bluuebunny/crossref_metadata_2025 huggingface dataset:
Urease-specific monoclonal antibodies prevent Helicobacter felis infection in mice
Authors: Blanchard T G , Czinn S J , Maurer R , Thomas W D , Soman G , Nedrud J G
Published: January 2020

Experiments were performed to determine the antigenic specificity of a monoclonal antibody (immunoglobulin A [IgA] 71) previously demonstrated to neutralize the ability of Helicobacter felis to colonize mice. Immunoprecipitation of radiolabeled H. felis outer membrane proteins with IgA 71 revealed specificity for a 62-kDa protein. Another of our monoclonal antibodies, IgG 40, precipitated a protein of similar molecular weight. IgA 71 but not IgG 40 also precipitated purified recombinant H. pylori urease. The antigenic specificity of both antibodies was confirmed to be urease by the ability of each to select Escherichia coli clones expressing the H. felis urease genes. The two antibodies were shown to bind nonoverlapping epitopes in a competition enzyme-linked immunosorbent assay. Both IgA 71 and IgG 40 could effectively neutralize H. felis infectivity by incubating the bacteria with the antibodies prior to oral administration to naive mice. The mechanism of protection does not appear to be inhibition of urease activity, as IgA 71 does not inhibit the conversion of urea to ammonia by H. pylori urease in vitro. These results support a protective role for the secretory humoral immune response in Helicobacter immunity and provide further evidence that the urease enzyme can serve as a protective antigen.
Comparison of the mechanisms of action of cholera toxin and the heat-stable enterotoxins of Escherichia coli
Authors: Peterson J W , Whipp S C
Published: January 2020

The mechanisms which enable cholera toxin (CT) and the Escherichia coli heat-stable enterotoxins (STa and STb) to stimulate intestinal secretion of water and electrolytes are only partially understood. CT evokes the synthesis of 3',5'-cyclic AMP (cAMP), and STa is known to elevate intestinal levels of 3',5'-cyclic GMP (cGMP). Neither of these recognized second messengers appears to mediate E. coli STb responses. We compared the secretory effects of CT, STa, and STb using the pig intestinal loop model and also measured the effects of toxin challenge on the synthesis of cAMP, cGMP, and prostaglandins (e.g., prostaglandin E2 [PGE2]), as well as on the release of 5-hydroxytryptamine (5-HT) from intestinal enterochromaffin cells. All three enterotoxins elicited fluid accumulation within a 2-h observation period. A combination of maximal doses of STa with STb yielded additive effects on fluid accumulation, which suggested different mechanisms of action for these toxins. Similarly, challenge of pig intestinal loops with a combination of CT and STb resulted in additive effects on fluid accumulation and luminal release of 5-HT. Unlike its effect on intestinal tissues from other animals, CT did not appear to elicit a dose-dependent cAMP response measurable in mucosal extracts from pig small intestine. In contrast, luminal fluid from CT-challenged pig intestinal loops contained dose-related amounts of cAMP and PGE2 that had been secreted from the mucosa. cAMP responses to STa or STb could not be demonstrated in either mucosal tissue or luminal fluid. In contrast, cGMP levels were increased in the intestinal fluid of loops challenged with STa but not in those challenged with STb. While the mechanisms of action of CT and STa are thought to involve impulse transmission via the enteric nervous system, we demonstrated significant stimulation of PGE2 synthesis and 5-HT release for CT and STb but very little for STa. We conclude from these data that the mechanisms of action of STa, STb, and CT are distinct, although the mode of action of STb may have some similarity to that of CT. Since STb stimulated the release of both PGE2 and 5-HT from the intestinal mucosa, the data suggested the potential for an effect of STb on the enteric nervous system.
Independent down-regulation of central and peripheral tumor necrosis factor production as a result of lipopolysaccharide tolerance in mice
Authors: Faggioni R , Fantuzzi G , Villa P , Buurman W , van Tits L J , Ghezzi P
Published: January 2020

