Reciprocal connections between the medial preoptic area and the midbrain periaqueductal gray in rat: a WGA-HRP and PHA-L study. The midbrain periaqueductal gray (PAG) participates in diverse functions such as analgesia, autonomic regulation, sexual behavior, and defense/escape responses. Anatomical studies of the circuits involved in such functions have largely focused on the connections of PAG with the medulla. Projections to PAG from forebrain structures are extensive, but their organization has received little attention. Previous anatomic studies indicate that the medial preoptic area (MPO), involved in a variety of physiological and behavioral functions, is a major source of afferent input to the periaqueductal gray. Here, we have examined the topography of reciprocal connections between these two structures in the rat by using wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) and Phaseolus vulgaris leucoagglutinin (PHA-L). Multiple WGA-HRP injections at several rostrocaudal levels of PAG retrogradely labeled large numbers of neurons in the medial preoptic area; labeled cells were primarily located in the medial preoptic nucleus, the median preoptic nucleus, and the region lateral to the medial preoptic nucleus. The distribution of labeled cells shifted medially to laterally along the rostral to caudal axis of the medial preoptic area. Rostrally, there was selective retrograde labeling in the central and lateral divisions of medial preoptic nucleus, whereas caudally, labeled cells were primarily located only in the lateral subdivision of medial preoptic nucleus. Tracer injections in PAG also produced strong anterograde labeling in MPO. WGA-HRP and PHA-L injections in the medial preoptic area resulted in dense anterograde labeling along the entire rostrocaudal axis of PAG. The terminal labeling in PAG from the medial preoptic area was not uniformly distributed throughout PAG, however. Instead, this projection formed one or two rostrocaudally oriented longitudinal columns that terminated in different subregions of PAG along the entire rostrocaudal axis of this structure. Rostrally, inputs from the medial preoptic area project heavily to dorsomedial PAG, and at mid-PAG levels, the projection becomes distinctly bipartite with two discrete longitudinal terminal columns in dorsomedial and lateral PAG; caudally, the heaviest labeling is in ventrolateral PAG. The projection also exhibited a central to peripheral (radial) gradient; labelled fibers and terminals were heaviest near the aqueduct and much lower in the peripheral parts of PAG. WGA-HRP injections in MPO also produced retrograde labeling of neurons at all rostrocaudal levels of PAG; more neurons were labeled in the rostral than the caudal half of PAG. The majority of labeled cells were located in dorsomedial and ventral/ventrolateral parts of PAG; only a few neurons in the dorsal raphe region appear to project to MPO.(ABSTRACT TRUNCATED AT 400 WORDS)


Organization of medullary adrenergic and noradrenergic projections to the periaqueductal gray matter in the rat. The periaqueductal or midbrain central gray matter (CG) in the rat contains a dense network of adrenergic and noradrenergic fibers. We examined the origin of this innervation by using retrograde and anterograde axonal tracers combined with immunohistochemistry for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT). Following injections of the fluorescent tracers Fast Blue or Fluorogold into the CG, double-labeled neurons in the medulla were identified mainly in the noradrenergic A1 group in the caudal ventrolateral medulla (VLM) and A2 group in the medial part of the nucleus of the solitary tract (NTS); and in the adrenergic C1 group in the rostral ventrolateral medulla and C3 group in the rostral dorsomedial medulla. Injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into these cell groups resulted in a distinct pattern of axonal labeling in various subdivisions of the CG. Anterogradely labeled fibers originating in the medial NTS were predominantly found in the lateral portion of the dorsal raphe nucleus and in the adjacent part of the lateroventral CG (CGlv). Following PHA-L injections into the C3 region the anterogradely labeled fibers were diffusely distributed in the CGlv and the dorsal raphe nucleus at caudal levels, but rostrally tended to be located laterally in the CGlv. In contrast, ascending fibers from the caudal and rostral VLM terminated in the rostral dorsal part of the CGlv and in the dorsal nucleus of the CG, whereas ventral parts of the CG, including the dorsal raphe nucleus, contained few afferent fibers. Double-label studies with antisera against DBH and PNMT confirmed that noradrenergic neurons in the A1 and A2 groups and adrenergic neurons in the C1 and C3 groups contributed to these innervation patterns in the CGlv. Noradrenergic and adrenergic projections from the medulla to the CG may play an important role in a variety of autonomic, sensory and behavioral processes.


Cerebellar nuclei and the nucleocortical projections in the rat: retrograde tracing coupled to GABA and glutamate immunohistochemistry. The amino acids GABA and glutamate (Glu) are thought to be the principal substances in the central nervous system responsible for neuronal inhibition and excitation. Their distributions among the different neurons in a defined pathway may thus be indicative of the contributions of the cells to pathway function. Examples of such neurons are those of the cerebellar nuclei which, while regulating output from the Purkinje cells of the cerebellar cortex, are also found to project back to the cerebellar cortex. Immunohistochemical experiments were done to identify GABA and glutamate (Glu) containing cells in the adult rat cerebellar nuclei. Consecutive semithin and serial vibratome sections were incubated with antisera raised in rabbit against GABA and Glu. In semithin sections, only small neurons were intensely GABA immunoreactive (GABA-IR) (31.7%), and the majority (80.5%) were Glu immunoreactive (Glu-IR) of different sizes. Consistent with Glu being a metabolic precursor for GABA, 75.4% of the GABA-IR population colocalized Glu. In vibratome sections GABA-IR neurons showed some local differences in number, whereas the Glu-IR were uniformly distributed in the three nuclei studied. Measured mean diameters for these neurons showed a distinct size difference for the GABA- and Glu-IR with little overlap. Cerebellar nuclei neurons projecting to the cortex (nucleocortical neurons, NCN) were identified by locally preinjecting the retrograde transported WGA-apoHRP-colloidal gold complex in the cerebellar cortex. Vibratome sections of these cerebellar were silver intensified for the retrograde tracer and double labeled for GABA and Glu. Of the total number of identified NCN, 8.7% were GABA-IR (10 animals) and 47.7% Glu-IR (5 animals). Many retrograde labeled NCN in the core of the thick sections were immunonegative for both amino acids due to poor antibody penetration, thus underestimating the proportions of cells containing GABA and Glu. The size distributions for the GABA-IR and Glu-IR NCN were similar to those measured in non-retrograde labeled nuclei in thick sections. The conclusions reached are that GABA-IR neurons of the cerebellar nuclei, including the NCN, use GABA as the presumed inhibitory neurotransmitter and that Glu-IR neurons may use Glu or another excitatory neurotransmitter.


Interconnections between the primate cerebellum and midbrain near-response regions. The goal of this study was to determine the pattern of the connections between the midbrain and cerebellum that may play a role in the modulation of the near-response in the macaque. Injection of the retrograde tracer wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) into the physiologically identified midbrain near-response region, which includes the supraoculomotor area, labelled cells throughout the deep cerebellar nuclei. However, labelled cells were particularly concentrated in the ventrolateral corner of the contralateral posterior interposed nucleus and in the contralateral and, to a lesser extent, the ipsilateral fastigial nuclei. Subsequently, injections of WGA-HRP were used to define the midbrain terminations of the deep cerebellar nuclei. Fastigial nucleus injections labelled terminals in a band along the border between the oculomotor nucleus and the supraoculomotor area that included the Edinger-Westphal nucleus. Injections of the posterior interposed nucleus labelled terminals in the portion of the supraoculomotor area dorsal to the fastigial projection and did not involve the Edinger-Westphal nucleus. In both cases, the terminal label was primarily found contralaterally. In contrast, retrogradely labelled cells were primarily found ipsilaterally within the supraoculomotor area following cerebellar injections. Retrogradely labelled cells projecting to the deep nuclei were also found bilaterally in the anteromedian nucleus, along with sparse terminal label. Taken as a whole, these results demonstrate the presence of a highly specific pattern of labelling in the supraoculomotor area, which may indicate that the posterior interposed nucleus and the fastigial nucleus play different roles in the control of the near-response. Alternatively, these projections may subserve other functions, such as modulating the pupillary light reflex. The fact that the projection from the deep nuclei is primarily contralateral, while the supraoculomotor projection to the deep nuclei is primarily ipsilateral, suggests that this may not be a simple feedback system, but may instead be involved in balancing the gains in the two eyes. In sum, physiological experiments have indicated the presence of near-response neurons in the midbrain supraoculomotor area and have indicated that the cerebellum may play a role in modulating the components of the near-response, as well as activity in the intrinsic eye muscles. The present experiments suggest a pattern of connections that might subserve this cerebellar modulation.


Ly-1 (CD5), a membrane glycoprotein of mouse T lymphocytes and a subset of B cells, is a natural ligand of the B cell surface protein Lyb-2 (CD72). CD5 is a 67-kDa glycoprotein expressed on the cell surface membrane of all T lymphocytes and on a small proportion of B lymphocytes. The physiologic role of this Ag is still unknown. Structural and functional studies of CD5 suggest that it might act as a receptor for a positive signal. CD5-specific mAb augment CD3- or mitogen-induced T cell proliferation, IL-2 secretion, and IL-2R expression and induce a rise in intracellular [Ca2+]. In this report, we describe the purification of mouse CD5 protein (mCD5) and its use as a probe to search for the ligand of CD5. We demonstrate that mCD5 specifically interacts with the mouse B cell differentiation Ag CD72/Lyb-2. Three serologically defined allelic forms of mouse CD72/Lyb-2 can all interact with mCD5. We further show that mCD5 can interact with human CD72/Lyb-2, and similarly, that human CD5 can interact with mouse CD72/Lyb-2. These studies may have major implications for the mechanisms of T-B cell communication.


Broad recognition of cytotoxic T cell epitopes from the HIV-1 envelope protein with multiple class I histocompatibility molecules. A few cases have been described of antigenic determinants that are broadly presented by multiple class II MHC molecules, especially murine I-E or human DR, in which polymorphism is limited to the beta chain, and the alpha chain is conserved. However, no similar cases have been studied for presentation by class I MHC molecules. Because both domains of the MHC peptide binding site are polymorphic in class I molecules, exploring permissiveness in class I presentation would be of interest, and also such broadly presented antigenic determinants would clearly be useful for vaccine development. We had defined an immunodominant determinant, P18, of the HIV-1 gp160 envelope protein recognized by human and murine CTL. To determine the range of class I MHC molecules that could present this peptide and to determine whether two HIV-1 gp160 Th cell determinants, T1 and HP53, could also be presented by class I MHC molecules, we attempted to generate CTL specific for these three peptides in 10 strains of B10 congenic mice, representing 10 MHC types, and BALB/c mice. P18 was presented by at least four different class I MHC molecules from independent haplotypes (H-2d, p, u, and q to CD8+ CTL. In H-2d and H-2q the presentation was mapped to the D-end class I molecule, and for Dd, a requirement for both the alpha 1 and alpha 2 domains of Dd, not Ld, was found. HP53 was also presented by the same four different class I MHC molecules to CD8+ CTL although at higher concentrations. T1 was presented by class I molecules in three different strains of distinct MHC types (B10.M, H-2f; B10.A, H-2a; and B10, H-2b) to CTL. The CTL specific for P18 and HP53 were shown to be CD8+ and CD4- and to kill targets expressing endogenously synthesized whole gp160 as well as targets pulsed with the corresponding peptide. To compare the site within each peptide presented by the different class I molecules, we used overlapping and substituted peptides and found that the critical regions of each peptide are the similar for all four MHC molecules. Thus, antigenic sites are broadly or permissively presented by class I MHC molecules even without a nonpolymorphic domain as found in DR and I-E, and these sequences may be of broad usefulness in a synthetic vaccine.


