bluuebunny/crossref_metadata_2025 huggingface dataset:
Complete development of Cryptosporidium parvum in bovine fallopian tube epithelial cells
Authors: Yang S , Healey M C , Du C , Zhang J
Published: January 2020

Cryptosporidium parvum is a coccidian parasite responsible for causing protracted and life-threatening diarrheal illness in immunocompromised humans, especially patients with AIDS. The lack of medications effective in treating people suffering from cryptosporidiosis has prompted the development of in vivo and in vitro models for this disease. This study is the first to demonstrate that C. parvum can complete its entire life cycle (from sporozoite to infective oocyst) in a primary culture of bovine fallopian tube epithelial (BFTE) cells. Scanning and transmission electron photomicrographs were used to detail the ultrastructure of individual parasitic stages. Successful infections were produced by inoculating cell cultures with either oocysts or purified sporozoites. Infection of BFTE cells with C. parvum close paralleled in vivo infections with regard to host cell location and chronology of parasite development. Infecting BFTE cells with sporulated oocysts provided a reproducible and quantitative cultivation system with significantly (P < or = 0.001) higher infection rates than in Madin-Darby canine kidney cells. Oocysts produced in BFTE cells were infective for immunosuppressed adult C57BL/6N mice. Cultivation of C. parvum in BFTE cells will facilitate the study of interactions between parasites and host cells as well as provide a reliable system for evaluating anticryptosporidial compound efficacy.
Invasin production by Yersinia pestis is abolished by insertion of an IS200-like element within the inv gene
Authors: Simonet M , Riot B , Fortineau N , Berche P
Published: January 2020

The two enteropathogens Yersinia pseudotuberculosis and Yersinia enterocolitica penetrate eukaryotic cells in vitro through invasin, a surface-exposed protein. In contrast, Yersinia pestis, the causative agent of plague, is unable to enter mammalian cell lines, although the inv gene is present on its chromosome. Although 99.3% identical to the inv gene of Y. pseudotuberculosis, the Y. pestis inv gene was disrupted in its central region by a 708-bp IS200-like element. Multiple copies of this insertion sequence element were found within the genome of the plague bacillus.
Vaccination with genetically modified Shiga-like toxin IIe prevents edema disease in swine
Authors: Bosworth B T , Samuel J E , Moon H W , O'Brien A D , Gordon V M , Whipp S C
Published: January 2020

Escherichia coli strains producing Shiga-like toxin II variant (SLT-IIe, formerly called SLT-IIv) cause edema disease in weaned pigs. Vaccination of pigs with a genetically modified form of Shiga-like toxin IIe, SLT-IIe(E167Q), has been previously shown to be nontoxic and to induce antibodies to SLT-IIe (V.M. Gordon. S.C. Whipp, H.W. Moon, A.D. O'Brien, and J.E. Samuel, Infect, Immun. 60:485-502, 1992). Fifty micrograms of SLT-IIe(E167Q) toxin was used to vaccinate suckling pigs at 1 and 2 weeks of age. Both vaccinated and nonvaccinated pigs were orally inoculated with an SLT-IIe-producing strain of E. coli after weaning (3 to 4 weeks of age). Pigs fed a low-protein diet that were not vaccinated with SLT-IIe(E167Q) developed subclinical edema disease, histologically evident as vascular necrosis. Pigs fed a high-protein diet that were not vaccinated with SLT-IIe(E167Q) developed clinical edema disease manifested as vascular necrosis, reduced weight gain, ataxia, palpebral edema, lateral recumbency, and death. Pigs vaccinated with SLT-IIe(E167Q) had a reduction in the incidence of subclinical edema disease and never developed clinical edema disease. These data demonstrate that vaccination with a genetically modified form of SLT-IIe prevents edema disease and are consistent with the notion that diet influences susceptibility to edema disease.
Cytolytic activity in the genus Leishmania: involvement of a putative pore-forming protein
Authors: Noronha F S , Ramalho-Pinto F J , Horta M F
Published: January 2020

We describe here that parasites of the genus Leishmania contain a cytolytic activity which acts optimally at pH 5.0 to 5.5 and at 37 degrees C in vitro. or the four species examined, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) major presented considerable hemolytic activity, whereas Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis showed little and no hemolytic activity, respectively. The cytolytic factor of L. amazonensis promastigotes was characterized as a protein with no protease-, phospholipase-, or detergent-like activity, probably localized inside membranous vesicles. The use of osmotic protectants revealed the colloid-osmotic nature of hemolysis, which is indicative of pore formation in the membranes of target cells. This putative pore-forming protein also damaged nucleated cells, including macrophages, causing an increase in their membrane permeability with leakage of cytoplasmic proteins. Both promastigotes and amastigotes express this lytic activity, suggesting that the cytolysin may have a function in both stages of this parasite. The pH and temperature required for optimal activity indicate that it might be more effective within the mammalian host, particularly inside the macrophage parasitophorous vacuole. In promastigotes of L. amazonensis, the expression of lytic activity seems to be regulated during their growth in vitro, being maximal at the early stationary phase.
Safety and immunogenicity of investigational Shigella conjugate vaccines in Israeli volunteers
Authors: Cohen D , Ashkenazi S , Green M , Lerman Y , Slepon R , Robin G , Orr N , Taylor D N , Sadoff J C , Chu C , Shiloach J , Schneerson R , Robbins J B
Published: January 2020

