bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibody-independent activation of the classical pathway of human serum complement by lipid A is restricted to re-chemotype lipopolysaccharide and purified lipid A
Authors: Vukajlovich S W
Published: January 2020

Incubation of most bacterial lipopolysaccharides (LPS) with normal human sera at 37 degrees C activates the serum complement system, resulting in decreased levels of hemolytic complement. A panel of R-chemotype LPS preparations isolated from Salmonella minnesota rough mutant strains, as well as smooth wild-type LPS from S. minnesota, Escherichia coli O55-B5, Serratia marcescens, and Yersinia enterolitica, were used to examine the effect of LPS polysaccharide chain length on LPS lipid (lipid A)-dependent activation of the classical pathway of complement (CPC). To examine specific lipid A-dependent activation of the CPC, sera deficient in alternative pathway of complement activity were prepared by the removal of factor D. Absorption of normal human sera with formalinized rabbit erythrocytes was found to remove natural antibodies, factors capable of forming LPS complexes which activate the CPC, or both. By using such factor D-depleted formalinized rabbit erythrocyte-absorbed normal human sera, only isolated lipid A and Re-chemotype LPS (R595 LPS) were found to activate the CPC. Thus, the presence of the additional monosaccharide L-glycero-D-mannoheptose in the Rd2 LPS oligosaccharide chain compared with the L-glycero-D-mannoheptose-deficient Re-chemotype LPS structure is sufficient to block lipid A-dependent activation of the CPC by LPS.
Cloning of a serotype-specific antigen from Pasteurella haemolytica A1
Authors: Gonzalez-Rayos C , Lo R Y , Shewen P E , Beveridge T J
Published: January 2020

Recombinant plasmids coding for a soluble (or surface) antigen of Pasteurella haemolytica A1 were identified. Two plasmids, both containing the same 5.4 kilobase pairs of insert DNA, were recovered independently by screening a clone band of P. haemolytica A1 genomic DNA in Escherichia coli for the expression of P. haemolytica A1 soluble antigens (R. Y. C. Lo and L.A. Cameron, Can. J. Biochem. Cell Biol. 64:73-76, 1986). E. coli cells carrying the plasmids were found to be agglutinated by an antiserum raised against the P. haemolytica A1 soluble antigens. Analysis of the E. coli clones by electron microscopy revealed patches of amorphous material on the surface of the cells which were not present on the controls. Further characterization with protein A-colloidal gold labeled both these patches and the outer membranes of these cloned cells pretreated with the specific antiserum. These results indicated that the cloned antigen was expressed on the surface of the E. coli cells. The cloned antigen was found to be specific for serotype 1 when tested by slide agglutination against a collection of P. haemolytica typing antisera. Southern blot hybridization, using the cloned DNA as a probe, labeled the genomic DNA from P. haemolytica serotype 1 as well as the cross-agglutinating serotypes 2 and 7, but not DNA from the non-cross-agglutinating serotypes 3 and 4 and Pasteurella multocida. These results demonstrated that serotype specificity could be attributed to the particular antigenic determinants in the genome of the organism.
Adjuvant activity of 6-O-acyl-muramyldipeptides to enhance primary cellular and humoral immune responses in guinea pigs: dose-response and local reactions observed with selected compounds
Authors: Tsujimoto M , Kotani S , Shiba T , Kusumoto S
Published: January 2020

6-O-(2-Tetradecylhexadecanoyl)-N-acetylmuramyl-L-alanyl-D-isoglutamine (B3O-MDP) and 6-O-(3-hydroxy-2-docosylhexacosanoyl)-MDP (BH48-MDP) were examined for immunopotentiating activity in stimulating the primary immune responses of guinea pigs to ovalbumin and for adverse reactions at the injection site and in the regional lymph nodes when administered in combination with several nonirritating vehicles. B30-MDP was found to potently stimulate both humoral and cellular immune responses, the latter by induction of delayed-type hypersensitivity, irrespective of the administration vehicle examined (liposomes, a squalene in water emulsion, Intralipid, phosphate-buffered saline, and Nikkol HCO-60 glucose solution), although the minimum effective dose was dependent on the vehicle used. Reactions in the footpad receiving the injection were negligible. Noticeable local reaction consisted of swelling of the lymph nodes in the region of the injection, but the swelling was only noted with higher doses and subsided 3 to 4 weeks after immunization, unlike the persistent swelling caused by the administration of water-in-mineral oil emulsions with or without B30-MDP. BH48-MDP and its L-serine analog, BH48-MDP(L-Ser), which has L-serine in place of L-alanine in MDP, in combination with the squalene-in-water emulsion, also intensely stimulated both cellular and humoral antiovalbumin immune responses, but the effects of these compounds seemed to be influenced to a greater degree by the vehicles than by B30-MDP. Thus, B30-MDP was chosen from a series of a 6-O-acyl derivatives of MDP as the most promising candidate for possible application in practical vaccines.
Interaction of purified lipoteichoic acid with the classical complement pathway
Authors: Loos M , Clas F , Fischer W
Published: January 2020

Glycerophosphate-containing lipoteichoic acids (LTAs) interact with the first component of the classical complement pathway (C1). This resulted in the activation of the classical complement pathway in serum, shown by the consumption of C1, C2, and C4. The dose-dependent interaction of LTAs with purified C1 and C1q was dependent on the negative charges of the phosphate groups of LTA. It was reduced by charge compensation through D-alanine ester substituents and by sterical hindrance through di- and trihexosyl residues linked to position 2 of the glycerol moieties. The charge density of LTA may also play a role: poly(digalactosylglycerophosphate) LTAs, in which the phosphate groups are in a greater distance from each other, were less effective, and the loss of micellar organization by deacylation of LTA drastically reduced the complement activation capacity.
Monoclonal antibody to a major glycoprotein immunogen mediates differential complement-independent lysis of Trichomonas vaginalis
Authors: Alderete J F , Kasmala L
Published: January 2020

