bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship of virulence-associated autoagglutination to hemagglutinin production in Yersinia enterocolitica and Yersinia enterocolitica-like bacteria
Authors: Kapperud G , Lassen J
Published: January 2020

A total of 339 Yersinia strains were tested for virulence-associated autoagglutination and hemagglutinin (HA) production. Autoagglutination was frequent among human clinical isolates belonging to Y. enterocolitica serogroups O:3, O:8, and O:9 and among porcine isolates of serogroups O:3 and O:9. Positive autoagglutination results were also observed in serogroups O:1 and O:2, associated with outbreaks among chinchillas, hares, and goats. All of 164 environmental isolates were negative in the autoagglutination assay. When subcultured serially in nutrient broth, none of the clinical isolates produced HA at either 22 or 37 degrees C, whereas 22.6% of the environmental yersiniae yielded mannose-resistant HA (MRHA). Six different MRHA patterns were recorded. Eighteen strains, all of which produced MRHA in nutrient broth, yielded similar MRHA patterns after cultivation on colonization factor agar. None of the autoagglutination-positive-(AA+) strains produced MRHA in either nutrient broth or on colonization factor agar. In contrast, a distinct autoagglutination-associated MRHA was detected after growth in Eagle minimal essential medium. Comparison of AA+ and autoagglutination-negative (AA-) derivatives of serogroups O:2, O:3, O:5, O:8, and O:9 showed that all AA+ cultures produced MRHA when grown in minimal essential medium at 37 degrees C overnight, whereas no MRHA was observed with any of the AA- derivatives of the same serogroups. This autoagglutination associated MRHA reacted with guinea pig erythrocytes, but not with erythrocytes from seven other species.
Molecular cloning and expression of Treponema pallidum DNA in Escherichia coli K-12
Authors: van Embden J D , van der Donk H J , van Eijk R V , van der Heide H G , de Jong J A , van Olderen M F , Osterhaus A B , Schouls L M
Published: January 2020

A gene bank of Treponema pallidum DNA in Escherichia coli K-12 was constructed by cloning SauI-cleaved T. pallidum DNA into the cosmid pHC79. Sixteen of 800 clones investigated produced one or more antigens that reacted with antibodies from syphilitic patients. According to the separation pattern of the antigens produced on sodium dodecyl sulfate-polyacrylamide gels, six different phenotypes were distinguished among these 16 clones. These antigens reacted also with anti-T. pallidum rabbit serum. No antibodies against the cloned antigens were found in normal rabbit serum and in nonsyphilitic human serum. The antigens produced by the E. coli K-12 recombinant DNA clones comigrated in sodium dodecyl sulfate-polyacrylamide gels with antigens extracted from T. pallidum bacteria, suggesting that the treponemal DNA is well expressed in E. coli K-12. Several of the cosmid recombinant plasmids have been subcloned, resulting in smaller T. pallidum recombinant plasmids which are more stably maintained in the cell and produce more treponemal antigen. Monoclonal antibodies were raised against T. pallidum, and one hybridoma produced antibodies that reacted not only with an antigen from T. pallidum but also with the antigen produced by one of the E. coli clones.
Enhancement of macrophage-mediated tumor cell killing by bacterial outer membrane proteins (porins)
Authors: Weinberg J B , Ribi E , Wheat R W
Published: January 2020

Various microbial products are known to influence the function of mouse peritoneal macrophages. Lipopolysaccharide (LPS) and certain lipid A-associated proteins are known to enhance the tumoricidal effects of macrophages. The purpose of this study was to determine whether porins (outer membrane proteins) of Salmonella typhimurium G30/C21 would influence the activity of macrophages from lipid A-responsive and -unresponsive mice. Porins, extracted by a combined sodium dodecyl sulfate-EDTA method from cell walls, were free of LPS as determined by Limulus amebocyte lysate assay and appeared as a band at approximately 36,000 molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In tumor cell killing assays done under LPS-free conditions, the porins in doses of 1 to 10 ng/ml enhanced the tumoricidal effect of macrophages from bacillus Calmette-Guérin-infected C3H/HeN or C3H/HeJ mice. Protein-free LPS enhanced the tumoricidal activity of macrophages from bacillus Calmette-Guérin-infected C3H/HeN but not C3H/HeJ mice. The tumoricidal-enhancing activity of protein-free LPS was blocked by the lipid A-binding antibiotic polymyxin B sulfate, but the effects of porins were not altered by the polymyxin B sulfate. These results suggest that porins, proteins known to alter membrane function, may alter macrophage function by interaction with macrophage membranes.
Molecular epidemiological studies of United States Gulf Coast Vibrio cholerae strains: integration site of mutator vibriophage VcA-3
Authors: Goldberg S , Murphy J R
Published: January 2020

Environmental and clinical Vibrio cholerae O-1 strains isolated from the U.S. Gulf Coast region were found to be lysogenic for a vibriophage which we have designated VcA-3. Comparison of VcA-3 with the previously described vibriophages VcA-1 and VcA-2 has shown that VcA-1 and VcA-3 are homoimmune, have extensive sequence homology, but have markedly different restriction endonuclease digestion patterns. VcA-3 was found to randomly integrate into the V. cholerae RV79 chromosome and to introduce stable auxotrophic mutations. We show that all U.S. Gulf Coast environmental and clinical isolates that are lysogenic for VcA-3, including both tox+ and tox- isolates, contain the prophage integrated at an identical chromosomal site. Given the known stability of temperate mutator bacteriophages, these results suggest that there is a clonal relationship among the V. cholerae O-1 strains examined in this study, including the tox+ and tox- isolates.
In vivo function of hemolysin in the nephropathogenicity of Escherichia coli
Authors: Waalwijk C , MacLaren D M , de Graaff J
Published: January 2020

