Ficoll Gradient Centrifugation Research Articles

Recombinant Human Thrombopoietin Reduces the Risk of Acute Graft-Versus-Host-Disease and Its Mechanisms

Objective: Acute graft-versus-host-disease (aGVHD) is an important complication that is observed after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We aimed to evaluate the effects of recombinant human thrombopoietin (TPO; rhTPO) on aGVHD using clinical data, mice models, and peripheral blood mononuclear cells (PBMCs). Methods: From 2016 to 2018, 162 cases of allo-HSCT in our hospital were recruited in this study. The correlation between TPO use and aGVHD was analyzed. After a GVHD model was constructed in Balb-C mice, HE staining was performed to compare the pathophysiological changes of the liver, intestine, lung, kidney, and other organs of the mice. The levels of lymphocyte subsets and cytokines in the mice spleens in the different groups were detected. PBMCs were isolated from the peripheral blood of healthy persons and patients with allo-HSCT via Ficoll gradient centrifugation and treated with different concentrations of rhTPO. The proliferation of PBMCs was detected via the CCK8 method, and the differentiation of PBMCs into lymphocytes was analyzed via flow cytometry. Results: 1) The occurrence of aGVHD was observed in 10 patients (47.6%) in the control group and 27 (23.1%) in the treatment group. The difference between the two groups was statistically significant (χ2=5.465, P=0.019). 2) The results of the aGVHD mice proved that rhTPO decreased the damage to the mouse liver, intestine, lung, and kidney tissues. In addition, splenic cell subpopulations CD4+cells, CD8+cells, T cells, and NK cells increased in the GVHD group but decreased due to rhTPO (P<0.05). After rhTPO treament, T cell subpopulations Th2 and Th17 cells decreased in the GVHD mice (P<0.05). Cytokines CD8+IL-17A+ and CD8+IL-4+IFNγ showed no difference between the four groups (P>0.05). Compared to GVHD mice, CD8+IL-4+ decreased in TPO-treated GVHD mice, and the effect of TPO at a high dose was more significant (P<0.05). 3) The results of the PMBCs suggested that a low dose of rhTPO could promote the proliferation of PMBCs, while a high dose could inhibit the proliferation. rhTPO promoted the differentiation of PBMCs into the CD3-PC5 subset. Conclusions: Altogether, this study indicates that rhTPO reduces the risk of aGVHD and is a protective factor against the occurrence of aGVHD.

Abstract 2995: Cross-presented serine proteases as immunotherapy targets for lung cancer and osteosarcoma

Abstract Background: Immunotherapy is the standard of care for many solid tumor malignancies. However, due to a paucity of known tumor antigens in non-small cell lung cancer (NSCLC) and osteosarcoma (OS), identification of novel immunogenic antigens is needed. Our group has experience with neutrophil serine proteases (NSP) derived from the azurophilic granules of polymorphonuclear leukocytes (PMN), specifically, cathepsin (CG), proteinase 3 (P3), and neutrophil elastase (NE). We have identified two HLA-A2 (HLA-A*0201)-restricted nonameric peptides: CG1 (FLLPTGAEA), derived from CG, and PR1 (VLQELNVTV), derived from NE and P3. We have previously shown that CG1 and PR1 are effective targets in hematological malignancies and that PR1 can be a target for some solid tumor malignancies. Here, we extend our findings to demonstrate that CG1 and PR1 can be immunotherapeutic targets for NSCLC and OS. Methods: Healthy donor PMN were isolated from buffy coats using double Ficoll gradient centrifugation. To evaluate uptake, malignant cells were pulsed with PMN lysates, harvested, permeabilized, and stained with anti-CG, anti-NE or anti-P3. To evaluate cross-presentation, cells were cultured with PMN lysates, harvested and stained with CG1 TCR-like antibody, or PR1 TCR-like antibody (8F4). Flow cytometry was performed using the Fortessa X-20 Cell Analyzer. CG1- or PR1-specific cytotoxic T-lymphocyte (CTL) were expanded from healthy donor peripheral blood mononuclear cells. Standard cytotoxicity assays were used to evaluate the lysis of target cells co-cultured with CG1- or PR1-specific CTL. Results: We have previously demonstrated that NE and P3 are taken up and cross-presented by NSCLC and become targets for PR1-CTL, thus in this study, we investigate if NSCLC cells take up CG, and whether OS cells take up NE and P3, resulting in enhanced cytotoxicity by peptide-specific CTLs. We first employed The Cancer Cell Line Encyclopedia to show the absence of NSP’s in NSCLC and OS cell lines. RT-PCR confirmed lack of endogenous CG mRNA expression in NSCLC and OS, and lack of NE and P3 in OS cell lines. Next, we analyzed antigen cross-presentation and showed that NSCLC cells take up and cross-present CG, leading a 13.3-fold increase in CG1-CTL killing of NSCLC cells pulsed with CG versus non-pulsed cells. In addition, OS cells take up and cross-present NE and P3, with a 13.1-fold increase in PR1-CTL killing of OS cells that were pulsed with NE and P3 vs. non-pulsed cells. Conclusion: Our data define two novel HLA-A2 restricted peptides that can be targeted in NSCLC and OS. Specifically, CG1 and PR1 are presented on the surfaces of NSCLC and OS, increasing their susceptibility to killing by CG1 and PR1 specific CTL. Our work represents a novel link between innate and adaptive immune responses in solid tumors and identifies potential antigen targets for CTL, peptide vaccines, and TCR-like antibodies in the setting of NSCLC and OS. Citation Format: Amber Gibson, Pariya Sukhumalchandra, Celine Kerros, Na Qiao, Anne Philips, Mao Zhang, Sami Alkoutami, Scott Semelsberger, Yoshimi Lu, Lauren Byers, Ze Tian, Chunhua Shi, Jun Yan, Dongxing Zha, Alejandro Marinos, Anne Sergeeva, Hong He, Lisa St. John, Jeffrey J. Molldrem, Gheath Alatrash. Cross-presented serine proteases as immunotherapy targets for lung cancer and osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2995.

