bluuebunny/crossref_metadata_2025 huggingface dataset:
Sodium dodecyl sulfate- and salt-extracted antigens from various Brucella species induce proliferation of bovine lymphocytes
Authors: Brooks-Worrell B M , Splitter G A
Published: January 2020

One- and two-dimensional gel electrophoresis and cellular immunoblotting were used to compare the protein profiles and immunogenic capabilities of salt- and sodium dodecyl sulfate-extracted components isolated from different Brucella species. Cellular immunoblotting demonstrated that freshly isolated bovine peripheral blood mononuclear cells proliferated to similar molecular mass components from the various Brucella extractions. One- and two-dimensional gel electrophoresis showed similarities in protein profiles among the different Brucella species that correlated with lymphocyte reactivity. The results presented in this study provide preliminary evidence that common proteins that induce lymphocyte proliferation are present among different Brucella species, suggesting that a genuswide subunit vaccine may be feasible.
Tyrosine protein kinase inhibitors block invasin-promoted bacterial uptake by epithelial cells
Authors: Rosenshine I , Duronio V , Finlay B B
Published: January 2020

The ability to enter into (invade) mammalian cells is an essential virulence determinant of many pathogenic bacteria and intracellular parasites. These organisms are internalized by host cells upon attachment to their surface. However, the mechanisms used by intracellular parasites to induce internalization into host cells have not been defined. We found that the protein kinase inhibitor staurosporine blocks invasion by some pathogenic bacteria, including Yersinia enterocolitica and Yersinia pseudotuberculosis. Using Escherichia coli containing the cloned Y. enterocolitica invasion gene inv (which codes for invasin, an integrin-binding protein), we found that staurosporine inhibits invasion by blocking bacterial internalization. Two specific tyrosine protein kinase inhibitors, genistein and tyrphostin, also block the internalization but not the binding of bacteria, suggesting that bacterial uptake may be dependent on the activity of this enzyme class in host HeLa cells. In contrast to invasion promoted by invasin, the invasion of HeLa cells by Salmonella typhimurium is not inhibited by any of these drugs.
The hbpA gene of Haemophilus influenzae type b encodes a heme-binding lipoprotein conserved among heme-dependent Haemophilus species
Authors: Hanson M S , Slaughter C , Hansen E J
Published: January 2020

A membrane-associated lipoprotein of Haemophilus influenzae type b has previously been shown to bind heme in vitro and to promote binding of this compound by Escherichia coli recombinants expressing this protein. The H. influenzae type b heme-binding protein A (HbpA) was found to be highly conserved with respect to both antigenicity and apparent molecular weight among heme-requiring Haemophilus species pathogenic for humans. To further the characterization of the structure and function of HbpA, the complete nucleotide sequence of its gene, hbpA, was determined. Analysis of the nucleotide sequence revealed a single large open reading frame of 1,638 bp encoding a protein of 546 amino acid residues, with a molecular weight of 60,695. The sequence of the amino-terminal end of this protein contained a potential site for lipid acylation and for cleavage by signal peptidase II, consistent with earlier biochemical evidence which indicated that HbpA is a lipoprotein. A search of GenBank for proteins with amino acid sequence similarity to HbpA revealed that the periplasmic dipeptide transport protein of E. coli, DppA, has 53% sequence identity to HbpA.
Isolation of two Giardia lamblia (WB strain) clones with distinct surface protein and antigenic profiles and differing infectivity and virulence
Authors: Udezulu I A , Visvesvara G S , Moss D M , Leitch G J
Published: January 2020

To determine the relationship between antigenic profiles and pathogenicity among Giardia lamblia clones (WB strain), trophozoites were cloned by the technique of limiting dilution. The phenotype of each clone was determined by an indirect immunofluorescence test using a polyclonal rabbit anti-G. lamblia trophozoite serum made against the parent strain. Two clones were chosen for further studies: a highly fluorescent clone, F+, in which more than 95% of the trophozoites fluoresced, and a low-fluorescence clone, F-, in which fewer than 5% fluoresced. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and enzyme-linked immunotransfer blot studies of the membrane fractions of the two clones and parent strain revealed differences in both the total protein and antigenic profiles. A serum cytotoxicity test with the polyclonal serum showed that the F+ clones were more susceptible to immobilization and killing, while the majority of cells of the F- clones were resistant to such killing. Assessment of the infectivity of the two clones in the Mongolian gerbil animal model indicated that the F- clone more readily initiated infections, produced more cysts, had a higher intestinal trophozoite load, and produced a more severe clinical syndrome, while the F+ clone was less phenotypically stable in vivo and in some cases took longer to be cleared from the intestine.
Cryptosporidium parvum in calves: kinetics and immunoblot analysis of specific serum and local antibody responses (immunoglobulin A [IgA], IgG, and IgM) after natural and experimental infections
Authors: Peeters J E , Villacorta I , Vanopdenbosch E , Vandergheynst D , Naciri M , Ares-Mazás E , Yvoré P
Published: January 2020