The role of hemolysin in the nephropathogenicity of Escherichia coli was studied in a hematogenous pyelonephritis model in mice. The nephropathogenicity of a nonhemolytic, avirulent E. coli strain was increased by simultaneous injection with its hemolytic, nephropathogenic parent. This helper mechanism could be attributed to hemolysin, since the simultaneous injection of partially purified hemolysin gave a similar enhancement of nephropathogenicity. Intraperitoneal injection of hemoglobin or iron sulfate before intravenous challenge with this avirulent strain also led to increased virulence. The nephropathogenicity-enhancing effect of hemolysin is therefore supposed to depend on increasing the level of available iron in the host. Under conditions of plentiful iron, hemolysin production was repressed, as shown by in vitro growth experiments in the presence of exogenous iron. These results suggest that the production of hemolysin is regulated by feedback inhibition.
Response of mice to rotaviruses of bovine or primate origin assessed by radioimmunoassay, radioimmunoprecipitation, and plaque reduction neutralization
Authors: Offit P A , Clark H F , Plotkin S A
Published: January 2020

Sera from (i) gnotobiotic BALB/c, CD-1, and CFW mice and (ii) conventional BALB/c mice were evaluated by radioimmunoassay, radioimmunoprecipitation, and plaque reduction neutralization, using the Wa, SA-11, and WC-3 (bovine) strains of rotavirus as the detecting antigens. The gnotobiotic mice had no antirotavirus antibody detectable by radioimmunoprecipitation and no neutralizing antibody at a dilution of 1:50 by plaque reduction neutralization. All sera from the conventional mice had rotavirus-specific antibodies detected by radioimmunoassay and by radioimmunoprecipitation at serum dilutions of 1:50 and 1:10,000, respectively. The antibodies were directed against viral proteins p116, p94, p88, and p84 of all three viruses, but had no neutralizing activity against heterologous rotaviruses at a dilution of 1:50. Conventional seropositive mice were parenterally immunized with the Wa, SA-11, or WC-3 strain of rotavirus. An approximate 100-fold increase in rotavirus-specific antibodies was detected by radioimmunoassay, and greater than 20-fold selective neutralization of the immunizing strain of virus was observed. Sera from the mice immunized with Wa virus had antibodies directed against inner and outer capsid proteins of all three rotaviruses. The mouse can be a useful model for studying the immune response to heterologous rotavirus infection; preexisting antibodies presumably directed towards murine rotavirus do not prevent the development of a type-specific immune response to a nonmurine rotavirus.
Interactions of preimplantation mouse embryos with reovirus serotypes 1 and 3
Authors: Abramczuk J , Vorbrodt A , Rubin D H , Fields B N , Koprowski H
Published: January 2020

Mouse two-cell embryos were infected in vitro with reovirus serotypes 1/Lang and 3/Dearing, and the embryos were either implanted into pseudopregnant mice or observed in vitro for cytopathic effects. The reovirus serotypes 1/Lang and 3/Dearing differed in their capacity to kill embryos in vitro and in vivo: when embryos were infected in vitro with reovirus serotype 1/Lang and then transferred to foster mothers, pups resulted only at multiplicities of infection of a few particles per embryo. In contrast, infection of embryos with as much as 6 X 10(4) reovirus type 3 particles per embryo resulted in viable pups. In vitro, reovirus serotype 1/Lang was more virulent than serotype 3/Dearing. The infection of ovum with reovirus offers a unique model for the study of congenital infection and should yield important information concerning the molecular basis of virus virulence to maturing fetuses.
Characterization of surfaces involved in adherence of Legionella pneumophila to Fischerella species
Authors: Bohach G A , Snyder I S
Published: January 2020

Legionella pneumophila adheres to the slime coat of Fischerella spp. This was shown by microscopic examination and by a decline in L. pneumophila CFU in samples removed from coincubation mixtures of both organisms. Binding of partially purified Fischerella slime by L. pneumophila was most efficient by young, less hydrophobic L. pneumophila cells than by older, more hydrophobic cells. Uptake of crystal violet and partitioning into hexadecane were used to measure hydrophobicity of L. pneumophila. Purified soluble Legionella antigen also bound to Fischerella slime, as shown by indirect immunofluorescence. Adherence was not specific for L. pneumophila, since a variety of gram-negative, gram-positive, and acid-fast bacteria also bound to Fischerella slime.
Renal deposition of alpha interferon in systemic lupus erythematosus
Authors: Panem S , Ordóñez N , Vilcek J
Published: January 2020

Earlier studies from several laboratories showed that interferon-alpha (IFN-alpha) is present in the sera of a large percentage of patients with systemic lupus erythematosus (SLE). We now report the detection of IFN-alpha by indirect immunofluorescence in renal sections of three patients with SLE but not in six control kidneys. The immunofluorescence reaction was mediated by three hyperimmune antisera to IFN-alpha raised in three different species, but not by any preimmune serum. The reaction was specifically blocked by absorption of the anti-IFN-alpha sera with purified IFN-alpha made by recombinant DNA techniques or with IFN-alpha isolated from the serum of an SLE patient, but not by bovine serum albumin or human immunoglobulin G. In contrast, antisera to IFN-beta or IFN-gamma did not mediate immunofluorescence. The pattern of IFN-alpha deposition resembled that seen with anti-human immunoglobulin G, suggesting association with immune complexes. Immune complexes were then preparatively eluted from the homogenate of an SLE kidney by treatment with buffer at pH 2.8. Biologically active IFN was found in this eluate and was demonstrated to be IFN-alpha by specific neutralization with IFN antisera. These results extend the specific association of IFN-alpha with SLE.
Adherence and penetration of vascular endothelium by Candida yeasts
Authors: Klotz S A , Drutz D J , Harrison J L , Huppert M
Published: January 2020

