bluuebunny/crossref_metadata_2025 huggingface dataset:
Augmented production of colony-stimulating factor in C3H/HeN mice immunized with Nocardia rubra cell wall skeleton
Authors: Hayashi S , Masuno T , Hosoe S , Kawase I , Sakatani M , Ogura T , Kishimoto S , Yamamura Y
Published: January 2020

C3H/HeN mice subcutaneously injected repeatedly with Nocardia rubra cell wall skeleton (N-CWS) acquired cellular immunity against N-CWS. In these N-CWS-immunized mice, the serum colony-stimulating factor (CSF) and granulocyte-macrophage colony-forming units in the bone marrow remarkably increased after an intraperitoneal (i.p.) injection of N-CWS compared with those in normal control mice given an single i.p. injection. To analyze the effects of secondary immune response to N-CWS on CSF production by splenocytes, whole mononuclear leukocytes (WMNL), nylon fiber-nonadherent splenocytes (NADC), and plastic-adherent splenocytes (ADC) were cultured in the presence of N-CWS, and then the supernatants of each cell fraction were assayed for CSF. A fraction of WMNL from immunized mice was found to produce more CSF than WMNL from control mice, and either fraction of NADC or ADC separated from WMNL produced markedly less potent CSF when cultured separately. However, when NADC from immunized mice were cultured with ADC from either immunized or normal mice, CSF production recovered to the level shown by WMNL. The role of ADC could be substituted for by cell-free culture medium of either ADC plus N-CWS or the J774 cell line, in which high interleukin-1 activity was detected. A surface marker study showed that depletion of either Lyt-1.1+ or Lyt-2.1+ cells caused a striking loss of CSF production. These data suggested that CSF in the sera of immunized mice challenged with an N-CWS i.p. injection is mainly produced by N-CWS-sensitized T lymphocytes with help of a macrophage-derived humoral factor(s).
Identification and characterization of mouse small intestine mucosal receptors for Escherichia coli K-12(K88ab)
Authors: Laux D C , McSweegan E F , Williams T J , Wadolkowski E A , Cohen P S
Published: January 2020

Adhesion of 3H-labeled Escherichia coli K-12(K88ab) to CD-1 mouse small intestine mucus and brush border preparations, immobilized on polystyrene, was studied. E. coli K12(K88ab) was shown to adhere readily to either crude mucus or brush border preparations, but not to bovine serum albumin. In contrast, the nearly isogenic E. coli K-12 strain, i.e., lacking the K88ab plasmid, did not bind well to either mucus, brush borders, or bovine serum albumin. The adhesion of E. coli K-12(K88ab) to both mucus and brush borders required pilus expression (i.e., growth at temperatures greater than 18 degrees C) and was inhibited by pretreatment of either mucus or brush borders with trypsin, pronase, or sodium metaperiodate and by the presence of D-galactosamine. Crude mucus was fractionated by gel filtration, and the proteins in receptor-containing fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Separated proteins were Western blotted to nitrocellulose. Adhesion of 35SO4-labeled E. coli K-12(K88ab) and 35SO4-labeled E. coli K-12 to Western blots followed by autoradiography revealed two E. coli K-12(K88ab)-specific mucus receptor proteins (57 and 64 kilodaltons). Brush borders contained the same two receptor proteins present in mucus and an additional 91-kilodalton receptor protein.
Occurrence of chromosome- or plasmid-mediated aerobactin iron transport systems and hemolysin production among clonal groups of human invasive strains of Escherichia coli K1
Authors: Valvano M A , Silver R P , Crosa J H
Published: January 2020

The incidence of the aerobactin system and the genetic location of aerobactin genes were investigated in Escherichia coli K1 neonatal isolates belonging to different clonal groups. A functional aerobactin system was found in all members of the O7 MP3, O1 MP5, O1 MP9, and O18 MP9 clonal groups examined and also in K1 strains having O6, O16, and O75 lipopolysaccharide types, which are less frequently associated with neonatal infections. In contrast, the aerobactin system was not detected in strains from the O18 MP6 clone. The combined results of plasmid and colony hybridization experiments showed that the aerobactin genes were located on the chromosome in the majority (75%) of the aerobactin-producing K1 isolates, the genetic location of the aerobactin genes was closely correlated with the outer membrane protein profile rather than the O lipopolysaccharide type, the K1 strains harboring a chromosome-mediated aerobactin system did not possess colicin V genes, and five of six K1 isolates possessing a plasmid-borne aerobactin system contained colicin V genes which were located on the same plasmids carrying the aerobactin genes. The comparison of hemolysin production with possession of the aerobactin system in virulent clones of E. coli K1 strains showed that all of the aerobactin-producing strains from the O18 MP9 and O7 MP3 clonal groups did not synthesize hemolysin, whereas 11 of 12 aerobactin-nonproducing O18 MP6 isolates were hemolytic. Of the K1 strains examined, 92.5% possessed either the aerobactin system or the ability to produce hemolysin or both.
Interleukin-1 released by blood-monocyte-derived macrophages from patients with leprosy
Authors: Ridel P R , Jamet P , Robin Y , Bach M A
Published: January 2020