Fecal and serum anti-Cryptosporidium parvum immunoglobulin A (IgA), IgM, and IgG were monitored by an enzyme-linked immunosorbent assay after experimental and natural infection of calves with C. parvum. Although all experimentally infected calves showed high levels of colostral antibodies in the feces, they acquired C. parvum infection. Three of five animals died. Calves which acquired natural infection showed only diarrhea. Levels of colostral coproantibodies dropped quickly. Experimental infection was followed by a rise in local anti-C. parvum IgM levels from day 5 postinfection (p.i.). IgM peaked at day 14 p.i. and then disappeared quickly. Anti-C. parvum IgA levels rose between days 7 and 14 p.i. and decreased slowly. Rising levels of coproantibodies coincided with falling oocyst output. Fecal anti-C. parvum IgG levels rose slightly during oocyst output, and IgG disappeared 3 weeks p.i. Similar kinetics were established in naturally infected calves. Although fecal anti-C. parvum IgA levels declined slowly, reinfections were established 5, 7, and 14 weeks after the primary contact. Serum anti-C. parvum IgG levels rose during maximal oocyst excretion, whereas serum anti-C. parvum IgA levels peaked later than did local IgA levels. Challenge reinfection of naturally infected calves at day 112 was not followed by clinical signs or oocyst output or by a secondary antibody response. Sequential Western immunoblotting with fecal extracts revealed up to 32 different parasite antigens. Convalescent-phase sera recognized up to 23 antigens. Fecal IgA reacted intensely with antigens with relative molecular weights (M(r)) of approximately 11,000 and 15,000. These antigens were not recognized by convalescent-phase serum IgG. Both local IgA and serum IgG also showed strong reactions with 23,000- and 44,000-M(r) antigens and with several antigens of between 66,200 and 200,000 M(r). Most bands remained detectable for at least 16 weeks p.i.
Antigens of Brucella abortus S19 immunodominant for bovine lymphocytes as identified by one- and two-dimensional cellular immunoblotting
Authors: Brooks-Worrell B M , Splitter G A
Published: January 2020

Cellular immune responses are influential for protection against intracellular bacteria such as brucellae. Therefore, identification of Brucella abortus antigens that activate primed bovine lymphocytes is fundamental for discerning the breadth of cellular response in bovine brucellosis. Potentially antigenic components of B. abortus S19 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by nitrocellulose blotting. Specific one-dimensional blot segments induced proliferation of peripheral blood lymphocytes from all 25 of the vaccinated cattle tested and were defined as immunodominant. Individual proteins that stimulated lymphocyte proliferation were further characterized by two-dimensional cellular immunoblotting by two different approaches. Individual one-dimensional stimulatory blot segments were eluted, concentrated, and then subjected to two-dimensional cellular immunoblotting. Alternatively, entire two-dimensional gels containing all of the B. abortus components were blotted and nitrocellulose sections containing individual proteins were assayed for lymphocyte activation. Thirty-eight Brucella proteins that induced lymphocyte proliferation were resolved by both procedures. Phenotypic analysis of the proliferating cell population demonstrated the presence of CD4+, CD8+, and immunoglobulin M+ lymphocytes. Two immunogenic proteins, 12 and 31 kDa, identified by two-dimensional cellular immunoblotting, were subjected to partial N-terminal amino acid analysis. The 12-kDa protein was within the area of greatest lymphocyte proliferation, while the 31-kDa protein was chosen for comparison with a 31-kDa protein previously reported by others. A search of the National Biomedical Research Foundation protein data bank showed that the sequences were not homologous with other known proteins. Identification of Brucella proteins immunogenic for bovine lymphocytes provides an important step in distinguishing the various proteins involved in pathogenicity and/or disease resistance.
Leukotriene B4 omega-oxidation by human polymorphonuclear leukocytes is inhibited by pyocyanin, a phenazine derivative produced by Pseudomonas aeruginosa
Authors: Muller M , Sorrell T C
Published: January 2020

Human polymorphonuclear leukocytes (PMNL) metabolize the potent chemotaxin leukotriene B4 (LTB4) by omega-oxidation to 20-hydroxyl-LTB4 and 20-carboxy-LTB4. The ability of unstimulated human PMNL to metabolize exogenous LTB4 was found to be inhibited by pyocyanin, a phenazine derivative produced by Pseudomonas aeruginosa, in a dose-dependent manner. 1-Hydroxyphenazine (1-OHP), a metabolite of pyocyanin, was not inhibitory under identical conditions. The initial enzymic step in the conversion of LTB4 is catalyzed by an NADPH-dependent cytochrome, P-450. Reduction of the phenazine derivatives by NADPH was measured spectrophotometrically. Pyocyanin was reduced by NADPH in vitro in a pH-dependent manner, while 1-OHP was poorly or negligibly reduced under similar conditions. Formation of NADP+ was 20.3 +/- 1.8 nmol min-1 for pyocyanin (10 microM) at pH 5.5, compared with 0.6 +/- 0.2 nmol min-1 for 1-OHP (10 microM), while at pH 7.5 a value of 2.2 +/- 1.3 nmol min-1 was obtained for pyocyanin, with no detectable activity for 1-OHP. This indicates that inhibition of LTB4 omega-hydroxylase activity by pyocyanin might be achieved by competition for NADPH. Incorporation of exogenous 5-hydroxyeicosatetraenoic acid by PMNL into lipid pools was not affected by either phenazine derivative. The ability of bacterial pyocyanin to limit the omega-oxidation of LTB4 may have important implications for PMNL LTB4 receptor status and chemotaxis in vivo.
Expression of receptors for enterotoxigenic Escherichia coli during enterocytic differentiation of human polarized intestinal epithelial cells in culture
Authors: Kernéis S , Chauvière G , Darfeuille-Michaud A , Aubel D , Coconnier M H , Joly B , Servin A L
Published: January 2020

