bluuebunny/crossref_metadata_2025 huggingface dataset:
Low levels of vitronectin and clusterin in acute meningococcal disease are closely associated with formation of the terminal-complement complex and the vitronectin-thrombin-antithrombin complex
Authors: Høgåsen K , Mollnes T E , Brandtzaeg P
Published: January 2020

Patients with terminal complement deficiencies and thus impaired lytic efficiency have a highly increased likelihood of contracting invasive meningococcal infections but generally experience a mild disease course. Deficiencies of lysis inhibitors might therefore be associated with severe disease. We have quantified the complement lysis inhibitors vitronectin and clusterin, as well as complexes containing the proteins, in plasma from patients with acute meningococcal disease. At hospital admission, the median vitronectin concentrations were 0.10 (range, 0.04 to 0.17) g/liter in 10 septic patients and 0.19 (0.09 to 0.47) g/liter in 14 nonseptic patients (P = 0.001). The corresponding clusterin concentrations were 0.09 (0.01 to 0.13) and 0.14 (0.06 to 0.29) g/liter (P = 0.005). The vitronectin-thrombin-antithrombin complex concentration was 1.8 (0.22 to 35.6) arbitrary units (AU)/ml in septic patients, but the complex was not detectable in most nonseptic patients (< 0.10 to 0.16 AU/ml) (P < 0.0001). The corresponding levels of the terminal complement complex (contains vitronectin and clusterin) were 4.4 (3.6 to 20.1) and 2.6 (1.6 to 4.7) AU/ml (P = 0.0005). We found no evidence of constitutively low levels of vitronectin or clusterin in patients contracting meningococcal disease. The low levels of the proteins may partly be explained by hemodilution, extravasation, and increased consumption due to incorporation into complexes which are quickly removed from circulation.
Entamoeba histolytica interactions with polarized human intestinal Caco-2 epithelial cells
Authors: Li E , Stenson W F , Kunz-Jenkins C , Swanson P E , Duncan R , Stanley S L
Published: January 2020

To model the initial pathogenic effects of Entamoeba histolytica trophozoites on intestinal epithelial cells, the interactions of E. histolytica HM1-IMSS trophozoites with polarized human intestinal Caco-2 cell monolayers grown on permeabilized filters were examined. Trophozoites, when incubated with the apical surface of the monolayers at 37 degrees C, induced a rapid decrease in transepithelial resistance over 15 to 60 min. The transmonolayer resistance response was not associated with changes in short-circuit current but was associated with an increase in mannitol flux, suggesting that the drop in resistance reflected a nonselective increase in epithelial permeability rather than stimulation of electrogenic ion transport. This response preceded the earliest detection of morphologic disruption of monolayer integrity by light or electron microscopy. Apical injury to the monolayer was detected by ultrastructural studies which revealed a loss of brush border in regions of contact between epithelial cells and amebas and by chromium release assays where a small increase in the apical release of 51Cr from the monolayer (6% over background) was observed. The transmonolayer resistance response was inhibited when the temperature was reduced to 4 degrees C and by addition of cytochalasin D (1 microgram/ml) to the medium at concentrations that did not directly affect transmonolayer resistance. Application of amebic lysates or medium conditioned by coincubation of amebas with Caco-2 monolayers failed to lower transmonolayer resistance, suggesting that this effect was not mediated by soluble amebic cytotoxins. Polarized Caco-2 monolayers grown on permeable filters provide a useful model for studying the initial interactions of E. histolytica trophozoites with intestinal epithelial cells.
Physiological basis of the low calcium response in Yersinia pestis
Authors: Fowler J M , Brubaker R R
Published: January 2020

It is established that duplication in vitro of that amount of Ca2+ (2.5 mM) and Mg2+ (1.5 mM) present in blood permits vegetative growth of Yersinia pestis with repression of virulence factors encoded by the Lcr plasmid (Lcr+); similar simulation of intracellular fluid (no Ca2+ and 20 mM Mg2+) promotes bacteriostasis with induction of these virulence determinants. However, proliferation of yersiniae in mice occurs primarily within necrotic focal lesions (supplied by Ca(2+)-deficient host cell cytoplasm) within visceral organs rather than in Ca(2+)-sufficient blood. The present study addressed this enigma by defining conditions necessary for achieving vegetative growth of Lcr+ yersiniae at 37 degrees C in simulated intracellular fluid. Maximum optical densities were increased by substitution of K+ for Na+ and elimination of Cl-; the combination of Na+ plus L-glutamate was selectively toxic to Lcr+ cells. This phenomenon was attributed in part to the absence of aspartase in Y. pestis (a lesion known to facilitate massive accumulation of L-aspartate via transamination of the oxalacetate pool by L-glutamate). Replacement of L-glutamate by exogenous L-aspartate or alpha-ketoglutarate reversed this toxicity by favoring retention of oxalacetate. Proliferation of Lcr+ cells in a medium containing K+ and L-aspartate but lacking added Ca2+ and Na+ was markedly enhanced by increasing the concentration of fermentable carbohydrate. Accordingly, in the worst-case scenario (i.e., added Na+, Cl-, and L-glutamate), Lcr+ yersiniae underwent restriction of growth after one doubling, and in the best-case scenario (i.e., added K+ and L-aspartate), the organisms completed more than five doublings, thereby achieving full-scale growth. Both of these Ca(2+)-deficient media promoted maximum induction of Mg(2+)-induced V antigen, a virulence factor encoded by the Lcr plasmid.
Pulmonary immunity to Pseudomonas aeruginosa in intestinally immunized rats roles of alveolar macrophages, tumor necrosis factor alpha, and interleukin-1 alpha
Authors: Buret A , Dunkley M L , Pang G , Clancy R L , Cripps A W
Published: January 2020

