bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of murine Peyer's patch immunoglobulin A response by enterotoxigenic Escherichia coli
Authors: Wess J A , Petro T M
Published: January 2020

Enterotoxigenic Escherichia coli (ETEC) may have profound effects on the capacity of gut-associated lymphoid tissue to mount a secretory immune response because of the potential ability of heat-stable toxin or heat-labile toxin to modulate the immune response. To examine the effects of ETEC or its purified enterotoxins upon the humoral immune response of murine small intestinal Peyer's patch lymphocytes, BDF1 (lipopolysaccharide-responder) and C3H/HeJ (lipopolysaccharide-nonresponder) mice were orally primed with sheep erythrocytes (SRBC) four times during a 2-week period to initiate differentiation of Peyer's patch B lymphocytes to cells committed to anti-SRBC immunoglobulin A (IgA) production. Halfway through the oral priming regimen the mice were gastrically intubated with 10(8) ETEC, 10(8) non-ETEC, or saline. ETEC persisted in the small intestine for at least 7 days at a level of 10(3) to 10(4) bacteria per mouse. Seven days after the last oral dosing with SRBC, Peyer's patch lymphocytes were removed from infected or saline-treated mice and incubated in vitro with SRBC. The ETEC infection had a small effect on the anti-SRBC IgM plaque-forming cell response of SRBC-primed mice but inhibited significantly the anti-SRBC IgA plaque-forming cell response in both BDF1 and C3H/HeJ mice as compared with uninfected controls. The non-ETEC, an isolate from normal mouse small intestine, had no significant effect on either IgM or IgA anti-SRBC plaque-forming cell response. Purified heat-labile toxin, not heat-stable toxin, alone in a dose-dependent manner significantly inhibited both the IgA and IgM plaque-forming cell response of Peyer's patch lymphocytes from primed mice. These data suggest that ETEC can inhibit the development of the gut-associated lymphoid tissue IgA immune response through the immunopharmacological effect of an enterotoxin, the heat-labile toxin.
Probing the surface of Neisseria gonorrhoeae: simultaneous localization of protein I and H.8 antigens
Authors: Robinson E N , McGee Z A , Buchanan T M , Blake M S , Hitchcock P J
Published: January 2020

Gonococcal outer membrane protein I and the neisserial antigen H.8 are being investigated for inclusion in a gonococcal vaccine. To determine the distribution of immunoaccessible protein I and H.8 molecules on the surface of viable gonococci and to approximate the accessibility of these antigens to vaccine-elicited antibodies, immunologic probes composed of protein I- and H.8-specific antibodies linked to gold spheres were developed. When whole gonococci were exposed to the protein I and H.8 immunologic probes and examined by transmission electron microscopy, gold spheres clearly marked the surface of some of the gonococci, but not the surface of other gonococci from the same culture. The immunologic accessibility of gonococcal protein I or neisserial H.8 varied among gonococci. This diversity may affect the efficacy of a vaccine composed of these surface antigens.
Conservation and diversity of Campylobacter pyloridis major antigens
Authors: Perez-Perez G I , Blaser M J
Published: January 2020

Infection with Campylobacter pyloridis has been strongly associated with gastritis in humans although its etiologic significance is currently undefined. We examined the structure and antigenicity of whole-cell, outer-membrane, acid-extractable surface protein, and proteinase K-treated whole cell lysate preparations from eight C. pyloridis strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with homologous and heterologous immune rabbit serum. Whole-cell and outer-membrane profiles observed in all strains of C. pyloridis were nearly identical; none were similar to those of C. jejuni and C. fetus. Major whole-cell bands migrated at 26,000, 29,000, 56,000, and 62,000 molecular weights. The acid-extracted protein profiles of all C. pyloridis strains also were similar to one another and showed similarities with acid-extracted proteins from C. jejuni, with major bands migrating at 29,000, 48,000 to 53,000, and 62,000. All proteinase K-treated lysates showed different lipopolysaccharide (LPS) profiles, ranging from rough to smooth with multiple repeating side chains. Immunoblots of whole-cell and proteinase K-treated preparations of the C. pyloridis strains showed that there was antigenic cross-reactivity of proteins migrating at 62,000 and 56,000, but not in other regions, and cross-reactivity between LPS core regions but not side chains. These results suggest that C. pyloridis has both protein and core LPS group antigens and strain-specific protein and LPS side chain antigens.
Maintenance of biological activity of pertussis toxin radioiodinated while bound to fetuin-agarose
Authors: Armstrong G D , Peppler M S
Published: January 2020

We developed a method to produce radioiodinated pertussis toxin (PT) which was active in the goose erythrocyte agglutination and CHO cell assay systems. The procedure used fetuin coupled to agarose to prevent inactivation of the toxin during the iodination reaction. Analysis of the labeled PT by affinity chromatography on fetuin-agarose and wheat germ agglutinin-agarose and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that there were minimal amounts of labeled fetuin or other contaminants in the labeled PT preparations. All five of the subunits of the toxin appeared to be labeled by the procedure. The labeling method will facilitate further investigations into the nature of the interaction and activity of PT in host tissues.
Partition of heat-labile-enterotoxin genes between human and animal Escherichia coli isolates
Authors: Vinal A C , Dallas W S
Published: January 2020