Characterization of the immune response to a secondary encephalitogenic epitope of basic protein in Lewis rats. I. T cell receptor peptide regulation of T cell clones expressing cross-reactive V beta genes. In Lewis rats, immunization with myelin basic protein induces two distinct encephalitogenic T cell populations, those responding to the immunodominant 72-89 epitope and those specific for a secondary epitope including residues 87-99. The 72-89 specific T cells were I-A restricted and preferentially expressed V beta 8.2 in their TCR. To determine the fine specificity, MHC restriction, and TCR V beta gene use in T cells reactive to the secondary epitope, we characterized 23 T cell clones from the lymph nodes (LN) and spinal cords (SC) of rats immunized with either whole basic protein or synthetic peptides S85-99 and S87-99 that were found to be functionally similar. The S85-99/S87-99 specific clones from LN and SC were all encephalitogenic despite differences in recognition of intact basic protein and class II MHC restriction. Unlike LN clones that overexpressed V beta 8 (46%+) and V beta 6 (31%+), however, SC clones were strongly biased (86%+) in their expression of V beta 6. This V gene bias raised the possibility of TCR peptide therapy using V beta 6 peptides. The V beta 6 sequence was similar to V beta 8.2 in the CDR2 region, and the corresponding peptides from this region were found to be cross-reactive in vivo. Moreover, both peptides were effective in the treatment of EAE induced with either S85-99, biased in V beta 6+ and V beta 8+ T cells, or guinea pig basic protein, biased only in V beta 8+ T cells. These data demonstrate the presence of common immunogenic epitopes among subsets of TCR V region gene families that possess important regulatory activity on effector T cell function.


Characterization of the immune response to a secondary encephalitogenic epitope of basic protein in Lewis rats. II. Biased T cell receptor V beta expression predominates in spinal cord infiltrating T cells. The immune response of Lewis rat lymph node T cells to guinea pig myelin basic protein (GP-BP) in experimental allergic encephalomyelitis is directed primarily against a region of basic protein encompassed by residues 72-89. T cells that respond to this epitope are restricted by the RT1.B class II molecule of the MHC and use V beta 8.2 exclusively in their TCR. A second region of GP-BP, residues 87-99, also induces experimental allergic encephalomyelitis in Lewis rats but this response is restricted primarily by RT1.D. Elsewhere we describe the biologic characteristics of T cell clones responding to the synthetic peptide, s87-99, and to a related peptide, s85-99. We present a detailed analysis of TCR V beta gene expression among these clones, derived from the lymph node and spinal cord of immunized animals, and among spinal cord derived T cell clones reactive to GP-BP 72-89. We find that spinal cord-derived clones, reactive to s85-99 and to s87-99, use V beta 6 predominantly. In contrast, T cell clones derived from lymph nodes and reactive to the same peptides express multiple V beta genes including V beta 6. This difference in heterogeneity of V beta usage at the clonal level is also seen in T cell lines derived from spinal cord and immune lymph node. DNA sequence comparison of the CDR3 regions in V beta 6+ spinal cord clones revealed a conserved amino acid motif also found in the majority of V beta 6 sequences from the spinal cord anti-s85-99 line. Although V beta 6 was expressed in some lymph node-derived clones, only one contained a CDR3 region similar to that seen in spinal cord isolates. All spinal cord-derived T cell clones reactive to GP-BP 72-89 used V beta 8.2 and most (five of six) contained the AspSer residues in CDR3 previously shown to be associated with V beta 8.2 receptors expressed by the majority of lymph node T cells responding to GP-BP 72-89. These data indicate that TCR V beta usage in peripheral T cells responding to an autoantigen does not always predict the V beta usage among T cells at the site of an autoimmune attack. Possible explantations for the relative homogeneity in TCR V beta expression seen in T cell clones derived from the spinal cord are discussed.


Recombinant oncostatin M stimulates the production of acute phase proteins in HepG2 cells and rat primary hepatocytes in vitro. Acute inflammation is characterized by increased liver output of acute phase proteins (APP). Several cytokines including IL-6, leukemia inhibitory factor, and IL-11 are capable of stimulating APP synthesis by hepatocytes and hepatoma cells. We have tested the activity of a separate and unique cytokine oncostatin M (OM) and have found potent APP-inducing activity of human recombinant OM on hepatocytes. OM acted in a dose-dependent fashion (ED50 5 to 10 ng/ml) in stimulating APP synthesis in human HepG2 cells, rat H35 cells, and primary rat hepatocyte cultures, but not human Hep3B cells. Human OM induced equivalent to or greater responses than IL-6 in HepG2 cells, however, it was less effective than human IL-6 in stimulating rat cells. Northern analysis showed that OM stimulated mRNA levels of haptoglobin and alpha 1-antichymotrypsin in HepG2 cells. OM induced CAT activity in HepG2 cells transfected with CAT constructs containing IL-6-responsive elements, suggesting that OM induces transcription of native proteins through mechanisms involving IL-6-responsive element-like sequences in gene promoters. OM was also shown to act additively with IL-6 or leukemia inhibitory factor and synergistically with glucocorticoid or IL-1 in the induction of specific APP. These results suggest that OM plays a role as a mediator of APP synthesis in inflammatory responses.


Gangliosides suppress tumor necrosis factor production in human monocytes. Both normal and malignant cells contain gangliosides as important cell membrane constituents that, after being shed, may influence cells of the immune system. We have studied the impact of gangliosides on the expression of TNF in blood monocytes and in the monocytic cell line Mono Mac 6. Although under standard culture conditions, bovine brain gangliosides (100 micrograms/ml) suppressed LPS-stimulated TNF production 5-fold in PBMC and 10-fold in Mono Mac 6 cells, suppression was more efficient under serum-free conditions. Looking at highly purified gangliosides, GD3, GD1a, GM3, GM2, and GM1 were all effective in reducing TNF production in PBMC, and in Mono Mac 6 by factor 10 to 50. The suppressive activity was lost in molecules, lacking the sugar moiety or the lipid moiety. Gangliosides appear to act at an early step of activation in that TNF transcripts were reduced and the mobilization of the nuclear factor kappa B was blocked. Furthermore, in time kinetics, gangliosides were effective for up to 30 min after addition of LPS, but not thereafter. However, the expression of the CD14 Ag, a receptor molecule for LPS-LPS binding protein complexes, was unaffected by gangliosides. Finally, when using Staphylococcus aureus or platelet activating factor as a stimulus, gangliosides were able to suppress TNF production in Mono Mac 6 cells by factor 5 to 10, as well. On the other hand, phorbol ester-induced production of O2- was similar in cells treated with and without gangliosides. Taken together, our data demonstrate that TNF gene expression in monocytes induced by different types of stimuli can be blocked by gangliosides at an early step of signal transduction.


Molecular heterogeneity of Fc alpha receptors detected by receptor-specific monoclonal antibodies. Fc alpha receptors (Fc alpha R) were isolated from a human monocytic cell line and used to raise four mAb with receptor specificity. The antibodies were used to identify the types of white blood cells that express Fc alpha R and the molecular heterogeneity of the receptor molecules. Nonpolymorphic epitopes, outside of the Fc alpha-binding site, were recognized only on blood cells of granulocyte and monocyte/macrophage lineages. The molecules identified, both by the antibodies and by the IgA ligand, were glycoproteins ranging in relative molecular mass from 55 to 75 kDa. However, one antibody detected a subpopulation of Fc alpha R molecules characterized by relatively restricted size heterogeneity. A complex glycosylation pattern was revealed by the resolution of discrete 32- and 36-kDa molecular species after removal of N-linked oligosaccharides and by evidence for O-linked carbohydrate moieties on at least a portion of the Fc alpha R molecules. In biosynthetic studies, all four anti-Fc alpha R antibodies and the IgA ligand bound a single 32-kDa core protein present in tunicamycin-treated cells, and the exceptional antibody again recognized molecules with relatively restricted glycosylation in the nontreated cells. These antibodies and native IgA ligands thus provide complementary reagents for definition of the complex structure and function of Fc alpha R in systemic IgA antibody responses.


Association of human lymph node homing receptor (Leu 8) with the TCR/CD3 complex. Cross-linking of the human homologue of the murine MEL-14 lymph node homing receptor (Selectin-1, LECAM-1, Leu 8) on both T and B cells results in modification of cell function. To investigate this phenomenon, we performed studies to determine if the Leu 8 molecule influences T cell activation via the TCR/CD3 complex. In initial studies, we treated T cells with immobilized anti-CD3 (OKT3 mAb) in the presence or absence of immobilized Leu 8 mAb. We found that although Leu 8 mAb alone had no effect on T cell proliferation, this antibody markedly augmented immobilized OKT3 mAb-induced proliferation. In further studies, we immunoprecipitated surface radioiodinated T cell lysates with OKT3 and Leu 8 mAb to determine if molecules in the TCR/CD3 complex associate with Leu 8 molecules. Although Leu 8 mAb immunoprecipitated only a single protein of approximately 80 kDa from T cell lysates treated with Nonidet P-40 under reducing condition, it coimmunoprecipitated additional proteins of 48, 42, 28, 24, and 22 kDa from T cell lysates treated with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. These additional proteins were identified as the alpha-, beta-, gamma-, delta-, and epsilon-chains of the TCR/CD3 complex by one-dimensional and two-dimensional diagonal SDS-PAGE. Densitometric scanning showed that, on average, 18% of the TCR/CD3 complex associates with Leu 8. In a final study, we showed by immunoblotting analysis using anti-zeta peptide antibody that Leu 8 mAb coimmunoprecipitates the zeta-chain of CD3. These results indicate that the human lymph node homing receptor homologue (Leu 8) participates in the activation of T cells, probably via its association with the TCR/CD3 complex.


