bluuebunny/crossref_metadata_2025 huggingface dataset:
Inflammatory effects of Salmonella typhimurium porins
Authors: Galdiero F , Tufano M A , Galdiero M , Masiello S , Di Rosa M
Published: January 2020

The inflammatory activity of porins purified from Salmonella typhimurium has been investigated. Porins (0.3 to 30 micrograms) injected into the rat paw induced a dose-related edema that was not due to lipopolysaccharide contamination and did not appear to be dependent on the activation of the complement system. The edema induced by 30 micrograms of porins was comparable to that caused by 1 mg of carrageenin and was inhibited by indomethacin (5 mg/kg) and dexamethasone (0.1 mg/kg). Porins (1 to 10 micrograms/ml) induced a concentration-related release of histamine from rat peritoneal cells. These results are discussed in the light of the possible pathogenic role of porins in infections.
Selection of alpha/beta interferon- and gamma interferon-resistant rickettsiae by passage of Rickettsia prowazekii in L929 cells
Authors: Turco J , Winkler H H
Published: January 2020

The ability of endogenously produced alpha/beta interferon (IFN-alpha/beta) to inhibit rickettsial growth in infected L929 cell cultures was evaluated by comparing the growth of Rickettsia prowazekii Madrid E in untreated cultures and cultures treated with anti-mouse IFN (alpha + beta) serum. The endogenously produced IFN was neutralized, and rickettsial growth was enhanced in the antiserum-treated cultures. This inhibitory effect of endogenously produced IFN-alpha/beta was used to select rickettsiae resistant to IFN-alpha/beta. Rickettsiae were screened for resistance to IFN-alpha/beta after being cultured in untreated L929 cells for several weeks to several months. Two isolates derived from R. prowazekii Madrid E and two isolates derived from plaque-purified R. prowazekii Madrid E were plaque-purified twice, grown in embryonated hen eggs, and evaluated for resistance to IFN-alpha/beta and IFN-gamma. Compared with the parental rickettsial strain, all four isolates were significantly resistant to IFN-alpha/beta and IFN-gamma. In addition, they were as resistant or more resistant to IFN-gamma when compared with two previously described IFN-gamma resistant isolates that were selected in IFN-gamma-treated L929 cells. One of the two isolates from IFN-gamma-treated L929 cells was also resistant to IFN-alpha/beta; the other isolate was similar to the parental Madrid E strain in sensitivity to IFN-alpha/beta.
Association of rns homologs with colonization factor antigens in clinical Escherichia coli isolates
Authors: Caron J , Maneval D R , Kaper J B , Scott J R
Published: January 2020

rns is a trans-acting positive regulatory factor required for expression of the colonization factor antigen II (CFA/II) antigens CS1 and CS2 (J. Caron, L. M. Coffield, and J. R. Scott, Proc. Natl. Acad. Sci. USA 86:963-967, 1989). All 35 CFA/II-positive strains hybridized with a rns gene probe, as did all 10 CFA/I strains and all 4 CS4 strains. Hybridization with rns was detected in 25% of non-enterotoxigenic Escherichia coli strains and was not detected in enteric pathogens with low G + C content.
Partial biochemical characterization of cell surface hydrophobicity and hydrophilicity of Candida albicans
Authors: Hazen K C , Lay J G , Hazen B W , Fu R C , Murthy S
Published: January 2020

Hydrophobic yeast cells of Candida albicans are more virulent than hydrophilic yeast cells in mice. Results of experiments performed in vitro suggest that surface hydrophobicity contributes to virulence in multiple ways. Before definitive studies in vivo concerning the contribution of fungal surface hydrophobicity to pathogenesis can be performed, biochemical, physiological, and immunochemical characterization of the macromolecules responsible for surface hydrophobicity must be accomplished. This report describes our initial progress toward this goal. When hydrophobic and hydrophilic yeast cells of C. albicans were exposed to various enzymes, only proteases caused any change in surface hydrophobicity. Hydrophobic cell surfaces were sensitive to trypsin, chymotrypsin, pronase E, and pepsin. This indicates that surface hydrophobicity is due to protein. Papain, however, had no significant effect. The hydrophobicity of hydrophilic cells was altered only by papain. The proteins responsible for surface hydrophobicity could be removed by exposure to lyticase, a beta 1-3 glucanase, for 30 to 60 min. When 60-min lyticase digests of hydrophobic and hydrophilic cell walls were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 12.5% resolving gel, each protein population contained a single unique protein that was not evident in the other protein population. However, when the cell wall surface proteins of hydrophobic and hydrophilic cells were first labeled with 125I and then removed by lyticase and analyzed by SDS-PAGE, at least four low-molecular-mass (less than 65 kilodaltons) proteins associated with hydrophobic cells were either absent or much less abundant in the hydrophilic cell digests. This result was seen for both C. albicans strains that we tested. When late-exponential-phase hydrophilic cells were treated with tunicamycin, high levels of surface hydrophobicity were obtained by stationary phase. These results indicate that the surface hydrophobicity of C. albicans reflects changes in external surface protein exposure and that protein mannosylation may influence exposure of hydrophobic surface proteins.
Hyperoxia potentiates Ureaplasma urealyticum pneumonia in newborn mice
Authors: Crouse D T , Cassell G H , Waites K B , Foster J M , Cassady G
Published: January 2020

