bluuebunny/crossref_metadata_2025 huggingface dataset:
Virulent Salmonella typhimurium-induced lymphocyte depletion and immunosuppression in chickens
Authors: Hassan J O , Curtiss R
Published: January 2020

The effect of experimental Salmonella infection on chicken lymphoid organs, immune responses, and fecal shedding of salmonellae were assessed following oral inoculation of 1-day-old chicks or intra-air-sac infection of 4-week-old chickens with virulent S. typhimurium wild-type chi 3761 or avirulent S. typhimurium delta cya delta crp vaccine strain chi 3985. Some 4-week-old chickens infected intra-air-sac with chi 3761 or chi 3985 were challenged with Bordetella avium to determine the effect of Salmonella infection on secondary infection by B. avium. S. typhimurium chi 3761 caused lymphocyte depletion, atrophy of lymphoid organs, and immunosuppression 2 days after infection in 1-day-old chicks and 4-week-old chickens. The observed lymphocyte depletion or atrophy of lymphoid organs was transient and dose dependent. Lymphocyte depletion and immunosuppression were associated with prolonged fecal shedding of S. typhimurium chi 3761. No lymphocyte depletion, immunosuppression, or prolonged Salmonella shedding was observed in groups of chickens infected orally or intra-air-sac with chi 3985. Infection of chickens with salmonellae before challenge with B. avium did not suppress the specific antibody response to B. avium. However, B. avium isolation was higher in visceral organs of chickens infected with chi 3761 and challenged with B. avium than in chickens infected with B. avium only. Infection of chickens with chi 3985 reduced B. avium colonization. We report a new factor in Salmonella pathogenesis and reveal a phenomenon which may play a critical role in the development of Salmonella carrier status in chickens. We also showed that 10(8) CFU of chi 3985, which is our established oral vaccination dose for chickens, did not cause immunosuppression or enhance the development of Salmonella carrier status in chickens.
Noradrenaline inhibits lipopolysaccharide-induced tumor necrosis factor and interleukin 6 production in human whole blood
Authors: van der Poll T , Jansen J , Endert E , Sauerwein H P , van Deventer S J
Published: January 2020

Sepsis and lipopolysaccharide (LPS) trigger the systemic release of both cytokines and catecholamines. Cytokines are known to be capable of eliciting a stress hormone response in vivo. The present study sought insight into the effect of noradrenaline on LPS-induced release of tumor necrosis factor alpha (TNF) and interleukin 6 (IL-6) in human whole blood. Whole blood was incubated with LPS for 4 h at 37 degrees C in the presence and absence of noradrenaline and/or specific alpha and beta antagonists and agonists. Noradrenaline caused a dose-dependent inhibition of LPS-induced TNF and IL-6 production. This effect could be completely prevented by addition of the specific beta 1, antagonist metoprolol, while it was not affected by the alpha antagonist phentolamine. Specific beta-adrenergic stimulation by isoprenaline mimicked the inhibiting effect of noradrenaline on LPS-evoked cytokine production, whereas alpha-adrenergic stimulation by phenylephrine had no effect. Fluorescence-activated cell sorter analysis demonstrated that beta-adrenergic stimulation had no effect on LPS binding to and internalization into mononuclear cells or on the expression of CD14, the major receptor for LPS on mononuclear cells. In acute sepsis, enhanced release of noradrenaline may be part of a negative feedback mechanism meant to inhibit ongoing TNF and IL-6 production.
Reduced virulence of Serpulina hyodysenteriae hemolysin-negative mutants in pigs and their potential to protect pigs against challenge with a virulent strain
Authors: Hyatt D R , ter Huurne A A , van der Zeijst B A , Joens L A
Published: January 2020

The role of the Serpulina hyodysenteriae hemolysin encoded by the tlyA gene in the pathogenesis of swine dysentery (SD) was studied. tlyA mutants of two S. hyodysenteriae strains (B204 and C5) were tested for virulence in pigs. None of the animals developed SD. However, after infection with wild-type strain B204 or C5, the incidence of SD was 100 or 60%, respectively. Thus, the tlyA-encoded hemolysin of S. hyodysenteriae is an important virulence factor in SD. The potential of these mutants to protect pigs against challenge with a virulent S. hyodysenteriae strain was also studied. After challenge with wild-type strain B204, 50% of pigs previously inoculated with the B204 tlyA mutant were protected, whereas all control pigs contracted SD. None of the pigs previously inoculated with the C5 tlyA mutant developed SD upon challenge with wild-type strain B204, whereas 40% of the control pigs developed SD in this experiment. Thus, previous colonization with S. hyodysenteriae tlyA mutants in pigs provides partial protection against challenge with a virulent S. hyodysenteriae strain.
Uroepithelial cells are part of a mucosal cytokine network
Authors: Hedges S , Agace W , Svensson M , Sjögren A C , Ceska M , Svanborg C
Published: January 2020