Heat-labile toxin (LT) genes from human and animal Escherichia coli isolates from a restricted geographical region (Thailand) exhibited a segregated pattern of dissemination that was revealed by a restriction enzyme site polymorphism. The results suggest an exclusion of LT genes from or a low transfer rate for LT genes between E. coli strains infectious for humans and those infectious for animals in natural settings.
Enterotoxigenic strains of Escherichia coli O128 are not restricted to subgroup ac but also belong to subgroups ab and abc
Authors: Thomas L V , Scotland S M , Rowe B
Published: January 2020

Ninety strains of Escherichia coli O128 isolated in many different countries were examined. The majority (77 strains) belonged to the antigenic subgroup O128ab, and 41 of these strains produced heat-stable enterotoxin (ST), heat-labile enterotoxin (LT), or both. Eight strains were of the antigenic subgroup O128ac; six produced ST only, and two were nontoxigenic. Five strains were of the antigenic subgroup O128abc; two produced ST and LT, and three were nontoxigenic. Ten of the strains studied produced Vero cytotoxin, and all belonged to the antigenic subgroup O128ab. It was concluded that, contrary to the report of Guth et al. (B. E. C. Guth, M. L. M. Silva, I. C. A. Scaletsky, M. R. F. Toledo, and L. R. Trabulsi, Infect. Immun. 47:338-340, 1985), enterotoxigenic strains of E. coli O128 are not restricted to subgroup ac but also belong to the most common subgroup, ab.
An early response to lipopolysaccharide is the elicitation of macrophages specialized for antigen degradation with negative regulatory effects on the induction of specific immune responses
Authors: Cluff C W , Ziegler H K
Published: January 2020

The ability of macrophages to catabolize antigens is relevant both as a means to process complex antigens before presentation to T cells and as a way to down-regulate immune responses by destroying the antigenicity of polypeptides. With these considerations in mind, we investigated the regulation of macrophage catabolic activity by lipopolysaccharide (LPS). Catabolic activity was quantitated by following the distribution and molecular form of 125I-labeled surface components of heat-killed Listeria monocytogenes after their uptake by macrophages. We compared the catabolic activity of macrophages from peritoneal exudates of mice injected intraperitoneally with saline or LPS and found that LPS-elicited macrophages displayed a greatly enhanced (threefold) rate of catabolism. This increase in catabolic activity peaked 3 days after LPS injection and slowly declined thereafter, approaching a base-line level after 3 weeks. The enhancement of catabolic activity was under Lps gene control. Macrophages that were elicited 3 days after intraperitoneal injection of LPS rapidly destroyed the antigenicity of bacterial antigens, expressed low levels of Ia molecules, and processed and presented antigen slowly when tested as antigen-presenting cells in vitro. We also showed that an injection of LPS before infection with L. monocytogenes resulted in diminished development of T-cell reactivity to this organism. These results suggest that LPS elicits a macrophage population specialized for antigen degradation functions, with negative regulatory effects on the induction of specific immune responses.
Morphogenesis and pathogenicity of Histoplasma capsulatum
Authors: Medoff G , Kobayashi G S , Painter A , Travis S
Published: January 2020

The sulfhydryl blocking agent p-chloromercuriphenylsulfonic acid (PCMS) irreversibly inhibited the mycelium-to-yeast transitions of two virulent strains of Histoplasma capsulatum, G184A and G222B, when the temperature of incubation was raised to 37 degrees C, and the block persisted even after the cultures were washed free of PCMS. Instead of transforming to yeast cells, PCMS-treated mycelia continued to grow as mycelia at the elevated temperatures. A less virulent strain (Downs) was more temperature sensitive, but it showed a similar irreversible effect at 34 degrees C. Therefore, the mycelium-to-yeast transition of H. capsulatum is not required for the adaptation of mycelia to elevated temperatures but probably results from the temperature-dependent activation of yeast-specific genes. The transition to yeast is inferred to be obligate for pathogenicity in mice because PCMS-treated mycelia failed to cause infection, and no fungi were seen in tissues after PCMS-treated mycelia were injected into mice.
Antigenic specificity of convalescent serum from cattle with haemophilus somnus-induced experimental abortion
Authors: Corbeil L B , Arthur J E , Widders P R , Smith J W , Barbet A F
Published: January 2020

The antigens of Haemophilus somnus recognized by convalescent bovine serum were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with a protein A-peroxidase conjugate. The same two 76K and 40K antigens were predominant in whole-bacterium preparations and in outer-membrane-enriched, Triton X-100-insoluble fractions. The surface location of these two antigens was confirmed by absorbing antiserum with whole, live bacteria. Absorption with H. somnus removed antibody reactivity for the 76K antigen and reduced reactivity for the 40K antigen. Absorption with Pasteurella multocida, Actinobacillus equuli, or Escherichia coli did not reduce reactivity, and results with Pasteurella haemolytica were equivocal. The two immunodominant antigens detected in this study were conserved in isolates of H. somnus from thromboembolic meningoencephalitis, pneumonia, reproductive failure, or asymptomatic carriers. Convalescent sera from nearly all 17 cattle studied recognized these two antigens. Other antigens were recognized less consistently. Although other antigens may also be involved, the 76K and 40K surface antigens of H. somnus appear to be important candidates for a subunit vaccine or an immunodiagnostic assay.
Protective ability and specificity of convalescent serum from calves with Haemophilus somnus pneumonia
Authors: Gogolewski R P , Kania S A , Inzana T J , Widders P R , Liggitt H D , Corbeil L B
Published: January 2020