In highly purified blood-monocyte-derived macrophages collected from patients with leprosy and from healthy individuals and cultured in vitro with mycobacterial antigens such as Mycobacterium bovis BCG or Mycobacterium leprae, we nonspecifically induced the synthesis of interleukin-1. Normally, all supernatants from cultured macrophages of all subjects tested produced similar amounts of interleukin-1. However, only in patients with lepromatous leprosy, M. leprae, but not BCG, induced high-level synthesis of prostaglandin E2, which acted as a suppressor factor in the mouse thymocyte proliferative assay used to measure the interleukin-1 content of the supernatants. Normal interleukin-1 content of those supernatants was demonstrated by blocking the prostaglandin E2 synthesis by the addition of indomethacin to the medium throughout the experimental procedure. We also tested the efficiency of a combination of BCG and M. leprae in reducing the prostaglandin E2 synthesis, but with the methodology used, we did not observe any beneficial effect of such a combination. These results demonstrate the possible role of M. leprae in the induction of at least one of the suppressive monokines and are additional arguments for the involvement of macrophages in the suppression of the specific cell-mediated immunity to M. leprae observed in lepromatous leprosy.
Localization of a determinant for HEp-2 adherence by enteropathogenic Escherichia coli
Authors: Baldini M M , Nataro J P , Kaper J B
Published: January 2020

pMAR2, a 60-megadalton plasmid encoding localized HEp-2 adherence in enteropathogenic Escherichia coli, was mapped with BamHI, HindIII, and SalI. Deletion and insertion mutants were constructed and used to define a potential DNA probe. Preliminary results indicate that this probe is sensitive and specific for the genes encoding the enteropathogenic E. coli adherence factor.
Escherichia coli hemolysin may damage target cell membranes by generating transmembrane pores
Authors: Bhakdi S , Mackman N , Nicaud J M , Holland I B
Published: January 2020

Escherichia coli hemolysin is secreted as a water-soluble polypeptide of Mr 107,000. After binding to target erythrocytes, the membrane-bound toxin resembled an integral membrane protein in that it was refractory towards extraction with salt solutions of low ionic strength. Toxin-induced hemolysis could be totally inhibited by addition of 30 mM dextran 4 (mean Mr, 4,000; molecular diameter approximately 3 nm) to the extracellular medium. Uncharged molecules of smaller size (e.g., sucrose, with a molecular diameter of 0.9 nm, or raffinose, with a molecular diameter of 1.2 to 1.3 nm) did not afford such protection. Treatment of erythrocytes suspended in dextran-containing buffer with the toxin induced rapid efflux of cellular K+ and influx of 45Ca2+, as well as influx of [14C]mannitol and [3H]sucrose. [3H]inulin only slowly permeated into toxin-treated cells, and [3H]dextran uptake was virtually nil. Membranes lysed with high doses of E. coli hemolysin exhibited no recognizable ultrastructural lesions when examined by negative-staining electron microscopy. Sucrose density gradient centrifugation of deoxycholate-solubilized target membranes led to recovery of the toxin exclusively in monomer form. Incubation of toxin-treated cells with trypsin caused limited proteolysis with the generation of membrane-bound, toxin-derived polypeptides of Mr approximately 80,000 without destroying the functional pore. We suggest that E. coli hemolysin may damage cell membranes by partial insertion into the lipid bilayer and generation of a discrete, hydrophilic transmembrane pore with an effective diameter of approximately 3 nm. In contrast to the structured pores generated by cytolysins of gram-positive bacteria such as staphylococcal alpha-toxin and streptolysin O, pore formation by E. coli hemolysin may be caused by the insertion of toxin monomers into the target lipid bilayers.
Physiological effects of a bactericidal protein from human polymorphonuclear leukocytes on Pseudomonas aeruginosa
Authors: Hovde C J , Gray B H
Published: January 2020

The physiological changes seen in Pseudomonas aeruginosa after exposure to a bactericidal protein (BP) from the granules of human polymorphonuclear leukocytes were studied. It was demonstrated, using radiolabeled proline or leucine, that both the rate of cellular uptake and amino acid incorporation into trichloroacetic acid-insoluble material were markedly decreased immediately after exposure to BP. The rate of O2 consumption by P. aeruginosa was decreased immediately after exposure to BP and continued to decline exponentially until it ceased completely 30 min after exposure to BP. In the presence of 30 mM CaCl2 or MgCl2, bacteria were protected from death due to BP and respiration rates were unaffected. The cellular ATP pool of P. aeruginosa remained constant for up to 2 h after exposure to BP. Membrane depolarization was measured by the influx of the lipophilic anion thiocyanate. It was shown that the cytoplasmic membrane of P. aeruginosa was partially depolarized after exposure to BP. Purified BP killed 95% of 5 X 10(6) CFU of P. aeruginosa at a concentration of 60 to 100 ng of protein per ml. Although the concentration of bacteria and BP varied with each type of experiment, the BP/bacteria ratio required to cause a 95 to 99% loss in viability remained constant. We propose that cytoplasmic membrane depolarization is the biochemical lesion responsible for the other physiological changes seen and ultimately for the death of P. aeruginosa induced by BP.
Chemotactic response to formate by Campylobacter concisus and its potential role in gingival colonization
Authors: Paster B J , Gibbons R J
Published: January 2020