This study compared the cytokine production of uroepithelial cell lines in response to gram-negative bacteria and inflammatory cytokines. Human kidney (A498) and bladder (J82) epithelial cell lines were stimulated with either Escherichia coli Hu734, interleukin 1 alpha (IL-1 alpha), or tumor necrosis factor alpha (TNF-alpha). Supernatant samples were removed, and the RNA was extracted from cells at 0, 2, 6, and 24 h. The secreted cytokine levels were determined by bioassay or immunoassay; mRNA was examined by reverse transcription-PCR. The two cell lines secreted IL-6 and IL-8 constitutively. IL-6 and IL-8 mRNA were constitutively produced in both cell lines; IL-1 beta mRNA was detected in J82 cells. IL-1 alpha induced significantly higher levels of IL-6 secretion than did E. coli Hu734 or TNF-alpha. IL-1 alpha and TNF-alpha induced significantly higher levels of IL-8 secretion than did E. coli Hu734. Secreted IL-1 beta was not detected; IL-1 alpha and TNF-alpha were not detected above the levels used for stimulation. IL-1 alpha, IL-1 beta, IL-6, and IL-8 mRNAs were detected in both cell lines after exposure to the stimulants. TNF-alpha mRNA was occasionally detected in the J82 cell line after TNF-alpha stimulation. Cytokine (IL-6 and IL-8) and control (glyceraldehyde 3-phosphate dehydrogenase [G3PDH] and beta-actin) mRNA concentrations were quantitated with internal PCR standards. Cytokine mRNA levels relative to beta-actin mRNA levels were the highest in E. coli-stimulated cells. In comparison, the cytokine mRNA levels relative to G3PDH mRNA levels were the highest in IL-1 alpha-stimulated cells. beta-Actin mRNA levels decreased after bacterial stimulation but not after cytokine stimulation, while G3PDH mRNA levels increased in response to all of the stimulants tested. These results suggested that E. coli Hu734 lowered the beta-actin mRNA levels in uroepithelial cells, thus distorting the IL-6 and IL-8 mRNA levels relative to this control. In summary, E. coli IL-1 alpha and TNF-alpha were found to activate the de novo synthesis and secretion of IL-6 and IL-8 in uroepithelial cells. These results emphasize the role of epithelial cells in cytokine-mediated responses during the early stages of infection.
Serum antibody immunoglobulin G of mice convalescent from Plasmodium yoelii infection inhibits growth of Plasmodium falciparum in vitro: blood stage antigens of P. falciparum involved in interspecies cross-reactive inhibition of parasite growth
Authors: Ray P , Sahoo N , Singh B , Kironde F A
Published: January 2020

We demonstrated that antibodies in the serum of BALB/c mice convalescent from Plasmodium yoelii infection inhibit the in vitro growth of Plasmodium falciparum. Blood stage P. falciparum antigens that cross-react with the convalescent-phase mouse serum antibodies were identified and partially characterized. Convalescent-phase mouse serum immunoglobulin G (IgG) reacted with P. falciparum lysates at up to a 1:15,000 dilution of the immune sera and bound to P. falciparum-parasitized erythrocytes at up to a 1:5,000 dilution of the sera. The cross-reactive moieties of antigens in parasite lysates were resistant to oxidation by periodate but sensitive to trypsinization. About 15 polypeptides (M(r)s of 15,000 to 110,000) of P. falciparum blood stages were recognized by the convalescent-phase mouse anti-P. yoelii sera; many of these antigens were metabolically 35S labeled and specifically immunoprecipitated. Also, virtually all of the cross-reactive antigens were recognized by human malaria-immune sera. The anti-P. yoelii serum antibodies bound, with high affinity, to at least five of the cross-reactive antigens (M(r)s of 107,000, 84,000, 53,000, 36,000, and 30,000). By phase separation in Triton X-114, eight interspecies cross-reactive antigens (M(r)s of 84,000, 76,000, 51,000, 31,000, 29,000, 28,000, 23,000, and 22,000) were found to be integral membrane proteins. Convalescent-phase mouse serum IgG strongly inhibited the differentiation of P. falciparum from schizonts to rings; 75 micrograms of IgG per ml caused 80% inhibition of release of merozoites and their invasion into erythrocytes. On the other hand, the anti-P. yoelii serum antibodies also inhibited intracellular development of P. falciparum from rings to schizonts; 25 micrograms of IgG per ml caused 50% inhibition. Inhibition of P. falciparum growth by anti-P. yoelii serum IgG suggests that some of the interspecies cross-reactive antigens contain important conserved epitopes and induce protective antibodies against P. falciparum.
Immunobiological activities of a 55-kilodalton cell surface protein of Prevotella intermedia ATCC 25611
Authors: Matsushita K , Nagaoka S , Arakaki R , Kawabata Y , Iki K , Kawagoe M , Takada H
Published: January 2020

A protein was extracted from whole cells of Prevotella intermedia ATCC 25611 with sodium lauroylsarcosine and purified by chromatography on a DEAE-Sepharose fast-flow column. The The apparent molecular weight of the protein was 55,000. A mouse polyclonal antibody specific for the protein recognized the cell surface structure of P. intermedia and also reacted with proteins in lysates of other black-pigmented anaerobic bacteria, such as Porphyromonas endodontalis and Prevotella melaninogenica, but not with those in lysates of Porphyromonas gingivalis or with the purified fimbriae of P. gingivalis 381. The N-terminal sequence of the 55-kDa protein showed only low homology with the cell surface proteins of any black-pigmented bacteria reported to date. The level of immunoglobulin G antibody to the antigen was higher in the sera of patients with periodontitis than in the sera of healthy volunteers. The protein induced interleukin-1 alpha, -1 beta, -6, and -8 and tumor necrosis factor alpha in human peripheral blood mononuclear cell cultures and interleukin-1 beta and -6 in human umbilical vascular endothelial cell and gingival fibroblast cultures. The protein induced interleukin-6 and tumor necrosis factor alpha activities in peritoneal macrophages from C3H/HeJ as well as from C3H/HeN mice and also induced cytokine activities in the sera of both strains of mice primed with muramyldipeptide.
Molecular cloning and characterization of the 78-kilodalton glucose-regulated protein of Trypanosoma cruzi
Authors: Tibbetts R S , Kim I Y , Olson C L , Barthel L M , Sullivan M A , Winquist A G , Miller S D , Engman D M
Published: January 2020

