bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein synthesis early in the developmental cycle of Chlamydia psittaci
Authors: Plaunt M R , Hatch T P
Published: January 2020

The incorporation of [35S]methionine into protein by intracellular and host-free Chlamydia psittaci 6BC was analyzed at intervals between 15 min and 28 h postinfection by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. The profiles of proteins synthesized in the two systems were similar at all times, indicating that the host-free system can be used to monitor the temporal expression of genes in chlamydiae. The host-free system permitted detection of synthesis of chlamydial proteins as early as 15 min postinfection. Some of the proteins synthesized during the initial phases of reorganization of elementary bodies to reticulate bodies either were not synthesized or were synthesized in greatly reduced amounts during the other phases of the developmental cycle. The effects of rifampin and actinomycin D indicated that host-free protein synthesis was at least partially dependent on the initiation and continuation of RNA synthesis in the isolated organisms.
Differential regulation of in vitro humoral and cellular immune responsiveness in Brugia pahangi-infected jirds
Authors: Prier R C , Lammie P J
Published: January 2020

Patent filarial infection is associated with the downregulation of parasite-specific immune reactivity. In the present study, the relationship between in vitro parasite antigen-specific humoral and cellular immune responsiveness was investigated in Brugia pahangi-infected jirds and in jirds immunized with soluble antigens. Spleen cells from B. pahangi-immunized jirds or from jirds with prepatent infections mounted significant in vitro proliferative and antibody responses to B. pahangi. The antigen concentration which elicited optimal antibody production was 10- to 10,000-fold lower than that required to stimulate optimal blastogenesis. Lymph node cells from both immunized and infected jirds consistently produced lower levels of parasite-specific antibody than spleen cells, yet generated higher proliferative responses to filarial antigen. A dissociation between in vitro antibody production and proliferation was also observed in experiments with spleen cells from microfilaremic jirds; spleen cells from patent animals did not proliferate when stimulated with B. pahangi antigen, but did produce significant levels of parasite-specific antibody. Depletion of adherent or histamine receptor-bearing cells restored the proliferative reactivity of spleen cells from microfilaremic jirds, but had limited effects on antibody production. In admixture experiments, spleen cells from microfilaremic animals suppressed the proliferative responsiveness of cells from keyhole limpet hemocyanin (KLH)-immunized jirds to KLH by 38%, but had no effect on KLH-specific antibody production. The present results support the hypothesis that parasite-specific cellular and humoral reactivity are differentially regulated in experimental filariasis.
Nearest neighbor analysis of outer membrane proteins of nontypeable Haemophilus influenzae
Authors: Klingman K L , Jansen E M , Murphy T F
Published: January 2020

The arrangement of outer membrane proteins on the surface of nontypeable Haemophilus influenzae was investigated with cleavable and noncleavable bis-imidate cross-linking agents. Whole organisms were subjected to cross-linking agents, and oligomers of proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis, and immunoblot assay, using monoclonal antibodies to outer membrane proteins. The major outer membrane protein (P2) formed dimers and trimers detected by all three methods. Oligomers of other outer membrane proteins were not detected. These data indicate that P2 exists as a trimer on the outer membrane and suggest that other outer membrane proteins exist as monomers on the outer membrane.
Tumor necrosis factor (cachectin) mediates induction of cachexia by cord factor from mycobacteria
Authors: Silva C L , Faccioli L H
Published: January 2020

The mechanism by which cord factor (CF), a toxic glycolipid from mycobacteria, induces cachexia was studied in BALB/c mice. Body weight was markedly reduced 48 h after CF administration; the animals became severely wasted and exhibited hypertriglyceridemia, hypoglycemia, and high levels of tumor necrosis factor (TNF) in plasma. After CF administration, a transferable factor which caused cachexia and hypertriglyceridemia in recipient mice was detected in the blood. Dexamethasone partially inhibited the cachexia-inducing action of CF. Conditioned medium from adherent peritoneal cell cultures incubated with CF produced the same wasting symptoms when inoculated intravenously into mice. These studies also demonstrated that adherent peritoneal cells produced a humoral factor in response to CF which was related to CF-induced cachexia. Antiserum to recombinant TNF-alpha prevented the cachectin action in passive-transfer experiments. Our findings indicate that cachectin (TNF) plays a role as a central mediator of the wasting induced by CF.
Nucleotide sequence of the gene encoding the major subunit of CS3 fimbriae of enterotoxigenic Escherichia coli
Authors: Boylan M , Smyth C J , Scott J R
Published: January 2020

The complete nucleotide sequence of a 612-base-pair DNA fragment containing the gene for the major fimbrial subunit of CS3 of enterotoxigenic Escherichia coli is presented. A possible promoter region, a ribosome-binding site, and two potential signal peptidase cleavage sites are indicated. Unlike the best-studied fimbrial proteins, the predicted CS3 sequence has no Cys residues.
Role of activation in alveolar macrophage-mediated suppression of the plaque-forming cell response
Authors: Mbawuike I N , Herscowitz H B
Published: January 2020