Immunoreactivity of a 10-kDa antigen of Mycobacterium tuberculosis. Identification of Ag of Mycobacterium tuberculosis recognized by T cells is essential to understanding the pathogenesis of tuberculosis and mechanism(s) of resistance to infection. Previous studies evaluating the immunoreactivity of nitrocellulose transfers of M. tuberculosis Ag separated by SDS-PAGE indicated that a high proportion of M. tuberculosis-reactive T cell lines proliferate in response to a 10-kDa Ag. We therefore purified this Ag from M. tuberculosis culture filtrates and evaluated its immunoreactivity in patients with tuberculous infection. Proliferative responses of PBMC to the 10-kDa Ag were similar to those induced by whole M. tuberculosis and greater than those elicited by other proteins isolated from culture filtrate. Furthermore, in patients with tuberculous pleuritis, proliferative responses to the 10-kDa Ag were higher in pleural fluid mononuclear cells than in PBMC, indicating that T cell reactivity to this Ag is enhanced at the site of disease. The first 15 amino acids of the 10-kDa Ag were identical to those defined previously for Bacillus Calmette-Guérin-a (BCG-a), and a T cell clone recognized the 10-kDa Ag and a peptide of BCG-a, indicating that the 10-kDa Ag corresponds to BCG-a. This Ag elicited IFN-gamma production by pleural fluid mononuclear cells and by PBMC from healthy tuberculin reactors, suggesting that the 10-kDa Ag can enhance macrophage activation and resistance to mycobacterial infection. Our findings indicate that the 10-kDa Ag of M. tuberculosis is highly immunoreactive and should be evaluated for its capacity to elicit protective immunity.


Regions of the CD4 molecule not involved in virus binding or syncytia formation are required for HIV-1 infection of lymphocytes. Cell surface-expressed CD4 binds to the envelope glycoprotein of HIV-1 and mediates syncytia formation through interacting with membrane expressed HIV-1 gp120. Further possible roles of the CD4 molecule in the process of cell infection by HIV-1 remain poorly understood. In our study we describe two mAb that recognize the V3/V4 domain of the CD4 molecule. Although these mAb do not inhibit gp120-CD4 binding or HIV-1-induced syncytia formation, they inhibit HIV-1 infection of human PBL. These findings suggest that discrete, definable domains of the CD4 molecule may be involved in interactions after HIV-1 envelope binding that lead to virus entry into the cell.


Mouse genotype affects inducible expression of cytokine genes. The levels of circulating IFN in mice infected with Newcastle disease virus (NDV) are regulated by the If-1 locus. In this study we show that in NDV-infected C57BL/6 mice, which carry the If-1h allele and produce high levels of IFN, high levels of both IFN-alpha and -beta mRNA can be detected in the spleen. In contrast, only very low levels of IFN mRNA could be detected in spleens of infected BALB/c mice containing the If-1l allele and producing low levels of IFN or in B6.C-H28c mice that are congenic for the If-1l allele. The relative levels of all individual IFN-alpha 1, alpha 4, and alpha 6 mRNA in spleens of infected BALB/c were lower than in spleens of infected C57BL/6 mice, indicating that the If-1 locus affects the expression of all IFN-alpha subtypes and is not associated with the deletion or inactivation of a specific IFN gene. The relative levels of IFN regulatory factor-1 mRNA in infected mice carrying the If-1l and If-1h loci were comparable, suggesting that the If-1 regulation is not associated with the altered expression of the IFN regulatory factor-1 gene. Quantitative difference in the expression of IFN-alpha and -beta genes was also observed in in vitro-infected peritoneal macrophages isolated from either C57BL/6 or BALB/c mice. A surprise finding was that the If-1 locus also affected the NDV-induced expression of two other cytokine genes, TNF-alpha and IL-6. Priming of the macrophage cultures with murine IFN enhanced the expression of all cytokine genes, and the relative levels of IFN, TNF-alpha, and IL-6 mRNA induced by NDV in macrophages derived from C57BL/6 and BALB/c mice were comparable. We propose that the If-1 locus affects the early stages of a signal transduction pathway which are common to the virus-mediated induction of IFN, TNF-alpha, and IL-6 genes.


Use of recombinant fusion proteins for generation and rapid characterization of monoclonal antibodies. Application to the Kunitz domain of human beta amyloid precursor protein. Production of peptides by recombinant DNA techniques is an efficient alternative to chemical synthesis of peptides. Proteins and peptides produced by recombinant DNA methods in E. coli are routinely used as antigens for the production of antibodies. However, most small peptides are rapidly degraded within the E. coli cell, and therefore, must initially be expressed as components of larger, more stable fusion proteins. The peptide of interest must be cleaved from the fusion protein, and purified prior to immunization to eliminate epitopes contributed by the fusion partner. We have now established methods for the production and characterization of monoclonal antibodies using partially purified, uncleaved fusion proteins. We have also described a method for efficient production and detection of the fusion protein, an EIA for rapid differential screening of hybridoma supernatants, and a strategy for epitope mapping of the antibodies. These methods have been applied to the production and characterization of monoclonal antibodies specific for a 75-amino-acid internal segment of the Alzheimer amyloid precursor protein, and should be applicable to a wide variety of other peptides and proteins.


Generation of species-specific, rat monoclonal antibodies that bind to the kappa chain of mouse immunoglobulin. Large numbers of mouse monoclonal antibodies (MAbs) with exquisite binding specificities have become available. However the study of these primary antibodies in biological fluids by second, anti-mouse antibody techniques, has been confounded by the large quantity of other animal immunoglobulin which is often present in these fluids. To overcome this problem, we have derived high affinity rat MAbs which bind specifically to the antigen binding fragments (Fab) of mouse antibody and display minimal or no crossreactivity with human or rabbit immunoglobulin at the concentrations which are usually found in plasma. Equilibrium binding studies demonstrated that these antibodies had binding affinity constants for mouse Fab that ranged from 4.3 x 10(8) to 4.1 x 10(9) M-1. All of these rat MAbs bound to the kappa chain of mouse immunoglobulin, though competition binding studies amongst these MAbs indicated that they probably recognized three different epitopes. Because of their specificity and affinity, these rat anti-mouse kappa MAbs may be useful in a wide variety of experimental biological systems both in vitro and in vivo.


A rapid staining procedure for two-color analysis of lymphocyte antigen expression. Two color immunofluorescence analysis of lymphocyte cell surface antigen expression using an unconjugated plus a biotinylated monoclonal antibody (mAb) requires four incubation steps: (1) unconjugated mAb; (2) fluorochrome-labelled goat anti-mouse Ig; (3) biotinylated mAb; (4) fluorochrome-labelled avidin or streptavidin. We describe a time-saving modification of this procedure which requires only two incubation steps: (1) simultaneous unconjugated and biotinylated mAbs; (2) fluorochrome-labelled avidin/streptavidin followed by fluorochrome-labelled goat anti-mouse Ig. The slightly delayed (5 min) addition of the goat anti-mouse Ig prevents it from binding to the mAb which has already interacted with avidin/streptavidin. Both procedures yield identical results with a variety of different mAbs.


Species differentiation of mycoplasmas by EF-Tu specific monoclonal antibodies. Ten mouse hybridoma cell lines producing IgG monoclonal antibodies to mycoplasmal elongation factor Tu (EF-Tu) were established. These mAbs showed different degrees of cross-reactivity between mollicutes and even other bacteria. This finding, indicating protein structure diversities of pan bacterial EF-Tu should permit species differentiation of mycoplasmas by epitope pattern analysis of a single protein. Epitope patterns of 23 mollicute type strains and of 40 M. hominis isolates were determined by ELISA. All M. hominis patterns were found to be closely related whereas intrageneric patterns differed in a species specific manner.


[A study on the proliferation of the uterine endometrium]. A proliferative activity study of the endometrium was evaluated in three different parts by means of an immunohistochemical approach and flow cytometry (FCM). The materials were 29 cases of hysterectomies due to uterine myoma. Twenty-one cases were in the proliferative phase, and 8 cases were in the secretory phase. The three parts of the endometrium obtained from the upper, middle and lower part of the uterine body were investigated by an immunohistochemical technique with Ki-67 and anti-DNA polymerase alpha antibody. Single staining with Propidium Iodide (PI) was used for cell cycle analysis in FCM. (1) The rate of positive Ki-67 cells was 25.9 +/- 6.3% in the upper part, 26.0 +/- 3.3% in the middle part, and 25.5 +/- 5.8% in the lower part. And the rate of positive DNA polymerase alpha cell was 27.2 +/- 5.8% in the upper part, 24.0 +/- 2.9% in the middle part, and 24.8 +/- 4.0% in the lower part. The growth potential of each part was not significant. (2) FCM did not detect a significant difference among the three parts of the endometrium in cell cycle analysis. (3) Hematoxylin-eosin staining showed that the upper part was thicker than the lower part and the number of glands in the upper part was more abundant than in the lower part. This difference was significant (p less than 0.01). (4) Ki-67 was recognized even in the early secretory phase. (5) And each segment of the endometrium proliferated evenly, not focussed.(ABSTRACT TRUNCATED AT 250 WORDS)


[Immunohistochemical study on keratin of squamous cell carcinoma of the uterine cervix]. An immunohistochemical study of squamous metaplasia (n = 10), dysplasia (n = 18), squamous cell carcinoma (n = 48) and 3 cases of adenosquamous carcinoma of the uterine cervix with anti-56KD keratin and 68KD keratin antibodies was performed. In the cases of squamous metaplasia, there were two types of staining of which one type had 56KD positive and 68KD negative and another type had both positive. In the cases of dysplasia, there were two types of staining the same as in squamous metaplasia. But in the cases of carcinoma in situ (CIS) (n = 25), there were three types of staining of which the first type had both 56KD and 68KD negative (n = 7), the second type had 56KD positive and 68KD negative (n = 15), and the third type had both 56KD and 68KD positive (n = 3). In invasive carcinoma (n = 23), there were two types of staining the same as in dysplasia of which one type had 56KD positive and 68KD negative (n = 17) and another type had both positive (n = 6). The keratin negative cases in CIS showed morphologically atypical reserve cell hyperplasia composed of atypical small cells with round nuclei and had a small lesion compared with other types. This result suggested that keratin negative CIS was an early form of CIS which was keratin positive. The results indicating that all dysplasia had 56KD keratin positive and CIS had not always 56KD keratin positive suggested that dysplasia was not always a precursor lesion of CIS.(ABSTRACT TRUNCATED AT 250 WORDS)


Albuminuria is not an aggravating factor in experimental focal glomerulosclerosis and hyalinosis. It is widely known that the severity of glomerular sclerosis is proportional to the degree and chronicity of proteinuria and that the degenerative changes of glomerular epithelial cells that are associated with overflow albuminuria can be experimentally induced by the injection of large quantities of heterologous albumin. Such evidence suggests that autologous albuminuria per se may have a harmful effect on the kidneys. To examine the cause and effect relationship between renal lesions and albuminuria, we produced Adriamycin-induced experimental focal glomerular sclerosis in Nagase analbuminemic (NA) rats and control Sprague-Dawley (SD) rats and observed both the renal functional and histologic changes for 20 weeks. At week 4 after injection of Adriamycin glomerular epithelial lesions including foot process fusion were similarly revealed by an electron microscopic study in both groups in spite of the presence of a large difference in the amount of proteinuria (SD rats: 491 +/- 84 mg/day, NA rats: 43 +/- 30 mg/day) and albuminuria (SD rats: 383 +/- 73 mg/day, NA rats: 2 +/- 1 mg/day). At week 20, a light microscopic study showed the same degree of glomerular sclerosis and hyalinosis and tubulointerstitial changes associated with a decrease in inulin clearance in both groups. The increased glomerular accumulation of immunoglobulin M or complement 3 and glomerular trapping of aggregated human immunoglobulin G were also similar between the SD and NA groups. In summary, renal destruction of Adriamycin-nephropathy was not dependent on the degree of albuminuria. These results suggest that albuminuria is not an aggravating factor in focal glomerulosclerosis.


