bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of human polymorphonuclear leukocyte oxidative metabolism by type 1 pili from Escherichia coli
Authors: Goetz M B , Silverblatt F J
Published: January 2020

We compared the degree to which Escherichia coli phase variants which do (T1P+ E. coli) or do not (T1P- E. coli) express type 1 pili (T1P) stimulate human polymorphonuclear leukocyte (PMN) oxidative activity. Unopsonized T1P+ E. coli stimulated the release of 0.20 to 0.24 nmol of H2O2 per 10(6) PMN per min and the consumption of 1.4 to 4.0 nmol of O2 per 10(6) PMN per min; no measurable PMN oxidative activity was stimulated by unopsonized T1P- E. coli. In the presence of serum opsonins, T1P+ E. coli stimulated the release of 1.12 to 1.16 nmol of H2O2 per 10(6) PMN per min and the consumption of 5.0 to 6.0 nmol of O2 per 10(6) PMN per min, whereas T1P- E. coli stimulated the release of 0.42 to 0.43 nmol of H2O2 per 10(6) PMN per min and the consumption of 0.6 to 2.0 nmol of O2 per 10(6) PMN per min. Although unaggregated T1P did not stimulate PMN, latex beads coated with T1P (T1P-latex) stimulated alpha-methylmannoside-inhibitable, opsonin-independent PMN oxidative activity. The activity stimulated by either T1P+ E. coli or T1P-latex was susceptible to inhibition by cytochalasin B. Latex particles coated with bovine serum albumin or mannose-resistant pili did not stimulate PMN. These data indicate that T1P+ E. coli stimulate PMN oxidative metabolism more effectively than do T1P- E. coli and that a similar PMN oxidative response follows cellular stimulation by either unopsonized T1P+ or opsonized T1P- E. coli. Furthermore, T1P-latex faithfully mimics the ability of T1P+ E. coli to stimulate PMN oxidative metabolism. Such particles may be useful in further analyses of cellular responses to T1P+ E. coli.
Fluid accumulation in infant mice caused by Vibrio hollisae and its extracellular enterotoxin
Authors: Kothary M H , Richardson S H
Published: January 2020

Vibrio hollisae, a halophilic bacterium isolated from patients with diarrhea, was examined for virulence factor production. Intragastric administration of 2 X 10(7) CFU per mouse elicited fluid accumulation which peaked at ca. 6 h postchallenge in infant mice. An enterotoxin which elongated Chinese hamster ovary (CHO) cells was detected in extracts of infected-mouse intestines and in culture fluids from various growth media. The yield of the enterotoxin was maximal beginning at the onset of the stationary phase of growth in heart infusion broth supplemented with 0.5% NaCl. A concentrated preparation obtained by ammonium sulfate precipitation of culture supernatant fluids induced intestinal fluid accumulation which peaked at 2 h postchallenge in infant mice. The abilities of the enterotoxin preparation to elongate CHO cells and to elicit fluid accumulation in infant mice were inseparable by gel filtration, isoelectric focusing, and hydrophobic interaction chromatography. The enterotoxin has a molecular weight of ca. 33,000 by gel filtration and an isoelectric point of ca. 4 and is sensitive to heat.
Induction of delayed-type hypersensitivity to Leishmania major and the concomitant acceleration of disease development in progressive murine cutaneous leishmaniasis
Authors: Dhaliwal J S , Liew F Y
Published: January 2020

BALB/c mice injected intradermally with 10(5) or higher doses of formaldehyde-fixed promastigotes (FFP) of Leishmania major developed strong delayed-type hypersensitivity (DTH) to leishmanial antigens injected into the hind footpad 3 to 10 days later. The DTH peaked 15 to 18 h after footpad injection and disappeared by 48 h. This specific DTH correlated with the homing of 51Cr-labeled syngeneic bone marrow cells and the infiltration of proliferating cells to the site of antigen administration. Spleen cells from FFP-sensitized mice also gave significant proliferative response to FFP in vitro. The DTH was adoptively transferable by Lyt-1+2-L3T4+ T cells and was H-2 restricted. DTH could be substantially enhanced by pretreatment with cyclophosphamide or pertussigen. Such DTH enhancement was accompanied by concomitant exacerbation of disease progression after L. major infection. Mice injected intravenously with FFP developed substantial immunity to cutaneous leishmaniasis but specifically suppressed DTH reactivity. Treatment of mice with pertussigen before intravenous immunization, however, abolished the protection and reversed the suppression of DTH. These results therefore demonstrate that the early-appearing type of DTH is not involved in host protection but that it actually facilitates disease progression in cutaneous leishmaniasis. Further evidence, which also shows the nonspecific nature of this disease exacerbation, is provided by local cell transfer experiments. Splenic T cells from mice sensitized to keyhole limpet hemocyanin or FFP induced significantly larger lesions compared with normal T cells when they were transferred into the footpad together with specific antigen and L. major promastigotes.
Detection of specific antibody in adult human periodontitis sera to surface antigens of Bacteroides gingivalis
Authors: Naito Y , Okuda K , Takazoe I
Published: January 2020

Sera from adult periodontitis patients were analyzed for antibody activity against surface antigens of Bacteroides gingivalis by immunoblotting analysis.
Presence of high concentrations of circulating Toxoplasma antigens during acute Toxoplasma infection in athymic nude mice
Authors: Suzuki Y , Kobayashi A
Published: January 2020

