bluuebunny/crossref_metadata_2025 huggingface dataset:
Liposomes, lipid A, and aluminum hydroxide enhance the immune response to a synthetic malaria sporozoite antigen
Authors: Richards R L , Hayre M D , Hockmeyer W T , Alving C R
Published: January 2020

A liposome-encapsulated cloned protein (R32tet32) containing sequences from the tetrapeptide repeat region of the circumsporozoite protein of Plasmodium falciparum sporozoites was examined for immunogenicity with rabbits and monkeys. Effects of adjuvants were tested by encapsulation of the antigen in liposomes either lacking or containing lipid A and adsorption with aluminum hydroxide (ALUM). When rabbits were immunized with R32tet32 alone, a primary antibody response was not seen and a secondary response did not appear until 32 to 36 weeks after boosting. Immunization with ALUM-adsorbed R32tet32 resulted in a minimal primary antibody response. A moderate secondary antibody response was detected within 2 weeks after boosting, but antibody levels decreased to preimmunization levels 8 weeks after boosting. When R32tet32 was encapsulated in liposomes containing lipid A, strong primary and secondary antibody responses were observed. Strong primary and secondary responses also were obtained when R32tet32 was encapsulated in liposomes either containing or lacking lipid A and the liposomes were adsorbed with ALUM. The strongest antibody response was obtained by immunization with ALUM-adsorbed liposomes containing lipid A and R32tet32, suggesting that the adjuvant effects of liposomes, lipid A, and ALUM were additive or synergistic.
Immunoelectron microscopic localization of outer membrane proteins II on the surface of Neisseria gonorrhoeae
Authors: Robinson E N , Clemens C M , McGee Z A , Cannon J G
Published: January 2020

To determine the ultrastructural distribution and immunological accessibility of proteins II (P.IIs) on the surfaces of whole gonococci, anti-P.II gold probes were developed and used in electron microscopic studies of viable P.II-expressing variants of Neisseria gonorrhoeae FA1090. Anti-P.II probes clearly marked the surfaces of organisms and their associated outer membrane blebs. The surface-exposed portion of P.II is antigenically variable. With the use of two different sizes of gold probes, it was demonstrated that individual gonococcal cells can express more than one antigenic type of P.II simultaneously.
Iron uptake from lactoferrin and transferrin by Neisseria gonorrhoeae
Authors: McKenna W R , Mickelsen P A , Sparling P F , Dyer D W
Published: January 2020

The major iron (Fe) sources available to Neisseria gonorrhoeae in the human host are probably transferrin (TF) and lactoferrin (LF). Although a number of studies have examined Fe uptake by Neisseria meningitidis, no comparable studies have been done on Fe uptake by the gonococcus from TF and LF. We found that, like meningococci, gonococci removed Fe from TF and LF in an energy-dependent manner; uptake was repressed by Fe and did not proceed by a siderophore-mediated uptake system. Unlike published studies examining meningococcal Fe uptake from TF, our study showed that gonococcal Fe uptake from both TF and LF was highly efficient; uptake saturated at 1 microM protein, and growth with 5% saturated TF and LF occurred at maximal rates when the protein was present in appreciable concentrations. We conclude that the availability of protein-bound Fe probably does not limit growth of N. gonorrhoeae in the human body.
Inhibitory monoclonal antibody against a (myristylated) small-molecular-weight antigen from Plasmodium falciparum associated with the parasitophorous vacuole membrane
Authors: Kara U A , Stenzel D J , Ingram L T , Bushell G R , Lopez J A , Kidson C
Published: January 2020

A small-molecular-weight antigen that occurs in asexual blood stages in synchronized cultures of Plasmodium falciparum was detected by a monoclonal antibody which inhibits parasite growth in vitro. This antigen, QF116, showed a molecular weight of 15,000 in parasite strain FCR-3K+ from The Gambia and 19,000 in strain FCQ-27 from Papua New Guinea. The protein did not show significant glycosylation by galactose or glucosamine labeling but was found to be acylated by myristic acid. By using immunogold labeling and electron microscopy, the location of the antigen could be attributed to the parasitophorous vacuole membrane and to inclusions and vesicles residing within the cytoplasm of the erythrocyte host cell.
Cloning of a species-specific antigen of Mycobacterium bovis
Authors: Radford A J , Duffield B J , Plackett P
Published: January 2020

A DNA library from a virulent strain of Mycobacterium bovis was constructed in the expression vector lambda gt11, and the library was probed with antisera to M. bovis. Clones expressing M. bovis antigens were isolated and characterized by using M. bovis-specific monoclonal antibodies that recognize a 22,000-molecular-weight protein (MPB70). MPB70 is a major protein antigen of the vaccine strain of M. bovis BCG and of virulent M. bovis, the causative agent of bovine tuberculosis. Of 32 clones selected by using polyclonal affinity-purified anti-M. bovis sera, 5 were recognized by the anti-MPB-70 monoclonal antibodies, and one monoclonal antibody, SB10, recognized all 5 clones. Characterization of these clones showed that one clone containing a 253-base-pair insert expressed a polypeptide bound by all of the MPB70-specific monoclonal antibodies. Western blots (immunoblots) showed that this cloned protein was recognized by sera from M. bovis-infected cattle, although not all cattle with bovine tuberculosis produced antibodies reactive to this clone. DNA sequencing of the clone showed that it coded for 84 amino acids from positions 17 to 114 of the 161-amino-acid protein, with a 16-peptide deletion between positions 79 and 94. Apart from this deletion, there were seven other variations between the cloned sequence and that deduced from M. bovis BCG MPB70.
A pleiotropic iron-uptake mutant of Neisseria meningitidis lacks a 70-kilodalton iron-regulated protein
Authors: Dyer D W , West E P , McKenna W , Thompson S A , Sparling P F
Published: January 2020

