bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple protein differences exist between Neisseria gonorrhoeae type 1 and type 4
Authors: Klimpel K W , Clark V L
Published: January 2020

Neisseria gonorrhoeae undergoes a spontaneous conversion from a form which is virulent, competent for DNA-mediated transformation, and piliated (type 1) to a form which is avirulent and neither piliated nor competent (type 4). This phase variation has become thought of as simply a conversion from piliated to nonpiliated. Using the techniques of cell fractionation, two-dimensional electrophoresis, and nonequilibrium pH gradient gel electrophoresis, we identified differences in the expression levels of multiple proteins between type 1 and type 4 cells. A total of 26 type 1-specific (T1S) and 23 type 4-specific (T4S) cytoplasmic or cytoplasmic membrane proteins were identified in O'Farrell two-dimensional gels. Using nonequilibrium pH gradient gel electrophoresis, we detected a minimum of eight T1S outer membrane proteins and four T4S outer membrane proteins which were not detected in the O'Farrell gels. Thus, the conversion from type 1 to type 4 is a complex event involving many different proteins of all cellular locations.
A basic replicon of virulence-associated plasmids of Shigella spp. and enteroinvasive Escherichia coli is homologous with a basic replicon in plasmids of IncF groups
Authors: Silva R M , Saadi S , Maas W K
Published: January 2020

Shigella species and enteroinvasive Escherichia coli strains carry a large (120- to 140-megadalton) plasmid called pINV, which contains genes essential for the invasiveness of these pathogens. Hybridization with specific probes derived from the RepFIC and RepFIB replicons of the IncF1 Ent plasmid P307 showed that pINVs present in 35 clinical isolates are homologous with RepFIC but not RepFIB, regardless of the serogroup of the Shigella or E. coli strain. RepFIC of P307, in turn, is very similar to RepFIIA replicons of IncFII R plasmids. These and other related replicons constitute the RepFIIA family. With one pINV, pWR110, a plasmid of Shigella flexneri 5, we demonstrated the existence of a functional replicon, RepINV, with a restriction map similar to that of RepFIIA of plasmid R1. We isolated the putative inc RNA coding region of RepINV, which is a major determinant of incompatibility. The nucleotide sequence of the RepINV-inc RNA-coding region was determined and compared with the corresponding sequences of RepFIC and RepFIIA. The differences were small, but apparently were sufficient to affect the target specificity of the inc RNAs, thus rendering the replicons compatible with each other. We conclude that pINVs present in Shigella spp. and enteroinvasive E. coli constitute a homogeneous group, containing one basic replicon that belongs to the RepFIIA family of replicons.
Vaccine-induced immunity against cutaneous leishmaniasis in BALB/c mice
Authors: Frommel D , Ogunkolade B W , Vouldoukis I , Monjour L
Published: January 2020

Partially purified antigens, derived from Leishmania infantum or L. major promastigotes and isolated under reducing conditions, were used to immunize BALB/c mice. Three subcutaneous injections of the 64- to 97-kilodalton preparation in conjunction with muramyl dipeptide conferred long-lasting immunity against L. mexicana subsp. mexicana and L. major infection; they led to the development of antibodies neutralizing the infectiousness of promastigotes, induced specific delayed-hypersensitivity reactions, and generated populations of peritoneal macrophages capable of killing amastigotes. Vaccination resulted in no harmful effects, since these antigen neither exacerbated preexisting Leishmania infection nor impeded the formation of antibodies to other antigens administered concomitantly.
Genetic control in the susceptibility of germfree inbred mice to infection by Escherichia coli O115a,c:K(B)
Authors: Itoh K , Matsui T , Tsuji K , Mitsuoka T , Ueda K
Published: January 2020

We studied the susceptibility of five germfree inbred strains of mice to oral infection by murine pathogenic Escherichia coli O115a,c:K(B) (MPEC), the causative agent of mouse megaenteron. Although MPEC colonized all strains of mice at 10(9)/g of feces, the mouse strains could be divided into three groups according to their intestinal lesions. In CF1 and C3H/He mice, intestinal lesions were produced in the cecum and colon with hyperplasia of epithelial cells accompanied by severe inflammatory reactions and erosion. The lesions in NC and C57BL/6 mice were restricted to the tip of the cecum, and hyperplasia of epithelial cells was more severe in these mice than in CF1 or C3H/He mice. BALB/c mice had no lesions. Analysis of F1 hybrids of CF1, NC, and BALB/c mice and offsprings from backcrosses of F1 mice to parental strains showed that susceptibility to MPEC seemed to be controlled genetically by a single locus which may be related to the receptors on epithelial cells for MPEC adherence. However, the differences in lesions between CF1 and NC mice suggest that a combination of this locus and another locus to which it may be related regulates the hyperplasia of intestinal epithelial cells.
Purification and characterization of a hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus and related to the thermostable direct hemolysin
Authors: Honda T , Ni Y X , Miwatani T
Published: January 2020

