bluuebunny/crossref_metadata_2025 huggingface dataset:
Persistent infection of L cells with an ovine abortion strain of Chlamydia psittaci
Authors: Perez-Martinez J A , Storz J
Published: January 2020

L cells inoculated at multiplicities of infection greater than or equal to 1 inclusion-forming unit of the abortigenic chlamydial strain B577 were destroyed within 10 to 15 days. Upon continued incubation in fresh medium, a few surviving cells repopulated the flasks, and the reemerging cultures remained persistently infected. The persistent state was characterized by cycles of repopulation with a low ratio of infected cells and cycles of extensive cytopathic changes in which greater than 90% of the cells had chlamydial inclusions and which could be delayed or even terminated by penicillin treatment. Immunofluorescence and superinfection during the period of repopulation revealed that the persistently infected cells could adsorb chlamydiae but their multiplication was arrested. This nonpermissive state could be terminated by the specific action of cycloheximide. L cells spontaneously cured from a persistent infection exhibited no change in susceptibility to chlamydiae when compared with normal L cells. However, chlamydiae derived from L cells after 7.5 months of persistence destroyed L-cell monolayers more rapidly and at lower multiplicities of infection than the wild type. This state of chlamydia-host cell interaction could not be established with the arthropathogenic strain LW613 because chlamydial infectivity was lost after the first cytolytic burst of infection in the cell cultures. The persistence described for the strain B577-L-cell system appears to differ from previously described models involving other chlamydial strains.
Comparative studies on the effect of growth conditions on adhesion, hydrophobicity, and extracellular protein profile of Streptococcus sanguis G9B
Authors: Knox K W , Hardy L N , Markevics L J , Evans J D , Wicken A J
Published: January 2020

Streptococcus sanguis G9B was grown in continuous culture at different generation times and pH values in media containing either glucose or fructose and differing in the concentrations of Na+ and K+. The growth pH, carbohydrate, and cation concentration each affected the yield of organisms, their ability to adhere to saliva-coated hydroxyapatite beads, and their hydrophobicity, as measured by adhesion to hexadecane. There was no correlation between adhesion to saliva-coated hydroxyapatite beads and hydrophobicity, the values for hydrophobicity varying between 44 and 83% for organisms that adhered poorly and between 24 and 75% for those that adhered effectively. For organisms grown in batch culture at pH 6.0 or 7.0 there was similarly no correlation between adhesion and hydrophobicity. The growth conditions also had a considerable influence on the production of extracellular protein. The total amount was greater at pH 7.5 than at other pH values, and there were also differences in the individual components in response to changes in generation time, pH, carbohydrate source, and cation concentration. Two protein bands were identified, namely, glucosyltransferase and protein P1 (also called antigen B or I/II). However, there was no correlation between a particular protein component and adhesion.
Activation of the alternative and classical complement pathways by Entamoeba histolytica
Authors: Calderon J , Schreiber R D
Published: January 2020

Entamoeba histolytica HM1 supported the activation of human alternative and classical complement pathways in the absence of ameba-reactive antibodies. Nonimmune serum depleted of C1q and factor D (NHS s C1q + D) and reconstituted with C1q was able to specifically deposit C3b onto trophozoites and produce lysis. This activity was not modified by the absorption of serum on E. histolytica. Serum depleted of factor B allowed C3b binding to amebae. Serum devoid of C4 effected only small amounts of C3 uptake. The kinetics of lysis of E. histolytica by serum in the presence of Mg-EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] (lacking classical pathway function) or by NHS s C1q + D and reconstituted with factor D was slow and only produced one-half the amount of lysis produced by NHS s C1q + D supplemented with C1q. These results indicate that the surface of the ameba can promote complement activation by the classical pathway, without the participation of specific antibodies, and that the magnitude of this activation is greater than that induced by the alternative pathway.
Relation between the hemolytic property and iron metabolism in Escherichia coli
Authors: Lebek G , Gruenig H M
Published: January 2020

The hemolytic activity of wild-type strains of Escherichia coli was measured by a standardized method in liquid broth. The system also allowed us to investigate the influence of various Fe3+ concentrations in the cultures on the amount of secreted hemolysin. We found that the hemolysin secretion of all strains was clearly reduced after addition of FeCl3. However, the influence of additional iron chelators showed remarkable differences. The hemolytic activity of Hly plasmid-containing strains isolated in Berne significantly increased. Most of the strains with a chromosomal hemolysin determinant showed a similar effect, but to a lesser degree. Contrary to this, Hly plasmid-containing strains isolated in Essex and some strains with chromosomal hemolysin determinant were either not affected or even showed reduced hemolysin secretion after limitation of free iron ions in the broth. Our results suggest that hemolysin secretion in E. coli is related to the bacterial iron metabolism, and hemolysin secretion is differentially regulated among E. coli strains.
Antibacterial resistance, macrophage influx, and activation induced by bacterial rRNA with dimethyldioctadecylammonium bromide
Authors: Gonggrijp R , Mullers W J , Dullens H F , van Boven C P
Published: January 2020