We isolated an iron-uptake mutant of Neisseria meningitidis M986-NCV-1 that was severely limited in the ability to use several sources of iron in the form of Fe3+. This mutant, FAM11, grew poorly or not at all with human transferrin (TF) or lactoferrin (LF) as the sole iron source in a defined medium but grew as well as wild-type meningococci with hemin or hemoglobin. Uptake of 55Fe bound to TF, LF, dicitrate complexes, aerobactin, or nitrilotriacetate was reduced to 0 to 4% of the wild-type level. FAM11 did not produce an iron-repressible outer membrane protein (FeRP) of 70 kilodaltons (kDa) found in membranes of iron-stressed M986-NCV-1. Western blot (immunoblot) analysis using rabbit antiserum against this protein revealed that at least 17 of 18 meningococcal and 10 of 14 gonococcal strains produced an FeRP of ca. 70 kDa. The 70-kDa FeRP was shown to be surface exposed by radioimmunoprecipitation with human immune sera. These data suggest that the 70-kDa FeRP is somehow involved in Fe uptake from TF and LF. However, we were unable to transform the iron-uptake phenotype from FAM11 into wild-type meningococci to confirm this. Revertants of FAM11 that grew with TF and LF did not regain the ability to make the 70-kDa FeRP but also did not completely regain the Fe-uptake phenotype of M986-NCV-1.
Limited diversity of the immunoglobulin A1 protease gene (iga) among Haemophilus influenzae serotype b strains
Authors: Poulsen K , Hjorth J P , Kilian M
Published: January 2020

Immunoglobulin A1 (IgA1) proteases are thought to be important virulence factors in certain bacterial infections, including meningitis, and may have potential usage in vaccines. In this study, we compared the locations of EcoRI, BamHI, and PstI restriction endonuclease sites in the IgA1 protease gene (iga) region of whole-cell DNA from 76 Haemophilus influenzae strains. The analysis was performed by using isolated fragments of the cloned iga gene, which encodes the IgA1 protease originating from a H. influenzae serotype d strain, as probes in Southern blot experiments. All strains, including three without detectable IgA1 protease activity, had DNA sequences with a high degree of homology to the iga probes. The numbers and sizes of the DNA fragments hybridizing with the probes indicated that only three strains, none of which was of serotype b, had more than one iga gene. The iga restriction fragment length patterns of 60 clinical isolates of serotype b were of only four distinct types, which correlated with previously observed clusters of multilocus genotypes (electrophoretic types). This correlation supports the concept of the clonal population structure of H. influenzae. Three of the iga gene restriction types, which appear to represent 98% of the H. influenzae serotype b population, encode IgA1 proteases that were inhibited by antisera to any one of these types and therefore could form the basis for the development of a vaccine against H. influenzae meningitis.
Colonization of the streptomycin-treated mouse large intestine by a human fecal Escherichia coli strain: role of growth in mucus
Authors: Wadolkowski E A , Laux D C , Cohen P S
Published: January 2020

The relative colonizing abilities of Escherichia coli F-18, isolated from the feces of a healthy human, and E. coli F-18col-, a strain derived from it which does not make the E. coli F-18 colicin, were studied. In a previous report, it was shown that when each strain was fed individually to streptomycin-treated mice, at approximately 10(10) CFU per mouse, each colonized the large intestine at between 10(7) and 10(8) CFU/g of feces indefinitely. However, when simultaneously fed to mice, although E. coli F-18 colonized at about 10(8) CFU/g of feces, E. coli F-18col- dropped to a level of 10(3) CFU/g of feces within 3 to 5 days. In the present investigation, we show that when given enough time to establish a state of colonization, E. coli F-18col- persists in feces in high numbers despite subsequent challenge by E. coli F-18. Therefore, a major defect in the ability of E. coli F-18col- to colonize in the presence of E. coli F-18 appears to be in initiating that state. In addition, when mucus was scraped from the cecal wall and, without further treatment, was inoculated with E. coli F-18 or F-18col-, both strains grew well. However, when cecal mucus was inoculated with both strains simultaneously, E. coli F-18 grew far more rapidly than E. coli F-18col-. Moreover, neither strain grew in cecal luminal contents. Together, these data suggest the possibility that both E. coli F-18 and F-18col- must grow in mucus to colonize the streptomycin-treated mouse large intestine, that E. coli F-18col- is eliminated by E. coli F-18 because it does not grow in mucus as well as E. coli F-18, and that E. coli F-18col- can resist elimination by E. coli F-18 if it is allowed enough time to establish itself within the mucus layer.
Capsular antibodies induce type-specific phagocytosis of capsulated Staphylococcus aureus by human polymorphonuclear leukocytes
Authors: Karakawa W W , Sutton A , Schneerson R , Karpas A , Vann W F
Published: January 2020

Capsular types 5 and 8, which account for about 70% of Staphylococcus aureus strains isolated from the blood of patients, resisted in vitro phagocytosis by human polymorphonuclear leukocytes (PMN). Antisera and monoclonal antibody to type 5 and 8 capsular polysaccharides (CPS) induced type-specific in vitro phagocytosis of capsulated organisms by PMN. Antibodies directed against the O-acetyl moiety of the type 8 CPS were more effective in inducing phagocytosis of type 8 organisms by PMN. Either type-specific antiserum or monoclonal antibody reactive with the native O-acetylated type 8 CPS was most effective in inducing in vitro phagocytosis of type 8 organisms by PMN. These results provide further evidence that CPS of S. aureus are associated with host immunity to this organism.
Effect of environmental pH on enzyme activity and growth of Bacteroides gingivalis W50
Authors: McDermid A S , McKee A S , Marsh P D
Published: January 2020