An immunoglobulin G type 2a (IgG2a) monoclonal antibody (MAb), designated C20A3, which reacts with a highly immunogenic trichomonad membrane glycoprotein (approximately 270,000 daltons), produced complement-independent cytolysis of Trichomonas vaginalis organisms. Time- and temperature-dependent lysis of parasites was observed following incubation of washed, live T. vaginalis with certain concentrations of C20A3 IgG. Differential killing of trichomonal isolates and clones of a given isolate by C20A3 was dependent on the presence of the glycoprotein antigen on the parasite surface.
Copurification of Leptospira interrogans serovar pomona hemolysin and sphingomyelinase C
Authors: Bernheimer A W , Bey R F
Published: January 2020

The hemolytic and sphingomyelinase C activities of supernatants of cultures of Leptospira interrogans serovar pomona tended to copurify when isoelectric fractionation was carried out. Both activities focused primarily at pH 8.1. Considered in conjunction with other circumstantial evidence, the results led to the conclusion that sphingomyelinase C is responsible for hemolysis.
Identification of the O-linked sialyloligosaccharides of glycophorin A as the erythrocyte receptors for S-fimbriated Escherichia coli
Authors: Parkkinen J , Rogers G N , Korhonen T , Dahr W , Finne J
Published: January 2020

The erythrocyte receptors for S-fimbriated Escherichia coli, which causes sepsis and meningitis in newborn infants, were investigated. Neuraminidase and trypsin treatments of erythrocytes abolished the hemagglutination ability of the bacteria. To identify the receptor glycoproteins, we separated erythrocyte membrane proteins by gel electrophoresis, blotted them to nitrocellulose, and incubated them with 125I-labeled bacteria. The only bacterium-binding bands identified corresponded to glycophorin A dimer and monomer, and the binding was abolished by neuraminidase treatment of the blot. Radiolabeled bacteria also bound to purified glycophorin A adsorbed to polyvinyl chloride microwells, and the binding was inhibited by other sialoglycoproteins and isolated sialyloligosaccharides containing the NeuAc alpha 2-3Gal sequence. Oligosaccharides which contain the NeuAc alpha 2-3Gal beta 1-3GalNAc and NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc sequence and which are identical to the O-linked saccharides of glycophorin A were twofold more effective inhibitors of binding than were other oligosaccharides containing the NeuAc alpha 2-3Gal sequence. The replacement of sialic acid in asialoerythrocytes with a purified Gal beta 1-3GalNAc alpha 2-3 sialyltransferase, which forms the O-linked NeuAc alpha 2-3Gal beta 1-3GalNAc sequence in asialoglycophorins, restored bacterial hemagglutination. These results indicated that the major erythrocyte receptor for S-fimbriated E. coli is the NeuAc alpha 2-3Gal beta 1-3GalNAc sequence of the O-linked oligosaccharide chains of glycophorin A.
Transformation of Streptococcus mutans with chromosomal and shuttle plasmid (pYA629) DNAs
Authors: Murchison H H , Barrett J F , Cardineau G A , Curtiss R
Published: January 2020

Transformation (i.e., DNase-sensitive genetic transfer) of strains of Streptococcus mutans representing serotypes c and e was accomplished by using chromosomal DNA from a Rifr Strr Spcr isolate of strain GS5 (UAB525) and a chimeric plasmid, pYA629. Shuttle plasmid pYA629 comprises the S. mutans plasmid pVA318, an inducible erythromycin resistance determinant originally isolated from a group A streptococcal strain, the tetracycline resistance gene and replication region of the Escherichia coli plasmid pBR322, and the promoter region of the S. mutans gene for aspartate beta-semialdehyde dehydrogenase. The strains examined for recipient ability included those known to lack a cryptic plasmid (GS5, UA130, UA159, and MT8148) and those known to contain a widely disseminated 5.8-kilobase cryptic plasmid (LM7, V318, UA101, UA174, and 3098791). The transformation frequencies in GS5 for GS5 chromosomal antibiotic resistance markers were comparable to those reported by others, but UA101, UA130, UA159 and UA174 were transformed with both chromosomal and plasmid markers at much higher efficiencies. In a larger strain survey, strains containing the 5.8-kilobase cryptic plasmid were more frequently transformable with both chromosomal and pYA629 DNAs than were strains lacking this cryptic plasmid. All plasmid-containing strains except LM7 lost their resident cryptic plasmids when transformed with pYA629. LM7 transformed with pYA629 retained pLM7. There are therefore at least two incompatibility groups among S. mutans cryptic plasmids. yPA629 DNA isolated from either E. coli or S. mutans transformed S. mutans with equal efficiency. pYA629 DNA isolated from S. mutans transformed both restriction-deficient and restriction-proficient E. coli recipients. Therefore, the strains of S. mutans used lack a restriction-modification system for pYA629 DNA sequences. S. mutans strains that are readily transformable, display maximal cariogenicity in gnotobiotic rats, and give high scores for in vitro measures of important virulence attributes have been identified to facilitate studies on the genetic basis and control of virulence.
Presence of a macrophage-mediated suppressor cell mechanism during cell-mediated immune response in experimental visceral leishmaniasis
Authors: Murray H W , Carriero S M , Donelly D M
Published: January 2020

