bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the physical state of Salmonella lipopolysaccharide in expression of biological and endotoxic properties
Authors: Shnyra A , Hultenby K , Lindberg A A
Published: January 2020

Lipopolysaccharide (LPS) extracted from three strains of Salmonella typhimurium, i.e., the rough Re mutant SL1102, the rough Ra mutant TV119, and the smooth strain SH4809, was first electrodialyzed (eLPS) and then divalent cation deprived by EDTA treatment and finally made monomeric by deoxycholate solubilization. The removal of excess detergent by extensive dialysis in the absence of mineral cations resulted in the reassociation of LPS subunits into monodisperse micelles of reduced aggregation number (dLPS) as estimated by electron microscopy and gel filtration chromatography. For all LPS chemotypes tested, the developed procedure reproducibly results in stable and clear solutions of dLPS in concentrations of up to 100 mg/ml. The dLPS and eLPS preparations possessed the same reactivity with monoclonal antibodies (MAbs) raised against different LPS domains. The 100% lethal dose in galactosamine-sensitized mice of 0.01 microgram for the smooth eLPS was from 10- to 100-fold lower than that of dLPS at 0.1 to 1.0 microgram. dLPS from both the smooth strain and the Ra mutant had a significantly reduced capacity to activate the proenzyme cascade in the Limulus amoebocyte lysate assay in comparison with the slightly reduced activity of dLPS from the Re mutant. In contrast, dLPS as well as the deoxycholate-dispersed and then diluted eLPS from the smooth strain had a higher mitogenic activity on splenocytes than eLPS. The results indicate that the biological and endotoxic properties of LPS are significantly influenced by the physical state of its aggregates in aqueous solutions. The approach developed for production of a stable and dispersed form of LPS should further assist in investigation of LPS properties and interpretation of the data of endotoxic research.
Gamma interferon-induced nitric oxide production reduces Chlamydia trachomatis infectivity in McCoy cells
Authors: Mayer J , Woods M L , Vavrin Z , Hibbs J B
Published: January 2020

McCoy cells, murine-derived cells commonly used for propagation of chlamydiae, were found to be efficient producers of nitric oxide (NO) when primed with murine gamma interferon (IFN-gamma) and then exposed to the second signals provided by Escherichia coli lipopolysaccharide, human interleukin-1 alpha, murine tumor necrosis factor alpha, or Chlamydia trachomatis type H. Murine recombinant IFN-gamma over a range of 0 to 50 U/ml inhibited infectivity of C. trachomatis type H in a dose-dependent fashion in McCoy cells while simultaneously inducing NO production. Quantitation of infectious chlamydia progeny remaining in McCoy cells 48 or 72 h postinfection revealed that IFN-gamma-primed McCoy cells reduced chlamydial inclusion-forming units (expressed as units per milliliter) by 4 log10 units at higher IFN-gamma concentrations (50 U/ml) compared with control values. The magnitude of this antichlamydial effect was directly related to increased synthesis of NO, the production of which was IFN-gamma dose dependent. The antichlamydial effects of IFN-gamma were blocked in a dose-dependent manner by the addition of N-guanidino-monomethyl L-arginine (MLA), an inhibitor of nitric oxide synthesis. These results suggest that although IFN-gamma priming of McCoy cells is required for antichlamydial activity, nitric oxide is a necessary effector molecule involved in the mechanism(s) of IFN-gamma-induced inhibition of chlamydial proliferation in this murine cell line. The ability to block the potent antichlamydial effects of IFN-gamma by inhibition of a specific enzyme, nitric oxide synthase, may give insights into mechanisms by which IFN-gamma and perhaps other cytokines are able to control proliferation of chlamydiae and other intracellular pathogens.
Suppression of T-cell proliferation by CD8+ T cells induced in the presence of protoscolices of Echinococcus multilocularis in vitro
Authors: Kizaki T , Ishige M , Kobayashi S , Bingyan W , Kumagai M , Day N K , Good R A , Onoé K
Published: January 2020

Immunoregulatory influences of protoscolices (PSC) of Echinococcus multilocularis on murine T-lymphocyte functions have been examined in an in vitro system. Proliferative responses of spleen cells stimulated with concanavalin A (ConA) or anti-CD3 monoclonal antibodies were depressed by the addition of PSC. In the presence of PSC, both interleukin-2 (IL-2) production and IL-2 receptor (IL-2R) expression by lymphocytes stimulated with ConA were significantly reduced. However, exogenous IL-2 reconstituted both the ConA-stimulated proliferative responses and IL-2R expression. These findings suggest that PSC of E. multilocularis can suppress lymphoid cell responses via influences on IL-2 production. Indeed, addition of CD(8+)-enriched cells from cultures stimulated with ConA plus PSC to fresh spleen cells showed marked suppression of the ConA responses. IL-2 production as well as IL-2R expression on the spleen cells so treated were suppressed. These findings reveal a suppressive immunologic function induced by E. multilocularis PSC that involves inhibition of IL-2 production and reduction of IL-2R expression. The PSC-induced CD8+ cells appear to play a key role in the suppressive regulation of host immune responses against E. multilocularis.
Sequencing of leucocidin R from Staphylococcus aureus P83 suggests that staphylococcal leucocidins and gamma-hemolysin are members of a single, two-component family of toxins
Authors: Supersac G , Prevost G , Piemont Y
Published: January 2020

