bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of recombinant lambda gt11 bacteriophages expressing four different Mycobacterium intracellulare antigens
Authors: Morris S L , Rouse D A , Hussong D , Chaparas S D
Published: January 2020

Four bacteriophages expressing different immunoreactive recombinant Mycobacterium intracellulare antigens were isolated from a lambda gt11 library with monoclonal antibodies to M. intracellulare. These four antibodies reacted with native M. intracellulare proteins of 54, 43, 40/38, and 22 kilodaltons. Southern blot hybridizations with DNA probes prepared from insert fragments of these bacteriophages confirmed the M. intracellulare derivation of the inserts. The physical maps of the immunoreactive phages were deduced by restriction enzyme digestions. The molecular weights of the expressed recombinant antigens were determined by Western (immuno-) blotting.
Characterization of murine monoclonal and murine, rabbit, and human polyclonal antibodies against chlamydial lipopolysaccharide
Authors: Brade L , Holst O , Kosma P , Zhang Y X , Paulsen H , Krausse R , Brade H
Published: January 2020

Murine monoclonal and rabbit, murine, and human polyclonal antibodies against chlamydial lipopolysaccharide (LPS) were characterized by the passive hemolysis and passive hemolysis inhibition assays and by absorption experiments with LPSs of Chlamydia psittaci, Chlamydia trachomatis, and a recombinant strain of Salmonella minnesota Re (r595-207) expressing the chlamydia-specific LPS epitope, as well as natural and synthetic partial structures of chlamydial LPS. Eleven monoclonal antibodies of the immunoglobulin M and G classes were characterized as chlamydia-specific by their failure to react with Re-type LPS, binding to a similar epitope for which the trisaccharide alpha-3-deoxy-D-manno-2-octulosonic acid (KDO)-(2-8)-alpha-KDO-(2-4)-alpha-KDO was an absolute prerequisite. For optimal binding, parts of the lipid A moiety were also involved; however, phosphoryl and ester-linked acyl groups and the reducing glucosamine residue of lipid A were dispensable. A similar antibody specificity was detected in lapine and murine hyperimmune sera after immunization with chlamydia, in addition to those recognizing more complex (e.g., those requiring the presence of phosphoryl residues) and less complex epitopes. Among the latter were those cross-reacting with Re-type LPS, which could be removed by absorption. The titers of different antibody specificities, in particular the ratio of chlamydia-specific to cross-reactive antibodies, present in murine polyclonal antisera depended on the immunization protocol. The preferential formation of chlamydia-specific antibodies was observed after immunization with liposome-incorporated immunogens. Human sera from patients with suspected genital chlamydial infections were also found to contain chlamydia-specific and cross-reactive antibodies, the latter of which could be removed by absorption with Re-type LPS.
Cloning and expression of the damselysin gene from Vibrio damsela
Authors: Cutter D L , Kreger A S
Published: January 2020

The gene encoding damselysin, an extracellular cytolysin produced by virulent Vibrio damsela strains, was cloned and expressed in Escherichia coli. DNA sequences homologous to that of the cloned gene were detected in hemolytic strains of V. damsela but not in other hemolytic Vibrio species.
Stimulation of antibacterial macrophage activities by B-cell stimulatory factor 2 (interleukin-6)
Authors: Flesch I E , Kaufmann S H
Published: January 2020

Mononuclear phagocytes provide the major habitat of intracellular bacteria, including Mycobacterium tuberculosis and Mycobacterium bovis. The capacity of B-cell stimulatory factor 2 (interleukin-6 [IL-6]) to activate tuberculostatic functions was investigated by using murine bone marrow-derived macrophages (BMM phi). BMM phi stimulated with recombinant IL-6 and subsequently infected with M. bovis organisms failed to inhibit mycobacterial growth. In contrast, marked tuberculostasis was induced by IL-6 in BMM phi that were already infected with M. bovis, indicating that IL-6 has a macrophage-activating function.
A major immunogenic 36,000-molecular-weight antigen from Mycobacterium leprae contains an immunoreactive region of proline-rich repeats
Authors: Thole J E , Stabel L F , Suykerbuyk M E , De Wit M Y , Klatser P R , Kolk A H , Hartskeerl R A
Published: January 2020

The 36,000-molecular-weight antigen (36K antigen) of Mycobacterium leprae is a major immunogenic protein carrying common and specific antigenic determinants recognized by antibodies and T cells in leprosy patients. Recombinant DNA clones containing the complete gene coding for the 36 K antigen, designated in this paper as PRA, were isolated from both lambda gt11 and cosmid libraries of the M. leprae genome. The DNA sequence of the pra gene coded for a polypeptide of 249 amino acids with a predicted molecular mass of 26,299 daltons. The deduced amino acid sequence revealed a proline-rich (42%) amino-terminal region containing a number of repeated sequences similar or identical to the sequence PGGSYPPPPP. The reactivity of four monoclonal antibodies (F47-9, F67-1, F67-5, and F126-5) was directed to this proline-rich region of the PRA protein. DNA sequence and immunological data indicated that the lambda gt11 recombinant Y3180, which was previously isolated by using antibody F47-9 (R. A. Young, V. Mehra, D. Sweetser, T. Buchanan, J. Clark-Curtiss, R. W. Davis, and B. R. Bloom, Nature (London) 316:450-452, 1985), specifies a fusion protein unrelated to PRA but containing a similar epitope recognized by F47-9.
Pneumococcal surface protein A (PspA) is serologically highly variable and is expressed by all clinically important capsular serotypes of Streptococcus pneumoniae
Authors: Crain M J , Waltman W D , Turner J S , Yother J , Talkington D F , McDaniel L S , Gray B M , Briles D E
Published: January 2020

