bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucan-binding domain of a glucosyltransferase from Streptococcus sobrinus: isolation of a 55-kilodalton peptide from a trypsin digest of glucosyltransferase prebound to insoluble glucan
Authors: Kobayashi S , Koga K , Hayashida O , Nakano Y , Hasegawa Y
Published: January 2020

We isolated a glucan-binding domain of water-insoluble glucan synthase (GTF-I) of Streptococcus sobrinus B13. Mild trypsin digestion of GTF-I bound to a water-insoluble glucan (IG) produced one predominant large fragment (55 kilodaltons). The fragment was easily recovered in IG precipitate. The isolated fragment had the same degree of affinity to IG as did the native GTF-I but no glucan synthesis activity. By the same method, a similar 55-kilodalton fragment was protected for GTF-Sd but not for GTF-Si. Immunological comparisons using specific antisera against the purified glucan-binding fragment of GTF-I from strain B13 indicated that GTF-I and GTF-S have a distinct glucan-binding domain.
Development of resistance to Trypanosoma cruzi in mice depends on a viable population of L3T4+ (CD4+) T lymphocytes
Authors: Araujo F G
Published: January 2020

Mice injected with monoclonal antibody to the L3T4+ (CD4+) cell membrane surface glycoprotein of T lymphocytes and immunized with antigens of Trypanosoma cruzi had reduced antibody and cell-mediated immune responses to the organism. Mortality in these mice was 100% following challenge with 1.5 x 10(5) trypanosomes, whereas controls had a 50% survival rate, indicating that T-cell activation and viable L3T4+ (CD4+) T lymphocytes are essential for the development of resistance.
Elimination of vaginal colonization with Escherichia coli by administration of indigenous flora
Authors: Herthelius M , Gorbach S L , Möllby R , Nord C E , Pettersson L , Winberg J
Published: January 2020

A persistent vaginal colonization with a pyelonephritogenic strain of Escherichia coli, induced by administration of amoxicillin, was established in four adult cynomolgus monkeys. This colonization mimicked the one seen in urinary tract infection-prone human females. Attempts to eliminate the E. coli colonization and restore normal conditions were made. Either suspensions of lactobacilli or vaginal fluid from a healthy unmanipulated monkey was administered as repeated vaginal flushes for 5 to 9 days. A total elimination of vaginal E. coli was observed in two of six experiments with lactobacilli, and a decrease was observed in the other four. A better result was obtained with flushes of vaginal fluid, which eliminated the E. coli colonization in eight of eight experiments. In two of these, a single flush was sufficient to obtain a decolonization. The ability of fresh vaginal fluid to eliminate E. coli from the vagina could be transferred from one monkey to another. This study demonstrates the role of the normal flora in the defense against genital colonization with potentially pathogenic adhering E. coli. The possible clinical relevance of these findings must be further examined.
The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity
Authors: Pinkney M , Beachey E , Kehoe M
Published: January 2020

Site-directed mutagenesis of the TGC codon in a cloned streptolysin O (SLO) gene exchanged the single Cys residue in SLO for either Ala or Ser. The parent wild-type SLO (SLO.Cys-530) and the SLO.Ala-530 and SLO.Ser-530 mutant toxins, expressed in Escherichia coli, were purified and analyzed. Wild-type SLO.Cys-530 and the SLO.Ala-530 mutant showed no significant differences in their specific hemolytic activities, while the SLO.Ser-530 mutant had a reduced (ca. 25%), but still considerable, specific hemolytic activity as compared with that of wild-type SLO. The parent and mutant toxins extracted from lysed erythrocyte membranes had similar sedimentation profiles on sucrose density gradients, suggesting that the mutations did not affect the ability of SLO to form oligomers in membranes. These results show that the widely held assumption that the in vitro cytolytic activity of SLO requires an essential Cys residue is not true.
Separation of chromosomes of Cryptococcus neoformans by pulsed field gel electrophoresis
Authors: Perfect J R , Magee B B , Magee P T
Published: January 2020

Chromosomes from Cryptococcus neoformans, an encapsulated yeast pathogen, were separated by contour-clamped homogeneous field gel electrophoresis. Seven strains representing all four serotypes were studied. It was found that each strain had a unique, reproducible pattern of chromosome bands which could potentially be used for strain polymorphism studies. There were between 10 and 12 chromosomes in the strains studied, with an approximate genomic size of 15,000 to 17,000 kilobases. Chromosome separation also could be used to assign locations for cloned genes, and the ribosomal DNA genes were found on one of the larger C. neoformans chromosomes. The technique of electrophoretic karyotyping should be helpful for genetic and molecular investigations into the biology of C. neoformans.
Monoclonal antibodies that define neutralizing epitopes of pertussis toxin: conformational dependence and epitope mapping
Authors: Lang A B , Ganss M T , Cryz S J
Published: January 2020

