Purification and characterization of human skin mast cells. Evidence for human mast cell heterogeneity

Our previous studies of human lung and intestinal mast cells failed to show the heterogeneity found among mast cells in murine species. Recently, we and others have developed techniques for the enzymatic dispersion of human neonatal skin mast cells. In addition, we are now able to make single cell suspensions of mast cells from adult skin and to purify these cells to near homogeneity. Comparative studies of mast cells from these several sources have uncovered several major differences among them. Adult and neonatal skin mast cells themselves differ in that the former are 10-fold less sensitive to goat anti-human IgE, with maximal release occurring at 3.0 and 0.3 microgram/ml, respectively. Skin mast cells also differ in optimal temperature for release: adult mast cells respond maximally at 23 to 30 degrees C and neonatal cells at 37 degrees C. Skin mast cells from both sources are dramatically different from lung and intestinal mast cells in two aspects. First, skin mast cells are quite responsive to several stimuli--morphine sulfate (10(-4) to 10(-6) M), substance P (10(-5) to 10(-7) M), compound 48/80 (10 to 0.1 microgram/ml), f-Met peptide (10(-6) M), and C5a (10(-8) M)--to which the other mast cells fail to respond. Second, although stimulated skin mast cells produce prostaglandin D2, little leikotriene C4, if any, is generated, unlike lung or intestinal mast cells. These differences in inflammatory potential among human mast cells from various sites have important implications for the management of allergic and inflammatory responses.     

Denaturation of covalently closed circular DNA. Kinetics, comparison of several DNAs, mechanism and ionic effects

The rates of the alkaline denaturation of the covalently closed, circular DNAs (form I) of the replicative forms (RF) of phages G4, phi X174, and fd, and of plasmid pBR322 and phage PM2 have been measured at 0 degrees C and some at higher temperatures. These rates are orders of magnitude slower than the denaturation of linear DNA because of the increased stability of the helix to deprotonation that results from the accumulating positive superhelicity during denaturation. Denaturation reactions were initiated by rapid, infrasonic mixing (Camien, M.N., and Warner, R.C. (1984) Anal. Biochem. 138, 329-334), and their progress was measured by analytical ultracentrifugal analysis for the amounts of form I and denatured (Id) DNA after neutralization of the alkaline reaction. The comparative rates of the five DNAs varied over a wide range; the fastest, G4-RF, denatured at 500-fold the rate of the slowest, fd-RF. The differences are accounted for by the interaction of positive superhelicity with the sequence-dependent regions of relative helix stability in the various DNAs. Renaturation rates of Id DNAs varied similarly for Ids prepared at 0 degrees C, but only a few-fold for Ids prepared at 50 degrees C. The rate of denaturation of G4-RF was determined over a wide range of NaOH and NaCl concentrations at 0 degrees C, and the pHm was determined as a function of ionic strength and temperature. The effects of ionic strength have been analyzed in an application of the Manning ion condensation-screening theory (Manning, G.S. (1978) Q. Rev. Biophys. 11, 179-246) which is shown to account for the large destablizing effect of salts on the helix. The pH region of transition at 50 degrees C from renaturation to denaturation was examined, and it was found that the maximum rate of renaturation occurred at a pH about 0.05 units below the pHm.     

Identification of histamine releasing factor(s) in the late phase of cutaneous IgE-mediated reactions

We have shown that fluids collected from antigen-challenged skin blisters during the late phase reaction cause the release of substantial amounts of histamine (means = 42%, n = 14) from human basophils in vitro. Control fluids collected either during the immediate phase or from an unchallenged blister released less than or equal to 10% histamine from both basophils and lung mast cells. Late phase blister fluids induced low levels of histamine release from human lung cells (means = 11%, n = 4) that were slightly but not significantly greater than levels induced by control blister fluids. The characteristics of basophil release were similar to IgE-mediated stimuli in dose dependence, calcium and temperature requirements, and kinetics. The IgE dependence of the late phase blister fluid was demonstrated by desensitization of the basophils to anti-IgE, which obviated the response to anti-IgE and blister fluid but did not affect a non-IgE-mediated stimulus. Removal of the cell surface IgE with lactic acid also abolished the response to both anti-IgE and late phase blister fluid. Incubation of the "stripped" cells with serum containing IgE myeloma restored the response to anti-IgE but failed to affect response to late phase blister fluid. The characteristics of release obtained with this factor closely resemble those of an IgE-dependent histamine releasing factor from cultured macrophages previously described by our group.     

