Anthony E. James

Preclinical ex vivo expansion of cord blood hematopoietic stem and progenitor cells: duration of culture; the media, serum supplements, and growth factors used; and engraftment in NOD/SCID mice.

BACKGROUND: Ex vivo expansion of cord blood (CB) hematopoietic stem and progenitor cells increases cell dose and may reduce the severity and duration of neutropenia and thrombocytopenia after transplantation. This studys purpose was to establish a clinically applicable culture system by investigating the use of cytokines, serum‐free media, and autologous plasma for the expansion of CB cells and the engraftment of expanded product in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice.

Promoting effects of serotonin on hematopoiesis : Ex vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis

Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5‐hydroxytryptamine (5‐HT)2 receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34+ cells, bone marrow (BM) stromal cell colony‐forming unit‐fibroblast (CFU‐F) formation, and antiapoptosis of megakaryoblastic M‐07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34+ cells to early stem/progenitors (CD34+ cells, colony‐forming unit‐mixed [CFU‐GEMM]) and multilineage committed progenitors (burst‐forming unit/colony‐forming unit‐erythroid [BFU/CFU‐E], colony‐forming unit‐granulocyte macrophage, colony‐forming unit‐megakaryocyte, CD61+CD41+ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45+, CD33+, CD14+ cells, BFU/CFU‐E, and CFU‐GEMM engraftment in BM of animals 6 weeks post‐transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet‐derived growth factor, or vascular endothelial growth factor stimulated BM CFU‐F formation. In M‐07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase‐3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5‐HT2 receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation.

Ketamine effects on the urogenital system--changes in the urinary bladder and sperm motility.

Different doses of ketamine (10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, and 60 mg/kg) were injected i.p. (I.P.), respectively, to male ICR mice to determine the optimal dosage for chronic administration. At and above 40 mg/kg I.P. injection, mice had almost no hindlimb movement during swimming test. Subsequently, 30 mg/kg was used as the dose for the study in the toxicity of long‐term ketamine administration on urinary bladder and sperm motility. The treatment group were subdivided into two (n = 10 each group); one received daily ketamine treatment i.p. for 3 months and another group for 6 months. Corresponding number of mice in control groups (n = 5 each group) received saline injection instead of ketamine. Terminal dUTP nick and labeling (TUNEL) study and Sirius red staining were carried out on the sectioned slides of the urinary bladders to study the degree of apoptosis in both epithelium and muscular layers of the urinary bladder and the relative thickness of the muscular layers in this organ was also computed. Apoptosis in the bladder epithelium was observed initially in the 3‐month ketamine treated mice and the number of apoptotic cells was significantly different (P < 0.05) between the 3‐month and 6‐month ketamine treated mice and the control. The relative thickness of muscular layers in the bladder wall also decreased significantly (P < 0.05) when the 6‐month treated mice and the control were compared. Sirius red staining revealed increase of collagen in the urinary bladder of the treated mice, most evidently 6 months after ketamine treatment. In addition, the sperm motility was studied and there was a statistically significant difference between the control and ketamine treated groups in the percentages of sperms which were motile (P < 0.05). This suggested that the chronic administration of ketamine affected the genital system as well. Microsc. Res. Tech., 2011.

Platelet‐derived growth factor promotes ex vivo expansion of CD34+ cells from human cord blood and enhances long‐term culture‐initiating cells, non‐obese diabetic/severe combined immunodeficient repopulating cells and formation of adherent cells

Summary. Platelet‐derived growth factor (PDGF) is a major mitogen for connective tissue cells. In this study, we investigated the effects and mechanism of PDGF on the ex vivo expansion of cord blood CD34+ cells. Our data demonstrated that among various cytokine combinations of thrombopoietin (TPO), interleukin 1 beta (IL‐1β), IL‐3, IL‐6 and Flt‐3 ligand (Flt‐3L), TPO + IL‐6 + Flt‐3L was most efficient in promoting the expansion of CD34+ cells, CD34+CD38– cells, mixed‐lineage colony‐forming units (CFU‐GEMM) and long‐term culture‐initiating cells (LTC‐IC) by 21·7 ± 5·00‐, 103 ± 27·9‐, 10·7 ± 7·94‐ and 6·52 ± 1·51‐fold, respectively, after 12–14 d of culture. The addition of PDGF increased the yield of these early progenitors by 45·0%, 66·5%, 45·1% and 79·8% respectively. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in non‐obese diabetic (NOD)/severe‐combined immunodeficient (SCID) mice. The expression of PDGF receptor (PDGFR)‐β was not detectable in fresh CD34+ cells but was upregulated after culture for 3 d. PDGF also enhanced the development of adherent cells/clusters that expressed the endothelial markers VE‐cadherin and CD31. These findings suggest that PDGF is an effective cytokine for the ex vivo expansion of early stem and progenitor cells. The mechanism could be mediated by PDGFR‐β on committed CD34+ progenitor cells and/or secondary to the stimulation of autologous, stromal feeder cells.

