Structures of the O1B and O1C lipopolysaccharide antigens of Escherichia coli. The O-specific moieties of the O1B antigen (lipopolysaccharide) from Escherichia coli O1B:K1 and the O1C antigen from E. coli O1C:K- both consist of L-rhamnose, D-galactose, N-acetyl-D-glucosamine, and N-acetyl-D-mannosamine in a molar ratio of 2:1:1:1. By using fragmentation procedures, methylation analysis, and one- and two-dimensional nuclear magnetic resonance spectroscopy, the structures of these polysaccharides were found to be [formula: see text] In the O1B polysaccharide X is 2, and in the O1C polysaccharide X is 3. With the recently published structure of the O1A polysaccharides (B. Jann, A. S. Shashkov, D. S. Gupta, S. M. Panasenko, and K. Jann, Carbohydr. Polym. 18:51-57 1992), three related O1 antigens are now known. Their common (O1-specific) epitope is suggested to be the side-chain N-acetyl-D-mannosamine residue.


Acholeplasma laidlawii has tRNA genes in the 16S-23S spacer of the rRNA operon. We amplified the 16S-23S rRNA intergenic spacer region of Acholeplasma laidlawii PG8 by polymerase chain reaction (PCR) and obtained two specific PCR products in different sizes. We have sequenced both PCR products and found that one of them has sequence homologous to the spacer tRNA genes in Bacillus subtilis. This is the first evidence of tRNA genes between the 16S-23S rRNA intergenic spacer regions in members of the class Mollicutes.


Nitric oxide-induced S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase inhibits enzymatic activity and increases endogenous ADP-ribosylation. Using conditions that produced chronic inflammation in rat liver, we were able to find a correlation between induction of nitric oxide production and inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). This enzyme is a tetramer composed of identical M(r) 37,000 subunits. The tetramer contains 16 thiol groups, four of which are essential for enzymatic activity. Our information indicates that four thiol groups are S-nitrosylated by exposure to authentic nitric oxide (NO) gas. Furthermore, NO decreased GAPDH activity while increasing its auto-ADP-ribosylation. Reduced nicotinamide adenine dinucleotide and dithiothreitol are required for the S-nitrosylation of GAPDH caused by the NO-generating compound sodium nitroprusside. Our results suggests that a new and important action of nitric oxide on cells is the S-nitrosylation and inactivation of GAPDH. S-Nitrosylation of GAPDH may be a key covalent modification of multiple regulatory consequences in chronic liver inflammation.


Phorbol esters induce nitric oxide synthase activity in rat hepatocytes. Antagonism with the induction elicited by lipopolysaccharide. The incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS) or biologically active phorbol esters promotes the release of nitric oxide to the incubation medium. This process is the result of the induction of the Ca(2+)-and calmodulin-independent form of nitric oxide synthase. Both the release of nitric oxide to the incubation medium and the expression of nitric oxide synthase activity exhibited a lag period of about 45-60 min after cell stimulation. Exposure of hepatocytes to both stimuli produced an antagonistic effect on nitric oxide release, with a half-maximal inhibition obtained with 14 nM phorbol 12,13-dibutyrate at saturating concentration of LPS. Incubation of cells with alpha-phorbol 12,13-didecanoate failed to counteract the effect of LPS or to induce nitric oxide synthase, suggesting that activation of protein kinase C was involved in this process.


Alternative splicing of the Oct-2 transcription factor RNA is differentially regulated in neuronal cells and B cells and results in protein isoforms with opposite effects on the activity of octamer/TAATGARAT-containing promoters. The RNA encoding the octamer-binding transcription factor Oct-2 is alternatively spliced in both neuronal cells and in B cells to yield multiple mRNAs encoding different isoforms of the protein. We show that, compared with B cells, neuronal cells overexpress the mRNAs encoding Oct 2.4 and 2.5 which differ from the other forms (Oct 2.1, 2.2, and 2.3) at the C terminus of the protein. When introduced into cells lacking endogenous Oct-2, the various Oct-2 isoforms have different effects on octamer-containing promoters. The Oct 2.1, 2.2, and 2.3 forms stimulate all octamer-containing promoters. However, the Oct 2.4 and 2.5 forms can repress some promoters and stimulate others, depending on the sequence of the octamer motif and its context within the promoter. In contrast, when introduced into neuronal cells which express a high endogenous level of the inhibitory Oct 2.4 and 2.5 forms, all the Oct-2 isoforms can repress octamer-mediated gene expression. The significance of these findings is discussed with regard to the known inhibitory effect of the octamer motif on viral and cellular gene expression in neuronal cells.


Beta-L-thymidine 5'-triphosphate analogs as DNA polymerase substrates. beta-L-3'-Deoxythymidine 5'-triphosphate (L-ddTTP) and beta-L-3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (L-d4TTP) were substrates for human immunodeficiency virus reverse transcriptase, Escherichia coli DNA polymerase I (Klenow), and Sequenase (modified T7 DNA polymerase). The beta-D- and beta-L-enantiomers of 5-methyluridine 5'-triphosphate (rTTP) were inhibitors but not substrates of reverse transcriptase. The steady-state Km values for L-ddTTP and L-d4TTP, with all three enzymes, were 12-70-fold larger than the Km values for the corresponding D-enantiomers. The Km value of reverse transcriptase for L-ddTTP was 50-fold larger than that for D-ddTTP because the Kd for L-ddTTP was 5-fold larger than that for D-ddTTP, and the first-order rate constant for incorporation of L-ddTMP into the template-primer was 10% that of the D-enantiomer. The D- and L-enantiomers had kcat values with reverse transcriptase and Sequenase that were similar to kcat for the natural substrate, thymidine 5'-triphosphate (dTTP). Thus, the rate determining step appeared to be dissociation of the enzyme-chain-terminated template-primer complex. In contrast, kcat values for the L-enantiomers with Klenow were only 0.1% that of dTTP, and the kcat values for the D-enantiomers were 15% the kcat for dTTP. The reduced kcat values were due to a change in rate determining step from dissociation of the Klenow-chain-terminated template-primer complex to an earlier step in the reaction mechanism, presumably catalysis. Thus, these DNA polymerases did not stereospecifically recognize D-nucleoside 5'-triphosphate analogs as substrates.


The dual role of the cAMP-dependent protein kinase C alpha subunit in T-cell receptor-triggered T-lymphocytes effector functions. In order to directly evaluate the role of the cAMP-dependent protein kinase (PKA) catalytic (C) subunit in T-cell receptor- (TCR) triggered cytotoxic T-lymphocytes (CTL) effector functions, cells were studied after pretreatment with antisense oligomers complementary to mRNA for the C alpha or C beta subunits. C alpha subunit is shown to be predominantly expressed in CTL. In some experiments the pretreatment of the CTL with the C alpha antisense, but not with the control or C beta antisense oligomers, resulted in the inhibition of cAMP-independent PKA activity without significantly affecting the level of total cAMP-inducible PKA activity. In parallel assays, CTL which were pretreated with the C alpha antisense oligomer had enhanced antigen-bearing target cell-triggered-, anti-TCR monoclonal antibody-triggered-, and phorbol 12-myristate 13-acetate/A23187-triggered exocytosis of granules, as well as enhanced antigen-specific cytotoxicity. In contrast, the TCR-triggered gamma-interferon mRNA expression and gamma-interferon secretion were inhibited in C alpha antisense-pretreated CTL. These results suggest that the C alpha subunit of PKA may have a dual role in regulation of T-lymphocytes effector functions: (i) it may down-regulate TCR-triggered protein-synthesis independent responses such as cytotoxicity and exocytosis, thereby counteracting TCR-triggered activation even in the absence of the second messenger, cAMP, and (ii) the C alpha subunit activity is likely to be required for the nuclear and/or cytoplasmic events in CTL's activation involved in lymphokine synthesis and secretion.


Post-translational processing of the leukocyte integrin alpha 4 beta 1. The leukocyte integrin alpha 4 beta 1 (VLA-4, CD49d/CD29) is a receptor for the extracellular matrix protein fibronectin and the endothelial adhesion protein VCAM-1. We have analyzed the biosynthesis and post-translational modifications of the two subunits of this receptor complex. The alpha 4 subunit was initially synthesized as a single-chain polypeptide that underwent the formation of complex endoglycosidase H-resistant oligosaccharide side chains and which could be proteolytically cleaved into two noncovalently associated fragments. The level and rate of alpha 4 subunit cleavage was dependent on the cell studied. The T cell tumor line HPB-ALL expressed both intact and fragmented alpha 4 on the cell surface. The interleukin-2-dependent natural killer line NK 3.3 and long term interleukin-2-dependent activated T lymphocytes cleaved the alpha 4 polypeptide earlier and more efficiently than did HPB-ALL cells and did not have detectable levels of intact alpha 4 on the cell surface. The proteolysis of alpha 4 was blocked by treating cells with either the lysosomotrophic amine NH4Cl or the carboxylic ionophore monensin. The presence of complex N-linked oligosaccharides did not seem to be necessary for alpha 4 cleavage or for binding of the alpha 4 beta 1 complex to a synthetic peptide corresponding to the binding site for this receptor on fibronectin.


Release of transforming growth factor-beta 1 from the pericellular matrix of cultured fibroblasts and fibrosarcoma cells by plasmin and thrombin. A sensitive immunoblotting assay was developed for the detection of transforming growth factor (TGF)-beta 1 from cell extracts and culture medium. HT-1080 human fibrosarcoma cells and human fibroblasts were used as models for the secretion and proteolytic release of pericellular matrix-associated TGF-beta 1. Analysis of the pericellular matrices of the cells indicated that the majority of cell-layer associated TGF-beta 1 was associated with the pericellular matrix. Treatment of the cells with plasmin or thrombin released the matrix-associated TGF-beta 1 to the culture medium. Assays for the biological activity of plasmin-released TGF-beta 1 by Mv1Lu cell growth inhibition assays indicated that the majority was in the latent form. Northern hybridization analyses indicated that the mRNA levels of TGF-beta 1 were not elevated during the proteinase treatment. Experiments using radiolabeled TGF-beta 1 indicated that exogenous active TGF-beta 1 associates mainly with the presumed TGF-beta 1 receptors that were not retained in the extracellular matrix preparations. These results indicate that a major fraction of latent TGF-beta 1 that is produced by the cells is deposited to and remains associated with the pericellular matrices of cultured fibroblasts and fibrosarcoma cells, and that matrix-associated TGF-beta 1 is very susceptible to release by various proteolytic enzymes.


