Corticoadrenal activity regulates in rat betaine-homocysteine <Emphasis Type="Italic">S</Emphasis>-methyltransferase expression with opposites effects in liver and kidney

Betaine-homocysteine S-methyltransferase (BHMT) is an enzyme that converts homocysteine (Hcy) to methionine using betaine as a methyl donor. Betaine also acts as osmolyte in kidney medulla, protecting cells from high extracellular osmolarity. Hepatic BHMT expression is regulated by salt intake. Hormones, particularly corticosteroids, also regulate BHMT expression in rat liver. We investigated to know whether the corticoadrenal activity plays a role in kidney BHMT expression. BHMT activity in rat kidneys is several orders of magnitude lower than in rat livers and only restricted to the renal cortex. This study confirms that corticosteroids stimulate BHMT activity in the liver and, for the first time in an animal model, also up-regulate the BHMT gene expression. Besides, unlike the liver, corticosteroids in rat kidney down-regulate BHMT expression and activity. Given that the classical effect of adrenocortical activity on the kidney is associated with sodium and water re-absorption by the distal tubule leading to volume expansion, by promoting lesser use of betaine as a methyl donor, corticosteroids would preserve betaine for its other role as osmoprotectant against changes in the extracellular osmotic conditions. We conclude that corticosteroids are, at least in part, responsible for the inhibition of BHMT expression and activity in rat kidneys.     

Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection

This protocol requires 2-4 h and presents a method for injecting tumor cells, cancer stem cells or dispersed biopsy material into subcutaneous or orthotopic locations within recipient mice. The tumor cells or biopsy are mixed with basement membrane matrix proteins (CultrexBME or Matrigel) at 4 °C and then injected into recipient animals at preferred anatomical sites. Tumor cells can also be co-injected with additional cell types, such as fibroblasts, stromal cells, endothelial cells and so on. Details are given on appropriate cell numbers, handling and concentration of the basement membrane proteins, recipient animals, injection location and techniques. This procedure enables the growth of tumors from cells or biopsy material (tumor graft) with greater efficiency of take and growth, and with retention of the primary tumor phenotype based on histology. Co-injection with additional cell types provides more physiological models of human cancers for use in drug screening and studying cancer biology.     

IL-10 induces CCR6 expression during Langerhans cell development while IL-4 and IFN-gamma suppress it.

Immune responses are initiated by dendritic cells (DC) that form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells colonizing epithelial surfaces. We have recently shown that macrophage-inflammatory protein-3alpha/CCL20, a chemokine secreted by epithelial cells, induces the selective migration of LC among DC populations. In this study, we investigated the effects of cytokines on the expression of the CCL20 receptor, CCR6, during differentiation of LC. We found that both IL-4 and IFN-gamma blocked the expression of CCR6 and CCL20 responsiveness at different stages of LC development. The effect of IL-4 was reversible and most likely due to the transient blockade of LC differentiation. In contrast, IFN-gamma-induced CCR6 loss was irreversible and was concomitant to the induction of DC maturation. When other cytokines involved in DC and T cell differentiation were tested, we found that IL-10, unlike IL-4 and IFN-gamma, maintained CCR6 expression. The effect of IL-10 was reversible and upon IL-10 withdrawn, CCR6 was lost concomitantly to final LC differentiation. In addition, IL-10 induced the expression of CCR6 and responsiveness to CCL20 in differentiated monocytes that preserve their ability to differentiate into mature DC. Finally, TGF-beta, which induces LC differentiation, did not alter early CCR6 expression, but triggered its irreversible down-regulation, in parallel to terminal LC differentiation. Taken together, these results suggest that the recruitment of LC at epithelial surface might be suppressed during Th1 and Th2 immune responses, and amplified during regulatory immune responses involving IL-10 and TGF-beta.     

Mannose receptor ligand-positive cells express the metalloprotease decysin in the B cell follicle.

Decysin, a gene encoding a disintegrin metalloprotease, is transcribed in human dendritic cells (DC) and germinal centers (GC). We have cloned its murine homologue and show that it is processed by the endoprotease furin before secretion of the catalytic domain. We have defined the cell types that express decysin in mouse spleen in the course of an immune response to T cell-dependent Ags. Like in humans, decysin is transcribed by activated CD11c(+) DC that enter the T cell zone from the marginal zone (MZ). In the GC, decysin is expressed by follicular DC and tingible body macrophages. In addition, a MZ cell population expresses decysin and appears to migrate into the B cell follicle. The majority of these follicle-homing cells express the mannose receptor ligand, a marker for the macrophage-like MZ metallophils. The follicle-homing cells are M-CSF dependent, as they are absent in op/op mice that lack functional M-CSF. This suggests that mannose receptor ligand(+) MZ metallophils differentiate into cells that migrate from the MZ into the B cell follicle. Decysin represents the first marker for this previously unrecognized cell population of the mouse spleen, which may represent a precursor for GCDC and may be specialized in the transport of unprocessed Ag from the MZ into developing GC.     