In susceptible BALB/c mice, systemic intracellular infection with Leishmania donovani provokes generation of adherent spleen cells which can suppress both mitogen- and specific-antigen-stimulated T-cell responses. To characterize the responsible suppressor cell, we irradiated (2,000 R) adherent spleen cells from L. donovani-infected mice or treated them with anti-Thy-1.2 antibody plus complement. Neither anti-T-cell treatment diminished the capacity to inhibit lymphocyte proliferative activity. In addition, as judged by morphologic and functional criteria, 80 to 90% of the adherent cells appeared to be macrophages. Four observations suggested an immunopathogenic role for these suppressor macrophages. (i) Their appearance and disappearance paralleled the establishment and resolution of L. donovani visceral infection in vivo. (ii) Suppressive effects included inhibition of production of the macrophage-activating lymphokine gamma interferon (IFN-gamma). (iii) On transfer into immune mice, suppressor macrophages impaired naturally acquired resistance to L. donovani. (iv) Inhibition of macrophage prostaglandin metabolism by indomethacin reduced suppressor activity in vitro and resulted in a 50% decrease in parasite visceral replication in vivo. In addition, prophylactic cyclophosphamide treatment inhibited the development of suppressor macrophages, and under these conditions visceral infection was rapidly controlled. These results suggested that disseminated L. donovani infection provokes a macrophage-mediated suppressor mechanism which appears to contribute to establishment of visceral leishmaniasis in a susceptible host.
Immune responses to the lipopolysaccharides and capsular polysaccharides of Haemophilus pleuropneumoniae in convalescent and immunized pigs
Authors: Fenwick B W , Osburn B I
Published: January 2020

The immunologic responses to a smooth-type lipopolysaccharide (LPS) (HpS-LPS), a rough-type LPS (HpR-LPS), and a capsular-enriched polysaccharide preparation (HpC-PS) purified from Haemophilus pleuropneumoniae were determined in pigs immunized with a commercial H. pleuropneumoniae cellular vaccine, in pigs experimentally infected with H. pleuropneumoniae, in control pigs, and in immunized rabbits. The ability of the preparations to induce lymphocyte blastogenesis and B-cell activation was determined in the pigs and compared with the responses induced by the LPS of Escherichia coli O111:B4 and the LPS of Salmonella minnesota Re595. All the LPS preparations acted to induce proliferation of peripheral blood lymphocytes (PBL) from all pigs. The blastogenic response of PBL from H. pleuropneumoniae-infected pigs to HpS-LPS and HpR-LPS was significantly (P less than 0.05) greater than that of PBL from immunized and control pigs. HpC-PS did not induce a blastogenic response in the PBL of control pigs but did in PBL from H. pleuropneumoniae-infected pigs and to a greater degree in immunized pigs. An increase in the response of PBL to the S. minnesota LPS occurred only in the H. pleuropneumoniae-infected pigs. Significantly more (P less than 0.05) immunoglobulin-secreting cells (ISC) were induced in a reverse hemolytic plaque assay by stimulation with HpS-LPS and HpC-PS of PBL isolated from pigs infected with H. pleuropneumoniae than of PBL from immunized pigs. Increasing the number of T cells increased the number of ISC induced by HpS-LPS in control and immunized pigs, but not in convalescent pigs. The presence of macrophages reduced activation of ISC by HpS-LPS in control pigs and to a lesser degree in immunized pigs, whereas in H. pleuropneumoniae-infected pigs macrophages enhanced the induction of ISC by HpS-LPS. In immunized pigs, macrophages acted to inhibit the ability of HpC-PS to induce ISC. Serologic studies indicate that HpC-PS contains strain- and serotype-specific antigens; that HpS-LPS has both serotype-specific and cross-reacting species-specific antigens; and that HpR-LPS does not contain detectable serotype-specific antigens but does have both non species- and species-specific antigens. These studies show that the serotype-specific protection provided by immunization of pigs with an H. pleuropneumoniae cellular vaccine is principally the result of immunity to capsular antigens and that a weak cellular immune response occurs as compared with that induced by infection with H. pleuropneumoniae.(ABSTRACT TRUNCATED AT 400 WORDS)
Antigenic interrelationship between endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum (Nichols): molecular characterization of endoflagellar proteins
Authors: Radolf J D , Blanco D R , Miller J N , Lovett M A
Published: January 2020

Purified endoflagella from nonpathogenic Treponema phagedenis biotype Reiter were characterized biochemically and compared antigenically with the endoflagellar proteins of Treponema pallidum. T. phagedenis biotype Reiter endoflagella were dissociated into constituent polypeptides by incubation under conditions which disrupt noncovalent bonds. Chymotrypsin peptide maps of T. phagedenis biotype Reiter endoflagellar proteins revealed that the 37- and 33-kilodalton (kDa) major components shared significant homology with the 27- and 30-kDa minor components, respectively. The peptide maps also suggested that the major components shared a lesser degree of structural similarity with each other. These relationships were confirmed by Western blots of T. phagedenis biotype Reiter endoflagellar proteins employing antibodies that were purified against the individual endoflagellar polypeptides. Western blots of T. pallidum with the purified antibodies also demonstrated strong cross-reactivity between the T. phagedenis biotype Reiter endoflagellar proteins and T. pallidum proteins of identical or similar molecular weights. A unique Western blotting technique that we called epitope bridging was used to determine that the 37-kDa subunit contains most of the external epitopes on T. phagedenis biotype Reiter endoflagella. Immunoelectron microscopy with human syphilitic serum and rabbit T. phagedenis biotype Reiter endoflagellar antiserum confirmed the presence of cross-reactive epitopes on the surface of intact T. phagedenis and T. pallidum endoflagella.
Interactions of Bacteroides gingivalis with fibrinogen
Authors: Lantz M S , Rowland R W , Switalski L M , Höök M
Published: January 2020