The protozoan Trypanosoma cruzi is the etiologic agent of Chagas' disease, an illness responsible for morbidity and death among millions of Latin Americans. Mice also develop this disease when infected with T. cruzi and are a useful model organism for the study of parasite-specific immune responses. To identify immunogenic T. cruzi antigens, serum from an infected mouse was used to isolate clones from a T. cruzi epimastigote cDNA expression library. One of these clones was found to encode the 78-kDa glucose-regulated protein (grp78), the endoplasmic reticular member of the 70-kDa heat shock protein (hsp70) family. Like the mammalian and yeast grp78s, the T. cruzi protein contains an endoplasmic reticular leader peptide and a carboxyl-terminal endoplasmic reticular retention sequence. T. cruzi grp78 is encoded by a tandemly arranged family of three genes located on a chromosome of 1.6 Mb. The effects on grp78 expression of heat shock and tunicamycin treatment, the latter of which specifically stimulates mammalian grp78, were investigated. While the level of the grp78 protein remained constant under all circumstances, grp78 mRNA was unaffected by heat shock but induced fivefold by tunicamycin. Finally, we found that grp78 is the most immunogenic of the T. cruzi heat shock proteins we have characterized, reacting strongly in immunoblots with sera from infected mice.
Recombinant Mycobacterium bovis BCG secreting functional interleukin-2 enhances gamma interferon production by splenocytes
Authors: O'Donnell M A , Aldovini A , Duda R B , Yang H , Szilvasi A , Young R A , DeWolf W C
Published: January 2020

Mycobacterium bovis BCG was genetically engineered to express and secrete mouse interleukin-2 (IL-2) and rat IL-2. Genes encoding IL-2 were inserted into an Escherichia coli-BCG shuttle plasmid under the control of the BCG HSP60 promoter. To facilitate study of proteins produced in this system, the IL-2 gene product was expressed (i) alone, (ii) with the mycobacterial alpha-antigen secretion signal sequence at the amino terminus, (iii) with an influenza virus hemagglutinin epitope tag at the amino terminus, and (iv) with both the secretion signal sequence and the epitope tag. When expressed with the alpha-antigen signal sequence, biologically active IL-2 was secreted into the extracellular medium. Western blot (immunoblot) analysis of the intracellular IL-2 and extracellular IL-2 revealed that the secretion signal was appropriately cleaved from the recombinant lymphokine upon secretion. To assess the ability of recombinant BCG to stimulate cytokine production in a splenocyte population, mouse splenocytes were cultured together with wild-type or IL-2-producing BCG. IL-2-secreting BCG clones stimulated substantial increases in gamma interferon production, which could be reproduced by the addition of exogenous IL-2 to BCG. Levels of IL-6, IL-10, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor were not significantly changed, while IL-4 and IL-5 remained undetectable (less than 50 pg/ml). The enhanced production of gamma interferon in response to IL-2-secreting BCG was strain independent. Recombinant BCG expressing mammalian cytokines provides a novel means to deliver cytokines and may augment the immunostimulatory properties of BCG in immunization and cancer therapy.
Purification and antigenicity of a novel glucan-binding protein of Streptococcus mutans
Authors: Smith D J , Akita H , King W F , Taubman M A
Published: January 2020

A novel glucan-binding protein (GBP) having an apparent molecular mass of 59 kDa (GBP59) has been purified from Streptococcus mutans SJ by a combination of affinity chromatography on alpha-1,6-linked glucan, gel filtration chromatography, and ion-exchange chromatography. GBP59 was distinct from the quantitatively predominant S. mutans GBP (GBP74) on the basis of size, elution position in a salt gradient, and antigenicity. Rat antisera to purified GBP59 and GBP74 did not cross-react. GBP59 is apparently immunogenic in humans, since immunoglobulin A (IgA) antibody in 20 of 24 adult parotid saliva samples was shown to react with GBP59 in an enzyme-linked immunosorbent assay. The glucan-binding activity of GBP59 was confirmed by anti-GBP59 immunogold labelling of Sephadex G-50 that had been preincubated with S. mutans culture supernatant. GBP59 could be detected in culture supernatants of all laboratory strains of S. mutans (e.g., Ingbritt), as well as all strains of S. mutans that had been recently isolated from young children. GBP59 was often the only component in protease inhibitor-containing 4-h S. mutans culture supernatants that reacted with human parotid salivary IgA antibody in Western blot (immunoblot) analyses. These studies suggest that GBP59 is a structurally and antigenically distinct S. mutans GBP that can elicit significant levels of salivary IgA antibody in humans.
The Vibrio cholerae toxin-coregulated-pilus gene tcpI encodes a homolog of methyl-accepting chemotaxis proteins
Authors: Harkey C W , Everiss K D , Peterson K M
Published: January 2020

Virulence gene activation in Vibrio cholerae is under the control of the ToxR-ToxT regulatory cascade. The ToxR regulon consists of genes required for toxin-coregulated-pilus (TCP) biogenesis, accessory colonization factor genes, cholera toxin genes, and ToxR-activated genes (tag) of unknown function. The tagB gene was isolated by using a tagB::TnphoA fusion junction to probe a V. cholerae )395 bacteriophage lambda library. Nucleotide sequence analysis revealed that tagB is identical to tcpI, a gene which encodes a protein that negatively regulates the synthesis of the major pilin subunit of TCP (TcpA). Our results show that the tcpI gene encodes a 620-amino-acid protein that shares extensive sequence similarity with the highly conserved signaling domain in methyl-accepting chemotaxis proteins. Expression of tcpI in Escherichia coli results in the synthesis of a 71-kDa polypeptide that becomes localized to the inner membrane. Similarly, TcpI-PhoA alkaline phosphatase activity is enriched in V. cholerae inner membrane preparations. Colonies of V. cholerae tcpI::TnphoA mutant cells display increased swarming on solid media when compared with those of the parental V. cholerae O395. Taken together, these observations suggest that TcpI may play a dual role in promoting vibrio colonization of the small bowel. In response to the appropriate environmental signal(s), TcpI permits maximum expression of tcpA while simultaneously reducing vibrio chemotaxis-directed motility. We believe coordinate regulation of colonization and motility determinants, in such a fashion, facilitates efficient V. cholerae microcolony formation.
Immune responses of young mice to pneumococcal type 9V polysaccharide-tetanus toxoid conjugate
Authors: Lu C H , Lee C J , Kind P
Published: January 2020