Blood Inflammatory-like and Lung Resident-like Eosinophils Affect Migration of Airway Smooth Muscle Cells and Their ECM-Related Proliferation in Asthma

Airway remodeling is a hallmark feature of asthma, and one of its key structural changes is increased airway smooth muscle (ASM) mass and disturbed extracellular matrix (ECM) homeostasis. Eosinophil functions in asthma are broadly defined; however, we lack knowledge about eosinophil subtypes' interaction with lung structural cells and their effect on the airway's local microenvironment. Therefore, we investigated the effect of blood inflammatory-like eosinophils (iEOS-like) and lung resident-like eosinophils (rEOS-like) on ASM cells via impact on their migration and ECM-related proliferation in asthma. A total of 17 non-severe steroid-free allergic asthma (AA), 15 severe eosinophilic asthma (SEA) patients, and 12 healthy control subjects (HS) were involved in this study. Peripheral blood eosinophils were enriched using Ficoll gradient centrifugation and magnetic separation, subtyped by using magnetic separation against CD62L. ASM cell proliferation was assessed by AlamarBlue assay, migration by wound healing assay, and gene expression by qRT-PCR analysis. We found that blood iEOS-like and rEOS-like cells from AA and SEA patients' upregulated genes expression of contractile apparatus proteins, COL1A1, FN, TGF-β1 in ASM cells (p < 0.05), and SEA eosinophil subtypes demonstrated the highest effect on sm-MHC, SM22, and COL1A1 gene expression. Moreover, AA and SEA patients' blood eosinophil subtypes promoted migration of ASM cells and their ECM-related proliferation, compared with HS (p < 0.05) with the higher effect of rEOS-like cells. In conclusion, blood eosinophil subtypes may contribute to airway remodeling by upregulating contractile apparatus and ECM component production in ASM cells, further promoting their migration and ECM-related proliferation, with a stronger effect of rEOS-like cells and in SEA.

The density of bone marrow mononuclear cells and CD34+ cells in patients with three neurologic conditions

BackgroundThis study aimed to identify the density of mononuclear cells (MNCs) and CD34+ cells in the bone marrow of patients with three neurologic conditions.MethodsThe study included 88 patients with three neurologic conditions: 40 with cerebral palsy (CP) due to oxygen deprivation (OD), 23 with CP related to neonatal icterus (NI), and 25 with neurological sequelae after traumatic brain injury. Bone marrow aspiration was conducted from the patients’ bilateral anterior iliac crest under general anesthesia in an operating theater. MNCs were isolated by Ficoll gradient centrifugation and then infused intrathecally.ResultsThere was a significant difference in the average MNC per ml and percentage of CD34+ cells by the type of disease, age group, and infusion time (p value < 0.05). The multivariable regression model showed the percentage of CD34+ association with the outcome (gross motor function 88 items- GMFM-88) in patients with CP.ConclusionsThe density of MNCs was 5.22 million cells per mL and 5.03% CD34+ cells in patients with three neurologic conditions. The highest density of MNCs in each ml of bone marrow was found in patients with CP due to OD, whereas the percentage of CD34+ cells was the highest among patients with CP related to NI.

Predictive value of TRPV2 expression from peripheral blood mononuclear cells on the early recurrence of atrial fibrillation after radiofrequency catheter ablation

BackgroundRecent study has shown that the transient receptor potential vanilloid 2 (TRPV2) channel was exclusively upregulated in patients with atrial fibrillation (AF), and that this overexpression might be detrimental for occurrence and maintenance of AF. We aimed to characterize the expression levels of TRPV2 mRNA in peripheral blood mononuclear cells (PBMCs) with/without early recurrence of atrial fibrillation (ERAF) after radiofrequency catheter ablation (RFCA), and to find a reliable predictor for ERAF.Methods65 patients of AF, who underwent RFCA successfully, then divided into two groups according to ERAF during following 3 months. PBMCs were isolated from whole blood by Ficoll gradient centrifugation before and after RFCA. Gene set enrichment analysis was performed to evaluate TRPV channels expression levels and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping was used for pathway enrichment analysis.ResultsThere was no significant difference in the TRPV2 mRNA expression level between the two groups before RFCA, while without ERAF group of TRPV2 expression was markedly reduced compared to ERAF group after RFCA. Moreover, the number of TRPV2 expression was confirmed as an independent predictor for the first time through receiver operating characteristic and Kaplan–Meier survival curve analysis. It should be pointed out that the above results were only used to predict ERAF, and have no predictive significance for late recurrence of atrial fibrillation according to the current data. Additionally, ERAF was inversely correlated with P wave dispersion. KEGG mapping further clustered 41 pathways, revealing that ‘‘cyclic guanosine monophosphate-protein kinase G signaling pathway’’ was significantly enriched.ConclusionsWe firstly assume that downregulated expression of peripheral TRPV2 appear in patients without ERAF after RFCA. TRPV2 may thus represent a novel predictor of early phase after successful radiofrequency ablation.