Results of previous studies from our laboratory have shown that a strain of Bacteroides intermedius isolated originally from a patient with acute necrotizing ulcerative gingivitis binds and degrades human fibrinogen (M.S. Lantz, L.M. Switalski, K.S. Kornman, and M. Hook, J. Bacteriol. 163:623-628, 1985). We report that strains of Bacteroides gingivalis, an organism implicated in the etiology of several forms of periodontitis, also bind and degrade fibrinogen. The binding is rapid, reversible, saturable, and specific. The number of fibrinogen-binding sites per cell varies from 500 to 1,500 in different batches of bacteria, and the dissociation constant for the complex is on the order of 10(-8) M. B. gingivalis possesses cell-associated fibrinogenolytic activity that is activated by dithiothreitol and blocked by thiol protease inhibitors. Interaction with fibrinogen may mediate colonization and establishment of these organisms in the periodontal microbiota.
Clearance of Giardia muris infection in mice deficient in natural killer cells
Authors: Heyworth M F , Kung J E , Eriksson E C
Published: January 2020

Immunocompetent C57BL/6J mice and beige mice (which are deficient in natural killer cells) were infected with Giardia muris. Both types of mice cleared G. muris infection at similar rates. This observation suggests that clearance of G. muris parasites from the mouse intestine is not mediated by natural killer cells.
Selective infection of astrocytes by Chlamydia trachomatis in primary mixed neuron-glial cell cultures
Authors: Levitt D , Danen R , Levitt P
Published: January 2020

Both human biovars of Chlamydia trachomatis were able to productively infect primary cultures of fetal rat brain cells. Infected brain cells released bacteria that reinfected McCoy cells well as other cultured brain cells. The chlamydiae infected cultured astrocytes but were never observed to grow inside neurons, suggesting a selective susceptibility of specific brain cells to chlamydial infections.
Inhibition of gingival fibroblast growth by Bacteroides gingivalis
Authors: Larjava H , Uitto V J , Eerola E , Haapasalo M
Published: January 2020

Human gingival fibroblasts were exposed in culture to cell extracts of different black-pigmented Bacteroides species, and their growth was monitored by determining thymidine uptake and counting cells. Of the Bacteroides species tested (B. gingivalis, B. asaccharolyticus, and B. intermedius), B. gingivalis gave the extract with the strongest inhibitory effect on fibroblast thymidine uptake. Linear inhibition reaching 80% of the control level was obtained with a dose of 100 micrograms of B. gingivalis extract protein per ml. The effect of B. asaccharolyticus resembled that of B. gingivalis, but even at the highest dose tested B. intermedius had only a slight inhibitory effect. When fibroblasts were counted after 2- and 4-day exposures to B. gingivalis extracts, a clear depression in the number of fibroblasts was found. The effects of extracts obtained from early and late growth phases of B. gingivalis cultures were similar. A fraction of B. gingivalis consisting essentially of lipopolysaccharides (LPSs) was obtained by degrading the extract proteins with proteinase K. Silver staining of polyacrylamide gels revealed a LPS pattern with a molecular mass ranging from 37 to 60 kilodaltons. This LPS-rich fraction caused inhibition of thymidine uptake by gingival fibroblasts similar to that caused by the native extract alone. Thus, the inhibition of gingival fibroblast growth by B. gingivalis appeared to be LPS mediated. This inhibitory effect of B. gingivalis on oral fibroblast growth may be a virulence factor of this bacterium.
Induction of a macrophage-suppressive lymphokine by soluble cryptococcal antigens and its association with models of immunologic tolerance
Authors: Blackstock R , McCormack J M , Hall N K
Published: January 2020

Soluble extracts of Cryptococcus neoformans were examined for their ability to induce a macrophage-regulatory T-suppressor cell known to appear in the spleens of mice infected with cryptococci. Suppressor cells were induced by injection of extracts of encapsulated or thinly encapsulated strains of cryptococci. Dose-response analysis showed that as little as 25 micrograms of soluble capsular polysaccharide antigen could induce significant suppressor cell activity, with maximum suppression occurring at a dose of 100 micrograms. The suppressor cells appeared within 1 week of injection of antigen and persisted for at least 2 months. Suppressor cells were induced in animals given tolerogenic doses of levan, human gamma globulin, and soluble capsular polysaccharide antigen. When these same antigens were administered in immunogenic form, no suppressor cell activity was detected. Therefore, the suppressive mechanism was common to models of immunologic tolerance and was not unique to cryptococcal disease or cryptococcal capsular polysaccharide antigen. The phagocytosis-inhibiting lymphokine produced by the suppressor cell population completely inhibited the phagocytic activity of only a portion of peritoneal exudate cells. Other macrophages in the population were not totally inhibited but exhibited a reduction in the number of yeast cells engulfed.
Purification, partial characterization, and identification of a skin-reactive protein antigen of Mycobacterium bovis BCG
Authors: De Bruyn J , Bosmans R , Turneer M , Weckx M , Nyabenda J , Van Vooren J P , Falmagne P , Wiker H G , Harboe M
Published: January 2020