A clinical isolate (strain 4037) of Kanagawa phenomenon-negative Vibrio parahaemolyticus was studied. Although the strain was judged to be Kanagawa phenomenon-negative by various conventional tests, it produced a new hemolysin (named Vp-TRH, for thermostable direct hemolysin [Vp-TDH]-related hemolysin) that was related to the Vp-TDH produced by ordinary Kanagawa phenomenon-positive V. parahaemolyticus. Vp-TRH was purified by ammonium sulfate fractionation and successive column chromatographies on DEAE-cellulose, hydroxyapatite, and Mono Q. The molecular weight of Vp-TRH was estimated as 48,000 by Sephadex G-100 gel filtration, and the molecular weight of the subunit was estimated to be 23,000 by sodium dodecyl sulfate-slab gel electrophoresis. Thus, like Vp-TDH, Vp-TRH seems to be composed of two subunits. The isoelectric point of Vp-TRH was determined to be 4.6. Vp-TRH showed lytic activities different from those of Vp-TDH on erythrocytes from various animals, especially those from calves, chickens, and sheep. The hemolytic activity of Vp-TRH was labile on heat treatment at 60 degrees C for 10 min, unlike that of Vp-TDH. The immunological similarities, but not the identities of Vp-TRH and Vp-TDH, were demonstrated by Ouchterlony, neutralization, and latex agglutination tests. Thus, we conclude that this clinical isolate of Kanagawa phenomenon-negative V. parahaemolyticus produces a new type of hemolysin that is similar, but not identical, to Vp-TDH, which is usually produced by Kanagawa phenomenon-positive V. parahaemolyticus.
Hyperadhesive mutant of type 1-fimbriated Escherichia coli associated with formation of FimH organelles (fimbriosomes)
Authors: Abraham S N , Goguen J D , Beachey E H
Published: January 2020

The relationships of the genes and gene-products mediating D-mannose-specific attachment of type 1 fimbriae of Escherichia coli to eucaryotic cells were investigated by deletion mutation analysis of recombinant plasmid pSH2, which carries the genetic information for the synthesis and expression of functional type 1 fimbriae. Mutant pUT2004 was derived by a deletion remote from the structural gene encoding the 17-kilodalton (kDa) subunit protein of type 1 fimbriae. Phenotypically, the mutant demonstrated an eightfold-higher mannose-specific hemagglutination titer than the parent strain. On electron microscopy, the mutant strain expressed the same number of fimbriae as the parent strain. However, numerous 10-nm-diameter rounded structures (fimbriosomes) were observed both closely associated with fimbriae and in the culture medium. Fimbriosomes isolated from the medium agglutinated guinea pig erythrocytes in a mannose-sensitive manner. Dissociation of the fimbriosomes yielded a single 29-kDa protein, as demonstrated by sodium dodecyl sulfate gel electrophoresis. Antibodies raised against fimbriosomes reacted with a 29-kDa protein on immunoelectroblots of dissociated type 1 fimbriae and also blocked the adherence of other strains of type 1 fimbriated E. coli to eucaryotic cells. These findings suggest that the enhanced adhesive properties of the mutant pUT2004 strain are associated with overproduction of the 29-kDa FimH in the form of fimbriosomes which contain the determinant of the D-mannose-sensitive adhesion of type 1 fimbriae.
Colonization of the streptomycin-treated mouse large intestine by a human fecal Escherichia coli strain: role of adhesion to mucosal receptors
Authors: Wadolkowski E A , Laux D C , Cohen P S
Published: January 2020

Escherichia coli F-18, a normal fecal isolate, was previously shown to be an excellent colonizer of the streptomycin-treated CD-1 mouse large intestine, whereas E. coli F-18col-, a derivative of E. coli F-18 that no longer makes the E. coli F-18 colicin, was shown to be a poor mouse colonizer. It was also shown that E. coli F-18 bound two to three times more soluble colonic mucus protein than did E. coli F-18col- and that a major receptor in CD-1 mouse colonic mucus was a 50.5-kilodalton glycoprotein. In the present investigation, an additional E. coli F-18 colonic mucus glycoprotein receptor (66 kilodaltons) and three cecal mucus glycoprotein receptors (94, 73, and 66 kilodaltons) were identified. Numerous colonic and cecal brush border protein receptors specific for E. coli F-18 were also identified. Furthermore, E. coli F-18col- was found to bind to the same mucus and brush border receptors as E. coli F-18, although to a far lesser extent. Adhesion of both E. coli F-18 and F-18col- was inhibited by D-mannose and alpha-methyl-D-mannoside, and both strains were shown to bind specifically to the mannose moiety of a mannose-bovine serum albumin glycoconjugate, although again E. coli F-18col- bound to a lesser extent. Finally, both E. coli F-18 and F-18col- were shown to be piliated. The possible role of pilus mediated adhesion in E. coli F-18 colonization of the streptomycin-treated mouse large intestine is discussed.
A hemagglutinin of uropathogenic Escherichia coli recognizes the Dr blood group antigen
Authors: Nowicki B , Moulds J , Hull R , Hull S
Published: January 2020

A receptor moiety and blood group substance recognized by the O75X adhesin was studied. Well-defined erythrocytes representing different blood group systems and bacterial derivatives carrying plasmid pBJN406 encoding the adhesin were used in a direct hemagglutination assay. We showed that Dr blood group antigen, a component of the IFC blood group complex, is the receptor for the O75X fimbrialike adhesin (Dr hemagglutinin) of uropathogenic Escherichia coli. The molecule recognized by the Dr hemagglutinin on Dr blood group substance is a chloramphenicol-like structure. The inhibitory effect of the active compounds indicates that a tyrosine-containing molecule could be a natural receptor for the Dr hemagglutinin. Dr blood group substance was found in tubular basement membrane and Bowman's capsule of the human kidney. Specific attachment of a Dr hemagglutinin-positive bacterial strain to the kidney substructures was inhibited by chloramphenicol.
Development and characterization of an anti-idiotype antibody to the capsular polysaccharide of Neisseria meningitidis serogroup C
Authors: Westerink M A , Campagnari A A , Wirth M A , Apicella M A
Published: January 2020

