Ficoll-Hypaque Density Gradient Research Articles

Interleukin (IL)-21 and IL-21 receptor expression in peripheral T and B cells of patients with breast cancer.

IL-21 is a cytokine with versatile antitumor and pro-tumorigenic activities. It is mainly produced by CD4+ T cells and B cells are one of its pivotal targets. In this study, we assessed and compared the expression of IL-21 by CD4+ T cells and the IL-21 receptor (IL-21R) on B cells in the peripheral blood of women with breast cancer and healthy individuals. Blood samples were taken from both patients and controls. Mononuclear cells were seperated using Ficoll-Hypaque density gradient centrifugation. These isolated cells were then stained with either anti-CD19/anti-IL-21R or anti-CD4/anti-IL-21 antibodies and analyzed using flow cytometry. The results showed that there was no significant difference in the percentage of IL-21R+ B cells and IL-21+CD4+ T cells between patients and controls. However, the percentage of CD4+ T cells decreased significantly in patients with breast cancer (P=0.003). This decline was observed from the early stage and before lymph node (LN) involvement. In comparison to the control group, IL-21R+ B cells were relatively lower in patients with stages I+II and those with fewer than 4 involved LNs. The intensity of IL-21 expression in T cells was associated with HER2 expression (P=0.029). Furthermore, we found that the majority of IL-21R+ B cells exhibited a naïve phenotype and most of IL-21+CD4+ T cells did not produce IFN-γ or IL-17. In conclusion, breast cancer from the early stages leads to a significant reduction in the proportion of peripheral CD4+ T cells. However, we did not find a significant change in IL-21 and its receptor expression during disease progression.

STMN1 promotes cell malignancy and bortezomib resistance of multiple myeloma cell lines via PI3K/AKT signaling

ABSTRACT Background This study investigates the biological functions of Stathmin1 (STMN1) involving drug resistance and cell proliferation in multiple myeloma (MM) and its related mechanisms. Methods Bone marrow aspirates were collected from 20 MM patients, and the bone marrow mononuclear cells (BMMCs) were separated by Ficoll-Hypaque density gradient centrifugation. Blood samples of 20 patients with monoclonal gammopathy of undetermined significance (MGUS) and 20 healthy donors were collected. Normal plasma cells sorted from the peripheral blood of MGUS patients and healthy subject as controls. Two bortezomib (BTZ)-resistant MM cell lines were established, namely NCI-H929/BTZ and KM3/BTZ cells, and then transfected with lentiviruses packaging sh-STMN1 to knock down STMN1 level in BTZ-resistant cells. Expression of STMN1 was assessed by RT-qPCR and western blotting. CCK-8 assays were performed to assess 50% growth inhibition (IC50) values. Green fluorescent protein in BTZ-resistant cells infected with lentiviruses was observed by fluorescence microscopy. Cell viability, proliferation, cell cycle, and apoptosis were evaluated through MTT assays, colony formation assays, flow cytometry analyses, and TUNEL staining. Results STMN1 was upregulated in MM cells and bone marrow aspirates of MM patients. Additionally, STMN1 depletion attenuated BTZ resistance in MM cells. Moreover, downregulation of STMN1 limited the malignant phenotypes of BTZ-resistant cells. Mechanistically, the PI3K/Akt signaling was inactivated by STMN1 downregulation in BTZ-resistant cells. Conclusion STMN1 silencing inhibits cell proliferation and BTZ resistance in MM by inactivating the PI3K/Akt signaling.

Effect of Hispidulin on Proliferation and Apoptosis of Leukemia K562 Cells by Up-Regulating IL-37

To investigate the effect and underlying mechanism of hispidulin on the proliferation and apoptosis of leukemia K562 cells. K562 cells were cultured in vitro and treated with 0, 5, 25 or 100 μmol/L hispidulin for 24 h. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively. Western blot was used to assess the expression of Bax, Bcl-2 and interleukin (IL)-37 proteins. Bone marrow mononuclear cells were extracted from 17 chronic myeloid leukemia patients and 21 healthy individuals by Ficoll-Hypaque density gradient method, and the expression of IL-37 protein was measured by Western blot. K562 cells with IL-37 overexpression or knockdown were constructed, and then treated with 0 or 100 μmol/L hispidulin for 24 h. Cell proliferation, apoptosis and protein expression of Bax and Bcl-2 were determined in the same way as above. After K562 cells were treated with hispidulin, the cell inhibition rate, apoptosis rate, and the protein expression of Bax and IL-37 were significantly increased (P <0.05), but the cell proliferation and expression of Bcl-2 protein were decreased (P <0.05). The expression of IL-37 protein in bone marrow mononuclear cells of the leukemia patient was 0.24±0.03, which was significantly lower than 0.91±0.05 of healthy controls (P <0.05). Overexpression of IL-37 significantly promoted inhibition rate, apoptosis rate, and expression of Bax protein in K562 cells (P <0.05), but suppressed the expression of Bcl-2 protein (P <0.05). In addition, knockdown of IL-37 could reverse the effects of hispidulin on proliferation and apoptosis of K562 cells. Hispidulin inhibits the proliferation and induces apoptosis of leukemia K562 cells, which may be related to the up-regulation of IL-37 protein in cells.