Comparison of mitogen-induced proliferation in child and adult healthy groups by flow cytometry revealed similarities.

The proliferation of antigen-specific lymphocyte clones, the initial step in acquired immunity, is vital for effector functions. Proliferation tests both in immunology research and diagnosis are gaining attendance gradually, while the use of adult healthy individuals as controls of pediatric patients is a question. This study aimed to investigate and compare mitogen-stimulated proliferation responses of total lymphocytes and T- and B-lymphocyte subsets in adult and children healthy donors. Nineteen children and 20 adult healthy donors were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) purified from peripheral blood samples of the donors, by Ficoll gradient centrifugation, were stained with CFSE and were cultured in a 37℃ CO2 incubator for 120h with the absence or existence of polyclonal activators: PHA and CD-Mix. After cell culture, PBMCs were stained with monoclonal antibodies against CD4 and CD19, and proliferation percentages of CD4+ T and CD19+ B cells, together with total lymphocytes were determined by flow cytometry. This study revealed similarities between children and adult age groups, concerning mitogenic stimulation of the lymphocytes. The only difference was a significantly high proliferation of pediatric CD4+ T cells in response to PHA. CD4+ T cell responses against PHA were inversely correlated with altering age. When pediatric individuals were distributed into age groups of 0-2years, 3-5years, and 6-18years, PHA responses of CD4+ cells were found to be diminished with advancing age. These findings propose the possibility of enrollment of adult healthy individuals as controls for pediatric patients.

Effect Of Exposure Of Monosadium Glutamate (MSG) on Viability Of Monocyte Cells

The consumption rate of Monosodium Glutamate (MSG) in Indonesia has increased every year. Uncontrolled use of MSG in Indonesia for a long period of time can cause toxic effects on the body. The free glutamate content produced by MSG can affect the work of the immune system, especially in the innate immune system and cause oxidative stress. To determine the effect of exposure to Monosodium Glutamate (MSG) on the viability of monocyte cells. This study is a laboratory experimental in vitro with a post test only control group design. A total of 10cc of peripheral venous blood was isolated using the ficoll gradient centrifugation method. The results of monocyte cell isolates were exposed to Monosodium Glutamate (MSG) according to groups. Group I: negative control, group II: monocyte cells + MSG 3%, group III: monocyte cells + MSG 6%, group IV: monocyte cells + MSG 9%. Subsequently incubated for 24 hours at 37 °C in 5% CO2. Then the viability test was carried out using trypan blue staining. Monocyte cell viability calculations were carried out under an inverted microscope with a magnification of 400x per 100 cells. The data obtained were analyzed statistically using the one-way Anova test followed by the LSD test. The average viability in each group was obtained as follows, monocyte cell viability in the control group was 63%, group II was 47%, group III was 45% and group IV was 35%. There is an effect of exposure to Monosodium Glutamate (MSG) on the viability of monocyte cells with the most significant effect being the 9% MSG concentration with an average viability of 35%.The consumption rate of Monosodium Glutamate (MSG) in Indonesia has increased every year. Uncontrolled use of MSG in Indonesia for a long period of time can cause toxic effects on the body. The free glutamate content produced by MSG can affect the work of the immune system, especially in the innate immune system and cause oxidative stress. To determine the effect of exposure to Monosodium Glutamate (MSG) on the viability of monocyte cells. This study is a laboratory experimental in vitro with a post test only control group design. A total of 10cc of peripheral venous blood was isolated using the ficoll gradient centrifugation method. The results of monocyte cell isolates were exposed to Monosodium Glutamate (MSG) according to groups. Group I: negative control, group II: monocyte cells + MSG 3%, group III: monocyte cells + MSG 6%, group IV: monocyte cells + MSG 9%. Subsequently incubated for 24 hours at 37 °C in 5% CO2. Then the viability test was carried out using trypan blue staining. Monocyte cell viability calculations were carried out under an inverted microscope with a magnification of 400x per 100 cells. The data obtained were analyzed statistically using the one-way Anova test followed by the LSD test. The average viability in each group was obtained as follows, monocyte cell viability in the control group was 63%, group II was 47%, group III was 45% and group IV was 35%. There is an effect of exposure to Monosodium Glutamate (MSG) on the viability of monocyte cells with the most significant effect being the 9% MSG concentration with an average viability of 35%.

Evidence for the effect of soluble uric acid in augmenting endoplasmic reticulum stress markers in human peripheral blood mononuclear cells.

