bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacterium avium-Mycobacterium intracellular complex-induced suppression of T-cell proliferation in vitro by regulation of monocyte accessory cell activity
Authors: Tsuyuguchi I , Kawasumi H , Takashima T , Tsuyuguchi T , Kishimoto S
Published: January 2020

Heat-killed whole Mycobacterium avium-Mycobacterium intracellulare complex (MAC) and its lipid component impaired the capacity of human peripheral blood mononuclear cells to proliferate in vitro in response to concanavalin A (ConA), purified protein derivative of tuberculin (PPD), and to a lesser degree, phytohemagglutinin stimulation. Inhibition by MAC was not contingent upon prior exposure of the donor to MAC or other mycobacteria and occurred with lymphocytes from tuberculin-negative as well as -positive subjects. The suppression was not due to the toxicity of MAC. The suppression by MAC was not blocked by indomethacin. Adherent cell depletion and cell mixing experiments with T cells indicated that monocytes and not T cells were a major contributor to the immunosuppression observed. However, neither interleukin-1 production nor the expression of HLA-DR (Ia antigen) by monocytes was suppressed by MAC treatment. On the other hand, treatment of monocytes with MAC or MAC-derived lipid resulted in significant decreases in CD11b, a member of the leukocyte function-associated molecule-1 and LeuM3 (CD14) molecule. Anti-CD18 (beta-chain of the leukocyte function-associated molecule-1 family) monoclonal antibody had suppressive effects on ConA- and PPD- but not phytohemagglutinin-induced in vitro lymphocyte blastogenesis. We suggest that MAC and MAC-derived lipid suppress the ConA- and PPD-induced T-cell proliferations by blocking the expression of accessory molecules on the surfaces of monocytes which might be involved in nonspecific monocyte-T-cell interactions and not by inhibiting either monocyte Ia antigen expression or interleukin-1 production by monocytes.
Antigenic relationships of the lipopolysaccharides of Escherichia hermannii strains with those of Escherichia coli O157:H7, Brucella melitensis, and Brucella abortus
Authors: Perry M B , Bundle D R
Published: January 2020

Clinical isolates of Escherichia hermannii which showed serological cross-reaction with polyclonal antisera to the O-polysaccharide portion of the lipopolysaccharide of E. coli O157 strains and with antisera to the O antigens of Brucella abortus and B. melitensis were found by chemical and nuclear magnetic resonance analyses to have lipopolysaccharide O chains composed of linear polymers containing 1,2- and 1,3-linked 4-acetamido-4,6-dideoxy-alpha-D-mannopyranosyl (alpha-D-Rhap4NAc) residues. Two O-antigen structures were identified; each had an unbranched pentasaccharide repeating unit, and one was composed of three 1,2- and two 1,3-linked alpha-D-Rhap4NAc residues and the other had two 1,2- and three 1,3-linked alpha-D-Rhap4NAc residues. The above-described cross-serological reactivities, which have led to false-positive identifications, are related to the common occurrence of epitopes involving the presence of N-acyl derivatives of 4-amino-4,6-dideoxy-D-mannopyranosyl residues in the O-polysaccharide portions of the respective lipopolysaccharides of the organisms. Strains of E. hermannii which did not show serological cross-reactions with E. coli O157 and Brucella antisera were found to have unique lipopolysaccharide O chains devoid of D-Rhap4NAc residues, demonstrating the existence of serotypes of E. hermannii that are distinct on the basis of their lipopolysaccharide components.
Human immune response directed against Plasmodium falciparum heat shock-related proteins
Authors: Kumar N , Zhao Y , Graves P , Perez Folgar J , Maloy L , Zheng H
Published: January 2020

Heat shock-related stress proteins present in all eucaryotes and procaryotes have been shown to be immune targets in a broad range of infections. We have analyzed sera from people exposed primarily to Plasmodium falciparum for specific antibodies against two heat shock-related proteins (proteins similar to the heat shock protein with a molecular weight of 75,000 [Pfhsp] and a glucose-regulated protein with a molecular weight of 72,000 [Pfgrp]). In an immunoprecipitation analysis with metabolically labeled parasites and synthetic peptides in an enzyme-linked immunosorbent assay, specific antibodies against Pfhsp and Pfgrp were detected in the sera of these individuals. Sera from people exposed to a different human malarial parasite, Plasmodium vivax, did not react with the peptides in an enzyme-linked immunosorbent assay. Southern blot analysis with DNA isolated from P. falciparum from different geographical locations showed a conservation of genes for these stress proteins; thus, they are likely to be immune targets in various endemic areas. Lymphocytes from two tested immune donors responded in proliferation assays to purified Pfhsp and Pfgrp and purified recombinant proteins. However, a similar response was also seen in lymphocytes from nonimmune individuals and has raised questions pertaining to a generalized responsiveness of lymphocytes to some common determinants present in heat shock-related proteins in various pathogens.
Low pH-induced changes in Pseudomonas exotoxin and its domains: increased binding of Triton X-114
Authors: Idziorek T , FitzGerald D , Pastan I
Published: January 2020