Metastatic infection after hematogenous dissemination of Candida species is presumably dependent on the fungus traversing the vascular endothelium. An in vitro model of the earliest events of metastatic Candida infection was developed with whole vascular strips. Freshly obtained porcine blood vessels were secured in a perforated Lucite template that allowed the application of yeasts directly to the endothelial surface. Multiple wells allowed experimental and control observations on the same vascular segments. Adherence to endothelium was greatest with Candida albicans and Candida tropicalis, less with Candida Krusei, and least with Candida parapsilosis, Candida pseudotropicalis, and Torulopsis glabrata. This hierarchy of adherence parallels that in other in vitro systems employing mucosal epithelial cells or fibrin-platelet matrixes and reflects the known virulence of the respective species and their potential for hematogenous dissemination. C. albicans and C. tropicalis yeasts that adhered were capable of directly traversing the endothelial surface before the production of germ tubes. Heat or Formalin-killed yeasts and viridans group streptococci, although adherent, were incapable of vascular penetration, a process presumably attributable to enzymatic digestion of host tissue. Loss of integrity of penetrated endothelial tissue was verified by loss of dye exclusion, lactic dehydrogenase release, and ultramicroscopic changes. These two steps, adherence and penetration, provide direct insight into the earliest events in hematogenous Candida species dissemination and suggest that C. albicans and C. tropicalis yeasts are capable of initiating tissue invasion before germ tubes have had the opportunity to form and participate in the invasive process.
Morphological aberrations of nutritionally deficient streptococci: association with pyridoxal (vitamin B6) concentration and potential role in antibiotic resistance
Authors: Clark R B , Gordon R E , Bottone E J , Reitano M
Published: January 2020

A strain of a nutritionally deficient streptococcus was shown to undergo morphological aberrations according to pyridoxal concentrations in the growth medium. Filamentous rod-shaped cells, observed by electron microscopy, predominated in the presence of decreasing concentrations. Multiple invaginations in the outer cell wall suggested inhibition of binary fission. Penicillin antimicrobial studies performed in the presence of similar pyridoxal concentrations indicated a relationship between filamentous forms and penicillin susceptibility.
Strain distribution in extents of lysozyme resistance and O-acetylation of gonococcal peptidoglycan determined by high-performance liquid chromatography
Authors: Swim S C , Gfell M A , Wilde C E , Rosenthal R S
Published: January 2020

The extent of lysozyme resistance and O-acetylation of purified peptidoglycan (PG) from 20 strains of Neisseria gonorrhoeae was examined to determine how widespread these properties are among various subsets of gonococcal isolates. To determine digestibility by lysozyme, we treated [3H]- or [14C]glucosamine-labeled PG with hen egg white lysozyme (HEW-LZ) and determined the size distribution of HEW-LZ soluble PG at the completion of the reaction by molecular-sieve high-performance liquid chromatography, using a Varian TSK SW2000 column, a method that proved considerably more efficient than traditional chromatography for fractionating low-molecular-weight PG fragments solely on the basis of size. The extent of HEW-LZ resistance was expressed as the percentage of PG that was larger in size than disaccharide peptide tetramers (including insoluble PG removed by centrifugation). The percent O-acetylation was determined by converting insoluble PG totally to uncross-linked monomers by the combined action of Chalaropsis B muramidase followed by Escherichia coli endopeptidase and then quantitating radioactivity in O-acetylated and non-O-acetylated monomers after paper chromatography. The PG of the vast majority (19 of 20) of gonococcal strains examined was extensively HEW-LZ resistant (range, 40 to 60% larger than tetramers) and extensively O-acetylated (range, 34 to 52%). Only the PG of strain RD5 (highest rate of PG turnover among gonococci so far examined and the prototype of gonococci having O-acetyl-deficient PG) had greatly reduced O-acetylation (15%) and exhibited virtually no HEW-LZ resistance (2% larger than tetramers). Extensive HEW-LZ resistance and O-acetylation were apparently not associated specifically with (i) a given type of colonial variant (piliated versus nonpiliated or opaque versus transparent), (ii) a given type of clinical isolate (local versus disseminated), (iii) the extent of laboratory passage, or (iv) (with the possible exception of penicillin-resistant strain FA102) the presence of one or more genetic loci governing antibiotic resistance among members of an isogenic set of gonococci. From this survey, we conclude that lysozyme resistance and extensive O-acetylation of PG are widespread among gonococci and, thus, that most strains are potential sources of hydrolase-resistant PG that conceivably could persist as macromolecular fragments in vivo.
Activation of murine B lymphocytes by Neisseria meningitidis and isolated meningococcal surface antigens
Authors: Melancon J , Murgita R A , Devoe I W
Published: January 2020

