RESUMO
INTRODUCTION/OBJECTIVES: Non- albicans Candida species such as Candida parapsilosis and Candida glabrata have emerged as prevalent pathogens in premature infants. The aim of this study was to systematically delineate the histopathologic findings in neonatal non- albicans candidiasis. METHODS: We performed a retrospective clinicopathologic analysis of extremely premature (23-28 weeks' gestation) infants diagnosed with invasive candidiasis. Archival autopsy tissues were subjected to periodic acid-Schiff, methenamine-silver and anti- Candida (immuno)histochemical stains, as well as dual anti- Candida and anti-cytokeratin or anti-CD31 immunofluorescence assays. In addition, we studied the prevalence of intestinal Candida colonization in a consecutive autopsy series of extremely premature infants. RESULTS: Based on positive postmortem blood and/or lung cultures, invasive candidiasis (3 non- albicans and 11 Candida albicans) was diagnosed in 14 of the 187 extremely premature infants examined between 1995 and 2017. In contrast to the well-known inflammatory and tissue-destructive phenotype of congenital C. albicans infection, invasive non- albicans candidiasis/candidemia caused by C. parapsilosis and C. glabrata was inconspicuous by routine hematoxylin-eosin-based histopathologic analysis despite a heavy fungal presence detected in intestines, lungs, and blood by targeted (immuno)histochemical assays. Intestinal colonization by Candida species was identified in 16 of the 26 (61%) extremely premature neonates who had lived for at least 1 week, as assessed by anti- Candida immunostaining. CONCLUSION: Invasive neonatal non- albicans candidiasis/candidemia appears to have no distinct histopathologic signature. Based on the notoriously low sensitivity of fungal blood cultures and the observed high frequency of Candida intestinal colonization (>50%), it is likely that non- albicans candidiasis/candidemia may be underdiagnosed in (deceased) preterm infants. Routine inclusion of targeted (immuno)histochemical fungal detection strategies in the perinatal autopsy may lead to deeper insight into the prevalence and clinical relevance of neonatal non- albicans candidiasis.
Assuntos
Candida glabrata/isolamento & purificação , Candida parapsilosis/isolamento & purificação , Candidíase Invasiva/patologia , Lactente Extremamente Prematuro , Doenças do Prematuro/patologia , Candidíase Invasiva/diagnóstico , Candidíase Invasiva/microbiologia , Humanos , Recém-Nascido , Doenças do Prematuro/diagnóstico , Doenças do Prematuro/microbiologia , Estudos RetrospectivosRESUMO
The yeast Candida parapsilosis is an increasingly common cause of systemic fungal infections among immunocompromised individuals, including premature infants. Adhesion to host surfaces is an important step in pathogenesis, but this process has not been extensively studied in this organism. A microfluidics assay was developed to test the ability of C. parapsilosis to adhere to immobilized host extracellular matrix proteins under physiological fluid shear conditions. Growth in mammalian tissue culture medium at 37°C for 3 to 6 h led to the induction of an adhesive phenotype at shear forces of 1 to 5 dynes/cm2 in some isolates of C. parapsilosis Glutamic acid, proline, and calcium appeared to be the minimally necessary requirements for increased adhesion in these assays. To determine whether genes homologous to the ALS gene family of C. albicans were important for the adhesive phenotype, the expression levels of 5 homologous C. parapsilosis genes were quantified by using quantitative PCR (qPCR) under conditions leading to increased adhesion. CPAR2_404800 (CpALS7) and CPAR2_404780 showed increased expression levels compared to those in control yeast. The extent of adhesion was variable among different isolates, and linear regression identified the expression of CpALS7 but not CPAR2_404780 as having a strong positive correlation with adhesion. A homozygous CpALS7 deletion strain was deficient in adhesion, whereas the expression of CpALS7 in Saccharomyces cerevisiae resulted in increased adhesion. Together, these data provide strong evidence that CpAls7 aids in the adherence of C. parapsilosis to the extracellular matrix under shear forces and support its previously reported role in virulence.