The ability of convalescent serum to passively protect calves against Haemophilus somnus-induced pneumonia was studied. Preimmune and convalescent serum were obtained from calves before or after recovery from experimental chronic H. somnus pneumonia. Passive protection was assessed in another group of calves by intrabronchial inoculation of H. somnus that had been incubated with preimmune or convalescent serum. Each calf was inoculated with each treatment in alternating caudal lung lobes. Twenty-four hours after inoculation almost no pneumonia was present in lungs inoculated with bacteria incubated with convalescent serum, whereas severe pneumonia was present in lungs inoculated with bacteria incubated with preimmune serum. Quantitation of calf pneumonia in both treatment groups indicated a significantly different protective capacity between convalescent serum and preimmune serum (P less than 0.0005). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting of purified H. somnus lipopolysaccharide resulted in intense reactivity with convalescent serum, but no reactivity was detected with preimmune serum. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of H. somnus outer membrane-enriched fractions, Western blots with convalescent serum gave intense reactions against H. somnus outer membrane antigens with apparent molecular masses of 78 and 40 kilodaltons and weaker reactions with 60-, 34-, 31-, 29-, 18-, and 15-kilodalton outer membrane antigens. No reactivity was detected with preimmune serum. Antibodies eluted from H. somnus after adsorption of convalescent serum reacted almost identically to unadsorbed convalescent serum in Western blots against bacterial outer membrane-enriched fractions. Thus, most of the antigens recognized by convalescent serum are likely to be on the bacterial surface and accessible to antibody. Surface antigens recognized by protective convalescent serum are candidate antigens for a subunit vaccine against H. somnus pneumonia.
Effect of crevicular fluid and lysosomal enzymes on the adherence of streptococci and bacteroides to hydroxyapatite
Authors: Cimasoni G , Song M , McBride B C
Published: January 2020

Samples of hydroxyapatite (HA) beads smaller than 1 mg were coated with 10 microliter of either saliva, serum, or human crevicular fluid before being added to a suspension of Streptococcus sanguis or Bacteroides gingivalis. In some assays, preparations of a granular fraction, elastase, or cathepsin G from human leukocytes were used to coat HA or to treat saliva-coated HA (SHA) before mixing with bacteria. The number of cells adhering to the beads was then counted under a scanning electron microscope by a standardized procedure. More cells were found to adhere to SHA in this assay than in the conventional large-scale assay. Human crevicular fluid, even when diluted up to three times, completely inhibited the adherence of S. sanguis to HA. A 100% inhibition of S. sanguis adherence was also observed when HA was coated with the granular fraction of leukocytes, and a 65% inhibition observed when SHA was treated with the enzyme preparation. When used to coat HA, elastase and cathepsin G reduced the adherence of S. sanguis by 30 and 50%, respectively. The binding of S. sanguis to elastase- or cathepsin G-treated SHA was also reduced. B. gingivalis 33277 was found to adhere in high numbers to SHA. Coating HA with crevicular fluid or with the lysosomal enzyme preparation had a limited negative effect. We postulate that crevicular fluid prevents the adherence of S. sanguis by virtue of either its enzyme content or its albumin content or both.
Ingestion and intracellular growth of Mycobacterium paratuberculosis within bovine blood monocytes and monocyte-derived macrophages
Authors: Zurbrick B G , Czuprynski C J
Published: January 2020

Cellular immunity is thought to be of major importance in resistance to infection with Mycobacterium paratuberculosis, the causative agent of Johne's disease of ruminants. The results of this study clarify the influence of bovine mononuclear phagocyte maturation on the ingestion and intracellular survival of M. paratuberculosis in vitro. Optimal phagocytosis of M. paratuberculosis by cultured bovine blood monocytes and monocyte-derived macrophages required the presence of 5 to 20% serum; few bacilli were phagocytized in the absence of serum. Monocyte-derived macrophages consistently demonstrated greater phagocytosis of M. paratuberculosis than did freshly adherent monocytes. Ingested M. paratuberculosis multiplied approximately 200 to 250% over a 7-day incubation period within bovine monocytes and monocyte-derived macrophages, as determined by microscopic counts of acid-fast-stained monocyte monolayers and by plate counts of viable organisms. These findings suggest that resident macrophages and recently emigrated blood monocytes within the intestinal mucosa may have considerable ability to ingest M. paratuberculosis, but they are unlikely to kill or markedly restrict the intracellular growth of the ingested bacilli. The ability of these mononuclear phagocytes to provide an intracellular niche for the growth of M. paratuberculosis and the immune response that eventually develops are likely to be important components in the development of the granulomatous lesions that are characteristic of Johne's disease.
Polymorphonuclear neutrophil chemotaxis modulated by Bacteroides fragilis peptidoglycan
Authors: Sperry J F , Burns J M
Published: January 2020

Peptidoglycan was isolated from Bacteroides fragilis with boiling sodium dodecyl sulfate, and some was treated with pronase to eliminate contaminating protein. This peptidoglycan was chemotactic for rabbit polymorphonuclear neutrophils and had even greater chemotactic activity along with some chemokinetic activity after it was partially hydrolyzed with lysozyme. Significant chemotaxis-inhibitory activity was observed for an acid-precipitable component of the lysozyme-treated crude peptidoglycan of B. fragilis.
Correlation of DNA, RNA, and protein content by flow cytometry in normal and Coxiella burnetii-infected L929 cells
Authors: Baca O G , Crissman H A
Published: January 2020

