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Objective: To compare the antimycobacterial activities of rifampicin (RFP) and rifabutin (RBT), and to evaluate the correlation between RBT resistance and genetic alterations in the rpoB gene., Methods: The microplate-based alamar blue assay (MABA) method was performed to detect the antimycobacterial activities of RFP and RBT in 168 strains of Mycobacterium tuberculosis (M. tuberculosis). Meanwhile, we also analyzed the 81 bp core region of rpoB gene by DNA sequencing. The rate of gene mutations was analyzed by chi-square test., Results: RBT was sensitive for all of the 66 RFP-sensitive strains with no mutations in 81 bp core region of rpoB gene. But of the 102 RFP-resistant strains, 76 strains were also resistant to RBT. Cross resistance between RFP and RBT was 74.5% (76/102). Alterations at codons 516, 526, 531 in the rpoB gene correlated with resistance to both RFP and RBT. While point mutations at codons 511 and 533 possibly influenced the susceptibility to RFP but not to RBT. The mutation rate (92.1%, 70/76) of rpoB gene of RBT-resistant strains was significantly higher than that (23.9%, 22/92) of RBT-sensitive strains (χ(2) = 78.12, P < 0.05)., Conclusions: RBT was more active against M. tuberculosis as compared to RFP. The RFP-resistant strains with MIC ≤ 4 mg/L were still susceptible to RBT. Our results suggest that analysis of genetic alterations in the rpoB gene is useful for predicting RFP-resistance, and may have implications for evaluating RBT-resistance.

Tn order to set up a mouse model of myelofibrosis (MF) induced with high dose recombinant human erythropoietin (rhEPO). 60 mice were collected and divided into EPO and control groups, the former was injected with rhEPO and the latter with normal saline intraperitoneally. 5 mice from each group were executed on day 6, 30, 60, 90, 120 and 150 respectively. Their WBC count, Hb level, MCV, RDW and platelet amount were measured by automatic blood cell analyzer; CD34(+) cell ratio in bone marrow were analyzed by flow cytometry; liver and spleen coefficients were measured; pathological changes of liver, spleen, femur were observed by HE staining and reticular fibers staining; cortex thickness, femoral canal diameter and lumbar spine density were determined by computerized tomography (CT). The results indicated that as compared with normal control group in EPO induced group, WBC count was increased slightly in whole period, but without statistic significance (p > 0.05), Hb level and RDW increased at day 6 and 30 significantly (p < 0.05), MCV increased at day 6 significantly (p < 0.05), but platelet amount decreased significantly at all time points (p < 0.05). Most mice in EPO-induced group had hepatomegalia and their liver and spleen coefficient increased significantly at day 60 (p < 0.05), while most mice had splenomegaly and its coefficient was increased significantly at all time-points (p < 0.05). CD43(+) cell ratio of EPO group increased significantly in whole period (p < 0.05). CT scanning displayed femoral cortical thickening, medulla canal narrowing and lumbar spine density increasing at day 150, meanwhile, HE staining and reticular fiber staining showed the fatty degeneration or vacuolization in liver, splenomegaly with megakaryocytic proliferation, femur bone marrow fibrosis and osteosclerosis. It is concluded that the mouse induced by high dose of rhEPO displays the myelofibrosis associated with splenic extramedullary hemopoiesis, and this study is useful to establish a practical MF model, and to explore its pathological mechanism.

The purpose of this study was to explore the effect of intra-bone marrow infusion (iBMI) of cord blood (CB)-derived hematopoietic stem/progenitor cells (HS/PCs) on human hematopoietic reconstitution in xenotransplanted NOD-SCID mouse model. Aliquots containing 5 x 10(5) CB CD34(+) cells were transplanted into sublethally irradiated NOD-SCID mouse via intravenous infusion (iVI) or iBMI routes. 64 female NOD-SCID mice were divided randomly into 3 groups: iBMI group, iVI group and negative control group. The engraftment levels of human hematopoietic cells at 3, 5 and 8 weeks after xenotransplantation were detected by fluorescence-activated cell sorter (FACS), polymerase chain reaction (PCR), immunohistochemistry and HPC colony formation assay, and long-term hematopoietic reconstitution capacity of HSC was tested by secondary transplantation. The results showed that the percentages of human CD45(+) cells in bone marrow, peripheral blood and spleen of recipient in iBMI group at 8 weeks after xenotransplantation were significantly higher than those in iVI group (p < 0.05). HS/PCs given through both iVI and iBMI methods had the ability of multilineage differentiation, the percentages of CD45(+)CD19(+) cells, CD45(+)CD33(+) cells, CD45(+)CD56(+) cells and CD45(+)CD34(+) cells of recipient bone marrow in iBMI group at 8 weeks after xenotransplantation were significantly higher than those in iVI group (p < 0.05), while other lineages in iBMI group were also higher than that in iVI group (p > 0.05). alpha-satellite-specific fragment of human chromosome 17 could be detected by PCR in liver, spleen, lung, peripheral blood and bone marrow cells of long-term survival recipients in both iVI and iBMI groups. Human CD45 antigen could be detected by immunohistochemical method in spleen, liver and lung of recipients in iBMI group at 8 weeks after xenotransplantation. Total colony count in iBMI group at 8 weeks after xenotransplantation was significantly higher than that in iVI group (p < 0.05). alpha-satellite-specific fragment of human chromosome 17 could be detected in all above organs of both group recipients at 6 weeks after secondary xenotransplantation. It is concluded that iBMI of CB CD34(+) cells improves hematopoietic reconstitution in xenotransplanted NOD-SCID mouse model.

