métodos: Participaram deste estudo transversal 311 universitários de graduação e pós-graduação de faculdades odontológicas públicas e privadas da região sudeste do Brasil. Os participantes foram contatados via WhatsApp e redes sociais através de amostragem em bola de neve para responder um questionário com avaliação sociodemográfica, presença, frequência e severidade de possível bruxismo do sono (PBS) e/ou em vigília (PBV) e a versão brasileira da fear scale of COVID-19. Análises descritivas e bivariadas foram realizadas através dos testes estatísticos de Mann-Whitney e Kruskall-Wallis (p<0,05). Resultados: A prevalência de PBS grave foi de 12,5% e PBV 23,8%. Não houve associação entre escores de medo COVID-19 nem com PBS (p=0,342) nem com PBV (p=0,912). Quando analisado o PBV, observou-se maior prevalência dentre os participantes que não trabalhavam (p=0,008), sendo maior percentual entre os universitários na segunda metade do curso (p=0,021) e, dentre os participantes da pós-graduação, o bruxismo foi mais prevalente entre alunos matriculados em programas stricto sensu comparados àqueles de programas lato sensu (p=0,036). Conclusão: Estar fora do mercado de trabalho, a fase final da graduação e a inserção em uma pós-graduação stricto sensu influenciaram na prevalência de PBV.

Introduction: The possible bruxism is a self-reported behavior characterized by grinding and/or clenching teeth and is associated with psychological factors. The COVID-19 pandemic has affected people's emotions and can be associated with this behavior. Objective: Analyze the association between level of fear of COVID-19, as well as other factors associated to the prevalence of possible bruxism among university students. Materials and methods: A total of 311 undergraduate and graduate students from public and private dental schools in Southeastern Brazil participated in this cross-sectional study. Participants were contacted via WhatsApp and social networks through snowball sampling to answer an online questionnaire with sociodemographic assessment, presence, frequency and severity of possible sleep bruxism (PSB) and/or awake bruxism (PAB) and Brazilian version of the fear scale of COVID-19. Descriptive and bivariate analyzes were performed using the Mann-Whitney and Kruskall-Wallis statistical tests (p<0.05). Results: The prevalence of severe PSB was 12.5% and PAB 23.8%. There was no association between COVID-19 fear scores or with PSB (p=0.342) or with PAB (p=0.912). When analyzing the PAB, there was a higher prevalence among participants who didn't work (p=0.008), with a higher percentage among university students in the second half of the course (p=0.021). Among graduate participants, possible bruxism was more prevalent among students enrolled in stricto sensu programs compared to those in lato sensu programs (p=0.036). There wasn't association between COVID-19 fear score and possible bruxism. Conclusion: Being out of the job market, final stage of graduation and entry into a stricto sensu graduate program influenced the prevalence of PAB.

Lack of IL-1 Receptor-Associated Kinase-4 Leads to Defective Th1 Cell Responses and Renders Mice Susceptible to Mycobacterial Infection.

The Toll-like and IL-1 family receptors play critical roles in innate and adaptive immunity against intracellular pathogens. Although previous data demonstrated the importance of TLRs and IL-1R signaling events for the establishment of an effective immune response to mycobacteria, the possible function of the adaptor molecule IL-1R-associated kinase (IRAK)-4 against this pathogen has not been addressed. In this study, we determined the role of IRAK-4 in signaling pathways responsible for controlling mycobacterial infections. This kinase is important for the production of IL-12 and TNF-α by macrophages and dendritic cells exposed to mycobacteria. Moreover, Mycobacterium bovis-infected IRAK-4-knockout macrophages displayed impaired MAPK and NF-κB activation. IL-1ß secretion and caspase-1 activation were also dependent on IRAK-4 signaling. Mice lacking IRAK-4 showed increased M. bovis burden in spleen, liver, and lungs and smaller liver granulomas during 60 d of infection compared with wild-type mice. Furthermore, 80% of IRAK-4(-/-) mice succumbed to virulent M. tuberculosis within 100 d following low-dose infection. This increased susceptibility to mycobacteria correlated with reduced IFN-γ/TNF-α recall responses by splenocytes, as well as fewer IL-12p70-producing APCs. Additionally, we observed that IRAK-4 is also important for the production of IFN-γ by CD4(+) T cells from infected mice. Finally, THP-1 cells treated with an IRAK-4 inhibitor and exposed to M. bovis showed reduced TNF-α and IL-12, suggesting that the results found in mice can be extended to humans. In summary, these data demonstrate that IRAK-4 is essential for innate and adaptive immunity and necessary for efficient control of mycobacterial infections.