A 2,813-bp HincII-ClaI DNA fragment encodes the two S and F components (LukS-R and LukF-R) of leucocidin R (Luk-R) which are secreted by Staphylococcus aureus P83. The two genes (lukS-R and lukF-R) belong to a single operon. Two peptidic sequences were deduced: LukS-R is a 35,721-Da polypeptide of 315 amino acids, including a signal sequence of 29 residues, and LukF-R is a 36,838-Da polypeptide of 325 amino acids, including a signal sequence of 25 residues. LukS-R and LukF-R were expressed in Escherichia coli and purified from the periplasmic space. Luk-R exerts biological activities on polymorphonuclear cells and on erythrocytes from various animals. Comparison of the amino acid sequence of LukF-R with that of the B component of gamma-hemolysin (HlgB), those of the F and S components of another recently sequenced staphylococcal leucocidin, and those of a few peptides of the F component from Panton-Valentine leucocidin suggests that all four toxins belong to a single, two-component family of toxins.
Localization of the meningococcal receptors for human transferrin
Authors: Ala'Aldeen D A , Powell N B , Wall R A , Borriello S P
Published: January 2020

The interaction between gold-labelled human transferrin (Au-HTF) with live meningococci after growth in vivo or in different in vitro conditions was examined by electron microscopy to localize and quantify the numbers of HTF-binding sites on the cell surface. It was clearly demonstrated that HTF binds to the surface of live meningococci (of different serogroups and serotypes) after growth in either iron-sufficient or iron-restricted cultures, although the degree of labelling was always higher (2- to 35-fold) in the latter case. The commensal Neisseria polysaccharea behaved similarly. Ultrathin sections showed that Au-HTF was localized predominantly on the outer membrane of the cells and vesicles, with hardly any internalization. Au-HTF labelling on meningococci was significantly reduced after incubation with unlabelled HTF or with rabbit antiserum containing antibodies against transferrin-binding proteins (TBPs), demonstrating the specificity of the interaction. These sera also blocked binding between HTF and outer membrane proteins on Western immunoblots. Direct evidence of the expression of the TBPs (Western blots) and localization of the HTF receptor (electron microscopy) on in vivo-grown meningococci was obtained from organisms derived without laboratory culturing from the cerebrospinal fluid of a patient. There was considerable cell-to-cell variation in the amount of labelling present on cells of the same sample (in vitro- or in vivo-grown organisms) and between different strains. The degree of binding varied with time of incubation of the cells with Au-HTF. The gold particles frequently formed discrete circles on the cell surfaces of the in vitro-grown organisms; these circles appear to be associated with outer membrane vesicle formation. The results show that the TBPs, which form part of the active components of the HTF receptor(s), are expressed in vivo and are surface exposed and immunogenic and that antibodies against them can interfere with the HTF binding of the meningococcal cells, which may affect iron utilization. This study further supports the concept of regarding the TBPs as future vaccine candidates.
Staphylococcus aureus toxic shock syndrome toxin 1 and Streptococcus pyogenes erythrogenic toxin A modulate inflammatory mediator release from human neutrophils
Authors: Hensler T , Köller M , Geoffroy C , Alouf J E , König W
Published: January 2020

We studied the influence of staphylococcal toxic shock syndrome toxin 1 and streptococcal erythrogenic (pyrogenic) toxin A (ETA) on intact and digitonin-permeabilized human polymorphonuclear granulocytes (PMNs). As was shown by reversed-phase high-performance liquid chromatography analysis, toxic shock syndrome toxin 1 or ETA alone, in the absence of any additional stimulus, did not induce the generation of the chemoattractant leukotriene B4 (LTB4) from PMNs in a wide range of concentrations. In addition, pretreatment of intact PMNs with either toxin potentiated formyl-methionyl-leucyl-phenylalanine (fMLP)- and washed Staphylococcus aureus cell-induced generation of LTB4 in a time- and dose-dependent manner. This increase included LTB4 as well as its inactive omega-oxidated compounds. Further studies revealed evidence that toxin exposure was accompanied by enhanced cellular receptor expression for fMLP as well as for LTB4. The intrinsic GTPase activity of membrane fractions was modulated by both toxins. Short-term incubation with ETA increased the GTPase activity of PMNs up to 141%. Inhibitory effects were obtained when GTP-binding protein functions were stimulated with sodium fluoride (NaF). In addition, specific binding of Gpp(NH)p to GTP-binding protein was inhibited by both toxins during the first 10 min of incubation and was restored at later times of incubation. Our data therefore suggest that both toxins significantly affect the signal transduction pathways of human PMNs, which results in immunomodulatory functions.
T-cell-independent resistance to infection and generation of immunity to Francisella tularensis
Authors: Elkins K L , Rhinehart-Jones T , Nacy C A , Winegar R K , Fortier A H
Published: January 2020

