Reduced ß2-GPI is associated with increased platelet aggregation and activation in patients with prolonged isolated thrombocytopenia after allo-HSCT.

We aimed to measure platelet function and its relationship with ß2-GPI in prolonged isolated thrombocytopenia (PT) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Fifty-six patients with PT and 60 allo-HSCT recipients without PT (non-PT controls) were enrolled. Platelet aggregation and activation, ß2-GPI and anti-ß2-GPI antibody levels, vWF antigen, and vWF activity were analyzed. The effect of ß2-GPI on platelet aggregation was also measured ex vivo. Results showed that ADP-induced platelet aggregation significantly increased (39%±7.5% vs. 23%±8.5%, P=0.032), and the platelet expression of both CD62p (33.6%±11.6% vs. 8.5%±3.5%, P<0.001) and PAC-1 (42.4%±7.6% vs. 6.8%±2.2%, P<0.001) was significantly higher in patients with PT than in those without PT. Significantly lower ß2-GPI levels (164.2±12 µg mL-1 vs. 234.2±16 µg mL-1, P<0.001), higher anti-ß2-GPI IgG levels (1.78±0.46 U mL-1 vs. 0.94±0.39 U mL-1, P<0.001), and increased vWF activity (133.06%±30.50% vs. 102.17%±25.90%, P<0.001) were observed in patients with PT than in those without PT. Both ADP-induced platelet aggregation (n=116, r2=-0.5042, P<0.001) and vWF activity (n=116, r2=-0.2872, P<0.001) were negatively correlated with ß2-GPI levels. In summary, our data suggested that platelet aggregation and activation were significantly higher in patients with PT than in those without PT, which might be associated with reduced ß2-GPI levels. The reduced ß2-GPI levels might be due to the existence of anti-ß2-GPI IgG.

Increased circulating CD4+CXCR5+FoxP3+ follicular regulatory T cells correlated with severity of systemic lupus erythematosus patients.

As one specialized subset of regulatory T cells (Tregs), follicular regulatory T cells (TFR) could suppress follicular helper T cells (TFH) and B cells in germinal centers to maintain immune homeostasis. The unbalance of TFR and TFH cells could result in abnormal germinal center responses and contribute to pathogenesis of autoimmune diseases. However, the role of TFR cells in systemic lupus erythematosus (SLE) remains unclear. This study revealed a significant increase of CD4+CXCR5+FOXP3+ TFR cells in peripheral blood of SLE patients compared with healthy controls. Meanwhile, the suppression ability of circulating TFR cells was not altered. The ratios of TFR/TFH were increased in SLE patients and the frequency of TFR was positively correlated with auto-antibodies and SLEDAI scores of SLE patients. Our results demonstrated that circulating TFR cells were increased during SLE, which suggested that elevated TFR might be a response to the pathogenesis of SLE to suppress TFH function and may provide novel insight for the pathogenesis of SLE.

Increased Circulating Follicular Treg Cells Are Associated With Lower Levels of Autoantibodies in Patients With Rheumatoid Arthritis in Stable Remission.

OBJECTIVE: To examine the expression and changes in function of circulating CD4+CXCR5+FoxP3+ follicular Treg (Tfr) cells in patients with active rheumatoid arthritis (RA) and in patients with RA in stable remission, and to clarify the role of Tfr cells in the pathogenesis of RA. METHODS: Levels of Tfr cells and follicular helper T (Tfh) cells in the peripheral blood of 39 patients with active RA, 39 patients with RA in stable remission, and 33 healthy controls were detected by flow cytometry. The function of Tfr cells was measured by coculturing them with Tfh cells and B cells. Activated CD45RA-FoxP3high Tfr cells were also analyzed. Clinical indicators, including serum Ig and autoantibody levels, were tested, and correlations with Tfr cells were systematically analyzed. The Disease Activity Score in 28 joints (DAS28) was calculated, and correlation analysis with Tfr cells was conducted. RESULTS: The level of CD4+CXCR5+FoxP3+ Tfr cells and the Tfr cell:Tfh cell ratio in peripheral blood from patients with RA in stable remission were significantly increased compared with the same measures in patients with active RA and in healthy controls. The function of Tfr cells was enhanced, and the activated CD45RA-FoxP3high Tfr cell subset was increased in patients with RA in stable remission compared with healthy controls. Furthermore, the number of Tfr cells in RA patients was inversely correlated with IgG, rheumatoid factor, and anti-cyclic citrullinated peptide as well as with the DAS28. CONCLUSION: Circulating Tfr cells are increased as patients with RA achieve stable remission of disease, and increased Tfr cells can suppress autoimmunity in RA patients to stabilize their condition. Our results provide novel insight into RA pathogenesis.

