bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of experimental genital mycoplasmosis on pregnancy outcome in Sprague-Dawley rats
Authors: Steiner D A , Brown M B
Published: January 2020

Specific-pathogen-free (SPF) female Sprague-Dawley rats were infected by intravaginal inoculation with 3 x 10(7) CFU of Mycoplasma pulmonis X1048 in 0.1 ml of Frey's broth or with an equal volume of sterile Frey's broth. A minimum of 10 days postinfection, rats were bred to noninfected males. Rats were necropsied at days 11, 14, and 18 of gestation and within 24 h of parturition. Throughout pregnancy, at least 50% of rats remained infected in the lower genital tract. At parturition, the major site of colonization was the respiratory tract (P = 0.02). M. pulmonis was not isolated from any site of any control rat. Pregnancy outcome was adversely affected by infection with M. pulmonis. Infected rats had significantly smaller litter sizes at day 18 of gestation (P < or = 0.01) and at term (P < or = 0.004). No statistically significant differences among the gestational stages in infected rats were noted for litter size. Total litter weight is a reflection of individual pup weight and of the number of pups born. Therefore, it was obvious that infected rats would have a significantly lower (P < or = 0.008) total litter weight than noninfected controls. However, when individual pup weights were considered, infected pups (n = 49) also had significantly lower (P < or = 0.0001) birth weights than did noninfected controls (n = 68). The incidence of an adverse pregnancy outcome at term (stillbirths, macerated fetuses, or resorptions) was higher (P < or = 0.01) in infected rats than in noninfected control rats. No stillborn pups or macerated fetuses were observed in any control term rats (n = 5). All control rats had live-born pups. Three infected rats had no live-born offspring. Resorptions were more common in infected rats than in control rats (P < or = 0.01). The mean number of resorptions per rat was greater in rats which went to term than in rats necropsied during gestation, indicating that the severity of disease was progressive. The rat is frequently the laboratory animal of choice for a wide variety of reproductive studies, and the experimental parameters that are most often measured (litter size, pup weight, and neonatal survival) were all adversely affected by genital mycoplasmosis. Genital mycoplasmosis is important as an animal model for the interaction of infectious agents and the host during pregnancy as well as in its own right as a confounding variable affecting research projects which use the rat as a model to study reproductive function and physiology.
Ability of Pseudomonas pseudomallei malleobactin to acquire transferrin-bound, lactoferrin-bound, and cell-derived iron
Authors: Yang H , Kooi C D , Sokol P A
Published: January 2020

The ability of malleobactin to mobilize iron from transferrin and lactoferrin was examined in an equilibrium dialysis assay in the absence of bacteria. Malleobactin was capable of removing iron from both transferrin and lactoferrin at pH values of 7.4, 6.0, and 5.0. However, the levels of iron mobilization were greater for transferrin than for lactoferrin at all the pH values used in the assay. The ability of Pseudomonas pseudomallei to acquire iron from 30% iron-saturated transferrin and K562 human erythroleukemic cells was compared in parallel cultures as described previously (J. H. Brock, P. H. Williams, J. Liceaga, and K. G. Woldridge, Infect. Immun. 59:3185-3190, 1991). P. pseudomallei U7 tended to acquire iron from transferrin. In contrast, P. aeruginosa PAO and P. cepacia Pc275C acquired iron from both sources. P. cepacia H1721, which does not produce detectable siderophores, but can utilize malleobactin, pyochelin, and azurechelin as iron sources, was used in a similar experiment. Addition of malleobactin resulted in iron uptake only from transferrin, whereas pyochelin and azurechelin promoted iron uptake from both sources. When the siderophores were incubated with K562 cells alone, malleobactin was less efficient at removing iron from cells than pyochelin and azurechelin. It was also determined that malleobactin was less effective in binding to or entering cells than pyochelin and azurechelin. These results suggest that malleobactin can acquire iron more effectively from host proteins than from cellular sources. Pyochelin and azurechelin can acquire cell-derived iron in addition to iron bound to host proteins.
Cellular and humoral immune responses induced in cattle by vaccination with Babesia divergens culture-derived exoantigens correlate with protection
Authors: Valentin A , Precigout E , L'Hostis M , Carcy B , Gorenflot A , Schrevel J
Published: January 2020

Previous results with the Babesia divergens gerbil vaccination model were extended in studies with cattle. Two calves were vaccinated with culture-derived B. divergens exoantigens, and two others were treated with control supernatant; both preparations were adjuvanted with Quil-A saponin. A parasite-specific humoral response was observed after the first vaccine injection and was boosted by two succeeding vaccine injections. Sera from the two vaccinated calves immunoprecipitated eight major parasitic proteins (with molecular masses ranging between 17 and 110 kDa) whose patterns were close to those observed in gerbil vaccine assays. The cellular immune response, monitored by lymphoproliferation assays, was slightly delayed in comparison with the humoral response; a significant proliferation occurred only after the second vaccine injection. Mononuclear cell proliferation was dose dependent in the presence of (i) lysates of B. divergens-parasitized erythrocytes, (ii) exoantigens of the whole supernatant, or (iii) protective exoantigens of two low-molecular-mass fractions obtained after supernatant gel filtration chromatography. An infectious challenge was administered 3 weeks after the third vaccine injection, with 3.6 x 10(10) B. divergens-parasitized erythrocytes. Erythrocyte count, rectal temperature, and parasitemia of the animals were monitored daily until they returned to initial values. All parameters indicated that the exoantigens induced protection from B. divergens infection for the two vaccinated calves.
Localization of myosin heavy chain A in the human pathogen Entamoeba histolytica
Authors: Rahim Z , Raymond-Denise A , Sansonetti P , Guillen N
Published: January 2020