Pseudomonas exotoxin (PE), which is composed of three structural domains, is a 66-kilodalton protein secreted by P. aeruginosa that is cytotoxic for mammalian cells. After binding to cell surface receptors and internalization into low-pH endocytic vesicles, PE or an active fragment kills mammalian cells by translocating across an intracellular membrane to the cytoplasm and shutting down protein synthesis. To investigate possible conformational changes associated with the translocation process, full-length PE or recombinant proteins containing the PE cell recognition domain, translocation domain, enzymatic domain, or translocation plus enzymatic domains were incubated with Triton X-114 at pH values ranging from 3.0 to 7.0. The truncated forms used were intact domains that had been expressed in Escherichia coli and subsequently purified. Previous studies (K. Sandvig and J. O. Moskaug, Biochem. J. 245:899-901, 1987) had shown that full-length PE bound more Triton X-114 at a low pH than at a physiologic pH. Therefore, we investigated whether this increased binding was due to a global change in PE or a change within a particular domain. Results showed that all the truncated toxin proteins displayed a similar pH-dependent entry into the detergent phase as native PE, with a transition point of 4.2 for PE and 4.4 to 4.5 for the truncated toxins. The isoelectric points of the recombinant proteins were measured and indicate that, at a low pH (5.0), the cell recognition domain bears a net positive charge, the translocation domain bears a net negative charge, and the enzymatic domain bears no charge. The results suggest that upon acidification in the endosome, PE becomes globally hydrophobic and is converted into a translocation-competent form.
Inhibition of Streptococcus mutans glucosyltransferase activity by antiserum to a subsequence peptide
Authors: Dertzbaugh M T , Macrina F L
Published: January 2020

An antigenic 15-amino-acid peptide sequence (gtfB.1) from the glucosyltransferase B enzyme of the cariogenic bacterium Streptococcus mutans GS-5 was identified previously from the genetic fusion of this sequence to the B subunit of cholera toxin. The resulting chimeric protein was used to raise antiserum in rabbits. This antiserum was shown to recognize the native glucosyltransferase enzyme and to inhibit its activity. The antiserum inhibited the synthesis of water-soluble glucan by approximately 40% and the synthesis of water-insoluble glucan by greater than 90%. The antiserum was shown to partially inhibit fructosyltransferase activity as well. The ability of this antipeptide antiserum to inhibit several enzymes from S. mutans suggests that these enzymes share an epitope related to enzymatic activity.
Microflora associated with experimental root surface caries in humans
Authors: Nyvad B , Kilian M
Published: January 2020

This study describes the microflora from actively progressing root surface caries lesions, in which mineral loss had been determined by quantitative microradiography. The caries lesions were produced experimentally in root surface specimens from human molars inserted in lower partial dentures carried for 3 months by six elderly individuals. A total of 780 bacterial isolates were identified from 13 plaque samples, collected with a punch technique, and six dentin samples. The composition of the microflora showed distinct individual differences. The microflora from plaque samples associated with the highest mineral loss was dominated by either Actinomyces viscosus or a combination of mutans streptococci (serotypes c, d, and f) and Lactobacillus species (L. casei and L. brevis). Plaque from root surfaces with less pronounced mineral loss harbored a more complex microflora comprising gram-positive rods, mutans streptococci, Streptococcus mitis biovar 1, Veillonella spp., gram-negative rods, and low numbers of lactobacilli. In the latter samples, individual variations in the proportions of mutans streptococci (serotypes c, d, and g), Actinomyces species (A. viscosus and A. naeslundii), and Veillonella parvula biotypes were observed. These findings suggest that certain species or combinations of species are more cariogenic than others and that dominance of single acidogenic species in particular is conducive to high caries activity.
Recombinant granulocyte colony-stimulating factor and lipopolysaccharide maintain the phenotype of and superoxide anion generation by neutrophils
Authors: Ichinose Y , Hara N , Ohta M , Aso H , Chikama H , Kawasaki M , Kubota I , Shimizu T , Yagawa K
Published: January 2020

Superoxide anion (O2-) generation by human blood neutrophils induced by phorbol myristate acetate, formyl-methionyl-leucyl-phenylalanine, and monoclonal antibody YI51 was measured 24 h after incubation in medium alone, medium with recombinant human granulocyte colony-stimulating factor (rG-CSF), and medium with lipopolysaccharide (LPS). Monoclonal antibody YI51 was able to bind to neutrophils and induce O2- generation after the addition of anti-mouse immunoglobulin antibody as a cross-linking agent. In the 24-h culture, there was no significant difference in neutrophil survival among the three cultures. The amount of O2- generated by neutrophils in control medium markedly decreased compared with that before culture. However, cells in medium with rG-CSF or LPS maintained the ability to generate O2- well or moderately, respectively. Thus, the activity maintained by rG-CSF and LPS was neutralized by the anti-G-CSF serum. Furthermore, significant amounts of G-CSF were detected in supernatants of neutrophils cultured with LPS for 24 h. It was not detectable, however, in control supernatants. To examine whether the phenotype of the plasma membrane of cells changed in the 24-h culture, expression of CD16 (FcR III) and YI51 antigens was analyzed by flow cytometry. The expression of CD16 and YI51 antigens on cells cultured with rG-CSF or LPS was maintained compared with that of control cells. These observations thus indicate that G-CSF is one of the factors essential to maintain the functioning and phenotype of mature neutrophils.
Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes
Authors: Boehm D F , Welch R A , Snyder I S
Published: January 2020