Halothane decreases both tissue and airway resistances in excised canine lungs

Studies of the anesthetic effects on the airway often use pulmonary resistance (RL) as an index of airway caliber. To determine the effects of the volatile anesthetic, halothane, on tissue and airway components of RL, we measured both components in excised canine lungs before and during halothane administration. Tissue resistance (Rti), airway resistance (Raw), and dynamic lung compliance (CL, dyn) were determined at constant tidal volume and at ventilatory frequencies ranging from 5 to 45 min-1 by an alveolar capsule technique. Halothane decreased RL at each breathing frequency by causing significant decreases in both Raw and Rti but did not change the relative contribution of Rti to RL at any frequency. Halothane increased CL,dyn at each breathing frequency, although there was little change in the static pressure-volume relationship. The administration of isoproterenol both airway and tissue components of RL; it may act by relaxing the contractile elements in the lung. Both components must be considered when the effects of volatile anesthetics on RL are interpreted.     

Lymphoma models for B-cell activation and tolerance. VII. Pathways in anti-Ig-mediated growth inhibition and its reversal

WEHI-231, CH33, and CH31 are B-cell lymphomas that are inhibited in their growth by crosslinking of surface Ig receptors during early G1. This "negative signaling" process can be prevented or reversed under certain conditions. In the present paper, we use a cell synchronization procedure to demonstrate that activation of protein kinase C (PKC) is not involved in the negative signal for growth, but rather that PKC activation prevents growth inhibition when present early in the cell cycle. Indeed, the prevention of negative signaling is only accomplished by active phorbol esters. Moreover, phorbol esters and a calcium ionophore fail to deliver a negative signal under conditions in which anti-Ig can significantly prevent cell cycle progression into S phase, thereby ruling out synergy between PKC and calcium in growth inhibition. Whether phorbol esters reverse negative signaling by preventing internalization of the immune complex or phosphorylation of a critical intracellular protein is discussed.     

Regional Distribution of Cytochrome P-450 in the Rat Brain: Spectral Quantitation and Contribution of P-450b,e and P-450c,d

The cytochrome P-450 (P-450) content of different regions of the rat brain was measured after partial purification of the enzyme from homogenates, and the quantitative contribution of P-450b,e and P-450c,d to brain P-450 was assessed by Western immunoblotting and immunohistochemistry using rabbit antibodies raised against purified hepatic P-450b and P-450c, respectively). P-450 could be quantitated by its reduced CO difference spectrum after chromatography of homogenates on p-chloroamphetamine-coupled Sepharose. The yield of P-450 from whole brain was 90 +/- 19 pmol/g of tissue, which is approximately 1% of the level in liver microsomes from control rats. The amount of P-450 recovered from homogenates of olfactory lobes, hypothalamus, thalamus, striatum, cerebral cortex, and brainstem varied between 40 and 100 pmol/g of tissue. The cerebellum was a region of exceptionally high P-450 content, with yields of up to 400 pmol/g whereas the substantia nigra yielded only 16-20 pmol/g. Immunohistochemical studies with anti-P-450b and anti-P-450c revealed intense staining of a limited number of cells in the cerebellum with both antibodies and in the thalamus only with anti-P-450c. In the cerebellum, both anti-P-450b and anti-P-450c stained the Bergmann glial cells together with their radial processes. Individual glial cells in the granular cell layer were also stained. There was no staining of Purkinje cells. In the thalamus, anti-P-450b gave weak staining of certain astroglia, but with anti-P-450c, there was intense staining of neuronal somata.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variable biotherapeutic effects of Lactobacillus acidophilus isolates on orogastric and systemic candidiasis in immunodeficient mice

Two commercially available isolates of Lactobacillus acidophilus (NCFM and LA-1) were compared for their capacities to protect immunodeficient bg/bg-nu/un and bg/bg-nu/+ mice from candidiasis. L. acidophilus NCFM prolonged survival of adult and neonatal bg/bg-nu/nu mice, inhibited disseminated candidiasis in both mouse strains, suppressed weight loss associated with Candida albicans infection in bg/bg-nu/nu females, but did not decrease the severity or the incidente of orogastric candidiasis in gnotobiotic mice. L. acidophilus LA-1 suppressed numbers of C. albicans in the alimentary tracts of bg/bg-nu/+ mice and reduced the severity of mucosal candidiasis in bg/bg-nu/nuand bg/bg-nu/+ mice; however, L. acidophilus LA-1 did not improve the survival of bg/bg-nu/nu mice after oral challenge (colonization) with C. albicansand it was associated with lethality in gnotobiotic adult female bg/bg-nu/nu mice. These results demonstrate that the two isolates of L. acidophilus differed in their capacity to protect immunodeficient mice from candidiasis.