Pneumococcal surface protein A (PspA) has been shown previously to elicit antibodies protective against pneumococcal infection and to be necessary for full pneumococcal virulence in mice. The protein was originally defined by the two mouse monoclonal antibodies Xi64 and Xi126, which together recognized PspA on 14% of pneumococcal isolates. Some PspA molecules reacted with both antibodies, but most reacted with only one or the other. In the present study we demonstrated that PspA is produced by all pneumococci, confirming our hypothesis that there are variants of PspA which are not detected by Xi64 and Xi126. We produced a rabbit antiserum and five additional monoclonal antibodies specific for PspA for these studies. The rabbit antiserum reacted with each of 95 pneumococcal isolated tested, comprising 16 capsular serotypes. One or more of the seven monoclonal anti-PspA antibodies reacted with 95% (53 of 57) of pneumococcal isolates tested. The specificity of the monoclonal and polyclonal antibodies to PspA was confirmed in two ways: (i) by detection of molecules on wild-type pneumococci that are identical in molecular weight to those detected in Western blots (immunoblots) with Xi64 and Xi126 and (ii) by the use of mutants of Streptococcus pneumoniae that fail to produce PspA or that produce a truncated form of PspA. By using the seven monoclonal antibodies, we observed 31 PspA types among the 57 isolates. When the 53 strains reactive with the monoclonal antibodies were analyzed by capsular type as well as by serologic type and molecular weight of PspA, we observed 50 different clonotypes of pneumococci.
Excystation of in vitro-derived Giardia lamblia cysts
Authors: Boucher S E , Gillin F D
Published: January 2020

This is the first in-depth analysis of the excystation of Giardia lamblia cysts prepared in vitro. Its goals were both to achieve efficient excystation and to gain insights into this crucial but poorly understood process. To identify the critical elements of excystation, we tested the sequential low-pH induction and protease treatments which had been reported to be important for excystation of fecal cysts. The optimal pH for induction of excystation was 4.0. Emergence was greatly (approximately 10-fold) stimulated by subsequent exposure of in vitro-derived cysts to chymotrypsin, trypsin, or human pancreatic fluid. The stimulatory activity of each was abolished by soybean trypsin inhibitor, demonstrating that the activity of pancreatic fluid was due to these proteases. Excystation of in vitro-derived cysts was approximately 10 to 38%. Although the walls of in vitro-derived cysts were partially digested by protease treatment, trophozoites emerged only from one pole, as observed with fecal cysts. The conditions of encystation also determined the efficiency of excystation. Specifically, encystation in the presence of lactic acid, a major metabolite of colonic bacteria, stimulated excystation approximately fourfold, although it did not increase the total numbers of cysts. These experiments have shown that excystation of in vitro-derived cysts reflects that of cysts purified from human feces in that it is dependent upon conditions which simulate the passage of cysts through the human stomach (low pH) and into the small intestine (pancreatic proteases).
Identification and characterization of a zinc metalloprotease associated with invasion by the fish pathogen Vibrio anguillarum
Authors: Norqvist A , Norrman B , Wolf-Watz H
Published: January 2020

An invasiveness-defective mutant of the fish-pathogenic bacterium Vibrio anguillarum was isolated. Compared with the wild type, this mutant had a 1,000-fold higher 50% lethal dose after immersion infection of rainbow trout, Oncorhynchus mykiss, while after intraperitoneal infection, the mutant had only a 10-fold higher 50% lethal dose. In addition, the mutant showed a lower level of protease activity. Two forms of the protease (Pa and Pb) were found after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonheated samples. Pa was found predominantly in protease preparations of the wild type, while Pb was the predominant form in the mutant. Conversion of Pb to Pa was observed in protease preparations after incubation at 4 degrees C. Characterization of the protease showed that it was an elastolytic enzyme which required Zn2+ for activity and Ca2+ for stability. The molecular mass of the protease was 36 kilodaltons. N-terminal amino acid sequence analysis of the protease of V. anguillarum revealed homology to the elastase of Pseudomonas aeruginosa and the protease of Legionella pneumophila.
Defect in the tissue cellular immune response: experimental visceral leishmaniasis in euthymic C57BL/6 ep/ep mice
Authors: Squires K E , Kirsch M , Silverstein S C , Acosta A , McElrath M J , Murray H W
Published: January 2020

In BALB/c mice, successful defense against visceral leishmaniasis is T cell dependent, expressed by tissue granuloma formation, and probably mediated by macrophages activated by cytokines, including gamma interferon (IFN-gamma). C57BL/6 ep/ep (pale ear) mice, which reportedly exhibit impaired IFN-gamma production, were challenged with Leishmania donovani to determine the outcome of infection in a euthymic host with an apparent defect in lymphokine secretion. In BALB/c and normal C57BL/6 mice, L. donovani liver burdens peaked at 2 weeks and were largely eliminated by 4 weeks. In contrast, in pale ear mice, infection progressed until after 4 weeks and persisted at high levels at 8 weeks. The failure to resolve hepatic infections was not related to deficiencies in (i) Thy-1+, L3T4+, or Lyt-2+ T cells; (ii) IFN-gamma secretion; (iii) liver tissue Ia expression; (iv) macrophage antimicrobial capacity; or (v) antileishmanial antibody production. However, despite the anticipated influx of mononuclear cells into livers, these cells were not properly focused on the parasitized Kupffer cells, the inflammatory infiltrate receded prematurely, and mature granulomas failed to develop. These results suggest that there is a cellular immune defect at the tissue level and emphasize the critical role of granuloma formation in successful resolution of systemic intracellular infections.
Nonhemolytic Listeria monocytogenes mutants that are also noninvasive for mammalian cells in culture: evidence for coordinate regulation of virulence
Authors: Kathariou S , Pine L , George V , Carlone G M , Holloway B P
Published: January 2020

