bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigenic analysis of Serratia marcescens fimbriae with monoclonal antibodies
Authors: Jingushi S , Mitsuyama M , Moriya T , Amako K
Published: January 2020

Monoclonal antibodies (MAbs) were raised against the purified fimbriae of Serratia marcescens US46, a strain expressing three morphologically distinct fimbriae. The widths of these fimbriae were 7, 4.5, and 3 nm, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fimbriae showed three bands with molecular weights of 21,000, 20,000, and 19,000, respectively. This strain had mannose-resistant (MR) hemagglutinating activity and was agglutinated by yeast cells. Therefore, strain US46 appeared to have both MR and mannose-sensitive fimbriae. In the immunoblot analysis, all MAbs reacted with the 20,000-molecular-weight subunit when given a choice of three differently sized subunits. Immunoelectron microscopy showed these MAbs attached to the MR fimbriae with the largest width (7 nm). The antigenic cross-reactivity of fimbriae was examined by an MAb-mediated agglutination test. All MR strains of S. marcescens and some mannose-sensitive strains were agglutinated by the MAbs. The serological homogeneity of MR fimbriae was confirmed by a spot test, using the crude purified fimbriae from several MR strains of S. marcescens. In other gram-negative rods, clinical isolates of Klebsiella spp. with hemagglutinating activity were agglutinated, but clinical isolates of Escherichia coli and Enterobacter spp. were not.
Biochemical studies on the effect of Clostridium difficile toxin B on actin in vivo and in vitro
Authors: Mitchell M J , Laughon B E , Lin S
Published: January 2020

We describe a simplified procedure for purification of Clostridium difficile toxin B. In this procedure, cytotoxicity is associated with a single protein band with a molecular mass of 230 kilodaltons. We used direct fluorescent staining of actin filaments to study the effect of this toxin on cultured cells. Morphologic changes were preceded by a decrease in the number and length of stress fibers followed by their disappearance with condensation of cellular actin around the nucleus. We then showed that cells treated with either cytochalasin B or toxin B had a significant increase in the monomeric actin pool as quantitated by DNase I inhibition. In contrast to the cytochalasins, toxin B had no direct effect on the rate or extent of actin polymerization or network formation in vitro. Cytoplasmic extracts of toxin B-treated cells had a significantly lower level of modulating activity on actin assembly and interactions in vitro compared with extracts of untreated cells. These results suggest that the action of toxin B on cells is due to direct or indirect effects on cellular proteins involved in controlling the state of actin assembly in the cells.
In vivo studies with two phospholipase C fractions from Pseudomonas aeruginosa
Authors: Berk R S , Brown D , Coutinho I , Meyers D
Published: January 2020

Two phospholipase C fractions were detected in culture supernatants from Pseudomonas aeruginosa ATCC 19660, PAO1, and D10C by preparative polyacrylamide gel electrophoresis. Both hemolytic fractions from strain ATCC 19660 were isolated by polyacrylamide gel electrophoresis and were found to cause paralysis, death, dermonecrosis, footpad swelling, and vascular permeability in mice. In vivo toxicity was directly associated with enzymatic activity.
Genetic association of defects in macrophage larvicidal activity and vaccine-induced resistance to Schistosoma mansoni in P strain mice
Authors: James S L , Correa-Oliveira R , Sher A , Medvitz L A , McCall R D
Published: January 2020

In contrast to most inbred strains, P mice fail to develop significant resistance to Schistosoma mansoni infection as a result of vaccination with irradiated cercariae. Vaccinated P mice also exhibit a defect in macrophage activation for killing of larval schistosomes upon specific-antigen challenge in vivo. To examine the genetic basis of these defects in vaccine-induced immunity, inheritance of the two traits was examined in (C57BL/6 X P)F1, F2, and reciprocal backcross generations. The defect in macrophage function which characterizes the P strain parent was found to be inherited in a fully recessive manner and to be controlled by only one or two major genetic loci. Moreover, a highly significant correlation (P less than 0.0025) was observed between the level of macrophage larvicidal activity and the level of resistance to challenge infection in segregating generations. Such an association is consistent with a cause-and-effect relationship, providing strong in vivo evidence implicating activated macrophages as immune effector cells of resistance to S. mansoni in the mouse-irradiated-vaccine model.
Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide
Authors: Eckert T F , Kozel T R
Published: January 2020

Cryptococcus neoformans is surrounded by a capsular polysaccharide. There are at least four known serotypes of the polysaccharide. The objective of this study was to produce monoclonal antibodies (MAbs) that could be used to study the distribution of epitopes among the serotypes of C. neoformans. BALB/c mice were immunized with cryptococcal polysaccharides of serotype A or D that were coupled to sheep erythrocytes. Splenocytes were isolated, and hybridomas secreting MAbs specific for cryptococcal polysaccharides were isolated. Two hybridomas, designated MAbs 439 and 1255, were produced from mice immunized with serotype A polysaccharide. One hybridoma, designated MAb 302, was produced from mice immunized with serotype D polysaccharide. All three antibodies were of the immunoglobulin G1 isotype. MAb 302 showed a specificity for serotypes A and D in Ouchterlony diffusion, agglutination, and opsonophagocytosis assays. MAb 1255 was reactive with polysaccharides and cells of serotypes A, B, and D. MAb 439 was reactive with polysaccharides and cells of serotypes A, B, C, and D. The reactivity of these MAbs closely matched the distribution of epitopes among cryptococcal polysaccharides predicted in previous studies of polyclonal antibodies reactive with cryptococcal polysaccharides. The ability to produce a MAb against an epitope shared by all four serotypes may have value for the detection of cryptococcal antigens in body fluids.
Isolation and characterization of an extracellular proteinase of Coccidioides immitis
Authors: Yuan L , Cole G T
Published: January 2020