Cells of Campylobacter concisus 288 were chemotactic toward formate, but not to any other compound tested. Chemicals that were not chemoattractants included 20 sugars, inorganic salts, amino acids, and their derivatives, purines and pyrimidines, fatty acids, and natural mixtures such as saliva, serum, crevicular fluid, and mucin. Chemotaxis was measured quantitatively by a modification of the capillary method. Cells were suspended in 0.01 M Tris buffer, pH 7.5, supplemented with 5 mM KCl, 0.1% Na2S2O3, and 0.1 mM dithiothreitol. Whole, parotid, and submandibular salivas were also suitable as chemotaxis buffers. Optimum response (0.4 X 10(6) to 1.5 X 10(6) cells per capillary) for chemotaxis occurred with 5 X 10(-2) M formate at 30 degrees C for 60 min. At 5 to 15 degrees C, cells were motile, but chemotaxis was not detected. Glutamine, asparagine, homoserine, and methionine inhibited formate chemotaxis at threshold concentrations of 0.1 to 1.0 mM, whereas the threshold for all other amino acids tested was 50 mM. Sugars and fatty acids at concentrations up to 0.1 M did not inhibit formate chemotaxis. C. concisus 484, Wolinella recta 371, W. curva VPI 9584, and W. succinogenes 602 were also chemotactic to formate, but C. fetus subsp. jejuni VPI H641 and C. fetus subsp. intestinalis VPI 1176 were not. Motile bacteria harvested directly from subgingival plaque were also chemotactic to formate and, to a lesser extent, lactate. Selected sugars, other fatty acids, and amino acids did not serve as chemoattractants for these plaque bacteria.
Chemiluminescent response of polymorphonuclear leukocytes to Streptococcus pneumoniae and Haemophilus influenzae in suspension and adhered to glass
Authors: Hayashi K , Lee D A , Quie P G
Published: January 2020

We measured the luminol- and lucigenin-enhanced chemiluminescent response of human polymorphonuclear leukocytes (PMN) stimulated by various strains of Streptococcus pneumoniae and Haemophilus influenzae. In the absence of opsonin, phagocytosis of either bacterial species elicited good PMN response when the bacteria were adhered to a surface but minimal PMN response when they were in suspension. When 10% pooled human serum was used as a source of opsonin, a moderate to excellent chemiluminescent PMN response was elicited during phagocytosis of opsonized bacteria both in suspension and adhered to surface. We conclude that opsonin significantly enhances PMN chemiluminescence when a suspension-type assay is used and that opsonin-independent mechanisms play a significant role in the chemiluminescent response of PMN during phagocytosis of adherent bacteria.
Oligomannoside-type glycopeptides inhibiting adhesion of Escherichia coli strains mediated by type 1 pili: preparation of potent inhibitors from plant glycoproteins
Authors: Neeser J R , Koellreutter B , Wuersch P
Published: January 2020

Various structurally defined glycopeptides of natural origin were tested as inhibitors of guinea pig erythrocyte agglutination by enteropathogenic Escherichia coli strains expressing type 1 pili. Besides hybrid-type glycoasparagines from ovalbumin which were not active, large oligomannoside-type carbohydrate chains from legume storage glycoproteins moderately inhibited hemagglutinations, whereas the short oligomannoside-type glycoasparagine from ovalbumin Man alpha(1----6) [Man alpha(1----3)]Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc beta(1----4)GlcNAc beta(1----N)Asn exhibited a potent activity. These results strongly suggested that the nonsubstitution of the alpha(1----3)-linked mannosyl residue from the N-linked glycopeptide core structure is the key determinant in the minimal structural requirement specific to this fimbrial lectin. Such Man5GlcNAc2-containing glycopeptides were obtained from larger N-linked carbohydrate chains, occurring abundantly in natural sources. The ability of jack bean alpha-mannosidase to cleave the alpha(1----2)-linked mannoses more rapidly than the others allowed the controlled digestion of large oligomannoside-type glycopeptides from legume storage glycoproteins. Such shortened glycopeptides of plant origin were prepared which strongly inhibited guinea pig erythrocyte agglutinations as well as bacterial adhesion on human buccal cells, thus confirming their similarity (if not identity) with the receptor of type 1 pili on mammalian cells. The importance of this preparation of a receptorlike compound that inhibits bacterial adhesion with regard to the research on the role of type 1 pili in E. coli pathogenicity is discussed.
Reduced temperature alters Pseudomonas exotoxin A entry into the mouse LM cell
Authors: Morris R E , Saelinger C B
Published: January 2020

The movement of Pseudomonas exotoxin A (PE) into the cytoplasm of mouse LM fibroblasts was followed by using inhibition of protein synthesis as a biochemical index of toxin activity; biotinyl-PE and avidin-gold colloids were used for electron microscopy. At 37 degrees C both specific antitoxin and pronase-trypsin protected cells against PE toxicity when added within seconds of warming cells, whereas methylamine was protective when added during the first 7 min of endocytosis. Lowering the temperature to 19 degrees C afforded protection when the temperature transition was accomplished within 15 min of the original endocytic event. These data suggest that PE enters an acidic compartment before reaching a step blocked by shifting cells from 37 to 19 degrees C. PE expressed toxicity for LM cells at 19 degrees C, but at a concentration 1 order of magnitude higher than that required at 37 degrees C. At 19 degrees C, antitoxin or trypsin-pronase protection was rapidly ablated. In contrast cells were fully protected by methylamine for 90 min. Using electron microscopy we demonstrated that toxin moved normally (30 s) to coated areas at 19 degrees C, but remained at this site for up to 20 min before being internalized. The majority of the toxin internalized at 19 degrees C remained in endosomes or in Golgi-associated vesicles and was not delivered to lysosomes. The results suggest that, under physiological conditions (37 degrees C), PE rapidly enters cells through coated areas, moves to an acidic compartment (i.e., the endosome), and then probably to the Golgi region en route to lysosomes. The evidence suggests that movement of toxin from endosomes or Golgi vesicles to lysosomes is blocked at 19 degrees C. We hypothesize that the active form of PE enters the cytosol, where it expresses its toxicity during fusion of Golgi-derived, toxin-laden vesicles with lysosomes.
The surface of virulent Treponema pallidum: resistance to antibody binding in the absence of complement and surface association of recombinant antigen 4D
Authors: Radolf J D , Fehniger T E , Silverblatt F J , Miller J N , Lovett M A
Published: January 2020