Assuntos
Candida parapsilosis/metabolismo , Matriz Extracelular/fisiologia , Proteínas Fúngicas/metabolismo , Resistência ao Cisalhamento , Fenômenos Biomecânicos , Adesão Celular , Clonagem Molecular , Meios de Cultura , Proteínas Fúngicas/genética , Ligação Proteica , Saccharomyces cerevisiaeRESUMO
PRKAG2 encodes the γ2-subunit isoform of 5'-AMP-activated protein kinase (AMPK), a heterotrimeric enzyme with major roles in the regulation of energy metabolism in response to cellular stress. Mutations in PRKAG2 have been implicated in a unique hypertrophic cardiomyopathy (HCM) characterized by cardiac glycogen overload, ventricular preexcitation, and hypertrophy. We identified a novel, de novo PRKAG2 mutation (K475E) in a neonate with prenatal onset of HCM. We aimed to investigate the cellular impact, signaling pathways involved, and therapeutic options for K475E mutation using cells stably expressing human wild-type (WT) or the K475E mutant. In human embryonic kidney-293 cells, the K475E mutation induced a marked increase in the basal phosphorylation of T172 and AMPK activity, reduced sensitivity to AMP in allosteric activation, and a loss of response to phenformin. In H9c2 cardiomyocytes, the K475E mutation induced inhibition of AMPK and reduced the response to phenformin and increases in the phosphorylation of p70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). Primary fibroblasts from the patient with the K475E mutation also showed marked increases in the phosphorylation of p70S6K and 4E-BP1 compared with those from age-matched, nondiseased controls. Moreover, overexpression of K475E induced hypertrophy in H9c2 cells, which was effectively reversed by treatment with rapamycin. Taken together, we have identified a novel, de novo infantile-onset PRKAG2 mutation causing HCM. Our study suggests the K475E mutation induces alteration in basal AMPK activity and results in a hypertrophy phenotype involving the mechanistic target of rapamycin signaling pathway, which can be reversed with rapamycin.NEW & NOTEWORTHY We identified a novel, de novo PRKAG2 mutation (K475E) in the cystathionine ß-synthase 3 repeat, a region critical for AMP binding but with no previous reported mutation. Our data suggest the mutation affects AMP-activated protein kinase activity, activates cell growth pathways, and results in cardiac hypertrophy, which can be reversed with rapamycin.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Cardiomiopatia Hipertrófica/genética , Mutação de Sentido Incorreto , Miócitos Cardíacos/enzimologia , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Monofosfato de Adenosina/metabolismo , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiomiopatia Hipertrófica/enzimologia , Cardiomiopatia Hipertrófica/fisiopatologia , Proteínas de Transporte/metabolismo , Estudos de Casos e Controles , Análise Mutacional de DNA , Ativação Enzimática , Fibroblastos/enzimologia , Fibroblastos/patologia , Predisposição Genética para Doença , Células HEK293 , Humanos , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fenformin/farmacologia , Fenótipo , Fosfoproteínas/metabolismo , Fosforilação , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , TransfecçãoRESUMO
BACKGROUND: Mechanical ventilation plays a central role in the injury of premature lungs. However, the mechanisms by which mechanical signals trigger an inflammatory cascade to promote lung injury are not well-characterized. Transient receptor potential vanilloid 4 (TRPV4), a calcium-permeable mechanoreceptor channel has been shown to be a major determinant of ventilator-induced acute lung injury in adult models. However, the role of these channels as modulators of inflammation in immature lungs is unknown. In this study, we tested the hypothesis that TRPV4 channels are important mechanotransducers in fetal lung injury. METHODS: Expression of TRPV4 in the mouse fetal lung was investigated by immunohistochemistry, Western blot and qRT-PCR. Isolated fetal epithelial cells were exposed to mechanical stimulation using the Flexcell Strain Unit and inflammation and differentiation were analyzed by ELISA and SP-C mRNA, respectively. RESULTS: TRPV4 is developmentally regulated in the fetal mouse lung; it is expressed in the lung epithelium and increases with advanced gestation. In contrast, in isolated epithelial cells, TRPV4 expression is maximal at E17-E18 of gestation. Mechanical stretch increases TRPV4 in isolated fetal epithelial cells only during the canalicular stage of lung development. Using the TRPV4 agonist GSK1016790A, the antagonist HC-067047, and the cytokine IL-6 as a marker of inflammation, we observed that TRPV4 regulates release of IL-6 via p38 and ERK pathways. Interestingly, stretch-induced differentiation of fetal epithelial cells was also modulated by TRPV4. CONCLUSION: These studies demonstrate that TRPV4 may play an important role in the transduction of mechanical signals in the fetal lung epithelium by modulating not only inflammation but also the differentiation of fetal epithelial cells.