A proteinase isolated from the respiratory pathogen, Coccidioides immitis, was shown to have collagenolytic and elastinolytic activity, as well as the ability to cleave human serum immunoglobulin G and secretory immunoglobulin A. Proteolytic activity was demonstrated with a bovine casein digestion assay in conidial culture exudates, mycelial and spherule culture filtrates, conidial and spherule wall material, and Sephacryl S-300 fractions of the isolated soluble conidial wall material described previously. One of the latter fractions (fraction 2) demonstrated high proteolytic activity. The proteinase was purified from this chromatographic fraction by cold acetone extraction followed by Sephadex G-50 gel filtration and was identified as a polypeptide band of 36,000 Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By means of tandem two-dimensional immunoelectrophoresis, the proteinase was identified as antigen 11 on the basis of its reaction in the coccidioidin/anticoccidioidin reference system. The proteinase is characterized by a broad substrate specificity, optimal activity at 35 to 40 degrees C (pH 8.0) in the presence of human collagen, elastin, or hemoglobin, an isoelectric point of pH 4.5, and inhibition by organofluorides, N-tosyl-L-phenylalanine chloromethyl ketone, chymostatin, and alpha-1-antitrypsin. These features of the enzyme are comparable to those of chymotrypsinlike serine proteinases. Demonstration that the proteinase can cleave human immunoglobulins and digest ubiquitous tissue structural proteins (e.g., collagen and elastin) suggests that it may play a role in the virulence of the fungal pathogen.
Molecular cloning and characterization of the glucosyltransferase C gene (gtfC) from Streptococcus mutans LM7
Authors: Pucci M J , Jones K R , Kuramitsu H K , Macrina F L
Published: January 2020

A glucosyltransferase (GTF) gene, designated gtfC, was cloned from Streptococcus mutans LM7. Its gene product was detected by screening a bacteriophage lambda library with rabbit antiserum raised against S. mutans LM7 extracellular proteins. DNA isolated from the immunopositive recombinant phage revealed two S. mutans chromosomal EcoRI fragment inserts, 8.1 and 4.7 kilobase pairs in size. Escherichia coli minicell analyses revealed the approximate position and direction of transcription of the gtfC gene. The gene product was determined to be a polypeptide of about 150 kilodaltons which synthesized a water-soluble glucan. Restriction endonuclease mapping and DNA hybridization indicated a repeated region of DNA corresponding to a portion of the coding region of gtfC immediately downstream from the intact gtfC locus on the chromosome. A 300-base-pair gtfC-specific probe showed that the gene and the putative duplicated sequence were present in S. mutans serotypes c, e, and f, but not in other related oral streptococci which had GTF activity. In addition, the gtfC determinant displayed homology to sequences corresponding to the carboxy-terminal coding region of a gene (gtfB) encoding a GTF activity that synthesized water-insoluble glucans. These data suggest that at least one class of GTF genes may be present in multiple copies in S. mutans and, further, that GTF genes may contain conserved sequences internal to their coding regions.
Absence of lipopolysaccharide in the Lyme disease spirochete, Borrelia burgdorferi
Authors: Takayama K , Rothenberg R J , Barbour A G
Published: January 2020

We were unable to demonstrate the presence of the classic enterobacterium-type lipopolysaccharide in the cells of the Lyme disease spirochete, Borrelia burgdorferi B31. This finding was primarily based on chemical analysis and the absence of free lipid A upon mild acid hydrolysis of the appropriate cell extracts. These results do not preclude the possible existence of an unusual lipopolysaccharide-like compound(s) in B. burgdorferi.
Characterization of monoclonal antibodies to fimbria-associated adhesins of Bacteroides loescheii PK1295
Authors: Weiss E I , London J , Kolenbrander P E , Andersen R N , Fischler C , Siraganian R P
Published: January 2020

Bacteroides loescheii PK1295 fimbriae, which mediate the lactose-sensitive coaggregation with Streptococcus sanguis 34 and the lactose-insensitive coaggregation with Actinomyces israelii PK14, were injected into mice to raise adhesin-specific monoclonal antibodies (MAbs). Supernatants of hybridomas were screened for the capacity to inhibit coaggregation and agglutinate intact bacteria. Of the 10 MAbs that were isolated, 4 were specific and potent inhibitors of the coaggregation between B. loescheii and S. sanguis and two other MAbs specifically inhibited the B. loescheii-A. israelii interaction. None of the six MAbs which inhibited adherence were capable of agglutinating whole cells of B. loescheii, whereas the four remaining MAbs agglutinated whole cells but had no effect on coaggregation. Fab fragments of two MAbs, one that inhibited the coaggregation with S. sanguis and another that inhibited the interaction with A. israelii, also were shown to inhibit the respective coaggregation interactions, suggesting that each of the immunoglobulins recognized its adhesin molecule at or near the active sites. By immunoblotting or immunoprecipitation, the S. sanguis adhesin-specific MAbs reacted with a 75-kilodalton polypeptide present in fimbria-enriched preparations, whereas the A. israelii adhesin-specific MAbs recognized a 45-kilodalton polypeptide in the same preparations. By screening hybridoma supernatants directly for their capacity to block coaggregation, we isolated MAbs which were used to establish that the B. loescheii-S. sanguis and the B. loescheii-A. israelii interactions were mediated by different adhesins.
Stimulation of superoxide and lactoferrin release from polymorphonuclear leukocytes by the type 2 fimbrial lectin of Actinomyces viscosus T14V
Authors: Sandberg A L , Mudrick L L , Cisar J O , Metcalf J A , Malech H L
Published: January 2020