Since the pH of the gingival crevice increases from below neutrality in health to above pH 8 in disease, we decided to investigate the effect of environmental pH on the growth and enzyme activity of Bacteroides gingivalis W50. Cells were grown in a chemostat under hemin-excess conditions over a range of pH values; stable growth was observed only between pH 6.7 and 8.3, with the maximum yields obtained between pH 7.0 and 8.0. The enzyme profile of cells varied markedly with pH. Enzymes with a specificity for gingival connective tissue (collagenase, hyaluronidase) were produced optimally at or below neutral pH, whereas trypsinlike activity increased with the growth pH and was maximal at pH 8.0. Chymotrypsinlike activity was generally low, although its activity was highest at the extremes of growth pH, i.e., at pH 6.7 and 8.3. Inhibitor studies provided evidence that the breakdown of collagen involved the concerted action of both a collagenase and the trypsinlike enzyme. The ratio of trypsin to collagenolytic activity rose from 1:1 during growth at neutral pH and below to almost 7:1 during growth at pH 8.3. Thus B. gingivalis appears to be uniquely adapted as a periodontopathic organism in that under environmental conditions likely to prevail during the initial stages of pocket development it produces maximally those enzymes with a tissue-damaging potential. Then, as the pH of the pocket rises during the host inflammatory response, the activity of the trypsinlike enzyme increases markedly, which may enable cells to inactivate key components of the host defenses such as immunoglobulins and complement.
Isolation and characterization of recombinant Escherichia coli clones secreting a 24-kilodalton antigen of Treponema pallidum
Authors: Hsu P L , Qin M , Norris S J , Sell S
Published: January 2020

Escherichia coli clones containing Treponema pallidum DNA in the pUC8 vector and secreting a 24-kilodalton antigen of T. pallidum have been isolated. Both syphilitic human and syphilis-immune rabbit sera reacted with the recombinant p24 antigen, indicating that an equivalent protein in T. pallidum is capable of eliciting antibody responses during natural infections. The p24 antigen of T. pallidum was identified by using two-dimensional gel electrophoresis and immunoblotting with monospecific anti-p24 serum. We tentatively concluded that this cloned antigen is a secreted protein or a labile or minor component of T. pallidum because (i) p24 was secreted by the recombinant E. coli cells; (ii) recombinant p24 in E. coli cells was processed into several smaller species with molecular masses ranging from 12 to 20 kilodaltons, which correlate well with the masses of secreted antigens described by others; and (iii) p24 protein appeared to be highly antigenic during natural infections, but only a very small amount of this antigen was associated with or retained by the purified organisms. The possible role of the p24 protein in determining the growth characteristics of T. pallidum is suggested by the ability of recombinant p24 to induce growth changes in E. coli cells. All E. coli colonies expressing the p24 polypeptide exhibited a flat and rough colony morphology and a filamentous growth pattern that were different from those of other E. coli cells. The DNA sequence coding for the p24 polypeptide is located on a 1.7-kilobase-pair BamHI fragment of the T. pallidum genomic DNA and is absent in the nonpathogenic Treponema phagedenis DNA. However, any possible relationship between the p24 antigen and the virulence of T. pallidum remains to be determined. In preliminary studies, rabbits immunized with the purified p24 were not protected from the infection with live T. pallidum organisms.
Clearance of Serratia marcescens from blood in mice: role of hydrophobic versus mannose-sensitive interactions
Authors: Rumelt S , Metzger Z , Kariv N , Rosenberg M
Published: January 2020

In the present study, we examined the potential roles of cell surface hydrophobicity and mannose-sensitive (MS) interactions in blood clearance of Serratia marcescens in mice. Hydrophobic strain RZ, partially hydrophobic mutant 3162, and nonhydrophobic mutant 3164 were coinoculated into BALB/c male mice, and blood samples were plated out at different time intervals; colonies of the three strains were distinguished by their different morphologies. All three strains were cleared from the blood stream at similar rates, despite their large relative differences in cell surface hydrophobicity. Clearance from blood was subsequently studied by coinoculating two clinical isolates which differ in their abilities to adhere via MS interactions. MS+ strain 1785 was cleared much more rapidly than MS- strain 3255; moreover, in the presence of D-mannose, clearance of strain 1785 was inhibited to a rate similar to that of MS- strain 3255. When D-glucose was substituted for D-mannose, inhibition was not observed. The results suggest that MS, rather than hydrophobic, interactions are primarily responsible for the rapid clearance of S. marcescens from blood observed.
Mycobacterial lipoarabinomannan inhibits gamma interferon-mediated activation of macrophages
Authors: Sibley L D , Hunter S W , Brennan P J , Krahenbuhl J L
Published: January 2020