Abstract 4858: Effect of mTOR activity in peripheral blood mononuclear cells with metformin in high-risk prostate cancer patients receiving external beam radiotherapy and androgen deprivation therapy

Abstract Introduction: There is considerable interest in the antineoplastic properties of metformin for prostate cancer treatment, however, the underlying mechanism of action of metformin in this setting is poorly understood. It is suspected that metformin’s antineoplastic activity is related to inhibition of mTOR (mammalian target of rapamycin), which ultimately inhibits cell proliferation. In this pilot study, we assessed the mTOR activity of peripheral blood mononuclear cells (PBMCs) from high-risk Prostate cancer (PCa) patients treated with androgen deprivation therapy (ADT) and external beam radiotherapy (EBRT) plus metformin versus placebo. We hypothesized decreased mTOR expression in PBMCs from patients receiving metformin versus placebo. Methods: Normoglycemic adult males with high-risk PCa receiving ADT &amp; EBRT were randomized to metformin 500 mg 3 times daily or placebo. Peripheral blood samples were collected from participants ≥ 6 months after starting study drug, buffy coats were extracted, and PBMCs were isolated ≤2 hours after specimen collection using ficoll-hypaque density gradient method. The mTOR expression was assessed by phosphorylation of serine 2448 (pmTOR; S2448). Activity of mTOR was measured in both unstimulated or stimulated (using CD3/CD28 antibodies) CD4+ and CD8+ T cells. Results: Samples from 15 patients were analyzed (11 placebo &amp; 4 metformin). We observed lower expression of pmTOR in PBMCs in patients receiving metformin compared to placebo indicating metformin demonstrated inhibition of mTOR activity in PBMCs. A general trend of mTOR inhibition was observed in the metformin group across the cell population and conditions (unstimulated vs. stimulated T cell populations). However, the most pronounced difference was observed in unstimulated CD4+ cells, which is of interest since they represent the closest physiological state to the study participants. Conclusion: In this pilot study, we observed mTOR inhibition in the PBMCs of PCa patients treated with metformin. Further assessment of mTOR activity amongst PCa patients receiving metformin is warranted in order to confirm these findings. Citation Format: Febin Antony, Anuraag Shrivastav, Arbind Dubey, Nawaid Usmani, Aldrich Ong, Rashmi Koul, Harvey Quon, Gokulan Sivananthan, William Hunter, Don Yee, Lindsay Rowe, Brita Danielson, Andrew Plesniarski, Ruey-Chyi Su, Julian O. Kim. Effect of mTOR activity in peripheral blood mononuclear cells with metformin in high-risk prostate cancer patients receiving external beam radiotherapy and androgen deprivation therapy. [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 4858.

Levels of base excision repair proteins in CD4+ T cells in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multi-systemic disease with the unknown pathogenic mechanism. DNA demethylation is involved in SLE pathogenesis. Growth arrest and DNA damage inducible 45 alpha (Gadd45a) takes part in the process of DNA demethylation. Gadd45a is a DNA repair-related protein. This study aims to investigate the expressions of some proteins [including activation-induced cytidine deaminase (AID), thymine DNA glycosylase (TDG), and methyl-CpG-binding domain protein 4 (MBD4)] involving in base excision repair (BER) process in CD4+ T cells in patients with SLE, and to analyze the correlations between the above BER proteins and lupus disease. From January 2019 to September 2020, 12 SLE patients and 12 healthy controls were recruited from Second Xiangya Hospital of Central South University. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation and then CD4+ T cells were isolated via positive selection using Miltenyi beads. We measured the messenger RNA (mRNA) and protein expressions of AID, TDG, and MBD4 by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. In contrast to controls, in SLE CD4+ T cells, the mRNA and protein expressions of AID were elevated (P=0.003, P=0.022, respectively); TDG protein expression was increased (P=0.017); and MBD4 protein level was reduced (P<0.001). No visible distinctions was found in the mRNA expressions of either TDG or MBD4 between the 2 groups (both P>0.05). The mRNA and protein expressions of AID and the protein levels of TDG were positively correlated with SLE disease activity index (SLEDAI). And the mRNA and protein expressions of MBD4 were negatively correlated with SLEDAI. In SLE CD4+ T cells, the increased expressions of AID and TDG and the decreased MBD4 expression may participate in SLE pathogenic mechanism.

Association between miRNA-145 and miRNA-155 expression in peripheral blood mononuclear cells of patients with multiple sclerosis: a case-control study

IntroductionMicroRNAs (miR or miRNA) are short regulatory RNAs, which modulate post-transcriptional gene expression. Dysregulation of these molecules contributes to pathogenicity of autoimmune disorders, such as multiple sclerosis (MS).AimsThis study was conducted to investigate changed expression pattern of miRNA-145 and miRNA-155 in MS.MethodsWe collected blood samples of 75 patients with relapsing-remitting MS patients and 75 healthy controls. Ficoll-Hypaque density gradient method was used to isolate peripheral blood mononuclear cells. Also, total RNA was extracted and subjected to RT-PCR analysis. We used the Mann–Whitney U test to evaluate the differences in expression levels of target miRNAs between the groups.ResultsWe found that expression of miRNA-145 (P = 0.012) and miRNA-155 (P = 0.005) were partly reduced in patients with relapse-remitting MS in comparison with healthy controls. The miRNA-145 had an area under curve (AUC) of 0.621 (P = 0.01) and miRNA-155 levels had an AUC of 0.625 (P = 0.008).ConclusionDecreased expression of miRNA-145 and miRNA-155 contributes to development of relapse-remitting MS, while further large scale observational studies and meta-analyses are required.

MicroRNAs expression in peripheral blood mononuclear cells of patients with multiple sclerosis propose.