Pneumococcal type 9V polysaccharide (PS), contained in the current pneumococcal vaccine, induces only a weak antibody response in young children and therefore is not an effective vaccine for young children. To increase its immunogenicity, a conjugate of PS to a protein carrier, tetanus toxoid (TT), was prepared. To quantify the immune response, mouse anti-9V PS immunoglobulin G (IgG) and IgM reference standards were established. Young mice immunized at 2 weeks of age produced IgM antibody in response to 9V PS alone or 9V PS conjugated to TT. However, only the 9V PS-TT conjugate induced an IgG antibody response and an anamnestic effect. Thus, a covalent linkage between TT and 9V PS was required for isotype switching from IgM to IgG. 9V PS-TT adsorbed with aluminum hydroxide adjuvant resulted in a fivefold or greater increase in the IgG antibody level. We also studied the effect of maternal immunization on the immune response of young mice to 9V PS-TT. Maternal immunization before mating or before mating and during gestation primed 2-week-old progeny given two injections of 9V PS-TT to produce more IgM antibody than progeny from unimmunized mothers. The IgG antibody level of neonates at birth was similar to that observed in the mothers and was probably passive antibody. These results indicate that maternal immunization with an optimum dose of a PS-protein conjugate before and/or during pregnancy, followed by immunization of the offspring with the conjugate, could provide young children with an enhanced IgM antibody response to pneumococcal PSs.
Neutralizing monoclonal antibodies to an extracellular Pseudomonas cepacia protease
Authors: Kooi C , Cox A , Darling P , Sokol P A
Published: January 2020

Pseudomonas cepacia produces at least two extracellular proteases with apparent molecular masses of 36,000 and 40,000 Da. The 36-kDa protease has high proteolytic activity and the 40-kDa protease has low proteolytic activity with hide powder azure as a substrate. Monoclonal antibodies (MAbs) were raised against the purified 36- and 40-kDa proteases. Several MAbs directed against the 36-kDa protease were found to recognize the 40-kDa protease by Western immunoblot analysis. Similarly, a MAb directed against the 40-kDa protease recognized the 36-kDa protease, suggesting that these two proteases may be immunologically related. A MAb directed against the 36-kDa protease, designated 36-6-8, and a MAb directed against the 40-kDa protease (MAb G-11) cross-reacted with other extracellular proteases, such as Pseudomonas aeruginosa elastase and alkaline protease, Pseudomonas pseudomallei protease, and the Vibrio cholerae hemagglutinin/protease. MAb 36-6-8 neutralized the P. cepacia 36-kDa protease, P. aeruginosa elastase, P. pseudomallei protease, and V. cholerae hemagglutinin/protease but did not affect P. aeruginosa alkaline protease activity. In contrast, MAb G-11 to the 40-kDa protease neutralized only the P. cepacia 36-kDa protease. This evidence suggests that the neutralizing MAb, 36-6-8, recognizes an epitope conserved among some metalloproteases. This epitope may lie at or near the active site of the P. cepacia 36-kDa protease and P. aeruginosa elastase.
Epitope specificity and isoforms of the mycobacterial 19-kilodalton antigen
Authors: Harris D P , Vordermeier H M , Brett S J , Pasvol G , Moreno C , Ivanyi J
Published: January 2020

The topography and specificity of B- and T-cell stimulatory epitopes from the 19-kDa protein of Mycobacterium tuberculosis were investigated by using overlapping synthetic peptides. Murine antisera identified two cryptic epitopes (residues 11 to 30 and 61 to 80) and one species-specific immunodominant epitope (residues 140 to 159). Immunoglobulins G1 and G2a antibody isotypes varied for the respective peptide immunogens but without relationship to the T-cell cytokine profiles which were characterized by high gamma interferon and low interleukin 5 levels. Antisera to recombinant M. tuberculosis 19-kDa protein (rGST-19) cross-reacted with homologous proteins of similar size from organisms of the Mycobacterium avium-intracellulare complex. Two-dimensional gel electrophoresis revealed differences in the number, relative mobility, and charge of isoforms of the 19-kDa protein, possibly reflecting posttranslational modifications. The immunodominant T-cell epitope from the M. tuberculosis 19-kDa protein (residues 61 to 80) and the corresponding peptide sequence from Mycobacterium avium subsp. intracellulare (residues 64 to 83), differing at five residues, were both recognized in a genetically permissive manner. Peptides 61-80 and 64-83 stimulated cross-reactive responses in BALB/c (H-2d) mice, while in the C57BL/10 (H-2b) strain, responses to peptide 61-80 were species specific. In purified protein derivative-positive healthy individuals, the M. avium subsp. intracellulare peptide stimulated stronger responses than did the M. tuberculosis peptide, whereas patients with active tuberculosis had enhanced in vitro T-cell responses to both peptides.
Pertussis toxin activates platelets through an interaction with platelet glycoprotein Ib
Authors: Sindt K A , Hewlett E L , Redpath G T , Rappuoli R , Gray L S , Vandenberg S R
Published: January 2020