The primary structure of Escherichia coli hemolysin (HlyA) contains a 9-amino-acid sequence which is tandemly repeated 13 times near the C terminus and which is essential for hemolytic activity. Hemolysin also requires an unknown modification by an accessory protein, HlyC, for hemolytic activity. The role of calcium in the interaction of HlyA with erythrocytes was investigated by using recombinant strains which produced inactive hemolysins unmodified by HlyC or deleted of the repeat sequences. 45Ca2+ autoradiography of the recombinant hemolysins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose showed that full-length, active hemolysin bound calcium. The domain involved in binding calcium was identified as the tandemly repeated sequences, since the deletion derivative missing 11 of the 13 repeats did not bind calcium. Inactive hemolysin, unmodified by HlyC, contained the repeated sequences and bound calcium as efficiently as the active, full-length toxin. The binding of the inactive toxins to erythrocytes was investigated by immunoblotting saline-washed, toxin-treated cells with monoclonal antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of membrane proteins. The binding of full-length, active hemolysin to erythrocytes was calcium dependent. Inactive hemolysin deleted of the repeat units did not bind to cells. The inactive hemolysin, unmodified by HlyC, bound calcium but did not bind to erythrocytes. These results highlight the importance of calcium in the binding of hemolysin to erythrocytes and suggest that the binding of hemolysin to cells requires an interaction between the calcium-binding repeat domain and the modification produced by the HlyC protein.
Helicobacter pylori urease activity is toxic to human gastric epithelial cells
Authors: Smoot D T , Mobley H L , Chippendale G R , Lewison J F , Resau J H
Published: January 2020

A human gastric adenocarcinoma cell line was used to evaluate the contribution of urease from Helicobacter (formerly Campylobacter) pylori to its cytotoxicity. Gastric cells cultured in medium supplemented with 20 mM urea were exposed to 5 x 10(6) CFU of H. pylori per ml with or without the addition of a urease inhibitor, acetohydroxamic acid. Viabilities of cells exposed to H. pylori for 2, 24, and 48 h, assessed by incorporation of neutral red dye, were 60, 27, and 16%, respectively; however, the viabilities of cells exposed to both H. pylori and acetohydroxamic acid were 92, 46, and 20% after 2, 24, and 48 h, respectively, (P less than 0.001). Therefore, the urease activity of H. pylori may play an important role in its pathogenicity, and inhibition of this enzyme activity may have therapeutic potential.
Proteolysis of Clostridium perfringens type A enterotoxin during purification
Authors: Park K B , Labbé R G
Published: January 2020

The small satellite bands of enterotoxin frequently seen in polyacrylamide gels following purification of Clostridium perfringens enterotoxin were found to be due to endogenous protease activity and were not present if phenylmethylsulfonyl fluoride (PMSF; 1 mM) and EDTA (10 mM) were used in the purification protocol. The use of PMSF was avoided by passing gel filtration-purified enterotoxin material through DEAE-Sephacel. This modified protocol resulted in an 11.4-fold purification of enterotoxin and a 26.8% yield. Contrary to previous reports (B. R. Dasgupta and M. W. Pariza, Infect. Immun. 38: 592-597, 1982), if PMSF and EDTA were included during purification, we were unable to detect the novel enterotoxin ET-1 produced by strain NCTC 10240. C. perfringens proteases cleaved homogeneous enterotoxin into two additional fragments, suggesting that ET-1 was a product of endogenous protease action during purification.
Two distinct outer membrane serotype subcomplexes of Neisseria meningitidis serogroup A
Authors: Bläsius R , Strittmatter W , Crowe B , Achtman M
Published: January 2020

A 350-kilodalton serotype outer membrane complex containing the class 1, 3, and 4 outer membrane proteins was isolated from serogroup A Neisseria meningitidis. Partial denaturation yielded two serotype subcomplexes containing the class 3 and 1 proteins (85 kilodaltons) and the class 3 and 4 proteins (94 kilodaltons), respectively.
Vaccination of humans against cutaneous leishmaniasis: cellular and humoral immune responses
Authors: Nascimento E , Mayrink W , da Costa C A , Michalick M S , Melo M N , Barros G C , Dias M , Antunes C M , Lima M S , Taboada D C
Published: January 2020

Brazilian army conscripts were vaccinated against American cutaneous leishmaniasis by using nonliving polyvalent promastigote Leish vaccine 5 or Leish vaccine 6 (vaccines with five or six Leishmania stocks) with or without Corynebacterium parvum. No statistically significant differences in lymphocyte stimulation indices were found between vaccinated groups with or without C. parvum, but lymphocyte stimulation indices of all vaccinees were significantly higher (P less than 0.001) than those of the placebo group. A correlation of 90% was found between positive skin test results and positive lymphocyte stimulation indices. Eight major antigens with estimated molecular masses of 13.5, 25, 40, 63, 73, 85, 97, and 160 kilodaltons were recognized by Leish vaccine 5 sera. Our finding also demonstrated the predominance of immunoglobulin M antibody in sera of vaccinated subjects and that a component of Leish vaccine 5, gp63, was immunogenic in humans both at the T-cell level and at the antibody level.
Penetration and intracellular growth of Brucella abortus in nonphagocytic cells in vitro
Authors: Detilleux P G , Deyoe B L , Cheville N F
Published: January 2020