The intraperitoneal 50% lethal dose (LD50) for Francisella tularensis LVS in both normal control heterozygote BALB/c nu/+ mice and BALB/c nu/nu mice was 2 x 10(0). Both nu/+ and nu/nu mice given 10(7) LVS bacteria or more intradermally (i.d.) died, with a mean time to death of about 7 to 8 days. On the other hand, nu/+ mice given 10(6) LVS bacteria or less survived for more than 60 days and cleared systemic bacteria, while nu/nu mice given 10(6) LVS bacteria or less survived for more than 10 days but died between days 25 and 30. Thus, the short-term (i.e., < 10-day) i.d. LD50 of both nu/nu and nu/+ mice was 3 x 10(6), but the long-term (i.e., > 10-day) i.d. LD50 of nu/nu mice was less than 7 x 10(0). The short-term survival of i.d. infection was dependent on tumor necrosis factor and gamma interferon: treatment of nu/nu mice with anti-tumor necrosis factor or anti-gamma interferon at the time of i.d. infection resulted in death from infection 7 to 8 days later, whereas control infected nu/nu mice survived for 26 days. nu/nu mice infected with LVS i.d. generated LVS-specific serum antibodies, which were predominantly immunoglobulin M: titers peaked 7 days after i.d. infection but declined sharply by day 21, after which mice died. Surprisingly, nu/nu mice given 10(3) LVS bacteria i.d. became resistant to a lethal challenge (5,000 LD50s) of LVS intraperitoneally within 2 days after i.d. infection; nu/nu mice similarly infected with LVS i.d. and challenged with Salmonella typhimurium (10 LD50s) were not protected. nu/nu mice given nu/+ spleen cells intravenously as a source of mature T cells survived i.d. infection for more than 60 days and cleared bacteria. Taken together, these studies demonstrate that i.d. infection of nu/nu mice with LVS rapidly generates T-cell-independent, short-term, specific protective immunity against lethal challenge, but T lymphocytes are essential for long-term survival.
Ultrastructural analysis of attachment to and penetration of capillaries in the murine pons, midbrain, thalamus, and hypothalamus by Nocardia asteroides
Authors: Beaman B L , Ogata S A
Published: January 2020

The attachment to and penetration of endothelial cells in the pons and midbrain (especially the substantia nigra) regions of the brains of BALB/c mice by log-phase Nocardia asteroides GUH-2 cells were determined by both scanning and transmission electron microscopic analysis. Within 15 min after exposure, the nocardiae attached to the surface of the endothelial cell membrane. This attachment occurred primarily at the growing tip of the nocardial filament, and the outermost layer of the nocardial cell wall had regions (electron-dense areas) that bound firmly to the cytoplasmic membrane of the host cell. There appeared to be specificity for this binding localized within the capillaries and arterioles because some regions had large numbers of bacteria bound, whereas adjacent areas had no bacterial cells. Nocardial filaments that attached by the apex induced a cuplike deformation of the endothelial cell membrane. This was followed by a rapid penetration of the endothelial cell so that within 25 min many of the bacteria were internalized within the host cell. These internalized bacteria remained within vesicles, and there was no ultrastructural evidence of damage to the nocardial cell during this process. Heat-killed GUH-2 cells still attached to endothelial surfaces (at a reduced frequency), but they did not penetrate into the endothelial cell. These data suggest that brain-invasive nocardiae possess both an adhesin for attachment to the membrane of endothelial cells and an invasion factor that promotes nocardial penetration of these cells.
Endogenous and exogenous glucocorticoids have different roles in modulating endotoxin lethality in D-galactosamine-sensitized mice
Authors: Gonzalez J C , Johnson D C , Morrison D C , Freudenberg M A , Galanos C , Silverstein R
Published: January 2020

Endotoxin sensitivity and dexamethasone protection have been assessed in mice that were adrenalectomized and also treated with D-galactosamine at the time of endotoxin challenge. Our data establish that adrenalectomy did not detectably alter the magnitude of the increased sensitivity induced by D-galactosamine alone. Furthermore, protection provided by acute exogenous glucocorticoid treatment was still demonstrable in these mice and was not influenced by chronic experimentally induced glucocorticoid deficiency. Our data confirm that the adrenalectomized mouse model of endotoxin lethality is characterized by increased sensitivity to endotoxin and establish that the magnitude of this sensitizing effect is more than 100-fold. We also show for the first time that adrenalectomy causes an appreciable kinetic shift in the endotoxic crisis and that dexamethasone, given at the time of endotoxin challenge, will significantly reverse the increased sensitivity to lethality. Our results indicate that the protective effects of corticosteroids may involve important chronic as well as acute responses. In particular, we conclude that endogenous glucocorticoid need not always increase host resistance to endotoxin, nor does such a circumstance eliminate the possibility for exogenous glucocorticoid-mediated protective effects.
Characterization of a catalase-deficient strain of Neisseria gonorrhoeae: evidence for the significance of catalase in the biology of N. gonorrhoeae
Authors: Johnson S R , Steiner B M , Cruce D D , Perkins G H , Arko R J
Published: January 2020

We obtained a catalase-deficient (Kat-) strain of Neisseria gonorrhoeae isolated from a patient who had been unsuccessfully treated with penicillin. Quantitative enzyme assays and electrophoresis of cell extracts on native polyacrylamide gels subsequently stained for catalase and peroxidase activities failed to detect both enzymes. The strain exhibited no growth anomalies or unusual requirements when grown under ordinary laboratory conditions. However, the Kat- strain proved extremely sensitive to exogenous hydrogen peroxide, and analysis of the bacterial DNA after such exposure showed extensive single-strand breakage in both chromosomal and plasmid DNAs. Partial characterization of the gonococcal catalase from a Kat+ laboratory strain revealed that the enzyme had the physical and chemical properties of both catalase and peroxidase.
Expression of gtfS is essential for normal insoluble glucan synthesis by Streptococcus downei
Authors: Gilmore K S , Russell R R , Ferretti J J
Published: January 2020