To study the expression of human intestinal receptors for enterotoxigenic Escherichia coli (ETEC), the human polarized intestinal epithelial cell line Caco-2 in culture and several subpopulations of HT-29 cells in culture--parental (mainly undifferentiated) HT-29 cells (HT-29 Std), an enterocytelike subpopulation obtained by selection through glucose deprivation (HT-29 Glc-), and an enterocytelike subpopulation obtained by selection through glucose deprivation which maintains its differentiation characteristics when switched back to standard glucose-containing medium (HT-29 Glc-/+)--were used. Since Caco-2 spontaneously differentiated in culture under standard culture conditions (in the presence of glucose) and HT-29 cells were undifferentiated when cultured under standard conditions (HT-29 Std) and differentiated when grown in a glucose-free medium (HT-29 Glc-), we studied the expression of the receptors for colonization factor antigens (CFA) I, II, and III and the 2230 antigen of ETEC in relation to enterocytic differentiation. We provide evidence that expression of ETEC CFA receptors develops in parallel with other differentiation functions of the cultured cells. The expression of ETEC-specific brush border receptors was studied by indirect immunofluorescence using antibodies raised against purified ETEC CFA. No ETEC receptors were detected in HT-29 Std or short-term-cultured Caco-2 cells. However, among the population of HT-29 Std cells, 2 to 4% of the cells were found to bind ETEC, and these cells expressed positive carcinoembryonic antigen immunoreactivity. This indicated that among the population of undifferentiated HT-29 cells, clusters of differentiated cells were present. ETEC CFA receptors were expressed in the apical and basolateral domains of differentiated HT-29 cells, whereas in differentiated Caco-2 cells only apical expression was observed. Both in HT-29 cells (HT-29 Glc-/+) and in Caco-2 cells cultured under standard conditions, ETEC CFA receptors develop as a function of day in culture. This indicated that the expression of the ETEC CFA receptors was a growth-related event. Indeed, ETEC CFA receptors developed in step with the apical expression of differentiation-associated proteins.
Inhibition of murine macrophage protein kinase C activity by nonviable Histoplasma capsulatum
Authors: Wolf J E , Massof S E , Sherwin J R , Considine R V
Published: January 2020

Ingestion of Histoplasma capsulatum yeast cells inhibits the oxidative burst response of murine macrophages (M phi's). Since protein kinase C (PKC) is believed to be an integral part of the signal transduction pathway involved in the production of reactive oxygen intermediates, we investigated the relationship between PKC activity and oxidative burst inhibition in H. capsulatum-containing murine peritoneal M phi's. An inhibitory effect on both basal and phorbol myristate acetate-mobilized membrane PKC activities was observed in M phi's which had ingested H. capsulatum but not latex spheres. These results suggest that one way in which H. capsulatum may disrupt the oxidative burst is through a PCK-dependent mechanism.
Oral vaccination of weaned rabbits against enteropathogenic Escherichia coli-like E. coli O103 infection: use of heterologous strains harboring lipopolysaccharide or adhesin of pathogenic strains
Authors: Milon A , Esslinger J , Camguilhem R
Published: January 2020

To test the importance of lipopolysaccharide (LPS) and adhesin as major antigens in vaccination against rabbit enteropathogenic Escherichia coli (EPEC)-like E. coli O103 infection, we used two nonpathogenic wild-type strains to immunize rabbits at weaning. One of these strains (C127) harbors the O103 LPS but does not express the 32,000-molecular-weight adhesin that characterizes the highly pathogenic O103 strains. The other (C6) belongs to the O128 serogroup, which does not cross-react with the O103 serogroup, but expresses the adhesin. These strains were administered orally, either live or after Formalin inactivation. After vaccination, the animals were challenged with highly pathogenic O103 strain B10. Compared with rabbits vaccinated with the Formalin-killed homologous strain, rabbits vaccinated with killed C127 or C6 showed partial but significant protection. When given live, these strains colonized more or less heavily the digestive tract of the animals and provided nearly complete (C127) or complete (C6) protection against challenge. They induced a quick local immune response, as judged by fecal immunoglobulin A anti-LPS kinetics. Furthermore, strain C6 induced an ecological effect of "resistance to colonization" against challenge strain B10. This effect may have been due to the adhesin that is shared by both strains and to the production of a colicin. Strain C6 could inhibit in vitro the growth of highly pathogenic O103 strains. On the whole, our results show that adhesins and LPS are important, although probably not exclusive, protection-inducing components in rabbit EPEC-like colibacillosis and provide insight into possible protection of rabbits against EPEC-like E. coli infection with live strains.
Molecular cloning and expression of ptxA, the gene encoding the 120-kilodalton cytotoxin of Actinobacillus pleuropneumoniae serotype 2
Authors: MacDonald J , Rycroft A N
Published: January 2020