Heat-killed Neisseria meningitidis was found to be a potent mitogen for mouse splenic lymphocytes. Results obtained with different cell separation techniques indicated that the bacteria acted to selectively induce proliferation of B lymphocytes. First, partial or total depletion of T lymphocytes by treatment with various anti-T-cell antisera plus complement did not affect the ability of the remaining spleen cells to proliferate in response to N. meningitidis. Second, T lymphocytes purified by affinity chromatography through an immunoglobulin-antiimmunoglobulin-coated glass bead column were unresponsive to meningococcal stimulation, even when provided with a source of macrophages (irradiated or mitomycin C-treated spleen cells). Finally, treatment of spleen cells with soy bean agglutinin showed that, whereas the soy bean agglutinin-positive population (B-enriched lymphocytes) was highly responsive to stimulation by N. meningitidis, the soy bean agglutinin-negative population (T-enriched lymphocytes) displayed only a background level of proliferation when exposed to the bacteria. Isolated meningococcal surface antigens such as lipopolysaccharide (LPS) and outer membranes also possessed mitogenic activity and induced proliferation of B lymphocytes in a dose-dependent manner. Both LPS and non-LPS components contributed to the mitogenicity of outer membranes since the addition of outer membrane preparations to spleen cells from the low LPS responder C3H/HeJ mouse strain gave rise to a high level of proliferative activity.
Antiactin and antitubulin antibodies in canine visceral leishmaniasis
Authors: Pateraki E , Portocala R , Labrousse H , Guesdon J L
Published: January 2020

Visceral leishmaniasis, a chronic and often fatal disease, is caused by the protozoan parasite Leishmania donovani. Both specific and nonspecific antibodies are produced in the course of the disease, and autoantibodies may be involved in pathogenesis. Tubulin and actin have been found to be associated with L. donovani. To learn whether antiactin and antitubulin antibodies are present in visceral leishmaniasis, we tested sera from 263 infected dogs by enzyme-linked immunosorbent assay for antibodies to the antigens L. donovani, actin, and tubulin. All samples reacted positively with L. donovani, and a high percentage reacted positively with all three antigens. Sera from 202 uninfected dogs were also tested, none reacted with L. donovani antigen, although positive reactions were observed for 8 of the samples with actin or tubulin. It was found that the antibody-antigen reaction occurred at the Fab portion of the immunoglobulin molecule. Competitive enzyme immunoassays showed that the reaction was inhibited if the positive serum was first incubated with L. donovani antigen, actin, or tubulin and then tested by enzyme-linked immunosorbent assay. These results suggest that antiactin and antitubulin antibodies are present in the sera of dogs infected with visceral leishmaniasis.
Extraction and isolation of a leukotoxin from Actinobacillus actinomycetemcomitans with polymyxin B
Authors: Tsai C C , Shenker B J , DiRienzo J M , Malamud D , Taichman N S
Published: January 2020

A leukotoxin from Actinobacillus actinomycetemcomitans was isolated by a procedure that includes polymyxin B extraction, ion-exchange chromatography, and gel filtration chromatography. The procedure resulted in the recovery of 48% of the toxin with a 99-fold increase in specific activity. The isolated toxin has a molecular mass of 180,000 daltons by gel filtration and 115,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It retains all the major biological characteristics previously documented for crude leukotoxin preparations, including susceptibility to heat and proteolytic enzymes and neutralization by sera from patients with juvenile periodontitis. The isolated leukotoxin destroys human but not rat or guinea pig polymorphonuclear leukocytes and has no apparent effect on human erythrocytes. The availability of the A. actinomycetemcomitans leukotoxin should facilitate studies on its chemistry and mode of action as well as its role in the pathogenesis of human periodontal disease.
Immunization with Pseudomonas aeruginosa high-molecular-weight polysaccharides prevents death from Pseudomonas burn infections in mice
Authors: Pollack M , Pier G B , Prescott R K
Published: January 2020

High-molecular-weight polysaccharides from the extracellular slime of Pseudomonas aeruginosa were evaluated as immunogens in Pseudomonas burn infections in mice. Immunization with immunotype 1 or 2 polysaccharides induced a strong immunotype-specific and weak cross-reactive antibody response but protected mice against burn infections caused by either immunotype. Passive protection was provided by rabbit antiserum to immunotype 1 polysaccharide against burn infection by the homologous organism. Pseudomonas high-molecular-weight polysaccharides are potentially effective vaccines in burn infections.
Lack of homology between the iron transport regions of two virulence-linked bacterial plasmids
Authors: Walter M A , Bindereif A , Neilands J B , Crosa J H
Published: January 2020

Two plasmids involved in bacterial virulence, the Escherichia coli plasmid pColV-K30 and the Vibrio anguillarum plasmid pJM1, have been studied with respect to the iron sequestering systems mediated by these two plasmids. Bioassay results show that the two systems are not related functionally because specific iron uptake-deficient mutants in each system cannot be cross-fed by the heterologous bacteria using culture supernatants from iron-proficient strains containing wild-type plasmids. DNA hybridization studies show an extensive lack of homology between regions involved in iron sequestration in both plasmids.
Chemotactic and phagocytic responses of human alveolar macrophages to activated complement components
Authors: Richards S W , Peterson P K , Verbrugh H A , Nelson R D , Hammerschmidt D E , Hoidal J R
Published: January 2020

Human alveolar macrophages (AMs) migrated toward and aggregated to C5a but not toward C5a-deficient serum. Human AMs from cigarette smokers migrated significantly farther than did AMs from nonsmokers. Human AMs phagocytized Escherichia coli opsonized with activated C3. Immunoglobulin G was not required for phagocytosis. Human AMs demonstrate chemotaxis and aggregation to C5a. Further, human AMs can phagocytize bacteria coated with activated C3.
Nonopsonic phagocytosis of strains of Pseudomonas aeruginosa from cystic fibrosis patients
Authors: Speert D P , Eftekhar F , Puterman M L
Published: January 2020