The epitope specificities of 13 hybridomas secreting monoclonal antibodies (MAbs) specific for pertussis toxin (PT) is described. Hybridoma lines were derived by the fusion of spleen cells from mice immunized with native PT, Formalin-detoxified PT, or isolated PT subunits (S1 to S5) with the myeloma line X63-Ag8.653. Five MAbs showed a toxin-neutralizing ability, which was demonstrated by use of a Chinese hamster ovary cell assay system and by a NAD glycohydrolase assay. All five toxin-neutralizing MAbs demonstrated high specificities for and reactivities with native PT but were unable to bind to denatured PT. One MAb was able to neutralize the enzymatic activity of PT. The other four neutralizing MAbs inhibited the binding of PT or PT subunits to the surface of Chinese hamster ovary cells, as shown by an immunofluorescence assay. All neutralizing MAbs reacted with purified S2-S4 or S3-S4 dimers but not with S4 alone. Three MAbs which recognized a common epitope shared by S2 and S3 (which are about 70% homologous at the DNA level) and one MAb which recognized S4 were not neutralizing. Isolated S2-S4 and S3-S4 dimers bound to Chinese hamster ovary cells. These results indicate that the majority of critical epitopes which elicit neutralizing antibody are conformation dependent.
Dynamics of dibutyryl cyclic AMP- and prostaglandin E2-mediated suppression of lipopolysaccharide-induced tumor necrosis factor alpha gene expression
Authors: Spengler R N , Spengler M L , Lincoln P , Remick D G , Strieter R M , Kunkel S L
Published: January 2020

The regulation of lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF) production by prostaglandin E2 (PGE2), forskolin, and dibutyryl cyclic AMP (cAMP) was examined at the cellular and molecular levels. The above three agents could suppress LPS (100 ng/ml)-stimulated TNF production by immunologically activated murine macrophages (M phi s) in a dose-dependent manner. The concomitant addition of PGE2, dibutyryl cAMP, or forskolin to LPS-challenged M phi s resulted in 50% inhibition of TNF production at 10(-7), 3 X 10(-6), and 3 X 10(-5) M, respectively. Interestingly, delaying the addition of PGE2 or dibutyryl cAMP by 1.5 h post-LPS stimulation was also effective in suppressing the production of TNF bioactivity, but only dibutyryl cAMP was effective when its addition was delayed by 3 h. Northern (RNA) blot analysis of mRNA isolated from LPS-challenged M phi s treated with PGE2 or dibutyryl cAMP corroborated the bioactivity data. The delayed addition of PGE2 or dibutyryl cAMP by 1.5 h post-LPS stimulation resulted in a suppression of TNF mRNA accumulation by 50 to 70%. These data support the concept that LPS is a potent stimulus for M phi-derived TNF production and that this mediator is a very proximal signal in LPS-mediated disease states. Thus, therapeutic approaches that target the suppression of TNF in LPS-dependent disease states may be limited by the rapid expression of this mediator.
Francisella tularensis-specific T-cell clones are human leukocyte antigen class II restricted, secrete interleukin-2 and gamma interferon, and induce immunoglobulin production
Authors: Surcel H M , Ilonen J , Poikonen K , Herva E
Published: January 2020

T-cell clones (TLC) were established from a Francisella tularensis-vaccinated subject in order to study the cells responsive for cell-mediated immunity against F. tularensis. All the clones were human leukocyte antigen (HLA) class II restricted to one of the HLA-DR specificities of the original donor. The TLC cells were CD4+ and produced interleukin-2 and gamma interferon after stimulation with specific antigen. Seven of the eight clones tested assisted in the production of immunoglobulin G (IgG), IgA, and IgM antibodies.
Fimbria-associated adhesin of Bacteroides loeschei that recognizes receptors on procaryotic and eucaryotic cells
Authors: Weiss E I , London J , Kolenbrander P E , Andersen R N
Published: January 2020

Inhibition studies with a set of adhesin-specific monoclonal antibodies and various sugars revealed that a fimbria-associated adhesin of Bacteroides loeschei recognizes receptors on both procaryotic and eucaryotic cells. These interactions permit this bacterium to attach to both types of cells, producing coaggregates in the presence of strains of Streptococcus sanguis and hemagglutination in the presence of neuraminidase-treated human erythrocytes.
Correlates of cell-mediated immunity in Candida albicans-colonized gnotobiotic mice
Authors: Balish E , Filutowicz H , Oberley T D
Published: January 2020

Germfree athymic (nu/nu) and euthymic (nu/+) mice were colonized with a pure culture of Candida albicans. Correlates of cell-mediated immunity (lymphocyte proliferation and footpad responses to C. albicans antigens) and in vivo clearance of mucosal infections were assessed at different time intervals after alimentary tract colonization. C. albicans hyphae infected the dorsal surface of the tongue and the cardial section of the stomach in both nu/nu and nu/+ mice within 1 week after colonization with a pure culture of C. albicans. With time after colonization and infection with C. albicans, nu/+ mice manifested positive lymphocyte proliferation and positive footpad responses to Candida antigens that appeared to correlate with the capacity to clear Candida hyphae from the dorsal surface of the tongue and in the stomach. Conversely, nu/nu mice could not clear mucosal candidosis (in the stomach and on the tongue) and did not manifest either lymphocyte proliferation or footpad swelling in response to C. albicans antigens. These studies indicated that T-cell-mediated immunity may play a role in the acquired resistance of mice to mucosal candidosis. Since neither nu/nu nor nu/+ mice developed a progressive systemic disease, T cells apparently do not play a prominent role in murine resistance to systemic candidosis of endogenous origin.
Binding of Pseudomonas aeruginosa to neutral glycosphingolipids of rabbit corneal epithelium
Authors: Panjwani N , Zaidi T S , Gigstad J E , Jungalwala F B , Barza M , Baum J
Published: January 2020