L929 cell populations persistently infected for more than 600 days exhibited cell cycle distributions and generation times similar to those of uninfected cells. The RNA and protein contents of these long-term-infected cells, as determined by flow cytometry and correlated with the cell cycle, were likewise approximately the same as those in normal uninfected cells. Also, the ratios of RNA to DNA and RNA to protein of infected cells were not significantly different from those of normal cells. Collectively, these data indicate that the long-term-infected cells remained in a state of balanced growth and maintained normal cycle progression and division capacity, allowing for growth and proliferation of host cells and thus ensuring the propagation and persistence of the parasite.
Influence of type and opsonization of ingested particle on intracellular free calcium distribution and superoxide production by human neutrophils
Authors: Murata T , Sullivan J A , Sawyer D W , Mandell G L
Published: January 2020

Intracellular free calcium concentration [( Ca2+]i) was measured with the fluorescent Ca2+ indicator Fura 2 within individual human neutrophils during the phagocytosis of several types of particles, including serum-treated zymosan (STZ), immunoglobulin G (IgG)-coated zymosan (IGZ), C3b-coated zymosan (C3Z), nontreated zymosan (Z), and serum-treated similarly sized latex particles (STL). STZ was coated with both IgG and C3b. IGZ was coated with only IgG, and C3Z was coated with only C3b. STL was coated with only C3b but to a lesser extent than C3Z. The ingestion of particles was greatest for STZ and somewhat lower for C3Z. Ingestion of IGZ and STL was much less than ingestion of C3Z. The relative efficiencies of the particles for inducing superoxide production were as follows: STZ greater than IGZ = C3Z greater than Z = STL. [Ca2+]i significantly increased from the resting level of approximately 70 to greater than 240 nM (P less than 0.01) during phagocytosis of the particles. The increment in [Ca2+]i was greater in the paraphagosomal region than in the cell body after the ingestion of STZ or IGZ. The mean peak [Ca2+]i values in the paraphagosomal cytoplasm of neutrophils ingesting one particle of STZ, IGZ, C3Z, Z and STL were 536.1 +/- 57.6, 424.7 +/- 55.8, 373.8 +/- 62.7, 272.3 +/- 31.5, and 270.8 +/- 38.0 nM, respectively, which showed good correlation (r = 0.97) with the efficiency of the particles for inducing superoxide production. Depletion of extracellular Ca2+ by EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid] attenuated both [Ca2+]i increase and superoxide production induced by particles. Thus [Ca2+]i increased after ingestion of several types of particles, and the subcellular pattern of [Ca2+]i was different depending on the type of particle ingested. Greater increases in paraphagosomal [Ca2+]i were closely associated with greater increases in superoxide production by neutrophils.
The principal protein antigens of isolates of Mycoplasma pneumoniae as measured by levels of immunoglobulin G in human serum are stable in strains collected over a 10-year period
Authors: Vu A C , Foy H M , Cartwright F D , Kenny G E
Published: January 2020

To determine whether antigenic variation in protein antigens of Mycoplasma pneumoniae occurred over time, 12 isolates obtained from pneumonia patients over a 10-year period (1964 to 1974) were compared by immunoblotting (Western blotting) against acute and convalescent human serum samples obtained from the same patients. The strains selected were isolated from patients who had low anti-lipid complement-fixing antibody titers in their acute-phase serum samples and high titers in their convalescent-phase serum samples. The polypeptide composition of the strains was closely similar by protein staining even when compared with prototype FH-Liu. On immunoblotting, all strains showed five bands (170, 130, 90, 45, and 35 kilodaltons [kDa]) which were stained more intensely by convalescent-phase than by acute-phase specimens. A sixth band (62 kDa) was detected by the conjugate alone. In FH-Liu, one band (110 kDa) was prominently stained by convalescent-phase specimens; this band was much less apparent in all of the clinical isolates. Two isolates possessed an additional band (92 kDa) which was stained more prominently by some but not all convalescent-phase specimens. Because of its known antigenic relationships and culture similarities, Mycoplasma genitalium was used for comparison. More polypeptides of M. genitalium than of M. pneumoniae were recognized by acute-phase serum samples, and 4 of 12 convalescent-phase serum samples showed increases in antibodies to certain M. genitalium polypeptides. However, these reactive polypeptides did not correspond in molecular mass to polypeptides recognized in M. pneumoniae; thus the signature profile of human convalescent-phase specimens with M. pneumoniae was distinct. These five polypeptides, individually or in combination, are especially promising for use in detection of human serum antibodies by enzyme-linked immunosorbent assay because they were found in all M. pneumoniae isolates tested.
Type II heat-labile enterotoxin of Escherichia coli activates adenylate cyclase in human fibroblasts by ADP ribosylation
Authors: Chang P P , Moss J , Twiddy E M , Holmes R K
Published: January 2020

