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 study's 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. STUDY DESIGN AND METHODS: Enriched CB CD34+ cells were cultured in four media (Iscove's modified Dulbecco's medium with FCS, Gibco; X-Vivo-10, BioWhittaker; QBSF-60, Quality Biological; and StemSpan SFEM, Stem Cell Technologies) with four cytokine combinations (thrombopoietin [TPO], SCF, Flt-3 ligand [FL] with and without G-CSF, and/or IL-6). The effect of autologous CB plasma was also investigated. The read-out measures were evaluated on Days 8 and 12. After expansion at the optimized condition, cultured cells were transplanted into sublethally irradiated NOD/SCID mice. The engraftment of human CD45+ cells and subsets in the bone marrow, spleen, and peripheral blood was determined. RESULTS: QBSF-60 or StemSpan SFEM supported high yields of early progenitors (CD34+ cells, 相似文献   

Pharmacology of the Thromboxane Receptor Antagonist and Thromboxane Synthase Inhibitor BM‐531

BM‐531 (N‐tert‐butyl‐N'‐[(2‐cyclohexylamino‐5‐nitrobenzene)sulfonyl]urea), a torasemide derivative, is a novel noncarboxylic thromboxane receptor antagonist and thromboxane synthase inhibitor. Indeed, its affinity for human washed platelet TXA₂ receptors labeled with [3H]SQ‐29548 (IC₅₀= 0.0078 μM) is higher than sulotroban (IC₅₀= 0.93 μM) and SQ‐29548 (IC₅₀= 0.021 μM). Moreover, BM‐531 is characterized by a potent antiaggregatory property. Indeed, on one hand, in human citrated platelet‐rich plasma BM‐531 prevents platelet aggregation induced by arachidonic acid (600 μM) (ED₁₀₀= 0.125 μM), U‐46619, a stable TXA₂ agonist (1 μM) (ED₅₀= 0.482 μM) or collagen (1 μg/mL) (percentage of inhibition: 42.9% at 10 μM) and inhibits the second wave of ADP (2 μM)‐induced aggregation. On the other hand, when BM‐531 is incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA‐100®) is significantly prolonged. In addition, at the concentrations of 10 and 1 μM, BM‐531 totally prevents the production of TXB₂ by human platelets activated by arachidonic acid. Finally, at 10 μM, BM‐531 significantly prevents rat fundus contractions induced by U‐46619 but not by prostacyclin. These results suggest that BM‐531, which is devoid of the diuretic property of torasemide, can be regarded as a promising antiplatelet agent.     

Making sense of regulatory T cell suppressive function

Several types of regulatory T cells maintain self-tolerance and control excessive immune responses to foreign antigens. The major regulatory T subsets described over the past decade and novel function in transplantation will be covered in this review with a focus on CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells. Multiple mechanisms have been proposed to explain how Treg cells inhibit effector cells but none can completely explain the observed effects in toto. Proposed mechanisms to explain suppressive activity of Treg cells include the generation of inhibitory cytokines, induced death of effector cells by cytokine deprivation or cytolysis, local metabolic perturbation of target cells mediated by changes in extracellular nucleotide/nucleoside fluxes with alterations in intracellular signaling molecules such as cyclic AMP, and finally inhibition of dendritic cell functions. A better understanding of how Treg cells operate at the molecular level could result in novel and safer therapeutic approaches in transplantation and immune-mediated diseases.     

