bluuebunny/crossref_metadata_2025 huggingface dataset:
Native surface association of a recombinant 38-kilodalton Treponema pallidum antigen isolated from the Escherichia coli outer membrane
Authors: Fehniger T E , Radolf J D , Walfield A M , Cunningham T M , Miller J N , Lovett M A
Published: January 2020

A recombinant plasmid designated pAW305, containing a 6-kilobase insert of Treponema pallidum DNA, directed the expression of a 38-kilodalton (kDa) treponemal antigen in Escherichia coli. The 38-kDa antigen copurified with the outer membrane fraction of the E. coli cell envelope after treatment with nonionic detergents or sucrose density gradient centrifugation. Rabbits immunized with the recombinant 38-kDa antigen developed antibodies which reacted specifically with a 38-kDa T. pallidum antigen on immunoblots, and 38-kDa antisera specifically immobilized T. pallidum in a complement-dependent manner in the T. pallidum immobilization test. Antisera to the 38-kDa recombinant antigen were also used to demonstrate its native surface association on T. pallidum by immunoelectron microscopy.
Evidence for lipid peroxidation in endotoxin-poisoned mice
Authors: Peavy D L , Fairchild E J
Published: January 2020

Ethane has been identified and quantitated in air exhaled by mice following intraperitoneal injection of 20, 40, or 200 mg of Escherichia coli O111:B4 lipopolysaccharide (LPS) per kg. Significant increases in ethane concentration occurred within 1 to 5 h after LPS administration. In addition, increased concentrations of malondialdehyde were found in crude homogenates of livers obtained from mice 16 h after administration of 20 mg of LPS per kg. These results suggest that lipid peroxidation may be an important mechanism responsible for LPS toxicity.
Synergistic activity of rabbit granulocyte peptides against Candida albicans
Authors: Lehrer R I , Szklarek D , Ganz T , Selsted M E
Published: January 2020

Rabbit granulocytes contain six antimicrobial peptides that are structurally homologous to the human neutrophil "defensins." NP-5, a rabbit defensin, lacks significant activity against Candida albicans. Nevertheless, its addition to submicromolar concentrations of rabbit NP-1, NP-2, or NP-3a potentiates their candidacidal effect. Thus, granulocyte defensins can act synergistically against potential pathogens.
Comparison of immunoblot analyses of spherule-endospore-phase extracellular protein and mycelial-phase antigen of Coccidioides immitis
Authors: Zimmer B L , Pappagianis D
Published: January 2020

The extracellular proteins produced by Coccidioides immitis during growth of the spherule-endospore-phase and mycelial-phase antigen (coccidioidin) were studied by polyacrylamide gel electrophoresis followed by immunoblot analysis to detect specific serologic function. Filtrates obtained from 28- and 120-h growth of the spherule-endospore phase were compared with each other and with coccidioidin by using negative, immunoglobulin M (IgM) precipitin-positive, or complement fixation-positive pooled and single human sera followed by peroxidase-labeled anti-human IgA, IgE, IgG, or IgM (heavy chain specific) or peroxidase-labeled concanavalin A to detect the reaction. A total of 35 bands was seen in the stained gels. Different patterns were noted among the two spherule-endospore preparations and unheated and heated coccidioidin. At least 15 electrophoretically separate antigens were detected with positive serum ranging in approximate molecular weight (Mr) from 100,000 to 18,000. Most were clustered between 45 and 60 kilodaltons (kDa). Common bands were noted at 48 and 18 kDa. At least one band at 48 kDa was strongly reactive with complement fixation-positive serum demonstrated by reaction with anti-IgG and anti-IgE. In contrast, doublet bands in the 50- to 65-kDa area were highly reactive with IgM precipitin-positive serum detected by anti-IgM. IgM antibodies present in both positive sera reacted with a band at 46 kDa which was not reactive with IgG. Heating the antigens altered the reactivity of many of the antigens, including the 48-kDa band, but not the 46-kDa band.
Concomitant cellular and humoral expression of a regulatory cross-reactive idiotype in acute Schistosoma japonicum infection
Authors: Kresina T F , Olds G R
Published: January 2020

In this study the expression of a regulatory cross-reactive idiotype (SJ-CRIM), which is associated with anti-soluble egg antigen (SEA) molecules in murine Schistosoma japonicum infection, is described. Both humoral and cellular components of the immune response were analyzed during the course of infection with S. japonicum. In the humoral immune response, the content of SJ-CRIM decreases as the titer of anti-SEA antibody increases throughout infection. Quantitatively, values for serum ranged from 13.8 +/- 0.3 micrograms of SJ-CRIM, which binds anti-idiotypic antibody per ml of serum at 6 weeks postinfection, to 1.3 +/- 1.8 micrograms/ml at 30 weeks postinfection. Analysis of splenic cell subpopulations for expression of SJ-CRIM revealed that only splenic B cells expressed SJ-CRIM during acute infection (5 to 10 weeks postinfection). On the other hand, thymic cells with a high expression of the SJ-CRIM and Ly-1 marker were observed in acute infections up to 15 weeks postinfection. These data indicate that SJ-CRIM-bearing T cells are selectively localized in acute infection. In addition, the disappearance of expression of SJ-CRIM in serum and cells of chronically infected animals parallels the modulation of granulomatous inflammation and portal hypertension. Results of this study suggest that expression of SJ-CRIM on anti-SEA molecules could represent a marker for acute infection, while its disappearance from serum serves as a marker for modulation of disease.
Molecular cloning and expression in Escherichia coli K-12 of the O antigens of the Inaba and Ogawa serotypes of the Vibrio cholerae O1 lipopolysaccharides and their potential for vaccine development
Authors: Manning P A , Heuzenroeder M W , Yeadon J , Leavesley D I , Reeves P R , Rowley D
Published: January 2020