An immunogenic and skin-reactive protein called P64 was purified from Sauton zinc-deficient culture filtrate of Mycobacterium bovis BCG by using successively hydrophobic chromatography on phenyl-Sepharose, ion exchange on DEAE-Sephacel, and molecular sieving on Sephadex G-200. The final P64 preparation was found to be homogeneous based on several analyses. Protein P64 was a constituent of BCG cells since it was present in soluble cellular extract from normally grown BCG cells. It represented 8 to 9% of the soluble proteins of the extract and appeared as the major soluble protein antigen of BCG. This protein was found to have a molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in molecular sieving it eluted at a volume corresponding to a molecular weight of 246,000. An abnormal UV spectrum was observed for this protein. Its amino acid composition showed an abundance of acidic amino acids (or their amides). Aromatic amino acids represented only 3% of the total amino acid residues. The NH2-terminal amino acid sequence of this protein (10 amino acids) was determined. Its sugar content measured with the phenol-sulfuric acid test was lower than 0.3% (wt/wt.) Isolated P64 was tested by various crossed-immunoelectrophoresis techniques and was shown to correspond to antigen 82 in the reference system for BCG antigens. The protein antigen P64 elicited a delayed cutaneous reaction in guinea pigs sensitized with either living or heat-killed BCG. Its potency in skin reaction was, respectively, two- and threefold that of the BCG purified protein derivative. The two types of sensitization used for skin test reactions promoted significant immunoglobulin G antibody production against the protein antigen P64 in guinea pigs 7 weeks after sensitization.
Characterization of protein I from serum-sensitive and serum-resistant transformants of Neisseria gonorrhoeae
Authors: Judd R C , Tam M , Joiner K
Published: January 2020

The protein IAs of serum-sensitive (FA635) and serum-resistant (FA638) transformants of Neisseria gonorrhoeae, which have identical pedigrees, have been shown to be different by the use of a monoclonal antibody and were also shown to be different by proteinase K cleavage and primary structural and surface peptide mapping. The difference in structure is within the surface-exposed region of the molecule. The only other difference observed between the two strains was a very slight difference in lipooligosaccharide silver staining in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that protein I alone or in combination with lipooligosaccharide may significantly contribute to serum resistance.
Characterization of the Cryptosporidium antigens from sporulated oocysts of Cryptosporidium parvum
Authors: Luft B J , Payne D , Woodmansee D , Kim C W
Published: January 2020

The antigenic constituents of sporulated Cryptosporidium parvum oocyst antigens were characterized with antisera from mice immunized against C. parvum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining defined the major proteins. Six of seven lectins used recognized as many as 15 bands. The lectins concanavalin A, Dolichos biflorus, and wheat germ agglutinin showed strong activity against the same eight bands with molecular weights ranging from 72,000 to greater than 100,000. An enzyme-linked immunosorbent assay was used to detect antibody to C. parvum. Antibody binding was significantly decreased by heat and enzymatic treatment with trypsin, protease, and mixed glycosidases. C. parvum antigens were further defined by the reactivity of immune sera with a C. parvum sonicate preparation separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose paper. Antisera from orally infected mice consistently recognized four antigens with molecular weights ranging from 72,000 to greater than 100,000. These antigens also bound concanavalin A. Treatment of the antigen preparation with mixed glycosidases reduced the reactivity of antisera with most antigens with molecular weights greater than 60,000. The data suggest that the antigenic composition of C. parvum is complex and that carbohydrates alone or in association with lipids or proteins may be important in the immune response to C. parvum.
Impairment of macrophage function by inhibitors of ornithine decarboxylase activity
Authors: Kierszenbaum F , Wirth J J , McCann P P , Sjoerdsma A
Published: January 2020

The effects of irreversible inhibition of ornithine decarboxylase on the capacity of murine macrophages to take up a protozoan organism (Trypanosoma cruzi) or inert particles were investigated. Incubation of macrophage cultures with four different ornithine decarboxylase inhibitors, namely, DL-alpha-difluoromethylornithine (DFMO, 0.5 to 20 mM), delta-methyl-acetylenic putrescine (1 to 5 mM), monofluoromethyldehydroornithine ethyl ester (1 to 5 mM), and monofluoromethyldehydroornithine methyl ester (1 to 5 mM), before the addition of the parasites significantly reduced the percentage of macrophages with parasites, indicating that some of the host cells were no longer capable of binding or ingesting the parasite. The average number of trypanosomes per 100 macrophages was also diminished, denoting a lesser phagocytic capacity as a consequence of the treatments. These effects were reversible within 2 h after removal of excess DFMO. No alteration in parasite-macrophage interaction was seen when the trypanosomes were treated with DFMO. That the effects of DFMO on the macrophages probably resulted from a reduction in polyamine levels caused by inhibition of ornithine decarboxylase was indicated by the fact that these effects were not seen when the macrophages were incubated with DFMO in the presence of putrescine, the product of ornithine decarboxylation by ornithine decarboxylase. DFMO treatment of macrophages also inhibited the capacity of these cells to ingest killed parasites or latex beads and thus appeared to generally affect phagocytosis. An effect of DFMO on the susceptibility of macrophages to penetration by the parasites seemed less likely because no significant alteration in cell-parasite association occurred when myoblasts--which, not being phagocytic, can be infected only by membrane penetration--were treated with DFMO. Taken together, these results emphasize a role of ornithine decarboxylase activity and polyamine biosynthesis in macrophage function.
Construction and characterization of Bordetella pertussis toxin mutants
Authors: Black W J , Falkow S
Published: January 2020