To recognize myosin II in trophozoites of the human pathogen Entamoeba histolytica, a specific antimyosin polyclonal serum was raised against a fusion protein consisting of a 146-amino-acid fragment of the myosin II heavy chain A of E. histolytica (MhcA) fused with beta-galactosidase. The hybrid protein was encoded by a chimera gene formed by a DNA fragment, from the mhcA gene, amplified by polymerase chain reaction and fused with the lacZ gene of Escherichia coli. Polymerase chain reaction-amplified DNA is located within the region encoding the tail domain of myosin. This antibody recognized a 250-kDa protein in extracts of E. histolytica trophozoites. Confocal microscope analysis of antibody-labelled trophozoites indicated that MhcA localizes at the posterior pole of locomoting cells and concentrates within the uroid. These results might indicate that MhcA is involved in movement and in the uroid formation which help amoebas to escape the host immune response. These data are the first evidence indicating that myosin exists in E. histolytica. In addition, two other peptides were found in myosin-enriched extracts of amoebas, indicating that other myosins may be present in this parasite.
Comparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or iron-regulated promoter systems
Authors: Acheson D W , Calderwood S B , Boyko S A , Lincicome L L , Kane A V , Donohue-Rolfe A , Keusch G T
Published: January 2020

Shiga-like toxin I (SLT-I) B-subunit expression was examined by using the trc promoter in two different constructs, pSBC32 and pSBC54, in which 710 bp of DNA downstream of the B subunit in pSBC32 was deleted. The trc promoter in pSBC54 was replaced also with the SLT-I iron-regulated promoter to create a third plasmid, pSBC61. SLT-I B-subunit expression was examined from all three plasmids following transfer into Escherichia coli JM105 and the cholera toxin A-subunit gene deletion mutant Vibrio cholerae 0395-N1. The SLT-I B subunit was expressed from all constructs. pSBC61 was regulated by elemental iron and produced equivalent amounts of SLT-I B subunit from both E. coli and V. cholerae. In contrast to the cholera toxin B subunit, virtually all released into the medium, the SLT-I B subunit was predominantly cell associated in the pSBC61 constructs. Both pSBC32 and pSBC54 were inducible with isopropyl-beta-D-thiogalactopyranoside (IPTG) in the E. coli background but not the V. cholerae background; however, when E. coli cultures were allowed to grow for 24 h, the yield of SLT-I B subunit was not increased by IPTG induction. Both pSBC32 and -54 expressed more SLT-I B subunit in the V. cholerae host than in the E. coli host. Scale-up to a 9.9-liter fermentor culture of V. cholerae 0395 N1 (pSBC32) resulted in the isolation of 220 mg of SLT-I B. The purified B subunit was identical, in terms of binding to Vero cells, stoichiometry after chemical cross-linking, and ability to inhibit cytotoxicity of intact Shiga toxin, to native SLT-I B subunit from E. coli O157:H7.
Listeria monocytogenes infection in beta 2 microglobulin-deficient mice
Authors: Roberts A D , Ordway D J , Orme I M
Published: January 2020

beta 2 microglobulin-deficient mice, in which the beta 2m gene has been disrupted by homologous recombination, lack functional CD8 T cells and are able to contain but not resolve an intravenous immunizing inoculum of Listeria monocytogenes. We present evidence that compensatory immunity in such immunodeficient mice was mediated by a population of gamma delta T-cell receptor-positive cells; in contrast, neither CD4 cells nor natural killer cells appeared to play any part in this process. These data further support the emerging hypothesis that immune cells other than those bearing the alpha beta T-cell receptor type can also play an important role in acquired resistance to listeriosis.
In vitro model of adhesion and invasion by Bacillus piliformis
Authors: Franklin C L , Kinden D A , Stogsdill P L , Riley L K
Published: January 2020

An in vitro model of Bacillus piliformis infection was developed to investigate the mechanisms of adhesion and internalization of this obligate intracellular bacterium. Adhesion and internalization events were examined by electron microscopic evaluation of infected Caco-2 cell monolayers. A few bacteria were identified in apical surface invaginations and in vacuoles subjacent to the apical surface, whereas the majority of bacteria were observed free within the cytoplasm, suggesting that B. piliformis entered epithelial cells via a phagocytic process and rapidly escaped the phagosome. To confirm that host cell phagocytosis was involved in entry of B. piliformis into mammalian cells, Intestine 407 cells were treated with the phagocytic inhibitor cytochalasin D, infected with B. piliformis, and evaluated for bacterial internalization by double-fluorescence labeling. The results showed decreased intracellular bacteria, suggesting that internalization was dependent on host cell microfilament function. To examine the role of B. piliformis in internalization, growth of live and Formalin-killed bacteria was compared. Dead bacteria were not internalized, suggesting that B. piliformis actively participates in internalization. B. piliformis appears to enter host cells by a bacterially directed phagocytic process. The in vitro system described should prove invaluable in further investigations of B. piliformis pathogenic mechanisms.
Temporal relationship of cytokine release by peripheral blood mononuclear cells stimulated by the streptococcal superantigen pep M5
Authors: Kotb M , Ohnishi H , Majumdar G , Hackett S , Bryant A , Higgins G , Stevens D
Published: January 2020