Tolerogenic dendritic cells and their role in transplantation

The pursuit of clinical transplant tolerance has led to enhanced understanding of mechanisms underlying immune regulation, including the characterization of immune regulatory cells, in particular antigen-presenting cells (APC) and regulatory T cells (Treg), that may play key roles in promoting operational tolerance. Dendritic cells (DC) are highly efficient APC that have been studied extensively in rodents and humans, and more recently in non-human primates. Owing to their ability to regulate both innate and adaptive immune responses, DC are considered to play crucial roles in directing the alloimmune response towards transplant tolerance or rejection. Mechanisms via which they can promote central and peripheral tolerance include clonal deletion, the induction of Treg, and inhibition of memory T cell responses. These properties have led to the use of tolerogenic DC as a therapeutic strategy to promote organ transplant tolerance. In rodents, infusion of donor- or recipient-derived tolerogenic DC can extensively prolong donor-specific allograft survival, in association with regulation of the host T cell response. In clinical transplantation, progress has been made in monitoring DC in relation to graft outcome, including studies in operational liver transplant tolerance. Although clinical trials involving immunotherapeutic DC for patients with cancer are ongoing, implementation of human DC therapy in clinical transplantation will require assessment of various critical issues. These include cell isolation and purification techniques, source, route and timing of administration, and combination immunosuppressive therapy. With ongoing non-human primate studies focused on DC therapy, these logistics can be investigated seeking the optimal approaches. The scientific rationale for implementation of tolerogenic DC therapy to promote clinical transplant tolerance is strong. Evaluation of technical and therapeutic logistic issues is an important next step prior to the application of tolerogenic DC in clinical organ transplantation.     

Evaluation of bone marrow examinations performed by an advanced nurse practitioner: An extended role within a haematology service

Purpose

Traditionally, medical personnel have undertaken bone marrow (BM) examination. However, specially trained nurses in advanced practice roles are increasingly undertaking this role. This paper presents the findings from an audit of BM examinations undertaken by an advanced nurse practitioner (ANP) at a regional haematology specialist centre.

Methods

The audit evaluated the quality of BM examinations performed by the ANP over the past two years (September 2007–September 2009). Over the two year period, 324 BM examinations were performed at the centre of which 156 (48.1%) were performed by the ANP. A random sample of 30 BM examinations undertaken by the ANP were analysed by the consultant haematologist.

Results

All 30 BM examinations undertaken by the ANP were sufficient for diagnosis.

Conclusions

The ANP is capable and competent to obtain BM samples which are of a sufficient quality to permit diagnosis.     

Long lasting effects of predator stress on pCREB expression in brain regions involved in fearful and anxious behavior

Predator stress is one animal model of posttraumatic stress disorder (PTSD). Neural plasticity in amygdala afferent and efferent pathways underlies anxiogenic effects of predator stress. Predator stress increases pCREB expression in these pathways 20 min after stress, implicating pCREB in stress-induced neural plasticity. Here we examined impact of predator stress on pCREB expression 6-24 h and 7 days after stress in amygdala pathways and in the supramammillary nucleus (SuM). Patterns of change in pCREB expression were complex, time dependent, column dependent in the periaqueductal gray (PAG), and AP plane dependent in the amygdala. In contrast to past work at 20 min after stress, there were no stress-induced increases in pCREB in the amygdala in the anterior AP plane or in the lateral PAG at 6 h onward after stress. However, dorsal PAG pCREB was increased bilaterally at 24 h and 7 days after stress. In the mid AP plane of all amygdala nuclei there were bilateral stress-induced increases in pCREB at 6 h followed by decreases at 24 h post stress. A similar pattern was observed in the posterior AP plane. In addition, we found a persistent increase (6 h to 7 days after stress) in pCREB expression in the SuM. Further study of this nucleus as a contributor to fear sensitization following predator stress is warranted. Overall, these data highlight persistent neuroplastic changes in key brain areas following traumatic stress. Identification of these changes may aid in understanding the neural mechanisms underlying acquired anxiety disorders such as PTSD.     

Bone marrow retaining colitogenic CD4+ T cells may be a pathogenic reservoir for chronic colitis