Alveolar macrophages (AM) are highly suppressive of the in vitro plaque-forming cell (PFC) response of spleen cells obtained from mice primed with sheep erythrocytes. Comparison of macrophage populations obtained from disparate anatomical sites revealed that although in both cases there was a cell-concentration-dependent suppression of the PFC response, resident AM or AM activated as a result of intravenous injection of Mycobacterium bovis BCG were equally suppressive at the doses examined. Although there was a similar dose-dependent suppression with peritoneal macrophages, BCG-activated cells were more suppressive of the PFC response than were resident cells. In contrast, splenic macrophages at comparable concentrations were not at all suppressive. Resident AM exhibited significantly lower levels of 5'-nucleotidase activity than did resident peritoneal macrophages. Macrophage-mediated suppression of the in vitro PFC response could not be attributed to the release of toxic oxygen metabolites (H2O2, O2- ,and .OH) or prostaglandins, since the addition of catalase, superoxide dismutase, 2-mercaptoethanol, or indomethacin did not completely reverse suppression. These results suggest that the lung microenvironment may maintain AM in an activated state which contributes to their potential immunoregulatory functions.
Autoantibodies to the mitotic spindle apparatus in Mycoplasma pneumoniae disease
Authors: Lind K , Høier-Madsen M , Wiik A
Published: January 2020

Antibodies to the mitotic spindle apparatus (MS) have been detected in only a very few patient sera studied for antinuclear antibodies by routine screening (G. A. McCarty, D. W. Velencia, and M. J. Fritzler, J. Rheumatol. 11:213-218, 1984). We found anti-MS antibodies to be quite common in sera from patients with Mycoplasma pneumoniae, especially in those who developed cold agglutinins (71%). Absorption experiments indicated that the formation of anti-MS antibodies is not triggered by epitopes on the M. pneumoniae surface. These antibodies were distinct from cold agglutinins, antibrain antibodies, and smooth-muscle antibodies. All patients previously reported to harbor anti-MS had clinical features compatible with a diagnosis of systemic lupus erythematosus, rheumatoid arthritis, or an evolving connective-tissue disorder. Such systemic disorders might develop in a small proportion of patients who have had M. pneumoniae illness.
Iron acquisition by Haemophilus influenzae
Authors: Pidcock K A , Wooten J A , Daley B A , Stull T L
Published: January 2020

The mechanisms for acquisition of iron by Haemophilus influenzae and their role in pathogenesis are not known. Heme and nonheme sources of iron were evaluated for their effect on growth of type b and nontypable strains of H. influenzae in an iron-restricted, defined medium. All 13 strains acquired iron from heme, hemoglobin, hemoglobin-haptoglobin, and heme-hemopexin. Among nonheme sources of protein-bound iron, growth of H. influenzae was enhanced by partially saturated human transferrin but not by lactoferrin or ferritin. Purified ferrienterochelin and ferridesferrioxamine failed to provide iron to H. influenzae, and the supernatants of H. influenzae E1a grown in iron-restricted medium failed to enhance iron-restricted growth of siderophore-dependent strains of Escherichia coli, Salmonella typhimurium, and Arthrobacter terregens. Marked alterations in the profile of outer membrane proteins of H. influenzae were observed when the level of free iron was varied between 1 microM and 1 mM. Catechols were not detected in the supernatants of strain E1a; however, iron-related hydroxamate production was detected by two biochemical assays. We conclude that the sources of iron for H. influenzae are diverse. The significance of hydroxamate production and iron-related outer membrane proteins to H. influenzae iron acquisition is not yet clear.
Cerebrospinal fluid macrophage response to experimental cryptococcal meningitis: relationship between in vivo and in vitro measurements of cytotoxicity
Authors: Perfect J R , Hobbs M M , Granger D L , Durack D T
Published: January 2020

The functional abilities of macrophages from cerebrospinal fluid (CSF) have so far been little studied. We examined the acquisition of activation characteristics by CSF macrophages during the course of experimental cryptococcal meningitis. CSF macrophages developed the ability for increased reactive oxidative intermediate (H2O2) production and tumor and fungal cytotoxicity. Despite having been activated, CSF macrophages could not inhibit the growth of Cryptococcus neoformans in vitro. Immunosuppression with cyclosporine, which eliminates the natural resistance of rabbits to cryptococcal meningitis, did not prevent or diminish H2O2 production by CSF macrophages but did reduce their tumoricidal activity. Activation of CSF macrophages appears to be an integral part of the central nervous system immune response to C. neoformans in this model, but alone is insufficient to eliminate C. neoformans from the central nervous system.
Identification of the major antigens of Treponema hyodysenteriae and comparison with those of Treponema innocens
Authors: Chatfield S N , Fernie D S , Penn C , Dougan G
Published: January 2020

Eleven strains of Treponema hyodysenteriae isolated from pigs with swine dysentery were examined by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. T. hyodysenteriae strains formed a homogeneous group with respect to sodium dodecyl sulfate-soluble proteins. However, immunoblotting with antiserum from rabbits immunized with T. hyodysenteriae CN8368 revealed heterogeneity among the lipopolysaccharide complexes of different strains. Polypeptides of molecular weights between 30,000 and 36,000 were the predominant T. hyodysenteriae polypeptides detected by porcine immune serum. In contrast, Treponema innocens did not form a homogeneous group with respect to sodium dodecyl sulfate-soluble proteins. Adsorption studies and immunoblotting identified polypeptide antigens present on cells of T. hyodysenteriae which were not detected on cells of T. innocens. These unique antigens may play a role in the virulence of T. hyodysenteriae.
Heterogeneity among strains and a high rate of variation within strains of a major surface antigen of Mycoplasma pulmonis
Authors: Watson H L , McDaniel L S , Blalock D K , Fallon M T , Cassell G H
Published: January 2020