There is evidence regarding the association of hyperuricemia with inflammatory disorders. Hence, it has been of particular interest to dissect the exact role of alteration in uric acid (UA) levels in the context of inflammation. Recently, the endoplasmic reticulum (ER) stress pathway has come into the forefront as a possible mechanism linking hyperuricemia to inflammation. Here, we intended to examine the role of UA in the presence or absence of a second stimulus, LPS, in human peripheral blood mononuclear cells (PBMCs), and analyzed ROS production as well as expression of ER stress markers: GRP78 and CHOP, and inflammatory cytokines.PBMCs were isolated using Ficoll gradient centrifugation from healthy volunteers. Cell viability was measured by MTT assay. PBMCs were treated with an increasing concentration of soluble UA (0, 5, 12, and 20mg/dl) for 20h, followed by the addition of 100ng/mL of LPS or vehicle for another 4h. Real time-PCR was performed to investigate the mRNA expression of GRP78, CHOP, TNF-α, IL-1β, and IL-6, and western blot was used to investigate the protein levels of GRP78 and CHOP. Moreover, ELISA was used to evaluate the protein levels of TNF-α, IL-1β, and IL-6. Finally, intracellular ROS production was determined using fluorescent probes (DCFH-DA).High concentrations of UA either alone or combined with LPS increased the protein levels of GRP78 and CHOP. On the other hand, LPS alone increased the protein levels of GRP78 and CHOP. However, there was no significant difference between the mRNA expression of GRP78, CHOP, TNF-α, IL-1β, and IL-6 when PBMCs were treated with UA. High concentrations of UA augmented LPS-stimulated IL-1β transcript and protein levels as well as TNF-α protein levels in PBMC culture. Moreover, high concentrations of UA along with LPS significantly increased intracellular ROS production.It seems that a high concentration of UA not only induces the protein levels of ER stress markers in PBMCs but also augments the impact of LPS on the levels of pro-inflammatory markers and ROS production.

Development of a Method to Detect Mycobacterium paratuberculosis in the Blood of Farmed Deer Using Actiphage® Rapid.

Mycobacterium avium subsp paratuberculosis (MAP) is the causative agent of Johne's disease, which is an economically and clinically relevant pathogen for commercial deer production. The purpose of this study was to develop a method that could be used to rapidly detect MAP infection in deer using the Actiphage Rapid blood test. This test has previously been used to detect MAP in cattle blood following the purification of buffy coat using Ficoll gradients, however this method is quite laborious and costly. The purpose of this study was to develop a simpler method of blood preparation that was also compatible with deer blood and the Actiphage test. Initially differential lysis of RBCs using Ammonium Chloride-Potassium (ACK) blood lysis buffer was compared with the Ficoll gradient centrifugation method using cattle blood samples for compatibility with the Actiphage reagents, and it was found that the simpler ACK method did not have an impact on the Actiphage test reagents, producing an equivalent sensitivity for detection of low levels of MAP. When the two methods were compared using clinical blood samples from farmed deer, the ACK lysis method resulted in a cleaner sample. When a blinded test of 132 animals from 4 different production groups was carried out, the majority of the positive test results were found to be from animals in just one group, with a small number identified in a second group. The test results were found to be reproducible when a small set of positive animals were tested again 1 month after their initial testing. Finally a set of negative animals which had been previously screened using an ELISA test, all animals gave a negative Actiphage result. This study shows that this improved sample preparation method and Actiphage blood testing can be used to test blood samples from deer, and the full diagnostic potential of the method can now be evaluated.

Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells

Abstract Objective: In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) is an important and reliable measure of immunocompetence. PBMC can be stimulated directly after isolation or frozen for later use. However, cryopreservation may affect cell recovery, viability and functionality. This study aims to investigate cytokine synthesis in ex-vivo stimulated fresh and cryopreserved CD4+ and CD4- T cells. Methods: PBMCs were obtained by Ficoll gradient centrifugation from heparinized peripheral blood of 6 middle-aged clinically healthy subjects. Half of these cells (labeled “Fresh”) was further processed and the other half (labeled “Cryo”) was cryopreserved at -140°C for up to 3 months. Fresh-PBMCs were activated with Phorbol-Myristate-Acetate/Ionomycin/Monensin for 5 hours immediately after isolation while Cryo-PBMCs were identically activated after thawing and cell resting. Activated cells were fixed, permeabilized and intracellular cytokine staining was performed using Phycoerythrin (PE)-conjugated antibodies for Interleukin-2 (IL-2), Tumor Necrosis Factor-alpha (TNF-a), and Interferon-gamma (IFN-g). All samples were analyzed within 24 hours by flow cytometry. Results: Both Fresh and Cryo CD3+CD4+/CD3+CD4- sub-populations partially produced each of the three cytokines. A higher percentage of CD4+ T cells produced IL-2 and TNF-a and a greater percentage of CD4- T cells were found to produce IFN-g. A significantly higher percentage of Cryo-lymphocytes was shown to produce TNF-a in both CD3+CD4+ (31.4% vs 24.9%, p=0.031) and CD3+CD4- (22.7% vs 17.9%, p=0.031) subpopulations. No notable difference was found for IL-2 and IFN-g production between Fresh and Cryo T cells. Conclusion: Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.