The safety and immunogenicity of investigational conjugates, composed of the O-specific polysaccharides of Shigella sonnei and Shigella flexneri type 2a covalently bound to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), were evaluated in 192 Israeli soldiers. None had significant local reactions or fever. Fourteen days after injection, 90% of S. sonnei-rEPA recipients and 73 to 77% of S. flexneri-rEPA recipients had a fourfold or greater increase in serum immunoglobulin G (IgG) and IgA anti-lipopolysaccharide (anti-LPS) levels; at 2 years, these remained higher than at prevaccination (P < 0.01). There was a fourfold or greater increase in IgM anti-LPS in 20% of vaccinees at 2 weeks, but levels returned to prevaccination values at 6 to 12 months. IgG was the highest and most sustained class of LPS antibodies. Reinjection at day 42 did not boost antibody levels. Eighteen of 23 (78%) who received S. sonnei-rEPA and 13 of 19 (68%) who received S. flexneri-rEPA. had significant IgA-secreting cell responses. Significant IgG antibody-secreting cell responses were detected in 19 of 23 (83%) and 11 of 19 (58%) volunteers following vaccination with S. sonnei-rEPA and S. flexneri 2a-rEPA, respectively. On the basis of these data, further evaluation of the Shigella conjugates for protective efficacy in field trials in Israel was started.
Cell surface polypeptide CshA mediates binding of Streptococcus gordonii to other oral bacteria and to immobilized fibronectin
Authors: McNab R , Holmes A R , Clarke J M , Tannock G W , Jenkinson H F
Published: January 2020

Isogenic mutants of Streptococcus gordonii DL1 (Challis) in which the genes encoding high-molecular-mass cell surface polypeptides CshA and/or CshB were inactivated were deficient in binding to four strains of Actinomyces naeslundii and two strains of Streptococcus oralis. Lactose-sensitive interactions of S. gordonii with A. naeslundii ATCC 12104 and PK606 were associated with expression of cshA but not of cshB. Lactose-insensitive interactions of S. gordonii with A. naeslundii T14V and WVU627, and with S. oralis C104 and 34, were dependent on expression of cshA and cshB. S. gordonii DL1 cells bound to immobilized human fibronectin (Fn), but not to soluble Fn, in a dose-dependent manner, and binding was noninhibitable by heparin. S. gordonii cshA and cshB mutants were also deficient in binding to immobilized human Fn. Antibodies to an NH2-terminal nonrepetitive region (amino acid residues 42 to 886) of recombinant CshA inhibited binding of S. gordonii DL1 cells to A. naeslundii T14V and PK606 and to immobilized Fn. Conversely, antibodies to an amino acid repeat block segment of the COOH-terminal domain (amino acid residues 2026 to 2508) were not inhibitory to adherence. Assays using CshA-specific antibodies revealed that surface expression of CshA was reduced in cshB mutants. The results suggest that CshA acts as a multifunctional adhesin in S. gordonii and that major adhesion-mediating sequences are specified within the nonrepetitive NH2-terminal region of the polypeptide.
Borrelia burgdorferi induces secretion of pro-urokinase-type plasminogen activator by human monocytes
Authors: Fuchs H , Simon M M , Wallich R , Bechtel M , Kramer M D
Published: January 2020

Borrelia burgdorferi is transmitted by infected ticks and causes Lyme disease. To infect distant organ sites, B. burgdorferi spirochetes must disseminate from the site of the tick bite. During dissemination from the dermal tissue, they breach tissue barriers, probably by proteolysis. The previous findings that spirochetes bind serum-derived plasminogen and that plasmin favors spirochetal invasiveness and infectivity suggested a role for plasmin in the pathogenicity of B. burgdorferi. Binding of plasminogen to spirochetes and activation into plasmin is favored in a microenvironment that is rich in plasminogen and plasminogen activators. Plasminogen is abundant in plasma and interstitial fluids, and it is increased in inflammatory exudates. Since B. burgdorferi does not express endogenous plasminogen activators, the conversion of spirochete-bound plasminogen depends on host-derived plasminogen activators. In this report, we show that both intact B. burgdorferi organisms and its recombinant outer surface lipoprotein A induce human monocytes to express and secrete urokinase-type plasminogen activator in its zymogen form (pro-uPA). Moreover, we demonstrate that the presence of B. burgdorferi accelerates the interaction between (pro-)uPA and plasmin(ogen), leading to spirochete-bound plasmin. In a pro-uPA-serum mixture, spirochete-bound plasmin activity is generated. Taken together, the data suggest that B. burgdorferi may induce pro-uPA in a monocyte-containing inflammatory site and that the spirochetal surface provides an appropriate milieu for subsequent interactions between (pro-)uPA and plasmin(ogen), which result in spirochete-bound plasmin even in the presence of inhibitors for plasminogen activators and plasmin.
V antigen-polyhistidine fusion peptide: binding to LcrH and active immunity against plague
Authors: Motin V L , Nedialkov Y A , Brubaker R R
Published: January 2020