Monoclonal and monospecific antibodies were used to characterize a major Mycoplasma pulmonis surface antigen complex, V-1. Heterogeneity of V-1 was detected among strains and a high frequency of variation was detected within subclones of single strains. Analysis of 18 different strains showed that no two displayed identical electrophoretic immunoblot patterns for V-1. Analysis of 50 filter clones from an individual strain (not previously filter cloned) revealed at least 10 different V-1 patterns. The two most frequently occurring patterns were expressed by 36% and 24%, respectively, of the total population. Serial subcloning (four separate series) of several of these original clones showed that the average rate of V-1 variation was 2 x 10(-3) per cell per generation. Immunoblots with different anti-V-1 monoclonal antibodies demonstrated that there were both structurally and antigenically different forms of this antigen. Also, two-dimensional polyacrylamide gel analyses showed that different forms of V-1 could vary in charge. This potential for variability in a major surface antigen of mycoplasmas could have important implications as to how the organism interacts with its host.
Nonionic block polymer surfactants enhance immunogenicity of pneumococcal hexasaccharide-protein vaccines
Authors: Zigterman G J , Snippe H , Jansze M , Ernste E B , De Reuver M J , Willers J M
Published: January 2020

Incorporated in oil-in-water emulsions, nonionic block polymer surfactants change the kinetics of generated antibody responses against pneumococcal hexasaccharide-protein conjugates: prolonged immunoglobulin M and immunoglobulin G responses are realized. Nonionic block polymer surfactants favor the immunogenicity of hexasaccharide-protein conjugates in young mice in such a way that a single injection yields long-lasting protection.
Natural killer cell activation and interferon production by peripheral blood lymphocytes after exposure to bacteria
Authors: Klimpel G R , Niesel D W , Asuncion M , Klimpel K D
Published: January 2020

We have previously shown that peripheral blood natural killer (NK) cells have significant levels of cytotoxic activity against Shigella flexneri-infected HeLa cells. In this report, we show that NK cell activity against K562 tumor cells and Shigella flexneri-infected HeLa cells can be greatly enhanced by preincubating peripheral blood lymphocytes (PBL) for 18 h with kanamycin-treated Shigella flexneri or Salmonella typhimurium. Cell-free supernatants obtained from PBL-bacteria cultures contained high levels of interferon (IFN) activity, which was characterized as a mixture of IFN-gamma and IFN-alpha. Cytotoxic activity associated with PBL precultured with shigellae was associated with predominantly CD16+ (Leu-11+) and CD2+ (OKT-11+) cells. Further, IFN production was dependent upon the presence of CD16+ and CD2+ cells at culture initiation. Enhancement of cytotoxic activity associated with PBL-bacteria cultures did not, however, appear to be dependent upon IFN production, since low numbers of bacteria which failed to stimulate IFN production induced high levels of NK cell activity. Lipopolysaccharide appeared not to be involved in bacteria-induced IFN production or enhanced NK cell activity, since Salmonella lipopolysaccharide failed to induce IFN production or enhance NK cell activity. These results suggest that IFN production by NK cells and the killing of bacteria-infected cells play an important role in host defense against facultative intracellular bacterial infections.
Ultrastructural study of endocytosis of Chlamydia trachomatis by McCoy cells
Authors: Hodinka R L , Davis C H , Choong J , Wyrick P B
Published: January 2020

The entry of Chlamydia trachomatis into McCoy cells (fibroblasts) was studied by transmission electron microscopy. On adsorption of elementary bodies (EBs) to host cells at 37 degrees C, the EBs were bound primarily to preexisting cell-surface microvilli. They were also observed in coated pits located at the bases of the microvilli and along smooth surfaces of the host cells and were internalized within coated vesicles at this temperature. Postembedding immunogold labeling on Lowicryl thin sections with anti-clathrin antibody as the primary reagent revealed the gold marker localized in pits and vesicles containing chlamydiae. Some EBs were present in smooth-surfaced invaginations at or near the bases of microvilli and in vesicles devoid of distinguishable coat material. A similar entry process was observed with centrifugation-assisted inoculation of EBs onto the McCoy cells. Individual EBs were initially internalized into tightly bound endocytic vesicles. However, within 1 to 3 h postinfection, multiple C. trachomatis EBs were observed in large, loosely bound vesicles. Evidence suggests that vesicles containing C. trachomatis may have fused with one another early in the infectious process. These results indicate that chlamydiae can exploit the specific process of adsorptive endocytosis for entry into host cells and for translocation to a given intracellular destination, which may be different for each species.
Endotoxin-induced shedding of viable uroepithelial cells is an antimicrobial defense mechanism
Authors: Aronson M , Medalia O , Amichay D , Nativ O
Published: January 2020

ICR mice were infected intravesically with a virulent (7343) or a nonvirulent (U+) Escherichia coli strain. The U+ strain induced considerably more shedding of uroepithelial cells than did the 7343 strain. The stimulus for this shedding was shown to be associated with lipopolysaccharide and was abrogated by pretreatment with aprotinin. Desquamation commenced within 1 h postinjection, and the cells that were shed proved to be viable. Comparison of C3H/HeJ and C3H mice revealed that only the latter responded to shedding inducers. However, C3H/HeJ mice succumbed to a systemic infection on injection of 10(6) U+ cells intravesically, whereas other mouse strains required a 100-fold dose of bacteria for this effect. Since the first stage of a bacterial infection entails adherence of the microbes to epithelial cells, inducible shedding is an antimicrobial defense mechanism.
Cloning and expression of secreted antigens of Clostridium difficile in Escherichia coli
Authors: Dailey D C , Schloemer R H
Published: January 2020