We undertook this study to determine the quality, quantity, and temporal relationship of pep M5-induced cytokine release. The ability of pep M5 to stimulate interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) production by a T-cell-depleted, monocyte- and B-cell-enriched cell population was dependent on the presence of T cells. The requirement for T cells could be met by addition of exogenous gamma interferon (IFN-gamma). In the presence of IFN-gamma, pep M5 induced the release of TNF-alpha, IL-1, and IL-6, TNF-alpha levels peaked at 24 h, while IL-1 and IL-6 levels peaked at 48 h. pep M5 induced T cells to produce IFN-gamma, which may have accounted for the ability of the super antigen to induce the production of IL-1, IL-6, TNF-alpha, and TNF-beta by peripheral blood mononuclear cells (PBMC). The addition of excess IFN-gamma to cultures of pep M5 and PBMC did not further increase the release of these cytokines at 24 and 48 h but resulted in sustained higher levels at 72 h. Interestingly, TNF-beta production occurred only in the presence of pep M5 and exogenous IFN-gamma. The ability of pep M5 to induce cytokine production was compared with that of a potent super antigen, staphylococcal enterotoxin B (SEB). SEB was a 2- to 14-fold-more-potent inducer of IFN-gamma production. Furthermore, the profile of cytokine released by PBMC in response to this super antigen mimicked that seen with pep M5 in the presence of exogenous IFN-gamma. In conclusion, pep M5 induces the production of cytokines that are involved in immune regulation and inflammation. These cytokines also play a major role in human T-cell responses to this super antigen.
Safety and immunogenicity in human volunteers of a chloroform-methanol residue vaccine for Q fever
Authors: Fries L F , Waag D M , Williams J C
Published: January 2020

Current Q fever vaccines, consisting of Formalin-inactivated phase I whole Coxiella burnetii, are highly efficacious in preventing disease in high-risk settings but are associated with a risk of unacceptable local reactions in previously immune individuals and require cumbersome preliminary immunologic evaluation of potential vaccinees. A vaccine prepared from the residue of chloroform-methanol extraction of phase I Henzerling strain C. burnetii (CMR) has been shown to be less reactogenic but still immunogenic and protective in small animals and sheep. In a placebo-controlled trial, we immunized 35 healthy adults unscreened for markers of prior C. burnetii immunity with a single subcutaneous CMR dose of 30, 60, 120, or 240 micrograms. None of those receiving the 30- or 60-micrograms CMR dose and none of the placebo recipients experienced any adverse effects. Five of 15 120-micrograms dose CMR recipients complained of transient discomfort in the inoculated arm; erythema or induration of > or = 100 mm2 was noted in three and four, respectively, and two had malaise and low-grade fever (< 101 degrees F, orally). No 240-micrograms dose vaccinee reported limb discomfort, but 7 of 10 had erythema and/or induration of > or = 100 mm2 (P < 0.001 versus placebo). Two reported malaise, and one had low-grade fever. All adverse effects were self-limited. Serum immunoglobulin M responses were optimally detected with CMR antigen and occurred in 50, 60, 73, and 90% of recipients of the 30-, 60-, 120-, and 240-micrograms doses, respectively; results with phase I whole-cell antigen were similar. Serum immunoglobulin G responses were best detected with phase II antigen and were seen in 20, 20, and 40% of those receiving the 60-, 120-, and 240-micrograms doses, respectively. Peripheral blood T-cell proliferative responses to C. burnetii recall antigens were transient and of low magnitude but were seen with CMR antigen in 33% of 120-micrograms dose recipients and 40% of 240-micrograms dose recipients. Data from this study and those from comparative-efficacy trials in primates should provide the basis for field trials of the CMR vaccine.
Toxicity of pneumolysin to pulmonary alveolar epithelial cells
Authors: Rubins J B , Duane P G , Clawson D , Charboneau D , Young J , Niewoehner D E
Published: January 2020

Mortality during the first several days of pneumococcal pneumonia has not decreased appreciably over the past 30 years, despite the widespread use of antibiotics. Disruption of the alveolar epithelial barrier is likely an initial step in the pathogenesis of pneumococcal pneumonia. We report that soluble factors from Streptococcus pneumoniae can directly injure isolated rat alveolar epithelial cells. Using biochemical and immunological techniques, we identified pneumolysin as a major soluble S. pneumoniae toxin for alveolar epithelial cells. Alveolar epithelial cells at 24 or 72 h after isolation were equally sensitive to injury by purified pneumolysin. Purified pneumolysin substantially increased alveolar permeability in an isolated perfused rat lung model. Electron microscopy revealed that instilled pneumolysin caused widespread lung injury, primarily to type I alveolar epithelial cells. Pneumolysin toxicity to alveolar epithelial cells may be important in the pathogenesis of acute lung injury during pneumococcal pneumonia and may facilitate pneumococcal bacteremia.
Isolation and biochemical characterization of the iC3b receptor of Candida albicans
Authors: Alaei S , Larcher C , Ebenbichler C , Prodinger W M , Janatova J , Dierich M P
Published: January 2020

In an effort to identify the protein structure on Candida albicans, pseudohyphal forms which had been shown earlier to bind human iC3b, a protein of about 42 kDa (p42), were obtained from lysates of pseudohyphal forms by absorption with C3(H2O)-Sepharose. An antiserum raised in rabbits against this protein effectively inhibited adherence of sheep erythrocytes carrying iC3b (EAC3bi) to pseudohyphal forms. p42 cross-reacted with OKM-1, a monoclonal antibody directed against the human complement receptor type 3 (CR3, CD11b). This protein, p42, was designated p42-CR3. The antiserum against p42-CR3 was used for further purification of lysates by affinity chromatography. Three proteins of 66, 55, and 42 kDa were isolated. All were recognized by OKM-1 in immunoblots (p66-, p55-, and p42-CR3). The different proteins were separated and treated with neuraminidase and endoglycosidase F. Almost complete deglycosylation of the p66-CR3 protein was obtained after treatment with neuraminidase, indicating a high degree of glycosylation. Neuraminidase also had an effect on p55-CR3, but not on p42-CR3. Endoglycosidase F did not alter any of the three proteins. In ligand blots, p42-CR3 bound C3(H2O), C3b, and iC3b but not C3d; p55-CR3 clearly reacted with C3(H2O) and weakly reacted with C3b and iC3b. p66-CR3 never showed reactivity. It is suggested that p55 and p66 represent glycosylated forms of p42-CR3. Although C. albicans CR3 and human CR3 cross-react and bind identical ligands, the two receptors differ in structure.
Roles of Ca2+ and F-actin in intracellular aggregation of Chlamydia trachomatis in eucaryotic cells
Authors: Majeed M , Gustafsson M , Kihlström E , Stendahl O
Published: January 2020

