Integrated analysis of different microarray studies to identify candidate genes in type 1 diabetes.

BACKGROUND: Type 1 diabetes (T1D), an autoimmune disease, occurs most commonly in children. Identifying altered gene expression in peripheral blood mononuclear cells (PBMCs) of T1D may lead to new strategies for preserving or improving ß-ell function in patients with T1D. METHODS: The Gene Expression Omnibus database was searched for microarray studies in PBMCs of T1D. Subsequently, gene expression datasets from multiple microarray studies were integrated to obtain differentially expressed genes (DEGs) between T1D and normal controls (NC). Gene function analysis was performed to determine the functions of the DEGs identified. RESULTS: Four microarray studies were available for analysis, including 199 T1D samples and 74 NC samples. Analysis revealed 695 genes that were significantly differentially expressed in PBMCs from T1D compared with NC samples, with 450 upregulated and 245 downregulated. Signal transduction (gene ontology [GO]: 0007165; false discovery rate [FDR] = 1.54 × 10-7 ) and protein binding (GO: 0005515; FDR = 2.93 × 10-24 ) were significantly enriched for the GO categories of biological processes and molecular functions, respectively. The most significant pathway in the Kyoto Encyclopedia of Genes and Genomes analysis was arachidonic acid metabolism (FDR = 1.44 × 10-3 ). Protein-protein interaction network analysis showed that the significant hub proteins contained immature colon carcinoma transcript 1 (ICT1; degree = 214; clustering coefficient [C] = 4.39 × 10-5 ), zinc finger and BTB domain containing 16 (ZBTB16; degree = 112; C = 8.04 × 10-4 ), and SERTA domain containing 1 (SERTAD1; degree = 38; C = 0.0014). CONCLUSIONS: This integrated analysis will help develop improved therapies and interventions for T1D by identifying novel drug targets.

Linezolid Induced Twice Pure Red Cell Aplasia in a Patient with Central Nervous System Infection after Allogeneic Stem Cell Transplantation.

Linezolid (LZD), severed as the first oxazolidinone antibiotic, was active against multidrug-resistant gram-positive strains. LZD can induce thrombocytopenia, anemia and leukocytopenia. Currently, reports on pure red cell aplasia (PRCA) cases induced by LZD are relatively rare (4-7). In this paper, we reported a patient with PRCA twice induced by LZD. A 37-year-old man was diagnosed with myelodysplatic syndrome (MDS) and underwent allo-HSCT from an unrelated donor with ABO blood type and leukocyte antigen (HLA)-matching. After HSCT for 2 years, the patient suffered from refractory fever and headache. He was first treated with empirical antifungal agent and antibiotics for central nervous system (CNS) infection, but then changed to LZD therapy for little effect. Twenty-eight days after LZD treatment, the symptom improved significantly but the hemoglobin declined to 70 g/L and the reticulocyte level was only 0.23%. The LZD therapy was stopped and the fever and headache symptoms reoccurred 1 week latter. Then, erythropoietin (EPO) and halved dosage of LZD were used for treatment. The CNS infection and the anemia symptom relieved gradually and the level of hemoglobin and reticulocyte declined again. After blood transfusion, the half dose of LZD was sustained without anaemia recovery. In summary, patients with anemia, myelosuppressants history or potential abnormal proliferation of T cells may suffer PRCA with long term LZD treatment. The monitoring of complete blood count and reticulocyte count were necessary during LZD therapy. If the clinical condition permits, LZD dosage reduction and blood transfusion should be considered.

Rapid Recovery of CD3+CD8+ T Cells on Day 90 Predicts Superior Survival after Unmanipulated Haploidentical Blood and Marrow Transplantation.

BACKGROUND: Rapid immune reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is significantly associated with lower infection, relapse and possibly secondary malignancy rates. The aim of this study was to investigate the role of peripheral lymphocyte subsets, especially CD3+CD8+ cytotoxic T cell recovery, in predicting transplant outcomes, including the overall survival (OS) and non-relapse mortality (NRM) rates after unmanipulated haploidentical blood and marrow transplantation (HBMT). METHODS: Peripheral blood samples were obtained from 214 HBMT recipients with hematological malignancies. The peripheral lymphocyte subsets (CD3+ T cells, CD3+CD4+ helper T cells, CD3+CD8+ cytotoxic T cells, and CD19+ B cells) were analyzed by flow cytometry at days 30, 60, 90, 180, 270 and 360 after HBMT. RESULTS: The CD3+CD8+ cytotoxic T cell recovery at day 90 (CD3+CD8+-90) was correlated with bacterial infection (P = 0.001), NRM (P = 0.001), leukemia-free survival (LFS, P = 0.005), and OS (P = 0.001) at a cutoff value of 375 cells/µL CD3+CD8+ T cells. The incidence of bacterial infection in patients with the CD3+CD8+-90 at ≥375 cells/µL was significantly lower than that of cases with the CD3+CD8+-90 at <375 cells/µL after HBMT (14.6% versus 41.6%, P<0.001). Multivariate analysis showed the rapid recovery of CD3+CD8+ T cells at day 90 after HBMT was strongly associated with a lower incidence of NRM (HR = 0.30; 95% CI: 0.15-0.60; P = 0.000) and superior LFS (HR = 0.51; 95% CI: 0.32-0.82; P = 0.005) and OS (HR = 0.38; 95% CI: 0.23-0.63; P = 0.000). CONCLUSION: The results suggest that the rapid recovery of CD3+CD8+ cytotoxic T cells at day 90 following HBMT could predict superior transplant outcomes.

