Shengui Sansheng Pulvis maintains blood-brain barrier integrity by vasoactive intestinal peptide after ischemic stroke.

Background Shengui Sansheng Pulvis (SSP) has about 300 years history used for stroke treatment, and evidences suggest it has beneficial effects on neuro-angiogenesis and cerebral energy metabolic amelioration post-stroke. However, its protective action and mechanisms on blood-brain barrier (BBB) is still unknown. Purpose Based on multiple neuroprotective properties of vasoactive intestinal peptide (VIP) in neurological disorders, we investigate if SSP maintaining BBB integrity is associated with VIP pathway in rat permanent middle cerebral artery occlusion (MCAo) model. Methods Three doses of SSP extraction were administered orally. Evaluations of motor and balance abilities and detection of brain edema were performed, and BBB permeability were assessed by Evans blue (EB) staining. Primary brain microvascular endothelial cells (BMECs) were subjected to oxygen-glucose deprivation, and incubated with high dose SSP drug-containing serum and VIP-antagonist respectively. Transendothelial electrical resistance (TEER) assay and Tetramethylrhodamine isothiocyanate (TRITC)-dextran (4.4 kDa) and fluorescein isothiocyanate (FITC)-dextran (70 kDa) were used to evaluate the features of paracellular junction. Western blot detected the expressions of Claudin-5, ZO-1, Occludin and VE-cadherin, matrix metalloproteinase (MMP) 2/9 and VIP receptors 1/2, and immunofluorescence staining tested VIP and Claudin-5 expressions. Results Our results show that SSP significantly reduces EB infiltration in dose-dependent manner in vivo and attenuates TRITC- dextran and FITC-dextran diffusion in vitro, and strengthens endothelial junctional complexes as represented by decreasing Claudin-5, ZO-1, Occludin and VE-cadherin degradations and MMP 2/9 expression, as well as promoting TEER in BMECs after ischemia. Moreover, it suggests that SSP notably enhances VIP and its receptors 1/2 expressions. VIP-antagonist exacerbates paracellular barrier of BMECs, while the result is reversed after incubation with high dose SSP drug-containing serum. Additionally, SSP also improve brain edema and motor and balance abilities after ischemic stroke. Conclusions we firstly demonstrate that the ameliorated efficacy of SSP on BBB permeability is related to the enhancements of VIP and its receptors, suggesting SSP might be an effective therapeutic agent on maintaining BBB integrity post-stroke.

Conditioning Regimen of 5-Day Decitabine Administration for Allogeneic Stem Cell Transplantation in Patients with Myelodysplastic Syndrome and Myeloproliferative Neoplasms.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment for patients with myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). However, post-HSCT relapse remains a major cause of treatment failure. Here we assessed the efficacy of a new conditioning regimen comprising decitabine (Dec), busulfan (Bu), cyclophosphamide (Cy), fludarabine (Flu), and cytarabine (Ara-c) for allo-HSCT in patients with MDS and MDS/MPN. A total of 48 patients were enrolled, including 44 with MDS and 4 with chronic myelomonocytic leukemia (CMML). Patients received Dec 20 mg/m2/day on days -9 to -5, combined with a Bu/Cy/Flu/Ara-c-modified preparative regimen. At a median follow-up of 522 days (range, 15 to 1313 days), the overall survival (OS) was 86%, relapse incidence was 12%, and nonrelapse mortality was 12%. The incidence of severe acute (grade III-IV) graft-versus-host disease (GVHD) was 23% and that of chronic GVHD was 15%. At 2 years, OS was 74% and 86%, respectively for high-risk and very-high-risk patients with MDS. Survival was promising in patients with poor-risk gene mutations, such as TP53 and ASXL1 (88%), and in those with ≥3 gene mutations (79%). Results of immunomonitoring studies revealed that proper natural killer cells made essential contributions to these favorable clinical outcomes. Overall, this new regimen was associated with a low relapse rate, low incidence and severity of GVHD, and satisfactory survival in allo-HSCT recipients with MDS and MDS/MPN.