MicroRNAs (miRs) are involved in the autoimmune and neurological diseases, including multiple sclerosis (MS), through modulating post-transcriptional gene regulation. Accumulating evidence indicates that miR-10, miR-24a, miR-124, and miR-21 play an imperative role in MS pathogenesis. Therefore, the current research aimed to analyze the expression of the selected miRNAs for MS in Iranian population. Blood sample of 75 relapsing-remitting MS (RRMS) patients and 75 healthy individuals suffering no neurodegenerative illness was collected. Subsequently, the isolation of peripheral blood mononuclear cells (PBMCs) was performed by employing Ficoll-Hypaque density gradient method. Afterward, total RNA was extracted and subjected to qRT-PCR analysis. The obtained results evidenced that the relative expression of miR-10 (P = 0.0002), miR-21 (P = 0.0014), and miR-124 (P = 0.0091) significantly decreased in RRMS patients compared to healthy participants. On the contrary, no notable change was observed between the studies groups regarding miR-24a expression levels (P = 0.107). ROC curve analysis estimated an area under the curve (AUC) value equal to 0.75 with P = 0.0006 for miR-10, while it was decreased for miR-21 (AUC = 0.67 and P = 0.0054) and miR-124 (AUC = 0.66 and P = 0.012). The change in miR-10, miR-124, and miR-21 expression patterns was implied to participate in MS development. Further large scale observational studies are recommended.

In Vitro Generation of BK polyomavirus-specific T cells for adoptive cell therapy in refractory cystitis hemorrhagic patients after hematopoietic stem cell transplantation

IntroductionBKPyV associated hemorrhagic cystitis (BKPyV-HC) is a major and prevalent outcome of hematopoietic stem cell transplantation (HSCT) with no standard treatment option. Adoptive T cell therapy (ACT) against transplant-associated viruses has shown promising potential. We sought to produce virus-specific T cells (VSTs) against BKPyV with the aim of treating refractory HSCT-associated HC.MethodsPeripheral blood mononuclear cells (PBMC) from healthy donors were isolated by Ficoll-Hypaque density gradient centrifugation. BKPyV-pulsed, monocyte-derived dendritic cells (mo-DCs) and T cells were co-cultured and expanded over 2–3 weeks with the addition of IL-2. The T cells were examined for various functional assays.ResultsComparison analysis of Carboxyfluorescein diacetate succinimidyl ester (CFSE) indicated that the percentage of proliferated cells were significantly higher in donors (49.62 ± 7.09%) than controls (7.96 ± 4.55%). Furthermore, expanded T cells exhibited specificity to BKPyV antigens by IFN-γ ELISPOT assay. The expanded cells showed cytotoxic function versus human lymphoblastoid cell line (LCL). Final VST products mainly comprised of CD8/CD69 double-positive T cells, which were significantly higher in donors (46.8 ± 7.1%) than controls (16.91 ± 3.40%).ConclusionIn this study we demonstrated the feasibility of producing functional BKPyV-specific T cells in healthy donors using BKPyV PepMixes. These functional cells were able to proliferate and produce IFN-γ cytokine in response to BKPyV PepMixes. In addition, these T cells had cytotoxic ability against BKPyV antigen-expressing target cells.

Simultaneous effect of gamma and Wi-Fi radiation on gamma-H2Ax expression in peripheral blood of rat: A radio-protection note

IntroductionNuclear medicine patients are isolated in a room after the injection of a radiopharmaceutical. They may be active Wi-Fi option of its smartphone mobile or other environmental radiofrequency waves. The hypothesis of this study was the evaluation of increased biological effects of the simultaneous exposure to gamma-ray and the Wi-Fi waves by measuring the level of the increased double strand-breaks DNA in peripheral blood lymphocyte in the rat. Materials and methodsFifty male Wistar rats were exposed for 2, 24, and 72 h only by Wi-Fi, 99m Tc, and simultaneously by Wi-Fi and 99m Tc. The power density levels of Wi-Fi emitter at 15 cm was 4.2nW/ cm2. An activity of 100 μCi of 99m Tc was injected intraperitoneally. Blood samples were taken by cardiac puncture following general anesthesia. Mononuclear cells are extraction by Ficoll-Hypaque density gradient centrifugation. The number of gamma-H2AX foci per nucleus was counted by flow cytometry. The statistical differences between experimental groups at 2, 24, and 72 h were determined with a repeated measure's analysis of variance. The significant difference between groups at the same time was analyzed with the Kruskal-Wallis Test. ResultsThe manner of gamma-H2AX expression was not the same for three groups in time. The number of gamma-H2AX foci between the three groups was a significant difference after 72 h. ConclusionSimultaneous Wi-Fi and gamma-ray exposures can increase the number of double-strand break DNA in peripheral blood lymphocytes to exposure of gamma-ray to 72 h after technetium injection in the rat.

Prognostic Relevance of Clonal Circulating Plasma Cells Detected By Multiparameter Flow Cytometry in Patients with Newly Diagnosed Multiple Myeloma Treated with Novel Agents: Comparative Investigation with the Revised International Staging System

