Reducing TGF-ß1 cooperated with StemRegenin 1 promoted the expansion ex vivo of cord blood CD34+ cells by inhibiting AhR signalling.

OBJECTIVE: As an inhibitor of the AhR signalling pathway, StemRegenin 1 (SR1) not only promotes the expansion of CD34+ cells but also increases CD34- cell numbers. These CD34- cells influenced the ex vivo expansion of CD34+ cells. In this work, the effects of periodically removing CD34- cells combined with SR1 addition on the ex vivo expansion and biological functions of HSCs were investigated. MATERIALS AND METHODS: CD34- cells were removed periodically with SR1 addition to investigate cell subpopulations, cell expansion, biological functions, expanded cell division mode and supernatant TGF-ß1 contents. RESULTS: After 10-day culture, the expansion of CD34+ cells in the CD34- cell removal plus SR1 group was significantly higher than that in the control group and the SR1 group. Moreover, periodically removing CD34- cells with SR1 addition improved the biological function of expanded CD34+ cells and significantly increased the percentage of self-renewal symmetric division of CD34+ cells. In addition, the concentration of total TGF-ß1 and activated TGF-ß1 in the supernatant was significantly lower than those in the control group and the SR1 group. RT-qPCR results showed that the periodic removal of CD34- cells with cooperation from SR1 further reduced the expression of AhR-related genes. CONCLUSIONS: Periodic removal of CD34- cells plus cooperation with SR1 improved the expansion of CD34+ cells, maintained better biological function of expanded CD34+ cells and reduced the TGF-ß1 contents by downregulating AhR signalling.

CD34 and Bcl-2 as predictors for the efficacy of neoadjuvant chemotherapy in cervical cancer.

BACKGROUND: Successful neoadjuvant chemotherapy (NACT) could improve the surgical resection rate and radical curability of patients with cervical cancer, but only a subset of patients benefits. Therefore, identifying predictive biomarkers are urgently needed. The aim of this study was to evaluate the predictive value of CD34 and Bcl-2 in the NACT effectiveness of cervical cancer. METHODS: Sixty-seven patients with locally advanced cervical cancer (FIGO stages IB3, IIA2 or IIB) were classified into two groups based on effective (n = 48) and ineffective (n = 19) response to NACT. Immunohistochemistry was employed to identify CD34 and Bcl-2 expression before and after NACT. We analyzed the associations between the pre-NACT expression of these two biomarkers and the response of NACT. The expression of these two biomarkers before and after NACT was also assessed and compared. RESULTS: More patients were CD34 positive expression before NACT in effective group compared to ineffective group (p = 0.005). However, no statistically significant difference in Bcl-2 expression before NACT were found between two groups (p = 0.084). In NACT effective group, the expression of both CD34 and Bcl-2 after NACT are down-regulated (p < 0.001 and p < 0.001, respectively), while there are no statistical differences between the pre- and post-NACT expression of CD34 and Bcl-2 in NACT ineffective group (p = 0.453 and p = 0.317, respectively). CONCLUSION: The positive CD34 expression before NACT may serve as a predictive biomarker for NACT of cervical cancer, but the pre-NACT expression of Bcl-2 is not an independent predictor. The down-regulated expression of these two indicators after NACT may indicate effective NACT.

Eltrombopag preferentially expands haematopoietic multipotent progenitors in human aplastic anaemia.

Eltrombopag has been added to first-line treatment of immune aplastic anaemia (AA), resulting in higher responses. We analysed marrow samples of AA patients who responded to immunosuppressive therapy (IST) alone or in combination with eltrombopag for the composition of the haematopoietic stem and progenitor cell (HSPC) compartment. The number of CD34+ cells and multipotent progenitors was higher in patients treated with eltrombopag (P < 0·005; P < 0·05; respectively), but not the number of stem cells. No aberrant phenotype was observed. These results indicate that eltrombopag augments CD34+ cells in vivo and preferentially expands multipotent progenitors, but not stem cells.

Paeoniflorin in experimental BALB/c mansoniasis: A novel anti-angiogenic therapy.