Elevated Plasma P-Selectin Autoantibodies in Primary Sjögren Syndrome Patients with Thrombocytopenia.

BACKGROUND Primary Sjögren's syndrome (pSS) is one of the most common chronic systemic autoimmune diseases, and thrombocytopenia is one of the hematological manifestations of pSS. When platelet and endothelial cells are activated, P-selectin is expressed on the cell surface. This study aimed to investigate the role of P-selectin autoantibodies in the pathogenesis of thrombocytopenia in pSS. MATERIAL AND METHODS P-selectin autoantibodies were measured by enzyme-linked immunosorbent assay (ELISA) in 38 pSS patients without thrombocytopenia and 32 pSS patients with thrombocytopenia, 32 idiopathic thrombocytopenic purpura (ITP) patients, and 35 healthy controls. RESULTS The plasma P-selectin autoantibodies (A490) in ITP patients and pSS patients with/without thrombocytopenia were significantly higher than those in healthy controls, but there were no significant differences between ITP patients and pSS patients with thrombocytopenia. The positive rate of P-selectin autoantibodies in pSS patients with thrombocytopenia was significantly higher than that in ITP patients. The platelet count was lower in P-selectin autoantibodies-positive patients, while among pSS patients with thrombocytopenia, the platelet count was lower in P-selectin autoantibodies-positive patients than in P-selectin autoantibodies-negative patients. In ITP patients and pSS patients with thrombocytopenia, the platelet count was lower in P-selectin autoantibodies-positive patients. CONCLUSIONS Elevated plasma P-selectin autoantibodies may play a role in the pathogenesis of thrombocytopenia in pSS patients.

Outcomes of Adults with Acute Lymphoblastic Leukemia After Autologous Hematopoietic Stem Cell Transplantation and the Significance of Pretransplantation Minimal Residual Disease: Analysis from a Single Center of China.

BACKGROUND: The postremission therapies for adult patients generally contain consolidation chemotherapy, allogeneic hematopoietic stem cell transplantation and autologous hematopoietic stem cell transplantation (auto-HSCT). Because of the various results from different centers, the optimal therapy for adult acute lymphoblastic leukemia (ALL) patients is still uncertain. This study aimed to better understand predictive factors and role of auto-HSCT in the postremission therapy for adult ALL patients. METHODS: The outcomes of 135 adult patients with ALL, who received the first auto-HSCT in Hematopoietic Stem Cell Transplantation Center of Blood Diseases Hospital, Chinese Academy of Medical Sciences from January 1, 1994 to February 28, 2014, were retrospectively analyzed. Survival curves were estimated using the Kaplan-Meier method and simultaneous effects of multiple covariates were estimated with the Cox model. RESULTS: Overall survival (OS) and disease-free survival (DFS) at 5 years for the whole cohort were 59.1 ± 4.5% and 59.0 ± 4.4%, respectively. The cumulative nonrelapse mortality and relapse rate at 5 years were 4.5 ± 0.03% and 36.6 ± 0.19%. For both OS and DFS, acute T-cell lymphoblastic leukemia, high lactate dehydrogenase (LDH) at diagnosis, blast cell proportion ≥5% on the 15 th day of induction therapy, and extramedullary infiltration before HSCT were the poor prognosis factors. In addition, age ≥35 years predicted poor DFS. Only T-ALL and high LDH were the independent undesirable factors associated with OS and DFS in Cox regression model. For 44 patients who had results of pretransplantation minimal residual disease (MRD), positive MRD (MRD ≥0.01%) indicated poor OS (P = 0.044) and DFS (P = 0.008). Furthermore, for the standard risk group, the patients with negative MRD (MRD <0.01%) had better results (OS at 18 months was 90.0 ± 9.5%, while for the patients with positive MRD OS was 50.0 ± 35.4%, P = 0.003; DFS at 18 months was 90.0 ± 9.5%, while for the positive MRD group DFS was 0%, P < 0.001). CONCLUSIONS: This study confirmed that auto-HSCT combined with posttransplantation maintenance chemotherapy could be an option for adult ALL patients and pretransplantation MRD may play a significant role in the direction of therapy for adult ALL patients.