The effect of intracellular free Ca2+ ([Ca2+]i) on the intracellular aggregation of Chlamydia trachomatis serovars L2 and E in McCoy and HeLa cells is investigated. Loading the cells with the Ca2+ chelator MAPT/AM (1,2-bis-5-methyl-amino-phenoxylethane-N,N-n'-tetra-acetoxymethyl acetate), thereby decreasing the [Ca2+]i from 67 to 19 nM, decreased the number of cells with a local aggregation of chlamydiae in a dose-dependent manner. Neither the attachment nor the uptake of elementary bodies (EBs) was, however, affected after depletion of Ca2+ from the cells. There was no significant difference in the level of measured [Ca2+]i between infected and uninfected cells. Reducing the [Ca2+]i also significantly inhibited chlamydial inclusion formation. Differences in the organization of the actin filament network were observed in response to [Ca2+]i depletion. In Ca(2+)-depleted cells, where few EB aggregates were formed, few local accumulations of F-actin were observed in the cytosol. These results suggest that the aggregation of EBs in eucaryotic cells requires a normal homeostasis of intracellular Ca2+. By affecting F-actin reorganization and putatively certain Ca(2+)-binding proteins, [Ca2+]i plays a vital role in the infectious process of chlamydiae.
Gamma interferon cooperates with lipopolysaccharide to activate mouse splenic macrophages to an antihistoplasma state
Authors: Lane T E , Wu-Hsieh B A , Howard D H
Published: January 2020

Inhibition of the intracellular growth of Histoplasma capsulatum by murine resident red pulp splenic macrophages was examined. Splenic macrophages, unlike resident peritoneal macrophages, required a prolonged preincubation (18 h) with recombinant murine gamma interferon (rMuIFN-gamma) for activation. To be fully activated, the splenic macrophages required incubation with rMuIFN-gamma in combination with 0.1 microgram of lipopolysaccharide (LPS) per ml. Splenic macrophages stimulated with rMuIFN-gamma, LPS, or rMuIFN-gamma and LPS produced tumor necrosis factor alpha (TNF-alpha), but recombinant murine TNF-alpha (rMuTNF-alpha) did not activate macrophages when used alone or as a second signal with rMuIFN-gamma. Anti-TNF-alpha antibody did not block IFN-gamma-LPS activation of splenic macrophages to any significant extent. One hundred micromolar ferrous sulfate antagonized IFN-gamma-LPS activation of splenic macrophages, indicating that iron was involved in the fungistatic activity of cytokine-stimulated phagocytes. Our results indicate that (i) splenic macrophages differ significantly from peritoneal macrophages in their requirements for activation and (ii) the mechanism by which splenic macrophages exert their antifungal effects involves iron.
Identification of a salivary agglutinin-binding domain within cell surface adhesin P1 of Streptococcus mutans
Authors: Crowley P J , Brady L J , Piacentini D A , Bleiweis A S
Published: January 2020

DNA encoding the alanine-rich region (A-region) of the cell surface adhesin, P1, from Streptococcus mutans was subcloned and expressed as a fusion protein with the maltose-binding protein (MBP) of Escherichia coli. The A-region fusion protein was shown to competitively inhibit both adherence of S. mutans to salivary agglutinin-coated hydroxyapatite and fluid-phase agglutinin-mediated aggregation of this organism. MBP alone or an MBP-paramyosin fusion protein was not inhibitory. Proteolytic cleavage of the fusion protein into its component moieties, MBP and A-region, resulted in breakdown of the A-region into three main fragments. Western immunoblot analysis of calcium-dependent agglutinin binding to this preparation revealed binding specificity for a 28-kDa fragment. Thus, the A-region of P1 is an important domain which interacts directly with salivary agglutinin, and this interaction interferes with both the aggregation and the adherence mechanisms in vitro.
Murine T-cell response to native and recombinant protein antigens of Rickettsia tsutsugamushi
Authors: Hickman C J , Stover C K , Joseph S W , Oaks E V
Published: January 2020

A polyclonal T-cell line with TH1 characteristics was used to assess the murine cellular immune response to native and recombinant Rickettsia tsutsugamushi antigens. Proliferation of this T-cell line was observed in response to numerous native antigen fractions, which indicates that the murine T-helper-cell response is directed at multiple scrub typhus antigens with no apparent antigenic immunodominance. Subsequent analysis of recombinant R. tsutsugamushi antigens made it possible to identify a 47-kDa scrub typhus antigen (Sta47) that was stimulatory for the polyclonal T-cell line. Recombinant clones encoding 56-, 58-, and 110-kDa antigens (Sta56, Sta58, and Sta110, respectively) were unable to induce proliferation of this T-cell line. DNA sequence analysis of the cloned rickettsial insert encoding the Sta47 protein revealed the presence of four open reading frames potentially encoding proteins of 47, 30, 18, and 13 kDa. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated and eluted fractions of lysates from the recombinant HB101(pRTS47B4.3) demonstrated that the fractions containing the 47-kDa protein as well as those containing proteins less than 18 kDa were stimulatory. Selected synthetic amphipathic peptides derived from the Sta47 antigen sequence identified a 20-amino-acid peptide that gave a 10-fold increase in T-cell proliferation over a control malarial peptide of similar length. Recognition of the 47-kDa antigen by a T-cell line with TH1 characteristics implicates this protein as one of potential importance in protection studies and future vaccine development.
Haemophilus ducreyi adheres to but does not invade cultured human foreskin cells
Authors: Alfa M J , Degagne P , Hollyer T
Published: January 2020