Endothelium-targeted human Delta-like 1 enhances the regeneration and homing of human cord blood stem and progenitor cells.

BACKGROUND: Umbilical cord blood (UCB) is becoming an alternative cell source for hematopoietic stem cell transplantation (HSCT). However, umbilical cord blood transplantation (UCBT) has been severely limited by low and finite numbers of hematopoietic stem cells and their delayed engraftment. New strategies are needed to improve ex vivo expansion efficiency and in vivo haematopoietic recovery. METHODS: We produced an endothelium-targeted soluble Notch ligand, the Delta-Serrate-Lag-2 (DSL) domain of human Delta-like 1 fused with a RGD motif (hD1R), and tested the effects of this protein on human umbilical cord blood hematopoietic stem and progenitor cell (UCB-HSPC) ex vivo and in vivo. RESULTS: hD1R-mediated ex vivo expansion system was able to significantly increase the absolute number of UCB-HSPCs. The hD1R-expanded cells had the enhanced homing and maintained long-term hematopoietic stem cell repopulation capacity in the bone marrow of immunodeficient nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice. Moreover, systemic administration of hD1R promoted the in vivo regeneration of donor cells in recipient mice and accelerated hematopoietic recovery, particularly in settings wherein the HSPCs dose was limiting. CONCLUSIONS: Our results indicated that hD1R might be applied in improving hematopoietic recovery and HSC engraftment in human UCBT.

[Effect of endothelial cell-targeted soluble Notch ligand hD1R protein on expansion and engraftment of cord blood hematopoietic stem/progenitor cells].

OBJECTIVE: To evaluate the effects of endothelial cell- targeted soluble Notch ligand hD1R protein on expansion and engraftment of cord blood hematopoietic stem/progenitor cell (CB HSPCs). METHODS: Recombinant hD1R protein was first induced and purified. Human cord blood CD34⁺ cells were co-cultured on human umbilical vein endothelial cells (HUVECs) supplemented with a cocktail containing 5 types of human cytokines including TPO, SCF, FL, IL-6, IL-3 (5GF) and soluble hD1R. The expansion of CD34⁺ cells was tested under different culture conditions including PBS group (PBS replaces HUVEC), hD1R group, sup group (HUVEC supernatant replaces HUVEC), fix group (fixed HUVEC replaces HUVEC), Day 0 group (Control). Cell cycle and apoptosis of cultured cells were also analyzed. Their progeny expanded in PBS or hD1R group were transplanted into sublethally irradiated NOD/SCID mice. The percentages of human CD45⁺ (hCD45⁺) cells in the marrow of recipient mice were determined by FACS 12 weeks later. RESULTS: hD1R induced more expansion in the total number of CD34⁺ cells cocultured with HUVECs plus 5GF, which was 87.50-fold increase compared to the Day 0 group, and 7.98-fold increase than that of PBS group. FACS analysis also showed that the percentage of CD34⁺ cells was 77.0% in G0/G1 phase in the hD1R group, which indicated that hD1R enhanced HSPCs expansion and inhibited apoptosis. Moreover, hD1R significantly promoted human HSPC engraftment after BM transplantation in irradiated mice. CONCLUSION: The Notch-mediated ex vivo expansion system has been established and hD1R promoted expansion and engraftment of human CB HSPCs, which provided the evidence for further clinical application.

Modified bortezomib, adriamycin and dexamethasone (PAD) regimen in advanced multiple myeloma.

The PAD regime, composed of bortezomib, adriamycin and dexamethasone, improves the outcomes of patients with advanced multiple myeloma (MM), but at the same time produces high frequency of serious toxic side effects. For the first time, we evaluated the efficacy and safety of a bortezomib-dose-reduced PAD regime in the treatment of relapsed/refractory MM in this clinical study. Forty-five patients were treated with two to six 21-day cycles of PAD, comprising bortezomib at 1.3 mg/m(2) (P1AD, n = 21) or 1.0 mg/m(2) (P2AD, n = 24) (days 1, 4, 8, 11), adriamycin at 9 mg/m(2) (days 1-4) and dexamethasone at 40 mg/day (days 1-4). Overall, 36 patients (80 %) showed at least partial remission (PR), in which 9 cases (20 %) showed complete remission (CR) and 10 cases (22 %) showed very good partial remission (VGPR). The efficacy of PAD regimen in advanced MM patients was not related to the traditional prognostic factors. There was no significant difference between P1AD and P2AD in the rates of PR, CR or VGPR, 1.5-year progression-free survival (PFS), and overall survival (OS) (81 % vs. 79 %, 48 % vs. 38 %, 64 % vs. 59 %, and 85 % vs. 73 %, respectively). However, the grade 3-4 toxic effects, including thrombocytopenia (13 % vs. 38 %), peripheral neuropathy (8 % vs. 33 %) and 3-4 grade gastrointestinal reaction (13 % vs. 43 %), were markedly inhibited after P2AD compared to P1AD (P < 0.05). The bortezomib-dose-reduced PAD regime reduced the incidence of adverse reactions without affecting the treatment efficacy in patients with advanced MM.

