Brief communication: Effects of conditioned media from human platelet lysate cultured MSC on osteogenic cell differentiation in vitro.

Culturing mesenchymal stromal cells (MSC) in human platelet lysate (HPL) supplemented media can enhance their osteogenic differentiation potential. The objective of this study was to test the hypothesis that conditioned media (CM) derived from HPL-cultured MSC also have pro-osteogenic effects. Pooled CM was prepared from HPL-cultured human bone marrow MSC (BMSC) of multiple donors and applied on BMSC of different donors (than those used for CM preparation), with or without additional supplementation [HPL, fetal bovine serum (FBS)] and osteogenic stimulation. At various time-points, cell proliferation, alkaline phosphatase (ALP) activity, osteogenic gene expression and in vitro mineralization were assessed. BMSC in standard unstimulated growth media served as controls. After 3-7 days, CM alone did not promote BMSC proliferation or ALP activity; supplementation of CM with HPL slightly improved these effects. After 2 and 7 days, CM alone, but not CM supplemented with HPL, promoted osteogenic gene expression. After 14 days, only CM supplemented with FBS and osteogenic stimulants supported in vitro BMSC mineralization; CM alone and CM supplemented with HPL did not support mineralization, regardless of osteogenic stimulation. In summary, CM from HPL-cultured BMSC promoted osteogenic gene expression but not in vitro mineralization in allogeneic BMSC even when supplemented with HPL and/or osteogenic stimulants. Future studies should investigate the role and relevance of supplementation and osteogenic induction in in vitro assays using CM from MSC

Proteomic Analysis of Mesenchymal Stromal Cells Secretome in Comparison to Leukocyte- and Platelet-Rich Fibrin.

Secretomes of mesenchymal stromal cells (MSCs) are emerging as a novel growth factor (GF)-based strategy for periodontal and bone regeneration. The objective of this study was to compare the secretome of human bone marrow MSC (BMSC) to that of leukocyte- and platelet-rich fibrin (L-PRF), an established GF-based therapy, in the context of wound healing and regeneration. Conditioned media from human BMSCs (BMSC-CM) and L-PRF (LPRF-CM) were subjected to quantitative proteomic analysis using liquid chromatography with tandem mass spectrometry. Global profiles, gene ontology (GO) categories, differentially expressed proteins (DEPs), and gene set enrichment (GSEA) were identified using bioinformatic methods. Concentrations of selected proteins were determined using a multiplex immunoassay. Among the proteins identified in BMSC-CM (2157 proteins) and LPRF-CM (1420 proteins), 1283 proteins were common. GO analysis revealed similarities between the groups in terms of biological processes (cellular organization, protein metabolism) and molecular functions (cellular/protein-binding). Notably, more DEPs were identified in BMSC-CM (n = 550) compared to LPRF-CM (n = 118); these included several key GF, cytokines, and extracellular matrix (ECM) proteins involved in wound healing. GSEA revealed enrichment of ECM (especially bone ECM)-related processes in BMSC-CM and immune-related processes in LPRF-CM. Similar trends for intergroup differences in protein detection were observed in the multiplex analysis. Thus, the secretome of BMSC is enriched for proteins/processes relevant for periodontal and bone regeneration. The in vivo efficacy of this therapy should be evaluated in future studies

Benefit from B-Lymphocyte Depletion Using the Anti-CD20 Antibody Rituximab in Chronic Fatigue Syndrome. A Double-Blind and Placebo-Controlled Study

Background: Chronic fatigue syndrome (CFS) is a disease of unknown aetiology. Major CFS symptom relief during cancer chemotherapy in a patient with synchronous CFS and lymphoma spurred a pilot study of B-lymphocyte depletion using the anti-CD20 antibody Rituximab, which demonstrated significant clinical response in three CFS patients. Methods and Findings: In this double-blind, placebo-controlled phase II study (NCT00848692), 30 CFS patients were randomised to either Rituximab 500 mg/m2 or saline, given twice two weeks apart, with follow-up for 12 months. Xenotropic murine leukemia virus-related virus (XMRV) was not detected in any of the patients. The responses generally affected all CFS symptoms. Major or moderate overall response, defined as lasting improvements in self-reported Fatigue score during follow-up, was seen in 10 out of 15 patients (67%) in the Rituximab group and in two out of 15 patients (13%) in the Placebo group (p = 0.003). Mean response duration within the follow-up period for the 10 responders to Rituximab was 25 weeks (range 8–44). Four Rituximab patients had clinical response durations past the study period. General linear models for repeated measures of Fatigue scores during follow-up showed a significant interaction between time and intervention group (p = 0.018 for self-reported, and p = 0.024 for physician-assessed), with differences between the Rituximab and Placebo groups between 6–10 months after intervention. The primary end-point, defined as effect on selfreported Fatigue score 3 months after intervention, was negative. There were no serious adverse events. Two patients in the Rituximab group with pre-existing psoriasis experienced moderate psoriasis worsening. Conclusion: The delayed responses starting from 2–7 months after Rituximab treatment, in spite of rapid B-cell depletion, suggests that CFS is an autoimmune disease and may be consistent with the gradual elimination of autoantibodies preceding clinical responses. The present findings will impact future research efforts in CFS