Intraperitoneally injected rRNA from Pseudomonas aeruginosa combined with dimethyldioctadecylammonium bromide (DDA) increased nonspecifically the resistance of mice against an intraperitoneal challenge with extracellular (P. aeruginosa, Escherichia coli) and intracellular (Listeria monocytogenes) bacteria. This study concerns the mechanism underlying the nonspecific resistance. RNA with DDA (RNA-DDA) induced a cell influx and activated peritoneal macrophages (M phi) as judged by the decreased 5'-nucleotidase and alkaline phosphodiesterase activities in M phi lysates, the enhanced O2- release, and the increased antitumor activity in comparison with unstimulated M phi. RNA without DDA did not enhance the resistance and did not influence the peritoneal cell numbers or M phi properties. DDA without RNA enhanced the resistance of mice only slightly; it induced a cell influx, yielding elicited M phi as judged by the decreased 5'-nucleotidase activity and increased alkaline phosphodiesterase activity, the slightly enhanced O2- release, and the absence of increased antitumor activity. Both RNA-DDA and DDA M phi showed an enhanced capacity to ingest and kill L. monocytogenes in vitro, DDA M phi being slightly less effective than RNA-DDA M phi with respect to killing. We conclude that the enhanced killing capacity of M phi for L. monocytogenes is characteristic of both elicited DDA M phi and activated RNA-DDA M phi. The relationship between nonspecific resistance, peritoneal cell numbers, and antibacterial M phi activity is discussed. In addition, it is shown that RNA and DDA retain their activity when they are injected apart, suggesting that they activate M phi by sequential action.
Natural emergence of antigen-reactive T cells in lepromatous leprosy patients during erythema nodosum leprosum
Authors: Laal S , Bhutani L K , Nath I
Published: January 2020

Fifteen lepromatous leprosy (LL) patients undergoing erythema nodosum leprosum (ENL) reactions were compared with 13 stable, uncomplicated, anergic individuals of the same leprosy background. ENL patients showed significant antigen-induced leukocyte migration inhibition (migration index = 0.058 +/- 0.01), paralleling the values obtained with a responder tuberculoid leprosy population (migration index = 0.04 +/- 0.004). Both phytohemagglutinin-induced general T-cell proliferation and, more significantly, antigen-induced lymphoproliferation were enhanced during the acute phase of the reaction. Suppressor cell activity, monitored by a costimulant assay, showed enhanced antigen-stimulated suppression of mitogen responses. Interestingly, the improvement in in vitro T-cell responses was not reflected in dermal reactivity, since 48-h delayed-type hypersensitivity responses after intradermal injection of soluble Mycobacterium leprae antigens continued to be poor. After subsidence of reactional lesions, leukocyte migration inhibition, lymphoproliferation, and suppressor cell activity were reduced to the unresponsive state seen in stable LL patients. Significantly, perturbations of T-cell reactivity are detectable in ENL reactions, indicating the natural but transient emergence of antigen-induced T cells in LL.
Production of listeriolysin by beta-hemolytic strains of Listeria monocytogenes
Authors: Parrisius J , Bhakdi S , Roth M , Tranum-Jensen J , Goebel W , Seeliger H P
Published: January 2020

Listeriolysin was isolated from target rabbit erythrocyte membranes after lysis of the cells with partially purified toxin derived from a culture supernatant of Listeria ivanovii. The membrane form of the toxin exhibited properties similar to those previously found for streptolysin O. Detergent-solubilized, delipidated listeriolysin was found to comprise a heterogeneous population of partially and fully circularized, amphiphilic oligomers whose embedment within the lipid bilayer generated large transmembrane pores. The molecular weight of the toxin monomer was estimated to be 55,000 to 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunological cross-reactions between the toxin and streptolysin O were demonstrable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. An immunoblot assay for detecting listeriolysin in agar-incorporated, lysed erythrocyte membranes was developed, and 28 defined, clinical isolates of Listeria monocytogenes were examined for toxin production. These isolates caused beta-hemolysis on the agar plates and had previously been regarded as listeriolysin producers. However, we found that only two isolates produced genuine listeriolysin, since the sensitive immunoblot assay entirely failed to detect the toxin in all other cases. We excluded that this finding derived from proteolytic degradation of membrane-bound toxin. Thus, the great majority of human pathogenic Listeria strains appear to produce one or several hemolysins that are immunologically and, by inference, molecularly distinct from the streptolysin O-related listeriolysin. We propose that the streptolysin O-related toxin be designated alpha-listeriolysin and that the other hemolysin(s) be termed beta-listeriolysin.
Collagenolytic activity of Coccidioides immitis
Authors: Lupan D M , Nziramasanga P
Published: January 2020

Coccidioides immitis appears to be unable to digest particulate collagen when cultured on collagen-containing semisolid culture media. However, all C. immitis strains solubilized collagen when the fungus was grown in liquid suspension cultures. Moreover, sterile culture filtrates were collagenolytic in collagen-buffer-agar plate assays.
Type-specific opsonic antibodies evoked with a synthetic peptide of streptococcal M protein conjugated to polylysine without adjuvant
Authors: Beachey E H
Published: January 2020

A chemically synthesized copy (S-CB7) of a fragment (35 amino acid residues) of type 24 streptococcal M protein was covalently linked to polylysine with carbodiimide and injected subcutaneously into rabbits without adjuvant. Although the primary immune responses as measured by enzyme-linked immunosorbent assays at biweekly intervals were weak, the secondary responses as measured by both enzyme-linked immunosorbent assays and opsonophagocytic assays were as high as those obtained previously in rabbits immunized with the peptide conjugate emulsified in complete Freund adjuvant. Injection of murabutide, a synthetic muramyl dipeptide derivative of bacterial peptidoglycan, with the initial immunizing dose of peptide conjugate had no apparent effect on the secondary immune responses. These results indicate that protective immune responses may be raised against polylysine conjugates of chemically synthesized peptide copies of streptococcal M protein without adjuvant.
Monoclonal antibodies against the nonhemagglutinating fimbrial antigen 1C (pseudotype 1) of Escherichia coli
Authors: Schmitz S , Abe C , Moser I , Orskov I , Orskov F , Jann B , Jann K
Published: January 2020