The binding of immunoglobulin G present in syphilitic immune rabbit serum, syphilitic human serum, and rabbit antiserum to purified recombinant Treponema pallidum antigen 4D by T. pallidum, Nichols strain, was studied by immunoelectron microscopy. Treponemes were incubated with antiserum under the conditions of the T. pallidum immobilization test, in which T. pallidum-specific antibody renders the organism nonmotile and avirulent only in the presence of complement after a 16-h incubation period in an anaerobic environment. Antibody was not demonstrable on the surface of T. pallidum incubated with nonimmune rabbit serum or normal human serum in the presence of complement. Similarly, in the absence of complement, little or no antibody was found on the treponemal surface after incubation with syphilitic immune rabbit serum, syphilitic human serum, or rabbit antiserum directed against the recombinant 4D antigen. The addition of complement to syphilitic immune rabbit serum, syphilitic human serum, and anti-4D antibody resulted in immobilization and the deposition of antibody on the entire surface of the immobilized organisms. These results corroborate earlier work by other investigators demonstrating the resistance of freshly isolated T. pallidum to antibody binding in a variety of serological tests. Detection of 4D antigen on the surface of immobilized T. pallidum strongly implies that the use of T. pallidum immobilization test conditions provides a means to demonstrate the association of individual surface antigens on virulent T. pallidum. The resistance of T. pallidum to antibody binding may be relevant to the pathogenesis of syphilis.
T-lymphocyte involvement in abscess formation in nonimmune mice
Authors: Nulsen M F , Finlay-Jones J J , McDonald P J
Published: January 2020

Athymic mice formed significantly smaller abscesses than euthymic mice in response to the intraperitoneal inoculation of an abscess-inducing mixture of Escherichia coli, Bacteroides fragilis, and autoclaved colonic contents, an abscess-potentiating agent. Adoptive transfer of nonimmune, Thy-1-positive spleen cells to athymic mice restored their ability to make abscesses of sizes similar to those in controls, indicating that T lymphocytes contribute to abscess formation in normal mice.
Interaction between Chlamydia spp. and human polymorphonuclear leukocytes in vitro
Authors: Register K B , Morgan P A , Wyrick P B
Published: January 2020

Chlamydia psittaci and Chlamydia trachomatis elementary bodies (EB) incubated in the presence of complement or specific antibody or both caused chemotaxis of human polymorphonuclear leukocytes (PMN) in vitro. Reticulate bodies and culture supernatants had no effect on these cells. The ability of chlamydiae to enter and survive in PMN under nonopsonizing conditions was investigated by measuring the association of 3H-labeled EB and of inclusion-forming units with these phagocytes. Both assays indicated that C. psittaci as well as C. trachomatis EB are efficiently internalized. The mechanism by which this is accomplished is distinct from classical phagocytosis in that it is not dependent upon the presence of complement or antibody. Furthermore, uptake of at least C. psittaci appeared to be rapid, with no additional increase occurring after 15 min. The majority of cell-associated chlamydiae were rendered acid soluble or noninfectious within 1 h. Subsequently, there was a small but steady loss of infectivity for up to 10 h, which may have been due to the conversion of EB to the noninfectious reticulate-body form of the organism. However, even at 10 h after entry a small percentage of bacteria was still capable of infecting a second target cell. This is noteworthy in that PMN are relatively short-lived cells, and after lysis, intracellular organisms may be free to infect adjacent tissue. Electron microscopic observations were consistent with the data on uptake and persistence. The ability of a small percentage of infecting chlamydiae to maintain infectivity in PMN for at least several hours may enable these organisms subsequently to establish productive infection in permissive host cells.
Progesterone binding and inhibition of growth in Trichophyton mentagrophytes
Authors: Schär G , Stover E P , Clemons K V , Feldman D , Stevens D A
Published: January 2020

Specific binding of [3H]progesterone to cytosol of Trichophyton mentagrophytes was demonstrated. Scatchard analysis of [3H]progesterone binding showed a single class of binding sites with a dissociation constant of 9.5 X 10(-8) [corrected] +/- 2.4 X 10(-8) M (standard deviation) and a maximal binding capacity of 4,979 +/- 3,489 fmol/mg of cytosol protein. Deoxycorticosterone and dihydrotestosterone competitively inhibited binding by 50% at molar ratios of 10:1 and 20:1, respectively. Other steroid hormones that were tested had minimal activity, indicating binding specificity. Steroid hormone actions in T. mentagrophytes were examined in growth studies. Growth was assessed by determination of cellular ATP content. Progesterone inhibited growth in a dose-responsive manner, with a 50% inhibition concentration of 5.5 X 10(-6) M. Partial recovery from inhibition occurred after 24 to 48 h; inhibition could be enhanced by dividing the amount of added progesterone every 24 h. In the same rank order as was their relationship to each other and progesterone in binding studies, deoxycorticosterone and dihydrotestosterone were less effective inhibitors; other steroid hormones that were tested showed no consistent effect. We hypothesize that the binder described, acting as a hormone receptor, is the molecular site of action for the functional effect of the hormone. The functional effect may be related to the observed resistance of females to dermatophytosis.
Natural and recombinant interferons inhibit epithelial cell invasion by Shigella spp
Authors: Niesel D W , Hess C B , Cho Y J , Klimpel K D , Klimpel G R
Published: January 2020