A monoclonal anti-idiotypic antibody (Ab2) whose antibody combining site contained a surrogate image of the meningococcal group C capsular polysaccharide was developed. To accomplish this, a monoclonal antibody against the group C capsular polysaccharide was developed by the fusion of splenocytes from mice immunized with Neisseria meningitidis group C strain MP13 with Sp2/0-Ag14 plasmacytoma cells. Monoclonal antibody 1E4, an immunoglobulin M isotype, demonstrated binding to the serogroup C polysaccharide in enzyme-linked immunosorbent assay (ELISA). Monoclonal antibody 1E4 reacted with 30 of 30 group C strains and 1 of 36 group B strains in immunodot assay, slide agglutination, inhibition ELISA, and bactericidal assay. This monoclonal antibody was selected as idiotype (Ab1) for the development of hybridomas producing an anti-idiotype antibody. One of the hybridomas developed, designated 6F9, was capable of over 70% inhibition of 1E4 in binding in the meningococcal C polysaccharide-specific ELISA. Studies with convalescent human serum demonstrated 100% inhibition of a serogroup C-specific ELISA with 200 micrograms of 6F9 per ml and 50% inhibition of this ELISA was achieved with 50 micrograms of 6F9 per ml. Monoclonal anti-idiotype antibodies (Ab3) with specificities similar to Ab1, 1E4 were generated from BALB/c mice immunized with the Ab2 (6F9). Immunization of rabbits with 6F9 resulted in an immunoglobulin G response which was significantly greater than that of control to a titer of 1:160. These studies indicate that monoclonal 6F9 contained a surrogate image on the combining antibody site which mimicked meningococcal C polysaccharide. This surrogate image is capable of evoking antibodies to the meningococcal C polysaccharide in syngenic and xenogenic species.
Role of gamma interferon in induction of natural killer activity by Legionella pneumophila in vitro and in an experimental murine infection model
Authors: Blanchard D K , Friedman H , Stewart W E , Klein T W , Djeu J Y
Published: January 2020

Legionella pneumophila has been shown to induce gamma interferon (IFN-gamma) both in vitro and in vivo during experimental infections of mice. With complement-mediated serologic depletion of murine splenocytes, the cellular sources of IFN-gamma following in vitro stimulation with L. pneumophila antigens were Thy-1.2+, Lyt-2-, L3T4-, and asialo-GM1+, which is consistent with the natural killer (NK) cell phenotype. Additionally, Percoll density discontinuous centrifugation demonstrated that maximal production of IFN coincided with high NK activity in fractions which were enriched for large granular lymphocytes. Furthermore, 18- to 24-h incubation of splenocytes with L. pneumophila whole-cell vaccine resulted in augmented NK cytotoxic activity against YAC-1 tumor target cells in a 51Cr release assay. The addition of macrophages to purified large granular lymphocyte populations augmented both IFN-gamma production and NK activity, suggesting that antigen is required for optimal responses. In an experimental infection model using an intratracheal inoculation route, NK activity was enhanced in the spleen, peripheral blood, and lung cells of infected mice, with maximal stimulation in the lung leukocytes at the site of infection. The results of the present study indicate that NK cells respond in vivo and in vitro to stimulation by L. pneumophila by producing IFN-gamma and by increased cytolytic activity.
Characteristics of heat-stable extracellular hemolysin from Pseudomonas aeruginosa
Authors: Fujita K , Akino T , Yoshioka H
Published: January 2020

Heat-stable hemolysin produced by the cellophane-agar plate method consisted of two major acidic glycolipids. One was composed of 2 mol of rhamnose and 2 mol of beta-hydroxydecanoic acid, and the other was composed of 1 mol of rhamnose and 2 mol of beta-hydroxydecanoic acid. The hemolysis-producing moiety of the hemolysin was shown to be the dimer of beta-hydroxydecanoic acid contained in the glycolipid molecule.
Antimicrobial properties of Kupffer cells
Authors: Filice G A
Published: January 2020

To characterize the antimicrobial activities of Kupffer cells, I harvested macrophages from livers with a technique involving perfusion with collagenase and DNase. Ninety-nine percent of glass-adherent cells had typical macrophage morphology, 99% were esterase positive, and 60% phagocytosed opsonized zymosan when challenged with four particles per macrophage. Toxoplasma gondii multiplied within Kupffer cells from unmanipulated mice, but multiplication was intermediate between that observed in highly permissive peritoneal macrophages and highly activated macrophages. Intravenous injection of heat-killed Propionibacterium acnes, a stimulus known to activate macrophages in other compartments, resulted in a uniform, highly activated population of liver macrophages. Kupffer cells from P. acnes-injected mice were capable of generating reactive oxygen intermediates as shown by reduction of Nitro Blue Tetrazolium during phagocytosis of T. gondii or opsonized zymosan. In contrast, intravenous P. acnes injection did not activate spleen macrophages. Intravenous injection of P. acnes into athymic mice activated Kupffer cells, which suggested that T cells were not essential for this response. Kupffer cells were not activated in mice with latent Toxoplasma infection or during acute Giardia muris infection. Ordinarily, Kupffer cells became highly permissive for T. gondii during 48 h in culture, but inclusion of recombinant murine gamma interferon maintained their moderate inhibitory activity.
Acidic fibroblast growth factor modulates Staphylococcus aureus adherence to human endothelial cells
Authors: Blumberg E A , Hatcher V B , Lowy F D
Published: January 2020