The principal efferent role of the macrophage in acquired resistance to intracellular pathogens depends on activation by T-cell lymphokines, primarily gamma interferon (IFN-gamma). However, mouse macrophages that are heavily burdened with Mycobacterium leprae are refractory to activation by IFN-gamma and are thus severely compromised in their capacity for both enhanced microbicidal and tumoricidal activities. We report here that lipoarabinomannan (LAM), a highly immunogenic lipopolysaccharide that is a prominent component of the cell walls of M. leprae and M. tuberculosis, was a potent inhibitor of IFN-gamma-mediated activation of mouse macrophages in vitro. Inhibition of macrophage activation by LAM required preincubation for approximately 24 h, resulting in uptake of LAM into cytoplasmic vacuoles of macrophages. Intact LAM was necessary to inhibit IFN-gamma-mediated activation, as this property was lost when the acyl side chains were removed from LAM by mild alkaline hydrolysis. In addition, LAM was an abundant constituent of macrophages isolated from lepromatous granulomas of M. leprae-infected nude mice and likely contributed to the defective activation of granuloma macrophages by IFN-gamma.
Association of electrophoretic karyotype of Candida stellatoidea with virulence for mice
Authors: Kwon-Chung K J , Wickes B L , Merz W G
Published: January 2020

Seven isolates of Candida stellatoidea were studied for their electrophoretic karyotype, virulence for mice, sensitivity to UV radiation, growth rate in vitro, reaction on cycloheximide-indicator medium, and proteinase activity. The isolates exhibited one of two distinct electrophoretic karyotypes as determined by orthogonal field alternating gel electrophoresis (OFAGE). Four isolates, including the type culture of C. stellatoidea, belonged to electrophoretic karyotype type I by OFAGE, showing eight to nine bands of which at least two bands were less than 1,000 kilobases in size as estimated by comparison with the DNA bands of Saccharomyces cerevisiae. These isolates failed to produce fatal infection in mice within 20 days when 5 X 10(5) cells were injected intravenously. The yeasts were cleared from the kidneys of two of three mice tested by day 30. Type I showed proteinase activity on bovine serum albumin agar at pH 3.8 and produced a negative reaction on cycloheximide-bromcresol green medium within 48 h. The three grouped in type II by OFAGE showed banding patterns similar to those of a well-characterized isolate of Candida albicans. The isolates of type II had an electrophoretic karyotype of six to seven bands approximately 1,200 kilobases or greater in size. All three type II isolates were highly virulent for mice, producing fatality curves similar to those of a previously studied C. albicans isolate. From 80 to 90% of the mice injected with 5 X 10(5) cells intravenously died within 20 days. The type II isolates produced a positive reaction on cycloheximide-bromcresol green agar and showed no proteinase activity on bovine serum albumin agar at the low pH. In addition, the type II isolates grew faster and were significantly more resistant to UV irradiation than the type I isolates. These results indicated that type II, but not type I, isolates can be considered simply as sucrose-negative C. albicans.
Clonal population structure of encapsulated Haemophilus influenzae
Authors: Musser J M , Kroll J S , Moxon E R , Selander R K
Published: January 2020

Chromosomal genotypes of 2,209 isolates of the six polysaccharide capsule types of Haemophilus influenzae recovered from human hosts worldwide were characterized by an analysis of electrophoretically demonstrable allelic profiles at 17 metabolic enzyme loci. For 222 representative isolates, restriction fragment length polymorphism patterns produced by digestion of cap region DNA were also determined. With few exceptions, isolates belonging to individual phylogenetic lines or groups of allied lineages identified by multilocus enzyme electrophoresis had characteristic cap region restriction fragment length polymorphism patterns and characteristic combinations of cap region patterns and outer membrane protein types. The occurrence of strong associations of characters and the recovery of isolates with identical genetic properties in widely separated geographic regions and over a 40-year period indicated that the population structure of encapsulated H. influenzae is clonal. Recombination of chromosomal genes, including those mediating capsule synthesis, apparently is not a major factor in the short-term evolution of these pathogenic organisms and, therefore, may be of minor clinical significance.
ADP-ribosyltransferase mutations in the catalytic S-1 subunit of pertussis toxin
Authors: Barbieri J T , Cortina G
Published: January 2020

The ADP-ribosyltransferase activity of pertussis toxin resides within the S-1 subunit of the toxin. Deletion mapping of a recombinant S-1 subunit produced in Escherichia coli showed that amino acids 2 through 180 are required for ADP-ribosylation of Gi protein. Mutants of the S-1 subunit which lacked either amino acids 2 through 22 or amino acids 153 through 180 failed to express enzyme activity, implicating a functional or structural role for these residues in catalysis. The catalytic carboxy-terminal S-1 deletion, C-180, was found to be more soluble than the recombinant S-1 subunit, making it a useful construct for future structure-function studies on enzyme catalysis. Four independent single-amino-acid substitutions which decreased ADP-ribosyltransferase activity were constructed in the recombinant S-1 subunit. Substitution of Asp-11 by Ser, Arg-13 by Leu, or Trp-26 by Ile decreased enzyme activity to below detectable levels (less than 1.0% of that of the recombinant S-1 subunit). The Glu-139-to-Ser substitution reduced ADP-ribosyltransferase activity to 15% of that of the recombinant S-1 subunit. Both the oxidized and reduced forms of the recombinant S-1 subunit and recombinant S-1 subunits containing single-amino-acid substitutions were degraded through identical immunoreactive tryptic peptides, suggesting that the conformations of the mutants are similar to that of the recombinant S-1 subunit. Identification of noncatalytic forms of the S-1 subunit of pertussis toxin which have conserved protein structure is an initial step in the generation of a recombinant noncatalytic form of pertussis toxin which may be tested as a candidate for an acellular vaccine against Bordetella pertussis.
Effect of lipopolysaccharide on proteoglycan synthesis by adult human gingival fibroblasts in vitro
Authors: Bartold P M , Millar S J
Published: January 2020