Type II heat-labile enterotoxin (LT-II) from Escherichia coli causes characteristic morphological changes and accumulation of cyclic AMP in Y-1 adrenal cells, but it is not neutralized by antisera against choleragen (CT) or the classical type I heat-labile enterotoxin (LT-1) from E. coli. The action of purified LT-II on CT- and LT-I-responsive human fibroblasts was investigated and compared with that of CT. Fibroblasts incubated with LT-II or CT had an increased cyclic AMP content as well as a fourfold elevation of membrane adenylate cyclase activity. In membranes, activation of cyclase by toxin was enhanced by NAD, GTP, and dithiothreitol. The effect of LT-II on intact fibroblasts or membranes was increased by trypsin treatment of toxin. Since activation of adenylate cyclase by LT-II was stimulated by NAD, the ability of LT-II to catalyze the [32P]ADP-ribosylation of membrane proteins in the presence of [32P]NAD from control and LT-II- and CT-treated fibroblasts was investigated. Similar proteins were [32P]ADP-ribosylated in membranes exposed to LT-II or CT; LT-II- and CT-specific labeling was significantly decreased in membranes prepared from cells preincubated with either LT-II or CT. These studies are consistent with the hypothesis that LT-II, similar to CT and LT-I, increases cyclic AMP by activating adenylate cyclase through the GTP-dependent ADP-ribosylation of specific membrane proteins.
Characterization of murine monoclonal antibodies that recognize neutralizing epitopes on human C5a
Authors: Larrick J W , Wang J , Fendly B M , Chenoweth D E , Kunkel S L , Deinhart T
Published: January 2020

We generated a panel of 10 murine monoclonal antibodies (MAbs) that recognize human complement fragment C5a. These MAbs were characterized for their ability to immunoprecipitate 125I-labeled C5a, bind C5a in solid-phase enzyme immunoassay, and block 125I-labeled C5a binding to polymorphonuclear leukocytes. Four of these MAbs had affinity constants for C5a in the 1 X 10(9) to 3 X 10(9) M-1 range. These MAbs blocked C5a-induced neutrophil polarization and chemiluminescence. They blocked the ability of passively administered C5a to cause neutropenia in rabbits. These anti-C5a neutralizing MAbs may have potential therapeutic use in states of complement activation.
Resistance to Paracoccidioides brasiliensis in mice is controlled by a single dominant autosomal gene
Authors: Calich V L , Burger E , Kashino S S , Fazioli R A , Singer-Vermes L M
Published: January 2020

In a previous report it was shown that there are resistant, susceptible, and intermediate strains of mice to intraperitoneal Paracoccidioides brasiliensis infection. In the present work, we investigated the type of inheritance and the number of genes that determine resistance to paracoccidioidomycosis. Parental and hybrid mice were inoculated intraperitoneally with 5 X 10(6) P. brasiliensis yeast cells, and mortality was scored daily. Analysis of susceptible and resistant parental strains and of F1, F2, and backcross mice showed that the resistance to P. brasiliensis seems to be controlled genetically by a single dominant gene, which we designated the Pbr locus. The mean survival times of susceptible F2 and backcross hybrids were very similar to that of the susceptible parent. Examination of the pathological changes observed in parental and F1 mice, 6 months after infection, showed that F1 offspring presented a similar number and distribution of lesions to those of the resistant strains. The Pbr gene is not linked to H-2, Hc, and albino genes. Furthermore, resistance to paracoccidioidomycosis is controlled by an autosomal gene.
Haemophilus influenzae type b lipooligosaccharide: stability of expression and association with virulence
Authors: Kimura A , Patrick C C , Miller E E , Cope L D , McCracken G H , Hansen E J
Published: January 2020

Spontaneous antigenic and phenotypic variations in the lipooligosaccharide (LOS) of two strains of Haemophilus influenzae type b (Hib) were previously shown to be associated with changes in virulence (A. Kimura and E.J. Hansen, Infect. Immun. 51:69-79, 1986). The goal of the present study was to define further the stability of LOS expression by this pathogen and the role of Hib LOS in virulence. Variation in LOS antigenic reactivity, as detected with LOS-specific monoclonal antibodies, was observed in 3 of 30 Hib strains after single-colony passage. When large numbers of individual colonies from seven other Hib strains were screened, however, spontaneous LOS antigenic variation was detected in all of the strains. Antigenic variation was not consistently associated with an altered LOS phenotype, as determined by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and silver staining of LOS preparations. Changes in the LOS antigenic phenotype were correlated with altered virulence potential in two strains. In these strains, acquisition of reactivity with certain LOS-directed monoclonal antibodies was associated with the synthesis of a higher-molecular-weight LOS, enhanced virulence, and increased resistance to serum killing involving the classical complement pathway.
Plasmid-encoded expression of lipopolysaccharide O-antigenic polysaccharide in enteropathogenic Escherichia coli
Authors: Riley L W , Junio L N , Libaek L B , Schoolnik G K
Published: January 2020

The role of a plasmid in the virulence activity of an enteropathogenic Escherichia coli (EPEC) strain belonging to serotype 0111:NM was examined. EPEC strain B171, which is resistant to chloramphenicol, streptomycin, sulfathiazole, and tetracycline, harbors a 54-megadalton plasmid, pYR111, and exhibits localized adherence (LA) with HeLa cells. Curing the plasmid yielded strain B171-4, which had lost the ability to exhibit LA, resistance to the antibiotics, and the lipopolysaccharide (LPS) O-antigenic polysaccharide. To confirm that these phenotypic characteristics were specified by pYR111, the plasmid was transferred by conjugation into a nalidixic acid-resistant strain of E. coli HB101. LA and antimicrobial resistance were expressed in most of the transconjugants examined. The O-polysaccharide side chains, antigenically reactive with O111-specific antiserum, were also expressed by the transconjugants. Although EPEC plasmids coding for both drug resistance and LA have been described, an EPEC plasmid encoding the expression of an LPS O antigen has not been previously reported. Similar findings described for some Shigella and Salmonella strains suggest that plasmid-encoded modification of the LPS in some enteric bacterial species may be more common than previously recognized and may contribute to the characteristic virulence activity of the organism.
In vivo adherence and colonization of Vibrio cholerae strains that differ in hemagglutinating activity and motility
Authors: Teppema J S , Guinée P A , Ibrahim A A , Pâques M , Ruitenberg E J
Published: January 2020

