bluuebunny/crossref_metadata_2025 huggingface dataset:
Adhesion to and invasion of HeLa cells by pathogenic Escherichia coli carrying the afa-3 gene cluster are mediated by the AfaE and AfaD proteins, respectively
Authors: Jouve M , Garcia M I , Courcoux P , Labigne A , Gounon P , Le Bouguénec C
Published: January 2020

The afa-3 gene cluster, expressed by uropathogenic and diarrhea-associated Escherichia coli strains, determines the formation of an afimbrial adhesive sheath composed of the AfaD and AfaE-III adhesins. The adherence to HeLa cells by recombinant HB101 strains producing both or only one of these two adhesins was investigated. Ultrastructural analyses of the interaction and gentamicin protection assays showed adherence to HeLa cells by HB101 producing both the AfaD and AfaE-III proteins and internalization of a subpopulation of the bacteria into the cells. The interactions of HeLa cells either with HB101 mutants producing AfaD or AfaE-III or with polystyrene beads coated with purified His6-tagged AfaD or His6-tagged AfaE-III proteins were studied. These experiments demonstrated that AfaE-III allows binding to HeLa cells and that AfaD mediates the internalization of the adherent bacteria. Ultrastructural analyses of the interaction of His6-AfaD-gold complexes with HeLa cells confirmed that AfaD is able to bind to the HeLa cell surface and indicated that it penetrates the cells via clathrin vesicles. These data demonstrate that the afa gene cluster is unique among bacteria, as alone it encodes both adhesion to and invasion of epithelial cells.
Enhancement of neutrophil-mediated killing of Plasmodium falciparum asexual blood forms by fatty acids: importance of fatty acid structure
Authors: Kumaratilake L M , Ferrante A , Robinson B S , Jaeger T , Poulos A
Published: January 2020

Effects of fatty acids on human neutrophil-mediated killing of Plasmodium falciparum asexual blood forms were investigated by using a quantitative radiometric assay. The results showed that the antiparasitic activity of neutrophils can be greatly increased (>threefold) by short-term treatment with fatty acids with 20 to 24 carbon atoms and at least three double bonds. In particular, the n-3 polyenoic fatty acids, eicosapentaenoic and docosahexaenoic acids, and the n-6 fatty acid, arachidonic acid, significantly enhanced neutrophil antiparasitic activity. This effect was >1.5-fold higher than that induced by an optical concentration of the known agonist cytokine tumor necrosis factor alpha (TNF-alpha). At suboptimal concentrations, the combination of arachidonic acid and TNF-alpha caused a synergistic increase in neutrophil-mediated parasite killing. The fatty acid-induced effect was independent of the availability of serum opsonins but dependent on the structure of the fatty acids. The length of the carbon chain, degree of unsaturation, and availability of a free carboxyl group were important determinants of fatty acid activity. The fatty acids which increased neutrophil-mediated killing primed the enhanced superoxide radical generation of neutrophils in response to P. falciparum as detected by chemiluminescence. Scavengers of oxygen radicals significantly reduced the fatty acid-enhanced parasite killing, but cyclooxygenase and lipoxygenase inhibitors had no effect. These findings have identified a new class of immunoenhancers that could be exploited to increase resistance against Plasmodium species.
Enterococcus faecalis antigens in human infections
Authors: Xu Y , Jiang L , Murray B E , Weinstock G M
Published: January 2020

Genomic libraries of two Enterococcus faecalis strains, OG1RF and TX52 (an isolate from an endocarditis patient), were constructed in cosmid vectors pBeloBAC11 and pLAFRx, and screened with a serum from a rabbit immunized with surface proteins of an E. faecalis endocarditis isolate and sera from four patients with enterococcal endocarditis. Seventy-five cosmid clones reacted with at least two of the sera. Thirty-eight of the 75 immunopositive clones were considered to contain distinct inserts based on their DNA restriction patterns and were chosen for further subcloning into a pBluescript vector. Each sublibrary was screened with one of the five sera, and the DNA sequence of the immunopositive subclones was determined. Analysis of these sequences revealed similarities to a range of proteins, including bacterial virulence factors, transporters, two-component regulators, metabolic enzymes, and membrane or cell surface proteins. Fourteen subclones did not show significant similarity to any sequence in the databases and may contain novel genes. Thirteen of the immunopositive cosmid clones did not yield immunopositive subclones, and one such cosmid clone produced a nonprotein antigen in Escherichia coli.
Interaction between Entamoeba histolytica and intestinal epithelial cells involves a CD44 cross-reactive protein expressed on the parasite surface
Authors: Renesto P , Sansonetti P J , Guillén N
Published: January 2020

This study shows that Entamoeba histolytica binds hyaluronic acid. The binding molecule was identified as an 80-kDa membrane protein and was recognized by anti-CD44 monoclonal antibodies. These data indicate that a CD44 cross-reacting adherence molecule is expressed on E. histolytica.
Interleukin-12 gene expression in human monocyte-derived macrophages stimulated with Mycobacterium bovis BCG: cytokine regulation and effect of NK cells
Authors: Matsumoto H , Suzuki K , Tsuyuguchi K , Tanaka E , Amitani R , Maeda A , Yamamoto K , Sasada M , Kuze F
Published: January 2020

