Cryopreservation and thawing of hematopoietic stem cell CD34-induced apoptosis through caspase pathway activation: Key role of granulocytes

IntroductionCell damage inescapably occurs during both the freezing and the thawing graft processes for autologous hematopoietic stem cell (HSC) transplantation. To estimate HSC injury, a quality control is performed including: (i) CD34⁺ quantification; (ii) percentage of CD34⁺ viability and (iii) evaluation of HSC functional ability to form colony forming unit–granulocyte macrophage (CFU-GM). Apoptosis involves complex pathways such as caspase enzymes. Here, we assess the extent of apoptosis that is caspase-dependent before and after cryoconservation of CD34⁺, using a Fluorescent Labeled Inhibitor of CAspases (FLICA).MethodsCaspase pathway activation status was evaluated in 46 patients (multiple myeloma [n = 24], lymphoma [n = 22]), by flow cytometry, using a 7-aminoactinomycin-D (7AAD)/FLICA staining test, in CD34⁺, CD3⁺, CD14⁺ and CD56⁺ cells. Viable 7AAD^?/FLICA⁺ cells were then correlated with various parameters.ResultsWe showed a significant caspase pathway activation, with 23% CD34⁺/7AAD^?/FLICA⁺ cells after thawing, compared with the 2% described in fresh CD34⁺ cells (P < 0.0001). Moreover, caspase pathway was significantly activated in thawing CD3⁺, CD56⁺ and CD14⁺ cells. We also report a significant correlation between the rate of CD34⁺/7AAD^?/FLICA⁺ cells and post-thawing granulocytes count (P = 0.042) and their potential to be differentiated into CFU-GM (P = 0.004).DiscussionOur results show substantial cell death, induced by the increase of caspase pathway activation, secondary to the thawing process, and across all study cell types. This observation may affect the immune response quality during recipient aplasia, without detecting a clinical impact. Moreover, caspase pathway activation through CD3⁺ and CD56⁺ subpopulations could modify the therapeutic result of donor lymphocytes infusion (DLI).     

How well are velocity effects on ∂13C signatures transmitted up the food web from algae to fish?

1. Benthic algae fractionate carbon isotopes less at low water velocities because of reduced boundary layer exchange, and this effect on δ¹³C is passed on to consumers via trophic transfer. This study examines the relationships between δ¹³C signatures of consumers (invertebrates and salmonid fishes) and water velocity in the Sainte Marguerite River, QC, Canada, and compares them to patterns for periphyton, both along the river main‐stem and in a small tributary. 2. Relationships of δ¹³C signatures of herbivore/grazers and collector/gatherers with water velocity were strong and similar to those of periphyton, but relationships for filter‐feeders were weak, probably reflecting the effect of spatial averaging of their food supply as a result of downstream transport. 3. Velocity effects on salmonid signatures were much weaker than those of lower trophic levels, being barely significant except in the small tributary where the fish were resident and isolated from the main river. In the river main‐stem, even when reach standardised (reach mean subtracted from each data point), fish signatures were only weakly related to water velocity. 4. The fidelity with which velocity effects are transmitted to consumers from benthic algae is highly variable, and depends on a combination of consumer and resource movements, in addition to the trophic position of the consumer.     

PRODUCT OPTIMIZATION AND THE ACCEPTOR SET SIZE

The acceptability of a product, measured by the acceptor set size (percentage of consumers rating the product acceptable), is a function of the perception of its attributes. The attributes are themselves a function of the inputs to the product (such as ingredients, processing or storage variables). These assumptions lead to the following model:
Acceptor set size = F (attribute₁, attribute₂, …, attribute_n)
Attribute j = f (input₁, input₂, …, input_m)
If we assume that these functions are differentiable, we can estimate the partial derivatives of the acceptor set size, with respect to the input variables. The gradient vector obtained indicates the fastest way to maximize the acceptor set size. The gradient search method, using the acceptor set size as an objective measure, can be applied in a variety of situations: to improve existing products, to maximize the acceptability of new products, and to study the relationship between shelf-life and acceptability.     

Gastropod biodiversity at the 'Evolution Canyon' microsite, lower Nahal Oren, Mount Carmel, Israel

Twenty-six species of gastropods (terrestrial, shell-bearing snails and slugs) were recorded at the 'Evolution Canyon' microsite, lower Nahal Oren, Mount Carmel, Israel. Twenty-five species were recorded at the temperate, mesic north-facing slope (NFS) and 20 species at the xeric south-facing slope (SFS). Out of these species, six were NFS specific ( Pilorcula raymondi hebraica , Euchondrus septemdentatus , Monacha crispulata , Pyramidula rupestris hierosolymitana , Truncatellina haasi , and Vitrea contracta ) and one was SFS specific ( Prolimax eustrictus ). The interslope difference was probably partly due to missing forest species at the SFS in comparison with the NFS. Twenty-two species were Levantine endemics (84.6%) and four species were more widely distributed in the Palaearctic region (15.4%). The Levantine species are inhabitants of the mesic and mainly mountainous regions, but four species ( Granopupa granum , Calaxis hierosolymarum , Cecilioides acicula , and Helix engaddensis ) also penetrate the deserts. Seven species ( Buliminus labrosus , H. engaddensis , Levantina spiriplana caesareana , Metafruticicola fourousi , Monacha syriaca , Sphincterochila cariosa , and Xeropicta vestalis joppensis ) were significantly more abundant on the SFS than on the NFS. The local physical microclimatic sharp divergence leads to gastropod adaptive interslope biotic divergence caused by natural selection.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 147–155.     

Comparison of TRPs From Murine and Human Malignant Melanocytes

Most of our knowledge of the mammalian tyrosinase related protein (TRP) activities is derived from studies using murine melanoma models, such as B16 or Cloudman S-91 melanocytes. Owing to the high degree of homology between the murine and human enzymes, it has been assumed that their kinetic behavior could be similar. However, the protein sequences at the metal binding sites of the murine and human enzymes show some differences of possible functional relevance. These differences are more significant in the metal-A site than in the metal-B site. By using three human melanoma cell lines (HBL, SCL, and BEU), we have studied the catalytic abilities of the human melanogenic enzymes in comparison to those obtained for the counterpart murine enzymes isolated from B16 melanoma. We have found that TRP2 extracted from all cell lines show dopachrome tautomerase activity, although the activity levels in human malignant melanocytes are much lower than in mouse cells. Reconstitution experiments of the human enzyme indicate that TRP2 has Zn at its metal binding-sites. Although mouse tyrosinase does not show DHICA oxidase activity, and this step of the melanogenesis pathway is specifically catalyzed by mouse TRP1, the human enzyme seems to recognize carboxylated indoles. Thus, human tyrosinase could display some residual DHICA oxidase activity, and the function of human TRP1 could differ from that of the murine protein. Attempts to clarify the nature of the metal cofactor in TRP1 were unsuccessful. The enzyme contains mostly Fe and Cu, but the reconstitution of the enzymatic activity from the apoprotein with these ions was not possible.