In mice infected with an avirulent strain of Toxoplasma gondii, circulating Toxoplasma antigens were detectable in the sera during weeks 1 to 3 of infection by a simple agglutination test that uses latex particles coated with anti-Toxoplasma antibodies. An infection in athymic nude mice resulted in high agglutination titers in the anti-Toxoplasma antibody-coated latex particle test and the absence of anti-Toxoplasma antibodies in sera during the acute phase, suggesting that the detection of circulating Toxoplasma antigens is a good tool for the diagnosis of acute toxoplasmosis, especially in severely immunocompromised hosts.
Triton X-114 phase fractionation of an integral membrane surface protein mediating monoclonal antibody killing of Mycoplasma hyorhinis
Authors: Riethman H C , Boyer M J , Wise K S
Published: January 2020

A previously defined immunoglobulin M(kappa) monoclonal antibody reacting with a surface epitope of Mycoplasma hyorhinis is shown in this report to mediate specific, complement-dependent mycoplasmacidal activity. Immunoblot analysis of mycoplasma components and their tryptic cleavage products showed that the epitope recognized was present on a protein with an apparent molecular weight of 23,000 (p23) and on a limit tryptic fragment of this protein with an apparent molecular weight of 18,000 (p18). Both p23 and p18 are shown by Triton X-114 phase fractionation to partition efficiently into the hydrophobic detergent phase. Other antigens bearing epitopes not expressed at the cell surface were present among the numerous hydrophilic proteins found in the aqueous phase. The external orientation and membrane association of the p23 antigen were further established by demonstrating that trypsin treatment of intact mycoplasmas generated the antigenic p18 fragment, which remained tightly associated with the organism. These results localize an epitope responsible for antibody-mediated mycoplasma killing onto a specific, surface-exposed region of an integral membrane protein of this organism. Since the monoclonal antibody used in this study does not bind to the surface of all strains of M. hyorhinis, the epitope identified also defines a structural marker of antigenic surface variation within this species, a feature previously observed during serological classification of the organism. Analysis of the antigenic and structural features of the p23 surface antigen may therefore be useful in establishing mechanisms of surface antigen variation among integral membrane proteins of mycoplasmas that could dictate important antigenic characteristics recognized during chronic disease caused by these agents.
Identification and purification of a recombinant Treponema pallidum basic membrane protein antigen expressed in Escherichia coli
Authors: Dallas W S , Ray P H , Leong J , Benedict C D , Stamm L V , Bassford P J
Published: January 2020

A recombinant plasmid designated pLVS3 previously was described that harbored a 14-kilobase insert of Treponema pallidum genomic DNA. Escherichia coli maxicells programmed with this plasmid synthesized three treponemal protein antigens of molecular weights 39,000, 35,000, and 25,000 (39K, 35K, and 25K proteins, respectively). In this study, a detailed deletion analysis of pLVS3 demonstrated that the genetic information for all three protein antigens is contained within a 1.5-kilobase EcoRI-HpaI restriction fragment. The DNA sequence of this fragment revealed a single open reading frame of 361 codons that most likely encodes a signal peptide-bearing precursor to the 39K protein that can be transiently detected in E. coli maxicells. Evidence indicated that the 35K and 25K protein antigens are derivatives of the larger protein and are only produced in maxicells. A significant elevation in expression of the 39K treponemal protein antigen in E. coli was obtained by using the E. coli lpp and lac promoters and a genetic construction in which the signal peptide and first four residues of the "mature" 39K protein were replaced by six amino acids encoded by the vector. This hybrid protein exhibited an unusually high pI, which greatly facilitated its purification to homogeneity. By using antibody prepared against the hybrid protein, the native treponemal protein counterpart, also of molecular weight 39,000, was identified as a membrane component of T. pallidum. Since the native protein also exhibited a net positive charge, it has been designated the T. pallidum basic membrane protein.
Immune response to the B oligomer of pertussis toxin
Authors: Arciniega J L , Burns D L , Garcia-Ortigoza E , Manclark C R
Published: January 2020

Immunization of mice with the B oligomer of pertussis toxin induced antibodies to the native toxin as measured by an enzyme-linked immunosorbent assay. These antibodies neutralized the ability of pertussis toxin to alter the morphology of Chinese hamster ovary cells. Furthermore, mice immunized with the B oligomer, when challenged with pertussis toxin, did not exhibit the leukocytosis normally associated with exposure to the toxin. These results demonstrate that the B oligomer, which does not contain the enzymatic activity of the holotoxin, can be used to induce a neutralizing antibody response and suggest that the B oligomer might be considered for use in acellular pertussis vaccines.
Cloning and expression in Escherichia coli of the Ibc protein genes of group B streptococci: binding of human immunoglobulin A to the beta antigen
Authors: Cleat P H , Timmis K N
Published: January 2020