Hybridoma-derived monoclonal antibodies were produced with fimbrial preparations from Escherichia coli 20025 (04:K12:H-) with fimbrial (F) antigens 1C, 13, one related to 12, and one preliminarily termed y and from E. coli 2980 (018ac:K5:H-) with F antigens 1C and 8. Two clones of subclonal hybrid cells were studied which produced monoclonal antibodies (mc-20025-F2b, immunoglobulin G2b [IgG2b]; mc-2980-F2, IgG1) that were reactive with E. coli 20025, 2980, and a number of additional strains which exhibited the F1C antigen. Results of enzyme-linked immunosorbent assay and Western blot analysis indicated that the antibodies had F1C specificity, and competitive enzyme-linked immunosorbent assay with 125I-labeled antibodies showed that they recognized different epitopes on the fimbrial subunit. Neither of the antibodies agglutinated F1C-fimbriated E. coli but bound to the bacteria. There was no binding to E. coli without F1C fimbriae.
Bactericidal effect of lactoferrin on Legionella pneumophila
Authors: Bortner C A , Miller R D , Arnold R R
Published: January 2020

Lactoferrin, an iron-binding protein found in mucosal secretions and in specific granules of polymorphonuclear leukocytes, has been shown to be bactericidal for a variety of organisms. In this study, the effect of lactoferrin on Legionella pneumophila was investigated. Purified human apolactoferrin was bactericidal for the Knoxville 1 strain (serogroup 1), with a 4-log decrease in viability within 2 h at 37 degrees C. Killing was dependent on the iron-free state since iron-saturated lactoferrin had no activity. Guinea pig passage of this strain did not affect its sensitivity to lactoferrin. Treatment of the cells with dilutions of the lactoferrin resulted in correspondingly reduced killing. Activity was temperature dependent; there was no loss of viability at 1 or 22 degrees C and slightly enhanced killing at 41 degrees C. Addition of Mg2+ blocked bactericidal activity. In addition, mature human milk, a lactoferrin-containing mucosal secretion, was also bactericidal for L. pneumophila. As demonstrated with the purified lactoferrin, bactericidal activity was lost when the milk was iron saturated.
Utilization of anion-exchange chromatography and monoclonal antibodies to characterize multiple pilus types on a uropathogenic Escherichia coli O6 isolate
Authors: Gander R M , Thomas V L
Published: January 2020

Multiple pilus types from a uropathogenic strain of Escherichia coli O6, strain 6260, were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high-pressure liquid chromatography, binding assays, and erythrocyte adsorption. In addition, monoclonal antibodies were raised against purified pili of E. coli 6260 and used for immunological characterization. SDS-PAGE analysis of the purified pili showed at least three different subunits with molecular weights of 15,700, 17,800, and 19,300. SDS-PAGE analysis of four protein peaks from anion-exchange chromatography of intact pili showed polypeptides with molecular weights of 19,300 (fraction 1), 15,700 (fraction 2), and 17,800 and 15,700 (both fractions 3 and 4). Erythrocyte adsorption of the whole-pilus preparation removed the 17,800-molecular-weight subunit (17.8K subunit) and reduced the 15.7K subunit. Pili from an isogenic hemagglutination-negative variant of E. coli 6260, showing only the 15.7K and 19.3K subunits by SDS-PAGE, lacked the 17.8K subunit of fractions 3 and 4 present in the parent high-pressure liquid chromatography profile. Our data suggest that two of the pilus subunits, the 15.7K and 17.8K subunits, mediate mannose-resistant agglutination of human erythrocytes. Pili in fractions 1 and 2 from the parent strain bound specifically to mannose residues, while pili in fraction 4 bound to P-coated horse erythrocytes; no receptor specificity was identified for pili in fraction 3. Immunological analysis by the immunoblot technique showed that monoclonal antibody 11-2 reacted with the 19.3K subunit, monoclonal antibodies 34-3 and 73-3 reacted with the 15.7K subunit, and monoclonal antibodies 81-1, 82-1, and 91-1 reacted with polymers of subunits retained in the stacking gel. Intact pili precipitated by any of the six monoclonal antibodies showed two polypeptides by SDS-PAGE: 15.7K and 19.3K polypeptides for monoclonal antibody 11-2, and 15.7K and 17.8K polypeptides for monoclonal antibodies 34-3, 73-3, 81-1, 82-1, and 91-1. The cross-reactivity of the monoclonal antibodies with purified pili from other E. coli strains was determined by enzyme-linked immunosorbent assay. Monoclonal antibody 11-2 showed no significant cross-reactivity with heterogeneous pili. In contrast, the other monoclonal antibodies showed equivalent or greater reactivity with P pili from heterologous strains as compared with reactivity with E. coli 6260 pili.(ABSTRACT TRUNCATED AT 400 WORDS)
Mycobacterium leprae fails to stimulate phagocytic cell superoxide anion generation
Authors: Holzer T J , Nelson K E , Crispen R G , Andersen B R
Published: January 2020