In pregnant ruminants, Brucella abortus localizes and replicates within the rough endoplasmic reticulum of trophoblastic epithelial cells. In this study, Vero cells were exposed to B. abortus to investigate its internalization and intracellular growth in nonphagocytic cells. A new double-fluorescence staining procedure to discriminate between extracellular and intracellular bacteria was developed. Studies with the double-fluorescence staining procedure and quantitative bacteriologic culture of disrupted host cells showed that various B. abortus strains replicated within Vero cells, including smooth virulent (strains 2308S and 544), smooth attenuated (strain 19), and rough (strains 45/20 and 2308R) strains. Rough brucellae were more adherent and entered a greater number of Vero cells. Intracellular replication occurred in a larger percentage of cells with smooth virulent (2308S and 544) strains than with smooth attenuated (19) or rough (45/20 and 2308R) strains. Differences in adhesiveness and invasiveness were correlated to hydrophobicity of the organism, as measured by hydrocarbon adherence. Ultrastructurally, intracellular smooth (2308S) and rough (45/20) brucellae were consistently found within cisternae of the rough endoplasmic reticulum and nuclear envelope. The results suggest that transfer to the rough endoplasmic reticulum is the limiting step in the infection of nonphagocytic cells by B. abortus.
Differential inhibition of lipopolysaccharide-induced phenomena by anti-tumor necrosis factor alpha antibody
Authors: Vogel S N , Havell E A
Published: January 2020

Tumor necrosis factor alpha (TNF alpha) has been implicated as a major mediator of lipopolysaccharide (LPS)-induced phenomena. Administration to mice of a polyclonal, monospecific antibody prepared against recombinant murine TNF alpha abolished detection of LPS-induced TNF alpha activity and significantly reduced levels of LPS-induced colony-stimulating factor but failed to reduce the production of LPS-induced interferon, corticosterone, or LPS-induced hypoglycemia.
Comparative study of attachment to and invasion of epithelial cell lines by Shigella dysenteriae
Authors: Sen A , Leon M A , Palchaudhuri S
Published: January 2020

Henle 407 and HeLa cells were compared as hosts for Shigella dysenteriae at a low multiplicity of infection. Efficiency of attachment and invasion without centrifugation, as well as selectivity for pathogenic over nonpathogenic S. dysenteriae without Congo red, were much greater for Henle 407 cells than for HeLa cells.
Characterization and identification of early proteins in Chlamydia trachomatis serovar L2 by two-dimensional gel electrophoresis
Authors: Lundemose A G , Birkelund S , Larsen P M , Fey S J , Christiansen G
Published: January 2020

The synthesis of early proteins from Chlamydia trachomatis serovar L2 was analyzed by two-dimensional gel electrophoresis. By pulse-label experiments, the synthesis of seven proteins was observed at 2 to 8 h postinfection before the major outer membrane protein was detected at 8 to 10 h after infection. The early proteins were synthesized throughout the 30-h period investigated, but the synthesis of three proteins of 75, 62, and 45 kilodaltons decreased from 26 to 30 h postinfection. Pulse-chase analysis showed that the signals from the same three proteins declined 26 to 30 h after infection. Three of the early proteins were identified as the S1 ribosomal protein, the GroEL-like protein, and DnaK-like protein, respectively.
Diversity of Francisella tularensis antigens recognized by human T lymphocytes
Authors: Surcel H M
Published: January 2020

The Francisella tularensis T-lymphocyte antigens, which may have a role in protection against tularemia, were investigated with vaccine-immunized subjects. Preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to fractionate the bacterial envelope preparation. The 23 fractions obtained represented membrane proteins of different apparent molecular masses ranging from 10 to 150 kilodaltons. Different fractions contained one to four separate protein bands stained with Coomassie blue. The lymphocyte blast transformation responses of five tularemia vaccine-immunized and three nonimmunized subjects were tested against bacterial material eluted out of SDS-PAGE fractions. Every fraction stimulated lymphocytes from at least one of the subjects. No clearly immunodominant or inhibitory antigens were detected among the envelope fractions. Expression of the HLA-DR antigen at the surface of CD4- and CD8-positive lymphocytes was also studied as a measure of cell activation. The numbers of CD4+ DR+ cells varied directly with the lymphocyte proliferation profiles, and very few CD8+ cells were found in the preparations stimulated with the different fractions. The diversity of the antigens recognized by immune T lymphocytes was confirmed by using F. tularensis-specific T-lymphocyte clones obtained from vaccinated subjects. Most of the 36 T-lymphocyte clones tested were stimulated by one SDS-PAGE fraction only.
Lymphocyte proliferation in Peyer's patches of Giardia muris-infected mice
Authors: Hill D R
Published: January 2020

Gastrointestinal immune events in giardiasis are important in controlling infection. In this study, Peyer's patch lymphocytes from mice infected with Giardia muris developed specific, proliferative responses to G. muris antigen. This proliferation correlated with clearance of infection. Further understanding of the gut immune response will be helpful in developing immunoprophylactic strategies in the control of giardiasis.
Comparison of candidacidal and candidastatic activities of human neutrophils
Authors: Sohnle P G , Collins-Lech C
Published: January 2020

Disruption of neutrophils causes the release of a cytoplasmic protein which can inhibit the growth of Candida albicans without killing the organisms. The present study was undertaken with a combined system in which candidacidal and candidastatic activities of human neutrophils could be compared. In this system it was found that disruption of either half or all of the neutrophils in the samples markedly improved the ability of the cells to handle an inoculum of Candida yeast cells over a 48-h period, even though the disrupted cells had primarily candidastatic activity, with very little candidacidal activity.
Cloning, partial sequence, expression, and antigenic analysis of the filamentous hemagglutinin gene of Bordetella pertussis
Authors: Delisse-Gathoye A M , Locht C , Jacob F , Raaschou-Nielsen M , Heron I , Ruelle J L , de Wilde M , Cabezon T
Published: January 2020