We identified nonhemolytic mutants of Listeria monocytogenes that were severely deficient in their ability to invade mammalian nonprofessional phagocytes. These mutants were generated spontaneously or by means of transposon Tn916 mutagenesis. In terms of their extracellular proteins, the noninvasive mutants were deficient not only in the sulfhydryl-activated hemolysin (listeriolysin) but also in an antigenically unrelated extracellular protein with an apparent molecular weight of 32,000 which could induce opacity in egg yolk and is considered to be a phospholipase. Our results suggest the existence of a common genetic control between the expression of listeriolysin and that of other determinants, including a phospholipase and determinants involved in the ability of L. monocytogenes to enter mammalian cells.
Functional activity of individual abscess neutrophils from mice
Authors: Kenny P A , Spencer L K , McDonald P J , Finlay-Jones J J
Published: January 2020

In the absence of antibiotic therapy, viable bacteria can persist within intra-abdominal abscesses in mice for at least 10 weeks. The mechanisms contributing to this survival are unknown, but abscess-derived neutrophils have impaired abilities to kill, in vitro, organisms engulfed in vivo. In order to determine whether subpopulations of abscess neutrophils might be discernible on the basis of phenotypic or functional criteria, cells from murine intra-abdominal abscesses were examined for phagocytic activity, CR3 expression, and H2O2 production in response to soluble and particulate stimuli. With respect to phagocytosis of Proteus mirabilis, abscess cells were no less efficient than peritoneal exudate neutrophils; no significant subpopulation of cells was incapable of phagocytosis in the presence of normal mouse serum. Using flow cytometry to examine abscess neutrophils for CR3 expression, we found that no subpopulations of cells were observed with unstimulated cells or with cells incubated with either phorbol 12-myristate 13-acetate or bacteria and serum. Intracellular H2O2 levels were measured by using the probe 2',7'-dichlorofluorescin diacetate. In general, incubation with phorbol 12-myristate 13-acetate resulted in similar increases in H2O2 production in all cells of the population. However, stimulation with bacteria and serum revealed a variable but consistent, poorly responsive subpopulation of neutrophils in abscess cell populations. Cell-sorting experiments showed that cells from the poorly responsive section of the FACS profile contained significantly higher numbers of abscess-derived bacteria, suggesting the presence of a subpopulation of viable abscess neutrophils harboring persisting viable bacteria.
Molecular analysis of hemolytic and phospholipase C activities of Pseudomonas cepacia
Authors: Vasil M L , Krieg D P , Kuhns J S , Ogle J W , Shortridge V D , Ostroff R M , Vasil A I
Published: January 2020

By using a gene-specific fragment from the hemolytic phospholipase C (PLC) gene of Pseudomonas aeruginosa as a probe and data from Southern hybridizations under reduced stringency conditions, we cloned a 4.2-kb restriction fragment from a beta-hemolytic Pseudomonas cepacia strain which expressed hemolytic and PLC activities in Escherichia coli under the control of the lac promoter. It was found, by using a T7 phage promoter-directed expression system, that this DNA fragment carries at least two genes. One gene which shares significant DNA homology with both PLC genes from P. aeruginosa encodes a 72-kDa protein, while the other gene encodes a 22-kDa protein. When both genes on the 4.2-kb fragment were expressed from the T7 promoter in the same cell, hemolytic and PLC activities could be detected in the cell lysate. In contrast, when each individual gene was expressed in different cells or when lysates containing the translated products of each separate gene were mixed, neither hemolytic activity nor PLC activity could be detected. Clinical and environmental isolates of P. cepacia were examined for beta-hemolytic activity, PLC activity, sphingomyelinase activity, and reactivity in Southern hybridizations with a probe from P. cepacia which is specific for the larger gene which encodes the 72-kDa protein. There were considerable differences in the ability of the different strains to express hemolytic and PLC activities, and the results of Southern DNA-DNA hybridizations of the genomic DNAs of these strains revealed considerable differences in the probe-reactive fragments between high- and medium-stringency conditions as well as remarkable variation in size and number of probe-reactive fragments among different strains. Analysis of the genomic DNAs from hemolytic and nonhemolytic variants of an individual strain (PC-69) by agarose gel electrophoresis. Southern hybridization, and transverse alternating pulsed field gel electrophoresis suggests that the conversion of the hemolytic phenotype to the nonhemolytic phenotype is associated with either the loss of a large plasmid (greater than 200 kb) or a large deletion of the chromosome of P. cepacia PC-69.
Platelet-interactive products of Streptococcus sanguis protoplasts
Authors: Herzberg M C , Erickson P R , Kane P K , Clawson D J , Clawson C C , Hoff F A
Published: January 2020