Total-cell DNA isolated from a highly virulent serotype Ic strain of a group B streptococcus was used to construct a gene bank with bacteriophage lambda L47.1 in Escherichia coli K-12. Recombinant phage plaques in the bank were immunoblotted by using anti-alpha- and anti-beta-specific antibodies directed towards the Ibc proteins purified from the streptococcal cell surface, and hybrid phages expressing the alpha protein (lambda alpha +) and the beta protein (lambda beta +) were identified. DNA inserts in these phages were subcloned into E. coli high-copy-number plasmid vectors to produce stable alpha + (pPHC10) and beta + (pPHC8 and pPHC33) recombinant plasmids, and restriction maps of the cloned streptococcal sequences were constructed. Antibodies against the two Streptococcus-derived proteins reacted with high-molecular-weight polypeptides made in E. coli cells carrying the corresponding hybrid plasmids and with several degradation peptides from them. A 190-kilodalton alpha protein, previously undetected, was identified; this species may be the native alpha protein or a precursor of it. In addition, mutagenesis of the cloned sequences was carried out by using the omega fragment to determine the direction of transcription. In E. coli, the beta protein, but not the alpha protein, bound human immunoglobulin A (IgA) in Western blots, and neither protein bound IgG or IgM.
Metabolism of Mycobacterium leprae in macrophages
Authors: Ramasesh N , Hastings R C , Krahenbuhl J L
Published: January 2020

The incorporation of 14C-labeled palmitic acid ( [U-14C]PA) into the phenolic glycolipid-I (PGL-I) fraction of Mycobacterium leprae was studied in a murine macrophage system in vitro. Peritoneal macrophages from Swiss Webster mice were infected with fresh viable or Formalin-killed M. leprae harvested from infected footpads of nu/nu mice, and [U-14C]PA was added to the culture medium. Labeled glycolipid synthesized by live M. leprae was fractionated on a Florisil-silicic acid column and identified as PGL-I by using thin-layer chromatography and localization on a polysulfone membrane with an anti-PGL-I monoclonal antibody. Increased incorporation of [U-14C]PA into the PGL-I fraction was observed in macrophages infected with only live M. leprae. Treatment of the infected macrophages with rifampin caused a significant reduction in the incorporation of palmitic acid into PGL-I. These preliminary studies suggest that PGL-I synthesis can be used to quantitate the metabolism of M. leprae in macrophages in vitro.
Expression of the S-1 catalytic subunit of pertussis toxin in Escherichia coli
Authors: Barbieri J T , Rappuoli R , Collier R J
Published: January 2020

The S-1 subunit of pertussis toxin was expressed as a fusion protein in a strain of Escherichia coli deficient in protein degradation. The fusion protein reacted with anti-pertussis toxin antibody, and, like authentic pertussis toxin, it ADP-ribosylated a 41,000-molecular-weight membrane protein from human erythrocytes.
Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type IV, V, and VI antigens, group 3,4 antigen, and an epitope common to all Shigella flexneri and Shigella dysenteriae type 1 stains
Authors: Carlin N I , Lindberg A A
Published: January 2020

Monoclonal antibodies reactive with Shigella flexneri O antigens were generated in both mouse and rat systems. Antibody-producing hybridomas were screened in an enzyme-linked immunosorbent assay using chemically defined lipopolysaccharides as antigens, and the epitope specificities were determined with a panel of lipopolysaccharides and synthetic O-antigen-specific glycoconjugates as antigens. To verify the specificity seen in the enzyme-linked immunosorbent assay, the antibodies were used in agglutination against a large number of S. flexneri strains. Monoclonal antibodies with the following specificities were identified: type, antigen IV (reactive with serotype 4a and 4b bacteria); type antigen V (reactive with serotype 5a and 5b bacteria); type antigen VI (reactive with serotype 6 bacteria); group antigen 3,4(reactive with serotype 1a, 2a, 3b, 4a, 5a, and Y bacteria); and group antigen 1 (reactive with an epitope present on all S. flexneri and Shigella dysenteriae type 1 bacteria). Furthermore, a monoclonal antibody defining a new O-antigenic epitope present on some S. flexneri strains of serotypes 4a, X, and Y was characterized (4X). The monoclonal antibodies analyzed in this study define epitopes described by polyclonal antisera (type antigens IV, V, and VI), define a hitherto uncharacterized epitope (group antigen 1), and finally identify new epitopes in what has previously been considered as one epitope (group antigen 3,4 and type antigen IV). These immunochemically characterized monoclonal antibodies may have a powerful potential in studies of the importance of humoral immunity in shigellosis.
Chemiluminescent responses of alveolar macrophages from normal and Mycobacterium bovis BCG-vaccinated rabbits as a function of age
Authors: Chida K , Myrvik Q N , Leake E S , Gordon M R , Wood P H , Ricardo M J
Published: January 2020

Luminol-dependent chemiluminescence (CL) responses of alveolar macrophages (AM) from normal and Mycobacterium bovis BCG-vaccinated infant and adult rabbits were compared. AM from 1-, 7-, and 14-day-old normal rabbits exhibited much lower peak CL responses than did AM from 28- and 42-day-old normal animals as well as rabbits 2 to 3 or 5 to 6 months and 1 to 2 years of age. The most striking differences among AM from infant and adult rabbits were noted when AM were obtained from 28-day-old and 5- to 6-month old rabbits 21 days after the rabbits were immunized with 200 micrograms of BCG intravenously. In this case, AM from 5- to 6-month-old animals gave peak counts per minute of 400,000 to 500,000 whereas AM from 28-day-old rabbits vaccinated with BCG (harvested at 49 days of age) gave peak counts per minute of only 40,715 +/- 2,688. These data reveal that AM from neonatal animals are grossly deficient as responders to phorbol myristate acetate-induced CL. This deficiency, which improved with age, is still apparent in AM from 28-day-old animals. The data also reveal that BCG vaccination of 28-day-old animals yields AM that are poor responders to phorbol myristate acetate compared with AM from BCG-vaccinated animals 2 to 3 and 5 to 6 months of age. AM from animals vaccinated with BCG at 28 days of age contained fewer and smaller electron-dense lysosomelike structures than did AM from adult rabbits similarly vaccinated. These findings provide an explanation for the difficulties infants have in developing effective cell-mediated immune responses against intracellular parasites.
Elimination of group A streptococcal cell walls from mammalian tissues
Authors: Gilbart J , Fox A
Published: January 2020