The effect of continuous exposure to 80% oxygen on newborn mice with Ureaplasma urealyticum pneumonia was determined. Mice were inoculated intranasally with either U. urealyticum or sterile broth and then housed in either 80% oxygen or room air (21% oxygen). The mice were sacrificed at either 7 or 14 days after inoculation. Significantly more mice in the U. urealyticum group housed in 80% O2 than in the room air-exposed group were culture positive 14 days after inoculation (P = 0.042), but no difference was found at 7 days. The presence of alveolar macrophages, neutrophils, and lymphocytes and alveolar wall thickness were determined. Overall, the group housed in 80% O2 and inoculated with U. urealyticum had severe pulmonary lesions at both time points, while the lesion severity in the room air-exposed group inoculated with U. urealyticum and the group housed in 80% O2 and inoculated with sterile broth was dependent on the time point. Mortality was significantly higher in the group housed in 80% O2 and inoculated with U. urealyticum than it was in all other groups (P less than 0.001). Our results indicate that hyperoxia causes the persistence of U. urealyticum in the lungs of newborn mice, acutely potentiates the inflammatory response, and turns an otherwise self-limited pneumonia into a lethal disease.
Activation of cholera toxin-specific T cells in vitro
Authors: Elson C O , Solomon S
Published: January 2020

Cholera toxin (CT) and its B subunit (CT-B) are potent oral immunogens in vivo, although both strongly inhibit polyclonal lymphocyte activation in vitro. In order to help understand this paradox, we have studied the activation and proliferation of CT-specific T cells in vitro, by using CT-B-primed lymph node T cells as responders, concanavalin A-stimulated peritoneal macrophages as antigen-presenting cells (APCs), and various forms of CT-B as antigen. The results indicate that in many ways CT-specific T cells respond in a manner similar to that of T cells specific for other protein antigens: the degree of proliferation was proportional to the dose of antigen and APCs in the cultures, was antigen specific, and was H-2 restricted. APCs from genetic high-responder strains to CT stimulated significantly more proliferation in F1 (high x low) responder T cells than did APCs from low responder strains. However, there was a marked difference in the activation of CT-specific T cells when different forms of CT-B were used. Native CT-B stimulated little or no T-cell proliferation, whereas denatured CT-B or CT-B blocked by its ligand, GM1 ganglioside, stimulated T cells well. Addition of native CT-B to cocultures of primed T cells, APCs, and these latter stimulatory forms of CT-B inhibited the specific proliferative response to CT-B to varying degrees, depending on the ratio of the two forms in culture. We conclude that the ability of CT-B to inhibit T cells extends even to T cells specific for CT itself. Because of these inhibitory properties, processing of CT to nonbinding molecular forms or fragments must be an important prerequisite for the immune response to CT to occur in vivo, and such processing is likely to be important in the immune response to a variety of other enterotoxins as well.
Humoral antibody response and protective immunity in swine following immunization with the 104-kilodalton hemolysin of Actinobacillus pleuropneumoniae
Authors: Devenish J , Rosendal S , Bossé J T
Published: January 2020

Five cesarean-derived, colostrum-deprived pigs were given three adjuvant-supplemented subcutaneous and one intravenous injection of the purified 104-kDa hemolysin from serotype 1 Actinobacillus pleuropneumoniae CM-5. Six control animals received phosphate-buffered saline only. Five of six control pigs died within 24 h after challenge. The sixth control pig was moribund and euthanized after 48 h. All six pigs had pleuropneumonia, and A. pleuropneumoniae was isolated from all six lungs. None of the vaccinated pigs died as a result of challenge. After being euthanized, two pigs in this group had no lung lesions but three had chronic pleuropneumonia involving 10, 20, and 40% of the lung tissue. A. pleuropneumoniae was isolated from lung lesions of these three animals but not from the two pigs without lesions. The prechallenge hemolysin-neutralizing antibody titers in the vaccinated pigs were 1:10,900, 1:10,600, 1:4,800, 1:3,900, and 1:3,000, in order of increasing lung involvement. None of the control pigs had neutralizing antibodies. Enzyme-linked immunosorbent assay (ELISA) antibodies to capsule, lipopolysaccharide, and hemolysin were not detected in serum samples collected from the control pigs. In the vaccinated group, prechallenge sera did not contain ELISA antibodies to capsule or lipopolysaccharide. ELISA antibodies to the hemolysin were detected only in the prechallenge and postchallenge serum samples. These results indicate that pigs immunized with the 104-kDa hemolysin of serotype 1 A. pleuropneumoniae are protected against challenge with virulent bacteria. The association between neutralizing antibodies and protection indicates indirectly that the hemolysin is an important virulence factor.
Lymphocyte receptors for pertussis toxin
Authors: Clark C G , Armstrong G D
Published: January 2020

We have investigated human T-lymphocyte receptors for pertussis toxin by affinity isolation and photoaffinity labeling procedures. T lymphocytes were obtained from peripheral human blood, surface iodinated, and solubilized in Triton X-100. The iodinated mixture was then passed through pertussis toxin-agarose, and the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of the fixed, dried gels revealed several bands in the pertussis toxin-bound fraction that were not observed in fractions obtained from histone or fetuin-agarose. Further investigations employed a photoaffinity labeling reagent, sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate, to identify pertussis toxin receptors in freshly isolated peripheral blood monocytic cells, T lymphocytes, and Jurkat cells. In all three cell systems, the pertussis toxin affinity probe specifically labeled a single protein species with an apparent molecular weight of 70,000 that was not observed when the procedure was performed in the presence of excess unmodified pertussis toxin. A protein comparable in molecular weight to the one detected by the photoaffinity labeling technique was also observed among the species that bound to pertussis toxin-agarose. The results suggest that pertussis toxin may bind to a 70,000-Da receptor in human T lymphocytes.
Establishment of a foreign antigen secretion system in mycobacteria
Authors: Matsuo K , Yamaguchi R , Yamazaki A , Tasaka H , Terasaka K , Totsuka M , Kobayashi K , Yukitake H , Yamada T
Published: January 2020

