Case Report: Evolution of a Severe Vascular Refractory Form of ECD Requiring Liver Transplantation Correlated With the Change in the Monocyte Subset Analysis

Erdheim–Chester disease is a rare histiocytosis characterized by iconic features associated with compatible histology. Most patients have somatic mutations in the MAP-kinase pathway gene, and the mutations occur in CD14+ monocytes. Differentiation of the myeloid lineage plays a central role in the pathogenesis of histiocytosis. Monocytes are myeloid-derived white blood cells, divided into three subsets, but only the CD14++CD16− “classical monocyte” can differentiate into dendritic cells and tissue macrophages. Since most mutations occur in CD14+ cells and since ECD patients have a particular monocytic phenotype resembling CMML, we studied the correlation between disease activity and monocytic subset distribution during the course of a severe vascular form of ECD requiring liver transplantation. During early follow-up, increased CD14++CD16− “classical monocyte” associated with decreased CD14lowCD16++ “non-classical monocyte” correlated with disease activity. Further studies are needed to confirm the use of monocyte as a marker of disease activity in patients with ECD.

Inter-center comparison of good manufacturing practices-compliant stromal vascular fraction and proposal for release acceptance criteria: a review of 364 productions

Background Even though the manufacturing processes of the stromal vascular fraction for clinical use are performed in compliance with the good manufacturing practices applying to advanced therapy medicinal products, specifications related to stromal vascular fraction quality remain poorly defined. We analyzed stromal vascular fraction clinical batches from two independent good manufacturing practices-compliant manufacturing facilities, the Swiss Stem Cell Foundation (SSCF) and Marseille University Hospitals (AP-HM), with the goal of defining appropriate and harmonized release acceptance criteria. Methods This retrospective analysis reviewed the biological characteristics of 364 batches of clinical-grade stromal vascular fraction. Collected data included cell viability, recovery yield, cell subset distribution of stromal vascular fraction, and microbiological quality. Results Stromal vascular fraction from SSCF cohort demonstrated a higher viability (89.33% ± 4.30%) and recovery yield (2.54 × 105 ± 1.22 × 105 viable nucleated cells (VNCs) per mL of adipose tissue) than stromal vascular fraction from AP-HM (84.20% ± 5.96% and 2.25 × 105 ± 1.11 × 105 VNCs per mL). AP-HM batches were significantly less contaminated (95.71% of sterile batches versus 74.15% for SSCF batches). The cell subset distribution was significantly different (higher proportion of endothelial cells and lower proportion of leukocytes and pericytes in SSCF cohort). Conclusions Both centers agreed that a good manufacturing practices-compliant stromal vascular fraction batch should exert a viability equal or superior to 80%, a minimum recovery yield of 1.50 × 105 VNCs per mL of adipose tissue, a proportion of adipose-derived stromal cells at least equal to 20%, and a proportion of leukocytes under 50%. In addition, a multiparameter gating strategy for stromal vascular fraction analysis is proposed.

Inter-center comparison of good-manufacturing-practices compliant stromal vascular fraction and proposal for release acceptance criteria: a review of 364 productions

