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).     

Polyethylene glycol-modified GM-CSF expands CD11b(high)CD11c(high) but notCD11b(low)CD11c(high) murine dendritic cells in vivo: a comparative analysis with Flt3 ligand

Dendritic cells (DC) are potent APCs that can be characterized in the murine spleen as CD11b(high)CD11c(high) or CD11b(low)CD11c(high). Daily injection of mice of Flt3 ligand (FL) into mice transiently expands both subsets of DC in vivo, but the effect of administration of GM-CSF on the expansion of DC in vivo is not well defined. To gain further insight into the role of GM-CSF in DC development and function in vivo, we treated mice with polyethylene glycol-modified GM-CSF (pGM-CSF) which has an increased half-life in vivo. Administration of pGM-CSF to mice for 5 days led to a 5- to 10-fold expansion of CD11b(high)CD11c(high) but not CD11b(low)CD11c(high) DC. DC from pGM-CSF-treated mice captured and processed Ag more efficiently than DC from FL-treated mice. Although both FL- and pGM-CSF-generated CD11b(high)CD11c(high) DC were CD8alpha-, a greater proportion of these DC from pGM-CSF-treated mice were 33D1+ than from FL-treated mice. CD11b(low)CD11c(high) DC from FL-treated mice expressed high levels of intracellular MHC class II. DC from both pGM-CSF- and FL-treated mice expressed high levels of surface class II, low levels of the costimulatory molecules CD40, CD80, and CD86 and were equally efficient at stimulating allogeneic and Ag-specific T cell proliferation in vitro. The data demonstrate that treatment with pGM-CSF in vivo preferentially expands CD11b(high)CD11c(high) DC that share phenotypic and functional characteristics with FL-generated CD11b(high)CD11c(high) DC but can be distinguished from FL-generated DC on the basis of Ag capture and surface expression of 33D1.     

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.     

The single-cell epigenomic and transcriptional landscape of immunity to influenza vaccination

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Comparative Evaluation of Porous Versus Nonporous Mucoadhesive Films as Buccal Delivery System of Glibenclamide

The present research work focused on the comparative assessment of porous versus nonporous films in order to develop a suitable buccoadhesive device for the delivery of glibenclamide. Both films were prepared by solvent casting technique using the 3² full factorial design, developing nine formulations (F1–F9). The films were evaluated for ex vivo mucoadhesive force, ex vivo mucoadhesion time, in vitro drug release (using a modified flow-through drug release apparatus), and ex vivo drug permeation. The mucoadhesive force, mucoadhesion time, swelling index, and tensile strength were observed to be directly proportional to the content of HPMC K4M. The optimized porous film (F4) showed an in vitro drug release of 84.47 ± 0.98%, ex vivo mucoadhesive force of 0.24 ± 0.04 N, and ex vivo mucoadhesion time of 539.11 ± 3.05 min, while the nonporous film (NF4) with the same polymer composition showed a release of 62.66 ± 0.87%, mucoadhesive force of 0.20 ± 0.05 N, and mucoadhesive time of 510 ± 2.00 min. The porous film showed significant differences for drug release and mucoadhesion time (p < 0.05) versus the nonporous film. The mechanism of drug release was observed to follow non-Fickian diffusion (0.1 < n < 0.5) for both porous and nonporous films. Ex vivo permeation studies through chicken buccal mucosa indicated improved drug permeation in porous films versus nonporous films. The present investigation established porous films to be a cost-effective buccoadhesive delivery system of glibenclamide.KEY WORDS: buccoadhesive drug delivery, glibenclamide, in vitro release and ex vivo permeation, porous film