In order to develop recombinant Mycobacterium bovis BCG into a useful multivaccine vehicle, we established a foreign antigen secretion system in mycobacteria in which an extracellular alpha antigen of Mycobacterium kansasii was utilized as a carrier. By using this system, a B-cell epitope (Glu-12-Leu-Asp-Arg-Trp-Glu-Lys-Ile-19) of human immunodeficiency virus type 1 p17gag, which was identified by a fusion protein-based method, has been successfully obtained from BCG along with the alpha antigen. This is the first report of expression and secretion of a foreign viral antigen from BCG. It is possible that the system can become a universal vaccination vehicle applicable to protection against various infectious diseases.
Two-step processing for activation of the cytolysin/hemolysin of Vibrio cholerae O1 biotype El Tor: nucleotide sequence of the structural gene (hlyA) and characterization of the processed products
Authors: Yamamoto K , Ichinose Y , Shinagawa H , Makino K , Nakata A , Iwanaga M , Honda T , Miwatani T
Published: January 2020

Vibrio cholerae O1 biotype El Tor produces and secretes a 65-kDa cytolysin/hemolysin into the culture medium. We cloned the structural gene (hlyA) for the cytolysin from the total DNA of a V. cholerae O1 El Tor strain, N86. Nucleotide sequence analysis of hlyA revealed an open reading frame consisting of 2,223 bp which can code for a protein of 741 amino acids with a molecular weight of 81,961. Consistent with this, a 79-kDa protein was identified as the product of hlyA by maxicell analysis in Escherichia coli. N-terminal amino acids of this 79-kDa HlyA protein and those of a 65-kDa El Tor cytolysin purified from V. cholerae were Asn-26 and Asn-158, respectively. The 82- and 79-kDa precursors of the 65-kDa mature cytolysin were found in V. cholerae by pulse-chase labeling and Western blot (immunoblot) analysis of hlyA products. Hemolytic activity of the 79-kDa HlyA protein from E. coli was less than 5% that for the 65-kDa cytolysin from V. cholerae. Our results suggest that in V. cholerae, the 82-kDa preprotoxin synthesized in the cytoplasm is secreted through the membranes into the culture medium as the 79-kDa inactive protoxin after cleavage of the signal peptide and is then further processed into the 65-kDa active cytolysin by release of the N-terminal 15-kDa fragment.
Conservation of Neisseria gonorrhoeae pilus expression regulatory genes pilA and pilB in the genus Neisseria
Authors: Taha M K , Marchal C
Published: January 2020

The pili of Neisseria gonorrhoeae mediate bacterial adhesion to the host-susceptible tissues. We have previously reported the identification of two genes, pilA and pilB, which act in trans to regulate pilus expression. Besides this regulatory function, pilA participates in an essential function for bacterial viability. Here we show that pilA and pilB homologs are also present in a variety of other members of the Neisseriaceae family of bacteria in contrast to the gonococcal pilin gene which hybridizes only to the pathogenic Neisseria species.
Purification and characterization of a protease produced by Vibrio mimicus
Authors: Chowdhury M A , Miyoshi S , Shinoda S
Published: January 2020

A protease produced by Vibrio mimicus was purified to apparent homogeneity by ammonium sulfate fractionation and successive column chromatography on Sephacryl S-100 and Mono Q Monobeads. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) of the final preparation of the enzyme revealed the homogeneity of the purified enzyme. Conventional PAGE showed that the purified protease migrated as a single band with protease activity. The molecular weight of the protease was estimated to be about 31,000 on the basis of its mobility on sodium dodecyl sulfate-PAGE. The purified protease had both proteolytic and hemagglutination (HA) activities. The proteolytic and HA activities were inhibited by metalloprotease inhibitors and heat treatment. V. mimicus protease therefore appeared as a heat-labile, bifunctional molecule capable of mediating proteolysis and HA. The immunodiffusion analysis showed that the proteases produced by Vibrio cholerae and V. mimicus are immunologically cross-reactive.
Colloidal gold immunolabeling of immunoglobulin-binding sites and beta antigen in group B streptococci
Authors: Coleman S E , Brady L J , Boyle M D
Published: January 2020

We have characterized the immunoglobulin A (IgA)-Fc-binding properties and beta-antigen expression of several strains of group B streptococci by using ultrastructural immunocytochemistry. Colloidal gold-labeled tracers were used with intact and sectioned bacteria in order to gain information regarding the location and distribution of cell surface and cytoplasmic IgA-Fc-binding molecules and beta antigen. Colloidal gold (5- or 15-nm particles) was conjugated to IgA to characterize IgA-binding properties and to IgG to test for IgG binding. Rabbit anti-beta antiserum was reacted with the bacteria and then with protein G labeled with 15-nm colloidal gold particles. A double-labeling technique was used for simultaneous localization of IgA-Fc- and anti-beta-antibody-binding properties on sectioned bacteria. The data corroborated previous results which indicated that (i) IgA-Fc-binding and IgA-Fc-nonbinding forms of beta antigen can be secreted by strains which do not express beta antigen on the cell surfaces (HG806, VC75); (ii) differences in levels of expression of beta antigen and/or IgA-Fc-binding proteins can be detected among various group B isolates; (iii) group B streptococci do not express human IgG-Fc-binding proteins; and (iv) not all forms of beta antigen are capable of binding human IgA.
The 34-kilodalton membrane immunogen of Treponema pallidum is a lipoprotein
Authors: Swancutt M A , Radolf J D , Norgard M V
Published: January 2020

