Zonghong Shao

Osteoblast inhibition by chemokine cytokine ligand3 in myeloma-induced bone disease

BackgroundMultiple myeloma is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow. A common manifestation of the disease is myeloma bone disease (MBD), which is caused by increased osteoclastic bone resorption and decreased bone formation. The chemokine cytokine ligand 3 (CCL3) is a pro-inflammatory protein and chemokine that stimulates osteoclasts in MBD. However, little is known about the effect of CCL3 on osteoblasts (OB).MethodsThe OBs are induced from patients with MBD and healthy donors, cultured in vitro, and identified by histochemistry. The effects of CCL3 and CCL3 antibody on the OBs in vitro are observed. The CCL3 receptor (CCR1), osteocalcin (OCN), runt-related transcription factor 2 (Runx2), and osterix (Osx) are detected using flow cytometry, enzyme-linked immunosorbent assay, and real-time PCR.ResultsProliferation and osteogenic potential of the OB in patients with MBD are suppressed. Moreover, the CCR1 expression is significantly higher in patients with MBD than in normal controls. The OCN level, quantity of calcium nodules, and Runx2 and Osx levels decrease after CCL3 stimulation, which indicates that CCL3 inhibits OB function. Furthermore, CCL3 antibody partially restores OB activity through the upregulation of the OCN, Runx2, and Osx.ConclusionsCCL3 contributes to the OB/OC imbalance by inhibiting OB differentiation and function in MBD.

Monocyte-Derived Macrophages Are Impaired in Myelodysplastic Syndrome

Background. The myelodysplastic syndrome (MDS) comprises a group of clonal hematopoietic stem cell diseases characterized by cytopenia, dysplasia in one or more of the major myeloid lineages, ineffective hematopoiesis, and increased risk of development of acute myeloid leukemia (AML). Macrophages are innate immune cells that ingest and degrade abnormal cells, debris, and foreign material and orchestrate inflammatory processes. We analyzed the role of macrophages from MDS patients in vitro. Methods. Macrophages were induced from peripheral blood of patients with MDS via granulocyte macrophage colony-stimulating factor (GM-CSF). Phagocytic capacity of macrophages was measured with carboxyfluorescein succinimidyl ester and fluorescent microspheres. CD206 and signal regulatory protein alpha (SIRPα) on macrophages were detected by flow cytometry. Inducible nitric oxide synthase (iNOS) was measured by ELISA method. Results. Compared with normal control group, the number of monocytes increased in MDS patients. However, the monocytes showed impaired ability to induce macrophages and the number of macrophages induced from MDS samples was lower. Further, we demonstrated that the ex vivo phagocytic function of macrophages from MDS patients was impaired and levels of reorganization receptors CD206 and SIRPα were lower. Levels of iNOS secreted by macrophages in MDS were increased. Conclusions. Monocyte-derived macrophages are impaired in myelodysplastic syndromes.

Erythroblastic Islands in the Bone Marrow of Patients with Immune-Related Pancytopenia

Background Immune-related pancytopenia (IRP) is characterized by pancytopenia caused by autoantibody-mediated bone marrow destruction or suppression. The bone marrows of IRP patients have remarkably increased erythroblastic islands (EIs). Methodology and Principal Findings We determined the immunoglobulin G (IgG) autoantibodies in some parts of EIs of IRP patients using immunofluorescence to investigate the biological function of EIs with IgG in the pathophysiology of IRP. The dominant class of autoantibodies detected in mononuclear cells was IgG (CD34 IgG, CD15 IgG, and GlycoA IgG), specifically IgG on GlycoA-positive cells (GlycoA IgG). Results show that extravascular hemolysis occurred in IRP through IgG autoantibodies in the EIs. These data included a high percentage of reticulocytes in the peripheral blood, hypererythrocytosis in the bone marrow, and high serum bilirubin. Furthermore, we examined the macrophages in the bone marrow of IRP patients. The results show that the number of activated macrophages relatively increased, and the phagocytic activity of macrophages significantly increased. Conclusions and Significance Increased EIs with IgG were the sites of erythroblast phagocytosis by the activated macrophages, rather than erythropoietic niches. The IgG autoantibodies in the EIs possibly functioned as adhesion molecules for a ring of erythroblasts around the macrophages, thereby forming morphologic EIs.