The gene clusters that determine the biosynthesis of both the Inaba and Ogawa serotypes of the O antigen of the lipopolysaccharide of Vibrio cholerae were cloned and expressed in Escherichia coli K-12. Restriction analysis of the clones demonstrated that about 15 kilobases were common to all clones and a further 5 kilobases were common to the Ogawa clones. The O antigens expressed by E. coli K-12 had the specificity of V. cholerae. Antibodies raised against E. coli K-12 that harbor one of these clones, pPM1001 (Inaba), were as highly protective in the infant mouse model system as were antibodies to V. cholerae itself. Introduction of such clones into suitable carrier strains could be expected to produce a good oral immunogen against cholera.
Endotoxin-inactivating activity in normal and pathological human blood samples
Authors: Obayashi T , Tamura H , Tanaka S , Ohki M , Takahashi S , Kawai T
Published: January 2020

The endotoxin-specific chromogenic test revealed that plasma endotoxin-inactivating activity was markedly diminished by endotoxemia, but not by fungemia or by dialysis with cellulose membranes, suggesting that fungal polysaccharides and other nonendotoxic, Limulus-reactive materials do not consume endotoxin-inactivating factors in the blood. There was a close negative correlation between plasma endotoxin concentration and endotoxin-inactivating activity. The specificity of the test was improved by fractionating amebocyte lysate and using only the factors that constitute the endotoxin-sensitive coagulation pathway of the horseshoe crab. This test was able to differentiate endotoxemia from fungemia and from contamination with other nonendotoxic, Limulus-reactive materials.
Arthropathic group A streptococcal cell walls require specific antibody for activation of human complement by both the classical and alternative pathways
Authors: Eisenberg R A , Schwab J H
Published: January 2020

The induction of acute arthritis in rats by a single intraperitoneal injection of group A streptococcal cell wall is associated with the activation of complement. We have therefore investigated the interaction of arthropathic peptidoglycan-polysaccharide complex of streptococcal cell walls and human complement. The incubation of cell wall in normal human serum results in the formation of complexes of cell wall and the C3 and C4 components of complement. Using agammaglobulinemic serum, we have further shown that the activation of complement and formation of complement-cell wall complexes absolutely requires the presence of a small quantity of specific antibody. This antibody is present in normal human immunoglobulin G and is effective as the Fab fragment (alternative pathway). Although antibodies specific for three cell wall epitopes were capable of inducing complement-cell wall complex formation by the classical complement pathway, only anti-A polysaccharide antibody (N-acetyl-D-glucosamine epitope) was effective by the alternative complement pathway. A complement consumption assay showed that anti-cell wall antibody was required not only for complement-cell wall complex formation, but also for activation of complement by streptococcal cell wall in human serum. These studies suggest that a minimal level of anti-cell wall antibody may be required for the induction of arthritis in rats by streptococcal cell wall.
Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin
Authors: Fuchs G , Mobassaleh M , Donohue-Rolfe A , Montgomery R K , Grand R J , Keusch G T
Published: January 2020

This study examined the binding of purified 125I-labeled shigella toxin to rabbit jejunal microvillus membranes (MVMs). Toxin binding was concentration dependent, saturable, reversible, and specifically inhibited by unlabeled toxin. The calculated number of toxin molecules bound at 4 degrees C was 7.9 X 10(10) (3 X 10(10) to 2 X 10(11))/micrograms of MVM protein or 1.2 X 10(6) per enterocyte. Scatchard analysis showed the binding site to be of a single class with an equilibrium association constant, K, of 4.7 X 10(9) M-1 at 4 degrees C. Binding was inversely related to the temperature of incubation. A total of 80% of the labeled toxin binding at 4 degrees C dissociated from MVM when the temperature was raised to 37 degrees C, but reassociated when the temperature was again brought to 4 degrees C. There was no structural or functional change of MVM due to toxin as monitored by electron microscopy or assay of MVM sucrase activity. These studies demonstrate a specific binding site for shigella toxin on rabbit MVMs. The physiological relevance of this receptor remains to be determined.
Binding of Streptococcus pyogenes to soluble and insoluble fibronectin
Authors: Courtney H S , Ofek I , Simpson W A , Hasty D L , Beachey E H
Published: January 2020

The interaction of soluble and insoluble fibronectin with Streptococcus pyogenes was investigated. Soluble fibronectin bound to S. pyogenes in a dose-dependent and irreversible manner. Lipoteichoic acid competitively inhibited the binding of fibronectin to S. pyogenes but had little effect on the binding of fibronectin to staphylococci or pneumococci. The phase of growth of the streptococci had a slight effect on binding of fibronectin, with optimal binding occurring in the late log phase. S. pyogenes cells bound to fibronectin immobilized on microtiter plates in a dose-dependent and saturable manner. Both soluble fibronectin and lipoteichoic acid inhibited the binding of streptococci to immobilized fibronectin, suggesting that streptococci interact with soluble and insoluble fibronectin in a similar manner. Antibodies to fibronectin blocked the attachment of streptococci to immobilized fibronectin, whereas normal serum had no effect. Adherence of streptococci to buccal epithelial cells was inhibited by antibodies to fibronectin, but not by normal sera or by antibodies to buccal epithelial cells. The data suggest that lipoteichoic acid on the surface of S. pyogenes binds to fibronectin exposed on the host cell and that such binding mediates the attachment of streptococci to host cells.
Pasteurellosis in laboratory rabbits: characterization of lipopolysaccharides of Pasteurella multocida by polyacrylamide gel electrophoresis, immunoblot techniques, and enzyme-linked immunosorbent assay
Authors: Manning P J , Naasz M A , DeLong D , Leary S L
Published: January 2020