Treponema pallidum subsp. pallidum and Escherichia coli incorporated exogenous [3H]palmitate into the 34-kilodalton (kDa) pathogen-specific antigen of T. pallidum. Radiolabeled fatty acid remained associated with the protein upon immunoprecipitation and after boiling in sodium dodecyl sulfate, acetone precipitation, and extensive extractions in organic solvents, suggesting that the fatty acid was covalently bound to the protein. Detection of [3H]palmitate after alkaline and acid hydrolyses confirmed the identity of the incorporated label. Globomycin inhibited maturation of the recombinant 34-kDa antigen, suggesting that E. coli uses the lipoprotein-specific signal peptidase II to process the treponemal antigen. Globomycin also inhibited processing of the 34-kDa antigen, as well as the 44.5- and 15-kDa antigens, in T. pallidum, implying that T. pallidum also possesses the lipoprotein export pathway common to both gram-negative and gram-positive bacteria. Ethanol inhibited processing of the 34-kDa antigen in minicells, suggesting that the 34-kDa antigen normally is translocated through the cytoplasmic membrane. Comparison of the Triton X-114 phase partitioning behavior of the 34-kDa antigen produced either by minicells or by a cell-free translation system indicated that the covalent attachment of fatty acid conferred hydrophobic biochemical properties to the 34-kDa antigen, consistent with the hypothesis that the attached lipid anchors the 34-kDa antigen into the membrane.
Experimental infection with Chlamydia pneumoniae in nonhuman primates
Authors: Holland S M , Taylor H R , Gaydos C A , Kappus E W , Quinn T C
Published: January 2020

To serially examine the immunopathogenesis and histopathology of infection with Chlamydia pneumoniae, we inoculated two cynomolgus monkeys in the conjunctival sac, nose, and nasopharynx with C. pneumoniae TWAR. After inoculation, C. pneumoniae was isolated from the inoculation sites and the rectums of both monkeys for a period of 5 weeks. After a second inoculation, C. pneumoniae was recovered from the inoculation sites and the rectums of both monkeys for 20 weeks. A third inoculation with C. pneumoniae caused very little productive infection at any site. Prior C. pneumoniae infection did not prevent subsequent C. trachomatis serovar E (Bour strain) infection. Clinical and histopathologic ocular responses to C. pneumoniae infection were mild compared with those to infection with C. trachomatis serovar E. Rectal infection, demonstrated by culture isolation and immunohistopathology, occurred without direct experimental inoculation. Both immunofluorescent staining of mucosal smears with monoclonal antibodies and tissue culture were able to detect C. pneumoniae infection. Experimental nonhuman primate infection with C. pneumoniae appears to be clinically and histopathologically mild and can occur at extrapulmonary sites.
Characterization of and human serologic response to proteins in Helicobacter pylori broth culture supernatants with vacuolizing cytotoxin activity
Authors: Cover T L , Dooley C P , Blaser M J
Published: January 2020

Helicobacter pylori infection is strongly associated with histologic gastritis and peptic ulcer disease. Broth culture supernatants from a subset of H. pylori strains induce vacuolization in cultured cells, a phenomenon that has been attributed to cytotoxin activity. Concentrated culture supernatants from 15 of 28 (53.6%) H. pylori strains tested induced vacuolization in HeLa cells in titers ranging from 1:10 to 1:180. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of supernatants from these 28 strains and 2 control strains demonstrated an 82-kilodalton (kDa) protein band in 3 of 16 supernatants with vacuolizing activity, but in none of 14 supernatants without vacuolizing activity. By immunoblotting with human sera, a 128-kDa band was recognized in all 16 supernatants with vacuolizing activity, compared with 9 of 14 (64%) supernatants without vacuolizing activity (P = 0.014). Serologic recognition of the 128-kDa band in H. pylori culture supernatants was more prevalent among persons infected with vacuolizing H. pylori strains than among persons infected with nonvacuolizing strains, but the difference was not statistically significant (80 versus 45%; P = 0.079); human serologic recognition of the 82-kDa band was less common. The 128-kDa band was recognized by 100% of 31 serum samples from H. pylori-infected patients with duodenal ulcer disease, compared with 60.8% of 74 serum samples from H. pylori-infected persons without peptic ulcer disease (P = 0.0001). These data indicate that antigenic 128- and 82-kDa proteins are present in H. pylori broth culture supernatants with vacuolizing activity and that serologic responses to the 128-kDa protein are more prevalent among H. pylori-infected persons with duodenal ulceration than among infected persons without peptic ulceration.
Mortality and bacteriology of sepsis following cecal ligation and puncture in aged mice
Authors: Hyde S R , Stith R D , McCallum R E
Published: January 2020

