The statistical analysis of survival in animal populations

Estimating, comparing and modelling survival rates are central to population biology. However, there are many difficulties in measuring these rates in animal populations in the wild. The most relevant information is based on samples of marked individuals, i.e. capture-recapture data. In recent years, a number of new statistical approaches to the analysis of such data have been developed, permitting more sophisticated and precise measurement of survival rates.     

Partial requirements forin vitro survival of human red blood cells

Some of the requirements for survival of human red blood cells were studied in vitro at 25 and 37 degrees C for 1--2 weeks. During the first week at 25 degrees C in Krebs-Ringer bicarbonate medium with glucose, the cells at 2--5% hematocrit (HCT) maintained normal K+, Na+, and water contents with negligible hemolysis. After six days ion gradients decreased, preceded by decline of ATP. With adenosine, ATP was maintained for 1--2 weeks. Sustained in vitro survival of human red blood cells at 25 or 37 degrees C requires constant pHo and sufficient substrates to support a glycolytic carbon flux as well as a nitrogen flux via nucleotide turnover. In Earle's salts buffered with HEPES and supplemented with glucose, Eagle's essential vitamins, albumin, and antibiotics, suspensions at 0.1% HCT exhibited constant pH at 7.39 +/- 0.03 for at least two weeks at 37 degrees C. With glucose alone, ATP declined steadily to negligible levels despite constant pHo, but 0.1 mM adenine supported ATP for one week. Also, several amino acids partially prevented the decline of reduced glutathione during the first week at 37 degrees C. These results and current knowledge of red cell metabolism suggest a new defined medium for experiments requiring long term incubations, and extend the characterization of human red cell in vitro survival to a time period not previously studied.     

An experimental study on the freezing of red blood cells with and without hydroxyethyl starch

Red blood cells were frozen in small capillaries down to ?196 °C at different linear cooling rates with or without the cryoadditive HES; the thawing rate was 3000 or 6500 °C/min. Hematocrit and hydroxyethyl starch concentration varied independently. The hemolysis of red blood cells was determined photometrically after 250-fold dilution and compared to totally hemolyzed samples. The typical U-shaped curves for hemolysis as a function of the cooling rate were obtained for all cell suspensions investigated. Relative optimum cooling rates were determined for the respective combinations of HES and hct. The results show that increasing hct causes an increased hemolysis; increased HES concentration C_HES reduces the optimum cooling rate B_opt; increased hct results in higher optimal cooling rates. The findings allow one to establish a linear correlation of the HES concentration and the optimum cooling rates when the dilution of the extracellular medium by the cell water efflux during freezing is taken into account. A comparison with results from larger volumes frozen (25 ml) shows that the established relationship between hematocrit, HES concentration, and optimal cooling rate remains valid.     

Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo

New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.     

Thermodynamic concepts in the study of microbial populations: age structure in Plasmodium falciparum infected red blood cells

Variability is a hallmark of microbial systems. On the one hand, microbes are subject to environmental heterogeneity and undergo changeable conditions in their immediate surroundings. On the other hand, microbial populations exhibit high cellular diversity. The relation between microbial diversity and variability of population dynamics is difficult to assess. This connection can be quantitatively studied from a perspective that combines in silico models and thermodynamic methods and interpretations. The infection process of Plasmodium falciparum parasitizing human red blood cells under laboratory cultivation conditions is used to illustrate the potential of Individual-based models in the context of predictive microbiology and parasitology. Experimental data from several in vitro cultures are compared to the outcome of an individual-based model and analysed from a thermodynamic perspective. This approach allows distinguishing between intrinsic and external constraints that give rise to the diversity in the infection forms, and it provides a criterion to quantitatively define transient and stationary regimes in the culture. Increasing the ability of models to discriminate between different states of microbial populations enhances their predictive capability which finally leads to a better the control over culture systems. The strategy here presented is of general application and it can substantially improve modelling of other types of microbial communities.     

Phospholipid vesicles increase the survival of freeze-dried human red blood cells

In a previous report [Z. T?r?k, G. Satpathy, M. Banerjee, R. Bali, E. Little, R. Novaes, H. Van Ly, D. Dwyre, A. Kheirolomoom, F. Tablin, J.H. Crowe, N.M. Tsvetkova, Preservation of trehalose loaded red blood cells by lyophilization, Cell Preservation Technol. 3 (2005) 96-111.], we presented a method for preserving human red blood cells (RBCs) by loading them with trehalose and then freeze-drying. We have now improved that method, based on the discovery that addition of phospholipid vesicles to the lyophilization buffer substantially reduces hemolysis of freeze-dried RBCs after rehydration. The surviving cells synthesize 2,3-DPG, have low levels of methemoglobin, and have preserved morphology. Among the lipid species we studied, unsaturated PCs were found to be most effective in suppressing hemoglobin leakage. RBC-vesicle interactions depend on vesicle size and structure; unilamellar liposomes with average diameter of less than 300 nm were more effective in reducing the hemolysis than multilamellar vesicles. Trehalose loaded RBCs demonstrated high survival and low levels of methemoglobin during 10 weeks of storage at 4 degrees C in the dry state when lyophilized in the presence of liposomes.     