The gtfI and gtfS genes of Streptococcus downei were investigated to determine the contribution of the respective enzymes to glucan production in the presence and absence of other glucosyltransferases. Extracts of Escherichia coli expressing cloned gtfS produced a short linear dextran from sucrose which could act as a primer for insoluble glucan synthesis when mixed with extracts of a strain expressing recombinant gtfI. To elucidate the contribution of gtfS to glucan production by S. downei, a mutant was constructed by insertionally inactivating gtfS. S. downei (gtfS mutant) colonies exhibited a marked phenotypic change on sucrose-containing media and a decreased ability to adhere to glass and produced no detectable water-insoluble glucan. These experiments confirm that expression of gtfS is essential for normal insoluble glucan synthesis by S. downei.
Characterization of a neutralizing monoclonal antibody directed at variable domain I of the major outer membrane protein of Chlamydia trachomatis C-complex serovars
Authors: Qu Z , Cheng X , de la Maza L M , Peterson E M
Published: January 2020

A monoclonal antibody (MAb), C10, that neutralized in vitro the infectivity of serovars C, I, J, and L3 (members of the C and C-related complexes) of Chlamydia trachomatis was identified. Of the 15 major serovars and the mouse pneumonitis strain of C. trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae, which were used as nontreated and heat-treated (56 degrees C, 30 min) antigens in a dot blot assay, only serovars C, I, J, and L3 were recognized with both the native and treated antigens. Western blot (immunoblot) results showed that MAb C10 recognized the major outer membrane protein of these four serovars. Overlapping hexameric peptides corresponding to variable domains (VDs) I, II, III, and IV of the major outer membrane protein of C. trachomatis serovar C were synthesized, and peptide screening showed that MAb C10 mapped to the VD I amino acid sequence VAGLQNDPT. Results of an in vitro neutralization assay correlated with those of the indirect immunofluorescence assay, Western blot, and dot blot assay in that only serovars C, I, J, and L3 were neutralized by MAb C10. In vitro competitive neutralization experiments, using a peptide representing VD I of serovar C to compete with C. trachomatis serovar C for MAb C10 binding, revealed that both serological and neutralizing activities of MAb C10 were inhibited by the VD I peptide. In an in vivo toxicity/infectivity assay using serovar L3 pretreated with MAb C10, there was 100% survival of mice infected with a lethal dose at 48 h. In contrast, the control group, consisting of mice injected with the same dose of L3 pretreated with a MAb that does not recognize L3, had no survivors during a 48-h observation period. In summary, since the surface-exposed contiguous epitope recognized by MAb C10 binds neutralizing antibodies that are subspecies specific for the C and C-related complexes, it should be considered for inclusion in the development of a chlamydial vaccine.
Superoxide dismutase and the resistance of Escherichia coli to phagocytic killing by human neutrophils
Authors: Papp-Szabò E , Sutherland C L , Josephy P D
Published: January 2020

Transformation of Escherichia coli K-12-derived strains with a plasmid carrying the genetic determinants for synthesis of lipopolysaccharide O antigen by Shigella dysenteriae allows the construction of phenotypically smooth derivatives. We show that such E. coli K-12 derivatives are highly resistant to killing by human serum. Isogenic wild-type and sodB mutant (Fe superoxide dismutase-deficient) strains were constructed. The results of experiments on phagocytic killing of these strains by human neutrophils are reported. We observed no difference between the sensitivities of wild-type and sodB mutant strains to phagocytic killing, in contrast to the results reported by other researchers who used species other than E. coli or strains other than K-12.
Localization of the virulence-associated genes pilA, pilR, rpoN, fliA, fliC, ent, and fbp on the physical map of Pseudomonas aeruginosa PAO1 by pulsed-field electrophoresis
Authors: Farinha M A , Ronald S L , Kropinski A M , Paranchych W
Published: January 2020

Seven virulence-associated genes have been placed on a genomic map of Pseudomonas aeruginosa PAO1, using pulsed-field electrophoresis, on the basis of the previous physical maps of Romling et al. (U. Romling, M. Duchene, D. Essar, D. Galloway, C. Guidi-Rontani, D. Hill, A. Lazdunski, R. Miller, K. Schleifer, D. Smith, H. Toschka, and B. Tummler, J. Bacteriol. 174:327-330, 1992; U. Romling, D. Grothues, W. Bautsch, and B. Tummler, EMBO J. 8:4081-4089, 1989) and Ratnaningsih et al. (E. Ratnaningsih, S. Dharmsthiti, V. Krishnapillai, A. Morgan, M. Sinclair, and B. W. Holloway, J. Gen. Microbiol. 136:2351-2357, 1990). The new locations for the outer membrane enterobactin iron-siderophore receptor ent gene (41 to 42 min) and the fliA gene (59 to 61 min), which encodes a minor sigma factor of RNA polymerase, are given. The pilA (the pilin structural gene), pilR (a pilin regulatory gene), and rpoN (encoding another minor sigma factor of RNA polymerase) genes map together at 71 to 75 min, locations correcting the previously reported values (V. Shortridge, M. Pato, A. Vasil, and M. Vasil, Infect. Immun. 59:3596-3603, 1990). The fbp gene (28 to 29 min), which encodes an outer membrane ferripyochelin-binding protein of low molecular weight, and the fliC gene (64 to 66 min), the flagellin structural gene, were determined to lie in the previously reported locations.
Interaction between human immunodeficiency virus and Toxoplasma gondii replication in dually infected monocytoid cells
Authors: Welker Y , Molina J M , Poirot C , Ferchal F , Decazes J M , Lagrange P , Derouin F
Published: January 2020

