bluuebunny/crossref_metadata_2025 huggingface dataset:
Penetration of host cells by Rickettsia rickettsii appears to be mediated by a phospholipase of rickettsial origin
Authors: Silverman D J , Santucci L A , Meyers N , Sekeyova Z
Published: January 2020

Internalization of obligate intracellular bacteria belonging to the genus Rickettsia by eukaryotic cells requires participation of both the parasitized host and the microorganism. The term "induced phagocytosis" has been used specifically to describe the entry of Rickettsia prowazekii, although a similar mechanism is likely for R. rickettsii. A role for a phospholipase in the internalization process has been proposed for both of these organisms, with the strongest supporting evidence provided for R. prowazekii. Despite general acceptance of the notion that phospholipase activity is involved in the internalization process of these bacteria, the origin of the enzyme is not known. The results of the study presented here, which used R. rickettsii and Vero cells, suggest that a rickettsial phospholipase, rather than a host cell phospholipase, mediates internalization of the organism. This conclusion is based upon results which show that pretreatment of R. rickettsii, but not of host cells, with a specific chemical inhibitor of phospholipase, and also antiserum to this enzyme, significantly reduces uptake of the organism and its ability to cause plaque formation.
Inhibition of adhesion of S-fimbriated Escherichia coli to buccal epithelial cells by human milk fat globule membrane components: a novel aspect of the protective function of mucins in the nonimmunoglobulin fraction
Authors: Schroten H , Hanisch F G , Plogmann R , Hacker J , Uhlenbruck G , Nobis-Bosch R , Wahn V
Published: January 2020

We investigated the presence of factors in human milk that inhibit invasion of pathogenic bacteria. The effect of human milk fat globule membrane (HMFGM) components on adhesion of cloned S-fimbriated Escherichia coli to human buccal epithelial cells was analyzed. S fimbriae are a common feature of E. coli strains causing sepsis and meningitis in newborns and are bound to epithelia via sialyl-(alpha-2-3)galactoside structures. Human milk fat globules (HMFG) could be agglutinated by the above-mentioned bacteria. Agglutination could be inhibited by fetuin, human glycophorin, and alpha 1-acid glycoprotein. In addition, pretreatment of HMFG with Vibrio cholerae neuraminidase markedly reduced bacterium-induced agglutinations, indicating the involvement of neuraminic acid-containing glycoproteins. In contrast, lipid droplets of infant formula or artificial lipid emulsions (Intralipid) could not be agglutinated. HMFG were present in stools of breast-fed neonates as shown by indirect immunofluorescence staining with a monoclonal antibody directed against carbohydrate residues present on HMFGM. These HMFG could be agglutinated by bacteria. HMFG inhibited E. coli adhesion to buccal epithelial cells. To further characterize relevant E. coli binding structures, HMFGM components were separated by gel chromatography. The mucin fraction showed the most pronounced inhibitory effect on adhesion of S-fimbriated E. coli to human buccal epithelial cells. Our data suggest that HMFG inhibit bacterial adhesion in the entire intestine and thereby may provide protection against bacterial infection.
Propionate induces polymorphonuclear leukocyte activation and inhibits formylmethionyl-leucyl-phenylalanine-stimulated activation
Authors: Brunkhorst B A , Kraus E , Coppi M , Budnick M , Niederman R
Published: January 2020

Short-chain carboxylic acids (SCCA) are metabolic by-products of bacterial pathogens which can alter cytoplasmic pH and inhibit a variety of polymorphonuclear leukocyte (PMN) motile functions. Since cytoskeletal F-actin alterations are central to PMN mobility, in this study we examined the effects of SCCA on cytoskeletal F-actin. Initially, we tested nine SCCA (formate, acetate, propionate, butyrate, valerate, caproate, lactate, succinate, and isobutyrate). We document here that while eight altered cytoplasmic pH, only six altered cytoskeletal F-actin. We then selected one SCCA that altered both F-actin and cytoplasmic pH (propionate) and one SCCA that altered only cytoplasmic pH (lactate) for further study. Propionate, but not lactate, caused an irregular cell shape and F-actin distribution. Furthermore, propionate, but not lactate, inhibited formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated PMN polarization, F-actin localization, and cytoplasmic pH oscillation. Propionate-induced changes in cytoskeletal F-actin and cytoplasmic acidification were not affected by the fMLP receptor antagonist N-t-BOC-1-methionyl-1-leucyl-1-phenylalanine; however, alkalinization was affected. Pertussis toxin treatment completely inhibited propionate-induced changes in F-actin but had no effect on propionate-induced cytoplasmic pH oscillation. These results indicate that propionate (i) bypasses the fMLP receptor and G protein(s) to induce cytoplasmic pH oscillation, (ii) operates through G protein(s) to induce actin oscillation, cell shape changes (to irregular), and F-actin localization, and (iii) inhibits fMLP-stimulated cytoplasmic pH and actin oscillation, PMN polarization, and F-actin localization.
Adhesion protein YadA of Yersinia species mediates binding of bacteria to fibronectin
Authors: Tertti R , Skurnik M , Vartio T , Kuusela P
Published: January 2020

The interaction between fibronectin and Yersinia strains was studied. Wild-type Y. enterocolitica strains expressing the virulence-plasmid-encoded adhesion protein YadA adhered strongly to fibronectin-coated coverslips, while their plasmid-cured variants expressed weaker binding. The cloned yadA gene of Y. enterocolitica or Y. pseudotuberculosis conferred fibronectin-binding ability both to Escherichia coli and to Y. psuedotuberculosis strains lacking the YadA protein. The YadA protein did not mediate binding to isolated fragments of fibronectin or to soluble fibronectin.
hsp70 synthesis in Schwann cells in response to heat shock and infection with Mycobacterium leprae
Authors: Mistry Y , Young D B , Mukherjee R
Published: January 2020

