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14-3-3sigma regulates B-cell homeostasis through stabilization of FOXO1.

14-3-3σ regulates cytokinesis and cell cycle arrest induced by DNA damage but its role in the immune system is unknown. Using gene-targeted 14-3-3σ-deficient (i.e., KO) mice, we studied the role of 14-3-3σ in B-cell functions. Total numbers of B cells were reduced by spontaneous apoptosis of peripheral B cells. Upon B-cell antigen receptor engagement in vitro, KO B cells did not proliferate properly or up-regulate CD86. In response to T cell-independent antigens, KO B cells showed poor secretion of antigen-specific IgM. This deficit led to increased lethality of KO mice after vesicular stomati

[Bifidobacterium DNA upregulates Th1 type response of umbilical cord blood mononuclear cell].

OBJECTIVE: To study the effect of bifidobacterium genomic DNA on umbilical cord blood mononuclear cell (CBMC), and investigate the immunoregulation of bifidobacterium DNA and explore possible mechanisms by which bifidobacterium acts against allergic reaction. METHODS: Bifidobacterium genomic DNA (bDNA) and human DNA (hDNA) were extracted with phenol/chloroform/isoamyl alcohol and stored at -20 degrees C for later use. Parts of bDNA were completely digested with DNaseI (d-bDNA) at 37 degrees C. CBMCs were isolated with Ficoll from umbilical cord blood and incubated at 37 degrees C in a 5% CO2 …

Innovative two-step negative selection of granulocyte colony-stimulating factor-mobilized circulating progenitor cells: adequacy for autologous and allogeneic transplantation.

A major obstacle in purifying either autologous or allogeneic hematopoietic stem cells from granulocyte colony-stimulating factor (G-CSF) mo … PC, rosetting cells (myeloid precursor cells, granulocytes, monocytes, and T cells) were removed by Ficoll-Hypaque density gradient centrifugation with a blood cell processor apparatus, COBE (Lakewood, CO) 2991. After this step, a significant reduction of the initial cellularity was consistently obtained (range, 72% to 97%), whereas the median absolute r …

Optimizing viral and non-viral gene transfer methods for genetic modification of porcine mesenchymal stem cells.

INTRODUCTION: Mesenchymal stem cells (MSCs) provide an excellent source of pluripotent progenitor cells for tissue-engineering applications … ied from bone marrow aspirates from the proximal tibiae of four 3-month-old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of reco …

Expression of thrombospondin receptor (CD36) in B-cell chronic lymphocytic leukemia as an indicator of tumor cell dissemination.

BACKGROUND AND OBJECTIVE: The expression of CD36 antigen has not been conclusively associated with human B-lymphocytes although CD36 was rec … and bone marrow samples from 24 patients affected by CD5+ B-CLL. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation, were labeled with fluorochrome-conjugated monoclonal antibodies under standard experimental conditions and were analyzed by flow cytometry. CD36 expression was quantified both in terms of frequency of CD19+CD …

T-bet regulates T-independent IgG2a class switching.

The IgG2a Ig subclass plays a critical role in the pathogenesis of humoral autoimmunity and protection against pathogens. The T-box transcription factor T-bet has been implicated as a critical mediator of class-switch recombination (CSR) to IgG2a, but its relative importance to this process in various immune contexts remains incompletely defined. We report here that, surprisingly, T-bet is selectively required for IgG2a class switching in response to T-independent, but not T-dependent, stimuli. Specifically, T-dependent signaling through CD40, in contrast to T-independent signaling via lipopol

[Effect of neonatal BCG vaccination on phenotype and function of splenic dendritic cells of BALB/c mice].

OBJECTIVE: The impact of dendritic cells (DCs) and regulatory T cells (Tr) on the pathogenesis of asthma have been investigated over the pas … urity of splenic DCs were analysed. Moreover, cells were further separated into mononuclear cell by Ficoll solution. The mononuclear cells were stimulated by 1 microg/ml lipopolysaccharide (LPS) for 18 or 10(5) CFU /ml BCG for 48 hours at 37 degrees C in a humidified atmosphere containing 5% CO2 and cytokines concentration was detected by ELISA. RESULTS …

[Effects of exogenous interleukin-2, interleukin-23 on differentiation of IRBP 1-20-specific T cells toward Th1 and Th17 and comparison of the pathogenicity of IRBP 1-20-specific T cells].

