Characterization of the effects and potential mechanisms leading to increased megakaryocytic differentiation under mild hyperthermia.

The physical culture parameters have important influences on the proliferation and differentiation fate of hematopoietic stem cells. Recently, we have demonstrated that CD34+ cord blood (CB) cells undergo accelerated and increased megakaryocyte (Mk) differentiation when incubated under mild hyperthermic conditions (i.e., 39 degrees C). In this study, we investigated in detail the impacts of mild hyperthermia on Mk differentiation and maturation, and explored potential mechanisms responsible for these phenomena. Our results demonstrate that the qualitative and quantitative effects on Mk differentiation at 39 degrees C appear rapidly within 7 days, and that early transient culture at 39 degrees C led to even greater Mk yields (p<0.03). Surprisingly, cell viability was only found to be significantly reduced in the early stages of culture, suggesting that CB cells are able with time to acclimatize themselves to 39 degrees C. Although mild hyperthermia accelerated differentiation and maturation of CB-derived Mks, it failed to promote their polyploidization further but rather led to a small reduction in the proportion of polyploid Mks (p=0.01). Conversely, gene arrays analysis demonstrated that Mks derived at 39 degrees C have a normal gene expression program consistent with an advanced maturation state. Finally, two independent mechanisms that could account for the accelerated Mk differentiation were investigated. Our results suggest that the accelerated and increased Mk differentiation induced by mild hyperthermia is not mediated by cell-secreted factors but could perhaps be mediated by the increased expression of Mk transcription factors.

Increased production of megakaryocytes near purity from cord blood CD34+ cells using a short two-phase culture system.

Expansion of hematopoietic progenitor cells (HPC) ex vivo remains an important focus in fundamental and clinical research. The aim of this study was to determine whether the implementation of such expansion phase in a two-phase culture strategy prior to the induction of megakaryocyte (Mk) differentiation would increase the yield of Mks produced in cultures. Toward this end, we first characterized the functional properties of five cytokine cocktails to be tested in the expansion phase on the growth and differentiation kinetics of CD34+-enriched cells, and on their capacity to expand clonogenic progenitors in cultures. Three of these cocktails were chosen based on their reported ability to induce HPC expansion ex vivo, while the other two represented new cytokine combinations. These analyses revealed that none of the cocktails tested could prevent the differentiation of CD34+ cells and the rapid expansion of lineage-positive cells. Hence, we sought to determine the optimum length of time for the expansion phase that would lead to the best final Mk yields. Despite greater expansion of CD34+ cells and overall cell growth with a longer expansion phase, the optimal length for the expansion phase that provided greater Mk yield at near maximal purity was found to be 5 days. Under such settings, two functionally divergent cocktails were found to significantly increase the final yield of Mks. Surprisingly, these cocktails were either deprived of thrombopoietin or of stem cell factor, two cytokines known to favor megakaryopoiesis and HPC expansion, respectively. Based on these results, a short resource-efficient two-phase culture protocol for the production of Mks near purity (>95%) from human CD34+ CB cells has been established.

Efficient in vitro megakaryocyte maturation using cytokine cocktails optimized by statistical experimental design.

OBJECTIVE: A multi-step statistical strategy was applied to quantify individual and interactive effects of cytokines on megakaryopoiesis and to determine the concentration of the selected cytokines that optimize ex vivo megakaryocyte (MK) expansion, maturation, and platelet production in stromal- and serum-free conditions. MATERIALS AND METHODS: Immature MK were first generated from human CD34(+)-enriched cord blood cells cultured for 7 days in conditions favoring MK commitment. Then, the effect of different combinations of cytokines at various concentrations on MK differentiation and platelet production was tested on the day-7 MK. RESULTS: A large-scale screening of 13 cytokines in the presence of thrombopoietin (TPO) using Placket-Burman designs (PBD) was initially performed to identify stimulators of MK maturation. Afterwards, a statistical analysis of the two-level factorial designs revealed that in the presence of TPO, MK maturation was significantly stimulated by stem cell factor (SCF), interleukin (IL)-6, and IL-9, whereas Flt-3 ligand (FL) had a positive effect only on the expansion of MK progenitors. In contrast, erythropoietin (EPO) and IL-8 were inhibitors of MK maturation. A response surface methodology was then used to optimize the concentrations of the selected cytokines (TPO, SCF, IL-6, and IL-9) and defined a new cytokine cocktail that maximized MK expansion and maturation. Importantly, the increased MK output was accompanied by a very high MK purity ( approximately 90%). Another optimum was also found at a higher SCF concentration, which further improved MK expansion and maturation, but reduced MK purity. CONCLUSION: These statistical methods provide an efficient tool to analyze complex systems of cytokines and to develop promising ex vivo MK culture systems for clinical applications.

Increased megakaryopoiesis in cultures of CD34-enriched cord blood cells maintained at 39 degrees C.

Based on previous evidence suggesting positive effects of fever on in vivo hematopoiesis, we tested the effect of hyperthermia on megakaryopoiesis (MK) in ex vivo cultures of CD34-enriched cord blood (CB) cells. The cells were cultured at 37 degrees C or 39 degrees C for 14 days in cytokine conditions optimized for megakaryocyte development and analyzed periodically. Compared to 37 degrees C, the cultures maintained at 39 degrees C produced significantly more (up to 10-fold) total cells, myeloid and MK progenitors, and total MKs, and showed accelerated and enhanced MK maturation with increased yields of proplatelets and platelets. This observation could facilitate clinical applications requiring ex vivo expansion of hematopoietic cells.