Induction of heat shock protein synthesis was monitored in murine and monkey Schwann cells exposed to elevated temperatures. Synthesis of the stress-inducible 70-kDa heat shock protein (hsp70) was detected in both murine and primate Schwann cells by metabolic labelling and by immunoblotting with a specific monoclonal antibody. hsp70 synthesis was also induced in Schwann cells after infection with Mycobacterium leprae and was detected from 24 h to 1 week postinfection. These results are discussed with respect to the possible role of heat shock proteins in immunopathological events associated with the clinical manifestations of leprosy.
A 16-kilodalton lipoprotein of the outer membrane of Serpulina (Treponema) hyodysenteriae
Authors: Thomas W , Sellwood R , Lysons R J
Published: January 2020

Serpulina (Treponema) hyodysenteriae P18A and VS1 were extracted by using the detergent Triton X-114 and separated into detergent and aqueous phases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis confirmed that a membrane-associated 16-kDa antigen was hydrophobic, since it was found in the detergent phase. A 45-kDa antigen partitioned into the aqueous phase, suggesting that it was hydrophilic and may be of periplasmic origin. When spirochetes were grown in the presence of [3H]palmitic acid, a predominant 16-kDa antigen was labeled; from the results of immunoprecipitation experiments, this antigen appeared to be the same as that recognized by both polyclonal and monoclonal antisera to a previously described 16-kDa antigen. This antigen was proteinase K sensitive and was not a component of the lipopolysaccharide, which, although [3H]palmitate labeled, was resistant to proteinase K digestion. The most probable explanation is that the 16-kDa antigen is a membrane-associated, surface-exposed, immunodominant lipoprotein.
Immune responses to Cryptosporidium muris and Cryptosporidium parvum in adult immunocompetent or immunocompromised (nude and SCID) mice
Authors: McDonald V , Deer R , Uni S , Iseki M , Bancroft G J
Published: January 2020

Adult murine models of Cryptosporidium infection involving Cryptosporidium muris and C. parvum were used to study immunity to cryptosporidiosis in the mammalian host. Immunocompetent BALB/c or C57BL/6 mice developed a highly patent infection with the RN 66 strain of C. muris but overcame the infection and were immune to reinfection. In contrast, severe combined immunodeficiency (SCID) mice or nude mice had a chronic infection lasting at least 109 days. The development of the C. muris infection appeared to be confined to the gastric epithelium in immunocompetent and immunocompromised mice. SCID mice injected intraperitoneally with histocompatible spleen or mesenteric lymph node cells from uninfected BALB/c mice were able to recover from the C. muris infection. The protective effect of donor spleen cells was not reduced by depletion of the B cell population but was significantly reduced by depletion of Thy.1 cells. Treatment of C57BL/6 or BALB/c mice during infection with a gamma interferon-neutralizing monoclonal antibody, but not a tumor necrosis factor-neutralizing monoclonal antibody, resulted in a significant increase in oocyst production. In the C. parvum model, a severe and eventually fatal chronic infection with a cervine isolate was established in SCID mice, with parasitization occurring in the ileum, cecum, and colon. SCID mice injected with unprimed BALB/c spleen cells prior to inoculation of C. parvum oocysts were resistant to infection. These results suggested that the two animal models should be valuable in the study of immunity to cryptosporidial infection.
Molecular population genetic evidence of horizontal spread of two alleles of the pyrogenic exotoxin C gene (speC) among pathogenic clones of Streptococcus pyogenes
Authors: Kapur V , Nelson K , Schlievert P M , Selander R K , Musser J M
Published: January 2020

It has recently been demonstrated that the bacteriophage-borne gene (speC) encoding pyrogenic exotoxin C is harbored by phylogenetic lineages representing virtually the entire breadth of genomic differentiation present in the species Streptococcus pyogenes (J. M. Musser, A. R. Hauser, M. H. Kim, P. M. Schlievert, K. Nelson, and R. K. Selander, Proc. Natl. Acad. Sci. USA 88:2668-2672, 1991). To determine whether the speC genes occurring in association with divergent chromosomal genotypes (clones) are identical or represent a group of allelic variants, we sequenced speC from 23 S. pyogenes strains representing 15 clones identified by multilocus enzyme electrophoresis. Two alleles of speC are present in natural populations, and each allele occurs in clones that are well differentiated in overall chromosomal character; in one case, isolates of a single clone had different speC alleles. We interpret these patterns of toxin allele-clone distribution as evidence of occasional episodes of speC horizontal dissemination, presumably by bacteriophage-mediated gene transfer and recombination.
Host cell actin assembly is necessary and likely to provide the propulsive force for intracellular movement of Listeria monocytogenes
Authors: Sanger J M , Sanger J W , Southwick F S
Published: January 2020