The lipopolysaccharides (LPSs) of five isolates of Pasteurella multocida from rabbits were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblots, and enzyme-linked immunosorbent assay. Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of purified unaggregated LPSs resembled those of semirough strains of gram-negative enterobacteria and consisted of one or two bands that migrated within an interval just ahead or slightly behind the migration of the Ra chemotype of "Salmonella minnesota," which has a molecular size of 4.3 kilodaltons. Polyclonal rabbit antisera to P. multocida whole cells used in Western blots and enzyme-linked immunosorbent assays of unabsorbed and LPS-absorbed antisera revealed that the LPS of these isolates of P. multocida contained at least two types of antigens: a nonserospecific antigen and a serospecific antigen. The LPSs of four isolates each had a different serospecific antigen. The nonserospecific antigen was expressed in two isolates and was the only demonstrable LPS antigen in one other isolate.
Early events in the action of staphylococcal alpha-toxin on the plasma membrane of adrenocortical Y1 tumor cells
Authors: Blomqvist L , Thelestam M
Published: January 2020

The early events in staphylococcal alpha-toxin action on mouse adrenocortical (Y1) tumor cells were studied. Cell-bound toxin could be partially neutralized by anti-alpha-toxin and inactivated by trypsin added within 10 min at 37 degrees C after the end of the binding step. Likewise, cell-bound toxin was capable of lysing rabbit erythrocytes (RRBC) added to the cells within 10 min after binding at 37 degrees C. After this time, the Y1 cells could not be rescued from intoxication by antibodies or trypsin, and the toxin was not accessible for lysis of RRBC. However, at 0 to 4 degrees C, the cell-bound toxin remained accessible to antibodies for at least 4 h. CaCl2 (30 mM) did not affect binding of the toxin to Y1 cells but completely prevented the intoxication if added within 10 min at 37 degrees C after the end of the binding step. The intoxication was independent of metabolic energy, active receptor clustering on the cell surface, and endocytosis of the toxin. Therefore, alpha-toxin interacted with the Y1 cell membrane in at least three separable steps: binding, a conformational change at the cell surface, and membrane damage. These early events appear to be similar to those occurring on RRBC treated with alpha-toxin.
Oral immunization of mice with attenuated Salmonella enteritidis containing a recombinant plasmid which codes for production of the B subunit of heat-labile Escherichia coli enterotoxin
Authors: Clements J D , Lyon F L , Lowe K L , Farrand A L , el-Morshidy S
Published: January 2020

We used Salmonella enteritidis serotype dublin strain SL1438, a nonreverting, aromatic-dependent, histidine-requiring mutant, as a recipient for a recombinant plasmid coding for production of the nontoxic B subunit of the heat-labile Escherichia coli enterotoxin. The S. enteritidis derivative EL23 produced heat-labile enterotoxin subunit B that was indistinguishable from heat-labile enterotoxin subunit B produced by strains of E. coli or Salmonella typhi harboring the same plasmid. Mice immunized orally with strain EL23 developed progressively increasing mucosal and serum antibody responses to both heat-labile enterotoxin subunit B and to the lipopolysaccharide of the vaccine strain. The mucosal antibody response was shown to be immunoglobulin A specific and to be capable of neutralizing the biological activities of both E. coli heat-labile enterotoxin and cholera enterotoxin in vitro.
Protection of C3H/HeJ mice from lethal Salmonella typhimurium LT2 infection by immunization with lipopolysaccharide-lipid A-associated protein complexes
Authors: Killion J W , Morrison D C
Published: January 2020

C3H/HeJ mice were immunized intraperitoneally (i.p.) with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes or with purified protein-free LPS prior to lethal i.p. or intravenous Salmonella typhimurium LT2 challenge. Our results demonstrated that these Salmonella-hypersusceptible mice can be effectively protected against 1,000 100% lethal doses of S. typhimurium LT2 (i.e., 1,000 viable bacteria) administered by intravenous challenge when previously immunized with LAP-LPS complexes. In contrast to these results, immunization with LPS afforded markedly less protection regardless of the route of challenge, thus suggesting that the LAP portion of LAP-LPS complexes may be necessary for inducing protection against Salmonella infections. For most experiments, antigens were emulsified in complete Freund adjuvant (CFA); however, the CFA portion of the vaccine was suggested not to be an essential component for the induction of immunity to Salmonella infections, since equivalent levels of protection were obtained when it was omitted from the vaccine. The induction of immunity to murine salmonellosis by prior immunization with CFA-LAP-LPS was demonstrated not to be a transient phenomenon, since C3H/HeJ mice were still protected against lethal S. typhimurium LT2 challenge as late as 225 days postimmunization.
The chicken embryo as a model for campylobacter invasion: comparative virulence of human isolates of Campylobacter jejuni and Campylobacter coli
Authors: Field L H , Headley V L , Underwood J L , Payne S M , Berry L J
Published: January 2020