Assuntos
Diferenciação Celular/fisiologia , Células Epiteliais/fisiologia , Mecanotransdução Celular/fisiologia , Canais de Cátion TRPV/biossíntese , Animais , Células Cultivadas , Células Epiteliais/patologia , Feminino , Feto , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Mucosa Respiratória/embriologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologiaRESUMO
Candida parapsilosis is a frequent cause of disseminated candidiasis and is associated with significant morbidity and mortality. Although important in pathogenesis, interactions of this organism with endothelial cells have received less attention than those of Candida albicans. Internalization of C. parapsilosis by monolayers of human endothelial cells was examined in an in vitro assay and compared to that of C. albicans. Both live and heat-killed yeast were efficiently internalized, with heat-killed yeast subsequently being detected in an acidic subcompartment. Internalization was marked by a process of engulfment by thin membrane extensions from the endothelium. Efficiency of internalization differed among different clinical isolates and species of yeast. Opsonization of C. parapsilosis by serum factors was not sufficient to cause endocytosis; instead, serum appeared to directly stimulate endothelial uptake. Colocalization of endothelial actin and N-WASP at sites of C. parapsilosis internalization was observed. A Förster-resonance energy transfer (FRET) probe for N-WASP activity showed active N-WASP at sites of internalization for both live and heat-killed C. parapsilosis and C. albicans. An actin nucleation inhibitor (cytochalasin D) and an N-WASP inhibitor (wiskostatin) both inhibited uptake of heat-killed C. parapsilosis, as did short interfering RNA-mediated ablation of N-WASP. Thus, endocytosis by endothelial cells may represent a means of traversal of the blood vessel wall by yeast during disseminated candidiasis, and N-WASP may play a key role in the process.
Assuntos
Candida , Candidíase/metabolismo , Endocitose/fisiologia , Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Western Blotting , Humanos , RNA Interferente Pequeno , TransfecçãoRESUMO
The COBRE for Perinatal Biology (CPB) was the third grant in Rhode Island to be funded by the COBRE mechanism. The CPB was based at Women & Infants Hospital, and housed in the Kilguss Research Institute, in the Jewelry District in Providence. The scientific focus of the CPB was on perinatal diseases such as preeclampsia and preterm birth, as well as more broadly on cardiopulmonary development and reproductive biology. Over the course of three phases and 17 years, the CPB funded 22 projects. CPB investigators, in turn, generated over $27.5 million in independent funding from federal and non-federal sources. Besides providing scientific and career mentorship to new investigators in Rhode Island, the CPB established a Research Core. The Kilguss Research Core remains active and flourishing and provides advanced imaging, molecular biology and flow cytometry abilities to researchers at Women & Infants as well as the greater Rhode Island scientific community.
Assuntos
Pesquisa Biomédica , Nascimento Prematuro , Biologia , Feminino , Organização do Financiamento , Humanos , Recém-Nascido , Mentores , GravidezRESUMO
C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The Candida clade, including Cp, is increasingly developing resistance to the first and the second line of antifungals. Cp is frequently isolated from hands and skin surfaces, as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to the body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and allow for the fluorescent quantification of individual isolates. We also demonstrate that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.
Assuntos
Candida parapsilosis , Soroalbumina Bovina , Animais , Candida , Mãos , Humanos , Reprodutibilidade dos TestesRESUMO
Candida parapsilosis has emerged as a frequent cause of invasive candidiasis with increasing evidence of unique biological features relative to C. albicans As it adapts to conditions within a mammalian host, rapid changes in gene expression are necessary to facilitate colonization and persistence in this environment. Adhesion of the organism to biological surfaces is a key first step in this process and is the focus of this study. Building on previous observations showing the importance of a member of the ALS gene family in C. parapsilosis adhesion, three clinical isolates were cultured under two conditions that mimic the mammalian host and promote adhesion, incubation at 37°C in tissue culture medium 199 or in human plasma. Transcriptional profiles using RNA-seq were obtained in these adhesion-inducing conditions and compared to profiles following growth in yeast media that suppress adhesion to identify gene expression profiles associated with adhesion. Overall gene expression profiles among the three strains were similar in both adhesion-inducing conditions and distinct from adhesion-suppressing conditions. Pairwise analysis among the three growth conditions identified 133 genes that were differentially expressed at a cutoff of ±4-fold, with the most upregulated genes significantly enriched in iron acquisition and transmembrane transport, while the most downregulated genes were enriched in oxidation-reduction processes. Gene family enrichment analysis identified gene families with diverse functions that may have an important role in this important step for colonization and disease.IMPORTANCE Invasive Candida infections are frequent complications of the immunocompromised and are associated with substantive morbidity and mortality. Although C. albicans is the best-studied species, emerging infections by non-albicans Candida species have led to increased efforts to understand aspects of their pathogenesis that are unique from C. albicansC. parapsilosis is a frequent cause of invasive infections, particularly among premature infants. Recent efforts have identified important virulence mechanisms that have features distinct from C. albicansC. parapsilosis can exist outside a host environment and therefore requires rapid modifications when it encounters a mammalian host to prevent its clearance. An important first step in the process is adhesion to host surfaces. This work takes a global, nonbiased approach to investigate broad changes in gene expression that accompany efficient adhesion. As such, biological pathways and individual protein targets are identified that may be amenable to manipulation to reduce colonization and disease from this organism.