Lipopolysaccharide (LPS) induces a variety of central and peripheral effects that are largely mediated by cytokines, including tumor necrosis factor (TNF). Peripheral (intravenous [i.v.]) administration of LPS (2.5 micrograms per mouse) induced TNF levels in the serum and spleen but not in the brain, while central (intracerebroventricular [i.c.v.]) administration of LPS induced TNF production both in the brain and in the periphery. Mice challenged with LPS after LPS pretreatment (35 micrograms per mouse, intraperitoneally, as a single dose on day -3 or as a 4-day treatment on days -5 to -2) were unresponsive in terms of induction of serum TNF. When peripherally LPS-tolerant mice (where LPS pretreatment was given intraperitoneally) were challenged with an i.c.v. dose of LPS, brain (but not serum) TNF was still produced, meaning that the LPS-tolerant state was confined to the periphery. However, if LPS pretreatment was given i.c.v. (35 micrograms, as a single dose), the brain, like the periphery, became LPS tolerant in terms of TNF production. We investigated how tolerance to LPS affected two of its actions, decrease in food intake and induction of serum corticosterone (CS). After an i.v. challenge in peripherally LPS-tolerant mice, no decrease in food intake was observed, but this response was still elicited by an i.c.v. challenge. LPS tolerance reduced the CS response to i.v. and i.c.v. challenge. These results suggest that LPS-induced decrease in food intake might be a fully central effect, while the increase of serum CS might be due to both central and peripheral actions.
Analysis of Neisseria meningitidis class 3 outer membrane protein gene variable regions and type identification using genetic techniques
Authors: Bash M C , Lesiak K B , Banks S D , Frasch C E
Published: January 2020

The class 3 porin proteins of Neisseria meningitidis stimulate bactericidal antibodies and express serotype-specific antigenic epitopes. Sequence analysis of porB genes for the class 3 proteins revealed regions of variability that map to surface-exposed loops. To evaluate the relationship between serotype and variable-region (VR) genotype, sequences from the 11 class 3-expressing serotype strains and 3 additional serotype 4 strains were analyzed by molecular techniques. Multiple-sequence alignment revealed a limited number of unique sequences at each of four VRs (VR1 to VR4), ranging from four unique sequences at VR1 to seven sequence patterns at VR2 and VR4. Serotype-specific VR sequences were found in each of the four VRs, suggesting that each VR has immunologic importance. Five serotypes had at least one VR sequence that was unique. Three serotypes which had sequences in common with other serotypes at each VR were distinguished by examining multiple VRs. Serotype 3 was identical to serotype 19 at each VR, and serotype 8 was identical to serotype 18 at each VR. Serotypes 4 and 21 were identical at VR1 and significantly different at VR3 and VR4. A subpopulation of serotype 4 strains with a unique VR2 sequence was identified. The serotypes which were grouped with closely related or identical sequences at one VR were grouped with different serotypes at other VRs consistent with the pattern of genetic mosaicism described for the porA (class 1 protein) gene. Hybridization assays demonstrated the ability to identify VR genotypes and distinguish serotypes using biotin-labelled oligonucleotide probes. This information may be useful in strain selection for vaccine development, in epidemiologic studies to determine the prevalence of the individual VR genotype (especially among nonserotypeable strains) and, combined with PCR, in the identification of culture-negative suspected meningococcal cases.
Serum antibodies to outer membrane proteins (OMPs) of Moraxella (Branhamella) catarrhalis in patients with bronchiectasis: identification of OMP B1 as an important antigen
Authors: Sethi S , Hill S L , Murphy T F
Published: January 2020

Moraxella (Branhamella) catarrhalis is a common cause of lower respiratory tract infections in adults and of otitis media in children. Little is known about the human immune response to this bacterium. In this study, immunoblot assays were performed to detect serum immunoglobulin G antibodies directed at purified outer membrane of M. catarrhalis. Twelve serum samples, two each from six patients with bronchiectasis who were persistently colonized with this organism, were tested with their homologous M. catarrhalis sputum isolates. In all the sera, the most prominent and consistent antibody response was to a minor 84-kDa outer membrane protein, OMP B1. Immunoblot adsorption assays show that these antibodies recognize surface exposed epitopes on OMP B1. Further analysis of human serum antibodies eluted from the surface of intact bacterial cells shows that these surface-exposed epitopes on OMP B1 are heterogeneous among strains of M. catarrhalis. OMP B1 is therefore an important OMP antigen on the surface of M. catarrhalis for the human immune response to infection by this bacterium.
Effects of immunization with Cryptococcus neoformans cells or cryptococcal culture filtrate antigen on direct anticryptococcal activities of murine T lymphocytes
Authors: Muth S M , Murphy J W
Published: January 2020