Immunohistochemical evaluation with Ki-67: an application to salivary gland tumours. Using Ki-67, a monoclonal antibody, the proliferating capacity of 15 salivary gland tumours, including nine pleomorphic adenomas, four adenoid cystic carcinomas, one mucoepidermoid carcinoma and one acinic cell carcinoma was determined immunohistochemically, using normal salivary gland tissue as a control. The frequency of Ki-67 positive cells was 4.7 percent in the normal salivary gland and one percent in pleomorphic adenomas, whereas the average frequency in malignant tumours was 18.3 percent. Among adenoid cystic carcinomas, the frequency was related to the morphological type; the solid sub-type had the highest frequency of Ki-67-positive cells. As this sub-type is recognized as the most aggressive of these tumours, this technique has the potential of providing an early indication of the clinical behaviour of a tumour.


Synergism of interleukin 6 and 1 alpha,25-dihydroxyvitamin D3 in induction of myeloid differentiation of human leukemic cell lines. Interleukin 6 (IL-6) is a multifunctional cytokine with an important role in immunity. We analyzed the effect of recombinant human IL-6 in combination with 1 alpha,25-dihydroxyvitamin D3 (Vit. D3) on differentiation of the human myeloid leukemic cell lines U937 and HL-60 with respect to alterations in antigen expression and functional activity. Of a panel of antigens analyzed, only CD11b (the alpha chain of CR3), and CD14 (a cell surface protein recognizing the lipopolysaccharide-binding protein-lipopolysaccharide complex) had significantly increased expression. Expression of ICAM-1 (CD54), a ligand for LFA-1, was also found to be enhanced with a concomitant increase of ICAM-1 mRNA levels. Enhanced nonspecific esterase levels and induction of respiratory burst activity confirmed that cell differentiation was induced. Furthermore, IL-6 and Vit. D3 had a profound effect on functional activities, as shown by enhancement of rosetting between sheep erythrocytes, sensitized with C3bi (EAC), and either U937 or HL-60 cells. Also, phorbol myristate acetate-induced homotypic adhesion of U937, which is ICAM-1 dependent, was markedly induced by these agents. These results indicate an important role of IL-6 and Vit. D3 in myeloid cell function and development.


Effect of adenosine analogues and cAMP-raising agents on TNF-, GM-CSF-, and chemotactic peptide-induced degranulation in single adherent neutrophils. Adenosine and adenosine analogues are potent inhibitors of the respiratory burst in neutrophils. Most investigators, however, have found little or no effect of these compounds on neutrophil degranulation from cytochalasin B-treated neutrophils in suspension. We have instead investigated the effect of adenosine and 2-chloroadenosine on degranulation in adherent neutrophils in the absence of cytochalasin B. Both adenosine and 2-chloroadenosine were effective inhibitors of lactoferrin secretion induced by the chemotactic peptide N-formyl-methionine-leucyl-phenylalanine (fMLP) [50% inhibitory concentration (IC50) of less than 10(-6) M]. Secretion induced by tumor necrosis factor (TNF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was inhibited only at high concentrations (IC50 of approximately 10(-4) M). In the presence of cytochalasin B no inhibitory effect of 2-chloroadenosine was seen. The effect of cAMP-raising agents on secretion from adherent neutrophils was also investigated. Dibutyryl cAMP at 0.2 mM reduced secretion in response to fMLP by 50% but did not inhibit TNF- and GM-CSF-induced degranulation. At a concentration of 2.0 mM dibutyryl cAMP also inhibited exocytosis in response to the two cytokines. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) at 300 microM reduced fMLP-induced degranulation, whereas a concentration of 1 mM was required to inhibit TNF- and GM-CSF-mediated secretion. The adenylate cyclase activator forskolin (50 microM) alone did not inhibit secretion in response to TNF or fMLP. However, in combination with IBMX (300 microM), forskolin (50 microM) reduced both TNF- and fMLP-induced secretion to less than 10%. PMA-induced exocytosis was unaffected by all these agents. In conclusion, adenosine appears to be an effective inhibitor of neutrophil granule protein secretion induced by fMLP but only a weak inhibitor of exocytosis in response to TNF or GM-CSF. Secretion in response to fMLP was also found to be more susceptible to a rise in cAMP than degranulation induced by TNF and GM-CSF.


Effects of deglycosylation of human thyroperoxidase on its enzymatic activity and immunoreactivity. Thyroid peroxidase (TPO) is a glycoprotein enzyme which catalyses the iodination of thyroglobulin and the coupling of iodinated tyrosines. Human TPO (hTPO) is the microsomal antigen recognized by the autoantibodies in the serum of patients with autoimmune thyroid disease. An active detergent-solubilized immunoaffinity-purified hTPO was deglycosylated, either by peptide N-glycosidase F (PNGase F) or by endo-beta-N-acetylglucosaminidase H (endo H), and the enzymatic activity and immunoreactivity of the native and deglycosylated forms were compared. Electrophoretic controls and affinoblotting with concanavalin A showed that deglycosylation was not total and that it was more pronounced with endo H than with PNGase F. The enzymatic activity of hTPO was inhibited by endo H deglycosylation, but not by PNGase F deglycosylation; this inhibition was not due to aggregation and/or insolubilization of the molecule subsequent to deglycosylation. Immunoreactivity was monitored by enzyme-linked immunosorbent assay (ELISA) with 13 mouse monoclonal antibodies, rabbit polyclonal antibodies and antibodies from serum of patients with Hashimoto's thyroiditis. In contrast with enzymatic activity, immunoreactivity was not modified or was slightly enhanced (with four monoclonal antibodies) by deglycosylation. The results indicate that strong, if not total, deglycosylation induces a modification of the tertiary structure of hTPO, which affects the enzymatic site but does not modify markedly the epitopes implicated in the recognition of the molecule by the antibodies tested.


Muscle siderosis in AIDS: a marker for macrophage dysfunction? We have observed numerous iron granules in muscle fibres, endothelial cells and macrophages of muscle biopsy specimens of 21 out of 41 AIDS patients with different patterns of muscle involvement. All patients were severely immunodepressed. We report on our findings and discuss the mechanism of muscle siderosis that may point to deterioration of some functions of macrophages at a late stage of HIV infection.


Cell culture as a tool for the study of poultry skeletal muscle development. Postnatal development of skeletal muscle is the responsibility of the myogenic satellite cells. Satellite cells, isolated from the pectoralis major muscle of young growing tom turkeys, have been cultured in vitro to provide a system for studying cellular and hormonal aspects of poultry skeletal muscle development. Satellite cell clones derived from primary cultures have been developed so that in vitro observations would not be confounded by the presence of nonmyogenic cells. Likewise, a serum-free medium that promotes proliferation of the turkey satellite cell has been developed to provide a hormonally controlled environment for in vitro developmental studies. These two techniques have enabled us to examine the following: 1) factors that influence satellite cell proliferation and differentiation, 2) the interaction of hormones with cellular receptors, 3) secretion of biologically important proteins from cells and 4) the expression of genes important to muscle development.


Fetal cleft lip repair in rabbits: histology and role of hyaluronic acid. This study examines the histologic and biochemical features of wound healing in a cleft lip model in the mid-third-trimester fetal rabbit. At days 1, 2, and 4 after the procedure, control, unrepaired, and repaired fetal heads were obtained, sectioned, and stained for histologic examination. The localization of hyaluronic acid in the wound was documented using a cartilage-derived hyaluronic acid-binding protein. In both repaired and unrepaired wounds, the fetal cleft healed without inflammatory cell infiltration or scar formation. Six months after birth, the repaired cleft showed complete regeneration of muscle across the wound and the collagen fibers were of normal density and orientation. Decreased hyaluronic acid deposition was observed in unrepaired clefts as compared with adjacent tissue; no such difference was detected in repaired clefts. Our findings support the hypothesis that a cleft lip repaired in utero heals without the scarring that accompanies postnatal repair. This may explain the lack of maxillary growth restriction after in utero cleft lip repair.


Methodological pitfalls in serum IgG2 level measurements by immunoenzymatic assays with monoclonal antibodies. Four anti-IgG2 monoclonal antibodies (Mabs) were evaluated for their reactivity with purified myeloma IgG2 of different light chain types and Gm allotypes in three distinct immunoenzymatic assays (ELISA). The reactivity of three Mabs with solid-phase antigens was similar whereas an anti-Fab antibody (clone HP 6114) predominantly bound IgG2 kappa. In competitive and immunometric (sandwich type) assays, the binding of the two anti-IgG2 Mabs (HP 6014 and HP 6114) reacting with epitopes located on the Fab fragment was strongly influenced by the light chain type of IgG2 and by other factors (probably including differences in the variable regions); the Mab HP 6114 reacted virtually only with IgG2 kappa whereas the Mab HP 6014 displayed a much stronger affinity for IgG2 lambda than for IgG2 kappa; for both anti-Fab Mabs, important differences were found in their binding to individual IgG2 proteins. In addition, the Mab HP 6014 seemed to show a slightly better affinity for IgG2 bearing the Gm(23) allotype. These results urge much caution in IgG2 level measurement, especially with commercial kits, most of which use the Mab 6014 as the single anti-IgG2 reagent.