Chronic hepatic schistosomiasis causes portal hypertension, fibrosis and lethal hepatosplenic complications. Previous studies focused mainly on schistosomicidal drugs and neglected the therapeutic approaches against the vascular complications after portal hypertension. Investigating a novel anti-angiogenic therapy is an urgent. The current study is to evaluate the performance of Paeoniflorin (PAE) as an anti-angiogenic therapy, being a powerful anti-fibrotic, compared to artemether (ART) and praziqantel (PZQ) in schistosomiasis mansoni BALB/c mice. Thirty two laboratory bred male BALB/c Swiss albino mice. The mice were classified into four groups (8 mice each), control infected (CI), PZQ (300 mg/kg/12 h), ART (0.1 ml/mg/d) and PAE (50 mg/kg/d) treated groups for one month. All mice groups were sacrificed 15 weeks post infection for assessment of the drugs' efficacy by parasitological, histopathological and immunohistochemical studies. Our results in PAE group showed marked reduction in the mean egg count/gram stool, worm burden, egg count/gram liver tissue, granuloma diameter and pro-angiogenic factors as vascular endothelial growth factor (VEGF), Proliferating cell nuclear antigen (PCNA), alpha-smooth muscle actin (α-SMA) and CD34; conversely, there was an augmentation of the tissue inhibitor metalloproteinases-2 (TIMP-2) as an anti-angiogenic expression that was exceeded ART and PZQ treated groups compared to CI group (p˂0.001). Conclusively, PAE has an anti-angiogenic impact with no vascular proliferative activity or recanalization, no micro-vessel density (MVD) changes, granuloma resolution and fibrosis regression. PAE is predicted to be a potential therapy for chronic hepatic diseases associated with fibrosis and angiogenesis, hopeful in protecting from advanced serious complications; cancer and metastasis.

Protective Effect of the c-mpl Agonist Romiplostim on Megakaryocytopoiesis of Human CD34+ Hematopoietic Progenitor Cells Exposed to Ionizing Radiation.

A thrombopoiesis-stimulating protein, the myeloproliferative leukemia virus protooncogene (Mpl) ligand romiplostim (RP), is currently approved as a therapeutic agent for idiopathic thrombocytopenic purpura in many countries. Although the action of the initial MPL ligand thrombopoietin (TPO) on human megakaryocytic regeneration from irradiated human hematopoietic stem cells has been examined, there are few reports on the action of RP. In the present study, freshly prepared nonirradiated and 2-Gy X-irradiated human CD34 positive (CD34+) cells from placental umbilical cord blood were cultured with a combination of RP and various cytokines. As a result, the effect of RP on cell proliferation of nonirradiated CD34+ cells was found to be comparable to that of TPO. However, the stimulating activity of RP on megakaryocytic progenitor-derived colony formation was markedly lower compared with TPO. Regarding the action of RP with various cytokines, the present results showed that a combination of RP with interleukin-3 (IL-3) or IL-3 plus stem cell factor (SCF) showed a high regenerative effect on cell proliferation, megakaryopoiesis, thrombopoiesis, and megakaryocyte colony formation from X-irradiated CD34+ cells. The present study showed that human recombinant RP has potential effects on human megakaryocytic regeneration from X-irradiated human CD34+ cells and synergistically acts with IL-3 and IL-3 plus SCF, just as observed with TPO.

Impact of prior melphalan exposure on stem cell collection in light chain amyloidosis.

Use of melphalan in multiple myeloma was observed to have a deleterious effect on stem cell collection in older studies. There is limited data on the impact of melphalan in light chain (AL) amyloidosis, especially in the plerixafor era. We retrospectively evaluated stem cell mobilization in 610 patients with AL amyloidosis, of which 79 had prior exposure to melphalan, 167 to other chemotherapeutics, while 364 had no chemotherapy exposure. Collection of CD34+ stem cells × 106/kg was lower in the melphalan group. Median total yields in the melphalan, non-melphalan, and no chemotherapy groups were 5.5, 7.7, and 7.8, respectively; p < 0.001. Day-1 yields were 2.7, 3.5, and 4.0 (p = 0.0003), respectively, and median yields per collection were 2.0, 3.3, and 4.0 (p < 0.001), respectively. Similar results were observed in the sub-group analysis after plerixafor was integrated in our collection algorithm (2009). Patients in the melphalan group had higher failure rate of 9% vs. 2% each in the other two groups (p = 0.006). Impact of melphalan was dose-dependent, with cumulative melphalan exposure of >150 mg (median: three cycles) resulting in lower yields. Therefore, duration of melphalan exposure prior to stem cell collection should be limited, ideally, not exceeding more than two cycles of treatment.