Listeria monocytogenes is able to escape from the phagolysosome and grow within the host cell cytoplasm. By 3 h after initiation of infection, actin filaments begin to concentrate at one end of the bacterium. Polarization of F-actin is associated with intracellular bacterial movement, long projections of actin filaments forming directly behind the moving bacteria. New actin monomers are added to the region of the projection in proximity to the bacterium. The rate of new actin filament growth correlates closely with the speed of bacterial migration. This actin structure is anchored within the cytoplasm, serving as a fixed platform for directional expansion of the actin filament network. The actin projection progressively lengthens as the bacterium migrates. Cytochalasin blocks both elongation of the projection and bacterial movement but does not result in complete depolymerization of the bacterially induced actin structure, residual actin and alpha-actinin persisting in proximity to one end of the bacterium. Bacteria initially migrate within the cortical cytoplasm but later move to the peripheral membrane, where they form filopodiumlike structures which pivot and undulate in the extracellular medium. In the filopodia, bacteria are occasionally seen to abruptly change direction, turn 180 degrees, and move back into the medullary region of the host cell. All filopodium movement ceases once the bacterium containing the F-actin projection returns to the cortical cytoplasm. These results indicate that host cell actin polymerization is necessary for intracellular migration of listeriae and suggest that directional actin assembly may in fact generate the propulsive force for bacterial and filopodial movement.
Group G streptococcal M protein exhibits structural features analogous to those of class I M protein of group A streptococci
Authors: Collins C M , Kimura A , Bisno A L
Published: January 2020

We have previously studied a collection of group G streptococcal strains isolated from bacteremic human infections and demonstrated that such strains resist phagocytosis by human polymorphonuclear leukocytes but are type specifically opsonized by homologous antiserum. We have now performed Southern hybridization analysis on genomic DNA from eight blood isolates. All eight isolates showed DNA homology to a group A emm24 gene probe. The M-protein gene of one of the isolates, strain 1750, has now been isolated. This gene (emmG1) encodes a polypeptide of 67 kDa (MG1) which is reactive with antibodies to the partially purified M protein of the parent strain. The predicted amino acid structure of MG1 demonstrates significant identity with the carboxy terminus (C, D, and anchor domains) of M6 and M24 but only limited identity with the amino terminus (variable portion) of these group A M proteins. Southern hybridization of genomic DNA of the eight group G blood isolates with an emmG1 gene probe indicated there were at least four emm alleles associated with these strains. These studies indicate that M proteins of group G streptococci, like those of group A, are genetically heterogeneous. Moreover, MG1 appears to conform to the recently proposed class I structure of M-protein molecules and thus shares certain distinct structural features with the M proteins of well-established rheumatogenic group A streptococcal serotypes. Further comparison of the structures of group G and group A M proteins of throat and skin isolates may cast light on those configurations of the M protein molecules which are and are not critical for the expression of rheumatogenicity.
Release of cytokines induced by Salmonella typhimurium porins
Authors: Galdiero F , de L'ero G C , Benedetto N , Galdiero M , Tufano M A
Published: January 2020

Salmonella typhimurium SH5014 porins induce the release of tumor necrosis factor alpha (TNF-alpha), interleukin 1 alpha (IL-1 alpha), and IL-6 by human monocytes and of gamma interferon (IFN-gamma) and IL-4 by human lymphocytes. Porins at 1 microgram/ml induce the greatest release of TNF-alpha, IL-1 alpha, and IL-6 by monocytes and of IL-4 by lymphocytes, while porins at 5 micrograms/ml induce the greatest release of IFN-gamma by lymphocytes. The R form of lipopolysaccharide (LPS-R) induces the greatest release of TNF-alpha and IL-1 alpha by monocytes when used at a low concentration (1 microgram/ml). At higher concentrations (5 and 10 micrograms/ml, respectively), LPS-R induces the maximal release of IL-6 from monocytes and the maximal release of IL-4 from lymphocytes. The S form of LPS (LPS-S) induces the greatest release of TNF-alpha, IL-1 alpha, and IL-6 by monocytes and that of IL-4 by lymphocytes when used at a concentration of 1 microgram/ml. After stimulation with LPS-S, the largest quantity of TNF-alpha and IL-1 alpha released was less than that obtained after stimulation with LPS-R at the same concentration, while the quantity of IL-6 released was found to be slightly higher than that obtained after stimulation with porins or LPS-R. LPS-S (1 microgram/ml) induces IFN-gamma release from lymphocytes in notably smaller quantities than that obtained with LPS-R and slightly larger quantities than that obtained with porins. The preparation of porins used was found to be contaminated with 10 pg of LPS per 10 micrograms of porins, a quantity which was found to have no biological effect; furthermore, porin preparations with the addition of polymyxin B gave the same results.
Polymorphism in genes for the enzyme arginine deiminase among Mycoplasma species
Authors: Sugimura K , Ohno T , Azuma I , Yamamoto K
Published: January 2020

The extent of restriction fragment length polymorphism in genes for the arginine deiminase enzyme among 28 species of mycoplasmas was assessed by Southern blot analysis of DNA digested with EcoRI or TaqI nuclease probed with a 725-bp internal fragment of the arginine deiminase gene from Mycoplasma arginini. The results indicated unexpected heterogeneity among species of a single genus.
Identification of murine T-cell epitopes on the S4 subunit of pertussis toxin
Authors: Petersen J W , Holm A , Ibsen P H , Hasløv K , Heron I
Published: January 2020

The aim of the present study was to identify murine T-cell epitopes on pertussis toxin subunit S4. Six mouse strains with five different haplotypes at the H-2 locus were immunized with the pertussis toxin B oligomer. Lymph node lymphocytes were isolated and stimulated in an in vitro proliferation assay with pertussis toxin components and 11 overlapping synthetic peptides synthesized on the basis of the primary sequence of S4. In vitro proliferative responses to the synthetic peptides revealed the presence of four distinct murine T-cell epitopes on subunit S4. The recognition of the peptides was major histocompatibility complex restricted. Immunizing four of the six mouse strains with the synthetic peptides showed that the peptides which were demonstrated to contain T-cell epitopes following immunization with the B oligomer were able to induce proliferative responses to detoxified pertussis toxin and pertussis toxin components containing subunit S4. One of the identified murine T-cell epitopes corresponded to one of the major human T-cell epitopes previously identified on subunit S4. It is hoped that this murine model system will facilitate the development of a synthetic immunogen mimicking the protective properties of pertussis toxin.
Purification of a factor which provides a costimulatory signal for gamma interferon production
Authors: Nakamura K , Okamura H , Nagata K , Komatsu T , Tamura T
Published: January 2020