Polymorphonuclear leukocyte (PMN)-dependent destruction of Actinomyces viscosus T14V is initiated by the recognition of galactose-containing receptors on sialidase-treated PMNs by the lectin associated with the type 2 fimbriae of these bacteria. A. viscosus T14V also stimulates the respiratory burst in PMNs as well as the release of contents of the secondary granules, as determined by the presence of lactoferrin in the culture supernatants. Under the experimental conditions employed, these bacteria do not induce the release of beta-glucuronidase, a constituent of primary granules. None of the three PMN responses studied occurs in cultures containing a mutant of A. viscosus T14V that lacks fimbriae. Activation of the PMNs is mediated by the lectin associated with the type 2 fimbriae, as demonstrated by the finding that beta-linked galactosides inhibit stimulation of the respiratory burst. Thus, the interaction of the Actinomyces fimbrial lectin with its complementary receptors on PMNs results not only in killing of these bacteria but also in the release of reactive oxygen intermediates and enzymes that may be detrimental to surrounding host tissues.
Identification and properties of Trichomonas vaginalis proteins involved in cytadherence
Authors: Alderete J F , Garza G E
Published: January 2020

Trichomonas vaginalis NYH286 surface proteins which are candidates for mediating parasite cytadherence (adhesins) were identified. At least four trichomonad protein ligands ranging in relative molecular mass from 65 to less than or equal to 21 kilodaltons were found to selectively bind to chemically stabilized HeLa cells. The proteins were present on the surfaces of 10 different isolates of T. vaginalis examined; however, the nonpathogenic trichomonad T. tenax did not possess similar HeLa cell-binding proteins under identical experimental conditions, suggesting that these proteins are unique to the pathogenic human trichomonads. The surface nature of the candidate adhesins was confirmed by the ability of the proteins on intact, live organisms to be radioiodinated and to be removed with trypsin treatment. Rabbit antiserum (immunoglobulin G fraction) generated against adhesin proteins electroeluted from acrylamide preparations inhibited cytadherence compared with control immunoglobulin G. An adherence-negative subpopulation of T. vaginalis NYH286 organisms was also isolated. These nonadherent trichomonads did not synthesize the adhesin proteins. Interestingly, absence of adhesins from these parasites paralleled expression of a major immunogen known to undergo phenotypic variation. Revertant organisms derived from the adherence-minus subpopulation synthesized the adhesins and attached to HeLa cells. The emergence of revertant adherent T. vaginalis organisms also corresponded with the appearance of parasites which were without the major immunogen on their surface. Finally, it was determined that only those parasites lacking the major surface immunogen were capable of adherence and toxicity to HeLa cells.
Determination of bovine lymphocyte responses to extracted proteins of Brucella abortus by using protein immunoblotting
Authors: Brooks-Alder B , Splitter G A
Published: January 2020

Isolation and identification of Brucella antigenic determinants important to cellular responses have been difficult. In this study, bovine peripheral blood mononuclear (PBM) cells from cattle vaccinated with Brucella abortus 19 proliferated to extracted bacterial proteins blotted onto nitrocellulose. Proteins were extracted from gamma-irradiated B. abortus 19 with a sodium dodecyl sulfate extraction buffer. The extracted proteins were separated electrophoretically by sodium dodecyl sulfate-polyacrylamide gel electrophoresis prior to electroblotting onto nitrocellulose. Nitrocellulose sections corresponding to individual lanes of the gel (containing all separated proteins) were then cultured with the PBM cells. Primary and secondary stimulation responses of the PBM cells with the whole protein blots were similar kinetically to the responses of the PBM cells stimulated with whole irradiated B. abortus 19 or with whole irradiated B. abortus 19 blotted onto nitrocellulose. Although lipopolysaccharide was determined to be associated with the extracted proteins and transferred onto the blots, the lipopolysaccharide did not stimulate cellular proliferation, as indicated by the antigen-specific secondary responses. Stimulating PBM cells with portions of the blot containing high (greater than 45,000)-, medium (25,000 to 45,000)- or low (25,000)-molecular-weight proteins demonstrated that the responding cells were specific only to the proteins of corresponding molecular weights. These results indicate that cellular responses to individual proteins can be studied without cloning the bacterial genes or purifying the individual proteins.
Isolation and morphology of an immunoreactive outer wall fraction produced by spherules of Coccidioides immitis
Authors: Cole G T , Seshan K R , Franco M , Bukownik E , Sun S H , Hearn V M
Published: January 2020