Impact of lenalidomide-based induction therapy on the mobilization of CD34+ cells, blood graft cellular composition, and post-transplant recovery in myeloma patients: a prospective multicenter study.

BACKGROUND: Lenalidomide is an immunomodulatory drug that is also currently used in transplant-eligible patients with multiple myeloma. Previous studies have suggested a negative impact of lenalidomide on the mobilization of CD34+ cells. No data are available regarding the more detailed composition of blood grafts after lenalidomide. STUDY DESIGN AND METHODS: In a multicenter, prospective study, we analyzed the mobilization of CD34+ cells, graft cellular composition, and post-transplant hematologic recovery in 26 patients with multiple myeloma after lenalidomide-based induction and in 34 lenalidomide-naive controls with multiple myeloma. All patients were mobilized with low-dose cyclophosphamide plus granulocyte-colony-stimulating factor. The cellular composition of the grafts was analyzed from thawed, cryopreserved samples with flow cytometry. Graft function was evaluated by engraftment data and by complete blood counts until 12 months after the graft infusion. RESULTS: Patients in the lenalidomide arm had lower median peak CD34+ counts and approximately 40% lower CD34+ cell yields from the first apheresis session, but these differences were not significant. The median total number of CD34+ cells collected was comparable (6.4 vs. 7.5 × 106 /kg). The number of apheresis sessions was higher in the lenalidomide group (2 vs. 1; p = 0.039). The blood graft composition was comparable between the groups. Hematologic recovery within 12 months post-transplant did not differ between the groups. CONCLUSION: Lenalidomide-based induction seems to have an impact on the number of aphereses performed, but not on the total yields of the CD34+ cells in the graft. Neither cellular composition of the grafts nor post-transplant recovery was affected by the limited pre-transplant exposure to lenalidomide.

Effects of Transendocardial CD34+ Cell Transplantation on Diastolic Parameters in Patients with Nonischemic Dilated Cardiomyopathy.

We sought to evaluate the physiological background and the effects of CD34+ cell transplantation on diastolic parameters in nonischemic dilated cardiomyopathy patients (DCM). We enrolled 38 DCM patients with NYHA class III and LVEF < 40% who underwent transendocardial CD34+ cell transplantation. Peripheral blood CD34+ cells were mobilized by G-CSF, collected via apheresis, and injected transendocardially in the areas of myocardial hibernation. Patients were followed for 1 year. At baseline, estimated filling pressures were significantly elevated (E/e' ≥ 15) in 18 patients (Group A), and moderately elevated (E/e '< 15) in 20 patients (Group B). The groups did not differ in age (54 ± 9 years vs. 52 ± 10 years; p = .62), gender (male: 85% vs. 78%; p = .57), or LVEF (31 ± 7% vs. 34 ± 6%; p = .37). When compared to Group B patients in Group A had more segments with myocardial scar (4.9 ± 2.7 vs. 2.7 ± 2.9; p = .03), myocardial hibernation (2.2 ± 1.6 vs. 0.9 ± 1.1; p = .02), and longer average local relaxation time on electroanatomical mapping (378 ± 41 ms vs. 333 ± 34 ms, p = .01). During follow-up there was an improvement in diastolic parameters in Group A (E/e': from 24.3 ± 12.1 to 16.3 ± 8.0; p = .005), but not in Group B (E/e': from 10.2 ± 3.7 to 13.2 ± 9.1; p = .19). Accordingly, in Group A, we found an increase in 6-minute walk distance (from 463 ± 83 m to 546 ± 91 m; p = .03), and a decrease in NT-proBNP (from 2140 ± 1743 pg/ml to 863 ± 836 pg/ml; p = .02). In nonischemic DCM, diastolic dysfunction appears to correlate with areas of myocardial scar and hibernation. Transendocardial CD34+ cell transplantation may improve diastolic parameters in this patient cohort. Stem Cells Translational Medicine 2017;6:1515-1521.