Epidemiologic data suggest that elderly adults are more susceptible to invasive bacterial infection by indigenous gut flora than are younger adults. The purpose of this investigation was to characterize a murine model of clinically encountered peritonitis in the aged. We subjected three different age groups (young, 16 weeks; mature, 12 months; senescent, 24 months) of C57BL/6NNia mice to surgically induced peritonitis by the cecal ligation and puncture procedure. Senescent mice died in a significantly shorter time following surgery than mature mice (median time to death, 24.4 versus 38.5 h, respectively; P less than or equal to 0.001). Blood, liver, spleen and occasionally, ceca were obtained at 2 and 12 h after the cecal ligation and puncture procedure and immediately following death, to characterize the bacterial kinetics of the model. Qualitative and quantitative aerobic, anaerobic, and coliform cultures were performed. No age-related differences were found in the types of bacteria isolated throughout the time course of progressive sepsis. In mice in the mature and senescent age groups, at 2 and 12 h postsurgery, gram-negative anaerobes and gram-positive aerobes predominated in all tissues that were cultured. At the time of death, however, blood and tissue isolates consisted predominantly of coliform bacteria. The shift from mixed infection during sepsis to predominantly gram-negative bacterial infection reflected a similar progressive shift in bacterial types found in the cecum. At death, senescent mice had 100-fold fewer coliform bacteria in the bloodstream than those found in mature mice (2.5 x 10(9) versus 4.6 x 10(11), respectively). The increased sensitivity of aged mice to invasive bacterial infection documented in this series of experiments accords well with human epidemiologic experience and demonstrates the appropriateness of the model for continued investigations of sepsis in the aged.
Surface antigens from Escherichia coli O2 and O78 strains of avian origin
Authors: Dho-Moulin M , van den Bosch J F , Girardeau J P , Brée A , Barat T , Lafont J P
Published: January 2020

Fimbriae from O2 and O78 virulent strains of avian Escherichia coli were compared with type 1A fimbriae with regard to the apparent molecular weights of their subunits and their antigenic relationships. Under static broth culture conditions, most O78 strains expressed fimbriae closely related to those of type 1A. Under the same culture conditions, another type of fimbriae, sharing some common properties with type 1A fimbriae, was observed only on O2 strains; however, these fimbriae differed from type 1A fimbriae in the apparent molecular weights of their subunits and in the expression of specific epitopes. They were called type 1-like fimbriae. Homologies in lipopolysaccharide and outer membrane protein profiles were also demonstrated among the strains expressing type 1-like fimbriae, which suggests the existence of a clonal relationship among O2:K1 avian E. coli strains. The O78 strains studied did not appear to be clonally related.
Mucosal and systemic candidiasis in congenitally immunodeficient mice
Authors: Cantorna M T , Balish E
Published: January 2020

Colony counts and light microscopy were used to assess the capacity of Candida albicans to colonize, infect the alimentary tract, and cause disseminated disease in athymic (nu/nu), euthymic (nu/+), beige (bg/bg), black (bg/+), beige athymic (bg/bg nu/nu), or beige euthymic (bg/bg nu/+) germfree mice. The alimentary tracts of all six genotypes of germfree mice were quickly colonized after exposure to yeast-phase C. albicans. Only bg/bg nu/nu mice showed obvious morbidity and mortality after mucosal colonization with C. albicans. Histopathology of C. albicans-colonized immunocompetent (nu/+, bg/+) and singly immunodeficient (nu/nu, bg/bg, bg/bg nu/+) mice showed minimal to moderate mucosal infections, whereas doubly immunodeficient (bg/bg nu/nu) mice showed extensive yeast and hyphal infection of the palate, tongue, esophagus, and stomach. A progressive systemic infection in C. albicans-colonized mice occurred only in bg/bg nu/nu mice 12 to 16 weeks after colonization and mucosal infection. Thus, it appears that a combination of defective cell-mediated immunity and phagocytic cell defects (polymorphonuclear leukocytes and/or macrophages) predisposed mice to severe mucosal and systemic candidiasis of endogenous origin. This is the first report of a mouse strain that is not only naturally susceptible to mucosal and systemic candidiasis of endogenous origin but also shows lethality at early (1 to 4 weeks) and late (12 to 16 weeks) times after alimentary tract colonization.
Effect of enteroviruses on adherence to and invasion of HEp-2 cells by Campylobacter isolates
Authors: Konkel M E , Joens L A
Published: January 2020

Coinfection of HEp-2 epithelial cells with coxsackievirus B3, echovirus 7, poliovirus (LSc type 1), porcine enterovirus, and Campylobacter isolates was performed to determine if a synergistic effect could be obtained. The invasiveness of Campylobacter jejuni ATCC 33560 was significantly increased for HEp-2 cells preinfected with echovirus 7, coxsackievirus B3, and UV-inactivated (noninfectious) coxsackievirus B3 particles. Additionally, the invasiveness of C. jejuni M96, a clinical isolate, was significantly increased for HEp-2 cells preinfected with coxsackievirus B3. Poliovirus and porcine enterovirus had no effect on C. jejuni ATCC 33560 adherence and invasiveness. Furthermore, poliovirus had no effect on the ability of C. jejuni M96 to adhere to and invade HEp-2 cells. Campylobacter hyointestinalis and Campylobacter mucosalis, two noninvasive isolates, did not invade virus-infected HEp-2 cells. The increase in the invasiveness of C. jejuni appeared to be the result of specific interactions between the virus and the HEp-2 cell membrane. The data suggest that the invasiveness of Campylobacter spp. is dependent upon the inherent properties of the organism. Virus-induced cell alterations can potentiate the invasiveness of virulent Campylobacter spp. but are not sufficient to allow internalization of noninvasive bacteria.
Trypanosoma cruzi-induced suppression of human peripheral blood lymphocytes activated via the alternative (CD2) pathway
Authors: Beltz L A , Kierszenbaum F , Sztein M B
Published: January 2020