Eleven-day-old chicken embryos were used to compare the relative virulence of minimally passaged human isolates of Campylobacter jejuni and Campylobacter coli. Graded doses of bacteria were inoculated onto the chorioallantoic membrane, and 50% lethal doses were calculated at 72 h postinfection. Strains varied markedly in their ability to invade the chorioallantoic membrane and kill the embryos. The 50% lethal doses varied by about 6 logs for 25 strains of C. jejuni, and by 2 logs for 5 strains of C. coli. Although both outbred and inbred embryos were employed in the study, the latter were found to be more susceptible to infection with most strains. All isolates were screened for plasmid DNA, but there was no apparent relationship between plasmid content and virulence of strains for the embryos. Neither could virulence be associated with the production of siderophores by the strains. The ability of selected strains of C. jejuni to invade the liver of embryos was also studied. The number of campylobacters culturable from the liver was found to be inversely related to the 50% lethal dose of the strain. By inoculating 11-day-old embryos intravenously, it was possible to demonstrate that a strain of C. jejuni which was poorly virulent after chorioallantoic inoculation was relatively noninvasive. Invasiveness alone, however, could not fully account for the lethality of two highly virulent strains of C. jejuni administered by the intravenous route. Finally, there was no correlation between motility and virulence in this model system.
Treponemal infection specifically enhances node T-cell regulation of macrophage activity
Authors: Tabor D R , Bagasra O , Jacobs R F
Published: January 2020

Hamsters experimentally inoculated in the inguinal region with Treponema pallidum subsp. endemicum develop considerable pathology at that site. We examined the cell populations from these inguinal lymph nodes to determine their intercellular responses to infection. In vitro, syphilitic-node T cells markedly suppressed C3b receptor-mediated ingestion (C3bMI) in syphilitic macrophages derived from sites both proximal and distal to the inoculation. This activity was more pronounced when node T cells rather than peritoneal T cells were used. When treponemal preparations or live treponemes were added to the coculture system, the suppression was specifically enhanced, whereas the addition of heterologous agents did not promote this effect. Syphilitic macrophages from either compartment cultured alone showed no significant inhibition of C3bMI. In parallel studies on syphilitic macrophages, we observed that the expression of Ia quickly became elevated and was sustained throughout the infection. Moreover, in vitro culturing of the syphilitic-node T cells with these macrophages did not alter this function. These observations suggest that the syphilitic node contains a subpopulation of T cells that can selectively suppress macrophage C3bMI activity and concurrently regulate their cellular response to treponemal infection.
Polyclonal response of human lymphocytes to Chlamydia trachomatis
Authors: Räsänen L , Lehtinen M , Lehto M , Paavonen J , Leinikki P
Published: January 2020

The mitogenicity of Chlamydia trachomatis was investigated. Umbilical cord blood cells from healthy newborns were used. These cells were stimulated in cultures with partially purified C. trachomatis elementary bodies. Proliferation of cultured lymphocytes and secreted immunoglobulins and leukocyte migration inhibitory factor were measured in the culture supernatants. Our results showed that C. trachomatis is able to elicit polyclonal antibody, lymphokine, and DNA synthesis.
Cloning and expression in Escherichia coli of the Streptococcus pneumoniae gene encoding pneumolysin
Authors: Paton J C , Berry A M , Lock R A , Hansman D , Manning P A
Published: January 2020

A gene bank of Sau3A1-generated Streptococcus pneumoniae DNA fragments was constructed in Escherichia coli K-12 by cloning into the BamHI site of the cosmid vector pHC79. Clones expressing the pneumolysin determinant were selected by testing for hemolytic activity which could be inhibited by antibody to purified pneumolysin and by cholesterol. Restriction analysis of pneumolysin-positive recombinant cosmid DNA indicated that the coding sequence for the toxin was located within a 2.9-kilobase-pair (kbp) ClaI DNA fragment. This fragment, which included 0.35 kbp of vector pHC79 DNA, was subcloned into the plasmid pBR322. E. coli cells harboring this recombinant plasmid (designated pJCP20) produced approximately one-third of the amount of pneumolysin found in the donor S. pneumoniae strain. Plasmid pJCP20 was stably maintained in E. coli and resulted in the accumulation of active pneumolysin in the cytoplasm. Western blot analysis showed that E. coli harboring pJCP20 produced two forms of the toxin with molecular weights of 54,000 and 52,000. The lower-molecular-weight form was indistinguishable from native pneumolysin. Subcloning the 2.9-kbp DNA fragment into the expression vector pEV31 allowed the determination of the direction of transcription of the pneumolysin gene. The pneumolysin-coding sequence (approximately 1.5 kbp) has been localized to within a 1.75-kbp segment of pneumococcal DNA.
Human T helper cells specific for antigens of typhus group rickettsiae enhance natural killer cell activity in vitro
Authors: Carl M , Martin E E , Dasch G A
Published: January 2020