Characteristics of germinal center-like structures in patients with Sjögren's syndrome.

AIM: To analyze the relationship between ectopic germinal centers (GCs) in the salivary glands and the clinical/laboratory characteristics of patients with Sjögren's syndrome (SS). METHODS: Retrospectively, 126 patients with primary SS (pSS) and 16 patients with secondary SS (sSS) were analyzed. Minor salivary gland biopsies were evaluated for the presence of GC-like morphology by hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining for CD21. Clinical and serological data were obtained from medical records. RESULTS: GC-like structures were observed in 36/126 (28.6%) pSS patients and 4/16 (25.0%) sSS patients. The mean inflammatory focus score of the gland was significantly higher in GC-positive samples than in GC-negative ones in both pSS and sSS patients (P = 0.007 and 0.024, respectively). In pSS, significantly elevated titers of rheumatoid factor (RF)-IgM (P = 0.023) and antinuclear antibodies (ANA) (P = 0.036), increased levels of IgA (P = 0.012) and IgG (P = 0.017) were encountered in GC-positive patients. The GC-positive group also presented higher prevalence of anti-SSA antibodies, lower levels of white blood cells, higher levels of erythrocyte sedimentation rate and γ-globulin, although not statistically significant. In sSS patients with ectopic GC formation, ANA titers were remarkably elevated. The anticyclic citrullinated peptide (anti-CCP)-IgG titers and the prevalence of antikeratin antibody (AKA)-IgG, antiperinuclear factor (APF)-IgG were also increased, yet not significantly. GCs were found to be associated with antibody and immunoglobulin production. CONCLUSION: This study indicates that SS patients with ectopic GCs have distinct features. Ectopic GC structures were particularly noted in patients with higher focus scores, and might play an essential role in sustaining antibody production as well as B cell activation.

Decreased ADAMTS-13 level is related to inflammation factors and risk stratification of acute lymphoblastic leukemia patients.

As a kind of metalloprotease of the ADAMTS family, ADAMTS-13 is crucial for maintaining the normal size of von Willebrand factor. Reduced ADAMTS-13 had been reported in patients with both localized and disseminated malignancies. However, the expression and potential role of ADAMTS-13 in hematological malignancies remain unclear. In this research, we measured and compared ADAMTS-13 levels in plasma of 35 acute lymphoblastic leukemia (ALL) patients and 30 healthy controls and found that ALL patients possessed lower level of ADAMTS-13 than controls. Correlations between ADAMTS-13 and inflammation factors were calculated and ADAMTS-13 was negatively correlated with C-reactive protein and interleukin-1ß. ALL patients with infections had lower level of ADAMTS-13 than patients without infections. In addition, high-risk ALL patients possessed lower ADAMTS-13 than patients at low risk. To conclude, ADAMTS-13 level is decreased in the plasma of ALL patients and the level of ADAMTS-13 is related to plasma inflammation factors and risk stratification of ALL patients, which could contribute to better understanding of the clinical significance of ADAMTS-13.

Reduced ADAMTS-13 level negatively correlates with inflammation factors in plasma of acute myeloid leukemia patients.