Increased regulatory T cells in peripheral blood of children with eosinophilic esophagitis.

Considering the allergic basis of Eosinophilic esophagitis (EoE), this study was conducted to evaluate peripheral blood Tregs in children with EoE. Eosinophilic esophagitis (EoE) is an allergic inflammatory disease of gastrointestinal tract. Regulatory T cells (Tregs) have a confirmed role in allergic disorders. Children with EoE, gastroesophageal reflux disease (GERD) and healthy controls (HC) (10 subjects in each group) were recruited after diagnosis by a pediatric gastroenterologist and allergist. After obtaining informed written consent, peripheral blood was obtained. Peripheral blood mononuclear cells were isolated by Ficoll gradient centrifugation. Flowcytometry was used to enumerate peripheral blood Tregs (CD4 +CD25 +FOXP3+ gated lymphocytes were considered as Tregs). CD4+ gated lymphocytes significantly increased in EoE and GERD groups compared to HC group (p= 0.018). Tregs also was significantly increased in EoE in comparison to HC group (p=0.016). There were no statistically significant differences in Tregs of EoE as compared to GERD subjects (p=0.085). Peripheral blood Tregs increase in patients with EoE as compared to healthy controls, which may be indicative of a feedback mechanism to regulate inflammatory responses.

Chromatin Accessibility Identifies Regulatory Elements Predictive of Oncogene Expression in Multiple Myeloma

Chromatin Accessibility Identifies Regulatory Elements Predictive of Oncogene Expression in Multiple Myeloma

Outcomes of Bone Marrow-Derived Mononuclear Cell Transplantation for Patients in Persistent Vegetative State After Drowning: Report of Five Cases.

Aim: Anoxic brain injury (ABI) due to non-fatal drowning may cause persistent vegetative state (VS) that is currently incurable. The aim of this paper is to present the safety and feasibility of autologous bone marrow-derived mononuclear cell (BMMNC) transplantation in five drowning children surviving in persistent VS.Methods: We used BMMNC as a novel candidate therapeutic tool in a pilot phase-I study for five patients affected by neurological sequelae after near-death drowning. Autologous BMMNCs were freshly isolated using Ficoll gradient centrifugation then infused intrathecally to five patients. The number of transplantation varied from two to four times depending on the motor function improvement of patient after transplantation. Clinical therapeutic effects were evaluated using gross motor function measure and muscle spasticity rating scales, cognitive assessments, and brain MRI before and after cell administrations.Results: Six months after BMMNC transplantation, no serious complications or adverse events were reported. All five patients displayed improvement across the major parameters of gross motor function, cognition, and muscle spasticity. Three patients displayed improved communication including the expression of words. In particular, one patient remarkably reduced cerebral atrophy, with nearly normal cerebral parenchyma after BMMNC transplantation.Conclusions: Autologous BMMNC transplantation for the treatment of children in persistent VS after drowning is safe, feasible, and can potentially improve motor function and cognition and reduce muscle spasticity. These results pave the way for a future phase II clinical trial to evaluate the efficacy of the therapy.

Effects of WNT5A signaling-mediated inflammation of macrophages upon cartilage homeostasis