OBJECTIVES: To explore the effects of exogenous interleukin (IL)-2 and IL-23 on differentiation of IRBP 1-20-specific T cells originated fro … cell and mouse recombinant cytokine IL-2 or IL-23. The IRBP 1-20-specific T cells were separated by Ficoll gradient centrifugation, the expressions of IFN-γ mRNA and IL-17 mRNA were assessed by RT-PCR, and IFN-γ and IL-17 in T cells supernatant were detected using a commercial ELISA kit. The T cells were cultured for another 48 hours, and then the propor …

Beta 2-microglobulin-deficient mice are protected from hypergammaglobulinemia and have defective antibody responses because of increased IgG catabolism.

The goal of this study was to determine whether class I proteins play an important role in the regulation of Ig and to elucidate the mechanism(s) involved. We analyzed the phenotype imposed by a null allele of beta 2-microglobulin (beta 2m). Serum Ig levels of several mouse strains showed a beta 2m dependence that was most evident in mice genetically predisposed to develop chronic systemic lupus erythematosus, was preferential to IgG isotypes, and was greatly exaggerated in aging mice that normally develop hypergammaglobulinemia. Beta 2m-deficient mice, regardless of genetic background, also d

PBMC-Humanized Mouse Model for Multical Sclerosis: Изучение неуменные изменения и участие CNS.

Гуманизированная иммунная система (HIS) мыши являются мощными инструментами для изучения функций и дисфункций иммунной системы человека и разработки иммунотерапевтической системы человека. Наличие изощренных штаммов супериммунодефицитной мыши позволило гуманизировать иммунную систему, используя трансплантацию человеческих периферийных моноядерных клеток крови (PBMC) или гематополиетических стволовых клеток. Его мыши широко используются в иммунной онкологии, в то время как проводится меньше исследований в области автономного иммунитета, особенно в области множественного склероза (MS). Используя

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… Fresh whole peripheral blood is diluted with an equal volume of PBS, gently layered over Ficoll-Histopaque ® -1077. The tube is placed in the centrifuge for gradient centrifugation. The PBMC buffy coat is located at the interface of Ficoll and plasma, as indicated (arrow). 6. … Add 15 mL of Ficoll-Histopaque ® -1077 to three separate clean 50 mL tubes. 7. Carefully overlay the diluted fresh blood over the Ficoll-Histopaque ® -1077. CAUTION: In this step, the overlay has to be done very carefully so that the Ficoll does not mix with the blood. 8. …

Gene expression signatures that predict radiation exposure in mice and humans.

BackgroundThe capacity to assess environmental inputs to biological phenotypes is limited by methods that can accurately and quantitatively measure these contributions. One such example can be seen in the context of exposure to ionizing radiation.Methods and findingsWe have made use of gene expression analysis of peripheral blood (PB) mononuclear cells to develop expression profiles that accurately reflect prior radiation exposure. We demonstrate that expression profiles can be developed that not only predict radiation exposure in mice but also distinguish the level of radiation exposure, rang

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… MNCs were collected from each patient's PB using the identical Ficoll Hypaque methodology described above for mouse MNC collection, and total RNA was isolated. … PB samples (500 μl) were collected from each mouse 6 h following irradiation, PB MNCs were isolated via Ficoll Hypaque centrifugation, and total RNA was extracted from the PB MNCs from each mouse in the experimental groups along with seven nonirradiated controls. …

Transcriptional analysis of BMP2 and BMP6 in patients with newly diagnosed multiple myeloma.

Multiple myeloma (MM) is a highly heterogenous disease with diverse outcomes across patients. Stratifying patients is a promising approach to predict their clinical outcomes. However, despite notable progress in stratification systems, there is still a need for further optimization. Combining the existing staging parameters with molecular approaches could offer a better understanding of 'high risk' disease and guide treatment decisions. Bone morphogenetic proteins (BMPs) represent a group of proteins, which are members of the TGF-β superfamily that are now considered to be multifunctional cyto

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… Bone marrow mononuclear cells (BMMNCs) were isolated using density gradient separation with a Ficoll Paque Plus kit (MilliporeSigma) ( Fig. S1 ). … Bone marrow was diluted 1:1 with DPBS (Biowest) and was filtered through a 100 µm pore to remove cell clumps, clots and bone fragments. 4 ml of diluted cell suspension was carefully layered over 4 ml of Ficoll Paque Plus and an immediate centrifugation step (400 × g at 20°C for 30 min without brake) …

Доклад по делу: Выяснение роли генетических аномалий в миэломатном плевральном эффюляции: случай с несколькими миеломами типа IgG Lambda.