ADAMTS-13 is crucial for maintaining the normal size of vWF. Besides thrombotic thrombocytopenic purpura (TTP), decreased ADAMTS-13 had also been reported in patients with malignancy. However, the knowledge of expression and potential role of ADAMTS-13 in hematological malignancies is still limited. We measured and compared ADAMTS-13 levels in the plasma of 82 acute myeloid leukemia (AML) patients and 34 healthy controls and found that AML patients possessed lower ADAMTS-13 than controls. AML patients with infections possessed lower level of ADAMTS-13 than patients without infections and ADAMTS-13 levels were negatively correlated with C-reactive protein(CRP), IL-6, TNFα and IL-1ß. Furthermore, high risk AML patients are with lower ADAMTS-13 than patients with low risk. ADAMTS-13 negatively correlated with ISTH scores and patients accompanying DIC possessed lower ADAMTS-13.Multivariate analyses proved that low level of ADAMTS-13 is an independent risk factor for AML outcome. To conclude, ADAMTS-13 levels are decreased in plasma of AML patients and the level of ADAMTS-13 is related to inflammation and infection of AML patients. Besides, low ADAMTS-13 level is one potential risk factor for AML patients.

Elevated D-dimer and fibrinogen levels in serum of preoperative bone fracture patients.

The changes of coagulation parameters in preoperative fracture patients reflect the coagulation status before surgery. We did retrospective assessment of preoperative fracture patients (n = 113) admitted to the hospital between September 2013 and September 2014. The control group were selected from healthy adults (n = 113) with matched age and gender. Platelet, PT INR, APTT, fibrinogen (FIB) and D-dimer values were collected and analyzed. PT INR level was 1.043 ± 0.119, APTT was 31.91 ± 7.56 s, FIB was 320.6 ± 71.8 mg/dl and D-dimer was 1283 ± 1582 ng/ml for the fracture patients. For the control group, PT INR level was 0.9976 ± 0.0602, APTT was 33.22 ± 2.55 s, FIB was 277.3 ± 44.7 mg/dl and D-dimer was 97.53 ± 63.90 ng/ml. Meanwhile, D-dimer levels of different sites of fractures were also measured: Femora 2448 ± 1961 ng/ml; Humerus 792.4 ± 691.2 ng/ml; Ulna/Radius 619.4 ± 843.7 ng/ml; Vertebra 647.7 ± 787.1 ng/ml; Tibia/Fibula 496.3 ± 268.8 ng/ml; Clavicle 260.9 ± 170.9 ng/ml; Ankle 415.4 ± 286.6 ng/ml. To conclude, D-dimer and fibrinogen levels get higher in preoperative fracture patients than controls. Besides, D-dimer levels are significantly different among different locations of fractures, and our data revealed that D-dimer levels of Femora fracture were higher than other sites.

Increased prostacyclin levels inhibit the aggregation and activation of platelets via the PI3K-AKT pathway in prolonged isolated thrombocytopenia after allogeneic hematopoietic stem cell transplantation.

OBJECTIVES: The aim of this study was to investigate the role of prostacyclin (PGI2) in prolonged isolated thrombocytopenia (PT) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and the effect of PGI2 on the activation and aggregation of platelets in PT. METHODS: We enrolled 37 patients with PT and 36 controls following allo-HSCT in this study. Platelet aggregation and activation and PGI2 levels were measured. Endothelial progenitor cells (EPCs) from either PT or control patients were cultured ex vivo with serum from either PT or control patients. PGI2 secretions were then measured. PGI2 was added to the platelets ex vivo, and platelet aggregation and activation and PI3K/Akt phosphorylation were analyzed. RESULTS: A higher PGI2 level was observed in the PT patients. The activation and aggregation of platelets were significantly lower in the PT patients. EPCs from PT patients cultured in PT serum secreted higher levels of PGI2, and PGI2 inhibited platelet activation and aggregation in a concentration-dependent manner ex vivo. PI3K/Akt phosphorylation of platelets was regulated by PGI2 after allo-HSCT. Disease status, serum PGI2 level and platelet aggregation were independent risk factors in patients with PT after allo-HSCT. CONCLUSIONS: Higher PGI2 levels and lower platelet activation and aggregation occurred simultaneously in PT patients. PGI2 inhibited platelet activation and aggregation, probably by regulating the phosphorylation of PI3K/Akt.