Haemophilus ducreyi is the etiologic agent of the localized genital ulcer disease known as chancroid. The pathogenesis of this organism is poorly understood. The role of attachment in the disease process has not been evaluated. In this study, 125I-H. ducreyi was used to quantitatively evaluate the interaction of virulent and avirulent H. ducreyi strains with human foreskin cells. Using this in vitro model system, we demonstrated that, at 22 and 35 degrees C, the attachment of virulent H. ducreyi 35000 to human foreskin cells was significantly more marked than that of avirulent H. ducreyi A77. Although H. ducreyi penetrated between human foreskin cells, internalization was not a major component. Our competition assay data suggest that the attachment mechanism of H. ducreyi may be similar to that of Neisseria gonorrhoeae. We speculate that the attachment and microcolony formation of virulent H. ducreyi may provide a mechanism for bacterial localization and evasion of host defenses.
Effects of in vitro and in vivo growth conditions on expression of type 8 capsular polysaccharide by Staphylococcus aureus
Authors: Lee J C , Takeda S , Livolsi P J , Paoletti L C
Published: January 2020

Type 8 capsular polysaccharide (CP8) is widely prevalent among clinical isolates of Staphylococcus aureus, but the role that the capsule plays in the pathogenesis of staphylococcal infections is unclear. This study was performed to identify growth conditions that would optimize the production of CP8 and to determine whether enhanced CP8 expression would influence staphylococcal virulence. S. aureus Becker grown in a chemically defined broth medium with < 1 microM ferric nitrate produced up to eightfold more CP8 per milligram of biomass than did bacteria cultivated in the same medium containing 20 microM ferric nitrate. The bacteria produced > 350-fold more cell-associated CP8 per milligram of biomass when grown on the surface of Columbia agar than when grown in Columbia broth. Most of the CP8 produced by broth-grown cells was secreted into the culture medium. S. aureus cultivated on the surface of nitrocellulose membranes floating on Columbia broth produced levels of CP8 similar to those produced by cells grown on Columbia agar. Similarly, bacteria harvested from endocardial vegetations of rabbits infected with S. aureus produced high levels of CP8. These results indicate that staphylococci grown on surfaces, both in vitro and in vivo, produce larger quantities of cell-associated CP8 than those grown in liquid cultures. However, no differences were observed in the 50% lethal dose for mice of strain Becker grown on solid medium (high levels of capsule expression) or in liquid medium (low levels of capsule expression).
Immune responses to Streptococcus sobrinus surface protein antigen A expressed by recombinant Salmonella typhimurium
Authors: Doggett T A , Jagusztyn-Krynicka E K , Curtiss R
Published: January 2020

In this study, we used a vaccine strain of Salmonella typhimurium to express antigenic determinants of the SpaA antigen of Streptococcus sobrinus, which is involved in the caries-forming process. We cloned either a single repeat (pYA2901) or three tandem repeats (pYA2905) of the 0.48-kb fragment of the spaA gene, which codes for an important component of the SpaA protein, plus a 1.2-kb minor antigenic determinant and measured the resulting immune responses to SpaA in orally immunized BALB/c mice. The single or triple repeat of the spaA gene fragment was inserted into the Asd+ vector pYA292 and was transformed into the S. typhimurium delta cya delta crp vaccine strain chi 4072 containing delta asd in the chromosome. Female BALB/c mice were then orally immunized with two doses of the S. typhimurium containing either of the two SpaA constructs, and the immune responses to the expressed SpaA protein were assessed. Significant serum immunoglobulin G (IgG) anti-SpaA titers were detected in mice immunized with chi 4072(pYA2905) but not chi 4072(pYA2901). Salivary anti-SpaA IgA titers were minimal and were only detected in mice immunized with S. typhimurium expressing the SpaA encoded by pYA2905. Intestinal anti-SpaA IgA titers, however, were detected in both groups of mice, particularly in mice immunized with chi 4072(pYA2905). An oral booster 26 weeks after the initial series of immunizations resulted in increased serum IgG titers in both chi 4072(pYA2901)- and chi 4072(pYA2905)-immunized animals, particularly in the chi 4072(pYA2905)-immunized animals. No anamnestic IgA response was detected in the saliva following the booster immunization.
Sequence divergence in two tandemly located pilin genes of Eikenella corrodens
Authors: Tønjum T , Weir S , Bøvre K , Progulske-Fox A , Marrs C F
Published: January 2020