A scanning electron microscopic study was carried out to compare the in vivo pathogenicity of two strains of Vibrio cholerae in an adult rabbit ligated-gut test model. V. cholerae C5 (serotype Ogawa, biotype El Tor), a motile strain possessing hemagglutinating activity in vitro, and C21 (serotype Ogawa, classical biotype), a nonmotile strain possessing no hemagglutinating activity, were tested. Tissue samples from small intestinal loops were examined 3, 6, 9, and 12 h postinoculation. Contradictory to most published data, neither hemagglutinating activity nor motility appeared to be essential prerequisites for the pathogenesis of cholera in the experimental animal model used: nonmotile hemagglutinin-negative strain C21 adhered to and colonized the small intestine at least to the same extent as did motile hemagglutinin-positive strain C5. Maximum colonization was seen at 9 h postinoculation for both strains. C5 and C21 vibrios caused comparable damage to the villi of the small intestine. The villous epithelium showed only mild changes during the first 9 h postinoculation. However, after 12 h the epithelium was seriously damaged concomitant with a decrease in the number of vibrios. Many villi showed partial or total denudation, owing to repelled epithelium, leaving a bare basal lamina with only some to moderate numbers of vibrios attached. Since similar changes were induced by pure cholera enterotoxin, these changes were likely the result of excessive fluid accumulation. From this study it is concluded that, at least in the animal model used, factors other than hemagglutinating activity and motility may also play a role in the association of V. cholerae with the small intestinal surface.
Growing tumors induce hypersensitivity to endotoxin and tumor necrosis factor
Authors: Bartholeyns J , Freudenberg M , Galanos C
Published: January 2020

Lewis lung carcinoma and EMT6 sarcoma growing as solid tumors in mice caused a gradual increase in the susceptibility of the animals to lethal toxicity of endotoxins (lipopolysaccharides [LPS]). By day 15 following inoculation of the tumors, the 50% lethal dose of LPS, which in normal mice was approximately 400 micrograms, decreased to 2 micrograms for the sarcoma-bearing mice and 0.1 microgram for the carcinoma-bearing mice. This sensitization to endotoxin was paralleled by a high sensitization to tumor necrosis factor (TNF). Human recombinant TNF given to normal mice was lethal at about 500 micrograms. It was lethal for 50% of the animals bearing EMT6 or Lewis lung carcinoma tumors in amounts of 4 and 0.01 micrograms, respectively, on day 15 following tumor inoculation. The sensitization of tumor-bearing animals to LPS and TNF was paralleled by marked granulocytosis.
Association of an abnormality of neutrophil chemotaxis in human periodontal disease with a cell surface protein
Authors: Van Dyke T E , Wilson-Burrows C , Offenbacher S , Henson P
Published: January 2020

Localized juvenile periodontitis (LJP) is characterized by severe, early-onset, molar and incisor bone loss; neutrophil chemotaxis disorders; and a high prevalence of Actinobacillus actinomycetemcomitans infection. LJP is further characterized by significant familial aggregation of the disease. Recent work in our laboratory has demonstrated the selective depletion of a surface glycoprotein of 110,000 Mr (GP110) from LJP neutrophils by using surface labeling with [14C]formaldehyde and autofluorography. The function of GP110 is unknown; however, it does not appear to be a chemotactic factor receptor. Rather, it is bound by a monoclonal antibody (NCD-1) that recognizes a neutrophil differentiation antigen and which itself alters neutrophil chemotactic and secreting functions. To quantify GP110 on LJP and normal neutrophils, fluorescein-labeled NCD-1 was bound to neutrophils and the amount of fluorescence was evaluated by using cytofluorography. Our results indicate that there is a quantifiable reduction (40%) of GP110 on the surface of LJP and GJP neutrophils, compared with controls. Other patients with neutrophil defects express normal quantities of GP110, suggesting disease specificity. Our data suggest that GP110 may be a useful disease marker for LJP and may provide a useful probe for the study of neutrophil chemotactic function and dysfunction.
Immune enhancement of pulmonary clearance of nontypable Haemophilus influenzae
Authors: Hansen E J , Hart D A , McGehee J L , Toews G B
Published: January 2020

BALB/c mice systemically immunized by intraperitoneal injection with whole, viable cells of two different strains of nontypable Haemophilus influenzae (NTHI) exhibited a markedly enhanced ability to clear the homologous strain of NTHI from the lower respiratory tract. Immunization did not influence the number of phagocytic cells recovered by bronchoalveolar lavage from mice before or after intrapulmonary challenge with NTHI. Immunization also induced the synthesis of relatively large quantities of NTHI-directed antibodies which were detectable in both the bloodstream and the alveolar spaces of the lung. Radioimmunoprecipitation and Western blot (immunoblot) analyses indicated that these antibodies were directed against both the proteins and lipooligosaccharide (LOS) in the NTHI outer membrane. Bactericidal and opsonophagocytic assays determined that the NTHI-directed antibodies in the serum were functional and able to kill or opsonize the homologous NTHI strain. Mice immunized with an NTHI major outer membrane protein-LOS complex also had an increased ability to effect pulmonary clearance of NTHI. Serum and bronchoalveolar lavage fluid collected from these animals immunized with the outer membrane protein-LOS complex contained relatively high levels of antibodies to both of these antigens. The serum from these animals also possessed bactericidal and opsonic activity against the homologous NTHI strain. These results indicate that systemic immunization can enhance the ability of experimental animals to clear NTHI from the lower respiratory tract and suggest that immunoprophylaxis of NTHI pulmonary disease may be feasible.
Lipopolysaccharide phase variation determines the complement-mediated serum susceptibility of Coxiella burnetii
Authors: Vishwanath S , Hackstadt T
Published: January 2020

