bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancing effect of cholera toxin on interleukin-6 secretion by IEC-6 intestinal epithelial cells: mode of action and augmenting effect of inflammatory cytokines
Authors: McGee D W , Elson C O , McGhee J R
Published: January 2020

Oral administration of cholera toxin (CT) induces a strong mucosal immune response to CT as well as having a potent adjuvant effect. Since one of the first cell types to encounter CT during cholera infection or after oral administration is the epithelial cell, we studied the effect of CT on interleukin-6 (IL-6) secretion by the rat intestinal epithelial cell line IEC-6. CT was found to rapidly enhance IL-6 secretion and IL-6 gene expression by these cells. The addition of dibutyryl cyclic AMP (cAMP) to cultures of IEC-6 cells had little effect on IL-6 secretion, yet mRNA levels were elevated, suggesting that the response may have been regulated by cAMP. Purified B subunit of CT did not significantly enhance IL-6 secretion or mRNA expression. CT and transforming growth factor beta 1 synergistically enhanced IL-6 secretion in IEC-6 cells. The addition of CT with either IL-1 beta or tumor necrosis factor alpha gave even greater synergistic enhancement of IL-6 secretion, and dibutyryl cAMP could mimic CT's synergy with IL-1 beta. These results indicate that the intestinal epithelial cell is capable of secreting high levels of IL-6 after encountering CT, especially in the presence of inflammatory cytokines. This high level of IL-6 secretion could be a very important component of the mucosal immune response to CT and may also account for a portion of the adjuvant effect of CT.
The eae gene of Citrobacter freundii biotype 4280 is necessary for colonization in transmissible murine colonic hyperplasia
Authors: Schauer D B , Falkow S
Published: January 2020

Transmissible murine colonic hyperplasia is characterized by proliferation of anchored stem cells in the mucosa of the descending colon of laboratory mice and is caused by Citrobacter freundii biotype 4280. This bacterium produces attaching and effacing lesions in the descending colon prior to the onset of gross hyperplasia. By mutational analysis, the chromosomal eae gene of C. freundii biotype 4280 was shown to be necessary for colonic colonization. Conversely, bacteria cured of a 65-kb plasmid, which was identified in C. freundii biotype 4280, were not attenuated for colonic colonization or for the induction of colonic hyperplasia.
Molecular characterization of the 98-kilodalton iron-regulated outer membrane protein of Neisseria meningitidis
Authors: Pettersson A , van der Ley P , Poolman J T , Tommassen J
Published: January 2020

When grown under iron limitation, Neisseria meningitidis expresses several additional outer membrane proteins (OMPs), which were studied to assess their vaccine potential. Two monoclonal antibodies were obtained against a 98-kDa OMP of strain 2996 (B:2b:P1.2). Cross-reactivity studies revealed that the two antibodies reacted with 44 and 42 of 74 meningococcal strains, respectively. The antibodies did not block the binding of transferrin or lactoferrin to intact cells. The structural gene for the protein, tentatively designated iroA, was isolated and sequenced. Computer analysis revealed homology to the ferric siderophore receptors in the outer membrane of Escherichia coli and to gonococcal transferrin-binding protein 1 (TbpA). The high degree of cross-reactivity and the results of Southern blot analyses, which showed that the iroA gene is also present in strains that did not react with the monoclonal antibodies, suggest that the 98-kDa OMP is well conserved among meningococci and that it is a suitable vaccine candidate. However, the antibodies were not bactericidal in an in vitro assay with human complement.
An 88-kilodalton antigen secreted by Aspergillus fumigatus
Authors: Kobayashi H , Debeaupuis J P , Bouchara J P , Latge J P
Published: January 2020

An 88-kDa component secreted in vitro by Aspergillus fumigatus has been purified by sequential chromatographic procedures. The molecule is a glycoprotein with an N-linked sugar moiety composed of mannose glucose, and galactose (16:10:1). It is recognized by antibodies from patients with aspergilloma and has potential for the immunodiagnosis of aspergilloma. The antigenicity is associated with the polypeptide part of the molecule (79 kDa).
Regulation of mycobacterial growth by the hypothalamus-pituitary-adrenal axis: differential responses of Mycobacterium bovis BCG-resistant and -susceptible mice
Authors: Brown D H , Sheridan J , Pearl D , Zwilling B S
Published: January 2020

The role of the hypothalamus-pituitary-adrenal (HPA) axis in regulating the growth of Mycobacterium avium in Mycobacterium bovis BCG-resistant and -susceptible congenic mice was evaluated. Restraint was used to activate the HPA axis, which resulted in an increase in the level of corticosterone in the plasma. Activation of the HPA axis increased the susceptibility of BALB/c.Bcgs mice to the growth of M. avium. In contrast, the growth of M. avium was not altered in BALB/c.Bcgr mice as a result of HPA activation. Adrenalectomy abolished the effect of HPA activation on mycobacterial growth, as did treatment of the mice with a glucocorticoid receptor antagonist, RU 486. Activation of the HPA axis also resulted in the increased susceptibility of splenic macrophages from Bcgs mice but not from Bcgr mice to M. avium growth in vitro. The production of tumor necrosis factor alpha and of reactive nitrogen intermediates by splenic macrophages from both strains of mice was suppressed as a result of HPA activation. The implications of these findings for resistance controlled by Bcg and for susceptibility to mycobacterial growth are discussed.
Association of Actinobacillus actinomycetemcomitans leukotoxin with nucleic acids on the bacterial cell surface
Authors: Ohta H , Hara H , Fukui K , Kurihara H , Murayama Y , Kato K
Published: January 2020