Platelets present a unique model to study the B-oligomer effects of pertussis toxin because they become activated in response to the B oligomer but are not susceptible to ADP-ribosylation by the holotoxin. In these studies, the B oligomer of pertussis toxin caused concentration-dependent platelet activation, as determined by increases in intracellular calcium concentration, dense granule secretion, and platelet aggregation. Stirring was required for pertussis toxin to increase intracellular calcium. A monoclonal antibody against platelet glycoprotein Ib abolished increases in intracellular calcium concentration and increased the latency and reduced the slope of the aggregation response elicited by the B oligomer. Pertussis toxin also evoked [14C]serotonin release from platelets, and this effect was inhibited, though not eliminated, by an antibody against platelet glycoprotein Ib. Binding of pertussis toxin to glycoprotein Ib was observed after nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that the B oligomer of pertussis toxin induces platelet activation mediated, at least in part, by an interaction with platelet glycoprotein Ib.
Inhibition of binding, entry, or intracellular proliferation of Ehrlichia risticii in P388D1 cells by anti-E. risticii serum, immunoglobulin G, or Fab fragment
Authors: Messick J B , Rikihisa Y
Published: January 2020

The effects of equine antiserum, immunoglobulin G (IgG) specific for Ehrlichia risticii, and its Fab fragment on E. risticii binding to, internalization into, and proliferation in P388D1 cells were studied by immunofluorescence flow cytometry. Anti-E. risticii equine serum or IgG inhibited E. risticii at a stage beyond binding and internalization. In contrast, monovalent anti-E. risticii equine Fab fragments inhibited E. risticii binding and internalization into P388D1 cells. In the presence of control equine serum, IgG, or its Fab fragment, E. risticii cells were bound, were internalized and subsequently grew within P388D1 cells, and eventually destroyed the host cells as effectively as was the case without equine serum, IgG, or Fab fragments. Anti-E. risticii IgG but not normal horse IgG inhibited L-[14C]glutamine metabolism in Percoll gradient-purified E. risticii. These findings suggest that the Fab fragment of intact anti-E. risticii IgG blocks the ligands on E. risticii responsible for non-IgG-mediated internalization and diverts them to bind via the Fc receptor. Following Fc-mediated entry of E. risticii, the antibody interfered with the metabolic activity of E. risticii cells, rendering them incapable of proliferation in P388D1 cells and resulting in the eventual destruction of the organisms.
T cells and macrophages in Trypanosoma brucei-related glomerulopathy
Authors: van Velthuysen M L , Mayen A E , van Rooijen N , Fleuren G J , de Heer E , Bruijn J A
Published: January 2020

In a previous study, susceptibility for Trypanosoma brucei-related glomerulopathy in mice was shown to be dependent on non-major histocompatibility complex genes. Glomerular disease in this model could not be explained by the production of autoantibodies alone. In order to analyze which part of the defense system, in addition to the B-cell compartment, is involved in the development of this infection-related glomerular disease, groups of athymic (BALB/c rnu/rnu), splenectomized, or macrophage-depleted BALB/c mice were inoculated with T. brucei parasites. Polyclonal B-cell activation, invariably observed in infected BALB/c mice, was absent in BALB/c rnu/rnu mice. Glomerular disease in athymic mice, however, as defined by albuminuria and deposition of immune complexes, was not different from that seen in euthymic infected BALB/c mice. Splenectomy prior to inoculation of parasites led to a decreased incidence of albuminuria in 40% of the animals, whereas splenectomy 21 days after inoculation reduced albuminuria significantly, suggesting a role for spleen cells in the induction of glomerular disease. After macrophage depletion with liposome-encapsulated dichlorodimethylene-diphosphonate, infected BALB/c mice developed significantly higher albuminuria levels for a period up to 2 weeks after depletion. Therefore, it was concluded that the development of T. brucei-related glomerular disease is independent of thymus-matured T cells, while the involvement of macrophages in the development of proteinuria is inhibitory rather than disease inducing. Spleen cells other than thymus-dependent T cells, B cells, and macrophages should be investigated for their role in the pathogenesis of this glomerulopathy.
Distinct binding properties of eaeA-negative verocytotoxin-producing Escherichia coli of serotype O113:H21
Authors: Dytoc M T , Ismaili A , Philpott D J , Soni R , Brunton J L , Sherman P M
Published: January 2020

Infection of humans with verotoxin-producing Escherichia coli (VTEC) O113:H21 is associated with clinical features comparable to those associated with infection with attaching and effacing VTEC strains including those of serotype O157:H7. We have shown previously that the adhesion phenotype of VTEC O157:H7 is influenced by the presence of a homolog of the chromosomal eaeA (for E. coli attaching and effacing) gene. In contrast, by colony blot hybridization, VTEC O113:H21 is negative for the eaeA gene. Therefore, the aim of this study was to define the adhesion phenotype of VTEC O113:H21 strain CL-15 to both cultured epithelial cells (HEp-2) and rabbit intestine in vivo. Under transmission electron microscopy, areas of microvillus effacement were observed in regions directly beneath the organism in CL-15-infected cells both in vitro and in vivo. However, F-actin adhesion pedestals on the host plasma membrane were absent. Failure of CL-15 to induce polymerization of actin was confirmed by using staining of F-actin with fluorescein-labeled phalloidin. Under indirect immunofluorescence microscopy, CL-15-infected HEp-2 cells also failed to demonstrate the recruitment of another cytoskeletal element, alpha-actinin, below foci of bacterial adhesion. In contrast, VTEC O157:H7 infection of HEp-2 cells was associated with increased alpha-actinin immunofluorescence. These findings suggest that bacterial factors distinct from those of EaeA are necessary for the adhesion phenotype of VTEC O113:H21.
Experimental Borrelia garinii infection of Japanese quail
Authors: Isogai E , Tanaka S , Braga I S , Itakura C , Isogai H , Kimura K , Fujii N
Published: January 2020