A previously undescribed, immunoreactive, membranous spherule outer wall (SOW) fraction produced by Coccidioides immitis (strains 634 and 735) grown in culture was isolated. Both this fraction and intact spherules were reactive with sera from coccidioidomycosis patients, as demonstrated by immunofluorescence microscopy. The serological activity of SOW was also demonstrated by its reactivity with human anti-C. immitis tube precipitin in a standardized immunodiffusion assay. Extraction of SOW with the nonionic detergent N-octyl-beta-D-glucopyranoside (OG) permitted the isolation of an OG-soluble fraction which was reactive in the immunodiffusion assay. Rabbit antisera raised against the OG-soluble fraction were used in immunofluorescence and immunoelectron-microscopic studies of the parasitic cycle to confirm that the immunoreactive components of the solubilized fraction of SOW were associated with the inner and outer layers of the spherule wall as well as with distinct cytoplasmic organelles observed in thin sections of spherules. The immunoreactivity of SOW with sera from patients suggested that infected individuals are exposed to this surface wall material isolated from in vitro-grown spherules.
Naturally occurring antibodies to an epitope on Plasmodium falciparum gametes detected by monoclonal antibody-based competitive enzyme-linked immunosorbent assay
Authors: Graves P M , Wirtz R A , Carter R , Burkot T R , Looker M , Targett G A
Published: January 2020

The antibody response to an epitope on gamete antigens of Plasmodium falciparum in persons naturally exposed to malaria has been investigated by competitive enzyme-linked immunosorbent assay. The assay detects antibodies to an epitope on the 48/45-kilodalton (kDa) gamete surface antigen by competition with horseradish peroxidase-labeled monoclonal antibody IIC5-B10. Five sera previously shown to immunoprecipitate the 230- and 48/45-kDa antigens significantly inhibited IIC5-B10 binding to an average of 24.2% of control. The one serum which precipitated only the 48/45-kDa antigen did not inhibit IIC5-B10 binding. For 26 sera which were negative by immunoprecipitation, mean binding in the assay was 112.7% of control (pooled London nonimmune sera). Recognition of both 230-kDa and 48/45-kDa antigens was associated with a titer of 1:9 or greater (reciprocal geometric mean titer, 27.6) for inhibition to more than 2 standard deviations from the mean of the negative sera. The results show that the IIC5-B10 binding site is a naturally immunogenic epitope recognized by the majority of persons who had antibodies to the 48/45-kDa protein. An additional finding was enhancement of binding of IIC5-B10 to an average of 154.4% of control by five sera which recognized only the 230-kDa antigen, presumably due to conformational alteration of the gamete antigen complex.
Evidence that in vitro adherence of Klebsiella pneumoniae to ciliated hamster tracheal cells is mediated by type 1 fimbriae
Authors: Fader R C , Gondesen K , Tolley B , Ritchie D G , Moller P
Published: January 2020

Clinical isolates of fimbriated and nonfimbriated Klebsiella pneumoniae were examined for the ability to adhere to hamster tracheal cells cultured in vitro. Fimbriated-phase K. pneumoniae adhered preferentially to ciliated cells, whereas nonfimbriated-phase organisms were not adherent. The adherence was inhibited by D-mannose but not D-glucose, suggesting that type 1 fimbriae serve as the adhesin in the attachment process.
Characterization of fibronectin-binding antigens released by Mycobacterium tuberculosis and Mycobacterium bovis BCG
Authors: Abou-Zeid C , Ratliff T L , Wiker H G , Harboe M , Bennedsen J , Rook G A
Published: January 2020

Fibronectin (FN)-binding antigens are prominent components of short-term culture supernatants of Mycobacterium tuberculosis. In 3-day-old supernatants, a 30-kilodalton (kDa) protein was identified as the major FN-binding molecule. In 21-day-old supernatants, FN bound to a double protein band of 30 and 31 kDa, as well as to a group of antigens of larger molecular mass (57 to 60 kDa). FN-binding molecules in this size range, but not of 30 to 31 kDa, were also found in sonicates. We showed that the 31- and 30-kDa FN-binding bands correspond to components A and B of the BCG85 complex, previously shown to be abundant in culture supernatants of Mycobacterium bovis BCG. Thus, a polyclonal antibody to the BCG85 complex bound to the 30- and 31-kDa antigens and inhibited binding of FN to them on immunoblots of the culture filtrates. Similarly, FN bound to the purified components of the BCG85 complex, and this binding was blocked by the antibody. A monoclonal antibody, HYT27, also bound both to the BCG85 components A and B and to the 30- and 31-kDa FN-binding molecules of M. tuberculosis, but it did not block the binding of FN. Related molecules appear to be present on the surface of BCG and to mediate the binding of BCG to FN-coated plastic surfaces, since this binding could also be blocked by the polyclonal anti-BCG85 antibody and by the purified components of BCG85, particularly component A, but not by monoclonal antibody HYT27. The binding of these mycobacterial antigens to FN appears to be of very high affinity, and we suggest that this property of major secreted antigens of M. tuberculosis indicates an important role in mycobacterial disease and in the binding of BCG to tumor cells during immunotherapy of bladder cancer.
Cloning and transposon insertion mutagenesis of virulence genes of the 100-kilobase plasmid of Salmonella typhimurium
Authors: Gulig P A , Curtiss R
Published: January 2020