35S-labeled Pseudomonas aeruginosa isolates were shown to bind to neutral glycosphingolipids (NGSLs) of rabbit corneal epithelia in culture by a thin-layer chromatogram overlay procedure. The lipids of the corneal epithelial cells grown in culture were extracted and partitioned into a chloroform-rich lower phase containing NGSLs and an aqueous upper phase containing gangliosides. By using a dot-blot assay, at least six times more radiolabeled P. aeruginosa isolates were shown to bind to the lipids in the lower phase compared with those in the upper phase. Thin-layer chromatography of the lower-phase lipids followed by staining with an orcinol spray revealed at least 10 NGSL components and several fast-migrating, nonglycosylated neutral lipid components (including cholesterol). 35S-labeled P. aeruginosa was shown to bind to NGSL components 1, 2, 5, 6, and 9. P. aeruginosa-reactive NGSL components 6 and 9 migrated with chromatographic mobilities similar to those of the standards ceramide trihexoside (CT) and ceramide monohexoside, respectively. Components 1 and 2 migrated slightly ahead of asialo GM1, and component 5 migrated faster than globoside but slower than CT. Among the various standards tested, P. aeruginosa bound to asialo GM1 and, to a lesser extent, to ceramide dihexoside and CT but not to GM1, GD1A, GM3, or ceramide monohexoside. It remains to be determined whether any of the five P. aeruginosa-reactive NGSL components of corneal epithelium identified in this study plays a role in the development of corneal infection. However, we have previously shown that component 9, one of the five P. aeruginosa-reactive NGSL components identified in this study, is present in significantly greater amounts in migrating epithelia than it is in nonmigrating epithelia (N. Panjwani, G. Michalopoulos, J. Song, G. Yogeeswaran, and J. Baum, Invest. Ophthalmol. Vis. Sci., in press). This may prove to be of biological significance because it is generally believed that traumatized (migrating) epithelia are more susceptible to infection than normal (nonmigrating) epithelia are.
Endotoxic induction of prostaglandin release from macrophages by nontoxic lipid A analogs synthesized chemically
Authors: Tanamoto K , Shade U , Rietschel E T , Kusumuto S , Shiba T
Published: January 2020

The ability of synthetic lipid A analogs to induce prostaglandin synthesis in macrophages was compared with that of native lipopolysaccharide. The synthetic preparations comprised monomeric or dimeric derivatives of D-glucosamine with different patterns of substitution by phosphate and tetradecanoic, (R)-3-hydroxytetradecanoic, and (R)-3-tetradecanoyloxytetradecanoic acid. All of these preparations are structurally distinct from native lipid A (principally regarding the position of fatty acid substitution) and hence have been previously shown to be endotoxically inactive in many biological tests. It was found that many of these synthetic samples exhibit strong activity in inducing prostaglandin E2 and prostaglandin F2 alpha, with some of them having activity comparable to that of lipopolysaccharide. Experiments with macrophages of C3H/HeJ mice enabled us to differentiate between endotoxin-specific (in the case of dimeric preparations) and endotoxin-nonspecific (for monomeric preparations) mechanisms for the induction of prostaglandins. These results indicate that there is a difference in the mechanism of induction of prostaglandin synthesis between monomeric lipid A's and dimeric or native lipid A structures.
Kinetics of Cryptosporidium parvum sporozoite neutralization by monoclonal antibodies, immune bovine serum, and immune bovine colostrum
Authors: Perryman L E , Riggs M W , Mason P H , Fayer R
Published: January 2020

Monoclonal antibodies, immune bovine serum, and immune bovine colostral whey neutralized infectivity of Cryptosporidium parvum sporozoites for mice in a time-dependent manner. Immune colostral whey neutralized sporozoites more rapidly and completely than immune serum, monoclonal antibody (MAb) 18.44, or a combination of MAb 18.44 and MAb 17.41. Mice were partially protected against oral challenge with C. parvum oocytes when treated with immune colostral whey, MAb 17.41, or a combination of MAb 17.41 and MAb 18.44.
Specificity of glycosphingolipid recognition by Entamoeba histolytica trophozoites
Authors: Bailey G B , Nudelman E D , Day D B , Harper C F , Gilmour J R
Published: January 2020

The ability of purified glycosphingolipids to enhance liposome-stimulated Entamoeba histolytica actin polymerization was assessed as a means of defining the specificity of mammalian cell membrane lipid glycan recognition by this parasite. Synthetic liposomes containing a variety of individual glycosphingolipids bearing neutral, straight-chain oligomeric glycans with galactose or N-acetylgalactosamine termini stimulated rapid (90-s) polymerization of amoeba actin. Glycans with terminal N-acetylglucosamine residues were not stimulatory at all or were only weakly stimulatory. Glycans with glucose, N-acetylglucosamine, galactose, and N-acetylgalactosamine as the penultimate residue were recognized. Attachment of N-acetylneuraminate to the terminal residue of a stimulatory glycosphingolipid eliminated activity; attachment of fucose to the penultimate sugar reduced activity. Glycans with a terminal beta 1-4 or 1-3 glycosidic bond were most effective; glycans with terminal alpha 1-4 or 1-3 glycosides were less effective. The activity of glycans with both beta- and alpha-linked terminal glycosides was inhibited by lactose, suggesting recognition of both configurations by a single amoeba protein. The ability of liposomes to stimulate actin polymerization reflected the extent of liposome phagocytosis.
Developmental cycle-specific host-free RNA synthesis in Chlamydia spp
Authors: Crenshaw R W , Fahr M J , Wichlan D G , Hatch T P
Published: January 2020