G-CSF mobilized peripheral blood stem cell collection for allogeneic transplantation in healthy donors: Analysis of factors affecting yield.

Mobilized PBSC are the main source for allogeneic HSCT. We aimed to evaluate factors that affect CD34+ cell yield including the donor's age, gender, BSA, processed blood volume and the method of G-CSF dose calculation. Data from 170 healthy donors were analyzed. The concentration of CD34+ cells in the peripheral blood (PB) and the processed volume of blood were significantly correlated to CD34+ cells yield (P < .00005 and P < .001, respectively). The G-CSF dose per m2 was significantly correlated to the concentration of CD34+ cells in the PB (P = .0003) and in the product (P = .01). Smaller BSA and less processed volume were found among female donors, who were given lesser G-CSF dose per m2 , and showed lower yield compared to men. However, multivariate analysis of the yield showed that only the concentration of CD34+ cells in the PB and the processed volume remained independent significant.

Mobilization of human immature hematopoietic progenitors through combinatory use of bortezomib and immunomodulatory drugs.

Combination use of the proteasome inhibitor bortezomib and the immunomodulatory drugs lenalidomide or thalidomide has provided superior outcomes in multiple myeloma over their single use; however, these combinations can produce significant toxicities. Unexpectedly, we found a small but significant increase in the population of immature granulocytes and erythrocytes/megakaryocytes in peripheral blood in 16 of 22 patients (73%) treated with dexamethasone in combination with bortezomib and immunomodulatory drugs (triplet), but not in any of 25 patients treated with either bortezomib or immunomodulatory drugs with dexamethasone (doublet). These immature cells gradually increased to a peak level (mean 2.6% per white blood cells) with triplet therapy, and disappeared immediately after therapy cessation. The numbers of circulating CD34+ cells and colony-forming cells derived from peripheral blood mononuclear cells increased after triplet therapy compared with those in patients treated by either bortezomib or immunomodulatory drugs plus dexamethasone. Furthermore, triplet regimen downregulated the expression of CXCR4, a chemokine receptor essential for bone marrow retention, on CD34+ cells, suggesting an unexpected effect on normal hematopoietic stem/progenitor cells through the reduced interaction with the bone marrow microenvironment. Our observations suggest that combination use should be carefully evaluated to exert synergistic anti-myeloma effects while avoiding unexpected adverse events.

Quantitation of Human Cells that Produce Neutrophils and Platelets in Vivo Obtained from Normal Donors Treated with Granulocyte Colony-Stimulating Factor and/or Plerixafor.

Plerixafor (P) together with granulocyte colony-stimulating factor (G) is now recognized as an important strategy for mobilizing hematopoietic cells for use in patients given myelosuppressive therapies. However, quantitative comparisons of their ability to mobilize human cells with different hematopoietic activities in vitro or in vivo (in immunodeficient mice) and their interrelationships have not been investigated. To address these questions, we collected samples from 5 normal adult volunteers before and after administering P alone and from another 5 before and after a 4-day course of G and again after a subsequent injection of P. Measurements of their blood content of CD34+ cells, in vitro myeloid colony-forming cells, 3- and 6-week long-term culture (LTC) cell outputs, and levels of circulating human platelets, as well as myeloid and lymphoid cells obtained in immunodeficient mice that received transplants, showed all activities were maximal 4 hours after P preceded by G, and 3-week LTC outputs showed the highest concordance with the 3-week circulating human neutrophil levels obtained in mice that received transplants. Thus, human cells capable of producing neutrophils rapidly in vivo were optimally mobilized by the G + P protocol, and the 3-week LTC assay appears to offer a more specific predictor of their levels than conventional CD34+ cell or colony-forming cell counts.

[Effect of bortezomib-based induction therapy on the peripheral blood stem cell harvest in multiple myeloma].