The structural gene for V antigen (lcrV) is known to be encoded within the lcrGVH-yopBD operon of the approximately 70-kb low-calcium-response or Lcr plasmid of Yersinia pestis. This 37-kDa monomeric peptide was reported to provide active immunity in mice, suppress inflammatory cytokines, and regulate expression of the low calcium response (Lcr+). Here we describe pVHB62, encoding a polyhistidine-V antigen fusion peptide (Vh) and linked LcrH. Vh underwent degradation from both the C terminus and N terminus during classical chromatographic fractionation but remained intact within two compartments during Ni2+ affinity chromatography. The first was homogeneous, capable of active immunization (mouse intravenous 50% lethal dose, > 10(7) bacteria), and stable at 4 degrees C. The second remained bound to the affinity column but could be eluted as a mixture of Vh, LcrH, and low-molecular-weight material by application of 6 M guanidine HCl. This mixture was dialyzed, denatured in 8 M urea, and again applied to the affinity column, which then hound Vh but not LcrH. The latter was recovered and renatured, and low-molecular-weight material was removed by biochemical fractionation. The resulting homogeneous LcrH bound protein AN antigen fusion peptide but not protein A in a sandwich enzyme-linked immunosorbent assay, and this reaction was inhibited by Vh. These observations indicate that LcrH normally binds V antigen in bacterial cytoplasm and suggest that only free LcrH down-regulates expression of the low calcium response.
CD4-mediated and CD8-mediated cytotoxic and proliferative immune responses to Toxoplasma gondii in seropositive humans
Authors: Purner M B , Berens R L , Nash P B , van Linden A , Ross E , Kruse C , Krug E C , Curiel T J
Published: January 2020

Both CD4+ and CD8+ cytotoxic T lymphocytes (CTL) are part of the human immune response to Toxoplasma gondii infection. To further our understanding of Toxoplasma immunity, we investigated factors influencing stimulation of CD4+ or CD8+ human T. gondii-specific immune cells. Both antigen-pulsed and Toxoplasma-infected antigen-presenting cells (APC) induced cell proliferation. Toxoplasma-infected APC elicited strong proliferation of CD4+ cells, but little or no proliferation of CD8+ cells, unless high antigen loads were used. Toxoplasma-infected APC stimulated specific cytotoxicity poorly or not at all, owing to death of stimulated cultures, whereas antigen-pulsed APC strongly elicited specific cytotoxicity. Cytotoxicity elicited by either type of APC resided exclusively in CD4+ T cells in polyclonal cultures. Thus, Toxoplasma-infected APC elicited stronger CD4-mediated than CD8-mediated cell proliferation and generated CD4+ CTL more readily than CD8+ CTL. Nonetheless, specific CD8+ memory cells were demonstrated, and rare CD8+ Toxoplasma-specific CTL were subcloned. Fixed Toxoplasma-infected APC (which induce CD8+ CTL) also elicited cell proliferation, but polyclonal cultures stimulated with these infected APC did not die. Unfixed Toxoplasma-infected APC strongly inhibited phytohemagglutinin-induced cell proliferation, whereas fixed APC did not. These data suggested that infected APC were inhibitory or lethal to some immune cells. Further investigations into interactions between immune cells and Toxoplasma-infected cells likely will help elucidate factors involved in the immunopathogenesis of Toxoplasma infection. As other intracellular parasites, including Plasmodium spp. and Leishmania spp., also elicit CD4+ CTL, such work may help establish paradigms governing immunity to intracellular parasites.
Proteus mirabilis ambient-temperature fimbriae: cloning and nucleotide sequence of the aft gene cluster
Authors: Massad G , Fulkerson J F , Watson D C , Mobley H L
Published: January 2020

Uropathogenic Proteus mirabilis produces at least four types of fimbriae. Amino acid sequences from two peptides, derived by tryptic digestion of the structural subunit of one type of these fimbriae, the ambient-temperature fimbriae, were determined: NVVPGQPSSTQ and LIEGENQLNYNA. PCR primers, based on these sequences and that of the N terminus, were used to amplify a 359-bp fragment. A cosmid clone, isolated from a P. mirabilis genomic library by hybridization with the 359-bp PCR product, was used to determine the nucleotide sequence of the atf gene cluster. A 3,903-bp region encodes three polypeptides: AtfA, the structural subunit; AtfB, the chaperone; and AtfC, the outer membrane molecular usher. No fimbria-related genes are evident either 5' or 3' to the three contiguous genes. AtfA demonstrates significant amino acid sequence identity with type 1 major fimbrial subunits of several enteric species. The 359-bp PCR product hybridized strongly with all Proteus isolates (n = 9) and 25% of 355 Escherichia coli isolates but failed to hybridize with any of 26 isolates among nine other uropathogenic species. Ambient-temperature fimbriae of P. mirabilis may represent a novel type of fimbriae of enteric species.
Attachment of a noninvasive enteric pathogen, enteropathogenic Escherichia coli, to cultured human intestinal epithelial monolayers induces transmigration of neutrophils
Authors: Savkovic S D , Koutsouris A , Hecht G
Published: January 2020

An intense inflammatory cell infiltrate, consisting primarily of polymorphonuclear leukocytes (PMN), accompanies enteric infection by enteropathogenic Escherichia coli (EPEC). The mechanism(s) by which this pathogen elicits PMN recruitment has not been studied. To determine whether EPEC infection of intestinal epithelial cells induces PMN to transmigrate, an in vitro model consisting of cultured human intestinal epithelial monolayers (T84), a human EPEC strain (E2348/69), and isolated human PMN was used. Results of these studies showed that EPEC attachment to T84 monolayers stimulated the transepithelial migration of PMN in a dose-dependent fashion. This event was not attributable to the classic bacterial chemoattractants, n-formylated peptides, or other soluble bacterial factors. A nonadherent EPEC strain, JPN15, was unable to cause PMN to cross the epithelial monolayer. Epithelial protein synthesis was required for maximum EPEC-induced PMN transmigration to occur. Transfer assays demonstrated the presence of a chemokine in sterilized medium from infected monolayers. Neutralizing antibodies to interleukin 8 ablated approximately 50% of the chemotactic activity. Studies with EPEC mutant strains revealed that the eaeB gene, required for the activation of signal transduction pathways in host cells, was crucial for eliciting PMN transmigration. These data show for the first time that attachment of a noninvasive enteric pathogen to intestinal epithelial cells induces PMN transmigration. These findings strongly suggest that EPEC attachment to target host cells activates a signal transduction cascade which ultimately leads to the expression and release of an epithelium-derived chemotactic factor(s) for PMN.
Contribution of TonB- and Feo-mediated iron uptake to growth of Salmonella typhimurium in the mouse
Authors: Tsolis R M , Bäumler A J , Heffron F , Stojiljkovic I
Published: January 2020