O processo de formação didático pedagógica em saúde: aprendizagens percebidas na voz dos pós-graduandos / The process of didatic-pedagogical training in health: learning perceived in the voice of postgraduate students

INTRODUÇÃO: O presente estudo integra o projeto "Formação de professor: estratégia dialógica problematizadora com uso de ambiente virtual de aprendizagem", desenvolvido na disciplina de Formação Didático Pedagógica em Saúde (FDPS), ofertada pelo Centro de Desenvolvimento do Ensino Superior em Saúde (CEDESS) da Universidade Federal de São Paulo (UNIFESP), no formato bimodal (presencial e a distância). OBJETIVO: Analisar as aprendizagens construídas pelos pós-graduandos no processo de formação docente ao longo da disciplina FDPS. MÉTODOS: Os sujeitos da pesquisa foram onze profissionais de diversas áreas da saúde. Para a coleta de dados, foi solicitada a cada pós-graduando, antes do início da disciplina, uma carta de apresentação, manifestando as suas expectativas e, ao término da disciplina, uma autoavaliação, sobre as aprendizagens construídas durante o processo. A análise temática partiu dos seguintes núcleos orientadores: expectativas Iniciais, aprendizagens construídas e perspectivas futuras na docência. RESULTADOS: A maioria dos participantes manifestou a intenção de conhecer técnicas de transmissão de conteúdos com centralidade no docente, em menor proporção também foi expresso o desejo de compartilhar informações e refletir sobre o cenário da educação e da saúde no país. No núcleo, aprendizagens construídas, os pós-graduandos valorizaram as estratégias utilizadas no decorrer da disciplina, destacando a construção e apresentação do planejamento educacional em grupos multiprofissionais. Nas perspectivas futuras, muitos demonstraram o reconhecimento de continuar sua formação docente para melhor exercício da profissão. CONCLUSÃO: A educação permanente do processo de formação docente parece ser uma demanda crescente, o que valoriza essa atividade e sua profissionalização.

INTRODUCTION: This study is part of the project "Teacher training: problem-dialogical strategy with virtual environment use of learning" developed in the discipline Didatic-Pedagogical Training in Health (DPTH), offered by the Higher Education Development Center on Health (CEDESS) in the Federal University of São Paulo (UNIFESP), in bimodal format (presence and distance). OBJECTIVE: To analyze the learning built by graduate students in the teacher education process throughout DPTH discipline. METHODS: The subjects of the research were eleven professionals from different areas of health. For the data collect, was requested to every postgraduate student, before the start of the course, an introduction letter,expressing their expectations and, in the end of the discipline, a self-assessment of the learning built during the process. The thematic analysis came from the following guiding cores: initial expectations, learning built and future perspectives. RESULTS: The expectations had manifested the intention to know techniques and tools to transmit the contents with centrality in the teacher, but even in minor proportion, was also expressed a desire to share information and reflect the education and health scenario in the country. In the core learning built, the students appreciated the strategies used in the discipline, emphasizing the construction and presentation of an education planning in small multiprofessional groups. In the future perspectives, many had shown the recognition to continue their teacher training to a better practice of the profession. CONCLUSION: the continuing education of the teacher training process appears to be a growing demand, which values this activity and its professionalisation.

House dust mites induce proliferation of severe asthmatic smooth muscle cells via an epithelium-dependent pathway.