Assuntos
Candida parapsilosis/genética , Candida parapsilosis/metabolismo , Adesão Celular/genética , Expressão Gênica , Transcrição Gênica , Candidíase/microbiologia , Humanos , Microfluídica/métodos , RNA-Seq , VirulênciaRESUMO
CD16+ monocytes represent 5-10% of peripheral blood monocytes in normal individuals and are dramatically expanded in several pathological conditions including sepsis, human immunodeficiency virus 1 infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines and may represent dendritic cell precursors in vivo. The mechanisms that mediate the recruitment of CD16+ monocytes into tissues remain unknown. Here we investigate molecular mechanisms of CD16+ monocyte trafficking and show that migration of CD16+ and CD16- monocytes is mediated by distinct combinations of adhesion molecules and chemokine receptors. In contrast to CD16- monocytes, CD16+ monocytes expressed high CX3CR1 and CXCR4 but low CCR2 and CD62L levels and underwent efficient transendothelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1 alpha (CXCL12) but not monocyte chemoattractant protein 1 (CCL2). CD16+ monocytes arrested on cell surface-expressed FKN under flow with higher frequency compared with CD16- monocytes. These results demonstrate that FKN preferentially mediates arrest and migration of CD16+ monocytes and suggest that recruitment of this proinflammatory monocyte subset to vessel walls via the CX3CR1-FKN pathway may contribute to vascular and tissue injury during pathological conditions.
Assuntos
Ciclo Celular/fisiologia , Movimento Celular/fisiologia , Quimiocinas CX3C/metabolismo , Proteínas de Membrana/metabolismo , Monócitos/metabolismo , Receptores de IgG/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Receptor 1 de Quimiocina CX3C , Quimiocina CX3CL1 , Quimiocinas CX3C/imunologia , Humanos , Subpopulações de Linfócitos , Proteínas de Membrana/imunologia , Monócitos/imunologia , Fenótipo , Ligação Proteica , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores de IgG/genética , Receptores de IgG/imunologiaRESUMO
Leukocyte trafficking to sites of inflammation follows a defined temporal pattern, and evidence suggests that initial neutrophil transendothelial migration modifies endothelial cell phenotype. We tested the hypothesis that preconditioning of human umbilical vein endothelial cells (HUVEC) by neutrophils would also modify the subsequent transendothelial migration of T lymphocytes across cytokine-stimulated HUVEC in an in vitro flow assay. Using fluorescence microscopy, preconditioning of HUVEC by neutrophils was observed to significantly reduce the extent of subsequent stromal cell-derived factor-1alpha (SDF-1alpha [CXCL12])-mediated T lymphocyte transendothelial migration, without reducing accumulation. In contrast, recruitment of a second wave of neutrophils was unaltered. Conditioned medium harvested after transendothelial migration of neutrophils or supernatants from stimulated neutrophils mediated a similar blocking effect, which was negated using a specific neutrophil elastase inhibitor. Furthermore, T lymphocyte transendothelial migration was inhibited by treatment of HUVEC with purified neutrophil elastase, which selectively cleaved the amino terminus of HUVEC-bound SDF-1alpha, which is required for its chemotactic activity. The reduction in T lymphocyte transendothelial migration was not observed using a different chemokine, ELC (CCL19), and was not reversed by replenishment of SDF-1alpha, indicating endothelial retention of the inactivated chemokine. In summary, transmigrating neutrophils secrete localized elastase that is protected from plasma inhibitors, and thereby modulate trafficking of other leukocyte subsets by altering the endothelial-associated chemotactic activities.