The effect of lipopolysaccharide preparations from Salmonella enteritidis, Bacteroides gingivalis, and Actinobacillus actinomycetemcomitans on human gingival fibroblasts was studied. Lipopolysaccharide from all sources inhibited fibroblast proliferation in the concentration range of 0.5 to 50 micrograms/ml, with the lipopolysaccharide from A. actinomycetemcomitans having the strongest inhibitory effect. Assessment of the effect of lipopolysaccharide on gingival fibroblast metabolism indicated both total protein and proteoglycan synthesis to be inhibited with increasing concentrations of lipopolysaccharide. As for the antiproliferative effect, lipopolysaccharide from A. actinomycetemcomitans had the greatest inhibitory effect on cell synthetic activity. This inhibitory effect was determined by pulse-chase experiments to be a true depression in synthesis. Furthermore, the effect was independent of lipopolysaccharide-induced changes in cell proliferation and prostaglandin synthesis. This study confirmed the toxic effect of lipopolysaccharide on fibroblasts and, in particular, indicated that various lipopolysaccharide preparations vary in their potency to influence cell proliferation and extracellular matrix synthesis.
Growth of Mycobacterium avium in activated macrophages harvested from inbred mice with differing innate susceptibilities to mycobacterial infection
Authors: Stokes R W , Collins F M
Published: January 2020

The growth of Mycobacterium avium in macrophages obtained from Mycobacterium bovis BCG-infected mice was compared with that in macrophages from uninfected mice. BCG vaccination resulted in substantial macrophage activation, measured as increased acid phosphatase and superoxide anion production, as well as enhanced leishmanicidal activity. However, the activated macrophages were only able to reduce the rate of intracellular growth by Listeria monocytogenes and M. avium in vivo and did not express detectable levels of mycobactericidal activity in vitro. Exposure of the macrophage monolayers to concanavalin A-stimulated spleen cell supernatant fluid and lipopolysaccharide did not further enhance the ability of the BCG-activated macrophages to control the intracellular replication of the M. avium. Macrophages from BCG-infected C57BL/6 (BCGs) mice were quantitatively better able to control the intracellular replication of the M. avium challenge than were similar phagocytes obtained from BCGr (A/J) mice. These findings have important implications with respect to the expression of acquired resistance to these atypical mycobacterial infections.
Characterization of antigenic determinants in histoplasmin that stimulate Histoplasma capsulatum-reactive T cells in vitro
Authors: Harris J E , Deepe G S
Published: January 2020

Although antigen-reactive T lymphocytes play a central role in the host response to Histoplasma capsulatum, little is known of the nature of Histoplasma antigens recognized by these cells in vitro. Employing a murine T-cell line and two clones that are reactive with histoplasmin, we examined whether activation of T cells by histoplasmin required the presence of carbohydrate or protein moieties. The approach taken was to modify carbohydrate or protein molecules in histoplasmin by chemical or enzymatic digestion or by lectin adsorption. In parallel, antigen was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to correlate alterations in functional activity with changes in the electrophoretic appearance of histoplasmin. Treatment of histoplasmin with periodate (0.1 M, 0.05 M, and 0.01 M) or with the endoglycosidases N-glycanase and endoglycosidase H sharply diminished the capacity of histoplasmin to trigger responses by T cells. Reactivity of T cells to histoplasmin that had been adsorbed with lectins binding mannose, glucose, or galactose was reduced by greater than 70%; conversely, the responses by T cells to antigen that had been adsorbed with lectins specific for fucose, N-acetylgalactosamine, or N-acetylglucosamine ranged from 82 to 91% of that to control antigen. Proliferative responses by T cells to histoplasmin that had been digested with chymotrypsin, protease, or trypsin were 2 to 43% of control values. The electrophoretic appearance of histoplasmin was modified by some but not all of the treatments. Partially purified H and M antigens triggered proliferation of T cells. Thus, both carbohydrates and proteins must be present to induce optimal responses by T cells. A portion of the carbohydrates is N linked to proteins, and alpha-D-mannose (or alpha-D-glucose) and beta-D-galactose are the sugar ligands of carbohydrate-containing antigens.
Genetic relationships among pathogenic Escherichia coli of serogroup O157
Authors: Whittam T S , Wilson R A
Published: January 2020

Escherichia coli strains of serotype O157:H7 are a newly described clonal pathogenic form associated with recent outbreaks of hemorrhagic colitis in humans. Although O157 strains of various H types have long been recognized as enterotoxigenic in animals, little is known about how these pathogenic animal strains are related to those of serotype O157:H7. To determine the genetic relatedness of O157:H7 isolates to animal O157 strains, we examined 194 O157 isolates, representing 12 distinct flagellar antigens (H serotypes), obtained from a variety of animal and human infections. To characterize isolates, we assayed allelic variation at 19 enzyme loci by multilocus enzyme electrophoresis. Genotypic comparisons of isolates revealed extensive variation among 33 distinct clonal genotypes that differed, on average, at 44% of the enzyme loci. K88 fimbriae were expressed in 72% of the isolates and occurred in a diversity of chromosomal genotypic backgrounds. Five major clonal groups were recognized; one group was clearly associated with porcine colibacillosis, and another was associated with human urinary tract infections. The O157:H7 genotype was not closely allied with any of the major groups of clones. The results indicate that O157 E. coli are genetically diverse and strongly suggest that the O157:H7 lineage was not recently derived from other pathogenic strains of the O157 serogroup.
Kinetics of murine delayed-type hypersensitivity response to Eimeria falciformis (Apicomplexa: Eimeriidae)
Authors: Shi Y F , Mahrt J L , Mogil R J
Published: January 2020