Identification of inducible calmodulin-dependent nitric oxide synthase in the liver of rats. A calmodulin-dependent nitric oxide synthase was significantly induced in the liver of rats treated intravenously with heat-killed Propionibacterium acnes and 5 days later with Escherichia coli lipopolysaccharide. The apparent calmodulin-dependent and -independent isozymes were separated by Mono Q column chromatography after their partial purification by 2',5'-ADP-agarose affinity chromatography. Both enzymes had a molecular weight of 125,000 as determined by SDS-polyacrylamide gel electrophoresis and required NADPH, tetrahydrobiopterin, and dithiothreitol as cofactors. Their activities were completely inhibited by the specific nitric oxide synthase inhibitors NG-monomethyl-L-arginine and N omega-nitro-L-arginine at 80 and 800 microM, respectively. The peptide maps of these two isozymes with lysylendopeptidase and their reverse-phase column chromatographic profiles were indistinguishable. In the presence of bovine calmodulin, the purified calmodulin-dependent isozyme behaved as a calmodulin-independent isozyme on Mono Q column chromatography. The purified calmodulin-independent isozyme was converted to a calmodulin-dependent isozyme by EDTA and EGTA. Calmodulin blot analysis using 125I-calmodulin showed that the two isozymes bound calmodulin equally efficiently.


Amino terminus of the interleukin-8 receptor is a major determinant of receptor subtype specificity. Interleukin-8 (IL-8) is a key mediator in the migration of neutrophils from the circulation to the site of inflammation in the tissue. IL-8 is secreted by many cell types in response to proinflammatory stimuli such as interleukin 1, tumor necrosis factor, and lipopolysaccharide and is a potent chemoattractant and activator of neutrophils. Neutrophil activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA/GRO) are structurally and functionally related to IL-8 and, like IL-8, bind to specific G protein-coupled receptors on neutrophils. In the present study two closely related cloned IL-8 receptor subtypes are characterized by expression of the cDNA clones in monkey kidney cells (COS-7) or chinese hamster ovary cells and analysis of their ligand binding profiles. Both receptor subtypes bind 125I-labeled IL-8 with similar high affinity, however, the F3R receptor binds IL-8 exclusively, while the 4Ab receptor binds both IL-8 and MGSA/GRO with high affinity and NAP-2 with lesser affinity. Furthermore, we demonstrate with the use of intersubtype chimeric receptors that the specificity of ligand binding to both IL-8 receptor subtypes is dictated by the heterogeneous NH2-terminal domain. The F3R receptor is representative of a restricted IL-8 receptor subtype, and 4Ab represents a nonrestricted receptor subtype. It is proposed that these subtypes be named IL-8 receptors alpha and beta, respectively.


The Na-K-Cl cotransporter of avian salt gland. Phosphorylation in response to cAMP-dependent and calcium-dependent secretogogues. The effect of a cAMP-dependent secretogogue (VIP) on the phosphorylation of an endogenous, membrane-bound protein (pp170) was assessed in an intact cell preparation from the avian salt gland. The addition of VIP, in the presence of 100 microM isobutylmethylxanthine, resulted in a concentration-dependent increase in phosphorylation of pp170. This effect was rapid and transient with a 3-5-fold increase in phosphorylation occurring 1 min after the addition of VIP. Under similar incubation conditions, VIP stimulated a 4.6-fold increase in cAMP accumulation that paralleled phosphorylation. Exposure of cells to either forskolin or 8-Br-cAMP resulted in a 5-8-fold increase in the phosphorylation of pp170. The effect of forskolin was dose dependent with an EC50 similar to that for stimulation of secretion (35 nM). These results implicate an involvement for a cAMP-dependent protein kinase in the phosphorylation of pp170. The identity of pp170 was assessed utilizing a monoclonal antibody (Q3) directed against pp170. Q3 recognized a single 170-kDa band on Western blots of salt gland membrane protein. Immunoprecipitation of pp170 from salt gland cells resulted in the selective extraction of a single protein whose phosphorylation state was increased approximately 5-fold in response to carbachol or VIP. The identity of pp170 was established using two criteria. First, Q3 recognized affinity-purified Na:K:Cl cotransporter preparations from shark rectal gland membranes. Second, pp170 was selectively immunoprecipitated by monoclonal antibodies (J3, J4, and J7) that recognize different epitopes of the shark transport protein. These results suggest that pp170 is homologous to the shark rectal gland Na-K-Cl cotransporter, and thus the proteins may be functionally similar.


Measurement of intracellular cadmium with fluorescent dyes. Further evidence for the role of calcium channels in cadmium uptake. Cellular uptake of Cd2+ has been monitored using intracellularly trapped dyes, Fura 2 and Quin 2, which bind Cd2+ with extremely high affinity, and digital fluorescence imaging has been used to visualize intracellular free Cd2+. The excitation spectrum of the Cd2+ complex of Fura 2 is similar to that of the Ca2+ complex, whereas Cd2+ displaces Ca2+ from Quin 2 and reduces fluorescence. Fluorescence of Fura 2-loaded cells increased when 50 microM extracellular Cd2+ was added and fluorescence of Quin 2-loaded cells decreased. Cd2+ uptake by GH3 pituitary cells, which occurs in part via voltage-sensitive L-type calcium channels, was increased by BAY K8644 and depolarization and decreased by nimodipine. When Fura 2 and Quin 2 were used to measure Cd2+ uptake by glial C6 cells, which have no L-channel activity, high K+ and BAY K8644 did not change the apparent rate of Cd2+ uptake. GH3 and C6 cells were incubated with Cd2+ for 24 h and loaded with Fura 2, and fluorescence was measured before and after addition of tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), a membrane permeant chelator with extremely high affinity for metals. TPEN had little effect on fluorescence of Fura 2-loaded GH3 and C6 cells not exposed to Cd2+ but decreased fluorescence of cells that had been incubated with 1-10 microM Cd2+. Fluorescence ratio imaging of Fura 2-loaded cells was used to image intracellular free Cd2+ for both GH3 and C6 cells. Cd2+ uptake over 30-180 min could be followed by the increase in 340/380 fluorescence ratio and the increase in fluorescence ratio was reversed within 5 min by TPEN. The results provide further evidence for the importance of voltage-gated calcium channels to Cd2+ uptake of certain cells.


Characterization of insulin-like growth factor (IGF) binding proteins and their role in modulating IGF-I action in BHK cells. We have found that over one-half of the total cell surface 125I-insulin-like growth factor I (IGF-I) binding to BHK cells represents binding to IGF binding proteins (IGFBPs) rather than to the IGF-I receptor. In addition to a number of secreted IGFBPs, we have now characterized two cell-associated IGFBPs with unique characteristics. The cell-associated IGFBPs have molecular weights of 30,000 (30K) and 25,000 (25K), as determined by the Western ligand blot technique. IGFBP-30K is located at the cell surface and can be readily labeled by affinity cross-linking with 125I-IGF-I. Surface expression of IGFBP-30K increases 5.4 +/- 1.2-fold (n = 11) with serum starvation. This induction is fully evident by 4 h, plateauing by 24 h, and is completely inhibitable by cycloheximide. The fasting-induced increase in IGFBP-30K is inhibited by IGF-I and by des-IGF-I and, to a lesser extent, by insulin. Unlike cell-associated IGFBP-30K, secretion of IGFBP was stimulated (6.8 +/- 0.5-fold, n = 2) by IGF-I, whereas IGFBP secretion was inhibited 54% by insulin. These results demonstrate coordinate regulation of IGFBP by serum starvation and IGF-I, such that at low concentrations of IGF-I, cell surface binding protein increases whereas binding protein secretion decreases. At high concentrations of IGF-I, IGFBP secretion increases and cell surface IGF-I receptor, as well as IGFBP, decreases. Taken together, these regulatory events regulate the availability of IGF-I for biologic signalling.


Release of amino-terminal fragments from amyloid precursor protein reporter and mutated derivatives in cultured cells. Abnormal proteolytic processing of amyloid precursor protein (APP) is thought to be central to the formation and deposition of beta amyloid peptide in Alzheimer's disease. A putative "secretase" activity normally releases an amino-terminal APP fragment by cleaving APP at residues within the beta amyloid peptide thereby precluding amyloidogenesis. In order to better understand the requirements for APP cleavage by secretase, we have expressed a modified cDNA construct representing the 751-amino acid isoform of APP (APP-REP) and mutated APP-REP proteins in cultured cells. Here, we show that: (a) APP-REP is predominantly associated with membranes; (b) intracellular turnover and processing of APP-REP is similar to that reported for the intact APP protein; (c) secretion appears unaltered by introduction of the glutamate to glutamine mutation found in the APP gene of patients suffering from hereditary cerebral hemorrhage with amyloidosis of Dutch origin; (d) a mutation in which the 18 juxtamembranous amino acids encompassing the secretase site are deleted also allows release of an amino-terminal fragment into the conditioned medium; and (e) kinetics of cleavage of APP-REP and its mutated derivatives are similar. These results indicate that the secretory cleavage of the extracellular amino-terminal fragments of APP-REP can occur in the presence of different novel juxtamembranous amino acid sequences.


Giant sarcoplasmic reticulum vesicles: a study of membrane morphogenesis. Rabbit sarcoplasmic reticulum vesicles were fused into giant proteoliposomes in a medium of 0.1 M KCl, 10 mM Tris-maleate, pH 7.0, 10 micrograms ml-1 antipain, 10 micrograms ml-1 leupeptin, 25 IU per ml Trasylol, 3 mM NaN3, 3.75% PEG 1500 and 3% DMSO by brief exposure to 37 degrees C, followed by incubation for 4 h at 25 degrees C. Approximately 5-10% of the sarcoplasmic reticulum elements underwent fusion, forming single-walled spherical vesicles of 1-25 microns diameter, in which the polarity of the native membrane was preserved. The Ca(2+)-stimulated ATPase activity remained essentially unchanged after fusion. On exposure to decavanadate in a Ca(2+)-free medium the spherical vesicles assumed a corrugated appearance with the formation of long ridges separated by deep furrows that eventually pinched off longitudinally and separated into numerous long crystalline tubules of uniform (approximately 0.1 microns) diameter. The vanadate-induced transformation of giant vesicles into tubules implies that the geometry of the sarcoplasmic reticulum membrane is determined by the conformation of the Ca(2+)-ATPase.


S-100ab increases Ca2+ release in purified sarcoplasmic reticulum vesicles of frog skeletal muscle. The S-100ab protein, a mixed isoform member of the S-100 family, stimulates Ca(2+)-induced Ca(2+)-release from sarcoplasmic reticulum vesicles purified from frog skeletal muscle cells. The effects of S-100ab appear to be specific and result from its peculiar characteristics rather than the fact that it is a calcium-binding protein. Moreover, the addition of S-100ab to the solution completely abolished the inhibition provoked when Ruthenium Red was added alone. Experiments that added labelled Ryanodine with and without S-100 indicated that the protein diminished the affinity of the alkaloid at its receptor site.