The effect of natural and recombinant interferons (IFNs) on the abilities of Shigella flexneri, S. sonnei, and Salmonella typhimurium to invade different human and murine cells was examined. Pretreatment of cell monolayers with natural and recombinant IFNs reduced the number of Shigella-infected cells in a dose-dependent manner. Establishment of an anti-invasive cellular state was time dependent, requiring 10 h for 50% inhibition of bacterial invasion. The inhibitory effect of IFN was species specific, with human or murine IFN effective against homologous but not heterologous cells. Gamma IFN was slightly more potent than alpha IFN at inhibiting bacterial invasion. Inhibition of Shigella invasion was dependent on the challenge dose of bacteria. Little inhibition of invasion was seen when cells were pretreated with low concentrations of IFN and challenged with high multiplicities of infection of Shigella sp. In contrast to Shigella invasion, the maximum inhibitory effect of IFN on Salmonella invasion of cells was observed at low levels (5 to 50 U) of IFN. These results suggest that Shigella and Salmonella invasions occur at unique sites on eucaryotic cells or by different penetration mechanisms. More importantly, these data suggest that IFN may play a significant role in host defense against Shigella and Salmonella infections.
A preclinical evaluation of aminopyridines as putative therapeutic agents in the treatment of botulism
Authors: Simpson L L
Published: January 2020

4-Aminopyridine and 3,4-diaminopyridine were evaluated for their abilities to delay the onset of paralysis due to botulinum neurotoxin types A, B, and E. Experiments were done on phrenic nerve-hemidiaphragm preparations excised from mice. At a concentration that produced an enhancement in muscle twitch amplitude, 4-aminopyridine and 3,4-diaminopyridine delayed the onset of paralysis due to botulinum toxin type A. Under the same conditions, the drugs did little to protect tissues against botulinum toxin types B and E. 3,4-Diaminopyridine was also evaluated for its ability to reverse the paralysis due to botulinum toxin. Experiments were done on rat phrenic nerve-hemidiaphragm preparations that had previously been poisoned in vivo. The drug produced transient increases in neuromuscular transmission, with the effect being greater for botulinum neurotoxin type A than for botulinum neurotoxin types B and E. Equivalent types of experiments were done with tetanus toxin. The results with 3,4-diaminopyridine showed that tetanus toxin resembled botulinum toxin types B and E. The data help to clarify the role of aminopyridines as therapeutic agents in the treatment of botulism. They also provide insights into the mechanism of action of clostridial neurotoxins.
Protection of mice against lethal endotoxemia by a lipid A precursor
Authors: Proctor R A , Will J A , Burhop K E , Raetz C R
Published: January 2020

Lipid X, the major biosynthetic precursor of lipid A, has recently been described. Although lipid X is a mitogen and coagulates the Limulus amebocyte lysate, we found that it is not lethal for mice, even when given in large doses (2 X 10(6) micrograms/kg). Furthermore, lipid X was found to give partial protection against a 100% lethal dose of endotoxin, even if the lipid X was given as late as 6 h after endotoxin challenge.
Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities
Authors: Strockbine N A , Marques L R , Newland J W , Smith H W , Holmes R K , O'Brien A D
Published: January 2020

Escherichia coli O157:H7 strain 933 contains two distinct toxin-converting phages (933J and 933W). The biologic activities and antigenic relationship between the toxins produced by 933J and 933W lysogens of E. coli K-12, as well as the homology of the genes that encode the two toxins, were examined in this study. The 933J and 933W toxins, like Shiga toxin produced by Shigella dysenteriae type 1, were cytotoxic for the same cell lines, caused paralysis and death in mice, and caused fluid accumulation in rabbit ileal segments. The cytotoxic activity of 933J toxin for HeLa cells was neutralized by anti-Shiga toxin, whereas the activity of 933W toxin was not neutralized by this antiserum. In contrast, an antiserum prepared against E. coli K-12(933W) neutralized 933W toxin but not 933J toxin or Shiga toxin. For E. coli 933, most of the cell-associated cytotoxin was neutralized by anti-Shiga toxin, whereas most of the extracellular cytotoxin was neutralized by anti-933W toxin. However, a mixture of these antisera indicated the presence of both toxins in cell lysates and culture supernatants. Among 50 elevated cytotoxin-producing strains of E. coli, we identified 11 strains isolated from cases of diarrhea, hemorrhagic colitis, or hemolytic uremic syndrome that produced cell-associated cytotoxins which were neutralized by the 933W antitoxin. Southern hybridization studies showed that the cloned toxin structural genes from phage 933J hybridized with DNA from phage 933W under conditions estimated to allow no more than 26% base-pair mismatch. These findings indicate that E. coli produces two genetically related but antigenically distinct cytotoxins with similar biologic activities which we propose to name Shiga-like toxins I and II. Strains of E. coli that produce elevated levels of Shiga-like toxin I or Shiga-like toxin II, or both, have been associated with the clinical syndromes of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome.
Serological characterization and gene localization of an Escherichia coli-expressed 37-kilodalton Treponema pallidum antigen
Authors: Rodgers G C , Laird W J , Coates S R , Mack D H , Huston M , Sninsky J J
Published: January 2020