RATIONALE: Asthma is a frequent airway disease, and asthma control determinants have been associated with indoor allergen sensitization. The most frequent allergens are house dust mites (HDM), which act in vivo on the bronchial epithelial layer. Severe asthma has also been associated with bronchial remodeling and more specifically with increased mass of bronchial smooth muscle (BSM). However, the relationship between HDM stimulation of the bronchial epithelial layer and BSM remodeling is unknown. OBJECTIVES: To evaluate whether epithelial stimulation with HDM induces BSM cell proliferation in subjects with severe asthma. METHODS: A total of 22 subjects with severe asthma and 27 subjects with no asthma were recruited. We have developed an in vitro culture model combining an epithelium layer in air-liquid interface (ALI) interacting with BSM. We assessed BSM proliferation using BrdU incorporation. We explored the role of epithelium-derived mediators using reverse-transcriptase polymerase chain reaction (RT-PCR) and ELISA in vitro and in vivo. Finally, leukotrienes receptor expression was assessed in vitro by flow cytometry and RT-PCR and ex vivo by laser microdissection and RT-PCR. MEASUREMENTS AND MAIN RESULTS: We found that epithelial stimulation by HDM selectively increased the proliferation of asthmatic BSM cells and not that of nonasthmatic cells. The mechanism involved epithelial protease-activated receptor-2-dependent production of leukotrienes C4 associated with an overexpression of leukotrienes receptor CysLTR1 by asthmatic BSM cells in vitro and ex vivo. CONCLUSIONS: This work demonstrates the selective role of HDM on BSM remodeling in patients with severe asthma and points out different therapeutic targets at epithelial and smooth muscle levels.

Protease activated receptor-2 expression and function in asthmatic bronchial smooth muscle.

Asthmatic bronchial smooth muscle (BSM) is characterized by structural remodeling associated with mast cell infiltration displaying features of chronic degranulation. Mast cell-derived tryptase can activate protease activated receptor type-2 (PAR-2) of BSM cells. The aims of the present study were (i) to evaluate the expression of PAR-2 in both asthmatic and non asthmatic BSM cells and, (ii) to analyze the effect of prolonged stimulation of PAR-2 in asthmatic BSM cells on cell signaling and proliferation. BSM cells were obtained from both 33 control subjects and 22 asthmatic patients. PAR-2 expression was assessed by flow cytometry, western blot and quantitative RT-PCR. Calcium response, transduction pathways and proliferation were evaluated before and following PAR-2 stimulation by SLIGKV-NH2 or trypsin for 1 to 3 days. Asthmatic BSM cells expressed higher basal levels of functional PAR-2 compared to controls in terms of mRNA, protein expression and calcium response. When PAR-2 expression was increased by means of lentivirus in control BSM cells to a level similar to that of asthmatic cells, PAR-2-induced calcium response was then similar in both types of cell. However, repeated PAR-2 stimulations increased the proliferation of asthmatic BSM cells but not that of control BSM cells even following lentiviral over-expression of PAR-2. Such an increased proliferation was related to an increased phosphorylation of ERK in asthmatic BSM cells. In conclusion, we have demonstrated that asthmatic BSM cells express increased baseline levels of functional PAR-2. This higher basal level of PAR-2 accounts for the increased calcium response to PAR-2 stimulation, whereas the increased proliferation to repeated PAR-2 stimulation is related to increased ERK phosphorylation.

The endoplasmic reticulum acts as a platform for ubiquitylated components of nuclear factor κB signaling.

The innate and adaptive immune responses involve the stimulation of nuclear factor κB (NF-κB) transcription factors through the Lys(63) (K(63))-linked ubiquitylation of specific components of NF-κB signaling pathways. We found that ubiquitylated components of the NF-κB pathway accumulated on the cytosolic leaflet of the endoplasmic reticulum (ER) membrane after the engagement of cell-surface, proinflammatory cytokine receptors or antigen receptors. Through mass spectrometric analysis, we found that the ER-anchored protein metadherin (MTDH) was a partner for these ubiquitylated activators of NF-κB and that it directly bound to K(63)-linked polyubiquitin chains. Knockdown of MTDH inhibited the accumulation of ubiquitylated NF-κB signaling components at the ER, reduced the extent of NF-κB activation, and decreased the amount of proinflammatory cytokines produced. Our observations highlight an unexpected facet of the ER as a key subcellular gateway for NF-κB activation.