Assuntos
Quimiocinas CXC/fisiologia , Células Endoteliais/citologia , Elastase de Leucócito/fisiologia , Neutrófilos/fisiologia , Linfócitos T/fisiologia , Comunicação Celular , Movimento Celular , Células Cultivadas , Quimiocina CXCL12 , Complemento C5a/farmacologia , Células Endoteliais/metabolismo , Humanos , Interleucina-1/farmacologia , Interleucina-8/farmacologiaRESUMO
The leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) and its endothelial ligand intercellular adhesion molecule (ICAM)-1 play an important role in transmigration as demonstrated by in vivo and in vitro models of inflammation. Despite the prominent role, little is known concerning the distribution and dynamic behavior of these adhesion molecules during leukocyte transmigration. Therefore, we examined the spatial and temporal distribution of LFA-1 on neutrophils actively transmigrating tumor necrosis factor-alpha-activated human umbilical vein endothelial monolayers under shear flow. Upon neutrophil arrest, LFA-1 was evenly distributed. However, once neutrophils initiated transmigration, LFA-1 rapidly redistributed to form a ringlike cluster at the neutrophil-endothelial junctional interface through which transmigration occurred. As transmigration was completed, LFA-1 redistributed to the neutrophil uropod. Endothelial ICAM-1 and JAM-A both colocalized with the ringlike LFA-1 cluster. Further analysis of PMA-stimulated neutrophils, which increase mobility of LFA-1, showed a rapid redistribution of LFA-1 and ICAM-1, but not endothelial JAM-A. Thus, endothelial JAM-A does not appear to contribute to adhesion or transmigration in this system. This is the first demonstration that neutrophil LFA-1 rapidly redistributes to form a ringlike structure that coclusters with endothelial ICAM-1 as the neutrophil transmigrates.
Assuntos
Movimento Celular/fisiologia , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Neutrófilos/fisiologia , Carcinógenos/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/fisiologia , Humanos , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/citologia , Veias Umbilicais/fisiologiaRESUMO
The mammalian genome encodes multiple Wiskott-Aldrich syndrome protein (WASP)/WASP-family Verprolin homologous (WAVE) proteins. Members of this family interact with the actin related protein (Arp) 2/3 complex to promote growth of a branched actin network near the plasma membrane or the surface of moving cargos. Arp2/3 mediated branching can further lead to formation of comet tails (actin rockets). Despite their similar domain structure, different WASP/WAVE family members fulfill unique functions that depend on their subcellular location and activity levels. We measured the relative efficiency of actin nucleation promotion of full-length WASP/WAVE proteins in a cytoplasmic extract from primary human umbilical vein endothelial cells (HUVEC). In this assay WAVE2 and WAVE3 complexes showed higher nucleation efficiency than WAVE1 and N-WASP, indicating distinct cellular controls for different family members. Previously, WASP and N-WASP were the only members that were known to stimulate comet formation. We observed that in addition to N-WASP, WAVE3 also induced short actin tails, and the other WAVEs induced formation of asymmetric actin shells. Differences in shape and structure of actin-based growth may reflect varying ability of WASP/WAVE proteins to break symmetry of the actin shell, possibly by differential recruitment of actin bundling or severing (pruning or debranching) factors.
Assuntos
Actinas/metabolismo , Endotélio Vascular/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Células Cultivadas , HumanosRESUMO
Dengue fever is an important tropical illness for which there is currently no virus-specific treatment. To shed light on mechanisms involved in the cellular response to dengue virus (DV), we assessed gene expression changes, using Affymetrix GeneChips (HG-U133A), of infected primary human cells and identified changes common to all cells. The common response genes included a set of 23 genes significantly induced upon DV infection of human umbilical vein endothelial cells (HUVECs), dendritic cells (DCs), monocytes, and B cells (analysis of variance, P < 0.05). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one of the common response genes, was identified as a key link between type I and type II interferon response genes. We found that DV induces TRAIL expression in immune cells and HUVECs at the mRNA and protein levels. The induction of TRAIL expression by DV was found to be dependent on an intact type I interferon signaling pathway. A significant increase in DV RNA accumulation was observed in anti-TRAIL antibody-treated monocytes, B cells, and HUVECs, and, conversely, a decrease in DV RNA was seen in recombinant TRAIL-treated monocytes. Furthermore, recombinant TRAIL inhibited DV titers in DV-infected DCs by an apoptosis-independent mechanism. These data suggest that TRAIL plays an important role in the antiviral response to DV infection and is a candidate for antiviral interventions against DV.