Phase variation of Coxiella burnetii is due to variation of the lipopolysaccharide (LPS), a phenomenon analogous to smooth-to-rough LPS variation of gram-negative enteric bacteria. Virulent enterobacteria usually have a smooth LPS and resist serum killing, whereas avirulent rough LPS mutants are sensitive to complement-mediated serum killing. Like gram-negative enterobacteria, smooth LPS phase variants of C. burnetii are virulent, whereas the rough LPS variants are avirulent. We therefore studied the effects of human serum on the LPS variants of the Nine Mile strain of C. burnetii. Analogous to gram-negative enterobacteria, the smooth and intermediate LPS C. burnetii phase variants were resistant to complement-mediated serum killing, whereas the rough LPS variants were killed by serum complement. Although the smooth and intermediate LPS variants were serum resistant, they differed in their interactions with the complement system. The smooth LPS variant activated complement poorly and did not bind C3b; however, the intermediate LPS variant activated complement and bound C3b. The rough LPS variant activated complement via the alternative pathway, whereas the intermediate LPS variant activated the classical pathway. These results provide an explanation for the avirulent nature of the rough LPS variant of C. burnetii and suggest that differences in C. burnetii LPS structure influence the interactions of the LPS phase variants with the complement system.
Killing of Proteus mirabilis by polymorphonuclear leukocyte granule proteins: evidence for species specificity by antimicrobial proteins
Authors: Shafer W M , Engle S A , Martin L E , Spitznagel J K
Published: January 2020

Low-molecular-weight (Mr, ca. 3,800) polypeptides containing human defensins HNP-1 and HNP-2 (T. Ganz, M. S. Selsted, D. Szlarek, S. L. Harwig, K. Daher, D. F. Bainton, and R. I. Lehrer, J. Clin. Invest. 76:1427-1434, 1985) prepared in our laboratory from acid extracts of human polymorphonuclear granulocyte granules and purified human defensins were found to exert potent bactericidal action against Proteus mirabilis. The antimicrobial action of the extracts of human polymorphonuclear leukocytes granules against P. mirabilis appears to be due to the presence of the defensins. Because P. mirabilis resists the antimicrobial action of other granule proteins, we interpret the present results to mean that the various antimicrobial proteins display species specificity in their microbicidal action.
Shigella flexneri invasion plasmid antigens B and C: epitope location and characterization with monoclonal antibodies
Authors: Mills J A , Buysse J M , Oaks E V
Published: January 2020

Invasion plasmid antigens B (IpaB) and C (IpaC) are associated with the ability of shigellae to invade cultured mammalian cells. Monoclonal antibodies against IpaB and IpaC polypeptides were produced and used in a whole-cell enzyme-linked immunosorbent assay to show that both IpaB and IpaC polypeptides were exposed on the surface of virulent shigellae. Moreover, these surface epitopes were shown to be highly conserved among different serotypes of Shigella spp. and enteroinvasive Escherichia coli. Cross-reactive epitopes were not found on noninvasive Shigella strains or on other enteric bacteria including Salmonella, Yersinia, Campylobacter, Vibrio, and Aeromonas spp. and various pathogenic strains of E. coli. The monoclonal antibodies were used in competitive binding assays to define three unique epitopes of the IpaB polypeptide and four unique epitopes of the IpaC polypeptide. Epitope locations and their corresponding DNA-encoding regions were defined by examining the IpaB and IpaC products expressed by lambda gt11 recombinants and by constructing a genetic map of the insert DNAs of these recombinants. Three IpaB epitopes (2F1, 1H4, 4C8) were found to be encoded on three contiguous DNA regions comprising a 700-base-pair (bp) segment that corresponded to the amino-terminal end of the IpaB polypeptide. Similarly, a 640-bp DNA segment that corresponded to the amino-terminal end of the IpaC polypeptide was found to encode three clustered IpaC epitopes (5H1, 9B6, 5B1). Approximately 50 bp downstream from this region a fourth IpaC epitope-encoding region (2G2) was found. The effect of the monoclonal antibodies on plaque formation by virulent Shigella flexneri on a monolayer of cultured mammalian cells (a sensitive measure of invasiveness) was determined. Only the IpaB-specific monoclonal antibody 2F1 was able to reduce the plaque-forming capacity by greater than 50%, suggesting that this epitope of the IpaB polypeptide is involved in the invasion process.
Iron regulation of Serratia marcescens hemolysin gene expression
Authors: Poole K , Braun V
Published: January 2020