Ion channel blockers inhibit B cell activation at a precise stage of the G1 phase of the cell cycle. Possible involvement of K+ channels

Lymphocytes express voltage-activated K+ channels in their membrane. Combining the patch-clamp techniques of recording with immunological methods, we have analyzed the expression and the involvement of these channels during defined steps of LPS-induced B cell activation. We show that the number of K+ channels increased strongly when B cells entered in the G1 phase of the cell cycle. The involvement of ion channels in B cell proliferation was assessed using channel blockers that inhibit the K+ current. It was first found that TEA, but not TMA, quinine and verapamil totally suppressed both K+ current and DNA synthesis by stimulated lymphocytes as measured by [3H]TdR uptake or propiedium iodide staining. The drugs affected neither the induction by LPS of activation markers such as Ag of the murine class II MHC and type II receptor for the Fc region of IgG nor the initial cell enlargement that occur early during activation. These data indicate that functional K+ channels are not essential for the transition from the G0 to the G1 phases. In contrast, the same channel antagonists blocked the induction of transferrin receptor expression, characteristic of the final stages of G1. These drugs acted on cells already in G1, because their addition 30 h after LPS still suppressed DNA synthesis, and because they inhibited the proliferation of purified B cell blasts. The effect of tetraethylammonium was reversible, a lag period of 12 h occurring before the cells start DNA synthesis after drug removal. Taken together, these data demonstrate that the proliferation of LPS-stimulated B cells requires functional ion channels at a critical period in the G1 phase, taking place before transferrin receptor expression and the entry into the S phase. The involvement of voltage dependent K+ channels at this particular point is suggested by the parallel effects of the drugs used on K+ currents and DNA synthesis.     

Characterization of suppressive immunoglobulin-binding factor (IBF). III. Biochemical and immunochemical characteristics of IBF produced by activated T cells.

The biochemical characteristics of immunoglobulin binding factor produced by activated cells (ATC) were investigated. For this purpose, supernatants of ATC were purified by affinity chromatography on insolubilized IgG and the eluted material was iodinated (125I), treated with mercaptoethanol; and run on SDS polyacrylamide gels. The radioactivity was found in two peaks corresponding to m.w. of 38,000 d and 18,000 d. This result extends and confirms our previous findings that IBF produced by ATC is identical to IBF produced by L-5178-Y internally labeled thymoma cells. The effect of various pH, temperatures, and proteolytic and glycolytic enzymes on the binding properties of 3H-leucine-or H-fucose-labeled IBF to IgG and on the polyacrylamide gel profiles was also studied. By all these criteria, IBF appeared to be a glycoprotein in which the presence of the 38,000 to 40,000 d chain is necessary for the binding to IgG. In the attempt to study the relationships between IBF and I-region products, purified IBF produced by ATC was incubated with anti-Ia immunoadsorbent, and the eluted material was iodinated and run on gels. The 38,000 d and 18,000 d chains characteristic of IBF were found to be specifically retained on the relevant immunoadsorbent. These data favor the hypothesis that IBF bears or is associated with Ia determinants.     

Immunobiological characteristics of impurities in corpuscular influenza vaccines

Admixtures of free antigens have been shown to play the main role in the anaphylactogenic danger of vaccines. The immunogenic and anaphylactogenic action of such antigenic admixtures in corpuscular influenza vaccine can be observed after the immunization of animals in 2 or 3 injections. Host antigens incorporated into viral particles induce no anaphylactic reaction in guinea pigs after their immunization in 3 injections.     

Inhibition of the in vitro 19S and 7S antibody response by immunoglobulin-binding factor (IBF) from alloantigen-activated T cells

A soluble factor secreted by alloantigen-activated mouse T cells which binds to the Fc fragment of IgG and inhibits complement activation by IgG (immunoglobulin-binding factor, IBF) suppressed the in vitro 19S and 7S antibody response by mouse spleen cells to T-dependent as well as T-independent antigens. IBF inhibited the 19S plaque response best when it was added late during PFC generation (between 48 and 72 hr). On the other hand, when it was left in cultures for up to 60 hr and then removed, antibody synthesis was not inhibited. However, its presence for only 2 hr starting after 72 hr of incubation was sufficient to inhibit PFC formation. The suppressive activity of IFB could be neutralized by adding aggregated mouse IgG prior to the critical stage around 72 hr. These data favour the view that IBF could be a suppressive T cell factor and point to the possibility that IBF may act on already triggered B cells during their final differentiation to active PFC.     

Compelling P1 substituent affect on metalloprotease binding profile enables the design of a novel cyclohexyl core scaffold with excellent MMP selectivity and HER-2 sheddase inhibition

A serendipitous discovery that the metalloprotease binding profile of a novel class of 2-carboxamide-3-hydroxamic acid piperidines could be significantly attenuated by the modification of the unexplored P1 substituent enabled the design and synthesis of a novel 2-carboxamide-1-hydroxamic acid cyclohexyl scaffold core that exhibited excellent HER-2 potency and unprecedented MMP-selectivity that we believe would not have been possible via conventional P1′ perturbations.