We examined the role of iron(II) and iron(III) uptake, mediated by FeoB and TonB, respectively, in infection of the mouse by Salmonella typhimurium. The S. typhimurium feoB gene, encoding a homolog of an Escherichia coli cytoplasmic membrane iron(II) permease, was cloned, and a mutant was generated by allelic exchange. In addition, an S. typhimurium tonB mutant was constructed. Together these two mutations inactivate all known iron uptake systems of S. typhimurium. We examined the abilities of these mutants to grow in vitro and in different compartments of the host. Mutants in feoB were outcompeted by the wild type during mixed colonization of the mouse intestine, but the feoB mutation did not attenuate S. typhimurium for oral or intraperitoneal infection of mice. The tonB mutation attenuated S. typhimurium for infection of mice by the intragastric route but not the intraperitoneal route, and the mutant was recovered in lower numbers from the Peyer's patches and mesenteric lymph nodes than the wild type. These results indicate that TonB-mediated iron uptake contributes to colonization of the Peyer's patches and mesenteric lymph nodes but not the liver and spleen of the mouse. The tonB feoB double mutant, given intraperitoneally, was able to infect the liver and spleen at wild-type doses, indicating that additional iron acquisition systems are used during growth at systemic sites of infection.
Lipopolysaccharide binding proteins on polymorphonuclear leukocytes: comparison of adult and neonatal cells
Authors: Qing G , Howlett S , Bortolussi R
Published: January 2020

We have previously shown that polymorphonuclear leukocytes (PMN) from cord blood of normal full-term infants have a decreased priming response to lipopolysaccharide (LPS) compared with PMN of adults. Because the reason for this difference is poorly understood, we compared LPS binding on PMN from adults and newborns by using a photoactivatable iodinated LPS (from Escherichia coli O111:B4), coupled to 2-(p-azidosalicylamido)-1,3'-dithopropionate (LPS-ASD) to covalently link LPS to the PMN membrane. We incubated 2 x 10(4) adult or neonatal PMN with 125I-ASD-LPS (100 ng/ml) together with unlabelled LPS (0 to 100,000 ng/ml) for 20 min at 4 degrees C. The maximum total 125I-ASD-LPS binding to newborn PMN (1,004 +/- 103 cpm) was lower than that binding to adult PMN (3,583 +/- 444 cpm; P < 0.01 with respect to newborn PMN). However, the concentration of unlabelled LPS that displaced 50% of the maximum specifically bound 125I-ASD-LPS was similar for PMN from adult and newborn infants (-4.85 +/- 0.04 and -5.13 +/- 0.14 log g of LPS per ml, respectively; P > 0.05). We further assessed the membrane binding of 125I-ASD-LPS to PMN by using membrane extracts analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. LPS binding proteins were found at approximately 73, 55 to 57, and 25 kDa in both adult and neonatal PMN. However, PMN from newborn infants had markedly lower membrane-associated 125I-ASD-LPS at the 55- to 57- and 25-kDa protein bands as indicated by the intensity of the autoradiograph. Binding of LPS at these bands was specific for the lipid A portion of LPS, since purified unlabelled lipid A displaced 125I-ASD-LPS in a dose-dependent manner. Thus, PMN from newborn infants bind less LPS than do PMN from adults, even though the sites for LPS membrane binding appear to be the same.
Candida albicans binding to the oral bacterium Streptococcus gordonii involves multiple adhesin-receptor interactions
Authors: Holmes A R , McNab R , Jenkinson H F
Published: January 2020

Candida albicans binds to several species of oral streptococci, in particular Streptococcus gordonii, through recognition of a streptococcal cell wall polysaccharide receptor (A. R. Holmes, P. K. Gopal, and H. F. Jenkinson, Infect. Immun. 63:1827-1834, 1995). We now show that isogenic cell surface protein mutants of S. gordonii DL1, unaltered in expression of cell wall polysaccharide, are reduced in ability to support adherence of C. albicans cells in a solid-phase assay. Inactivation of the S. gordonii cshA and cshB genes, encoding high-molecular-mass cell surface polypeptides, and inactivation of the sspA and sspB genes, encoding antigen I/II salivary adhesins, resulted in 40 and 79% reductions, respectively, in adherence of C. albicans cells. Inactivation of the S. gordonii scaA gene encoding a cell surface lipoprotein had no effect on C. albicans adherence. Polyclonal antiserum to streptococcal antigen I/II protein SpaP and antibodies specific to the amino-terminal nonrepetitive (NR) domain of CshA both inhibited adherence of C. albicans to S. gordonii cells. Conversely antibodies to the amino acid repeat block repetitive (R) domain of CshA, or to ScaA, did not inhibit C. albicans adherence. Immobilized recombinant polypeptide fragments of CshA comprising NR domain or R domain sequences both supported adherence of C. albicans cells. Expression of S. gordonii SspB protein on the surface of Enterococcus faecalis conferred on the enterococcal cells the ability to bind C. albicans, and this was ablated by antigen I/II antiserum. Collectively the results suggest that interaction of C. albicans with S. gordonii is mediated by a complement of adhesin-receptor interactions that involves two families of streptococcal multifunctional polypeptide adhesins, bacterial cell wall polysaccharide, and as yet unidentified yeast cell surface components.
Adhesion of enteroaggregative Escherichia coli to pediatric intestinal mucosa in vitro
Authors: Hicks S , Candy D C , Phillips A D
Published: January 2020