Eikenella corrodens normally inhabits the human respiratory and gastrointestinal tracts but is frequently the cause of abscesses at various sites. Using the N-terminal portion of the Moraxella nonliquefaciens pilin gene as a hybridization probe, we cloned two tandemly located pilin genes of E. corrodens 31745, ecpC and ecpD, and expressed the two pilin genes separately in Escherichia coli. A comparison of the predicted amino acid sequences of E. corrodens 31745 EcpC and EcpD revealed considerable divergence between the sequences of these two pilins and even less similarity to EcpA and EcpB of E. corrodens type strain ATCC 23834. EcpC from E. corrodens 31745 displayed high degrees of homology to the pilins of Neisseria gonorrhoeae and Pseudomonas aeruginosa. EcpD from E. corrodens 31745 showed the highest homologies with the pilin of one of the three P. aeruginosa classes, whereas EcpA and EcpB of strain ATCC 23834 most closely resemble Moraxella bovis pilins. These findings raise interesting questions about potential genetic transfer between different bacterial species, as opposed to convergent evolution.
Listeria monocytogenes-induced gamma interferon secretion by intestinal intraepithelial gamma/delta T lymphocytes
Authors: Yamamoto S , Russ F , Teixeira H C , Conradt P , Kaufmann S H
Published: January 2020

gamma/delta T cells represent a major proportion of intestinal intraepithelial lymphocytes (IEL), and it has been suggested that these IEL serve as a first immune barrier against microbial invasion and that they do so by destroying infected epithelial cells. In the present study, we confirm that both alpha/beta and gamma/delta IEL from naive mice express potent cytotoxicity and produce gamma interferon (IFN-gamma) after T-cell receptor (TCR) engagement by specific monoclonal antibodies (MAb). Intraperitoneal administration of the anti-gamma/delta TCR MAb GL3 caused downregulation of the gamma/delta TCR in IEL, and IEL from gamma/delta TCR-modulated mice failed to express cytotoxic activity and to secrete IFN-gamma after gamma/delta TCR engagement. In contrast, alpha/beta IEL from such mice were still cytolytic and secreted IFN-gamma. Mice were infected orally with virulent Listeria monocytogenes at doses which caused bacterial invasion through the intestinal epithelia. Although alpha/beta and gamma/delta IEL from these mice expressed high cytolytic activities in antibody-redirected killer assays, target cells pulsed with listerial antigens were not lysed. In contrast, IFN-gamma secretion by IEL from L. monocytogenes-infected mice was induced not only by anti-TCR MAb but also by target cells pulsed with listerial antigens, whereas irrelevant antigens, including heat shock protein 60, did not induce IFN-gamma secretion. Furthermore, the number of IFN-gamma-secreting IEL, as assessed by the enzyme-linked immunospot technique, was increased during listeriosis. gamma/delta TCR modulation by GL3 administration abrogated antigen-induced IFN-gamma secretion by IEL from infected mice. These findings suggest that L. monocytogenes induced IFN-gamma secretion by gamma/delta IEL from mice suffering from intestinal L. monocytogenes infection and invasion. Thus, the data provide evidence for a role of IFN-gamma-secreting IEL in local resistance against listeriosis and perhaps other food-borne diseases.
Lipopolysaccharide restores anti-Candida albicans growth inhibition activity of polymorphonuclear neutrophils from retrovirus-immunosuppressed mice
Authors: Yamamoto Y , Specter S , Friedman H
Published: January 2020

It has been documented that the immune function of leukocytes may be markedly suppressed after infection of mice with the murine retrovirus Friend leukemia virus (FLV). Antimicrobial activity of polymorphonuclear neutrophils (PMNs) against Candida albicans is impaired after retrovirus infection of mice, and this occurs as early as 3 days after infection of genetically susceptible BALB/c mice. By 2 weeks after infection, there was essentially very little growth inhibition of C. albicans by PMNs from the FLV-infected mice. However, when bacterial lipopolysaccharide (LPS), a known activator of macrophages and PMNs, was added to PMNs from the FLV-infected mice, anti-C. albicans activity was restored to normal levels. This restoration of anti-C. albicans activity of FLV-infected mouse PMNs was observed after stimulation with as little as 0.01 micrograms of LPS per ml. The data obtained show that the impaired antimicrobial function of PMNs from retrovirus-infected mice can be readily restored by a biological response modifier such as bacterial LPS.
Inhibition of human neutrophil migration in vitro by low-molecular-mass products of nontypeable Haemophilus influenzae
Authors: Cundell D R , Taylor G W , Kanthakumar K , Wilks M , Tabaqchali S , Dorey E , Devalia J L , Roberts D E , Davies R J , Wilson R
Published: January 2020

Nontypeable Haemophilus influenzae commonly causes infections in the lower and upper respiratory tract, although the mechanisms of its colonization and persistence in the airways are unclear. Culture filtrates from six clinical isolates of this bacterium were assessed for their abilities to influence neutrophil function in vitro. Each culture filtrate was assessed on six separate occasions with neutrophils obtained from six different donors. During the log and early stationary phases of growth (0 to 18 h), culture filtrates contained primarily neutrophil chemokinetic activity but no activity affecting neutrophil migration toward the chemotactic factors N-formyl-L-methionyl-L-leucyl-L-phenylalanine and leukotriene B4. In contrast, filtrates obtained after 24 h of culture contained factors which inhibited neutrophil migration toward both of these chemotactic factors. This chemotaxis-inhibitory activity persisted between 24 and 72 h of bacterial culture, and it was not associated with the presence of either chemotactic or chemokinetic activity as assessed by checkerboard analysis. Gel filtration of pooled 72-h filtrates yielded three major peaks of chemotaxis-inhibitory activity. Endotoxin was present together with two other low-molecular-mass hydrophobic factors of approximately 8 and 2 kDa. These low-molecular-mass factors are chloroform insoluble and heat stable, and they are inactivated by protease, periodate, and diborane reduction. Activity was completely retained on a wheat germ agglutinin column, and it could be eluted with N-acetyl-D-glucosamine. These data suggest that inhibitory activity is associated with N-acetyl-D-glucosamine-containing glycopeptides, possibly derived from the bacterial cell wall. The production of these compounds may contribute to the persistence of this bacterium in vivo by inhibiting neutrophil chemotaxis in the microenvironment of the respiratory mucosa.
Effects of Listeria monocytogenes and Yersinia enterocolitica on cytokine gene expression and release from human polymorphonuclear granulocytes and epithelial (HEp-2) cells
Authors: Arnold R , Scheffer J , König B , König W
Published: January 2020

