Intracellular S1P Generation Is Essential for S1P-Induced Motility of Human Lung Endothelial Cells: Role of Sphingosine Kinase 1 and S1P Lyase

Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P(1) receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility

ЛИТИЙ КАК ФАКТОР СОПРЯЖЕНИЯ НАРУШЕНИЙ МИНЕРАЛЬНОГО И УГЛЕВОДНОГО ГОМЕОСТАЗА ПРИ ЗЛОКАЧЕСТВЕННЫХ НОВООБРАЗОВАНИЯХ ЭПИТЕЛИАЛЬНЫХ ТКАНЕЙ

The impact of changes in orientation of the metabolism of carbohydrates and minerals in the cell malignancy has been demonstrated in several studies. The aim of this study was to analyze the molecular mechanisms and relationship of carbohydrate and mineral homeostasis with the processes of carcinogenesis. Parameters of carbohydrate and mineral metabolism of blood were defined in 73 patients with malignant tumors of epithelial tissues and 31 healthy subjects. In the presence of malignant tumors of epithelial tissues there was a statistically significant increase in the levels of glucose and glycated hemoglobin in the early stages of the disease and the absence of them at stage IV of the disease. There were no statistically significant differences in the levels of C-peptide and immunoreactive insulin in blood samples of cancer patients, although they tended to increase compared with the control group. Analysis of the composition of macroelements at the early stages of carcinogenesis revealed a statistically significant reduction of sodium level in plasma which wasn’t observed at the terminal stage of the disease. The concentrations of potassium and chlorine tend to increase in cancer patients, but the differences between these parameters were not statistically significant. Concentrations of calcium and magnesium significantly increased in the presence of malignant tumors. Analysis of microelements in the blood plasma showed a decrease in the concentration of cuprum and lithium (in 2.5-5 times) and the growth of strontium concentrations. Lithium has multiple effects on the life of cells, affecting a number of elements of messengers, as well as being the link between the carbohydrate metabolism and cell malignancy. Disorders of mineral homeostasis are important element in the disintegration of the metabolic processes in carcinogenesisВлияние изменения направленности метаболизма углеводов и минерального обмена на малигнизацию клеток было наглядно показано в ряде работ. Целью данного исследования стал анализ молекулярных механизмов взаимосвязи углеводного и минерального гомеостаза с процессами канцерогенеза. Определяли параметры углеводного и минерального обменов крови у 73 больных злокачественными новообразованиями эпителиальных тканей и 31 практически здоровых лиц. При злокачественных новообразованиях эпителиальных тканей выявлено статистически значимое повышение уровней глюкозы и гликилированного гемоглобина на начальных стадиях заболевания при отсутствии такового при IV стадии заболевания. Статистически значимых отличий по уровням С-пептида и иммунореактивного инсулина в крови онкологических больных выявлено не было, хотя и наблюдалась тенденция к их повышению по сравнению с контрольной группой. При анализе содержания макроэлементов уже на начальных стадиях канцерогенеза обнаружено статистически значимое снижение уровня Na в плазме крови, не наблюдающееся при терминальной стадии. Концентрации K и Сl имеет тенденцию к повышению у онкологических больных, но различия этих показателей статистически не значимы. При злокачественных новообразованиях значимо повышается содержание Ca, Р, Mg. Анализ уровня микроэлементов в плазме крови показал снижение концентрации Cu, Li (в 2,5–5 раз), рост содержания Sr. Литий оказывает множественные эффекты на жизнедеятельность клеток, влияя на ряд элементов систем мессенджеров, а также являясь сопрягающим звеном между углеводным обменом и малигнизацией клеток. Нарушение минерального гомеостаза является значимым звеном в дезинтеграции метаболических процессов при канцерогенезе.

Non-Small Cell Lung Carcinoma Cell Motility, Rac Activation and Metastatic Dissemination Are Mediated by Protein Kinase C Epsilon

Background: Protein kinase C (PKC) e, a key signaling transducer implicated in mitogenesis, survival, and cancer progression, is overexpressed in human primary non-small cell lung cancer (NSCLC). The role of PKCe in lung cancer metastasis has not yet been established. Principal Findings: Here we show that RNAi-mediated knockdown of PKCe in H358, H1299, H322, and A549 NSCLC impairs activation of the small GTPase Rac1 in response to phorbol 12-myristate 13-acetate (PMA), serum, or epidermal growth factor (EGF). PKCe depletion markedly impaired the ability of NSCLC cells to form membrane ruffles and migrate. Similar results were observed by pharmacological inhibition of PKCe with eV1-2, a specific PKCe inhibitor. PKCe was also required for invasiveness of NSCLC cells and modulated the secretion of extracellular matrix proteases and protease inhibitors. Finally, we found that PKCe-depleted NSCLC cells fail to disseminate to lungs in a mouse model of metastasis. Conclusions: Our results implicate PKCe as a key mediator of Rac signaling and motility of lung cancer cells, highlighting its potential as a therapeutic target