Macrophage-derived interleukin-12 (IL-12) is essential for the activation of a protective immune response against intracellular pathogens. In this study, we examined the regulation of IL-12 mRNA expression by monocyte-derived macrophages (MDM) in response to Mycobacterium bovis BCG stimulation. A reverse transcription-PCR assay detected p40 mRNA of IL-12 at 3 h and showed a peak at 6 to 12 h with a subsequent decline. Semiquantitation of mRNA levels by competitive PCR revealed that pretreatment with gamma interferon (IFN-gamma) amplified the expression approximately 100-fold, while pretreatment with tumor necrosis factor alpha (TNF-alpha) or granulocyte-macrophage colony-stimulating factor augmented this expression about 10-fold. In contrast, pretreatment with IL-10 and IL-4 inhibited IL-12 mRNA expression. These results were further confirmed by measuring the p70 bioactive protein level in each conditioned medium by an enzyme-linked immunosorbent assay. Since IL-12 mRNA expression was weak without cytokine pretreatment and IFN-gamma strongly augmented production, we speculated that IFN-gamma might have a role in BCG stimulation of IL-12 mRNA expression. Unexpectedly, the addition of three different kinds of anti-IFN-gamma antibodies and anti-IFN-gamma receptor antibody and the coaddition of anti-TNF-alpha antibody with anti-IFN-gamma receptor antibody all failed to inhibit IL-12 mRNA expression. However, the MiniMACS method used to remove NK cells from a mononuclear cell suspension inhibited the expression of p40 mRNA but not the expression of mRNA of TNF-alpha or IL-1beta. We concluded that the coexistence of NK cells was essential for the induction of IL-12 in MDM stimulated with BCG rather than through the secretion of IFN-gamma.
Regions of Yersinia pestis V antigen that contribute to protection against plague identified by passive and active immunization
Authors: Hill J , Leary S E , Griffin K F , Williamson E D , Titball R W
Published: January 2020

V antigen of Yersinia pestis is a multifunctional protein that has been implicated as a protective antigen, a virulence factor, and a regulatory protein. A series of V-antigen truncates expressed as glutathione S-transferase (GST) fusion proteins (GST-V truncates) have been cloned and purified to support immunogenicity and functionality studies of V antigen. Immunization studies with GST-V truncates have identified two regions of V antigen that confer protection against Y. pestis 9B (a fully virulent human pneumonic plague isolate) in a mouse model for plague. A minor protective region is located from amino acids 2 to 135 (region I), and a major protective region is found between amino acids 135 and 275 (region II). In addition, analysis of IgG titers following immunization suggested that the major antigenic region of V antigen is located between amino acids 135 and 245. A panel of monoclonal antibodies raised against recombinant V antigen was characterized by Western blotting against GST-V truncates, and epitopes of most of the monoclonal antibodies were mapped to region I or II. Monoclonal antibody 7.3, which recognizes an epitope in region II, passively protected mice against challenge with 12 median lethal doses of Y. pestis GB, indicating that region II encodes a protective epitope. This is the first report of a V-antigen-specific monoclonal antibody that will protect mice against a fully virulent strain of Y. pestis. The combined approach of passive and active immunization has therefore confirmed the importance of the central region of the protein for protection and also identified a previously unknown protective region at the N terminus of V antigen.
Immunization with heat-killed Mycobacterium vaccae stimulates CD8+ cytotoxic T cells specific for macrophages infected with Mycobacterium tuberculosis
Authors: Skinner M A , Yuan S , Prestidge R , Chuk D , Watson J D , Tan P L
Published: January 2020

Immune responses to Mycobacterium tuberculosis are analyzed in mice which have been immunized with Mycobacterium vaccae to examine novel ways of altering protective immunity against M. tuberculosis. The spleen cells of mice immunized with M. vaccae proliferate and secrete gamma interferon (IFN-gamma) in response to challenge with live M. tuberculosis in vitro. Immunization with M. vaccae results in the generation of CD8+ T cells which kill syngeneic macrophages infected with M. tuberculosis. These effector cytotoxic T cells (CTL) are detectable in the spleen at 2 weeks after immunization with M. vaccae but cannot be found in splenocytes 3 to 6 weeks postimmunization. However, M. tuberculosis-specific CTL are revealed following restimulation in vitro with heat-killed M. vaccae or M. tuberculosis, consistent with the activation of memory cells. These CD8+ T cells secrete IFN-gamma and enhance the production of interleukin 12 when cocultured with M. tuberculosis-infected macrophages. It is suggested that CD8+ T cells with a cytokine secretion profile of the Tc1 class may themselves maintain the dominance of a Th1-type cytokine response following immunization with M. vaccae. Heat-killed M. vaccae deserves attention as an alternative to attenuated live mycobacterial vaccines.
Chemokine secretion by human polymorphonuclear granulocytes after stimulation with Mycobacterium tuberculosis and lipoarabinomannan
Authors: Riedel D D , Kaufmann S H
Published: January 2020

Macrophages (MAC) and polymorphonuclear granulocytes (PNG) are professional phagocytes which perform essential functions in antibacterial defense. The intracellular bacterium Mycobacterium tuberculosis persists and replicates in resting macrophages. Although it is generally assumed that activated MAC are central to protection against M. tuberculosis, PNG may also contribute to defense. We wondered whether PNG produce proinflammatory chemokines after stimulation by M. tuberculosis or its major cell wall component, lipoarabinomannan (LAM). In this study, we showed that M. tuberculosis- and LAM-activated human PNG secrete the leukocyte attractant interleukin-8 (IL-8) and the PNG-specific chemokine GRO-alpha in a dose-dependent manner. Treatment of PNG with the leukotriene-B4 inhibitor MK-886 prior to stimulation with M. tuberculosis or LAM partially blocked IL-8 and GRO-alpha induction, suggesting involvement of the 5-lipoxygenase pathway in the secretion of these chemokines. We conclude that PNG contribute to early resistance to M. tuberculosis via chemokine secretion.
Borrelia burgdorferi strain-specific Osp C-mediated immunity in mice
Authors: Bockenstedt L K , Hodzic E , Feng S , Bourrel K W , de Silva A , Montgomery R R , Fikrig E , Radolf J D , Barthold S W
Published: January 2020