Morphology of glutaraldehyde-fixed preserved red blood cells and 24-hr post-transfusion survival

Liquid-stored red blood cells and washed, previously frozen red blood cells were studied to determine whether a correlation existed between morphology and post-transfusion survival. Red cell concentrates were stored at 4 °C in citrate-phosphate-dextrose (CPD) for 21 days or in CPD-adenine (CPDA-1, CPDA-2, or CPDA-3) for as long as 35 days as liquid-preserved red cells. Both nonrejuvenated and rejuvenated red blood cells were frozen with 40%$\text{w}\text{v}$ glycerol at ?80 °C and were washed prior to testing.Samples of fresh, liquid-stored, and washed, previously frozen red blood cells were fixed with a 2% veronal glutaraldehyde solution. Phase, light, and electron microscopy were used to measure the numbers of discocytes, discoechinocytes, echinocytes, echinospherocytes, and spherocytes in each sample. A morphology score was assigned, with 100 representing all discocytes and 500 all spherocytes. In all samples phase and light microscopy gave nearly identical scores (r = 0.94), and phase and electron microscopy gave highly similar scores (r = 0.83).The morphology score proved to be a good indicator of 24-hr post-transfusion survival in liquid-stored red blood cells but not in washed, previously frozen red blood cells. Red blood cells stored in the liquid state at 4 °C in CPD, CPDA-1, CPDA-2, or CPDA-3 showed a significant inverse correlation between morphology and 24-hr post-transfusion survival (r = ?0.611) and a significant correlation between red cell ATP and 24-hr post-transfusion survival (r = 0.742). We saw no significant correlation between morphology scores and 24-hr post-transfusion values or between ATP levels and post-transfusion survival values in nonrejuvenated or rejuvenated washed, previously frozen red blood cells.     

Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells

We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34(+) stem cells (up to 1.95 x 10(6)-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.     

Oxidative state of glutathione in red blood cells and plasma of diabetic patients: in vivo and in vitro study

The oxidative state of glutathione in red blood cells (RBC) and plasma of diabetic patients and of age-matched volunteers has been studied. Oxidized glutathione (GSSG) levels in plasma from diabetic subjects were higher than those from controls (17.2 +/- 2.5 and 3.3 +/- 0.4 micrograms/ml, respectively). This phenomenon was evident also in in vitro experiments: incubated RBC from diabetic patients released very high amounts of GSSG in medium. Thus, erythrocytes are responsible for the enhanced amounts of GSSG found in plasma from diabetic patients. The fall in the conversion of GSSG to reduced glutathione in RBC could be due to a reduced activity of the glucose-6-phosphate dehydrogenase (G6PDH) enzyme which has been observed in diabetic patients. In this way, G6PDH supplies reduced amounts of NADPH to the glutathione reductase enzyme affecting the integrity of the glutathione system; on the other hand, the activation by glucose of the polyol pathway also reduces the levels of NADPH for the glutathione reductase enzyme.     

Refractoriness of Callithrix penicillata red blood cells to Plasmodium falciparum in vivo and in vitro

Attempts to infect the New World marmot Callithrix penicillata with Plasmodium falciparum were unsuccessful. Attempts were also made to infect red blood cells of C. penicillata and Saimiri sciureus with P. falciparum in vitro, and these too were unsuccessful due to a high rate of hemolysis produced by apparently adverse culture conditions. It is concluded that modifications to the existing culture conditions will need to be made before successful parasitemia can be induced in vitro in simian erythrocytes.     

An NMR study of the oxidation state of Cu,Zn superoxide dismutase in human red blood cells

The oxidation state of Cu,Zn superoxide dismutase was investigated by 19F-NMR spectroscopy in intact red blood cells and in their lysates. The superoxide dismutase concentration was determined in the red cells both by activity and by F- nuclear relaxation rate measurements and the results obtained showed that the high relaxation rate of F- in erythrocytes is mainly due to the presence of superoxide dismutase. The relaxation rate of F- was unaffected or slightly increased by the addition of a superoxide ion generating system to the cells or to their lysates so indicating that superoxide dismutase is fundamentally in steady state. The results are discussed in terms of the possible reactions of the enzyme in erythrocytes.     

Proteases in malaria-infected red blood cells

The discrimination between erythrocyte and Plasmodium proteases is now made easier by using synthetic fluorogenic substrates, high-pressure liquid chromatography, reliable methods of cell preparation, as well as radiolabeled extracts from in vitro cultures of P. falciparum. The reinvasion process of an erythrocyte by a merozoite involves specific proteinases, which were recently identified using fluorogenic peptidyl-AEC substrates and by analysis of schizont and merozoite extracts with the gelatin-SDS-PAGE method. The biological targets of both host and parasite proteinases are not yet well characterized because Plasmodium-infected red blood cells contain at least four compartments with different pH values, which could modulate the proteinase activities according to their pH range activity. The processing of the precursor for the major merozoite surface antigens involves cleavage of very specific peptidic bonds by, so far unknown, proteinases. The depletion of the erythrocyte cytoskeleton could depend on a 37 kD proteinase, which cleaves spectrin and the 4.1 component, as shown in P. berghei and P. falciparum species. In contrast to leupeptin, which inhibits the merozoite release from schizont-infected erythrocytes, the structural inhibitor analogous to the Val-Leu-Gly-Lys (or Arg) P. falciparum neutral proteinase substrates appears to block the invasion step of erythrocytes by merozoites and may open new trends in chemotherapeutical strategies.