The aims of this study were to assess the role played by alveolar macrophages, tumor necrosis factor alpha (TNF-alpha), and interleukin-1 alpha (IL-1 alpha) in pulmonary immunity against Pseudomonas aeruginosa in animals that have been immunized via the gut-associated lymphoid tissue. Following intra-Peyer's patch immunization and subsequent intratracheal challenge with live bacteria, significantly enhanced bacterial clearance from the lungs correlated with an increase in bronchoalveolar neutrophils, increased recruitment and phagocytic activity of alveolar macrophages, and accelerated production of TNF-alpha in the bronchoalveolar space, while levels of IL-1 alpha remained low. Administration of recombinant TNF-alpha in physiological concentrations did not affect the proliferation of P. aeruginosa in vitro, but when given intratracheally to rats at the time of infection, recombinant TNF-alpha significantly increased bacterial clearance from the lungs. In these animals, phagocytic activity of bronchoalveolar neutrophils was enhanced, while the recruitment of alveolar macrophages and neutrophils remained unchanged. In acutely infected nonimmune animals, bronchoalveolar concentrations of soluble IL-1 alpha and TNF-alpha increased until the time of death. Levels of prostaglandin E2 and thromboxane B2 were similar in each experimental group. These results indicate that infection in immune animals enhanced both recruitment and phagocytic activity of alveolar macrophages as well as induced an accelerated production of TNF-alpha. In immune challenged animals, this cytokine enhanced the phagocytic activity of neutrophils and improved bacterial clearance from the lung. Levels of soluble IL-1 alpha and TNF-alpha in nonimmune rats increased consistently following infection until the time of death, thus implicating these cytokines in the pathogenesis of acute P. aeruginosa pneumonia.
Mouse interleukin-1 receptor antagonist induced by Actinobacillus actinomycetemcomitans lipopolysaccharide blocks the effects of interleukin-1 on bone resorption and osteoclast-like cell formation
Authors: Nishihara T , Ohsaki Y , Ueda N , Saito N , Mundy G R
Published: January 2020

We have reported that P388D1 cell line murine macrophages stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans release interleukin-1 (IL-1) inhibitor. The IL-1 inhibitor was purified from conditioned media of P388D1 cells stimulated with A. actinomycetemcomitans LPS for 72 h to homogeneity by a four-step procedure: acetic acid extraction from conditioned media; Bio-Gel P-60 gel filtration chromatography; DEAE-Sepharose CL-6B column chromatography; and reverse-phase high-performance liquid chromatography on a C18 hydrophobic support. The purified IL-1 inhibitor gave a single band of protein with a molecular mass of 26 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified IL-1 inhibitor was a heat- and acid-stable protein that was inactivated by digestion with trypsin and reduction with dithiothreitol. This inhibitory factor suppressed the proliferation of C3H/HeJ mouse thymocytes and the proliferation of IL-1-dependent cell lines, D10.G4.1 and RPMI 1788, induced by IL-1. However, this inhibitor did not affect the proliferation of IL-2-dependent CTLL-2 cells induced by IL-2, the proliferation of C3H/HeJ mouse thymocytes stimulated with a mitogenic dose of concanavalin A, and the proliferation of IL-6-dependent B9 cells induced by IL-6. Furthermore, the IL-1 inhibitor significantly blocked stimulation of bone resorption in organ cultures of newborn mouse calvaria and inhibited the osteoclast-like cell formation in mouse marrow cultures. A monoclonal antibody prepared against the purified IL-1 inhibitor reacted with mouse recombinant IL-1 receptor antagonist (rIL-1ra), and a polyclonal antibody to mouse rIL-1ra reacted with the IL-1 inhibitor by Western blot (immunoblot) analysis. These results indicate that the IL-1 inhibitor is an identical molecule to rIL-1ra, suggesting that the IL-1 inhibitor (IL-1ra) released by macrophages stimulated with LPS from A. actinomycetemcomitans may play an important mediative role in the development of periodontal disease.
Septicemia-inducing Escherichia coli O115:K"V165"F165(1) resists killing by porcine polymorphonuclear leukocytes in vitro: role of F165(1) fimbriae and K"V165" O-antigen capsule
Authors: Ngeleka M , Martineau-Doizé B , Fairbrother J M
Published: January 2020

Escherichia coli O115:K"V165":F165(1) wild-type strain 5131 survives in the bloodstream of experimentally inoculated gnotobiotic pigs and induces septicemia, whereas its afimbriate (F165(1)-negative) TnphoA mutant M48 and its acapsular (K"V165"-negative) spontaneous mutant 5131a are both nonpathogenic. We evaluated the role of the mannose-resistant F165(1) fimbrial system and of the O-antigen K"V165" capsule in resistance to phagocytosis by porcine polymorphonuclear leucocytes (PMNLs) in vitro. F165(1)-positive strains (5131 and 5131a) attached to and were ingested by PMNLs at a significantly higher level than afimbrial mutant M48 (P < 0.001) after 1 h of incubation. During incubation of these strains with PMNLs for up to 6 h, parental strain 5131 resisted killing whereas afimbriate mutant M48 and acapsular mutant 5131a were gradually killed and were found at significantly lower numbers than the parental strain 5131 at 2 (P < 0.05) and 6 (P < 0.001) h. When bacteria were opsonized with normal pig serum, the afimbriate and acapsular mutants survived less well than when the bacteria were nonopsonized. Upon examination by electron microscopy of PMNLs after 2 h of incubation with bacteria, structurally normal bacteria were observed more often within phagosomes of PMNLs incubated with the parental strain than within phagosomes of PMNLs incubated with the afimbriate or the acapsular mutant. The extracellular oxidative response (as tested by release of hydrogen peroxide) of PMNLs stimulated by phorbol myristate acetate was completely inhibited by F165(1)-positive strains but only partially inhibited by the afimbriate mutant. These results suggest that the F165(1) fimbrial system may mediate adherence of E. coli O115 to PMNLs. Survival of the parental strain in the presence of PMNLs, which may be intracellular, is at least partially due to the presence of the F165(1) fimbrial system and of the O-antigen capsule K"V165". Furthermore, the presence of the F165(1) fimbrial system may contribute to the bacterial inhibition of the oxidative response of porcine PMNLs.
Serum antibodies from patients with ankylosing spondylitis and Reiter's syndrome are reactive with HLA-B27 cells transfected with the Mycobacterium tuberculosis hsp60 gene
Authors: Kellner H , Wen J , Wang J , Raybourne R B , Williams K M , Yu D T
Published: January 2020