Pertussis toxin is one of the major virulence determinants produced by Bordetella pertussis. The DNA encoding the structural genes for pertussis toxin was cloned in Escherichia coli, and pertussis toxin subunit S4 was expressed under the control of the tac promoter. Mutations were introduced into the cloned toxin genes, and a conjugative shuttle vector system was devised for delivering the mutations from E. coli back into B. pertussis. The mutations were introduced by allelic exchange into the chromosome of B. pertussis resulting in a series of B. pertussis strains which were isogenic except at the loci encoding the structural genes for pertussis toxin. These B. pertussis strains were utilized to study the biogenesis of pertussis toxin. Polar mutations in the S1 gene led to a lack of detectable S2 or S4 subunits in whole-cell lysates, suggesting a polycistronic arrangement for these genes. Mutations in the S5 subunit gene resulted in a truncated S1 subunit, while mutations in the S4 gene resulted in a lack of detectable S2 subunit, suggesting that physical relationships among the toxin subunits are directly reflected in the stable biogenesis of the subunits.
Degradation of gonococcal peptidoglycan by granule extract from human neutrophils: demonstration of N-acetylglucosaminidase activity that utilizes peptidoglycan substrates
Authors: Striker R , Kline M E , Haak R A , Rest R F , Rosenthal R S
Published: January 2020

The degradation of purified Neisseria gonorrhoeae peptidoglycan (PG) by granule extract derived from normal human polymorphonuclear leukocytes was examined. Hen egg lysozyme-resistant, extensively O-acetylated [3H]PG (O-PG) from strain FA19 and lysozyme-sensitive, non-O-acetylated [14C]PG (non-O-PG) from strain RD5 (each containing label in both glucosamine and muramic acid) were mixed and incubated with granule extract at pHs 4.5, 5.5, and 6.5. The rate of degradation of O-PG was uniformly slower than that of non-O-PG in the same tube, but ultimately, even the O-PG was rendered completely soluble. Molecular-sieve high-performance liquid chromatography revealed that both PGs were degraded by granule extract at the pH values tested to disaccharide peptide monomers and peptide-cross-linked oligomers, reflecting the action of human lysozyme. Of particular interest was the appearance of a peak containing free N-acetylglucosamine which was quite prominent in reaction mixtures at pH 4.5, less prominent at pH 5.5, and not detectable at pH 6.5. Free N-acetylglucosamine was not released from control PG samples at any pH in the absence of granule extract. Treatment of purified gonococcal PG monomers with granule extract at pH 4.5 yielded exclusively free N-acetylglucosamine and muramyl peptides with no N-acetylglucosamine. These data suggest that granule extract contains a previously undescribed pH-dependent N-acetylglucosaminidase with specificity for PG as well as an N-acetylmuramidase activity that degrades O-PG less efficiently than it does non-O-PG.
Fluoride inhibition of proton-translocating ATPases of oral bacteria
Authors: Sutton S V , Bender G R , Marquis R E
Published: January 2020

The ATPases of isolated membranes of lactic acid bacteria were found to be inhibited by fluoride in a complex manner. Among the enzymes tested, that of Streptococcus mutans GS-5 was the most sensitive to fluoride, and the initial rate of hydrolysis of ATP was reduced 50% by approximately 3 mM fluoride. The enzyme of Lactobacillus casei ATCC 4646 was the most resistant, and about 25 mM fluoride was required for 50% inhibition. The response to fluoride appeared to involve reversible, noncompetitive inhibition during short exposure to low levels of fluoride and nonreversible inhibition at higher fluoride levels. In addition, kinetic studies of the effects of fluoride on the enzymes of membranes of S. mutans and L. casei indicated that reversible inhibition was at least partly overcome at high levels of either ATP or Mg. The effects of pH on fluoride inhibition of ATPases were markedly different from the effects of pH on inhibition of acid/base regulation of intact cells by fluoride. It appeared that formation of HF was not required for inhibition of the ATPases. F1 ATPases isolated from the membranes by washing with buffers of low ionic strength proved to be less sensitive to fluoride than the membrane-associated F1F0 holoenzymes, and it was concluded that the F0 or membrane sector of the holoenzyme is involved in fluoride inhibition.
Role of anaerobic flora in the translocation of aerobic and facultatively anaerobic intestinal bacteria
Authors: Wells C L , Maddaus M A , Reynolds C M , Jechorek R P , Simmons R L
Published: January 2020

It is thought that the normal enteric microflora acts not only to prevent intestinal colonization but also to prevent subsequent systemic dissemination of ingested, potentially pathogenic bacteria. To determine the relative roles of specific components of the intestinal bacterial flora in bacterial translocation out of the gut, mice were given various antimicrobial agents to selectively eliminate specific groups of intestinal bacteria. The cecal flora and the translocating bacteria in mesenteric lymph nodes were monitored both before and after oral inoculation with antibiotic-resistant Escherichia coli C25. Orally administered streptomycin selectively eliminated cecal facultative gram-negative bacilli, orally administered bacitracin-streptomycin eliminated all cecal bacterial species except low numbers of aerobic sporeformers, and parenterally administered metronidazole selectively eliminated cecal anaerobic bacteria. Compared with control mice, only metronidazole-treated mice had significantly increased rates of dissemination of intestinal bacteria into mesenteric lymph nodes, indicating that the exclusive absence of anaerobic bacteria facilitated the translocation of the intestinal facultative bacteria. In a parallel experiment with streptomycin-resistant E. coli C25 as a marker, parallel results were obtained. Metronidazole increased the translocation of the marker strain and the indigenous strains of intestinal bacteria. Thus, anaerobes appeared to play a key role in confining indigenous bacteria to the gut. However, intestinal colonization and translocation of E. coli C25 occurred most readily after bacitracin-streptomycin treatment, suggesting that in addition to anaerobic bacteria, other bacterial groups may play a role in limiting the intestinal colonization and extraintestinal dissemination of E. coli C25.
Molecular cloning and expression of Neisseria meningitidis class 1 outer membrane protein in Escherichia coli K-12
Authors: Barlow A K , Heckels J E , Clarke I N
Published: January 2020