Antibodies to the outer surface proteins (Osps) A, B, and C of the spirochete Borrelia burgdorferi can prevent infection in animal models of Lyme borreliosis. We have previously demonstrated that immune serum from mice infected with B. burgdorferi N40 can also prevent challenge infection and induce disease regression in infected mice. The antigens targeted by protective and disease-modulating antibodies are presently unknown, but they do not include Osp A or Osp B. Because Osp C antibodies are present in immune mouse serum, we investigated the ability of hyperimmune serum to recombinant Osp C (N40) to protect mice against challenge infection with N40 spirochetes. In both active and passive immunization studies, Osp C (N40) antiserum failed to protect mice from challenge infection with cultured organisms. Mice actively immunized with recombinant Osp C (N40) were susceptible to tick-borne challenge infection, and nymphal ticks remained infected after feeding on Osp C-hyperimmunized mice. In contrast, similar immunization studies performed with Osp C (PKo) antiserum prevented challenge infection of mice with a clone of PKo spirochetes pathogenic for mice. Both Osp C (N40) and Osp C (PKo) antisera showed minimal in vitro borreliacidal activity, and immunofluorescence studies localized Osp C beneath the outer membrane of both N40 and PKo spirochetes. We conclude that Osp C antibody-mediated immunity is strain specific and propose that differences in Osp C surface expression by spirochetes in vivo may account for strain-specific immunity.
Development of a model of low-inoculum Streptococcus pneumoniae intrapulmonary infection in infant rats
Authors: Saladino R A , Stack A M , Fleisher G R , Thompson C M , Briles D E , Kobzik L , Siber G R
Published: January 2020

We have developed a model of low-inoculum Streptococcus pneumoniae infection in infant rats. We challenged 4-day-old Sprague-Dawley pups via intraperitoneal or intrapulmonary injection of S. pneumoniae serotypes 1, 3, 4, 5, 6b, 7f, 9v, 14, 19f, and 23f. To achieve bacteremia with low inocula, it was necessary to passage the isolates in rats. Inocula of the 10 S. pneumoniae serotypes producing bacteremia in 50% or more animals ranged from 1 to 400 CFU. Virulence was similar by intraperitoneal and intrapulmonary routes. Lung specimens from animals challenged by the intrapulmonary route grew S. pneumoniae and demonstrated histologic evidence of focal infection. Meningitis was detected in 20 to 50% of bacteremic animals, and mortality invariably followed bacteremia within 24 to 48 h. This model of intrapulmonary infection uses low inocula of S. pneumoniae and results in bacteremia, meningitis, and death in infant rats.
Differences in coughing and other responses to intrabronchial infection with Bordetella pertussis among strains of rats
Authors: Hall E , Parton R , Wardlaw A C
Published: January 2020

Four strains of rats were each infected intrabronchially with approximately 10(8) CFU of Bordetella pertussis 18-323 encased in fine agarose beads. After 8 days, Sprague-Dawley rats developed the highest incidence of coughing paroxysms, as monitored with voice-activated tape recorders; Brown Norway, Lewis, and Hooded Lister rats coughed significantly less frequently. Marked leukocytosis, with counts up to four times the normal levels, and retardation of normal weight gain occurred in all four rat strains. Both coughing and leukocytosis were greater in animals that were infected at 4 weeks of age than in those infected at 6 weeks of age. Total serum immunoglobulin E (IgE) rose in all four rat strains 9- to 244-fold by day 8 after infection and returned to near preinfection levels at 6 weeks. Sprague-Dawley and Lewis rats, which had the lowest basal levels of total IgE in serum, showed the greatest degrees of elevation. All four rat strains had IgG to B. pertussis whole-cell sonicate and to filamentous hemagglutinin in 6-week-postinfection sera. However, the strains differed in production of IgG to pertussis toxin, with Sprague-Dawley rats having the highest titers and Hooded Lister and Lewis rats being nonresponders. These studies highlight the importance of rat strain as a variable in the coughing-rat model of pertussis and validate the choice of the Sprague-Dawley rats in previous studies.
Cloning and disruption of the antigenic catalase gene of Aspergillus fumigatus
Authors: Calera J A , Paris S , Monod M , Hamilton A J , Debeaupuis J P , Diaquin M , López-Medrano R , Leal F , Latgé J P
Published: January 2020