THP-1 monocytoid cells, either not infected or chronically infected with human immunodeficiency virus type 1 (HIV-1), were challenged with Toxoplasma gondii. Parasitic growth, as assessed by trophozoite counting and measurement of supernatant p30 membrane antigen, was similar in HIV-infected and noninfected THP-1 cells. Also, T. gondii did not affect HIV replication. These experiments therefore failed to demonstrate any interaction between HIV-1 and T. gondii replication in concurrently infected monocytoid cells.
Endotoxin-induced tumor necrosis factor alpha synthesis in murine embryo fibroblasts
Authors: Havell E A , Rogerson B J
Published: January 2020

Murine embryo fibroblasts (MEF) were found to secrete tumor necrosis factor (TNF) in response to stimulation with endotoxin. Endotoxin-induced TNF production by MEF was inhibited by cycloheximide. However, reversal of the effect of this inhibitor on protein synthesis results in TNF being secreted in amounts equivalent to those produced by endotoxin-induced MEF not treated with cycloheximide. Actinomycin D treatment of MEF blocked the production of endotoxin-induced TNF. Maximal production of TNF required MEF gene transcription during the first 6 h of incubation with endotoxin. To determine whether endotoxin-induced TNF alpha (TNF-alpha) and/or TNF beta were produced by MEF, cDNA was synthesized from the total RNA isolated from endotoxin-induced MEF and amplified by the polymerase chain reaction in the presence of oligonucleotide primers specific for each cytokine. On the basis of the polymerase chain reaction analysis, it was determined that TNF-alpha mRNA levels were increased in endotoxin-induced MEF. Thus, production of TNF-alpha by fibroblasts in response to the endotoxin component of bacterial cell walls is likely to contribute to the expression of TNF-mediated effects occurring in fibroblast-rich tissues infected with gram-negative bacteria.
The alkaline protease of Aspergillus fumigatus is not a virulence determinant in two murine models of invasive pulmonary aspergillosis
Authors: Tang C M , Cohen J , Krausz T , Van Noorden S , Holden D W
Published: January 2020

Little is known of the pathophysiology of invasive pulmonary aspergillosis (IPA), an opportunistic fungal infection usually caused by Aspergillus fumigatus. It has been suggested that the ability of the fungus to degrade elastin may aid its invasion and growth in lung tissue. We have described previously the construction of a strain of A. fumigatus in which the gene encoding an alkaline protease, AFAlp, had been disrupted (C.M. Tang, J. Cohen, and D.W. Holden, Mol. Microbiol. 6:1663-1671, 1992); this mutant is deficient in extracellular proteolytic and elastinolytic activity over a broad pH range. In this study, we compared the pathogenicity of this and another AFAlp disruptant with their isogenic, elastase-producing parental strains in two murine models of IPA. In both models, animals were inoculated via the respiratory tract. In the first model, the inoculum was delivered as airborne conidia and animals developed signs of respiratory distress within 2 to 4 days. In the second model, conidia were administered intranasally as a suspension and the disease developed over a 2-week period. No difference was observed between the wild-type and AFAlp disruptants in terms of mortality, and elastin breakdown was detected in lung tissue from animals inoculated with all four strains. We conclude that AFAlp is not a virulence determinant in these models of IPA.
Induction of macrophage-mediated production of tumor necrosis factor alpha by an L-form derived from Staphylococcus aureus
Authors: Kuwano K , Akashi A , Matsu-ura I , Nishimoto M , Arai S
Published: January 2020

We investigated the capability of an L-form derived from Staphylococcus aureus to induce tumor necrosis factor alpha (TNF-alpha) production in murine peritoneal macrophages. The activity for TNF-alpha induction was found in the membrane fraction of the L-form but not in the cytoplasmal fraction purified by the sucrose step gradient centrifugation. TNF-alpha mRNA was also detected in macrophages stimulated with L-form membranes. L-form induced TNF-alpha production in macrophages from both lipopolysaccharide-responsive and -unresponsive mouse strains. Regardless of the presence of polymyxin B, the activity of TNF-alpha induction of L-form was mostly found in the phenol layer, but not in the aqueous layer, both of which were prepared by phenol extraction method. Fractions of L-form membranes representing molecular masses of approximately between 29 and 36 kDa were primarily responsible for inducing the production of TNF-alpha consistently. Moreover, this stimulatory effect was abolished by digestion with Streptomyces griseus protease. In Western blot (immunoblot) analysis with anti-lipoteichoic acid antibody, two bands (65 and 45 kDa) were observed in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phenol layer, whereas one band (14 kDa) was observed in either the aqueous layer or lipoteichoic acid of S. aureus. These results suggest that the component in the membrane of the L-form, distinct from cell wall components such as teichoic acid or lipopolysaccharide, possesses the capability to stimulate TNF-alpha production by macrophages.
Characterization of a heat-modifiable outer membrane protein of Haemophilus somnus
Authors: Tagawa Y , Haritani M , Ishikawa H , Yuasa N
Published: January 2020