BRE is an antiapoptotic protein in vivo and overexpressed in human hepatocellular carcinoma

BRE binds to the cytoplasmic domains of tumor necrosis factor receptor-1 and Fas, and in cell lines can attenuate death receptor-initiated apoptosis by inhibiting t-BID-induced activation of the mitochondrial apoptotic pathway. Overexpression of BRE by transfection can also attenuate intrinsic apoptosis and promote growth of the transfected Lewis lung carcinoma line in mice. There is, however, a complete lack of in vivo data about the protein. Here, we report that by using our BRE-specific monoclonal antibody on the immunohistochemistry of 123 specimens of human hepatocellular carcinoma (HCC), significant differences in BRE expression levels between the paired tumoral and non-tumoral regions (P<2.2e−16) were found. Marked overexpression of BRE was detected in majority of the tumors, whereas most non-tumoral regions expressed the same low level of the protein as in normal livers. To investigate whether BRE overexpression could promote cell survival in vivo, liver-specific transgenic BRE mice were generated and found to be significantly resistant to Fas-mediated lethal hepatic apoptosis. The transgenic model also revealed post-transcriptional regulation of Bre level in the liver, which was not observed in HCC and non-HCC cell lines. Indeed, all cell lines analysed express high levels of BRE. In conclusion, BRE is antiapoptotic in vivo, and may promote tumorigenesis when overexpressed.

The role of inducible nitric oxide synthase in gamete interaction and fertilization: a comparative study on knockout mice of three NOS isoforms.

Nitric oxide (NO), which is produced from l‐arginine by three isoforms of NO synthase (NOS), has been implicated in reproductive functions. However, the specific role of NOS isoforms in gamete function and fertilization is not clear. Three types of NOS knockout mice were super ovulated and fertilized in vitro and in vivo. The sperm count and motility, in vivo and in vitro fertilization rate as indicated by two‐cell embryos and blastocyst rate were examined. The sperm count and motility from all three knockout mice were not significantly different from that of the wild type. Inducible NOS (iNOS) knockout mice were found to have the largest number of two‐cell embryos/mouse collected after fertilization in vivo (P < 0.01), but the rate of blastocyst formation from two‐cell embryos in vitro was similar for all three knockouts. The rate of in vitro fertilization using either iNOS‐deficient sperm or oocytes, but not those deficient in the other two NOS isoforms, was significantly elevated when compared to that in the wild type (P < 0.001). While all three types of NOS do not seem to play a significant role in pre‐ejaculated sperm function, iNOS may play an inhibitory role in sperm and oocyte functions affecting the process of fertilization and early embryo development.

The effect of peripheral resistance on impedance cardiography measurements in the anesthetized dog

In the vasodilated and septic patient, the impedance method of measuring cardiac output (CO) may underestimate the true value. In this study, we sought to determine whether impedance CO (COIC) measurements are influenced by total peripheral resistance (TPR). In eight anesthetized and ventilated dogs, a high-precision flowprobe was placed on the ascending aorta, and direct CO was measured (CO flowprobe (COFP)). Mean arterial blood pressure was measured from the femoral artery. Simultaneous COIC measurements were made. TPR (mean arterial blood pressure × 80/COFP) was varied over 1–2 h by using infusions of phenylephrine and adrenaline and inhaled halothane. The bias between methods of CO measurement (COIC − COFP) was calculated and compared with TPR by using correlation and regression analysis. A total of 547 pairs of CO measurements were collected from the 8 dogs as TPR was varied. COFP changed by a mean of 190% (range, 89%–425%), and TPR changed by a mean of 266% (range, 94%–580%) during the experiment. The impedance method underestimated CO when TPR was low and overestimated CO when TPR was high. There was a logarithmic relationship between the CO bias and TPR. Correlation coefficients (r) between the CO bias and TPR ranged from 0.46 to 0.89 (P < 0.0001). The bias changed by 0.62 ± 1.8 L/min, or by 34%, every time TPR halved or doubled. This finding explains the poor agreement between COIC and other methods of CO measurement found in validation studies involving critically ill patients.