A genomic library of meningococcal DNA from a clinical isolate of Neisseria meningitidis was constructed in the expression vector lambda gt11. Outer membrane complex was prepared from the same strain and used to immunize rabbits to raise polyclonal anti-outer membrane complex serum. The amplified library was probed with this polyclonal serum, and seven expressing recombinants were isolated; further investigations indicated these to be identical. The expressed meningococcal gene in these recombinants was fused to vector beta-galactosidase and shown to encode epitopes present on the 42-kilodalton class 1 outer membrane protein. Estimation of the size of the recombinant fusion protein suggests that up to 40 kilodaltons of protein-coding sequence is present. The lambda gt11 recombinant contains a 3.4-kilobase DNA insert, which has been recloned into a plasmid and characterized by restriction endonuclease analysis. A restriction fragment from the insert, representing the protein-coding region hybridizes to a single 2.2-kilobase XbaI fragment from the homologous strain and to similar-sized XbaI fragments in other strains of meningococci, expressing antigenically distinct class 1 proteins.
Effect of complement depletion on anticapsular-antibody-mediated immunity to experimental infection with Haemophilus influenzae type b
Authors: Schreiber J R , Basker C J , Siber G R
Published: January 2020

Antibody to the capsular polysaccharide of Haemophilus influenzae type b (Hib) was not protective in infant rats depleted of complement by cobra venom factor (CoVF) even when serum antibody levels were many times the minimum protective level. Partial protection from Hib infection was achieved in CoVF-treated rats only if they were passively hyperimmunized with large doses of immunoglobulin G Hib capsular polysaccharide antibody. In addition, nonimmune CoVF-treated rats had higher mortality and blood bacterial density than nonimmune rats with intact complement systems.
Synergistic induction of interleukin-1 by endotoxin and toxic shock syndrome toxin-1 using rat macrophages
Authors: Beezhold D H , Best G K , Bonventre P F , Thompson M
Published: January 2020

We studied interleukin-1 (IL-1) secretion by rat peritoneal exudate macrophages stimulated with purified toxic shock syndrome toxin-1 (TSST-1). TSST-1 was observed to be a more potent inducer of IL-1 than was endotoxin. The induction of IL-1 secretion by TSST-1 was not blocked by polymyxin B but could be blocked by monoclonal antibodies directed against TSST-1. Synergistic induction of IL-1 was observed when the cells were stimulated with TSST-1 and endotoxin. The sequence of addition was found to be important for the synergistic response. Enhanced IL-1 production was observed only when macrophages were exposed to endotoxin before or simultaneously with TSST-1. Prior exposure of macrophages to TSST-1 had no enhancing effect on endotoxin-induced IL-1 secretion. We conclude that stimulation of the macrophage by endotoxin enhances the responsiveness of the cells to TSST-1 and may thereby play a role in the pathogenesis of toxic shock syndrome.
Comparative studies of endotoxin uptake by isolated rat Kupffer and peritoneal cells
Authors: Fox E S , Thomas P , Broitman S A
Published: January 2020

The process of uptake of endotoxin by cells of the reticuloendothelial system is of current interest. Rabbit peritoneal macrophages have been used to study macrophage-endotoxin interactions and have suggested a receptor-mediated process. It is generally believed that the site of in vivo endotoxin clearance is the liver and that this clearance involves the Kupffer cell population. In the current report, the uptake characteristics of iodine-125-labeled Salmonella minnesota lipopolysaccharide (LPS) were compared in both isolated rat Kupffer cells and elicited rat peritoneal cells. Both types of cells were isolated from male Sprague-Dawley rats fed a semisynthetic AIN-76 5% saturated-fat diet either by peritoneal lavage for peritoneal cells or by collagenase perfusion followed by purification on a 17.5% metrizamide gradient for Kupffer cells. Hot phenol water-extracted S. minnesota LPS was labeled with iodine by the chloramine-T method following a reaction with methyl-p-hydroxybenzimidate. The in vitro uptake of [125I]LPS by Kupffer cells was unsaturable up to concentrations of 33.33 micrograms/ml, while peritoneal cells became saturated at between 16.67 and 25 micrograms of LPS per ml. Uptake by both types of cells could be inhibited by a 10-fold excess of unlabeled LPS. Kinetic experiments demonstrated that Kupffer cells were unsaturable after 60 min of incubation, while peritoneal cells were saturable after 40 min of incubation. Pretreatment with 75 mM colchicine inhibited uptake by peritoneal cells but not Kupffer cells, while pretreatment with 12 mM 2-deoxyglucose inhibited uptake by Kupffer cells but not peritoneal cells. These results are consistent with a process of receptor-mediated endocytosis for peritoneal cells, while Kupffer cells may internalize endotoxins by absorptive pinocytosis. These results suggest that studies of peritoneal cell-endotoxin interactions do not accurately describe the physiologic process within the liver, the major site for the clearance of gut-derived endotoxins.
A recombinant molecule from a disseminating strain of Neisseria gonorrhoeae that confers serum bactericidal resistance
Authors: McShan W M , Williams R P , Hull R A
Published: January 2020