Organ cultures of small- and large-intestinal mucosa from children were used to examine the interactions of enteroaggregative Escherichia coli (EAEC) with human intestine. Mucosae from patients aged between 3 and 190 months were cultured with five EAEC strains isolated from infants with diarrhea in the United Kingdom and with two well-described prototype EAEC strains, 17-2 and 221. The prototype strains adhered to jejunal, ileal, and colonic mucosae. The wild-type strains also adhered to this tissue but showed a variable pattern of adhesion: two adhered to all intestinal levels, one adhered to jejunum and ileum, one adhered to ileum only, and one adhered to ileum and colon. Adherence was in an aggregative or stacked-brick pattern, resembling that seen on HEp-2 cells. Electron microscopy of infected small intestinal mucosa revealed bacteria in association with a thick mucus layer above an intact enterocyte brush border, which contained extruded cell fragments. This mucus layer was not present on controls. EAEC adherence to colonic mucosa was associated with cytotoxic effects including microvillous vesiculation (but without evidence of an attaching/effacing lesion), enlarged crypt openings, the presence of intercrypt crevices, and increased epithelial cell extrusion. These results demonstrate that in vitro organ culture of intestinal mucosa from children can be used to investigate EAEC pathogenesis in childhood directly. EAEC strains appear able to colonize many regions of the gastrointestinal tract, without overt changes to small intestinal mucosa but with cytotoxic effects on colonic mucosa.
Proteins in the cell wall and membrane of Cryptococcus neoformans stimulate lymphocytes from both adults and fetal cord blood to proliferate
Authors: Mody C H , Sims K L , Wood C J , Syme R M , Spurrell J C , Sexton M M
Published: January 2020

Cryptococcus neoformans is an encapsulated yeast that infects patients who have defective cell-mediated immunity, including AIDS, but rarely infects individuals who have intact cell-mediated immunity. Studies of the immune response to C. neoformans have been hampered by a paucity of defined T-lymphocyte antigens, and hence, the understanding of the T-cell response is incomplete. The goal of this study was to separate C. neoformans into its component parts, determine whether those components stimulate lymphocyte proliferation, perform preliminary characterization of the proteins, and establish the potential mechanism of lymphocyte proliferation. The lymphocyte response to fungal culture medium, whole organisms, disrupted organisms, and the yeast intracellular fraction or cell wall and membrane was studied by determining thymidine incorporation and by determining the number of lymphocytes at various times after stimulation. The cell wall and membrane of C. neoformans stimulated lymphocyte proliferation, while the intracellular fraction and culture filtrate did not. The optimal response occurred on day 7 of incubation, with 4 x 10(5) peripheral blood mononuclear cells per well and with 13 microg of cryptococcal protein per ml. The number of lymphocytes increased with time in culture, indicating that thymidine incorporation was accompanied by proliferation. Proteinase K treatment of the cell wall and membrane abrogated lymphocyte proliferation, indicating that the molecule was a protein. [35S]methionine labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography were performed to analyze the proteins contained in the cell wall and membrane, intracellular fraction, and culture filtrate. At least 18 discrete bands were resolved from the cell wall and membrane. Since a large percentage of healthy adults responded to the cryptococcal cell wall and membrane, a mitogenic effect was investigated by testing proliferation of fetal cord blood lymphocytes. The percentage of fetal samples that proliferated in response to the cell wall and membrane was similar to the percentage of fetal samples that proliferated in response to Staphylococcus enterotoxin B, a microbial mitogen. Thus, a protein in the cell wall and membrane of C. neoformans is a potent stimulant of lymphocyte proliferation and has mitogenic properties, which may have important implications for cell-mediated immunity to C. neoformans.
Secretion of extracellular proteins by enterohemorrhagic Escherichia coli via a putative type III secretion system
Authors: Jarvis K G , Kaper J B
Published: January 2020

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) infections result in attaching and effacing lesions on intestinal epithelial cells. Secretion of extracellular proteins via a type III secretion apparatus is necessary for the formation of attaching and effacing lesions by EPEC. We now show that EHEC also secretes polypeptides via a putative type III secretion system. The secreted EHEC proteins are recognized by rabbit antiserum raised against the proteins secreted from EPEC and by human serum from a patient infected with an EHEC O157:H7 strain.
The HtrA stress response protease contributes to resistance of Brucella abortus to killing by murine phagocytes
Authors: Elzer P H , Phillips R W , Robertson G T , Roop R M
Published: January 2020

Compared with virulent Brucella abortus 2308, the isogenic htrA mutant PHE1 shows decreased resistance to killing by cultured murine neutrophils and macrophages and significant attenuation during the early stages of infection in the BALB/c mouse model. These findings further define the contributions of the htrA gene product to the pathogenesis of B. abortus infections.
Growth of Chlamydia pneumoniae induces cytokine production and expression of CD14 in a human monocytic cell line
Authors: Heinemann M , Susa M , Simnacher U , Marre R , Essig A
Published: January 2020

Chlamydia pneumoniae was able to survive and to multiply in the human monocytic cell line Mono Mac 6. Growth of C. pneumoniae induced production of tumor necrosis factor alpha, interleukin 1beta, and interleukin 6, as well as up-regulation of the CD14 molecule in a time-dependent manner. Infection of monocytic cells and a proinflammatory cytokine response may be important in C. pneumoniae pathogenesis.
Antiendotoxin activity of cationic peptide antimicrobial agents
Authors: Gough M , Hancock R E , Kelly N M
Published: January 2020