The peripheral blood mononuclear cells (PBMC) from 5 individuals immune to typhus group rickettsiae and from 13 nonimmune individuals were stimulated in vitro for 7 days with typhus group rickettsial antigen (TGRA). At the end of day 7, lysis of the natural killer (NK)-susceptible target K562 by these PBMC was determined. As controls, PBMC from both groups of donors were cultured in vitro for 7 days without antigen or were freshly isolated, and lysis of the K562 target was determined. There was no significant difference between the level of NK activity in freshly isolated PBMC from immune and nonimmune donors. PBMC from immune donors which were stimulated with antigen for 7 days exhibited significantly greater NK activity than did the control population, which was cultured for 7 days without antigen. PBMC from immune donors which were stimulated with TGRA demonstrated significantly higher NK activity than the same PBMC stimulated with antigen derived from an antigenically unrelated rickettsia, Coxiella burnetii. There was no significant difference, however, in the level of NK activity of nonimmune antigen-stimulated PBMC compared with that of the same PBMC population cultured without antigen. Most of the antigen-stimulated NK activity was mediated by Leu-11-positive cells as determined by electronic cell sorting. The ability of TGRA to sustain the NK activity of PBMC from immune donors was abolished when the T4/Leu-3-positive population of lymphocytes was eliminated by positive or negative selection prior to antigen stimulation. The ability of TGRA to sustain the NK activity of PBMC from immune donors was also significantly decreased in the presence of antibodies against human interleukin-2. The results suggest that the activity of human NK cells can be sustained in vitro by antigen-specific T helper cells and that the effect of the T helper cell is mediated, at least in part, by interleukin-2.
Entry of Bartonella bacilliformis into erythrocytes
Authors: Benson L A , Kar S , McLaughlin G , Ihler G M
Published: January 2020

Bartonella bacilliformis, which causes the human diseases Oroya fever and verruga peruana, binds to human erythrocytes in vitro and produces substantial and long-lasting deformations in erythrocyte membranes, including cone-shaped depressions, trenches, and deep invaginations. The deforming force is probably provided by the polar flagella of these highly motile bacteria. Deep invaginations containing bacteria are commonly seen, and membrane fusion at the necks of the invaginations leads to the formation of intracellular vacuoles containing bacteria. Fluorescent compounds present externally render the vacuoles fluorescent and, occasionally, lightly fluorescent cells are seen, suggesting that the vacuoles sometimes rupture to admit the bacteria to the cytoplasm. Vacuoles present in fluorescent erythrocytes prepared by preloading the erythrocytes with fluorescent compounds are seen as dark areas from which the fluorescent marker is excluded. Entry of the bacteria appears to be the result of a process of forced endocytosis.
Studies on the genetic and cellular control of sensitivity to enterotoxins in the sealed adult mouse model
Authors: Richardson S H , Kuhn R E
Published: January 2020

A sealed adult mouse (SAM) model was developed for studies on the effects of cholera enterotoxin (CT). With this system, 38 strains of outbred, inbred, congenic, recombinant, and mutant mice were starved for 24 h, anorectally occluded with cyanoacrylamide ester glue, given CT per os, and sacrificed at 6 h. Fluid accumulation (FA) values were calculated as gut weight to body weight ratios. At a saturating dose of CT (24 micrograms per mouse), FA responses were found to be independent of body weight and gut length. It was found, using recombinant and congenic mice, that mice which possess the H-2k haplotype (homozygous or heterozygous) are 2.5 to 3 times less responsive to CT than animals with the H-2b haplotype. The allele(s) responsible for this affect is located near the K end of the H-2 complex. Inbred and congenic mice given CT intravenously exhibited the same (b = responder, k = nonresponder) pattern in terms of weight loss and death, thus indicating that the H-2 effect is not limited just to the small intestinal epithelium. Mice given sublethal doses of CT intravenously and challenged after conversion to SAM 14 days later showed an immune response inversely related to weight loss (i.e., b haplotypes lost 10 to 15% body weight, recovered, but were not protected against challenge; k haplotypes lost little or no weight but were protected). To examine the possibility of a cellular basis for control of innate responses to CT, responder C57BL/10 (B10) mice were irradiated with 950 rads and immediately reconstituted with bone marrow from (B10 X B10.BR)F1 (nonresponder) mice. The chimeras became nonresponsive to CT when challenged 5 weeks after reconstitution. Reconstituted B10 controls responded normally. Outbred and inbred nude athymic mice also were nonresponsive when compared with normal responder controls. These data demonstrate a genetic basis for resistance to CT and that response and nonresponse is mediated, at least in part, by cells derived from bone marrow.
Effect of gamma interferon on hydrogen peroxide production by cultured mouse peritoneal macrophages
Authors: Sharp A K , Banerjee D K
Published: January 2020

Macrophage activation is thought to be mediated via a number of T-lymphocyte products, including gamma interferon (IFN-gamma). However, our studies indicate that IFN-gamma acts as a regulator molecule rather than solely as an activator. This depends upon the status of the macrophage. IFN-gamma treatment of resting macrophages and those activated or elicited by sodium caseinate, lipopolysaccharide, or Mycobacterium bovis BCG did not result in activation, as measured by hydrogen peroxide production; however, when thioglycolate was used as an eliciting agent, incubation with IFN-gamma resulted in a dramatic increase in hydrogen peroxide production compared with that by untreated controls.
Interaction of complement with serum-sensitive and serum-resistant strains of Pseudomonas aeruginosa
Authors: Schiller N L , Joiner K A
Published: January 2020