Elevated levels of CXCL10 in the Periodic Fever, Aphthous stomatitis, Pharyngitis and cervical Adenitis syndrome (PFAPA) during and between febrile episodes; an indication of a persistent activation of the innate immune system

Background: The Periodic Fever, Aphthous stomatitis, Pharyngitis and cervical Adenitis syndrome (PFAPA) is the most common periodic fever syndrome in childhood. Clinically, PFAPA may resemble autoinflammatory diseases, but the etiology is not fully understood. Methods: We measured inflammatory proteins in plasma and hematologic parameters in children with PFAPA during and between febrile episodes, and in a control group with suspected bacterial pneumonia. In children with PFAPA, a first blood sample was taken within 24 hours of a febrile episode and a second sample between episodes. In children with pneumonia, the first sample was taken shortly after admission and a second sample after full recovery. Results: A total of 22 children with PFAPA and 14 children with pneumonia were included. In children with PFAPA, levels of interleukin (IL) 6, CXCL10 and CCL4 were significantly increased during febrile episodes. The levels of IL-6 and CXCL10 were higher in children with PFAPA during febrile episodes than in children with pneumonia. The levels of CXCL10 remained higher in children with PFAPA between febrile episodes compared to children with pneumonia after recovery. Children with PFAPA had a relative eosinopenia and lymphocytopenia with reduced numbers of both CD4+ and CD8+ T cells during febrile episodes. This pattern was not observed in the children with pneumonia. Conclusions: The results indicate an innate immune response as the initial step in PFAPA, and a subsequent adaptive response with activation and redistribution of T cells. Moreover, an activation of the innate immune system involving CXCL10 may persist between febrile episodes. CXCL10 may be a possibly clinical marker in children with PFAPA

Peripheral Blood Stem Cell Mobilization in Healthy Donors by Granulocyte Colony-Stimulating Factor Causes Preferential Mobilization of Lymphocyte Subsets

Background: Allogeneic hematopoietic stem cell transplantation is associated with a high risk of immune-mediated post-transplant complications. Graft depletion of immunocompetent cell subsets is regarded as a possible strategy to reduce this risk without reducing antileukemic immune reactivity. Study design and methods: We investigated the effect of hematopoietic stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) on peripheral blood and stem cell graft levels of various T, B, and NK cell subsets in healthy donors. The results from flow cytometric cell quantification were examined by bioinformatics analyses. Results: The G-CSF-induced mobilization of lymphocytes was a non-random process with preferential mobilization of naïve CD4+ and CD8+ T cells together with T cell receptor αβ+ T cells, naïve T regulatory cells, type 1 T regulatory cells, mature and memory B cells, and cytokine-producing NK cells. Analysis of circulating lymphoid cell capacity to release various cytokines (IFNγ, IL10, TGFβ, IL4, IL9, IL17, and IL22) showed preferential mobilization of IL10 releasing CD4+ T cells and CD3−19− cells. During G-CSF treatment, the healthy donors formed two subsets with generally strong and weaker mobilization of immunocompetent cells, respectively; hence the donors differed in their G-CSF responsiveness with regard to mobilization of immunocompetent cells. The different responsiveness was not reflected in the graft levels of various immunocompetent cell subsets. Furthermore, differences in donor G-CSF responsiveness were associated with time until platelet engraftment. Finally, strong G-CSF-induced mobilization of various T cell subsets seemed to increase the risk of recipient acute graft versus host disease, and this was independent of the graft T cell levels. Conclusion: Healthy donors differ in their G-CSF responsiveness and preferential mobilization of immunocompetent cells. This difference seems to influence post-transplant recipient outcomes.publishedVersio

Data_Sheet_1_Peripheral Blood Stem Cell Mobilization in Healthy Donors by Granulocyte Colony-Stimulating Factor Causes Preferential Mobilization of Lymphocyte Subsets.zip