A 73-year-old Мужчина с многократной миеломой, с постепенно ухудшающейся болью в левом плече, которая не реагировала на консервативное управление. После проведения всеобъемлющих диагностических процедур, включая съемку и генетическое тестирование, у него был диагностирован множественный миэлома типа IgG Lambda, осложненный развитием миэломатного плеврального эффюля (MPE). Лечение пациента состояло из сочетания химиотерапии PDD, включая Бортезомиб, Доксорубицин и Дексаметазон, наряду с адъюнктивными терапиями, направленными на контроль плевральной эфлуции. Оценка после обработки выявила разреше

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… Flow cytometry analysis of bone marrow cell immunophenotype Mononuclear cells were isolated from patient bone marrow samples using Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation. … This figure presents the flow cytometry immunophenotype analysis of abnormal plasma cells from a patient with Multiple Myeloma (MM) using a bone marrow aspirate sample separated into mononuclear cells via Ficoll. Each subplot represents different combinations of immune markers tested. …

Цирсадианская ритмичность симптомов фенотипов при множественном склерозе: протокол исследования CircaMS и осуществимость сбора биомаркеров.

ВВЕДЕНИЕ: Множественный склероз (MS) представляет собой хроническое аутоиммунное неврологическое заболевание с переменным прогнозом и непредсказуемым курсом. Усталость, боль и слабое настроение являются обычными симптомами, которые, как правило, колеблются в людях с MS (PwMS). Вариации симптомов могут быть связаны с нарушением циркадианских ритмов. Различие межиндивидуальных различий и временных ежедневных колебаний симптомов MS может помочь определить конкретные симптоматические фенотипы. Понимание того, каким образом эти фенотипы связаны с качеством жизни и их иммунологическими основами - им

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… On arrival, human PBMCs and PMNs are isolated using a Ficoll gradient following published protocols. 39 Briefly, the Ficoll gradient is performed following the manufacturer’s instruction; the buffy coat is collected and PMNs are isolated by treating the granulocyte–erythrocyte layer using a red blood …

Regulatory Effect and Mechanism of Erythroblastic Island Macrophages on Anemia in Patients with Newly Diagnosed Multiple Myeloma.

ObjectiveTo examine the clinical characteristics and anemia-related factors in patients with newly diagnosed multiple myeloma (NDMM), as well as the effect and mechanism of erythroblastic islands (EBIs) and EBI macrophages in NDMM patients with anemia.MethodsWe collected and analyzed clinical data to find anemia-related factors. Using flow cytometry, the numbers and ratios of erythroblasts and EBI macrophages were determined. RNA sequencing (RNA-seq) was used to determine the differences of EBI macrophages in NDMM patients with or without anemia.ResultsBased on the clinical characteristics of

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… Using a lymphocyte separation medium (human, Ficoll-Paque), mononuclear cells were obtained according to the manufacturer’s instructions. 14 The bone marrow was diluted with 5 mL of phosphate buffered saline (PBS) and 5 mL of Ficoll-Paque was added in a 15 mL tube, which was then centrifuged at 400 g …

Daratumumab plus bortezomib and dexamethasone versus bortezomib and dexamethasone in relapsed or refractory multiple myeloma: updated analysis of CASTOR.

Daratumumab, a CD38 human monoclonal antibody, demonstrated significant clinical activity in combination with bortezomib and dexamethasone versus bortezomib and dexamethasone alone in the primary analysis of CASTOR, a phase 3 study in relapsed and/or refractory multiple myeloma. A post hoc analysis based on treatment history and longer follow up is presented. After 19.4 (range: 0-27.7) months of median follow up, daratumumab plus bortezomib and dexamethasone prolonged progression-free survival (median: 16.7 versus 7.1 months; hazard ratio, 0.31; 95% confidence interval, 0.24-0.39; Pversus 63.2

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… MRD-negative status was evaluated at a sensitivity threshold of 10 −5 using bone marrow aspirate samples that were prepared using Ficoll and analyzed by the clonoSEQ ® assay. MRD: minimal residual disease; D-Vd: daratumumab plus bortezomib and dexamethasone; Vd: bortezomib and dexamethasone. … MRD-negative status was evaluated over time at a sensitivity threshold of 10 −5 using bone marrow aspirate samples that were prepared using Ficoll and analyzed by the clonoSEQ ® assay. …

Identification of the Distinct Immune Microenvironment Features Associated with Progression Following High-Dose Melphalan and Autologous Stem Cell Transplant in Multiple Myeloma.