To isolate a more native, platelet-interactive macromolecule (class II antigen) of Streptococcus sanguis, cultured protoplasts were used as a source. Protoplasts were optimally prepared from fresh washed cells by digestion with 80 U of mutanolysin per ml for 75 min at 37 degrees C while osmotically stabilized in 26% (wt/vol) raffinose. Osmotically stabilized forms were surrounded by a 9-nm bilaminar membrane, as shown by transmission electron microscopy. Protoplasts were cultured in chemically defined synthetic medium and osmotically stabilized by ammonium chloride. Spent culture media were harvested daily for 7 days. Each day, soluble proteins were isolated from media, preincubated with platelet-rich plasma, and tested for inhibition of platelet aggregation induced by S. sanguis cells. Products released from S. sanguis protoplasts and reactive with an anti-class II antigen immunoaffinity matrix were able to inhibit S. sanguis-induced platelet aggregation. As resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, anti-class II-reactive protoplast products included silver-stained bands of 67, 79, 115, 216, and 248 kDa. The 115-kDa protein fraction was isolated by gel filtration and ion-exchange chromatography. This form of the class II antigen contained N-formylmethionine at its amino terminus. Rhamnose constituted 18.2% of the total residual dry weight and nearly half of its carbohydrate content. Diester phosphorus constituted 1% of this fraction. After trypsinization of the protoplast products from either preparation, a 65-kDa protein fragment was recovered. This protoplast protein fragment and the S. sanguis cell-derived 65-kDa class II antigen, previously implicated in the induction of platelet aggregation, were shown to be functionally and immunologically identical.
Caveats in the investigation of form-specific molecules of Candida albicans
Authors: Brawner D L , Cutler J E , Beatty W L
Published: January 2020

Numerous reports purporting the existence of form-specific antigens of Candida albicans have been published, but it is generally unclear whether antigenic variability is an acceptable alternative interpretation. In this study, we used indirect immunofluorescence and immunogold electron microscopy to determine the distribution and form specificities of two antigens during yeast and hyphal growth in several defined and complex media. The results confirmed that antigen expression varies with length of incubation, nutrition, and serotype and indicate that the form specificities of antigens may be misinterpreted when conclusions are based exclusively on indirect immunofluorescence and extraction procedures. We therefore suggest that investigations be designed to include serotype A and B isolates grown in both complex and chemically defined media and that agglutination, immunofluorescence, or enzyme-linked immunosorbent assays on whole cells or cell extracts be used as presumptive tests. Confirmation of form-specific antigens should be done by appropriate immunoelectron microscopic evaluation.
Binding of collagens to an enterotoxigenic strain of Escherichia coli
Authors: Visai L , Speziale P , Bozzini S
Published: January 2020

An enterotoxigenic strain of Escherichia coli, B34289c, has been shown to bind the N-terminal region of fibronectin with high affinity (G. Fröman, L. M. Switalski, A. Faris, T. Wadström, and M. Höök, J. Biol. Chem. 259:14899-14905, 1984). We now report that this strain also binds collagen. The binding of 125I-labeled type II collagen to bacteria was time dependent and reversible. Bacteria expressed a limited number of collagen receptors (2.2 x 10(4) per cell) and bound collagen with a Kd of 20 nM. All collagen types tested (I to V) as well as all tested cyanogen bromide-generated peptides [alpha 1(I)CB2, alpha 1(I)CB3, alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB4] were recognized by bacterial receptors, as demonstrated by the ability of these proteins to inhibit the binding of 125I-labeled collagen to bacteria. Of several unlabeled proteins tested in competition experiments, fibronectin and its N-terminal region strongly inhibited binding of the radiolabeled collagen to E. coli cells. Conversely, collagen competed with an 125I-labeled 28-kilodalton fibronectin fragment for bacterial binding. Collagen bound to bacteria could be displaced by excess amounts of either unlabeled fibronectin or its N-terminal fragment. Similarly, collagen could displace 125I-labeled N-terminal peptide of fibronectin bound to the bacterial cell surface. Bacteria grown at 41 degrees C or in the presence of glucose did not express collagen or fibronectin receptors. These results indicate the presence of specific binding sites for collagen on the surface of E. coli cells and furthermore that the collagen and fibronectin binding sites are located in close proximity, possibly on the same structure.
Localization of host immunoglobulin G to the surface of Pneumocystis carinii
Authors: Blumenfeld W , Mandrell R E , Jarvis G A , Griffiss J M
Published: January 2020

In immunoblotting studies of Pneumocystis carinii surface proteins, we found that a secondary antibody, anti-human immunoglobulin G (IgG), recognized a 52-kilodalton (kDa) band in homogenates of P. carinii purified from human autopsy lungs and bronchoalveolar lavage fluids, even when serum as a source of primary antibody was omitted. The electrophoretic mobility of the 52-kDa band is identical to that of IgG heavy chains. In addition to affinity-purified, anti-human IgG, monoclonal antibodies specific for the Fab and Fc regions of human IgG recognized the 52-kDa band. To determine whether the 52-kDa band represents IgG bound to the surface of P. carinii, we treated intact organisms with Triton X-100 and acid in order to elute immunoglobulin from the surface of P. carinii. After purification over a protein G column, the eluate comigrated with human IgG, was recognized by anti-IgG, and bound to discrete bands with molecular sizes of 65 to 70, 60, 50, and 35 kDa in purified, rat-derived P. carinii. To confirm the presence of human IgG on the surface of P. carinii, we performed immunocytochemical and immunoelectron microscopic studies. Staining of intact P. carinii aggregates by anti-human IgG was pronounced and was abolished by acid treatment. IgA was also present. Ultrastructural studies showed the presence of IgG on the cyst wall and on fine membranous structures and vesicles adjacent to cysts. We conclude that the surface of P. carinii is coated with human IgG. The close association of human IgG with P. carinii may have implications for the pathogenesis of P. carinii pneumonia in acquired immunodeficiency syndrome.
Chlamydia trachomatis-host cell interactions: role of the chlamydial major outer membrane protein as an adhesin
Authors: Su H , Watkins N G , Zhang Y X , Caldwell H D
Published: January 2020