Immunizing CBA/J mice with intact Cryptococcus neoformans cells or with a cryptococcal culture filtrate antigen (CneF) induces an anticryptococcal delayed-type hypersensitivity response. Recently, it has been shown that two phenotypically different T-cell populations are responsible for delayed-type hypersensitivity reactivity in mice immunized with intact cryptococcal cells, whereas only one of those populations is present in mice immunized with soluble cryptococcal antigens in complete Freund's adjuvant (CFA). The purpose of this study was to determine if differences occur with regard to direct anticryptococcal activity between T-lymphocyte-enriched populations from mice immunized with intact viable or dead cryptococcal cells and similar cell populations from mice immunized with the soluble cryptococcal culture filtrate antigen, CneF, emulsified in CFA. The percentage of lymphocytes which form conjugates with C. neoformans and the percentage of cryptococcal growth inhibition in vitro are greater with T-lymphocyte-enriched populations from mice sublethally infected with C. neoformans or from mice immunized with intact heat-killed cryptococcal cells in the presence or absence of CFA than with lymphocyte populations from mice immunized with CneF-CFA. Enhanced anticryptococcal activity of T lymphocytes could be induced by immunizing mice with heat-killed C. neoformans cells of serotype A, B, C, or D as well as by immunizing with a similar preparation of an acapsular C. neoformans mutant but not by immunizing with CFA emulsified with CneF prepared from any one of the C. neoformans isolates. These data indicate that the soluble cryptococcal culture filtrate antigens do not induce the same array of functional T lymphocytes as whole cryptococcal cells.
Selection of variant Borrelia burgdorferi isolates from mice immunized with outer surface protein A or B
Authors: Fikrig E , Tao H , Barthold S W , Flavell R A
Published: January 2020

A nonclonal population of Borrelia burgdorferi N40 (passage 3) that survived protective immunity following challenge inoculation of outer surface protein (Osp) A- or B-hyperimmunized mice were characterized for the molecular basis of evasion of immunity. Two of six B. burgdorferi isolates, cultured from OspA-immunized mice, had antigenic diversity in the carboxyl terminus of OspA and did not bind to the protective OspA monoclonal antibody designated IXDII. However, OspA-immunized mice challenged with these variants were fully protected. Moreover, B. burgdorferi isolates with a point mutation in ospB, which results in a truncated OspB that does not bind to protective OspB monoclonal antibody 7E6C, were frequently enriched after infection of OspB-immunized mice. These studies suggest that the incomplete efficacy of an OspA- or OspB-based vaccine may be partly due to immunomediated in vivo selective pressure, resulting in the persistence of some spirochetes that do not bind to protective antibodies.
Potentiation of interferon-mediated inhibition of Chlamydia infection by interleukin-1 in human macrophage cultures
Authors: Carlin J M , Weller J B
Published: January 2020

One mechanism by which interferons (IFNs) can inhibit chlamydial infection is by the induction of the enzyme indoleamine 2,3-dioxygenase (IDO), which restricts the availability of tryptophan, which is required for chlamydial growth. Other immunomodulating agents, including interleukin-1 (IL-1), can interact synergistically with IFNs, resulting in increased IDO activity in macrophages. The objectives of this study were to establish that IL-1 can enhance IFN-mediated inhibition of chlamydial growth by increasing the amount of IDO activity induced by IFNs and to identify immunomodulatory agents in culture supernatants from chlamydia-infected macrophages that interact synergistically with IFNs in restricting chlamydial growth. Monocyte-derived macrophages were treated with IL-1 combined with gamma IFN (IFN-gamma) or IFN-beta. The ability of treated cells to support the growth of Chlamydia psittaci was directly related to the amount of IDO activity induced; as IDO activity increased, so did inhibition of chlamydial growth. Furthermore, concentrations of IFNs were identified at which little IDO activity was induced and chlamydial growth was permitted yet which in the presence of IL-1 resulted in increased IDO activity and restriction of chlamydial growth. The addition of exogenous tryptophan reversed the effect of combined IFN and IL-1 treatment, indicating that IDO activity induced by combined cytokine treatment was responsible for chlamydial inhibition. Supernatants from chlamydia-infected macrophages were capable of potentiating IDO induction by IFN-gamma and of restricting the growth of C. psittaci. Antibody to IL-1 beta neutralized the potentiating effects of supernatants from chlamydia-infected cells on both IDO induction and chlamydial inhibition. Thus, IL-1 produced in response to chlamydial infection may contribute to the elimination of the infection.
Lipoarabinomannans derived from different strains of Mycobacterium tuberculosis differentially stimulate the activation of NF-kappa B and KBF1 in murine macrophages
Authors: Brown M C , Taffet S M
Published: January 2020

The inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) is rapidly induced in macrophages after exposure to Mycobacterium tuberculosis. Recently it was shown that lipoarabinomannan (LAM) derived from an attenuated (H37Ra) strain of M. tuberculosis (AraLAM) was capable of macrophage activation and induction of TNF-alpha production, whereas LAM derived from the virulent Erdman strain (ManLAM) was considerably reduced in this activity. A critical component in the regulation of many genes central to immune function is the transcription factor NF-kappa B. Lipopolysaccharide (LPS)-mediated induction of TNF-alpha expression in murine macrophages has been demonstrated to be regulated in part by NF-kappa B. In this study, we demonstrate that AraLAM is capable of rapid activation of NF-kappa B- and KBF1-binding activities in C3H/HeN bone marrow-derived macrophages and the J774.A and RAW264.7 murine macrophagelike cell lines, whereas ManLAM is considerably less potent at stimulating NF-kappa B. Treatment of RAW264.7 cells with AraLAM or LPS results in the stimulation of DNA binding of both forms within 7.5 min, which peaks within 30 min and 1 h, respectively. Interestingly, treatment of RAW264.7 macrophage-like cells with AraLAM, LPS, or ManLAM for greater than 2 h resulted in significant accumulation of KBF1. Inhibition of protein synthesis blocked the transient nature of NF-kappa B activation as well as the accumulation of KBF1. Using Western immunodetection of the NF kappa B1 p50 subunit, we also show that AraLAM and LPS stimulate the loss of the NF kappa B1 p105 precursor. These results demonstrate that NF-kappa B and KBF1 are rapidly induced in response to AraLAM and may play a role in avirulent M. tuberculosis activation of TNF-alpha expression in macrophages. The differential temporal regulation of kappa B element DNA-binding activities and the transient stimulation of NF kappa B followed by the sustained accumulation of KBF1 may serve as a feedback switch ensuring transient induction of TNF-alpha transcription.
A mutant pertussis toxin molecule that lacks ADP-ribosyltransferase activity, PT-9K/129G, is an effective mucosal adjuvant for intranasally delivered proteins
Authors: Roberts M , Bacon A , Rappuoli R , Pizza M , Cropley I , Douce G , Dougan G , Marinaro M , McGhee J , Chatfield S
Published: January 2020

We examined the capacity of a genetically detoxified derivative of pertussis toxin (PTX), PT-9K/129G, to act as a mucosal adjuvant for an intranasally (i.n.) administered tetanus vaccine. Groups of mice were immunized i.n. with the nontoxic C-terminal 50-kDa portion of tetanus toxin (fragment C [Frg C]) either alone or mixed with PT-9K/129G, PTX, or cholera toxin (CT) or were immunized subcutaneously (s.c.) with an equivalent amount of Frg C adsorbed to alhydrogel. In response to a single immunization, mice receiving Frg C plus PT-9K/129G or CT i.n. and parenterally immunized mice developed high-titer (> 20,000) anti-Frg C antibodies, whereas mice immunized i.n. with Frg C plus PTX or with Frg C alone seroconverted only after being boosted. The serum anti-Frg C response was dominated by immunoglobulin G1 (IgG1) in mice immunized with Frg C plus PT-9K/129G, with Frg C plus PTX, or s.c. In contrast, IgG1, IgG2a, and IgG2b contributed almost equally to the Frg C response when CT was the adjuvant. Anti-Frg C IgE was detected only in the sera of mice immunized i.n. with Frg C plus PTX and immunized s.c. with Frg C plus alhydrogel. High levels of IgA antibodies were present in nasal lavage fluid from mice immunized i.n. with Frg C plus PT-9K/129G, PTX, or CT but not in that from mice given Frg C alone i.n. or parenterally. The mucosal adjuvanticity of PT-9K/129G was manifested in inbred as well as outbred mice. A single i.n. dose of Frg C plus either PT-9K/129G or PTX (with high specific activity) was sufficient to protect all immunized mice from tetanus toxin challenge, in contrast to the case for mice that received Frg C alone i.n. We conclude that the pertussis toxin analog PT-9K/129G, which is devoid of ADP-ribosyltransferase activity, is a potent mucosal adjuvant for vaccines delivered via the respiratory tract.
Release of tumor necrosis factor alpha and interleukin 6 during antibiotic killing of Escherichia coli in whole blood: influence of antibiotic class, antibiotic concentration, and presence of septic serum
Authors: Prins J M , Kuijper E J , Mevissen M L , Speelman P , van Deventer S J
Published: January 2020