Actinobacillus actinomycetemcomitans, a periodontopathic gram-negative bacterium, produces a leukotoxin that is a member of the RTX cytotoxin family. Although genes may function in toxin secretion, the leukotoxin is not secreted extracellularly but remains associated with the bacterial cell surface. We report here that this toxin-cell surface association is mediated by nucleic acids and directly demonstrate that the extracellular secretion of toxin occurs in growing cultures with increased ionic strength of medium. All examinations were performed with freshly harvested A. actinomycetemcomitans 301-b from anaerobic fructose-limited chemostat cultures. The occurrence of cell surface-localized DNA was shown by directly digesting whole cells with the restriction endonuclease EcoRI or HindIII, which yielded many DNA fragments. The cell surface DNA constituted about 20% of the total cellular DNA. The leukotoxin was released from the whole cells by digestion with DNase I as well as restriction endonucleases. Because the leukotoxin binds ionically to DNA, it is dependent on the ionic strength of buffers or media. Accordingly, the toxin was released from cells suspended in saline at pH 7.5 in the presence of increasing amounts of MgCl2 (0 to 10 mM) or NaCl (0 to 50 mM). Moreover, a considerable quantity of leukotoxin was detected in the culture supernatant of fructose-limited chemostat cultures when sodium succinate solution was pumped into the steady state as an additional salt (30 and then 50 mM). This toxin-DNA association was also found in well-characterized strains including not only the leukotoxin-producing ATCC 29522 but also the toxin production-variable ATCC 29523 and the non-leukotoxin-producing ATCC 33384 when these strains were grown in the chemostat culture.
Roles of leukotriene B4, prostaglandin E2, and cyclic AMP in Campylobacter jejuni-induced intestinal fluid secretion
Authors: Everest P H , Cole A T , Hawkey C J , Knutton S , Goossens H , Butzler J P , Ketley J M , Williams P H
Published: January 2020

Infection of rabbit ileal loops with inflammatory Campylobacter jejuni strains caused elevation of cyclic AMP, prostaglandin E2, and leukotriene B4 levels in tissue and fluids. Incubation of cultured Caco-2 cells with loop fluids caused elevated cellular cyclic AMP levels, an effect which was inhibited by antiserum against prostaglandin E2.
Interleukin 10 protects mice against staphylococcal enterotoxin B-induced lethal shock
Authors: Bean A G , Freiberg R A , Andrade S , Menon S , Zlotnik A
Published: January 2020

We investigated the ability of interleukin 10 (IL-10) to protect mice against lethal shock induced by staphylococcal enterotoxin B (SEB). Treatment of mice with IL-10 prevented the death of mice injected with SEB in a dose-dependent manner. IL-10-mediated protection was apparent when administered either prior to or concurrent with SEB but was less effective when administered following SEB injection. This observation indicates that IL-10 is capable of regulating T-cell activation in vivo.
Characterization of an avirulent pleiotropic mutant of the insect pathogen Bacillus thuringiensis: reduced expression of flagellin and phospholipases
Authors: Zhang M Y , Lövgren A , Low M G , Landén R
Published: January 2020

An avirulent pleiotropic mutant of the insect pathogen Bacillus thuringiensis subsp. gelechiae, isolated by Heierson et al. (A. Heierson, I. Sidén, A. Kivaisi, and H. G. Boman, J. Bacteriol. 167:18-24, 1986) as a spontaneous phage-resistant mutant, was further characterized and found to lack the expression of phosphatidylcholine- and phosphatidylinositol-degrading phospholipase C, beta-lactamase, and flagellin because of the absence of corresponding mRNAs. The avirulent mutant was also found to be less efficient in killing insect cells in vitro than the wild type and to have altered behavior in vivo; wild-type B. thuringiensis does not circulate in the insect hemolymph after injection, whereas the avirulent mutant and nonpathogenic control bacteria remain in circulation. Flagella and motility may be important for virulence in the early stages of an infection; mutants with decreased motility appear less virulent when fed to Trichoplusia ni but not when injected. The 50% lethal doses of wild-type strain Bt13 and avirulent mutant strain Bt1302 were estimated to be 0.52 +/- 0.25 and 2,600 +/- 1,300 CFU per injected larva, respectively.
Evidence for asialo GM1 as a corneal glycolipid receptor for Pseudomonas aeruginosa adhesion
Authors: Hazlett L D , Masinick S , Barrett R , Rosol K
Published: January 2020

