ALBERT

Description: Introduction: Endothelial cells (ECs) provide a fertile niche for hematopoietic stem cell (HSC) maintenance, differentiation, and migration.Several studies have indicated that bone marrow (BM) vascular niche was impaired after HSC transplantation and severely inhibited hematopoietic reconstruction. Pigment epithelium-derived factor (PEDF) is an important potential cytoprotection and therapeutic agent for injured cells. The direct role of the injured endothelial cells on hematopoietic stem cells and whether PEDF has protective effect in this system remain unknown. This study aims to observe the influence of enjured ECs on HSCs and to explore the role of PEDF in endothelial-HSC coculture system. Methods: Injury of Endothelial cells by two important preparative regimenconditioning radiation and Busulfan respectively was evaluated with CCK8 assay. The expression of endothelial tight junctions(TJs),adherent junctions related molecules and endothelial to Mesenchymal Transition molecules such as ZO-1, Occludin,VE-cadherin, ICAM, α-SMA, CD31 and VCAM were detected by RT-qPCR, flow cytometry, immunofluorescence and western blot. The effects of injured endothelial cells on HSC self-renewal, differentiation, cell cycle and apoptosis were evaluated by flow cytometry, photography, viable cell count and clone formation assay. Hematopoiesis regulation factors SCF, IL-6, TGF-β and TNF-α were detected by ELISA. The protective effect of PEDF was also explored. Results: Both radiation and Busulfan could decrease cell viability of endothelial cells. The expression level of ZO-1, Occludin, VE-cadherin, ICAM, CD31 and VCAM were decreased and α-SMA was increased when EC exposed to radiation or Busulfan suggesting endothelial activation, impaired EC permeability and endothelial to Mesenchymal Transition after EC injured. Compared with normal endothelial cells and hematopoietic stem cell co-culture group, the HSC% of injured endothelial cells and hematopoietic stem cells co-cultured group were significantly decreased, the cell colony formation ability was decreased, the proportion of mature cells increased, and the damage of endothelial cells could not maintain the characteristics of HSC, weakened the self-renewal and multidirectional differentiation potential of HSC and promoted the maturation of HSC. After the administration of PEDF, endothelial to Mesenchymal Transition of EC was suppressed and the EC permeability was improved. Most importantly, the proportion of HSC was significantly increased, and the proportion of mature cells decreased in the coculture system. Conclusion: Injured endothelial cells can inhibit proliferation of hematopoietic stem cells, self-renewal and promote HSC differentiation. PEDF could ameliorate endothelial injury and promote HSC expansion by suppressing endothelial-mesenchymal transition and protecting TJs and AJs. Disclosures No relevant conflicts of interest to declare.

Description: Pre-transplant chemoradiotherapy can impair intestinal barrier function and disrupt immune homeostasis, and thus increase the incidence of infection and other related complications. Mesenchymal stem cells (MSCs) have the potential to rescue Inflammatory Bowel Disease and intestinal graft versus host disease owing to their immunosuppressive capabilities. However, limited studies regarding MSC administration has been reported to aggravate T cell-mediated tissue injury. Plus, endothelial progenitor cells (EPCs) could improve endothelium repair, facilitate hematopoietic reconstitution and alleviate complications associated with hematopoietic cell transplantation (HCT). The key goals of this study were to i) identify the role of MSC derived soluble and contact dependent factors and how these affect intestinal injury; ii) investigate the effect of MSC combined EPC therapy for repairing the injured intestine. In this study, BALB/c mice were randomly divided into five groups, namely, total body irradiation only, bone marrow transplantation (BMT), MSC(BMT with 1 × 106MSC infusion), EPC(BMT with 5 × 105 EPCs infusion), and MSC+EPC (BMT with 1 × 106 MSC and 5 × 105 EPCs infusion). Results showed that the best performance of intestine was found in the MSC+EPC treated group, which showed more epithelial and goblet cells, and less apoptosis cells and adjacent