Synergy between encephalitogenic T cells and myelin basic protein-specific antibodies in the induction of experimental autoimmune encephalomyelitis. Experimental autoimmune encephalomyelitis (EAE) is an experimentally induced demyelinating disease mediated by CD4+ T cells specific for various myelin proteins including myelin basic protein (MBP) and myelin proteolipid protein (PLP). Although myelin- and other CNS-specific antibodies are produced in EAE, B cells and antibodies are thought by most not to play a decisive role in the induction of EAE. In this report we show that B cells serve as the major antigen-presenting cells (APC) during the T cell activation stage in lymph nodes, and that MBP-specific antibodies can greatly enhance the induction of EAE. The role of B cells as APC is demonstrated in B cell-depleted mice. EAE cannot be induced by antigen/complete Freund's adjuvant immunization unless these mice are locally reconstituted with B cells prior to immunization. The enhancing effect of antibodies is demonstrated in experiments in which EAE is induced by the adoptive transfer of encephalitogenic T cells. The adoptive transfer of large numbers of encephalitogenic T cells induces EAE in 90% of normal recipient mice, but only 33% of B cell-depleted mice get EAE at the same cell dose. The efficiency of EAE induction in B cell-depleted mice can be enhanced if MBP-specific antibodies are simultaneously administered. A similar enhancement is also seen in normal mice when the number of adoptively transferred T cells is limiting. We propose that MBP-specific antibodies enhance the presentation of myelin-derived antigens by APC in the CNS to the adoptively transferred encephalitogenic T cells.


Novel LCMV-specific H-2k restricted CTL clones recognize internal viral gene products and cause CNS disease. H-2k (C3H/Hej) cytotoxic T lymphocytes (CTL) specific for lymphocytic choriomeningitis virus (LCMV) were cloned. Three clones recognizing internal viral antigens were studied. One such CTL clone recognized neither the glycoprotein nor nucleoprotein encoded by the viral short RNA segment, but reacted with a protein encoded by the long RNA segment, either the viral polymerase, or the Z protein. This one clone, in addition to primary CTL harvested from immunized C3H mice, failed to lyse target cells expressing the Z protein, suggesting recognition was to the viral polymerase. Two other clones recognized the viral nucleoprotein, amino acids 93-100, as determined by protein deletion and peptide mapping studies. When introduced directly into the central nervous systems of LCMV-infected histocompatible mice, all clones were active in vivo and capable of causing immunopathologically mediated death.


Peripheral nervous system (PNS) expression of mRNAs encoding myelin proteins and Fc gamma RIII during experimental allergic neuritis. Experimental allergic neuritis (EAN) was induced in Lewis rats by injection of 'SP26', a peptide homologous to amino acids 53-78 of bovine myelin P2 protein, in complete Freund's adjuvant. The rats developed signs of EAN which began on day 14, were maximal on day 18, and had subsided by day 30. RNA content of cauda equina and sciatic nerves increased more than 2-fold at the height of EAN. Expression of myelin P0 and P1 mRNAs did not fall during EAN, nor rise during recovery. Fc gamma R mRNA, which encodes Fc gamma RIII, an immunoglobulin-binding protein mediating activation of natural killer cells and macrophages by immune complexes, was transiently, but markedly induced in scattered endoneural cells, presumably macrophages, in cauda equina and sciatic nerves during the period of increasing weakness.


Autoimmunoregulation: differential modulation of CD10/neutral endopeptidase 24.11 by tumor necrosis factor and neuropeptides. Neutral endopeptidase (NEP) 24.11 appears to be an important enzyme in both vertebrate and invertebrate autoimmunoregulation. Activation of human or invertebrate immunocytes that express NEP with substrates such as monokines and neuropeptides results in its increased expression, in other words, upregulation. However, since certain neuropeptides are also substrates for NEP, these activated immunocytes will respond to neuropeptides only at higher concentrations, thus downregulating the response. Specifically, in tumor necrosis factor (TNF)-treated immunocytes, we demonstrate the effects of increased NEP expression on altering the stimulatory activities of the neuropeptides met-enkephalin, melanocyte-stimulating hormone and substance P. We demonstrate the significance of NEP in modulating these responses through the use of specific enzyme inhibitors such as phosphoramidon, thiorphan and captopril. Furthermore, we present evidence suggesting that the individual variations seen in immunocytes from both different and the same donors to activating substances may reflect fluctuating levels of NEP expressed in response to endogenous stimuli. These results indicate that NEP is a highly significant factor in controlling the response(s) of certain immunocytes in man and higher invertebrates to the influence of biologically active substances such as monokines and neuropeptides.


Evidence for target tissue regulation of resistance to the induction of experimental allergic encephalomyelitis in AO rats. A myelin basic protein (MBP)-specific T cell line derived from F1 hybrids between experimental allergic encephalomyelitis (EAE)-susceptible DA (RT1avl) strain and EAE-resistant AO (RT1u) strain was capable of inducing clinical EAE in F1 hybrids and DA, but not in AO rats. In vitro restimulation with MBP presented by AO antigen-presenting cells (APC) resulted in the generation of a MBP-specific subline restricted by RT1u MHC products which induced clinical EAE in F1 hybrids but not in the AO parental strain. Deletion of hosts' leukocytes using sublethal irradiation and cytotoxic drugs did not abrogate the resistance of AO rats, which argues against the involvement of hosts' lymphoid cells in the regulation of autoagression. Thus, mechanism(s) regulating the activity of autoagressive T cells on functional elements in the target tissue might be responsible for differences in susceptibility to EAE.


Afferent connections of the laterodorsal and the pedunculopontine tegmental nuclei in the rat: a retro- and antero-grade transport and immunohistochemical study. Increasingly strong evidence suggests that cholinergic neurons in the mesopontine tegmentum play important roles in the control of wakefulness and sleep. To understand better how the activity of these neurons is regulated, the potential afferent connections of the laterodorsal (LDT) and pedunculopontine tegmental nuclei (PPT) were investigated in the rat. This was accomplished by using retrograde and anterograde axonal transport methods and NADPH-diaphorase histochemistry. Immunohistochemistry was also used to identify the transmitter content of some of the retrogradely identified afferents. Following injections of the retrograde tracer wheatgerm agglutinin-conjugated horseradish peroxidase (WGA-HRP) into either the LDT or the PPT, labelled neurons were seen in a number of limbic forebrain structures. The medial prefrontal cortex and lateral habenula contained more retrogradely labelled neurons from the LDT, whereas in the bed nucleus of the stria terminalis and central nucleus of the amygdala, more cells were labelled from the PPT. Moderate numbers of neurons were seen in the magnocellular regions of the basal forebrain, and many labelled neurons were observed in the lateral hypothalamus, the zona incerta, and the midbrain central gray from both the LDT and the PPT. Accessory oculomotor nuclei in the midbrain as well as eye movement-related structures in the lower brainstem contained some neurons labelled from the LDT, and fewer neurons from the PPT. A few labelled neurons were seen in somatosensory and other sensory relay nuclei in the brainstem and the spinal cord. Retrograde labelling was seen in a number of extrapyramidal structures, including the globus pallidus, entopenduncular and subthalamic nuclei, and substantia nigra following PPT injections; with LDT injections, labelling was similar in density in the substantia nigra but virtually absent in the entopeduncular and subthalamic nuclei. Data with the fluorescent retrograde tracer fluorogold combined with immunofluorescence indicated that many neurons in the zona incerta-lateral hypothalamic region that were retrogradely labelled from the LDT contained alpha-melanocyte-stimulating hormone. Numerous neurons were labelled throughout the reticular formation of the brainstem following either LDT or PPT injections. Many neurons retrogradely labelled in the LDT and PPT, the dorsal and median raphe nuclei, and the locus ceruleus contained choline acetyltransferase, serotonin, and tyrosine hydroxylase, respectively. The anterograde tracers WGA-HRP and phaseolus vulgaris leucoagglutinin were used to confirm some of the projections indicated by the retrograde labelling data; anterograde labelling was seen in the LDT and PPT following injections of one of these tracers into the medial prefrontal cortex, lateral hypothalamus, and the contralateral LDT.(ABSTRACT TRUNCATED AT 400 WORDS)


Developmental differentiation of MAP2 expression in the central versus the peripheral and efferent projections of the inner ear. The goal of this study was to extend our knowledge of MAP2 localization in the peripheral nervous system of mammals, since most results on MAP2 distribution are obtained in the central nervous system (CNS). This study shows the presence of microtubule-associated protein 2b (MAP2b) and MAP2c in the inner ear and describes the immunocytochemical distribution of MAP in adult and developing spiral ganglion of the rat by using a well-characterized antibody for MAP2a and MAP2b. (This antibody does not recognize the immature MAP2c). MAP2 labeling is already present in spiral ganglion neurons at 16 days of gestation. From this stage and up to the first postnatal week, MAP2 labeling was strong in all spiral ganglion neurons and their central processes. Double immunostaining at the 16-day stage with anti-MAP2 and anti-neurofilament (NF) antibodies mainly showed NF labeling in central branches that corresponded to anatomically and functionally described axons of spiral neurons. The peripheral branches lacked MAP2 labeling. In neonatal and postnatal stages, MAP2 reactivity was located in spiral ganglion perikarya and their neurites. The intensity of adult labeling was, however, lower than in younger animals. The antibody used in this study did not label axons originating in the CNS as seen by a negative response in efferent fibers from the intraganglionic spiral bundle of the cochlea. Our results suggest that during ontogenesis, MAP2 is highly expressed in the central projection of spiral ganglion neurons, and then is reduced to lower quantities in the central branch after the first postnatal week and persists into adulthood.(ABSTRACT TRUNCATED AT 250 WORDS)


Physiology and morphology of neurons in the dorsal motor nucleus of the vagus and the nucleus of the solitary tract that are sensitive to distension of the small intestine. We have recently shown that distension-sensitive vagal afferents are part of a neural circuit affecting absorption of water in the rat small intestine. Our results indicated that vagal afferent activity directly or indirectly influences the activity of neurons in the dorsal motor nucleus of the vagus (DMNV). In the present study we pursued this interaction by examining the structure and function of neurons in the DMNV and nucleus of the solitary tract (NST) that responded to moderate distension of the small intestine. Distension-sensitive cells were filled by intracellular iontophoretic injection of horseradish peroxidase. A total of 43 distension-sensitive brainstem neurons were successfully characterized and labeled. Sixteen of the 17 NST neurons were excited by distension of the small intestine. Ten of the seventeen were restricted to the ipsilateral NST. Only two NST neurons possessed axons that terminated in the subjacent DMNV. In contrast to the response profile of the NST neurons, 24 of 26 DMNV neurons were inhibited by intestinal distension. Fourteen of the DMNV neurons appeared to contribute to the vagus nerve and 15 extended dendrites into the overlying NST. We propose that distension-induced inhibition of DMNV activity is accomplished by inhibitory NST neurons, which synapse on the dendrites of DMNV neurons in the NST.