HLA-B27-related arthritis is probably mediated by an immune response against HLA-B27 complexed with peptides derived from proteins of arthritis-causing bacteria. Immunogenic proteins with a high degree of homology among bacteria, such as in the hsp60 family, are likely candidates. To create such complexes experimentally, we transfected an HLA-B27 cell line with the Mycobacterium tuberculosis hsp60 gene. Because of previous observations that HLA-B27-peptide complexes can be distinguished by antibodies, we tested the transfected cell line with a panel of sera from 24 HLA-B27+ arthritis patients. Significant antibodies were detected in at least eight of the sera. Several cell lines and peptides were used as negative controls to ensure that the antibody reactivity was specific to HLA-B27-peptide complexes. A panel of nine peptides derived from the sequence of the Mycobacterium hsp60 were synthesized and tested. At least three were identified as being responsible for the serological activities.
Proteus mirabilis fimbriae: construction of an isogenic pmfA mutant and analysis of virulence in a CBA mouse model of ascending urinary tract infection
Authors: Massad G , Lockatell C V , Johnson D E , Mobley H L
Published: January 2020

Proteus mirabilis, a cause of urinary tract infection and acute pyelonephritis, produces a number of different fimbriae. An isogenic fimbrial mutant of P. mirabilis HI4320 was constructed by marker exchange with delta pmfA::aphA to determine the role of the P. mirabilis fimbriae (PMF) in hemagglutination and in virulence in the CBA mouse model of ascending urinary tract infection. The pmfA mutant, which did not express the 19,500-Da major subunit of PMF, colonized the bladders of transurethrally challenged CBA mice (n = 20 in each group) in numbers 83-fold lower than those of the wild-type strain (mutant, log10 4.87 CFU/g; wild-type strain, log10 6.79 CFU/g; P = 0.023). However, the mutant colonized the kidneys in numbers similar to those of the wild-type strain. Hemagglutination patterns of the mutant ruled out the involvement of PMF in both mannose-resistant, Proteus-like and mannose-resistant, Klebsiella-like hemagglutination. Similarly, PMF does not appear to be involved in adherence to uroepithelial cells (UEC), since the mutant was as adherent as the wild-type strain (mutant, 14.1 +/- 11.7 mean bacteria per UEC, 60% of UEC with > or = 10 bacteria; wild-type strain, 18.1 +/- 16.2 mean bacteria per UEC, 68% of UEC with > or = 10 bacteria; not significantly different). These data suggest a role for PMF in colonization of the bladder but not in colonization of kidney tissue. PMF appear not to be responsible for mannose-resistant, Proteus-like or mannose-resistant, Klebsiella-like hemagglutination.
Analysis of toxinogenic functions associated with the RTX repeat region and monoclonal antibody D12 epitope of Escherichia coli hemolysin
Authors: Rowe G E , Pellett S , Welch R A
Published: January 2020

Amino acids (aa) 550 through 850 of the Escherichia coli hemolysin (HlyA) contain sequences important for several steps in cytolysis. These include the Ca(2+)-binding glycine-rich tandem repeats recognized by the monoclonal antibody A10, the putative HlyC-dependent acylation site that corresponds to the monoclonal antibody D12 epitope, and the erythrocyte specificity domain which confers erythrolytic activity to the Pasteurella haemolytica leukotoxin. To further investigate the toxinogenic functions associated with this region of HlyA, we constructed mutants in the hlyA sequences coding for the repeat region and the D12 epitope. Mutants were analyzed for anti-HlyA antibody reactivity, cytolytic activities, target cell binding, Ca2+ requirements, and virulence. The D12 epitope was mapped to aa 673 through 726, with portions of the epitope both amino terminal and carboxy terminal to aa 700. This region was necessary, but not sufficient, for toxin binding to erythrocytes. A substitution at aa 684 resulted in loss of the D12 epitope, while cytolytic activity was retained. The nature of the D12 epitope and its associated functions are discussed. The A10 epitope mapped to residues 745 through 829, corresponding to repeats 4 through 11. Insertions within the glycine-rich repeats resulted in mutant forms of HlyA which retained A10 reactivity but required increased Ca2+ for lytic activity. These in vitro effects on cytolysis corresponded to a significant decrease in HlyA-mediated virulence in mice. HlyA from one insertion mutant was able to associate with leukocyte membranes under conditions that were Ca2+ deficient for cytolysis. The role of the glycine-rich repeats and Ca2+ in HlyA activity are discussed.
Bacterium-host cell interactions at the cellular level: fluorescent labeling of bacteria and analysis of short-term bacterium-phagocyte interaction by flow cytometry
Authors: Raybourne R B , Bunning V K
Published: January 2020