The feasibility of the cloning and expression of Clostridium difficile antigens in Escherichia coli was investigated. The expression of a limited number of cloned clostridial antigens under the control of clostridial promoter elements in E. coli was observed.
Developmental-form-specific DNA-binding proteins in Chlamydia spp
Authors: Wagar E A , Stephens R S
Published: January 2020

We identified DNA-binding proteins specific to the elementary body (EB) developmental form of Chlamydia spp. Chlamydial proteins from whole lysates were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with nick-translated chlamydial DNA. By this method, C. trachomatis serovar L2 EBs had three unique protein bands of 58,000, 25,700, and 17,000 molecular weight not seen in the reticulate bodies. The 17,000-molecular-weight protein and the 25,700-molecular-weight protein were identified in an acid-soluble protein fraction and were resistant to high-salt elution, similar to other procaryotic nucleoproteins. The 17,000-molecular-weight protein was also detected in preparations with isolated chromosomes from EBs. Preliminary characterization indicated that the protein-DNA interaction was principally charge related.
Interaction between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of the human flora
Authors: Galili U , Mandrell R E , Hamadeh R M , Shohet S B , Griffiss J M
Published: January 2020

Anti-alpha-galactosyl immunoglobulin G (anti-Gal) is a natural antibody present in unusually high amounts in human sera. It constitutes as much as 1% of circulating immunoglobulin G in humans and displays a distinct specificity for the carbohydrate epitope galactosyl alpha(1----3) galactosyl (Gal alpha 1----3Gal). Recently, it has been suggested by various investigators that anti-Gal may be related to some autoimmune phenomena, since marked elevation of its titer was found in sera of patients with autoimmune thyroid disorders, rheumatoid arthritis, glomerulonephritis, and Chagas' disease. In view of the ubiquitous presence of anti-Gal in high titers in humans, throughout life, we hypothesized that, analogous with synthesis of anti-blood group antibodies against bacterial antigens, bacteria within normal intestinal flora may provide constant antigenic stimulation for the synthesis of anti-Gal. This hypothesis would imply that anti-Gal may bind to a variety of bacterial strains of human flora. In the present study, the interaction between affinity chromatography-purified anti-Gal and various bacterial strains was studied. By the use of a direct immunostaining assay and an enzyme-linked immunosorbent assay, anti-Gal was found to interact with a variety of Escherichia coli, Klebsiella, and Salmonella strains, some of which were isolates from normal stool. Furthermore, the anti-Gal-binding sites in some strains were found to be present on the carbohydrate portion of bacterial lipopolysaccharides. It is thus suggested that Gal alpha 1----3Gal epitopes in the outer membranes of normal flora enterobacteria may provide a continuous source for antigenic stimulation. Since there is no immune tolerance to the Gal alpha 1----3Gal carbohydrate structure in humans, anti-Gal seems to be constantly produced in response to these enterobacteria. In addition, bacteria which express Gal alpha----3Gal epitopes and which may adhere to various cells mediated binding of anti-Gal to human cell lines. These findings raise the possibility that anti-Gal may damage normal human tissues via inflammatory processes facilitated by bacterial Gal alpha 1----3Gal epitopes.
Production and characterization of monoclonal antibodies to the protective antigen component of Bacillus anthracis toxin
Authors: Little S F , Leppla S H , Cora E
Published: January 2020

Thirty-six monoclonal antibodies to the protective antigen protein of Bacillus anthracis exotoxin have been characterized for affinity, antibody subtype, competitive binding to antigenic regions, and ability to neutralize lethal and edema toxin activities. At least 23 antigenic regions were detected on protective antigen by a blocking, enzyme-linked immunosorbent assay. Two clones, 3B6 and 14B7, competed for a single antigenic region and neutralized the activity of both the lethal toxin in vivo (Fisher 344 rat) and the edema toxin in vitro (CHO cells). These two antibodies blocked the binding of 125I-labeled protective antigen to FRL-103 cells. Our results support the proposal that binding of protective antigen to cell receptors is required for expression of toxicity.
Pathogen specificity of Treponema pallidum subsp. pallidum integral membrane proteins identified by phase partitioning with Triton X-114
Authors: Radolf J D , Norgard M V
Published: January 2020

The antigenically conserved proteins of Treponema pallidum subsp. pallidum and four nonpathogenic cultivatable treponemes were investigated by phase partitioning with the nonionic detergent Triton X-114 and immunoblot analysis. None of the T. pallidum integral membrane proteins identified by phase partitioning (detergent-phase proteins) appeared to be antigenically related to proteins of the nonpathogens. Protease-resistant material similar to lipopolysaccharide was identified in the detergent phase from T. phagedenis biotype Reiter but was not detected in T. pallidum.
Polyclonal B-cell stimulation by L3T4+ T cells in experimental leishmaniasis
Authors: Lohoff M , Matzner C , Röllinghoff M
Published: January 2020