Mice recovering from a primary infection with an intestinal protozoan parasite, Eimeria falciformis (Apicomplexa: Eimeriidae), showed a classic delayed-type hypersensitivity (DTH) reaction to oocyst antigen challenge. This reaction was characterized by a biphasic pattern of footpad swelling. The first swelling peaked at 2 h after antigen challenge, whereas the second swelling peaked at 24 to 48 h after challenge. The DTH reaction was transferable with a T-cell-enriched spleen cell population from mice that had recovered from E. falciformis infection. Cytotoxic depletion of immune T cells with anti-L3T4 antibody and complement abrogated DTH transfer, indicating that L3T4-positive T cells were required. A T-cell-enriched spleen cell population from acutely infected mice suppressed the transfer of DTH with immune cells from recovered animals, implicating the existence of infection-induced immunoregulatory cells controlling the parasite-specific immune response during infection. Immune spleen cells also transferred resistance to infection as measured by oocyst production and death rate of recipients. Together, these results indicate that the DTH reaction, induced by infection with E. falciformis, is mediated by L3T4-positive T cells and is associated with resistance to infection.
T-cell modulation of the antibody response to bacterial polysaccharide antigens
Authors: Taylor C E , Bright R
Published: January 2020

Pretreatment of mice with subimmunogenic doses of meningococcal polysaccharide (MP), Pseudomonas aeruginosa lipopolysaccharide (PA), or Streptococcus mutans polysaccharide (SM) resulted in suppression of antibody response. The transfer of putative suppressor T cells (Ts cells) from donor mice primed with a subimmunogenic dose of MP to naive recipients at the time of immunization with MP substantially reduced the magnitude of the antibody response. Also, the infusion of B cells taken from animals immunized with either MP or PA suppressed the antibody response of naive recipients to MP or PA, respectively, relative to controls, suggesting that Ts cells respond to determinants on immune B cells. We observed that the injection of concanavalin A or phytohemagglutinin (two lectins known to augment the activity of amplifier T cells [Ta cells]) 2 days postimmunization enhanced the antibody response to MP and SM. In addition, Ta-cell activity was transferred to naive animals by using spleen cells. Although the administration of phytohemagglutinin at the time of immunization with MP also resulted in increased antibody response, the injection of concanavalin A simultaneous with immunization resulted in a suppression of the antibody response to MP. Although Ts cells generated in response to pneumococcal polysaccharide type III were found to respond to monoclonal antibody Ly-m22, Ta cells responded to monoclonal antibodies L3T4 and Ia but not to Ly-m22. These studies suggest that Ta and Ts cells can modulate the antibody response to MP, SM, and PA in a positive and negative manner, respectively.
Experimental production of proliferative ileitis in Syrian hamsters (Mesocricetus auratus) by using an ileal homogenate free of Campylobacter jejuni
Authors: Stills H F , Hook R R
Published: January 2020

The role of Campylobacter jejuni in the pathogenesis of proliferative ileitis of Syrian hamsters (Mesocricetus auratus) has been uncertain. C. jejuni has been implicated as the etiologic agent on the basis of the campylobacter-type morphology of the intracellular organism and the repeated microbiologic isolation of C. jejuni from hamsters with proliferative ileitis. The inability to reproduce the disease with pure culture inocula, coupled with immunohistochemical studies, however, has suggested that although C. jejuni may be present in the ilea of infected hamsters, its involvement in the pathogenesis of proliferative ileitis is questionable. In this study hamsters were inoculated with infective ileal homogenates prepared from ilea which were extensively washed to remove the ileal contents before grinding. The ilea from hamsters inoculated with this homogenate were also washed before being ground and used to experimentally inoculate a second group of hamsters. Of the 20 hamsters from this second group, 12 developed lesions typical of proliferative ileitis. Extensive microbiologic cultures from these hamsters were negative for C. jejuni. Immunofluorescence studies with a C. jejuni-specific monoclonal antibody were also negative. The use of a Campylobacter genus-specific monoclonal antibody, however, revealed numerous campylobacter-type organisms within the ileal epithelial cells of the crypts and villi. The presence of C. jejuni is therefore apparently not necessary for the production of proliferative ileitis in hamsters, and the intracellular campylobacter-type organism present in the ileal epithelial cells of infected hamsters is probably not C. jejuni.
Surface-exposed and antigenically conserved determinants of outer membrane proteins of Branhamella catarrhalis
Authors: Murphy T F , Bartos L C
Published: January 2020

The outer membrane proteins (OMPs) of Branhamella catarrhalis were studied in an effort to identify surface-exposed determinants that are conserved among strains of the bacterium. Aliquots of polyclonal antiserum were absorbed individually by strains of B. catarrhalis. The absorbed antisera were tested in comparison with unabsorbed antiserum in an immunoblot assay against OMPs of the homologous strain. The absence of a band recognized by antibodies in the absorbed antiserum compared with the unabsorbed antiserum indicated that surface-exposed determinants of the absorbing strain cross-reacted with determinants on the homologous strain. Two antisera were absorbed individually by 20 strains of B. catarrhalis, and the absorbed sera were studied in this way in immunoblot assays. OMP E (molecular weight, ca. 56,000) expresses surface-exposed determinants that are shared among 17 of the 20 strains studied. Antibodies to OMP G (molecular weight, 28,000) were absorbed from both antisera by 14 of the 20 strains. These studies demonstrate that OMP E and OMP G express determinants that are exposed on the surface of the intact bacterium. Furthermore, these determinants are antigenically conserved among a majority of strains of B. catarrhalis. On the basis of these observations, OMPs E and G should be considered when bacterial antigens are evaluated as potential vaccine candidates.
Identification of antigens recognized by T cells in human leishmaniasis: analysis of T-cell clones by immunoblotting
Authors: Melby P C , Sacks D L
Published: January 2020