The major outer membrane protein (MOMP) of Chlamydia trachomatis is characterized by four symmetrically spaced variable domains (VDs I to IV) whose sequences vary among serotypes. The surface-exposed portions of these VDs contain contiguous sequences that are both serotyping determinants and in vivo target sites for neutralizing antibodies. Previous studies using surface proteolysis of C. trachomatis B implicated VDs II and IV of the MOMP of this serotype in the attachment of chlamydiae to host cells. In this study, we used monoclonal antibodies (MAbs) specific to antigenic determinants located in VDs II and IV of the MOMP of serotype B to further investigate the role of the MOMP in the attachment of chlamydiae to host cells. MABs specific to serotype- and subspecies-specific epitopes located in exposed VDs II and IV, respectively, neutralized chlamydial infectivity for hamster kidney cells by blocking chlamydial attachment. We radioiodinated these MAbs and used them to determine the number and topology of the surface-exposed VDs II and IV epitopes on chlamydial elementary bodies. VDs II and IV each comprised approximately 2.86 x 10(4) negatively charged sites and were in proximity on the chlamydial cell surface. These studies suggest that the MAbs blocked chlamydial attachment by inhibiting electrostatic interactions with host cells. We examined the effects of thermal inactivation on both chlamydial attachment and conformation of the MOMP. Heat-inactivated chlamydiae failed to attach to host cells and exhibited a conformational change in an inaccessible invariant hydrophobic nonapeptide sequence located within VD IV of the MOMPs of C. trachomatis serotypes. These findings suggest that in addition to electrostatic interactions, a common hydrophobic component of the MOMP also contributes to the binding of chlamydiae to host cells. Thus, we propose that the MOMP functions as a chlamydial adhesin by promoting nonspecific (electrostatic and hydrophobic) interactions with host cells. Surface-accessible negatively charged VDs appear to be important in electrostatic binding, while the invariant region of VD IV may provide a subsurface hydrophobic depression which further promotes binding of chlamydiae to host cells through hydrophobic interactions.
Differences in susceptibilities of the lymphogranuloma venereum and trachoma biovars of Chlamydia trachomatis to neutralization by immune sera
Authors: Peterson E M , Hoshiko M , Markoff B A , Lauermann M W , de la Maza L M
Published: January 2020

Sera from seven patients from whom a C. trachomatis serovar L2 strain was isolated were tested in vitro for their ability to neutralize the infectivity of this organism. In one patient an inguinal lymph node was culture positive, whereas the remaining six patients had positive rectal biopsies. Sera from four of the patients, including the patient with the lymph node isolate, failed to neutralize serovar L2(434). In addition, the homologous strain recovered from the inguinal lymph node was available and was resistant to neutralization by the homologous sera. However, the same sera effectively neutralized a trachoma serovar, E(Bour). All four sera had inclusion immunofluorescent-antibody titers to C. trachomatis serovar L2 of 2,048 to 16,384 and microimmunofluorescent-antibody titers to the lymphogranuloma venereum biovar were equal or higher in all cases than to the 12 serovars of the trachoma biovar. The three remaining sera, while neutralizing the infectivity of the L2 strains tested, neutralized serovar E to a greater extent. These sera had the same inclusion immunofluorescent antibody titers as the sera that failed to neutralize serovar L2. To see whether this difference in the sensitivity of the biovars toward neutralization could be characterized, sera were obtained from mice immunized with different doses of both serovars L2 and E. Sera obtained from mice immunized with serovar E were able to effectively neutralize the homologous strain. In contrast, neutralization of the immunizing strain, L2(UCI-20), was not seen with sera obtained on days 7, 14, and 21 after immunization from animals receiving 8 x 10(5) and 8 x 10(4) inclusion-forming units of L2(UCI-20); however, these same sera neutralized serovar E. However, with a higher immunizing dose of L2 (10(7) IFUs), both E and L2 were neutralized with sera obtained 7 and 14 days after immunization. Therefore, the relative resistance to neutralization by serovar L2 compared with that of serovar E in the mouse model was inoculum dependent.
Difference in the induction of macrophage interleukin-1 production between viable and killed cells of Listeria monocytogenes
Authors: Mitsuyama M , Igarashi K , Kawamura I , Ohmori T , Nomoto K
Published: January 2020