A protein factor which induces high levels of gamma interferon (IFN-gamma) in resting splenic nonadherent cells was isolated from the sera of mice with generalized inflammation caused by endotoxic shock. The factor was highly purified by ammonium sulfate precipitation followed by ion-exchange column chromatography on DEAE-Sepharose, molecular sieving on Ultrogel AcA 44, and hydrophobic column chromatography with phenyl-Sepharose. It was further purified to apparent homogeneity by polyacrylamide gel electrophoresis. It induced IFN-gamma production in a dose-dependent manner in the presence of interleukin-2, monoclonal anti-CD3 antibody (anti-CD3 MAb), or concanavalin A (ConA) in spleen cells deprived of plastic plate- and nylon wool-adherent cells. Anti-CD3 MAb induced the highest level of production of the three. The factor, interleukin-2, anti-CD3 MAb, or ConA alone induced a trace of or no detectable IFN-gamma in these cells. The factor also exhibited an accessory function during proliferation in these cells in the presence of a suboptimal dose of ConA. However, the factor failed to stimulate IFN-gamma production when staphylococcal enterotoxin A, a superantigenic T-cell mitogen, was employed. Treatment with pronase or heat abolished these activities. These studies confirm the existence of a soluble protein factor which is able to exhibit a novel accessory function in IFN-gamma production in resting T or natural killer cells. It will be of interest to compare this factor with the recently cloned human natural killer stimulatory factor (NKSF/IL-12).
Cytokine gene expression in human peripheral blood mononuclear cells stimulated by mannoprotein constituents from Candida albicans
Authors: Ausiello C M , Urbani F , Gessani S , Spagnoli G C , Gomez M J , Cassone A
Published: January 2020

The expression of cytokine genes in cultures of human peripheral blood mononuclear cells (PBMC) stimulated with mannoprotein constituents (MP) of Candida albicans has been studied by means of S1 nuclease mapping analysis, polymerase chain reaction, and enzyme-linked immunosorbent assay. MP induced early, consistent, and long-lasting production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, and IL-6 mRNAs. Similar results were obtained when the same PBMC cultures were stimulated with the purified protein derivative (PPD) from Mycobacterium tuberculosis or with IL-2, although lower levels of IL-6 mRNA were detected in IL-2-stimulated cells than in MP- or PPD-stimulated cells. MP, PPD, and IL-2 induced appreciable levels of granulocyte-macrophage colony-stimulating factor and gamma interferon, but only MP and PPD were able to induce IL-2 mRNA. MP were unable to stimulate a consistent expression of the genes encoding for IL-4, IL-5, and IL-10, while low, sometimes barely detectable levels of these cytokine mRNAs were observed in PPD- or IL-2-stimulated PBMC cultures. When protein synthesis of MP-stimulated PBMC was inhibited by cycloheximide, a superinduction of mRNAs for IL-4 and IL-10 and, more markedly, gamma interferon was observed. Overall, these results highlight the powerful, selective induction of cytokine gene expression by MP constituents of C. albicans in human PBMC cultures, thus providing some functional clues to explain the efficient state of the anticandidal response in normal human subjects.
Collagen mediates adhesion of Streptococcus mutans to human dentin
Authors: Switalski L M , Butcher W G , Caufield P C , Lantz M S
Published: January 2020

Some strains of Streptococcus mutans were found to recognize and bind collagen type I. Binding of 125I-labeled collagen type I was specific in that collagen types I and II, but not unrelated proteins, were able to inhibit binding of the labeled ligand to bacteria. Collagen binding to S. mutans was partially reversible and involved a limited number of bacterial binding sites per cell. S. mutans UA 140 cells bound collagen type I with high affinity (Kd = 8 x 10(-8) M). The number of binding sites per cell was 4 x 10(4). Collagen-binding strains of S. mutans were found to adhere to collagen-coated surfaces as well as to pulverized root tissue. S. mutans strains that did not bind the soluble ligand were unable to adhere to these substrata. Adherence to collagen-coated surfaces could be inhibited with collagen or clostridial collagenase-derived collagen peptides. Adherence of S. mutans to dentin was enhanced by collagen types I and II but inhibited by collagen peptides. S. mutans UA 140 bound significantly less 125I-collagen type I following treatment with peptidoglycan-degrading enzymes. These enzymes released a collagen-binding protein (collagen receptor) with a relative molecular size of 16 kDa. The results of this study suggest that collagen mediates adhesion of S. mutans to dentin. This interaction may target collagen-binding strains of S. mutans to dentin in the oral cavity and may play a role in the pathogenesis of root surface caries.
Transfer of antibody against Borrelia duttonii from mother to young in ddY mice
Authors: Morshed M G , Yokota M , Nakazawa T , Konishi H
Published: January 2020