Decreased histamine release by luteinizing hormone-releasing hormone antagonists obtained upon translocation of the cationic amino acid from position 8 to position 7. We report analogues of N-Ac-D-Nal-D-Cpa-D-Pal-Ser-Lys(Pic)-D-Lys(Pic)-Leu-Ilys-Pro-D-Ala- NH2, the parent antagonist (PA), which is a potent antagonist of LHRH. To simplify future radioactive labeling we prepared N-Ac-D-Nal-D-Cpa-D-Pal-Ser-Lys(Pic)-D-Lys(Pic)-Leu-Arg-Pro-D-Ala-NH2 (4), [Arg8]PA, which had good activity in the antiovulatory assay (AOA). Other analogues were designed at first by substituting with Arg at positions 5, 6, 7, 9, and 10, and Trp or Leu at position 8. Subsequent analogues were prepared in attempts to improve the AOA of the initial ones. Substitutions with Arg9 or Arg10 led to analogues 1-3 with no AOA activity at 5 micrograms/rat. However, substitution with Arg7 gave 9, [Arg7,Leu8]PA, with significant activity in the AOA at 5 micrograms/rat and borderline activity at 2.5 micrograms/rat, and substitution with Ilys7 gave 13, [Ilys7,Leu8]PA, with borderline activity at 2 micrograms/rat, both analogues showing much weaker activity than PA in the histamine release assay (HRA) and therefore being potentially safer. Substitutions with D-Arg6 or Arg5 led to analogues with either good AO activity at 5 micrograms/rat (analogue 7) or with borderline activity at 5 micrograms/rat (analogue 8), although both were more potent than 6 in the HRA. Combinations of Ilys or Arg at positions 7 and 8 led to 10 and 11, both of which were tested at 2 micrograms/rat and found to have either good AO activity (analogue 10) or borderline activity (analogue 11) but unsuitably potent in HR. Substitutions using Ilys7 and neutral amino acids at position 8 led to 14-17 which were inactive in the AOA. Of great significance is the substitution with Arg7 yielding analogue 9, which was much safer in the HRA than analogue 4, [Arg8]PA. Analogues 9 and 13, featuring substitutions with the Arg7-Leu8 or Ilys7-Leu8 sequences were even safer than PA or 6 in the HRA. Analogue 12, [D-Trp3,Tyr5,D-Arg6,Arg7,Leu8]PA, featuring the Arg7-Leu8 sequence, had much lower potency in the HRA than [D-Trp3,Tyr5,D-Arg6,Leu7,Arg8]PA, which has the normal Leu7-Arg8 sequence. Ilys7 together with neutral amino acids at position 8 led to analogues 14-17 which were also very weak (safer) in the HRA, with the smaller amino acids Ala8 and Abu8 being the weakest of all analogues prepared.(ABSTRACT TRUNCATED AT 400 WORDS)


Effects of irreversible and reversible cholinesterase inhibitors on single acetylcholine-activated channels. The use of cholinesterase (CHE) inhibitors provided valuable information about the mechanism(s) of neuromuscular transmission, but questions on side effects at the level of ACh-activated channels were raised. Patch-clamp recording was used to study the effects of prostigmine (PST) and methanesulfonyl fluoride (MSF), a reversible and an irreversible cholinesterase inhibitor, respectively, on ACh-activated channels. We found that these drugs diminish the average dwell time of elementary currents from around 5 msec (control) to less than 1 msec in the presence of PST (20 microM) or MSF (5 mM) (at room temperature). With MSF the ACh-activated channel conductance of the most frequently observed amplitude class decreased from 45 pS (control) to 30 pS, but not in the presence of PST. In control conditions there were also amplitude classes of 60 and 24 pS, with probabilities of occurrence less than 10%. In the presence of 1.5 mM MSF, where current dwell time was not affected, additional subconductance states of 19 and 36 pS were observed and may be due to partial blockade of the open channel. We conclude that the drug of choice to be used in studies on the role of CHE in the neuromuscular transmission is MSF, because at 20 microM PST, where blockade of ACh-activated channels is significant, cholinesterase was reported to be partially inhibited, whereas at 1 mM MSF it is fully inhibited and the dwell time of ACh-activated channels is not affected.


Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural RNA. The in vitro fidelity of reverse transcriptase from human immunodeficiency virus type I (HIV-1 RT) upon copying an RNA template was measured using the phi Xam 16 reversion assay. A phi X174 sequence harboring the amber 16 codon was cloned into a transcription vector. RNA obtained from transcription by bacteriophage T7 RNA polymerase was used as a template for RNA-directed DNA synthesis by HIV-1 RT. An imbalance of dNTP concentrations during the reverse transcription step served to distinguish between errors that arose from the transcription step and errors from reverse transcription. The frequency of dGTP.U mismatches was determined to be 1/360, while dGTP.rA mismatches formed at a rate of 1/4600. These are 20-fold and sevenfold higher, respectively, than the error rates determined for the same sequence with a DNA template. Due to a high background of errors in the RNA template originating from the transcription step only upper limits for the frequency of three other mismatches can be given. The data indicate that the reverse transcription step of the HIV-1 replication cycle contributes significantly to the generation of mutant viruses.


Coexpression of vimentin and keratins by human melanoma tumor cells: correlation with invasive and metastatic potential. Several protein markers, including vimentin, have been used to diagnose human melanoma. Because melanoma often has metastasized by the time of diagnosis, early markers prognostic for metastatic potential need to be identified. Commonly, vimentin is found in mesenchymal cells, and keratins are present in epithelial cells, but recent studies report coexpression of vimentin and keratin(s) in epithelial and nonepithelial neoplasms, including some melanomas. Our purpose was to determine whether coexpression of vimentin and keratin(s) is correlated with tumor cell invasion and metastatic behavior. We evaluated nine human melanoma cell lines expressing vimentin and other markers of aggressive tumor behavior (HMB-45, S-100, HLA-ABC class I and HLA-DR class II histocompatibility antigens, and K8 and K18 keratins). Levels of K8 and K18 keratins were determined in the highly metastatic C8161 cell line, the poorly metastatic A375P line, and the moderately metastatic A375M line. To determine whether the presence of keratin affects migratory ability, we altered the conformational structure of keratin filaments in C8161 cells by transfection with a mutant K18 complementary DNA. We also determined messenger RNA levels of human type IV collagenase, an enzyme marker for invasion and metastasis. In A375P cells, two-dimensional electrophoresis with Coomassie-stained gels, immunoblotting, and immunofluorescence staining showed no detectable levels of K8 or K18. A375M cells showed low levels of K8 and K18 by Western and Northern blotting, with a distinctive fluorescent subpopulation of cells. In comparison, K8 and K18 levels in C8161 cells were high in all cells. Type IV collagenase messenger RNA levels were lowest in A375P cells and highest in C8161 cells, correlating with invasive ability in vitro and metastatic potential in athymic nude mice. The transfectant clones C1070-10 and C1070-14 derived from the C8161 parent line showed dramatic morphological changes, disrupted keratin filaments, and decreased invasive and metastatic potential directly correlated with a reduction in migratory activity. These findings show a correlation between the coexpression of vimentin with K8 and K18 keratins and the invasive and metastatic behavior of three representative human melanoma cell lines.


[Relapse of peptic ulcer after quick healing induced by proton pump inhibitors]. Peptic ulcers treated with a proton pump inhibitor heal more quickly than those treated with a histamine H2 receptor antagonist. Although satisfactory healing without relapse is desirable in the medical treatment of peptic ulcers, the relapse rate after treatment with proton pump inhibitors has not been sufficiently studied. Up to now, several reports have suggested that peptic ulcers treated with omeprazole recur less frequently than those treated with H2 antagonists. Our experimental studies on angiogenesis in granulation tissue of acetic acid-induced gastric ulcers, gastric mucosal collagen synthesis etc., show that H2 antagonists have an inhibitory effect on wound healing, but that proton pump inhibitors do not. It is suggested, therefore, that proton pump inhibitors may at least have no undesirable effect on the natural history of peptic ulcer.


[Intracellular killing mechanisms of alveolar macrophages against Mycobacterium avium complex]. To clarify the intracellular killing mechanisms of alveolar macrophages against Mycobacterium avium complex, effects of cytokines on O2- and NO2- production from normal and BCG-induced alveolar macrophages were studied. Intracellular growth of M. avium complex was inhibited in the alveolar macrophages stimulated by TNF, but not IFN. Enhancement of O2- production by normal alveolar macrophages stimulated by cytokines, was associated with the inhibition of intracellular growth of M. avium complex. However, when NO2- production by the alveolar macrophages was enhanced by the stimulating of IFN or IFN+TNF, in the presence L-arginine in the culture medium, their defense activity against M. avium complex decreased.


Effect of peripheral-blood progenitor cells mobilised by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy. The haemopoietic growth factor granulocyte colony-stimulating factor (G-CSF; filgrastim) substantially shortens the period of severe neutropenia that follows high-dose chemotherapy and autologous bone-marrow infusion by stimulating granulopoiesis. Filgrastim also increases numbers of circulating progenitor cells. We have studied the ability of filgrastim to mobilise peripheral-blood progenitor cells and assessed their efficacy when infused after chemotherapy on recovery of neutrophil and platelet counts. 17 patients with non-myeloid malignant disorders received filgrastim (12 micrograms/kg daily for 6 days) by continuous subcutaneous infusion. Numbers of granulocyte-macrophage progenitors in peripheral blood increased a median of 58-fold over pretreatment values, and numbers of erythroid progenitors increased a median of 24-fold. Three leucapheresis procedures collected a mean total of 33 (SEM 5.7) x 10(4) granulocyte-macrophage progenitors per kg body weight. After high-dose chemotherapy in 14 of the patients (busulphan and cyclophosphamide), these cells were used to augment autologous bone-marrow rescue and post-transplant filgrastim treatment. Platelet recovery was significantly faster in these patients than in controls who received the same treatment apart from the infusion of peripheral-blood progenitors; the platelet count reached 50 x 10(9)/l a median of 15 days after infusion of haemopoietic cells in the study patients compared with 39 days in controls (p = 0.0006). The accelerated neutrophil recovery associated with filgrastim treatment after chemotherapy was maintained. This method may be widely applicable to aid both neutrophil and platelet recovery after high-dose chemotherapy; it will allow investigation of peripheral-blood progenitor-cell allotransplantation.


Artificial viral envelopes containing recombinant human immunodeficiency virus (HIV) gp160. An artificial viral envelope was constructed, resembling the human immunodeficiency virus (HIV) envelope with respect to ultrastructure, size, phospholipid profile and lipid:cholesterol ratio. Recombinant HIV surface protein gp160 was anchored in the outer surface of the envelope membrane using a double detergent dialysis. The envelopes remained physically stable for several months. Immunolabeling with anti-gp160/41 monoclonal antibody revealed surface insertion and availability of gp160 for binding. Cell fusion and cytosolic transfer of the encapsulated fluorescent marker FITC-dextran was demonstrated. Flow cytometry indicated more efficient transfer of the fluorescent marker to cells which were approximately 60% CD4+ (REX-1B), relative to cells which were only approximately 18% CD4+ (KG-1). However, plain lipid envelopes without gp160 fused very efficiently with both cell types, indicating their potential usefulness as "fusogenic liposomes". Complete artificial viral envelopes may serve as subunit vaccines, and receptor-targeted delivery systems for drugs, toxins and genetic constructs.