The precise levels in mammalian tissues of rhamnose (a constituent of the group-specific polysaccharide) and muramic acid (a constituent of peptidoglycan) at various times after systemic administration of streptococcal cell walls were noted for the first time. The extreme resistance of the streptococcal cell wall to degradation by mammalian enzymes can now be clearly appreciated. The results help explain the chronic nature of certain inflammatory diseases elicited by streptococcal cell wall debris.
Response of inbred mice to aerosol challenge with Mycobacterium tuberculosis
Authors: Musa S A , Kim Y , Hashim R , Wang G Z , Dimmer C , Smith D W
Published: January 2020

An autosomal dominant gene (Bcg), which maps to mouse chromosome 1, has been shown to confer on mice resistance to attenuated Mycobacterium bovis BCG Montreal, Salmonella typhimurium, and Leishmania donovani. Most animal models used for the study of the Bcg gene have involved intravenous injection of a large number of microorganisms (greater than 10(4) CFU). The present study examines the effect of the Bcg gene on the resistance of inbred mice to challenge via the respiratory route with 5 to 10 CFU of virulent Mycobacterium tuberculosis. The number of tubercle bacilli recovered from the lung lobes indicates that the growth kinetics of the microorganism did not differ between BCG-resistant and BCG-susceptible strains of mice. The number of tubercle bacilli recovered from the spleen was also similar among strains. Although there were reproducible differences in the time of first recovery of bacilli from the spleen, these differences appeared to be unrelated to the expression of the Bcg gene. When mice were challenged with purified protein derivative, all strains responded similarly as observed by measurements of footpad swelling.
Comparison of the genetic determinant coding for the S-fimbrial adhesin (sfa) of Escherichia coli to other chromosomally encoded fimbrial determinants
Authors: Ott M , Schmoll T , Goebel W , Van Die I , Hacker J
Published: January 2020

DNA probes specific for different regions of the S-fimbrial adhesin (sfa) determinant were constructed and hybridized with DNA sequences coding for P (F8 and F13), mannose-sensitive hemagglutinating type 1 (F1A), and F1C fimbriae. While the sfa and F1C DNA determinants exhibited homology along their entire lengths, the P-fimbrial and type 1-fimbrial determinants exhibited homology to regions of the sfa cluster responsible for the control of transcription and, to a minor extent, to regions coding for proteins involved in biogenesis and/or adhesion of the fimbriae and for the N-terminal part of the fimbrillin subunit.
Antigenic and structural differences among six proteins II expressed by a single strain of Neisseria gonorrhoeae
Authors: Barritt D S , Schwalbe R S , Klapper D G , Cannon J G
Published: January 2020

Gonococci express a family of related outer membrane proteins designated protein II (P.II), which undergo both phase and antigenic variation. Six P.II proteins have been identified in strain FA1090. We developed monoclonal antibodies specific for each P.II protein. Using these antibodies as probes, we purified the six different P.II proteins of this strain. Despite the relatedness of the proteins, we could not purify all of them by a single purification scheme. Four P.II proteins were purified by chromatofocusing, and the remaining two proteins were purified by hydrophobic interaction chromatography on phenyl-Sepharose. The N-terminal amino acid sequence of the proteins showed a high degree of sequence conservation. However, there was variability at specific amino acid residues, giving each P.II protein a unique N-terminal amino acid sequence. Thus P.II proteins of one strain differ among themselves not only in antigenic determinants and primary structure, but also in other characteristics affecting their properties in different chromatographic systems.
Infectivity and neutralization of Cryptosporidium parvum sporozoites
Authors: Riggs M W , Perryman L E
Published: January 2020

Cryptosporidiosis, a diarrheal disease of calves and humans caused by the coccidian parasite Cryptosporidium parvum, is terminated in hosts with normal immune systems. To assess the mechanisms of immunity in cryptosporidiosis, it is necessary to isolate and quantitate sporozoites, the infective stage of Cryptosporidium spp. Here we report the (i) separation of infective C. parvum oocysts from calf feces by ether extraction, sieving, and hypochlorite treatment; (ii) separation of viable C. parvum sporozoites from intact and excysted oocysts by anion-exchange chromatography; and (iii) quantitation of sporozoite infectivity in vivo by direct intraintestinal injection of isolated sporozoites in 7-day-old BALB/c mice. When isolated sporozoites were incubated with heat-inactivated immune bovine serum, 25 times the 50% infective dose for 7-day-old mice was completely neutralized. Sporozoites incubated with preimmune bovine serum were infectious for 7-day-old mice.
Substitutions of cysteine residues of Escherichia coli heat-stable enterotoxin by oligonucleotide-directed mutagenesis
Authors: Okamoto K , Okamoto K , Yukitake J , Kawamoto Y , Miyama A
Published: January 2020