Alteration of human endothelial cells may increase their susceptibility to staphylococcal invasion and thus may contribute to the development of intravascular staphylococcal disease. Acidic fibroblast growth factor, a potent regulator of endothelial cell function, had a significant effect on Staphylococcus aureus infection of cultured human endothelial cells. Three of four S. aureus strains had diminished adherence to endothelial cells when the latter were grown in the presence of acidic fibroblast growth factor (P less than 0.05). The diminished adherence was time dependent, maximal at 72 h, and independent of the initial bacterial inoculum. A twofold enhancement of S. aureus adherence was observed when endothelial cells were pretreated with heparitinase. Adherence was unaffected by endothelial cell activation by interleukin-1 or endotoxin. Thus, acidic fibroblast growth factor exerted a protective effect, deterring S. aureus adherence to cultured endothelial cells. Endothelial cell heparan sulfate was also directly involved in the adherence process. Subtle modulations of endothelial cells can significantly affect the ability of S. aureus to adhere to and then infect these cells. Similar alterations may contribute to the ability of S. aureus to infect endovascular tissue in vivo.
In vivo-activated mononuclear phagocytes and protective immunity to chlamydiae in mice
Authors: Huebner R E , Byrne G I
Published: January 2020

Peritoneal macrophages (M phi s) collected from Chlamydia psittaci 6BC-immune mice after intraperitoneal challenge with 10(6) 6BC (immune-boosted [IB] M phi s) were compared by various functional criteria with other in vivo- and in vitro-activated M phi populations. While casein-, protease peptone-, and thioglycolate (Thio)-elicited M phi s were equally susceptible to in vitro infection with 6BC, IB M phi s did not support chlamydial growth and M phi s from Mycobacterium tuberculosis BCG- or Listeria monocytogenes-sensitized mice exhibited intermediate susceptibility to infection. The resistance of IB M phi s was not due to the ingestion of fewer 6BC organisms, nor were these cells persistently infected, since chlamydiae could not be recovered from infected IB M phi s after in vitro infection, even after extended incubation times. In contrast, Thio M phi s stimulated in vitro with gamma interferon (IFN-gamma), with or without lipopolysaccharide, resulted in cells that exhibited chlamydiastatic activity which was lost shortly after IFN-gamma was removed from the culture medium. Conversely, the antichlamydial activity of IB M phi s was stable over time but not through the production of autostimulatory cytokines, as evidenced by the lack of stimulation of Thio M phi s to restrict 6BC replication in coculture experiments. IB M phi s exhibited enhanced oxidative activity, but anti-IFN-gamma antibody did not abrogate this response. IB M phi s were recovered only from immunized mice that survived an otherwise lethal 6BC intraperitoneal challenge. These cells appear to be important for development of protective immunity to chlamydiae, and evidence suggests that stimulation by cytokines other than IFN-gamma (with or without lipopolysaccharide) is required for the observed heightened in vivo activation.
Immunization of cattle with a 36-kilodalton surface protein induces protection against homologous and heterologous Anaplasma marginale challenge
Authors: Palmer G H , Oberle S M , Barbet A F , Goff W L , Davis W C , McGuire T C
Published: January 2020

Immunization of cattle with a purified Anaplasma marginale major surface protein, AmF36, induced protection against homologous challenge with the Florida isolate. Similarly, immunized cattle were protected from challenge with the antigenically and structurally distinct Washington-O isolate of A. marginale. The degree of protection in AmF36-immunized cattle varied from complete prevention of rickettsemia to significant delay in the onset of rickettsemia compared with control immunized cattle. A single AmF36 vaccinate was not protected against homologous challenge despite development of a strong antibody response. Immunoprecipitation of A. marginale proteins with a monoclonal antibody to AmF36 identified minor molecular size heterogeneity in this protein from different isolates, including the Florida and Washington-O isolates. The apparent molecular size of this surface protein in the Florida isolate was 36 kilodaltons, whereas the analogous proteins in Washington-O and four other isolates of A. marginale from the United States had molecular masses of 33 to 34 kilodaltons. Significantly, the surface-exposed peptides of these proteins appear to be conserved among the different isolates. These results demonstrate the potential of AmF36 as a subunit immunogen for bovine anaplasmosis and indicate a structural basis for its cross-protective ability.
Chemotactic behavior of Campylobacter jejuni
Authors: Hugdahl M B , Beery J T , Doyle M P
Published: January 2020

The chemotactic behavior of Campylobacter jejuni was determined in the presence of different amino acids, carbohydrates, organic acids, and preparations and constituents of mucin and bile. L-Fucose was the only carbohydrate and L-aspartate, L-cysteine, L-glutamate, and L-serine were the only amino acids producing a chemotactic (positive) response. Several salts of organic acids, including pyruvate, succinate, fumarate, citrate, malate, and alpha-ketoglutarate, were also chemoattractants, as were bile (beef, chicken, and oxgall) and mucin (bovine gallbladder and hog gastric). Most constituents of bile tested individually were chemorepellents, but the mucin component was chemoattractant. The chemotactic behavior of C. jejuni toward L-fucose, a constituent of both bile and mucin, may be an important factor in the affinity of the organism for the gallbladder and intestinal tract.
Lipopolysaccharide structure determines ionic and hydrophobic binding of a cationic antimicrobial neutrophil granule protein
Authors: Farley M M , Shafer W M , Spitznagel J K
Published: January 2020