The incorporation of radiolabeled GTP into RNA in host-free Chlamydia trachomatis serovar L2 organisms was investigated. The incorporation was partially inhibited by rifampin and dactinomycin and hydrolyzed by RNase. RNA made by host-free chlamydiae consisted mainly of species of fewer than 800 bases in size, although 16S and 23S species were noted by agarose-gel electrophoresis. The hybridization of radiolabeled host-free RNA to restriction fragments of the gene encoding the major outer membrane protein was analyzed; all regions of the gene were transcribed. The relative intensity of hybridization of host-free RNA made by chlamydiae isolated during the middle and late stages of the developmental cycle to the DNA of clones encoding gene products known to be made at these times in vivo indicated that the temporal patterns of host-free and in vivo transcription were similar. Radiolabeled RNA from 1- and 24-h host-free Chlamydia psittaci 6BC organisms hybridized to many of the same EcoRI and BamHI restriction fragments of C. psittaci genomic DNA, although some differences could be noted. When these RNAs were used to screen a partial C. psittaci genomic library in lambda gt11, plaques were identified that reacted mainly either with 1-h RNA or with 24-h RNA. Because RNA synthesized by host-free chlamydiae appears to be developmental cycle stage specific, transcripts made by host-free chlamydiae may be convenient probes that can be used to clone developmental stage-specific chlamydial genes.
Pseudomonas aeruginosa alginate in cystic fibrosis sputum and the inflammatory response
Authors: Pedersen S S , Kharazmi A , Espersen F , Høiby N
Published: January 2020

Alginate, a viscous polysaccharide from mucoid Pseudomonas aeruginosa, may interfere with the host defenses in patients with cystic fibrosis and chronic P. aeruginosa lung infection. The alginate concentration in the sol phase of expectorated sputum was quantitated by a biochemical method and a newly developed enzyme-linked immunosorbent assay. There was a high degree of correlation between the methods, and the concentration of alginate ranged from 4 to 101 micrograms/ml with a median of 35.5 micrograms/ml when measured by enzyme-linked immunosorbent assay. Alginate could not be detected in the bronchial secretions from patients without P. aeruginosa infection. In vitro investigation of alginate did not show any activation of the alternative pathway of complement, as determined by a hemolytic kinetic assay and by testing for neutrophil chemotaxis. At a high concentration, P. aeruginosa alginate caused a slight activation of the classical pathway of complement. Alginate did not cause neutrophil chemotaxis by itself but was able to reduce the neutrophil chemotactic response to N-formylmethionylleucylphenylalanine and for zymosan-activated serum. P. aeruginosa and seaweed alginates were able to prime neutrophils for increased N-formylmethionylleucylphenylalanine-induced neutrophil oxidative burst, as determined by chemiluminescence. Because of its ability to prevent attraction of neutrophils to the site of infection, lack of complement activation, and ability to enhance neutrophil oxidative burst, alginate from P. aeruginosa may contribute to the persistence and pathogenesis of chronic P. aeruginosa infection in cystic fibrosis.
Immunization of mice against Plasmodium vinckei with a combination of attenuated Salmonella typhimurium and malarial antigen
Authors: Kumar S , Gorden J , Flynn J L , Berzofsky J A , Miller L H
Published: January 2020

Infection with the blood stage of the malaria parasite Plasmodium vinckei is uniformly lethal in mice. We found that immunization of BALB/c mice with a combination of killed P. vinckei antigens and an attenuated (aroA) Salmonella typhimurium strain induces high levels of protection against challenge with live P. vinckei. This is especially significant because, in our previous studies, immunization of mice with killed P. vinckei antigens and adjuvants such as Bordetella pertussis, complete Freund adjuvant, and saponin failed to induce protective immunity. Immunization with attenuated S. typhimurium alone did not provide any nonspecific immunity. In vivo depletion of CD4+ T cells in the mice immunized with attenuated S. typhimurium and P. vinckei antigens caused the loss of their immunity. Expression of this immunity required the presence of a spleen. These results support our previous hypothesis that a blood stage malaria vaccine may need both induction of CD4+ T cells specific for the parasite and modification of the spleen with a vaccine vehicle. Therefore, attenuated Salmonella strains such as the one used in this study, when expressing recombinant malarial antigens, might fulfill this requirement.
Immunological cross-reactivity between Streptococcus mutans and human heart tissue examined by cross-immunization experiments
Authors: Wu H Y , Russell M W
Published: January 2020

Hyperimmunization of rabbits with Streptococcus mutans or other related cariogenic streptococci sometimes induces serum antibodies that react with human heart muscle. To determine whether antigen I/II (AgI/II), a major surface protein antigen present in most human isolates of these organisms, was responsible for inducing cross-reactive antibodies, we tested it for antigenic similarity to heart components, exploiting the ability of immune systems to mount anamnestic responses to antigens previously encountered. Mice immunized with a strain of Streptococcus pyogenes type M6, known to be heart cross-reactive, or with intact S. mutans cells developed antibodies that could be detected on a human heart sarcolemmal preparation. However, mice immunized with AgI/II and boosted with sarcolemma were unable to develop significant antisarcolemmal antibodies attributable to prior sensitization by AgI/II. Similarly, AgI/II was unable to recall antisarcolemmal responses in mice previously immunized with sarcolemma. Nevertheless, strong immunoglobulin G antibody responses to AgI/II were detected at the single-cell level in spleens and as circulating antibodies in all mice immunized with AgI/II or AgI/II-bearing S. mutans. We conclude that the ability of S. mutans to induce heart-reactive antibodies is not due to antigenic similarity between AgI/II and components of human heart but may be caused by other cross-reactive antigens in the bacterial cells or by nonspecific stimulation of the immune system.
Synthesis of species-specific stress proteins by virulent strains of Listeria monocytogenes
Authors: Sokolovic Z , Fuchs A , Goebel W
Published: January 2020