Coculture of blood forms of Trypanosoma cruzi with human peripheral blood mononuclear cells stimulated with anti-CD2(2) and anti-CD2(3) monoclonal antibodies, i.e., via an antigen-independent pathway of T-lymphocyte activation, resulted in marked immunosuppression compared with that in parallel cultures in which parasites were absent. This effect was evidenced by a decreased lymphoproliferative capacity, a significant reduction in the proportion of cells expressing interleukin-2 receptors, and a significant diminution in the cell surface density of this receptor.
Antibody enhances killing of Tritrichomonas foetus by the alternative bovine complement pathway
Authors: Aydintug M K , Leid R W , Widders P R
Published: January 2020

The role of bovine antibody and complement in host defense against Tritrichomonas foetus was measured by using an assay of trichomonad viability based on protozoal uptake of tritiated adenine. Moderate killing was measured in the absence of antibody only with high concentrations of complement-preserved hypogammaglobulinemic bovine serum. However, very low concentrations of hyperimmune serum promoted significant enhancement (P less than 0.05) of killing by complement. Heat inactivation of complement (56 degrees C for 30 min) eliminated antibody-dependent and -independent killing. Similarly, depletion of bovine factor B in serum by heat treatment (50 degrees C for 45 min) abolished antibody-dependent and -independent killing. However, selective inactivation of the classical complement pathway with magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not affect antibody-dependent or -independent killing by complement. These findings demonstrate antibody enhancement of complement-mediated killing of T. foetus by the alternative pathway of bovine complement.
Identification of a pathogenic isolate-specific 30,000-Mr antigen of Entamoeba histolytica by using a monoclonal antibody
Authors: Tachibana H , Kobayashi S , Kato Y , Nagakura K , Kaneda Y , Takeuchi T
Published: January 2020

A monoclonal antibody (MAb) produced against trophozoites of Entamoeba histolytica strain HM-1:IMSS, reacted with all of 42 isolates and 4 clones showing pathogenic zymodeme (Z) patterns, i.e., Z-II, Z-II alpha-, Z-II (glucose phosphate isomerase: gamma +), Z-VII, Z-VII (glucose phosphate isomerase: alpha lack, gamma +), Z-XI, Z-XIV, and Z-XIX, regardless of culture conditions, geographical origins, or host symptoms in an indirect fluorescence antibody test. In contrast, the MAb failed to react with 14 isolates possessing nonpathogenic zymodemes Z-I and Z-VIII and did not react with other enteric protozoan parasites, such as E. histolytica-like Laredo, Entamoeba hartmanni, Entamoeba coli, Endolimax nana, Dientamoeba fragilis, Trichomonas hominis, and Giardia lamblia. Western immunoblotting analysis showed that the molecular weight of the antigenic component recognized by the MAb was exclusively 30,000 in pathogenic isolates of different zymodemes. These results suggest that the 30,000-molecular-weight antigen is a marker of pathogenic isolates and that the indirect fluorescent-antibody test with the MAb is useful for the accurate discrimination of pathogenic amebae.
Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins
Authors: Brandt M E , Riley B S , Radolf J D , Norgard M V
Published: January 2020

The pathogenic spirochete Borrelia burgdorferi contains a set of integral membrane proteins which were selectively extracted into the detergent phase upon solubilization of intact B. burgdorferi with the nonionic detergent Triton X-114. Virtually all of these hydrophobic proteins were recognized by antibodies in pooled sera from patients with chronic Lyme arthritis, demonstrating that proteins partitioning into the detergent phase of Triton X-114 encompass the major B. burgdorferi immunogens. Furthermore, most of these immunogenic proteins, including the previously characterized OspA and OspB membrane antigens, could be biosynthetically labeled when B. burgdorferi was incubated in vitro with [3H]palmitate. The OspA and OspB antigens were radioimmunoprecipitated from [3H]palmitate-labeled detergent-phase proteins with monoclonal antibodies, and [3H]palmitate was recovered unaltered from these proteins after sequential alkaline and acid hydrolyses. The combined results provide formal confirmation that the major B. burgdorferi immunogens extracted by Triton X-114 are lipoproteins. The demonstration that B. burgdorferi integral membrane antigens are lipoproteins may explain the basis of their immunogenicity and may help to improve our understanding of the surface topology of B. burgdorferi.
Purification and N-terminal analysis of urease from Helicobacter pylori
Authors: Hu L T , Mobley H L
Published: January 2020

Urease of Helicobacter pylori (formerly Campylobacter pylori) is believed to represent a critical virulence determinant for this species. Ammonia generated by hydrolysis of urea may protect the acid-sensitive bacterium as it colonizes human gastric mucosa. An H. pylori strain, cultured from a gastric biopsy of a patient with complaints of abdominal pain and a history of peptic ulcer disease, was isolated on selective medium and cultured in Mueller-Hinton broth supplemented with 4% fetal calf serum. Whole cells were ruptured by French pressure cell lysis, and soluble protein was chromatographed on DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 resins. Purified urease represented 6% of the soluble protein of crude extract, was estimated to have a native molecular size of 550 kilodaltons (kDa), and was composed of two distinct subunits of apparent molecular sizes of 66 and 29.5 kDa. On the basis of subunit size, a 1:1 subunit ratio as measured by scanning densitometry of Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels, and estimated native molecular size, the data are consistent with a stoichiometry of (29.5 kDa-66 kDa)6 for the structure of the native enzyme. Km for urea was estimated at 0.2 mM. By N-terminal analysis, the 29.5-kDa subunit of H. pylori urease was found to share significant amino acid sequence similarity with the smallest of three subunits of the Proteus mirabilis and Morganella morganii ureases, as well as to the amino terminus of the unique jack bean subunit. The 66-kDa subunit also shared up to 80% similarity with the largest of three subunits of P. mirabilis, M. morganii, and Klebsiella aerogenes ureases and to internal sequences (amino acids 271 to 285) of the jack bean urease subunit. Thus, the amino acid sequence is conserved among ureases with one, two, and three distinct subunits, suggesting a common ancestral urease gene. Also, urease subunits of M. morganii and jack bean were specifically recognized by antisera raised against the 66-kDa subunit of H. pylori urease, demonstrating that at least some antigenic determinants were conserved among ureases from different species.
T-cell hybridomas reveal two distinct mechanisms of antileishmanial defense
Authors: Sypek J P , Wyler D J
Published: January 2020