The route of transfer of anti-Borrelia duttonii antibody subclasses from mother to young and their role in protection against borrelial challenge infection in ddY mice were investigated. Offspring from infected and noninfected mice were segregated and nursed by noninfected or infected mothers. Enzyme-linked immunosorbent assay analysis of antibodies of the cross-suckled offspring revealed that anti-B. duttonii immunoglobulin G1 (IgG1) is transferred exclusively in milk and that IgG2a is transferred mainly in milk but also slightly through the yolk sac route. On the other hand, IgG3 is transferred mainly through the yolk sac route but also slightly in milk, whereas IgG2b is transferred through both routes but to a lesser extent. Anti-borrelial IgM was not detected in any offspring. The protective role of transferred IgG subclasses was examined by challenge infection with B. duttonii. Offspring from noninfected mice fed by infected mothers had IgG1, IgG2a, and IgG3 at challenge and were completely protected against the challenge infection. On the other hand, offspring from infected mice fed by noninfected mothers had only IgG3, and 8 of 10 were completely protected from challenge infection whereas the other 2 contracted slight and transient spirochetemia. These findings suggested that anti-borrelial IgG3 alone has considerable protective activity and that IgG1, IgG2a, or both, either by themselves or together with IgG3, have a complete protective activity against borrelial infection.
Identification of hydrogen peroxide as a Streptococcus pneumoniae toxin for rat alveolar epithelial cells
Authors: Duane P G , Rubins J B , Weisel H R , Janoff E N
Published: January 2020

Streptococcus pneumoniae infections of the lung are associated with significant damage to the alveolar epithelium. Host phagocytes and pneumolysin, a cytolytic toxin of S. pneumoniae, are believed to contribute to this cellular damage, yet experiments in which these elements are absent demonstrate the presence of an additional soluble S. pneumoniae factor that is toxic to alveolar epithelium. We examined the effects of S. pneumoniae-associated alveolar epithelial cell injury by factors other than S. pneumoniae-derived pneumolysin or phagocyte products by exposing cultured rat type II alveolar epithelial cells (RAEC) to S. pneumoniae mutants that lacked pneumolysin activity. We found that mutant pneumolysin-deficient strains of S. pneumoniae produced injury to RAEC similar to that produced by the parent strains. A toxin of type 14 S. pneumoniae was distinguished from pneumolysin by physiochemical (i.e., molecular mass and heat stability) and functional (i.e., hemolytic activity and cytotoxic activity) properties and was identified as hydrogen peroxide. All S. pneumoniae strains tested produced hydrogen peroxide, and in many strains hydrogen peroxide production was comparable to that of activated neutrophils. We conclude that S. pneumoniae produces hydrogen peroxide in concentrations that are cytotoxic to RAEC in vitro and that alveolar epithelial damage due to hydrogen peroxide may be involved in the pathogenesis of host cellular injury in pneumococcal pneumonia.
A poliovirus hybrid expressing a neutralization epitope from the major outer membrane protein of Chlamydia trachomatis is highly immunogenic
Authors: Murdin A D , Su H , Manning D S , Klein M H , Parnell M J , Caldwell H D
Published: January 2020

Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes on the major outer membrane protein (MOMP) of C. trachomatis have been identified as important targets for the development of vaccines. In order to examine the immunogenicity of a recombinant vector expressing a chlamydial epitope, a poliovirus hybrid was constructed in which part of neutralization antigenic site I of poliovirus type 1 Mahoney (PV1-M) was replaced by a sequence from variable domain I of the MOMP of C. trachomatis serovar A. The chlamydial sequence included the neutralization epitope VAGLEK. This hybrid was viable, grew very well compared with PV1-M, and expressed both poliovirus and chlamydial antigenic determinants. When inoculated into rabbits, this hybrid was highly immunogenic, inducing a strong response against both PV1-M and C. trachomatis serovar A. Antichlamydia titers were 10- to 100-fold higher than the titers induced by equimolar amounts of either purified MOMP or a synthetic peptide expressing the VAGLEK epitope. Furthermore, rabbit antisera raised against this hybrid neutralized chlamydial infectivity both in vitro, for hamster kidney cells, and passively in vivo, for conjunctival epithelia of cynomolgus monkeys. Because poliovirus infection induces a strong mucosal immune response in primates and humans, these results indicate that poliovirus-chlamydia hybrids could become powerful tools for the study of mucosal immunity to chlamydial infection and for the development of recombinant chlamydial vaccines.
Epithelial cells secrete the chemokine interleukin-8 in response to bacterial entry
Authors: Eckmann L , Kagnoff M F , Fierer J
Published: January 2020

Bacterial invasion of mucosal surfaces results in a rapid influx of polymorphonuclear leukocytes. The chemotactic stimulus responsible for this response is not known. Since epithelial cells are among the first cells entered by many enteric pathogens, we investigated the ability of epithelial cells to provide an early signal for the mucosal inflammatory response through the release of chemotactic cytokines. As shown herein, the chemokine interleukin-8 (IL-8), a potent chemoattractant and activator of polymorphonuclear leukocytes, was secreted by intestinal and cervical epithelial cells in response to bacterial entry. Moreover, a variety of different bacteria, including those that remain inside phagosomal vacuoles, e.g., Salmonella spp., and those that enter the cytoplasm, e.g., Listeria monocytogenes, stimulated this response. Increased IL-8 mRNA levels could be detected within 90 min after infection. Neither bacterial lipopolysaccharide nor noninvasive bacteria, including Escherichia coli and Enterococcus faecium, induced an IL-8 response. Moreover, tumor necrosis factor alpha, which is known to be expressed by some epithelial cells, was not detected in the culture supernatants after bacterial entry, and addition of anti-tumor necrosis factor alpha antibodies had no effect on the IL-8 response following bacterial entry. These data suggest the novel concept that epithelial cells serve as an early signaling system to host immune and inflammatory cells in the underlying mucosa following bacterial entry.
Passive immunizing activity of sera from mice infected with Borrelia burgdorferi
Authors: Barthold S W , Bockenstedt L K
Published: January 2020