T-cell-mediated immunity to Listeria monocytogenes in mice, as determined by delayed-type hypersensitivity and acquired resistance, was induced by immunization with viable bacteria but not with killed bacteria, even when killed cells were injected in a high dose or repeatedly. T cells obtained from mice immunized with viable L. monocytogenes were readily stimulated with killed-bacterial antigens, resulting in T-cell proliferation in vitro and expression of a delayed footpad reaction in vivo. After immunization with killed-bacterial vaccine, T-cell responsiveness to interleukin 2 (IL-2) never developed but a lower level of responsiveness to IL-1 appeared later than with T cells from mice immunized with viable bacteria. When IL-1 production by macrophages was examined in vitro, viable L. monocytogenes stimulated a high level of IL-1 release while killed bacteria did not. Avirulent strains which were ineffective in the induction of T-cell mediated immunity were incapable of inducing IL-1 production as well. The impaired ability of killed bacteria to stimulate IL-1 production was confirmed by the level of IL-1 mRNA expression. These results suggested that the ineffectiveness of killed L. monocytogenes vaccine is not due to loss or lack of antigenic epitopes but may be ascribed to insufficient induction of IL-1 production in the initial stage of the immune response in vivo.
Mycoplasma fermentans-derived high-molecular-weight material induces interleukin-6 release in cultures of murine macrophages and human monocytes
Authors: Quentmeier H , Schmitt E , Kirchhoff H , Grote W , Mühlradt P F
Published: January 2020

A Mycoplasma fermentans-derived high-molecular-weight material (MDHM) is described which causes differentiation of concanavalin A-stimulated CBA/J or C57BL/6 mouse thymocytes to cytolytic effector T cells (CTLs). The effect of MDHM was inhibited by addition of monoclonal anti-interleukin-6 (IL-6) antibody. It could also be abolished after removal of adherent cells. However, adherent cell-depleted thymocytes could still form CTLs after addition of IL-6. The action of MDHM could thus be explained by the capacity of MDHM to stimulate IL-6 release from adherent cells. MDHM was active on macrophages from CBA/J and C3H/HeJ endotoxin nonresponder mice and was also capable of stimulating IL-6 release from human monocytes. On gel chromatography, MDHM had an apparent molecular size of 1.5 x 10(6) daltons. Treatment with RNase and DNase had no effect on either size or biological activity. Proteinase K did not abolish activity but reduced the apparent molecular size of MDHM. MDHM production by M. fermentans required either coculture with eucaryotic cell lines in RPMI 1640 medium with fetal calf serum or addition of eucaryotic cell sonic extracts to this medium. The biological activity of MDHM is not identical to that of a mitogen for murine spleen cells derived from M. arthritidis; MDHM caused only slight proliferation in this system compared with the mitogen from M. arthritidis, and the latter did not elicit IL-6 release from macrophages. The results are discussed in relation to mycoplasmas as putative etiological agents for rheumatoid arthritis, since high IL-6 titers were reported for synovial fluid from patients with this disease.
Oxygen-dependent up-regulation of mucoid exopolysaccharide (alginate) production in Pseudomonas aeruginosa
Authors: Bayer A S , Eftekhar F , Tu J , Nast C C , Speert D P
Published: January 2020

We previously showed substantial differences in Pseudomonas aeruginosa exopolysaccharide production in vitro at oxygen tensions reflective of the right versus left cardiac circuits in vivo (40 versus 80 mm Hg, respectively; A. S. Bayer, T. O'Brien, D. C. Norman, and C. C. Nast, J. Antimicrob. Chemother. 23:21-35, 1989). However, those studies did not specifically confirm this exopolysaccharide to be the characteristic P. aeruginosa mucoid alginate seen in patients with cystic fibrosis. With a murine monoclonal antibody prepared against P. aeruginosa alginate, strongly positive immunofluorescence (IF) staining of a nonmucoid P. aeruginosa strain (PA-96) was seen after its exposure in vitro to oxygen tensions (pO2) of approximately 80 mm Hg; the intensity of the IF staining under these conditions was similar to that observed with a phenotypically mucoid P. aeruginosa strain (C1712M) from a cystic fibrosis patient. In contrast, the same nonmucoid strain (PA-96), after exposure to pO2 of approximately 40 mm Hg, showed little IF staining for alginate. Following enzyme treatment with alginase, PA-96 cells previously exposed to the higher pO2 and exhibiting enhanced alginate production, as determined by IF staining, now showed no IF staining. Moreover, treatment of the oxygen-up-regulated PA-96 cells with alginase released amounts of unsaturated alginate breakdown products (uronic acids) quantitatively similar to those released by typically mucoid strains treated with the same enzyme. These data indicated that the P. aeruginosa exopolysaccharide in our studies was, indeed, mucoid alginate and that variations in oxygen tensions represent one of the trigger mechanisms for the up-regulation of mucoid exopolysaccharide production.
Phorbol esters enhance the cyclic GMP response of T84 cells to the heat-stable enterotoxin of Escherichia coli (STa)
Authors: Weikel C S , Spann C L , Chambers C P , Crane J K , Linden J , Hewlett E L
Published: January 2020

We examined the effect of protein kinase C (PKC) activation on the cyclic GMP response to heat-stable enterotoxin (STa) in a colonic carcinoma intestinal epithelial cell line, T84 cells. Our results demonstrate that the active phorbol ester analog, phorbol dibutyrate, but not the inactive alpha-phorbol dibutyrate, acts synergistically with STa to elevate cyclic GMP in intact T84 cells. The effect is observed in the absence or presence of the phosphodiesterase inhibitor, isobutylmethylxanthine, which suggests that phorbol dibutyrate modifies cyclic GMP synthesis rather than cyclic GMP degradation. In contrast to several systems in which prolonged treatment with phorbol ester desensitizes PKC-mediated responses, the cyclic GMP response in T84 cells is not diminished by prolonged treatment of T84 cells with phorbol dibutyrate. Also, transient treatment of T84 cells with phorbol dibutyrate enhances subsequent STa-stimulated cyclic GMP accumulation. These observations suggest that PKC activation produces a long-lived signal in T84 cells which enhances cyclic GMP accumulation in response to STa. Second messenger "cross talk" [T. Yoshimasa, D. R. Sibley, M. Bouvier, R. J. Lefkowitz, and M. G. Caron, Nature (London) 327:67-70, 1987] may be important in the pathogenesis of diarrheal disease.
Diminished immunogenicity of a recombination-deficient derivative of Vibrio cholerae vaccine strain CVD103
Authors: Ketley J M , Kaper J B , Herrington D A , Losonsky G , Levine M M
Published: January 2020