The genetic determinants of the 120-kDa cytotoxin of Actinobacillus pleuropneumoniae serotype 2 were isolated from a lambda DNA library by a plaque immunoblot technique. Expression of the 120-kDa polypeptide was confirmed by Western immunoblot analysis of infected Escherichia coli cell lysates, which were shown to be toxic for porcine alveolar macrophages in vitro. The genetic determinants of the toxin were subcloned into the plasmid vector pUC18. This plasmid (pPTX1) directed the synthesis and secretion of the active 120-kDa cytotoxin in E. coli. The recombinant toxin was indistinguishable from native cytotoxin from A. pleuropneumoniae serotype 2 with respect to molecular size, reaction in Western blot analysis, heat lability, cytotoxic activity, and neutralization by serum antibody. A restriction endonuclease cleavage map of pPTX1 was prepared, and deletion mutants were used to locate the minimal region of DNA required for production of intracellular toxin; this gene was termed ptxA. Southern hybridization analysis with a 1.7-kb PvuII fragment located within the ptxA gene revealed sequences with a high degree of homology in serotype reference strains 2, 3, 4, 6, and 8. Other reference strains did not contain sequences that were recognized by this probe. However, related sequences (greater than 71% homology) were detected in Actinobacillus actinomycetemcomitans and A. equuli. Weak hybridization was observed between the ptxA probe and pLKT5, which carries the lktAC genes of Pasteurella haemolytica, and between the ptxA probe and pAPH1, which carries the structural gene for type II hemolysin from A. pleuropneumoniae. The isolation of the genetic determinants of this cytotoxin will enable investigations of the structure and organization of the ptx DNA region and further analysis of its role in the pathogenesis of pleuropneumonia.
Characterization of the Shigella serotype D (S. sonnei) O polysaccharide and the enterobacterial R1 lipopolysaccharide core by use of mouse monoclonal antibodies
Authors: Viret J F , Bruderer U , Lang A B
Published: January 2020

In the course of developing a live vaccine, we generated three murine monoclonal antibodies (MAb) specific for Shigella sonnei. The specificities of these MAb were determined by enzyme-linked immunosorbent assay and immunoblot analyses with whole cells or purified lipopolysaccharides (LPSs) as antigens. Two of them are specific for the Shigella serotype D O-polysaccharide determinant, whereas one specifically binds to the core hexose region of R1-type LPSs. With these MAb, it was possible to analyze clinical isolates and a hybrid Salmonella typhi strain for their expression of the corresponding LPS moieties. In addition to their use in the screening of candidate vaccine strains, the new MAb provide a powerful tool for epidemiological and phylogenetic studies of natural enterobacterial populations.
Epitope mapping of hemagglutinating adhesion HA-Ag2 of Bacteroides (Porphyromonas) gingivalis
Authors: Deslauriers M , Mouton C
Published: January 2020

Thirteen monoclonal antibodies (MAbs) directed against hemagglutinating adhesion HA-Ag2 of Bacteroides (Porphyromonas) gingivalis were produced by immunizing mice with the relevant immunoprecipitate from crossed immunoelectrophoresis (CIE). Crossed immuno-affinoelectrophoresis and hemagglutination experiments confirmed that our MAbs recognized a molecule able to bind erythrocytes and involved in the hemagglutination process. In immunoelectron microscopy, these MAbs labelled amorphous material as novel cell-bound appendages distinct from fimbriae. CIE experiments allowed differentiation of the MAbs according to reactivity with immunoprecipitates Ag2, Ag8a, and Ag8c, which define HA-Ag2. The epitopes recognized by nine MAbs were mapped on three main antigenic domains (I, II, and III) by competition experiments and further grouped according to chemical composition and distribution on CIE immunoprecipitate. Domain I, defined by two MAbs, comprises an epitope with protein and carbohydrate determinants and distributed on Ag2 only. Epitopes of domain IIA, defined by four MAbs, are distributed on Ag8a, Ag8c, and Ag2 and are essentially composed of protein determinants but also have carbohydrate determinants that enhance the binding of the MAbs but are not essential. Epitopes of domain IIB, defined by two MAbs, and of domain III, defined by a single MAb, have a composition similar to that of domain IIA epitopes but are distributed on Ag8a and Ag8c only. A competition enzyme-linked immunosorbent assay with serum from normal subjects and patients with periodontitis suggested that domain I is more immunogenic than domain II.
Passive protection of suckling infant mice against F41-positive enterotoxigenic Escherichia coli strains by intravenous inoculation of the dams with monoclonal antibodies against F41
Authors: Duchet-Suchaux M , Menanteau P , van Zijderveld F G
Published: January 2020

Ten monoclonal antibodies (MAbs) against five different epitope clusters of adhesion factor F41 (two MAbs per cluster) were tested for protection of infant mice against an oral challenge with F41-positive enterotoxigenic Escherichia coli (ETEC) B2C and B41M. Infant mice suckling dams intravenously inoculated with MAbs were orally challenged, and the survival rates were measured for 12 days after inoculation and challenge. Irrespective of their epitope specificity, all F41 MAbs given in a single dose of 4 mg per dam had a protective effect against both ETEC strains. In contrast, one K99 MAb of the same isotype and given in the same dose as the F41 MAbs did not protect infant mice at all. A reduction in the dose of F41 MAbs to 0.032 mg per dam resulted in a decrease in protection. Two different MAbs against the same epitope cluster were not necessarily equally protective. Combining MAbs two by two, whether the MAbs recognized the same epitope cluster or not, resulted in protective activity essentially similar to that obtained with each MAb separately, without any improvement. Therefore, one MAb against any epitope may be sufficient for protection. Enzyme-linked immunosorbent assay (ELISA) titers of MAbs in the serum of dams were similar, irrespective of the epitope specificity of the MAbs, and gradually decreased from day 1 to day 12 after inoculation. We found a good correlation between colostrum and milk ELISA titers of MAbs and serum ELISA titers of MAbs. Colostrum and milk MAb titers were 10-fold lower than corresponding serum MAb titers and stayed high until day 5 after inoculation. The most protective MAb had the highest ELISA titers in colostrum and milk for the first 5 days after inoculation. ETEC strain B2C colonized the intestines of infant mice suckling MAb-inoculated mothers until day 12 after challenge. Intestinal levels of the challenge strain were high on day 2 but never reached the very high numbers (10(9) to 10(10)) described previously in a diarrheic infant mouse model. MAbs did not eliminate the challenge ETEC strain from the intestines of infant mice.
Spleen cell cytokine secretion in Mycobacterium bovis BCG-infected mice
Authors: Huygen K , Abramowicz D , Vandenbussche P , Jacobs F , De Bruyn J , Kentos A , Drowart A , Van Vooren J P , Goldman M
Published: January 2020