[Analyses of risk factors for intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To investigate the risk factors of intestinal acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: The clinical data of 534 cases of 533 patients undergoing allo-HSCT during Jan 2004 and Sep 2012 were retrospectively analyzed. The effects of donor-recipient HLA mismatching, recipient age, donor age, donor-recipient sex combination, donor-recipient relationship, HSC source, conditioning regimen with or without total body irradiation (TBI) and HLA loci on intestinal aGVHD with different severity were analyzed by Logistic regression. RESULTS: Intestinal aGVHD occurred in 123(23.0%) cases, with 86(16.1%) cases of stage 1 intestinal aGVHD(16.1%) and 37(6.9%) cases of stage 2 to 4 intestinal aGVHD. Multivariate analysis showed that donor-recipient HLA mismatching (OR=2.519, P=0.002), increasing donor age (OR=1.034, P=0.003), female donor for male recipient (OR=1.855, P=0.007) were risk factors for intestinal aGVHD, HLA-B38 (OR=0.256, P=0.032) was its protective factor. Donor-recipient HLA mismatching (OR=2.799, P=0.011), increasing donor age (OR=1.045, P=0.012), HLA-A1 (OR=4.157, P=0.002), A30 (OR=3.143, P=0.005) were risk factors for stage 2 to 4 intestinal aGVHD. CONCLUSION: Occurrence of intestinal aGVHD and its severity are associated with donor-recipient HLA mismatching, donor age, donor-recipient sex relationships and some HLA loci.

[Prophylaxis of invasive fungal infection with different administration regimens of itraconazole in patients with acute myeloid leukemia: a report from a randomized, controlled trial].

OBJECTIVE: To evaluate the efficacy and safety of antifungal prophylaxis of itraconazole in patients with acute myeloid leukemia (AML) to probe the relationship of the antifungal effect and the adverse events with serum concentration. METHODS: From April 2009 to May 2011, a total of 310 courses from 112 patients referred to our institute were enrolled in this study; of them, 297 courses were eligible for analysis. Eligible cases were randomized into oral group and injection/oral group according to different chemotherapy of induction and consolidation. Blood samples were collected at different time points for measurements of serum itraconazole levels. The morbidity of IFI and the adverse events were analyzed. RESULTS: The morbidities of IFI in injection/oral and oral groups were 10.1% and 20.9%, respectively (P=0.010). 7 and 9 cases in injection/oral and oral groups, respectively were withdrawn from the study because of adverse events, and the difference between these two groups was of no significance. Serum itraconazole levels of injection/oral and oral groups were 672(299-1097) µg/L and 534(210-936) µg/L, respectively (P<0.01). CONCLUSION: Antifungal prophylaxis with itraconazole in AML patients was effective and safe. Prophylactic effect with injection/oral itraconazole was superior to oral itraconazole solution; moreover, prophylactic effect of itraconazole was highly correlated with its serum level.

[Efficient isolation of mesenchymal stem cells from human bone marrow by direct plating method combined with modified primary explant culture].