To address potential concerns over the release of genetically engineered live bacterial vaccines, we constructed a recombination-deficient derivative of the Vibrio cholerae O1 vaccine strain CVD103 (CVD103RM). Oral immunization of adult volunteers with CVD103RM showed that the recA mutation significantly diminished colonization ability and immunogenicity of the vaccine strain.
Influence of Pasteurella haemolytica A1 crude leukotoxin on bovine neutrophil chemiluminescence
Authors: Czuprynski C J , Noel E J
Published: January 2020

Pasteurella haemolytica A1 crude leukotoxin (25%, vol/vol) rapidly diminished the bovine neutrophil chemiluminescence response to opsonized zymosan. This inhibition was neither prevented nor reversed by 75 mM sucrose. Dilute leukotoxin did not directly stimulate neutrophil chemiluminescence nor did it alter the chemiluminescence response of the neutrophils to opsonized zymosan.
Effect of in vivo T-cell depletion on the effector T-cell function of immunity to Eimeria falciformis
Authors: Stiff M I , Vasilakos J P
Published: January 2020

BALB/c mice were exposed to the enteric parasite Eimeria falciformis to produce a natural acquired immunity. The mice were then depleted of their effector T-cell function by in vivo administration of a cytotoxic Thy-1.2 mouse monoclonal antibody. T-cell depletion was demonstrated by a reduction in concanavalin A-induced proliferation of splenic lymphocytes in treated mice compared with that in controls. Twenty-four hours following T-cell depletion, the mice were challenged with 5,000 oocysts of E. falciformis. Daily total oocyst counts were done for each mouse from days 6 to 21 following challenge. Our studies demonstrated that depleting mice of their effector T-cell function following establishment of immunity caused an abrogation of protective immunity to this parasite.
Cloning and expression in Escherichia coli of a Haemophilus influenzae type b lipooligosaccharide synthesis gene(s) that encodes a 2-keto-3-deoxyoctulosonic acid epitope
Authors: Spinola S M , Kwaik Y A , Lesse A J , Campagnari A A , Apicella M A
Published: January 2020

The composition of lipooligosaccharide (LOS) can modify the virulence of Haemophilus influenzae type b (Hib). A genomic library of Hib strain A2 was constructed in the lambda bacteriophage EMBL3. Twenty-six phage clones expressed a Hib LOS oligosaccharide epitope in Escherichia coli that was detected by the monoclonal antibody (MAb) 6E4. None of the clones bound a polyclonal sera specific for Hib A2 LOS or an anti-H. influenzae lipid A MAb. One clone, designated EMBLOS-1, assembled an oligosaccharide with an apparent molecular weight of 1,400 (the 1.4K oligosaccharide) on a 4.1K lipopolysaccharide (LPS) species in E. coli LE392 and produced a novel 5.5K LPS that bound 6E4. Binding of 6E4 to the 5.5K EMBLOS-1 LPS band was abolished by treatment with sodium metaperiodate but was not affected by digestion with proteinase K, confirming the carbohydrate nature of the epitope. The EMBLOS-1 Haemophilus insert hybridized to similar restriction fragments in type b and nontypeable strains regardless of whether they expressed the 6E4 epitope. The 6E4 epitope did not undergo phase variation in Hib strain A2 at a frequency of greater than 10(-3). The oligosaccharide of the Salmonella minnesota Re mutant and 2-keto-3-deoxyoctulosonic acid (KDO) inhibited binding of 6E4 to Hib A2 LOS. We conclude that a gene(s) encoding an enzyme(s) that assembles a stable Hib LOS epitope containing KDO is conserved in H. influenzae and that the cloned Hib LOS synthesis gene products assemble a Hib LOS epitope on an E. coli K-12 LPS core.
Expression of antigens from chromosomal and linear plasmid DNA of Borrelia coriaceae
Authors: Perng G C , LeFebvre R B
Published: January 2020

Three recombinant plasmids containing DNA from Borrelia coriaceae, the putative agent of epizootic bovine abortion, expressed antigens in Escherichia coli that reacted with antibodies specific for B. coriaceae. Two of the recombinants each expressed a single high-molecular-weight antigen. The third recombinant expressed three smaller antigens. The DNA inserts were sized and mapped. Hybridization of the cloned inserts to pulsed-field electrophoresis samples of B. coriaceae whole-cell DNA revealed the origin of two of the inserts to be located in linear plasmids. One of these, expressing three antigens, was located in a 210-kilobase linear plasmid. A second recombinant expressed a single antigen but hybridized to at least three distinct linear plasmids. The third clone also expressed a single antigen but was demonstrated to be chromosomal in origin.
Identification of Campylobacter jejuni surface proteins that bind to Eucaryotic cells in vitro
Authors: de Melo M A , Pechère J C
Published: January 2020