The concentration and accessibility of endotoxin can increase following antibiotic killing of gram-negative bacteria. There are indications that antibiotics may differ in this respect. We measured endotoxin levels in RPMI 1640 and tumor necrosis factor alpha (TNF-alpha) and interleukin-6 production in whole blood ex vivo after exposure of log-phase Escherichia coli to antibiotics belonging to different classes, in a final concentration of 0.5, 5, or 50 times the MIC. After 4 h of incubation at 50 times the MIC, ceftazidime and ciprofloxacin treatment resulted in levels of endotoxin, TNF-alpha, and interleukin-6 significantly higher than those of imipenem and gentamicin (P < 0.001). Similar differences in cytokine induction were measured after 8 h of incubation. At 0.5 times the MIC, the differences between the antibiotics in measured endotoxin and cytokine levels were small, with levels comparable to the levels in untreated cultures. Polymyxin B and, to a lesser degree, recombinant bactericidal/permeability-increasing protein 21 (rBPI-21) were found to be potent inhibitors of TNF-alpha release, supporting the concept that the differences between the antibiotics in cytokine production were indeed due to differences in amounts of biologically active endotoxin. The presence of serum from patients suffering from untreated sepsis decreased TNF-alpha production significantly, in a concentration-dependent manner.
Human microvascular endothelial tissue culture cell model for studying pathogenesis of Brazilian purpuric fever
Authors: Quinn F D , Weyant R S , Worley M J , White E H , Utt E A , Ades E A
Published: January 2020

Brazilian purpuric fever (BPF) is a fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. All known BPF cases have been caused by three clones of Haemophilus influenzae biogroup aegyptius and have occurred in either Brazil or Australia. Using an immortalized line of human vascular endothelial cells, we developed an in vitro assay that identifies all known BPF-causing H. influenzae biogroup aegyptius strains (R. S. Weyant, F. D. Quinn, E. A. Utt, M. Worley, V. G. George, F. J. Candal, and E. W. Ades, J. Infect. Dis. 169:430-433, 1994). With multiplicities of infection (MOIs) as low as one bacterium per 1,000 tissue culture cells, BPF-associated strains produce a unique cytotoxic effect in which the tissue culture cells detach and aggregate in large floating masses after 48 h of incubation. In this study, using a BPF-associated strain and a non-BPF-associated control, we demonstrated that strains which produce the cytotoxic phenotype were able to replicate intracellularly whereas non-BPF-associated strains, with MOIs of > or = 1,000 did not replicate and did not produce the phenotype. We also showed that this phenotype is not caused by the activity of an endotoxin or the release of some other compound from the bacterial cell, since neither gamma irradiation-killed whole BPF clone bacteria nor bacterial cell fractions at MOIs of > 1,000 produced the cytotoxic effect. Furthermore, bacteria in numbers equal to MOIs of > 1,000 treated with chloramphenicol did not produce the cytotoxic phenotype, suggesting a requirement for bacterial protein synthesis. In addition, viable bacteria separated from the tissue culture monolayer by a 0.2-micron-pore-size membrane also failed to produce the phenotype. The ability of the bacterium to invade, replicate, and produce the phenotype appears to be primarily parasite directed since phagocytosis, pinocytosis, and eukaryotic protein synthesis inhibitors, including cycloheximide, cytochalasin D, and methylamine, had no effect on the ability of the bacterium to invade and cause a cytotoxic response. Understanding the basic mechanisms involved in this tissue-destructive process should enhance our knowledge of the general pathogenesis of BPF.
Isolation and characterization of a Clostridium botulinum C2 toxin-resistant cell line: evidence for possible involvement of the cellular C2II receptor in growth regulation
Authors: Fritz G , Schroeder P , Aktories K
Published: January 2020

Clostridium botulinum C2 toxin, which consists of the binding component C2II and the enzyme component C2I, acts on eukaryotic cells by selective ADP-ribosylation of G-actin. To obtain C2 toxin-resistant cells, we mutagenized CHO-K1 cells with N-nitroso-N-methylurea and selected for C2 resistance. Cells which survived the selection procedure with 50 ng of C2I and 100 ng of C2II per ml were obtained with a frequency of 30 x 10(-6). The colony-forming ability of CHO wild-type cells was reduced to 50% with 10 ng of C2I and 20 ng of C2II per ml. In contrast, the colony-forming ability of the isolated CHO mutant cells was not influenced by up to 200 ng of C2I and 400 ng of C2II per ml. Toxin-induced ADP-ribosylation of G-actin was not impaired in lysates of mutant cells. The C2 toxin-resistant phenotype remained sensitive to the cell-rounding activities of cytotoxins from C. perfringens (iota-toxin), C. novyi, C. difficile, and C. botulinum (C3) and to cytochalasin D. Binding of component C2II was impaired in resistant CHO cells, suggesting mutation of the toxin cell surface receptor. Serum factors protected wild-type cells against the cytotoxic effect of C2 toxin. Furthermore, the C2-resistant phenotype correlated with an increased serum dependency. The data suggest that the action of C. botulinum C2 toxin is mediated by its binding and uptake via a cell surface receptor which might be involved in growth regulation.
Convalescent pigs are protected completely against infection with a homologous Actinobacillus pleuropneumoniae strain but incompletely against a heterologous-serotype strain
Authors: Cruijsen T , van Leengoed L A , Ham-Hoffies M , Verheijden J H
Published: January 2020