Hinge region of human IgG2 protein: conformational studies with monoclonal antibodies. We previously produced three anti-human IgG2 mAbs with high specificity and found that they recognize distinct epitopes in the hinge region and neighboring residues in human IgG2: HG2-6A was reactive with the hinge region (Glu216-Pro230); HG2-56F with the Pro234 residue and HG2-30F with the Val235 residue. In this study, we evaluated the reactivities of those three mAbs with human IgG2 protein under various conditions. The results obtained using HG2-6A mAb indicated that the hinge region was concealed in the native form, but exposed after heat treatment at 63 degrees C, or chemical treatment with 3 M KSCN, 3 M guanidine, 30% CH3CN, 8 M urea or acid at pH 2.0 as well as by adsorption onto polystyrene beads. The IgG2 hinge region was also exposed after binding to specific antigens. The Pro234 residue recognized by HG2-56F mAb was exposed under all conditions studied. The neighboring Val235 residue recognized by HG2-30F, however, was completely concealed in the native and antigen-bound states. Only treatment with 3 M guanidine and acid at pH 2.0, or physical adsorption induced conformational changes to partially expose the Val235 residue.


Biochemical characterization of CD26 (dipeptidyl peptidase IV): functional comparison of distinct epitopes recognized by various anti-CD26 monoclonal antibodies. In this paper, we performed further biochemical characterization of the CD26 antigen, as defined by the mAbs in anti-1F7 and anti-Ta1, in order to clarify the observed functional differences among these mAbs. For this purpose, we developed a mAb, anti-5F8, which recognizes yet another epitope on the CD26 antigen different from that recognized by anti-1F7 and anti-Ta1 and compared their respective effect on T cell activation as well as the structures recognized by these mAbs. Functionally, anti-5F8 did not exhibit a comitogenic effect on T cell activation via the CD3 and CD2 pathways. Peptide mapping studies suggested that the 110 kDa molecules precipitated by these mAbs are identical. We showed that the 110 kDa CD26 structure on human T cells is composed of a family of heterogeneous molecules, as determined by isoelectric focusing studies. In addition, we demonstrated that the CD26 antigen has a DPPIV enzyme activity and this enzyme activity is found only on the principal basic structure of CD26 but not on the additional acidic structures. Biochemical studies also revealed that these mAbs recognized distinct epitopes on the CD26 antigen. Pulse-chase studies showed the the 1F7 epitope was found on both the immature (100 kDa) and mature (110 kDa) forms of the CD26 antigen. On the other hand, the Ta1 and 5F8 epitopes were expressed mainly on the mature form of the CD26 antigen. Moreover, anti-IF7 consistently precipitated an additional 43 kDa molecule in association with the principal 110 kDa molecule. Taken together, these data suggested that the additional 43 kDa structure or the distinct epitope recognized by anti-IF7 may play a role in human T cell activation via the CD3 and CD2 pathways.


Identification of allergenic epitopes on Der f I, a major allergen of Dermatophagoides farinae, using monoclonal antibodies. The antigenic and allergenic structure of Der f I, a major allergen of the house dust mite Dermatophagoides farinae (Df) was investigated by means of a panel of 11 selected monoclonal antibodies (mAb) obtained from BALB/c mice immunized with purified Der f I. The species specificity of these mAb, tested with Der f I and Der p I--the homologous allergen from Dermatophagoides pteronyssinus--was generally restricted to Der f I since 10 out of 11 mAb reacted only with this allergen. Epitope specificity of the mAb was determined by both competitive inhibition and sandwich ELISA experiments. The results indicated the presence of at least four non-overlapping, non-repeated antigenic sites on Der f I, which were recognized by one or several mAb (sites A, B, C and D). Comparative epitope specificity studies between human IgE antibodies and mice mAb were performed, on sera and basophils of Df sensitive patients, using different inhibition assays (ELISA and histamine release experiments). The degree of inhibition varied between the patients and upon the assay design. Most of the mAb tested were found to significantly inhibit the binding of human IgE to Der f I (p less than 0.01) when compared with Der p I specific mAb as a control. The mAb reacting with site A was found to be the most potent inhibitor, presenting a mean inhibition of up to 56% in ELISA as well as in histamine release experiments. The results show that both human IgE antibodies and mAb can be directed against identical or closely related epitopes of Der f I. Therefore anti-Der f I mAb constitute immunologic probes in further allergenic epitope and peptide analysis of this major mite allergen.


Epitope mapping of the carcinoembryonic antigen with various related recombinant proteins expressed in Chinese hamster ovary cells and 25 distinct monoclonal antibodies. Antigenic epitopes of carcinoembryonic antigen (CEA) and non-specific cross-reacting antigen (NCA) were analysed in relation to their domain structures [domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M for CEA and domains N, I (A1-B1), and M for NCA]. We reconstructed cDNAs for CEA-N, CEA-N-I, CEA-N-I-II, CEA-N-I-II-III-M (CEA-whole), NCA-N, NCA-N-I and NCA-N-I-M (NCA-whole), which were expressed in Chinese Hamster Ovary (CHO) cells. The recombinant proteins were purified by immunoadsorption and gel filtration. Their mol. wts judged from Western blotting were 17,000-26,000 for CEA-N, 70,000 for CEA-N-I, 150,000 for CEA-N-I-II, 165,000 for s-CEA-whole which was spontaneously released from cells into culture medium, 180,000 for p-CEA-whole which was solubilized with phosphatidylinositol specific phospholipase C (PI-PLC) from cells, 18,000-25,000 for NCA-N, 63,000 for NCA-N-I, and 96,000 for p-NCA-whole which was solubilized with PI-PLC from cells. The divergence of the observed mol. wts from those calculated from cDNA sequences seems to indicate that these recombinant proteins are highly N-glycosylated. By enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 25 distinct anti-CEA monoclonal antibodies (MAbs), each representative of 25 different subgroups within five groups (Groups 1-5) previously classified by us in terms of the reactivity with CEA and CEA-related antigens. Twenty-one MAbs previously shown to react with different protein epitopes of the CEA molecule allow to define six groups (A-F) of epitopes according to their expression by different domains of the CEA and NCA molecules. Among four epitopes common to CEA and NCA, two were found to be present on domain N (Group A) and two on domain I (Group B). Among 15 epitopes absent from NCA but expressed by CEA and normal fecal antigens (NFAs), four were on domain N (Group C), five on domain I (Group D) and six on domain II (Group E). Two epitopes were previously described as "CEA distinctive", because they were recognized by MAbs reacting with CEA but not with the NFAs. These two epitopes (Group F) were found to be expressed by p-CEA-whole but not by s-CEA-whole. The latter results suggest that the Group F epitopes are located on a part of the domain III close to the anchoring device of the CEA molecule.(ABSTRACT TRUNCATED AT 400 WORDS)


IgE binding structures of the major house dust mite allergen Der p I. The group I allergens Der p I and Der f I are potent allergens of mites from the genus Dermatophagoides. IgE radioimmune dot blots and immunoabsorption with recombinant peptides have been used to define areas of antigenicity. Four linear binding regions comprising residues 15-33, 60-80, 81-94 and 101-111 were found in the N terminal domain and one, 155-187, in the C-terminal domain, but direct evidence for their discontinuous nature is shown. Firstly, the binding activity of residues 60-80 required either C- or N-terminal flanking sequences to express reactivity and secondly a discontinuous determinant was directly demonstrated by the two non-overlapping peptides 53-99 and 101-154 which significantly cross absorbed specificities to one another. This also indicates considerable homogeneity in the antibodies recognising these peptides. The IgE binding peptides could be located to equivalent residues on the X-ray crystallographic structure of the homologous proteins actinidin and papain. The residues 81-94 and 101-111 which gave strong reactivity were located on a flexible loop connecting the domains and represent areas in which synthetic peptides could be expected to retain activity.


New monoclonal antibodies as probes for human cardiac troponin I: epitopic analysis with synthetic peptides. Forty monoclonal antibodies (MAbs) specific for human cardiac troponin I (TnI) were selected to develop a new alternative for specific biological diagnosis of acute myocardial infarction. Using an immunoenzymatic sandwich assay, these MAbs were employed in the mapping of human cardiac TnI and showed six different epitopes. Parts of the TnI peptide sequences were synthesised; the sequences were chosen from the published sequences of mammalian TnI. Immunological assays showed that 8 out of 40 MAbs recognised a RAYATEPHAK (P2) N-terminus cardiac-specific sequence of human TnI. The information obtained from epitopic mapping of TnI and the properties of the peptides allowed pairs of MAbs to be selected for the development of a future specific TnI assay.


Collaborative study using the preincubation Salmonella typhimurium mutation assay for airborne particulate matter in Japan. A trial to minimize interlaboratory variation. A collaborative study has been performed over a period of 3 years to develop a suitable method for monitoring the mutagenicity of airborne particulate matter. The study was organized with 8 laboratories and performed in the following steps: (1) selection of a suitable technique for each process involved in the mutagenicity monitoring, (2) developing a tentative protocol by combining systematically the selected techniques, (3) evaluation of the protocol by intra- and inter-laboratory studies, (4) modification of the protocol according to the evaluation, and (5) evaluation of the modified protocol by conducting an interlaboratory study. We found a suitable method for mutagenicity monitoring of particles in the atmosphere. Airborne particles were sampled with a high-volume sampler, the samples were stored at -80 degrees C, extracted by sonication using dichloromethane, solvent-exchanged, and assayed by the preincubation method using Salmonella typhimurium TA98 and TA100. The observed mutagenic activity was normalized with that of an internal standard. Round robin tests revealed that the method resulted in excellent reproducibility. The coefficient of variation for mutagenic activities of airborne particulate samples collected in various districts of Japan were in the range of 14.7 +/- 6.6% to 19.6 +/- 4.0% for strains TA98 and TA100 with and without metabolic activation. We also found that the plate incorporation method was equivalent to the preincubation method for airborne particulate extracts.


Discrimination of aneuploidogens from clastogens by C-banding, DNA and area measurements of micronuclei from mouse bone marrow. Micronuclei (MN) obtained from mouse bone marrow cells, in vivo exposed to 3 typical clastogens (procarbazine, azathioprine, ethyl methanesulfonate) and 3 typical aneuploidogens (vinblastine, tubulazole, colchicine), were examined for C-band, area and DNA content. C-banding allows a clear discrimination between clastogens and aneuploidogens: the clastogens do not exceed 50% C-band-positive MN and the aneuploidogens all 3 produce 65-75% C-band-positive MN. Concerning the DNA content the percentages of MN containing more DNA than an average chromosome (chr) are lower than 12% for the clastogens and 38-60% for the aneuploidogens. As far as the area of the MN is concerned the percentages of MN which have a larger area than chr are lower than 23% for the clastogens and range from 47% to 71% for the aneuploidogens. Additionally 3 other mutagens were studied. Hydroquinone induces 43% C-band-positive MN with DNA content far below the content of chr; considering the area measurements, however, hydroquinone behaves as an aneuploidogen (65% of the MN are larger than chr). Mitomycin C lies between the clastogens and the aneuploidogens for all 3 criteria but 5-azacytidine is comparable to the model aneuploidogens.