Immunity in human leishmaniasis is mediated by sensitized T lymphocytes; however, the antigens involved in eliciting this immunity have not been defined. We describe the generation of human T-lymphocyte clones derived from two patients with healed leishmaniasis. By use of one- and two-dimensional cellular immunoblotting techniques, we directly identified the parasite antigens recognized by these clones. To our knowledge, these are the first leishmanial antigens identified to which CD4+, gamma interferon-producing T cells from immune individuals have been shown to respond, the strategy may be of general use for the identification of antigens involved in immunity in this disease.
Purification and characterization of a 27,000-Mr extracellular proteinase from Trichophyton rubrum
Authors: Apodaca G , McKerrow J H
Published: January 2020

A proteinase of Mr 27,000 with a possible role in the metabolism and invasion of host tissues was purified from the conditioned medium of Trichophyton rubrum by concanavalin A and anion-exchange chromatography. Peaks of proteolytic activity were analyzed by substrate gel electrophoresis. The 27,000-Mr proteinase had a pH optimum of 8.0, a calcium dependence of 2 mM, and was inhibited by serine proteinase inhibitors, especially phenylmethylsulfonyl fluoride and Phe-Gly-Ala-Leu-chloromethyl ketone. By polyacrylamide gel electrophoresis, the 27,000-Mr proteinase had a reduced molecular weight of 44,000 and reacted with [3H]diisopropyl fluorophosphate. The proteinase degraded azocoll, type III collagen, type IV procollagen, laminin, fibronectin, and the peptide substrates succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (1,573 M-1 s-1) and t-butyloxy carbonyl-Ala-Ala-Leu-p-nitroanilide (1,614 M-1 s-1).
Regulation of Trichophyton rubrum proteolytic activity
Authors: Apodaca G , McKerrow J H
Published: January 2020

Trichophyton rubrum is the most common dermatophyte of humans and normally colonizes the superficial layers of the epidermis (stratum corneum). Several proteinases with a possible role in the metabolism of host proteins have been purified from this fungus. The regulation of these enzymes and their role in fungal metabolism were studied at the biochemical level. General proteolytic (azocollytic) activity was repressed when log-phase cultures of T. rubrum were grown in a minimal medium that contained readily metabolized sources of carbon, nitrogen, sulfur, and phosphorus. When either carbon, nitrogen, or sulfur was deleted from this minimal medium, azocollytic activity was derepressed. In all cases a high-molecular-weight activity (Mr, greater than 200,000) was expressed. A 71,000-Mr proteinase was observed in nitrogen-depleted cultures, and proteolytic species of Mr 124,000 and 27,000 were secreted in sulfur-depleted cultures. The addition of either inorganic (MgSO4, Na2SO3, NaS2O3) or organic (methionine, cysteine) sulfur to the sulfur-depleted medium repressed the expression of azocollytic activity. In contrast, keratinolytic activity was not repressed by carbon, nitrogen, or sulfur but instead was induced when a protein source was included in the minimal medium. Stationary-phase cultures of T. rubrum secreted all proteolytic activities constitutively. Unlike log-phase cultures, the stationary-phase cultures secreted azocollytic, elastinolytic, and keratinolytic activity in minimal medium. These activities fell in the carbon-, nitrogen-, and phosphorous-depleted media but remained high in sulfur-depleted medium. The following model is proposed for the regulation of T. rubrum proteolytic activity. In the initial stages of infection, T. rubrum grows logarithmically. In this state, proteolytic activity is derepressed whenever carbon, nitrogen, or sulfur is lacking in the fungal milieu. The general proteinases produced would act on the nonkeratinous proteins in the stratum corneum. There are probably peptidases, as yet unidentified, that would cleave the peptides generated by the initial proteolysis into amino acids. These amino acids would provide the cell with a source of carbon, nitrogen, and sulfur. Under these conditions, the expression of general proteinases would be repressed, whereas specific keratinases would be induced in this nutrient-rich environment. Disease may occur when the fungus reaches stationary phase, when proteinases are secreted constitutively. These enzymes may directly or indirectly incite a host response, resulting in the inflammatory manifestations of dermatophytosis.
Immunological responsiveness of chinchillas to outer membrane and isolated fimbrial proteins of nontypeable Haemophilus influenzae
Authors: Bakaletz L O , Tallan B M , Andrzejewski W J , DeMaria T F , Lim D J
Published: January 2020

Thin, nonhemagglutinating fimbriae have been demonstrated on 100% of the clinical isolates of nontypeable Haemophilus influenzae recovered from children with chronic otitis media tested in this laboratory (L. O. Bakaletz, B.M. Tallan, T.M. Hoepf, T.F. DeMaria, H.G. Birck, and D.J. Lim, Infect. Immun. 56:331-335, 1988). Chinchillas with induced otitis media responded to this surface-located antigen of both the infecting and a heterologous strain. Antibodies were found in both serum and middle ear fluids.
Pseudomonas elastase acts as a virulence factor in burned hosts by Hageman factor-dependent activation of the host kinin cascade
Authors: Holder I A , Neely A N
Published: January 2020