BackgroundAlthough previous studies demonstrated the presence and prognostic significance of clonal circulating plasma cells (cPCs) detected by multiparameter flow cytometry (MFC) in patients with multiple myeloma (MM), monocle gammopathy of undetermined significance (MGUS), and smoldering MM (sMM), most were conducted during the pre-novel agent era. The relationship between cPC quantification and the revised international staging system (R-ISS) has not been elucidated thus far. We retrospectively analyzed the prognostic significance of cPCs in patients with newly diagnosed MM (NDMM) receiving novel agent-containing induction therapies and their relationship with R-ISS.Patients and methodsFrom November 2007 to April 2017, we evaluated clonal cPCs of newly diagnosed, symptomatic MM patients admitted to Kameda Medical Center, Kamogowa-shi, Japan. We retrospectively analyzed clonal cPCs at diagnosis. Diagnosis and assessment of response to treatment were made according to IMWG criteria. Survival analysis was performed using Kaplan-Meier curve and differences were assessed by log-rank test. Two-tube 6- or 7-color MFC was performed on peripheral blood mononuclear cells (MNCs), isolated by the Ficoll-Hypaque density gradient]. We stained MNCs with monoclonal antibodies to CD45, CD38, CD19, CD138, CD56, and cytoplasmic kappa and lambda light chains. To achieve a sensitivity of 0.01%, we required 500,000 events. Negative cPC was defined as <1 clonal cPC detected by MFC among 10,000 events. Clonal cPCs were expressed as the number per 500,000 total events. Receiver operating characteristics (ROC) analysis determined the optimal cutoff levels of cPCs for predicting the highest risk of disease progression within 3 years for all patients and patients with R-ISS stage Ⅱ, and disease progression within 2 years for patients with R-ISS stage Ⅲ.ResultsClonal cPCs were measured in 132 patients and detected in 95 patients (72.0%). The median number of clonal cPCs was 228 [interquartile range (IQR), 28-1,362] per 500,000 total MNCs. ROC analysis showed the optimal cutoff for predicting the highest risk for disease progression within 3 years, in all cohorts, to be 500 cPCs per 500,000 MNCs, yielding an area under the curve (AUC) of 0.649 with a sensitivity of 65.0% and a specificity of 64.1%. All 132 patients were divided into patients with higher (≥500) and lower (<500) cPCs. Comparing the higher and lower clonal cPCs groups, male sex, patients with advanced clinical stage (ISS, R-ISS, and Durie-Salmon stage), albumin, β2-microglobulin, hemoglobin, lactate dehydrogenase, and presence of high-risk cytogenetic abnormality (CA) were significantly associated with the higher clonal cPC group. Survival analysis showed that patients in the higher clonal cPC group had significantly shorter progression-free survival (PFS) [median survival; 52.2 vs 21.9 months (m), P=0.001] and overall survival (OS; median survival; not reached vs 40.7 m, P=0.02). These survival differences remained significant when separately analyzed in patients with R-ISS stage II and stage III (median PFS, 17.9 vs. 41.2 m; P=0.002, and median OS, 40.4 m vs. not reached; P=0.040, in patients with R-ISS stage II, and median PFS, 6.6 vs. 44.9 m; P=0.004, and median OS, 35.8 m vs. not reached; P=0.025, in patients with R-ISS stage III). We used univariate analysis to assess clinical variables and determine their survival effects. High-risk CA, ISS ≥stage II, and higher cPCs were significantly associated with a shorter PFS and ISS. For patients with R-ISS ≥stage II, higher cPCs were associated with OS. Multivariate analysis showed higher cPCs to be independent predictors of poor PFS after adjusting for high-risk CA, ISS ≥stage II, R-ISS ≥stage II and maintenance therapy (P=0.001).cPCs also independently predicted poor OS after adjusting for age, ISS ≥stage II, and R-ISS ≥stage II (P=0.017).ConclusionQuantification of cPCs by MFC predicts PFS and OS in patients with NDMM, treated with novel agents. It also showed prognostic significance in patients with the same R-ISS categories (both stage Ⅱ and Ⅲ,), suggesting that it is an independent prognostic predictor of R-ISS and has potential for refining the current prognostic system, including R-ISS. [Display omitted] DisclosuresKitadate:Toyama kagaku: Research Funding; Otsuka: Research Funding; Eisai: Research Funding; Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Asahi Kasei: Research Funding; Chugai: Research Funding.

MSCs Derived from Human Amnion Possess a Higher Proliferation Activity and Clearer Stem Cell Properties Than from Bone Marrows and They Exhibit Similar Immunosuppressive Ability In Vitro

Objectives: Human bone marrow mesenchymal stem cells (hBMSCs) have been used for the prevention and treatment of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cells transplantation (allo-HSCT). In this study, we compared the biological characteristics and immunosuppressive activity of human amniotic mesenchymal stem cells (hAMSCs) and hBMSCs to provide experimental evidence for the potential use of hAMSCs for treating aGVHD, thus solving the problem of insufficient hBMSCs sources. Methods: hAMSCs and hBMSCs were isolated by using enzymatic digestion and Ficoll-Hypaque density gradient centrifugation, respectively, were culture-expanded in vitro to obtain the 4th-generation cells. The biological characteristics of both stem cell types were compared by morphological analysis, analysis of cell growth, cell cycle profiling, immunophenotyping, and immunofluorescence assays. An in vitro co-culture of MSCs and peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin (PHA-PBMSC) was performed. The proliferation and changes of T lymphocyte subsets were measured using the Cell Counting Kit-8(CCK-8) assay and flow cytomety, respectively. The production of Interferon-γ (IFN-γ), interleukin-2 (IL-2) and IL-10 in the co-culture supernatant was determined using enzyme-linked immunosorbent assay (ELISA). Results: Both hAMSCs and hBMSCs had fibroblast-like morphology. hAMSCs could be maintained for at least 15 culture passages, but hBMSCs started to show signs of aging and remarkably reduced proliferation at 6-7 passages. There was no significant difference in the proportion of cells in G2/M phase between hAMSCs and hBMSCs (P > 0.05). Immunophenotyping revealed positive expression of CD105, CD90, and CD73 and negative expression of CD34, CD45, CD11b, CD19, and HLA-DR on the surface of both hAMSCs and hBMSCs. hAMSCs were positive for Oct-3/4, but hBMSCs were not. Both hAMSCs and hBMSCs expressed vimentin. PHA-stimulated PBMCs proliferation was inhibited by hAMSCs and hBMSCs. There were not significant differences between the inhibitory effects of the two MSCs types on PBMCs proliferation (P > 0.05). Co-culture with either hAMSCs or hBMSCs significantly increased the proportions of Treg, Th2 and Tc2 and decreased Th1 and Tc1 cell subsets in the PBMCs as compared with the PBMCs cultured alone (P < 0.05), and the changes in the PBMCs were similar between the two co-culture systems (P>0.05). In both of the two co-culture systems, IFN-γ, IL-2 production by the lymphocytes was significantly lowered (P < 0.05) and IL-10 production was significantly increased (P < 0.05) as compared with their levels in the PBMCs cultured alone; no significant difference was found in IFN-γ, IL-2 or IL-10 levels between the two co-culture systems (P > 0.05).Conclusion: The results of this study demonstrated that MSCs derived from human amnion and bone marrows have similar immunomodulatory effects on the T lymphocytes, however, hAMSCs have higher proliferation activity and clearer stem cell properties than hBMSCs, suggesting the possibility of using hAMSCs in the treatment of aGVHD after allo-HSCT. DisclosuresNo relevant conflicts of interest to declare.