The gene expression and cytokine release of the proinflammatory cytokines interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) after infection of human epithelial cells (HEp-2 cells) and polymorphonuclear granulocytes (PMNs) were investigated by using isogenic pairs of Listeria monocytogenes and Yersinia enterocolitica strains. By polymerase chain reaction-assisted mRNA amplification and RNA dot blot analysis, we showed that PMNs and HEp-2 cells expressed enhanced levels of mRNA encoding IL-1 beta, IL-6, and TNF-alpha after bacterial infection. Concomitant with the enhanced mRNA level, an increased secretion rate of IL-1 beta, IL-6, and TNF-alpha from PMNs as assessed by enzyme-linked immunosorbent assay was observed. HEp-2 cells after infection also released IL-6 and TNF-alpha into the cell supernatant, while no IL-1 beta release was detected. Cellular coincubation experiments were carried out with Transwell chambers. Our studies revealed that the coculture of PMNs and HEp-2 cells led to an increased IL-1 beta and IL-6 release. In contrast, after infection with the invasive bacteria, reduced levels of TNF-alpha were measured. Our data show that PMNs secrete the proinflammatory cytokines IL-1 beta, IL-6, and TNF-alpha within some hours after infection with L. monocytogenes and Y. enterocolitica and that cellular interactions with epithelial cells alone via soluble mediators influence the net amount of released proinflammatory cytokines.
Evidence that mannans of Candida albicans are responsible for adherence of yeast forms to spleen and lymph node tissue
Authors: Kanbe T , Han Y , Redgrave B , Riesselman M H , Cutler J E
Published: January 2020

We have described a unique binding system between Candida albicans yeast-form cells and the marginal zone of mouse spleen (16). The chemical nature of the fungal adhesin(s) involved in this binding phenomenon was examined. A fraction obtained by 2-mercaptoethanol extraction (2-ME extract) of fungal cells caused a dose-response inhibition of yeast cell adherence to splenic marginal zone sites and also to subcapsular and medullary sinuses of mouse popliteal lymph nodes. Latex beads coated with the 2-ME extract showed a pattern of spleen and lymph node tissue binding identical to that observed with yeast cells. The extracted adhesins retained their binding activity in vivo. When 0.5 mg of the 2-ME extract was given intravenously to mice, spleen tissue removed up to 3 h later showed over 80% inhibition of yeast cell binding to the spleen marginal zone, and over 50% inhibition was retained for at least 24 h. The adhesins bound to a concanavalin A affinity column and were eluted by 0.5 M alpha-methyl-D-mannopyranoside, and the eluted adhesins were designated Fr.II. Fr.II was further fractioned by DEAE-Sephacel ion-exchange column chromatography, and one especially active and abundant fraction was designated Fr.IIa. The adhesin moiety appeared to be carbohydrate, because the activity of Fr.IIa was destroyed by 20 mM sodium periodate or by 5 U of alpha-mannosidase, but boiling (30 min) or proteinase K (100 micrograms/ml) treatments had no effect. Chemically, whereas the 2-ME extract contained significant amounts of protein and mannose, Fr.IIa consisted of over 98% mannose and less than 0.5% protein. These data strongly suggest that the mannan portion within a mannoprotein is responsible for the binding of yeast cells to splenic marginal zone and to subcapsular and medullary sinuses of mouse lymph node tissue.
A siderophore production mutant of Bordetella bronchiseptica cannot use lactoferrin as an iron source
Authors: Foster L A , Dyer D W
Published: January 2020

Bordetella bronchiseptica secreted a hydroxamate siderophore when grown in Fe-depleted medium. A Tn5lac insertion mutant of B. bronchiseptica, DBB22, did not produce this hydroxamate siderophore and was incapable of using lactoferrin as an Fe source. Our data suggest that B. bronchiseptica uses a siderophore for removal of Fe from lactoferrin and transferrin rather than relying upon a receptor for these host Fe-binding proteins.
Relationship between loss of pigmentation and deletion of the chromosomal iron-regulated irp2 gene in Yersinia pestis: evidence for separate but related events
Authors: Iteman I , Guiyoule A , de Almeida A M , Guilvout I , Baranton G , Carniel E
Published: January 2020

The irp2 gene, coding for a 190-kDa iron-regulated protein (HMWP2), and the hemin storage locus (hms), which determines Yersinia pestis pigmentation, are each located on a large chromosomal fragment which carries virulence genes and deletes spontaneously. To determine whether the two loci are located on one unstable fragment or on two different excisable DNA segments, the pigmentation status and the presence of irp2 in 43 strains of Y. pestis isolated in various parts of the world were examined. Three different types were observed: Pgm+ Irp2+ (39.5%), Pgm- Irp2- (44.2%), and Pgm- Irp2+ (16.3%). No Pgm+ Irp2- strain was found. These three types were also recovered in vitro from the parental strain Saigon 55-12-39 (Pgm+ Irp2+), but again, no Pgm+ Irp2- colony was observed. Pgm- Irp2- derivatives were obtained from a single Pgm- Irp2+ colony, indicating sequential loss of the two traits. The fact that the genomic SpeI restriction patterns obtained by pulsed-field gel electrophoresis were specific for each of the three variants suggested that distinct large-scale chromosomal rearrangements had occurred in the Pgm- Irp2+ and Pgm- Irp2- derivatives. The virulence of Pgm- Irp2+ bacteria in mice was ca. 10(7)-fold lower than that of the Pgm+ Irp2+ strains injected subcutaneously but was not significantly decreased when injected intravenously. In contrast, the Pgm- Irp2- microorganisms were markedly less pathogenic (10(6)-fold) than the Pgm+ Irp2+ strains injected intravenously and were 100 times less virulent than the Pgm- Irp2+ strains injected subcutaneously.
An anti-CD3 monoclonal antibody protects mice against a lethal infection with Listeria monocytogenes through induction of endogenous cytokines
Authors: Nakane A , Okamoto M , Asano M , Kohanawa M , Minagawa T
Published: January 2020