Fine structure of rat septohippocampal neurons: I. Identification of septohippocampal projection neurons by retrograde tracing combined with electron microscopic immunocytochemistry and intracellular staining. In this report the normal dendritic organization and fine structure of identified septohippocampal projection neurons is described as a prerequisite for a time course analysis of retrograde changes in these neurons following axotomy (see Naumann et al., J. Comp. Neurol. 325:219-242, 1992). Septohippocampal projection neurons were retrogradely labeled by injection of the fluorescent tracer Fluoro-Gold into the hippocampus. Next, retrogradely labeled cells in Vibratome sections of the medial septum/diagonal band complex were intracellularly stained with the fluorescent dye Lucifer Yellow (LY). Photooxidation of LY resulted in a stable electron-dense reaction product, which allowed us to study these double-labeled neurons by electron microscopy. Another series of sections containing retrogradely labeled neurons were immunostained for choline acetyltransferase (ChAT) or parvalbumin (PARV). In this way the fine structure of two different chemically characterized subpopulations of septohippocampal neurons could be compared with that of the LY-injected neurons. Intracellular filling of retrogradely labeled neurons with LY stained the cell body and the entire dendritic arbor. Essentially, three classes of neurons could be distinguished, i.e., bipolar cells, multipolar neurons, and an intermediate group. All these neurons displayed smooth, often varicose dendrites lacking spines. Mainly located close to the midline, there was a group of cells with only very few if any LY-stained dendrites. In the electron microscope, the double-labeled neurons were easily identified by numerous electron-dense lysosomes associated with transported Fluoro-Gold and the diffuse reaction product resulting from photooxidation. They displayed fine-structural characteristics as previously described for cholinergic neurons. In fact, our fine-structural analysis of ChAT-positive Fluoro-Gold-labeled neurons, but also of back-filled PARV-positive cells, gave very similar results. All these neurons had infolded nuclei, abundant cytoplasmic organelles, and a few axosomatic synapses. Thus, a plain electron microscopic study does not allow one to distinguish between subpopulations of septohippocampal projection neurons.


Distribution of the 275 kD hair cell antigen and cell surface specialisations on auditory and vestibular hair bundles in the chicken inner ear. The 275 kD hair cell antigen (HCA) is a protein that is specifically associated with the apical surface of sensory hair cells in the chick inner ear. A comparative study of the vestibular and auditory organs of the inner ear, using both wholemounts and cryosections double labelled for the HCA and F-actin, reveals that two distinct types of hair cells can be distinguished on the basis of antibody staining in each of the vestibular epithelia. One type of hair cell has the HCA restricted to the base of the stereocilia bundle and is found in the striolae of the maculae and in a large, centrally located region of each ampulla. The other type of hair cell is found in the extrastriolar regions of the maculae and the peripheral regions of the ampullae and has the HCA distributed over the entire surface of the stereocilia bundle. In the basilar papilla, the auditory epithelium of the chick inner ear, the HCA is, as in the striolar regions of the maculae, restricted to the base of the hair bundles. In all sensory epithelia the HCA is also present on the apical, nonstereociliary surface of the hair cells. Ultrastructural examination of the basilar papilla and the striolar and the extrastriolar regions of the lagenar macula after staining with ruthenium red and tannic acid shows that there are four morphologically different types of interstereociliary connectors (oblique tip connectors, horizontal tip connectors, shaft connectors and basal connectors) associated with the hair bundles. Oblique tip connectors and basal connectors are found on hair cells from all regions and have a similar distribution. Horizontal tip connectors are seen only on hair cells in the basilar papilla and the striolar region of the lagenar macula. Shaft connectors extend all the way to the tips of extrastriolar hair cell bundles, but extend only a short way up the bundles of hair cells in the basilar papilla and striolar region of the lagenar macula. Immunogold labelling confirms the results obtained with immunofluorescence microscopy and demonstrates that the distribution of the HCA on the surface of adjacent stereocilia correlates closely with that of the shaft connectors; i.e., immunostaining is observed up to the tips of the extrastriolar hair cell bundles, but is restricted to the lower regions of hair cell bundles in the striolar region and basilar papilla.(ABSTRACT TRUNCATED AT 400 WORDS)


Cytoarchitecture and visual field representation in area 17 of the tammar wallaby (Macropus eugenii). Tritiated proline was injected into one eye in the tammar wallaby and transported label was studied in the cortex after transneuronal passage through the lateral geniculate nucleus. The autoradiographic label and cytoarchitecture were used to anatomically demarcate the borders of area 17. Electrophysiological recordings from single units were done to obtain a retinotopic map of area 17. Single units in area 17 were found to have orientation sensitivity comparable to those seen in placental mammals such as cat and monkey. They could also be classified as simple, complex, and hypercomplex cells. Changes in the cortical areal magnification factor with eccentricity were found to match the drop off in retinal ganglion cell density only along the vertical meridian representation. Along the horizontal meridian, the cortical magnification falls off significantly with eccentricity, whereas the ganglion cell density shows only a mild reduction. Thus central vision, especially the binocular segment, is heavily represented at the cost of the periphery.


Butyrylcholinesterase-rich neurons in rat brain demonstrated by a sensitive histochemical method. Butyrylcholinesterase (BChE) is a highly active enzyme in brain, but little is known about its physiological functions. One obstacle has been the lack of a sensitive and specific method for determining its cellular localization. We report here on a histochemical technique that has permitted BChE to be detected in neuronal, glial, and vascular structures. The method, which utilizes butyrylthiocholine iodide as the substrate, is a modification of our previously described method for acetylcholinesterase (AChE) histochemistry. BChE-rich neuronal somata stained much more intensely than capillaries or glia. Prominent neuronal groups were located in the anterodorsal, laterodorsal, anteroventral, reuniens, centrolateral, paratenial, and periventricular thalamic nuclei, the laterodorsal tegmental nucleus, the pedunculopontine tegmental nucleus, and the dorsal motor nucleus of vagus. Several other areas of the forebrain and brainstem showed modest numbers of positive cells. No positive cells were detected in the striatum, hippocampus, and most parts of the hypothalamus, which are regions containing numerous AChE-rich neurons. Although the distribution pattern of BChE-rich neurons differed from that of AChE-rich neurons, some neuronal groups contained both esterases. The results suggest that BChE may play a unique role in neuronal function, particularly since many BChE-rich neurons have not been identified as to neurotransmitter type.


Mast cells and eosinophils in the allergic mucosal response to allergen challenge: changes in distribution and signs of activation in relation to symptoms. An allergen challenge was performed in 10 asymptomatic patients with strictly seasonal allergic rhinitis. For comparison; seven nonallergic subjects were challenged with allergen, and seven allergic patients were challenged with diluent. Cell samples, obtained with use of a brush technique to recover cells from within the epithelium and nasal lavage to collect cells from the epithelial surface, and symptom scores were taken before challenge and at 2-hour intervals during 12 hours. The cell suspensions were cytocentrifuged onto object slides for light microscopy. Histamine was determined in the cell pellets. In brush samples from the allergic patients challenged with allergen, eosinophils, expressed as a percentage of the total granulocytes, increased from 4.3% +/- 2.7% (mean +/- SEM) to 10.3% +/- 3.8% (p < 0.05) 4 hours after challenge. This level was maintained for up to 12 hours. A similar increase was noted in the lavage specimens 2, 6, and 8 hours after the challenge. In the brush samples the proportion of eosinophils containing two or more cytoplasmic vacuoles, taken as a sign of activation, increased from 20% to 72% (p < 0.05) 8 hours after provocation. In brush samples from the allergic patients challenged with allergen, the numbers of metachromatic cells increased to a maximum of eightfold at 10 hours. In the lavage specimens, no metachromatic cells were observed before provocation, but they progressively increased in number 2 to 12 hours after provocation. Cell pellet histamine content decreased temporarily 2 to 4 hours after challenge (p < 0.05) in brush samples from allergen-challenged allergic patients. The local metachromatic cell density before challenge, as reflected in the brush specimens, correlated with nasal congestion, sneezing, and the degree of eosinophilia.


Identification of common allergenic structures in hazel pollen and hazelnuts: a possible explanation for sensitivity to hazelnuts in patients allergic to tree pollen. It is known that most patients with type I allergy to tree pollens also suffer from intolerance to nuts. To identify allergenic structures common to hazel pollen and hazelnuts, cross-reactivity of patients' IgE was investigated. With use of immunoblotting,.serum IgE from 25 patients displaying type I allergic reactions to tree pollens and intolerance to hazelnuts (group I) bound to the 17 kd major hazel pollen allergen Cor a I (100%) and to the 14 kd hazel pollen profilin (16%). IgE binding to proteins of comparable molecular weights in hazelnut extracts was found (18 kd and 14 kd), suggesting that proteins similar to Cor a I and hazel profilin might be also expressed in hazelnuts. In contrast, only four sera (22%) from 18 patients (group II) with tree pollen allergy but without any case history of nut hypersensitivity showed IgE binding to the 18 kd protein of hazelnut extract, and none of these sera exhibited IgE reactivity to the hazelnut profilin. To characterize the hazel pollen and hazelnut allergens, purified recombinant Bet v I (major birch pollen allergen) and purified recombinant Bet v II (birch profilin), respectively, were used for IgE-inhibition experiments. Binding of IgE from patients (with nut allergy) to the blotted hazelnut allergens could be blocked by preincubation of patients' sera with the recombinant proteins. Furthermore, the 18 kd protein of hazelnut extract was purified and induced specific release of histamine from basophils of a patient suffering nut hypersensitivity but not from a healthy control donor. A rabbit antibody raised against celery profilin identified the 14 kd proteins in hazel pollen and hazelnuts as profilin. Our experiments suggest a protein with IgE binding properties similar to the major allergens from pollens of hazel, Cor a I, and of birch, Bet v I, as predominant allergens in hazelnuts, and show that the plant pan-allergen profilin can be detected in both hazel pollen and hazelnut extracts.


Localization of inhaled trimellitic anhydride to lung with a respiratory lymph node antibody secreting cell response. Male Sprague-Dawley rats were exposed to trimellitic anhydride (TMA) by inhalation (500 micrograms/m3), 4 hours a day, for 1 to 10 days. TMA was localized to lung cells by immunoelectron microscopy. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis were used in an attempt to localize TMA to lung lavage proteins. The lung-associated lymph node (LALN) B-lymphocyte response was measured by quantitation of immunoglobulin (Ig)G, IgA, and IgM antibody secreting cells specific for TMA rat serum albumin (TM-RSA) by use of the enzyme-linked immunospot assay (ELISPOT) method. The IgG, IgM, and IgA antibody response to TM-RSA in serum and lavage fluid was quantitated by ELISA. Lung injury was assessed by the number of external lung hemorrhagic foci and lung weight. Immunoelectron microscopy localized TMA to alveolar and bronchial cells on all exposure days. ELISA detected trace amounts of TMA haptenized lavage proteins that could not be detected by Western blot analysis. A marked increase occurred in lung injury from day 7 to 10. The LALN IgG, IgA, and IgM antibody secreting cell response to TM-RSA paralleled measures of lung injury. IgG, IgM, and IgA serum and lavage antibody to TM-RSA were correlated with lung injury measures. Lavage and serum IgG antibody levels had the highest correlation with lung injury.