Anti-gangliotetraosylceramide (anti-asialo GM1) and antiparagloboside monoclonal antibodies (MAbs) were used in immunofluorescence, immunoelectron-microscopic, and in vitro binding inhibition assays to determine whether either of the glycolipids was detectable in the normal cornea, whether levels changed following corneal scarification and either trypsin treatment or incubation in vitro with Pseudomonas aeruginosa, and whether either of the MAbs could competitively inhibit P. aeruginosa binding to cornea. No immunostaining above background for either glycolipid was observed in frozen, unfixed sections or in lightly fixed, K4M-embedded antibody-gold-labeled thin sections of normal cornea. In frozen sections of organ-cultured scarified cornea, no increased immunostaining for anti-asialo GM1 or antiparagloboside reactivity was noted immediately or 60 min after corneal scarification. However, at 60 min after scarification and in vitro incubation of the eye with either trypsin or P. aeruginosa, enhanced immunostaining for both glycolipids was associated with cells within or immediately adjacent to the wound site. Trypsin increased immunoreactivity in the wound site more markedly compared with incubation with P. aeruginosa, but immunostaining was similarly localized with either treatment. No staining above background was seen in control sections. Similarly, with immunoelectron microscopy, increased immunogold-MAb staining for both glycolipids was seen on the plasma membranes of the wound-site cells of eyes incubated with either trypsin or P. aeruginosa compared with controls that were similarly immunostained but with the primary antibody either omitted or substituted with a nonspecific MAb. Competitive binding inhibition assays, in which the bacterial inoculum or the eye in organ culture was incubated with anti-asialo GM1 MAb prior to topical ocular application of the bacteria, showed significantly decreased P. aeruginosa adhesion compared with preparations similarly treated with phosphate-buffered saline or antiparagloboside MAb. These data provide evidence to support the hypothesis that asialo GM1, not paragloboside, serves as a receptor for P. aeruginosa binding to the scarified cornea of the adult mouse and spatially localizes both glycolipids in the wound site.
Expression of the Salmonella virulence plasmid gene spvB in cultured macrophages and nonphagocytic cells
Authors: Fierer J , Eckmann L , Fang F , Pfeifer C , Finlay B B , Guiney D
Published: January 2020

Certain serotypes of salmonellae carry virulence plasmids that greatly enhance the pathogenicity of these bacteria in experimentally infected mice. This phenotype is largely attributable to the 8-kb spv regulon. However, spv genes are not expressed while bacteria grow in vitro. We now show that spvB, which is required for virulence, is expressed rapidly after Salmonella dublin is ingested by cultured J774 and murine peritoneal macrophages and that expression is not affected by the alkalinization of intracellular vesicles. The level of induction of spvB is reduced when macrophages are pretreated with gamma interferon. spvB is also expressed in human and canine epithelial cell lines and a human hepatoma cell line. In all cases, spvB expression is dependent on the spvR gene, just as it is in stationary-phase cultures in vitro. These data suggest that spv virulence genes are expressed by intracellular salmonellae in vivo in response to a signal that is common to the intracellular compartments of cells that are invaded by salmonellae.
Effect of polysaccharide capsule and methods of preparation on human lymphocyte proliferation in response to Cryptococcus neoformans
Authors: Mody C H , Syme R M
Published: January 2020

Cryptococcus neoformans is a pathogenic encapsulated yeast that infects patients that have defective cell-mediated immunity, including AIDS patients. Whole cryptococcal organisms that are killed by heating stimulate normal human lymphocytes to proliferate. However, strains of C. neoformans vary widely in virulence and therefore in their ability to cause disease in humans. To determine the effect of virulence factors such as the cryptococcal capsule, serotype, and the state of the organisms on the lymphocyte response to C. neoformans, human peripheral blood mononuclear cells were stimulated with C. neoformans in vitro and lymphocyte proliferation was determined. The major determinant of the lymphocyte response to C. neoformans was the amount of polysaccharide present. The response was greater after stimulation by minimally encapsulated strains (strains C3D, 68, and 613) than by heavily encapsulated strains (strains 6 and 145). A heavily encapsulated strain (strain 6) did not suppress the response to an acapsular mutant (strain 67). However, the response to an acapsular strain was suppressed by the addition of purified polysaccharide. Human lymphocytes responded to both serotypes of C. neoformans var. neoformans. The antigen responsible for lymphocyte stimulation was preserved despite various techniques of inactivation, including heat, paraformaldehyde fixation, irradiation, and mechanical disruption. Finally, lymphocytes responded equally to live and killed organisms. These results suggest that capsular polysaccharide, a known virulence factor, may suppress the human lymphocyte response to C. neoformans during an infection. Lymphocytes could respond to C. neoformans regardless of the viability of the organism, and they could also respond to disrupted organisms. We speculate that lymphocyte proliferation in vitro could be related to the protective immune response in host defense to C. neoformans and that it is suppressed by virulence factors of C. neoformans.
Stress induction of the virulence proteins (SpvA, -B, and -C) from native plasmid pSDL2 of Salmonella dublin
Authors: Valone S E , Chikami G K , Miller V L
Published: January 2020

The virulence region of the wild-type plasmid pSDL2 contained in Salmonella dublin is highly conserved among plasmids from several nontyphoid Salmonella serotypes and is essential for the development of systemic infection in BALB/c mice. Polyclonal antibodies against three proteins (SpvA, -B, and -C) expressed from a 4.1-kb EcoRI subclone of the plasmid virulence region were generated. These antibodies were used to detect expression of the Spv proteins when S. dublin was grown in vitro under stress-inducing conditions, such as nutrient deprivation and increased temperature, that the bacteria may encounter during the course of infection within the host. Glucose starvation resulted in expression of all three proteins shortly after the lag phase. When the bacteria were grown to the late-log phase without glucose, heat shock strongly induced expression of SpvA but not SpvB or SpvC. The addition of 0.2% glucose to the medium resulted in loss of expression of the proteins until the late-log to stationary phase. Iron limitation or lowered pH induced expression of the proteins during exponential growth even in the presence of glucose. Insertion mutations into the positive regulator gene spvR upstream from spvABC and insertions into spvA and spvC resulted in loss of expression of SpvA, -B, and -C, suggesting a complex regulation of expression. These studies define a variety of environmental conditions that induce expression of the Spv virulence proteins from the wild-type plasmid pSDL2 in S. dublin in vitro.
Transfer of protective immunity in murine histoplasmosis by a CD4+ T-cell clone
Authors: Allendoerfer R , Magee D M , Deepe G S , Graybill J R
Published: January 2020

