172 research outputs found
Activation of RhoA and ROCK Are Essential for Detachment of Migrating Leukocytesh
Detachment of the rear of the cell from its substratum is an important aspect of locomotion. The
signaling routes involved in this adhesive release are largely unknown. One of the few candidate
proteins to play a role is RhoA, because activation of RhoA in many cell types leads to contraction,
a mechanism probably involved in detachment. To study the role of RhoA in detachment
regulation, we analyzed several subsets of expert migratory leukocytes by video microscopy. In
contrast to fast-migrating neutrophils, eosinophils do not detach the rear of the cell unless
stimulated with serum. When measuring the amount of active RhoA, with the use of a GSTRhotekin
pulldown assay, we found that serum is an excellent activator of RhoA in granulocytes.
Inhibition of RhoA or one of Rhos target proteins, the kinase ROCK, in neutrophils leads to the
phenotype seen in eosinophils: the rear of the cell is firmly attached to the substratum, whereas
the cell body is highly motile. ROCK-inhibition leads to impaired migration of granulocytes in
filters, on glass, and through endothelial monolayers. Also, the ROCK signaling pathway is
involved in changes of integrin-mediated adhesion. Eosinophil transduction by a tat-fusion
construct containing active RhoA resulted in detachment stimulation in the presence of chemoattractant.
From these results we conclude that activation of the RhoA-ROCK pathway is essential
for detachment of migratory leukocytes
Transduction of a dominant-negative H-Ras into human eosinophils attenuates extracellular signal-regulated kinase activation and interleukin-5-mediated cell viability
Inhibition of eosinophil apoptosis by exposure
to interleukin-5 (IL-5) is associated
with the development of tissue eosinophilia
and may contribute to the
inflammation characteristic of asthma.
Analysis of the signaling events associated
with this process has been hampered
by the inability to efficiently manipulate
eosinophils by the introduction of
active or inhibitory effector molecules.
Evidence is provided, using a dominantnegative
N17 H-Ras protein (dn-H-Ras)
and MEK inhibitor U0126, that activation
of the Ras-Raf-MEK-ERK pathway plays a
determining role in the prolongation of
eosinophil survival by IL-5. For these
studies, a small region of the human
immunodeficiency virus Tat protein, a protein
transduction domain known to enter
mammalian cells efficiently, was fused to
the N-terminus of dn-H-Ras. The Tat-dn-HRas
protein generated from this construct
transduced isolated human blood
eosinophils at more than 95% efficiency.
When Tat-dn-H-Ras-transduced eosinophils
were treated with IL-5, they exhibited
a time- and dosage-dependent reduction
in extracellular regulated kinase 1
and 2 activation and an inhibition of p90
Rsk1 phosphorylation and IL-5-mediated
eosinophil survival in vitro. In contrast,
Tat-dn-H-Ras did not inhibit CD11b upregulation
or STAT5 tyrosine phosphorylation.