To study whether Actinobacillus pleuropneumoniae induces a species-specific immunity, we infected pigs in the left lung with serotype 3 or 9 and after 3 weeks we infected their right lungs with serotype 9. Convalescent pigs were protected against homologous strain reinfection, but after heterologous strain reinfection the degree of protection varied. Neutralizing antibodies were not essential for protection.
Increased gamma-delta T-lymphocyte response to Mycobacterium bovis BCG in major histocompatibility complex class I-deficient mice
Authors: Muller D , Pakpreo P , Filla J , Pederson K , Cigel F , Malkovska V
Published: January 2020

Mice with a homologous deletion of the beta 2-microglobulin gene (beta 2m-) are deficient in class I major histocompatibility complex molecules (MHC) and consequently are deficient in CD8+ T cells. These beta 2m- mutant mice control the intraperitoneal growth of an avirulent vaccine strain of mycobacteria, Mycobacterium bovis BCG, after intraperitoneal infection similarly to normal mice. We show that beta 2m- mice have an increased gamma-delta (gamma delta) T-cell response after infection with live avirulent mycobacteria. beta 2m- mice have an earlier and more sustained rise in the proportion of intraperitoneal gamma delta T cells, averaging 17% of T cells, compared with 6% in normal mice, at 28 days after infection. Compared with the population in normal mice, gamma delta T cells in the spleens of beta 2m- mice averaged a higher proportion of the total T-cell population of the spleen on days 5, 8, and 14 after intraperitoneal infection. These data document the kinetics of gamma delta T cells reactive to mycobacterial antigens in vivo without class I MHC restriction and support a role for class I MHC and CD8+ T cells in the in vivo regulation of gamma delta T cells.
In vitro evaluation of the role of humoral immunity against Bartonella henselae
Authors: Rodriguez-Barradas M C , Bandres J C , Hamill R J , Trial J , Clarridge J E , Baughn R E , Rossen R D
Published: January 2020

The contribution of humoral immunity against Bartonella henselae was evaluated by examining the in vitro bactericidal activity of sera and the ability of these microorganisms to activate complement and stimulate phagocytosis and an oxidative burst in polymorphonuclear leukocytes. The organism was killed by complement-mediated cytolysis. Complement activation preferentially proceeded by the alternative pathway. The presence of specific antibodies did not increase the serum bactericidal activity or complement activation. However, phagocytosis and the subsequent production of oxygen radicals, evaluated by flow cytometry, were significantly enhanced in the presence of bacteria previously opsonized with immune sera.
Mucosal immunogenicity of polysaccharides conjugated to a peptide or multiple-antigen peptide containing T- and B-cell epitopes
Authors: Lett E , Klopfenstein C , Klein J P , Schöller M , Wachsmann D
Published: January 2020

In this study we investigated the mucosal and systemic responses to two T-cell-independent polysaccharides, a serogroup f polysaccharide (formed of rhamnose glucose polymers [RGPs]) from Streptococcus mutans OMZ 175 and a mannan from Saccharomyces cerevisiae, covalently conjugated either to a linear peptide (peptide 3) or to a multiple-antigen peptide (MAP), both derived from S. mutans protein SR, an adhesin of the I/II protein antigen family of oral streptococci. Peptide 3 and MAP, which contained at least one B- and one T-cell epitope, were tested as carriers for the polysaccharides and as protective immunogens. Intragastric intubation of rats with the conjugates (RGPs-peptide 3, RGPs-MAP, mannan-peptide 3, and mannan-MAP) associated with liposomes produced salivary immunoglobulin A (IgA) antibodies which reacted with RGPs or mannan, peptide 3 or MAP, protein SR, and S. mutans or S. cerevisiae cells. Administration of conjugate boosters to the animals showed that both carriers conjugated to the polysaccharides were able to induce, in immunized animals, a salivary antipolysaccharide IgA memory. In contrast, animals primed and challenged with unconjugated polysaccharide showed no anamnestic response. Rats orally immunized with the conjugates also developed systemic primary antipolysaccharide and antipeptide IgM antibody responses which were characterized by a switch from IgM to IgG during the course of the secondary response. Data presented here demonstrated that both peptide 3 and the MAP construct can act as good carriers for orally administered polysaccharides. Unexpectedly, the use of a MAP did not further improve the immunogenicity of polysaccharides at the mucosal level; nevertheless, such a construct should be of great interest in overcoming the problem of genetic restriction induced by linear peptides.
Alternate routes of invasion may affect pathogenesis of Salmonella typhimurium in swine
Authors: Fedorka-Cray P J , Kelley L C , Stabel T J , Gray J T , Laufer J A
Published: January 2020