Low-Dose Interleukin-2 Restores Human Regulatory T Cell Function, in Vitro, As a Potential in the Handle of Graft Versus Host Disease (GvHD)

BackgroundCurrently, isolation of natural regulatory T cells (nTregs) and in vitro -expanded nTregs was shown to be an effective therapy to GVHD patients. However, shortage of nTregs in peripheral blood and time consumption of expansion in vitro may eventually limit the clinical application. Conversely, induction regulatory T cells (iTregs) can be generated in vitro from naïve T cells and to a large number of iTregs in short time. The activity of iTregs to suppress the responder T cells should be maintained for longer time, then may improve the GVHD therapeutic efficiency. So far, paracrine uptake of soluble mediators as would intensify the Tregs activity on T cell suppression, especially the interleukin-2 (IL-2) is essential for CD4+CD25+ Tregs function. In clinical, low-dose IL-2 administration was suggested to induce the functional Tregs in vivo, and improve the therapy of cGVHD. Therefore, IL-2 may improve iTregs function and expansion during short time, in vitro, then to ensure the successful acute GVHD therapy.AimAs we know, iTreg cells would decay after a period of time, in vivo or in vitro . If we want to maintain a certain number of iTregs for the GVHD treatment, it is best to provide iTregs to the patient more than once. In order to provide more number of effective iTregs for clinical use, we try to develop a method for induction and expansion of more iTregs via IL-2 restores Tregs in vitro .MethodsHuman PBSC were prepared from peripheral blood of health donor by Ficoll-Hypaque density gradient centrifugation. Naïve T cells were isolated by negative selection. The harvested naïve T cells were cultured and stimulated under cytokines-containing RPMI1640 medium. The flow chart was shown in figure. For cell proliferation and activity maintenance, the cells were cultured under IL-2 supplemented medium. The harvested cells were analyzed by flow cytometry with fluorescence-conjugated CD antibodies, including CD4, CD25, CD127 and FoxP3. The iTregs suppression function were analyzed via MLR assay.ResultsWith our purpose, provide more effective iTreg cells should be efficient for clinical application. Amplification of the T cell number can obtain more iTreg cells; therefore, cultivation of the T cells under IL-2-containing medium would stimulate the cell proliferation to about 4-fold on the 10th day. After the first cytokines [IL-2, TGFb-1 and retinoic acid (RA)] stimulation, we harvested the iTregs, and then, keep the cells in low-dose IL-2 only medium for another 5 days to expand and maintain activity of the T cells. On the 10th day, naïve T cell would be performed with anti-CD3/CD28 antibodies and with cytokines supplement for the further induction. The IL-2 expanded-T cells would be further induced to iTregs. FoxP3 gene expression was as a functional iTregs indicator, and showed the highly expression on the 5th- and 15th- day iTergs but lower on the 10th-day. It indicated that the iTregs would be restored for application within a short period under low-dose IL-2 medium. Besides, we could harvest more iTregs after the low-dose IL-2 incubation, because of the cell expansion during IL-2 medium incubation for 5 days. Using MLR assay, we have confirmed all of these functional iTregs to suppress the responder T cells.ConclusionOur study showed that after the activation and induction of naïve T cells, we could maintain the cells under IL-2 containing medium. Then, after the second induction, we could restore the iTreg cells. Therefore, we may provide more of the functional iTregs to GVHD patients, continuously. The result should develop a novel-cell based approach for potentially reducing the risk of acute GVHD within short time. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effect of LPS on Cytokine Secretion from Peripheral Blood Monocytes in Juvenile Idiopathic Arthritis-Associated Uveitis Patients with Positive Antinuclear Antibody.