Listeriolysin is a virulence factor that appears to be necessary for the intracellular survival of Listeria monocytogenes. As shown in this investigation, listeriolysin is produced in only small amounts by clinical isolates of L. monocytogenes belonging to the serogroup 1/2a, but its synthesis can be induced by heat shock and to a lesser extent by oxidative stress. In addition to about 15 heat shock proteins that appear to be common to L. monocytogenes and Listeria species that are nonpathogenic for humans, at least five heat shock proteins are specifically coinduced with listeriolysin in all L. monocytogenes strains under heat shock conditions but not in the other Listeria species. One type of L. monocytogenes mutant blocked in the expression of listeriolysin failed to synthesize several of these specific heat shock proteins.
Recognition of binding sites on Candida albicans by monoclonal antibodies to human leukocyte antigens
Authors: Mayer C L , Diamond R D , Edwards J E
Published: January 2020

Candida albicans exhibits examples of human molecular mimicry, including receptors resembling human steroid receptors and human complement receptor (CR)-like receptors that bind the complement fragments C3d and iC3b. To further characterize the epitopes of the Candida human CR-like molecules, a panel of monoclonal antibodies (MAbs) against epitopes within the human CR3 was used. MAbs Mo1, M1/70HL, and 7C3 bound to the germ tube, as assessed by immunofluorescence. MAbs Leu15, 60.1, and 95G8 did not bind. Miscellaneous MAbs against other antigens on human leukocytes (B2, 3D9, and OKT4) did not bind. However, MY9, which recognizes a monocyte antigen, bound extensively to the germ tube. The binding of certain anti-CR3 MAbs suggested limited identity between the Candida CR3-like receptor and the human CR3. The binding of MY9 identified an antigen recognized by a MAb to a human cell antigen not previously known to exist on C. albicans.
Fab fragments from a monoclonal antibody against a germ tube mannoprotein block the yeast-to-mycelium transition in Candida albicans
Authors: Casanova M , Martínez J P , Chaffin W L
Published: January 2020

Fab fragments prepared from the immunoglobulin G monoclonal antibody (MAb) 4C12, which reacts with a determinant expressed on the hyphal extension of germ tubes of Candida albicans, inhibited germ tube formation, but intact MAb 4C12 did not. Indirect immunofluorescence showed a punctate binding pattern on cells incubated with Fab fragments but a confluent binding on cells incubated with intact MAb 4C12.
Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata
Authors: Dobbelaere D A , Prospero T D , Roditi I J , Kelke C , Baumann I , Eichhorn M , Williams R O , Ahmed J S , Baldwin C L , Clevers H
Published: January 2020

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed.
Comparison of the Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase
Authors: Häse C C , Finkelstein R A
Published: January 2020

The soluble hemagglutinin/protease (HA/protease) produced by Vibrio cholerae and the elastase of Pseudomonas aeruginosa are both zinc/calcium-dependent proteases. In the present study the two enzymes are compared immunologically and functionally. The N-terminal amino acid sequences of the proteins had 65% identity within the first 20 amino acids. Polyclonal antisera against each purified protein recognized the enzyme of the other species in enzyme-linked immunosorbent assay, checkerboard immunoblot, and Western blot analyses and inhibited the protease activity of both enzymes in milk and elastin agars. Like the HA/protease, the elastase hemagglutinated "responder" but not "nonresponder" chicken erythrocytes, degraded ovomucin, lactoferrin, and fibronectin, and nicked the A subunit of the cholera toxin-related heat-labile enterotoxin from Escherichia coli. Whereas none of the three proteases tested (elastase, HA/protease, or pronase E) had any obvious effect in ileal loop tests in rabbits at doses up to 50 micrograms, all three produced some detectable skin reactions at a dose of 0.1 micrograms and necrosis at a higher dose (i.e., 5 micrograms). We conclude that the V. cholerae HA/protease and the P. aeruginosa elastase are structurally, functionally, and immunologically related.
Susceptibility of inbred mice to Leishmania major infection: genetic analysis of macrophage activation and innate resistance to disease in individual progeny of P/J (susceptible) and C3H/HeN (resistant) mice
Authors: Fortier A H , Tong A , Nacy C A
Published: January 2020

We tested the possibility that two phenotypic traits, defective activation of macrophage antileishmanial activities and susceptibility to infection with Leishmania major, were controlled by the same gene. We used P/J (susceptible) and C3H/HeN (resistant) mice to breed F1, backcross (Bx), and F2 mice that were tested individually for both traits, each of which is known to be controlled by a single autosomal gene. We found no correlation between the macrophage defect and cutaneous disease. There was a correlation between development of systemic disease and defective macrophage activation in Bx mice; this correlation, however, was not confirmed in the F2 population.
Effects of gastrointestinal candidiasis, antibiotics, dietary arabinitol, and cortisone acetate on levels of the Candida metabolite D-arabinitol in rat serum and urine
Authors: Wong B , Brauer K L , Clemens J R , Beggs S
Published: January 2020