The Escherichia coli 18-amino-acid, heat-stable enterotoxin STp has six cysteine residues linked intramolecularly by three disulfide bonds. These disulfide bonds are important for toxic activity, but the precise role of each bond is not clear. We substituted cysteine residues of STp in vivo by oligonucleotide-directed site-specific mutagenesis to dissociate each disulfide bond and examined the biological activities of the resulting mutants. The Cys-6----Ala and Cys-17----Ala mutations caused a complete loss of toxic activity. The Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17 mutations caused a large decrease in toxic activity. These results mean that all three disulfide bonds formed at fixed positions are required for full expression of the biological activity of STp. However, a weak but significant toxicity still remained after three mutations, Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17. This indicates that STp has some flexibilities in its conformation to exert toxic activity and that the role of each disulfide bond exerting toxic activity is not quite the same.
Localization of stx, a determinant essential for high-level production of shiga toxin by Shigella dysenteriae serotype 1, near pyrF and generation of stx transposon mutants
Authors: Sekizaki T , Harayama S , Brazil G M , Timmis K N
Published: January 2020

Hfr strains of Shigella dysenteriae serotype 1 were constructed by transient integration of an RP4 plasmid derivative carrying transposon Tn501 into the Shigella chromosome through Tn501-mediated cointegration. The Hfr strains were mated with Escherichia coli K-12 recipients carrying various auxotrophic markers, and E. coli recombinants which had received prototrophic Shigella genes were selected. Some of the E. coli transconjugants produced high levels of a cytotoxin which was neutralized by both polyclonal and monoclonal anti-Shiga toxin sera. The determinant for Shiga toxin production, designated stx, was first transferred to E. coli K-12 and then mapped by Hfr crosses to the trp-pyrF region located at 30 min on the E. coli chromosome. Bacteriophage P1-mediated transduction analysis of stx gave the following gene order: trp-pyrF-stx. The level of Shiga toxin production in E. coli Stx+ transconjugants and transductants was as high as that of the parental S. dysenteriae 1 strain. Stx- mutants of an Stx+ E. coli transductant were generated by random in vivo insertion mutagenesis with a Tn10 derivative transposon, Tn-mini-kan, followed by P1 cotransduction of the kanamycin resistance and PyrF+ markers into a pyrF Stx+ E. coli K-12 recipient. One stx::Tn-mini-kan transposon mutation was transferred by P1 transduction from this E. coli Stx- mutant to an E. coli K-12 Hfr strain and in turn transferred by conjugation to the original S. dysenteriae 1 strain plus two others. All kanamycin-resistant recombinants of S. dysenteriae 1 had lost their ability to produce high levels of Shiga toxin. A gene that specifies high-level Shiga toxin production is thus located near pyrF on the chromosome of S. dysenteriae 1. Stx- mutants of S. dysenteriae 1 exhibited full virulence in the Serény test.
Direct activation of human monocyte-derived macrophages by a bacterial glycoprotein extract inhibits the intracellular multiplication of virulent Legionella pneumophila serogroup 1
Authors: Rajagopalan P , Dournon E , Vildé J L , Pocidalo J J
Published: January 2020

Intracellular multiplication of virulent Legionella pneumophila serogroup 1 was inhibited by human monocyte-derived macrophages activated by a glycoprotein extract of Klebsiella pneumoniae, RU 41.740. Macrophage cultures were infected with L. pneumophila in the presence of immune antibodies on day 7 of culture. Extracellular bacteria were removed an hour after infection, and various concentrations of RU 41.740 or an antibiotic, erythromycin, were added. Intracellular multiplication in the presence of RU 41.740 was significantly slowed down compared with that of cultures without RU 41.740. The reduction was, however, significantly less than that effected by erythromycin, which was used as a positive control for inhibition of intracellular multiplication. Cultures incubated with RU 41.740 before infection also demonstrated a significant reduction in the intracellular multiplication of L. pneumophila. In addition, RU 41.740 increased superoxide anion production from human monocytes in suspension in the presence of L. pneumophila. These results show that direct nonspecific activation of macrophages by a bacterial glycoprotein inhibits the intracellular multiplication of L. pneumophila and may suggest a role for activated macrophages in host defense against intracellular pathogens.
Changes in the cell surface properties of Treponema pallidum that occur during in vitro incubation of freshly extracted organisms
Authors: Stamm L V , Hodinka R L , Wyrick P B , Bassford P J
Published: January 2020

We previously reported that a number of Treponema pallidum membrane proteins appear to reside on the cell surface, since intact treponemes radiolabeled by overnight incubation in medium containing [35S]methionine bind immunoglobulin G (IgG) antibodies directed against these proteins. In the present study, it was found that freshly extracted organisms radiolabeled in vitro for only 2 h inefficiently bound IgG antibodies directed against just two proteins of molecular weights 40,000 and 34,000. An in vitro incubation period of greater than 8 h was required before IgG antibodies present in rabbit syphilitic serum could recognize additional protein antigens on the cell surface. Treatment of aged treponemes, but not freshly extracted organisms, with 0.04% sodium dodecyl sulfate selectively removed a membranous layer from the treponemal surface. Only three treponemal proteins were found associated with this structure, including the same 40,000- and 34,000-molecular-weight proteins mentioned above. These two proteins most likely represent endoflagellar subunits, since they were precipitated with rabbit antisera prepared against purified endoflagellar subunits of the cultivable treponemal strain Treponema phagedenis. Further evidence also was obtained that cells of T. pallidum actively secrete into their extracellular environment a unique class of low-molecular-weight proteins.
Gamma interferon-mediated increase in the number of Ia-bearing macrophages during infection with Listeria monocytogenes
Authors: Koga T , Mitsuyama M , Handa T , Watanabe Y , Nomoto K
Published: January 2020