Objectives Antinuclear antibody (ANA) positivity is a key finding in JIA-associated uveitis (JIAU), but there are quite a few patients with negative ANA. There is no relevant report on the difference of their clinical manifestations. Previous animal model studies have found that the occurrence of uveitis is related to macrophage activation. In this article, our goal is to investigate changes in the morphology and cytokines of peripheral blood mononuclear cells (PBMCs) in uveitis patients testing positive or negative for ANAs after lipopolysaccharide (LPS) stimulation. Methods A total of 30 patients were included in this study (10 in each group). They were divided into three groups (the ANA-positive [ANA+] group, ANA-negative [ANA-] group, and control group). There were ten patients (6 females and 4 males) in each group. Peripheral venous blood was collected into a heparinized tube, and PBMCs were isolated as soon as possible by the Ficoll-Hypaque density gradient separation method. Isolated cells were mixed with RPMI-1640 medium, and the cell concentration was adjusted to ensure that each patient had the same number of cells entering the study. After putting the extracted PBMC into the culture plate, LPS was added carefully to the plate. The cell culture supernatants were collected at 0 h, 3 h, 6 h, 12 h, and 24 h after LPS stimulation to detect the concentrations of IL-6, IL-1, TNF-α, and IL-10. Immunofluorescence was used to discover the deformation of macrophages after LPS stimulation. Results The newly isolated cells were approximately round. 6 h after LPS stimulation, the ratio of noncircular cells/circular cells was the highest in the ANA+ group. Unlike IL-10 that has been rising during the observation period, IL-6, IL-1, and TNF-α peaked at 6 h after LPS stimulation. Conclusion With LPS motivation, cytokines in the ANA+ group increased the most violently.

Effect of High-Density Lipoprotein from Healthy Subjects and Chronic Kidney Disease Patients on the CD14 Expression on Polymorphonuclear Leukocytes.

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-β-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.

Effects of storage time and temperature on highly multiparametric flow analysis of peripheral blood samples; implications for clinical trial samples.

We sought to determine the effect of time and temperature of blood sample storage before preparation of human peripheral blood mononuclear cells (PBMCs) by Ficoll-hypaque density gradient centrifugation. Blood samples from healthy donors were stored at room temperature (RT) or refrigerated at 4°C before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved samples. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4+ and CD8+ T cells and CD45RA-positive terminal effector CD8+ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.

Flow Cytometric Evaluation of CD4+ and CD8+ T-cell Immune Response in SPF Chickens Induced by Fowlpox Vaccine.

Fowlpox (FP) is a viral disease that is widely distributed throughout the world. The disease has an economic impact on the poultry industry, and its prevalence has even been reported in vaccinated flocks. The present study used flow cytometry to evaluate the CD4+ and CD8+ T-cell immune response of chicks induced by FP vaccine. 120 specific pathogen-free (SPF) 21-day-old chicks were randomly divided into three groups of 40. One group was used as negative control with PBS inoculation, the other two groups were inoculated with the local fowlpox vaccine produced by Razi Institute and commercial FP vaccines, and they were kept for five weeks. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque density gradients and the percentages of CD3+, CD3+, CD4+, and CD3+CD8+ T lymphocytes were analyzed with flow cytometry. Seven days post-immunization, a maximum (90-100%) swelling formation ("take") on the vaccination site was observed. The ratios of CD4+ to CD8+ T-lymphocytes in both vaccinated groups were significantly higher (p < 0.05) than the control group inoculated with PBS. The percentages of CD3+, CD3+CD4+, and CD3+CD8+ T-lymphocytes were increased in chickens vaccinated with commercial and local FP vaccines. There were no significant differences between the groups receiving commercial and local fowl pox vaccines. The present study showed that protective immunity could be associated with increased cellular immune responses, which has been interpreted as enhancing T-cell proliferation and increasing CD4+ to CD8+ ratios through vaccination with the FP vaccine. This study further suggests that the induction of enhanced immune responses is due mainly to the Th1-type response.

Heterogeneity of Hematopoietic Stem and Progenitor Cell (HSPC) Composition in Αβ T-Cell/CD19 B-Cell Depleted Peripheral Blood Cell Stem Cell (PBSC) Transplant Grafts and Correlation with Immune and Hematopoietic Recovery