We have reported that a murine Histoplasma capsulatum-reactive CD4+ T-cell line and clones thereof did not adoptively transfer protection against H. capsulatum infection in normal or cyclophosphamide-treated C57BL/6 mice. One explanation for the results was that the T cells failed to traffic to lymphoid organs in these animals. In this study, we have sought to determine whether one of these clones, 2.3H3, could mediate protection in nude (C57BL/10) or irradiated (5 Gy) heterozygous nude (nu/+) C57BL/6 mice. Mice were inoculated intravenously with 10(7) resting 2.3H3 cells or with an equal number of cells of the ovalbumin-reactive clone 1S6; 2 h later, the mice were challenged intranasally with 5 x 10(6) yeast cells. By day 5 of infection, lungs, livers, and spleens of nude and irradiated nu/+ mice given 2.3H3 contained significantly fewer (P < 0.05) CFU than the same organs from mice inoculated with 1S6. This effect was specific for H. capsulatum, since 2.3H3 did not reduce the number of Coccidioides immitis CFU in lungs, livers, and spleens of irradiated nu/+ mice. By day 10, the amounts of H. capsulatum CFU in lungs, livers, or spleens of nude and irradiated nu/+ mice inoculated with 2.3H3 were smaller than those in 1S6-inoculated mice, but these differences did not reach statistical significance (P > 0.05). The mortality rate of mice inoculated with 2.3H3 and that of mice inoculated with 1S6 were similar. Histopathological examination of tissues from 2.3H3- and 1S6-inoculated mice demonstrated the presence of granulomatous inflammation in organs from both groups. Tissues from 2.3H3-treated mice contained fewer yeasts per high-power field than tissues from 1S6-treated mice. Thus, irradiated or nude mice are permissive for the expression of protective immunity by a CD4+ T-cell clone. Although the protective capacity of T cells in these animals is transient, these animals will be useful for differentiating protective from nonprotective T-cell clones.
Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment
Authors: Yamauchi K , Tomita M , Giehl T J , Ellison R T
Published: January 2020

Although the antimicrobial activity of lactoferrin has been well described, its mechanism of action has been poorly characterized. Recent work has indicated that in addition to binding iron, human lactoferrin damages the outer membrane of gram-negative bacteria. In this study, we determined whether bovine lactoferrin and a pepsin-derived bovine lactoferrin peptide (lactoferricin) fragment have similar activities. We found that both 20 microM bovine lactoferrin and 20 microM lactoferricin release intrinsically labeled [3H]lipopolysaccharide ([3H]LPS) from three bacterial strains, Escherichia coli CL99 1-2, Salmonella typhimurium SL696, and Salmonella montevideo SL5222. Under most conditions, more LPS is released by the peptide fragment than by whole bovine lactoferrin. In the presence of either lactoferrin or lactoferricin there is increased killing of E. coli CL99 1-2 by lysozyme. Like human lactoferrin, bovine lactoferrin and lactoferricin have the ability to bind to free intrinsically labeled [3H]LPS molecules. In addition to these effects, whereas bovine lactoferrin was at most bacteriostatic, lactoferricin demonstrated consistent bactericidal activity against gram-negative bacteria. This bactericidal effect is modulated by the cations Ca2+, Mg2+, and Fe3+ but is independent of the osmolarity of the medium. Transmission electron microscopy of bacterial cells exposed to lactoferricin show the immediate development of electron-dense "membrane blisters." These experiments offer evidence that bovine lactoferrin and lactoferricin damage the outer membrane of gram-negative bacteria. Moreover, the peptide fragment lactoferricin has direct bactericidal activity. As lactoferrin is exposed to proteolytic factors in vivo which could cleave the lactoferricin fragment, the effects of this peptide are of both mechanistic and physiologic relevance.
Assessment of enterotoxin production by Yersinia enterocolitica and identification of a novel heat-stable enterotoxin produced by a noninvasive Y. enterocolitica strain isolated from clinical material
Authors: Robins-Browne R M , Takeda T , Fasano A , Bordun A M , Dohi S , Kasuga H , Fang G , Prado V , Guerrant R L , Fong G
Published: January 2020

Twenty-eight clinical isolates of Yersinia enterocolitica were investigated for their abilities to produce heat-stable enterotoxin (YST). All 21 invasive strains (serogroup O3 biotype 4) carried the previously described gene for YST (yst), with toxin detectable in culture supernatants from 20 strains. One of seven noninvasive, biotype 1A strains also had enterotoxic activity, despite failure to hybridize with a probe for yst. The toxin produced by this noninvasive (serogroup O6) strain resembled YST in terms of molecular size, heat stability, and solubility in methanol. It differed from YST, however, with respect to regulation of its production by temperature and its mechanism of action, which did not appear to involve cyclic GMP.
Probable presence of beta(1-2)-linked oligomannosides that act as human immunoglobulin G3 epitopes and are distributed over a Candida albicans 14- to 18-kilodalton antigen
Authors: Poulain D , Faille C , Delaunoy C , Jacquinot P M , Trinel P A , Camus D
Published: January 2020