We have cloned regions of the 100-kilobase (kb) plasmid, pStSR100, of Salmonella typhimurium SR-11 that confer virulence to plasmid-cured S. typhimurium. Cells carrying recombinant plasmids that conferred virulence were selected by inoculating mice orally with recombinant libraries in virulence plasmid-cured S. typhimurium and harvesting isolates that infected spleens. Three plasmids, pYA401, pYA402, and pYA403, constructed with the cosmid vector pCVD305 conferred wild-type levels of virulence to plasmid-cured S. typhimurium and had a common 14-kb DNA insert sequence. Another recombinant plasmid, pYA422, constructed with the vector pACYC184, conferred to plasmid-cured S. typhimurium a wild-type 50% lethal dose (LD50) level, but mice died more slowly than when infected with wild-type S. typhimurium. Furthermore, pYA422 conferred the ability to cause a higher, but not a wild-type, level of splenic infection on plasmid-cured S. typhimurium. pYA422 had a 3.2-kb insert sequence which mapped to the center of the 14-kb common sequence of the cosmid clones. Transposon Tn5 insertion mutations in pYA403 inhibited virulence to various degrees, and when transduced into the native virulence plasmid of S. typhimurium, these Tn5 insertions decreased virulence to degrees similar to those observed when the Tn5 insertions were present in pYA403. vir-22::Tn5 in pStSR100 greatly lowered infection of spleens relative to unmutagenized virulence plasmid, while vir-26::Tn5 and vir-27::Tn5 lowered splenic infection to lesser degrees. At least three proteins were encoded by pYA403 containing 23 kb of insert sequence and subclone pYA420, containing the 14-kb common insert sequence present in all of the cosmid clones. One of these proteins, with an apparent molecular weight of 28,000, was also encoded by pYA422. The Tn5 insertion that most attenuated virulence, vir-22::Tn5, inhibited synthesis of the 28,000-molecular-weight protein. The vir-22::Tn5 insertion was complemented by recombinant plasmids encoding only the 28,000-molecular-weight protein, suggesting a role of this protein in virulence. However, recombinant plasmids, exemplified by pYA422, that encoded only the 28,000-molecular-weight protein did not confer full virulence.
Membrane glycoprotein M-2 protects against Leishmania amazonensis infection
Authors: Champsi J , McMahon-Pratt D
Published: January 2020

Previous passive antibody transfer experiments have indicated that immunity to a 46-kilodalton membrane glycoprotein (M-2) of Leishmania amazonensis may protect against infection with this parasite. In the studies described in this paper, we investigated the ability of the purified M-2 molecule to elicit a protective immune response in conjunction with Freund incomplete and complete adjuvants, saponin, and Corynebacterium parvum. Both relatively susceptible (BALB/c and CBA) and resistant (C57BL/6) strains of mice were examined. C. parvum appeared to be the most effective adjuvant in the three mouse strains tested. The level of protection varied with the mouse strain, although all animals received identical preparations of antigen and adjuvant. Immunization of CBA mice with the M-2 glycoprotein and C. parvum resulted in complete protection against a challenge infection of 10(4) and 10(6) late log-phase promastigotes of L. amazonensis. In the BALB/c strain, complete protection was observed in some of the immunized animals (28 to 50%); in the rest of the mice the onset of infection was significantly delayed. Protective immunity for C57BL/6 mice was observed only at the low infecting dose (10(4) L. amazonensis organisms). The level of protection observed is reflected by increased antibody response (immunoglobulins G1 and G2) developed to the M-2 molecule. The relationship of pure T-cell (nonantibody) immunity to this protection remains to be elucidated.
Induction of nonspecific acquired resistance and delayed-type hypersensitivity, but not specific acquired resistance in mice inoculated with killed mycobacterial vaccines
Authors: Orme I M
Published: January 2020

A number of nonliving mycobacterial preparations were tested in vivo for their capacity to generate various relevant parameters of cellular immunity. All preparations tested had some detectable activity in raising resistance to challenge with Mycobacterium tuberculosis or with Listeria monocytogenes and in conferring the ability to mount a delayed-type hypersensitivity response to tuberculin. This report presents the first evidence, however, that none of these preparations were able to generate protective T cells capable of adoptive immunization against virulent tuberculosis. These data are discussed in terms of the use of these preparations in generating M. tuberculosis-reactive T-cell lines and the application of these lines in the continuing search for an improved vaccine against tuberculosis.
Hormonal modulation of sex differences in resistance to Leishmania major systemic infections
Authors: Mock B A , Nacy C A
Published: January 2020

Differences in susceptibility to intravenously inoculated Leishmania major were observed in male and female mice of the BALB/cAnPt, DBA/2N, and DBA/2J strains and (BALB/cAnPt x DBA/2N)F1 hybrids. In all cases, males had significantly higher liver parasite burdens than females. Orchidectomy of BALB/c males resulted in a 20% decrease in the number of parasites in the liver compared with either normal or sham-gonadectomized controls. Additionally, testosterone treatment of female BALB/c mice resulted in an 88% increase in the number of liver amastigotes. These results suggest that the hormone testosterone can modulate systemic L. major infections in BALB/c mice.
Genetic control of Salmonella typhimurium-induced depression of delayed-type hypersensitivity to sheep erythrocytes in mice
Authors: Nauciel C , Ronco E , Guenet J L
Published: January 2020

Infection of mice with a temperature-sensitive mutant of Salmonella typhimurium C5TS allowed the survival of genetically susceptible mice. The ability to mount a delayed-type hypersensitivity (DTH) response to sheep erythrocytes during infection with C5TS was studied in various inbred mouse strains, recombinant inbred strains derived from C57BL/6 (susceptible) and A/J (resistant) mice, and C3H congenic mice. Suppression of the DTH response to sheep erythrocytes was found in mice that carried the Itys allele, the H-2b haplotype, or both. These genes are known to increase susceptibility to S. typhimurium infection. In contrast, no DTH response suppression was observed in mouse strains that carried other genes that increased susceptibility to S. typhimurium, e.g., DBA/2 and C3H/HeJ. Apart from a transient suppression in A/J mice, the DTH responses of resistant mice (A/J and CBA) were normal or increased. The DTH response to sheep erythrocytes could be restored in immunodepressed mice by increasing the immunizing dose, suggesting the possible role of activated macrophages in depression of the DTH response.
H-2 restriction in acquired cell-mediated immunity to infection with Francisella tularensis LVS
Authors: Anthony L S , Kongshavn P A
Published: January 2020