Mycobacterium leprae is an intracellular pathogen that is ingested by and proliferates within cells of the monocyte/macrophage series. Mechanisms by which intracellular pathogens resist destruction may involve failure to elicit a phagocyte "respiratory burst" or resistance to toxic oxygen derivatives and lysosomal enzymes. We have studied the ability of M. leprae and Mycobacterium bovis BCG to stimulate the generation of superoxide anion (O2-) in vitro by human blood neutrophils and monocytes and murine peritoneal macrophages. M. leprae bacteria failed to stimulate significant O2- release except at high bacteria-to-cell ratios (greater than 50:1) whether or not they were pretreated with normal serum or serum from patients with lepromatous leprosy. Either viable or irradiated BCG; on the other hand, stimulated the three cell types to release significant amounts of O2- when challenged with as few as 10 organisms per cell. Serum pretreatment enhanced the release of O2- by the three cell types. Preincubation for 18 h with viable M. leprae did not inhibit the ability of monocytes to respond with an oxidative burst to phagocytic stimuli. The failure of M. leprae to stimulate phagocyte O2- generation may be an important factor in its pathogenicity.
Molecular characterization of Chlamydia trachomatis and Chlamydia psittaci plasmids
Authors: Joseph T , Nano F E , Garon C F , Caldwell H D
Published: January 2020

Plasmids from Chlamydia trachomatis LGV-434 (serotype L2) and Chlamydia psittaci meningopneumonitis strain Cal-10 were cloned into the BamHI and EcoRI sites of pBR322, respectively. The recombinant plasmids pCTL2 and pCPMn, each containing an entire respective chlamydial plasmid, were transformed into Escherichia coli. The sizes of the plasmids of C. trachomatis and C. psittaci were 7.3 and 6.2 kilobases, respectively. The two plasmids were found to be distinct by restriction endonuclease analysis, DNA-DNA hybridization, and electron microscopic heteroduplex analysis. However, partial homology was observed between restriction fragments of pCTL2 and pCPMn by Southern blot analysis. Polypeptide products encoded by these plasmids were synthesized in vitro by an E. coli-directed transcription-translation system and in vivo in E. coli maxicells and minicells. None of these polypeptides was immunoreactive with anti-chlamydial sera by immunoblotting or immunoprecipitation. Based on the comparative analysis data, the C. trachomatis and C. psittaci plasmids were found to share little genetic relatedness.
Plasmids coding for drug resistance and localized adherence to HeLa cells in enteropathogenic Escherichia coli O55:H- and O55:H6
Authors: Laporta M Z , Silva M L , Scaletsky I C , Trabulsi L R
Published: January 2020

Plasmids coding for drug resistance and localized adherence (LA) to HeLa cells were found in two enteropathogenic Escherichia coli strains belonging to serotypes O55:H- and O55:H6. Strain 49-81 HSJ (O55:H-) carries two plasmids, one coding for both ampicillin resistance (Apr) and LA (pMS49). Strain 71-82 HSJ (O55:H6) harbors only one plasmid, coding for resistance to sulfadiazine, chloramphenicol, kanamycin, ampicillin, and LA (pMS71). Plasmids pMS49 and pMS71 were transferred to E. coli K-12 711 and from this strain to E. coli K-12 J53. Curing with acridine orange of an Apr LA+ transconjugant showed that both characteristics were lost simultaneously. The plasmids have a molecular weight of approximately 55 X 10(6) and are the first naturally recombinant plasmids coding for adherence and drug resistance described in enteropathogenic E. coli.
Role of immune serum and complement in stimulation of the metabolic burst of human neutrophils by Plasmodium falciparum
Authors: Salmon D , Vilde J L , Andrieu B , Simonovic R , Lebras J
Published: January 2020

Interaction between human neutrophils (polymorphonuclear leukocytes [PMN]) and Plasmodium falciparum in the natural defense of the host remains to be elucidated. In patients with acute malaria, oxygen consumption (QO2) of PMN at rest and after stimulation by zymosan was significantly increased compared with that in the controls. With 10% immune serum, both QO2 and chemiluminescence of normal PMN were significantly increased after stimulation by a P. falciparum erythrocyte culture. This activation was not observed with a nonparasitized erythrocyte culture and was correlated with parasitemia. Immune serum and complement were required to trigger this metabolic activation of normal PMN. With normal serum or heat-inactivated immune serum, a parasitized erythrocyte culture did not significantly stimulate QO2 or chemiluminescence of normal PMN. The classical complement pathway was essential for this stimulation, whereas the alternate pathway was less involved. Hyperimmune sera from subjects residing in endemic areas were more able to trigger the metabolic burst than were immune sera from subjects from other sources. The use of synchronous cultures showed that PMN were more stimulated by cultures rich in merozoites than by the same cultures which contained only intraerythrocytic forms. Giemsa staining showed granules of hemozoin and occasional merozoites or parasitized erythrocytes within PMN. This increase in production of activated oxygen radicals could damage intra-or extraphagocytic parasitic forms. As P. falciparum is sensitive to oxidant stress and PMN is the phagocyte with the most intense metabolic burst, the role of PMN in defense against malaria should be considered.
Cross-reactive lymphocyte responses and protective immunity against other spotted fever group rickettsiae in mice immunized with Rickettsia conorii
Authors: Jerrells T R , Jarboe D L , Eisemann C S
Published: January 2020