The role of gamma interferon (IFN-gamma) in an increase in Ia-bearing macrophages during Listeria monocytogenes infection was studied. The peritoneal macrophages from L. monocytogenes-infected mice contained a high proportion of Ia. Intraperitoneal injection of the supernatant from a culture of spleen cells from L. monocytogenes-infected mice induced Ia-rich exudates in normal mice. The Ia-inducing activity in the culture supernatant was abrogated by the pretreatment of spleen cells with anti-Thy-1.2 antibody plus complement. Immunoadsorption of the culture supernatant with anti-recombinant IFN-gamma antibody and protein A-Sepharose CL-4B completely abrogated its Ia-inducing activity. These results suggested that an increase in Ia-bearing macrophages during L. monocytogenes infection was attributable to T-cell-derived IFN-gamma.
Induction of murine cytolytic T lymphocytes by Pseudomonas aeruginosa exotoxin A
Authors: Zehavi-Willner T
Published: January 2020

Pseudomonas aeruginosa exotoxin A (PA), a potent protein synthesis inhibitor, was found to be a weak T-cell mitogen for murine splenocytes. Maximal stimulation of [3H]thymidine incorporation was obtained with 10 to 100 ng of toxin per ml following a 4-day induction. PA was also shown to be a polyclonal activator of cytolytic T lymphocytes (CTL), effective against concanavalin A-treated target cells. The effective PA dose for CTL induction was the same as that for mitogenic stimulation, only with a prolonged priming time (7 days). In contrast to other mitogens, PA could not reactivate memory CTL into secondary CTL. The stimulation of CTL by subcytotoxic doses of PA may be relevant to its modulatory effect on the immunocellular system.
Isolation and comparison of Escherichia coli strains from canine and human patients with urinary tract infections
Authors: Low D A , Braaten B A , Ling G V , Johnson D L , Ruby A L
Published: January 2020

We analyzed Escherichia coli strains isolated from dogs with urinary tract infections (UTIs) in an attempt to determine if any of these strains were similar to E. coli isolated from humans with UTIs. Using genotypic and phenotypic traits, we identified four canine and six human E. coli UTI isolates that all appeared to be closely related or identical. All isolates shared similar DNA sequences for pyelonephritis-associated pili (pap), alpha-hemolysin (hly), and insertion sequence 5 (IS5), on the basis of Southern blot analysis. Similar outer membrane protein, pilin, and plasmid profiles were obtained for each of the isolates, although minor heterogeneity was observed. All of these isolates expressed a neuraminidase-sensitive binding phenotype in contrast to the majority of human isolates, which are known to express an adhesin that recognizes terminal digalactoside residues. Taken together, these results suggest that similar E. coli uropathogens may be capable of infecting both dogs and humans. To determine if the intestinal tracts of dogs were a reservoir for uropathogenic E. coli, eight paired rectal and urine pap+ E. coli strains were cultured from dogs with UTIs. By using the same genotypic and phenotypic criteria described above as a basis for strain identity, seven of eight urine-rectal pairs showed intrapair identity. However, each urine-rectal pair displayed a unique overall profile and could be distinguished from the other pairs. We conclude that the uropathogen colonizing the bladders of dog can also be the predominant strain colonizing the intestinal tracts.
Association of toxic shock toxin-1 determinant with a heterologous insertion at multiple loci in the Staphylococcus aureus chromosome
Authors: Chu M C , Kreiswirth B N , Pattee P A , Novick R P , Melish M E , James J F
Published: January 2020

Most Staphylococcus aureus strains associated with toxic shock syndrome and producing toxic shock syndrome toxin 1 (TSST-1) require tryptophan because of a genetic defect in tryptophan biosynthesis. The association between TSST-1 production and tryptophan auxotrophy was not correlated with the phage type, the colonization site, or the disease status of the patient from whom the isolate came. Protoplast fusion and transformation mapping located the genetic determinant of TSST-1 production (tst) very close to the trp operon in such strains and very close to tyrB in a Trp+ TSST-1+ strain. Southern blot hybridization of ClaI-restricted chromosomal DNA with a tst-specific probe revealed a common homologous segment in all of the Trp+ strains with tst linked to tyrB. These results confirmed that the tst determinant in Trp- strains is located at one site, whereas in Trp+ TSST-1+ strains the determinant is located elsewhere on the S. aureus chromosome. It is suggested that the TSST-1 determinant is associated with the insertion of a transposonlike segment into several sites on the S. aureus chromosome.
Electron microscopic study of Vibrio cholerae O1 adherence to the mucus coat and villus surface in the human small intestine
Authors: Yamamoto T , Yokota T
Published: January 2020

Vibrio cholerae O1, irrespective of the biotype or serotype, adhered to and was entrapped in the mucus coat covering the mucosal surface of isolated human ileal segments. The evidence for such mucus coat adherence was obtained by treatment of the ileal segments with 10% Formalin. In any case, adherence to the mucus coat was much more prominent than adherence to the epithelial cell surface of the small intestinal villi. Mucus coat adherence was affected by sugars and by the growth phase of the bacterial culture and was diminished by the heating of V. cholerae O1. We conclude that the small intestinal mucus coat is a primary adherence target for V. cholerae O1 in human infection and that the cell-associated hemagglutinin of V. cholerae O1 plays a role, at least in part, in adherence.
Glucan-binding factor in saliva
Authors: Cowan M M , Parrish K , Kessler R E , Pyle C , Taylor K G , Ciardi J E , Doyle R J
Published: January 2020