Kinetic analysis of candidosis patients' immunoglobulin G3 response has shown that reactivity towards beta(1-2)-linked mannan-derived oligomannosides was associated with the recognition through metaperiodate-sensitive epitopes of a 14- to 18-kDa Candida albicans antigen unreactive with concanavalin A.
Proteus mirabilis fimbriae: N-terminal amino acid sequence of a major fimbrial subunit and nucleotide sequences of the genes from two strains
Authors: Bahrani F K , Cook S , Hull R A , Massad G , Mobley H L
Published: January 2020

Proteus mirabilis, a common cause of urinary tract infection in hospitalized and catheterized patients, produces mannose-resistant/klebsiella-like (MR/K) and mannose-resistant/proteus-like (MR/P) hemagglutinins. The gene encoding the major structural subunit of a fimbria, possibly MR/K, was identified in two strains. A degenerate oligonucleotide probe based on the N terminus of the Proteus uroepithelial cell adhesin and antiserum raised against the denatured polypeptide were used to screen a cosmid gene bank of strain HU1069. A cosmid clone that reacted with the probe and antiserum was identified, and a fimbria-like open reading frame was determined by nucleotide sequencing. The predicted N-terminal amino acid sequence of the processed polypeptide, ENETPAPKVSSTKGEIQLKG (residues 23 to 42), did not match the uroepithelial cell adhesin N terminus but, rather, matched exactly the N-terminal amino acid sequence of a polypeptide with an apparent molecular size of 19.5 kDa isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a fimbrial preparation from strain HI4320 expressing MR/K hemagglutinin. By using an oligonucleotide from the HU1069 open reading frame, the fimbrial gene was isolated and sequenced from a cosmid gene bank clone of strain HI4320. A 552-bp open reading frame predicts a 184-amino-acid polypeptide including a 22-amino-acid hydrophobic leader sequence. The unprocessed polypeptide is predicted to be 18,921 Da; the processed polypeptide is predicted to be 16,749 Da. The predicted amino acid sequence of the polypeptide encoded by the gene, designated pmfA, displayed 36% exact matches with the mannose-resistant fimbrial subunit encoded by smfA of Serratia marcescens but only 15% exact matches with the predicted sequence encoded by mrkA of Klebsiella pneumoniae.
Phagocytosis of Staphylococcus aureus induces a selective stress response in human monocytes-macrophages (M phi): modulation by M phi differentiation and by iron
Authors: Kantengwa S , Polla B S
Published: January 2020

Phagocytosis of microorganisms represents a stress not only for the phagocytosed agent but also for the host cell. We have investigated the stress response induced in human monocytes-macrophages (M phi) phagocytosing inactivated Staphylococcus aureus. Exposure of human M phi to S. aureus induced in these cells (i) a threefold increase in superoxide dismutase activity, (ii) a selective and differentiation-dependent induction of host heat shock protein synthesis (HSP70 but not HSP65), and (iii) de novo synthesis of heme oxygenase, but only when exogenous iron was added to the cultures. The coordinate upregulation of two scavenging enzymes and of HSP70 suggests that all three are part of cellular protective mechanisms against phagocytosis-related oxidative injury to host cells.
Schistosoma japonicum-infected mice show reduced hepatic fibrosis and eosinophilia and selective inhibition of interleukin-5 secretion by CD4+ cells after treatment with anti-interleukin-2 antibodies
Authors: Cheever A W , Xu Y , Sher A , Finkelman F D , Cox T M , Macedonia J G
Published: January 2020

Schistosoma japonicum-infected mice were injected with antibodies to interleukin-2 (IL-2) and/or IL-2 receptor to clarify the role of IL-2 on the granulomatous reaction around schistosome eggs in the liver. Granulomas were of normal or slightly increased size in animals subjected to IL-2 blockade, but hepatic fibrosis was markedly decreased in treated animals 10 weeks after infection. Anti-IL-2 treatment significantly decreased the in vitro secretion of IL-5 by antigen-stimulated spleen cells, and peripheral eosinophilia and tissue eosinophilia were diminished. Secretion of IL-2, IL-4, and gamma interferon was unaffected. Our results indicate that IL-2 is not an essential determinant of granuloma size in S. japonicum-infected mice but that, as in Schistosoma mansoni infection, the development of hepatic fibrosis is critically dependent on IL-2 levels and granuloma size and hepatic fibrosis are differentially regulated.
Evidence that lipopolysaccharide and pertussis toxin bind to different domains on the same p73 receptor on murine splenocytes
Authors: Lei M G , Morrison D C
Published: January 2020