The H-2 restriction imposed on the T-lymphocyte-macrophage interaction leading to the expression of acquired cellular immunity was evaluated in an experimental model of infection with the live vaccine strain of Francisella tularensis. Restriction between T cells and macrophages was examined in vitro in cultures containing macrophages from C57BL/10 (B10) mice, T cells from immune B10 H-2 congenic mice, and F. tularensis antigen. The cellular interaction was assayed by the production in the cultures of factors which stimulate thymocyte DNA synthesis. It was observed that homology at the I-A region of the H-2 complex was required for productive T-cell-macrophage cooperation to occur. Restriction was also investigated in an in vivo passive cell transfer system. Spleen cells from immunized B10 mice were injected into naive B10 H-2 congenic mice, which were then challenged with F. tularensis. Enhanced resistance to the challenge infection in recipient mice was used as a marker of a successful T-cell-macrophage interaction. It was found that when the recipient strain shared H-2 I-A region homology with the donor strain, enhanced antitularemic resistance was expressed, whereas homology at the H-2 K or D region was insufficient. Thus, macrophage--T-cell cooperation in immunity to experimental tularemia was restricted at the level of class II determinants.
Effect of nutritional constraints on the biosynthesis of the components of the phosphoenolpyruvate: sugar phosphotransferase system in a fresh isolate of Streptococcus mutans
Authors: Rodrigue L , Lacoste L , Trahan L , Vadeboncoeur C
Published: January 2020

A procedure for the purification of enzyme I (EI) and the protein HPr, the general components of the phosphoenolpyruvate:sugar phosphotransferase system, from Streptococcus mutans serotype c is presented. The method was also applied successfully to the purification of EI and HPr from Streptococcus salivarius, Streptococcus sobrinus, and Streptococcus sanguis. Using specific antibodies obtained against the proteins purified from S. mutans DR0001, we determined quantitatively by rocket electrophoresis the cellular levels of EI and HPr in a freshly isolated strain of S. mutans grown under various conditions in continuous culture. The activity of a few specific EIIs was also determined by an in vitro phosphorylation test. Results indicated that maximum EII activities for glucose, mannose, and 2-deoxyglucose were obtained under conditions of glucose limitation, at pH 7.0 and low dilution rate (D = 0.057/h). Increasing the amount of glucose or the dilution rate (D = 0.40/h) or decreasing the pH from 7.0 to 5.5 resulted in a 1.4- to 24-fold decrease in these activities. The EII activity for fructose was not influenced by the growth conditions in the same way as the other EIIs. The fructose EII was highest at pH 5.5 and at high dilution rate under conditions of glucose or nitrogen limitation and was always repressed at pH 7.0 and at low dilution rates. The intracellular levels of EI were also dependent on the growth conditions. The highest concentration (0.65 nmol/mg of protein) was observed in cells grown under glucose limitation at pH 7.0 and high dilution rate, and the lowest concentration (0.12 nmol/mg of protein) was found in cells grown under glucose excess at pH 7.0 and high dilution rate. The other general component of the phosphoenolpyruvate:sugar phosphotransferase system, the protein HPr, was not influenced significantly by varying growth conditions.
Interactions between human natural killer (NK) lymphocytes and yeast cells: human NK cells do not kill Candida albicans, although C. albicans blocks NK lysis of K562 cells
Authors: Zunino S J , Hudig D
Published: January 2020

Rodent natural killer (NK) lymphocytes are cytotoxic to certain fungi. We investigated whether human NK cells are cytotoxic to the yeast Candida albicans. We found that human peripheral blood lymphocytes possessing NK cell activity had little or no effect on the viability of the yeast. Unopsonized C. albicans, however, were able to block NK cell-mediated cytotoxicity at a ratio of 100 yeast to one K562 erythroleukemia cell. C. albicans was not toxic to the lymphocytes nor did it take up isotope released by the K562 cells. Furthermore, C. albicans that was pretreated with human serum blocked NK cell activity more than did untreated C. albicans. Binding of the yeasts to NK cells could account for the blocking effect of serum-treated yeasts, but not for that of the untreated yeasts. Flow cytometry indicated that there was preferential binding of C. albicans to NK lymphocytes but not to T cells when the yeasts were pretreated with human serum. In this report we affirm the results of the study by Vecchiarelli et al. (A. Vecchiarelli, F. Bistoni, E. Cenci, S. Perito, and A. Cassone, Sabouraudia 23:377-387, 1985), that the first report of rodent NK cell activity against the yeast Cryptococcus neoformans (J. W. Murphy and D. O. McDaniel, J. Immunol. 128:1577-1583, 1982) cannot be extrapolated to a general phenomenon of unprimed lymphocyte-mediated destruction of all species of yeast. Our data extend the observations to humans and also suggest that in vivo interactions between NK lymphocytes and opportunistic fungal pathogens may affect NK cell function.
Interaction of macrophage cationic proteins with the outer membrane of Pseudomonas aeruginosa
Authors: Sawyer J G , Martin N L , Hancock R E
Published: January 2020