The endotoxin from gram-negative bacteria consists of a molecule lipopolysaccharide (LPS) which can be shed by bacteria during antimicrobial therapy. A resulting syndrome, endotoxic shock, is a leading cause of death in the developed world. Thus, there is great interest in the development of antimicrobial agents which can reverse rather than promote sepsis, especially given the recent disappointing clinical performance of antiendotoxin therapies. We describe here two small cationic peptides, MBI-27 and MBI-28, which have both antiendotoxic and antibacterial activities in vitro and in vivo in animal models. We had previously demonstrated that these peptides bind to LPS with an affinity equivalent to that of polymyxin B. Consistent with this, the peptides blocked the ability of LPS and intact cells to induce the endotoxic shock mediator, tumor necrosis factor (TNF), upon incubation with the RAW 264.7 murine macrophage cell line. MBI-28 was equivalent to polymyxin B in its ability to block LPS induction of TNF by this cell line, even when added 60 min after the TNF stimulus. Furthermore, MBI-28 offered significant protection in a galactosamine-sensitized mouse model of lethal endotoxic shock. This protection correlated with the ability of MBI-28 to reduce LPS-induced circulating TNF by nearly 90% in this mouse model. Both MBI-27 and MBI-28 demonstrated antibacterial activity against gram-negative bacteria in vitro and in vivo against Pseudomonas aeruginosa infections in neutropenic mice.
Differential influence of the two Bacillus anthracis plasmids on regulation of virulence gene expression
Authors: Fouet A , Mock M
Published: January 2020

Fully virulent Bacillus anthracis bacilli are encapsulated and toxinogenic. These bacteria contain two plasmids, pXO1 and pXO2, carrying genes coding for toxins (pag, lef, and cya) and for capsule synthetic enzymes (capB, capC, capA, and dep), respectively. A transcriptional fusion between the capB regulatory region and the lacZ reporter gene was constructed to study the regulation of capsule synthesis. A single copy of this fusion was inserted into the cap region of pXO2. The influence of environmental factors on the capB-lacZ fusion expression was initially analyzed in a pXO1-negative background: bicarbonate but not temperature induced the transcription from the capB promoter. A strain carrying the recombinant pXO2 and (delta)pag pXO1 was constructed for transregulatory studies. The pXO1 plasmid strongly enhanced capsule formation without modifying the bicarbonate-dependent induction level. A (delta)cap pXO2 was transduced into a strain containing pXO1 harboring a pag-lacZ transcriptional fusion (19). pXO2 showed no influence on the toxin gene transcription.
In vitro attachment and invasion of chicken ovarian granulosa cells by Salmonella enteritidis phage type 8
Authors: Thiagarajan D , Saeed M , Turek J , Asem E
Published: January 2020

The attachment and invasion of chicken ovarian granulosa cells by Salmonella enteritidis was examined in vitro. The attachment was inhibited by preincubation of granulosa cells with anti-chicken fibronectin antibody (approximately 70% reduction in attachment) or preincubation with a 14-kDa fimbrial protein isolated from S. enteritidis (68% reduction in attachment). Treatment of bacterial cells with the tetrapeptide RGDS before addition to granulosa cells resulted in inhibition of attachment (60% inhibition when 2 x 10(7) CFU of bacteria was treated with 500 microg of peptide). Treatment with the peptide GRGD resulted in similar magnitude of inhibition, indicating that extracellular matrix proteins play significant roles in the interaction of S. enteritidis with granulosa cells. In contrast, treatment of the bacterial cells with the peptide GRAD did not result in significant inhibition of attachment to the granulosa cells. S. enteritidis was found to attach specifically to fibronectin, collagen IV, and laminin-coated microtiter plate wells, with the rank order of attachment as follows: fibronectin > laminin > collagen IV. Light and transmission electron micrographs of S. enteritidis invasion of granulosa cells showed organisms with or without a surrounding membrane in the cytoplasm of granulosa cells. In some instances, dividing bacterial cells were observed in the cytoplasm. Results of this study demonstrated that S. enteritidis interacts with granulosa cells in a specific manner and can invade and multiply in these cells. The granulosa cell layer of the preovulatory follicles may be a preferred site for the colonization of the chicken ovaries by invasive strains of S. enteritidis.
Roles of the bacterial cell wall and capsule in induction of tumor necrosis factor alpha by type III group B streptococci
Authors: Vallejo J G , Baker C J , Edwards M S
Published: January 2020

Group B streptococci (GBS) are the major cause of sepsis and fatal shock in neonates in the United States. The precise role of tumor necrosis factor alpha (TNF-alpha) in the development of human GBS sepsis has not been defined; however, whole GBS have been shown to induce the production of this inflammatory cytokine. We sought to determine which bacterial cell wall components of GBS are responsible for triggering TNF-alpha production. Human cord blood monocytes were stimulated with encapsulated (COH1) or unencapsulated (COH1-13) whole type III GBS or with purified bacterial components, including type III capsular polysaccharide (III-PS), group B polysaccharide (GB-PS), lipoteichoic acid (LTA), or peptidoglycan (PG). Lipopolysaccharide from Escherichia coli served as a control. Supernatants were harvested at specific timed intervals, and TNF-alpha levels were measured by enzyme-linked immunosorbent assay. Monocytes exposed to COH1 and COH1-13 induced similar amounts of TNF-alpha. III-PS, GB-PS, LTA, and PG each induced TNF-alpha in a time- and concentration-dependent manner. However, TNF-alpha release was significantly greater after stimulation by the GB-PS or PG than after stimulation by III-PS or LTA (P < 0.05). Our findings indicate that GB-PS and PG are the bacterial cell wall components primarily evoking TNF-alpha release. These, alone or in concert with other factors, may be responsible for septic shock accompanying GBS sepsis.
A 35-kilodalton protein is a major target of the human immune response to Mycobacterium leprae
Authors: Triccas J A , Roche P W , Winter N , Feng C G , Butlin C R , Britton W J
Published: January 2020