A single injection of serum from C3H mice at 90 days after intradermal inoculation with 10(4) Borrelia burgdorferi spirochetes protected naive mice when administered subcutaneously at -18 h relative to intradermal challenge inoculation with 10(4) B. burgdorferi spirochetes. When immune serum was given at intervals (-18, 0, +24, +48, and +96 h) relative to intradermal challenge with 10(4) B. burgdorferi spirochetes, it was protective if given before or at the time of challenge but not at later times. Protection with 90-day serum given at -18 h was effective at dilutions up to 1:32, but not 1:64, on the basis of culture or disease at either 5 or 15 days after challenge. Passive immunizing activity was also present in sera from mice at 21 days after intradermal challenge with 10(4), 10(2), or 10(1) spirochetes, indicating that the immunizing component was not dose dependent and probably not related to antibody to outer surface protein A. Passive immunizing titers of sera from mice at days 1, 15, 30, 90, 180, and 360 after intradermal B. burgdorferi inoculation appeared as early as day 15, were highest on day 30, and then declined progressively on days 90, 180, and 360. Immunizing titers of sera from mice at 360 days after intradermal B. burgdorferi inoculation were identical in passively immunized mice challenged with the original inoculum or with B. burgdorferi isolated at 360 days after inoculation, suggesting that there was no antigenic discrimination between the original inoculum and late isolates. These results suggest that protective antibody is produced early in the course of B. burgdorferi infection and is unrelated to antibody to outer surface protein A. In addition, the decline of protective serum titers over time despite persistent infection suggests that the antigens eliciting the protective response are either modified or suppressed, but antigenic modification could not be demonstrated.
Prevention of corticosteroid-induced suppression of human polymorphonuclear leukocyte-induced damage of Aspergillus fumigatus hyphae by granulocyte colony-stimulating factor and gamma interferon
Authors: Roilides E , Uhlig K , Venzon D , Pizzo P A , Walsh T J
Published: January 2020

Neutrophils (PMNs) are a critical line of defense against Aspergillus fumigatus infection. Increased frequency of invasive aspergillosis has been observed in patients receiving corticosteroids, suggesting a deleterious effect of these compounds on PMN antifungal function. To investigate this hypothesis and to determine the potential preventive utility of granulocyte colony-stimulating factor (G-CSF) and gamma interferon (IFN-gamma), the effects of hydrocortisone (HCS) and dexamethasone (DXS) on PMN-induced damage of Aspergillus fumigatus hyphae were studied with or without pretreatment of PMNs with G-CSF and IFN-gamma. PMNs treated with HCS (> or = 3,000 microM) or DXS (> or = 10 microM) during a 2-h colorimetric tetrazolium metabolic assay (using methylthiotetrazolium) showed suppressed percentage of hyphal damage (P < 0.02). In addition, both HCS (> or = 30 microM) and DXS (> or = 1 microM) significantly suppressed oxidative burst measured as superoxide anion release by PMNs in response to opsonized and nonopsonized hyphae as well as to N-formylmethionyl leucyl phenylalanine. Pretreatment of PMNs with G-CSF (4,000 U/ml) and/or IFN-gamma (100 and 1,000 U/ml) for 90 min prevented the suppression of hyphal damage that occurred in the presence of HCS (3,000 microM; P < 0.01) or DXS (10 microM; P < or = 0.001). G-CSF (4,000 U/ml) and IFN-gamma (100 U/ml) combined had an additive effect on increasing the antifungal activity of HCS-treated but not of DXS-treated PMNs compared with IFN-gamma alone (P = 0.015). Thus, these findings reveal that corticosteroids impair PMN function in response to A. fumigatus and that G-CSF and IFN-gamma prevent this impairment.
Isolation and nucleotide sequence of the gene encoding cytotoxic necrotizing factor 1 of Escherichia coli
Authors: Falbo V , Pace T , Picci L , Pizzi E , Caprioli A
Published: January 2020

Cytotoxic necrotizing factors (CNFs) are dermonecrotic protein toxins produced by human and animal clinical isolates of Escherichia coli. In this study, the CNF1 determinant was isolated and sequenced, showing that expression of biologically active toxin is governed by a unique open reading frame encoding a protein of 1,014 amino acids with a predicted molecular mass of 113.7 kDa. Nucleotide and protein data base searches showed significant homology between CNF1 and the dermonecrotic toxin of Pasteurella multocida. In particular, the two toxins were found to share a hydrophobic region of about 220 amino acids which is a potential membrane-spanning domain.
Molecular analysis of the leukotoxin determinants from Pasteurella haemolytica serotypes 1 to 16
Authors: Burrows L L , Olah-Winfield E , Lo R Y
Published: January 2020

All sixteen serotypes of Pasteurella haemolytica were shown to produce a leukotoxin protein which is immunologically related to the well-characterized serotype 1 leukotoxin. All of the leukotoxins were weakly hemolytic and were able to bind to BL-3 target cells. The leukotoxin determinants were characterized by Southern blot hybridization by use of the previously cloned serotype 1 determinant as the probe, and a number of distinct classes were identified. The leukotoxin determinants from serotypes 2, 3, and 11 were cloned. Nucleotide sequence analysis of the lktC and lktA genes of the serotype 3 and 11 determinants revealed nucleotide substitutions throughout the coding sequences. A comparison of the lktC and lktA genes and deduced proteins of serotypes 1, 3, and 11 showed that they are highly homologous.
Identification of coccoid Escherichia coli BJ4 cells in the large intestine of streptomycin-treated mice
Authors: Krogfelt K A , Poulsen L K , Molin S
Published: January 2020