Distinguishing immunorelated haemocytopenia from idiopathic cytopenia of undetermined significance (ICUS): a bone marrow abnormality mediated by autoantibodies

In recent years we have observed that some patients with idiopathic cytopenia of undetermined significance (ICUS) responded well to corticosteroid and high‐dose intravenous immunoglobulin treatment, indicating that some cytopenia in ICUS might be mediated by autoantibodies. In this study, we analysed 166 ICUS cases retrospectively, some of which were autoantibodies detected on haemopoietic cells in bone marrow (BM) by BM mononuclear cell (BMMNC)‐Coombs test, flow cytometry (FCM), Western blot and immunofluorescence (IF). We found that 25·9% (43 of 166) of the cases had autoantibodies positive verified with BMMNC‐Coombs test or FCM analysis, 72·1% (31 of 43) of whom had immunoglobulin (Ig)G autoantibody positive by Western blot. IgG could be detected in the erythroblastic islands on the BM smear of nine (32·1%, nine of 28) ICUS patients with autoantibodies by IF. Of these 43 patients, the median percentage of reticulocytes was 1·79%. More than half the patients had hyper‐BM cellularity with a higher percentage of nucleated erythroid cells in the sternum. Total response rates to immunosuppressive therapy at 6, 12, 24 and > 36 months were 46·5% (20 of 43), 75% (30 of 40), 77·4% (24 of 31) and 66·7% (16 of 24), respectively. We termed this group of ICUS cases with autoantibodies as immunorelated haemocytopenia (or BMMNC‐Coombs test‐positive haemocytopenia).

Efficacy of ALK5 inhibition in myelofibrosis

Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF.

Lower level of IL‑35 and its reduced inhibition in Th17 cells in patients with bone marrow mononuclear cells Coombs test‑positive hemocytopenia

Interleukin (IL)-35 is the latest member of IL-12 family, which plays an important role in other autoimmune diseases. Bone marrow mononuclear cells Coombs test-positive hemocytopenia, also termed immunorelated hemocytopenia (IRH) is a type of autoimmune-associated diseases. The present study investigated the relationship of IL-35 in patients with IRH. A total of 43 patients with IRH and 19 normal controls were enrolled in the current study. Serum levels of IL-35 and IL-17 in peripheral blood were evaluated by ELISA. Regulatory T cells (Tregs) level was detected by flow cytometry and IL-35 subunits mRNA in Treg was determined using reverse transcription-quantitative polymerase chain reaction: Epstein-Barr virus induced 3 (EBI3) and IL-12α chain p35. Effect of IL-35 on T helper 17 cells (Th17) cells was determined by mix-culture of IL-35 with CD4+ T lymphocytes. Serum level of IL-35 was decreased in untreated patients with IRH compared with remission patients (P<0.01) and was significantly associated with clinical indexes. Frequency of IL-35 produced Tregs was lower and IL-35 subunits mRNA in CD4+CD25+ Tregs were decreased in patients with IRH compared with health controls (P<0.01). Serum level of IL-17 was increased in patients with IRH (P<0.01) and there was a negative correlation between IL-35 and IL-17 (r=−0.553; P<0.01). The production of Th17 cells and IL-17A mRNA expression were reduced (P<0.05) after mix-culture of CD4+ T lymphocytes with IL-35 compared with mix-culture of CD4+ T lymphocytes without IL-35. In conclusion, the present study revealed that IL-35 may be a monitoring indicator of IRH occurrence and progression. IL-35 level was lower and the inhibition on Th17 cells was reduced in the patients with IRH.

Transforming growth factor 15 increased in severe aplastic anemia patients

ABSTRACT Objectives: The patients with severe aplastic anemia (SAA) usually rely on red cell transfusion which lead to secondary iron overload. Transforming growth differentiation factor-15 (GDF-15) plays an important role in erythropoiesis and iron regulation. In this study, we investigated the level of GDF-15 and other indexes of iron metabolism in SAA patients to explore the correlation with GDF-15 and iron overload in SAA. Methods: The levels of serum GDF-15, hepcidin (Hepc), and erythropoietin (EPO) were determined by ELISA. The levels of serum iron (SI), ferritin, TIBC, and transferrin saturation (TS) were measured by an auto analyzer. Iron staining of bone marrow cells was used for testing extracellular and intracellular iron. Results: The GDF-15 level in the experimental group was higher than that of the case–control group and normal control group (all p < 0.05). The Hepc level in the experimental group and case–control group were both higher than that of healthy controls (all p < 0.05). The Hepc level was significantly lower in the experimental group patients who had excessive GDF-15 (r = −0.766, p = 0.000). There was a positive correlation between the level of GDF15 and EPO in the experimental group (r = 0.68, p < 0.000). The level of GDF15 in SAA patients was positively correlated with SI levels (r = 0.537, p = 0.008), TS levels (r = 0.466, p = 0.025), and sideroblasts (%) (r = 0.463, p = 0.026). Moreover, there was a positive correlation between GDF-15 level and blood transfusion-dependent time (r = 0.739, p = 0.000). Discussion: Our data indicated that GDF-15 plays an important role in iron metabolism in SAA. GDF-15 might be a novel target for SAA therapy.