crypts fusion in intestine morphology. EPC or MSC only group improved the intestinal injuries slightly compared with BMT group. The higher MECA-32 expression level was found in the intestinal tissue of MSC+EPC and MSC groups compared with other treated groups. The tight junction molecules occludin had highest expression level in the intestinal epithelial cells of MSC+EPC group, followed by MSC group and then EPC groups, but the expression level in BMT group was extremely low. Further study demonstrated that in MSC+EPC group the phosphorylated P38 enhanced heat shock protein HSP27 activation, which promoted cytoskeleton reconstruction and intestinal epithelial cells proliferation; and phosphorylated P38 also down-regulated the expression of apoptosis-related molecule caspase3. Moreover, the multiple effects of MSCs on cellular immunity may reflect their diverse influences on the different T-cell subpopulations. MSC+EPC infusion increased the number of IL-17A secreting cells (Th17 and Tc17) in mesenteric lymph nodes early after HCT, and decreased Th1 cells at day 10 and delayed expanding of Tc1 from day 10 to day 15. The soluble IL-17A in intestinal tissue was significantly increased after MSC+EPC infusion in according with IL-17A secreting cells in mesenteric lymph nodes. Furthermore, the gut bacterial information analyzed by high throughput sequencing uncovered that MSC and MSC+EPC groups had higher bacterial diversity and richness compared with other groups, and the bacterial community structures exhibited big changes regarding different treatment. Co-occurrence analysis demonstrated that the genus Akkermansia inducing PD-1 expression of T cells had significant correlation with phosphorylation of P38 and HSP27 in MSC+EPC and EPC treated groups, which indicated Akkermansia may be a key bacteria participanting the intestinal repairing in MSC and MSC+EPC groups. In conclusion, this study confirmed that the MSC+EPC infusion can obviously repair injured intestine, and gut bacteria Akkermansia correlated with phosphorylation of P38 and HSP27 expression and participated in intestinal repairing. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Successful homing, engrafment and effective hematopoietic recovery after hematopoietic stem cell transplantation (HSCT) are strictly regulated by various hematopoietic microenvironment cells. Increasing evidence shows that macrophages (MФs), one of the most important component niche cells are crucial for the haematopoietic regulation. MФs depletion can enhance hematopoietic stem cell mobilization. Our previous study showed that MФs ameliorate bone marrow inflammatory injury and promote hematopoiesis in mice after allo-HSCT, but its role in syngeneic HSCT and acute bone marrow injury is still unknown. Our aim is to explore the role of macrophages in acute bone marrow injury and hematopoietic reconstitution after isogenic hematopoietic stem cell transplantation and sublethal dose irradiation in vivo. Methods BALB/c male mice at 8-10 weeks were irradiated with 60 Co 7.5 Gy and 3.0 Gy, respectively, and then isogenic hematopoietic stem cell transplantation model and sublethal-dose bone marrow injury model were constructed. The transplantation model mice were randomly divided into total body irradiation group (TBI group), bone marrow cell transplantation group (BMT group), bone marrow cell transplantation + Clodronate Liposomes injection group (BMT+Clod-Lip group), bone marrow cell transplantation + PBS Liposomes injection group ( BMT + PBS-Lip group), and normal control group (Normal group). The sublethal-dose experimental mice were randomly divided into the total body irradiation group (TBI group), the whole body irradiation + Clodronate Liposomes injection group (TBI+Clod-Lip group), the whole body irradiation + PBS Liposomes injection group (TBI+PBS-Lip group), and normal control group (Normal group). Mice in Clod-Lip group were injected with Clodronate Liposomes for several specific times to deplete macrophages until the specimens were obtained. Mice in PBS-Lip group were injected PBS Liposomes as controls.Then, the living conditions and body weight changes of the mice were observed and the survival rates of mice in different experimental groups were recorded. Peripheral blood and bone marrow in each group were collected at