Human bone marrow is the major source of mesenchymal stem cells (MSC). It was reported that the standard density gradient centrifugation method was not efficient in isolating MSC and it may be caused by the existing of bone marrow particles. In previous study, a lot of MSC were obtained by culturing bone marrow particles alone combined with standard method. However, it is time- and labor-consuming to obtain bone marrow particles by filtering and to isolate MNC by density gradient centrifugation. This study was purposed to explore the more simple and efficient method to isolate MSC from bone marrow. Seven normal bone marrow aspirates were collected and centrifugated. The bone marrow particles floated on surface layers were cultured by modified primary explant culture, whereas the bone marrow aspirates deposited were cultured by direct plating method, then the immun phenotype and differentiation capability of isolated cells were analyzed. The results showed that in 3 of 7 aspirates, bone marrow particles were floated on surface layers, whereas the other bone marrow cells and some particles were deposited after centrifugation. The MSC were reliably isolated from the floating layers or deposited aspirates by modified primary explant culture and direct plating method separately. After 3 passages the isolated MSC did not express CD45 and CD34, but expressed CD105, CD73, CD44, CD90, CD49e and they could differentiate into chondrocytes and adipocytes. It is concluded that normal human bone marrow MSC can be isolated simply and efficiently by direct plating method in combination with modified primary explant culture.

[Synergistic immunomodulatory effects of interferon-gamma and bone marrow mesenchymal stem cells].

OBJECTIVE: To investigate mesenchymal stem cells (MSCs) immunosuppressive activity in the presence of interferon-gamma (IFN-γ) to reveal synergistic immunomodulatory effects of IFN-γ and MSCs. METHODS: ① MSCs were cultured in the presence or absence of IFN-γ（100 ng/ml）, the supernatants were collected for measurements of PGE2、HGF and TGF-ß1 by ELISA kits. ② MSCs were cultured in the presence or absence of IFN-γ （100 ng/ml）for 48 h. The cDNA was analysed for the expression of human indoleamine 2, 3-dioxygenase（IDO）mRNA by semiquantitative RT-PCR. ③ Mononuclear cells (MNCs) were extracted from peripheral blood of healthy donors. The T cell proliferation was tested in the co-culture system added with MSCs, recombinant human IFN-γ (100 ng/ml) and anti-IFN-γ mAb (5 µg/ml) by BrdU ELISA kit. RESULTS: ①The immunosuppressive cytokines PGE2、HGF and TGF-ß1 were detectable within 24-48 h in the supernatants. Their expressions were significantly up-regulated in the presence of IFN-γ. Concentrations of these cytokines were as of (1715.5±628.6) pg/ml vs (1344.5±709.4) pg/ml (P=0.001);(4031.8±1496.8) pg/ml vs (2452.4±1375.3) pg/ml（P=0.011）;(1753.5±413.8) pg/ml vs (1026.6±450.5) pg/ml（P<0.001）,respectively. ②The expression of IDO mRNA was undetectable when MSCs were cultured alone. In contrast, The IDO mRNA expression was remarkably enhanced in the presence of IFN-γ. ③Bone marrow-derived MSCs remarkably suppressed allogeneic T cell proliferation in vitro. Addition of exogenous IFN-γ had no significant effect on the inhibitory capacity of MSCs, the inhibitory ratios of T cell proliferation were （40.4±10.9）% vs（36.7±7.4）% (P=0.272). By contrast, the inhibitory ratio of T cell proliferation was significantly decreased in the presence of anti-IFN-γ mAb[(40.4±10.9)% vs (23.9±7.6)%,P=0.002]. CONCLUSION: ①Human MSCs constitutively expressed immunosuppressive concentrations of PGE2, HGF and TGF-ß1, and their expressions were significantly up-regulated by IFN-γ. ②IFN-γ-induced expression of IDO on MSCs involved in tryptophan catabolism. ③MSCs notably suppressed allogeneic T cell proliferation in vitro. IFN-γ promoted the immunosuppressive capacity of human MSCs, indicating the synergistic immunomodulatory effect of IFN-γ and MSCs.

[Autologous stem cell transplantation for adult patients with acute lymphoblastic leukemia and related prognostic factors].