The gene coding for the filamentous hemagglutinin (FHA), one of the main factors involved in mediating adherence of Bordetella pertussis to ciliated host cells, was cloned in Escherichia coli, and the 3,500-base-pair nucleotide sequence encoding the amino-terminal region was determined. Molecular cloning, together with the characterization of recombinant FHA-related proteins produced in E. coli, revealed that the primary translation product is a protein of about 370 kilodaltons (kDa). The mature 220-kDa FHA polypeptide secreted by B. pertussis is most probably generated by proteolytic processing that eliminates a carboxy-terminal portion of about 150 kDa. The 1,087 amino-terminal residues of the predicted FHA sequence showed a number of remarkable features. Extensive homology to the Serratia marcescens and Proteus mirabilis hemolysin proteins was found between amino acids 91 and 205 of the FHA sequence, suggesting involvement of this FHA domain in host cell binding or secretion of FHA from B. pertussis. In addition, two regions containing repetitive amino acid sequences were identified. One region, extending from residues 382 to 664, was formed by six repeats, and a second, extending from residues 701 to 912, contained three repeats. The reactivities of several recombinant FHA-derived proteins with a panel of monoclonal antibodies identified at least four epitopes composing an immunoreactive domain present in the carboxy-terminal moiety of the mature FHA.
Identification of an antigenic marker of slime production for Staphylococcus epidermidis
Authors: Christensen G D , Barker L P , Mawhinney T P , Baddour L M , Simpson W A
Published: January 2020

The pathogenic Staphylococcus epidermidis strain RP62A (ATCC 35984) adheres to smooth surfaces by forming a tenacious bacterial film known as slime. The mechanism of slime production is not known; however, workers in the laboratory of G. Pier (Harvard Medical School, Boston, Mass.) have isolated from RP62A a galactose-rich capsular polysaccharide adhesin (CPA) which mediates the attachment of the organism to smooth surfaces. We have obtained two daughter strains from RP62A that no longer produce slime. One daughter strain, H4A, was obtained by selection for a spontaneous variant; the other strain, HAM892, was obtained by treating growing cultures of RP62A with acriflavin. Using an antiserum generated against whole cells of RP62A, we have examined lysozyme-lysostaphin digests of RP62A, H4A, and HAM892 by double immunodiffusion. The two strains that no longer produced slime no longer produced a particular antigen, which we refer to as the slime-associated antigen (SAA). SAA was also produced by unrelated strains of slime-producing S. epidermidis. SAA was heat and protease stable, had a molecular weight of greater than 50,000, and could be partially purified by chromatographing trypsin-digested material over a Sephadex G-200 column. Chemical analysis of partially purified SAA by gas-liquid chromatography found SAA to be glucose rich (59%) and galactose poor (1.4%). This analysis chemically distinguished SAA from CPA. When tested together by double immunodiffusion with anti-RP62A and anti-CPA antisera, partially purified SAA did not cross-react with CPA. Kinetic studies suggested that SAA is a marker for surface accumulation whereas CPA mediates initial adherence.
Monoclonal antibodies against the 70-kilodalton iron-regulated protein of Neisseria meningitidis are bactericidal and strain specific
Authors: Pettersson A , Kuipers B , Pelzer M , Verhagen E , Tiesjema R H , Tommassen J , Poolman J T
Published: January 2020

When grown under iron limitation, Neisseria meningitidis expresses a number of outer membrane proteins (OMPs), one of which is a 70-kilodalton (kDa) major OMP. After immunization of mice with outer membrane preparations of iron-depleted cells of strain H44/76 (B:15:P1.7,16), hybridoma cell lines producing monoclonal antibodies against the 70-kDa OMP were obtained. Some of these monoclonal antibodies demonstrated strong bactericidal activity against the homologous strain H44/76 in the presence of human complement, suggesting potential application of the 70-kDa OMP as a vaccine component. However, none of the 10 selected monoclonal antibodies was able to recognize the corresponding protein from five heterologous strains of various serosubtyping characteristics. A polyclonal anti-70-kDa OMP serum also did not react with the other strains. This result shows that immunodominant surface-exposed epitopes of the meningococcal 70-kDa iron-limitation-inducible OMP are strain specific.
Monoclonal antibodies demonstrate that superoxide dismutase contributes to protection of Nocardia asteroides within the intact host
Authors: Beaman L , Beaman B L
Published: January 2020

The importance of superoxide dismutase (SOD) in protecting cells of Nocardia asteroides from the oxidative killing mechanisms within the intact murine host was determined. Murine monoclonal antibodies specific for nocardial SOD and for another nocardial antigen were prepared. Both antibodies adhered to cell surface antigens, as shown by fluorescence-labeled-antibody staining. The anti-nocardial SOD antibody inhibited the effect of nocardial SOD on superoxide generated in vitro. Cells of N. asteroides GUH-2 in log phase of growth were incubated with monoclonal anti-nocardial SOD, another monoclonal antinocardial antibody (not reactive with SOD), or phosphate-buffered saline and then injected intravenously into mice. Total recovery of CFU and inhibition of growth were determined at 3, 24, and 48 h after infection. The brains, kidneys, spleens, lungs, and livers were weighed, homogenized, and plated in order to quantitate the number of organisms in each organ at each time period. There was an initial killing followed by enhanced clearance of N. asteroides from the lungs and livers of mice which had received anti-SOD antibody-treated nocardiae. There was also enhanced early killing in the spleen. At 48 h, there were fewer organisms recovered from the brains, kidneys, and livers of mice which had received anti-SOD antibody-treated nocardia. This was not true for mice which had received antinocardial antibody not specific for SOD. The data demonstrate that surface-associated SOD protects N. asteroides for oxidative killing in vivo during all stages of infection.
Protein D, an immunoglobulin D-binding protein of Haemophilus influenzae: cloning, nucleotide sequence, and expression in Escherichia coli
Authors: Janson H , Hedén L O , Grubb A , Ruan M R , Forsgren A
Published: January 2020