In previous studies, we used a photoactivable, radioiodinated lipopolysaccharide (LPS) derivative to define and characterize a specific bacterial endotoxic LPS-binding protein (p73) on mammalian lymphoreticular cells, including B and T lymphocytes and macrophages. More recently, using the same methodology, we characterized a specific interaction of LPS with the S2 subunit of Bordetella pertussis pertussis toxin (PT) in the fluid phase (M.-G. Lei and D. C. Morrison, J. Biol. Chem., 268:1488-1493, 1993). Furthermore, we showed that lysozyme (LZM) but not polymyxin B can compete with PT for binding to LPS in the fluid phase, a result suggesting that these two molecules compete for the same binding site on LPS. In this report, we demonstrate that the binding of PT to murine splenocytes (cell-bound PT) reduces the ability of the LPS photo-cross-linking probe to bind to the p73 receptor. The reduction can also be demonstrated with the PT B oligomer, a result indicating that the observed reduction of LPS binding to the p73 receptor by PT is A-protomer (S1-subunit) independent. More importantly, our studies document that cell-bound PT can be radiolabelled by the LPS probe, coincident with the observed reduction in p73 photoaffinity labelling. The preferential interaction of LPS with the PT S2 subunit in the fluid phase was, however, not observed with cell-bound PT. The reduction in radiolabelling of the p73 receptor by the LPS probe and in radiolabelling of cell-bound PT was shown to be concentration dependent. The data presented here document, however, that LZM does not reduce the ability of the LPS probe to bind to the p73 receptor on mouse splenocytes, nor does the presence of LZM bound to LPS influence the observed reduction in photoaffinity labelling of p73 by the LPS probe or radiolabelling of cell-bound PT by the LPS probe. Collectively, these results support the concept that the ability of LPS to interact with PT in the fluid phase is not responsible for the ability of cell-bound PT to influence the binding of the LPS probe to the p73 receptor. Thus, it is suggested that PT and LPS bind to different sites on the p73 molecule and that this same p73 protein may recognize both LPS and PT.
Tick salivary gland extract and interleukin-2 stimulation enhance susceptibility of lymphocytes to infection by Theileria parva sporozoites
Authors: Shaw M K , Tilney L G , McKeever D J
Published: January 2020

Intracellular parasites show host cell specificity, and precise information on the range of host cells is a prerequisite for the identification of host molecules that account for the specificity and are involved in entry processes. The sporozoite stage of the tick-borne protozoan parasite Theileria parva binds to and enters bovine lymphocytes, but precise information on the susceptibility of other cell types present at the tick attachment site is unavailable. We quantitatively examined the susceptibility of cell types known to be present at the tick attachment site by a previously established in vitro assay. Apart from lymphocytes, sporozoites also bind to and enter macrophages and afferent lymph veiled cells; they do not bind to or enter fibroblasts, granulocytes, or erythrocytes. Sporozoites are not phagocytosed by the macrophages or veiled cells but enter them as they do lymphocytes. Since the tick attachment site is a region of cellular inflammation, we also examined the effects of agents known to be present in this area on lymphocyte susceptibility. Short-term preincubation of lymphocytes with tick salivary gland extract, with compounds that induce lymphocyte proliferation, or with interleukin-2 (IL-2), a cytokine produced by activated lymphocytes, increased host cell susceptibility by between 30 and 60%. The IL-2-induced increase in host cell susceptibility could be prevented by treating the lymphocytes with the monoclonal antibody IL-A 111, which reacts with the bovine IL-2 receptor alpha chain and inhibits IL-2-driven cell proliferation. The changes induced by tick salivary gland extract and IL-2 occurred in less than 90 min. Similarly, peripheral blood mononuclear cells from an animal previously immunized with a nonrelated antigen (trypanosome variant surface glycoprotein) and stimulated in vitro with the same antigen showed increases in host cell susceptibility of between 70 and 125%. In contrast, treatment of lymphocytes with gamma interferon did not induce any increase in host cell susceptibility.
Comparison of receptors required for entry of Leishmania major amastigotes into macrophages
Authors: Guy R A , Belosevic M
Published: January 2020

We investigated the mechanisms of entry of amastigotes of Leishmania major from two different sources into macrophages by comparing their use of the Fc receptor (FcR), complement receptor type 3 (CR3), and mannose-fucose receptor (MFR). Amastigotes were obtained from BALB/c mice and SCID mice. FcR involvement was examined by opsonizing L. major with parasite-specific immunoglobulin G (IgG). Antiparasite IgG did not alter the uptake of amastigotes from BALB/c mice since these amastigotes had antibody bound to their surface: IgG1 was the most predominant antibody, followed by IgG2b, IgM, and IgG2a. However, opsonization with antiparasite IgG enhanced the entry of amastigotes that lacked antibody on their surface, namely, amastigotes obtained from SCID mice or from macrophages infected in vitro. These results indicate that the FcR is important for amastigote entry into macrophages. Down-modulation of FcRs onto immune complexes, however, did not reduce the entry of amastigotes containing surface-bound IgG into macrophages. Monoclonal antibodies against the CR3 inhibited the entry of amastigotes from either BALB/c or SCID mice into J774A.1 macrophage-like cells. Simultaneous blocking of FcR and CR3 further increased the inhibition of phagocytosis. Treatment of macrophages with soluble mannan or down-modulating the MFR onto mannan-coated coverslips had no effect on the entry of amastigotes from BALB/c or SCID mice. Thus, the MFR does not appear to be used by amastigotes of L. major. We show that ingestion of amastigotes appears to occur primarily through the FcR and CR3; however, additional receptors may also participate in the uptake of amastigotes.
Complement resistance of pathogenic Entamoeba histolytica mediated by trypsin-sensitive surface component(s)
Authors: Hamelmann C , Urban B , Foerster B , Horstmann R D
Published: January 2020