Lymphocyte proliferation in response to antigens on spotted fever group rickettsiae was used as a method to investigate the group-specific protective immunity to rechallenge characteristic of this group of rickettsiae at the T-cell receptor level. Spleen cells from Rickettsia conorii-immune C3H/HeJ mice proliferated in response to R. rickettsii Sheila Smith, R. sibirica 246, R. australis, and all tested strains of R. conorii (Casablanca, Moroccan, and Malish). Spleen cells from these mice, however, responded poorly or not at all to antigens prepared from the Kaplan or Hartford strain of R. akari. Proliferation of immune T cells maintained as in vitro cell lines showed a similar pattern of reactivity to these antigens; however, response to R. akari was consistently demonstrable. Spleen cells from C3H/HeJ mice immunized with R. akari responded to R. akari and R. conorii antigens as well as antigens from the other spotted fever group rickettsiae. Lymphocytes obtained from lymph nodes draining foot pads infected with R. conorii or R. akari demonstrated cross-reactivity similar to that found with immune spleen cells. If immunization was accomplished with R. conorii antigen emulsified in Freund complete adjuvant, the resulting lymph node cells were able to respond to R. akari antigens. These data suggest that infection with R. conorii induces a population of T lymphocytes that recognize an antigen(s) that also is found on other spotted fever rickettsiae and that may be responsible for cross-protective immunity. This antigen probably is not a major antigen on R. akari.
Characterization of a phase I Coxiella burnetii chloroform-methanol residue vaccine that induces active immunity against Q fever in C57BL/10 ScN mice
Authors: Williams J C , Damrow T A , Waag D M , Amano K
Published: January 2020

The effect of phase I Coxiella burnetii chloroform-methanol residue vaccine (CMRV) on the response of murine splenic lymphocytes to mitogenic and antigenic stimuli was evaluated in C57BL/10 ScN endotoxinnonresponder mice with an in vitro lymphocyte proliferation assay. Intraperitoneal injection of phase I CMRV resulted in antibody production against phases I and II antigens. Lymphocytes were responsive in vitro to concanavalin A, phytohemagglutinin, pokeweed mitogen, and specific recall antigens. Antibodies against phases I and II antigens were not detected after intraperitoneal injection of chloroform-methanol extract (CME). Lymphocytes also were only slightly hyporesponsive to mitogens. Reconstitution of the CMRV with the CME of phase I whole cells restored the immunopathological reactions that were associated with the phase I whole cell vaccine (WCV). The CMRV was more mitogenic than the WCV for lymphocytes from mice injected with saline. Lymphocytes from phase I WCV-injected mice were negatively modulated with nontoxic concentrations of homologous WCV or CMRV. Lymphocytes from phase I CMRV-injected mice were only slightly hyporesponsive to mitogens and were significantly stimulated by antigens of either WCV or CMRV as recall antigens. Vaccination of mice with 100 micrograms of CMRV, CME, or WCV provided 80, 0, or 50% protection, respectively, against a lethal intraperitoneal challenge with viable phase I C. burnetii. The epitopes which induce immunological hyporesponsiveness, negative modulation, and the death of lymphocytes were fractionated into the CMRV and CME. The CMRV provides at least one of the determinants which induce immunosuppression, whereas CME contains specific or nonspecific components or both. Collectively, these results show that the CMRV may be a potential candidate to replace the WCV currently used for human vaccination.
Humoral immune response to Rocky Mountain spotted fever in experimentally infected guinea pigs: immunoprecipitation of lactoperoxidase 125I-labeled proteins and detection of soluble antigens of Rickettsia rickettsii
Authors: Williams J C , Walker D H , Peacock M G , Stewart S T
Published: January 2020

Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, purified from infected L-929 cells by density gradient banding were extrinsically radioiodinated with lactoperoxidase. Immunodominant 125I-labeled antigens were identified by radioimmunoprecipitation of detergent-solubilized antigens with protein A-Sepharose and anti-R. rickettsii sera collected 0, 3, 7, 11, 32, and 163 days after infection of guinea pigs. The average fever greater than or equal to 40 degrees C was detected by days 3 and 4 after infection with 6 X 10(7) and 6 X 10(6) PFU, respectively. By microagglutination and complement fixation assays, anti-R. rickettsii antibodies were detected as early as day 3 after infection, with titers increasing markedly between days 7 and 163. Convalescent sera, collected on day 163, from infected guinea pigs were used to identify seven 125I-labeled antigens with apparent molecular sizes of 186,000 (I), 145,000 (II), 49,000 (III), 32,000 (IV), 27,500 (V), 17,500 (VI), and 16,500 (VII) daltons. Differences in antibody reactivity and specificity against the seven antigens were demonstrated with serially obtained sera. Sera from a guinea pig infected with 6 X 10(7) PFU exhibited antibody-antigen interactions with all seven 125I-labeled antigens by day 7, whereas the same antibody activity required 32 days for an animal infected with 6 X 10(6) PFU. Prominent antibody activities toward proteins II and IV were demonstrated both early and late after infection. The fluids obtained from infected L-929 cells contained three soluble antigens which were detected with the 11-, 32-, and 163-day sera by an immunodiffusion assay. The soluble and 125I-labeled antigens of R. rickettsii identified in this study may be important candidates for vaccines against Rocky Mountain spotted fever.
Genetic relationships and clonal structure of strains of Escherichia coli causing neonatal septicemia and meningitis
Authors: Selander R K , Korhonen T K , Väisänen-Rhen V , Williams P H , Pattison P E , Caugant D A
Published: January 2020