A cosmid gene library was prepared from Neisseria gonorrhoeae JC1, a serum-resistant clinical isolate from a patient with disseminated gonococcal infection. From this library a recombinant molecule, pWM3, was isolated which had the ability to transform F62, a serum-sensitive strain of N. gonorrhoeae, to serum resistance. This plasmid contained 2.2 kilobases of insert gonococcal DNA that coded for two peptides, one of 29 kilodaltons (kDa) and one of 17.5 kDa. Deletion of the region coding for the 29-kDa peptide resulted in the loss of the ability of the plasmid to transform F62 to serum resistance. N. gonorrhoeae F62 acquired the ability to bind blocking antibody when transformed with pWM3 or subclones that code for only the 29-kDa protein. Although similar in size, the cloned 29-kDa protein and protein III are antigenically distinct.
Antigenic structure of Clostridium botulinum type B neurotoxin and its interaction with gangliosides, cerebroside, and free fatty acids
Authors: Kozaki S , Ogasawara J , Shimote Y , Kamata Y , Sakaguchi G
Published: January 2020

A fragment distinct from the heavy and light chains was obtained by treatment of Clostridium botulinum type B neurotoxin with chymotrypsin. Enzyme-linked immunosorbent assay and immunoblotting analysis with monoclonal antibodies showed that the fragment consisted of the light chain and part of the heavy chain (H-1 fragment) linked together by a disulfide bond. Monoclonal antibodies reacting to the heavy chain but not to the fragment were thought to recognize the epitopes on the remaining portion (H-2 fragment) of the heavy chain, being easily digested by chymotrypsin. Thus, the antigenic structure of type B neurotoxin resembles those of type A and E neurotoxins. The chymotrypsin-induced fragment bound to cerebroside and free fatty acids but not to gangliosides. The manner of binding of type B neurotoxin to gangliosides and free fatty acids was different from those of type A and E neurotoxins. Such differences in the reactivities to lipids may be related to the finding that each neurotoxin binds to a type-specific site on the neural membrane.
Virulence markers of mesophilic aeromonads: association of the autoagglutination phenomenon with mouse pathogenicity and the presence of a peripheral cell-associated layer
Authors: Janda J M , Oshiro L S , Abbott S L , Duffey P S
Published: January 2020

Autoagglutination (AA phenotype) of mesophilic aeromonads in broth was found to be a virulence-associated marker. There were two kinds of AA+ strains: those that spontaneously pelleted (SP+), and those that pelleted only after boiling (PAB+). Of 79 strains tested, 24 (30%) were AA+, and 18 of these were recovered from clinical specimens. Most of the AA+ strains (n = 21) were identified as either Aeromonas sobria or Aeromonas hydrophila. Of the well-documented clinical isolates of A. sobria and A. hydrophila available, 5 (46%) of 11 from invasive disease and 4 (14%) of 29 from noninvasive disease were SP- PAB+. The SP- PAB+ phenotype was significantly associated with invasive infections (e.g., bacteremia and peritonitis [chi 2, P less than 0.05]). All seven of the SP- PAB+ A. sobria and A. hydrophila strains tested killed mice within 48 h after intraperitoneal infection with 1 x 10(7) to 3 x 10(7) CFU, whereas only two of four SP+ PAB+ strains tested were lethal. All of the SP- PAB+ A. sobria and A. hydrophila isolates examined shared common O somatic antigens and possessed an external layer peripheral to the cell wall as determined by thin-section electron micrography. The LL1 strain of A. hydrophila used by Dooley et al. (J. S. G. Dooley, R. Lallier, and T. J. Trust, Vet. Immunol. Immunopathol. 12:339-344, 1986) to demonstrate an S membrane protein component in aeromonads virulent for fish also was SP- PAB+ and possessed the peripheral membrane, suggesting an association between these two components. Seven AA- and three SP+ strains tested lacked this layer; furthermore, 22 (71%) of 31 such isolates did not kill mice. The AA phenotype was a stable characteristic upon long-term passage of isolates in vitro. Study of SP+ and PAB+ aeromonads by surface charge and hydrophobicity analyses indicated that neither property correlated with either virulence or the presence of an external layer.
Virulence of protein A-deficient and alpha-toxin-deficient mutants of Staphylococcus aureus isolated by allele replacement
Authors: Patel A H , Nowlan P , Weavers E D , Foster T
Published: January 2020

The gene coding for protein A (spa) of Staphylococcus aureus 8325-4 has been inactivated by substituting part of the spa coding sequence for a DNA fragment specifying resistance to ethidium bromide. The in vitro-constructed spa::EtBrr substitution mutation was introduced into the S. aureus chromosome by recombinational allele replacement. Southern blot hybridization showed that the in vitro-constructed mutation was present in the chromosomal spa locus. We have previously reported the inactivation of the alpha-toxin gene (hly) by allele replacement with an in vitro-constructed hly::Emr (erythromycin resistance) mutation (M. O'Reilly, J.C.S. de Azavedo, S. Kennedy, and T.J. Foster, Microb. Pathogen. 1:125-138, 1986). A double Spa- Hly- mutant was constructed by transduction. The virulence of Spa- and Hly- mutants was tested by experimental infection of mice. When subcutaneous injections were given, Hly- mutants formed a flat, darkened lesion, whereas Hly+ strains caused a raised, cream lesion. Alpha-toxin was shown to be a major factor in forming subcutaneous lesions and in causing the death of mice injected intraperitoneally. Spa- mutants were slightly less virulent than their Spa+ counterparts, which suggests that protein A is also a virulence factor of S. aureus.