We studied the effects of gastrointestinal (GI) colonization by Candida albicans, dietary arabinitol, intragastric antibiotics, and cortisone on levels of the Candida metabolite D-arabinitol in rat serum and urine. Rats given conventional laboratory chow, intragastric gentamicin and chloramphenicol, and 6.0 x 10(8) live C. albicans B311 blastoconidia by gavage had minimal invasive GI disease and no more DL-arabinitol in the urine than controls given killed C. albicans. However, colonized and uncolonized rats given intragastric antibiotics had transiently higher urine arabinitol levels than the corresponding controls given saline. Rats given conventional laboratory chow (which contained 50 micrograms of arabinitol per g) had higher serum and urine arabinitol levels than rats given no dietary arabinitol, but the differences were less than expected. Moreover, intragastric antibiotics did not cause increased arabinitol excretion in rats given no dietary arabinitol. Rats given intragastric antibiotics and live C. albicans but no dietary arabinitol had no more arabinitol in their serum or urine than controls given antibiotics and killed C. albicans or saline and live or killed C. albicans. Lastly, cortisone acetate (10 mg/kg of body weight per day intramuscularly for 10 days) did not cause increased serum or urine arabinitol levels. We conclude that neither GI colonization by C. albicans nor cortisone should interfere with the usefulness of arabinitol as a marker for invasive candidiasis; antibiotics appear to increase arabinitol excretion by suppressing GI bacteria capable of consuming dietary arabinitol.
Purification and characterization of the extracellular C3d-binding protein of Candida albicans
Authors: Saxena A , Calderone R
Published: January 2020

A C3d-binding glycoprotein was purified from the culture filtrate of Candida albicans by preparative isoelectric focusing. The protein possessed a pI of 3.9 to 4.1 and could inhibit rosetting of EAC3d (sheep erythrocytes conjugated to C3d) by pseudohyphae of C. albicans. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol, the protein migrated as a doublet with apparent molecular masses of 55 and 60 kilodaltons (kDa) and as a 50-kDa band in nonreducing gels. These results were observed with Aurodye stain for proteins. Western immunoblot, and concanavalin A stain, which indicates that both bands contain carbohydrate as well as antigenic determinants. The treatment of purified glycoprotein with endoglycosidase F but not endoglycosidases H, N, and O resulted in a complete conversion of the doublet into a faster-migrating broad band with an apparent molecular mass of 45 kDa. When the amino acid analysis of the C3d-binding protein was compared with that of the CR2 from B lymphocytes, significant differences were observed. These data indicate that C. albicans secretes a C3d-binding protein during growth in vitro which appears to be different from the mammalian C3d receptor.
Synthesis and characterization of Escherichia coli O18 O-polysaccharide conjugate vaccines
Authors: Cryz S J , Cross A S , Sadoff J C , Fürer E
Published: January 2020

Nontoxic, serologically reactive O polysaccharide was derived from Escherichia coli O18 lipopolysaccharide by acid hydrolysis, extraction with organic solvents, and gel filtration chromatography. Oxidized O polysaccharide was covalently coupled to either Pseudomonas aeruginosa toxin A or cholera toxin by using adipic acid dihydrazide as a spacer molecule in the presence of carbodiimide. The resulting conjugates were composed of approximately equal amounts of O polysaccharide and protein and were nontoxic and nonpyrogenic. Both conjugates engendered an immunoglobulin G antibody response in rabbits that recognized native O18 lipopolysaccharide. Such antibody was able to promote the uptake and killing of an E. coli O18 strain bearing the K1 capsule by human polymorphonuclear leukocytes. Immunoglobulin G isolated from the sera of rabbits immunized with either conjugate afforded protection against an E. coli O18 challenge when passively transferred to mice.
Characterization of a cell population which amplifies the anticryptococcal delayed-type hypersensitivity response
Authors: Fidel P L , Murphy J W
Published: January 2020

Cell-mediated immunity to Cryptococcus neoformans can be detected by delayed-type hypersensitivity (DTH) to a culture filtrate antigen of C. neoformans. Recently, we have identified a population of cells in spleens of mice immunized with cryptococcal antigen that, when transferred to recipient mice at the time of immunization, amplifies the anticryptococcal DTH response. If the cell donor mice are treated with cyclosporin A during induction of the anticryptococcal DTH response, the amplifier cells are not induced, whereas the cells which transfer DTH (TDH cells) are induced. The purpose of this study was to characterize the amplifier cells with respect to their surface and functional properties and, in so doing, determine whether or not the amplifier cells are analogous to long-lived memory cells. We demonstrated that the amplifier cells were nylon-wool-nonadherent, antigen-specific, CD4 (L3T4+ Lyt-2-) T lymphocytes which appear in the spleens of mice 5 days postimmunization with cryptococcal culture filtrate antigen in complete Freund adjuvant. The amplifier T (Tamp) cells are not considered to be memory cells because they are relatively short-lived, being present 14 but not 18 days after the stimulating immunization. Moreover, the amplified anticryptococcal DTH response does not fulfill the criteria of the typical secondary immune (anamnestic) response in that the amplified response does not appear early relative to the appearance of the primary anticryptococcal DTH response, and it does not persist longer than the primary DTH response. We speculate that Tamp cells are not long-lived memory cells but rather act in a T-helper cell capacity to amplify the anticryptococcal DTH response.
Contribution of superoxide dismutase and catalase activities to Shigella flexneri pathogenesis
Authors: Franzon V L , Arondel J , Sansonetti P J
Published: January 2020