Flow cytometry is a potentially powerful tool for analyzing the interactions of facultative intracellular bacteria and macrophages on a cellular level, particularly when fluorochromes are used to label the bacteria. We labeled Listeria monocytogenes and Salmonella typhimurium with a lipophilic dye, PKH-2, and used flow cytometry to investigate phagocytosis by J774A.1 cells and short-term bacterial survival. Labeled and unlabeled bacteria were identical in terms of viability, growth kinetics, and survival within macrophages, although recovery per macrophage was much greater for L. monocytogenes than for S. typhimurium. Using L. monocytogenes as a prototypical facultative intracellular bacterium, we estimated bacterial survival during phagocytosis on the basis of linear fluorescence measurements of infected J774A.1 cells and recovery of L. monocytogenes from sorted cells. The lower percentage of surviving L. monocytogenes in macrophages containing higher bacterial loads indicated the accumulation of nonviable bacteria within phagocytes. Removal of the external source of viable bacteria by washes and gentamicin treatment reduced the percentage of surviving intracellular L. monocytogenes to a baseline level, and all baseline levels were similar, regardless of bacterial load. Listeria enrichment recoveries, derived from individually sorted J774A.1 cells, demonstrated the heterogeneity of macrophages in intracellular bacterial survival, especially within heavily infected cells. These results indicated that survival of L. monocytogenes was dependent on the adaptations of a small fraction of bacteria within a population of macrophages which permit intracellular growth.
Heterogeneous surface distribution of the fibrinogen-binding protein on Candida albicans
Authors: Martínez J P , López-Ribot J L , Chaffin W L
Published: January 2020

As detected by indirect immunofluorescence and confocal microscopy, fibrinogen binding was heterogeneously distributed on the surface of Candida albicans. A low level of binding was generally observed homogeneously distributed on some yeast and most hyphal extensions of germ tubes. However, on most hyphal extensions, there were randomly distributed areas of increased expression, as revealed by patches of greater fluorescence intensity.
Molecular cloning of an Onchocerca volvulus extracellular Cu-Zn superoxide dismutase
Authors: James E R , McLean D C , Perler F
Published: January 2020

Onchocerca volvulus, a human parasitic nematode, is the third leading cause of preventable blindness worldwide. This study describes the molecular cloning of a novel superoxide dismutase (SOD) from the parasite. This putative O. volvulus extracellular SOD (OvEcSOD) is 628 nucleotides (nt) long, including a 22-nt 5' spliced leader (SL1) and a portion encoding an N-terminal hydrophobic 42-amino-acid signal peptide. The remainder of the cDNA shares 71% identity with an O. volvulus cytosolic SOD sequence and is 3 nt longer. All residues involved in metal ion binding, active site formation, folding, and dimer formation in SODs are conserved. Data indicate the OvEcSOD and O. volvulus cytosolic SOD are separate gene products and that the OvEcSOD appears to possess the characteristics of a membrane-bound or secreted enzyme which may be involved in the parasite defense against phagocyte-generated reactive oxygen species.
Monoclonal antibodies to Cryptococcus neoformans capsular polysaccharide modify the course of intravenous infection in mice
Authors: Mukherjee S , Lee S , Mukherjee J , Scharff M D , Casadevall A
Published: January 2020

Immunoglobulin G1 (IgG1) monoclonal antibodies (MAbs) to the capsular glucuronoxylomannan (GXM) were studied for their ability to modify the course of intravenous Cryptococcus neoformans infection in mice. A/J mice were given intraperitoneal injection of 1.0 mg of either a GXM-binding IgG1 MAb (2H1 or 2D10 gamma 1) or the irrelevant isotype-matched control MAb 36-65 prior to intravenous infection. Parameters used to study antibody efficacy were lung and brain tissue fungal burden, lung and brain weights, serum GXM levels, and histopathological examination of lung, brain, heart, kidney, and spleen tissues. Mice given GXM-binding MAb had significantly reduced lung tissue fungal burden as measured by CFU. In contrast to the reduction in lung tissue burden, the reduction in brain tissue burden was small and did not achieve statistical significance. Serum GXM levels were reduced in mice receiving GXM-binding MAb. Histopathological examination revealed reduced numbers of granulomas and C. neoformans organisms in the lungs, brains, and kidneys of MAb 2H1-treated mice relative to control mice. The lungs and brains of mice receiving GXM-binding MAb weighed significantly less than those of control animals, consistent with the reduced inflammation noted histologically. Subendocardial inflammation and kidney cortical infarctions were present in control infected mice but not in MAb 2H1-treated mice. Immunocytochemical staining for polysaccharide antigen revealed a marked reduction in the amount of tissue polysaccharide in mice treated with MAb 2H1 relative to control mice. The results support an useful role for passive antibody administration in C. neoformans infections.
Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli
Authors: Berry A M , Lock R A , Thomas S M , Rajan D P , Hansman D , Paton J C
Published: January 2020

A gene bank of Sau3A1-generated Streptococcus pneumoniae type 23 DNA fragments was constructed in Escherichia coli K-12 with the low-copy-number cosmid vector pOU61cos. Clone lysates were screened by immunoblotting using a mouse antiserum raised against a crude pneumococcal hyaluronidase preparation. One immunoreactive clone was isolated, and it produced high level of hyaluronidase activity. This clone contained a recombinant cosmid (designated pJCP800) with an approximately 35-kb DNA insert, and the putative hyaluronidase coding sequence was subcloned into pBluescript SK as a 3.8-kb PstI-ClaI fragment (designated pJCP802). The complete nucleotide sequence of this insert was determined. The region included an open reading frame sufficient to encode a polypeptide with an M(r) of 107,751. An active hyaluronidase with an M(r) of approximately 89,000 was purified to homogeneity from E. coli DH5 alpha(pJCP802). N-terminal amino acid sequence analysis of the purified protein suggested that translation initiation was occurring primarily at a TTG codon within the major open reading frame. However, immunoblot analysis using antiserum raised against the purified 89-kDa hyaluronidase indicated that E. coli DH5 alpha(pJCP802) also expressed the 107-kDa form of the enzyme. This antiserum labelled a 107-kDa protein in partially purified hyaluronidase preparations from S. pneumoniae. The hyaluronidase activity in this pneumococcal extract was also neutralized by the antiserum.
Protection of gerbils from amebic liver abscess by immunization with a recombinant Entamoeba histolytica antigen
Authors: Zhang T , Cieslak P R , Stanley S L
Published: January 2020