ВЛИЯНИЕ ЖЕНСКОЙ АУТОСЫВОРОТКИ КРОВИ НА АЛЛОГЕННЫЕ ВЗАИМОДЕЙСТВИЯ В КРАТКОСРОЧНОЙ КУЛЬТУРЕ ЛИМФОЦИТОВ СУПРУГОВ, ИМЕЮЩИХ ДЕТЕЙ С КОНОТРУНКАЛЬНЫМИ ПОРОКАМИ

Highlights The findings of this original study ensure the detection of violations in the humoral regulation of the maternal immune interactions with semiallogeneic fetus, considered as a risk factor for developing sporadic conotruncal heart malformations in the next generation.Aim To study the role of female autoserum blood in limiting allogeneic interactions in short-term lymphocyte cultures of parents having children with conotruncal heart malformations.Methods 21 married couples (the study group) with children suffering from conotrucnal heart malformations (Tetralogy of Fallot) without chromosomal diseases were examined. The control group consisted of 21 families with three or more healthy children. The immune response in a mixed lymphocyte culture of parents was assessed by the increase in HLA-DR expression in the mixed culture with respect to spontaneous lymphocyte cultures. Primary staining of female and male lymphocytes with monoclonal antibodies to CD45, conjugated with various fluorescent dyes (PC-5 and PC-7), allowed assessing the immune response of female lymphocytes to male and vice versa.Results The effects of female autoserum on the mixed lymphocyte culture of parents were assessed. The obtained results reported that the birth of children with conotruncal heart malformations is associated with the interfering effect of female autoserum on HLA-DR expression on subpopulations of female lymphocytes (CD3+, HLA-DR+) and the activating effect on subpopulations of female lymphocytes (CD3-, HLA-DR+). The observed role of female autoserum in the study group may be associated with the absence of HLA-DR-blocking autoantibodies and high synthesis of cytokines by T2 and T3 helper lymphocytes.Conclusion The effects of female autoserum on allogeneic lymphocyte interactions of parents may be observed in short-term mixed lymphocyte cultures. The evaluation of the activating and interfering effects ensures timely identification of any violations in the humoral regulation of the maternal immune interactions with the HLA semiallogenic fetus, considered as a risk factor for developing sporadic conotruncal heart malformations in the next generation.Основные положения Полученные данные оригинального исследования позволят выявлять нарушения в гуморальной регуляции иммунных взаимодействий матери и полуаллогенного по HLA эмбриона/плода, как фактора риска формирования спорадических пороков конотрункуса в последующем поколении.Цель Изучение роли женской аутосыворотки крови в ограничении аллогенных взаимодействий в краткосрочной культуре лимфоцитов супругов, имеющих детей с пороками конотрункуса.Материалы и методы Для выполнения поставленной цели обследована 21 семейная пара (основная группа), имеющая детей с пороками конотрункуса (тетрада Фалло) без хромосомных заболеваний. Контрольную группу составила 21 семья, имеющая трех и более здоровых детей. Иммунный ответ в смешанной культуре лимфоцитов (СКЛ) супругов оценивали по увеличению экспрессии HLA-DR в смешанной культуре по отношению к спонтанным культурам лимфоцитов. Первичная окраска женских и мужских лимфоцитов моноклональными антителами к CD45, конъюгированными с различными флуоресцентными красителями (PC-5 и PC-7), позволила оценить иммунный ответ женских лимфоцитов на мужские и наоборот.Результаты Проведена оценка эффекта женской аутосыворотки на СКЛ супругов. Результаты исследования показали, что рождение детей с пороками конотрункуса ассоциировано с блокирующим эффектом женской аутосыворотки в отношении экспрессии HLA-DR на субпопуляции женских лимфоцитов (CD3+, HLA-DR+) и активирующим эффектом на эти реакции в отношении субпопуляции женских лимфоцитов (CD3-, HLA-DR+). Эти эффекты женской аутосыворотки в опытной группе могут быть связаны с отсутствием блокирующих HLA-DR аутоантител и высоким синтезом цитокинов Т2- и Т3-хелперными лимфоцитами.Заключение В краткосрочной СКЛ отражаются эффекты женской аутосыворотки крови на аллогенные взаимодействия лимфоцитов супругов. Исследование коэффициента прироста и блокирующего коэффициента позволит выявлять нарушения в гуморальной регуляции иммунных взаимодействий матери и полуаллогенного по HLA эмбриона/плода, как фактора риска формирования спорадических пороков конотрункуса в последующем поколении.