The well-established polyclonal B-cell stimulation in the lymphoid organs in mice infected with Leishmania major is thought to be dependent on T cells. Here we present clear experimental evidence that this is indeed the case by showing that BALB/c-derived, L3T4-positive L. major-specific T cells induce syngeneic B cells to polyclonal proliferation and immunoglobulin production.
Saliva inhibits the chemiluminescence response, phagocytosis, and killing of Staphylococcus epidermidis by polymorphonuclear leukocytes
Authors: Saito K , Kato C , Teshigawara H
Published: January 2020

Saliva inhibited several functional properties of polymorphonuclear leukocytes (PMNs) from murine peritoneal exudate, namely, luminol-mediated chemiluminescence (CL) induced by either Staphylococcus epidermidis or formylmethionyl-leucyl-phenylalanine (FMLP), phagocytosis, and killing of bacteria in vitro. The concentration of saliva in the reaction mixture that caused a complete inhibition of the CL response of PMNs to both S. epidermidis and FMLP was 25%. However, there was no catalase or superoxide dismutase activity in saliva that could influence the CL response of PMNs. The production of superoxide by PMNs stimulated with S. epidermidis was assayed in the presence or absence of saliva by inhibition of the reduction of cytochrome c by superoxide dismutase. In the presence of 50% saliva, O2- generation by PMNs was only 7.3% of that observed in the absence of saliva. After gel filtration of salivary material through Sephadex G-25 or Sephacryl S-200, several fractions were obtained that inhibited the CL response of PMNs to either FMLP or S. epidermidis or to both. Two inhibitory fractions were analyzed. One contained immunoglobulin A, and the other contained a peptide which was composed of 14 different amino acids. The two fractions of high molecular weight included in the first protein peak of Sephacryl S-200 gel filtration were able to inhibit the CL response to S. epidermidis and to inhibit phagocytic activity, while fractions of low molecular weight (under 12,500 Mr) inhibited the CL response to FMLP and to S. epidermidis but did not inhibit phagocytic activity.
Expression of K99 adhesion antigen controlled by the Escherichia coli tryptophan operon promoter
Authors: Baecker P A , Shelton E R , Bursztyn-Pettegrew H , Salazar F H , Osen E G , Stoufer S D , Lee S W , Chan H W
Published: January 2020

The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 [O101:K99] has been cloned as a 7.0-kilobase BamHI-generated DNA fragment into the vector pBR322 by us and others (J. D. A. van Embden, F. K. de Graaf, L. M. Schouls, and J. S. Teppma, Infect. Immun. 29:1125-1133, 1980). Cells harboring one such construction, known as pK99-64, are capable of expressing K99 antigen on the cell surface. We replaced the natural promoter sequence for the gene encoding the K99 pilus subunit with a strong, inducible exogenous promoter, the E. coli tryptophan (trp) operon promoter, to construct the plasmid pBR-TrpK99. E. coli cells harboring pBR-TrpK99 or a similar construction in the plasmid pDR540, known as pKO-TrpK99, upon induction with 3-beta-indoleacrylic acid, produced about fourfold more K99 antigen than did cells bearing pK99-64 with the natural promoter. Expression of the pilus antigen was found to be under control of the tryptophan promoter. Plasmid instability was encountered, however, in cells bearing pKO-TrpK99 when the trp promoter was derepressed. Introduction of the aminoglycoside 3'-phosphotransferase gene of transposable element Tn5 into pKO-TrpK99 to generate pKON-TrpK99 effectively stabilized the plasmid in cells grown under identical conditions in medium containing kanamycin.
Detection of pilus subunits (pilins) and filaments by using anti-P pilin antisera
Authors: Salit I E , Hanley J , Clubb L , Fanning S
Published: January 2020

P pilus filaments are important in binding to globoside through an adhesin located at the tip of the pilus. There is considerable antigenic variation among P pili, and the immunologic response is usually serotype specific. We purified denatured pilin subunits and used them as immunogens to prepare more broadly cross-reactive antisera. Although antifilament antisera (AFA) detected predominantly the homologous strain, antisubunit antisera (ASA) prepared from two different strains detected P pili in 16 of 16 and 14 of 16 P-piliated strains by Western blotting (immunoblotting). The binding of ASA to the homologous pilus filament was inhibited by only 3 of 17 strains. ASA agglutinated only two of nine heterologous strains and immunoprecipitated pili from one of three heterologous strains. By immunoelectron microscopy ASA was seen to bind to pilus filaments but not as strongly as AFA. Antiserum raised to the denatured pilin subunit was not substantially more reactive with pilus filaments derived from heterologous strains than was AFA. ASA was, however, a very useful probe for detecting most P pilins.
Modulation of infection by gamma interferon treatment following trauma
Authors: Hershman M J , Polk H C , Pietsch J D , Shields R E , Wellhausen S R , Sonnenfeld G
Published: January 2020

Gamma interferon (IFN-gamma) has been shown to be able to modulate several microbiol infections, perhaps as a result of the immunoregulatory properties of this interferon. The present study was designed to determine the efficacy of IFN-gamma treatment in a mouse model of infection that simulates clinical conditions occurring following abdominal trauma. In this model, mice were first infected intraperitoneally with Escherichia coli and then underwent immediate surgical laparotomy. Finally the mice were secondarily infected intramuscularly with Klebsiella pneumoniae. Groups of CBA/J mice received either IFN-gamma or RPMI 1640 medium (controls) subcutaneously. IFN-gamma was administered daily at a dose of 7,500 U, commencing 1 h postlaparotomy and continuing until the second bacterial challenge. Mice treated with IFN-gamma survived significantly longer than controls. The Ia antigen expression of peripheral blood monocytes was severely reduced in animals for 3 days after laparotomy and for 5 days after laparotomy and infection. This drop in Ia antigen expression was prevented in animals treated with IFN-gamma. These data indicate that IFN-gamma had a beneficial effect in a model simulating bacterial infection after trauma and that maintenance of Ia antigen expression in interferon-treated mice may have contributed to the observed therapeutic effect.
Cloning and expression of a Streptococcus sanguis surface antigen that interacts with a human salivary agglutinin
Authors: Demuth D R , Davis C A , Corner A M , Lamont R J , Leboy P S , Malamud D
Published: January 2020