Aspergillus fumigatus possesses two catalases (described as fast and slow on the basis of their electrophoretic mobility). The slow catalase has been recognized as a diagnostic antigen for aspergillosis in immunocompetent patients. The antigenic catalase has been purified. The enzyme is a tetrameric protein composed of 90-kDa subunits. The corresponding cat1 gene was cloned, and sequencing data show that the cat1 gene codes for a 728-amino-acid polypeptide. A recombinant protein expressed in Pichia pastoris is enzymatically active and has biochemical and antigenic properties that are similar to those of the wild-type catalase. Molecular experiments reveal that CAT1 contains a signal peptide and a propeptide of 15 and 12 amino acid residues, respectively. cat1-disrupted mutants that were unable to produce the slow catalase were as sensitive to H2O2 and polymorphonuclear cells as the wild-type strain. In addition, there was no difference in pathogenicity between the cat1 mutant and its parental cat1+ strain in a murine model of aspergillosis.
Serum antibodies to Porphyromonas gingivalis block the prostaglandin E2 response to lipopolysaccharide by mononuclear cells
Authors: Bainbridge B W , Page R C , Darveau R P
Published: January 2020

The ability of rabbit and monkey immune sera to neutralize prostaglandin E2 (PGE2) production by human monocytes stimulated with lipopolysaccharide (LPS) was examined. CD14-dependent LPS activation of PGE2 was examined under assay conditions which allowed the comparison of preimmune and immune sera. Serum obtained from rabbits immunized with formalin-fixed Porphyromonas gingivalis cells dramatically reduced the amount of PGE2 produced in response to LPS obtained from three different strains of P. gingivalis but not that from Escherichia coli or Bacteroides fragilis. In addition, a significant reduction in the mean PGE2 level was observed in the presence of sera from immunized but not control monkeys employed in a vaccine trial. Immune serum samples from five of nine immunized monkeys were able to reduce LPS-induced production of PGE2 by greater than 50% compared to that in the corresponding preimmune sera. Immune monkey serum, similar to immune rabbit serum, blocked PGE2 production in response to P. gingivalis LPS but not E. coli LPS. These data demonstrate that immunization with P. gingivalis whole cells can elicit an antibody response that is able to block the PGE2 response to LPS. Neutralization of LPS-mediated inflammatory mediator production may account in part for the observed suppression of alveolar bone loss in immunized monkeys.
Lethal tuberculosis in interleukin-6-deficient mutant mice
Authors: Ladel C H , Blum C , Dreher A , Reifenberg K , Kopf M , Kaufmann S H
Published: January 2020

Tuberculosis is a chronic infectious disease which causes major health problems globally. Acquired resistance is mediated by T lymphocytes and executed by activated macrophages. In vitro studies have emphasized the importance of macrophage activation for mycobacterial growth inhibition. In vivo, the protective host response is focused on granulomatous lesions in which Mycobacterium tuberculosis is contained. A cellular immune response of the T helper 1 (Th1) type is considered central for control of tuberculosis. Using interleukin-6 (IL-6)-deficient mice, we here demonstrate a crucial role of this pluripotent cytokine in protection against M. tuberculosis but not against Mycobacterium bovis BCG. Infection with M. tuberculosis was lethal for the IL-6-deficient mice at inocula that were still controlled by IL-6-competent mice. Spleen cells from M. tuberculosis-infected IL-6-/- mouse mutants produced elevated levels of IL-4 and reduced levels of gamma interferon compared to the control levels. Cytofluorometric analyses of spleen cells from M. tuberculosis-infected mice revealed more-profound alterations in T-cell ratios in IL-6-/- mice than in control mice. We assume that IL-6 contributes to host resistance by its proinflammatory activity and by its influence on cytokine secretion.
Identification of superoxide dismutase activity in Borrelia burgdorferi
Authors: Whitehouse C A , Williams L R , Austin F E
Published: January 2020

Infective and noninfective strains of Borrelia burgdorferi, along with Borrelia afzelii and Borrelia garinii, possessed a single iron-containing superoxide dismutase (SOD). None of the Lyme disease spirochetes tested possessed catalase or peroxidase activities. The borrelial SOD was not inducible by growth with increased oxygen concentrations and thus appeared to be produced constitutively.
Intrapulmonary Hartmannella vermiformis: a potential niche for Legionella pneumophila replication in a murine model of legionellosis
Authors: Brieland J , McClain M , LeGendre M , Engleberg C
Published: January 2020

The potential role of inhaled protozoa as a niche for intrapulmonary replication of Legionella pneumophila was investigated in vivo with mutant strains of L. pneumophila which have reduced virulence for the amoeba Hartmannella vermiformis. L. pneumophila AA488 and AA502 were derived from wild-type strain AA100 after transposon mutagenesis. These mutants have reduced virulence for H. vermiformis but are fully virulent for mononuclear phagocytic cells. A/J mice, which are susceptible to replicative L. pneumophila lung infections, were inoculated intratracheally with L. pneumophila AA100, AA488, or AA502 (10[6] bacteria per mouse) or were coinoculated with one of the L. pneumophila strains (10[6] bacteria per mouse) and uninfected H. vermiformis (10[6] amoebae per mouse). The effect of coinoculation with H. vermiformis on intrapulmonary growth of each L. pneumophila strain was subsequently assessed. In agreement with our previous studies, coinoculation with H. vermiformis significantly enhanced intrapulmonary growth of the parent L. pneumophila strain (AA100). In contrast, intrapulmonary growth of L. pneumophila AA488 or AA502 was not significantly enhanced by coinoculation of mice with H. vermiformis. These studies demonstrate that L. pneumophila virulence for amoebae is required for maximal intrapulmonary growth of the bacteria in mice coinoculated with H. vermiformis and support the hypothesis that inhaled amoebae may potentiate intrapulmonary growth of L. pneumophila by providing a niche for bacterial replication.
Role of gamma interferon in natural clearance of Bordetella pertussis infection
Authors: Barbic J , Leef M F , Burns D L , Shahin R D
Published: January 2020