Amebiasis, infection by the intestinal protozoan parasite Entamoeba histolytica, is a leading parasitic cause of death. As a step in the development of a recombinant antigen vaccine to prevent E. histolytica infection, we looked at the ability of a recombinant version of the serine-rich E. histolytica protein (SREHP) to elicit a protective immune response against invasive amebic disease. Gerbils, a standard model for amebic liver abscess, were immunized with either a recombinant SREHP/maltose-binding protein (MBP) fusion, recombinant MBP alone, or phosphate-buffered saline (PBS), all combined with complete Freund's adjuvant. In the first trial (group 1), gerbils received a primary and two booster immunizations intraperitoneally; in the second trial (group 2), gerbils were immunized by a single intradermal injection. SREHP/MBP-immunized gerbils in both groups produced antibody to native SHEHP and developed delayed-type hypersensitivity responses to recombinant SREHP. All gerbils were challenged by an intrahepatic injection with 5 x 10(4) virulent E. histolytica HM1-IMSS trophozoites. Complete protection from amebic liver abscess was seen in 64% of the SHEHP/MBP-immunized gerbils in group 1 and in 100% of the SREHP/MBP-immunized gerbils in group 2. There was no protection observed in MBP- or PBS-immunized gerbils in either group. Our results indicate that the SREHP molecule has potential as a vaccine to prevent amebic infection and demonstrate that successful vaccination of animals with recombinant E. histolytica antigen vaccines is possible.
Induction of early-response genes KC and JE by mycobacterial lipoarabinomannans: regulation of KC expression in murine macrophages by Lsh/Ity/Bcg (candidate Nramp)
Authors: Roach T I , Chatterjee D , Blackwell J M
Published: January 2020

The murine chromosome 1 gene Lsh/Ity/Bcg (candidate Nramp) regulates macrophage activation for antimicrobial activity against Salmonella typhimurium, Leishmania donovani, and Mycobacterium spp. To determine early events in the activation pathway, the ability of mycobacterial lipoarabinomannan (LAM) to induce early gene (KC and JE) expression in macrophages from susceptible (S) C57BL/10ScSn (Lshs) and congenic resistant (R) B10.L-Lshr mice was investigated. Stimulation with 1.8 microgram of arabinofuranosyl-terminated LAM (AraLAM) per ml resulted in similar kinetics for KC or JE expression in S and R macrophages. However, whereas JE/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA ratios remained equivalent, R macrophages consistently showed enhanced KC/GAPDH ratios within 30 to 40 min of stimulation compared with S macrophages. Significant differences in KC/GAPDH ratios were observed throughout the peak period (0.5 to 6 h) of the KC response and with doses of AraLAM ranging from 0.01 to 2.5 micrograms/ml. Heavily mannosylated LAM from virulent Mycobacterium tuberculosis Erdman, in doses of up to 2.5 micrograms/ml, failed to stimulate KC or JE in S or R macrophages. Gamma interferon alone (25 U/ml) stimulated equivalent JE expression in S and R macrophages and synergized with AraLAM to enhance JE in both. In contrast, AraLAM-induced KC expression was inhibited in the presence of gamma interferon. Agonist/inhibitor studies were undertaken to determine the signal transduction pathways mediating KC expression. The protein kinase C (PKC) inhibitor Calphostin C (200 nM) inhibited AraLAM-induced KC by 34% +/- 4% in S macrophages and 43% +/- 5% in R macrophages; the cyclic AMP-dependent PKA inhibitor KT5720 (2 microM) inhibited AraLAM-induced KC by 33% +/- 4% (S) and 25% +/- 5% (R). A role for Ca2+ was indicated because ionophore alone stimulated KC expression and synergized with AraLAM to give a dramatically enhanced response. Induction of KC was also inhibited by (i) blocking constitutive nitric oxide (NO) production by preincubation of macrophages with NG-monomethyl-L-arginine (400 microM) (48% +/- 8% [S] and 40% +/- 11% [R]) and (ii) incubation of macrophages with the cyclic GMP-dependent kinase inhibitor KT5823 (4 microM) (65% +/- 4% [S] and 72% +/- 6% [R]). The manner in which these PKC-, PKA-, and Ca(2+)-dependent, NO-mediated cyclic GMP-dependent kinase signal transduction pathways may relate to function of the candidate Lsh/Ity/Bcg gene Nramp is discussed.
Role of human major histocompatibility complex DQ molecules in superantigenicity of streptococcus-derived protein
Authors: Esaki Y , Fukui Y , Sudo T , Yamamoto K , Inamitsu T , Nishimura Y , Hirokawa K , Kimura A , Sasazuki T
Published: January 2020