The interaction of complement with the following two strains of Pseudomonas aeruginosa was examined: 144M, a mucoid, serum-sensitive strain bearing short lipopolysaccharide O chains, and 144M-SR, a mucoid, serum-resistant strain bearing long lipopolysaccharide O chains isolated by repeated passage of 144M in increasing concentrations of pooled normal human serum (PNHS). While significant killing of 144M occurred in 5 to 40% PNHS, no killing of 144M-SR was observed. Both strains activated complement, especially 144M-SR which consumed 88.7, 96.4, and 100% of the available complement 3 (C3), C5, and C9, respectively, in 10% PNHS during a 60-min incubation at 37 degrees C. Although it activated more C3 than did 144M (54.9% consumption), 144M-SR bound only half as much C3 as 144M. Similarly, although 144M-SR activated more C9 than did 144M (50.0% consumption in 60 min), there was considerably less C9 attached to 144M-SR (2,990 molecules of C9 per bacterium) than to 144M (13,700 molecules per bacterium) after 60 min of incubation. Furthermore, only 162 molecules of the C9 bound to 144M-SR remained bound after treatment with 0.1% trypsin, while 5,692 molecules of the C9 bound to 144M remained bound under similar conditions. These results show that the serum resistance of 144M-SR does not represent a failure to activate complement efficiently, but instead reflects failure of the assembled terminal complement complex C5b-9 to insert stably into the outer membrane of this strain.
Role of mononuclear phagocytes in expression of resistance and susceptibility to Mycobacterium avium infections in mice
Authors: Stokes R W , Orme I M , Collins F M
Published: January 2020

The growth of Mycobacterium avium 702 in the spleens and livers of four inbred strains of mice varied such that the mice could be separated into naturally susceptible (BALB/c and C57BL/6) and naturally resistant (A/Tru and DBA/2) strains. This phenomenon was independent of the size of the infecting inoculum of bacteria in that both low (10(4))- and high (10(7))-dose inocula of M. avium grew progressively in susceptible strains and were eliminated from the target organs of resistant strains. Resistance and susceptibility were also demonstrated in in vitro preparations of macrophages from these strains of mice. Over a 7-day period, replication of M. avium in susceptible mouse macrophages was far greater than that in resistant macrophages. Evidence was obtained to suggest that toxic oxygen metabolites were not responsible for this difference. Though no difference was found in the rate of clearance of M. avium from the blood of susceptible or resistant mice, resident macrophages from susceptible mice ingested more M. avium in vitro than did resident macrophages from resistant animals. Growth of M. avium in spleens of susceptible mice induced a large influx of phagocytes, whereas this was not observed in resistant mice. In contrast to this it was found that, after injection of a variety of inflammatory agents, influx of leukocytes into the peritoneal cavity could not be used to distinguish susceptible and resistant strains of mice.
Brucella outer membrane lipoprotein shares antigenic determinants with Escherichia coli Braun lipoprotein and is exposed on the cell surface
Authors: Gómez-Miguel M J , Moriyón I , López J
Published: January 2020

In an enzyme-linked immunosorbent assay (ELISA), purified Brucella abortus and Escherichia coli peptidoglycan-linked lipoproteins gave a strong cross-reaction with sera from rabbits hyperimmunized with the heterologous lipoprotein. When smooth E. coli cells were used as ELISA antigens, the immunological cross-reaction was not observed unless the cells were treated to remove lipopolysaccharide and other outer membrane components. In contrast, intact cells from smooth strains of B. abortus and Brucella melitensis bound anti-lipoprotein immunoglobulin G, and the controls performed by ELISA showed that this reaction was not due to antibodies to the lipopolysaccharide, group 3 outer membrane proteins, or porins. Electron microscopy of cells labeled with antilipoprotein serum and protein A-colloidal gold showed specific labeling of smooth cells from both B. abortus and B. melitensis, even though unspecific labeling by nonimmune serum was observed with rough B. abortus. These results confirm the close similarity between E. coli and Brucella peptidoglycan-linked lipoproteins and show that, in contrast to E. coli, the lipoprotein of B. abortus and B. melitensis is partially exposed on the surface of smooth cells.
Identification of a new fimbrial structure in enterotoxigenic Escherichia coli (ETEC) serotype O148:H28 which adheres to human intestinal mucosa: a potentially new human ETEC colonization factor
Authors: Knutton S , Lloyd D R , McNeish A S
Published: January 2020

Three important fimbrial colonization factor antigens (CFAs) designated CFA/I, CFA/II, and E8775 were identified originally in some human enterotoxigenic Escherichia coli (ETEC) strains because of their mannose-resistant hemagglutination properties. To identify CFA, in strains lacking mannose-resistant hemagglutination properties we exploited the ability of human ETEC strains to adhere to human proximal small intestinal mucosa. ETEC strain B7A (O148:H28) was selected for study because it belongs to an epidemiologically important serotype and does not produce a known CFA, and yet it is known to be pathogenic and cause diarrheal disease in human volunteers. Results of an human enterocyte adhesion assay indicated that some bacteria in cultures of B7A produced adhesive factors. To select for such bacteria, cultured human duodenal mucosal biopsy samples were infected with B7A for up to 12 h, after which time a large percentage of the mucosal surface became colonized by bacteria. A new fimbrial structure morphologically distinct from CFA/I, CFA/II, and E8775 fimbriae and consisting of curly fibrils (approximately 3 nm in diameter) was readily identified when bacteria were subcultured from the mucosa and examined by electron microscopy. Identical fimbriae were produced by ETEC strain 1782-77 of the same serotype. Identification of these fimbriae only on bacteria subcultured from human intestinal mucosa strongly suggests that they promote mucosal adhesion of ETEC serotype O148:H28 and thus represent a potentially new human ETEC CFA.
Characterization of plasmids encoding the adherence factor of enteropathogenic Escherichia coli
Authors: Nataro J P , Maher K O , Mackie P , Kaper J B
Published: January 2020