Genetic diversity and relationships among 63 isolates of Escherichia coli from infants in Finland with septicemia or meningitis were assessed by analyzing electrophoretic variation in 21 enzymes encoded by chromosomal genes. Thirty-nine multilocus genotypes (electrophoretic types) were distinguished, 23 of which formed a closely related, distinctive subset (group 1) of five or six clones represented by 40 (63%) of the isolates. The remaining isolates represented a second subset of 16 electrophoretic types (group 2) that were, on the average, rather more distantly related to one another. Although the number of electrophoretic types causing neonatal systemic disease is smaller than that occurring in healthy intestinal floras, the pathogenic electrophoretic types are only slightly less diverse genetically. Isolates of group 1 were characterized by relatively high incidences of hemolysin production and S, type 1, type 1C, and P fimbriae. However, because phenotypic characters, considered individually or in combination, did not adequately reflect the overall genetic relationships of isolates, it is recommended that the genetic structure of populations be defined on the basis of multilocus chromosomal genotypes.
Molecular cloning and expression in Escherichia coli K-12 of the gene for a hemagglutinin from Vibrio cholerae
Authors: Franzon V L , Manning P A
Published: January 2020

Using antiserum to the purified soluble hemagglutinin we isolated an Escherichia coli K-12 clone expressing the gene for a hemagglutinin from Vibrio cholerae 569B. The plasmid present in this clone was designated pPM471. By deletion analysis with both specific restriction endonucleases and Bal 31 nuclease, we localized the gene, to a 0.72-kilobase region of DNA, implying a molecular weight of less than 27,000 for the protein. Analysis in E. coli K-12 minicells of plasmids containing the cloned gene and deletion derivatives of these plasmids identified a protein of 24,000 daltons correlating with hemagglutinating activity. Using the cloned gene as a probe, we demonstrated the presence of homologous DNA in a variety of V. cholerae strains including both biotypes. Furthermore, by screening gene banks in E. coli K-12 of V. cholerae El Tor O17, we isolated several El Tor clones containing this region of DNA and also expressing hemagglutinating activity.
Properties of proteins MPB64, MPB70, and MPB80 of Mycobacterium bovis BCG
Authors: Harboe M , Nagai S , Patarroyo M E , Torres M L , Ramirez C , Cruz N
Published: January 2020

The immunogenic proteins MPB64 and MPB80 of Mycobacterium bovis BCG were purified to homogeneity and compared with MPB70. MPB70 and MPB80 showed a similar distribution in substrains of BCG, both being present in high concentrations in culture fluids of BCG substrain Tokyo, BCG Moreau, BCG Russia, and BCG Sweden and in only very small amounts in BCG Glaxo, BCG Tice, BCG Copenhagen, and BCG Pasteur. In various physicochemical properties MPB70 and MPB80 were closely similar, but MPB80 had a distinctly lower pI value. The N-terminal amino acid sequence was identical for the first 30 residues. In reactions with anti-MPB70 antibodies and delayed-type hypersensitivity skin reactions, MPB70 and MPB80 also had very similar properties. These results show that MPB70 and MPB80 are two closely similar forms of the same gene product, and postsynthetic changes probably explain the observed differences. By contrast, MPB64 had a higher molecular weight. The N-terminal amino acid sequence showed no homology with MPB70, and these two proteins showed no immunologic similarity. MPB64 and MPB70 showed only very restricted cross-reactivity with other species of mycobacteria but cross-reacted with Nocardia asteroides. The similar occurrence in eight different substrains of BCG indicated that the two proteins are influenced by similar control mechanisms, but in contrast to MPB70, MPB64 occurred in sufficient concentration in two strains of Mycobacterium tuberculosis to give a distinct spot in two-dimensional polyacrylamide gel electrophoresis of their culture fluids.
Production and characterization of monoclonal antibodies to Clostridium perfringens enterotoxin
Authors: Horiguchi Y , Uemura T , Kamata Y , Kozaki S , Sakaguchi G
Published: January 2020

Four hybridoma cell lines producing monoclonal antibodies to Clostridium perfringens enterotoxin were established by fusion of mouse myeloma and spleen cells obtained from mice immunized with the enterotoxin and its toxoid. An enzyme-linked immunosorbent assay indicated that the two antibodies, 2-B-4 and 3-G-10, bound to those regions that were located close each other; the others, 3-B-2 and 2-H-2, bound to other independent regions on the enterotoxin. Release of 51Cr from Vero cells with the enterotoxin was inhibited by either 2-B-4 or 3-G-10, both of which inhibited the binding of 125I-labeled enterotoxin to the cells. Neither binding nor cytotoxicity of the enterotoxin was affected by 2-H-2; 3-B-2 only barely inhibited the binding but neutralized the enterotoxin shown by 51Cr release. It seems justified to conclude that 3-B-2 blocks the toxic action after the enterotoxin has bound to Vero cells.
Antigenic variation in the phase I lipopolysaccharide of Coxiella burnetii isolates
Authors: Hackstadt T
Published: January 2020

Coxiella burnetii isolates from a variety of clinical and geographical sources were screened for antigenic variation of lipopolysaccharides (LPSs) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis coupled with silver staining or immunoblotting. All isolates from chronic Q fever or other sources possessed a phase I-type LPS. These LPSs appeared to fall into three groups based on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile or on reactivity with rabbit anti-C. burnetti antisera. The LPS of one group was identified on isolates from milk, ticks, or primary Q fever. The two remaining groups were found almost exclusively on isolates from human cases of chronic Q fever.
Release of interleukin-1 by peripheral blood mononuclear cells in patients with tuberculosis and active inflammation
Authors: Chensue S W , Davey M P , Remick D G , Kunkel S L
Published: January 2020