Ca(2+)-activated K(+) channel-3.1 blocker TRAM-34 attenuates airway remodeling and eosinophilia in a murine asthma model.

Key features of asthma include bronchial hyperresponsiveness (BHR), eosinophilic airway inflammation, and bronchial remodeling, characterized by subepithelial collagen deposition, airway fibrosis, and increased bronchial smooth muscle (BSM) mass. The calcium-activated K(+) channel K(Ca)3.1 is expressed by many cells implicated in the pathogenesis of asthma, and is involved in both inflammatory and remodeling responses in a number of tissues. The specific K(Ca)3.1 blocker 5-[(2-chlorophenyl)(diphenyl)methyl]-1H-pyrazole (TRAM-34) attenuates BSM cell proliferation, and both mast cell and fibrocyte recruitment in vitro. We aimed to examine the effects of K(Ca)3.1 blockade on BSM remodeling, airway inflammation, and BHR in a murine model of chronic asthma. BALB/c mice were sensitized with intraperitoneal ovalbumin (OVA) on Days 0 and 14, and then challenged with intranasal OVA during Days 14-75. OVA-sensitized/challenged mice received TRAM-34 (120 mg/kg/day, subcutaneous) from Days -7 to 75 (combined treatment), Days -7 to 20 (preventive treatment), or Days 21 to 75 (curative treatment). Untreated mice received daily injections of vehicle (n = 8 per group). Bronchial remodeling was assessed by histological and immunohistochemical analyses. Inflammation was evaluated using bronchoalveolar lavage and flow cytometry. We also determined BHR in both conscious and anesthetized mice via plethysmography. We demonstrated that curative treatment with TRAM-34 abolishes BSM remodeling and subbasement collagen deposition, and attenuates airway eosinophilia. Although curative treatment alone did not significantly reduce BHR, the combined treatment attenuated nonspecific BHR to methacholine. This study indicates that K(Ca)3.1 blockade could provide a new therapeutic strategy in asthma.

Role of YKL-40 in bronchial smooth muscle remodeling in asthma.

RATIONALE: Bronchial remodeling, including increased bronchial smooth muscle (BSM) mass, contributes to bronchial obstruction in asthma. However, its mechanisms are complex and remain controversial. Recently, a role of the chitinase 3-like 1 protein (YKL-40) has been evoked in asthma. Indeed, YKL-40 concentration was increased in asthmatic serum, and correlated with asthma severity and subepithelial membrane thickness. Nevertheless, the role of YKL-40 on BSM cells remains to be investigated. OBJECTIVES: To evaluate whether YKL-40 altered the physiologic properties of BSM cells in asthma in vitro and ex vivo. METHODS: We enrolled 40 subjects with asthma, 13 nonsmokers, and 16 smokers. BSM cells were derived from bronchial specimens obtained by either fiberoptic bronchoscopy or lobectomy. We assessed cell proliferation using BrdU, flow cytometry, and cell count; signaling intermediates using Western blot; cell migration using inserts, wound healing, and phalloidin staining; and cell synthesis using ELISA and Western blot. The involvement of protease activated receptor (PAR)-2 was evaluated using blocking antibody and dedicated lentiviral small hairpin RNA. We also determined the BSM area and the YKL-40 staining ex vivo using immunohistochemistry on biopsies from subjects with asthma and control subjects. MEASUREMENTS AND MAIN RESULTS: We demonstrated that YKL-40 increased BSM cell proliferation and migration through PAR-2-, AKT-, ERK-, and p38-dependent mechanisms. The increased cell migration was higher in BSM cells of subjects with asthma than that of control subjects. Furthermore, YKL-40 epithelial expression was positively correlated with BSM mass in asthma. CONCLUSIONS: This study indicates that YKL-40 promotes BSM cell proliferation and migration through a PAR-2-dependent mechanism.