Japanese quail inoculated subcutaneously with Borrelia garinii responded to infection. B. garinii was reisolated mainly from skin and randomly from several organs between 7 and 56 days postinoculation. Skin lesions were occasionally observed in association with spirochete recovery. All birds were positive for antibody at 53 to 56 days postinoculation. These results suggest that the Japanese quail is a model for experimental infection with B. garinii and that birds are reservoirs for Lyme disease spirochetes.
Distribution of 3-hydroxy iC17:0 in subgingival plaque and gingival tissue samples: relationship to adult periodontitis
Authors: Nichols F C
Published: January 2020

Gram-negative organisms incorporate hydroxy fatty acids into the lipid A moiety of lipopolysaccharide (LPS), and in the case of some members of the family Enterobacteriaceae, hydroxy fatty acids are incorporated exclusively into lipid A. However, a limited number of Bacteroides species have been shown to incorporate several classes of 3-hydroxy fatty acids, particularly 3-hydroxy iC17:0, into constitutive lipids as well as LPS. The present study examined the distribution of hydroxy fatty acids in two periodontal pathogens, Prevotella intermedia and Porphyromonas gingivalis, by employing a phospholipid extraction procedure (E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37:911-917, 1959) which partitioned constitutive lipids into the organic solvent phase and LPS into the aqueous phase. The distribution of hydroxy fatty acids within organic solvent and aqueous extracts of these bacterial species was then compared with the distribution in subgingival plaque samples isolated from either gingivitis or severe periodontitis sites as well as the distribution in gingival tissue samples. The organic solvent and aqueous extracts were hydrolyzed under strong alkaline conditions, and the free fatty acids were treated to form pentafluorobenzyl-ester, trimethylsilyl-ether derivatives. Hydroxy fatty acid levels were quantified by using gas chromatography-negative-ion chemical ionization-mass spectrometry. By using this approach, the mean values of the 3-hydroxy iC17:0 recovered within organic solvent extracts of P. gingivalis strains ranged from 56 to 63% of total 3-hydroxy iC17:0. Substantially less 3-hydroxy iC17:0 (< 5%) was recovered in organic solvent extracts of P. intermedia. By comparison, 75% of the 3-hydroxy iC17:0 in periodontitis subgingival plaque samples was recovered in organic solvent extracts, while only 43% of the 3-hydroxy iC17:0 in gingivitis plaque samples from the same patients was recovered in organic solvent extracts. However, 3-hydroxy iC17:0 was recovered essentially only in organic solvent extracts of both healthy or mildly inflamed and periodontitis gingival tissue samples. The preferential recovery of 3-hydroxy iC17:0 in tissue lipids indicates that gingival tissues do not harbor significant levels of subgingival plaque organisms which contain 3-hydroxy iC17:0. Furthermore, these results indicate that LPS from these organisms is not prevalent in gingival tissues. Finally, these results indicate either selective penetration of certain bacterial lipids into gingival tissues or that 3-hydroxy iC17:0 is metabolically transferred from bacterial lipids into gingival tissue lipids.
Variable region sequences of a protective human monoclonal antibody specific for the Haemophilus influenzae type b capsular polysaccharide
Authors: Lucas A H , Larrick J W , Reason D C
Published: January 2020

A hybridoma secreting a human immunoglobulin G2 kappa monoclonal antibody (MAb) specific for the capsular polysaccharide of Haemophilus influenzae type b (Hib) was isolated. This MAb, designated CA4, was bactericidal to Hib in vitro and protected infant rats from Hib bacteremia. Nucleotide sequence analysis of CA4 variable (V) region cDNA showed that the heavy (H)-chain V region was of subgroup III and was 96% identical to the VH germ line gene segment DP77 (V3-21). The light (L)-chain V region was of the kappa subgroup III and was 94% identical to the A27 (Humkv325) germ line gene, which is commonly used by rheumatoid factors and other autoantibodies. MAb CA4 did not have rheumatoid factor activity and did not react with histones, DNA, or chromatin. These findings identify an additional VHIII gene segment which can contribute to the anti-Hib capsular polysaccharide repertoire and demonstrate that a VL gene commonly encoding autoantibodies can be utilized for protective immunity.
Cloning, sequencing, and expression of a fibronectin/fibrinogen-binding protein from group A streptococci
Authors: Courtney H S , Li Y , Dale J B , Hasty D L
Published: January 2020

Lipoteichoic acid and several streptococcal proteins have been reported to bind fibronectin (Fn) or fibrinogen (Fgn), which may serve as host receptors. We searched for such proteins by screening a library of genes from M type 5 group A streptococci cloned into Escherichia coli. Lysates of clones were probed with biotinylated Fn and biotinylated Fgn. One clone expressed a 54-kDa protein that reacted with Fn and Fgn. The protein, termed FBP54, was purified and used to immunize rabbits. Anti-FBP54 serum reacted with purified, recombinant FBP54 and with a protein of similar electrophoretic mobility in extracts of M type 5, 6, and 24 streptococci. Anti-FBP54 serum also reacted with 5 of 15 strains of intact, live streptococci, suggesting that FBP54 may be a surface antigen. Southern blot analysis confirmed that the gene is found in group A streptococci but not in Staphylococcus aureus or E. coli. The cloned gene was sequenced and contained an open reading frame encoding a protein with a calculated molecular weight of 54,186. Partial amino acid sequencing of purified FBP54 confirmed that this open reading frame encoded the protein. As determined by utilizing fusion proteins containing truncated forms of FBP54, the primary Fn/Fgn-binding domain appears to be contained in residues 1 to 89. These data suggest that FBP54 may be a surface protein of streptococci that reacts with both Fn and Fgn and therefore may participate in the adhesion of group A streptococci to host cells.
Iron release from transferrin by pyoverdin and elastase from Pseudomonas aeruginosa
Authors: Wolz C , Hohloch K , Ocaktan A , Poole K , Evans R W , Rochel N , Albrecht-Gary A M , Abdallah M A , Döring G
Published: January 2020