Proteinase 3 expression on the neutrophils of patients with paroxysmal nocturnal hemoglobinuria

Proteinase 3 (PR3) is released from neutrophils and regulates platelet activity, which is associated with cluster of differentiation (CD)177 antigen (NB1), a glycosylphosphatidylinositol-linked protein. In the present study, the effect of PR3 on thrombosis in paroxysmal nocturnal hemoglobinuria (PNH) and PNH-aplastic anemia (AA) syndrome was explored. The expression of PR3 and NB1 on CD59- neutrophils was detected by flow cytometry, immunofluorescence (IF), reverse transcription-quantitative polymerase chain reaction analysis and western blotting. Serum levels of PR3, proteinase-activated receptor 1 (PAR1) and D-Dimer were measured using ELISAs. The expression of PR3 and NB1 on the plasma membrane of CD59- neutrophils in patients with PNH/PNH-AA was significantly lower compared with their expression on CD59+ neutrophils in patients and controls (P=0.001). However, no correlation between PR3 and NB1 expression was identified. IF staining further demonstrated partially positive PR3 expression on CD59- neutrophils. The serum level of PR3 in patients was identified to be significantly decreased compared with healthy controls (P<0.0001), and significantly negatively correlated with PAR1 (r=-0.456; P=0.043) and D-Dimer (r=-0.503; P=0.028) levels. The mRNA and protein levels of PR3 on PNH clones did not change significantly compared with the control group. In conclusion, PR3 expression on the plasma membrane of neutrophils and in the serum of patients with PNH/PNH-AA decreased, which may result in increased PAR1 expression and increased clotting. The present study provides the basis for further study on platelets in PNH.

High serum levels of complements C3 and C4 as novel markers for myeloma bone disease

Dear Editor, Myeloma bone disease (MBD) is the most common complication of multiple myeloma (MM), which can cause high mortality [1]. More sensitive biochemical markers need to be discovered for detecting clinical bone lesion because of limits of imaging examinations. Several studies have shown a tight interaction between complement systems and bone homeostasis, specifically with osteoblast and osteoclast activities [2]. However, there were few investigations of complements in MBD. In order to explore clinical significance of complements C3, C4, and C4a in MBD, 108 newly diagnosed MM patients and 48 healthy controls (HCs) were enrolled in this study. According to X-ray scanning data obtained before treatment, bone morbidity was graded into three stages: stage A no osteolytic lesions or osteoporosis alone; stage B one to three osteolytic lesions; stage C more than three osteolytic lesions and/or a pathological fracture [3]. There were 50 patients in stage A, 26 in stage B, and 32 in stage C. All data are presented as mean ± standard deviation (SD). We found that patients in stage C had significantly higher levels of C3 (101.89 ± 30.87 mg/dl) and C4 (31.77 ± 13.65 mg/dl) than patients in stage A (76.66 ± 20 . 4 2 and 17 . 7 6 ± 10 . 3 2 mg / d l ) ( p < 0 . 0 01 , p < 0 .001 ) , s t a g e B (84 . 21 ± 28 .62 and 21 . 11 ± 11.62 mg/dl) (p = 0.008, p = 0.013), and HCs (79.17 ± 21.81 and 16.09 ± 5.95 mg/dl) (p < 0.001, p < 0.001) (Fig. 1 A1, A2). Moreover, patients in stages B and C had significantly higher C4a levels (1016.57 ± 130.46 and 1006.01 ± 207.94 ng/ml) than patients in stage A (831.65 ± 176.89 ng/ml) (p = 0.016, p = 0.005) and HCs (867.85 ± 198.05 ng/ml) (p = 0.049, p = 0.022) (Fig. 1 A3). Furthermore, we explored that the levels of C3 (r = 0.307, p < 0.001), C4 (r = 0.466, p < 0.001), and C4a (r = 0.329, p = 0.005) were highly related with the severity of bone disease (Fig. 1b). And there were significant correlations between C3 (r = 0.392, p = 0.008), C4 (r = 0.553, p < 0.001), or C4a (r = 0.586, p < 0.001) and the percentage of osteoclast precursors (OCPs) detected by flow cytometry [4] (Fig. 1c). Serum levels of complement C3 (r = 0.215, p = 0.026, and r = 0.361, p = 0.028) and C4 (r = 0.409, p < 0.001, and r = 0.489, p = 0.002) were significantly correlated with the level of bone resorption metabolites (CTX and TRACP-5b) [4]. However, therewere no significant differences between complements and the percentage of osteoblast precursors (OBPs), bone formation markers (OCN and PINP) [4]. Several studies have indicated that C3 is required for osteoclast formation. Sato et al. elucidated that the C3 is somehow involved in osteoclast development by potentiating M-CSF-dependent proliferation of bone Fengjuan Jiang and Hui Liu contributed equally to this work.

Deep Sequencing of whole genome exon in Paroxysmal Nocturnal Hemoglobinuria

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