Respiratory allergy to Aspergillus-derived enzymes in bakers' asthma. Baking and food industry workers are exposed to several powdered Aspergillus-derived enzymes with carbohydrate-cleaving activity that are commonly used to enhance baked products. We describe a retrospective study of sensitization to fungal alpha-amylase and cellulase on bakers. Five bakers in whom respiratory allergy symptoms developed when they were exposed to bread "improvers" that contained fungal alpha-amylase and cellulase were investigated by in vivo and in vitro tests. Type I hypersensitivity to these enzymes was demonstrated in the five patients by means of skin testing, histamine release test, positive reverse enzyme-immunoassay for specific IgE antibodies, and bronchial provocation test response to alpha-amylase or cellulase or both. Isolated immediate and dual responses to the bronchial challenge tests with these enzymes were observed. Immunoblot analysis with use of a pooled serum identified IgE-binding components in both enzymes. In the reverse-enzyme immunoassay-inhibition assays cross-reactivity between alpha-amylase and cellulase was not found, but some degree of cross-reactivity between alpha-amylase and A. oryzae, and between cellulase and A. niger was demonstrated. Four of the patients were also sensitized to cereal flour. Aspergillus-derived enzymes used as flour additives can elicit IgE-mediated respiratory allergy, and this fact has to be considered in the diagnosis and clinical management of bakers' asthma.


Presence of galanin in rat vagal sensory neurons: evidence from immunohistochemistry and in situ hybridization. Galanin (GAL), a 29 amino acid peptide originally isolated from the porcine upper small intestine, is widely distributed in the rat central nervous system, including the area postrema (AP) and nucleus of the solitary tract (NTS). Although vagal sensory neurons terminate in the AP/NTS, it is not known whether these neurons contain GAL in the rat. Therefore, we examined the presence and distribution of GAL in the rat nodose ganglia which contain the cell bodies of vagal sensory neurons. We used avidin-biotin-peroxidase immunohistochemistry and in situ hybridization histochemistry with a 35S-labeled oligonucleotide probe. Results with both techniques revealed the presence of GAL-containing cell bodies and fibers in the nodose ganglion. GAL-like immunoreactive cell bodies, mostly between 25 and 40 microns in diameter, were unevenly scattered throughout the nodose ganglia. The distribution and cell diameter range of GAL mRNA-labeled neurons appeared similar to those of GAL-like immunoreactive cells. These findings suggest a role for GAL in the transmission of visceral sensory information by the vagus nerve in rats.


Brain oxygen transport related to levels of fetal haemoglobin in stable preterm infants. The relative amount of regional cerebral oxygen transport was compared between different preterm infants by performing measurements of cerebral blood flow velocity, mean arterial blood pressure, whole blood viscosity and haemoglobin content for each individual. In addition the percentage of fetal haemoglobin was determined. On 25 occasions measurements of fetal haemoglobin and cerebral oxygen transport have been performed prior to and following a blood transfusion with adult red blood cells. Comparison of the data for cerebral oxygen transport suggests that the actual amount of cerebral oxygen transport is lowest at fetal haemoglobin levels below 30% and will increase progressively as soon as the percentage of fetal haemoglobin rises about 30%. Thus, at increasing fetal haemoglobin levels, cerebral haemodynamic mechanisms in the human neonate cause elevations of regional cerebral blood flow and oxygen transport. The found increase of cerebral blood flow and oxygen transport at high fetal haemoglobin levels will minimize the impeded dissociation and delivery of oxygen to brain tissues.


Corticotrophin-releasing factors in the hypothalamus of the developing fetal sheep. Corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) are secreted from the hypothalamic median eminence to elicit the secretion of ACTH from the pituitary corticotrophs. During fetal development there is progressive maturation of the hypothalamic-pituitary-adrenal axis, manifest as increasing plasma ACTH and cortisol concentrations, which in species such as sheep culminates in the onset of birth. However, the precise nature of the hypothalamic signal controlling fetal pituitary ACTH secretion remains poorly understood. To investigate the ontogeny of this hypothalamic signal, the present study examined immunoreactive and bioactive ACTH-releasing factors in the developing fetal sheep hypothalamus. Immunoreactive CRH and AVP were measured by radioimmunoassay in extracts of hypothalami taken at day 70, day 100, and day 130 gestation (term = 145 days). There was a progressive and significant (P < 0.01) increase in hypothalamic CRH and AVP concentrations which was particularly marked between d100 and d130 gestation. AVP was always present in higher concentrations that CRH, although this difference was significantly reduced by day 130 gestation as the result of a large increase in the content of CRH relative to AVP. Sephadex G50 chromatography revealed that immunoreactive CRH and AVP in hypothalamic extracts existed as single molecular forms corresponding to synthetic peptides at each gestational age. In addition, these immunoreactive forms of CRH and AVP possessed significant ACTH-releasing bioactivity as measured in primary cultures of adult sheep anterior pituitary cells. Furthermore, significant bioactivity was present in high and low molecular weight fractions eluted after chromatography which did not contain any CRH or AVP immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)


Diagnostic techniques.

Diagnostic techniques. Use of the ocular microbiology techniques described in this article when inoculating specimens greatly reduces, if not eliminates, the confusion that occurs when an organism that is part of the normal flora or that is common in the environment is found in specimens of ocular material. The growth of microorganisms can be quantified. The results of lid and conjunctival cultures can be compared with growth from the cornea or anterior chamber and vitreous fluids to provide clues to the likely significance of the isolate.


Evidence for voltage modulation of IL-2 production in mitogen-stimulated human peripheral blood lymphocytes. The membrane potential of human PBMC was modulated in culture by isotonic high extracellular K+ (K+e), or the K+ channel blocker, charybdotoxin (ChTX), to determine the effect of depolarization on stimulated proliferation, IL-2 elaboration, and gene expression. In serum-free cultures, ChTX and high [K+]e induced a specific dose-dependent decrease in IL-2 production. ChTX inhibited proliferation of PBMC and purified T cells, decreased IL-2 elaboration 15 h after stimulation by 78.4 +/- 5.3% (n = 5), and decreased IL-2 mRNA steady-state levels by 80% between 8 and 10 h after stimulation. The IC50 for ChTX-inhibition of IL-2 elaboration and IL-2 mRNA were both 1 nM. Similarly, high [K+]e inhibited proliferation with an IC50 of 38.9 +/- 1.1 mM (n = 13), decreased IL-2 elaboration with an IC50 of 21.3 +/- 1.2 mM (n = 6), and decreased IL-2 mRNA steady-state levels with an IC50 of 18 mM. The sensitivities of both IL-2 production and proliferation to depolarization were substantially reduced by calcium, serum, and exogenous rIL-2. From these findings we conclude that membrane potential may contribute to the control of immune responsiveness in vivo.


CTLA-4 and CD28 mRNA are coexpressed in most T cells after activation. Expression of CTLA-4 and CD28 mRNA does not correlate with the pattern of lymphokine production. Ag-presenting cells provide at least two distinct signals for T cell activation. T cell receptor-dependent stimulation is provided by presentation of a specific peptide Ag in association with MHC molecules. In addition, APC also supply costimulatory signals required for T cell activation that are neither Ag- nor MHC restricted. One such costimulatory signal is mediated via the interaction of B7 on APC with the CD28 receptor on T cells. Recently, CTLA-4 has been shown to be a second B7 receptor on T cells. In the present report, we have examined the expression of CD28 and CTLA-4 on a panel of resting and activated normal T cell subsets and T cell clones by RNA blot analysis in an attempt to determine whether their expression defines reciprocal or overlapping subsets. CD28 was detected in resting T cells, whereas CTLA-4 was not. After stimulation with PHA and PMA for 24 h, CTLA-4 mRNA was expressed in both the CD4+ and CD8+ subsets as well as in CD28+ T cells. We examined 37 human and six murine T cell clones that had been previously characterized for their cytokine production. After activation, CTLA-4 and CD28 mRNA were coexpressed in 36 of 37 human T cell clones and all six murine T cell clones. These included T cells of CD4+8-, CD4-8+, and CD4-8- phenotypes as well as clones with Th1 and Th2 cytokine profiles. In contrast, CD28 but not CTLA-4 mRNA was detected in leukemic T cell lines and myelomas. CTLA-4 and B7 mRNA but not CD28 mRNA was detected in two long term HTLV-I-transformed T cell lines. These data demonstrate that CD28 and CTLA-4 mRNA are coexpressed in most activated T cells and T cell clones, providing evidence that they do not define reciprocal subsets. Moreover, they are consistent with the hypothesis that B7 transmits its signal through a single receptor, CD28, on resting T cells, and multiple receptors, CD28 and CTLA-4, on activated T cells.


Murine B7 antigen provides a sufficient costimulatory signal for antigen-specific and MHC-restricted T cell activation. We have previously shown that the murine B7 (mB7) molecule, when expressed in Chinese hamster ovary cells in stable fashion, can costimulate with anti-CD3 mAb or Con A to induce T cell activation. We have now derived, by gene transfection, Chinese hamster ovary cell lines that express the I-Ad molecule, either alone or in context with mB7. We have analyzed these transfectants for their capacity to present Ag to murine CD4+ T lymphocytes. I-Ad/mB7-double transfectants were able to stimulate mixed lymphocyte reactions and to present peptide Ag to specific T cells. Chinese hamster ovary cells that expressed only the I-Ad molecule were not able to stimulate T cell proliferation in these systems. Thus, the mB7 protein is a sufficient costimulatory molecule for the physiologic, Ag-dependent/MHC-restricted activation of murine CD4+ T cells. Stimulation of T cell bulk cultures resulted predominantly in the production of IL-2 and not of IL-4. The costimulatory activity of mB7 is not, however, restricted to the IL-2-secreting subset. We have identified one IL-4-secreting T cell clone, CDC35, which is responsive to mB7 triggering. Finally, we present experiments that suggest that mB7 and peptide/MHC complexes need to be expressed on the same cell for optimal induction of T cell activation.


Bromelain treatment of human T cells removes CD44, CD45RA, E2/MIC2, CD6, CD7, CD8, and Leu 8/LAM1 surface molecules and markedly enhances CD2-mediated T cell activation. Treatment of T cells with the cysteine protease bromelain has been widely used to enhance the binding of human T cells to human E (autologous E rosettes) and has been shown to remove surface T cell CD44 molecules. Ligand binding to CD44 has been shown to markedly augment T cell activation. To study the activation potential of bromelain-treated CD44 T cells, we have compared the proliferation of sham- and bromelain-treated normal human PBMC to mitogenic CD2 mAb. We found that bromelain not only removed T cell CD44, but also removed the CD45RA isoform of CD45 as well as E2/MIC2, CD6, CD7, CD8, and Leu 8/LAM1 molecules. T cell proliferation in response to CD2 mAb was increased 325% in bromelain-treated PBMC compared to sham-treated PBMC (p < 0.005). Reciprocal treatment experiments using purified T cells and monocytes demonstrated that the enhancement of T cell CD2 activation by bromelain occurred only when T cells were treated with bromelain and was accompanied by increased adhesion of T cells to monocytes. These data demonstrate that expression of portions of the extracellular domains of the CD44, CD45RA, E2/MIC2, CD6, CD7, CD8, and Leu 8/LAM1 surface molecules are not required for CD2 activation of human T cells. Rather, the removal of these surface molecules by bromelain is associated with enhanced T cell-monocyte aggregation and enhanced CD2-mediated T cell activation. Taken together with data that CD44, E2/MIC2, CD6, and CD7 mAb inhibit CD2/lymphocyte function-associated Ag-3-mediated cellular interactions and also augment CD2-mediated triggering of T cells, these data suggest that members of the bromelain-sensitive group of surface molecules may comprise a set of CD2-associated adhesion ligands that acts in concert to modulate human T cell activation.