BRE over-expression promotes growth of hepatocellular carcinoma

BRE, also known as TNFRSF1A modulator and BRCC45, is an evolutionarily highly conserved protein. It is a death receptor-associated protein in cytoplasm and a component of BRCA1/2-containing DNA repair complex in nucleus. BRE was found to have anti-apoptotic activity. Over-expression of BRE by transfection promoted survival of cell lines against apoptotic induction; whereas depletion of the protein by siRNA resulted in the opposite. In vivo anti-apoptotic activity of BRE was demonstrated by significant attenuation of Fas-induced acute fulminant hepatitis in transgenic mice expressing the human protein specifically in the liver. BRE was also implicated in tumor promotion by the accelerated tumor growth of Lewis Lung carcinoma transfected with human BRE; and by high expression of BRE specifically in the tumoral regions of human hepatocellular carcinoma (HCC). The present study was to test directly if transgenic expression of BRE in livers could promote HCC development in neonatal diethylnitrosamine model. By 8months after tumor induction, the maximal sizes of tumor nodules of transgenic mice were significantly larger than those of the non-transgenic controls, although the numbers of tumor nodules between the two groups did not significantly differ. Importantly, as in human HCC, the mouse endogenous BRE level was up-regulated in mouse HCC nodules. These results show that BRE over-expression can indeed promote growth, though not initiation, of liver tumors. Furthermore, the common occurrence of BRE over-expression in human and mouse HCC suggests that up-regulation of BRE is functionally important in liver tumor development.

Comparison between phytoestrogens and estradiol in the preventionof atheroma in ovariectomized cholesterol-fed rabbits

Objectives There is increasing interest in the role of complementary and alternative medicines for the treatment of menopause-related problems. This study compared the preventive effect on atheroma formation of a commercially available mixed phytoestrogen concentrate with that of estradiol. Methods An ovariectomized cholesterol-fed rabbit model of atheroma formation was used. Rabbits were ovariectomized before the commencement of the 12-week treatment period. There were two control groups. Control Group 1 received isoflavone-free rabbit chow whilst Control Group 2 received 1% cholesterol-enriched isoflavone-free rabbit chow. Rabbits in Group 3 received 1% cholesterol-enriched isoflavone-free rabbit chow plus a 500 mg tablet containing a concentrated extract of Trifolium pretense (red clover). Rabbits in Group 4 received 1% cholesterol-enriched isoflavone-free rabbit chow plus a 0.5 mg tablet of oral estradiol. Atheroma formation was measured by, first, calculation of the area of atheroma on the intimal surface, and, second, measuring the cholesterol content in the aorta. Results There were no significant differences in serum cholesterol between the cholesterol-fed control Group 2 and the treatment Groups 3 and 4. However, there was significantly less staining for atheroma and significantly less cholesterol accumulation in the aorta in Group 4 (estradiol-treated) rabbits compared with either control Group 2 or Group 3 (phytoestrogen-treated) rabbits. Conclusion In this study, only estradiol was shown to have a significant protective effect against atheroma formation.

Preclinical ex vivo expansion of G‐CSF‐mobilized peripheral blood stem cells: effects of serum‐free media, cytokine combinations and chemotherapy

Abstract:  Objectives: Ex vivo expansion of granulocyte‐colony stimulating factor (G‐CSF)‐mobilized peripheral blood stem cells (PBSC) is a promising approach for overcoming the developmental delay of bone marrow (BM) reconstitution after transplantation. This project investigated the effects of culture duration, serum‐free media, cytokine combinations, and chemotherapy on the outcomes of expansion. Methods: Enriched CD34+ cells were cultured for 8 or 10 d in serum‐free media (QBSF‐60 or X‐Vivo 10) and four combinations of cytokines consisting of recombinant human pegylated‐megakaryocyte growth and development factor, stem cell factor, flt‐3 ligand, G‐CSF, interleukin (IL)‐6, platelet‐derived growth factor (PDGF), and IL‐1β. Results: Eight days of culture in QBSF‐60 significantly supported efficient expansions of CD34+ cells, CD34+ CD38− cells, colony‐forming units (CFU) of myeloid, erythroid, megakaryocytic, and mixed lineages to 3.76‐, 14.4‐, 28.3‐, 24.0‐, 38.1‐, and 15.7‐fold, respectively. Whilst PDGF or IL‐6 enhanced the expansion of early, myeloid, and erythroid progenitors, IL‐1β specifically promoted the megakaryocytic lineage. Engraftment of human CD45+ cells were detectable in all non‐obese diabetic/severe‐combined immunodeficient mice transplanted with expanded PBSC from donor samples, being 5.80 ± 3.34% of mouse BM cells. The expansion and engraftment capacity of CD34+ cells from subjects postchemotherapy were significantly compromised across the panel of progenitor cells. Conclusion: Our results provided an optimized protocol for PBSC expansion, applicable to ameliorating neutropenia and thrombocytopenia in post‐BM transplant patients by the prompt provision of progenitor cells. For postchemotherapy patients, expansion products might provide committed progenitors for improving short‐term engraftment, but not self‐renewable stem cells.