A key treatment for patients with multiple myeloma is high-dose melphalan followed by autologous stem cell transplant (ASCT). It can provide a deep response with long-term remission. However, some patients progress quickly, and it is not clear why. In this study, we performed single-cell RNA and T-cell receptor sequencing of the immune microenvironment of 40 patients before and after ASCT to determine if differences in the immune composition could define those who would progress. Clear differences in cell populations were identified in progressors, including increased T-cell infiltration, decr

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… Granulocytes were removed from the analysis for comparison with single-cell data in which samples had undergone Ficoll separation. … Granulocytes were removed from the flow panel data to match the single-cell samples, which had undergone Ficoll separation. …

GBA1 as a risk gene for osteoporosis in the specific populations and its role in the development of Gaucher disease.

BACKGROUND: Osteoporosis and its primary complication, fragility fractures, contribute to substantial global morbidity and mortality. Gaucher disease (GD) is caused by glucocerebrosidase (GBA1) deficiency, leading to skeletal complications. This study aimed to investigate the impact of the GBA1 gene on osteoporosis progression in GD patients and the specific populations. METHODS: We selected 8115 patients with osteoporosis (T-score ≤ - 2.5) and 55,942 healthy individuals (T-score > - 1) from a clinical database (N = 95,223). Monocytes from GD patients were evaluated in relation to endoplasmic

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… Peripheral blood mononuclear cells (PBMCs) were isolated from samples collected in EDTA tubes from patients with GD and HDs using a gradient centrifugation procedure with Ficoll-Paque (GE Healthcare Life Sciences)[ 16 ]. … The Ficoll-Paque–plasma interface was used to isolate PBMCs, which were then washed in RPMI-1640 medium containing 10% fetal calf serum (FCS) and resuspended in RPMI-FCS (500× g , 10 min, and 4 °C). …

Unraveling potential gene biomarkers for dengue infection through RNA sequencing.

Dengue virus hijacks host cell mechanisms and immune responses in order to replicate efficiently. The interaction between the host and the virus affects the host's gene expression, which remains largely unexplored. This pilot study aimed to profile the host transcriptome as a potential strategy for identifying specific biomarkers for dengue prediction and detection. High-throughput RNA sequencing (RNA-seq) was employed to generate host transcriptome profiles in 16 dengue patients and 10 healthy controls. Differentially expressed genes (DEGs) were identified in patients with severe dengue and t

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… PBMC isolation and total RNA extraction Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples using Ficoll-Paque density gradient centrifugation (GE Healthcare) as outlined by Boyum [ 3 ] with some modifications. … Next, 8 ml of Ficoll-Paque (GE Healthcare) which has been brought to room temperature was pipetted carefully to the diluted blood without mixing the solution and subjected to centrifugation for 20 min at 400 g using the “soft” mode and inactivated brake. …

Novel_circ_003686 regulates the osteogenic differentiation of MSCs in patients with myeloma bone disease through miR-142-5p/IGF1 axis.

ObjectivesCirc_003686 is a novel_circRNA with abnormally low expression found in the samples of multiple myeloma bone disease (MBD) patients. The current research intended to investigate the effects of novel_circ_003686 in osteogenesis-induced differentiation of bone marrow mesenchymal stem cells (BMSCs) in MBD.MethodsBMSCs were extracted from MBD patients and normal participants, the pcDNA3.1 encoding the circ_003686 (ov-circ_003686), miR-142-5p-mimic/inhibitor and siRNA oligonucleotides targeting insulin like growth factor 1 (IGF1, si-IGF1) were applied to intervene circ_003686, miR-142-5p a

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… Isolation and identification of BMSCs Ficoll (Sigma, USA) was used to isolate BMSCs from MBD patients (MBD-BMSC) or normal participants (normal-BMSC). … The bone marrow fluid was collected, and the same amount of Ficoll was added, centrifuged at room temperature for 20 min at 450 × g , the upper layer of separation fluid was removed to obtain monocytes. …