These data demonstrate that Tat
dominant-negative protein transduction
can serve as an important and novel tool
in studying primary myeloid cell signal
transduction in primary leukocytes and
can implicate the Ras-Raf-MEK-ERK pathway
in IL-5-initiated eosinophil survival
Characteristics, causes and treatment of postpartum haemorrhage in first and second pregnancies
Leptin is an adipokine that is thought to be important in many inflammatory diseases, and is known to influence the function of several leukocyte types. However, no clear consensus is present regarding the responsiveness of neutrophils for this adipokine. In this study a 2D DIGE proteomics approach was used as an unbiased approach to identify leptin-induced effects on neutrophils. Additionally chemotaxis and survival experiments were performed to reproduce results from literature showing putative effects of leptin on these neutrophil responses. Leptin did not induce any significant changes in the proteome provided leptin was added at physiologically relevant concentrations (250 ng). Our leptin batches were biologically active as they induced proliferation in LeptinR expressing Ba/F3 cells. At high concentrations (25000 ng) leptin induced a change in neutrophil proteome. Seventeen differently regulated spots were identified of which twelve could be characterized by mass spectrometry. Two of these identified proteins, SerpinB1 and p40 phox, were chosen for further analysis but leptin-induced expression analyzed by western blot were highly variable. Additionally leptin also induced neutrophil survival at these high concentrations. No leptin-induced chemotaxis of human neutrophils was detected at any concentration. In conclusion, physiological concentrations of leptin do not affect neutrophils. High leptin concentrations induced survival and changes in the neutrophils proteome, but this was most likely mediated by an indirect effect. However, it cannot be ruled out that the effects were mediated by a yet not-identified leptin receptor on human neutrophils
Interleukin-5 Potentiates Sulfidopeptide Leukotriene Production by Human Eosinophils
Interleukin-5 (IL-5) has been shown to be a selective eosinophil
growth and differentiation factor. In the present study, the effect
of recombinant human IL-5 on human eosinophil sulfidopeptide
leukotriene production was investigated. IL-5 did not affect
leukotriene synthesis in unstimulated eosinophils. However, IL-5
potentiated leukotriene synthesis by eosinophils stimulated with
serum treated zymosan (STZ) or the calcium ionophore A23187 by
69% and 135%, respectively. The priming effect of IL-5
was dose dependent, with significant stimulation occurring at 1 000
U/ml for STZ and 100-1 000 U/ml for A23187. Pre-incubation with IL-5
did not increase leukotriene synthesis further
Tyrosine phosphorylation-dependent activation of phosphatidylinositide 3-kinase occurs upstream of Ca^(2+)-signalling induced by Fcy receptor cross-linking in human neutrophils
The effect of wortmannin on IgG-receptor (FcyR)-mediated
stimulation of human neutrophils was investigated. The Ca^(2+)
influx induced by clustering of both Fcy receptors was inhibited
by wortmannin, as was the release of Ca^(2+) from intracellular
stores. Wortmannin also inhibited, with the same efficacy, the
accumulation of Ins(1,4,5)P3 observed after FcyR stimulation,
but did not affect the increase in Ins(1,4,5)P3 induced by the
chemotactic peptide, formyl-methionine-leucine-phenylalanine.
Because wortmannin is, in the concentrations used here, an
inhibitor of PtdIns 3-kinase, these results suggested a role for
PtdIns 3-kinase upstream of Ca^(2+) signalling, induced by FcyR
cross-linking. Support for this notion was obtained by investigating
the effect of another inhibitor of PtdIns 3-kinase, LY
294002, and by studying the kinetics of PtdIns 3-kinase activation.
We found translocation of PtdIns 3-kinase to the plasma
membrane and increased PtdIns 3-kinase activity in the membrane
as soon as 5 s after FccR cross-linking, even before the
onset of the Ca^(2+) response. Moreover, the translocation of
PtdIns 3-kinase to the plasma membrane was inhibited by cocross-
linking of either FcyRIIa and FcyRIIIb with the tyrosine
phosphatase, CD45, indicating a requirement for protein tyrosine
phosphorylation in the recruitment of PtdIns 3-kinase to the
plasma membrane. Taken together, our results suggest a role for
PtdIns 3-kinase in early signal transduction events after FcyR
cross-linking in human neutrophils
Constitutive cytoplasmic localization of p21Waf1/Cip1 affects the apoptotic process in monocytic leukaemia
In the present study, we analysed the expression and localization of p21Waf1/Cip1 in normal and malignant haematopoietic cells. We demonstrate that in normal monocytic cells, protein kinase C (PKC)-induced p21 gene activation, which is nuclear factor-κB (NF-κB) independent, results in predominantly cytoplasmic localized p21 protein. In acute monocytic leukaemia (M4, M5), monocytic blasts (N=12) show constitutive cytoplasmic p21 expression in 75% of the cases, while in myeloid leukaemic blasts (N=10), low nuclear and cytoplasmic localization of p21 could be detected, which is also PKC dependent. Constitutive p21 expression in monocytic leukaemia might have important antiapoptotic functions. This is supported by the finding that in U937 cells overexpressing p21, VP16-induced apoptosis is significantly reduced (20.0±0.9 vs 55.8±3.8%, P<0.01, N=5), reflected by a reduced phosphorylation of p38 and JNK. Similarly, AML blasts with high cytoplasmic p21 were less sensitive to VP16-induced apoptosis as compared to AML cases with low or undetectable p21 expression (42.25 vs 12.3%, P<0.01). Moreover, complex formation between p21 and ASK1 could be demonstrated in AML cells, by means of coimmunoprecipitation. In summary, these results indicate that p21 has an antiapoptotic role in monocytic leukaemia, and that p21 expression is regulated in a PKC-dependent and NF-κB independent manner.