In immunoblot analysis, a murine monoclonal antibody (MAb), 27-1, which was produced to an outer membrane protein (OMP) of Haemophilus somnus, showed that a major OMP is heat modifiable, having a molecular mass of 28 kDa when the N-lauroylsarcosine-insoluble OMP preparation was solubilized at 60 degrees C and a mass of 37 kDa when the OMP preparation was solubilized at 100 degrees C. The heat-modifiable OMP reacted intensely with convalescent sera obtained from calves with experimental H. somnus pneumonia in immunoblot analysis. Immunoelectron microscopic and antibody absorption studies revealed that the MAb 27-1 epitope was not surface exposed on the intact bacterium. However, a decrease in antibody reactivity to the heat-modifiable OMP in immunoblot analysis after absorption of convalescent serum with intact bacterial cells of H. somnus suggests that a surface-exposed portion of the heat-modifiable OMP is expressed on the intact bacterium. MAb 27-1 reacted with 45 of 45 strains of H. somnus tested in immunoblot analysis. The apparent molecular mass of the antigen varied among strains, and five reactivity patterns demonstrated by MAb 27-1 were observed. MAb 27-1 also reacted with six species in the family Pasteurellaceae, Escherichia coli, and Salmonella dublin, but not with the other eight species of gram-negative bacteria. The heat-modifiable OMP of H. somnus showed immunological cross-reactivity with the OmpA protein of E. coli K-12 and significant N-terminal amino acid sequence homology with the OmpA proteins of gram-negative bacteria. We conclude that a major, 37-kDa heat-modifiable OMP of H. somnus, which elicits an antibody response in H. somnus-infected animals, is a common antigen among H. somnus strains tested and is structurally related to the OmpA protein of E. coli.
Increased C3 production in human monocytes after stimulation with Candida albicans is suppressed by granulocyte-macrophage colony-stimulating factor
Authors: Høgåsen A K , Abrahamsen T G
Published: January 2020

Activation of the complement system is an important part of host resistance against fungal infections. When human monocytes, cultured for 2 days or more, were treated in vitro with Candida albicans for 24 h, an enhancement of their biosynthesis of the complement components C3 and factor B was found. However, when C. albicans was administered to freshly isolated monocytes, a consistent stimulation of factor B biosynthesis occurred, while the C3 production was increased in about 50% of the donors. C. albicans also induced the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) from the cultured cells, apparently in larger amounts in the donors in whom no stimulation of C3 production was found. An antibody to GM-CSF administered with the yeast at the initiation of the monocyte culture caused an increase in the C3 production. Furthermore, when monocytes were treated with recombinant human GM-CSF either at the same time as or 4 days prior to the addition of C. albicans, the increase in C3 production was suppressed or neutralized, while factor B biosynthesis was unaffected. Taken together, these results indicate that monocytes respond to C. albicans with an increased production of complement factors. This may be an important mechanism both for opsonization of the fungus and for initiation of an inflammatory reaction. At an inflammatory site, this complement response may be suppressed by locally produced GM-CSF.
Kupffer cell elimination enhances development of liver schizonts of Plasmodium berghei in rats
Authors: Vreden S G , Sauerwein R W , Verhave J P , Van Rooijen N , Meuwissen J H , Van Den Broek M F
Published: January 2020

We investigated the development of exoerythrocytic forms (EEF) of Plasmodium berghei in livers of normal and macrophage-depleted Brown Norway rats. Macrophages were depleted by use of liposome-encapsulated dichloromethylene diphosphonate. Upon inoculation of sporozoites, macrophage-depleted rats had significantly larger numbers of EEF than untreated rats. We also investigated the effect of macrophage impairment by silica treatment on the development of EEF and confirmed that silica induces a significant reduction of EEF development. Intravenous administration of silica induced high levels of interleukin-6 in plasma within a few hours. The seemingly contradictory results for EEF development may be explained by our previous observation that interleukin-6 strongly inhibits sporozoite penetration and EEF development in vivo. We conclude that in experimental infections with sporozoites, Kupffer cells inhibit rather than enhance EEF development.
Lipopolysaccharide and granulocyte colony-stimulating factor delay neutrophil apoptosis and ingestion by guinea pig macrophages
Authors: Yamamoto C , Yoshida S , Taniguchi H , Qin M H , Miyamoto H , Mizuguchi Y
Published: January 2020

In previous studies, neutrophil-ingesting macrophages were clearly and easily observed in the peritoneal cavity of guinea pigs after intraperitoneal injection of thioglycolate medium, and phagocytosis of neutrophils by macrophages could be detected in in vitro cultures of peritoneal exudate cells. Using an in vitro system, we examined the effect of bacterial lipopolysaccharide and recombinant human granulocyte colony-stimulating factor on the apoptosis (programmed cell death) of neutrophils and their subsequent ingestion by macrophages. Lipopolysaccharide delayed karyopyknosis and apoptosis of neutrophils, as shown by endogenous endonuclease activity and a high proportion of trypan blue-excluding cells, and subsequent ingestion by autologous macrophages. Granulocyte colony-stimulating factor also delayed neutrophil karyopyknosis and ingestion by macrophages. When a thioglycolate medium was coinjected intraperitoneally with lipopolysaccharide into guinea pigs in the in vivo system, delays in neutrophil disappearance and ingestion by macrophages in the peritoneal cavity were also observed. We suggest that bacterial products and cytokines regulate neutrophil apoptosis and subsequent ingestion by macrophages at inflamed sites.
Resistance to nitric oxide in Mycobacterium avium complex and its implication in pathogenesis
Authors: Doi T , Ando M , Akaike T , Suga M , Sato K , Maeda H
Published: January 2020