The control of leprosy will be facilitated by the identification of major Mycobacterium leprae-specific antigens which mirror the immune response to the organism across the leprosy spectrum. We have investigated the host response to a 35-kDa protein of M. leprae. Recombinant 35-kDa protein purified from Mycobacterium smegmatis resembled the native antigen in the formation of multimeric complexes and binding by monoclonal antibodies and sera from leprosy patients. These properties were not shared by two forms of 35-kDa protein purified from Escherichia coli. The M. smegmatis-derived 35-kDa protein stimulated a gamma interferon-secreting T-cell proliferative response in the majority of paucibacillary leprosy patients and healthy contacts of leprosy patients tested. Cellular responses to the protein in patients with multibacillary leprosy were weak or absent, consistent with hyporesponsiveness to M. leprae characteristic of this form of the disease. Almost all leprosy patients and contacts recognized the 35-kDa protein by either a T-cell proliferative or an immunoglobulin G antibody response, whereas few tuberculosis patients recognized the antigen. This specificity was confirmed in guinea pigs, with the 35-kDa protein eliciting strong delayed-type hypersensitivity in M. leprae-sensitized animals but not in those sensitized with Mycobacterium tuberculosis or Mycobacterium bovis BCG. Therefore, the M. leprae 35-kDa protein appears to be a major and relatively specific target of the human immune response to M. leprae and is a potential component of a diagnostic test to detect exposure to leprosy or a vaccine to combat the disease.
Tumor necrosis factor-inducing activities of Cryptococcus neoformans components
Authors: Delfino D , Cianci L , Migliardo M , Mancuso G , Cusumano V , Corradini C , Teti G
Published: January 2020

Cryptococcus neoformans-induced tumor necrosis factor alpha (TNF-alpha) production may lead to increased human immunodeficiency virus replication in patients with AIDS. In order to identify cryptococcal components that are predominantly responsible for stimulating TNF production, various concentrations of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), mannoproteins (MP), and alpha(1-3) [corrected] glucan were added to whole-blood cultures. All of the cryptococcal components tested, as well as whole heat-killed cryptococci, were capable of inducing TNF-alpha release in a dose-dependent manner. MP were significantly more potent than any of the other cryptococcal components tested or heat-killed cryptococci in stimulating TNF-alpha production (P < 0.05). GXM, in contrast, was significantly less potent in this activity than either GalXM or MP (P < 0.05). As little as 0.5 microg of MP per ml was sufficient to produce moderate but significant elevations of TNF-alpha release. Maximal MP-induced TNF-alpha levels were similar to those induced by Salmonella enteritidis lipopolysaccharide, our positive control. Further experiments using isolated leukocytes suggested that monocytes were the cell population mainly responsible for TNF-alpha production, although the participation of other cell types could not be excluded. The presence of complement-sufficient plasma was a necessary requirement for TNF-alpha induction by GXM, GalXM, and low doses of MP. High MP concentrations (100 microg/ml) were also capable of stimulating TNF-alpha production in the absence of plasma. These data indicate that soluble products released by C. neoformans are capable of inducing TNF-alpha secretion in human leukocytes. This may be clinically relevant, since high concentrations of such products are frequently found in the body fluids of AIDS patients infected with C. neoformans.
Evidence for the presence of streptococcal-superantigen-neutralizing antibodies in normal polyspecific immunoglobulin G
Authors: Norrby-Teglund A , Kaul R , Low D E , McGeer A , Andersson J , Andersson U , Kotb M
Published: January 2020

Recently we demonstrated that normal polyspecific immunoglobulin given intravenously (IVIG) and plasma samples from patients treated with IVIG neutralize the mitogenic and cytokine-inducing activities of group A streptococcal (GAS) superantigens. Here we investigated whether this neutralizing activity is mediated by antibodies to these superantigens. IVIG and plasma samples collected from a patient with GAS necrotizing fasciitis post-IVIG infusions markedly inhibited the mitogenic activity elicited by the streptococcal pyrogenic exotoxins SpeB and SpeC, as well as by GAS culture supernatant. Immunoblot analysis showed marked increases in the levels of antibodies to SpeC and proteins in the GAS culture supernatant in post-IVIG over those of pre-IVIG plasma samples. Removal of antisuperantigen antibodies in IVIG by adsorption to SpeC- and GAS culture supernatant-coupled Sepharose markedly reduced the neutralizing ability of IVIG against respective stimuli. The neutralizing activity was totally recovered in the eluted antibodies. By contrast, although pre- and post-IVIG plasma samples contained antibodies to SpeA, these antibodies did not block the activity of this superantigen. Nonspecific immunomodulatory activity of IVIG was ruled out because neither the IVIG nor the affinity-purified antibodies significantly inhibited the response to the polyclonal T-cell mitogen phytohemagglutinin A. These data provide direct evidence that the neutralizing activity in IVIG, and in patient plasma samples following IVIG treatment is mediated by antibodies to superantigens and indicate that the quality rather than the quantity of these antibodies may be more clinically relevant.
Molecular definition and identification of new proteins of Mycobacterium leprae
Authors: Pessolani M C , Brennan P J
Published: January 2020