OBJECTIVE: This study was aimed to observe the efficacy of autologous stem cell transplantation (ASCT) for adult patients with acute lymphoblastic leukemia (ALL), and investigate related prognostic factors. METHODS: A total of 86 adult ALL patients underwent ASCT in Institute of Hematology and Blood Disease Hospital from November 2001 to January 2012 were followed up. Clinical characteristics and outcomes of all patients were retrospectively analyzed. Survival and univariate prognosis were analyzed by the Kaplan-Meier method and multivariate analysis by COX regression model. RESULTS: Outcomes were assessed in 81 cases, including 47 standard-risk and 34 high-risk patients. 1-, 3-, 5-, and 10-year leukemia-free survival (LFS) of standard-risk patients were (82.3±5.7)%, (76.9±6.5)%, (74.1±6.8)%, (67.4±8.9)% respectively,and relapse rates (RR) were as of (13.6±5.2)%, (21.6±6.4)%, (24.5±6.8)%, (31.3±9.0)% respectively. 1-, 3-, 5-, and 10-year LFS of high-risk patients were (55.8±8.9)%, (39.8±9.3)%, (39.8±9.3)%, (39.8±9.3)% respectively, and relapse rates (RR) were (38.8±9.2)%, (56.4±10.0)%, (56.4±10.0)%, (56.4±10.0)% respectively. T-ALL, white blood cell count(WBC) more than 30×109/L when first visited, increased LDH, positive fusion gene of TCR and bone marrow transplantation were the adverse prognostic factors. Multivariate analysis showed bone marrow transplantation was an independent adverse prognostic factor. CONCLUSION: ASCT was a choice for adult ALL patients when suitable donors were unavailable.

[Isolation of mesenchymal stem cells from bone marrow filters by primary explant culture].

This study was aimed to investigate whether mesenchymal stem cells (MSC) can be isolated from bone marrow filters which have always been discarded. The bone marrow (BM) particles from BM filters of 2 healthy donors were cultivated by primary explant culture. After expansion, the number of MSC was counted and their immunophenotype and differentiation potential were detected. The results indicated that many MSC were found in bone marrow particles from filters, and nearly 10(7) MSC were obtained at 3 passages of expansion. They not only possessed the characteristics of morphology and immunophenotype of MSC, but also could differentiate into osteoblasts, chondrocytes and adipocytes. In conclusion, a large amount of MSC can be obtained from BM filters if the BM particles were cultivated by primary explant culture.

[Risk factors and prognosis of invasive fungal infections in patients with hematological diseases].

OBJECTIVE: To investigate the incidence, risk factors, prognosis and high risk patients of invasive fungal infections (IFI) in patients with hematological diseases. METHODS: : Over 2-week hospitalized patients from January 2007 to December 2008 were retrospectively reviewed. Logistic regression was used to analyze the risk factors of IFI, and recursive partitioning to reveal high risk patients. Incidence of IFI was estimated by cumulative incidence function, and the prognosis by Kaplan-Meier method. RESULTS: A total of 1048 assessable treatment cycles were recorded and 93 cases of IFI were diagnosed, with an incidence of 8.87 per 100 treatment cycles. Multivariate logistic regression revealed the following risk factors: age (OR 1.025, 95% CI 1.010-1.041, P = 0.002), duration of neutropenia (OR 1.028, 95% CI 1.014-1.042, P < 0.0001) and uncontrolled underlying diseases (OR 2.620, 95% CI 1.608-4.268, P = 0.0001). Recursive partitioning found two groups of high risk patients: (1) patients with uncontrolled underlying diseases and neutropenia duration > or = 58 days (7/12, 58.3%), (2) patients with uncontrolled underlying diseases and age > or = 33 years (40/208, 19.2%). At the end of follow-up, 111 cases of IFI were recorded in 451 patients, with a 1-year cumulative incidence of 27.1%. In patients with established IFI, overall survival rate and IFI related mortality rate at 12 weeks after diagnosis were 83.4% and 13.5% respectively. CONCLUSION: Age, duration of neutropenia and uncontrolled underlying diseases are risk factors of IFI; patients with uncontrolled underlying diseases and age > or = 33 years were at high risk of IFI and need major concern. IFI has a better prognosis and a lower related mortality in this study.