the corresponding detection time, blood routine analyzer was used to detect blood routine changes, HE staining was used to observe bone marrow damage, and flow cytometry was used to analyze changes in macrophages, hematopoietic stem/progenitor cells and their subgroups such as myeloid cells, megakaryocytes, and nucleated red blood cells in bone marrow. Results Depletion of bone marrow macrophages could reduce the survival rate of hematopoietic stem cell transplantation mice. The pathological results of bone marrow showed that bone marrow injury were heaviest on the 7th day in all three transplantation groups, and then gradually alleviated. The recovery of the BMT+Clod-Lip group was inferior to that of the BMT+PBS-Lip group and the BMT group at the corresponding time point. Depletion of macrophages increased the percentage of myeloid cells in the bone marrow and the number of white blood cells in the peripheral blood, reduced the total number of bone marrow cells, the proportion of hematopoietic stem cells and megakaryocytes in the bone marrow, and delayed recovery of red blood cells and platelets in peripheral blood; Depletion of bone marrow macrophages could also reduce survival rate of sublethal dose irradiation mice, delayed the repair of pathological damage of bone marrow, and increase the proportion of progenitor cells, CMP, GMP, myeloid cells and the number of peripheral white blood cells ,increase the proportion of hematopoietic stem cell apoptosis, reduce the total number of bone marrow cells, the proportion of hematopoietic stem cells, MEP, megakaryocytes and nucleated red blood cells in the bone marrow, delayed peripheral blood recovery of red blood cells and platelets. Conclusion In the isogenic hematopoietic stem cell transplantation model and the sublethal dose irradiation mouse model, the removal of mouse bone marrow macrophages could affect the survival rate of transplanted mice, aggravate the pathological damage of bone marrow, increase the number of GMP and white blood cells, and reduce the total number of bone marrow cells, the number of hematopoietic stem cells and MEP cells. Macrophage depletion was not conducive to the recovery of peripheral blood red blood cells and platelets. Disclosures No relevant conflicts of interest to declare.

Description: Chimeric Antigen Receptor T cell( CAR-T) is an effective treatment for refractory recurrent hematologic malignancies nowdays. However, CAR T cells can potentially damage normal tissues by specifically targeting a tumor-associated antigen that is also expressed on those tissues. Cytokine release syndrome (CRS), a systemic inflammatory response caused by cytokines, is the most common type of toxicity, including fever, hyoxemia, pain and so on. In our study, we found 6 cases abnormal coagulation function which was not very common complication after CAR-T infusion. 4 cases relapsed/refractory acute B lymphoblastic leukemia(B-ALL), including 2 female 53 and 45 years old, 2 male 20 and 6 years old. 1 case multiple myeloma(MM), female 65 years old and 1 case relapsed/refractory diffuse large B-cell lymphoma (DLBCL), male 17 years old. After conditioning chemotherapy with fludarabine and cyclophosphamide, CAR-T cells were infused into patients. In these cases, female 65 years old B-ALL patient had the most severe cytokine release reactions, multiple organ function damage, and abnormal coagulation function that prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged and reached a peak (Too long to detect) on the 14th day after CAR-T infusion, while fibrinogen (FIB) was reduce d (peak on the 14 day, 0.328g/L) and D-Dimer, fibrinogen degradation products (FDP) significantly increased (peak on the 12th day, 28.7ug/mL and more than 10mg/L respectively). After positive hemostasis and other treatments with amperenic acid, vitamin K1 and so on, the level of PT, APTT, FIB, etc were returned to normal on the 19thday. The other 5 patients went through the same process, only slightly. Patients with severe abnormal coagulation function were prone to bleed and even life-threatening, and giving active treatment, finally they all recovered. For the first time, we reported the abnormal coagulation function after CAR-T infusion in hematologic malignancies. Disclosures No relevant conflicts of interest to declare.