Antigenicity of peptic extract from type 12 group A streptococci (PEAST12) for T cells was examined in major histocompatibility complex (MHC) class II transgenic mice. PEAST12 was mitogenic for murine T cells when antigen-presenting cells were obtained from human MHC (HLA)-DQ4 alpha beta transgenic mice or from DQ6 alpha beta transgenic mice but was not mitogenic in DR alpha transgenic, DR51 alpha beta transgenic, E alpha transgenic, or nontransgenic mice. In addition, PEAST12 showed mitogenicity for murine T cells in DQ4 alpha singly transgenic mice but not in DQ4 beta singly transgenic mice. T-cell stimulation by PEAST12 was unrestricted by but dependent on the expression of HLA-DQ molecules on antigen-presenting cells, and PEAST12 selectively activated T-cell receptor V beta 11-, V beta 15-, and V beta 18-positive T cells in mice. We propose that PEAST12 contains a superantigen which binds preferentially to the alpha-chain of HLA-DQ molecules. The well-known phenomenon that peptic extracts from group A streptococci are mitogenic in humans but not in mice is likely due to structural differences in MHC class II molecules between these two species of mammals.
Analysis of genes encoding two unique type IIa immunoglobulin G-binding proteins expressed by a single group A streptococcal isolate
Authors: Boyle M D , Hawlitzky J , Raeder R , Podbielski A
Published: January 2020

An emm-like gene (emmL) and a fcrA gene from group A streptococcal strain 64/14 (emmL64/14 and fcrA64/14) were amplified by PCR and force cloned into the heat-inducible expression vector pJLA 602. The emmL gene encoded a recombinant protein that bound human IgG1, IgG2, and IgG4 in a nonimmune fashion. This is the reactivity profile of a type IIa IgG-binding protein. The emmL64/14 gene product was antigenically similar to the previously identified high-molecular-weight type IIa IgG-binding protein of strain 64/14 and had an N-terminal sequence identical to that of the wild-type protein. The fcrA gene also encoded a recombinant protein with type IIa functional activity. This protein was similar to the lower-molecular-weight type IIa IgG-binding protein previously isolated from strain 64/14 and was antigenically distinct from the higher-molecular-weight type IIa protein encoded by the emmL64/14 gene. The sequences for both genes including the intervening regions are presented. The emmL gene demonstrates significant homology to other class I emm and emmL genes expressed by opacity factor-negative group A streptococcal isolates. The fcrA gene was found to be homologous to other fcrA genes normally present in opacity factor-positive group A isolates. The sequence upstream of the fcrA gene and the intervening sequence between the end of the fcrA gene and the start of the emmL gene were similar to those reported for other fcrA genes.
Immunobiological activities of Helicobacter pylori porins
Authors: Tufano M A , Rossano F , Catalanotti P , Liguori G , Capasso C , Ceccarelli M T , Marinelli P
Published: January 2020

Studies were carried out on some biological activities of Helicobacter pylori porins in vitro. We extracted and purified a porin with an apparent molecular mass of 30 kDa. Human polymorphonuclear leukocytes preincubated with H. pylori porins showed a decrease of chemotaxis, of adherence to nylon wool, and of chemiluminescence. Used as chemotaxins in place of zymosan-activated serum or as chemotaxinogens in place of zymosan, the porins induced polymorphonuclear leukocyte migration. Human monocytes and lymphocytes cultivated in the presence of H. pylori porins released cytokines. Release of the various cytokines studied was obtained with differentiated kinetics and at various porin concentrations. Starting only 3 h after culture, tumor necrosis factor alpha is released quickly, reaching a peak at 18 h, at a porin concentration of 1 microgram/ml/10(6) cells. Interleukin-6 (IL-6) appears later, with a peak at 10 micrograms/ml/10(6) cells, while IL-8 is released after 6 h of culture, with a peak at 24 h, at a porin concentration of 10 micrograms/ml/10(6) cells, while IL-8 is released after 6 h of culture, with a peak at 24 h, at a porin concentration of 10 micrograms/ml/10(6) cells. Lymphocytes stimulated by H. pylori porins release gamma interferon after 18 h of culture at higher concentrations of porins (20 micrograms/ml/10(6) cells). Granulocyte macrophage colony-stimulating factor is released from 6 to 48 h at a concentration of 1 microgram/ml/10(6) cells, while both IL-3 and IL-4 are released after 18 h of culture at different porin concentrations (0.1 and 1 microgram/ml/10(6) cells, respectively). Our results lead us to think that during H. pylori infection, surface components, porins in particular, are able to induce a series of chain reactions ranging from the inflammatory to the immunological responses.
Conserved sequence homology of cysteine-rich regions in genes encoding glycoprotein A in Pneumocystis carinii derived from different host species
Authors: Wright T W , Simpson-Haidaris P J , Gigliotti F , Harmsen A G , Haidaris C G
Published: January 2020

Pneumocystis carinii surface glycoprotein A (gpA) exhibits host species-specific phenotypic and genotypic variation. Despite this heterogeneity, the gpAs of P. carinii isolated from different host species appear to be homologous molecules sharing certain biochemical and antigenic characteristics. Using two degenerate oligodeoxyribonucleotide primers corresponding to conserved cysteine regions from ferret and rat P. carinii gpAs, a PCR product of approximately 300 bp was amplified from ferret, rat, and SCID mouse P. carinii-infected lung genomic DNA. Northern (RNA) hybridization revealed a transcript of 3,450 nucleotides in P. carinii-infected SCID mouse lung mRNA, which is similar in size to the transcripts for ferret and rat P. carinii gpAs. Nucleotide sequence analysis of SCID mouse P. carinii gpA subclones derived from the PCR products identified two isoforms, which were 89% identical to each other in the amplified region and 73 and 54% identical to the rat- and ferret-derived P. carinii gpA genes, respectively. Comparison of the deduced amino acid sequences of mouse, ferret, and rat P. carinii gpAs revealed striking similarity in residues adjacent to and including the conserved cysteines. Furthermore, the spacing of two proline residues is invariant, and a potential N-linked glycosylation site is found at a similar position in all of the gpAs. Despite the heterogeneity observed in P. carinii gpAs, the conservation of cysteine residues and adjacent sequences implies similar secondary structure and, most likely, similar function for the gpAs of P. carinii isolated from different host species.
Characterization of the C-terminal domains of intimin-like proteins of enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii, and Hafnia alvei
Authors: Frankel G , Candy D C , Everest P , Dougan G
Published: January 2020