A Shigella flexneri serotype 5 strain deficient in the production of the iron-containing superoxide dismutase FeSOD (sodB) and a catalase-negative (katFG) S. flexneri serotype 5 strain were isolated. Both strains were examined for increased sensitivity to oxygen stress by using assays involving killing by mouse peritoneal macrophages and human polymorphonuclear leukocytes as well as infection of rabbit ileal loops. The sodB mutant was extremely sensitive to killing by phagocytes when compared with the wild-type parent, M90T. The catalase mutant also showed an increased sensitivity to killing, but to a much lesser extent. Upon infection of rabbit ileal loops and subsequent histopathological examination, the sodB mutant caused very little detectable damage to intestinal villi. The pattern of infection was roughly similar to that of BS176, an avirulent plasmidless derivative of M90T. The katFG mutant, on the other hand, showed a high degree of destruction, similar to that caused by M90T. This evidence suggests that the superoxide dismutase encoded by sodB may play an important role in the pathogenesis of S. flexneri. In contrast, catalases appear to make a limited contribution to virulence.
Characterization of phospholipase C from Pseudomonas aeruginosa as a potent inflammatory agent
Authors: Meyers D J , Berk R S
Published: January 2020

Phospholipase C (PLC) from Pseudomonas aeruginosa induced a marked inflammatory response when injected intraperitoneally in C3H/HeJ mice. This inflammation was characterized by the accumulation of inflammatory cells and plasma protein and the release of arachidonic acid metabolites (6-trans-12-epi-leukotriene B4 [LTB4], 6-trans-LTB4, LTB4, 5-HETE (5-hydroxyeicosatetraenoic acid), LTC4, LTD4, LTE4, prostaglandin E2 [PGE2], PGF2-alpha, and thromboxane B2 [TxB2]) in the peritoneal cavity of the mice. Heat-inactivated PLC did not evoke any of these effects, suggesting that enzyme activity is necessary for PLC-induced inflammation. When human granulocytes were incubated with PLC in vitro, 6-trans-12-epi-LTB4, 6-trans-LTB4, LTB4, 5-HETE, and PGE2 were generated. Mouse peritoneal cells stimulated with PLC released 6-trans-LTB4, LTB4, PGE2, PGF2-alpha, and TxB2. Both human granulocytes and mouse peritoneal cells stimulated with PLC generated significantly increased levels of arachidonic acid metabolites as compared with cells incubated with heat-inactivated PLC. Leukotriene production by both populations of cells was inhibited when the cells were preincubated with nordihydroguaiaretic acid and subsequently stimulated with PLC. Similarly, both cell types released significantly lower amounts of cyclooxygenase pathway products when they were preincubated with indomethacin and subsequently stimulated with PLC.
Synthesis and immunological properties of conjugates composed of group B streptococcus type III capsular polysaccharide covalently bound to tetanus toxoid
Authors: Lagergard T , Shiloach J , Robbins J B , Schneerson R
Published: January 2020

A synthetic scheme for covalently binding group B streptococcus type III to tetanus toxoid (TT), using adipic acid dihydrazide as a spacer, is described. Type III alone or as a conjugate with TT was injected subcutaneously into laboratory mice, and the type-specific and TT antibody responses elicited by these immunogens were assayed. Type III-TT elicited significantly higher levels of type-specific antibodies after each immunization than did the type III alone. These levels were related to the dosage of the conjugate, enhanced by Freund adjuvant, and exhibited booster responses. Type III alone elicited only immunoglobulin M (IgM) antibodies in Swiss albino mice and mostly IgM and low levels of IgG antibodies of the IgG3 subclass in BALB/c mice. Type III-TT conjugates, in contrast, elicited mostly IgG antibodies in both strains of mice. IgA type III antibodies were not detected. The first two immunizations with the conjugates elicited type III antibodies in the IgG1 and in the IgG3 subclasses. Low levels of IgG2a type III antibodies were detected after a third injection of type III-TT. Conjugate-induced antibodies facilitated opsonization of group B streptococcus type III organisms and did not react with the structurally related pneumococcus type 14. TT alone or as a component of type III-TT induced mostly antibodies of the IgG class: IgG1 levels were the highest of the four subclasses. No IgA TT antibodies were detected. The conjugation procedure, therefore, enhanced the immunogenicity of and conferred T-cell dependent properties to the type III while preserving the immunogenicity of the TT component. The T-cell dependent properties of the conjugates were responsible for stimulating IgG type III antibodies which could be boosted. Evaluation of type III-TT conjugates in antibody-negative women of child-bearing age is planned.
Phosphorylase-cross-reactive antibodies evoked by streptococcal M protein
Authors: Dale J B , Courtney H S , Kotb M , Schifferli D
Published: January 2020