Background : Even though the manufacturing processes of the stromal vascular fraction for clinical use are performed in compliance with the good-manufacturing practices applying to advanced therapy medicinal products, specifications related to stromal vascular fraction quality remain poorly defined. We analyzed stromal vascular fraction clinical batches from two independent good-manufacturing practices-compliant manufacturing facilities, the Swiss Stem Cell Foundation (SSCF) and Marseille University Hospitals (AP-HM), with the goal of defining appropriate and harmonized release acceptance criteria.Methods: This retrospective analysis reviewed the biological characteristics of 364 batches of clinical-grade stromal vascular fraction. Collected data included cell viability, recovery yield, cell subset distribution of stromal vascular fraction and microbiological quality. Results: stromal vascular fraction from SSCF cohort demonstrated a higher viability (89.33 % ± 4.30 %) and recovery yield (2.54 × 105 ± 1.22 × 105 viable nucleated cells (VNCs) per mL of adipose tissue) than stromal vascular fraction from AP-HM (84.20% ± 5.96% and 2.25 × 105 ± 1.11 × 105 VNCs). AP-HM batches were significantly less contaminated (95.71 % of sterile batches versus 74.15 %) The cell subset distribution was significantly different (higher proportion of endothelial cells and lower proportion of leukocytes and pericytes in SSCF cohort). Conclusions: Both centers agreed that a good-manufacturing practices-compliant stromal vascular fraction batch should exert a viability equal or superior to 80 %; a minimum recovery yield of 1.50 × 105 VNCs per mL of adipose tissue; a proportion of adipose-derived stromal cells at least equal to 20 %; and a proportion of leukocytes under 50 %. In addition, a multiparameter gating strategy for stromal vascular fraction analyze is proposed.

Systemic Inflammation and the Breakdown of Intestinal Homeostasis Are Key Events in Chronic Spinal Cord Injury Patients

Our aim was to investigate the subset distribution and function of circulating monocytes, proinflammatory cytokine levels, gut barrier damage, and bacterial translocation in chronic spinal cord injury (SCI) patients. Thus, 56 SCI patients and 28 healthy donors were studied. The levels of circulating CD14+highCD16−, CD14+highCD16+, and CD14+lowCD16+ monocytes, membrane TLR2, TLR4, and TLR9, phagocytic activity, ROS generation, and intracytoplasmic TNF-α, IL-1, IL-6, and IL-10 after lipopolysaccharide (LPS) stimulation were analyzed by polychromatic flow cytometry. Serum TNF-α, IL-1, IL-6 and IL-10 levels were measured by Luminex and LPS-binding protein (LBP), intestinal fatty acid-binding protein (I-FABP) and zonulin by ELISA. SCI patients had normal monocyte counts and subset distribution. CD14+highCD16− and CD14+highCD16+ monocytes exhibited decreased TLR4, normal TLR2 and increased TLR9 expression. CD14+highCD16− monocytes had increased LPS-induced TNF-α but normal IL-1, IL-6, and IL-10 production. Monocytes exhibited defective phagocytosis but normal ROS production. Patients had enhanced serum TNF-α and IL-6 levels, normal IL-1 and IL-10 levels, and increased circulating LBP, I-FABP, and zonulin levels. Chronic SCI patients displayed impaired circulating monocyte function. These patients exhibited a systemic proinflammatory state characterized by enhanced serum TNF-α and IL-6 levels. These patients also had increased bacterial translocation and gut barrier damage.

Abstract 15905: Monocyte Subsets Predict Mortality After Cardiac Arrest

Background: After successful cardiopulmonary resuscitation (CPR) with return of spontaneous circulation (ROSC), many patients show signs of an overactive immune activation. Monocytes are a heterogeneous cell population that can be distinguished into three subsets. The aim of this prospective, observational study was to analyse whether monocyte subset distribution is associated with mortality at 6 months in patients after cardiac arrest (CA). Methods: We included 53 patients admitted to our medical intensive care unit (ICU) after CA. Blood was taken on admission and after 72 hours and monocyte subset distribution was analysed by flow cytometry and distinguished into classical monocytes (CD14++CD16-), intermediate monocytes (CD14++CD16+CCR2+) and non-classical monocytes (CD14+CD16++CCR2-). Results: Median age was 64.5 (interquartile range (IQR) 49.8-74.3) years and 75.5% were male. Six-month mortality was 50.9% and survival with good neurological outcome was 37.7%. Monocyte subset distribution upon admission to the ICU did not differ according to survival. However, patients who died within 6 months showed a more pronounced increase in the pro-inflammatory subset of intermediate monocytes (8.3% (3.8-14.6)% vs. 4.1% (1.5-8.2)%; p=0.025), and a decrease of classical monocytes (87.5% (79.9-89.0)% vs. 90.8% (85.9-92.7)%; p=0.036) 72 hours after admission. In addition, intermediate monocytes were predictive of outcome independent of time to ROSC and witnessed cardiac arrest, and correlated with the cerebral performance category (CPC)-score at 6 months (R=0.32; p=0.043). Discussion: Monocyte subset distribution is associated with outcome in patients surviving a CA. The activation of the innate immune system may play a significant role in CA patients.