Pseudomonas aeruginosa is the predominant respiratory pathogen in patients with cystic fibrosis, but its mechanism of persisting in pulmonary secretions is poorly understood. We observed that three nonmucoid cystic fibrosis P. aeruginosa strains were phagocytized and one strain resisted phagocytosis by human polymorphonuclear leukocytes in the absence of serum. Phagocytosis was assessed by luminol-enhanced chemiluminescence, inspection of stained smears, bactericidal assay, reduction of nitroblue tetrazolium dye, and electron microscopy. Phagocytosis, determined by visual inspection, occurred at 35 degrees C but not at 4 degrees C. Nonopsonic phagocytosis was inhibited most efficiently by D-mannose, mannose-containing saccharides, and D-fructose. Opsonin-dependent phagocytosis of P. aeruginosa and of zymosan was not markedly inhibited by mannose, suggesting different leukocyte receptors for nonopsonic and opsonic phagocytosis.
Production and characterization of three monoclonal antibodies to Candida albicans proteins
Authors: Strockbine N A , Largen M T , Buckley H R
Published: January 2020

Three monoclonal antibodies, designated A2C7, C2C7, and F19, were produced which recognize proteins from Candida albicans. All are of the immunoglobulin G1 heavy chain and kappa light chain class. A2C7 and C2C7 immunoprecipitated three proteins contained in a partially purified fraction (region A) of a mycelial cytoplasmic extract of C. albicans. The apparent molecular weights of these proteins are 120,000 (120K) to 135K, 44K to 52K, to 38K. Monoclonal antibody F19 was reactive with proteins of 42K, 43K, and 50K in immunoblotting experiments. F19 was also able to form a precipitin band in agarose gel with protein(s) contained in region A. Limited proteolytic digestion of the three proteins immunoprecipitated by A2C7 and C2C7 demonstrated that both monoclonal antibodies recognized the same three Candida proteins and that there exists a significant degree of relatedness in primary structure among the three proteins. Proteins with apparent molecular weights of 120K to 135K, 44K to 52K, and 35K to 38K that were immunoprecipitated by sera from two patients with invasive candidiasis and by the serum from a rabbit immunized against a 48K (44K to 52K) Candida protein were also analyzed by limited proteolysis. Patterns of peptide fragments generated by enzymatic digestion of these proteins showed that the proteins recognized by the monoclonal antibodies are the same proteins recognized by antibodies in the sera of patients during an invasive Candida infection and by antibodies in the serum of the immune rabbit.
Characterization of monoclonal antibodies to heat-labile enterotoxin encoded by a plasmid from a clinical isolate of Escherichia coli
Authors: Belisle B W , Twiddy E M , Holmes R K
Published: January 2020

Eight selected hybridoma cell lines that produced monoclonal antibodies against heat-labile enterotoxin from an Escherichia coli strain of human origin (LTh) were characterized. Antibodies produced by these cell lines were tested for binding specificity in a series of solid-phase radioimmunoassays and Western blots by using as test antigens LTh, the A, A1, A2, and B polypeptides of LTh, the heat-labile enterotoxin from an E. coli strain of porcine origin, and cholera toxin. The monoclonal antibodies were also tested for isotype and ability to neutralize LTh. Two of the anti-LTh monoclonal antibodies cross-reacted with cholera toxin, and six were specific for determinants of LTh that were not present on cholera toxin. One was specific for a unique epitope of LTh that was not shared by the heat-labile enterotoxin from an E. coli strain of porcine origin or cholera toxin. Four antibodies specific for epitopes on the B subunit of LTh (LTh-B) reacted with pentameric LTh-B but did not react in Western blots with monomeric LTh-B. The remaining four antibodies were specific for epitopes on LTh-A; two of these antibodies bound to A1, one reacted with A2, and one recognized only intact LTh-A. Only one monoclonal antibody had detectable neutralizing activity, and it was specific for LTh-A.
Frequency of gene sequences necessary for pyelonephritis-associated pili expression among isolates of Enterobacteriaceae from human extraintestinal infections
Authors: Hull R A , Hull S I , Falkow S
Published: January 2020

Bacteria representing eight genera of the Enterobacteriaceae were collected from human extraintestinal infections and examined to determine whether they contained gene sequences necessary for expression of a pili type often associated with bacteria causing human pyelonephritis. Escherichia coli isolated from most of the extraintestinal sites were frequently found to possess pap-related DNA sequences. Isolates which possessed these sequences were often found to exhibit D-mannose-resistant hemagglutination of human erythrocytes and to have surface antigens related to P pili.
Vero cell invasiveness of Proteus mirabilis
Authors: Peerbooms P G , Verweij A M , MacLaren D M
Published: January 2020

Vero cell invasiveness was studied for a group of Proteus mirabilis strains isolated from the urinary tract and feces and for a limited group of urinary isolates of Escherichia coli. Experimental conditions affecting this invasiveness were studied. All of the P. mirabilis strains tested were capable of cell invasion, whereas none of the E. coli strains was. Correlation between the hemolytic activity of the P. mirabilis strains and their invasive ability suggested that the bacterial hemolysin may be involved in the invasion process. Other experimental evidence supporting this hypothesis is discussed. The differences in the invasive capacities of P. mirabilis and of E. coli may be important for the apparent differences in the pathogenesis of urinary tract infection by both species.
Oxygen-independent killing of Bacteroides fragilis by granule extracts from human polymorphonuclear leukocytes
Authors: Wetherall B L , Pruul H , McDonald P J
Published: January 2020