Micronucleated reticulocyte induction by ethylating agents in mice. Six model ethylating agents were tested for clastogenic potency by means of a new technique of the micronucleus assay with mouse peripheral blood cells using acridine orange (AO)-coated slides, to evaluate the test. The alkylating agents were: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), diethylsulfate (DES), ethyl methanesulfonate (EMS), epichlorohydrin (ECH) and ethylene dibromide (EDB). The animals were given a single intraperitoneal injection of the following doses of the chemicals: ENNG and ENU, 25, 50 and 100 mg/kg; EMS and DES, 100, 200 and 400 mg/kg body weight. For EDB and ECH, the doses were 50, 100 and 200 mg/kg, given twice, 24 h apart. Before and after the injection, blood samples were taken from the tails at 24-h intervals up to 72 h and preparations were made on AO-coated slides. For each dose group, 4 animals were used and 1000 reticulocytes were examined per slide for the presence of micronuclei. At the optimum induction time of 48 h, ENU induced micronucleated reticulocytes (MNRETs) at all 3 doses. ENNG and EMS induced MNRETs significantly at 2 dose levels each and DES only at the highest dose. ECH and EDB failed to induce MNRETs. On the basis of the dose of chemical needed to double the spontaneous frequency, the order of clastogenic potency was ENU greater than ENNG greater than EMS greater than DES. The results obtained compared favorably with those from other in vivo methods. The present technique proves to be simple, flexible and relatively sensitive. Shifts in the optimum induction peak in individual animals and by some chemicals can be picked up easily which is important when testing weak mutagens and chemicals with an unknown mechanism of action.


Contributions to the design and statistical analysis of in vivo SCE experiments. Two issues that arise in the design and statistical analysis of in vivo SCE and similar experiments are considered. First, with regard to analysis, the merits of various methods of data transformation are explored in depth. The conclusion drawn is that common transformations of the type studied here seemingly offer little advantage in the assessment of whether a test agent induces SCE in a dose-related manner. Second, a proposal is made for a method to determine, subject to budgetary constraints, the desired numbers of animals/dose group and cells scored/animal. The approach advocated also lends itself to discussions weighing the gains and losses from possible reductions in the number of animals below the 'desired' levels.


Species differences in the genotoxicity of cyclophosphamide and styrene in three in vivo assays. Species differences in dispositional factors such as distribution, metabolism and excretion may often account for species differences in the toxic responses to foreign chemicals. In this study we compared the genotoxic responses of cyclophosphamide (CP) and styrene (ST) between Porton rats and LACA Swiss mice in three in vivo assays (bone marrow micronucleus (MN), sperm morphology (SM) and sister-chromatid exchange (SCE) assays). The sensitivities of the three assays were compared by the doses of the compounds required to elicit a significant genotoxic response. The baseline levels for the MN, SCE and SM assays were 1.1-1.4 and 1.2-1.3 MNPCEs/1000 PCEs, 0.23-0.24 and 0.20-0.21 SCEs/chromosome, 3.5-5.7% and 1.6-1.9% abnormal sperm in mice and rats, respectively. CP was a potent genotoxin in the MN and SCE assays but weakly genotoxic in the SM assay. At comparable doses, the rat was approximately 3-, 2.5- and 1.8-fold more sensitive to CP than mice in the MN, SM and SCE assays, respectively. ST produced weak genotoxic responses in all assays in mice and only in the SM and SCE assays in rats. The mice were more sensitive to ST in the MN and SM assays, while it was difficult to compare the species in the SCE assay. For both compounds the sensitivity of the three assays, in decreasing order, were SCE greater than MN much greater than SM. For CP the relative responses in the Porton rats and LACA Swiss mice were qualitatively similar to previous reports. Although the use of different strains may explain differences between the studies in the magnitude of the responses observed. The results for ST in the rat shows that the choice of genotoxic endpoint can determine whether a response is detectable. Moreover, the discrepancies between the results for ST in this study and others, suggest that as well as using a battery of in vivo tests, it may be prudent to select more that one strain or species to fully assess a compound's ability to produce DNA damage.


Improved high bioactivation cross for the wing somatic mutation and recombination test in Drosophila melanogaster. The two tester strains of the high bioactivation (HB) cross for the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster developed by Frölich and Würgler possess high metabolic capacity to activate promutagens. These strains contain chromosomes 1 and 2 of the DDT-resistant stock Oregon R(R) which exhibits a high constitutive level of cytochrome P450. However, they show several disadvantages for routine application, such as disturbed wing hair patterns in certain areas of the wing, making spot classification difficult, and a delay in development of the larvae. We have established and evaluated an improved HB cross (ORR; flr3 females and mwh males) producing ORR heterozygous individuals. These develop normally and have a normal, undisturbed wing hair pattern while exhibiting high bioactivation. The hybrid larvae of the improved HB cross show P450-dependent bioactivation capacity equal to or even slightly higher than those of the original HB cross. This was demonstrated by measuring the genotoxic activity of the promutagens diethylnitrosamine, 7,12-dimethylbenz[a]anthracene, N-nitrosopyrrolidine, and urethane. In addition, the improved HB cross has a sensitivity to the direct-acting alkylating agent ethyl nitrosourea equal to that of the standard cross. The main advantage of the improved HB cross is to combine the high bioactivation capacity with the ease of scoring the wings using the same criteria as for the standard cross.


Micronuclei and other nuclear anomalies in buccal smears: methods development. Laboratory work aimed at improving the epidemiologic utility of an innovative genotoxicity assay is described. The exfoliated cell micronucleus assay involves microscopic analysis of epithelial smears to determine the prevalence of micronucleation, an indicator of structural or numerical chromosome aberrations. While the assay holds promise for the study of epithelial carcinogens, it is hampered by the fact that exfoliated cells are moribund and undergo degenerative phenomena that can produce extranuclear objects difficult to distinguish from classical micronuclei. Modifications in the protocol were assessed in sample buccal smears from several study populations: radiotherapy patients, nonusers of tobacco, and snuff users. Refinements in micronucleus scoring criteria and the inclusion of other nuclear anomalies in the scoring system are proposed. We demonstrate that our criteria are successful in detecting excess micronucleation in positive controls. We also provide evidence that other nuclear anomalies are at least as common as micronucleation and that therefore there is the potential for extensive misclassification. Reliability was assessed in duplicate readings.


Application of an epithelial liver cell line, metabolically competent, for mutation studies of promutagens. Recently numerous attempts have been made to reduce the use of vertebrate animals in laboratory experiments to evaluate general and acute toxicity, mutagenesis and teratogenesis of new drugs or chemicals. One common approach is to use established, proliferating cell lines that preserve differentiated functions such as the competence to metabolize xenobiotics. To this end a continuous Chinese hamster epithelial liver cell line (CHEL cells) was established, cultured as used for mutagenesis studies. Structurally different promutagens, such as 7,12-dimethylbenz[a]anthracene (7,12-DMBA), benzo[a]pyrene (B(a)P), aflatoxin B1 (AB1) and cyclophosphamide (CP), were used in order to check and validate the test system. anti-Chrysene-1,2-diol 3,4-epoxide (CDE) and mitomycin C (MMC) were taken as representatives of direct mutagens. The genetic change induced by the mutagens was quantified by measuring mutation frequencies at the HGPRT locus. Several parameters, such as mutant expression time for each chemical, cell density for selection of mutants and enzymatic characterization for HGPRT phenotype, were examined to establish the optimal assay conditions. All promutagens analyzed significantly affected either the cloning efficiency and/or the mutant frequency of CHEL cells after 24 h of exposure. In addition, various enzyme activities involved in the metabolism of the promutagens were determined in CHEL cells, under the experimental conditions of chemical exposure used in the mutagenesis assay. The enzyme activities were compared with those found in uninduced Chinese hamster liver.


Mutagenicity assay for nitroarenes of air pollutants held in leaves of woody plants. A new method was developed for the detection of mutagenic nitroarenes held in the leaves of woody plants using a small amount of leaves. This method consists of extraction of mutagen from fresh leaves (15-30 g) by ultrasonication with ethyl acetate and purification by elution with benzene on a silica gel column to remove such inhibitory components for mutagenesis as chlorophyll. The mutation assay uses the new Salmonella typhimurium strains YG1021 (a nitroreductase-overproducing strain of TA98) and YG1024 (an O-acetyltransferase-overproducing strain of TA98), which are very sensitive to some nitroarenes in the absence of S9, and standard strain YG1020 (TA98 containing pBR322-Aps) for comparison. This method was applied to woody plants growing on various sites in the Tokyo metropolitan area and the suburbs. It was shown that the leaves of all woody plants tested contained different amounts of mutagens, probably mutagenic nitroarenes, depending upon their growing sites.


Biphenyl and fluorinated derivatives: liver enzyme-mediated mutagenicity detected in Salmonella typhimurium and Chinese hamster V79 cells. Hepatocarcinogenic polychlorinated and polybrominated biphenyls usually show negative results in in vitro mutagenicity assays. Problems in their testing result from their low water solubility and their slow rate of metabolism. We therefore investigated better soluble model compounds, namely biphenyl and its 3 possible monofluorinated derivatives. In the direct test, these compounds proved to be nonmutagenic in Salmonella typhimurium TA98 and TA100 (reversion to histidine prototrophy) and in Chinese hamster V79 cells (acquisition of resistance to 6-thioguanine). However, when the exposure was carried out in the presence of NADPH-fortified postmitochondrial fraction of liver homogenate from Aroclor 1254-treated rats, all 4 compounds showed mutagenic activity in V79 cells. 3-Fluorobiphenyl produced strong mutagenic effects in S. typhimurium TA100 as well, whereas the other biphenyls were inactive. In strain TA98, 3- and 4-fluorobiphenyl showed mutagenic activity. This mutagenicity was enhanced in the presence of 1,1,1-trichloropropene 2,3-oxide, an inhibitor of microsomal epoxide hydrolase, thus suggesting that epoxides may be active metabolites.


Differential effect of the amino acid cystine in cultured mammalian and bacterial cells exposed to oxidative stress. The effect of cystine in the cytotoxic response of cultured Chinese hamster ovary and Escherichia coli cells to challenge with hydrogen peroxide has been investigated. It was found that this amino acid could either protect or sensitize cells, depending on the cellular system. In fact, although a reduction in the growth-inhibitory effect of hydrogen peroxide was observed in mammalian cells, a marked increase in the susceptibility to oxidative stress was induced by cystine in bacteria. None of the amino acid precursors of glutathione, e.g., glutamate, glycine or cysteine, afforded protection in the mammalian cell system, whereas cysteine, but not glycine or glutamate, markedly sensitized bacteria to hydrogen peroxide-induced cell killing. In mammalian cells, methionine, an amino acid which is converted to cysteine, was also unable to modify the oxidative response. The results presented indicate that cystine displays differential effects in oxidatively injured mammalian or bacterial cells and suggest that the mechanism whereby the amino acid modulates the lethal action of hydrogen peroxide differs in the two cellular systems.