Surface proteins called intimins (Int), which are homologous to the invasin protein (Inv) of Yersinia spp., play a role in inducing brush border damage, termed attachment and effacement, which follows infection by enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii biotype 4280, and Hafnia alvei. Maltose-binding protein (MBP) fusions containing the C-terminal 280 amino acids of Int-like proteins of strains of enteropathogenic E. coli, enterohemorrhagic E. coli, H. alvei, and C. freundii biotype 4280 and of Yersinia pseudotuberculosis Inv were constructed and purified. The 3' end of the gene for the H. alvei Int-like protein was sequenced and showed homology to corresponding regions of other Int-encoding genes. Binding of MBP-Int-like and MBP-Inv fusion proteins to HEp-2 cells was demonstrated by immunofluorescence microscopy and by fluorescence-activated cell sorting. MBP-Inv induced attachment and spreading of HEp-2 cells to plastic-coated wells, but MBP-Int-like fusion proteins did not. Preincubation of HEp-2 cells with MBP-Inv, but not with MBP-Int-like fusion proteins, inhibited MBP-Inv-induced cell attachment. Fixed staphylococci and fluorescent polymer microspheres coated with both MBP-Int-like and MBP-Inv fusion proteins showed enhanced adhesion to HEp-2 cells. These fusion proteins will facilitate studies of the role of intimin in the pathogenesis of diarrhea associated with members of the family Enterobacteriaeceae that induce attachment and effacement.
Immunoglobulin G antibody responses to polyvalent pneumococcal vaccine in children in the highlands of Papua New Guinea
Authors: Pomat W S , Lehmann D , Sanders R C , Lewis D J , Wilson J , Rogers S , Dyke T , Alpers M P
Published: January 2020

The immunoglobulin G (IgG) antibody responses to a pneumococcal polysaccharide vaccine were examined for 480 children aged 3 months to 5 years and living in Tari, Southern Highlands Province, Papua New Guinea. Antipneumococcal IgG to the seven serotypes most frequently causing invasive disease (types 2, 5, 6B, 7F, 14, 19F, and 23F) was measured by an enzyme-linked immunosorbent assay in serum collected before vaccination and 1 and 6 months after vaccination. Prevaccination antibody levels fell rapidly after 3 months of age and remained low throughout the first 2 years of life. One month after vaccination, geometric mean titers of antipneumococcal IgG to serotypes 2, 7F, 23F, and 5 were at least twice those of antibodies in nonvaccinated children of the same age from the ages of 5, 6, 9, and 12 months onwards, respectively; postvaccination antibody responses to serotypes 6B, 14, and 19F rose gradually during the second year of life. Elevated antibody titers to serotypes 2 and 7F were maintained 6 months after vaccination. Thus, young Papua New Guinean children are capable of mounting a good immune response to some pneumococcal capsular polysaccharides from a young age, and the antibody responses to capsular polysaccharides are consistent with studies in developed countries. However, in Papua New Guinea, the serogroup distribution of invasive disease matches the immunogenic components of the pneumococcal polysaccharide vaccine more closely than in developed countries, a fact which helps to explain the results of controlled trials in Papua New Guinea, in which this vaccine prevented death and severe morbidity from pneumonia in young children.
Protection of C3H/HeN mice from challenge with Borrelia burgdorferi through active immunization with OspA, OspB, or OspC, but not with OspD or the 83-kilodalton antigen
Authors: Probert W S , LeFebvre R B
Published: January 2020

Recent advances in the development of animal models for Lyme borreliosis have provided means of identifying potential targets for the design of a subunit vaccine to prevent this disease. The C3H/HeN mouse model was used to study several Borrelia burgdorferi antigens from a single isolate for their ability to elicit borreliacidal and protective antibodies. The ospA, ospB, ospC, ospD, and 83-kDa genes from a California isolate, SON 188, were cloned and expressed in Escherichia coli as proteins fused to the C-terminal end of maltose-binding protein. Active immunization of mice with these fusion proteins elicited high titers of antibodies that recognized the homologous SON 188 antigens upon immunoblotting. Antibodies generated to the OspA and OspB fusion proteins, but not to the OspC, OspD, and the 83-kDa fusion proteins, demonstrated in vitro borreliacidal activity. Challenge of all actively immunized mice with 10(7) SON 188 spirochetes resulted in infection in all mice receiving the OspD or 83-kDa immunogens but not in any mice receiving the OspA, OspB, or OspC fusion proteins. These results demonstrate the potential of OspA, OspB, and OspC as components of a subunit vaccine for the prevention of Lyme borreliosis.
Mitogen- and antigen-specific proliferation of T cells in murine toxoplasmosis is inhibited by reactive nitrogen intermediates
Authors: Candolfi E , Hunter C A , Remington J S
Published: January 2020