Purified Pseudomonas elastase injected subcutaneously into the skin of an Evans blue dye-injected (intravenously) guinea pig caused dye leakage similar to that observed when histamine or bradykinin was injected in the same animal. The histamine-induced dye leakage was ablated in antihistamine-treated guinea pigs, but elastase- and bradykinin-induced dye leakages were not. Local injections of specific inhibitors of the host Hageman factor-dependent bradykinin-generating pathway given immediately prior to elastase injection reduced dye leakage in a dose-related manner. Elastase-related dye release was enhanced when angiotension-converting enzyme inhibitor, a substance which prevents host enzymes from breaking down bradykinin, was injected prior to elastase injection. We conclude that Pseudomonas elastase generates bradykinin in the infected host via a Hageman factor-dependent pathway.
Amastigote stage-specific monoclonal antibodies against Leishmania major
Authors: Jaffe C L , Rachamim N
Published: January 2020

Monoclonal antibodies were produced against gamma-irradiated amastigotes of Leishmania major. Five antibodies (T16 through T20) were selected which reacted in enzyme-linked immunoassays with the intracellular stage of the parasite. These antibodies did not react with promastigotes of L. major or Leishmania donovani. One of the monoclonal antibodies (T16) reacted with amastigotes of Leishmania mexicana amazonensis and L. donovani. Western blotting (immunoblotting) and immunoprecipitation of [35S]methionine-labeled amastigotes demonstrates that T16 reacted with multiple L. major amastigote components between 12 and 180 kilodaltons. Antibody T20 was shown to recognize a low-molecular-mass doublet (less than 26 kilodaltons) in both [14C]leucine- and [35S]methionine-labeled amastigotes. A protein of less than 180 kilodaltons was also weakly recognized by T17, T19, and T20 in metabolically labeled amastigotes. This protein reacted strongly with T16. The reactive antigens could be identified on the surface of amastigotes isolated from the lesions of infected mice and on newly transformed amastigotes within 24 h after the infection of mouse peritoneal macrophages by promastigotes. These monoclonal antibodies should prove useful for the diagnosis of L. major in human tissue biopsies.
A method for allelic replacement that uses the conjugative transposon Tn916: deletion of the emm6.1 allele in Streptococcus pyogenes JRS4
Authors: Norgren M , Caparon M G , Scott J R
Published: January 2020

The emm6.1 allele of Streptococcus pyogenes JRS4 was deleted by using the conjugative transposon Tn916. The aphA-3 gene, conferring resistance to kanamycin, was cloned between the sequences flanking the structural gene for the type 6 M protein (emm6.1) and inserted into the BstXI site of Tn916 to generate the chimeric transposon Tn916-5K3. Because the BstXI site lies in a nonessential region of Tn916, the chimeric transposon could transfer by conjugation from Bacillus subtilis into JRS4. In some of the transconjugants, Tn916-5K3 replaced the emm6.1 locus of JRS4 by homologous recombination between the cloned emm6.1-flanking regions and the resident chromosome. One recombinant studied in detail, JRS75, was kanamycin resistant and tetracycline sensitive and lacked immunologically detectable M6 protein. Furthermore, by Southern blot analysis, the DNA region encompassing the emm6.1 structural gene was found to have been replaced by aphA-3.
Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and production of cross-reactive monoclonal antibodies
Authors: Donohue-Rolfe A , Acheson D W , Kane A V , Keusch G T
Published: January 2020

Shiga toxin from Shigella dysenteriae 60R was purified to homogeneity by a novel one-step receptor analog affinity chromatography method. The method was based on the binding affinity of Shiga toxin for a specific disaccharide, Gal alpha 1----4Gal, which was also present in glycoproteins with P1 blood group seroreactivity produced in hydatid cysts from sheep infected with Echinococcus granulosus. Having shown that cyst fluid P1 glycoprotein bound Shiga toxin on a solid phase, a P1 glycoprotein affinity column was made by coupling P1-active substance to Sepharose 4B. Shiga toxin was purified by this method in large quantities (5 to 10 mg/20-liter batch) with a consistently good yield (greater than 80% of starting toxin). Shiga-like toxins I and II (SLT-I and -II, respectively) from Escherichia coli were also purified by the same method. A preparation containing SLT-II and SLT-I purified by receptor analog affinity chromatography was used to raise four monoclonal antibodies (MAbs) that were reactive with SLT-II by enzyme-linked immunosorbent assay. Three of these antibodies also reacted with Shiga toxin, which was the first clear demonstration of cross-reactivity between these toxins. One MAb, 4D1, which was specific for the B subunit of SLT-II and Shiga toxin, neutralized both toxins in a HeLa cell cytotoxicity assay. Two MAbs recognized the A subunit of both SLT-II and Shiga toxin by Western blot (immunoblot) analysis but were unable to neutralize either toxin. In addition, one B-subunit-specific MAb neutralized SLT-II alone, and a previously described Shiga toxin B-subunit-specific MAb was shown to be specific for Shiga toxin but not SLT-II.
Spontaneous switching of the sucrose-promoted colony phenotype in Streptococcus sanguis
Authors: Tardif G , Sulavik M C , Jones G W , Clewell D B
Published: January 2020

Streptococcus sanguis on media containing 3% sucrose gives rise to characteristic hard cohesive colonies (designated Spp+). Populations of Spp+ bacteria (strain Challis) on sucrose media switch to a soft noncohesive phenotype (designated Spp-) at a frequency of 10(-4) to 10(-3). Spp- bacteria switch back to Spp+ bacteria at a similar frequency. Successive rounds of Spp variation were observed. The Spp phenotypic switch was associated with changes in extracellular glucosyltransferase activity.