Using lymph node lymphocytes of Leishmania major-infected mice, we constructed and cloned two T-cell hybridomas that could activate macrophages to exert antileishmanial defense in vitro. One clone, 1D5, produced lymphokines (including gamma interferon) that induced these effects. Production of the macrophage-activating lymphokines and the protective effect of 1D5 were suppressed by the addition of cyclosporine A to cultures. The other clone, 1B6, produced no detectable macrophage-activating lymphokines, and its protective ability was not suppressed by cyclosporine A. Granulocyte-macrophage colony-stimulating factor (a lymphokine also known to induce antileishmanial effects in macrophages) was not detectable in culture supernatants of either clone. Furthermore, neither clone was cytotoxic to infected macrophages. Antileishmanial defense induced by 1B6 was genetically restricted; that is, infected macrophages and hybridoma cells had to be syngeneic for an antileishmanial effect to occur. In contrast, such restriction was not a property of clone 1D5, a clone that was responsive to alloantigens as well as leishmanial antigens. When incubated at a temperature (34 degrees C) at which lymphokines are relatively ineffective for antileishmanial defense, 1B6 but not 1D5 retained its antileishmanial properties. These observations provide clear evidence for the existence of two distinct mechanisms of macrophage activation: one that is lymphokine dependent, and one that is apparently lymphokine independent. The expression of these two mechanisms by cloned cells strongly suggests that they are properties of different T-cell subpopulations, extending our prior conclusions based on studies of heterogeneous T-cell populations. We hypothesize that the latter macrophage activation process involves a cell contact-dependent mechanism which might involve the interaction of a lymphocyte membrane-associated macrophage-activating factor (such as tumor necrosis factor) with its receptor on the macrophage, resulting in activation of antileishmanial effects but not host cell cytotoxicity.
Construction of a nontoxic fusion peptide for immunization against Escherichia coli strains that produce heat-labile and heat-stable enterotoxins
Authors: Clements J D
Published: January 2020

The 5' terminus of the gene that codes for the heat-stable enterotoxin of Escherichia coli (ST) was genetically fused to the 3' terminus of the gene that codes for the binding subunit of the heat-labile enterotoxin of E. coli (LT-B). The ST-encoding gene used for these studies was constructed synthetically with appropriate restriction sites to permit in-frame, downstream insertion of the oligomer. For this construction, maximum expression of ST antigenicity was obtained when a seven-amino-acid, proline-containing linker was included between the LT-B and ST moieties. The LT-B-ST fusion peptide was purified by affinity chromatography and consisted of a single polypeptide chain with an apparent molecular weight of 18,000 when examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. There was no evidence of multimer formation and no change in the mobility of the fusion peptide when it was boiled in SDS or in SDS with dithiothreitol. The LT-B-ST fusion peptide was nontoxic, and immunologic determinants of both LT and ST were recognized by antibodies to the native toxins. More importantly, the LT-B-ST fusion peptide was immunogenic. Animals immunized with crude or purified preparations containing the hybrid molecule produced antibodies that were able to recognize native toxin in vitro. Significantly, these antibodies were able to neutralize the biological activity of native ST.
Antiphagocytic effect of the capsule of Staphylococcus simulans
Authors: Ohshima Y , Schumacher-Perdreau F , Peters G , Quie P G , Pulverer G
Published: January 2020

An encapsulated strain of Staphylococcus simulans was observed to be more resistant to phagocytosis by human granulocytes than was a nonencapsulated strain. Phagocytosis of the encapsulated strain was enhanced by antisera to S. simulans, but opsonic activity of antisera was removed by absorption with S. simulans capsular material. The encapsulated strain of S. simulans was also more invasive than the nonencapsulated S. simulans in vivo. More encapsulated than nonencapsulated S. simulans were found in heart blood when equal numbers of organisms were injected intraperitoneally into mice. Invasion of the bloodstreams of mice by encapsulated S. simulans was prevented by passive immunization (rabbit antiserum). Thus, the capsule of S. simulans inhibited phagocytosis in vitro and contributed to virulence in vivo.
Suppression of tumor necrosis factor alpha production by a human immunoglobulin preparation for intravenous use
Authors: Shimozato T , Iwata M , Tamura N
Published: January 2020