High-molecular-weight polymers of alpha-1,6-linked D-glucans are insoluble in alcohol solutions. Whole, but not parotid, saliva prevented the precipitation of D-glucans by 80% (vol/vol) ethanol, showing that the whole saliva contained a factor which complexed with the glucan to render it alcohol soluble. The glucan-binding factor was retained on a column of Sephacryl S-200 which had been preequilibrated with 80% ethanol. The factor was then eluted with water. Passive hemagglutination assays revealed that the glucan-binding factor could sensitize erythrocytes to agglutination with anti-poly(glycerolphosphate), suggesting that the active glucan-binding component with lipoteichoic acid. The glucan-solubilizing factor was resistant to heat (100 degrees C), proteases, sialidase, lysozyme, lactoperoxidase, trichloroacetic acid, and Triton X-100. When sucrose was added to saliva, a suspension of Streptococcus cricetus AHT, or a suspension of Streptococcus sanguis 10556, relatively large amounts of glucan-binding factor were released in a soluble form. In addition, penicillin G caused the release of the glucan-solubilizing component from a suspension of S. cricetus AHT. It is suggested that whole saliva contains a component, tentatively identified as lipoteichoic acid, which can complex with glucans in a relatively hydrophobic solvent. This type of complex formation may be important in the adhesion of oral streptococci to saliva-coated surfaces.
In vitro model for Campylobacter pylori adherence properties
Authors: Neman-Simha V , Mégraud F
Published: January 2020

The adherence of 12 strains of Campylobacter pylori was studied on four cell lines. Immunofluorescence and scanning and transmission electron microscopy were used to visualize the bacteria. A heavy adherence to the epithelial cell line HEp-2 and to the intestinal cell line Int-407 was noted. By transmission electron microscopy, a close association between bacteria and cells in the form of cup-like structures was observed, but pedestals were not present.
Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis
Authors: Bricker T M , Boyer M J , Keith J , Watson-McKown R , Wise K S
Published: January 2020

Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 [35S]methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of [3H]palmitate-labeled cells showed that approximately 20 proteins of this organism were associated with lipid, all of which also efficiently partitioned as integral membrane components into the detergent phase. Immunoblotting and immunoprecipitation of TX-114-phase proteins from 125I-surface-labeled cells with four monoclonal antibodies to distinct surface epitopes of M. hyorhinis identified surface proteins p120, p70, p42, and p23 as intrinsic membrane components. Immunoprecipitation of [3H]palmitate-labeled TX-114-phase proteins further established that surface proteins p120, p70, and p23 (a molecule that mediates complement-dependent mycoplasmacidal monoclonal antibody activity) were among the lipid-associated proteins of this organism. Two of these proteins, p120 and p123, were acidic (pI less than or equal to 4.5), as shown by two-dimensional isoelectric focusing. This study established that M. hyorhinis contains an abundance of integral membrane proteins tightly associated with lipids and that many of these proteins are exposed at the external surface of the single limiting plasma membrane. Monoclonal antibodies are reported that will allow detailed analysis of the structure and processing of lipid-associated mycoplasma proteins.
Role of macrophages in serum colony-stimulating factor induction by Lactobacillus casei in mice
Authors: Nanno M , Shimizu T , Mike A , Ohwaki M , Mutai M
Published: January 2020

Heat-killed Lactobacillus casei YIT9018 (LC9018), when injected intravenously into mice at a dose of 4 to 40 mg/kg, induced the production of serum colony-stimulating factor (CSF). Since this induction was observed in both C3H/HeJ and C3H/HeN mice, LC9018 was considered to act differently from lipopolysaccharide. The amount of serum CSF induced by LC9018 in nude mice and whole-body-X-ray-irradiated mice was similar to that in control mice, but the induction of serum CSF was suppressed by the previous administration of carrageenan, indicating that macrophages, but not T cells, were responsible for serum CSF induction by LC9018. To determine whether macrophages themselves produce CSF or help other cells produce CSF in response to LC9018, we prepared adherent cells from the peritoneal cavity of normal mice and examined CSF activity in their conditioned media. Peritoneal adherent cells did not produce CSF without LC9018, but when cultivated with 1 mg of LC9018 per ml, they produced CSF at the same time that serum CSF was induced after the intravenous administration of LC9018. Additionally, in vitro-induced CSF formed macrophage, granulocyte, and mixed colonies, as serum CSF did. CSF production by peritoneal adherent cells was completely inhibited by cycloheximide (50 micrograms/ml), and neither the elimination of T cells from the peritoneal adherent cells by treating them with anti-Thy-1.2 antibody plus complement nor the addition of T cells affected CSF production. These results suggest that heat-killed LC9018 induces serum CSF in mice via direct stimulation of macrophages to produce CSF de novo.
Roles of CR3 and mannose receptors in the attachment and ingestion of Leishmania donovani by human mononuclear phagocytes
Authors: Wilson M E , Pearson R D
Published: January 2020