The gene for protein D, a membrane-associated protein with specific affinity for human immunoglobulin D, was cloned from a nontypeable strain of Haemophilus influenzae. The gene was expressed in Escherichia coli from an endogenous promoter, and the gene product has an apparent molecular weight equal to that of H. influenzae protein D (42,000). The complete nucleotide sequence of the gene for protein D was determined, and the deduced amino acid sequence of 364 residues includes a putative signal sequence of 18 amino acids containing a consensus sequence, Leu-Ala-Gly-Cys, for bacterial lipoproteins. The sequence of protein D shows no similarity to those of other immunoglobulin-binding proteins. Protein D is the first example of immunoglobulin receptors from gram-negative bacteria that has been cloned and sequenced.
Heat-labile enterotoxin can be released from Escherichia coli cells by host intestinal factors
Authors: Hunt P D , Hardy S J
Published: January 2020

The heat-labile enterotoxin (LT) of Escherichia coli is localized in the periplasm of the bacterial cell. Growth of an LT-producing strain of E. coli in the presence of bile salts, in concentrations within the range found in the human small intestine, caused leakage of LT into the culture medium. This leakage could be augmented by the presence in the medium of trypsin and by minimizing the concentration of free iron.
A monoclonal antibody identifies a protective C-protein alpha-antigen epitope in group B streptococci
Authors: Madoff L C , Michel J L , Kasper D L
Published: January 2020

Group B streptococci (GBS) are the leading causes of neonatal sepsis and meningitis in the United States, with a high rate of fatality and serious morbidity despite appropriate therapy. The C-protein antigens of GBS appear to be important in immunity to experimental infection, yet these antigens remain incompletely characterized with respect to their number, structure, and function. None of these proteins has yet been purified to homogeneity. We have developed a novel method for extraction of surface proteins from the A909 (Ia/c) strain of GBS by using mutanolysin. Antibodies raised in rabbits against these partially purified proteins conferred passive protection to lethal GBS infection in mice challenged with a GBS strain expressing C proteins with a heterologous capsule type. In addition, mouse monoclonal antibodies were produced and identified by reactivity with the mutanolysin-extracted proteins. One of these monoclonal antibodies (4G8) identifies an epitope on the alpha-antigen of the GBS C proteins (identified by protease susceptibility and mouse protection). On sodium dodecyl sulfate-polyacrylamide gels, this epitope appears as a series of regularly spaced bands ranging in apparent molecular mass from 160,000 to 30,000 Da. The monoclonal antibody 4G8 induces opsonic killing of GBS and protects mice from lethal challenge with GBS. Thus, the 4G8 monoclonal antibody identifies a fully protective epitope on the C-protein alpha-antigen of GBS.
Molecular detection of persistent Borrelia burgdorferi in the urine of patients with active Lyme disease
Authors: Goodman J L , Jurkovich P , Kramber J M , Johnson R C
Published: January 2020

Current diagnostic tests for Lyme disease (LD) are dependent upon the host serologic response and are insensitive early in infection and, possibly, following antibiotic therapy. We cloned a library of Borrelia burgdorferi 297 DNA and studied one clone, Ly-1, for its potential in diagnostic and pathogenic studies. Using pulsed-field electrophoresis, we demonstrated that Ly-1 is of chromosomal origin and estimated that the B. burgdorferi chromosome is approximately 1,100 kb in size. The 3.7-kb Ly-1 clone hybridizes with geographically diverse strains of B. burgdorferi. No cross hybridization occurs with DNA from human cells, Escherichia coli, Staphylococcus aureus, Clostridium difficile, or the closely related B. hermsii. We used a dot blot assay to detect 100 pg of B. burgdorferi DNA. We partially determined the nucleotide sequence of Ly-1 and used it to select and synthesize oligonucleotides for use in the polymerase chain reaction (PCR). Two different primer pairs were found to amplify DNA from nine geographically diverse isolates. We could detect 10 fg (less than 10 molecules) of B. burgdorferi or less than five spirochetes added to human urine. Finally, we were able to use the PCR to detect B. burgdorferi DNA in the urine of four of eight patients with suspected active LD (three with arthritis and one with neurologic manifestations), all of whom responded to antibiotic treatment. In contrast, those patients who were PCR negative either had inactive disease or had been appropriately treated and did not respond to additional antibiotics, and all four control urine specimens were PCR negative. We conclude that B. burgdorferi DNA can be sensitively detected by the PCR with the primers and methods we describe and that the urinary tract is a site of persistent infection in some cases of human LD, an observation of potential diagnostic and pathogenic importance.
Nucleotide sequence of the gelatinase gene (gelE) from Enterococcus faecalis subsp. liquefaciens
Authors: Su Y A , Sulavik M C , He P , Makinen K K , Makinen P L , Fiedler S , Wirth R , Clewell D B
Published: January 2020