We investigated the effect of a pH 4-treated human immunoglobulin preparation for intravenous use (pH4-G) on the production of tumor necrosis factor alpha (TNF-alpha) in vivo. The level of TNF-alpha in the sera of rabbits receiving pH4-G before lipopolysaccharide (LPS) injection was lower than that in rabbits receiving only LPS. Similarly, the in vitro production of TNF-alpha was suppressed by pH4-G. Rabbit peritoneal exudate cells stimulated with LPS in the presence of pH4-G produced less TNF-alpha than did those stimulated only with LPS. pH4-G, however, had no effect on various TNF-alpha activities, such as cytotoxicity against tumorigenic murine fibroblasts (L929 cells), induction of interleukin-1 production, or fever induction. These results indicate that pH4-G suppresses TNF-alpha production without affecting TNF-alpha activities. A suppressive effect on the expression of TNF-alpha mRNA was also observed.
Production and characterization of monoclonal antibodies against the lethal factor component of Bacillus anthracis lethal toxin
Authors: Little S F , Leppla S H , Friedlander A M
Published: January 2020

The lethal toxin of Bacillus anthracis consists of two components, protective antigen and lethal factor. Protective antigen is cleaved after binding to cell receptors, yielding a receptor-bound fragment that binds lethal factor. Sixty-one monoclonal antibodies to the lethal factor protein have been characterized for specificity, antibody subtype, and ability to neutralize lethal toxin. Three monoclonal antibodies (10G3, 2E7, and 3F6) neutralized lethal toxin in Fisher 344 rats. However, in a macrophage cytolysis assay, monoclonal antibodies 10G3, 2E7, 10G4, 10D4, 13D10, and 1D8, but not 3F6, were found to neutralize lethal toxin. Binding studies showed that five of the monoclonal antibodies that neutralized lethal toxin in the macrophage assay (10G3, 2E7, 10G4, 10D4, and 13D10) did so by inhibiting the binding of lethal factor to the protective antigen fragment bound to cells. Monoclonal antibody 1D8, which was also able to neutralize lethal toxin activity after lethal factor was prebound to cell-bound protective antigen, only partially inhibited binding of lethal factor to protective antigen. Monoclonal antibody 3F6 did not inhibit the binding of lethal factor to protective antigen. A competitive-binding enzyme-linked immunosorbent assay showed that at least four different antigenic regions on lethal factor were recognized by these seven neutralizing hybridomas. The anomalous behavior of 3F6 suggests that it may induce a conformational change in lethal factor. Differences in neutralizing activity of monoclonal antibodies were related to their relative affinity and epitope specificity and the type of assay.
Safety, immunogenicity, and efficacy against cholera challenge in humans of a typhoid-cholera hybrid vaccine derived from Salmonella typhi Ty21a
Authors: Tacket C O , Forrest B , Morona R , Attridge S R , LaBrooy J , Tall B D , Reymann M , Rowley D , Levine M M
Published: January 2020

A live oral vaccine consisting of attenuated Salmonella typhi Ty21a expressing Vibrio cholerae O1 Inaba lipopolysaccharide (LPS) O antigen was constructed and tested in volunteers for safety, immunogenicity, and efficacy. Fourteen adults ingested three doses of 10(10) viable organisms with buffer. One month later, 8 vaccinees and 13 unimmunized controls were challenged with 10(6) pathogenic V. cholerae O1 E1 T or Inaba organisms. No significant adverse reactions to vaccination were observed. All volunteers had significant rises in serum immunoglobulin G (IgG) antibody to S. typhi LPS. Only 2 (14%) of 14 had significant rises in serum IgA or IgG antibody to Inaba LPS, and 5 (36%) of 14 had fourfold rises in vibriocidal antibody. In the challenge study, diarrhea occurred in 13 of 13 controls and 6 of 8 vaccinees (vaccine efficacy, 25%; P = 0.13). The vaccine significantly reduced the severity of the clinical illness (P less than 0.05) and caused decreased excretion of challenge vibrios (P less than 0.05). Although the typhoid-cholera hybrid vaccine did not provide significant protection overall against experimental cholera, this study demonstrates the importance of antibody to V. cholerae O antigen in ameliorating clinical illness and illustrates the use of an S. typhi carrier vaccine strain expressing a foreign antigen.
Production of the hexitol D-mannitol by Cryptococcus neoformans in vitro and in rabbits with experimental meningitis
Authors: Wong B , Perfect J R , Beggs S , Wright K A
Published: January 2020

We studied the ability of Cryptococcus neoformans to produce the hexitol D-mannitol in vitro and in rabbits with experimental meningitis. Twelve of twelve human isolates of C. neoformans produced D-mannitol in yeast nitrogen base plus 1% glucose and released D-mannitol into the medium. In a pilot study, pooled cerebrospinal fluid (CSF) from cortisone-treated rabbits given 3 x 10(7) C. neoformans H99 intracisternally contained more D-mannitol (identified by gas chromatography and enzymatically) than CSF from normal controls or cortisone-untreated rabbits with self-limited meningitis. In a second experiment, cortisone-treated rabbits given C. neoformans intracisternally had significantly higher CSF D-mannitol concentrations than controls given cortisone alone at 4, 6, and 8 days after infection. Moreover, log10 CSF D-mannitol correlated well with log10 CSF CFU (r = 0.81) and log10 CSF cryptococcal antigen titers (r = 0.78). Lastly, the initial volume of distribution and elimination half-life of D-mannitol given intracisternally to normal rabbits suggested that D-mannitol was distributed in total CSF and was removed by CSF bulk flow. Thus, C. neoformans produces D-mannitol in vitro and in vivo, and D-mannitol is a quantitative marker for experimental cryptococcal meningitis. D-Mannitol produced by C. neoformans may also contribute to brain edema and interfere with phagocyte killing by scavenging hydroxyl radicals.