A recombinant plasmid containing a 5.6-kilobase-pair DNA fragment of the Treponema pallidum genome was characterized by endonuclease mapping, and the encoded proteins were expressed in Escherichia coli and analyzed by use of in vitro transcription and translation. One of the proteins, identified as having a molecular weight of 37,000 (37K protein), was selected for further study. Initially, the seroreactivity of the partially purified 37K antigen was demonstrated by immunoblotting. After its purification to near homogeneity, the cloned T. pallidum protein was assessed for diagnostic significance by radioimmunoassay. Although first identified as seroreactive by screening with secondary syphilitic sera (T. E. Fehniger, A. M. Walfield, T. M. Cunningham, J. D. Radolf, J. N. Miller, and M. A. Lovett, Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, B156, p. 44), the antigen was shown to be serologically reactive with antibodies in serum from all stages of syphilis but was not recognized by serum from controls by both immunoblotting and radioimmune assay. Further, a monospecific polyclonal rabbit antiserum generated to the 37K antigen recognized a polypeptide of the same molecular weight from T. pallidum but did not efficiently recognize proteins from five nonpathogenic treponemes tested. Therefore, because of reactivity with and specificity for T. pallidum antibodies, the 37K antigen may be of serodiagnostic value in the detection of syphilis.
Yersinia enterocolitica infection in resistant and susceptible strains of mice
Authors: Hancock G E , Schaedler R W , MacDonald T T
Published: January 2020

We investigated natural resistance in mice to Yersinia enterocolitica, an enteric bacterial pathogen of humans, with a view to determine host genetic factors that are important in resistance. Most mouse strains studied (C3H/HeN, BALB/c, BALB.B, DBA/2, A, Swiss, and SWR) were highly susceptible to infection (50% lethal dose [LD50], 2 X 10(2) to 6 X 10(2) Y. enterocolitica administered intravenously [i.v.]). In contrast, C57BL/6 mice were highly resistant (LD50, 2 X 10(5) Y. enterocolitica administered i.v.). Resistance to i.v. Yersinia infection did not appear to be related to the Ity locus (which codes for resistance to Salmonella typhimurium and other pathogens) because Ityr mice (C3H/HeN, DBA/2, A, and SWR) were more susceptible to Y. enterocolitica than were Itys (C57BL/6) mice. In addition, because BALB.B mice (congenic to C57BL/6 mice at the H-2 locus) were susceptible, resistance was probably not H-2 linked. BALB/c X C57BL/6 F1 mice were intermediate in their resistance to Y. enterocolitica infection (LD50, 3 X 10(4) organisms administered i.v.), suggesting that resistance to Y. enterocolitica depends on a gene dosage affect or a resistance gene(s) interaction between susceptible and resistant parents. Further studies with C57BL/6 and BALB/c mice as prototype resistant and susceptible strains were undertaken. A time course study of Y. enterocolitica growth in various organs following i.v. infection revealed no strain difference in bacterial growth during the first 48 h of infection. Thereafter, however, C57BL/6 mice were capable of restricting Y. enterocolitica growth in all tissues (liver, lung, spleen, kidneys), whereas extensive bacterial proliferation occurred in BALB/c mice tissues. BALB/c mice were also more susceptible to oral Y. enterocolitica infection than were C57BL/6 mice, demonstrating increased mortality and greater numbers of bacteria in the Peyer's patches. Finally, whereas thymus-bearing C57BL/6 X BALB/c F1 mice were resistant to infection, athymic (nude) C57BL/6 X BALB/c F1 mice were susceptible. These studies provide a model to investigate natural immunity to enteric pathogens at mucosal surfaces, as well as provide the basis for clarifying the role of host genotype in Y. enterocolitica resistance.
Antigenicity of Campylobacter jejuni flagella
Authors: Blaser M J , Hopkins J A , Perez-Perez G I , Cody H J , Newell D G
Published: January 2020

We studied the antigenicity of a wild-type flagellate and motile (F+M+) Campylobacter jejuni strain (81116) and two daughter mutants, one flagellate and immotile (F+M-) and one aflagellate and immotile (F-M-). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis of acid-extracted surface proteins, a 63-kilodalton (kDa) band identified from sheared flagella as the flagellar protein was present in the F+M+ and F+M- strains but not in the F-M- strain. No other differences in protein profile among the three strains were noted. By Western blotting, serum from rabbits immunized with either the F+M+ or F-M- strain detected a 63-kDa protein in the F+M+ and F+M- strains but not in the F-M- strain. That the F-M- antiserum recognized the 63-kDa band suggests that small amounts of this protein or a cross-reacting antigen is present on the F-M- strain. By counterimmunoelectrophoresis of the acid-extracted preparations with immune sera, all three strains were found to share three major antigens, but a fourth antigen with a net positive charge was present only in the F+M+ and F+M- strains. Antisera to five C. jejuni and two Campylobacter fetus strains recognized the 63-kDa protein of purified F+M+ flagella in Western blots, demonstrating a common antigen is present, but enzyme-linked immunosorbent assay results suggest that the sharing of this antigen among Campylobacter strains is variable.
Comparison of living and nonliving vaccines for Brucella abortus in BALB/c mice
Authors: Montaraz J A , Winter A J
Published: January 2020