The gene coding for gelatinase (also called metalloendopeptidase II; microbial proteinase, EC 3.4.24.4) of Enterococcus faecalis subsp. liquefaciens strain OG1-10 was cloned in an Escherichia coli-Enterococcus shuttle vector, and its nucleotide sequence was determined. The DNA sequence encodes one large open reading frame (ORF) with 509 amino acid residues. The ORF contains a signal sequence in its N-terminal region, whereas the N-terminal amino acid sequence determined from the purified extracellular proteinase starts at residue 192 deduced from the ORF. This implies that the gelatinase is synthesized as a prepropolypeptide or prezymogen. The mature gelatinase contains 318 amino acid residues (molecular weight, 34,582) and has significant homology with neutral proteinases from Bacillus species and elastase from Pseudomonas aeruginosa.
Characterization of murine B-cell epitopes on the Mycobacterium leprae proline-rich antigen by use of synthetic peptides
Authors: Klatser P R , De Wit M Y , Kolk A H , Hartskeerl R A
Published: January 2020

Using synthetic peptides representing overlapping sequences of the 100-amino-acid-long N-terminal region of the proline-rich antigen of Mycobacterium leprae (PRA), we have mapped the epitopes in the primary structure of PRA recognized by four monoclonal antibodies. The M. leprae-specific monoclonal antibody F47-9 recognized the amino acid sequence LGSAYP (residues 34 to 39). Both monoclonal antibodies F67-1 and F67-5 recognized the sequence YPPP within the repeated sequence of PRA at four sites (residues 38 to 41, 50 to 53, 60 to 63, and 70 to 73). Monoclonal antibody F126-5 recognized the sequence SYPPP, also within the repeat, at three sites (residues 49 to 53, 59 to 63, and 69 to 73). All three epitopes appeared to be linear as far as can be determined by this approach.
Anaerobic growth of Salmonella typhimurium results in increased uptake by Henle 407 epithelial and mouse peritoneal cells in vitro and repression of a major outer membrane protein
Authors: Schiemann D A , Shope S R
Published: January 2020

The growth of Salmonella typhimurium under anaerobic conditions resulted in its greater ability to invade Henle 407 epithelial cells and in greater uptake by mouse peritoneal cells in vitro. Anaerobic growth also resulted in the repression of at least one major outer membrane protein.
Adherence of Ureaplasma urealyticum to human erythrocytes
Authors: Saada A B , Terespolski Y , Adoni A , Kahane I
Published: January 2020

Ureaplasma urealyticum (four serotypes and two clinical isolates) were metabolically labeled with radioactive methionine to a high specific activity. Labeling allowed the study of the mechanism of adherence to human erythrocytes. The adherence mechanism was complex and partially mediated by proteinaceous surface components. The binding sites on the erythrocytes were partially sensitive to neuraminidase treatment, and adherence was inhibited by glycophorin and dextran sulfate, indicating recognition of sialyl residues and sulfated compounds.
Characterization and identification of a porcine small intestine mucus receptor for the K88ab fimbrial adhesin
Authors: Metcalfe J W , Krogfelt K A , Krivan H C , Cohen P S , Laux D C
Published: January 2020

The ability of Escherichia coli K-12(K88ab) to adhere to immobilized porcine small intestine mucus was examined. E. coli K-12(K88ab) but not the isogenic E. coli K-12 strain was found to adhere readily to immobilized crude mucus but not to bovine serum albumin. The adhesion of E. coli K-12(K88ab) was inhibited in a specific fashion by anti-K88 antiserum. Adhesion was also inhibited by pretreatment of receptor-containing crude mucus preparations with sodium metaperiodate or proteolytic enzymes. Removal of glycolipids from crude mucus by chloroform-methanol extraction did not affect the ability of E. coli K-12(K88ab) to bind to mucus preparations. Adsorption of crude mucus preparations with K88ab fimbriae but not type 1 fimbriae resulted in the removal of K88-specific receptors. Analysis of the pelleted fimbriae-receptor complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, together with gel filtration chromatography of crude mucus preparations, suggest that the K88-specific receptor present in porcine small intestine mucus is a 40- to 42-kDa glycoprotein.
The immunodominant outer membrane antigen of Actinobacillus actinomycetemcomitans is located in the serotype-specific high-molecular-mass carbohydrate moiety of lipopolysaccharide
Authors: Page R C , Sims T J , Engel L D , Moncla B J , Bainbridge B , Stray J , Darveau R P
Published: January 2020

Most patients with juvenile periodontitis manifest serum antibodies, sometimes at very high titers, to antigens of Actinobacillus actinomycetemcomitans, but the antigens inducing the immune response have been only partly characterized. We separated A. actinomycetemcomitans serotype b cells into protein, lipopolysaccharide (LPS), and soluble polysaccharide fractions and characterized them. Coomassie blue- and silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were used to detect protein and LPS components, and gas-liquid chromatography was used to determine their carbohydrate and fatty acid composition. Western blots, dot blots, and enzyme-linked immunosorbent assay inhibition with high-titer sera from juvenile periodontitis patients revealed which components were highest in antibody binding activity. These results showed that the major portion of the immunoglobulin G binding activity resides in the purified mannan-free LPS, with lesser amounts in the total protein fraction. Using Sephacryl S-300 chromatography, we separated LPS into high-molecular-mass components with high carbohydrate contents by gas-liquid chromatography and a low-molecular-mass component consisting mainly of lipid A and the inner core sugar heptulose. The results of quantitative dot blot assays and enzyme-linked immunosorbent assay inhibition show that the serotype-specific antibody binding activity is highly concentrated in the high-molecular-mass carbohydrate-rich LPS fraction and is almost completely absent in the low-molecular-weight lipid-rich fraction. Our observations contrast with previous reports that the predominant serotype antigen of A. actinomycetemcomitans resides in a mannan-rich polysaccharide isolated from spent culture medium. These observations support the conclusion that the immunodominant antigen of the outer membrane is the O antigen of the LPS.
Suppression by Trypanosoma brucei rhodesiense of the capacities of human T lymphocytes to express interleukin-2 receptors and proliferate after mitogenic stimulation
Authors: Kierszenbaum F , Muthukkumar S , Beltz L A , Sztein M B
Published: January 2020