Leishmania donovani is an obligate intracellular parasite of mammalian macrophages. Two macrophage receptors, the mannose-fucose receptor (MFR) and the receptor for complement component C3bi, CR3, were examined for their roles in the attachment and ingestion of L. donovani by human monocyte-derived macrophages. Two monoclonal antibodies which bind to the human CR3, anti-Mo1 and anti-Mac-1, inhibited both attachment and ingestion of L. donovani promastigotes after preincubation with human monocyte-derived macrophages; attachment was inhibited by 40 and 62% by anti-Mo1 and anti-Mac-1, respectively, and ingestion was inhibited by 34 and 51% by anti-Mo1 and anti-Mac-1, respectively. The interaction between promastigotes and CR3 may not have involved the C3bi-binding site on CR3, however, because a monoclonal antibody which exhibits specificity for this site, OKM10, inhibited promastigote attachment by only 18%. In contrast, OKM1, which is believed to react with the alternate lectinlike binding site on CR3, inhibited ingestion by 65%. MFR activity was inhibited using the soluble MFR ligands, mannan and mannosylated bovine serum albumin, which also inhibited promastigote attachment by 40 and 37%, respectively. The simultaneous inhibition of both CR3 (by anti-Mac-1) and the MFR (by either mannan or mannosylated bovine serum albumin) resulted in a greater decrease in promastigote attachment than inhibition of either receptor alone. Additionally, the reduction of MFR activity by allowing macrophages to adhere to a mannan-coated surface followed by the addition of anti-CR3 antibodies resulted in an 81% inhibition of promastigote ingestion, a greater decrease than was obtained by manipulation of either receptor alone. The results suggest that the MFR and CR3 independently participate in the attachment and ingestion of L. donovani promastigotes by human macrophages.
A block of urovirulence genes encoding multiple fimbriae and hemolysin in Escherichia coli O4:K12:H-
Authors: High N J , Hales B A , Jann K , Boulnois G J
Published: January 2020

Cosmid gene libraries were constructed from a uropathogenic isolate of Escherichia coli O4:K12:H- that secretes alpha-hemolysin and produces the F14, F12-rel, F1C, and F13 fimbrial antigens. A series of overlapping clones was generated, and individual cosmid clones were found to express various combinations of fimbriae and hemolysin, suggesting that the genes for these potential virulence factors are closely linked. By using Southern hybridization analysis and restriction endonuclease mapping, it was demonstrated that the cosmid clones carried a nested set of overlapping, cloned, genomic DNA fragments. A comparison of the phenotypic properties of individual cosmid clones and subclones allowed the order of the gene clusters encoding these factors to be deduced. The cloning also revealed the presence of a fifth fimbria that had P-adhesin specificity.
Levels of Listeria monocytogenes hemolysin are not directly proportional to virulence in experimental infections of mice
Authors: Kathariou S , Rocourt J , Hof H , Goebel W
Published: January 2020

The sulfhydryl-activated hemolysin of Listeria monocytogenes has been implicated in the virulence of the bacteria. Although loss of hemolytic activity by means of transposon mutagenesis is accompanied by loss of virulence in the mouse infection model, a direct relationship between in vitro production of hemolysin and virulence was not observed. Noticeable deviations in the extent of hemolysin production appeared to leave virulence unaffected.
Inhibition of superoxide production in human polymorphonuclear leukocytes by oral treponemal factors
Authors: Sela M N , Weinberg A , Borinsky R , Holt S C , Dishon T
Published: January 2020

The inhibition of superoxide (O2-) production by human peripheral blood polymorphonuclear leukocytes (PMNs) in the presence of oral treponemes, their cellular components, and their culture supernatants was investigated. Superoxide production was inhibited 56% by a 25-microgram/ml phenol extract of a human clinical isolate. Inhibition by culture supernatants of both the clinical isolate and a reference strain was related to the bacterial phase of growth and viability, though inhibition also persisted in the decline phase. Inhibition of superoxide production was not evident when either opsonized or nonopsonized whole spirochetes were reacted with PMNs. The suppressive activity depended, therefore, on the treponemes either being disrupted or growing and releasing the inhibitory factor into the culture medium. These results suggest that oral treponemes possess factors which interfere with the activity of PMNs and thereby alter the inflammatory process in the diseased periodontal pocket.
Chemical cross-linking of Chlamydia trachomatis
Authors: Birkelund S , Lundemose A G , Christiansen G
Published: January 2020

Purified elementary bodies (EBs) of Chlamydia trachomatis serovar L2 were analyzed by chemical cross-linking with disuccinimidyl selenodipropionate. The effect of the cross-linking was analyzed by immunoblotting sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated components which were reacted with monoclonal antibodies against major outer membrane protein (MOMP) and lipopolysaccharide (LPS). It was shown that in EBs, MOMP was cross-linked to the LPS component of the outer membrane. Migration analysis of the cross-linked components showed that with extensive cross-linking, most of the MOMP became cross-linked to LPS, changing the migration rate from 40 to 42.5 kilodaltons. A small fraction of MOMP associated with LPS was shown to be present in bands with migration rates of 100 and 110 kilodaltons. No association of MOMP or LPS to other proteins, or to dimer or multimer forms of MOMP without LPS, was observed. A totally different membrane structure must be present in reticulate bodies, since there, MOMP was so heavily cross-linked that it did not enter the polyacrylamide gel and thus became impossible to analyze. Furthermore, the monoclonal antibody, which reacted with LPS associated with MOMP in the cross-linked EBs, did not react with reticulate bodies.