Granule proteins from human neutrophils were prepared by extraction with acetate, and their antibacterial activity against Bacteroides fragilis was determined. Activity was highly dependent on pH; greatest killing occurred at the most acid pH tested (pH 5.0). Optimum activity was observed at physiological ionic strength and low bacterial numbers. Killing was inhibited by incubation temperatures of less than 37 degrees C. Eight times more extract was required to kill 50% of stationary-phase bacteria, compared with those growing in logarithmic phase. The antibacterial effect of granule extract was destroyed by boiling, but some activity was retained after heating to 56 degrees C and 80 degrees C. Granule extract activity was tested under conditions in which oxygen-dependent antibacterial systems were inhibited. The rate and extent of killing was not affected by anaerobiosis, sodium azide, or cysteine hydrochloride. These results suggest that the activity of granule extract is independent of oxidative antibacterial systems, and therefore, under conditions that occur in anaerobic infections, potent leukocyte granule-associated mechanisms exist for the destruction of B. fragilis.
Absence of Mycoplasma pneumoniae cytadsorption protein P1 in Mycoplasma genitalium and Mycoplasma gallisepticum
Authors: Baseman J B , Drouillard D L , Leith D K , Tully J G
Published: January 2020

Polyclonal and monoclonal antibodies to Mycoplasma pneumoniae protein P1 were nonreactive with whole-cell or soluble preparations of M. genitalium and M. gallisepticum. However, radioimmunoprecipitation performed with hyperimmune rabbit sera raised against each mycoplasma species indicated antigenic cross-reactivity between M. pneumoniae and M. genitalium.
Production of diphtheria toxin by selected isolates of Corynebacterium ulcerans and Corynebacterium pseudotuberculosis
Authors: Wong T P , Groman N
Published: January 2020

We determined the diphtheria toxin phenotype of specially selected isolates of Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (C. ovis). All produced proteins similar in size and immunological structure to diphtheria toxin. As with diphtheria toxin, they exhibited ADP-ribosylating activity, and their synthesis was regulated by iron.
Cloning and expression in Escherichia coli of the streptolysin O determinant from Streptococcus pyogenes: characterization of the cloned streptolysin O determinant and demonstration of the absence of substantial homology with determinants of other thiol-activated toxins
Authors: Kehoe M , Timmis K N
Published: January 2020

A gene bank of Streptococcus pyogenes Richards was constructed in Escherichia coli by using the bacteriophage replacement vector lambda L47.1, and hybrid phage expressing streptolysin O (SLO) were identified among the recombinants. DNA sequences encoding SLO were subcloned from an slo+ hybrid phage into a low-copy-number vector plasmid to yield an slo+ hybrid plasmid, pMK157. This plasmid contains 5.6 kilobase pairs of cloned streptococcal DNA sequences, is stable, and expresses SLO at easily detectable levels in E. coli. Transposon gamma delta insertion mutants and in vitro-generated deletion mutants of pMK157 were isolated and analyzed. This analysis showed that a single gene is sufficient for production of SLO in E. coli and allowed this slo gene to be mapped to within +/- 100 base pairs. Two forms of the slo gene product, with molecular weights of 68,000 and 61,000, were detected in E. coli minicells harboring slo+ plasmids and by immunoblotting of E. coli whole cells harboring slo+ plasmids. Southern blotting hybridization experiments with the cloned SLO DNA sequences as probes failed to demonstrate homology between the cloned SLO determinant and DNA isolated from bacteria expressing thiol-activated cytolysins related to SLO.
Extracellular metabolism of thyroid hormones by stimulated granulocytes
Authors: Rao M K , Sagone A L
Published: January 2020

Stimulated polymorphonuclear leukocytes have been shown by this laboratory to release a reactive-oxygen species (ROS) which is detectable in the supernatant and is capable of oxidizing reduced glutathione and reacting with methionine (Sagone et al., Blood 63:96-104, 1984). This ROS is dependent on H2O2 and heme for its production and is postulated to be a stable oxidant derived from hypochlorous acid, such as a chloramine. Further, this ROS was also shown to be able to oxidize and fix iodide to protein. This latter characteristic was the theoretical basis for our present study in which the same ROS was shown to be able to carry out the iodination of 3,3,5'-triiodothyronine to thyroxine in the presence of I-. Our results provide further support that granulocytes have a role in the peripheral utilization of thyroid hormones in patients with infectious diseases or other illnesses in which granulocytes may be activated, and our results indicate that the reactions may occur extracellularly.
Characterization of antigens specific to the surface of germ tubes of Candida albicans by immunofluorescence
Authors: Sundstrom P M , Kenny G E
Published: January 2020