Bactericidal activity and binding of a 57,000-dalton cationic antimicrobial neutrophil granule protein (CAP57) are determined by the presence on bacteria of O-antigen polysaccharide chains and the availability of negatively charged groups in the lipid A region, the inner core region, or both regions of lipopolysaccharide. Polymyxin B (PMB)-resistant mutants with well-defined alterations in lipid A structure and charge (pmrA) are also more resistant to CAP57. We used biologically active radioiodinated CAP57 to study the characteristics and kinetics of binding to a sensitive Rb lipopolysaccharide chemotype, Salmonella typhimurium SH9178, and the relatively resistant pmrA mutant strain SH7426. Binding occurred rapidly and was specific and saturable. Because CAP57 appears to be bound in a manner similar to that of PMB, competition binding studies were performed. Excess PMB did compete with CAP57 for binding to SH9178. Nonapeptide, a polycationic derivative of PMB that has lost its hydrophobic portions, demonstrated a marked decrease in ability to compete for binding with CAP57 compared with PMB. This demonstrated the importance of hydrophobic binding in the interaction of CAP57 with the microbial surface. Thus, we have shown that binding of CAP57 to SH9178 is specific, saturable, and similar to binding of PMB. Both hydrophobic and ionic properties of CAP57 appear to be necessary for binding.
Endotoxin-associated protein: interleukin-1-like activity on serum amyloid A synthesis and T-lymphocyte activation
Authors: Johns M A , Sipe J D , Melton L B , Strom T B , McCabe W R
Published: January 2020

Bacterial endotoxins or lipopolysaccharides (LPS) elicit a variety of biologic activities in intact animals and various in vitro systems. LPS from most gram-negative bacteria have appeared to have similar biologic activities regardless of the species of origin or method of preparation of the LPS. More recent studies have suggested differences in the effects of protein-rich as opposed to protein-free LPS in inducing mitogenesis of lymphocytes from endotoxin-resistant C3H/HeJ mice. These studies examine other activities of endotoxin-associated protein (EAP), purified to less than 0.007% contamination with LPS, and demonstrate that this material has activity mimicking some of the effects of interleukin-1 (IL-1). EAP proved to be as potent as LPS in eliciting rises in concentrations of serum amyloid A (SAA) and was active in both endotoxin-sensitive (CF1) and endotoxin-resistant (C3H/HeJ) mice. In contrast to LPS, which mediates its SAA-inducing activity by release of an inducer (IL-1) from LPS-stimulated macrophages, EAP appeared to act directly to induce SAA production, in that incubation with macrophages failed to increase its activity. EAP also exhibited IL-1-like activity in the lymphocyte-activating factor assay when both CF1 and C3H/HeJ thymocytes and macrophages were tested. The lymphocyte-activating factor activity of EAP was not blocked by addition of polymyxin B. In addition, EAP exerted stimulatory activity on resting human T lymphocytes, costimulated with Sepharose-bound anti-CD3 monoclonal antibody 64.1, comparable to that observed with purified human monocyte IL-1. These studies indicate that proteins from procaryotic cells may act as cytokines for some eucaryotic cells.
Endotoxin-associated protein: a potent stimulus for human granulocytopoietic activity which may be accessory cell independent
Authors: Bjornson B H , Agura E , Harvey J M , Johns M , Andrews R G , McCabe W R
Published: January 2020

Proteins coextracted with endotoxin, termed endotoxin-associated protein (EAP), have been shown to exert interleukin 1-like activities. The present studies demonstrate that EAP also exerts potent granulopoietic colony-stimulating activity (CSA) on human peripheral blood and bone marrow progenitor cells, comparable to that seen with various types of conditioned media. The CSA observed with EAP appeared to be heat (100 degrees C, 30 min) and trypsin resistant and partially pronase resistant. Similar resistance was observed with the porin proteins of the outer membrane of gram-negative bacteria, and similar CSA activity was observed with a purified porin preparation of Neisseria gonorrhoeae. The CSA of EAP could be demonstrated in human peripheral blood and bone marrow leukocytes rigorously depleted of monocytes, T lymphocytes, and B lymphocytes by treatment with specific monoclonal antibodies and complement.
Interaction of Mycoplasma pneumoniae with HeLa cells
Authors: Krause D C , Chen Y Y
Published: January 2020

The susceptibility of HeLa cells to Mycoplasma pneumoniae-induced injury was examined. Infections were initiated with relatively low mycoplasma doses, carried out in a culture medium incapable of supporting M. pneumoniae replication in the absence of host cells, and monitored for up to 10 days. Under these conditions, a time- and dose-dependent decline in the number of viable host cells compared with that of uninfected controls was observed. The effect of M. pneumoniae infection on host cell macromolecular synthesis was also evaluated. At high doses of infection, synthesis of both protein and RNA declined rapidly relative to that in control cells. At lower doses there was a biphasic response in protein synthesis, which was substantially lower than that in the uninfected control by day 1 postinfection, returned to control levels by day 4 postinfection, and was again less than that in control cells by day 7 postinfection. In contrast, no transient recovery was observed in RNA synthesis, which declined very gradually over 7 days in infected HeLa cells compared with that in uninfected control cells. The ability of HeLa cells to support the proliferation of M. pneumoniae under these experimental conditions was demonstrated by quantitation of mycoplasma CFU in the nonpermissive medium in the presence or absence of HeLa cells. A negligible increase in the number of M. pneumoniae was observed over 4 days when HeLa cells were absent, while CFU increased by almost 20-fold when M. pneumoniae was cultured in the presence of HeLa cells. The susceptibility and response in macromolecular synthesis in M. pneumoniae-infected HeLa cells differed from that recently described for a nontransformed culture of hamster trachea epithelial cells under the same experimental conditions (Y.-Y. Chen and D.C. Krause, Infect. Immun. 56: 570-576, 1988), underscoring the importance of the choice of host cell for in vitro modeling of M. pneumoniae pathogenesis.
Membrane-associated antigens of blood stages of Plasmodium, brasilianum, a quartan malaria parasite
Authors: Cochrane A H , Matsumoto Y , Kamboj K K , Maracic M , Nussenzweig R S , Aikawa M
Published: January 2020