Using a mouse model of Bordetella pertussis infection, we have analyzed the role of gamma interferon (IFN-gamma) in bacterial clearance from the respiratory tract. Adult BALB/c mice began to clear a respiratory infection within 3 weeks postinfection, with complete resolution of infection 6 to 8 weeks postinfection. In contrast, neither adult SCID mice (which lack mature B and T lymphocytes) nor adult nude mice (which lack mature T lymphocytes) controlled B. pertussis infection, and both strains died within 3 to 5 weeks postinfection. Short-term T-cell lines generated from the draining lymph nodes of the lungs of infected BALB/c mice were found to be CD4+ and produced IFN-gamma but no detectable interleukin-4. Analyses of IFN-gamma mRNA induction in the lungs of mice following B. pertussis infection showed that in both BALB/c and C57BL/6 mice, IFN-gamma mRNA levels increased sharply by 1 week postinfection and then subsequently declined. Further exploration of a potential role for IFN-gamma demonstrated that infection of adult BALB/c mice depleted of IFN-gamma in vivo with anti-IFN-gamma monoclonal antibodies resulted in greater numbers of bacteria recovered from the lungs than in infected, control BALB/c mice, although IFN-gamma-depleted mice could subsequently clear the infection. Infection of mice which have a disrupted IFN-gamma gene resulted in bacterial clearance with a time course similar to those seen with IFN-gamma-depleted mice. These results indicate that IFN-gamma plays a role in controlling B. pertussis infection.
A strategy for rational design of fully synthetic glycopeptide conjugate vaccines
Authors: Chong P , Chan N , Kandil A , Tripet B , James O , Yang Y P , Shi S P , Klein M
Published: January 2020

The present study describes a strategy to rationally design fully synthetic glycopeptide conjugate vaccines. Glycopeptide immunogens were constructed by coupling synthetic oligosaccharides comprising repeating units of synthetic 3-beta-D-ribose-(1-1)-D-ribitol-5-phosphate (sPRP) to synthetic peptides containing potent T-helper cell determinants and B-cell epitopes of the Haemophilus influenzae type b (Hib) outer membrane proteins (OMPs) P1, P2, and P6. Rabbit immunogenicity studies revealed that some of these fully synthetic glycoconjugates were capable of eliciting high titers of both anti-PRP and anti-OMP immunoglobulin G antibodies. In addition, we systematically investigated the factors which could influence their immunogenicity. We observed that the magnitude of the anti-PRP antibody response markedly depended on the relative spatial orientation of sPRP and T-cell epitopes, the anti-PRP antibody response was enhanced when a multiple antigenic peptide was used as a carrier, the anti-PRP antibody response was optimal for three PRP repeating units, and lipidation of peptide-PRP conjugates had a minimal effect on the magnitude of the anti-PRP antibody response. The results of this study clearly demonstrate that coupling a carbohydrate hapten to a peptide can provide T-cell help and convert it into a T-cell-dependent antigen. The antisera raised against these conjugates were also found to be protective against Hib infection in the infant rat model of bacteremia.
Characterization of B-cell responses to Chlamydia trachomatis antigens in humans with trachoma
Authors: Ghaem-Maghami S , Bailey R L , Mabey D C , Hay P E , Mahdi O S , Joof H M , Whittle H C , Ward M E , Lewis D J
Published: January 2020

The circulating B-cell responses to Chlamydia trachomatis of 60 children and 34 adults in The Gambia were characterized in a cross-sectional study of different grades of trachoma, using the enzyme-linked immunospot (ELISPOT) assay. Antibody-secreting cells (ASCs) specific to chlamydial major outer membrane protein (MOMP), heat shock protein 60, and whole elementary bodies were detected in children with no evidence of ocular disease, and the immunoglobulin (IgA) response was significantly increased in those with follicular trachoma. In marked contrast, children with the most intense ocular inflammation paradoxically had an almost completely absent B-cell response of all isotypes and to all chlamydial antigens, but with normal serum IgG and IgA responses, which was even lower than in the group with no ocular inflammation. Adults with or without evidence of trachomatous scarring had equivalent numbers of circulating B cells, principally IgA, to all chlamydial antigens. Plasmablasts secreting antibodies to MOMP were present in the urine of children in the absence of urogenital infection detectable by PCR, and relative numbers were 8 to 25 times higher than in blood, suggesting site-specific homing within a common mucosal immune system. These results suggest that ELISPOT assay of ongoing B-cell responses detects suppression of chlamydia-specific IgA ASCs during the proinflammatory response to ocular chlamydial infection seen in intense trachoma, which may play a role in tissue damage leading to trachomatous scarring.
Isolation and characterization of a pigmentless-conidium mutant of Aspergillus fumigatus with altered conidial surface and reduced virulence
Authors: Jahn B , Koch A , Schmidt A , Wanner G , Gehringer H , Bhakdi S , Brakhage A A
Published: January 2020