Susceptibility of three different strains of Mycobacterium avium complex (MAC), i.e., one strain of M. avium (Mino) and two strains of M. intracellulare (31F093T and KUMS 9007), to nitric oxide (NO) generated by rat alveolar macrophages (M phi) or NO generated chemically by acidification of NO2- was examined in vitro. We also investigated the effects of NO on phagocytosis and superoxide anion (O2-) generation by M phi. The intracellular growth of M. avium Mino was significantly suppressed by NO generated by gamma interferon (IFN-gamma)-stimulated M phi, whereas that of two strains of M. intracellulare (31F093T and KUMS 9007) was not. M. avium Mino was also more susceptible to NO generated chemically by acidification of NO2- than the two M. intracellulare strains. In L-arginine (1 mM)-containing medium, NO release from the M phi assessed by measuring NO2- increased as the concentration of IFN-gamma increased. The enhancing potential of IFN-gamma for NO release became more pronounced when M phi were infected with 31F093T, an NO-resistant strain. A large amount of NO generated by IFN-gamma-stimulated M phi suppressed both phagocytosis and O2- generation by the M phi, especially after infection of the M phi with strain 31F093T. These results indicate that the intracellular growth of MAC is not always inhibited by NO generated by immunologically activated M phi; rather, NO generation induced by infection with an NO-resistant MAC strain suppresses phagocytosis of the M phi, which may allow extracellular spreading of such NO-resistant mycobacteria. Therefore, the pathogenic potential of MAC may be partly attributed to its resistance to NO.
Staphylococcal enterotoxin type A internal deletion mutants: serological activity and induction of T-cell proliferation
Authors: Harris T O , Hufnagle W O , Betley M J
Published: January 2020

Previous findings indicate that the N-terminal region of staphylococcal enterotoxin type A (SEA) is required for its ability to induce T-cell proliferation. To better localize internal peptides of SEA that are important for induction of murine T-cell proliferation, SEA mutants that had internal deletions in their N-terminal third were constructed. A series of unique restriction enzyme sites were first engineered into sea; only one of these changes resulted in an amino acid substitution (the aspartic acid residue at position 60 of mature SEA was changed to a glycine [D60G]). Because the D60G substitution had no discernible effect on serological or biological activity, the sea allele encoding this mutant SEA was used to construct a panel of mutant SEAs lacking residues 3 to 17, 19 to 23, 24 to 28, 29 to 49, 50 to 55, 56 to 59, 61 to 73, 68 to 74, or 74 to 85. Recombinant plasmids with the desired mutations were constructed in Escherichia coli and transferred to Staphylococcus aureus. Staphylococcal culture supernatants containing the mutant SEAs were examined. Western immunoblot analysis with polyclonal anti-SEA antiserum revealed that each of the recombinant S. aureus strains produced a mutant SEA of the predicted size. All the mutant SEAs exhibited increased sensitivity to monkey stomach lavage fluid in vitro, which is consistent with these mutants having conformations unlike that of wild-type SEA or the SEA D60G mutant. In general, deletion of internal peptides had a deleterious effect on the ability to induce T-cell proliferation; only SEA mutants lacking either residues 3 to 17 or 56 to 59 consistently produced a statistically significant increase in the incorporation of [3H]thymidine. In the course of this work, two monoclonal antibodies that had different requirements for binding to SEA in Western blots were identified. The epitope for one monoclonal antibody was contained within residues 108 to 230 of mature SEA. Binding of the other monoclonal antibody to SEA appeared to be dependent on the conformation of SEA.
Antigenic analysis of the major outer membrane protein of Haemophilus somnus with monoclonal antibodies
Authors: Tagawa Y , Haritani M , Ishikawa H , Yuasa N
Published: January 2020

The major outer membrane protein of Haemophilus somnus possesses at least five distinct epitopes. Three surface-exposed epitopes on the major outer membrane protein include a conserved epitope with potential for development of a vaccine and a diagnostic test and two variable epitopes responsible for antigenic differences among strains; the remaining two epitopes are well preserved among strains but not exposed on the cell surface.
Facilitation of complement-dependent killing of the Lyme disease spirochete, Borrelia burgdorferi, by specific immunoglobulin G Fab antibody fragments
Authors: Kochi S K , Johnson R C , Dalmasso A P
Published: January 2020

In the absence of specific antibody, Borrelia burgdorferi is resistant to the bactericidal action of complement, despite the capacity of the spirochete to activate complement. Complement-mediated killing of B. burgdorferi requires the presence of antiborrelial immunoglobulin G (IgG). The effect of bactericidal IgG takes place after formation of the C5 convertase. Therefore, we examined the ability of Fab fragments from bactericidal IgG to mediate killing of B. burgdorferi by complement. The complement-activating domain of IgG, the Fc fragment, was not required for killing of borreliae, as monovalent Fab fragments prepared from immune IgG were also able to mediate killing. However, the killing efficiency of the Fab fragments was less than that of intact IgG, suggesting that the bactericidal activity of IgG is enhanced by divalency. IgG Fab-mediated killing occurred without increased complement activation or C3 fluid-phase consumption. Cell killing proceeded via the classical complement pathway, as no killing of Fab fragment-sensitized cells was observed in human serum deficient in C2. These results demonstrate directly that the bactericidal effect of anti-B. burgdorferi IgG is independent of the complement-activating properties of the antibody.
Variability of osp genes and gene products among species of Lyme disease spirochetes
Authors: Marconi R T , Konkel M E , Garon C F
Published: January 2020