Принципы организации объектно-ориентированных систем обработки неформализованной информации
Рассматривается класс логико-аналитических систем, использующих специальные лингвистические процессоры и базы знаний (БЗ) для обработки потоков неформализованных документов с целью решения пользовательских задач. На первом этапе формализации текста документа извлекаются информационные объекты и связи, которые образуют структуры знаний и запоминаются в БЗ. На уровне БЗ организуются различные виды анализа и объектных поисков: поиск похожих объектов и ситуаций, поиск по связям и другие. Рассматриваются основные компоненты подобных систем, называемых объектно-ориентированными, их особенности при использовании в различных приложениях: при обработке криминальной информации, при автоматической формализации резюме (заявок на работу), в системах обработки СМИ с выделением террористических групп и их деяний.A class of the logical-analytical systems using special linguistic processors and knowledge bases is considered. Such systems are called object-oriented. These systems are employed for processing of the unstructured documents flow for the user problems decision. At the first stage the document text is formalized: information objects and links are extracted and transferred into the knowledge structures which are stored in the knowledge base (KB). At the level of KB various kinds of analysis and object search are organized: the search for similar objects and situations, the search on the basis of links and other types of search. The basic components of these systems, their main features and the particular use in different applications are considered.The system operation in the subject areas of criminal information processing, automatic formalization of summary texts (applications for work), mass media analysis for extracting information about terrorist formations and their activities are presented
Increased activation of blood neutrophils after cigarette smoking in young individuals susceptible to COPD
Background: Cigarette smoking is the most important risk factor for Chronic Obstructive Pulmonary Disease (COPD). Only a subgroup of smokers develops COPD and it is unclear why these individuals are more susceptible to the detrimental effects of cigarette smoking. The risk to develop COPD is known to be higher in individuals with familial aggregation of COPD. This study aimed to investigate if acute systemic and local immune responses to cigarette smoke differentiate between individuals susceptible or non-susceptible to develop COPD, both at young (18-40 years) and old (40-75 years) age. Methods: All participants smoked three cigarettes in one hour. Changes in inflammatory markers in peripheral blood (at 0 and 3 hours) and in bronchial biopsies (at 0 and 24 hours) were investigated. Acute effects of smoking were analyzed within and between susceptible and non-susceptible individuals, and by multiple regression analysis. Results: Young susceptible individuals showed significantly higher increases in the expression of Fc gamma RII (CD32) in its active forms (A17 and A27) on neutrophils after smoking (p = 0.016 and 0.028 respectively), independently of age, smoking status and expression of the respective markers at baseline. Smoking had no significant effect on mediators in blood or inflammatory cell counts in bronchial biopsies. In the old group, acute effects of smoking were comparable between healthy controls and COPD patients. Conclusions: We show for the first time that COPD susceptibility at young age associates with an increased systemic innate immune response to cigarette smoking. This suggests a role of systemic inflammation in the early induction phase of COPD
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