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. The factors contributing to the predominance of A. fumigatus as an opportunistic pathogen are largely unknown. Since the survival of conidia in the host is a prerequisite for establishing disease, we have been attempting to identify factors which are associated with conidia and, simultaneously, important for infection. Therefore, an A. fumigatus mutant strain (white [W]) lacking conidial pigmentation was isolated. Scanning electron microscopy revealed that conidia of the W mutant also differed in their surface morphology from those of the wild type (WT). Mutant (W) and WT conidia were compared with respect to their capacities to stimulate an oxidative response in human phagocytes, their intracellular survival in human monocytes, and virulence in a murine animal model. Luminol-dependent chemiluminescence was 10-fold higher when human neutrophils or monocytes were challenged with W conidia compared with WT conidia. Furthermore, mutant conidia were more susceptible to killing by oxidants in vitro and were more efficiently damaged by human monocytes in vitro than WT conidia. In a murine animal model, the W mutant strain showed reduced virulence compared with the WT. A reversion analysis of the W mutant demonstrated that all phenotypes associated with the W mutant, i.e., altered conidial surface, amount of reactive oxygen species release, susceptibility to hydrogen peroxide, and reduced virulence in an murine animal model, coreverted in revertants which had regained the ability to produce green spores. This finding strongly suggests that the A. fumigatus mutant described here carries a single mutation which caused all of the observed phenotypes. Our results suggest that the conidium pigment or a structural feature related to it contributes to fungal resistance against host defense mechanisms in A. fumigatus infections.
Induction of gamma interferon production in human alveolar macrophages by Mycobacterium tuberculosis
Authors: Fenton M J , Vermeulen M W , Kim S , Burdick M , Strieter R M , Kornfeld H
Published: January 2020

Gamma interferon (IFN-gamma) is a cytokine which plays a critical role in resistance to Mycobacterium tuberculosis infection. While T lymphocytes and natural killer cells are a major source of IFN-gamma, previous demonstrations that it can be produced by murine macrophages prompted us to examine the capacity of human alveolar macrophages to express IFN-gamma. Here we report that in vitro infection of alveolar macrophages with M. tuberculosis induces both the release of IFN-gamma protein and a transient increase in IFN-gamma mRNA levels. The IFN-producing cells were shown to be macrophages by reverse transcription-in situ PCR. We also observed that M. tuberculosis stimulation resulted in IFN-gamma-dependent expression of the chemokines IFN-gamma-inducible protein 10 and monokine induced by IFN-gamma, suggesting that macrophage-derived IFN-gamma can function in an autocrine and/or paracrine manner. The existence of a positive regulatory loop was suggested by the observation that exogenous IFN-gamma protein could induce IFN-gamma mRNA expression in uninfected alveolar macrophages. Interleukin-12 was also found to be a potent inducer of IFN-gamma production, and M. tuberculosis-induced IFN-gamma production appears to be mediated, at least in part, by IL-12. In contrast, M. tuberculosis-induced IFN-gamma production by alveolar macrophages could be blocked by exogenous interleukin-10. These studies are the first to demonstrate an autoregulatory role for IFN-gamma produced by alveolar macrophages infected in vitro with M. tuberculosis.
Altered cytokine production by cystic fibrosis tracheal gland serous cells
Authors: Kammouni W , Figarella C , Marchand S , Merten M
Published: January 2020

Human submucosal tracheal glands are now believed to play a major role in the physiopathology of cystic fibrosis (CF). We successfully developed techniques for culturing human tracheal gland serous cells from normal individuals (HTGS cells) and from CF patients (CF-HTGS cells) and have shown that the cultured cells have retained most of their in vivo epithelial and secretory characteristics. In order to determine to what extent the serous cells may participate in the lung defense against infection, we examined the effects of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa on HTGS and CF-HTGS cells, with special reference to tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-8 secretion. HTGS cells showed a daily basal secretion of IL-6 (1.68 +/- 0.14 ng/10(6) cells) and IL-8 (9.6 +/- 1.3 ng/10(6) cells) and no constitutive secretion of TNF-alpha. Treatment with P. aeruginosa LPS resulted in a significant increase in the basal production of IL-6 (increase of 200% +/- 12%) and IL-8 (525% +/- 40%) as well as a rapid production of TNF-alpha (250 +/- 38 pg/10(6) cells). The LPS-induced secretion of IL-6 and IL-8, but not that of TNF-alpha, was inhibited by glucocorticoids. CF-HTGS cells showed a much higher basal secretion of IL-6 (13.2 +/- 0.5 ng/10(6) cells) and IL-8 (45.6 +/- 7.2 ng/10(6) cells) than normal cells. Treatment with the LPS of P. aeruginosa induced increased production of IL-6 (increase of 100% +/- 8%) and IL-8 (55% +/- 18%) but did not induce the secretion of TNF-alpha. Neither intracellular TNF-alpha nor TNF-alpha transcripts were found in CF-HTGS cells, whereas they were found in normal HTGS cells. In addition, dexamethasone was found to stimulate IL-6 and IL-8 secretion (in the presence or absence of LPS) but did not induce any secretion of TNF-alpha. All these data indicate that HTGS cells are responsive to P. aeruginosa LPS, which results in an increased secretion of IL-6, IL-8, and TNF-alpha, the secretion of which appeared to be impaired in CF-HTGS cells.
Identification of a Fusobacterium nucleatum PK1594 galactose-binding adhesin which mediates coaggregation with periopathogenic bacteria and hemagglutination
Authors: Shaniztki B , Hurwitz D , Smorodinsky N , Ganeshkumar N , Weiss E I
Published: January 2020