INTRODUCTION. While total CD34 counts in PBSC graft products have correlated with overall likelihood of hematopoietic recovery after allogeneic hematopoietic stem cell transplantation (HSCT), analyses of the HSPC composition and its relationship to relevant post-transplant clinical outcomes are lacking. In fact, the biological basis for different dynamics of hematopoietic/immune recovery, the risk of infection, and graft-versus-host disease (GvHD) is not fully understood. We have performed the first analysis of HSPC graft composition in 6 αβ T-cell/CD19 B-cell depleted haploidentical (αβhaplo) HSCT. Additionally, we correlated the HSPC graft composition with the distribution of the same HSPC subsets in serial post-HSCT bone marrow aspirates performed at Days 30, 60, and 90, with the peripheral blood counts [neutrophils, monocytes, platelet (PLT)] and with the immune recovery (CD3+, CD3+CD4+, CD3+CD8+, αβT, γδT, NK cells) at the same time points. The patients were divided in two groups: 3 patients had a robust and sustained hematopoietic recovery (Group 1) while 3 patients experienced mild cytopenia after Day 60 (Group 2). All patients were transplanted for acute leukemia and received a myeloablative TBI-based conditioning regimen. See Table 1 for details about patients and graft composition. METHODS. All patients were enrolled in the Stanford IRB approved BMT Protocols 179/351/361 and had peripheral blood (PB) and bone marrow (BM) evaluated at Day 30, 60 and 90 post HSCT for the primitive CD34+ Lin- HSPC subsets: HSC (CD38-CD45RA-CD90+), MPP (CD38+CD45RA+), CMP (CD38+CD45RA-CD123+), GMP (CD38+CD45RA+CD123+), MEP (CD38+CD45RA-CD123+) and CLP (CD38+CD127+). Aliquots of αβhaplo-HSCT grafts were cryopreserved for later analyses. Mononuclear cells were isolated from PBSC, PB and BM by Ficoll-Hypaque (Sigma-Aldrich) density gradient centrifugation. FACS analyses were performed on either fresh or frozen cells on Becton Dickinson (BD) Aria II flow cytometer. At least 5x105 events were acquired and analyzed using FlowJo software (BD). RESULTS. Despite consistent levels of αβT-cell depletion and CD34 enrichment, the frequency of the HSPC subsets varied between the grafts. Notably, while CMP and GMP were very consistent across the 6 grafts, the frequency of HSC, CLP, MEP and MPP showed a 2-fold range of variation (Fig1A). No significant correlation was observed between the HSC frequency in the graft and the hematopoietic/immune recovery. However, the frequency of HSC in the BM at Day 30 is statistically correlated (P=0.027) with the PLT at Day 90 (Fig1B). In these preliminary results, the different distribution of CMP, GMP, MEP and MPP did not impact on the hematopoietic/immune recovery. However, there was a significant correlation (P=0.02) between CLP and γδ T cells reconstitution at Day 90 in Group 1 patients (Fig1C). Additionally, the neutrophils, monocytes and phagocytic cells recovery at Day 90 is statistically correlated with the GMP frequency in the BM at Day 30 (P=0.017; P=0.018; P=0.0132, respectively) (Fig1D). Interestingly, the same strong correlation is observed between the CMP in the BM at Day 60 and the recovery of neutrophils and phagocytic cells (P=0.016, P=0.019) at Day 90 (Fig1E), but the CMP at day 30 are already predictive of a robust engraftment in the Group 1 patients (data not shown). CONCLUSIONS. In αβhaplo-HSCT, previously identified factors influencing hematopoietic recovery have been mainly limited to the enumeration of bulk CD34 counts and of mature effector cells, such as αβ/γδ T and NK cells. On the other hand, the presence of GvHD and thymic injury have been correlated to the kinetics of immune reconstitution. We hypothesized that the HSPC composition of the graft would impact lymphohematopoietic recovery in αβhaplo-HSCT recipients. Although preliminary, our data indicate that even with a consistent method of graft manipulation, the HSPC graft composition is heterogeneous. Variations in HSPC subsets frequency and number can contribute to significant differences in lymphohematopoietic recovery and, therefore, clinical outcome. The evaluation of a larger number of patients with longer follow up after HSCT are required. Comparative studies with unmanipulated T-cell replete and cord blood grafts are ongoing. Such analyses will be instrumental not only for prediction of clinical outcome, but also for optimization of novel graft engineering strategies. Disclosures No relevant conflicts of interest to declare.

Comparison of Fixed Vs ALC-Based Doses of Thymoglobulin® (ATG) in Pediatric Patients with Acute Leukemia Given αβhaplo-HSCT: Impact on Immune Reconstitution at Day 90

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (HSCT) remains the treatment of choice for pediatric patients with high risk or refractory leukemia. In the absence of related or unrelated HLA-matched donors, alternative approaches such as HLA-haploidentical HSCT have been implemented. Our group has developed one such approach, αβ T-cell/CD19 B-cell depletion (αβhaplo-HSCT), and demonstrated its clinical efficacy (Bertaina A, Blood 2018). In αβhaplo-HSCT, anti-thymocyte globulin (ATG) is used for preventing graft rejection and graft-versus-host disease (GvHD). However, the optimal dose still needs to be elucidated. Here, we present the first analysis comparing 2 different Thymoglobulin® ATG doses: one fixed at 3.75 mg/Kg, as established in previous European studies with the closely related ATG Grafalon®, and one based on a newly developed algorithm that also integrates absolute lymphocyte count (ALC). METHODS: Between March 2017 and April 2020, 27 pediatric patients (median age 12 years) with hematological malignancies were transplanted at Stanford University's Lucile Packard Children's Hospital. Importantly, 60% of these patients were in CR2 or greater and 2/27 had active disease at the time of HSCT. All children received a fully myeloablative conditioning regimen. On days -9/-8/-7 before αβhaplo-HSCT, patients were given a regimen of Thymoglobulin® ATG. No patient received post-HSCT pharmacological GvHD prophylaxis. The fixed dosage was given to 14 patients in cohort 1 (ATG: 3.75 mg/Kg) and the novel ALC-based dosage to 13 in cohort 2 (ATG ranging between 3-6 mg/Kg). All patients enrolled in this study (BMT Protocol 179 and 351 approved from our IRB) had a minimum follow-up of 100 days, evaluated at Day 30 and 90. Following Admiraal R (Lancet Hematology 2015), we defined CD4 immune reconstitution (IR) as more than 50 CD4 T-cells/ul twice within the first 100 days after HSCT. PBMC were enriched by Ficoll-Hypaque (Sigma Aldrich) density gradient centrifugation. Flow analyses were performed on fresh cells resuspended in PBS 2% FBS on Cytek DxP 10 flow-cytometer. At least 5x104 events of total cells were acquired and analyzed using FloJo software. RESULTS: With a median follow-up of 555 and 124 days for cohort 1 and 2 respectively, 12 patients (85.7%) in cohort 1 and 7 patients (54%) in cohort 2 achieved CD4 immune reconstitution. In cohort 2, CD3 αβ T cells were significantly lower at both Day 30 and Day 90 (P=0.0003, Figure 1A). At Day 90, both the CD4 and CD8 subpopulations were significantly depressed (P=0.01 and P=0.056, respectively, Figure 1C). In both subpopulations, the memory compartment was the most reduced (Figure 1D). The absolute numbers of CD3 γδ T cells did not differ between the cohorts at either Day 30 or 90 (Figure 1B). Viral reactivations were higher in cohort 1 (10/14, 71%) than in cohort 2 (6/13, 46%, P=NS). Half of the reactivations were CMV reactivations, but no patient developed organ disease. There was no statistically significant difference in overall survival and the incidence of relapse in the two cohorts. Three patients developed grade III-IV aGvHD: 2 in cohort 1 (14%) and 1 in cohort 2 (8%, P=NS). Remarkably, the only patient, who developed grade IV aGvHD in cohort 2, did not experience symptoms until Adenovirus reactivation 138 days after HSCT. CONCLUSION: Our analysis confirms that the optimal dose of ATG Thymoglobulin® before αβhaplo-HSCT remains elusive. There were no significant clinical differences between the 2 ATG regimens. However, the ALC-based regimen resulted in the more pronounced reduction of donor-derived memory T cells. Our analysis suggests two intriguing explanations for the observed pattern of results. First, the selective depletion of the memory compartment in both CD4 and CD8 T cells may well be due to a priming effect of ATG Thymoglobulin® on the few αβ T cells left over in the graft. Second, the equivalent reconstitution of naive T cells in the 2 cohorts is likely because the ATG has no impact on the thymus-dependent IR. Remarkably, in our overall cohort, 70% of the patients achieved CD4 IR by 90 days after αβhaplo-HSCT. This result is superior to the best results from other ex vivo T-cell depleted approaches 54%, recently reported by Van Roessel (Cytotherapy 2020). In vivo studies of the pharmacokinetics of ATG in αβhaplo-HSCT recipients and a comparison with the use of Grafalon® are required to shed more light on this crucial topic. Disclosures No relevant conflicts of interest to declare.