Purpose: To construct and comparehumanbone marrow-derived mesenchymal stem cells (MSCs) two dimension (2D)and bone marrow aspirates in vitrtranswell separated co-cultural models with recombinant adeno-associated virus (rAAV)-Wnt5a-modified activated macrophages and investigate the effect of Wnt5a signaling upon cartilage homeostasis through the regulation of macrophage pro-inflammatory responses. Methods: Macrophages, MSCs,and bone marrow aspirates specimens were extracted from 6 patients with severe knee deformities undergoing total knee arthroplasty (2 males, 4 females, 58-71 years old). The synovial tissues of knee joints were exposed to type II collagenase and obtained single cell suspensions, and the purity of macrophages was determined by Ficoll gradient centrifugation and anti-CD14 antibody flow cytometry. The macrophages were transduced with rAAV-lacZ or Wnt5a respectively for 48 hours prior to the stimulation of IFN-γ combined TNF-α for 24 hours, and subsequently transferred to the upper compartment of the 96-well transwell plate, with hMSCs 2D monolayer, and bone marrow aspirates at the lower compartment, respectively (Figure 1 & 2). Cell morphology and proliferation (cellularity), viral transfection efficiency and secretion of proinflammatory cytokine were estimated by Haemotoxylin and Eosin (H&E) staining, toluidine blue staining, X-gal staining, anti-Wnt5a, anti-II and X collagen immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) assay, respectively. Results: The isolated synovial macrophages were positive against anti-CD14 primary antibody, with a percentage of 90.31%, indicating that the cells were macrophages. The level of monocyte chemotactic protein-1 in supernatants was significantly increased after stimulation with IFN-γ and TNF-α, indicating that the macrophages were activated and at proinflammatory condition (Figure 3). After transduction with rAAV-lacZ for 3 days, X-gal staining indicated that lacZ gene transfer could efficiently transducethe activated macrophages, with the efficiency over 97.50% for at least 21 days. rAAV-Wnt5a-modified macrophages enhanced the intrinsic expression of Wnt5a, stimulated the extrinsic expression of Wnt5a in the above two models and inhibited the secretion of MCP-1. The expression of MCP-1 in Wnt5a group was 14.76 and 61.51 pg/ml, respectively (F = 6.78 and F = 4.67, p<0.05 or 0.01). In addition, rAAV-Wnt5a gene transfer could promote cell proliferation (F = 42.15 and F = 99.38, p<0.05 or 0.01) and chondrogenic differentiation and cartilage matrix synthesis,with a statistical significance in toluidine blue (F = 5.05, p<0.05 or 0.01) and immunohistochemical staining against type II and type X collagen (F = 20.65 and 26.47, p<0.05 or 0.01). In the 2D single-layer transwell co-culture model, the cell density of the Wnt5a group was 37.75 / μm2, with 2.40, 2.19 and 2.16 times comparing the remaining 3 groups respectively (Figure 4), while the cell proliferation capacity of the bone marrow aspirates transwell co-culture model in Wnt5a group was significantly enhanced to 99.28/ μm2, with 1.59, 1.73 and 1.91 times comparing the remaining 3 groups respectively. Moreover, Wnt5a transfection also promotes the expression of type II and Type X collagen in the bone marrow aspirates transwell co-culture model, with semi-quantitative analysis of 93.50 and 61.50, respectively, with 1.45, 1.56, 1.41 and 3.24, 3.08, 2.80 times comparing to lacZ and lacZ + KN93 and Wnt5a + KN93 group respectively (Figure 5). Conclusions: Under the condition of macrophage and MSCs 2D monolayer or aspirates transwell co-culture, rAAV-mediated overexpression of Wnt5a can promote the maintenance of cartilage homeostasis and chondrogenic differentiation of MSCs via macrophage inflammatory response,macrophages may affect cartilage homeostasis and MSCs chondrogenesis through Wnt5a signaling pathway.Fig. 2View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Variability of ex-vivo stimulated T-cells secretory profile in healthy subjects

Abstract Peripheral blood lymphocytes (PBL) are able to synthesize various cytokines that play key roles in the immune response and intercellular signaling. Since alterations in cytokine production and/or activity occur in many pathological processes, the study of cytokine synthetic capacity of PBL is a valuable tool for assessing the immune profile. In this paper, we aimed to investigate the variability of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ) synthetic capacity of CD4+/CD8+ T-cells stimulated ex-vivo in healthy subjects, by means of a commercial intracellular cytokine staining (ICS) protocol. Peripheral blood mononuclear cells were isolated from 16 healthy subjects by Ficoll gradient centrifugation and activated ex-vivo with PMA/Ionomycin/Brefeldin-A for 4 hours. Activated PBL were surface-stained for CD3/CD4/CD8, fixed and permeabilized. ICS was performed using anti-human IL-2/TNF-α/IFN-γ and samples were analyzed on a BD-FACSAria-III flow cytometer. We recorded high post-isolation and post-activation mean viabilities: 82.1% and 82.4% respectively, p=0.84. Both CD4+/CD8+ subpopulations were found to partially produce each of the three cytokines, but in different proportions. On average, a significantly greater percentage of CD4+ cells was shown to produce IL-2 and TNF-α, compared with CD8+ cells (61.5%+/-5.8 vs. 25%+/-5.6 and 26.9%+/-11 vs. 7.5%+/-3.3 respectively, p---lt---0.0001 for both). Contrarily, IFN-γ was produced by a higher proportion of CD8+ cells (8.4%+/-3.9 vs. 6.8%+/-3.2, p=0.01). These results show that the employed ICS protocol elicits a satisfactory and consistent cytokine response from PBL of healthy subjects. The collected data may be used to outline a preliminary reference range for future studies on both healthy/pathological subjects.

Effects of macrophage with Wnt5a over-expression on inflammation and chondrogenic differentiation in co-culture system