Assuntos
Vírus da Dengue/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Linfócitos B/virologia , Linhagem Celular , Células Cultivadas , Células Dendríticas/virologia , Células Endoteliais/virologia , Perfilação da Expressão Gênica , Humanos , Monócitos/virologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ligante Indutor de Apoptose Relacionado a TNF/biossínteseRESUMO
OBJECTIVE: The purpose of this study was to determine in a mouse model whether uterine natural killer (uNK) cell cytotoxic activation induces infection/inflammation-associated preterm labor and delivery. STUDY DESIGN: Wild type or interleukin (IL)-10(-/-) mice were injected intraperitoneally with lipopolysaccharide on gestational day 14. Mice were either killed for collection of uteroplacental tissue, spleen, and serum or allowed to deliver. Uteroplacental tissue was used for histology and characterization of uNK cells. RESULTS: Low-dose lipopolysaccharide treatment triggered preterm labor and delivery in IL-10(-/-), but not wild type mice, in a manner independent of progesterone levels. Preterm labor and delivery in IL-10(-/-) mice was associated with an increased number and placental infiltration of cytotoxic uNK cells and placental cell death. Depletion of NK cells or tumor necrosis factor (TNF)-alpha neutralization in these mice restored term delivery. Furthermore, TNF-alpha neutralization prevented uNK cell infiltration and placental cell apoptosis. CONCLUSION: The uNK cell-TNF-alpha-IL-10 axis plays an important role in the genesis of infection/inflammation-induced preterm labor/delivery.
Assuntos
Inflamação/imunologia , Células Matadoras Naturais/fisiologia , Trabalho de Parto Prematuro/imunologia , Placenta/imunologia , Útero/imunologia , Animais , Movimento Celular/imunologia , Feminino , Idade Gestacional , Inflamação/complicações , Interleucina-10/genética , Interleucina-10/farmacologia , Células Matadoras Naturais/citologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Mutantes , Trabalho de Parto Prematuro/tratamento farmacológico , Trabalho de Parto Prematuro/prevenção & controle , Placenta/citologia , Gravidez , Fator de Necrose Tumoral alfa/imunologia , Útero/citologiaRESUMO
OBJECTIVE: A novel indole ethyl isothiocyanate derivative (7Me-IEITC) was defined as a potent growth-suppressing agent to cell lines derived from ovarian cancers. Key mechanisms of the cellular response in vitro were studied and suggest a potential of 7Me-IEITC as a therapeutic drug. METHODS: The viability of ovarian cancer cell lines (SKOV-3, OVCAR-3) in comparison to pancreatic and prostate cancer cell lines, primary fibroblast and immortalized trophoblasts after treatment with 7Me-IEITC was analyzed. Morphological and apoptotic responses of SKOV-3 were studied by fluorescence microscopy (DAPI staining, TUNEL assay). SKOV-3 proliferation was estimated by a standardized BrdU incorporation assay. The phosphorylation of MAP-Kinases, pro-survival factors and the activation of caspases and PARP-1 were analyzed by western blotting. Changes of the mitochondrial transmembrane-potential and in cell-cycle progression were studied by FACS analysis. MAP-Kinase and caspase inhibitors were employed in cytotoxicity studies. RESULTS: 7Me-IEITC selectively reduced the viability of SKOV-3, OVCAR-3, BXPC-3 and PC-3 cells (IC(50) values < or = 5 microM), while the viability of fibroblasts or trophoblasts remained un-affected at concentrations below 20 microM. 7Me-IEITC treatment down-regulated pro-survival kinases and transcription factors (STAT-3, IKKalpha and NF-kappaB), caused rapid loss of the mitochondrial transmembrane-potential and inactivation of PARP-1 along with activation of caspases. The use of p38 MAP-Kinase-and caspase inhibitors suppressed the cytotoxicity of the drug. 7Me-IEITC acted as an anti-proliferative agent and arrested the cell-cycle progression of SKOV-3 in G2/M phase. CONCLUSION: 7Me-IEITC is a potent and growth-suppressing agent to cell lines derived from ovarian cancers by causing deactivation of survival signals, apoptosis, and cell-cycle arrest.