D and C Red No. 9: genotoxic or non-genotoxic carcinogen? The azo-compound, D and C Red No. 9 was assayed for genotoxicity in vivo using the rat micronucleus test and the rat ex vivo liver UDS assay. Uniformly negative results were obtained in both assays, even though large oral doses were used (2 g/kg). These results suggest that the tumorigenic effects of this compound in rats are mediated through non-genotoxic rather than a genotoxic mechanism. Further experiments using additional end-points such as 32P-post-labelling would further substantiate this conclusion.


Sister-chromatid exchange analysis in a rural population of Mexico exposed to pesticides. Cytogenetic damage was evaluated by means of the analysis of sister-chromatid exchange (SCE) in a rural population of Tlaxcala, Mexico, in occupational contact with pesticides. We studied 170 men, 94 exposed and 76 not exposed. It was shown that SCE followed a normal distribution and Student's t test did not present differences between the two groups (P = 0.4). The frequency of SCE was not correlated with the duration of exposure of the rural workers (r = -0.06), the multiple covariance analysis applied to the data of duration of exposure, tobacco intake and alcohol ingestion demonstrated a lack of statistical significance. In the exposed people we observed no symptoms provoked by these compounds.


The correlation between the frequency of micronuclei and specific chromosome aberrations in human lymphocytes exposed to microwave radiation in vitro. Human whole-blood samples were exposed to continuous microwave radiation, frequency 7.7 GHz, power density 0.5, 10 and 30 mW/cm2 for 10, 30 and 60 min. A correlation between specific chromosomal aberrations and the incidence of micronuclei after in vitro exposure was observed. In all experimental conditions, the frequency of all types of chromosomal aberrations was significantly higher than in the control samples. In the irradiated samples the presence of dicentric and ring chromosomes was established. The incidence of micronuclei was also higher in the exposed samples. The results of the structural chromosome aberration test and of the micronucleus test were comparatively analyzed. The values obtained showed a positive correlation between micronuclei and specific chromosomal aberrations (acentric fragments and dicentric chromosomes). The results of the study indicate that microwave radiation causes changes in the genome of somatic human cells and that the applied tests are equally sensitive for the detection of the genotoxicity of microwaves.


Detection of aneuploidy in male germ cells of mice by means of a meiotic micronucleus assay. The possibility of using a micronucleus (MN) assay in mouse germ cells for the identification of aneuploidogenic agents was evaluated by comparing the pattern of effects induced by 4 chemicals with different mechanisms of action (adriamycin, ADM; mitomycin C, MMC; chloral hydrate, CH; ethylenedinitrilotetraacetic acid, EDTA). The results obtained after treatment of spermatocytes at the premeiotic S-phase (preleptotene) indicated that only clastogenic agents (ADM and MMC) were able to induce MN at this cell stage. Previous data obtained with the same compounds demonstrated by contrast that the micronucleus spermatid assay may detect both clastogenic and aneuploidogenic effects after treatment of diakinesis/MI/MII cells. Analysis of MN size distributions, in the present and previous spermatid samples, revealed that the clastogens ADM and MMC produced relatively more small MN than CH and EDTA. These data are in agreement with the proposed mechanism of action of the chemicals tested.


Mutagenicity of nitric oxide and its inhibition by antioxidants. Nitric oxide (NO) is produced both by macrophages in vivo as a physiological response to infection and by a variety of cell types as an intercellular messenger. In addition, NO and nitrogen dioxide (NO2) are significant components of many combustion processes. The ubiquitous exposure of humans to nitrogen oxides (NOx), both endogenously and exogenously, may play a significant role in the carcinogenic process due to nitrosation of amines by NOx. We report here that exposure to low concentrations of NO, alone or in combination with NO2, results in significantly enhanced mutation in Salmonella typhimurium TA1535 using a modified Ames Salmonella reversion assay. The observed mutagenicity requires that the bacteria be actively dividing at the time of exposure to NO or NO2, suggesting that the nitrogen oxides, or their reaction products, function as direct-acting mutagens and that the induced lesion is easily repairable by non-dividing cells. Exposure to NO resulted in a time- and dose-dependent increase in the number of revertants approximately proportional to the square of the NO concentration from 0 to 20 ppm. NO was a more effective mutagen relative to NO2, however, the observed requirement for O2 suggests limited oxidation of NO (presumably to NO2) is necessary. Numerous lipid- and aqueous-phase inhibitors of nitrosation, as well as a number of other general antioxidants and free-radical trapping agents, were examined for their effectiveness in blocking the mutagenic effects of NO. The mutagenic activity of NO was most effectively inhibited by beta-carotene and tocopherols. BHT, dimethyl sulfoxide and mannitol also blocked the mutagenic effects of NOx but appeared less effective than beta-carotene or vitamin E, while ascorbate was ineffective as an inhibitor of mutation resulting from NO exposure.


Ozone is not mutagenic in the Tradescantia and tobacco mutagenicity assays. Ozone fumigation of a double heterozygous chlorophyll mutant Nicotiana tabacum var. xanthi n.c. with concentrations up to 300 nl/l and of a heterozygous Tradescantia clone 4430 with concentrations up to 800 nl/l did not increase the frequency of somatic mutations above the spontaneous levels. However, ozone fumigation at these concentrations led to distinct physiological damage to plant tissues.


The role of the excision and error-prone repair systems in mutagenesis by fluorinated quinolones in Salmonella typhimurium. Patterns of reversion produced by ciprofloxacin, enoxacin and ofloxacin in Salmonella typhimurium strains carrying the hisG428 ochre mutation have been studied. These fluorinated quinolones produce a significant increase in reversion of this mutation, even when it is located on the chromosome. Nevertheless, reversion is higher when the hisG428 mutation is on the multicopy plasmid pAQ1 than when it is on the chromosome. Reversion of hisG428 induced by fluorinated quinolones is abolished both in a uvrB genetic background and in the absence of the plasmid pKM101. Therefore, mutagenesis produced by fluorinated quinolones in the Salmonella mutagenicity assay is significantly affected by both the excision repair and the error-prone repair systems. Furthermore, fluorinated quinolones are also detected as moderate mutagens with the base substitution hisG46 mutation when both repair systems are functional in the tester strain.


N-methyl-N'-nitro-N-nitrosoguanidine-induced light emission in Chinese hamster cell cultures: correlation with enhancement of chromosomal aberrations. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) was found to induce an ultraweak photon emission in cultures of Chinese hamster fibroblasts (CHL). Measurements suggest that the light emission is due to a reaction between MNNG and cellular metabolites. The light emission depended on the concentration of MNNG and was oxygen-dependent, disappearing in a nitrogen atmosphere. Superoxide dismutase (SOD) or sodium azide decreased the emission intensity. The production of chromosomal aberrations in CHL by MNNG was correlated with the light emission intensity and was inhibited in the presence of SOD.


Construction of a umuDC operon substitution mutation in Escherichia coli. Using a specialized transducing lambda phage, the umuDC operon of Escherichia coli was deleted and replaced with the chloramphenicol acetyltransferase gene. The delta (umuDC)595::cat mutation was subsequently transferred by generalized P1 transduction into a variety of genetic backgrounds. It is concluded that the UmuDC proteins, which are normally required for inducible mutagenesis, are not essential for cell survival.


The movement of fluorescent endocytic tracers in Plasmodium falciparum infected erythrocytes. The fluorescent lipophilic probe 1,1'-dihexadecyl-3-3'-3-3'- tetramethylindocarbocyanine (diIC16) inserted in the red cell surface, functioned as a non-exchangeable lipid marker which was not metabolised or toxic in plasmodial cultures. Invasion by Plasmodium falciparum resulted in the internalisation of the lipid, suggesting the uptake of red cell membrane components during parasite entry. The fluorescent lipid was not transported from red cell to parasite membranes at subsequent stages of development, but label in the erythrocyte-derived parasitophorous vacuole was eventually detected in daughter merozoites. Fluorescent dextrans of 10 kDa in the extracellular medium were also not internalised during intraerythrocytic parasite growth. The results support that with the exception of the invasion step, plasmodial infection does not induce endocytosis in the erythrocyte membrane. Despite the lack of endocytosis, both D and L stereoisomers of the head group blocked phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) inserted in the erythrocyte membrane, were internalised by mature infected erythrocytes. Lipid internalisation occurred by a non head group dependent parasite mechanism, which could account for the stage-specific uptake of phospholipids observed in mature infected erythrocytes. We were unable to detect the transport of carbocyanine dyes and N-NBD-PE from intracellular parasites back to the erythrocyte membrane. Additionally, the carbocyanine dyes were not transferred between adjacent organisms in a double infected red cell. The data argue for the absence of bulk membrane lipid transport between individual parasites and their host cell bilayer in an infected erythrocyte.


[Spinal epidural stimulation for central pain caused by spinal cord lesion]. Epidural spinal cord stimulation was carried out in 4 patients with denervation caused by spinal cord lesion, and we reviewed previously reported cases. Initial result showed at 1 week in 100% of our cases, but about 1/3 of the cases, even those with the same denervation caused by spinal cord lesion, had no pain relief at this stage in previously reported cases. In our cases, excellent pain relief was gained temporarily, even though the painful area and the spinal cord lesion were separated somatotopically in 2 cases (case 3, 4). Temporary success bore no relationship to quality and duration of pain. In all cases except case 1, a rapidly decreasing effectiveness was noted, and finally no pain relief was gained at all after 4, 3 and 5 months, respectively. In case 1 there was persistent pain relief estimated at 70-80% after 19 months, only when the spinal cord was stimulated. Epidural stimulation also produced sensations in the painful area. Spinal cord stimulation would suppress at least the dorsal horn neurons which were destroyed by various kinds of diseases. A decline in effectiveness with time would occur due to essential causes of the deafferentation pain, such as anatomical and regeneration factors.


[AFP producing gastric cancer manifested by metastasis to the tentorium cerebelli; case report and review of the literature]. We report a case of AFP producing gastric cancer manifested by metastasis to the tentorium cerebelli. A 66-year-old male patient was admitted with dysarthria, occipital headache and nausea on May 1, 1990. Neurological examination revealed signs of increased intracranial pressure and the right-sided cerebellar hemispheric signs. CT and MRI showed a round tumor shadow 3cm in diameter, which originated in the right-side tentorium cerebelli and grew in the posterior fossa. Tumor stains fed by the right tentorial artery were recognized by angiography. Serum AFP level was 503.5ng/ml. The patient underwent an operation under general anesthesia in the prone position. The tumor was totally removed via the suboccipital transtentorial approach. Histological examination revealed AFP producing adenocarcinoma. The patient was found to have a gastric cancer after neurosurgical operation, and underwent subtotal gastrectomy by surgeons. Serum AFP level was 254.5ng/ml after removal of metastatic brain tumor, and 5.0ng/ml after subtotal gastrectomy.