Inhibition of tumor angiogenesis and tumor growth by the DSL domain of human Delta-like 1 targeted to vascular endothelial cells.

The growth of solid tumors depends on neovascularization. Several therapies targeting tumor angiogenesis have been developed. However, poor response in some tumors and emerging resistance necessitate further investigations of new drug targets. Notch signal pathway plays a pivotal role in vascular development and tumor angiogenesis. Either blockade or forced activation of this pathway can inhibit angiogenesis. As blocking Notch pathway results in the formation of vascular neoplasm, activation of Notch pathway to prevent tumor angiogenesis might be an alternative choice. However, an in vivo deliverable reagent with highly efficient Notch-activating capacity has not been developed. Here, we generated a polypeptide, hD1R, which consists of the Delta-Serrate-Lag-2 fragment of the human Notch ligand Delta-like 1 and an arginine-glycine-aspartate (RGD) motif targeting endothelial cells (ECs). We showed that hD1R could bind to ECs specifically through its RGD motif and effectively triggered Notch signaling in ECs. We demonstrated both in vitro and in vivo that hD1R inhibited angiogenic sprouting and EC proliferation. In tumor-bearing mice, the injection of hD1R effectively repressed tumor growth, most likely through increasing tumor hypoxia and tissue necrosis. The amount and width of vessels reduced remarkably in tumors of mice treated with hD1R. Moreover, vessels in tumors of mice treated with hD1R recruited more NG2(+) perivascular cells and were better perfused. Combined application of hD1R and chemotherapy with cisplatin and teniposide revealed that these two treatments had additive antitumor effects. Our study provided a new strategy for antiangiogenic tumor therapy.

Endothelium-targeted Delta-like 1 promotes hematopoietic stem cell expansion ex vivo and engraftment in hematopoietic tissues in vivo.

BACKGROUND: Notch ligands enhance ex vivo expansion of hematopoietic stem cells (HSCs). But to use Notch ligands in HSC therapies of human diseases, efforts are required to improve ex vivo expansion efficiency and in vivo transplant engraftment. DESIGN AND METHODS: We designed and produced an endothelium-targeted soluble Notch ligand, the DSL domain of Delta-like 1 fused with a RGD motif (D1R), and examined the effects of this protein on HSCs ex vivo and in vivo. RESULTS: D1R efficiently promoted ex vivo expansion of both mouse bone marrow (BM) and human umbilical cord blood HSCs. HSCs expanded with D1R up-regulated many of the stemness-related genes, and showed high BM engraftment efficacy with long-term repopulation capacity after transplantation. Moreover, in vivo administration of D1R increased the number of BM HSCs in mice, and facilitated BM recovery of mice after irradiation. Injection of D1R significantly improved HSC engraftment and myeloid recovery after BM transplantation in irradiated mice. D1R enhanced HSC engraftment not only in BM, but also in the liver and spleen after BM transplantation in mice. D1R induced the formation of compact cell clusters containing the transplanted HSCs in close contact with endothelial cells, reminiscent of HSC niches, in the liver and spleen. CONCLUSIONS: D1R might be applied in improving both HSC expansion ex vivo and HSC engraftment in vivo in transplantation.

Roles of plasma interleukin-6 and tumor necrosis factor-alpha and FFA and TG in the development of insulin resistance induced by high-fat diet.

The role of adipokines in development of insulin resistance still remains controversial. The purpose of the present study was to examine the dynamic changes of fasting plasma levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), free fatty acids (FFA) and insulin in a Sprague-Dawley rat insulin resistant model induced by high-fat diet. Heterotopic deposition of triglycerides (TG) in liver, skeletal muscles and pancreatic islet was also investigated. The fasting plasma level of insulin in rats in the high-fat diet group was significantly higher than that in the normal diet group on day 21 (P<0.01), suggesting that an increased insulin resistance developed in the high-fat diet group. However, no significant difference in the plasma IL-6 level was observed between the two groups (P>0.05), although in both groups, the plasma IL-6 level was significantly higher on day 21 than that of the day 0 (P<0.05). The plasma FFA level in the high-fat diet group began to increase significantly on day 21 (P<0.05), and elevated markedly on day 28, was positively correlated to the fasting plasma insulin level. Histological study revealed a more abundant TG deposition in liver and skeletal muscles (from quadriceps femoris) in the high-fat diet group than in the normal diet group on day 21, and the liver deposition was even higher on day 28. However, no deposition was observed in pancreatic islets. The plasma TNF-alpha level remained unchanged throughout the duration of the experiment. These results indicate that the progression of insulin resistance in high-fat diet rats is closely related to the plasma FFA elevation and the heterotopic deposition of TG in liver and skeletal muscles, but is unrelated to the plasma TNF-alpha and IL-6 levels.