Peripheral blood monocytes from patients with active tuberculosis and acute inflammatory disease showed spontaneous interleukin-1 production when compared with those from control patients or healthy controls. Moreover, interleukin-1 production appeared to be a more specific indicator of active disease than were other commonly used indices, such as the erythrocyte sedimentation rate and serum C-reactive protein levels.
Variables which affect suppression of the immune response induced by Pseudomonas aeruginosa exotoxin A
Authors: Holt P S , Misfeldt M L
Published: January 2020

Pseudomonas exotoxin A has been shown previously to induce suppression of the murine immune response. In the present study, various parameters were examined which may have an effect on immunosuppression. The addition of 10(-4) ng of exotoxin A induced suppression of the immune response to trinitrophenylated Ficoll from days 3 to 10, while 10 ng of toxin exerted no suppressive effect over the same examination periods. When the toxin was administered 1 or 2 days before antigen stimulation, suppression of the response was observed with both 10 and 10(-4) ng. Priming splenocytes with toxin either in vivo or in vitro for 1 or 2 days suppressed the response of fresh cultured splenocytes to antigenic stimulation. Heated toxin, photoaffinity-labeled toxin, or preincubation of the toxin with rabbit anti-exotoxin A antiserum eliminated the toxin-induced suppression. These results suggest that Pseudomonas exotoxin A can generate multiple biological effects.
Mechanisms of in vivo modulation of granulomatous inflammation in murine schistosomiasis japonicum
Authors: Olds G R , Stavitsky A B
Published: January 2020

In schistosomiasis japonicum, the major pathologic lesion is the granulomatous inflammation that occurs around parasite eggs trapped in the liver. The size of these granulomas and their major sequela, a rise in portal pressure, both peak between 8 and 10 weeks after infection and then spontaneously decrease. We have shown that the adoptive transfer of the serum, but not lymphoid cells, of 30-week-infected mice caused decreases in both the size of the hepatic granulomas and the portal pressure of acutely infected recipient mice. The present study examines the role of both humoral (serum) and cellular immune mechanisms of modulation throughout the course of murine infection. Pools of serum (0.3 ml), spleen cells (5 X 10(7)), or splenic T cells (2 X 10(7)) from mice infected for 10, 20, and 30 weeks were adoptively transferred into mice at 4 and 5 weeks of infection. One week later (6 weeks postinfection), the portal pressure and size of hepatic granulomas in all recipient mice were determined. The 10-week-infected mouse serum occasionally lowered these values, but serum from 20- and 30-week-infected animals was consistently suppressive. The active component of 30-week-infected mouse serum coeluted with immunoglobulin G1. In contrast, 10-week-infected spleen cells or T cells consistently lowered portal pressure and granulomatous inflammation, but 20- and 30-week spleen cells did not. The phenotype of these suppressive T cells was Lyt-1-2+. These in vivo observations confirm earlier in vitro studies on cellular and humoral immune modulation of egg antigen-induced spleen cell blastogenesis. The current study demonstrates that both cellular and humoral regulation of granulomatous inflammation occur in murine schistosomiasis japonicum but with different kinetics: cellular mechanisms are maximal early (10 weeks) while humoral mechanisms predominate late during the chronic stage of infection (20 and 30 weeks).
Immunity to heat-labile enterotoxins of porcine and human Escherichia coli strains achieved with synthetic cholera toxin peptides
Authors: Jacob C O , Arnon R , Finkelstein R A
Published: January 2020

Antiserum to a synthetic peptide, CTP3, consisting of residues 50 to 64 of the B subunit of cholera enterotoxin (CT), a sequence which is conserved in heat-labile enterotoxins from Escherichia coli strains of human (H-LT) or porcine (P-LT) origin, cross-reacted with and neutralized all three enterotoxins. It also cross-reacted with genetically engineered chimeric B subunit proteins in which H-LT amino acids had been substituted for the corresponding residues in P-LT. Antisera against another CT peptide, CTP1, consisting of residues 8 to 20, were weakly cross-reactive with H-LT but not with P-LT, whereas antisera against four other CT peptides were not cross-reactive with the heterologous enterotoxins. A single dose of CTP3-linked tetanus toxoid, by itself nonimmunogenic, primed rabbits to a vigorous immune response to subsequent administration of single, small, subimmunizing doses of any of the three enterotoxins.
T-cell immunity in murine malaria: adoptive transfer of resistance to Plasmodium chabaudi adami in nude mice with splenic T cells
Authors: Cavacini L A , Long C A , Weidanz W P
Published: January 2020