Mice were protected against a lethal infection with Listeria monocytogenes when treated with low doses of an anti-CD3 monoclonal antibody (MAb). Injection of anti-CD3 MAb induced rapid production of endogenous tumor necrosis factor (TNF) in the spleens and endogenous gamma interferon (IFN-gamma) in the bloodstreams and spleens of mice. Administration of anti-Thy1.2 MAb or a combination of anti-CD4 MAb and anti-CD8 MAb resulted in suppression of anti-CD3 MAb-induced endogenous cytokine production and antilisterial resistance. Alternatively, in vivo depletion of anti-CD3 MAb-induced TNF and IFN-gamma by the simultaneous administration of antibodies against TNF and IFN-gamma suppressed anti-CD3 MAb-induced antilisterial resistance. Moreover, injection of anti-complement receptor type 3 (Mac-1, CD11b) resulted in inhibition of anti-CD3 MAb-induced antilisterial resistance. These results suggest that the preventive effect of anti-CD3 MAb might be due to activation of phagocytes by TNF and IFN-gamma induced by stimulating CD4+ T cells and CD8+ T cells with the MAb. Furthermore, treatment with anti-CD3 MAb did not inhibit establishment of acquired resistance against secondary infection with L. monocytogenes.
Bovine polymorphonuclear leukocyte killing of Tritrichomonas foetus
Authors: Aydintug M K , Widders P R , Leid R W
Published: January 2020

The role of bovine antibody and complement in bovine neutrophil-mediated killing of Tritrichomonas foetus was investigated. No neutrophil-mediated trichomonacidal activity was detected when Hanks' balanced salt solution, a widely utilized and weakly buffered medium, was used. This lack of neutrophil activity was evident even in the presence of specific bovine antibody and bovine complement. Moreover, the pH of the weakly buffered Hanks' balanced salt solution was observed to fall from pH 7.0 to 5.8 in 4 h at 37 degrees C in the presence of T. foetus. The pH of 5.8 inhibited the bactericidal activity of bovine neutrophils for Staphylococcus epidermidis by 53.2% and may have contributed to the lack of neutrophil-mediated trichomonacidal activity in the weakly buffered salt solution. However, T. foetus was susceptible to bovine neutrophil-mediated destruction when a HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered Hanks' balanced salt solution was used (21.8% killing by neutrophils alone). Neither specific bovine immune serum nor purified immune bovine immunoglobulin G2 alone enhanced bovine neutrophil-mediated killing. When complement-sensitized trichomonads were incubated with bovine neutrophils, killing of T. foetus was observed, a result which represented the additive effects of each treatment. Significant (P < 0.05) killing of trichomonads was observed when antibody- and complement-opsonized trichomonads were exposed to bovine neutrophils (> 70% parasite destruction), an effect which reflected the additive nature of each treatment.
Amino-terminal sequence analysis of the Coccidioides immitis chitinase/immunodiffusion-complement fixation protein
Authors: Johnson S M , Zimmermann C R , Pappagianis D
Published: January 2020

A chitinase isolated from Coccidioides immitis was subjected to amino-terminal protein sequence analysis. The resulting 18-amino-acid sequence was compared with the previously reported amino acid sequence of coccidioidal immunodiffusion-complement fixation (IDCF) antigen. From the homology of the two sequences, the results support the identification of the IDCF antigen with a chitinase.
Inhibition of heat-labile cholera and Escherichia coli enterotoxins by brefeldin A
Authors: Donta S T , Beristain S , Tomicic T K
Published: January 2020

Cholera enterotoxin and the related heat-labile enterotoxins of Escherichia coli enter their target cells through noncoated vesicles, but how the toxins are processed intracellularly and how they get to their targeted enzyme, adenylate cyclase, remain to be defined. Brefeldin A, an inhibitor of the trans-Golgi network, is shown herein to transiently block the morphologic and enzymatic effects of the toxin at a step distal to the initial binding process but prior to activation of adenylate cyclase by the toxin. It is likely, therefore, that these toxins are processed by the Golgi apparatus before trafficking to the membrane adenylate cyclase.
Mechanisms of nonopsonic phagocytosis of Pseudomonas aeruginosa
Authors: Mork T , Hancock R E
Published: January 2020

The interaction of the macrophage cell line P388D1 with Pseudomonas aeruginosa in the absence of stimulators or opsonins led to substantial association of bacteria, as judged by visual counting and FACScan assays. This association was observable within 5 min of addition of bacteria, could not be disturbed by exhaustive washing, and occurred with pilus- or flagellum-deficient mutants but not with rpoN mutants, which have been proposed to lack a secondary adhesin. In contrast, specific antibody was capable of causing similar enhancement of bacterial uptake regardless of the rpoN phenotype. Fibronectin stimulated uptake of bacteria with the pilus as an adhesin, and stimulation was observable within 5 min. Both fibronectin-enhanced and antibody-opsonized uptake were susceptible to inhibition by pertussis toxin but not by cholera toxin. The influence of fibronectin on P388D1 cells was distinguishable from that of lipopolysaccharide, which caused substantial morphological changes in cells. Although lipopolysaccharide stimulated bacterial uptake, it actually suppressed fibronectin-mediated enhancement of uptake at high concentrations.