Pathogenic forms of the protozoan parasite Entamoeba histolytica were reported previously to resist the cytolytic effect of the alternative complement pathway (AP) only temporarily during exposure to complement. In contrast, nonpathogenic forms of E. histolytica had been found to show AP resistance as a stable property. We studied the mechanisms of AP resistance of the two forms. Upon exposure to AP activity, resistant pathogenic or nonpathogenic forms bound significantly less C3 products than complement-sensitive pathogenic amebae, indicating that the two resistant forms both inhibited AP amplification. Various enzymatic treatments and inhibition of membrane mobility by cytochalasin B and glutaraldehyde fixation showed that the mechanisms of AP inhibition differed between pathogenic and nonpathogenic forms; in contrast to nonpathogenic forms, pathogenic amebae required intact membrane mobility and a trypsin-sensitive surface component(s) to inhibit AP activation.
Neutralizing monoclonal antibody epitopes of the Entamoeba histolytica galactose adhesin map to the cysteine-rich extracellular domain of the 170-kilodalton subunit
Authors: Mann B J , Chung C Y , Dodson J M , Ashley L S , Braga L L , Snodgrass T L
Published: January 2020

Entamoeba histolytica adheres to human colonic mucins and colonic epithelial cells via a galactose-binding adhesin. The adhesin is a heterodimeric glycoprotein composed of 170- and 35-kDa subunits. Fragments of the hgl1 gene encoding the 170-kDa subunit were expressed as recombinant fusion proteins in Escherichia coli and reacted with anti-adhesin monoclonal antibodies (MAbs) or pooled human immune sera. The MAbs tested recognize seven distinct epitopes on the 170-kDa subunit and have distinct effects on the adherence and complement-inhibitory activities of the adhesin. All seven MAbs reacted with a fusion protein containing the cysteine-rich domain of the protein. Pooled human immune sera reacted with the same cysteine-rich domain as the MAbs and also with a construct containing the first 596 amino acids. Reactivity of three MAbs with the surface of intact trophozoites confirmed that the cysteine-rich domain was located extracellularly. The location of individual epitopes was fine mapped by constructing carboxy-terminal deletions in the cysteine-rich region of the fusion protein. The locations of adherence-enhancing and -inhibiting epitopes were partially distinguished, and the epitopes where complement-inhibitory MAbs bound were demonstrated to be near the adhesin's area of sequence identity with the human complement inhibitor CD59.
TnphoA Salmonella abortusovis mutants unable to adhere to epithelial cells and with reduced virulence in mice
Authors: Rubino S , Leori G , Rizzu P , Erre G , Colombo M M , Uzzau S , Masala G , Cappuccinelli P
Published: January 2020

Salmonella abortusovis is a pathogenic bacterium highly specific to sheep, causing spontaneous abortion. In order to understand the role of genes involved in pathogenicity, we investigated S. abortusovis with the random mutagenic TnphoA transposon. A total of 95 S. abortusovis TnphoA mutants yielding alkaline phosphatase active fusion protein were obtained. In this way we created a bank of strains in order to identify any phenotypic modification which could affect the periplasmic and/or exported proteins involved in virulence. The TnphoA mutants were screened for the ability to adhere to epithelial cells: a total of 23 mutant strains lost this phenotypic feature. To detect the chromosomal TnphoA insertions, DNA was restricted by the enzyme EcoRV, which does not cleave the TnphoA sequence. Southern blotting analysis revealed the existence of four classes of integration. Colonies of adhesiveless mutants appear to be as smooth as the S. abortusovis wild type, and electrophoretic analysis indicates a normal lipopolysaccharide profile. To identify mutations affecting genes encoding for outer membrane proteins (OMPs), the alkaline phosphatase portion of the fusion proteins was revealed in TnphoA mutants by immunoblotting with specific antibodies. A mutation in OMPs was detected in seven mutants. Restriction analysis identified in four mutants a common region of 2 kb where alterations in genes coding for OMPs occur. We suggested that this region is involved in pathogenicity in mice, since a group of mutant strains has shown reduced virulence in mice and one mutant is completely avirulent. Furthermore, after mice were exposed orally to these mutants, significant protection against oral challenge with the parental virulent strain resulted.
Molecular cloning, nucleotide sequence, and characterization of lppB, encoding an antigenic 40-kilodalton lipoprotein of Haemophilus somnus
Authors: Theisen M , Rioux C R , Potter A A
Published: January 2020

Haemophilus somnus is a facultative intracellular pathogen which causes a wide range of diseases in cattle. To identify putative virulence determinants, a genomic library of H. somnus in Escherichia coli was screened for Congo red binding, a property associated with virulence in pathogenic bacteria, and subsequently with bovine hyperimmune sera raised against H. somnus HS25. A Congo red-binding clone carrying a 1.8-kb DNA insert was found to encode a strongly seroreactive LppB protein with an apparent molecular weight of 40,000. The nucleotide sequence of the entire DNA insert was determined. Two open reading frames coding for polypeptides with calculated molecular weights of 21,893 and 30,721 were identified. The larger open reading frame encoded LppB, while the smaller reading frame encoded a nonseroreactive protein with a relative molecular mass of approximately 18 kDa. The 16 amino-terminal amino acids of the deduced LppB polypeptide showed strong sequence homology to the signal peptide of secreted bacterial proteins, and the sequence Leu-Ala-Ala-Cys at the putative cleavage site corresponds to the consensus cleavage sequence of bacterial lipoproteins. Synthesis of the mature LppB lipoprotein in H. somnus was inhibited by globomycin, a specific inhibitor of signal peptidase II. LppB was localized to the outer membrane of H. somnus.
Similar mechanisms of action of defined polysaccharides and lipopolysaccharides: characterization of binding and tumor necrosis factor alpha induction
Authors: Otterlei M , Sundan A , Skjåk-Braek G , Ryan L , Smidsrød O , Espevik T
Published: January 2020