The interaction of the polycationic rabbit alveolar macrophage cationic proteins MCP-1 and MCP-2 (or their identical neutrophil equivalents NP-1 and NP-2) with the surface of Pseudomonas aeruginosa was investigated. Both proteins bound avidly to purified lipopolysaccharide, as judged by their ability to competitively displace the probe dansyl polymyxin with 50% inhibition (I50) values of 2 to 3 microM. Similar I50 were measured with dansyl polymyxin as a probe for cell surface binding, suggesting that the initial binding site for MCP-1 and MCP-2 on the surface of cells was lipopolysaccharide. Both MCP-1 and MCP-2 permeabilized outer membranes to the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN). The initial rate of NPN uptake plotted against the concentration of MCP-1 or MCP-2 gave sigmoidal curves, suggesting cooperative permeabilization of the outer membrane. Replotting the data as a Hill plot gave an affinity parameter, S0.5, the concentration of MCP giving a half-maximal increase in the rate of NPN uptake, of 5 and 25 microM for MCP-1 and MCP-2, respectively, and thus subsequent studies concentrated on the more active permeabilizer MCP-1. Permeabilization of outer membranes to NPN was a function of buffer pH, with lower pH considerably favoring the permeabilizing effects of MCP-1. Thin-section electron microscopic visualization of MCP-1-treated cells showed production of extended blebs. Further evidence of an altered cell surface after MCP-1 treatment was obtained by demonstrating that treated unopsonized cells were more efficiently phagocytosed by unelicited rabbit alveolar macrophages. The data overall suggest that macrophage cationic proteins interact with the P. aeruginosa outer membrane in a manner typical of other polycations and suggest that one of their major functions may be to permeabilize the outer membrane.
Small-intestinal factors promote encystation of Giardia lamblia in vitro
Authors: Gillin F D , Reiner D S , Boucher S E
Published: January 2020

Bile salts and fatty acids stimulated differentiation of cultured Giardia lamblia trophozoites into water-resistant cysts at the slightly alkaline pH of the small intestinal lumen. Maximum encystation occurred at pH 7.8. Thus, specific small-intestinal factors may influence encystation in vivo as well as in vitro.
Genetic complementation of defects in vaccine-induced immunity against Schistosoma mansoni in P- and A-strain inbred mice
Authors: Correa-Oliveira R , James S L , Sher A
Published: January 2020

Inbred P- and A-strain mice are deficient in their capacity to develop resistance to challenge infection in response to vaccination with irradiated cercariae of Schistosoma mansoni and are also defective in their cell-mediated response as assessed by the activity of antigen-elicited macrophages in killing schistosome larvae in vitro. In contrast, vaccinated (P x A)F1 mice displayed high levels of both immunity to challenge and macrophage larvicidal activity, indicating that the P- and A-strain defects in the vaccine-induced response are controlled by distinct genetic loci.
Purification and characterization of H.8 antigen from group B Neisseria meningitidis
Authors: Bhattacharjee A K , Moran E E , Ray J S , Zollinger W D
Published: January 2020

The surface antigen (H.8) common to the pathogenic Neisseria species was purified by a simple procedure by use of high-performance liquid chromatography. The purified H.8 antigen was characterized as to its amino acid composition, susceptibilities to several proteolytic enzymes, isoelectric point, and susceptibilities to an acid and a base. The amino acid composition of purified H.8 antigen from two strains of Neisseria meningitidis group B, namely, 44/76 and 8047, were compared. It was found that glutamic acid, alanine, and proline accounted for about 80% of the total amino acids in each case. A preliminary analysis of the lipid content of this protein was made. It showed the presence of a lipid component that moves between C9 and C11 straight-chain fatty acids in the gas chromatograph. Limited amino acid sequence data were obtained by sequencing a fragment of the H.8 antigen that was isolated after partial acid hydrolysis. The H.8 antigen epitope was found to be labile to treatment with both a mild acid and a mild base.
Factors influencing the interaction of Candida albicans with fibroblast cell cultures
Authors: Merkel G J , Phelps C L
Published: January 2020

The interaction of Candida albicans clinical isolates with primary and established fibroblast cultures was studied. The intent was to determine whether yeast adherence and invasion of nonendothelial cell monolayer cultures could be quantitated reproducibly and whether this system could be used for future studies on yeast pathogenesis. Our results demonstrated that specific interactions between the yeast cells and fibroblasts only occurred at 37 degrees C and correlated with the germination process. Fluorescent-antibody staining indicated that invasion or tight associations between the germinating yeast cells and mammalian cells occurred after less than 3 h of incubation. Yeast adherence was estimated radiometrically and trypsin-resistant interaction with individual mammalian cells (infection) was measured microscopically after inoculated monolayer cells were detached with trypsin. We demonstrated that both types of association were time dependent at 37 degrees C; neither was affected by the concentration of glucose used to grow the yeast cells. Primary and established fibroblast cell lines were equally susceptible to infection, but primary cells appeared to have more yeast-binding sites. Fibroblasts maintained in confluent culture for an extended period of time also appeared to have more binding sites, and while not quantitatively more susceptible to infection, the older cells were more susceptible to infection-related cell death. An established kidney epithelial cell line (MDCK) was not susceptible to either type of yeast interaction, indicating that the yeast-fibroblast associations were specific.
Antibody synthesis specific for nonoral antigens in inflamed gingiva
Authors: Mallison S M , Szakal A K , Ranney R R , Tew J G
Published: January 2020

