Cell culture bags allow a large extent of ex vivo expansion of LTC-IC and functional mature cells which can subsequently be frozen: interest for a large-scale clinical applications

The aim of this study was to evaluate the ex vivo expansion of normal CD34+ cells in gas-permeable polypropylene bags suitable for clinical use. Cells were cultured for 14 days in serum-free medium supplemented with SCF, IL3, IL6, FLT3-1, G-CSF + MGDF or Epo. The bags supported the expansion of hematopoietic cells in a similar manner to small scale well or flask systems, allowing mean expansions of up to 2193-fold for total nucleated cells, 140-fold for CFU-GM and 66-fold for LTC-IC. Increasing the initial cell concentration from 5 x 10(3) to 1 x 10(5)CD34+ cells/ml induced the production of granulocytic cells with terminal differentiation while simultaneously decreasing the overall extent of expansion of the white blood cells produced. We tested the phagocytic activity and oxidative metabolism of the white blood cells produced. The percentage of phagocytic cells was 39+/-0.5% in expanded cultures derived from fractions initiated at 5 x 10(3), 10(4) or 10(5) cells/ml and 45+/-6% in cultured cells obtained from starting fractions containing 5 x 10(4) cells/ml, as compared to 58+/-4% in normal controls. A study of the potential for oxygen-dependent microbe killing showed that the expanded cells produced H2O2, although in lesser quantities than control cells. We subsequently investigated the possibility of freezing expanded cells. Total cell recovery after thawing was 45+/-4%, while recoveries of progenitors and stem cells ranged from 65 to 90%, without any influence of the initial cell concentration. This new approach could be of major interest for clinical practice, as it would allow evaluation of the quality of a graft prior to its infusion and employs experimental conditions which meet the criteria for potential clinical use.     

Clinico-epidemiological analysis of imported malaria in Donetsk region (1979-1997)

AIM: The study of possibility of reappearance of malaria foci in the Donets region. MATERIALS AND METHODS: 322 cases of imported malaria (IM) in the Donets region registered in 1979-1997 have been analysed clinically and epidemiologically. Malaria was diagnosed in immigrants from and visitors to Africa, Asia and Latin America, malaria-endemic Azerbaidjan, Tadzhikistan, Afghanistan. RESULTS: Late diagnosis of malaria occurred in immigrants and businessmen who got infected during their business trips. Causative agents of malaria imported from Azerbaijan and Asia were resistant to chemoprophylaxis with delagil and primaxine. Clinical forms of IM are described. CONCLUSION: There is a real danger of malaria reappearance in the Donetsk region as the residents are at risk of being infected from late-diagnosis foreign tourists, immigrants, refugees.     

Effects of injurious compression on matrix turnover around individual cells in calf articular cartilage explants

The effects of mechanical injury on the metabolism of cartilage matrix are of interest for understanding the pathogenesis of osteoarthrosis and the development of strategies for cartilage repair. The purpose of the present study was to examine the effects of injury on matrix turnover in a calf articular cartilage explant system for which the effects of mechanical loading on cell activity and the cell-mediated pathways of matrix metabolism are already well characterized. New methods of quantitative autoradiography were used in combination with established biochemical and biomechanical techniques for the analysis of cell and matrix responses to acute mechanical injury, with particular attention to the processes of localized matrix turnover in the cell-associated matrices of individual chondrocytes. Matrix deposition and turnover around cells in control explants was spatially dependent, with the highest rates of proteoglycan deposition and turnover and the lowest rates of collagen deposition (as indicated by [3H]proline autoradiography) occurring in the pericellular matrix. Injurious compression was associated with (a) an abrupt decrease in the tensile load-carrying capacity of the collagen matrix, apparently associated with mechanical failure of the tissue, (b) a considerable but subtotal decrease in cell viability, marked by the emergence of an apparently inactive cell population interspersed within catabolically active but abnormally large cells, and (c) sustained, elevated rates of proteoglycan turnover, particularly in the cell-associated matrices of apparently viable cells, which involved the increased release of aggregating species in addition to a spectrum of degradation fragments that were also in controls. These results may represent an in vitro model for the responses of chondrocytes and the cartilage extracellular matrix to mechanical injury.     

Effect of caffeine on the human small intestine

Methylxanthines produce intracellular accumulation of cyclic 3'5'-AMP (cAMP) by inhibition of phosphodiesterase and mucosal cAMP accumulation. Cyclic AMP is thought to mediate small intestinal secretion caused by some enterotoxins, hormones, and methylxanthines. These studies were designed to evaluate the effect of caffeine on small intestinal net fluid movement and transit times. The administration of caffeine in amounts ordinarily contained in many beverages and medications (75 to 300 mg) resulted in striking net secretion in the jejunum which lasted at least 15 minutes. This occurred in six of seven studies. Baseline net absorption of 0.5 ml per cm per hr was reversed to net secretion of 6.0 +/- 2.2 ml per cm per hr after oral caffeine ingestion (P less than 0.01). Net secretion also occurred in the ileum in seven of eight studies, but the onset of secretion appeared 35 min later than in the jejunum. These patterns of secretion correlated best with the passage of the intestinal bolus of caffeine rather than plasma caffeine levels. In contrast to other net secretory conditions, which increase the speed of transit, small intestinal transit times, as determined by dye dilution methods, were unchanged by caffeine. It is possible that methylxanthine-induced small intestinal secretion may play a role in the symptoms experienced by some patients with functional diarrhea.     

Cloning and localization of RPE65 mRNA in salamander cone photoreceptor cells1

RPE65 is a potential retinoid-processing protein expressed in the retinal pigment epithelium. Mutations in the RPE65 gene have been shown to cause certain inherited retinal dystrophies. Previous studies have shown that salamander cone photoreceptor cells have a unique retinoid processing mechanism which is distinct from that of rods. To determine whether RPE65 is expressed in photoreceptors, the RPE65 cDNA was cloned from a salamander retinal cDNA library. The deduced protein consists of 533 amino acids and is 85% identical to human and bovine RPE65. The RPE65 mRNA was detected in all of the single cone cells isolated from the salamander retina, as well as in the retinal pigment epithelium by RT-PCR, but not in the isolated rods. The RT-PCR products have been confirmed to be RPE65 by DNA sequencing. The results indicate that this potential retinoid processing protein is expressed in the cone photoreceptor cells but not in rods. Therefore, this protein may contribute to the unique retinoid processing capabilities in salamander cones.