Acute and chronic infections with Toxoplasma gondii result in a nonspecific suppression of immunologic function in mice and humans. Proliferation of spleen cells in response to concanavalin A (ConA) and toxoplasma lysate antigen (TLA) was studied during the course of infection in mice susceptible (CBA/Ca) and resistant (BALB/c) to development of toxoplasmic encephalitis to determine if reactive nitrogen intermediates (RNI) are involved in the suppression of the proliferative responses. Maximal suppression of proliferation of spleen cells in response to ConA and TLA was observed on days 7 and 14 after infection and correlated with elevated levels of nitrite in spleen cell culture supernatants. By day 68 postinfection in BALB/c mice, proliferative responses returned to normal levels, whereas in CBA/Ca mice, they remained suppressed. The addition of an inhibitor of production of RNI (NG-monomethyl-L-arginine) increased proliferation of spleen cells in response to both ConA and TLA at days 7, 14, and 21 after infection. Depletion of adherent cells from spleen cell preparations obtained from acutely infected mice followed by their repletion with adherent spleen cells from uninfected mice resulted in increased proliferation of spleen cells from infected mice and a significant decrease in nitrite in the cultures. These results indicate that production of RNI by macrophages contributes significantly to the suppression of the spleen cell proliferation observed in the acute stage of toxoplasmosis.
Mouse sialoadhesin is not responsible for Candida albicans yeast cell binding to splenic marginal zone macrophages
Authors: Han Y , Kelm S , Riesselman M H , Crocker P R , Cutler J E
Published: January 2020

Candida albicans hydrophilic yeast cells specifically adhere to mouse macrophages in the splenic marginal zone and in lymph node subcapsular and medullary sinuses. These macrophages express sialoadhesin that binds erythrocytes, but binding of yeast cells is not mediated by sialoadhesin because (i) erythrocytes did not block yeast cell binding, (ii) yeast cell adherence was unaffected by sialoadhesin-specific monoclonal antibodies, (iii) the purified sialoadhesin did not bind to yeast cells, and (iv) an Fc-sialoadhesin chimera immobilized on plastic did not bind yeast cells. These data give further support for a unique macrophage adhesion system that binds C. albicans.
Inhibition of nitric oxide synthesis leads to reduced parasitemia in murine Trypanosoma brucei infection
Authors: Sternberg J , Mabbott N , Sutherland I , Liew F Y
Published: January 2020

In murine Trypanosoma brucei infection, macrophage activation and nitric oxide (NO) production lead to suppressed splenic T-cell responses (J. Sternberg and F. McGuigan, Eur. J. Immunol. 22:2741-2744, 1992). In this study, evidence is presented that NO has no detectable trypanocidal activity under simulated in vivo conditions and that inhibition of NO production in vivo results in reduced parasitemia.
Intracellular expression of toxic shock syndrome toxin 1 in Saccharomyces cerevisiae
Authors: Deresiewicz R L , Flaxenburg J A , Chan M , Finberg R W , Kasper D L
Published: January 2020

In order to search for an occult cytotoxic enzymatic activity of the toxic shock syndrome toxin 1 (TSST-1), we placed the gene encoding TSST-1 (tstH) under the control of an inducible promoter in the eukaryotic yeast Saccharomyces cerevisiae. Under similar circumstances, the known bacterial enzymatic cytotoxins Shiga-like toxin and diphtheria toxin are both highly lethal to the yeast host. Although full-length stable TSST-1 was demonstrated within the yeast cells and although it retained mitogenicity for human T cells, it had no apparent effect on the yeast cells' growth kinetics or on their gross morphology. Retrieval and sequencing of the toxin gene revealed the wild-type sequence throughout, thus demonstrating that the apparent lack of toxicity for the yeast cells was not due to a serendipitous attenuating mutation within the coding region of the toxin gene. Similar results obtained after a second transformation of the same strain and after transformation of an unrelated strain demonstrate that neither chance permissive host mutation nor intrinsic host resistance was likely to have obscured an existing cytotoxic property of TSST-1. We conclude that TSST-1 probably does not possess a discrete enzymatic property cytotoxic for eukaryotic cells.
Comparison of nonhuman primate antibodies against Haemophilus influenzae type b polysaccharide with human antibodies in oligoclonality and in vivo protective potency
Authors: Kim K H , Park M K , Peeters C C , Poolman J T , Shearer M H , Kennedy R C , Nahm M H
Published: January 2020

Nonhuman primates are often used as a model for studying vaccines for humans. However, it is not always clear how closely the antibody responses in these species mimic human responses. Recent studies have characterized the human antibody response to Haemophilus influenzae type b (Hib) in great detail. In this study, we have compared the antibody response to Hib of humans with those of other primates. Studies of isoelectric points and V kappa subgroup usage show that, like humans, nonhuman primates produce oligoclonal antibodies. Also, monkey antibodies to the Hib polysaccharide are as protective as human antibodies in an in vivo model of Hib infection. Thus, we conclude that nonhuman primates produce antibodies to Hib polysaccharide that are structurally and functionally similar to human antibodies and are a good model for testing human vaccines.
Phase variation in pneumococcal opacity: relationship between colonial morphology and nasopharyngeal colonization
Authors: Weiser J N , Austrian R , Sreenivasan P K , Masure H R
Published: January 2020

When colonies of encapsulated isolates of Streptococcus pneumoniae are viewed with oblique, transmitted light on a transparent surface, they are heterogeneous in appearance because of variation in opacity. There is spontaneous phase variation among at least three discernible phenotypes at frequencies from 10(-3) to 10(-6). The ability to detect differences in opacity varies according to serotype, but variation is independent of capsule expression. Electron microscopy shows no difference in chain length but suggests that autolysis occurs earlier in the growth of the transparent variant. There was no identifiable difference in membrane protein profiles of opaque and transparent variants of the same strain. In an infant rat model of nasopharyngeal carriage, there was no significant colonization by opaque variants. Efficient and stable colonization by the transparent variants was observed, suggesting a selective advantage for this phenotype in the nasopharynx. In contrast, there was no difference in the incidence of bacteremia or in the 50% lethal dose among the variants following their intraperitoneal inoculation. These results suggest that phase variation which is marked by differences in colonial morphology may provide insight into the interaction of the pneumococcus with its host.