Molecular interactions mediating T-B lymphocyte collaboration in human lymphoid follicles. Roles of T cell-B-cell-activating molecule (5c8 antigen) and CD40 in contact-dependent help. In lymphoid follicles, CD4+ T lymphocytes provide contact-dependent stimuli to B cells that are critical for the generation of specific antibody responses in a process termed Th function. The CD4+ T cell-restricted surface activation protein, 5c8 Ag (T-BAM), has recently been shown to be a component of the contact-dependent helper signal to B cells. To further dissect this process, we utilized a Jurkat T cell lymphoma clone, termed D1.1, that constitutively expresses T-BAM and activates peripheral B cells to express surface CD23 in a contact-dependent mechanism that is inhibited by mAb anti-T-BAM (5c8). Similar to its effect on peripheral B cells, Jurkat D1.1 activates B cells from lymphoid organs, as well as a B cell lymphoma clone, RAMOS 266,4CN 3F10 (RAMOS 266), to up-regulate surface CD23. Interestingly, mAb to the B cell surface molecule, CD40 (mAb G28-5 and B-B20), inhibit D1.1 induced activation of RAMOS 266 and peripheral and lymphoid B cells. In contrast, mAb to CR2 or the adhesion molecules, LFA1, LFA3, or ICAM-1, have little effect. The inhibitory effect of anti-CD40 mAb on B cell activation induced by D1.1 is specific because anti-CD40 potentiates, rather than inhibits, the up-regulation of CD23 on B cells induced by rIL-4. Moreover, cross-linking CD40 molecules by anti-CD40 mAb bound to Fc gamma RII+ (CD32) L cells induces B cell CD23 expression. In vivo, T-BAM-expressing cells are CD4+ T cells that are restricted to lymphoid organs and are localized in the mantle and centrocytic zones of lymphoid follicles and the spleen periarteriolar lymphoid sheath in association with CD40+ B cells. Taken together, these data demonstrate that T-BAM on T cells and CD40 on B cells are involved in contact-dependent T-B help interactions that occur in lymphoid follicles.


Role of antigen-specific T cell help in the generation of in vivo antibody responses. I. Antigen-specific T cell help is required to generate a polyclonal IgG1 response in anti-IgD antibody-injected mice. A system in which injection of mice with an antibody to mouse IgD that they recognize as foreign stimulates a large, T cell-dependent IgG response was used to study whether Ag-specific T cell help is required to stimulate polyclonal (non-Ag-specific) IgG production in vivo. Igha x Ighb allotype heterozygous mice were injected with a conjugate of a foreign Ag coupled to a mAb specific for one of the two IgD allotypes expressed in these mice. This conjugate cross-links mIgD on B cells that express the recognized allotype. These cells process the conjugate and present the foreign Ag to Ag-specific T lymphocytes, which become activated. Thus, B cells of the recognized allotype can be stimulated by cross-linking of their mIgD, Ag-specific T cell help, non-Ag-specific cytokines, and non-Ag-specific contact with activated T cells. In contrast, B cells that express the Igh allotype not recognized by the Ag-anti-IgD antibody conjugate (bystander B cells) can be stimulated in this system only by non-Ag-specific cytokines and non-Ag-specific contact with activated T cells. Although both recognized and bystander B cells in conjugate-injected mice demonstrated substantial increases in size and Ia expression, only the recognized B cells were induced to synthesize DNA and to make a substantial polyclonal Ig response. Bystander B cells still failed to secrete IgG when mice were injected with an anti-IgD-Ag conjugate specific for the other Igh allotype as well as a mAb that cross-linked IgD of the bystander B cell allotype. These observations demonstrate that although non-Ag-specific cytokine and contact-mediated T cell help are sufficient to induce B cells to increase in size and Ia expression in anti-IgD antibody-injected mice, Ag-specific T cell help is required to stimulate the generation of an IgG response in these mice.


Role of antigen-specific T cell help in the generation of in vivo antibody responses. II. Sustained antigen-specific T cell help is required to induce a specific antibody response. The injection of mice with a goat or rabbit antibody to mouse IgD stimulates a large polyclonal IgG response, approximately 10% of which is specific for antigenic determinants on the anti-IgD antibody molecule. The large goat IgG (GIgG)-specific antibody response in mice injected with goat antibody to mouse IgD requires that GIgG-specific B cells undergo much greater clonal expansion than B cells specific for other Ag. One possible explanation for the greater clonal expansion of GIgG-specific B cells is that B cells that lack GIgG specificity can only be stimulated with GIgG-specific T help during the relatively short time that anti-IgD binds to, and is processed and presented by, these B cells before they cease to express membrane mIgD. In contrast, GIgG-specific B cells can continue to bind, process, and present GIgG through mIgM after they lose mIgD. To test the hypothesis that extended stimulation with Ag-specific T help is required to generate a specific antibody response, we determined time requirements for Ag-specific T cell help for the development of such a response. Mice were injected with rabbit antibody to mouse IgD plus one or more daily injections of FITC conjugated to a F(ab')2 fragment of rabbit IgG (FITC-(Fab')2), which has a short in vivo half-life, and IgG1 anti-FITC antibody production was analyzed. In this system, each additional injection of FITC-F(ab')2 extends the period during which FITC-specific B cells can process this Ag and present it to rabbit IgG-specific T cells. Each additional injection of FITC-F(ab')2 stimulated a several-fold increase in IgG1 anti-FITC antibody levels, and injections on 5 consecutive days were required to induce a maximal anti-FITC response. These observations provide evidence that sustained Ag-specific T cell help is required to stimulate the degree of B cell clonal expansion that characterizes a specific antibody response.


CD5+ B cells predominate in peripheral tissues of rabbit. Restricted usage of VH genes is observed in rabbit B lymphocytes and in human and murine CD5 B lymphocytes. This observation raised the possibility that most rabbit B lymphocytes were CD5+. To investigate this we cloned the CD5 gene from a rabbit cosmid library, using a probe derived from human CD5 cDNA. The rabbit CD5 gene was transfected into a murine T cell line and then we used the transfectants to develop anti-rabbit CD5 mAb. By Western blot analysis, the mAb reacted with a 67-kDa protein in lysates prepared from mesenteric lymph node and spleen cells. We determined the frequency of CD5+ B lymphocytes in peripheral lymphoid tissues of adult rabbits by two-color immunofluorescence analysis using anti-CD5 mAb and anti-L chain antibodies. The analysis showed that essentially all peripheral B lymphocytes in adult rabbits express CD5. The observation that CD5 is expressed on nearly all rabbit B lymphocytes contrasts markedly to mouse and human, where only a small number of B lymphocytes express CD5. We propose that most peripheral B lymphocytes in rabbit, as in chicken, develop early in ontogeny and are maintained throughout life by a self-renewing process.


Expression of surface lymphotoxin and tumor necrosis factor on activated T, B, and natural killer cells. The expression of membrane-associated forms of lymphotoxin (LT) and TNF were examined on cell lines of T, B, and myeloid origin, IL-2 dependent T cell clones, and peripheral blood lymphocytes. Inducible and constitutive patterns of surface LT expression were found on T cells as exemplified by the II-23.D7, a CD4+T cell hybridoma, and HUT-78, a T cell lymphoma. Phorbol ester induced surface LT expression on Ramos, an EBV transformed B cell line, but at a slower rate of appearance when compared to the II-23.D7. Secretion of LT was rapidly inducible by phorbol ester in II-23.D7 and also in HUT-78 but with slower kinetics; surface LT expression continued in both lines after secretion had ceased. Low levels of membrane TNF were transiently induced on II-23.D7 and HUT-78, but none was observed on Ramos. Peripheral blood monocytes and some myeloid tumor lines did not express surface LT. Several T cell clones expressed surface LT after Ag-specific stimulation, and expression persisted several days. Stimulation through the TCR or by IL-2 rapidly induced surface LT on resting peripheral T cells and CD56+ NK cells; pokeweed mitogen activation induced expression on CD20+ B cells. Consistent with previous results, immunoprecipitation with anti-LT mAb showed that LT was complexed with a distinct 33 kDa glycoprotein (p33) on cells that expressed surface LT, whereas secreted LT was not associated with p33. Surface and secreted modes of LT expression by activated T, B, and NK cells suggests that LT can be utilized as either a localized or diffusible mediator in immune responses.


Genomic structure and chromosomal mapping of the murine CD40 gene. The B cell-associated surface molecule, CD40, is likely to play a central role in the expansion of Ag-stimulated B cells, and their interaction with activated Th cells. In our study we have isolated genomic clones of murine CD40 from a mouse liver genomic DNA library. Comparison with the murine CD40 cDNA sequence revealed the presence of nine exons that together contain the entire murine CD40 coding region, and span approximately 16.3 kb of genomic DNA. The intron/exon structure of the CD40 gene resembles that of the low affinity nerve growth factor receptor gene, a close homolog of both human and murine CD40. In both cases the functional domains of the receptor molecules are separated onto different exons throughout the genes. Southern blot analysis demonstrated that murine CD40 is a single copy gene that maps in the distal region of mouse chromosome 2.


Deletion mapping of Ig VH gene segments expressed in human CD5 B cell lines. JH proximity is not the sole determinant of the restricted fetal VH gene repertoire. VH gene segments expressed in a panel of monoclonal human CD5 B cell lines have been positioned on the IgH locus by deletion mapping. The analysis yielded a relative order of VH fragments of the VH2, VH4, VH5, and VH6 gene families that was consistent with, and provided a further refinement of existing maps of the human IgH locus. We demonstrate that four of six VH gene segments expressed in the CD5 B cell lines map > 500 kb from the cluster of JH segments. Two of the gene segments, positioned at approximately 850 kb (58p2) and approximately 500 kb (1-9III) from the JH segments, respectively, belong to the previously identified small cohort of second trimester fetal VH gene segments. The data show that JH proximity is not the sole determinant of restricted VH gene utilization in early human ontogeny.


Highly diverse T cell recognition of a single Plasmodium berghei peptide presented by a series of mutant H-2Kd molecules. We have tested 21 independent CTL clones for recognition of a single peptide derived from the Plasmodium berghei circumsporozoite protein in the context of 13 mutants of the murine MHC class I molecule H-2Kd. In this series of Kd mutants, amino acid residues located on the upper surface of the alpha-helices were individually substituted by alanine. Remarkably, most clones displayed individual recognition patterns on the Kd mutants. We had previously found that this series of CTL clones was likewise highly diverse in terms of both TCR primary structure and peptide fine specificity. Our data thus reinforce the concept that multiple T cell epitopes are available on the surface of a single peptide-MHC class I complex for recognition by specific TCR.