Volunteer studies have shown that a 60-megadalton plasmid is required for full virulence of the human enteropathogenic Escherichia coli (EPEC) strain E2348/69 (O127:H6). The plasmid, designated pMAR2, encodes localized adherence to HEp-2 cells in tissue culture via the adhesin known as the EPEC adherence factor (EAF). Using a DNA probe for the EAF, we have previously shown that these genes are specific for EPEC and are usually encoded on plasmids ranging from 55 to 65 megadaltons. In this study, Southern blot analysis and S1 nuclease homology determination reveal a high degree of sequence conservation among these plasmids, despite some variation in restriction maps. Phenotypic characterization of the prototype EAF plasmid pMAR2 reveals that the plasmid belongs to the group IncFII and is negative for alpha-hemolysin, colicin, and aerobactin synthesis, as well as biochemical markers and antibiotic resistance. Regions encoding adherence to HEp-2 cells were localized by Tn801 insertion mutagenesis. Adherence genes were then cloned as two distinct plasmid regions which confer the adherence phenotype only when complementing each other in trans.
Production of a unique cytotoxin by Campylobacter jejuni
Authors: Guerrant R L , Wanke C A , Pennie R A , Barrett L J , Lima A A , O'Brien A D
Published: January 2020

Campylobacter jejuni is an important diarrheal pathogen worldwide; the mechanisms by which it causes disease remain unclear. Because of its association with inflammatory diarrhea, we postulated that C. jejuni might produce a cytotoxin similar to that produced by Shigella sp., enterohemorrhagic Escherichia coli O157, or Clostridium difficile. Filtrates of 12 polymyxin-treated isolates of C. jejuni were placed on HeLa cells (sensitive to Shiga toxin cytotoxicity) and Chinese hamster ovary (CHO) cells. Of 12 isolates of C. jejuni tested, 5 killed 50% of the cells at greater than or equal to 1:4 dilutions of filtered suspensions of 10(9) bacteria per ml; killing was similar in HeLa and CHO cells (the CHO cells being insensitive to Shiga cytotoxin). One isolate produced a titer of 1:32 to 1:128. The relative potency in HeLa cells was comparable to that of E. coli strains that produce intermediate amounts of Shiga-like toxin. The other seven strains showed no cytotoxic effect, nor did the control diluents, polymyxin B, or supernatants of C. jejuni not treated with polymyxin B. Sonication also released active cytotoxin, but slightly less well than did polymyxin. The cytotoxic effect was dose dependent. Concentration of the C. jejuni in suspension by 10-fold before treatment with polymyxin B resulted in a 10-fold increase in the 50% cytotoxic dose. The cytotoxin effect was not neutralized by Shiga toxin immune serum against either Shiga-like toxin I or II or by anti-Clostridium difficile antiserum. The C jejuni cytotoxin was partially labile to trypsin (0.25%) and to heating to greater than or equal to 60 degrees C. Cytotoxicity was retained in Scientific Products dialysis tubing D1615-1 (Mr cutoff, 12,000 to 14,000). Some isolates of C. jejuni release a substance lethal to HeLa or CHO cells in vitro that is distinct from Shiga-like or Clostridium difficile toxin. This cytotoxin may contribute to the colonic mucosal invasive process that characterizes C. jejuni enteritis.
The antichlamydial, antiviral, and antiproliferative activities of human gamma interferon are dependent on the integrity of the C terminus of the interferon molecule
Authors: de la Maza L M , Peterson E M , Burton L E , Gray P W , Rinderknecht E , Czarniecki C W
Published: January 2020

The effects of recombinant human gamma interferon (rHuIFN-gamma; two identical monomers of 140 residues in length) and of two re-engineered C-terminal variants, rHuIFN-gamma Tetra-Ser (residues 129 to 132 replaced by serine) and rHuIFN-gamma 125 (two identical monomers of 125 residues each with the last 14 residues plus an additional alanine from the C terminus deleted), were compared in terms of several in vitro biological activities. By using three different human cell lines (HeLa 229, HEp-2, and A549), the interferons were tested for their ability to inhibit: (i) growth of Chlamydia trachomatis; (ii) replication of encephalomyocarditis virus; and (iii) cell growth. rHuIFN-gamma restricted the growth of chlamydiae to 50% of the non-IFN-treated control at concentrations ranging from 0.01 to 0.05 ng/ml, depending on the cell type assayed. One of the modified proteins, rHuIFN-gamma Tetra-Ser, also decreased the growth of chlamydiae, but it required a concentration of approximately 0.5 ng/ml to produce 50% inhibition. rHuIFN-gamma 125 had the lowest antichlamydial activity of the three IFN-gamma variants tested; concentrations of 1 to 20 ng/ml were needed to reduce the growth of C. trachomatis to 50% of that of the control. The relative antiviral and antiproliferative activities of the three IFN-gamma preparations paralleled their antichlamydial activities in these three cell lines. The antiencephalomyocarditis virus activities of rHuIFN-gamma Tetra-Ser and rHuIFN-gamma 125 were reduced by approximately 10-fold and 10(2)- to 10(3)-fold, respectively, compared with the antiviral activity of rHuIFN-gamma. Proliferation of the three cell lines was restricted to approximately 50% of the control with 0.5 to 10 ng of rHuIFN-gamma per ml. Inhibition of cell growth by rHuIFN-gamma Tetra-Ser was significant only at concentrations equal to or greater than 30 ng/ml, and the rHuIFN-gamma 125 variant did not significantly decrease the growth of any of the three cell lines at the concentrations tested. These results suggest that the C-terminal portion of rHuIFN-gamma is critical for maintaining the conformation necessary for inducing the antichlamydial, antiviral, and antiproliferative activities of the molecule.
Ingestion of Giardia lamblia trophozoites by human mononuclear phagocytes
Authors: Hill D R , Pearson R D
Published: January 2020