To construct and compare macrophage by gene modification of Wnt5a which co-cultured by human bone marrow-derived mesenchymal stem cells (MSCs) with two dimension (2D) and bone marrow aspirates in vitr transwell, in order to investigate the effect of Wnt5a signaling on cartilage homeostasis through regulation of macrophage pro-inflammatory responses. Macrophages, MSCs and bone marrow aspirates specimens were extracted from 6 patients with severe knee deformities undergoing total knee arthroplasty(2 males, 4 females, aged from 58 to 71 years old) from September 2015 to December 2018. The synovial tissues of knee joints were exposed to type II collagenase and obtained single cell suspensions, and the purity of macrophages was determined by Ficoll gradient centrifugation and anti-CD14 antibody flow cytometry. The macrophages were transduced with IFN-γ combined with TNF-α for 48 h, and rAAV-lacZ or Wnt5a transfected for 24 h, then co-cultured model by human bone marrow-derived mesenchymal stem cells (MSCs) with two dimension (2D) and bone marrow aspirates in vitro transwell. HE staining, toluidine blue staining, X-gal staining and anti-Wnt5a, anti-II, X collagen immunohistochemical staining and enzyme-linked immunosorbent assay were applied to detect cell morphology and proliferation (cellularity), viral transfection efficiency respectively. Results of anti-CD68 immunohistochemistry showed macrophage of patients with osteoarthritis increased obviously. Anti-CD 14 flow cytometry confirmed that percentage of isolated synovial macrophages was 90.31%. The level of monocyte chemotactic protein-1 in supernatants was significantly increased after stimulation with IFN-γ and TNF-α, indicating that the macrophages were activated and at proinflammatory condition. After transduction with rAAV-lacZ for 3 days, X-gal staining indicated that lacZ gene transfer could efficiently transduce the activated macrophages with the efficiency over 97.50% for at least 21 days. After transfection macrophages by rAAV-Wnt5a stimulated expression of Wnt5a, and enhanced expression of Wnt5a between two models and inhibited the secretion of MCP-1. The expression of MCP-1 in Wnt5a group was 14.76 and 61.51 pg/ml respectively. In addition, rAAV-Wnt5a gene transfer could promote cell proliferation, chondrogenic differentiation and cartilage matrix synthesis. Under the condition of macrophage and MSCs 2D monolayer or aspirates transwe ll co-culture, rAAV-mediated over-expression of Wnt5a could promote maintenance of cartilage homeostasis and chondrogenic differentiation of MSCs via macrophage inflammatory response, macrophages may affect cartilage homeostasis and MSCs chondrogenesis through Wnt5a signaling pathway.

Leukemic Stem Cell Phenotype Is Associated with Mutational Profile in Acute Myeloid Leukemia

Background: Understanding leukemic stem cell (LSC) is important for acute myeloid leukemia (AML) treatment. As such, understanding the relationship between LSC and genetically defined sub-clones can, in turn, help to understand the heterogeneity of AML. However, to date, there are only a few reports specifically focusing on this topic. To this end, we conducted this study to (1) examine the phenotypic diversity of AML-LSC, (2) explore the association between AML-LSC phenotypes and gene mutations, and (3) investigate the prognostic implications of AML-LSCs. Methods: Mononuclear cells (MNCs) were isolated from the patient's bone marrow aspirates by ficoll gradient centrifugation and cryopreserved in serum-free medium. Stored cells were thawed to Iscove's Modified Dulbecco's Medium (IMDM) and washed with fluorescence-activated cell sorting (FACS) buffer [1% FBS, Dulbecco's Phosphate-Buffered Saline (DPBS)]. Cells were stained with following anti-human monoclonal antibodies: CD45-APC/cy7, CD34-APC, CD38-BV421, CD90-PE, CD123-PE/Cy7, CD45RA-PerCP/Cy5.5. Analyses were performed on a FACSCanto II (HTS) (BD Bioscience) and FlowJo V 10.0 (BD Bioscience) program. For sequencing, the DNA capture probes for 76 target genes were designed using the Agilent SureDesign web-based application. The target regions included protein coding exons with 10 bp intron flanking regions and hot spot regions of the 20 genes involved in recurrent translocations. DNA was extracted on a Chemagic 360 instrument (Perkin Elmer, Baesweiler, Germany). The genomic DNA was sheared using Covaris S220 focused‐ultrasonicator (Covaris, Woburn, MA). We used 50ng of total input genomic DNA. A library preparation was performed according to Agilent's SureSelectQXT Target Enrichment protocol. Paired-end 150-bp sequencing was using NextSeq 550 Dx platform (Illumina, San Diego, CA). Targeted sequencing raw data was obtained in FASTQ format. Results: In secondary AML patients, MPP-like LSC was significantly higher than de-novo AML (p=0.0037), and was higher in MPN-AML than in MDS-AML (p=0.0485). There was no correlation between age and LSC phenotype, though CD34+CD38- subpopulation was enriched in younger patients (&lt;65 yrs). Mutations of KRAS and NRAS were frequently observed in MPP-like LSC dominant patients (3/14 and 4/14), TP53 and ASXL1 mutations in LMPP-like LSC dominant patients (4/12 and 4/12) , and CEBPA, DNMT3A and IDH1 (6/12, 4/12, and 3/12) mutations in GMP-like LSC dominant patients. Furthermore, as shown in Figure, KRAS mutation was significantly associated with the percentage of MPP-like LSC phenotype (p=0.0540), and TP53 mutation with the percentage of LMPP-like LSC phenotype (p=0.0276). When the patients were separated according to the combined risk including next generation sequencing data, the poorer the prognosis, the higher the LMPP-like LSC expression (p=0.0052). The importance of our study lies in that we showed for a given AML patients there is a dominant LSC phenotype and LSCs are associated with clinical outcomes, supporting the significance of cancer stem cell model for human AML. First of all, based on detailed characterization of the surface immunophenotype of AML LSCs we found that AML show evidence of a hierarchical cellular organization. We also recognized that the composition of LSC phenotypes is associated with AML phenotypes. For example, secondary AML patients showed higher fraction of MPP-like LSCs compared to de novo AML patients. In this regard, the higher expression of MPP-like LSCs could explain the poor response to standard treatments traditionally associated with secondary AML. Furthermore, the higher expression of MPP-like LSCs in post-MPN AML compared to post-MDS AML could explain the dismal prognosis associated with post-MPN AML, despite the relative indolent clinical course in their chronic phase and the presence of druggable target. Conclusion: In conclusion, our findings provide better insights into the characteristics and clinical implications of LSC. Although in a small scale, we provide evidence that specific LSC phenotypes are associated with certain mutations thus should be in the AML risk stratification process. Figure Disclosures Yoon: Janssen: Consultancy; Kyowa Hako Kirin: Research Funding; Genentech, Inc.: Research Funding; Yuhan Pharma: Research Funding; MSD: Consultancy; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Mechanism of delayed apoptosis of peripheral blood neutrophils in severely burned patients