Rabbit antisera evoked by type 5 streptococcal M protein (M5) were screened by enzyme-linked immunosorbent assay (ELISA) for immunological cross-reactivity with purified rabbit muscle phosphorylases a and b. Of 10 pep M5 antisera tested, 3 showed significant cross-reactivity with both forms of the enzyme. ELISA inhibition studies using one of the pep M5 antisera showed that all of the phosphorylase b antibodies were inhibited by pep M5, the immunogen, and phosphorylase b, the ELISA antigen. All of the antibodies were also inhibited by pep M6 and pep M19, but not by pep M24, indicating that the cross-reactive epitopes were shared by multiple serotypes of M protein. Western blot (immunoblot) analyses showed that pep M5 antisera reacted strongly with the subunit of phosphorylase b. In addition, purified phosphorylase partially inhibited the binding of pep M5 antibodies to a 95-kilodalton protein of human myocardium. One of the three cross-reactive pep M5 antisera inhibited the enzymatic activity of phosphorylase a in a dose-related fashion, reaching a maximum inhibition of 75%. The enzymatic activity in the presence of antibody was totally restored when the antiserum was first incubated with pep M5.
Murine gamma interferon fails to inhibit Toxoplasma gondii growth in murine fibroblasts
Authors: Schwartzman J D , Gonias S L , Pfefferkorn E R
Published: January 2020

Although treatment of human macrophages or fibroblasts with human gamma interferon results in the inhibition of intracellular Toxoplasma gondii, murine gamma interferon stimulated only murine macrophages, not murine fibroblasts, to inhibit T. gondii. This species difference may be important in understanding the control of acute and chronic toxoplasmosis.
Cloning, expression, and sequence analysis of the Haemophilus influenzae type b strain M43p+ pilin gene
Authors: Gilsdorf J R , Marrs C F , McCrea K W , Forney L J
Published: January 2020

By using antiserum against Haemophilus influenzae type b (Hib) strain M43p+ denatured pilin, we screened a genomic library of Hib strain M43p+ and identified a clone that expressed pilin, but not assembled pili, on its surface. Southern blot analysis revealed the presence of one structural gene, which was also present in strain M42p-, a nonpiliated variant. Five exonuclease III deletion mutants, two of which had deletions that extended into the structural gene and failed to express pilin, were used to obtain the nucleotide sequence of the structural gene. The amino acid sequence of the open reading frame agrees with 38 of 40 amino acids from the published sequence of purified Hib M43p+ pilin. The pilin gene coded for a mature protein of 193 amino acids, with a calculated molecular mass of 21,101 daltons. Comparison of the Hib M43p+ pilin amino acid sequence with those of pilins of other bacteria revealed strong conservation of amino- and carboxy-terminal regions in M43p+ and Escherichia coli F17, type 1C, and several members of the P pili family, as well as Klebsiella pneumoniae type 3 MR/K, Bordetella pertussis serotype 2, and Serratia marcescens US46 fimbriae.
Adherence pili of avian strains of Escherichia coli O78
Authors: Yerushalmi Z , Smorodinsky N I , Naveh M W , Ron E Z
Published: January 2020

Avian colisepticemia starts with the invasion of the trachea by virulent strains of Escherichia coli, with strains of serotype O78 responsible for about 50% of the cases. Several O78 strains isolated from poultry with colisepticemia produced pili with a subunit of molecular weight of 18,000 that mediated adherence to avian epithelial tissues in vitro and in vivo.
Genomic structure of Candida stellatoidea: extra chromosomes and gene duplication
Authors: Rikkerink E H , Magee B B , Magee P T
Published: January 2020

Candida albicans and Candida stellatoidea are two closely related imperfect yeasts. Some isolates characterized as C. stellatoidea are in fact C. albicans, while others differ with respect to virulence and to karyotype, containing extra small chromosomes. Experiments in this study allowed us to infer that a typical C. stellatoidea isolate, Y2360, has 12 chromosomes rather than the 7 previously shown for C. albicans. The majority of cloned sequences tested hybridized to analogous chromosomes in C. albicans and in C. stellatoidea, although there were exceptions, and a repeated element isolated as specific for C. albicans hybridized to most of the chromosomes of C. stellatoidea. Several genes tested hybridized to one of the smaller, C. stellatoidea-specific chromosomes as well as to a larger one. The arrangement of restriction enzyme sites around the gene was the same in both the large and small chromosomes. For ADE2 and LYS2, the arrangements were identical to those of a typical C. albicans strain, FC18, suggesting a high degree of sequence conservation between the two species. Spheroplast fusion and segregation experiments showed that the ADE2 genes on both the large and small chromosomes of C. stellatoidea are active, implying that the organism is functionally at least triploid for this gene and probably for any others duplicated on the smaller chromosomes.
Biochemical and biological activities of recombinant S1 subunit of pertussis toxin
Authors: Barbieri J T , Pizza M , Cortina G , Rappuoli R
Published: January 2020

degP-deficient strains of Escherichia coli grown in M-9 medium supplemented with ZnCl2 expressed the recombinant S1 subunit of pertussis toxin (rS1) in a form electrophoretically identical to the authentic S1 subunit. Subcellular fractionation showed that the full-length form of rS1 was membrane associated, while proteolytic fragments of rS1 were present in the periplasm. rS1 was extracted from outer membrane preparations with 8 M urea and purified by gel filtration chromatography. Purified rS1 ADP-ribosylated transducin at a similar molar efficiency relative to authentic pertussis toxin and, when associated with the native B oligomer of pertussis toxin, elicited Chinese hamster ovary cell clustering.