Acute infections caused by the murine malarial parasite Plasmodium chabaudi adami are resolved by antibody-independent mechanisms of immunity. The fact that athymic nude mice developed high-grade unrelenting malaria and died when infected with this parasite suggested a significant role for T lymphocytes. Using adoptive transfer techniques, we demonstrated that spleen cells from either nonimmune or immune donor BALB/c mice eventually suppressed P. chabaudi adami infections in histocompatible recipient nude mice in a dose-dependent manner. Infections in recipients of "immune" spleen cells were less severe, demonstrating a depressed peak parasitemia and a shortened duration of patent infection, than was observed in recipients of normal spleen cells. Also, when sufficient numbers of immune spleen cells were transferred, the second wave of parasitemia (characteristic of this infection in nonimmune mice) failed to occur. T lymphocytes mediated protection in recipient mice, since T-cell-enriched, but not B-cell-enriched, spleen cell fractions suppressed P. chabaudi adami infections in nude mice. Protection was best achieved with T cells that bore the L3T4 phenotype. Patent parasitemias developed in all recipient mice, suggesting that the grafted cells did not limit parasite growth directly but achieved this end by activating other as yet unidentified inhibiting cell systems.
Differential contribution of chemotaxins and opsonins to neutrophil-mediated killing of Schistosoma mansoni larvae
Authors: King C H , Spagnuolo P J , Ellner J J
Published: January 2020

In neutrophil-mediated killing of schistosomula, antibody and serum complement serve two functions: (i) opsonization of the target pathogen and (ii) generation of cell-activating, chemotactic peptides. We studied neutrophil-mediated schistosomulum killing in experiments designed to isolate these functions of antibody and complement in the cell activation response of neutrophils to external pathogens. Schistosomula opsonized by antibody or antibody plus complement induced neutrophil-mediated killing, while complement-opsonized larvae did not. Antibody enhancement of cell-mediated killing correlated with increased cell-to-parasite adherence, while cell-mediated killing in the presence of fresh normal human serum (providing chemotactic factor stimulation and complement opsonization of larvae) demonstrated a dissociation between neutrophil adhesive and killing responses. Exogenous chemotactic stimuli were found to enhance significantly neutrophil-mediated killing of both opsonized and unopsonized larvae. Experiments involving parabiotic chambers confirmed that chemotactic-factor-stimulated neutrophils may cause significant parasite mortality without direct cell-parasite contact. In further analysis of the neutrophil response to surface and fluid-phase stimulation, release of neutrophil cytotoxic mediators was found to vary according to the type of stimulus provided: cell exposure to either larvae opsonized by antibody plus complement or to chemotactic factors provoked low-to-moderate cell release of superoxide anion, granule enzymes, and arginase; when opsonic and chemotactic stimuli were combined, a greater-than-additive secretory response was noted. Under such maximal stimulation, oxidative and nonoxidative mediators were synergistic in effecting neutrophil-mediated parasite killing. The primary function of complement in cell-mediated parasite killing appears to be to promote chemotactic-factor-mediated cellular release of toxic agents and not cell-target linkage, whereas antibody-mediated adherence is associated with concurrent cellular activation. Both neutrophils adherent to the antibody-opsonized schistosomulum and chemotactic-factor-stimulated cells (adherent and not adherent to the parasite) appear to contribute significantly to its demise.
Modulation of complement fixation and the phlogistic capacity of group A, B, and D streptococci by human lysozyme acting on their cell walls
Authors: Spitznagel J K , Goodrum K J , Warejcka D J , Weaver J L , Miller H L , Babcock L
Published: January 2020

Streptococci and streptococcal cell wall fragments induce arthritis in rats, with the severity and duration depending on the capacity of the cells or cell fragments to resist degradation by tissue enzymes. Their phlogogenic effects are apparently related to their ability to activate the alternate complement pathway (ACP). The in vitro activation of the ACP by lysozyme-treated cells and cell walls of group A, B, and D streptococci suggests that both rat and human lysozyme can modulate this activity, i.e., increasing it, decreasing it, or doing both in that order. The effects of the lysozymes also correlated with the degree to which they can unmask the aminosugar-reducing groups detectable in a given amount of cell wall, which suggests that partial depolymerization of the cell wall is critical for ACP activation. The effects of mutanolysin and C phage lysin on ACP activation were found to be correlated with their action on streptococcal cell walls. Neuraminidase had relatively little effect on ACP activation by most streptococcal strains tested. We conclude that the participation of tissue enzymes, including but not necessarily limited to lysozyme, is an important determinant for the clinical arthritis induced by group A, B, or D streptococci. Experimental arthritis induced in rats with whole (or disrupted) streptococci may depend both on the capacities of the cell walls to activate the ACP and on the capacities of the host tissue enzymes to modulate this activation. Great severity and long durations of the disease were determined by the capacity of the enzymes to degrade cell wall antigens to a degree sufficient to ensure efficient activation of the ACP without completely degrading the material so that it no longer activates complement. In this model, the limited resistance of group B peptidoglycan to lysozyme was a critical pathogenic factor.
Molecular cloning of DNA coding for outer membrane proteins of Haemophilus influenzae type b
Authors: Thomas W R , Rossi A A
Published: January 2020

DNA from Haemophilus influenzae type b was cloned in Escherichia coli with a vector lambda gt11 Amp1. Clones producing antigens reactive with hyperimmune rabbit antisera were identified by colony radioimmune assay. A second screening with hyperimmune serum adsorbed with intact H. influenzae type b bacteria was used to identify those clones producing surface-exposed outer membrane proteins. The proteins expressed in E. coli were coupled to Sepharose and used to affinity purify antibodies which were tested for reactivity with outer membrane vesicles. It was found by Western blotting that the clones were producing antigens corresponding to Mr 49,000, 39,500, and 35,000 major outer membrane protein or antigens of H. influenzae type b. Additional clones could be detected by human serum, but their reactivity was not removed when serum was adsorbed with intact bacteria. One of these studied in more detail was found to produce an antigen present in H. influenzae type b lysate but not in outer membrane vesicle preparations.