A high dose of melphalan followed by autologous stem cell transplantation (ASCT) is considered as the standard therapy for multiple myeloma. For induction therapy, 78 patients received conventional regimens (control group) and 32 patients received bortezomib-containing regimens (bortezomib group). We retrospectively compared the yield of harvested CD34+ cells between the two groups. In order to mobilize CD34+ cells, 83% of the control group and 63% of the bortezomib group received a high dose of cyclophosphamide followed by G-CSF, and 12% of the control group received a high dose of etoposide instead of cyclophosphamide. Furthermore, 5% of the control group and 38% of the bortezomib group received G-CSF alone for CD34+ cell mobilization. Overall, the yield of CD34+ cells was higher in the control group than in the bortezomib group (7.4 vs. 5.2×10(6)/kg, P=0.004). Regarding the patients mobilized by a high dose of cyclophosphamide followed by G-CSF, the rate of achieving CD34+ cells >2.0×10(6) cells/kg was similar. Bortezomib did not significantly affect the successful collection of at least CD34+ cells > 2.0×10(6) cells/kg after mobilization with a high dose of cyclophosphamide followed by G-CSF.

Beta-elemene inhibits melanoma growth and metastasis via suppressing vascular endothelial growth factor-mediated angiogenesis.

PURPOSE: It was to assess antiangiogenic effect of ß-elemene in vitro and in vivo, and it was involved in inhibiting melanoma growth and metastasis, as well as to elucidate its intrinsic mechanism. METHODS: Inhibitive effect of ß-elemene on B16F10 cells was performed by cell proliferation assay. Angiogenesis assays in vitro including rat aortic ring and chick embryo chorioallantoic membrane were used, as well as melanoma growth and metastasis assay in C57BL/6 mice. Vascular endothelial growth factor (VEGF) expression in vitro and in vivo was measured respectively by western blot analysis and enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry analysis of CD34 and VEGF expression in primary melanoma was also presented. RESULTS: ß-Elemene inhibited B16BF10 cell proliferation starting from 200 µM, but VEGF from 20 µM. Both 20 and 50 µM ß-elemene in vitro inhibited VEGF-induced sprouting vessel of rat aortic ring and microvessel formation of chick embryo chorioallantoic membrane. In vivo, tumor size of primary melanoma in mice intraperitoneally treated with ß-elemene was significantly smaller than that of the control; CD34 expression of primary melanoma was also suppressed; and the metastatic melanoma colonies and content of melanin in lung were detected obviously decreased in mice of ß-elemene-treated groups. Furthermore, results of VEGF expressing in primary melanoma, serum and lung of mice also disclosed that VEGF was inhibited in vivo. CONCLUSIONS: ß-Elemene inhibited melanoma growth and metastasis through suppressing VEGF-mediated angiogenesis. It is a natural potential antiangiogenic agent.

Effects of raloxifene on Ki-67 and CD34 antigen expression in breast cancer.

BACKGROUND: The aim of this study was to evaluate the effect of raloxifene on CD34 and Ki-67 antigen expression in breast cancer specimens from postmenopausal women. METHODS: Sixteen postmenopausal patients with operable, stage II (>or=3 cm), estrogen receptor-positive breast cancer, who took 60 mg of raloxifene daily for 28 days, participated in this study. Immunohistochemistry was carried out in tumor samples prior to and following raloxifene treatment to evaluate CD34 and Ki-67 protein expression. Angiogenesis was quantified in 10 randomly selected fields per slide, and Ki-67-stained nuclei were counted in 1,000 cells per slide using an image capture and analysis system with 400x magnification. Student's t test for paired samples was used for the statistical analysis of data. Statistical significance was established at p < 0.05. RESULTS: The mean number of microvessels was 44.44 +/- 3.54 prior to raloxifene therapy and 22.63 +/- 1.61 following therapy (p < 0.001), and the mean percentage of Ki-67-stained nuclei was 19.28 +/- 1.61 and 12.13 +/- 1.48 prior to and following raloxifene treatment, respectively (p < 0.001). CONCLUSION: Raloxifene significantly reduces CD34 and Ki-67 protein expression in breast carcinoma in postmenopausal women.

Treatment with anti-CC chemokine receptor 3 monoclonal antibody or dexamethasone inhibits the migration and differentiation of bone marrow CD34 progenitor cells in an allergic mouse model.