Escherichia coli BJ4, a rat isolate, was used to examine the growth and differentiation of the microorganism in its natural habitat, the intestine. Growth of E. coli BJ4 in the large intestine of streptomycin-treated mice was compared with its growth in laboratory media. By a number of methods, it was shown that E. coli BJ4 differentiates, during growth in the intestine, into two distinct populations, one that has the characteristics of the laboratory-grown strain and one that appears as a coccoid cell. Furthermore, it was shown that there is a natural selection for the coccoid-type cell in the intestine, while in laboratory media growth of rod-shaped E. coli BJ4 is enhanced.
Induction of cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein by immunization with soluble recombinant protein without adjuvant
Authors: Malik A , Gross M , Ulrich T , Hoffman S L
Published: January 2020

Immunization of mice with irradiated malaria sporozoites induces protection that is dependent on CD8+ T cells, and adoptive transfer of CD8+ cytotoxic T lymphocyte (CTL) clones against rodent malaria circumsporozoite (CS) protein and sporozoite surface protein 2 completely protects against sporozoite challenge. Thus, there are now efforts to develop vaccines that induce CTL against the CS protein and sporozoite surface protein 2. Until recently, it was thought that induction of CTL required production of target proteins within cells, breakdown of the proteins to peptides in the cytoplasm, and transport of the peptides to the cell surface in combination with class I major histocompatibility complex molecules. It has now been shown that immunization with peptides in Freund's complete adjuvant and with soluble protein in liposomes can induce CTL. To determine whether we could induce CTL against the Plasmodium falciparum CS protein by immunization with soluble protein, B10.BR mice were immunized intravenously, intraperitoneally, or intramuscularly with a recombinant P. falciparum CS protein called RLF mixed with the adjuvant DETOX (monophosphoryl lipid A, cell wall skeleton of Mycobacteria phlei, and squalane). Two weeks after the last dose, spleen cells from mice immunized intravenously, but not intraperitoneally or intramuscularly, had peptide-specific, major histocompatibility complex-restricted, CD8+ T-cell-dependent cytolytic activity against peptide 368-390 from the 7G8 P. falciparum CS protein. To determine whether the adjuvant was required for induction of the cytolytic activity, mice were immunized with RLF without adjuvant, and similar cytolytic activity was demonstrated. The finding that we could induce CTL by administration of soluble protein without adjuvant markedly broadens the possibilities for vaccinologists working to develop methods of inducing CTL in humans.
Proteins with molecular masses of 50 and 80 kilodaltons encoded by genes downstream from the fimbrilin gene (fimA) are components associated with fimbriae in the oral anaerobe Porphyromonas gingivalis
Authors: Yoshimura F , Takahashi Y , Hibi E , Takasawa T , Kato H , Dickinson D P
Published: January 2020

Flanking DNA regions of the fimbrilin gene (designated fimA), which encodes the major subunit protein of Porphyromonas (Bacteroides) gingivalis fimbriae, were cloned in several manners from the P. gingivalis chromosome into Escherichia coli by screening with probes derived from a 2.5-kb SacI DNA fragment previously cloned. A total of 10.4 kb of DNA fragments from the P. gingivalis genome was cloned in the pUC plasmid. Expression of the fimA gene and possible flanking genes in the fragments cloned was examined in a pUC plasmid vector system and in a bacteriophage T7 RNA polymerase-promoter expression vector system. The results show that in the pUC plasmid system, a 45-kDa protein, a product of fimA, was only poorly expressed as a precursor of the fimbrilin protein (FimA) and could be detected from cell extracts in Western blotting (immunoblotting) analysis as a sharp band but not in colony immunoblotting analysis. On the other hand, in the T7 RNA polymerase-promoter system, the product of fimA and products of the possible flanking genes responsible for fimbriation were overproduced as thick bands of the 45-kDa protein and as 63-, 50-, and 80-kDa proteins, respectively, in stained electrophoresis gels. All of the recombinant proteins were insoluble and seemed to be expressed as precursors with leader peptides. The 63-kDa, 45-k*Da (a truncated protein of the 50-kDa protein), and 80-kDa proteins were purified after solubilization with sodium dodecyl sulfate. N-terminal amino acid sequences of the 45-k*Da and 80-kDa proteins were analyzed up to the first 35 residues with a gas-phase sequencer. Monospecific antibodies directed to the recombinant proteins, i.e., the 63-kDa, 45-k*Da, and 80-kDa proteins, were raised in rabbits. By using the antibodies, localization of their matured proteins in P. gingivalis was investigated by Western blotting analysis. Immunoblotting analysis suggests that at least the 50- and 80-kDa proteins, encoded by genes downstream from the fimA gene, are minor components associated with fimbriae.
Human T cells recognize mycobacterial heat shock proteins in the context of multiple HLA-DR molecules: studies with healthy subjects vaccinated with Mycobacterium bovis BCG and Mycobacterium leprae
Authors: Mustafa A S , Lundin K E , Oftung F
Published: January 2020