Three susceptible mouse strains, i.e., BALB/c (H-2d), C57BL/6 (H-2b), and major histocompatibility complex-congenic BALB.B10 (H-2b), were infected intravenously with 4 x 10(6) CFU of live Mycobacterium bovis BCG and analyzed 4 weeks later for in vitro spleen cell cytokine secretion in response to purified protein derivative (PPD), BCG culture filtrate (CF), BCG cellular extract, total BCG, the purified extracellular 30-32-kDa antigen (the fibronectin-binding antigen 85), or the intracellular 65-kDa heat shock protein. C57BL/6 and BALB.B10 mice produced 5- to 10-fold more gamma interferon and interleukin-2 (IL-2) when stimulated with CF, PPD, and antigen 85 than BALB/c mice did. When stimulated with BCG extract and whole BCG, gamma interferon and IL-2 levels were generally lower and comparable in the three strains. IL-4 was detected in spleen cell culture supernatants from infected BALB/c mice but not from C57BL/6 or BALB.B10 mice. IL-5 could not be detected. C57BL/6 and BALB.B10 spleen cells also produced more tumor necrosis factor alpha and IL-6 after stimulation with PPD and CF than BALB/c cells did. Finally, BCG vaccination generated efficient protective immunity in C57BL/6 and BALB.B10 mice but not in BALB/c mice. These data suggest that secreted mycobacterial CF antigens selectively induce a strong TH1 response in BCG-infected C57BL/6 and BALB.B10 mice, whereas in BALB/c mice this response is partly counterbalanced by TH2 cells.
Morphological and biochemical study of cytoskeletal changes in cultured cells after extracellular application of Clostridium novyi alpha-toxin
Authors: Oksche A , Nakov R , Habermann E
Published: January 2020

Clostridium novyi alpha-toxin caused retraction and rounding of cultured endothelial cells from porcine pulmonary arteries; nevertheless, the endothelial cells firmly adhered to their supports. F-actin stained with fluorescein-labeled phalloidin was condensed around the nucleus, whereas intermediate filaments and microtubules appeared unchanged. The content of F-actin and myosin was decreased, but that of G-actin or vimentin was not. A predominant role of the microfilament system in C. novyi alpha-toxin cytopathic action is suggested.
Expression of interleukin-8 by lipopolysaccharide-stimulated bone marrow-derived mononuclear cells
Authors: Dibb C R , Strieter R M , Burdick M , Kunkel S L
Published: January 2020

Interleukin-8 (IL-8) is a potent neutrophil chemoattractant, produced by a variety of immune and nonimmune cells in response to exogenous and host-derived inflammatory stimuli. We demonstrate here that a suspension of normal bone marrow mononuclear cells, consisting principally of myeloid precursors, produces IL-8 in response to stimulation with lipopolysaccharide (LPS). IL-8-specific mRNA is rapidly induced, being detected first 30 min after stimulation. IL-8 is detected by enzyme-linked immunosorbent assay within 2 h of stimulation, with steady a increase in its level through 72 h. Further studies demonstrated that LPS could serve as a primary stimulus for the expression of IL-8, since LPS challenge in the presence of cycloheximide resulted in superinduction of bone marrow mononuclear cell-derived IL-8 mRNA. These investigations suggest that the stimulatory effect of LPS is independent of other cytokines such as IL-1 beta. When compared with LPS, IL-1 beta proved to be a weak signal for the expression of IL-8 by bone marrow mononuclear cells. In a dose-response study, the maximum stimulatory concentration of IL-1 beta (300 pg/ml) resulted in the production of 500 pg of IL-8 per 10(6) cells, whereas 1 microgram of LPS resulted in the production of 5.5 ng/10(6) cells. Although IL-1 beta was not a particularly potent stimulus for IL-8 production by bone marrow mononuclear cells, peripheral blood mononuclear cells were highly susceptible to IL-1 beta challenge. In addition, the potential dependence of LPS-induced marrow-derived IL-8 production on the intermediate synthesis of IL-1 beta was further investigated. Results of studies assessing kinetics, addition of cycloheximide, and blocking with IL-1 beta neutralizing antibody were all consistent with the ability of LPS to directly induce bone marrow-derived IL-8 independently of IL-1 beta. These investigations demonstrate that bone marrow may be a significant source of IL-8 and may play a significant role in acute infectious, inflammatory responses.
Functional analysis of the Ca(2+)-regulated hemolysin I operon of Actinobacillus pleuropneumoniae serotype 1
Authors: Gygi D , Nicolet J , Hughes C , Frey J
Published: January 2020