Transmission of Salmonella typhimurium in swine is traditionally believed to occur by the fecal-oral route, with invasion through the intestinal wall and Peyer's patches. However, involvement of the upper respiratory tract may be equally important. An esophagotomy was performed on 6- to 8-week-old pigs. Esophagotomized pigs were challenged intranasally with 10(9) CFU of S. typhimurium cells and necropsied at 3, 6, 12, and 18 h postinoculation (p.i.). By 3 h p.i., S. typhimurium was recovered from cecum, colon, head, and thoracic tissues and from the middle ileum involving a large number of Peyer's patches. The ileocolic lymph nodes and ileocolic junction were not positive for S. typhimurium until 6 and 12 h p.i., respectively. Additional pigs were inoculated transthoracically with 10(9) CFU of S. typhimurium and necropsied at 3 and 18 h p.i. By 3 h p.i., all tissues were positive for S. typhimurium. Tonsil explants seeded with 10(9) CFU of S. typhimurium indicated that within 6 h p.i., S. typhimurium was located within the tonsilar crypts. These data show that after intranasal inoculation, S. typhimurium rapidly appears in the gut tissues and suggest that the tonsils and lung may be important sites for invasion and dissemination of Salmonella species.
Protein Hpn: cloning and characterization of a histidine-rich metal-binding polypeptide in Helicobacter pylori and Helicobacter mustelae
Authors: Gilbert J V , Ramakrishna J , Sunderman F W , Wright A , Plaut A G
Published: January 2020

Helicobacter pylori is a human gastrointestinal pathogen involved in gastritis, duodenal ulcers, and gastric neoplasia. This microorganism produces large amounts of a urease which, like all known ureases, has nickel in the active site. We have identified a protein in clinical isolates of H. pylori and an identical protein in the ferret pathogen Helicobacter mustelae that strongly binds Ni2+ and Zn2+. This protein has been named Hpn to emphasize its origins in H. pylori and its affinity for nickel. The encoding hpn gene, cloned and expressed in Escherichia coli ER1793, has an open reading frame (180 bp) that specifies a protein with a calculated molecular mass of 7,077 Da and with the same amino-terminal sequence as that of wild-type Hpn. The deduced sequence of Hpn consists of 60 amino acids, of which 28 (47%) are histidines. The hpn gene does not map with the urease gene cluster on the H. pylori chromosome. An Hpn-negative, isogenic H. pylori strain, generated by hpn gene deletion and grown on blood agar, had the same urease activity that wild-type cells did. Thus, the role of Hpn in helicobacters is unknown.
Protective vaccination with a recombinant fragment of Clostridium botulinum neurotoxin serotype A expressed from a synthetic gene in Escherichia coli
Authors: Clayton M A , Clayton J M , Brown D R , Middlebrook J L
Published: January 2020

A completely synthetic gene encoding fragment C, a approximately 50-kDa fragment, of botulinum neurotoxin serotype A was constructed from oligonucleotides. The gene was expressed in Escherichia coli, and full-sized product was produced as judged by Western blot (immunoblot) analysis. Crude extracts of E. coli expressing the gene were used to vaccinate mice and evaluate their survival against challenge with active toxin. Mice given three subcutaneous vaccinations were protected against an intraperitoneal administration of 10(6) 50% lethal doses (ID50) of serotype A toxin. The same mice survived when challenged with 3 LD50 of botulinum toxin serotype E but died when challenged with 10 LD50 of serotype E or 3 LD50 of serotype B. Purified fragment C was compared with the botulinum toxoid vaccine in a vaccination and challenge study. Fragment C was as efficacious in protecting against challenge with active botulinum neurotoxin serotype A as the toxoid vaccine. This recombinant protein product has many properties that make it a good candidate for human use to protect against botulinum toxin.
Role of interleukin-6 in T-cell activation during primary and secondary infection with Listeria monocytogenes
Authors: Liu Z , Simpson R J , Cheers C
Published: January 2020