Preferential ex vivo expansion of megakaryocytes from human cord blood CD34+-enriched cells in the presence of thrombopoietin and limiting amounts of stem cell factor and Flt-3 ligand.

The high proliferative potential of cord blood (CB) stem cells and the identification of the key factor of megakaryopoiesis, thrombopoietin (TPO), permit the ex vivo expansion of megakaryocytes (MKs) for possible use in early post-transplant support of patients and the production of functional platelets for transfusion. However, culture conditions for the generation of adequate MKs for this purpose are not yet optimized. Therefore, we sought to define the mixture of early-acting cytokines and TPO that would promote the expansion of MK progenitors over other lineages and result in overall better MK expansion and platelet yields. CB CD34(+)-enriched cells were cultured in serum-free medium for 17 days in presence of TPO alone or in various combinations with early-acting cytokines used at different concentrations and addition times. MK expansion and polyploidy and platelet production were monitored by flow cytometry analysis using specific surface markers (CD41 and CD42b) and propidium iodide labeling. Our results showed that the use of high concentrations of stem cell factor (SCF) and Flt-3 ligand (FL) in early CB TPO-supplemented cultures was more favorable to monocytic and granulocytic cell expansion. However, we observed that their presence in limiting amounts was required for the preferential expansion of MK progenitors. The addition of SCF, FL, TPO, and interleukin-6 (IL-6) at high concentrations in secondary cultures of these expanded MKs resulted in optimal MK proportion (approximately 25% of MKs) and expansion (>300 MK per seeded cell), highest proportions of polyploid MKs (22% of mature MKs > or = 8N), and best platelet yields. Our results indicate that TPO-induced MK progenitors are more sensitive to early-acting cytokines than non-MK cells. We propose that MKs generated in the optimized conditions, in combination with immature stem/progenitor cells, could prove useful for the short-term platelet recovery following CB transplantation.

Modulations of anti-D affinity following promiscuous binding of the heavy chain with naïve light chains.

BACKGROUND: It is generally accepted that the antibody heavy (H) chain is more important than the light (L) chain for determining antigen specificity. In the case of anti-D, the predominant role of H chains in antigen binding is well recognized, but much less is known about the function of L chains. In this work, the contribution of L chains from non-D-immunized donors to the specificity and reactivity of anti-Ds was studied with L-chain shuffling. STUDY DESIGN AND METHODS: A kappa L chain library was recombined with the H chain of the 43F10 anti-D in a phagemid vector system (pComb3H, Scripps Institute). D-specific F(ab) phages were selected by panning on RBCs. Soluble F(ab)s were prepared, and their reactivity was assessed by RBC agglutination. The nucleotide and amino acid sequences of the variable region of the L chains were analyzed. RESULTS: The L chains of the six D-specific 43F10 F(ab) clones studied used five different germline genes from three Vkappa families and three different Jkappa segments. The L chains were all cationic with isoelectric points ranging from 8.1 to 10.2. CONCLUSION: The 43F10 anti-D H chain could bind promiscuously to a diversity of L chains from non-D-immunized donors without losing the D-antigen specificity. Relationships between the anti-D affinity and the cationic charge of the L chain as well as with the presence of an arginine residue in the L-chain complementarity-determining region 1 were observed.

[Benzodiazepine use in the elderly: state of the knowledge]. / Usage des benzodiazépines chez les personnes âgées: état des connaissances.

This article reviews the literature concerning addiction to benzodiazepine (BZD) in the elderly population of 65 and over. While the negative consequences related to a prolonged use of BZD are well known, much less is known on the phenomenon of dependency to these substances in this population. There is still no consensus on the definition of the problem. The continuous use of BZD may be more problematic in the elderly for whom tolerance to chemical agents diminishes with age. The risk factors involved in the chronic use are an interactive combination between the characteristics of the elderly as well as the physicians who prescribe such medication. Paths of research to support a paradigmatic change are presented in order to improve the research in this field.

Higher affinity human D MoAb prepared by light-chain shuffling and selected by phage display.

BACKGROUND: In blood banks, D MoAbs are routinely used to phenotype donors and patients. However, most D MoAbs do not agglutinate RBCs that weakly express D. The use of higher affinity MoAbs could overcome this problem. In this work, an attempt has been made to increase the affinity of the human clone 43F10, an IgG anti-D, by light (L)-chain shuffling followed by selection using phage display. STUDY DESIGN AND METHODS: PBMNCs of three polyimmunized individuals were used to construct the kappa L-chain repertoire that was recombined with the 43F10 heavy chain in a phagemid vector system (pComb3H, Scripps Institute, La Jolla, CA). L-chain-shuffled 43F10-F(ab) phages were selected on intact RBCs and characterized by ELISA, indirect agglutination, and sequence analysis. RESULTS: L-chain shuffling combined with phage display permitted the selection of a 43F10 MoAb variant (p3.17) with improved reactivity with weak D RBCs in agglutination assays. Nucleic acid sequence analysis showed that p3.17 and wild-type (wt) 43F10 L chains are encoded by different VL segments of the Vk1 family and different J segments, thus showing a relatively low degree of homology (86.4%). CONCLUSION: The use of a variant such as p3.17 could permit a further increase of the potency of existing anti-D reagents. The low homology between p3.17 and wt 43F10 sequences further exemplifies the predominant role of the heavy chain in determining the specificity of the anti-D.