The localization of Plasmodium brasilianum-derived antigens in short and long clefts within the cytoplasm of infected erythrocytes and in association with knobs of the host cell membrane was demonstrated by immunoelectron microscopy with monoclonal antibodies. Our results document that malaria-induced short and long clefts, previously distinguishable only by morphology, differ also in antigenic composition. Another parasite-derived antigen was found to be associated with the parasitophorous vacuole space in schizonts. In segmenters, this antigen was present in large amounts between merozoites and in the cytoplasm of infected cells. These antigens were characterized by biosynthetic labeling and gel electrophoresis.
Transfer of murine host protection by using interleukin-2-dependent T-lymphocyte lines
Authors: Paul C C , Norris K , Warren R , Smith R A
Published: January 2020

We have demonstrated in this study that long-term, interleukin-2 (IL-2)-dependent, salmonella antigen-specific T-lymphocyte lines, as well as peritoneal exudate-enriched T cells, could be developed from both the antigen-sensitized inguinal and periaortic lymph nodes. Only those lines (salmonella-specific lymph node cells or peritoneal exudate T cells) were capable of adoptively transferring significant host protection (P less than 0.01) compared with the immune reactions of lethally challenged naive controls or of mice that had ovalbumin-specific T-cell lines transferred. Of particular interest was the finding that IL-2-dependent T-cell lines derived from the lymph nodes could only confer host protection to naive mice when both the transfer and challenge dose were administered via the intravenous route. Likewise, those T-cell lines derived from the peritoneal exudate were only capable of adoptively transferring significant protection when the cells and challenge dose of salmonellae were administered intraperitoneally. These studies indicate that systemic host protection can be transferred to naive mice, but depending on the source, the IL-2-dependent T-cell lines (lymph node or peritoneally isolated) functioned differentially upon challenge. Also, the results of this study indicate that the administration of greater numbers of IL-2-specific T cells may result in decreased, rather than enhanced, host protection. This may be due to the fact that the IL-2-dependent T-cell population consisted of 20 to 25% Lyt-2,3+ cells, indicating that cells of the suppressor/cytotoxic phenotype were present. Thus, increasing the number of cells transferred may result in an abrogation of protection.
Expression of the glucan-binding lectin of Streptococcus cricetus requires manganous ion
Authors: Drake D , Taylor K G , Doyle R J
Published: January 2020

Streptococcus cricetus AHT exhibited a requirement for manganese for growth and expression of the glucan-binding lectin. While low concentrations of manganese (0.1 to 10 microM) were able to support growth, higher concentrations (greater than 100 microM) were required for full expression of the glucan-binding lectin. The manganous-aquo ion may be important in cellular adhesion and accumulation processes in dental plaque.
Purification, cloning, and sequence of outer membrane protein P1 of Haemophilus influenzae type b
Authors: Munson R , Grass S
Published: January 2020

Outer membrane protein P1 from Haemophilus influenzae type b MinnA was purified and partially characterized. Antiserum was generated against the purified protein and was used to immunologically screen a lamba EMBL3 genomic library prepared from strain MinnA DNA. A 4.2-kilobase-pair EcoRI-BamHI fragment containing the P1 gene was subcloned into pBR322. The recombinant protein was synthesized by Escherichia coli K-12, in which it localized to the outer membrane. The N-terminal sequence of the purified protein was determined and found to correspond to residues 23 through 36. The 22-amino-acid leader peptide had a typical structure, with two lysine residues near the amino terminus, a stretch of hydrophobic residues, and alanine residues at positions 20 and 22. The Mr of the processed protein was 47,752, which is in good agreement with the estimate of 50,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Putative -35 and -10 promoter sequences were identified upstream from the translational start site. Codon usage was examined and determined to be substantially different than the codon preference in E. coli.
Depression of immunity to Naegleria fowleri in mice by selective depletion of neutrophils with a monoclonal antibody
Authors: Ferrante A , Carter R F , Lopez A F , Rowan-Kelly B , Hill N L , Vadas M A
Published: January 2020

In an attempt to define the role of neutrophils in immunity to Naegleria fowleri in vivo, we examined the effects of treating immunized (with amoeba culture supernatant antigen) mice with the monoclonal antibody NIMP-R10, which binds to neutrophil complement receptor type 3bi (CR3) and causes selective neutrophil depletion in mice. Mice in the nonimmunized group challenged with amoebae all died by day 12, while 97% in the immunized group survived. By contrast, the immunized group treated with NIMP-R10 showed only 25% survival. The immunized group treated with "control" mouse ascites, WEM-G11, was highly resistant (90% survival). There was a significant neutrophil response in the nasal mucosa and olfactory lobes of immunized, NIMP-R10-treated mice, despite a marked degree of neutropenia similar to that seen in immunized, untreated mice. Nonimmunized mice showed virtually no neutrophil response. Despite this response in the NIMP-R10-treated mice, amoebic proliferation was not depressed, and there was no evidence of neutrophil degranulation or amoebic killing, despite the close apposition of large numbers of neutrophils to amoebae. The results indicate that neutrophils are necessary for the expression of immunity to N. fowleri.
Iron regulation of the cloned diphtheria toxin promoter in Escherichia coli
Authors: Tai S P , Holmes R K
Published: January 2020