Monocyte subsets predict mortality after cardiac arrest

Background After successful cardiopulmonary resuscitation with return of spontaneous circulation (ROSC), many patients show signs of an overactive immune activation. Monocytes are a heterogenous cell population that can be distinguished into three subsets. Purpose The aim of this prospective, observational study was to analyze whether monocyte subset distribution is associated with mortality at 6 months in patients after cardiac arrest. Methods We included 53 patients admitted to our medical ICU after cardiac arrest. Blood was taken on admission and monocyte subset distribution was analyzed by flow cytometry and distinguished into classical monocytes (CM; CD14++CD16-), intermediate monocytes (IM; CD14++CD16+CCR2+) and non-classical monocytes (NCM; CD14+CD16++CCR2-). Results Median age was 64.5 (IQR 49.8–74.3) years and 75.5% of patients were male. Mortality at 6 months was 50.9% and survival with good neurological outcome was 37.7%. Of interest, monocyte subset distribution upon admission to the ICU did not differ according to survival. However, patients that died within 6 months showed a strong increase in the pro-inflammatory subset of intermediate monocytes (8.3% (3.8–14.6)% vs. 4.1% (1.5–8.2)%; p=0.025), and a decrease of classical monocytes (87.5% (79.9–89.0)% vs. 90.8% (85.9–92.7)%; p=0.036) 72 hours after admission. In addition, intermediate monocytes were predictive of outcome independent of initial rhythm and time to ROSC and correlated with the CPC-score at 6 months (R=0.32; p=0.043). Discussion Monocyte subset distribution is associated with outcome in patients surviving a cardiac arrest. This suggests that activation of the innate immune system may play a significant role in patient outcome after cardiac arrest. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): FWF - Fonds zur Förderung der wissenschaftlichen Forschung

The effects of tumor resection and adjuvant therapy on the peripheral blood immune cell profile in patients with colon carcinoma

Objective The subset distribution and immunophenotype of circulating immune cells (“peripheral blood immune cell profile”) may reflect tumor development and response to cancer treatment. In order to use the peripheral blood immune cell profile as biomarker to monitor patients over time, it is crucial to know how immune cell subsets respond to therapeutic interventions. In this study, we investigated the effects of tumor resection and adjuvant therapy on the peripheral blood immune cell profile in patients with colon carcinoma (CC). Methods The subset distribution and immunophenotype of T cells (CD3+CD56−), CD56dim NK cells (CD3−CD56dim), CD56bright NK cells (CD3−CD56bright) and NKT-like cells (CD3+CD56+) were studied in preoperative and postoperative peripheral blood mononuclear cell (PBMC) samples of 24 patients with CC by multiparameter flow cytometry. Changes in immunophenotype of circulating immune cells after tumor resection were studied in patients treated with and without (capecitabine-based) adjuvant therapy. Results The NKT-like cell (% of total PBMCs) and CD8+ T cell (% of total T cells) populations expanded in the peripheral blood of non-adjuvant-treated CC patients after surgery. NK- and NKT-like cells showed upregulation of activating receptors and downregulation of inhibitory receptors in non-adjuvant-treated CC patients after surgery. These changes were not observed in the peripheral blood of adjuvant-treated CC patients. Conclusions Our results suggest tumor-induced suppression of NK- and NKT-like cells in CC patients, an effect that could not be detected after tumor resection. In contrast, adjuvant therapy maintained tumor-induced immunosuppression of NK- and NKT-like cells in CC patients.