T and B cell epitope mapping of SM23, an integral membrane protein of Schistosoma mansoni. SM23 is an integral membrane protein of the blood-vessel dwelling parasitic worm Schistosoma mansoni. This protein has been detected with antibodies in all stages of the parasite found in the human host, notably the lung stage, and therefore is of interest as a vaccine candidate. In addition SM23 has been shown to be a member of a proposed new superfamily of membrane proteins whose structures do not conform to the previously known classifications. To date there are 13 members including ME491 (CD63, Pltgp40), CD9 (p23), TAPA-1, CD37, CD53, MRC OX-44, CO-029, MRP-1, L6, the gene product of TI-1, the target of mAb AD-1, SM23, and SJ23 (the Schistosoma japonicum homologue). Most of these molecules except for those in the two blood vessel-dwelling parasites are found in membranes of hemopoietic and/or malignant cells and all have unknown function. In this study we used recombinantly expressed full-length and partial molecules as well as synthesized peptides to map T cell and B cell epitopes of SM23. The two predicted external hydrophilic domains were found to be highly immunogenic and contained several B cell epitopes. There were at least four T cell epitopes in the large hydrophilic domain. One segment of 23 amino acids contained both a T cell and B cell epitope as well as the putative glycosylation site. This particular segment was recognized by immune sera and cells of every mouse strain tested. The elucidation of these epitopes demonstrates the immunogenic nature of this molecule and raises questions as to the role of SM23 in the host/parasite relationship.


Role of colony-stimulating factor-1 in macrophage activation in tumor-bearing mice. We previously reported a dramatically increased number of macrophages in tumor-bearing mice. In this study, we investigated the involvement of CSF in that phenomenon. CSF-1 responding cells as macrophages precursors increased significantly in number in the spleens of tumor-bearing mice as compared with those in normal mice. Splenic cells and sera from the tumor-bearing mice respectively expressed CSF-1 in mRNA and serum protein levels, but failed to express the other CSF (granulocyte-macrophage-CSF or IL-3). Nonadherent splenic mononuclear cells (< 0.5% macrophages) from normal mice proliferated and differentiated into mature macrophages in culture within 7 days with recombinant mouse CSF-1 (rCSF-1). Both macrophages harvested from tumor-bearing mice and those activated in vitro with rCSF-1 expressed mostly Mac-1, -2 (and -3) Ag, showed yeast phagocytosis, produced IL-1 but not IL-2 or IL-3, and displayed potent cytotoxicity against NK cell resistant Meth-A tumor cells. These macrophages also expressed lipocortin I mRNA and secreted lipocortin I protein, and suppressed mitogenic responses of splenic lymphocytes. rCSF-1-activated macrophages derived from nonadherent splenic cells expressed both CSF-1 and CSF-1 receptor (c-fms) mRNA. Administration of rCSF-1 into normal mice induced hemopoietic and immunologic alternations similar to those observed in tumor-bearing mice. These results suggest that CSF-1 is involved in the dramatic increase of macrophages in tumor-bearing mice, possibly through an autocrine or paracrine loop.


Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells. One of the key features associated with programmed cell death in many tissues is the phagocytosis of apoptotic bodies by macrophages. Removal of apoptotic cells occurs before their lysis, indicating that these cells, during the development of apoptosis, express specific surface changes recognized by macrophages. We have compared the mechanisms by which four different macrophage populations recognize apoptotic cells. Murine macrophages elicited into the peritoneal cavity with either of two different phlogistic agents were able to phagocytose apoptotic cells. This phagocytosis was inhibited by phosphatidylserine (PS), regardless of the species (human or murine) or type (lymphocyte or neutrophil) of the apoptotic cell. In contrast, the murine bone marrow macrophage, like the human monocyte-derived macrophage, utilized the vitronectin receptor, an alpha v beta 3 integrin, for the removal of apoptotic cells, regardless of their species or type. That human macrophages are capable, under some circumstances, of recognizing PS on apoptotic cells was suggested by the observation that PS liposomes inhibited phagocytosis by phorbol ester-treated THP-1 cells. These results suggest that the mechanism by which apoptotic cells are recognized and phagocytosed by macrophages is determined by the subpopulation of macrophages studied.


IFN enhance expression of Sp100, an autoantigen in primary biliary cirrhosis. About 30% of patients suffering from the chronic autoimmune liver disease primary biliary cirrhosis produce autoantibodies against Sp100, a protein migrating in SDS-PAGE at a position corresponding to 100 kDa and located on discrete dot-shaped nuclear structures. The human Sp100 cDNA has recently been cloned and the deduced amino acid sequence was found to contain similarities to several transcriptional regulatory proteins; the biologic function of the Sp100 protein, however, is still unknown. In this study we present data which show that infection of HEp2 cells with influenza A virus, transformation of glial cells with SV40 DNA, and stimulation of PBL with mitogens affect the expression of the Sp100 autoantigen. These observations prompted us to investigate whether expression of the Sp100 protein is modulated by the action of IFN. Immunofluorescence staining of HEp2 and HeLa cells grown in the presence of IFN-alpha, IFN-beta, or IFN-gamma revealed an increase both in size and number of the Sp100 protein-containing nuclear dots, whereas no such effect was observed with cells treated with TNF-alpha. As measured by an immunoblot-based ELISA the amount of Sp100 protein in INF-beta-treated cells (1000 IU/ml, 18 h) was eight to nine times higher than in untreated cells. The enhanced protein expression was accompanied by an accumulation of the Sp100-specific mRNA (13-fold increase of the normal level after 10 h of INF-beta treatment of HEp2 cells). These findings characterize the Sp100 protein as a new member of IFN-modulated proteins and raise the question whether cytokine-mediated increase of Sp100 protein expression plays a role in induction of anti-Sp100 autoantibodies.


Human bone marrow stromal cell lines from myeloma and rheumatoid arthritis that can support murine pre-B cell growth. In order to elucidate the pathologic significance of the bone marrow (BM) microenvironment in multiple myeloma (MM) and rheumatoid arthritis (RA), we established patient- or healthy donor (HD)-derived BM stromal cell lines by transfecting the plasmid for expression of SV40 large T Ag and examined their ability to support the stromal cell-dependent growth of a pre-B cell line, DW34. The means of recovered cell numbers of DW34 co-cultured with MM- and RA-derived BM stromal cell lines ranged from 6- to 10-fold more than those with HD-derived ones. Their enhanced ability to support DW34 cell growth was not caused by cytokines, including IL-6, IL-7, and c-kit ligand, although exogenous IL-7 could augment the growth-supporting ability. DW34 cell growth on the stromal cell lines was abolished by inhibiting cell-to-cell interaction with a membrane filter. FACS analysis revealed that the stromal cell lines did not express LFA-1 alpha, beta, NCAM, or ELAM-1. Both patient and HD BM stromal cell lines variably expressed ICAM-1, VCAM-1, and CD44. However, surface expression levels of these molecules did not correlate with the ability of the stromal cell lines to support DW34 cell growth. Taken together, these results suggested that BM microenvironment might play important roles in the pathogenesis of MM and RA.


CD4 activation of HIV fusion.

CD4 activation of HIV fusion. The primary cellular receptor for the human immunodeficiency viruses type 1 (HIV-1) and type 2 (HIV-2) is the CD4 antigen. HIV infection of CD4+ cells is initiated by binding of the virus to the cell surface, via a high affinity interaction between CD4 and the HIV outer envelope glycoprotein, gp120. The development of model systems using soluble recombinant forms of CD4 (sCD4) has allowed kinetic and thermodynamic analyses of CD4 binding to gp120, and study of the post-binding events leading to virus-cell membrane fusion. It has thus been demonstrated that the affinity of sCD4 for gp120 on virions or HIV-infected cells depends on both the primary sequence and the tertiary structure of gp120 in the membrane. With cell-line adapted isolates of HIV-1, sCD4 binding induces conformational changes in gp120, leading to the complete dissociation of gp120 from the transmembrane glycoprotein, gp41, and exposing cryptic epitopes of gp41. Similar observations have been made with cell-anchored CD4; exposure of cryptic gp41 epitopes occurs at the fusion interface between clusters of CD4-expressing and HIV-infected cells. Thus, for HIV-1, CD4 induces exposure of fusogenic components of gp41 which triggers virus-cell membrane coalescence. This is termed receptor-mediated activation of fusion. With primary isolates of HIV-1 and the related lentiviruses, HIV-2 and simian immunodeficiency virus (SIV), the CD4-induced molecular rearrangements in gp120 are more subtle, implying that there is a spectrum of responses to sCD4 binding. The high-affinity binding site on CD4 for gp120 is necessary and probably sufficient for activation of HIV fusion, although other regions of CD4 may indirectly influence viral entry. There are two regions on the envelope glycoproteins which are recognized as playing a role in HIV entry: the N-terminus of gp41 and the gp120 V3 loop. The roles of these domains are discussed.


Histochemical and immunocytochemical characterizations of laminated bodies in the pancreas acinar lumen. Combined adrenalectomy and castration cause numerous morphological alterations in the exocrine pancreas of the rat. Accumulation of laminated bodies (LB) in the acinar lumen is one of these alterations. A series of classical histological stains was applied to identify the components of these structures. A positive reaction was observed with periodic acid-Schiff, Congo red, and Alcian blue (pH 2.5). The first stain reacts with neutral and some acid mucosubstances and the second with amyloid, whereas the last one reacts with sulfated and nonsulfated acid mucosubstances. The LB also responded to Luxol fast blue, indicating the presence of lipids, an observation that is in agreement with the osmiophilic properties of these structures. A more specific identification of LB components was carried out with the immunocytochemical protein A-gold technique. Presence of gamma-glutamyltranspeptidase (gamma-GT) and GP2, two glycoproteins known to be secreted by the pancreas, was tested. The gamma-GT was associated with LB whereas GP2 was found in the lumen but not associated with these structures. Amylase was undetectable when LB occupied the lumen, suggesting that the process leading to production of LB also blocks secretory activity. To determine if diet influences LB accumulation in the pancreas acinar lumen, their frequency was compared in rats fed Purina Lab Chow or a lipid-free synthetic diet. A significant increase was observed in castrated-adrenalectomized rats fed the latter diet. This increase corresponded to a pronounced reduction in the number of zymogen granules (ZG) in the acinar cell. Our results show that LB are made of lipids, neutral mucosubstances, and nonsulfated acid mucosubstances, and that hormonal (steroids) and dietary factors (lipids) influence their accumulation.


Protective effect of nafamostat mesilate on cellular and lysosomal fragility of acinar cells in rat cerulein pancreatitis. This in vivo and in vitro study demonstrates the protective effects of a new synthetic protease inhibitor--nafamostat mesilate, FUT-175--on increased cellular and lysosomal fragility within acinar cells during the early stage of cerulein-induced acute pancreatitis in rats. FUT-175 prevented hyperamylasemia, pancreatic edema, congestion owing to amylase, and lactic dehydrogenase (LDH) discharge from acini as well as cathepsin-B leakage from lysosomes dose-dependently in doses of 1-10 mg/kg.h. These results suggest that FUT-175 can protect against pancreatitis at subcellular levels in lysosomes and cellular or organelle membranes. Proteases may well play the important role in the pathogenesis of acute pancreatitis, and such a low molecular protease inhibitor may be useful clinically in the treatment of acute pancreatitis.