Attachment of Fusobacterium nucleatum to various oral surfaces is mediated by several adhesins anchored on its outer surface. Monoclonal antibodies (MAbs) were prepared and used to identify the putative galactose-binding adhesin of F. nucleatum PK1594. Four unique MAbs, 8G7, 26B9, 28G11, and 29D4, were isolated on the basis of their ability to inhibit coaggregation of F. nucleatum PK1594 with Porphyromonas gingivalis PK1924. All four MAbs were also capable of inhibiting galactose-inhibitable interactions of F. nucleatum PK1594 with other oral gram-negative bacteria and with erythrocytes. Preincubation of F. nucleatum PK1594 with MAb 26B9 or its Fab fragments at concentrations lower than 1 microg/ml resulted in complete inhibition of coaggregation with P. gingivalis PK1924 or hemagglutination. F. nucleatum PK1594 surface components prepared by mild sonication or by extracting whole cells with detergents were subjected to Western blot analysis. None of the MAbs were able to recognize any polypeptide in these experiments. Therefore, detergent extracts of F. nucleatum PK1594 surface components were subjected to three experimental procedures: (i) separation by ion-exchange chromatography and testing of fractions for reaction with MAb 26B9 in an enzyme-linked immunosorbent assay (ELISA), (ii) lactose-Sepharose affinity chromatography and testing of the lactose eluate in ELISA with MAb 26B9, and (iii) immunoseparation with either MAb 26B9 or 8G7. Collectively, the results suggest that the putative adhesin is a 30-kDa outer membrane polypeptide which mediates the coaggregation with P. gingivalis PK1924 as well as other galactose-sensitive interactions of F. nucleatum PK1594.
Calcium dependence and binding in cultures of Histoplasma capsulatum
Authors: Batanghari J W , Goldman W E
Published: January 2020

Histoplasma capsulatum is a pathogenic fungus with two distinct morphologies and lifestyles. The saprophytic form of this organism, a mold, thrives in soil and is especially abundant in the Ohio and Mississippi River valleys. Its parasitic counterpart, a yeast, colonizes phagolysosomes of mammalian macrophages. We have observed a major difference in the calcium requirements of the two forms of Histoplasma, potentially implicating the phagolysosome as a calcium-limiting compartment. Deprivation of calcium by the addition of EGTA to culture media inhibited the growth of mycelial H. capsulatum but had no effect on yeast growth in vitro. In addition, yeasts released a calcium-binding protein (CBP) detectable by a 45CaCl2 blotting technique. CBP was a major component of yeast culture supernatant and was also detectable by ruthenium red staining, another assay for calcium-binding activity. Conversely, mycelial H. capsulatum did not produce CBP, a finding that correlates with the dependence of mycelia on calcium for growth. We also describe here the purification of CBP from yeast culture supernatant by reversed-phase high-pressure liquid chromatography.
The transcriptional regulator SoxS is required for resistance of Salmonella typhimurium to paraquat but not for virulence in mice
Authors: Fang F C , Vazquez-Torres A , Xu Y
Published: January 2020

In Escherichia coli, the SoxRS regulon is required for resistance to redox-cycling agents which elevate cytosolic superoxide levels, as well as for resistance to nitric oxide-dependent macrophage killing. In Salmonella typhimurium, SoxS is also required for enhanced expression of Mn-superoxide dismutase and resistance to paraquat, but not for resistance to nitric oxide donor compounds in vitro, resistance to macrophage killing, or virulence in mice. Differences in other antioxidant defense systems or compensation by homologous regulons may account for species-specific differences in the role of SoxS.
A new putative fimbrial colonization factor, CS19, of human enterotoxigenic Escherichia coli
Authors: Grewal H M , Valvatne H , Bhan M K , van Dijk L , Gaastra W , Sommerfelt H
Published: January 2020

A gene probe derived from the colonization factor antigen I (CFA/I) operon cross-hybridized at very low stringency to plasmid DNA from coli surface antigen 17 (CS17)-producing enterotoxigenic Escherichia coli (ETEC) and from the ETEC strain F595C, which was negative for previously described CFAs, CSs, and putative colonization factors (PCFs). A 16-kDa protein was identified in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heat extracts prepared after growth of strain F595C at 37 degrees C on CFA agar containing bile salts. Transmission electron microscopy revealed bile salt- and temperature-dependent expression of fimbriae with a diameter of 7 nm. After transformation with a recombinant plasmid harboring the cfaR gene, which encodes a positive regulator of several CFAs, PCFs, and CSs, the 16-kDa protein was hyperexpressed. Polyclonal antibodies raised against this protein bound to the fimbriae and inhibited the adhesion of F595C bacteria to tissue-cultured Caco-2 cells. Nucleotide sequence determination of the gene encoding the 16-kDa fimbrial subunit revealed a high degree of amino acid sequence homology to the CFA/I, CS1, CS2, CS4, CS14, and CS17 polypeptides. The term CS19 is proposed for the new fimbria.
A 140-kilodalton extracellular protein is essential for the accumulation of Staphylococcus epidermidis strains on surfaces
Authors: Hussain M , Herrmann M , von Eiff C , Perdreau-Remington F , Peters G
Published: January 2020