Little has been reported about the effects of different polysaccharides on cytokine production from human monocytes. In this study, we show that several well-defined polysaccharides, including polymers with different sizes of beta 1-4-linked D-mannuronic acid (poly-M, high-M alginate, and M-blocks) and cellulose oxidized in the C-6 position, induced human monocytes to produce tumor necrosis factor alpha (TNF-alpha). Poly-M was the most efficient polysaccharide tested and, on a weight basis, was approximately as efficient as lipopolysaccharide (LPS) from Escherichia coli. TNF-alpha production was shown to depend strongly on the molecular weights of poly-M and high-M alginate, with maximal TNF-alpha production occurring at molecular weights above 50,000 and 200,000, respectively. G-blocks, alpha 1-4-linked L-guluronic acid polymers that did not induce cytokine production from monocytes, reduced the cytokine production induced by the beta 1-4-linked polyuronic acids and LPS. Furthermore, both G-blocks and LPS were found to inhibit the binding of poly-M to monocytes, as measured by flow cytometry. In addition, we found that the binding of LPS to monocytes was inhibited by G-blocks, M-blocks, and poly-M. Our results indicate that beta 1-4-linked polyuronic acids and LPS may stimulate monocytes to produce TNF-alpha by similar mechanisms and may bind to a common receptor.
Immunogenicity of Vibrio vulnificus capsular polysaccharides and polysaccharide-protein conjugates
Authors: Simonson J G , Siebeling R J
Published: January 2020

Opaque colony morphology has been correlated to Vibrio vulnificus virulence. However, the number of capsular serotypes expressed by virulent isolates is unknown. In an effort to produce anticapsule sera, capsular polysaccharide (CPS) from three opaque V. vulnificus strains was purified and characterized. Purified CPSs were acidic and contained considerable amounts of hexosamine and trace quantities of protein and nucleic acid. CPS purified from strain C7184 was poorly immunogenic for rabbits and mice, since repeated injection produced little detectable anticapsular antibody. To improve immunogenicity, CPS-protein conjugates were prepared from adipic acid hydrazide derivatives of CPS purified from each strain and carbodiimide as a coupling reagent. The immunogenicity of C7184 CPS was enhanced by conjugation to keyhole limpet hemocyanin, since injection into mice elicited production of anticapsular antibodies, the level of which was dependent on the dose and time since initial immunization. Injection of rabbits with CPS-protein conjugates also produced anticapsular antibodies. Staphylococcus aureus cells armed with each of the three anticapsular antibodies coagglutinated only the homologous opaque strain, indicating the existence of at least three capsular types. Further screening of 32 opaque and translucent V. vulnificus isolates revealed only three cross-reacting strains. These results suggest the presence of numerous V. vulnificus capsular types.
Antigenic and functional differences in adhesion of Plasmodium falciparum-infected erythrocytes to human and bovine CD36
Authors: Ockenhouse C F , Tandon N N , Jamieson G A , Greenwalt D E
Published: January 2020

Cytoadherence by Plasmodium falciparum-infected erythrocytes (PRBC) to microvascular endothelium is, in part, mediated by the specific interaction between a parasite-derived erythrocyte surface ligand and a specific binding site on human CD36. We describe the selection for increased adhesion of PRBC to bovine CD36 and demonstrate that the molecular interaction between PRBC and bovine CD36 is independent of and distinct from the OKM5/8 monoclonal antibody epitopes which block PRBC-human CD36 binding.
Identification of plasmid and chromosomal copies of 987P pilus genes in enterotoxigenic Escherichia coli 987
Authors: Casey T A , Schneider R A , Dean-Nystrom E A
Published: January 2020

A DNA probe derived from the subunit gene of a cloned 987P determinant was used to characterize the locations of the 987P genes in several Escherichia coli strains. We examined E. coli 987, a nonpiliated mutant of strain 987 (I36) that does not express 987P in vitro, and a variant of I36 that expressed 987P following growth in vivo. We found that plasmid and chromosomal copies of the 987P subunit gene could be differentiated in strain 987 by restriction endonuclease analysis and Southern blot hybridization. The nonpiliated mutant I36 had lost the plasmid copy but retained the chromosomal copy of the 987P gene. A 987P-piliated variant of I36, which did not contain the 987P plasmid, colonized and caused diarrhea in neonatal pigs similarly to wild-type 987. The plasmid that hybridized with the 987P probe was transferred from strain 987 to an E. coli K-12 strain by conjugation. We were unable to demonstrate expression of 987P by these transconjugants. The data suggest that the chromosomal and plasmid copies of the 987P genes may interact to influence 987P expression.
Attaching and effacing locus of a Citrobacter freundii biotype that causes transmissible murine colonic hyperplasia
Authors: Schauer D B , Falkow S
Published: January 2020

Citrobacter freundii biotype 4280 produces attaching and effacing (AE) lesions in the large intestine of laboratory mice and is the causative agent of transmissible murine colonic hyperplasia. AE lesions are also produced by enteropathogenic Escherichia coli in humans. Southern analysis revealed that biotype 4280, but not 20 other strains of C. freundii, contained DNA homologous to the eae (E. coli attaching and effacing) gene which is necessary for AE activity by enteropathogenic E. coli in vitro. We have cloned and determined the nucleotide sequence of the C. freundii eae homolog. Our findings suggest that the eae locus of C. freundii biotype 4280 is necessary for AE activity and has a role in the pathogenesis of transmissible murine colonic hyperplasia.