Participation of the cell polarity protein PALS1 to T-cell receptor-mediated NF-κB activation.

BACKGROUND: Beside their established function in shaping cell architecture, some cell polarity proteins were proposed to participate to lymphocyte migration, homing, scanning, as well as activation following antigen receptor stimulation. Although PALS1 is a central component of the cell polarity network, its expression and function in lymphocytes remains unknown. Here we investigated whether PALS1 is present in T cells and whether it contributes to T Cell-Receptor (TCR)-mediated activation. METHODOLOGY/PRINCIPAL FINDINGS: By combining RT-PCR and immunoblot assays, we found that PALS1 is constitutively expressed in human T lymphocytes as well as in Jurkat T cells. siRNA-based knockdown of PALS1 hampered TCR-induced activation and optimal proliferation of lymphocyte. We further provide evidence that PALS1 depletion selectively hindered TCR-driven activation of the transcription factor NF-κB. CONCLUSIONS: The cell polarity protein PALS1 is expressed in T lymphocytes and participates to the optimal activation of NF-κB following TCR stimulation.

Interplay between BCL10, MALT1 and IkappaBalpha during T-cell-receptor-mediated NFkappaB activation.

T-cell-receptor (TCR) signalling to NFkappaB requires the assembly of a large multiprotein complex containing the serine/threonine kinase CK1alpha, the scaffold protein CARMA1, the heterodimer BCL10-MALT1 (the CBM complex) and the IkappaB kinase complex (IKK). Although the mechanisms regulating recruitment and activation of IKK within the CBM microenvironment have been extensively studied, there is little understanding of how IKK subsequently binds and phosphorylates IkappaBalpha, the inhibitor of NFkappaB, to promote IkappaBalpha ubiquitylation and proteasomal degradation. Here, we show that BCL10, MALT1 and IKK inducibly associate with IkappaBalpha in a complex that is physically distinct from the early CK1alpha-CBM signalosome. This IkappaBalpha-containing complex probably maturates from the CBM, because siRNA-based knockdown of CARMA1, CK1alpha and BCL10 hampered its assembly, leading to a reduction in NFkappaB activation. By contrast, CK1alpha normally recruited both BCL10 and ubiquitylated species of MALT1 when IkappaBalpha levels were reduced. However, knockdown of IkappaBalpha led to an altered ubiquitylation profile of BCL10-MALT1 combined with a defect in MALT1 reorganisation within large cytoplasmic structures, suggesting that, following stimulation, IkappaBalpha might also participate in MALT1 recycling. Altogether, our data suggest a two-step mechanism to connect active IKK to IkappaBalpha, and further unveil a potential role for IkappaBalpha in resetting TCR-mediated signalling.

The adaptor protein CIKS/Act1 is essential for IL-25-mediated allergic airway inflammation.

IL-17 is the signature cytokine of recently discovered Th type 17 (Th17) cells, which are prominent in defense against extracellular bacteria and fungi as well as in autoimmune diseases, such as rheumatoid arthritis and experimental autoimmune encephalomyelitis in animal models. IL-25 is a member of the IL-17 family of cytokines, but has been associated with Th2 responses instead and may negatively cross-regulate Th17/IL-17 responses. IL-25 can initiate an allergic asthma-like inflammation in the airways, which includes recruitment of eosinophils, mucus hypersecretion, Th2 cytokine production, and airways hyperreactivity. We demonstrate that these effects of IL-25 are entirely dependent on the adaptor protein CIKS (also known as Act1). Surprisingly, this adaptor is necessary to transmit IL-17 signals as well, despite the very distinct biologic responses that these two cytokines elicit. We identify CD11c(+) macrophage-like lung cells as physiologic relevant targets of IL-25 in vivo.

Differentiating megakaryocytes in myelodysplastic syndromes succumb to mitochondrial derangement without caspase activation.