Pseudomonas aeruginosa produces the siderophores pyoverdin and pyochelin as well as receptors for siderophores in response to iron deprivation. Previously, it has been shown in vitro that at neutral pH purified pyoverdin acquires iron from transferrin only in the presence of P. aeruginosa elastase (LasB), which proteolytically degrades transferrin. We constructed a LasB-negative mutant, PAO1E, by insertional mutagenesis to investigate whether this mutant differs in growth from the parental strain PAO1 in an iron-depleted medium supplemented with transferrin or human serum. PAO1 and PAO1E did not differ in growth with 1.25 microM Fe2-transferrin as the only iron source. Urea gel electrophoresis indicated iron release from intact transferrin during the logarithmic growth phase of PAO1 and PAO1E. A total of 333 microM LasB was synthesized from PAO1 after onset of stationary-phase growth. Quantification of pyoverdin by spectroscopy revealed that up to 900 microM pyroverdin was produced during growth of the strains in medium supplemented with Fe2-transferrin or 10% human serum. Incubation of Fe2-transferrin and purified pyoverdin in concentrations similar to those found in the culture supernatant resulted in release iron from transferrin after 10 h at 37 degrees C. However, LasB significantly enhanced the rate constant for iron acquisition of pyoverdin from transferrin. We conclude that P. aeruginosa can use transferrin as an iron source without further need of LasB or pH changes. This is further supported by experiments with P. aeruginosa K437, which has a defective iron uptake system, and its LasB-negative mutant, K437E. Though K437 and K437E did not differ in growth with Fe2-transferrin as the only iron source, their growth was significantly reduced relative to that of PAO1 and PAO1E.
High-molecular-weight proteins of nontypeable Haemophilus influenzae mediate bacterial adhesion to cellular proteoglycans
Authors: Noel G J , Love D C , Mosser D M
Published: January 2020

A family of high-molecular-weight (HMW) surface-exposed proteins of nontypeable Haemophilus influenzae (NT H. influenzae) mediated adherence of these organisms to human epithelium. To better understand the molecular basis for this adherence, the role of glycosaminoglycans (GAGs), substances commonly expressed on cell surfaces, was examined. Bacterial adherence to cells with specific deficiencies in GAG biosynthesis was measured. HMW protein-dependent bacterial adherence to normal cells was significantly greater than adherence to cells deficient in sulfated GAGs or to cells deficient in heparan sulfate but overexpressing chondroitin sulfate. Cells expressing undersulfated heparan sulfate exhibited intermediate levels of bacterial adherence. The addition of exogenous dextran sulfate or heparin inhibited over 70% of the adherence of NT H. influenzae to normal cells, whereas hyaluronic acid and chondroitin sulfate tested at the same concentration (100 micrograms/ml) inhibited bacterial adherence by less than 11%. Treatment of cells with heparinase significantly reduced bacterial adherence. Following electrophoretic separation, HMW proteins were shown to bind directly to radiolabeled heparin. These results indicate that HMW protein-dependent adherence of NT H. influenzae is mediated by cellular sulfated GAGs and that heparan sulfate may be the predominant GAG involved in this process. However, the decreased adherence of bacteria to cells expressing undersulfated heparan sulfate and the inhibition of bacterial adherence by the addition of exogenous dextran sulfate suggest that bacterial adhesion to mammalian cells is likely to be influenced by a variety of factors, including the degree of sulfation and the specificity of the carbohydrate moieties contained in the cellular proteoglycans.
Nitric oxide-mediated antiplasmodial activity in human and murine hepatocytes induced by gamma interferon and the parasite itself: enhancement by exogenous tetrahydrobiopterin
Authors: Mellouk S , Hoffman S L , Liu Z Z , de la Vega P , Billiar T R , Nussler A K
Published: January 2020

Expression of inducible nitric oxide (NO) synthase has been shown to inhibit the development of several pathogens, including fungi, bacteria, parasites, and viruses. However, there is still controversy as to whether this effector mechanism can inhibit the development of human pathogens. We now report that gamma interferon (IFN-gamma) induces the elimination of Plasmodium falciparum-infected primary human hepatocytes from cultures and that the antimalarial activity is dependent on NO. Infection with the parasite alone in the absence of added IFN-gamma caused a 10-fold increase in NO formation. Both spontaneous inhibition and IFN-gamma-induced inhibition of Plasmodium yoelii-infected murine hepatocytes were increased with the addition of the NO synthase cofactor tetrahydrobiopterin, or sepiapterin, which is converted to tetrahydrobiopterin. These results indicate that under in vitro conditions the parasite itself provides a signal that triggers induction of the NO pathway in human and murine hepatocytes and that NO formation in infected hepatocytes is limited by tetrahydrobiopterin availability.
Tolerance to staphylococcal enterotoxin B initiated Th1 cell differentiation in mice infected with Candida albicans
Authors: Romani L , Puccetti P , Mencacci A , Spaccapelo R , Cenci E , Tonnetti L , Bistoni F
Published: January 2020