To observe the changes of peripheral blood neutrophil apoptosis rate and the effect of p38 mitogen-activated protein kinase (p38MAPK) inhibitor on peripheral blood neutrophil apoptosis in severely burned patients. Severe burn patients [burn area ≥ 30% total body surface area (TBSA), deep II to III degrees, burn index ≥ 20%, age > 20 years old] admitted to the department of burn and plastic surgery of Affiliated Suzhou Hospital of Nanjing Medical University from January to May in 2019 and health examination volunteers during the same period were enrolled. The peripheral blood neutrophils were isolated by Ficoll gradient centrifugation. The neutrophils of severely burned patients were divided into burn group and p38MAPK inhibitor SB203580 group (after 30 minutes incubation at room temperature and 24 hours incubation in incubator); the neutrophils of healthy volunteers were used as control group. The apoptosis of neutrophils was detected by flow cytometry; the level of reactive oxygen species (ROS) in neutrophils was detected by fluorescence probe; the expression of p38MAPK protein and its phosphorylation (p-p38MAPK) level were detected by Western Blot. Compared with the control group, the apoptosis rate of neutrophils in burn group was significantly decreased [(37.42±3.14)% vs. (50.20±9.87)%, P < 0.05], and the ROS production level in neutrophils was significantly increased (fluorescence intensity: 83.28±0.29 vs. 75.23±0.34, P < 0.05). The apoptosis rate of neutrophils in SB203580 group was further lower than that in burn group [(25.51±1.56)% vs. (37.42±3.14)%, P < 0.05], and the level of ROS production was further higher than that in burn group (fluorescence intensity: 95.56±3.67 vs. 83.28±0.29, P < 0.05). There was no significant difference in the protein expression of p38MAPK among the three groups. The p-p38MAPK protein level in burn group was significantly lower than that in control group (p-p38MAPK/p38MAPK: 0.45±0.06 vs. 0.91±0.09, P < 0.05), while the expression level of p-p38MAPK in SB203580 group was further lower than that in burn group (p-p38MAPK/p38MAPK: 0.22±0.04 vs. 0.45±0.06, P < 0.05). Peripheral blood neutrophil apoptosis delayed and ROS production were increased in severely burned patients. The mechanism may be related to p38MAPK pathway inhibitor.

Akciğer kanseri hastalarında dolaşımdaki tümör hücrelerini nasıl tespit ederiz? akım sitometrisi ile yapılan kısa bir çalışma

Circulating tumor cells (CTCs) play a crucial role in the metastatic spread of carcinoma. Therefore, CTC has been interest of a subject in the past few decades in terms of prognosis and response to the therapy in several cancer diseases. Recent improvements in technical approaches maintain to identify CTCs from whole blood have demonstrated the potential value of CTC detection as a liquid biopsy especially in those tumors where tissue accessibility is often challenging as in lung cancer. Lung cancer is the most common cause of death from cancer worldwide in both men and women which is commonly metastasize before it is diagnosed. The aim of this study is to enumerate of CTCs in peripheral blood sample (7.5 mL) of lung cancer patients by flow cytometry. Our modified method which consists of enrichment and detection steps get involved in 9 patients with lung cancer and 9 healthy volunteers. We performed a density-based ficoll gradient centrifugation and a immunomagnetic separation technique (CD45 negative selection) for the enrichment step. Next, multi-parameter flow cytometry based on the expression of anti-epithelial cell adhesion molecule and cytokeratins was used to detect circulating tumor cells among enriched cells. According to our results, circulating tumor cells were not detected on healthy volunteers but circulating tumor cells were found in all of patients with lung cancer (Z=3.823; p&amp;lt;0.001). We demonstrate that circulating tumor cells were detectable in peripheral blood sample of lung cancer patients by our modified method.

Separation and Optimisation of a Sucrose Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells (PBMC)

One step centrifugation procedure used commonly for separation of blood cells is the ficoll gradient centrifugation. In this method, after centrifugation, the peripheral blood mononuclear cells (PBMCs) are located on the top of the separation fluid, whereas other blood cells erythrocytes and granulocytes sediment to the bottom. In the present study 75% of lymphocyte suspension could be separated by using a one-step density gradient centrifugation of sodium heparin blood with Sucrose. Sucrose was diluted into different concentrations using miliQ water (10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%,). 4 mL of diluted blood was layered on 4 mL of each sucrose solution and centrifuged for 45 minutes at 1000 rpm. Clear separation of PBMCs could be observed in solution with 40% sucrose. The separated PBMCs were analysed in haeme analyser which showed 75% lymphocytes, 23% monocytes and 2% of other cells.