The BALB/c mouse was selected as a model for infection with Brucella abortus on the basis of protracted nonclinical infection produced by strain 2308, virulent for cattle, and relatively rapid clearance of strain 19, an attenuated strain used to vaccinate cattle. Protection in mice vaccinated with strain 19 was compared with that obtained with nonliving vaccines at early (1 week) and later (4 weeks) intervals after challenge with strain 2308 and assessed by enumeration of B. abortus organisms in the spleen. Mice challenged 4 weeks after vaccination with strain 19 exhibited significant protection at 1 and 4 weeks postinfection (p.i.), with an increased magnitude of protection at the later time. When challenged 6 weeks after vaccination with strain 19, the level of protection diminished between 1 and 4 weeks p.i. and at the later time was not always significantly different from controls. Mice immunized 4 weeks earlier with nonliving vaccines in mineral oil with t trehalose dimycolate (TDM) and muramyl dipeptide (MDP) demonstrated patterns of protection similar to those obtained following the 6 week vaccination-challenge interval with strain 19. Vaccination with cell envelopes derived from strain 2308 produced equivalent protection at 1 week p.i. whether administered in phosphate-buffered saline, incomplete Freund adjuvant, or the TDM and MDP adjuvant. Equivalent protection also followed vaccination with strain 2308 killed whole cells, cell envelopes, or outer membrane proteins in phosphate-buffered saline or in the TDM and MDP adjuvant. The TDM and MDP adjuvant alone induced nonspecific resistance, which peaked at 1 day p.i. and was still present at 1 week p.i., although by this time its magnitude was significantly less than the protection induced by antigen combined with the adjuvant. These data, together with the results of antibody assays and passive and adoptive transfer studies, suggested that protection at 1 week p.i. could be accounted for largely by an effect of O antibodies, with T cell-mediated immune responses having a subsidiary role.
Mechanisms involved in protection provided by immunization against core lipopolysaccharides of Escherichia coli J5 from lethal Haemophilus pleuropneumoniae infections in swine
Authors: Fenwick B W , Cullor J S , Osburn B I , Olander H J
Published: January 2020

In an investigation of the potential protective effects of immunity against common lipopolysaccharide core antigens of gram-negative bacteria during a severe gram-negative infection in the natural host, we induced Haemophilus pleuropneumoniae infections in weanling pigs immunized with a vaccine of an Rc mutant of Escherichia coli (strain J5). To help define the mechanism involved in J5-mediated protection, we compared the clinical, hematologic, bacteriologic, and serologic responses following an H. pleuropneumoniae infection in J5-immunized pigs with those following an H. pleuropneumoniae infection in nonimmunized control animals. As a result of an intranasal inoculation, all of the control animals and the J5-immunized animals were infected with H. pleuropneumoniae. However, while 80% (4 of 5) of the nonimmunized pigs died within 24 h as a result of the infection, no deaths occurred in the J5-immunized animals. In the immunized group, J5 titers dropped during the acute stages of the infection and rebounded to well above the prechallenge levels during convalescence. The J5 titer also increased in the single surviving control animal. These findings suggest that antibodies against common subsurface components of gram-negative bacterial cell walls correlate with protection from an otherwise lethal challenge of H. pleuropneumoniae but do not prevent infection. Important growth-phase-dependent antigenic changes have been recognized to occur during the growth of H. pleuropneumoniae in cultures (R. Nielson, Nord. Veterinaermed. 28:337-348, 1976). In a study of these changes and during an inquiry into the mechanism of J5 antibody-mediated protection, measured quantities of H. pleuropneumoniae were removed from a broth culture at hourly intervals and used to absorb hyperimmune equine J5 antiserum. Significantly greater amounts of J5-specific antibodies were absorbed during the log phase of bacterial growth than during the early or late phase. The availability of epitopes recognized by J5 antibodies appears to be closely related to the rate of bacterial multiplication. The results of these experiments suggest a mechanism of protection provided by increased immunity to E. coli J5 during gram-negative infections.
Detection and specificity of antibodies secreted by spleen cells in mice immunized with Streptococcus mutans
Authors: Russell M W , Czerkinsky C , Moldoveanu Z
Published: January 2020

Immune responses of mice to Streptococcus mutans serotype c were analyzed by means of the enzyme-linked immunospot assay to determine the predominant specificities of the antibodies developed. In general, the numbers of splenic antibody-secreting cells correlated with serum antibody levels. A low dose (10(8) CFU) of killed whole cells injected twice intraperitoneally induced antibodies mainly against surface protein antigen I/II. A higher dose (10(9) CFU) given two to six times also resulted in a predominance of antigen I/II antibody-secreting cells and, in addition, antibody responses to surface protein antigen III and lipoteichoic acid occurred. Cells producing antibodies to serotype c polysaccharide were elicited only on repeated immunization. These results agreed with the development of antibodies in rabbits repeatedly immunized intravenously with killed whole cells of S. mutans, S. rattus, and S. sobrinus, which induced specific antibodies in accordance with the surface antigens that they express. Mice immunized twice with the same dose of purified antigens I/II and III developed greater numbers of antigen I/II splenic antibody-forming cells than antigen III splenic antibody-forming cells and higher serum antibody levels to antigen I/II than to antigen III. Furthermore, a single injection of antigen I/II but not of antigen III was sufficient to induce a strong specific-antibody response. Some evidence was also obtained for weak polyclonal stimulation of spleen cells by S. mutans cells and by antigen I/II, a result which could be relevant to the induction by S. mutans of antibodies reactive with mammalian tissues. It was concluded that for the antigens examined, S. mutans elicited the strongest antibody response against antigen I/II, which was also highly immunogenic in purified form.
Effect of perorally administered lactobacilli on macrophage activation in mice
Authors: Perdigon G , de Macias M E , Alvarez S , Oliver G , de Ruiz Holgado A A
Published: January 2020