The hemolytic activity of Serratia marcescens was examined as a function of iron availability. Restriction of iron by the nonmetabolizable chelator 2,2'-dipyridyl or the iron-binding protein transferrin produced a marked increase in hemolytic activity. The hemolytic activity of S. marcescens is determined by two adjacent genes, 5'-shlB-shlA-3', where shlA encodes the hemolysin which requires the ShlB protein for activity. A gene fusion between the promoter-proximal portion of shlA and phoA, the Escherichia coli alkaline phosphatase gene, was subcloned into a medium-copy-number vector, and the recombinant plasmid was introduced into S. marcescens. The expression of shlA was measured as a function of alkaline phosphatase activity, which increased threefold under iron-restricted conditions. Removal of the 5' noncoding region upstream of shlB in the fusion vector resulted in a 10-fold decrease in alkaline phosphatase activity under iron-sufficient conditions, with no effect of iron limitation on this residual activity. This suggested that the site mediating iron regulation of shlA expression occurs upstream of shlB. Consistent with this, we observed iron-regulated synthesis of the ShlB protein in Western immunoblots of isolated outer membranes. The hemolysin determinant was subsequently expressed on a medium-copy-number vector in fur+/fur isogenic strains of E. coli K-12, where a 10-fold-higher activity was observed in the mutant strain compared with the wild type. A sequence exhibiting some homology to the Fur-binding consensus sequence was identified upstream of the shlB coding region, overlapping the -35 region of a putative promoter.
Role of Shiga toxin in the pathogenesis of bacillary dysentery, studied by using a Tox- mutant of Shigella dysenteriae 1
Authors: Fontaine A , Arondel J , Sansonetti P J
Published: January 2020

A Tox- mutant of Shigella dysenteriae 1, SC501, was genetically engineered by cloning the Shiga toxin operon, inserting a cassette into the A subunit gene, and exchanging this in vitro-mutagenized sequence with the wild-type gene. SC501 produced a low amount of residual cytotoxicity which was not neutralized by a rabbit immune serum directed against Shiga toxin. Invasion of cultured cells demonstrated that Shiga toxin had no effect on the rate of intracellular growth of bacteria or on the rapid killing of invaded host cells. On the other hand, several significant differences were observed in macaque monkeys infected intragastrically with either the wild-type strain or its mutant. The production of Shiga toxin by the invading strain was correlated with the presence of blood within stools, a sharp drop in blood polymorphonuclear cells, and histopathological alterations, such as the destruction of capillary vessels within the connective tissue of the colonic mucosa, severe inflammatory vasculitis of the peritoneal mesothelium, and major efflux of inflammatory cells to the intestinal lumen. It is proposed that Shiga toxin influences the severity of bacillary dysentery by inducing colonic vascular damage, which accounts for bloody stools, intestinal ischemia, and inflation of a polymorphonuclear intestinal compartment during the infectious process.
Virulence conversion of Legionella pneumophila: a one-way phenomenon
Authors: Catrenich C E , Johnson W
Published: January 2020

Previous investigations have shown that Legionella pneumophila converts from virulence to avirulence after passage on supplemented Mueller-Hinton (SMH) agar and may convert back to virulence after passage in guinea pigs. However, there is no additional information concerning the apparent interconversion of virulent and avirulent derivatives of L. pneumophila cultures. We investigated the stability of a parental virulent culture and its avirulent derivatives and the growth and viability of these cultures on charcoal-yeast extract (CYE) and SMH agars. Avirulent derivatives of a highly virulent L. pneumophila culture were obtained by passage of the virulent parent culture on SMH agar. The only time a virulent L. pneumophila culture was recoverable from an avirulent culture was when the avirulent culture was derived from a saline suspension of a virulent culture which had been passaged only five times on SMH agar. When an avirulent culture was derived from a virulent culture passaged 25 times on SMH agar or from an isolated colony which grew on a SMH agar plate, we were unable to recover a virulent culture after successive passage through guinea pigs. These results suggest that the conversion process which occurs between virulent and avirulent forms of L. pneumophila is a one-way phenomenon from virulence to avirulence and that stable avirulent derivatives can be isolated. Furthermore, our findings suggest that SMH agar acts as a selective medium for the growth of avirulent L. pneumophila, and growth on SMH agar may be a phenotypic marker for avirulence. Virulent cells, although unable to grow on SMH agar, may remain viable for several passages on SMH agar and propagate when inoculated into guinea pigs.
In vitro and in vivo studies of macrophage functions in amebiasis
Authors: Denis M , Chadee K
Published: January 2020

Experimental intrahepatic inoculation of the gerbil with Entamoeba histolytica trophozoites was used as a model of liver amebiasis to study the cellular immune response elicited by the parasite. It was shown that abscess-derived macrophages (5 to 20 days old) were deficient in their capacity to develop a respiratory burst, to secrete and express membrane-bound interleukin-1-like activity, and to kill E. histolytica trophozoites as well as to respond to lymphokines in vitro. However, macrophages isolated from the spleen and peritoneal cavities from the same infected animals were not significantly down regulated in these functions. Splenocytes from infected gerbils were shown to develop a strong responsiveness to amebic antigen, whereas their response to concanavalin A was suppressed. Crude E. histolytica extracts or conditioned medium down regulated murine BALB/c macrophage accessory and effector cell functions in vitro in a manner similar to abscess-derived macrophages, whereas crude extracts of the nonvirulent E. histolytica-like Laredo strain did not. Our results indicate that intrinsic or secreted products or both from E. histolytica are actively regulating macrophage functions at the abscess site and can possibly mediate other immunoregulatory mechanisms at distant targets.