Mononuclear phagocytes may be important effector cells against Giardia lamblia. Human monocyte-derived macrophages were incubated with G. lamblia trophozoites in 13% heat-inactivated autologous serum. At a G. lamblia/macrophage ratio of 1:1, the number of trophozoites ingested per 100 macrophages ranged from 1 to 12 at 0.5 h and increased for all donors (n = 6) to 10 to 92 at 8 h. Ingestion was confirmed by electron microscopy. Increasing the parasite/phagocyte ratio to 5:1 increased the percentage of macrophages with adherent but not ingested trophozoites. Incubating Giardia cells and macrophages with 20% immune serum increased ingestion of parasites eightfold, indicating that anti-G. lamblia antibody can enhance ingestion. Both phase-contrast microscopy and electron microscopy documented trophozoite destruction within macrophages. Ingestion of parasites elicited an oxidative burst as measured by luminol-enhanced chemiluminescence. In vitro, Giardia trophozoites were killed by greater than or equal to 5 X 10(-5) M H2O2. Fusion of lysosomes with parasite-containing phagosomes was suggested by acridine orange-stained preparations. Human macrophages have the capacity to ingest Giardia trophozoites and to kill intracellular parasites, possibly by oxidative microbicidal mechanisms.
Cholesterol metabolism by Treponema hyodysenteriae
Authors: Stanton T B
Published: January 2020

The sterol content of cellular lipids of Treponema hyodysenteriae, the agent of swine dysentery, was determined. When cultured in lipid-depleted brain heart infusion broth containing vesicles made from [4-14C]cholesterol and phosphatidylcholine, T. hyodysenteriae cells incorporated radioactive label. Most (95%) of this radioactivity was associated with bacterial membrane preparations. Lipids were extracted from radiolabeled cells and fractionated by silicic acid column chromatography. Components of the neutral lipid fraction were separated by reversed-phase high-performance liquid chromatography and were detected by monitoring both radioactivity and UV absorption (210 nm) of the column effluent. Cholesterol represented only about 5% of the total radioactivity in the bacterial neutral lipids. The remaining radioactivity was associated with a compound that did not absorb light at 210 nm. This lipid was purified and, on the basis of results from thin-layer chromatography and mass spectrometry, was identified as cholesterol (5 alpha-cholestan-3 beta-ol), a sterol lacking the unsaturated bond of cholesterol. Cholestanol was also present in cell-free culture broth, but only after growth of the spirochete. These results are evidence that cholesterol is used by T. hyodysenteriae for membrane synthesis. Cholesterol is converted to cholestanol in T. hyodysenteriae cultures and cholestanol is a major component (approximately 9% by weight) of T. hyodysenteriae cell lipids.
Relationship of type 1 pilus expression in Escherichia coli to ascending urinary tract infections in mice
Authors: Schaeffer A J , Schwan W R , Hultgren S J , Duncan J L
Published: January 2020

The role of type 1 pili and P adhesins during the in vivo growth of Escherichia coli inoculated into the urethras of BALB/c mice was studied. Strains which produced type 1 pili when grown in broth but lost this trait when grown on agar (regulated variants) were tested. Broth-grown organisms colonized the bladder of every animal tested, with counts of 10(3) to 10(4) viable organisms recovered from bladder homogenates. Agar-grown organisms gave lower rates of infection and the number of viable organisms recovered from bladders was significantly reduced. The degree of inoculum piliation influenced bladder colonization in a direct way: as piliation increased, the number of bacteria recovered from bladders also increased. After intraurethral inoculation, all of the bladders and 44% of the kidneys were colonized on day 1, and by day 5, 94% of the bladders and 16% of the kidneys were positive. Hemagglutination titers remained high for the bladder isolates, but the organisms colonizing the kidneys became significantly less piliated with time. Bacteriuria was unrelated to bladder or renal colonization. Strains that demonstrated random phase variation of type 1 pili during growth on agar produced similar colonizations of the urinary tract with broth- and agar-grown inocula. Strains that produced only P adhesins were less effective in colonizing the urinary tract than were type 1 piliated organisms. Other strains which did not produce pili only minimally colonized the bladder. The results suggest that type 1 pili play an essential role in ascending infections of the urinary tract.
Isolation and characterization of spontaneous avirulent variants of Histoplasma capsulatum
Authors: Klimpel K R , Goldman W E
Published: January 2020

A selection procedure was developed which allowed us to isolate spontaneous isogenic avirulent clones from virulent strains of Histoplasma capsulatum. The avirulent yeasts had a unique phenotype: they did not aggregate like the parental strains but grew as dispersed budded and unbudded single cells in liquid medium. On solid medium, the avirulent variant strains grew as smooth-textured colonies, whereas the virulent parental strains grew as rough convoluted colonies. Virulence testing in mice demonstrated that the smooth variants gave 50% lethal dose values similar to those of the avirulent Downs strain. Growth curves for the paired rough and smooth strains were similar. Furthermore, they had the same protein profiles when crude cell fractions were separated on one-dimensional polyacrylamide gels or when whole-cell extracts were separated by two-dimensional gel electrophoresis. Electrophoresis of culture supernatants, however, revealed a difference in a released low-molecular-weight peptide that may be related to virulence. In addition to their usefulness in comparative virulence studies, these avirulent strains should prove valuable for H. capsulatum genetic experiments because of the unique ability of these yeasts to grow without clumping.