Human saliva contains a high-molecular-weight glycoprotein (agglutinin) which binds to specific streptococci in a calcium-dependent reaction leading to the formation of bacterial aggregates. We report the cloning of a gene encoding a surface antigen from Streptococcus sanguis M5 and show that the expressed protein inhibits agglutinin-mediated aggregation and specifically binds the salivary agglutinin in a calcium-dependent fashion. Clones isolated from the immunological screening of S. sanguis M5 genomic libraries with polyclonal antibodies against whole cells were assayed for the ability to compete with S. sanguis for agglutinin. One clone, pSSP-5, expressed antigens of 165 and 130 kilodaltons (kDa) possessing this activity. A 3-kilobase-pair (kbp) insert fragment from this clone was used to screen a genomic library in lambda EMBL3 which resulted in the isolation of clone SSP-5A. This clone contained an insert of 17 kb and expressed proteins of 170 to 205 kDa that reacted with the anti-S. sanguis antibodies. Subcloning of a 5.3-kbp EcoRI-BamHI fragment from SSP-5A produced pEB-5, which expressed streptococcal components that were indistinguishable from SSP-5A. The streptococcal antigen was purified by gel permeation and ion exchange chromatography and shown to potently compete with S. sanguis M5 cells for agglutinin. The antigen also bound purified salivary agglutinin in the presence of 1 mM CaCl2. This binding was inhibited by EDTA. Both the SSP-5 antigen and a 205-kDa protein in surface protein extracts from S. sanguis M5 cross-reacted with antibodies directed against antigen B from S. mutans and SpaA from S. sobrinus 6715. These results indicate that a 205-kDa surface protein that is antigenically related to SpaA and antigen B is involved in the binding of salivary agglutinin to S. sanguis M5.
Administration of purified anti-L3T4 monoclonal antibody impairs the resistance of mice to Listeria monocytogenes infection
Authors: Czuprynski C J , Brown J F , Young K M , Cooley A J
Published: January 2020

Anti-L3T4 monoclonal antibody (GK1.5) treatment significantly impaired the antilisteria resistance of mice as manifested by increased recovery of listeriae from the spleens and livers of anti-L3T4-treated mice and by greater severity of damage to the liver and other organs. Anti-L3T4-treated mice demonstrated a profound decrease in their T cell-mediated responses to Listeria monocytogenes and its products; they failed to develop delayed type hypersensitivity to soluble listeria antigens in vivo, and their spleen cells proliferated poorly in response to stimulation by either mitogens or listeria antigens in vitro. Spleen cells from control listeria-infected mice produced significant amounts of gamma interferon when stimulated with listeria antigens in vitro, whereas under the same conditions spleen cells from anti-L3T4-treated listeria-infected mice failed to produce detectable gamma interferon. Anti-L3T4 treatment resulted in a slight increase in serum colony-stimulating activity as compared with control listeria-infected mice, probably as a result of the increased bacterial burden in these animals. Despite the dramatic decrease in T-cell activities, anti-L3T4-treated mice succeeded in clearing L. monocytogenes from the spleen and liver in a manner that was only slightly delayed as compared with control listeria-infected mice. In addition, anti-L3T4-treated listeria-immunized mice exhibited a moderate degree of enhanced resistance to rechallenge with L. monocytogenes, and their spleen cells were able to transfer a limited degree of antilisteria resistance to naive recipient mice. Both of these responses, however, were diminished as compared with those of control listeria-immunized mice in the same experiments. Although these observations establish a critical role for L3T4+ cells in the development of maximal resistance to listeriosis, they also suggest that compensatory mechanisms may allow mice to develop considerable antilisteria resistance even when L3T4+ cell activities are substantially reduced.
Isolation of a gene encoding a Chlamydia sp. strain TWAR protein that is recognized during infection of humans
Authors: Campbell L A , Kuo C C , Thissen R W , Grayston J T
Published: January 2020

Chlamydia sp. strain TWAR is a unique Chlamydia sp. that causes acute respiratory disease. A gene bank consisting of TWAR isolate AR-39 DNA in pUC19 was screened with anti-AR-39 rabbit immune sera. Two positive clones were isolated that contained 7.3-kilobase (pLC1) and 14.9-kilobase (pLC2) plasmids. Restriction mapping and hybridization studies showed that both pLC1 and pLC2 contained a common 4.2-kilobase PstI fragment. Plasmids were used as templates of in vitro transcription-translation. All three plasmids had a novel protein product of ca. 75 kilodaltons not found in the vector alone. Western blots showed that this protein reacted with anti-TWAR rabbit immune sera and with human immune serum from an individual who had proven TWAR infection. Whole-cell lysates of TWAR demonstrated a protein having the same molecular weight and immunoreactivity as the recombinant gene product. This protein was also recognized by rabbit immune serum against Chlamydia psittaci or Chlamydia trachomatis. Southern hybridizations with the cloned fragment as a probe of digests of other Chlamydia spp. showed weakly hybridizing fragments. These results suggest that we have isolated a gene encoding a protein recognized during human TWAR infection that contains some sequences shared among Chlamydia spp.
Human hyperimmune globulin protects against the cytotoxic action of staphylococcal alpha-toxin in vitro and in vivo
Authors: Bhakdi S , Mannhardt U , Muhly M , Hugo F , Ronneberger H , Hungerer K D
Published: January 2020