Myelodysplastic syndromes (MDS) constitute a preneoplastic condition in which potentially malignant cancer stem cells continuously die during differentiation. This MDS-associated cell death often involves caspase-3 activation, yet can also occur without caspase activation, for instance in differentiating megakaryocytes (MK). We investigated, the mechanisms through which MK from MDS patients undergo premature cell death. While polyploid, mature MK from healthy subjects or MDS patients manifested caspase-3 activation during terminal differentiation, freshly isolated, immature MK from MDS died without caspase-3 activation. Similarly, purified bone marrow CD34(+) cells from MDS patients that were driven into MK differentiation in vitro died without caspase-3 activation at an immature stage, before polyploidization. The premature death of MDS MK was accompanied by the mitochondrial release of cytochrome c, Smac/DIABLO and endonuclease G, a caspase-independent death effector, as well loss of the mitochondrial membrane potential and plasma membrane phosphatidylserine exposure before definitive loss of viability. Thus, a stereotyped pattern of mitochondrial alterations accompanies differentiation-associated MK death in MDS.

Enrichment of non-synchronized cells in the G1, S and G2 phases of the cell cycle for the study of apoptosis.

The susceptibility of cells to apoptosis induction is deeply influenced by their position in the cell cycle. Unfortunately, however, current methods for the enrichment of cells in defined phases of the cell cycle are mostly based on the synchronization of cells by agents or conditions that are intrinsically toxic and induce apoptosis on their own. We developed a novel procedure for the purification of cells in distinct phases of the cell cycle. This method is based on the stable transfection of cells with a chimeric protein made up by histone H2B and green fluorescent protein (GFP). Cytofluorometric purification of cells defined by their size and their H2B-GFP-dependent fluorescence (which reflects chromatin and hence DNA content) allowed for the efficient separation of diploid and tetraploid cells in the fluorescence-activated cell sorter (FACS). Moreover, when applied to diploid cells, this method allowed for the enrichment of live, functional cells in the G1, S and G2 phases of the cell cycle. FACS-purified cells were viable and readily resumed the cell cycle upon reculture. While staurosporine was equally toxic for cells in any phase of the cell cycle, camptothecin was particularly toxic for cells in the S phase. Moreover, BAY11-7082, a specific inhibitor of the IKK complex required for NF-kappaB activation, exhibited a particular cell cycle-specific profile of toxicity (G2>S>G1). These results delineate a novel procedure for studying the intersection between cell cycle regulation and cell death mechanisms.

NF-kappaB constitutes a potential therapeutic target in high-risk myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a preneoplastic condition that frequently develops into overt acute myeloid leukemia (AML). The P39 MDS/AML cell line manifested constitutive NF-kappaB activation. In this cell line, NF-kappaB inhibition by small interfering RNAs specific for p65 or chemical inhibitors including bortezomib resulted in the down-regulation of apoptosis-inhibitory NF-kappaB target genes and subsequent cell death accompanied by loss of mitochondrial transmembrane potential as well as by the mitochondrial release of the caspase activator cytochrome c and the caspase-independent death effectors endonuclease G and apoptosis-inducing factor (AIF). Bone marrow cells from high-risk MDS patients also exhibited constitutive NF-kappaB activation similar to bone marrow samples from MDS/AML patients. Purified hematopoietic stem cells (CD34+) and immature myeloid cells (CD33+) from high-risk MDS patients demonstrated the nuclear translocation of the p65 NF-kappaB subunit. The frequency of cells with nuclear p65 correlated with blast counts, apoptosis suppression, and disease progression. NF-kappaB activation was confined to those cells that carried MDS-associated cytogenetic alterations. Since NF-kappaB inhibition induced rapid apoptosis of bone marrow cells from high-risk MDS patients, we postulate that NF-kappaB activation is responsible for the progressive suppression of apoptosis affecting differentiating MDS cells and thus contributes to malignant transformation. NF-kappaB inhibition may constitute a novel therapeutic strategy if apoptosis induction of MDS stem cells is the goal.