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Improved risk stratification of patients with atrial fibrillation: an integrated GARFIELD-AF tool for the prediction of mortality, stroke and bleed in patients with and without anticoagulation.

OBJECTIVES: To provide an accurate, web-based tool for stratifying patients with atrial fibrillation to facilitate decisions on the potential benefits/risks of anticoagulation, based on mortality, stroke and bleeding risks. DESIGN: The new tool was developed, using stepwise regression, for all and then applied to lower risk patients. C-statistics were compared with CHA2DS2-VASc using 30-fold cross-validation to control for overfitting. External validation was undertaken in an independent dataset, Outcome Registry for Better Informed Treatment of Atrial Fibrillation (ORBIT-AF). PARTICIPANTS: Data from 39 898 patients enrolled in the prospective GARFIELD-AF registry provided the basis for deriving and validating an integrated risk tool to predict stroke risk, mortality and bleeding risk. RESULTS: The discriminatory value of the GARFIELD-AF risk model was superior to CHA2DS2-VASc for patients with or without anticoagulation. C-statistics (95% CI) for all-cause mortality, ischaemic stroke/systemic embolism and haemorrhagic stroke/major bleeding (treated patients) were: 0.77 (0.76 to 0.78), 0.69 (0.67 to 0.71) and 0.66 (0.62 to 0.69), respectively, for the GARFIELD-AF risk models, and 0.66 (0.64-0.67), 0.64 (0.61-0.66) and 0.64 (0.61-0.68), respectively, for CHA2DS2-VASc (or HAS-BLED for bleeding). In very low to low risk patients (CHA2DS2-VASc 0 or 1 (men) and 1 or 2 (women)), the CHA2DS2-VASc and HAS-BLED (for bleeding) scores offered weak discriminatory value for mortality, stroke/systemic embolism and major bleeding. C-statistics for the GARFIELD-AF risk tool were 0.69 (0.64 to 0.75), 0.65 (0.56 to 0.73) and 0.60 (0.47 to 0.73) for each end point, respectively, versus 0.50 (0.45 to 0.55), 0.59 (0.50 to 0.67) and 0.55 (0.53 to 0.56) for CHA2DS2-VASc (or HAS-BLED for bleeding). Upon validation in the ORBIT-AF population, C-statistics showed that the GARFIELD-AF risk tool was effective for predicting 1-year all-cause mortality using the full and simplified model for all-cause mortality: C-statistics 0.75 (0.73 to 0.77) and 0.75 (0.73 to 0.77), respectively, and for predicting for any stroke or systemic embolism over 1 year, C-statistics 0.68 (0.62 to 0.74). CONCLUSIONS: Performance of the GARFIELD-AF risk tool was superior to CHA2DS2-VASc in predicting stroke and mortality and superior to HAS-BLED for bleeding, overall and in lower risk patients. The GARFIELD-AF tool has the potential for incorporation in routine electronic systems, and for the first time, permits simultaneous evaluation of ischaemic stroke, mortality and bleeding risks. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier for GARFIELD-AF (NCT01090362) and for ORBIT-AF (NCT01165710)

Two-year outcomes of patients with newly diagnosed atrial fibrillation: results from GARFIELD-AF.

AIMS: The relationship between outcomes and time after diagnosis for patients with non-valvular atrial fibrillation (NVAF) is poorly defined, especially beyond the first year. METHODS AND RESULTS: GARFIELD-AF is an ongoing, global observational study of adults with newly diagnosed NVAF. Two-year outcomes of 17 162 patients prospectively enrolled in GARFIELD-AF were analysed in light of baseline characteristics, risk profiles for stroke/systemic embolism (SE), and antithrombotic therapy. The mean (standard deviation) age was 69.8 (11.4) years, 43.8% were women, and the mean CHA2DS2-VASc score was 3.3 (1.6); 60.8% of patients were prescribed anticoagulant therapy with/without antiplatelet (AP) therapy, 27.4% AP monotherapy, and 11.8% no antithrombotic therapy. At 2-year follow-up, all-cause mortality, stroke/SE, and major bleeding had occurred at a rate (95% confidence interval) of 3.83 (3.62; 4.05), 1.25 (1.13; 1.38), and 0.70 (0.62; 0.81) per 100 person-years, respectively. Rates for all three major events were highest during the first 4 months. Congestive heart failure, acute coronary syndromes, sudden/unwitnessed death, malignancy, respiratory failure, and infection/sepsis accounted for 65% of all known causes of death and strokes for <10%. Anticoagulant treatment was associated with a 35% lower risk of death. CONCLUSION: The most frequent of the three major outcome measures was death, whose most common causes are not known to be significantly influenced by anticoagulation. This suggests that a more comprehensive approach to the management of NVAF may be needed to improve outcome. This could include, in addition to anticoagulation, interventions targeting modifiable, cause-specific risk factors for death. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362