Regulation of the diphtheria toxin promoter by iron was studied in Escherichia coli by using a galK transcriptional fusion. A fragment of the toxin (tox) operon containing the regulatory region was cloned from corynephage beta into a galK transcription vector such that expression of galK activity was controlled by the tox promoter. When E. coli N100 (a galK mutant) harboring this tox-galK fusion plasmid was grown in Luria broth, the specific activity of galactokinase remained constant throughout the exponential phase of growth. When bacteria were shifted from such high-iron medium into low-iron Luria broth, the specific activity of galactokinase increased rapidly, but induction of galactokinase was prevented by the addition of iron to the medium. Measurement of tox-specific mRNA by dot blot hybridization showed that this regulation occurred at the level of transcription. When the plasmid containing the tox-galK fusion was introduced into a fur mutant of E. coli, expression of galK was maximal in both high-iron and low-iron media; but repressibility of galK by iron in this strain was restored by complementation with the fur+ allele. The tox promoter has significant homology with the consensus sequence for other iron-regulated promoters of E. coli that are controlled by fur. These data indicate that the product of the fur gene can function in E. coli as an iron-dependent repressor for the tox promoter from corynephage beta.
Hamster challenge potency assay for evaluation of Mycoplasma pneumoniae vaccines
Authors: Barile M F , Chandler D K , Yoshida H , Grabowski M W , Razin S
Published: January 2020

A hamster immunization challenge assay described in the accompanying paper (M. F. Barile, D. K. F. Chandler, H. Yoshida, M. W. Grabowski, R. Harasawa, and S. Razin, Infect. Immun. 56:2443-2449, 1988) was used to examine protection against Mycoplasma pneumoniae disease by passive immunization and to evaluate the protective potency of a Formalin-inactivated whole-cell and a cell extract M. pneumoniae vaccine. Passive immunization with a globulin fraction of hyperimmune mule antiserum to M. pneumoniae provided hamsters some protection against the challenge. When hamsters were actively immunized, a single dose of Formalin-inactivated vaccine provided only minimal protection, whereas multiple doses of this vaccine, particularly when combined with adjuvant, provided good protection. A single dose of the cell extract vaccine did not protect animals, but two doses caused a marked reduction of disease when a priming dose was given intraperitoneally, followed by a booster dose intratracheally. The correlation between the level of metabolism inhibition antibodies to M. pneumoniae in the sera of vaccinated hamsters and the degree of protection as measured by reduction of lung pathological scores and colonization was poor, indicating that seroconversion rates for metabolism inhibition antibodies are not by themselves adequate to measure the potency of M. pneumoniae vaccines.
Identification of wall-specific antigens synthesized during germ tube formation by Candida albicans
Authors: Casanova M , Gil M L , Cardeñoso L , Martinez J P , Sentandreu R
Published: January 2020

Walls of the two cellular forms (blastoconidia and mycelia) of Candida albicans ATCC 26555 were obtained from cells metabolically labeled (6-h pulse) with 14C-protein hydrolysate and [3H]threonine. Walls were purified by thorough washings with buffered and sodium dodecyl sulfate solutions and digested with Zymolyase 20T. The enzymatic treatment released four major high-molecular-weight mannoproteins (HMWM), with apparent molecular masses of 650, 500, 340, and 200 kilodaltons (HMWM-650, HMWM-500, HMWM-340, and HMWM-200, respectively), from yeast cells, whereas two high-molecular-mass mannoproteins (HMWM-260 and HMWM-180) were solubilized from mycelial cells. Some additional minor low-molecular-weight species were also detected in the enzymatic digests of walls from both types of cell. Single and dual pulse-chase experiments indicated that the HMWM-260 and HMWM-180 species reflect de novo synthesis of new proteins specific for the mycelia and do not represent a topological rearrangement of blastoconidium wall components. Monoclonal antibodies were raised against the HMWM-260 species (quantitatively the predominant component in the mycelial walls), and polyclonal rabbit antibodies were obtained against yeast or mycelial cell walls. Anti-mycelial cell wall polyclonal antibodies were adsorbed to whole killed blastoconidia to remove antibodies against common blastoconidium and mycelial wall antigens. Titration by enzyme-linked immunosorbent assay revealed that the monoclonal antibodies could recognize an epitope of the protein moiety of the HMWM-260 mannoprotein. Immunoblotting and immunofluorescence techniques using these monoclonal and polyclonal antibodies confirmed that the HMWM-260 and HMWM-180 species are specific components of the envelope of the mycelial cell walls.
Isolation and characterization of the alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) from fimbriated Escherichia coli
Authors: Hoschützky H , Lottspeich F , Jann K
Published: January 2020

The alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) was isolated from the fimbria-adhesin complex (FAC) of recombinant Escherichia coli strains expressing the F7(1), F8, or F13 fimbrial antigens. Separation into fimbriae and adhesin was achieved by heating the FAC to 80 degrees C in the presence of Zwittergent 3-16. After removal of the fimbriae by precipitation with lithium chloride, the adhesin was purified by anion-exchange fast protein liquid chromatography in the presence of 4 M urea. The purified adhesins from the three strains had pIs of 4.8 to 5.0 and molecular weights of approximately 35,000. The fimbrillins were smaller, their molecular weights being different with different F antigens. The amino-terminal amino acid sequence of the F7(1)- and F13-derived adhesins were different, that of the F13-derived adhesin being identical to that extrapolated from the DNA sequence of the papG gene (B. Lund, G. Lindberg, B.-I. Marklund, and S. Normark, Proc. Natl. Acad. Sci. USA 84:5898-5902). An antiadhesive monoclonal antibody which reacted with the three P adhesins was prepared. The FAC and the purified adhesins but not the fimbriae from which the adhesins had been removed agglutinated erythrocytes and galactose-galactose-coated latex beads. The adhesion of erythrocytes to the surface-fixed adhesins could be specifically inhibited with alpha-galactosyl-1,4-beta-galactosyl-1,4-glucosyl. The results indicate that the P adhesin(s) of uropathogenic E. coli represents a group of related proteins with conserved receptor recognition domains. The F13-derived P adhesin is the PapG protein postulated by Normark and his colleagues (Lund et al., Proc. Natl. Acad. Sci. USA 84:5898-5902; B. Lund, F. Lindberg, and S. Normark, J. Bacteriol. 170:1887-1894).
Characterization of the macrophage subset affected and its response to a T suppressor factor (TsFmp) found in cryptococcosis
Authors: Blackstock R , Hernandez N C
Published: January 2020

Previous reports from our laboratory described the detection of a suppressor factor which inhibited the phagocytic activity of a macrophage subset in murine cryptococcosis and in classical models of immune tolerance. The suppressor factor was originally named PIL (phagocytosis-inhibiting lymphokine) but has recently been renamed TsFmp (T suppressor factor for macrophage phagocytosis) because it was found to resemble the antigen-specific I-J-restricted suppressor factors described by others. The current investigation revealed that TsFmp acted rapidly upon the macrophage (15 min or less) to exert its effect of inhibiting the phagocytic process. The time for the macrophage to recover from the effects of TsFmp was likewise very rapid. The ability of TsFmp to inhibit phagocytosis was limited to engulfment of particles by Fc and mannan receptors and did not extent to phagocytosis via complement receptors or by nonspecific mechanisms. The macrophage subset that responded to TsFmp was determined to be in the I-A+ and I-J-IM+ subset.
Regulation of serum tumor necrosis factor in glucocorticoid-sensitive and -resistant rodent endotoxin shock models
Authors: Zuckerman S H , Bendele A M
Published: January 2020

Bolus injection of lethal or sublethal doses of endotoxin or lipopolysaccharide (LPS) results in the rapid and transient rise in tumor necrosis factor (TNF) levels in serum in mammals. TNF levels peak between 1 and 2 h after LPS injection in mice and guinea pigs and approach basal levels by 6 h. Although the kinetics of TNF in serum appear similar between these two species, guinea pigs respond to a lethal dose of LPS of 20 mg/kg by producing approximately 10-fold more TNF than mice do. These two endotoxin shock models also differ in their sensitivity to glucocorticoids. TNF levels in serum are not reduced in the lethal endotoxin shock model in guinea pigs after treatment with dexamethasone at 25 mg/kg. In contrast, TNF levels in mouse serum are inhibited by more than 90% after treatment with dexamethasone at 3 mg/kg. Coincident with the TNF peak in serum is a leukopenia which approaches control levels by 6 h in dexamethasone-treated mice, while remaining depressed in dexamethasone-treated guinea pigs. Treatment with dexamethasone at 25 mg/kg did not save guinea pigs from endotoxin lethality, whereas long-term survival of mice under identical conditions was apparent. These results suggest that the relative glucocorticoid resistance observed in guinea pigs is also apparent in a lethal endotoxin shock model in which dexamethasone does not modulate TNF levels or result in increased survival as occurs in mice. The lack of clear efficacy for steroid therapy in human clinical septic shock trials would suggest that the guinea pig endotoxin model may be a more predictive system than the mouse model for the identification of novel agents useful in the treatment of endotoxin shock.
Chemical differences in lipopolysaccharides from Actinobacillus (Haemophilus) actinomycetemcomitans and Haemophilus aphrophilus: clues to differences in periodontopathogenic potential and taxonomic distinction
Authors: Brondz I , Olsen I
Published: January 2020

While Actinobacillus actinomycetemcomitans has been associated with rapidly progressive periodontal destruction in man, the closely related Haemophilus aphrophilus has not been related to periodontal disease. This may be due to differences in composition and structure of the lipopolysaccharides (LPS) of these dental-plaque bacteria, since LPS probably exerts a series of detrimental effects on the periodontium. LPS was prepared by the phenol-water procedure from the type strains of A. actinomycetemcomitans and H. aphrophilus, purified by hexane extraction and ultracentrifugation, and analyzed with gas chromatography and gas chromatography-mass spectrometry. While the lipid content of LPS from A. actinomycetemcomitans constituted 35.4%, it was only 18.4% in H. aphrophilus: 3-hydroxytetradecanoic and tetradecanoic acids were 21.1 and 14.3% in A. actinomycetemcomitans and 10.9 and 7.5% in H. aphrophilus. There were qualitative and quantitative differences in the polysaccharide portions of their LPS. A actinomycetemcomitans contained both D-glycero-D-mannoheptose and L-glycero-D-mannoheptose (7.8 and 11.3%); H. aphrophilus contained only L-glycero-D-mannoheptose (17.4%). The rhamnose, fucose, galactose, glucose, and glucosamine/galactosamine contents in A. actinomycetemcomitans were 2.6, 5.2, 10.1, 22.4, and 5.2%, respectively; in H. aphrophilus, they were 2.1, 2.6, 19.4, 36.4, and 3.7%. Chemical differences in LPS from A. actinomycetemcomitans and H. aphrophilus may contribute to the divergence in periodontopathogenic potential of these organisms and help taxonomic differentiation.