This report describes N-terminal group analysis of six new proteins isolated from in vivo-grown Mycobacterium leprae, three of which correspond to products of the cysA, ahpC, and rpIL genes, which were recently defined through the M. leprae genome project and which encode a putative sulfate sulfurtransferase, an antioxidant enzyme, and the L7/L12 ribosomal protein, respectively.
Bacterial enzymes can add galactose alpha 1,3 to human erythrocytes and creates a senescence-associated epitope
Authors: Hamadeh R M , Jarvis G A , Zhou P , Cotleur A C , Griffiss J M
Published: January 2020

Humans have abundant circulating anti-alpha (1,3-di)-galactosyl (alpha Gal) antibodies (anti-Gal). Anti-Gal has been implicated in the clearance of senescent human erythrocytes (RBCs). The nature of the anti-Gal-binding RBC epitope has defied explanation, given that humans repress expression of the alpha 1,3 galactosyltransferase (alpha 1,3 GT) enzyme. This study explored whether alpha Gal epitopes on human RBCs might be synthesized by alpha 1,3 GTs of bacterial origin that are translocated into the circulation during commensal colonization of the gut by gram-negative bacteria. We found that an acellular Klebsiella pneumoniae sonicate could add 3H-UDP-Gal to human RBCs in the alpha configuration at 37 degrees C in the presence of 6 mM MnCl2 (pH 7.6). Gradient anion-exchange chromatography of the Klebsiella sonicate yielded four fractions that could catalyze the addition of 3H-Gal to human RBCs. Size-exclusion chromatography of these anion-exchange fractions yielded peaks of high GT activity for each, but only those derived from the first, third, and last anion-exchange fractions incorporated Gal such that the RBCs bound anti-Gal by fluorescence-activated cell sorter, suggesting that these three GTs are alpha 1,3 GTs. Thus, Klebsiella spp. make at least four GTs that can add an alpha Gal to human cell surface acceptor structures. Three of these GTs can form alpha 1,3 Gal structures on human RBCs that bind anti-Gal, thereby creating "autoimmune" senescence-associated RBC epitopes.
The N-terminal half of membrane CD14 is a functional cellular lipopolysaccharide receptor
Authors: Viriyakosol S , Kirkland T N
Published: January 2020

CD14, a glycosylphosphatidylinositol-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, is a receptor for lipopolysaccharide (LPS). It was recently reported that an N-terminal 152-amino-acid fragment of soluble CD14 was an active soluble lipopolysaccharide receptor (T. S. -C. Juan, M. J. Kelley, D. A. Johnson, L. A. Busse, E. Hailman, S. D. Wright, and H. S. Lichenstein, J. Biol. Chem. 270:1382-1387, 1995). To determine whether the N-terminal half of the membrane CD14 was a functional LPS receptor on the cell membrane, we engineered a chimeric gene coding for amino acids 1 to 151 of CD14 fused to the C-terminal region of decay-accelerating factor and expressed it in Chinese hamster ovary cells and 70Z/3 cells. We found that the chimeric, truncated CD14 is a fully functional LPS receptor in both cell lines.
Listeriolysin is a potent inducer of the phosphatidylinositol response and lipid mediator generation in human endothelial cells
Authors: Sibelius U , Rose F , Chakraborty T , Darji A , Wehland J , Weiss S , Seeger W , Grimminger F
Published: January 2020

The impact of Listeria monocytogenes listeriolysin O (LLO) secretion on phosphoinositide metabolism and mediator (platelet-activating factor and prostaglandin I2) generation was investigated in human umbilical vein endothelial cells. Wild-type L. monocytogenes, purified LLO, and an L. innocua strain engineered to secrete LLO all elicited a strong response, whereas mutant strains defective in LLO production were ineffective. Thus, human umbilical vein endothelial cell stimulation by listeriae is linked to production of LLO.
Plasmodium falciparum induces apoptosis in human mononuclear cells
Authors: Toure-Balde A , Sarthou J L , Aribot G , Michel P , Trape J F , Rogier C , Roussilhon C
Published: January 2020

The level of spontaneous apoptosis in short-term lymphocyte cultures was evaluated in different human immunodeficiency virus-negative groups of either healthy control individuals or patients with clinical malaria. The mean percentage of spontaneous apoptosis found in patients during a malaria attack was significantly higher than in sex- and age-matched healthy controls. The healthy asymptomatic controls were individuals with different degrees of exposure to Plasmodium falciparum as reflected by their various mean levels of specific anti-P. falciparum (immunoglobulin G and M) antibodies. The percentages of apoptotic nuclei were found to be significantly higher in lymphocytes from subjects living in an area where malaria is holoendemic than in lymphocytes from subjects less exposed. Concentrations of soluble plasma interleukin-2 receptor were also higher in subjects from areas where malaria is endemic than in other groups, revealing different levels of lymphocyte activation. Of particular relevance to the in vivo situation, a P. falciparum schizont-rich extract induced a systematic and significant elevation of apoptotic nuclei at day 6 in 87.5% (35 of 40) of the subjects tested. In additional studies with different concentrations of extract, [3H]thymidine incorporation was concomitant with a low or limited level of apoptosis. Taken together, our results strongly suggest that acute as well as chronic asymptomatic P. falciparum infections were consistently associated with a marked increase in the level of mononuclear cell apoptosis. This process could be implicated in some of the alterations reported for the proliferative T-cell responses in areas where malaria is endemic.