BACKGROUND & AIMS: Although bone marrow (BM) is known as a primary lymphoid organ, it also is known to harbor memory T cells, suggesting that this compartment is a preferential site for migration and/or selective retention of memory T cells. We here report the existence and the potential ability to induce colitis of the colitogenic BM CD4+ memory T cells in murine colitis models. METHODS: We isolated BM CD4+ T cells obtained from colitic severe combined immunodeficient mice induced by the adoptive transfer of CD4+ CD45RB(high) T cells and colitic interleukin (IL)-10(-/-) mice that develop colitis spontaneously, and analyzed the surface phenotype, cytokine production, and potential activity to induce colitis. Furthermore, we assessed the role of IL-7 to maintain the colitogenic BM CD4+ T cells. RESULTS: A high number of CD4+ T cells reside in the BM of colitic severe combined immunodeficient mice and diseased IL-10(-/-) mice, and they retain significant potential to induce type-1 T helper-mediated colitis in an IL-7-dependent manner. These resident BM CD4+ T cells have an effector memory (T(EM); CD44(high)CD62L(-)IL-7R(high)) phenotype and preferentially are attached to IL-7-producing BM cells. Furthermore, the accumulation of BM CD4+ T(EM) cells was decreased significantly in IL-7-deficient recipients reconstituted with the colitogenic lamina propria CD4+ T(EM) cells. CONCLUSIONS: Collectively, these findings suggest that BM-retaining colitogenic CD4+ memory T cells in colitic mice play a critical role as a reservoir for persisting lifelong colitis.     

Induction of ovalbumin-specific tolerance by oral administration of Lactococcus lactis secreting ovalbumin

BACKGROUND AND AIMS: Obtaining antigen-specific immune suppression is an important goal in developing treatments of autoimmune, inflammatory, and allergic gastrointestinal diseases. Oral tolerance is a powerful means for inducing tolerance to a particular antigen, but implementing this strategy in humans has been difficult. Active delivery of recombinant autoantigens or allergens at the intestinal mucosa by genetically modified Lactococcus lactis (L lactis) provides a novel therapeutic approach for inducing tolerance. METHODS: We engineered the food grade bacterium L lactis to secrete ovalbumin (OVA) and evaluated its ability to induce OVA-specific tolerance in OVA T-cell receptor (TCR) transgenic mice (DO11.10). Tolerance induction was assessed by analysis of delayed-type hypersensitivity responses, measurement of cytokines and OVA-specific proliferation, phenotypic analysis, and adoptive transfer experiments. RESULTS: Intragastric administration of OVA-secreting L lactis led to active delivery of OVA at the mucosa and suppression of local and systemic OVA-specific T-cell responses in DO11.10 mice. This suppression was mediated by induction of CD4(+)CD25(-) regulatory T cells that function through a transforming growth factor beta-dependent mechanism. Restimulation of splenocytes and gut-associated lymph node tissue from these mice resulted in a significant OVA-specific decrease in interferon gamma and a significant increase in interleukin-10 production. Furthermore, Foxp3 and CTLA-4 were significantly up-regulated in the CD4(+)CD25(-) population. CONCLUSIONS: Mucosal antigen delivery by oral administration of genetically engineered L lactis leads to antigen-specific tolerance. This approach can be used to develop effective therapeutics for systemic and intestinal immune-mediated inflammatory diseases.     

Actions and therapeutic potential of G-CSF and GM-CSF in cardiovascular disease

Despite their names, the cytokines granulocyte- and granulocyte-macrophage-colony stimulating factor (G-CSF and GM-CSF respectively) have actions far beyond simply stimulating the proliferation of neutrophil and monocyte lineage cells. A comprehensive body of evidence now exists demonstrating that G-CSF and GM-CSF effectively mobilize bone-marrow-derived progenitor cells into the peripheral circulation. These mobilized progenitor cells can be conveniently harvested for use in reconstituting bone marrow by transplantation after myelo-ablative treatment of hematological malignancies. In addition, much evidence has recently emerged to suggest that these cytokines may have multiple direct and indirect beneficial cardiovascular effects--including neovascularization of ischemic myocardium and reducing the extent of myocardial damage after infarction. Based on this knowledge and a strong safety record in hematological applications, a number of early clinical trials have evaluated the use of G-CSF or GM-CSF in patients with both acute and chronic myocardial ischemia. Although the interpretation of these trials is complicated by heterogeneity in study design, small patient numbers and methodological concerns related to appropriate selection and blinding of patients, the results of ongoing larger phase II/III trials should soon be available to determine if these agents will be useful additions to the cardiovascular armamentarium.