Loading red blood cells with trehalose: a step towards biostabilization

A method for freeze-drying red blood cells (RBCs) while maintaining a high degree of viability has important implications in blood transfusion and clinical medicine. The disaccharide trehalose, found in animals capable of surviving dehydration can aid in this process. As a first step toward RBC preservation, we present a method for loading RBCs with trehalose. The method is based on the thermal properties of the RBC plasma membranes and provides efficient uptake of the sugar at 37 degrees C in a time span of 7 h. The data show that RBCs can be loaded with trehalose from the extracellular medium through a combination of osmotic imbalance and the phospholipid phase transition, resulting in intracellular trehalose concentrations of about 40 mM. During the loading period, the levels of ATP and 2,3-DPG are maintained close to the levels of fresh RBCs. Increasing the membrane fluidity through the use of a benzyl alcohol results in a higher concentration of intracellular trehalose, suggesting the importance of the membrane physical state for the uptake of the sugar. Osmotic fragility data show that trehalose exerts osmotic protection on RBCs. Flow cytometry data demonstrate that incubation of RBCs in a hypertonic trehalose solution results in a fraction of cells with different complexity and that it can be removed by washing and resuspending the RBCs in an iso-osmotic medium. The data provide an important first step in long-term preservation of RBCs.     

Cutting edge: impairment of dendritic cells and adaptive immunity by Ebola and Lassa viruses

Acute infection of humans with Ebola and Lassa viruses, two principal etiologic agents of hemorrhagic fevers, often results in a paradoxical pattern of immune responses: early infection, characterized by an outpouring of inflammatory mediators such as TNF-alpha, IL-1 beta, and IL-6, vs late stage infections, which are associated with poor immune responses. The mechanisms underlying these diverse outcomes are poorly understood. In particular, the role played by cells of the innate immune system, such as dendritic cells (DC), is not known. In this study, we show that Ebola and Lassa viruses infect human monocyte-derived DC and impair their function. Monocyte-derived DC exposed to either virus fail to secrete proinflammatory cytokines, do not up-regulate costimulatory molecules, and are poor stimulators of T cells. These data represent the first evidence for a mechanism by which Ebola and Lassa viruses target DC to impair adaptive immunity.     

Distribution of ClC-2 chloride channel in rat and human epithelial tissues

The ubiquitous ClC-2 Clchannel is thought to contribute to epithelial Clsecretion, but the distribution of the ClC-2 protein in human epitheliahas not been investigated. We have studied the distribution of ClC-2 inadult human and rat intestine and airways by immunoblotting andconfocal microscopy. In the rat, ClC-2 was present in the lateralmembranes of villus enterocytes and was predominant at the basolateralmembranes of luminal colon enterocytes. The expression pattern of ClC-2in the human intestine differed significantly, because ClC-2 was mainlydetected in a supranuclear compartment of colon cells. We foundsignificant expression of ClC-2 at the apex of ciliated cells in bothrat and human airways. These results show that the distribution ofClC-2 in airways is consistent with participation of ClC-2 channels inCl secretion and indicate that extrapolation of resultsfrom studies of ClC-2 function in rat intestine to human intestine isnot straightforward.

     

Biosynthesis of methionine in mouse cells cultured in vitro

In the mouse cell-lines cultured in vitro, viz. L-cells and mouse embryo fibroblasts, the methylation of homocysteine to methionine is carried out by vitamin B12-dependent 5-methyltetrahydrofolate:L-homocysteine methyltransferase only. In these cells grown in the standard Eagle medium, the activity of another methyltransferase, which utilizes betaine as the methyl donor, was not detected. The high activity of the vitamin B12-dependent methionine synthetase is typical for mouse cells from the logarithmic phase of growth. In L-cells 60%, and in the mouse fibroblasts 30% of the enzyme exist in the holo-form; the ratio between the holo- and apoenzyme activity remains stable in cells from logarithmic and stationary cultures. The level of the activity of methionine synthetase strongly depends on the presence of vitamin B12, folate and methionine in the culture medium and is greater after prolonged contact of the cells with these agents.     

Viral infection of the thymus.

We have examined infection of the thymus during congenitally acquired chronic lymphocytic choriomeningitis virus (LCMV) infection of mice, a classic model of antigen-specific T-cell tolerance. Our results show that (i) infection starts at the fetal stage and is maintained throughout adulthood, and (ii) this chronic infection of the thymus can be eliminated by transfer of virus-specific cytotoxic T lymphocytes (CTL) that infiltrate the thymus and clear all viral products from both medullary and cortical regions. Elimination of virus from the thymus results in abrogation of tolerance. During the fetal stage, the predominant cell type infected is the earliest precursor of T cells with a surface phenotype of Thy1+ CD4- CD8- J11d+. In the adult thymus, infection is confined primarily to the cortisone-resistant thymocytes present in the medullary region. The infected cells are CD4+ and J11d+. The presence of J11d, a marker usually associated with immature thymocytes, on infected single positive CD4+ "mature" thymocytes is intriguing and suggests that infection by this noncytolytic virus may affect development of T cells. There is minimal infection of the CD8+ medullary thymocytes or of the double positive (CD4+ CD8+) cells present in the cortex. Infection within the cortex is confined to the stromal cells. Interestingly, there is infection of the double negative (CD4- CD8-) thymocytes in the adult thymus, showing that even during adulthood the newly developing T cells are susceptible to infection by LCMV. Virus can be eliminated from the thymuses of these carrier mice by adoptive transfer of medullary region first and then from the thymic cortex. This result clearly shows the need to reevaluate the widely held notion that mature T cells are unable to reenter the thymus. In fact, in our experiments the donor T cells made up to 20 to 30% of the total cells in the thymus at 5 to 7 days after the transfer. The number of donor T cells declined as virus was eliminated from the thymus, and at 1 month posttransfer, the donor T cells were hardly detectable. The results of this study examining the dynamics of viral infection and clearance from the thymus, the primary site of T-cell development, have implications for understanding tolerance induction in chronic viral infections.     