The genetic determinant encoding the synthesis and secretion of hemolysin I (HlyI; gene designation, hlyI) by Actinobacillus pleuropneumoniae serotype 1 4074T was cloned in the lambda vector EMBL4. A 10.2-kb fragment that encoded hemolytic activity in the phage lysate was aligned by Southern blot hybridization to genes hlyC, hlyA, hlyB, and hlyD of the Escherichia coli hemolysin operon, and expression of the A. pleuropneumoniae genes in E. coli revealed that they have the same functions as their E. coli analogs: hlyIC encodes a protein that activates inactive 105-kDa prohemolysin I (encoded by hlyIA) to active hemolysin I, while hlyIB and hlyID are necessary for HlyIA secretion. Northern (RNA) hybridization of A. pleuropneumoniae RNA revealed that the gene cluster is transcribed as two RNA species, a major one of 3.5 kb, corresponding to hlyICA, and a second, minor one of 7.5 kb, corresponding to the whole operon, hlyICABD. The level of hlyI mRNA was substantially higher in A. pleuropneumoniae 4074T cells grown in the presence of Ca2+, supporting the view that the expression of the hlyI determinant is Ca2+ regulated. Parallel RNA hybridization with random gene probes suggested that this Ca2+ regulation is specific for the hlyI determinant.
Selection of an escape variant of Borrelia burgdorferi by use of bactericidal monoclonal antibodies to OspB
Authors: Coleman J L , Rogers R C , Benach J L
Published: January 2020

Two immunoglobulin G (IgG) monoclonal antibodies (MAbs) to outer surface protein B (CB2 and CB6), affinity purified from mouse ascitic fluid, exhibited concentration-dependent inhibitory and bactericidal properties against Borrelia burgdorferi after a 24-h incubation period in spirochete medium. Fab fragments derived from these MAbs showed the same effects, indicating that they were not caused by agglutination of the organisms by the intact MAbs. The inhibition of spirochete growth in cultures containing MAbs was also detected by spectrophotometric analysis of the media. CB2 did not inhibit the growth of Borrelia hermsii or the BEP4 strain of B. burgdorferi, neither of which is recognized by the MAb. Affinity-purified IgG from hybridoma supernatants had similar effects on B. burgdorferi as the ascitic-fluid-derived IgG did, indicating that the inhibitory and bactericidal properties were not due to nonspecific toxic contaminants. The bactericidal properties of the MAbs were not complement dependent as there was none in the serum-free system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of B. burgdorferi organisms surviving after exposure to CB2 revealed an escape variant which failed to express OspB. The continued presence of OspA in these escape variants indicates that the lack of OspB was not due to the loss of the plasmid which contains the genes for both of these proteins.
Kinetic analysis of the amplification phase for activation and binding of C3 to encapsulated and nonencapsulated Cryptococcus neoformans
Authors: Kozel T R , Wilson M A , Welch W H
Published: January 2020

Encapsulated and nonencapsulated cryptococci exhibit quantitative and qualitative differences in their activation of the complement system. We examined the kinetics for the rapid amplification phase in which C3 was activated and bound to encapsulated cryptococci, nonencapsulated cryptococci, and zymosan particles. Yeast cells were incubated in normal human serum containing 125I-labeled C3, and bound C3 fragments were measured after 1 to 64 min of incubation. A kinetic analysis showed that the apparent first-order rate constant (k') for binding of C3 to nonencapsulated cryptococci did not differ significantly from k' for binding of C3 to zymosan particles (P greater than 0.05). However, the rate constant for binding of C3 to encapsulated cryptococci was significantly (P less than 0.001) greater than k' for binding of C3 to nonencapsulated cryptococci and zymosan particles. A plot of C3 molecules bound to encapsulated cryptococci versus time cubed was nearly linear, suggesting that accumulation of C3 in the cryptococcal capsule follows the kinetics predicted by an expanding sphere. In contrast, the plot of C3 molecules bound to nonencapsulated cryptococci or zymosan particles against time was nearly linear, but those plots against time squared or time cubed were not. This result indicates that the rate-limiting step for the addition of C3 fragments to these latter yeast cells follows the kinetics of neither the perimeter of an expanding circle nor the surface of an expanding sphere. Taken together, the results indicate that the high rate of accumulation of C3 in the cryptococcal capsule is consistent with the expected geometry of an expanding sphere of bound C3 within the three-dimensional matrix of the capsule.
Induction of tumor necrosis factor (TNF) and interleukin-1 (IL-1) by Pseudomonas aeruginosa and exotoxin A-induced suppression of lymphoproliferation and TNF, lymphotoxin, gamma interferon, and IL-1 production in human leukocytes
Authors: Staugas R E , Harvey D P , Ferrante A , Nandoskar M , Allison A C
Published: January 2020

Pseudomonas aeruginosa is a dominant pathogen in infection in cystic fibrosis. This bacterium is thought to play a major role in the chronic bronchial infection-induced pathophysiology. Our data showed that whole formalin-fixed heat-killed P. aeruginosa was mitogenic for human lymphocytes and induced production of substantial amounts of tumor necrosis factor alpha (TNF) in peripheral blood mononuclear leukocytes in cultures. Significant amounts of TNF were produced at 10(3) bacteria per 2 x 10(5) mononuclear leukocytes. Treatment of P. aeruginosa with polymixin B did not affect its ability to stimulate TNF production, suggesting that bacterial lipopolysaccharide is not involved. P. aeruginosa, however, did not stimulate production of the T-cell lymphokine lymphotoxin (TNF beta). Exotoxin A, considered to be an important virulence factor produced by P. aeruginosa, did not stimulate either lymphoproliferation or production of TNF. In fact, this toxin, at nontoxic concentrations, was found to depress lymphoproliferation induced by phytohemagglutinin and Staphylococcus aureus and decreased production of TNF, lymphotoxin, and gamma interferon in either lymphocytes or macrophages. This toxin similarly inhibited the production of interleukin-1 beta (IL-1 beta) and IL-1 alpha, but for the inhibition of the latter, 25-fold-less toxin was required than for inhibition of the former. Inhibition of production of TNF was as sensitive as the IL-1 alpha to exotoxin A. The effects of exotoxin A on lymphoproliferation and cytokine production could be neutralized by the addition of anti-exotoxin A antibodies. These results suggest that two mechanisms by which P. aeruginosa could contribute to the chronic bronchial infection-induced pathophysiology are the nonspecific stimulation of TNF and IL-1 and the release of exotoxin A, a toxin which depresses immune responses.
A novel murine model of disseminated trichosporonosis
Authors: Gokaslan A , Anaissie E
Published: January 2020