Objective: To investigate the role of tumor necrosis factor (TNF) alpha on the homing efficiency of hematopoietic stem/progenitor cells (HS/PC) into bone marrow and its mechanism., Methods: CFSE-labeled umbilical cord blood (UCB) CD34+ cells were transplanted into irradiated (control group) or combined with TNF alpha prepared (experimental group) BALB/c recipient mice. The distribution in peripheral blood, liver, lung and homing characteristics in bone marrow and spleen of UCB CD34+ cells, in BALB/c recipient mice were determined 20 hours after xenotransplantation by flow cytometry (FACS) and their homing efficiency was calculated. ELISA was used to measure serum SDF-1 alpha level. CXCR4 expression levels of on UCB CD34+ cells were assessed by FACS pre-/post-manipulation with TNF alpha. SDF-1 alpha expression level in bone marrow and spleen was tested by immunohistochemistry., Results: UCB CD34+ cells mainly home into recipient mice bone marrow and spleen; The homing efficiency in experimental group bone marrow [(0.65 +/- 0.13)%] was significantly higher than that in control ones [(0.30 +/- 0.09)%, P < 0.01], whereas the homing efficiency in experimental group spleen was dramatically lower than that in control ones (P < 0.01); Treatment with TNF alpha did not affect recipient serum SDF-1 alpha level; After 18 hours co-cultured with TNF alpha, the CXCR4e expression level on UCB CD34+ cells was similar to that on fresh ones; TNF alpha treatment induced significantly higher SDF-1 alpha expression on osteoblastic and stromal cells in bone marrow, and reversed spleen SDF-1 alpha gradient that was originally favorable for CD34+ cells homing., Conclusion: TNF alpha enhances the homing efficiency of HS/PC via up-regulating SDF-1 alpha gradient in bone marrow, and might be an useful enhancer for HS/PC homing in clinical practice.

Objective: To explore whether intra-bone marrow injection strategy could promote the engraftment of human umbilical cord blood derived hematopoietic stem/progenitor cells (HS/PC) in xenotransplanted NOD/SCID mouse model., Methods: Aliquots containing 1 x 10(3), 1 x 10(4), 0.5 x 10(5), 1 x 10(5) and 5 x 10(5) human umbilical cord blood (hUCB) CD34+ cells were transplanted into sublethally irradiated NOD/SCID mice via intra-venous (i.v.) and intra-bone marrow (iBM) injection. The homing and long-term engraftment capabilities of hUCB CD34+ cells from right tibia, right femur, left tibia, left femur and spleen were detected by PCR 24h after xenotransplantation and by FACS 8-week after xenotransplantation., Results: Tissues of liver, spleen, lungs, or cells from peripheral blood, right tibia, right femur, left tibia and left femur 24 hours after xenotransplantation in iBM injecting 5 x 10(5) CD34+ cells recipients expressed human chromosome 17 specific alpha-satellite fragment. 8-week engraftment of human cells was observed and engraftment level indicated dose-dependent effect in injected bone (right tibia) as well as non-injected bones (including right femur, left tibia and left femur), spleen and peripheral blood in all iBM recipients. 8-week engraftment levels of human cells were (44.063 +/- 20.095)% and (45.881 +/- 22.316)% for i.v. and iBM groups respectively, when transplanted with 1 x 10(5) hUCB CD34+ cells, being no statistical difference (P >0.05). More superior 8-week engraftment levels of human cells were observed in iBM recipients [(54.019 +/- 31.338)%] than in i.v. recipients [(12.197 +/- 10.350)%] when transplanted with 1.0 x 10(4) CD34 cells (P<0.01). Human cell engraftment was observed in iBM but not in i.v. recipients when transplanted with 1.0 x 10(3) CD34+ cells, and was usually observed in non-injected bones., Conclusion: Intra-bone marrow strategy can efficiently increase the engraftment of umbilical cord blood derived hematopoietic stem/progenitor cells in xenotransplanted NOD/SCID mouse model.