Alpha-toxin, the major cytolysin of Staphylococcus aureus, preferentially attacks human platelets and cultured monocytes, thereby promoting coagulation and the release of interleukin-1 and tumor necrosis factor. Titers of naturally occurring antibodies in human blood are not high enough to substantially inhibit these pathological reactions. In the present study, F(ab')2 fragment preparations from hyperimmune globulin obtained from immunized volunteers were tested for their capacity to inhibit the cytotoxic action of alpha-toxin in vitro and in vivo. These antibody preparations exhibited neutralizing anti-alpha-toxin titers of 80 to 120 IU/ml, whereas titers in commercial immunoglobulin preparations were 1 to 4 IU/ml. In vitro, the presence of 2 to 4 mg of hyperimmune globulin per ml protected human platelets against the action of 1 to 2 micrograms of alpha-toxin per ml. Similarly, these antibodies fully protected human monocytes against the ATP-depleting and cytokine-liberating effects of 0.1 to 1 microgram of alpha-toxin per ml. Intravenous application of 0.5 mg (85 to 120 micrograms/kg of body weight) of alpha-toxin in cynomolgus monkeys elicited acute pathophysiological reactions which were heralded by a selective drop in blood platelet counts. Toxin doses of 1 to 2 mg (170 to 425 micrograms/kg) had a rapid lethal effect, the animals presenting with signs of cardiovascular collapse and pulmonary edema. Prior intravenous application of 4 ml of hyperimmune globulins per kg inhibited the systemic toxic and lethal effects of 1 mg (200 micrograms/kg) of alpha-toxin. In contrast, normal human immunoglobulins exhibited no substantial protective efficacy in vitro and only marginal effects in vivo. It is concluded that high-titered anti-alpha-toxin antibodies effectively protect against the cytotoxic actions of alpha-toxin.
Spontaneous mutation results in loss of the cytadhesin (P1) of Mycoplasma pneumoniae
Authors: Su C J , Chavoya A , Baseman J B
Published: January 2020

The cytadhesin (P1) structural gene of a spontaneous mutant of Mycoplasma pneumoniae which displayed a P1-negative phenotype was analyzed. An extra adenine was discovered in a stretch of normally seven adenines near the N-terminal region of the mutant P1 structural gene. The frameshift mutation resulted in the early termination of protein translation. Possible causes of the mutation are discussed.
Beta interferon inhibits Toxoplasma gondii growth in human monocyte-derived macrophages
Authors: Schmitz J L , Carlin J M , Borden E C , Byrne G I
Published: January 2020

Gamma interferon (IFN-gamma) previously has been shown to block the replication of Toxoplasma gondii in fibroblasts by the induction of indoleamine 2,3-dioxygenase (IDO) activity. IFN-beta also is known to induce IDO activity in monocyte-derived macrophages, but its ability to block the growth of T. gondii has not been demonstrated. We found not only that the combination of IFN-beta and lipopolysaccharide induced greater IDO activity in monocyte-derived macrophages than did IFN-beta alone but that this combination also was effective in inhibiting the growth of T. gondii. In addition, the inhibition was reversed by the addition of exogenous tryptophan, thus demonstrating that a mechanism by which IFN-beta inhibited T. gondii replication was by the induction of IDO.
An orally administered bacterial immunomodulator primes rabbit neutrophils for increased oxidative burst in response to opsonized zymosan
Authors: Helmberg A , Böck G , Wolf H , Wick G
Published: January 2020

To assess the potential effect of an orally administered immunomodulator, consisting of a lysate of seven different bacteria, on polymorphonuclear leukocyte (PMN) function, rabbits were fed this preparation for five consecutive days via a gastric tube. On day 6, PMN were separated from peripheral blood and oxidative burst was triggered by opsonized zymosan or 12-O-tetradecanoylphorbol-13-acetate and quantitated on a single-cell basis. This study presents the extension of an existing flow cytometric method, leading to the possibility of quantitating single-cell oxidative burst triggered by particulate (instead of only soluble) stimuli. By this means, treated animals showed statistically significant increased oxidative burst reactions compared with the control group. The data provide evidence that oral application of a bacterial immunomodulator leads to a primed state in PMN for increased oxidative activity in response to a particulate stimulus. This offers the possibility that the beneficial effect of similar treatment in humans may in part be due to comparable mechanisms.
Endotoxicosis induced by Coxiella burnetii lipopolysaccharide stimulates a ribosomal protein S6 kinase: some properties of the partially purified enzyme
Authors: Picking W D , Hackstadt T , Paretsky D
Published: January 2020