Serious infections caused by Trichosporon beigelii have been noted with increasing frequency in immuno-compromised patients. Progress in understanding the pathogenesis of this emerging infection has been limited by the lack of an animal model. We developed a CF1 mouse intravenous inoculation model of disseminated trichosporonosis to evaluate the pathogenicity of T. beigelii in transiently immunosuppressed mice. Four inocula (1 x 10(6), 1 x 10(7), 2 x 10(7), and 4 x 10(7) CFU per animal) of one clinical strain of T. beigelii 3001 were tested. Mice in groups of 10 were each injected with a single intravenous dose of one inoculum. Mortality correlated with inoculum size, as survival time was significantly shorter in mice injected with 4 x 10(7) or 2 x 10(7) CFU than in mice that received 1 x 10(7) or 1 x 10(6) CFU (P less than 0.01). Necrotizing abscesses with conidial and hyphal elements and neutrophil and macrophage infiltration were observed in all major organs examined. Resistance to infection was markedly lowered by immunosuppression with either cyclophosphamide or cortisone acetate, with a significantly shorter survival time and a greater fungal burden per organ in immunosuppressed animals than in normal animals (P less than 0.01). Nine additional strains were inoculated intravenously with around 5 x 10(6) CFU. Injection of each of these strains caused 100% mortality, in a pattern similar to that observed with strain 3001.
Lymphokine-activated killer cell regulation of T-cell-mediated immunity to Candida albicans
Authors: Wei S , Blanchard D K , McMillen S , Djeu J Y
Published: January 2020

Monocytes are important accessory cells in the activation of T cells for specific antigen recognition yet little is known of their regulation. We demonstrated here that interleukin-2 (IL-2)-induced human lymphokine-activated killer (LAK) cells can inhibit monocyte antigen presentation, depending on the state of differentiation of the monocytes. Adherent monocytes cultured for 4 days in medium or granulocyte-macrophage colony-stimulating factor (GM-CSF) were found to equally process and present intact Candida albicans to autologous Percoll gradient-isolated T cells, as measured by [3H]thymidine uptake. However, only the GM-CSF-cultured monocytes were functionally inhibited by autologous 4-day IL-2-induced LAK cells. Even soluble candidal cell wall mannoprotein antigens could not be presented by these monocytes after exposure to LAK cells. Pretreatment of these monocytes with LAK cells for 1 h, followed by subsequent removal of the nonadherent LAK cells, was sufficient to cause significant inhibition, with maximal inhibition observed after 4 h. Northern (RNA) blot analysis indicated that mRNA expression for IL-1 alpha and IL-1 beta in response to C. albicans stimulation was also down-regulated in GM-CSF-cultured monocytes exposed to LAK cells. Interestingly, freshly isolated, Percoll gradient-purified large granular lymphocytes did not suppress antigen presentation in GM-CSF-treated monocytes. Another important finding was the inability of LAK cells to suppress the ability of freshly isolated or gamma interferon-cultured monocytes, which are resistant to LAK cell-mediated lysis, to present antigen to T cells. In contrast, IL-3 was similar to GM-CSF in inducing LAK cell susceptibility in monocytes. Taken together, these results indicated that IL-2 can induce LAK cells to down-regulate antigen presentation function in a select set of monocytes that have been activated by colony-stimulating factor (GM-CSF and IL-3) but not by gamma interferon. LAK cells may therefore play an important role in regulation of monocytes and their function, depending on their differentiation state.
Molecular analysis of the serotyping antigens of Neisseria meningitidis
Authors: Feavers I M , Suker J , McKenna A J , Heath A B , Maiden M C
Published: January 2020

Molecular approaches to the rapid analysis of the serotyping antigens of Neisseria meningitidis, the class 2 and 3 outer membrane proteins (OMPs), were developed, evaluated, and used to study 12 antigenic variants of these proteins. A primer set for the polymerase chain reaction (PCR) amplification of the genes encoding these antigens was devised. Low-stringency amplification of meningococcal chromosomal DNA with this primer set resulted in the amplification of two products from each strain, whereas at higher stringencies only one product was amplified in most strains. Southern hybridization techniques and restriction analyses were used to differentiate the PCR products amplified at high stringencies from strains expressing class 2 or class 3 OMPs; these PCR products were further characterized by the determination of their nucleotide sequences, confirming that they represented the amplified class 2 and class 3 OMP genes. Analyses of these and other nucleotide sequences enabled the construction of a phenogram illustrating the interrelationships between Neisseria OMP genes. The comparative analysis of deduced amino acid sequences revealed conserved and variable regions of the proteins; the latter probably correspond to surface loops on the protein and hence are potentially exposed to the immune system. Further analyses of the primary structures of these related porins from Neisseria species enabled construction of models of the secondary structure of these antigens and comparison of these models with those previously published. The methods reported in the present work are rapid reproducible procedures for the analysis of antigenic variants of these proteins.
Experimental model of intracerebral infection with Cryptococcus neoformans: roles of phagocytes and opsonization
Authors: Blasi E , Barluzzi R , Mazzolla R , Mosci P , Bistoni F
Published: January 2020