We studied the suppressive effects induced in phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) by purified blood forms of Trypanosoma brucei rhodesiense. The parasite was found to markedly impair lymphocyte proliferation (measured in terms of [3H]thymidine incorporation). The extent of this effect increased with parasite concentration and was not due to mitogen absorption, depletion of medium nutrients, or PBMC killing by the parasite. Significant reductions in interleukin-2 receptor (IL-2R) expression, determined by flow cytometric analysis, were also observed in PHA-stimulated PBMC cultured in the presence of T. b. rhodesiense as evidenced by marked decreases in the surface density of the receptor. Concomitant decreases in the percentage of IL-2R+ cells were recorded in approximately half of the experiments. A discrete, dimly stained subpopulation of IL-2R+ cells were consistently demonstrable whether or not a reduction in the percentage of IL-2R+ cells occurred. Living, but not glutaraldehyde-fixed, parasites suppressed IL-2R expression. In kinetic studies, a low but reproducible level of suppression of IL-2R was demonstrable as early as 6 h after PHA stimulation; the extent of this effect became considerably more pronounced as additional culture time elapsed. Levels of IL-2 biological activity in cocultures of T. b. rhodesiense with PHA-stimulated PBMC were comparable with or higher than those present in control cultures lacking the parasite. Therefore, insufficient levels of this cytokine would be an unlikely explanation for the noted suppression of IL-2R expression and lymphoproliferation. These effects of T. b. rhodesiense could represent an important component of the mechanism by which immunosuppression develops in African sleeping sickness.
Alterations in cysteine proteinase content of rat lung associated with development of Pneumocystis carinii infection
Authors: Hayes D J , Stubberfield C R , McBride J D , Wilson D L
Published: January 2020

The rate of hydrolysis of three cysteine-type proteinase substrates, N-benzyloxycarbonyl-Arg-Arg-4-methyl-7-coumarylamide (AMC) (cathepsin B), Arg-AMC (cathepsin H), and N-benzyloxycarbonyl-Phe-Arg-AMC (cathepsin L), were determined in rat lung throughout the time course of the induction of Pneumocystis carinii infection by immunosuppression. Cathepsin B-like and cathepsin L-like activities fell below control values initially, but from week 8 of the immunosuppressive treatment significant increases above the control were noted. Cathepsin H-like activity was greater than control levels from week 3, and by week 12 it was 7,600% of the mean control value. When compared with the relative degree of infection, as assessed from the number of cysts present in lung impression smears, cathepsin B-like and cathepsin L-like activities were significantly increased only at heavy parasite burdens while cathepsin H-like activity displayed a close correlation with parasite number (r = 0.884; P less than 0.001). Activity was detected in lysates of purified P. carinii with all three substrates. Treatment of heavily infected animals with co-trimoxazole cleared the lungs of P. carinii, and this was accompanied by a marked reduction in proteinase activity, in particular, cathepsin H-like activity, which fell from 108- to 3-fold the mean control value following drug treatment. Analysis of cathepsin H isozyme patterns by fluorography following isoelectric focusing revealed differences between treated and control lung samples. In the immunosuppressed group, there was a time-dependent increase in the intensity of some of the bands observed in the controls and an appearance of several novel bands which corresponded to bands observed in lysates of P. carinii. It is likely, therefore, that the increased proteinase activity observed in the treated group is due, at least in part, to isozymes from P. carinii; consequently, cathepsin H-like activity might be of use diagnostically in the identification of P. carinii infection and in the estimation of parasite burden.
Protection of rat intestine against cholera toxin challenge by monoclonal anti-idiotypic antibody immunization via enteral and parenteral routes
Authors: Lucas G P , Cambiaso C L , Vaerman J P
Published: January 2020

A mouse monoclonal anti-idiotypic (anti-id) immunoglobulin M (IgM) antibody, called MAb2, was raised against a mouse monoclonal anti-cholera toxin (anti-CT) antibody (MAb1). The MAb2 was shown, by competition with CT for MAb1, to bear the internal image of an epitope of CT. MAb2 immunization of rats was performed via the intraperitoneal, intragastric, and intrajejunal routes and compared with immunization of rats with either a control, isotype- and allotype-matched MAb or with CT via the same routes. Both serum IgG and bile IgA anti-CT Ab3's were detected by enzyme-linked immunosorbent assay in anti-id MAb2-immunized rats, although their titers were lower than those in CT-immunized rats. No anti-CT antibodies were detected in sera and bile of rats immunized with the control MAb. When tested for degree of gut protection against a CT challenge, rats immunized with MAb2 by the intrajejunal route showed a rather high degree of protection, which was only slightly lower than that of rats immunized with CT via the same route; all rats but one immunized with the control MAb were unprotected. There was, however, no correlation between serum or bile anti-CT titers and degree of gut protection in MAb2-immunized rats. Their serum anti-CT Ab3's were purified by adsorption and elution from a CT immunosorbent and resembled anti-CT MAb1 in their unique reactivity with MAb2. This constitutes to our knowledge the second report of protection against a pathogen by anti-id immunization via the enteric route.