Assuntos
Adenocarcinoma/fisiopatologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Indóis/farmacologia , Isocianatos/farmacologia , Neoplasias Ovarianas/fisiopatologia , Platina/farmacologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Biomarcadores/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/fisiopatologia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/fisiopatologiaRESUMO
Mechanical forces are critical for normal fetal lung development. However, the signaling events that promote lung maturation are not fully understood. In this study, the authors analyzed the role of Rho family guanidine triphosphatases (GTPases) in isolated embryonic day 19 (E19) fetal type II epithelial cells exposed to 5% cyclic stretch. The results showed that mechanical strain stimulated RhoA within 5 minutes of initiation of force. Rac1 was also activated, but not Cdc42. After 6 hours of equibiaxial stretch, actin filaments were oriented parallel to the long axis of the cells. By 16 hours, actin fibers still maintained the same orientation, but their intensity decreased when compared to 6 hours. These findings temporally correlated with a decrease in RhoA stimulation. Using adenoviruses encoding dominant negative mutants of RhoA and Rac1, the authors observed that both GTPases are important for strain-induced stress fiber formation, cell alignment, and extracellular signal-regulated kinase (ERK) phosphorylation. However, whereas inhibition of Rho increased surfactant protein C (SP-C) mRNA expression (a marker of type II cell differentiation), suppression of Rac had no effects. These studies suggest that RhoA and Rac1 regulate actin remodeling and cell alignment in fetal type II cells exposed to mechanical stretch. RhoA is a negative regulator of stretch-induced type II cell maturation.
Assuntos
Actinas/metabolismo , Células Epiteliais/citologia , Pulmão/embriologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Diferenciação Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fosforilação , Gravidez , Ratos , Ratos Sprague-Dawley , Estresse MecânicoRESUMO
Endothelial cells are known to respond to flow onset by increasing actin turnover rate. Current models assume that an increase in the actin turnover rate should result in a rise in cell crawling speed. Here we report that confluent endothelial monolayer shows an unexpected behavior: cell crawling speed decreases by approximately 40% within the first 30 min of flow onset. A drop in crawling speed has not been observed in either subconfluent endothelial cells or in VE-cadherin-deficient cells. We found that flow onset caused an increase in the number of VE-cadherin-GFP molecules in the junctions and elicited changes in the cytoskeleton-associated fractions of alpha, beta -catenins and VE-cadherin. Flow application also increased the strength of interactions of endothelial cells with surfaces coated with recombinant VE-cadherin. These observations suggest that endothelial cell junctional proteins respond to flow transiently by increasing the strength of intercellular attachments early after flow onset and support the view on the active role of intercellular adhesions in mechanotransduction.
Assuntos
Citoesqueleto de Actina/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Junções Intercelulares/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Cateninas/metabolismo , Bovinos , Comunicação Celular/fisiologia , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Células Endoteliais/ultraestrutura , Adesões Focais/metabolismo , Adesões Focais/ultraestrutura , Proteínas de Fluorescência Verde , Humanos , Junções Intercelulares/ultraestrutura , Mecanotransdução Celular/fisiologia , Proteínas Recombinantes de Fusão , Estresse MecânicoRESUMO
Candida parapsilosis is a fungal pathogen that is associated with hematogenously disseminated disease in premature neonates, acutely ill or immunocompromised patients. In cell culture, C. parapsilosis cells are actively and avidly endocytosed by endothelial cells via actin polymerization mediated by N-WASP. Here we present evidence that C. parapsilosis that were internalized by endothelial cells remained alive, and avoided being acidified or otherwise damaged via the host cell. Internalized fungal cells reproduced intracellularly and eventually burst out of the host endothelial cell. When neutrophils were added to endothelium and C. parapsilosis, they patrolled the endothelial surface and efficiently killed most adherent fungal cells prior to endocytosis. But after endocytosis by endothelial cells, internalized fungal cells evaded neutrophil killing. Silencing endothelial N-WASP blocked endocytosis of C. parapsilosis and left fungal cells stranded on the cell surface, where they were susceptible to neutrophil killing. These observations suggest that for C. parapsilosis to escape from the bloodstream, fungi may adhere to and be internalized by endothelial cells before being confronted and phagocytosed by a patrolling leukocyte. Once internalized by endothelial cells, C. parapsilosis may safely replicate to cause further rounds of infection. Immunosurveillance of the intravascular lumen by leukocytes crawling on the endothelial surface and rapid killing of adherent yeast may play a major role in controlling C. parapsilosis dissemination and infected endothelial cells may be a significant reservoir for fungal persistence.