Adipose-Derived Mesenchymal Stem Cells (ADSCs) With the Potential to Ameliorate Platelet Recovery, Enhance Megakaryopoiesis, and Inhibit Apoptosis of Bone Marrow Cells in a Mouse Model of Radiation-Induced Thrombocytopenia.

Substantial damage to the bone marrow can be caused by exposure to radiation, which can then develop into severe thrombocytopenia. In this study, we investigated the in vivo impact of adipose-derived mesenchymal stem cells (ADSCs) on megakaryopoiesis and platelet recovery in irradiated mice. Radiation markedly reduced peripheral blood counts. Recovery of both platelets and WBCs was better in the ADSC-treated group compared with the saline group and the fibroblast group 21 days after irradiation. A significant increase in the total CFU and MK-CFU after irradiation was observed in the ADSC group compared with the saline group and the fibroblast group. Further, the proportion of CD41(+) cells in the ADSC group was significantly higher than that in the saline group and the fibroblast group. ADSC treatment significantly improved the cellularity and decreased the apoptotic cells in the bone marrow while normal fibroblasts did not. Administration of ADSCs upregulated protein expression of phosphorylated Akt and Bcl-xL, whereas the expression of Bax, a protein related to apoptosis, was significantly lower in the ADSC group. In conclusion, this study suggests that ADSCs were capable of promoting platelet recovery, improving megakaryopoiesis, and inhibiting apoptosis of bone marrow cells in irradiated mice. The antiapoptotic effect of ADSCs is likely to be mediated via the PI3K/Akt pathway. These findings may provide a scientific basis for using ADSCs as a new therapy after irradiation.

Reduced IL-35 levels are associated with increased platelet aggregation and activation in patients with acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Acute graft-versus-host disease (aGVHD) is a major complication associated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Interleukin (IL)-35 is a novel anti-inflammatory cytokine that suppresses the immune response. This prospective study explored IL-35 plasma levels in 65 patients after HSCT. The results revealed that the peripheral blood of patients with grades III-IV aGVHD (23.46 ng/ml) had reduced IL-35 compared to transplanted patients with grades I-II aGVHD (40.26 ng/ml, p < 0.01) or patients without aGVHD (41.40 ng/ml, p < 0.05). Allografts, including granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cell (PBPC) and G-CSF-primed bone marrow (GBM), from 38 patients were analyzed for IL-35 levels with respect to aGVHD. The patients who received lower levels of IL-35 cells in the GBM (28.0 ng/ml, p = 0.551) or lower levels of IL-35 in PBPC (53.46 ng/ml, p = 0.03) exhibited a higher incidence of aGVHD. Patients with aGVHD have increased platelet aggregation. IL-35 was added to patient blood in vitro, and platelet aggregation was inhibited by IL-35 in a dose-dependent manner. The markers of platelet activation (CD62P/PAC-1) can also be inhibited by IL-35. The results indicate that IL-35 may affect the development of aGVHD by inhibiting platelet activation and aggregation. Our data suggests that IL-35 represents a potentially effective therapeutic agent against aGVHD after allo-HSCT.

Focused ion beam lithography and anodization combined nanopore patterning.

In this study, focused ion beam lithography and anodization are combined to create different nanopore patterns. Uniform-, alternating-, and gradient-sized shallow nanopore arrays are first made on high purity aluminum by focused ion beam lithography. These shallow pore arrays are then used as pore initiation sites during anodization by different electrolytes. Depending on the nature of the anodization electrolyte, the nanopore patterns by focused ion beam lithography play different roles in further pore development during anodization. The pore-to-pore distance by focused ion beam lithography should match with that by anodization for guided pore development to be effective. Ordered and heterogeneous nanopore arrays are obtained by the focused ion beam lithography and anodization combined approach.