The effect of perorally (p.o) administered Lactobacillus casei and L. bulgaricus on macrophage activation in mice was studied. L. casei and L. bulgaricus were administered p.o. to mice for 8 days. The macrophage activation was measured on days 2, 3, 5, and 8 of lactobacillus administration by using biochemical and functional criteria. We measured the release of lysosomal hydrolases, the level of a nonlysosomal enzyme, and in vitro phagocytic activity of mouse peritoneal macrophages. All the assays were performed comparatively with mice inoculated with L. casei and L. bulgaricus (viable and nonviable cells) intraperitoneally (i.p.) at the same dose as for p.o. administration. The phagocytic activity was significantly higher in mice treated i.p. than in control mice. For p.o. administration, there was an increase only when L. casei was used. L. bulgaricus had little effect. No differences were found between viable and nonviable cells. The phagocytic function of the reticuloendothelial system was tested by the carbon clearance test, which showed that L. casei and L. bulgaricus accelerate the phagocytic function in mice treated p.o and i.p., from day 2 onward. These observations show that L. casei and L. bulgaricus given by p.o. administration are able to activate macrophages in mice and suggest that these bacteria, when passing through the intestinal tract, may be responsible for the enhanced host immune response. This fact is very significant because the diet includes fermented and manufactured products containing lactobacilli.
Immunological protection of rabbits infected with Staphylococcus aureus isolates from patients with toxic shock syndrome
Authors: Scott D F , Kling J M , Best G K
Published: January 2020

Toxic shock syndrome toxin-1 (TSST-1) isolated from the growth medium of Staphylococcus aureus 1169 and 555 was used to immunize male rabbits before infection with either a TSST-1+ or a TSST-1- strain of S. aureus isolated from cases of TSS. None of the immunized rabbits died as a result of the infections, whereas 50% of the nonimmunized rabbits infected with the TSST-1- strain, D4508, and 75% of those infected with the TSST-1+ strain, 555, died. Western blots of crude extracellular protein preparations probed with sera from immunized rabbits indicated that the TSST-1- strain produces a 30,000-molecular-weight protein that cross-reacts with antiserum to TSST-1. Because both organisms caused similar diseases in rabbits, we propose to designate the cross-reacting protein as TSST-2.
Isolation of a coccidioidin component that reacts with immunoglobulin M precipitin antibody
Authors: Cox R A , Britt L A
Published: January 2020

Detection of immunoglobulin M (IgM) precipitin antibody to coccidioidin, the autolysate of mycelial-phase cells of Coccidioides immitis, is an important serologic aid in establishing a diagnosis of primary coccidioidomycosis. In the present study, the component of coccidioidin that reacts with IgM precipitin antibody was isolated by a combination of immunoaffinity and anion-exchange chromatography. Antigenic analysis of the purified antigen in two-dimensional immunoelectrophoresis against goat anti-coccidioidin revealed a precipitinogen characterized by a complete cathodal leg and a partial anodal leg. The reactivity of this incomplete precipitating antigen with anti-C. immitis IgM was established by serologic assays and by the adsorption of reference IgM precipitin antibody on solid-phase immunosorbents containing the purified precipitinogen. The isolation of the coccidioidin component that reacts with IgM precipitin antibody and the production of monospecific antibody will provide the necessary reagents for the development of a sensitive immunoassay for detecting this serodiagnostic response.
Class and subclass antibodies to Haemophilus influenzae type b capsule: comparison of invasive disease and natural exposure
Authors: Ramadas K , Petersen G M , Heiner D C , Ward J I
Published: January 2020

Recently, the role of immunoglobulin G (IgG) subclasses in the immune responses to organisms with polysaccharide capsules, particularly Haemophilus influenzae type b, has been of interest. We developed assays to measure IgG2- and IgG4-specific antibodies to the polyribosylribitol phosphate (PRP) polysaccharide antigen of H. influenzae type b and demonstrated that these assays were subclass specific. Relative levels of subclass-specific antibody were assayed in serum from 30 Alaskan Eskimo children who had invasive H. influenzae type b disease and 30 healthy controls that were matched for age and village of residence. We also measured total PRP antibody and total serum IgG4. The group with invasive H. influenzae type b disease had a significantly higher mean level of IgG4-specific PRP antibody than did the controls (P = 0.0006). However, we found no significant difference between cases and controls for IgG2-specific PRP antibody, total IgG4, or total PRP antibody. The data suggest that IgG4-specific PRP antibody is elicited by invasive H. influenzae type b disease, independent of age. The IgG4 subclass thus may be a critical determinant of the immune response to invasive infection caused by H. influenzae type b, especially for young infants who generally have a weak immune response to this organism.
Monoclonal antibodies against the M-protein and carbohydrate antigens of histoplasmin characterized by the enzyme-linked immunoelectrotransfer blot method
Authors: Reiss E , Knowles J B , Bragg S L , Kaufman L
Published: January 2020

Monoclonal antibodies (MAbs) of two different specificities were produced by immunizing mice with the semipurified M antigen of histoplasmin. One type, from clone CB4, was an immunoglobulin M that precipitated a polysaccharide present in histoplasmin and also formed immunoprecipitates with a cross-reactive polysaccharide present in extracts of Blastomyces dermatitidis and Coccidioides immitis. The second type of MAb, from clone EC2, was an immunoglobulin G that reacted in the enzyme-linked immunoelectrotransfer blot (EITB) assay with a doublet of proteins with an apparent molecular size of 70 to 75 kilodaltons. This molecule is proposed as the authentic M protein antigen that is recognized by M antibodies in sera from mice and rabbits immunized with Histoplasma capsulatum and from persons with histoplasmosis. The M factor also occurs in an abundant disulfide-bridged dimer which has a molecular size of 150 kilodaltons and is nonimmunoreactive under the conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.