Isolation and functional characterization of a cyst(e)amine-sensitive protein protecting methyl-dithio groups

We have previously shown that S-methylthio cysteine mixed disulfide and cystamine potently stimulate thiol production and glutathione synthesis of a human T-cell line in SH-poor medium. Here, we describe a simple photometric method for the determination of methylthio-mixed disulfides (MT-groups) and show that liver contains relatively large amount of MT-groups mainly associated with the globulin fractions. At least a part of methylthio (MT-) globulins is in a complex with a heat-stable protein protecting methylthio-groups against reduction and was designated as anti-methylthio factor (AMTF). Similar complexes are present in some animal sera. AMTF isolated from mouse liver was shown to specifically inhibit redox interaction of methyldithio-groups of various origin with thiols but loses this ability in the presence of some agents such as cyst(e)amine, 2-mercaptoethanol and dimethyl sulfoxide abrogating the MT-binding activity of AMTF (= cystamine-sensitive protein-CSP). AMTF purified by heat treatment and isopropanol fractionation was chromatographed on Superose-12 column. Preliminary results showed that the molecular mass of the active component is about 34 kDa consisting of two identical subunits. The possible biological role of MT-AMT complexes was discussed.     

Cultured gastrointestinal smooth muscle cells: cell response to contractile agonists depends on their phenotypic state

In the digestive tract, the transit of ingested food induces a local contraction-relaxation reflex of which the smooth muscle cell (SMC) represents the functional unit. Although freshly isolated SMCs have been extensively used for in vitro studies, in specific cases cultured cells appear necessary. Because conventionally cultured SMCs lose their contractile properties, we have developed: (1) differentiated, contractile rabbit gastric SMCs (D-stim cells), cultured in a medium supplemented with insulin, and (2) proliferative, dedifferentiated rabbit gastric SMCs (P-stim cells), cultured in a medium supplemented with insulin, fetal serum, EGF and b-FGF. The proliferative index was 5±4% and 82±10%, respectively, for D-stim and P-stim cells. Expression of SM-myosin heavy chain was observed in 90% of D-stim cells, whereas it was progressively lost in P-stim cells. Carbachol (1–100 nM), glicentin (2 nM) and gastrin-17 (100 nM) induced contraction of D-stim cells cultured for 3 or 6 days, whereas they did not induce the contraction of P-stim cells; in contrast, gastrin-17 (10 nM) was able to stimulate DNA synthesis (1.86±0.09-fold increase) in P-stim cells. The coupling of muscarinic receptors to intracellular transduction pathways was evaluated in D-stim cells: at day 3, carbachol (100 nM) induced a twofold increase in the production of inositol tri-tetra-phosphates; in parallel, a phosphorylation of ERK MAP kinases occurred within 1 min of carbachol stimulation. In conclusion, cultured functional myocytes derived from mature tissue may be used for long-term studies concerning the events coupled either to proliferation or to motility regulation of differentiated SMCs due to the activation of G-protein-coupled receptors.This study was supported in part by grants from the AFM (Association Française contre les Myopathies).     

Design, synthesis, and evaluation of novelly substituted benzimidazole compounds as angiotensin II receptor antagonists

5-Nitrobenzimidazole derivatives with varying substituents at 2-position have been designed, synthesized, and evaluated for angiotensin II antagonistic activity. A drug-receptor interaction model has been proposed.     

Variegation of the immune response with dendritic cells and pathogen recognition receptors

One of the most fundamental questions in biology is: "How do cells differentiate in the right place, at the right time, into the right kinds?" Understanding the phenomenon of cell differentiation in its spatial and temporal framework is a prelude to understanding the development and physiology of all multicellular systems, including the immune system. Insights over the past 2300 years, since Aristotle, suggest that biological differentiation is guided by the interplay between genetic programs and specific environmental signals. This is exemplified by the mammalian immune response to pathogens, where qualitatively different types can emerge. Although it is appreciated that this type immunity is critical for optimal defense against different pathogens, the early "decision-making mechanisms" are largely obscure. Recent developments in innate immunity and genomics, especially in the biology of dendritic cells (DCs) and pathogen recognition receptors, have stimulated intense research in understanding the mechanisms guiding the differentiation of Th1, Th2, and T regulatory responses. In this study, I summarize recent findings which suggest that activation of DCs via distinct pathogen recognition receptors stimulate different gene expression programs and signaling networks in DCs that guide the variegation of immune responses.