To understand the role of Campylobacter jejuni surface proteins in the interaction of C. jejuni with cultured mammalian cell lines in vitro, we developed a ligand-binding assay. This procedure allowed us to antigenically identify C. jejuni outer membrane proteins (OMPs) that attach to intact host cell membranes. OMPs isolated from an invasive strain and a less invasive strain were antigenically indistinguishable. However, we found that proteins with molecular masses of 28 and 32 kilodaltons (kDa) from just the invasive strain bound to HEp-2 cell monolayers. Binding of the 32-kDa OMP was cell line specific and correlated directly with the ability of the invasive C. jejuni strain to penetrate. Such a correlation was probably also true for the 28-kDa OMP. We also investigated the binding of glycine acid extracts with cell line HEp-2. We identified four proteins with apparent molecular masses of 28, 32, 36, and 42 kDa in the invasive strain extracts that bound to HEp-2 cells. In contrast, only the 36-kDa protein from the less invasive strain bound to HEp-2 cells. Our data suggest that binding of these surface exposed proteins may play a key role in C. jejuni-host cell interactions and ultimate invasion.
Erythrocyte receptor (CD2)-bearing T lymphocytes are affected by diet in experimental pulmonary tuberculosis
Authors: Bartow R A , McMurray D N
Published: January 2020

Outbred (Hartley) and inbred (strain 2 and 13) guinea pigs were vaccinated with Mycobacterium bovis BCG and placed on isocaloric, purified diets containing either 10 or 30% ovalbumin or on commercial guinea pig chow. Six weeks later, the animals were challenged by the respiratory route with virulent M. tuberculosis H37Rv. At intervals postchallenge, groups were tuberculin tested and sacrificed. Thymus-dependent (T) lymphocytes were enumerated either by rosette formation with rabbit erythrocytes or by the indirect immunofluorescence assay (IFA) with a guinea pig pan-T-cell monoclonal antibody, 8BE6. Protein-deficient guinea pigs of all three strains had significantly fewer erythrocyte rosette-forming (CD2+) T cells in the peripheral blood, and malnourished strain 2 and Hartley guinea pigs exhibited reduced levels of CD2+ T cells in the thymus. In contrast, animals of all three strains fed the low-protein diet harbored more CD2+ T cells in the bronchotracheal lymph nodes than did their control-fed counterparts. A larger proportion of lymphocytes from the blood and lymph nodes of all three strains were IFA positive than formed erythrocyte rosettes regardless of diet treatment. Diet had no effect on IFA-positive lymphocytes in those organs. Protein deficiency is associated with significant alterations in the number and/or distribution of T lymphocytes expressing functional CD2-receptors in BCG-vaccinated animals exposed to virulent mycobacteria by the pulmonary route. These alterations may contribute to the reduction in BCG vaccine efficacy observed in this model.
Expression in Escherichia coli of the 37-kilodalton endoflagellar sheath protein of Treponema pallidum by use of the polymerase chain reaction and a T7 expression system
Authors: Isaacs R D , Radolf J D
Published: January 2020

We previously reported the complete primary structure of the 37-kilodalton endoflagellar sheath protein (FlaA) of Treponema pallidum. However, we were unable to determine the nucleotide sequence of flaA upstream of amino acid 10. The desired nucleotide sequence was obtained by use of a strategy based upon the polymerase chain reaction and was found to contain a consensus Escherichia coli promoter, a ribosomal binding site, and a 20-amino-acid signal peptide. Expression of FlaA in E. coli was achieved by cloning polymerase chain reaction-derived constructs lacking the native T. pallidum promoter into a temperature-inducible T7 expression system. Pulse-chase and ethanol inhibition analyses of protein processing in E. coli cells and minicells, respectively, indicated that processing of the FlaA precursor was incomplete. Native and recombinant FlaA were identical as assessed by antibody reactivity and sodium dodecyl sulfate- and two-dimensional polyacrylamide gel electrophoretic mobilities. Soluble FlaA was not detected in either the cytoplasmic or the periplasmic fractions of E. coli transformants. Fractionation of E. coli cell envelopes unexpectedly revealed that FlaA precursor and FlaA were associated with both the cytoplasmic and outer membranes. This is the first report of expression in E. coli of a T. pallidum protein which could not be cloned or expressed with its native promoter. Our data also indicate that information obtained in E. coli regarding the subcellular location of cloned treponemal proteins must be cautiously extrapolated to T. pallidum.
Evidence of reinfection with multiple strains of Campylobacter jejuni and Campylobacter coli in Macaca nemestrina housed under hyperendemic conditions
Authors: Russell R G , Sarmiento J I , Fox J , Panigrahi P
Published: January 2020

A prospective bacteriologic study of 18 infant pig-tailed macaques (Macaca nemestrina) housed in a nursery facility in which Campylobacter spp. are endemic was undertaken to determine the epidemiology of infection and reinfection. The isolates of Campylobacter jejuni and C. coli cultured from 8 of the 18 infants were characterized by serotyping, DNA hybridization, and polyacrylamide gel electrophoresis protein profiles. The chronology of infection was indicative of multiple reinfections with different strains of C. jejuni and C. coli during the 12-month study of each infant. The duration of infection with a particular strain was 3 to 4 weeks. Infants were also infected with nalidixic acid-resistant campylobacters. These observations indicated that long-term infections under endemic conditions are caused by continual reinfection. C. jejuni or C. coli infection correlated with diarrhea in 5 of the 18 infants at 1 to 4 months of age.