Two distinct pathogenic mechanisms, adhesion to polymer surfaces and subsequent accumulation of sessile bacterial cells, are considered important pathogenic steps in foreign body infections caused by Staphylococcus epidermidis. By using mitomycin mutagenesis, we have recently generated a mutant, strain M7, from S. epidermidis RP62A which is unaffected in adhesion but deficient in accumulation on glass or polystyrene surfaces and lacks a 115-kDa extracellular protein (designated the 140-kDa antigen; F. Schumacher-Perdreau, C. Heilmann, G. Peters, F. Götz, and G. Pulverer, FEMS Microbiol. Lett. 117:71-78, 1994). To evaluate the role of this protein in accumulation, we harvested extracellular proteins from S. epidermidis RP62A grown on dialysis membranes placed over chemically defined medium, purified the protein by using ion-exchange chromatography, determined its N-terminal amino acid sequence, and raised antiserum in rabbits. The antibody recognized only a single band in a Western immunoblot of the crude extracellular extract. With the microtiter biofilm test, antiserum at a dilution of < or =1:1,000 blocked accumulation of RP62A up to 98% whereas preimmune serum did not. The 140-kDa antigen was found only in extracellular products from bacteria grown under sessile conditions. Of 58 coagulase-negative clinical isolates, 32 strains were 140-kDa antigen positive and produced significantly larger amounts of biofilm than the 26 strains that were 140-kDa antigen negative. The 140-kDa protein appears to be biochemically and functionally unrelated to any previously described factors associated with biofilm formation. Thus, the 140-kDa antigen, referred to as accumulation-associated protein, may be a factor essential in S. epidermidis accumulation and, due to its immunogenicity, may allow the development of novel immunotherapeutic strategies for prevention of foreign body infection.
Oral immunization with PspA elicits protective humoral immunity against Streptococcus pneumoniae infection
Authors: Yamamoto M , McDaniel L S , Kawabata K , Briles D E , Jackson R J , McGhee J R , Kiyono H
Published: January 2020

Streptococcus pneumoniae is a major respiratory mucosal pathogen affecting infants and children. Although a polysaccharide-based vaccine has been useful in adult populations, it does not elicit protective immunity in infants and young children. Pneumococcal surface protein A (PspA) is a highly immunogenic surface protein produced by all strains of Streptococcus pneumoniae. Previous studies have shown that systemic immunization of mice with PspA can elicit protective immunity against fatal pneumococcal infection. In this study, we demonstrated that oral immunization with PspA could elicit protective immune responses against pneumococcal infection. When mice were orally immunized with PspA alone, low levels of PspA-specific immunoglobulin G (IgG) responses were induced in serum; none was induced in secretion. On the other hand, when PspA was given orally with the mucosal adjuvant cholera toxin (CT), significant levels of IgG and IgA anti-PspA responses were induced in serum. The major IgG subclass was IgG1, followed by IgG2b, a profile of antibody response supported by Th2-type cells. In addition, all mice orally immunized with PspA and CT were protected from the lethal challenge with capsular serotype 3 S. pneumoniae A66. These results suggested that an oral PspA vaccine may be a useful means of preventing pneumococcal disease.
Protection against infection in mice vaccinated with a Brucella abortus mutant
Authors: Boschiroli M L , Cravero S L , Arese A I , Campos E , Rossetti O L
Published: January 2020

This study determines whether a genetically engineered mutant of Brucella abortus, strain M-1, possesses differences in protective properties compared to the parental strain, vaccine S19. M-1 is a mutant unable to express BP26, a periplasmic protein with potential use in diagnosis. Mice vaccinated with S19 developed antibodies against BP26, while those vaccinated with M-1 did not. However, mice vaccinated with S19 or M-1 were similarly protected against challenge with pathogenic strain 2308, suggesting that the lack of BP26 does not affect the induction of the protective immune response exerted by S19. These and previous results showing that bacterial invasion and growth or replication in mouse spleens were indistinguishable between strains M-1 and S19 could indicate that the mutant is an attenuated strain which maintains the same protective properties as S19.
Production of monoclonal antibodies to the non-membrane-damaging cytotoxin (NMDCY) purified from Vibrio cholerae O26 and distribution of NMDCY among strains of Vibrio cholerae and other enteric bacteria determined by monoclonal-polyclonal sandwich enzyme-linked immunosorbent assay
Authors: Saha P K , Nair G B
Published: January 2020

The distribution of a newly described secretogenic non-membrane-damaging cytotoxin (NMDCY) among strains of Vibrio cholerae and other enteric bacteria was determined. To accomplish this, monoclonal antibodies against NMDCY were prepared and a sandwich monoclonal-polyclonal enzyme-linked immunosorbent assay (ELISA) was developed. By the sandwich ELISA, it was determined that 55.6% of the 412 strains of V. cholerae examined produced NMDCY at varying concentrations while 76, 37.9, and 15.6% of the clinical strains of Vibrio parahaemolyticus, Aeromonas spp., and Shigella spp., respectively, produced NMDCY. Because of its enterotoxigenic potential and based on its widespread distribution among strains of V. cholerae, we believe that NMDCY may constitute an important virulence determinant in the cascade of events which enable the organism to precipitate the disease.
Development of a murine model of chronic Salmonella infection
Authors: Sukupolvi S , Edelstein A , Rhen M , Normark S J , Pfeifer J D
Published: January 2020

The invasive disease caused by Salmonella typhimurium in mice resembles the acute phase of human typhoid fever caused by Salmonella typhi, and experimental murine salmonellosis is a widely used experimental model for systemic salmonellosis. In this paper we demonstrate that murine S. typhimurium infection can also be used to model the development of the chronic carrier state that develops in humans after infection with S. typhi. We describe a virulent variant of S. typhimurium that has decreased expression of AgfA fibers under all environmental conditions studied and that causes a chronic carrier state in BALB/c mice after peroral inoculation. The chronic carrier state is associated with persistence of bacteria in the small intestine, spleen, and liver, and chronic infection continues despite the development of protective immunity to challenge with virulent Salmonella.