Study on the expression of CD14 + CD16 + monocytes and VEGF in peripheral blood of patients with type 2 diabetes mellitus and diabetic macroangiopathy

To study the expression of CD14 + CD16 + monocytes and VEGF and the levels of serum adiponectin and MCP-1 in peripheral blood of patients with type 2 diabetes mellitus (T2DM) and diabetic macroangiopathy to understand the possible mechanism of inflammatory immune response in T2DM and diabetic macroangiopathy. Peripheral blood CD14 + CD16 + monocytes were detected by flow cytometry in 50 T2DM patients, 50 patients with diabetic macroangiopathy, and 20 healthy controls or normal controls who participated in outpatient physical examination, and used the Ficoll-Hypaque density gradient centrifugation isolated PBMC and quantitative PCR technology comparison between groups research object in the peripheral blood PBMC VEGF mRNA expression level. Serum levels of adiponectin and MCP-1 were measured by ELISA. Compared with normal control group (NGT), the fluorescence intensity of proinflammatory CD14 + CD16 + monocytes in simple T2DM group and T2DM combined with macroangiopathy group were significantly increased (p T2DM patients> healthy volunteers, the differences were significant (p < 0.05). Compared with NGT group, the levels of serum adiponectin in T2DM group and T2DM combined with macroangiopathy group were significantly lower than those in NGT group (p < 0.01), and the levels of serum adiponectin, simple T2DM Group were lower than T2DM combined with macroangiopathy group (p < 0.05). The level of MCP-1 in serum compared with simple T2DM group and NGT group, T2DM combined with macroangiopathy group had statistically significant difference (p < 0.05). The serum level of MCP-1 in T2DM group was also higher than that in NGT group (p < 0.05). At the same time, we also found that the increase of CD14 + CD16 + monocytes was positively correlated with serum MCP-1 levels. T2DM patients and T2DM combined with macroangiopathy patients have increased expression of VEGF and MCP-1 concentration in peripheral blood mononuclear cells. The increase of MCP-1 may increase the number of CD14 + CD16 + monocytes, which is involved in the chronic inflammation in patients with T2DM and T2DM combined with macroangiopathy, resulting in the occurrence and development of T2DM and its complications.

Effect of morphine and a low dose of ketamine on the T cells of patients with refractory cancer pain in vitro.

The combination of morphine and ketamine is considered safe and efficacious in many patients. However, a considerable number of immunomodulatory effects have been reported to be produced by both morphine and ketamine. The aim of the present study was to assess the direct effect of morphine and a low dose of ketamine on the T cells of patients with refractory cancer pain in vitro. Venous blood was obtained from patients with refractory cancer pain and peripheral blood mononuclear cells were isolated using the Ficoll-Hypaque density gradient method. Anti-CD3 beads were used to isolate T cells by positive selection. Subsequently, the T cells were treated with vehicle, 200 ng/ml of morphine or 200 ng/ml of morphine + 100 ng/ml ketamine for 24 h, following which the cells were stimulated with anti-CD3 and anti-CD28. Flow cytometric analysis of CD3+ T cells, and interleukin (IL)-2 and interferon (IFN)-γ in the supernatant, reverse transcription-quantitative PCR analysis for the detection of IL-2 and IFN-γ and western blotting for the detection of p65 nuclear factor (NF)-κB were performed. In vitro, the CD4+ and CD8+ T cell counts, CD4+/CD8+ ratio, secretion of IL-2 and IFN-γ in the supernatant, mRNA expression levels of IL-2 and IFN-γ and expression of p65 NF-κB were significantly decreased following treatment with morphine and morphine + ketamine, compared with results in the control group (all P<0.05). However, there was no significant difference between treatment with morphine and that with morphine + ketamine. Treatment with morphine + ketamine in vitro decreased the immune functions of patients with refractory cancer pain, although the effect of treatment with morphine and a low dose of ketamine did not differ significantly from that with morphine treatment alone.