To characterize germ tube-specific antigens of Candida albicans, rabbit antiserum prepared to Formalin-treated yeast possessing germ tubes was adsorbed with stationary-phase blastospores. By immunofluorescence and enzyme-linked immunosorbent assay, this antibody did not react with blastospores but detected germ tube-specific antigens in hyphal forms. Germ tube-specific antigens appeared 30 min after placing blastospores in appropriate conditions for germ tube formation. Hyphae, formed by allowing yeast to germinate for 24 h, still retained germ tube-specific antigens, but blastospores budding off these hyphae were unstained, as were log-phase blastospores. Germ tube-specific antigens were sensitive to heat, sodium metaperiodate oxidation, dithiothreitol reduction, and proteolysis with pronase, trypsin, or chymotrypsin, whereas antigens common to blastospores and germ tubes were stable to boiling, treatment with proteolytic enzymes, and dithiothreitol reduction. Thus, surfaces of germ tubes can be distinguished from those of blastospores not only immunologically, but also by the sensitivity of germ tube-specific antigens to proteolytic treatments.
Prevention of Epstein-Barr virus-induced B-cell outgrowth by interferon alpha
Authors: Garner J G , Hirsch M S , Schooley R T
Published: January 2020

An in vitro system for determining the efficacy of interferon alpha (IFN-alpha) in preventing B-cell outgrowth due to Epstein-Barr virus (EBV) was developed. Unfractionated cord blood mononuclear cells, T-cell-depleted cord blood mononuclear cells, or adult T-cell-depleted mononuclear cells were exposed to IFN-alpha for 18 to 20 h followed by incubation with the B95-8 strain of EBV for 2 h. B-cell outgrowth was monitored by microscopic examination, [3H]thymidine incorporation, and Epstein-Barr nuclear antigen detection. Cell density and viral inoculum both affected the sensitivity of outgrowth to IFN-alpha. IFN-alpha was most effective when added at each feeding after infection as well as before infection with EBV. The mean of the lowest IFN-alpha concentration tested at which transformation failed to occur after infection with the B95-8 strain of EBV at a 50% transforming dose of 10(2.0) to 10(3.0)/ml was similar for unfractionated cord blood mononuclear cells, T-cell-depleted cord blood mononuclear cells, and adult T-cell-depleted mononuclear cells. The B95-8 strain and clinical EBV isolates required similar IFN-alpha concentrations to prevent outgrowth. In this system, IFN-alpha at pharmacologically achievable concentrations prevented EBV-induced B-cell outgrowth. These data indicate that IFN-alpha deserves further study as a potential therapeutic agent for EBV-induced syndromes.
Campylobacter jejuni diarrhea model in infant chickens
Authors: Sanyal S C , Islam K M , Neogy P K , Islam M , Speelman P , Huq M I
Published: January 2020

To study the pathogenic mechanisms of Campylobacter jejuni infection, 36- to 72-h-old chickens were fed 10(3) to 10(6) live cells, using strains isolated from 40 patients with watery diarrhea and 6 with bloody mucoid diarrhea from whom no other known enteropathogen was detected. Chickens of Starbro strain were more likely to develop C. jejuni-induced diarrhea than were White Leghorn chickens. Diarrhea was defined on the basis of amounts of gut fluid in 288 chicks fed with live C. jejuni versus 183 saline-fed control as an accumulation greater than or equal to 0.4 ml of fluid in the guts (excluding ceca) of chickens. Twenty-five percent of the chickens developed diarrhea on day 2, 49% on day 4, and 81% on day 5. The intestines, including ceca, were distended with watery fluid. The majority of the strains, irrespective of whether they were isolated from watery or bloody mucoid enteritis patients, caused watery diarrhea in chickens, and a few caused mucoid diarrhea. No correlation was observed between the source of a strain and the outcome in the experimental model. Bloody diarrhea was never observed in chickens. The peak incidence of diarrhea on day 5 coincided with the mean of maximum fluid accumulation. The organisms multiplied by 3 to 4 logs in all parts of the intestine, with a steady increase in number until day 5. Systemic invasion occurred frequently: C. jejuni could be recovered from the spleen in 47% of the chickens on day 5, in 25% from the liver on day 6, and in 11% from heart blood on day 4. Histopathological examination of gut tissue of the chickens having watery diarrhea did not reveal any abnormality except slight submucosal edema. However, in chickens with mucoid diarrhea, the organisms were found to adhere to brush borders and penetrate into the epithelial cells with formation of a breach in continuity of the brush border lining. The electrolyte composition of the intestinal fluid from chickens infected with C. jejuni and from saline-fed controls did not show significant differences, except for depletion of K+ in the test group. The results obtained in this highly reproducible chicken diarrhea model indicate that (i) most chickens develop nonexudative watery diarrhea 2 to 5 days after oral feeding of 10(3) to 10(6) live cells of C. jejuni; (ii) the organism multiples in all parts of a chicken intestine, (iii) systemic invasion is common, and (iv) local invasion is sometimes observed.
Qualitative detection of muramic acid in normal mammalian tissues
Authors: Sen Z , Karnovsky M L
Published: January 2020

Rat tissues were extracted with 8% trichloroacetic acid, and the products were hydrolyzed with hydrochloric acid and purified. Fluorescamine derivatives were made and subjected to thin-layer chromatography; material with an Rf corresponding to the authentic muramyl derivative was obtained. It was oxidized with periodate, and the resulting formaldehyde was identified fluorimetrically. Alternatively, treatment with base released D-lactate (beta-elimination), which was identified fluorimetrically by reduction of NAD to NADH with D-lactate dehydrogenase. The data indicate that small muramyl compounds, presumably peptides of bacterial origin, are normally present in rat liver, brain, and kidney. The functions of muramyl compounds are suggested by much recent work.
Serum resistance encoded by colicin V plasmids in Escherichia coli and its relationship to the plasmid transfer system
Authors: Nilius A M , Savage D C
Published: January 2020