A comparison of the osp operon in 24 Lyme disease isolates, including representatives from each of the three established species, Borrelia burgdorferi, Borrelia garinii, and group VS461, was conducted. Several properties were assessed to determine whether the variability observed in this operon was reflective of the species of the isolate. At the transcriptional level, start site and Northern (RNA) blot analyses were conducted. B. garinii and VS461 group isolates were found to possess an untranslated leader sequence 6 nucleotides longer than that observed in B. burgdorferi isolates. By Northern blot analyses all Lyme disease isolates, except the B. garinii isolate VS102, were found to produce a polycistronic full-length ospAB message. Isolate VS102 produced a truncated message lacking the ospB portion of the transcript. Southern blot analyses suggest that the deletion occurred at the DNA level and was not due to a posttranscriptional event. Analysis of the outer surface proteins by two-dimensional gel electrophoresis demonstrated that the OspB isoelectric points were variable, with the OspB of B. garinii isolates exhibiting a pronounced acidic shift. The reactivity of different isolates to OspA and -B monoclonal antibodies and to a hyperimmune anti-ospAB serum was also variable. The results presented here demonstrate genotypic and phenotypic heterogeneity in the osp operon at both the inter- and intraspecies levels. The results have implications concerning the use of the osp genes or their gene products in the development of a Lyme disease vaccine, as diagnostic markers of Lyme disease, and in subtyping of Lyme disease isolates.
Human submandibular-sublingual saliva promotes adhesion of Candida albicans to polymethylmethacrylate
Authors: Edgerton M , Scannapieco F A , Reddy M S , Levine M J
Published: January 2020

The purpose of this study was to identify components of saliva that interact with Candida albicans in solution and that may modulate adhesion to dental acrylic (polymethylmethacrylate [PMMA]) surfaces. Saliva-derived pellicles extracted from C. albicans blastoconidia and hyphal-form cells mixed with fresh human submandibular-sublingual saliva (HSMSL) contained predominantly high- and low-molecular-weight mucins (MG1 and MG2, respectively). In contrast, few components from fresh human parotid saliva were adsorbed to yeast cells. Coating PMMA beads with HSMSL significantly enhanced (10-fold) adhesion of both growth forms of C. albicans compared with human parotid saliva (2-fold), suggesting a role for mucins in adhesion. HSMSL-enhanced adhesion was completely abolished by preadsorbing HSMSL with either blastoconidia or hyphal-form cells prior to coating PMMA. However, coating PMMA with purified salivary mucins or the addition of mucin to preadsorbed saliva did not enhance or restore adhesion to levels found with fresh HSMSL. Adhesion assays employing guanidine-treated fresh HSMSL showed a complete lack of Candida binding, suggesting that subjecting HSMSL to dissociating conditions may alter a property of salivary mucins crucial for C. albicans adhesion. Protease and glycosidase treatment of yeast cells significantly reduced adhesion to HSMSL-coated PMMA. In addition, preincubation of C. albicans with mannose and galactose inhibited adhesion to HSMSL-coated PMMA. These results suggest that mucins may play a role in C. albicans adhesion to saliva-coated PMMA and that a glycoprotein on the yeast surface may be involved in these events.
Specificity of Cryptococcus neoformans factor sera determined by enzyme-linked immunosorbent assay and dot enzyme assay
Authors: Belay T , Cherniak R , Shinoda T
Published: January 2020

An indirect enzyme-linked immunosorbent assay (ELISA) and a dot enzyme assay (DEA) were used to determine the specificities of Cryptococcus neoformans factor sera to serotype type-specific capsular polysaccharides, glucuronoxylomannans (GXMs). Pure and chemically characterized GXMs were obtained from representative isolates of C. neoformans serotypes A, B, C, and D. Distinctive specificity patterns and quantitative differences were observed for each factor serum when the selected GXMs were studied by ELISA. The specificity patterns for each factor serum determined by DEA almost completely paralleled the ELISA results. The serotype specificities demonstrated by ELISA and DEA were similar to previously reported results that were obtained by slide agglutination studies of whole cells. On the basis of the ELISA and DEA activity patterns, factor sera 5, 6, and 8 were specific for serotypes B, C, and D, respectively; factor serum 1 was strongly reactive to all serotypes; factor serum 2 was specific for serotypes A, B, and D; factor serum 3 was specific for serotypes A and D; and factor serum 4 was specific for serotypes B and C. The specificity of factor serum 7 for serotype A was demonstrated by DEA only. Structural variation was indicated among the serotype C isolates studied because a unique activity pattern versus factor serum 6 was observed for each isolate. The quantitative differences in the activity of the GXMs from five serotype C isolates suggest that mannopyranoside residues substituted O-2 and O-4 with xylose are essential elements of the determinant responsible for the observed activity of factor 6. No significant differences in activity patterns and specificities of factor serum 6 were observed when O-deacetylated GXMs were substituted for the native GXMs. Our results show that ELISA and DEA are valuable techniques for the serological analysis of cryptococcal factor sera and GXMs.
The RTX cytotoxin-related FrpA protein of Neisseria meningitidis is secreted extracellularly by meningococci and by HlyBD+ Escherichia coli
Authors: Thompson S A , Sparling P F
Published: January 2020

Neisseria meningitidis produces proteins (FrpA and FrpC) related to the RTX cytotoxin family. In meningococcal strain FAM20 these proteins were both localized in the outer membrane and secreted into the extracellular medium. An Escherichia coli strain with wild-type hemolysin secretion genes hlyB and hlyD and containing a cloned frpA gene secreted FrpA, whereas an isogenic hlyBD mutant strain did not. Low-stringency DNA hybridization revealed hlyBD-like sequences in N. meningitidis FAM20, suggesting that a similar RTX secretion system exists in meningococci. Structural features found at the C termini of other RTX proteins and thought to be important for their secretion were also found at the C terminus of FrpA. The secretion of FrpA from E. coli by heterologous RTX transport proteins further demonstrates the relation of the FrpA protein to RTX toxins.