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that specifically activates T cells bearing V beta 8 T-cell receptor domains, which eventually leads to a long-lasting state of clonal anergy accompanied by selective cell death in the targeted CD4+ subset. Because the superantigen is known to promote Th1 cell differentiation in vitro, we have investigated the effect of SEB treatment on the course of Th2-associated progressive disease in mice infected systemically with Candida albicans. On the basis of the kinetics of SEB-induced changes in CD4+ cells and production in sera of interleukin 4 (IL-4), IL-10, and gamma interferon, we obtained evidence that V beta 8+ cell anergy concomitant with infection abolished the early IL-4/IL-10 response of the host to the yeast, ultimately leading to a state of resistance characterized by gamma interferon secretion in vitro by antigen-specific CD4+ cells. In contrast, SEB administered near the time of challenge resulted in accelerated mortality. Significant resistance to infection was also afforded by exposure of mice to a retrovirally encoded endogenous superantigen. These data suggest that CD4+ V beta 8+ T cells play an important role in vivo in the initiation of a Th2 response to C. albicans and that suppression of their activity may alter the qualitative development of the T-cell response and the outcome of infection.
Immunoelectron-microscopic quantitation of differential levels of chlamydial proteins in a cell culture model of persistent Chlamydia trachomatis infection
Authors: Beatty W L , Morrison R P , Byrne G I
Published: January 2020

Electron immunolabeling techniques were used for quantitative evaluation of alterations in the steady-state levels of chlamydial antigens in persistent Chlamydia trachomatis cultures. Gamma interferon-mediated persistent chlamydial development correlated with an increase in the levels of the chlamydial heat shock protein (hsp60) and with a significant reduction in the levels of the major outer membrane protein.
Chemotactic response to mucin by Serpulina hyodysenteriae and other porcine spirochetes: potential role in intestinal colonization
Authors: Milner J A , Sellwood R
Published: January 2020

Chemotaxis of porcine spirochetes towards a variety of mucins was measured quantitatively by a capillary method. A chemotaxis buffer consisting of 0.01 M potassium phosphate buffer (pH 7.0) and 0.2 mM L-cysteine hydrochloride was necessary for chemotaxis of spirochetes. The optimum incubation time and incubation temperature were 1 h and 40 degrees C, respectively. The mucin concentration also affected the chemotaxis observed, and a concentration of 1% (wt/vol) was near the optimum. Virulent Serpulina hyodysenteriae strains were chemotactic towards 1% (wt/vol) hog gastric mucin and 1% (wt/vol) porcine colonic mucin but not towards 1% (wt/vol) bovine submaxillary mucin. Virulent S. hyodysenteriae strains were significantly more chemotactic than avirulent strains of S. hyodysenteriae (SA3 and VS1), Serpulina intermedius, and Serpulina innocens. Other spirochetes belonging to the proposed group of spirochetes Anguillina coli were also not chemotactic. Pathogenicity of S. hyodysenteriae strains that cause swine dysentery may, in part, be attributed to their attraction to porcine intestinal mucus.
Low-Temperature-Adapted Influenza A2/AA/6/60 Virus Vaccine in Man
Authors: Kitayama Toru , Togo Yasushi , Hornick Richard B. , Friedwald William T.
Published: January 2020

Volunteers inoculated nasopharyngeally with liver A2/AA/6/60 virus grown in primary bovine kidney cell cultures at 25 C were asymptomatic but developed significant serum and respiratory secretory antibody responses. Viruses were not recovered from these volunteers who had a low or moderate level of prevaccination antibody titers.
Creatine Phosphokinase Activity in Rabbit Alveolar Macrophages
Authors: DeChatelet Lawrence R. , McCall Charles E. , Shirley Pamela S.
Published: January 2020

An enzyme is present in extracts of rabbit alveolar macrophage which can catalyze the reaction of creatine phosphate with adenosine diphosphate to form adenosine triphosphate and creatine. The enzyme is moderately activated by reduced glutathione, has a
p
H optimum between
p
H 6.5 and 7.0, and shows an absolute requirement for Mg
2+
. The
K
m
for creatine phosphate is approximately 3.6 m
m
while the
K
m
for adenosine diphosphate is about 1.1 m
m
. The enzyme may play a role in the energy balance of the cell by creating a reserve of energy in the form of creatine phosphate.
Studies of the Role of
Dermacentor occidentalis
in the Transmission of Bovine Chlamydial Abortion
Authors: Caldwell Harlan D. , Belden E. Lee
Published: January 2020

Dermacentor occidentalis
nymphal ticks were successfully infected with the agent of bovine chlamydial abortion. Multiplication of the agent was shown to occur during nymphal engorgement, but chlamydial isolations were not made from nymphal ticks after detachment or during subsequent metamorphosis. Primary tissue cultures of nymphal viscera infected in vitro or in vivo failed to demonstrate chlamydial replication.
Inhibition of Glucosyltransferase Activity by Antisera to Known Serotypes of
Streptococcus mutans
Authors: Evans R. T. , Genco R. J.
Published: January 2020

Using a recently developed assay for glucosyltransferase activity based on
14
C-glucose incorporation into an alcohol-insoluble polysaccharide, we were able to study inhibition by antibody of this enzyme activity. Rabbit antibody was relatively specific for the strain of
Streptoccus mutans
from which the enzyme was obtained. Absorption studies showed that neither removal of antibodies directed to dextran nor absorption with intact bacteria offset the enzyme-inhibitory capacity of these sera, whereas absorption with partially purified enzyme did result in removal of the inhibitory capacity.