In vitro experimentation indicates that periodontitis-associated bacteria contain potent polyclonal B-cell activators (PBA). We reasoned that if PBA were operative in vivo, plasma cells specific for nonoral antigens should be present in the inflamed gingival tissues, which are characterized by a plasma cell infiltrate. To test this, rabbits with experimental periodontitis were immunized in the hind legs with the histochemically detectable antigen horseradish peroxidase (HRP) or glucose oxidase (GO). At various times after secondary immunization, inflamed gingival tissue was removed, sectioned, and treated histochemically to reveal plasma cells that specifically bound HRP or GO. Remarkably, by 9 days after secondary immunization, hundreds of HRP- or GO-binding plasma cells were found in the inflamed gingival tissue of immunized rabbits. The presence of these plasma cells, observed 7 to 10 days after booster immunization, was further substantiated by the presence of large amounts of locally produced HRP- or GO-specific antibody in gingival crevicular fluid. By 1 month after secondary immunization, the number of antigen-binding plasma cells had decreased dramatically, but a small number of antigen-specific plasma cells were detected for as long as 9 months after secondary immunization. The large number of HRP- or GO-specific plasma cells observed 9 days after immunization led us to see whether recently stimulated cells were more susceptible to PBA. Peripheral blood lymphocytes (PBL) were obtained at different times after booster immunization and cultured in the presence or absence of a PBA from Fusobacterium nucleatum. At 7 days after immunization, PBL spontaneously differentiated into antibody-forming cells in culture, and this process was enhanced by PBA. In contrast, PBL taken months after immunization produced little antibody in culture, and enhancement by PBA was difficult to detect. Compared with resting B cells, the recently stimulated B cells clearly differentiated more readily into antibody-forming cells. In conclusion, antibody synthesis specific for nonoral antigens did occur in inflamed gingival tissue, and a number of mechanisms, including PBA, probably contributed to this phenomenon.
Anticapsular antibody requirements for protection against experimental Haemophilus influenzae type b bacteremia after splenectomy
Authors: Rubin L G
Published: January 2020

Although asplenic individuals are at higher risk for infection with encapsulated bacteria, it is not known whether they require a higher concentration of anticapsular antibody than normal individuals do for protection against invasive disease caused by Haemophilus influenzae type b. At 21 days of age, rats were passively immunized with human hyperimmune serum globulin against H. influenzae b polysaccharide (or saline) after recovery from splenectomy or a sham operation. Starting at 18 h after immunization, rats received three intranasal inoculations of 10(7) CFU of H. influenzae b over the next 24 h. Of sham-operated rats given 0.75 or 3.0 micrograms of anticapsular antibody, 91 or 96%, respectively, were protected from bacteremia, whereas only 59 and 67% of similarly treated asplenic rats were protected (P less than 0.004, control versus asplenic rats). A 12-microgram antibody dose resulted in the complete protection of both groups. The magnitude of bacteria was significantly higher in the asplenic group at each dose of antibody. Thus, asplenic hosts may require a higher concentration of anticapsular antibody than normal individuals do for protection against invasive H. influenzae b disease.
Anti-idiotypic antibody with potential use as an Eimeria tenella sporozoite antigen surrogate for vaccination of chickens against coccidiosis
Authors: Bhogal B S , Nollstadt K H , Karkhanis Y D , Schmatz D M , Jacobson E B
Published: January 2020

Anti-idiotypic antibodies were raised in rabbits against four monoclonal antibodies with specificity for the surface antigenic determinants of Eimeria tenella sporozoites, the infective stage of the coccidial parasite. Two of the monoclonal antibodies (1073 and 15-1) transferred passive protection in chickens against E. tenella infection. The polyclonal anti-idiotype antibody preparations against protective monoclonal antibodies contained specificities for the paratope-associated idiotypes of these monoclonal antibodies, as assessed by the competitive inhibition of binding of the homologous idiotype-anti-idiotype by the sporozoite antigen. Competitive inhibition of binding of homologous idiotype-anti-idiotype by the parasite antigen was not observed when the anti-idiotype antibody preparations against monoclonal antibodies 1546 and 1096 were tested. The anti-idiotype 1073 and 15-1 antibodies functioned as surrogate antigens in vivo when used for vaccination of young chickens, as evidenced by the induction of partial protective immunity against subsequent challenge infection with virulent parasites and induction of antisporozoite antibodies. These data clearly support the view that anti-idiotypic antibodies raised against the paratope-associated idiotypes can mimic pathogen antigens and therefore can provide a possible alternative approach for the vaccination of chickens against coccidiosis.
Killing of human myelomonocytic leukemia and lymphocytic cell lines by Actinobacillus actinomycetemcomitans leukotoxin
Authors: Simpson D L , Berthold P , Taichman N S
Published: January 2020

The purified leukotoxin of Actinobacillus actinomycetemcomitans kills human leukemic cell lines (e.g., HL-60, U937, and KG-1) and human T- and B-cell lines (e.g., JURKAT, MOLT-4, Daudi, and Raji) in a dose- and time-dependent manner. The 50% effective doses for these cell lines are similar to those established for human polymorphonuclear leukocytes and monocytes. In contrast, other human and nonhuman tumor cell lines are not susceptible to the leukotoxin. These human leukemia and lymphoid cell lines will serve as useful model systems with which to study the molecular specificity and mechanism(s) of action of the actinobacillus leukotoxin.