Assuntos
Candida/imunologia , Candida/patogenicidade , Endocitose , Células Endoteliais/microbiologia , Neutrófilos/imunologia , Adesividade , Candida/crescimento & desenvolvimento , Candida/ultraestrutura , Células Cultivadas , Células Endoteliais/fisiologia , Células Endoteliais/ultraestrutura , Células Endoteliais da Veia Umbilical Humana , Humanos , Viabilidade Microbiana , Neutrófilos/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologiaRESUMO
Aldosterone induces fibrotic changes in cardiovascular tissues but its effects have usually been demonstrated in models of pre-existing renal injury and/or hypertension. This study tests the hypothesis that aldosterone can directly induce vascular fibrotic changes in the absence of prior renal injury or hypertension. Experiments were conducted in intact or adrenalectomized (ADX) mice. Mice were divided into groups and treated for 1 week with vehicle or aldosterone (8 µg/kg/day)± inhibitor (800 µg/kg/day): CONTROLS, mice treated with aldosterone, ADX-CONTROLS, ADX+corticosterone (CORT 8 µg/kg/day), ADX with aldosterone, ADX with aldosterone plus the mineralocorticoid receptor (MR) antagonist RU-318, ADX with aldosterone+CORT (CORT inhibitor dose), and ADX with aldosterone+11-dehydro-CORT. Aortic smooth muscle to collagen ratio, aorta intimal thickness (µm), heart weight/body weight ratio (mg/gm), and left ventricular collagen (%) were measured. Prior to sacrifice, blood pressures were normal in all animals. Lower dose CORT alone had no effect on any of the variables examined. Aldosterone exposure was associated with extra-cellular matrix accumulation in cardiovascular tissues in intact mice and adrenalectomy exacerbated these effects. RU-318, CORT (inhibitor dose), and 11-deydro-CORT each attenuated the early fibrotic changes induced by aldosterone. In the heart, aldosterone exposure affected all the parameters measured and caused intimal hypercellularity with monocytes adhering to endothelial cells lining coronary vessels. Cultured endothelial cells exposed to aldosterone (10nM) released E-selectin, produced collagen, and promoted monocyte adhesion. These effects were inhibited by RU-318 and 11-deydro-CORT but not by CORT. Thus, adrenalectomy enhances aldosterone induced early fibrotic changes in heart and aorta. Aldosterone initially targets vascular endothelial cells. MR antagonists and 11-dehydro-CORT, an 11ß-HSD dehydrogenase end-product, directly attenuate these effects.
Assuntos
Adrenalectomia , Aldosterona/farmacologia , Aorta/efeitos dos fármacos , Coração/efeitos dos fármacos , Túnica Íntima/efeitos dos fármacos , Glândulas Suprarrenais/cirurgia , Aldosterona/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal , Adesão Celular/efeitos dos fármacos , Colágeno/genética , Colágeno/metabolismo , Corticosterona/análogos & derivados , Corticosterona/farmacologia , Selectina E/genética , Selectina E/metabolismo , Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Túnica Íntima/metabolismoRESUMO
Hypercalcemia remains a major impediment to the clinical use of vitamin D in cancer treatment. Approaches to remove hypercalcemia and development of nonhypercalcemic agents can lead to the development of vitamin D-based therapies for treatment of various cancers. In this report, in vitro and in vivo anticancer efficacy, safety, and details of vitamin D receptor (VDR) interactions of PT19c, a novel nonhypercalcemic vitamin D derived anticancer agent, are described. PT19c was synthesized by bromoacetylation of PTAD-ergocalciferol adduct. Broader growth inhibitory potential of PT19c was evaluated in a panel of chemoresistant breast, renal, ovarian, lung, colon, leukemia, prostate, melanoma, and central nervous system cancers cell line types of NCI60 cell line panel. Interactions of PT19c with VDR were determined by a VDR transactivation assay in a VDR overexpressing VDR-UAS-bla-HEK293 cells, in vitro VDR-coregulator binding, and molecular docking with VDR-ligand binding domain (VDR-LBD) in comparison with calcitriol. Acute toxicity of PT19c was determined in nontumored mice. In vivo antitumor efficacy of PT19c was determined via ovarian and endometrial cancer xenograft experiments. Effect of PT19c on actin filament organization and focal adhesion formation was examined by microscopy. PT19c treatment inhibited growth of chemoresistant NCI60 cell lines (log10GI50 ~ -4.05 to -6.73). PT19c (10 mg/kg, 35 days) reduced growth of ovarian and endometrial xenograft tumor without hypercalcemia. PT19c exerted no acute toxicity up to 400 mg/kg (QDx1) in animals. PT19c showed weak VDR antagonism, lack of VDR binding, and inverted spatial accommodation in VDR-LBD. PT19c caused actin filament dysfunction and inhibited focal adhesion in SKOV-3 cells. PT19c is a VDR independent nonhypercalcemic vitamin D-derived agent that showed noteworthy safety and efficacy in ovarian and endometrial cancer animal models and inhibited actin organization and focal adhesion in ovarian cancer cells.