Background<p>Allogeneic hematopoietic stem cell transplantation is associated with a high risk of immune-mediated post-transplant complications. Graft depletion of immunocompetent cell subsets is regarded as a possible strategy to reduce this risk without reducing antileukemic immune reactivity.</p>Study design and methods<p>We investigated the effect of hematopoietic stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) on peripheral blood and stem cell graft levels of various T, B, and NK cell subsets in healthy donors. The results from flow cytometric cell quantification were examined by bioinformatics analyses.</p>Results<p>The G-CSF-induced mobilization of lymphocytes was a non-random process with preferential mobilization of naïve CD4⁺ and CD8⁺ T cells together with T cell receptor αβ⁺ T cells, naïve T regulatory cells, type 1 T regulatory cells, mature and memory B cells, and cytokine-producing NK cells. Analysis of circulating lymphoid cell capacity to release various cytokines (IFNγ, IL10, TGFβ, IL4, IL9, IL17, and IL22) showed preferential mobilization of IL10 releasing CD4⁺ T cells and CD3⁻19⁻ cells. During G-CSF treatment, the healthy donors formed two subsets with generally strong and weaker mobilization of immunocompetent cells, respectively; hence the donors differed in their G-CSF responsiveness with regard to mobilization of immunocompetent cells. The different responsiveness was not reflected in the graft levels of various immunocompetent cell subsets. Furthermore, differences in donor G-CSF responsiveness were associated with time until platelet engraftment. Finally, strong G-CSF-induced mobilization of various T cell subsets seemed to increase the risk of recipient acute graft versus host disease, and this was independent of the graft T cell levels.</p>Conclusion<p>Healthy donors differ in their G-CSF responsiveness and preferential mobilization of immunocompetent cells. This difference seems to influence post-transplant recipient outcomes.</p

Lymphocyte subsets during follow-up in the KTS-1-2008 and KTS-2-2010 trials.

<p>Panels A-D: Lymphocyte subsets among patients included in the randomized KTS-1-2008 study, in the rituximab group (red) and the placebo group (black), determined by flowcytometry and shown as million cells per liter at baseline (0 months), and at 1, 2, 3, 4, 6, 8, 10, and 12 months follow-up (n = 30). Panel A: CD3+ T-cells, panel B: CD4+ T helper cells, panel C: CD8+ cytotoxic T-cells, panel D: CD56+/16+ NK-cells. For 29 patients, data on lymphocyte populations were missing for 16 out of 261 (6.1%) time points. For General linear model (GLM) for repeated measures these were replaced by interpolation between preceding and succeeding values. The missing values were not replaced in the plots. Panels E-J: T-cell activation markers among patients included in the KTS-1-2008 trial, at baseline and through 12-months follow-up, and shown as percentage of the CD4+ T helper cell population, as determined by flowcytometry. Panel E: Regulatory T-cells, panel F: CD69+ cells, panel G: CD154+ cells, panel H: CD278+ (ICOS) cells, panel I: HLA-DR+ cells. Panel J shows the percentage of HLA-DR+ cells among the CD8+ cytotoxic T-cells. For 29 patients, data on T-regulatory cells and T-cell activation parameters were missing for 30 out of 261 (11.5%) time points. For GLM, missing values were replaced by interpolation between the preceding and succeeding analyses (not replaced in the plots). Panels K-O: Lymphocyte subsets among patients in the KTS-2-2010 study receiving rituximab maintenance therapy, determined by flowcytometry and shown as million cells per liter at baseline (0 months), and 3, 6, 12, 15, 20, and 24-months follow-up. Responders to B-cell depletion therapy are shown in red and non-responders in blue. Panel K: Ratio of CD4+/CD8+ T-cells, panel L: CD3+T- cells, panel M: CD4+ T helper cells, panel N: CD8+ cytotoxic T-cells, panel O: CD56/16+ NK-cells. In three patients, data on lymphocyte subpopulations for at least three time points were missing, these three were omitted leaving 23 patients for GLM analyses to assess the interaction between time and response group. In these 23 patients, 15/161 (9.3%) data were missing; these were replaced by interpolation between preceding and succeeding values (not replaced in the plots). Analyses for interaction between time and intervention group, i.e. assessing for difference in course of the variables over time, between the rituximab and placebo groups (panels A-J), or between responders and nonresponders to rituximab maintenance treatment (panels K-O), were performed using GLM for repeated measures. Error bars denote 95% confidence intervals (CI) for the mean values. The dotted lines indicate lower and upper normal reference values as established at Haukeland University Hospital.</p

Serum BAFF levels at baseline.

<p>Panel A: Baseline serum BAFF levels (pg/mL) in 70 ME/CFS patients and in 56 healthy controls. Serum samples before treatment in the KTS-1-2008 and KTS-2-2010 trials (n = 38), and in addition 32 samples from ME/CFS patients fulfilling Canadian diagnostic criteria, were included. Panel B: Baseline APRIL serum concentrations (ng/mL), in ME/CFS patients included in KTS-1-2008 study (n = 28) and in healthy controls (n = 22). P-values from unpaired t-test (equal variances not assumed). Error bars denote mean and 95% confidence intervals (CI).</p