Guinea pig endotoxicosis induced by lipopolysaccharide from Coxiella burnetii Nine Mile phase I stimulates phosphorylation of liver ribosomal protein S6, with a 50% increase at 12 h postinoculation. The responsible protein kinase (S6PK) has been partially purified from liver; its activity is independent of cyclic AMP and of Ca2+ plus phosphatidyl serine or diacylglycerol. The preparation has an apparent optimum concentration of 20 mM Mg2+, while Ca2+ and Mn2+ are each inhibitory at 2 mM. The apparent Km for ATP is 30 microM with intact ribosomes. Because of the central role of phosphorylation in metabolic regulation and a purported role of phosphorylated S6 in protein synthesis, the lipopolysaccharide-induced stimulation of S6PK suggests a significant regulatory role of such enzymes in the pathobiochemistry of Q fever infection and endotoxicosis.
A rabbit model for study of Mycobacterium paratuberculosis infection
Authors: Mokresh A H , Czuprynski C J , Butler D G
Published: January 2020

Of 21 newborn rabbits inoculated orally with Mycobacterium paratuberculosis ATCC 19698, 13 (62%) became infected, as determined by histopathology and culture. Of the 21 inoculated rabbits, 14 (67%) experienced episodes of intermittent diarrhea, sometimes as early as 5 months after inoculation. Feces varied in consistency from soft-semisolid to watery. The organism was isolated from the sacculus rotundus, vermiform appendix of the cecum, ileum, mesenteric lymph node, and feces of 9 of 21 (43%) M. paratuberculosis-inoculated rabbits 8 to 10 months after inoculation. One infected rabbit gradually became severely emaciated; advanced paratuberculosis was confirmed by culture and histopathology. Of 21 rabbits, 9 (43%) developed multifocal, well-demarcated granulomatous enteritis in the sacculus rotundus and the vermiform appendix of the cecum. There was no significant difference in the rate of infection when the organisms were administered daily for 5 or 10 days in cow milk or broth. There was no discernible effect of pregnancy, parturition, or lactation on the severity of intestinal lesions, clinical signs, or the number of rabbits infected. Complement fixation and delayed-type hypersensitivity skin tests failed to detect infection. The results of this study suggest that newborn rabbits inoculated orally with M. paratuberculosis constitute a useful animal model for the study of paratuberculosis infection.
Environmental alteration and phenotypic regulation of Candida albicans adhesion to plastic
Authors: Kennedy M J , Rogers A L , Yancey R J
Published: January 2020

The adhesion of Candida albicans to plastic was examined after growth in two chemically defined media, Lee-Buckley-Campbell (LBC) and yeast nitrogen base (YNB), by binding isotherms, Langmuir isotherms, and Scatchard plots, and the number of binding sites (N) and the affinity constants (K) were calculated. K and N were twofold and fourfold higher, respectively, after growth in LBC compared with that in YNB. A comparison of adhesion in different assay solutions gave similar results, with the solution given to dehydrated patients (5% glucose in 0.45% NaCl [D5.45]) allowing for the highest K and the largest N. Scatchard curves for both LBC- and YNB-grown cells had negative slopes, which is supportive evidence for the view that negative cooperativity is involved in the binding process. Additional experiments to examine the role of cell surface hydrophobicity in adhesion to plastic were conducted with the white and opaque phenotypes of C. albicans. There was no significant difference in the adhesion of these phenotypes to plastic, although the opaque phenotype was significantly more hydrophobic. Adhesion, but not cell surface hydrophobicity, of both phenotypes was significantly greater in D5.45. Moreover, relatively hydrophilic mycelial forms of C. albicans were found to attach only when D5.45 was used as the assay medium and, in contrast to yeast-phase cells, were insensitive to reduced adhesion by nonionic detergents. These results suggest that the adhesion of C. albicans to plastic is regulated by environmental circumstances and the phenotypic state of the organism.
Interrelationships between digestive proteolytic activities and production and quantitation of toxins in pseudomembranous colitis induced by Clostridium difficile in gnotobiotic mice
Authors: Corthier G , Muller M C , Elmer G W , Lucas F , Dubos-Ramaré F
Published: January 2020

Clostridium difficile pathogenicity is related to in vivo production of toxins, and it is of great interest to detect toxins produced in biological samples. Several reports have shown that proteases in stools interfere with immunological methods for quantitation of toxin A. The purpose of this work was to estimate the relationship between the proteases and the C. difficile toxins produced in a gnotobiotic mouse model of pseudomembranous cecitis. Cecal proteolytic activities hydrolyzed toxin A, and immunoglobulin G bound to the microtiter plate used in immunoassays. This interference could be blocked by the addition of trypsin inhibitor to the samples. The ability of soluble toxin A to bind to bound antibodies in an enzyme-linked immunosorbent assay was not affected by the proteases, but the biological activity was reduced 100-fold. The cytotoxicity of toxin B was not modified by proteolytic activity treatment. Mice inoculated with a low toxin A-producing strain of C. difficile did not died, and no modulation of proteolytic activities occurred. After inoculation with the lethal VPI strain of C. difficile, toxins A and B reached maximum levels in the ceca at 12 h postinfection. At this time, the proteolytic activities did not decrease from the levels seen at zero time. Mice died within 2 days. At this time (about 32 postinfection), proteolytic activities were sharply decreased in the lower parts of the digestive tracts. The findings that serum inhibited the proteases and that there was a 100-fold increase in serum-derived mouse immunoglobulins in the lumen as the C. difficile infection progressed suggest that the decrease in protease activity in the lower digestive tract may be related to the exudation of serum from the inflammation process.