Eight colicin V plasmids were conjugated into a plasmidless Escherichia coli (K-12) strain that was susceptible to the bactericidal effects of normal rabbit serum. The resulting colicin V-positive strains were examined for their capacity to resist the lethal effects of serum. Serum resistance was assessed as growth of the bacterial strain in medium containing 5% normal rabbit serum inoculated from a culture in the early exponential phase. Only three of the eight colicin V plasmids were found to confer the serum resistance phenotype on the host strain. Derepression of the transfer system was associated with serum resistance in two of the plasmids. Two other derepressed plasmids did not confer serum resistance on the host bacterium. Therefore, such derepression alone was insufficient to produce serum resistance. The factor(s) encoded by colicin V plasmids and responsible for the serum resistance of the bacterial strains bearing the plasmids was shown to be a property associated with the cell and not an extracellular factor excreted into the growth medium.
Monoclonal antibody that recognizes an outer membrane antigen common to the pathogenic Neisseria species but not to most nonpathogenic Neisseria species
Authors: Cannon J G , Black W J , Nachamkin I , Stewart P W
Published: January 2020

A hybridoma derived from a mouse immunized with gonococcal outer membranes produced an antibody, designated H.8, that bound to all strains of Neisseria gonorrhoeae and N. meningitidis tested, and to N. lactamica and N. cinerea, but only rarely to other nonpathogenic Neisseria species. Studies with the gonococcal strain used in production of the antibody showed that the antibody bound to a surface-exposed, protease-sensitive, and heat-modifiable outer membrane antigen that we believe is distinct from previously described gonococcal outer membrane proteins.
Type-specific capsular antigen is associated with virulence in late-onset group B Streptococcal type III disease
Authors: Klegerman M E , Boyer K M , Papierniak C K , Levine L , Gotoff S P
Published: January 2020

Strain differences have been postulated to explain the observation that group B Streptococcus type III (GBS III) late-onset disease occurs in only a fraction of colonized infants. To determine the distribution of type-specific polysaccharide antigen (Ag) in GBS III, Ag was measured by rocket immunoelectrophoresis in both supernatant fluids and EDTA extracts and by radial immunodiffusion in multiple HCl extracts of the pellet from cultures of 10 strains of GBS III. Capsular Ag was defined as the sum of Ag in EDTA extracts + Ag in multiple HCl extracts. Both Ag in EDTA extracts and Ag in supernatant fluids correlated with capsular Ag (r = 0.94). GBS III strains were obtained from the blood of 19 infants with late-onset sepsis, from the cerebrospinal fluid or blood of 22 infants with late-onset meningitis, and from mucosal surfaces of both 18 infants and 12 mothers of infants with low levels of type-specific antibody and asymptomatic colonization. Mean values of Ag in supernatant fluids in strains from infants with late-onset sepsis (1.50 +/- 0.08 micrograms/ml) and late-onset meningitis (1.67 +/- 0.09 micrograms/ml) were significantly greater than those in asymptomatic colonization strains (1.14 +/- 0.05 micrograms/ml; P less than 0.001). The number of organisms required for a 50% lethal dose in the chick embryo, determined in 29 strains, was inversely related to Ag in supernatant fluids (r = -0.60). The demonstration that the quantity of capsular Ag produced by GBS III strains is related to their virulence in chick embryos and to their invasiveness in susceptible infants supports the hypothesis that Ag is a virulence factor in humans.
Plasmodium knowlesi: persistence of transmission blocking immunity in monkeys immunized with gamete antigens
Authors: Gwadz R W , Koontz L C
Published: January 2020

Eight rhesus monkeys immunized with a partially purified preparation of Plasmodium knowlesi gametes were monitored for over 6 years to determine the extent of transmission blocking immunity. Monkeys were challenged regularly, and anti-gamete antibodies were assayed by in vivo and in vitro mosquito feedings. Transmission blocking immunity persisted at high levels in most of the monkeys. In those animals in which protection waned between challenges, a challenge infection provided a sufficient booster effect to prevent infection of mosquitoes. Immunity to other stages of malaria (i.e., sporozoites and asexual erythrocyte forms) failed to induce immunity against gametes.
Immunogenic proteins in cell-free culture supernatants of Haemophilus influenzae type b
Authors: Gulig P A , McCracken G H , Holmans P L , Hansen E J
Published: January 2020

Cell-free culture supernatant (CFCS) prepared from Haemophilus influenzae type b (Hib) was examined for the presence of soluble Hib proteins. Two proteins with apparent molecular weights of 100,000 (100K) and 116K were predominant in the CFCS, and antibodies directed against these proteins could be detected by radioimmunoprecipitation or Western blot analyses of serum from adult rats immunized with Hib. Radioimmunoprecipitation analyses and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these two proteins demonstrated that the 100K CFCS protein was also present on the cell surface of Hib, whereas the 116K CFCS protein was only detectable in culture supernatants. Both the 100K and 116K CFCS proteins were immunogenic in human infants with Hib meningitis and in infant rats systemically infected with Hib. In addition, the first detectable antibodies produced in these Hib-infected rats against Hib proteins were specific for the 100K protein in both its CFCS and cell-associated forms. These two CFCS proteins were also immunogenic in rats immunized with CFCS in the absence of Hib infection. Monoclonal antibody directed against the 100K protein reacted with 34 of 55 Hib strains examined by using a colony blot radioimmunoassay. The immunogenicity of the 100K and 116K CFCS proteins suggests that one or both of these proteins may have potential for vaccine development, either by themselves or covalently coupled to Hib capsular polysaccharide.