BACKGROUND: The migration and in situ differentiation of CD34(+) progenitors contribute to inflammatory eosinophilia in asthma and corticosteroids have been widely used in asthma. However, little is know about whether and how corticosteroids modulate the migration and differentiation of CD34(+) progenitors. This study was aimed to investigate the impact of anti-CC chemokine receptor 3 (CCR3) or dexamethasone on inflammatory eosinophilia in asthma and possible mechanism(s) underlying the action of dexamethasone or anti-CCR3 on migration and differentiation of CD34(+) progenitors in asthmatic context. METHODS: Using an asthmatic mouse model, airway inflammation of anti-CCR3- or dexamethasone-treated mice and that of controls were characterized. And the migration and differentiation of CD34(+) progenitor cells were analyzed in vivo, ex vivo or in vitro. RESULTS: Treatment with anti-CCR3 or dexamethasone significantly inhibited allergen-induced eosinophilia and CD34(+) progenitor cell infiltration in the lung, which was accompanied by lower levels of airway hyper-responsiveness and mucus production. Moreover, anti-CCR3 inhibited the eotaxin-mediated migration and IL-5/eotaxin-induced differentiation of CD34(+) progenitors in vitro. Dexamethasone was also shown to mitigate eotaxin-mediated migration and IL-5 or eotaxin-promoted differentiation of CD34(+) progenitor cells ex vivo, which were associated with the down-regulation of CCR3 expression on bone marrow progenitor cells. CONCLUSIONS: Treatment with anti-CCR3 or dexamethasone can inhibit the migration and differentiation of CD34(+) progenitor cells by regulating the eotaxin/CCR3 axis in asthmatic mice. Our findings provide new insights into understanding the mechanism(s) underlying the action of dexamethasone and CCR3-mediated signaling in allergic inflammation and aid in the design of new immunotherapy for intervention of human asthma.

High-grade glioma before and after treatment with radiation and Avastin: initial observations.

We evaluate the effects of adjuvant treatment with the angiogenesis inhibitor Avastin (bevacizumab) on pathological tissue specimens of high-grade glioma. Tissue from five patients before and after treatment with Avastin was subjected to histological evaluation and compared to four control cases of glioma before and after similar treatment protocols not including bevacizumab. Clinical and radiographic data were reviewed. Histological analysis focused on microvessel density and vascular morphology, and expression patterns of vascular endothelial growth factor-A (VEGF-A) and the hematopoietic stem cell, mesenchymal, and cell motility markers CD34, smooth muscle actin, D2-40, and fascin. All patients with a decrease in microvessel density had a radiographic response, whereas no response was seen in the patients with increased microvessel density. Vascular morphology showed apparent "normalization" after Avastin treatment in two cases, with thin-walled and evenly distributed vessels. VEGF-A expression in tumor cells was increased in two cases and decreased in three and did not correlate with treatment response. There was a trend toward a relative increase of CD34, smooth muscle actin, D2-40, and fascin immunostaining following treatment with Avastin. Specimens from four patients with recurrent malignant gliomas before and after adjuvant treatment (not including bevacizumab) had features dissimilar from our study cases. We conclude that a change in vascular morphology can be observed following antiangiogenic treatment. There seems to be no correlation between VEGF-A expression and clinical parameters. While the phenomena we describe may not be specific to Avastin, they demonstrate the potential of tissue-based analysis for the discovery of clinically relevant treatment response biomarkers.

Stem cell mobilization with cyclophosphamide overcomes the suppressive effect of lenalidomide therapy on stem cell collection in multiple myeloma.

A total of 28 treatment-naïve patients with stage II or III multiple myeloma (MM) were treated with the combination of clarithromycin, lenalidomide, and dexamethasone (BiRD). Stem cells were collected following granulocyte-colony stimulating factor (G-CSF) or cyclophosphamide (Cy) plus G-CSF mobilization at maximum response. Sufficient stem cells for 2 autologous stem cell transplants were collected from all patients mobilized with Cy plus G-CSF, versus 33% mobilized with G-CSF alone (P < .0001). The duration of prior lenalidomide therapy did not correlate with success of stem cell harvests (P = .91). In conclusion, Cy can be added to G-CSF for stem cell mobilization to successfully overcome the suppressive effect of prior treatment with lenalidomide.