Haemodilution in patients with peripheral arterial occlusive disease. Haemodilution is an efficient conservative therapy of peripheral arterial occlusive disease. Already a single isovolaemic haemodilution (replacement of 500 ml blood for Haes* 0.5, 10%) increases the pain-free walking distance by 85%. These effects can be maintained by a constant therapy over six weeks and following haemodilution once or twice per month. The haematocrit values should be between 38 and 42%. The haemodilution should be done hyper- or isovolaemically. Not more than 250 ml blood and 500 ml Haes should be infused during one session in order to avoid hypovolaemia. This means an infusion of 250 ml Haes, venesection of 250 ml blood via the same access and then infusion of the remaining 250 ml. The whole procedure should not last more than one hour. Blood pressure, heart rate, lung auscultation and percussion as well as creatinine values has to be controlled during an intensive therapy. If the hydroxyethyl starch concentration exceeds 150 g per week pruritus may occur in singular cases, if the concentration exceeds 700 g per week it is observed in 50% of the cases. Provided the preventive measures are observed haemodilution is an efficient and good therapy which also increases the compliance to practice vascular exercise.


RANTES and macrophage inflammatory protein 1 alpha induce the migration and activation of normal human eosinophil granulocytes. The cellular infiltrates of certain inflammatory processes found in parasitic infection or in allergic diseases consist predominantly of eosinophilic granulocytes, often in association with activated T cells. This suggests the existence of chemotactic agonists specific for eosinophils and lymphocyte subsets devoid of neutrophil-activating properties. We therefore examined four members of the intercrine/chemokine superfamily of cytokines (monocyte chemotactic peptide 1 [MCP-1], RANTES, macrophage inflammatory protein 1 alpha [MIP-1 alpha], and MIP-1 beta), which do not activate neutrophils, for their ability to affect different eosinophil effector functions. RANTES strongly attracted normal human eosinophils by a chemotactic rather than a chemokinetic mechanism with a similar efficacy as the most potent chemotactic myeloid cell agonist, C5a. MIP-1 alpha also induced eosinophil migration, however, with lower efficacy. RANTES and MIP-1 alpha induced eosinophil cationic protein release in cytochalasin B-treated eosinophils, but did not promote leukotriene C4 formation by eosinophils, even after preincubation with interleukin 3 (IL-3), in contrast to other chemotactic agonists such as C5a and formyl-methionyl-leucyl-phenylalanine (FMLP). RANTES, but not MIP-1 alpha, induced a biphasic chemiluminescence response, however, of lower magnitude than C5a. RANTES and MIP-1 alpha both promoted identical transient changes in intracellular free calcium concentration ([Ca2+]i), with kinetics similar to those induced by chemotactic peptides known to interact with G protein-coupled receptors. No cross-desensitization towards other peptide agonists (e.g., C5a, IL-8, FMLP) was observed, suggesting the presence of specific receptors. Despite its weaker eosinophil-activating properties, MIP-1 alpha was at least 10 times more potent on a molar basis than RANTES at inducing [Ca2+]i changes. Interestingly, RANTES deactivated the MIP-1 alpha-induced [Ca2+]i changes, while the RANTES response was preserved after MIP-1 alpha stimulation. MCP-1, a potent monocyte chemoattractant and basophil agonist, as well as MIP-1 beta, a peptide with pronounced homology to MIP-1 alpha, did not activate the eosinophil functions tested. Our results indicate that RANTES and MIP-1 alpha are crucial mediators of inflammatory processes in which eosinophils predominate.


Thymus reconstitution by c-kit-expressing hematopoietic stem cells purified from adult mouse bone marrow. The introduction of clonal assays and long-term culture systems has resulted in considerable progress in the understanding of the early events that control self-renewal and commitment to differentiation of pluripotent hematopoietic stem cells (PHSC). Relatively little is known about the factors that control the commitment of PHSC to the lymphoid lineages, especially the T cell lineage. In the present study, the expression of the proto-oncogene c-kit was used to isolate and study the capacity of highly purified day 14 colony-forming units-spleen (CFU-S) to reconstitute the thymus of sublethally irradiated Thy-1 congenic recipient mice. We demonstrate here that one c-kit positive (c-kitpos) stem cell upon intrathymic transfer can effectively reconstitute the thymus of a sublethally irradiated recipient. After a lag phase of 15 d, high levels of donor-derived thymocytes (Thy-1.1pos) could be detected until 65 d after transplantation in Thy-1.2pos host mice. Donor-derived cells were only detected in the lobe of the thymus in which cells were previously injected and not in the noninjected lobe. These data suggest that c-kitpos stem cells do not migrate from one lobe to another and that they do not re-seed the thymus after having migrated to the bone marrow. The level and duration of reconstitution was found to be cell dose dependent, suggesting that, over time, endogenous stem cells compete with donor stem cells for available sites in the thymus microenvironment. The data presented in this paper demonstrate that commitment of purified adult bone marrow-derived c-kitpos stem cells to the T cell differentiation pathway can occur in the thymus and does not have to happen in the bone marrow.


Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion. Signaling through the cell surface molecule, CD40, is known to play an important role in the proliferation and differentiation of B lymphocytes. Using the thymoma cell line EL4, we recently identified and cloned a cDNA encoding a murine ligand for the CD40 molecule (mCD40-L) and showed that it has biological activity in vitro. A cDNA encoding a human homologue of the mCD40-L was isolated using crosshybridization techniques from an activated peripheral blood T cell library. The predicted amino acid sequence indicates that this human ligand for CD40 (hCD40-L) is a 261 amino acid type II membrane protein that exhibits 78% amino acid identity with its murine counterpart. Northern blot and FACS analyses suggest that the hCD40-L is restricted in its expression to T lymphocytes, and that it is most abundant on the CD4+ T cell subpopulation. Cells transfected with hCD40-L caused the proliferation of human tonsil B cells in the absence of costimuli and, in the presence of interleukin 4, induced immunoglobulin E secretion from purified human B cells. A comparison of the efficacy of the hCD40-L and mCD40-L in these assays is presented.


The contribution of increased collagen synthesis to human glomerulosclerosis: a quantitative analysis of alpha 2IV collagen mRNA expression by competitive polymerase chain reaction. We previously reported that one of the main components of the sclerotic material in human glomerular diseases was type IV collagen. In this study we examined the contribution of increased synthesis to this process at the gene expression level. Sufficient material has not been available to study type IV collagen synthesis by normal or sclerotic glomeruli in humans. We took advantage of the availability of nephrectomy specimens from patients with renal carcinoma, and of the observation that approximately 50% of these patients develop varying degrees of glomerulosclerosis. We microdissected glomeruli from 10 patients and analyzed them using in situ reverse transcription coupled with polymerase chain reaction (PCR) analyses (in situ RT-PCR). alpha 2IV collagen mRNA, after reverse transcription into cDNA, was detected in all patients and appeared to be increased in those with glomerulosclerosis (n = 5). A competitive PCR assay was developed to quantitate this change. There was an average 3.7-fold increase in glomerular type IV collagen cDNA in patients with significant sclerosis. This change was not due to an increased number of glomerular cells. Thus, glomerulosclerosis in humans is associated with an elevation of glomerular type IV collagen gene expression, suggesting that increased synthesis of type IV collagen may represent one component of this process.


Functional analysis of the human vascular cell adhesion molecule 1 promoter. The vascular cell adhesion molecule 1 (VCAM-1) is a 110-kD member of the immunoglobulin gene superfamily expressed on the surface of interleukin 1 beta- or tumor necrosis factor alpha (TNF)-stimulated endothelial cells. The cell surface protein functions as an inducible adhesion receptor for circulating mononuclear leukocytes and some tumor cells. We have previously characterized the genomic organization of the VCAM1 gene and described its chromosomal localization. In this report, the promoter of the VCAM1 gene is characterized. New transcription of the VCAM1 gene occurred when endothelial cells were treated with TNF. Fusion plasmids containing the 5' flanking sequence of the VCAM1 gene and the chloramphenicol acetyltransferase reporter gene were used to identify cis-acting sequences that direct the cytokine-induced transcription. When transfected into bovine aortic endothelial cells, constructs containing 755 bp of the 5' flanking sequence were induced by TNF. Within the cytokine-responsive region of the core promoter were functional NF-kappa B and GATA elements. Upstream of the core promoter, the VCAM1 5' flanking sequence contained a negative regulatory activity. NF-kappa B-mediated activation of VCAM1 gene expression may lead to endothelial expression of a mononuclear leukocyte adhesion molecule associated with initial events in the development of an atherosclerotic lesion.


Major histocompatibility complex conformational epitopes are peptide specific. Serologically distinct forms of H-2Kb are stabilized by loading cells expressing "empty" class I major histocompatibility complex (MHC) molecules with different H-2Kb binding peptides. The H-2Kb epitope recognized by monoclonal antibody (mAb) 28.8.6 was stabilized by ovalbumin (OVA) (257-264) and murine cytomegalovirus (MCMV) pp89 (168-176) peptides, but not by vesicular stomatic virus nucleoprotein (VSV NP) (52-59) and influenza NP (Y345-360) peptides. The H-2Kb epitope recognized by mAb 34.4.20 was stabilized by VSV NP (52-59) peptide but not by OVA (257-264), MCMV pp89 (168-176), or influenza NP (Y345-360) peptides. Immunoprecipitation of H-2Kb molecules from normal cells showed that 28.8.6 and 34.4.20 epitopes were only present on a subset of all conformationally reactive H-2Kb molecules. Using alanine-substituted derivatives of the VSV peptide, the 28.8.6 epitope was completely stabilized by substitution of the first residue and partially stabilized by substitution of the third or the fifth residues in the peptides. These results indicate that distinct conformational MHC epitopes are dependent on the specific peptide that occupies the antigenic peptide binding groove on individual MHC molecules. The changes in MHC epitopes observed may also be important in understanding the diversity of T cell receptors used in an immune response and the influence of peptides on development of the T cell repertoire.


The Yersinia tyrosine phosphatase: specificity of a bacterial virulence determinant for phosphoproteins in the J774A.1 macrophage. YopH is a plasmid-encoded protein tyrosine phosphatase (PTPase) secreted by pathogenic Yersinia. Although the enzyme likely acts to dephosphorylate eukaryotic proteins during Yersinia infection of the mammalian host, the targets of YopH have not been identified. We infected the murine macrophage-like cell line J774A.1 with Yersinia pseudotuberculosis and investigated the specificity of YopH and YopHC403A, a catalytically inactive mutant derivative, for eukaryotic phosphoproteins. Upon infection, YopH specifically and rapidly dephosphorylated a macrophage protein of 120 kD. The 120-kD protein and a previously detected 55-kD substrate of YopH coprecipitated with YopHC403A. Coprecipitation of these proteins required tyrosine phosphorylation and could be competitively inhibited with excess phosphotyrosine. The 120- and 55-kD proteins that coprecipitate with YopHC403A exhibited the in vitro activity of protein tyrosine kinases (PTKases), suggesting that YopH dephosphorylates activated tyrosine kinases in vivo.