A murine model of intracerebral (i.c.) infection with Cryptococcus neoformans in which naive mice receiving an i.c. fungal inoculation developed a severe disease has been established. The effect was strictly dependent on the number of microorganisms injected and evolved as lethal meningoencephalitis. Murine susceptibility to i.c. infection with C. neoformans was enhanced by treatment with chloroquine and colchicine, agents known to greatly affect the host phagocytic compartment. Furthermore, the life spans of both naive and drug-treated mice were significantly augmented when opsonized fungi were injected. Therefore, phagocyte-mediated mechanisms are likely involved in local resistance to i.c. infection with C. neoformans. Further support for this conclusion was supplied by in vitro data showing that microglial cells were proficient anticryptococcal effectors, provided opsonized microorganisms were used.
Immune cell lethality induced by streptococcal pyrogenic exotoxin A and endotoxin
Authors: Leonard B A , Schlievert P M
Published: January 2020

Streptococcal pyrogenic exotoxin (SPE) A has many effects on the immune system, including immunolethality, which is characterized by a significant decrease in circulating immune cells as well as depletion of the spleen and lymph nodes prior to death of experimental animals. In this report, characterization of the mechanism of immunolethality has been undertaken. Synergistic induction of immunolethality was observed in vitro when human lymphocytes were treated with both SPE A and lipopolysaccharide (LPS). The same effect was demonstrated in the absence of a mitogenic response with the murine T-cell receptor, as well as in the absence of antigen-presenting cells and their secreted cytokines. The addition of antigen-presenting cells did not significantly affect lethality. SPE A directly interacted with LPS through interaction with ketodeoxyoctonate as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and iodinated exotoxin overlays. This interaction was demonstrated to be important for immunolethality, since simultaneous addition of SPE A and LPS was required, whereas sequential addition of SPE A and LPS did not result in lethality. LPS appeared to be acting, in part, to enhance the cell-binding ability of SPE A, since SPE A could only be detected in A.E7 cell membrane preparations after simultaneous incubation with SPE A and LPS.
An unusual pagC::TnphoA mutation leads to an invasion- and virulence-defective phenotype in Salmonellae
Authors: Miller V L , Beer K B , Loomis W P , Olson J A , Miller S I
Published: January 2020

Two phenotypes believed to contribute to the pathogenesis of Salmonella infections are macrophage survival and invasion of epithelial cells. It was recently observed that the Salmonella macrophage survival factor PagC has significant amino acid similarity to the Yersinia invasion factor Ail. This observation raised the possibilities that macrophage survival is in part determined by the pathway of entry and that PagC confers an entry mechanism that does not trigger the microbicidal activities of the macrophage. Thus, we sought to investigate the role of PagC in invasion by examining (i) the invasion phenotype of pagC mutants and (ii) the invasion phenotype of Escherichia coli carrying pagC. A previously identified invasion-defective TnphoA insertion mutant of Salmonella enteritidis was found to have TnphoA inserted into the signal sequence-encoding region of pagC; the pagC allele from this mutant, SM5T, was designated pagC64. In contrast, Salmonella typhimurium carrying the pagC1 allele (a TnphoA insertion mutation, downstream of the region encoding the signal sequence) was not defective for invasion. Further analysis of these two pagC alleles suggested that the invasion-defective phenotype associated with pagC64 is not due to the loss of PagC function but rather is due to the synthesis of a hybrid PagC-alkaline phosphatase protein that is aberrantly localized, most likely to the inner membrane, and thus may prevent proper localization or function of a factor(s) required for efficient invasion. The observation that pagC did not confer an invasive phenotype to E. coli further suggests that PagC is not an invasion factor. A cloned pagC gene complemented the macrophage survival defect of S. typhimurium pagC1 mutants, but the cloned ail gene did not. Together these results suggest that the structural similarity between PagC and Ail may not extend to a similarity in function. Interestingly, S. enteritidis carrying the pagC64 allele that results in both an invasion defect and a macrophage survival defect was less virulent for mice infected intragastrically or intraperitoneally than was S. enteritidis carrying the pagC1 allele that results only in a macrophage survival defect.
Recombinant Salmonella typhimurium strains that invade nonphagocytic cells are resistant to recognition by antigen-specific cytotoxic T lymphocytes
Authors: Gao X M , Tite J P , Lipscombe M , Rowland-Jones S , Ferguson D J , McMichael A J
Published: January 2020

To address the question of whether Salmonella-infected nonphagocytic cells could serve as target cells for recognition by antigen-specific, major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL), four recombinant Salmonella typhimurium constructs that expressed full-length, or fragments of, influenza A virus nucleoprotein (NP) were made. The bacteria were shown to infect Chinese hamster ovary (CHO) cells. Appropriate major histocompatibility complex restriction molecules, HLA-B27 and H-2 Db, were transfected into CHO cells, which were then infected with recombinant S. typhimurium and used as targets for NP-specific CTL. The cells in which NP was expressed by intracellularly replicating bacteria were not lysed by NP-specific CTL, although they were killed when appropriate influenza A virus or peptides were used. Thus, S.typhimurium bacteria within nonphagocytic cells were resistant to CTL recognition. In contrast to these results, mice infected with recombinant S.typhimurium that expressed fragments of NP in the periplasm were primed for NP-specific CTL responses. The results indicate that CTL responses specific to Salmonella antigens can be generated, but the bacteria may be safe from the CTL attack once they have entered the nonphagocytic cells.