Heat shock proteins (HSP) are considered to be important targets of the immune response to mycobacteria and, as such, relevant to subunit vaccine design. If HSP are major antigens in cell-mediated immunity, they should be recognized in the context of most of the HLA-DR molecules required for presentation of mycobacterial antigens to T cells. We tested peripheral blood mononuclear cells (PBMC) and T-cell lines from Mycobacterium leprae- and M. bovis BCG-vaccinated subjects for proliferation in response to the 18- and 65-kDa HSP of M. leprae, the 65-kDa HSP of M. bovis BCG, and the 70-kDa HSP of M. tuberculosis. Irrespective of HLA types, PBMC showing a strong response to M. leprae proliferated in response to mycobacterial HSP. HLA restriction analysis with T-cell lines showed that the M. leprae 18-kDa HSP was recognized in the context of HLA-DR4, HLA-Dw4, and HLA-DR1 molecules. The T-cell lines recognized the M. leprae 65-kDa HSP in the context of all of the HLA-DR molecules expressed by autologous antigen-presenting cells, i.e., HLA-DR1, HLA-DR2, HLA-DR5, HLA-DR7, and importantly HLA-DR4 (HLA-Dw4 and HLA-Dw14), which is relevant to autoimmunity. The M. tuberculosis 70-kDa antigen was also presented to the T-cell lines by HLA-DR1, HLA-DR2, HLA-DR5, and HLA-DR7 molecules. In addition, this HSP was recognized in the context of the HLA-DRw53 molecule, which is frequently expressed in many regions where leprosy is endemic. The T-cell lines proliferating in response to a given HSP lysed autologous monocytes-macrophages pulsed with that HSP. The results demonstrate that PBMC from individuals immunized with M. leprae respond to mycobacterial HSP and that these HSP are presented to T cells by multiple HLA-DR molecules, a prerequisite for their application in the next generation of subunit vaccines.
A T cell-independent protective host response against Cryptococcus neoformans expressed at the primary site of infection in the lung
Authors: Hill J O , Dunn P L
Published: January 2020

T cell-independent host resistance expressed against a primary lung infection with Cryptococcus neoformans was investigated. Following intratracheal inoculation of the yeast, BALB/cBy scid/scid mice or CD4+ plus CD8+ T cell-depleted BALB/cBy mice developed a primary lung infection that remained stable for several weeks before progressing and disseminating to kill the host. By contrast, normal BALB/cBy hosts resolved the infection after 4 to 8 weeks. Thy+ CD4- CD8- cells were found to accumulate in the pulmonary alveoli of infected scid/scid or normal mice. Depletion of these cells caused the infection to progress more rapidly and resulted 4 weeks later in a 30- to 70-fold increase in yeast numbers in the lungs and dissemination to extrapulmonary sites. Cytofluorometric studies revealed that the Thy+ CD4- CD8- cells responsible were negative for the CD3 T cell marker. A small percentage of these Thy+ CD3- cells expressed asialo-Gm1, but treatment with asialo-Gm1 antibody did not have the same infection-enhancing effect as Thy-1 monoclonal antibody treatment. Further experiments revealed that Thy-1 monoclonal antibody treatment had no effect on the establishment of infectious foci in the brain or liver following intravenous inoculation of the yeast. The data point to the existence of an early resistance mechanism for which Thy+ CD3- CD4- CD8- cells are essential. This mechanism of host defense, while insufficient for complete protection, may be capable of delaying the development of cryptococcal meningoencephalitis by restricting the growth of the yeast at primary sites of infection in the lungs, even in immunodeficient mice.
Virulence of two Streptococcus pyogenes strains (types M1 and M3) associated with toxic-shock-like syndrome depends on an intact mry-like gene
Authors: Perez-Casal J F , Dillon H F , Husmann L K , Graham B , Scott J R
Published: January 2020

The major virulence factor of Streptococcus pyogenes, the M protein, is positively regulated at the transcriptional level by mry in the M type 6 strain studied. We show here that in two S. pyogenes strains isolated from cases of toxic-shock-like syndrome, a type M1 strain and a type M3 strain, an mry-like gene is also required for resistance to phagocytosis.
Immunity and responses of circulating leukocytes and lymphocytes in monkeys to aerosolized staphylococcal enterotoxin B
Authors: Tseng J , Komisar J L , Chen J Y , Hunt R E , Johnson A J , Pitt L , Rivera J , Ruble D L , Trout R , Vega A
Published: January 2020

Rhesus monkeys immunized intramuscularly or orally with staphylococcal enterotoxin B (SEB) toxoid or SEB toxoid incorporated in microspheres made of poly(DL-lactide-co-glycolide) were challenged with a lethal dose of aerosolized SEB to study their immunity and cellular responses in the circulation. It was found that circulating antibodies play a critical role in preventing SEB from triggering toxicosis. Monkeys with high levels of antibodies survived, while those with low levels underwent 2 to 3 days of toxicosis and died. Intramuscular immunization induced high levels and oral immunization induced low levels of antibodies. The circulating antibodies in surviving monkeys decreased dramatically within 20 min and started to rebound at 90 min after SEB challenge. At 90 min, the dying monkeys showed in the circulation a dramatic increase of polymorphonuclear leukocytes and decreases of NK cells and monocytes (CD16 and CD56 markers) as well as of lymphocytes with HLA-DR, CD2, CD8, and IL2R alpha (CD25) markers. The number of polymorphonuclear leukocytes showed an inverse correlation with the numbers of monocytes and various lymphocyte subpopulations which, except for IL-2R, CD16, and CD56(+) cells, showed a direct correlation with one another. The changes in the populations of leukocytes, monocytes, NK cells, and lymphocytes seem to be an indication of initial toxicosis; however, the roles of these cells in toxicosis and death remain to be defined.