Injection of recombinant interleukin-6 (IL-6) into mice enhances recovery from infection with Listeria monocytogenes. In this study, the role of IL-6 during primary and secondary Listeria infection was further tested. Neutralization of IL-6 by polyclonal antibody exacerbated primary infection and significantly delayed gamma interferon production by cultured spleen cells. In contrast, administration of anti-IL-6 antibody at the time of secondary infection did not affect the recovery of mice from infection or gamma interferon production, showing that activated T cells are not dependent on IL-6.
Cytokine-mediated activation of macrophages from Mycobacterium bovis BCG-resistant and -susceptible mice: differential effects of corticosterone on antimycobacterial activity and expression of the Bcg gene (Candidate Nramp)
Authors: Brown D H , LaFuse W , Zwilling B S
Published: January 2020

Previous work in our laboratory has shown that corticosterone increases the susceptibility of macrophages from Bcgs mice to the growth of Mycobacterium avium. The innate antimycobacterial activity of macrophages from Bcgr mice was not affected by corticosterone. In contrast to the differential effect of corticosterone on the antimycobacterial activity of the macrophages, corticosterone suppressed the production of tumor necrosis factor alpha and nitric oxide by macrophages from both Bcgr and Bcgs mice. The purpose of this investigation was to compare the effects of corticosterone on the antimycobacterial activity of macrophages from Bcgr and Bcgs mice that have been activated in vitro with recombinant gamma interferon or granulocyte-macrophage colony-stimulating factor. We found that macrophages from both strains of congenic mice responded equally to the activation stimuli. The capacity of the activated macrophages from Bcgs mice to suppress the growth of M. avium was inhibited by the addition of corticosterone to the cultures. The addition of NG-monomethyl-L-arginine to the cultures did not affect the capacity of resident splenic macrophages from Bcgr mice to limit the growth of M. avium. However, NG-monomethyl-L-arginine reduced the capacity of gamma interferon-activated, but not granulocyte-macrophage colony-stimulating factor-activated, macrophages to limit the growth of M. avium by macrophages from both Bcgr and Bcgs mice. The addition of corticosterone suppressed Nramp expression by macrophages from Bcgs mice. Nramp expression by macrophages from Bcgr mice was not affected by corticosterone.
A conserved Aeromonas salmonicida porin provides protective immunity to rainbow trout
Authors: Lutwyche P , Exner M M , Hancock R E , Trust T J
Published: January 2020

A protein with an apparent M(r) of 28,000 was isolated from outer membrane preparations of Aeromonas salmonicida A440. The protein was tested for the ability to form pores, using a planar lipid bilayer model membrane system. The protein appeared to be a monomer with a single-channel conductance in 1.0 M KCl of 1.96 nS and a cation/anion permeability ratio of 2.91 +/- 0.68. These data show that the porin channel is comparable in size to OmpC and OmpF of Escherichia coli and is relatively nonselective, having some preference for cations over anions. The porin was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a polyclonal antibody was raised. Immunoblot analysis showed that an immunologically cross-reactive protein was present in other Aeromonas strains but not in strains of Vibrio or Yersinia. The N-terminal amino acid sequence of the porin was determined and was found to show some homology to an Aeromonas hydrophila outer membrane protein. This is the second porin species of A. salmonicida to be described, and it differs from the other in subunit molecular weight, aggregation properties, peptidoglycan association, pore size, and antigenicity. Rainbow trout (Oncorhynchus mykiss) immunized intraperitoneally with the purified porin protein were significantly protected from experimental A. salmonicida challenge. This is the first report of successful vaccination against A. salmonicida with a purified outer membrane component.
Exogenous tumor necrosis factor alpha and interleukin-1 alpha increase resistance to Salmonella typhimurium: efficacy is influenced by the Ity and Lps loci
Authors: Morrissey P J , Charrier K , Vogel S N
Published: January 2020

Interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF-alpha) administered prior to infection with Salmonella typhimurium increases survival in mice that are Ityr, not in susceptible Lpsd or Itys mice. Combined IL-1 alpha and TNF-alpha pretreatment results in greater survival than that seen with either cytokine alone in Ityr mice. Treatment after infection with TNF-alpha and/or IL-1 alpha increases the mean time to death but not the survival fraction of Lpsd mice and was ineffective in either Ityr or Itys mice.
Hemolytic activity of the Pasteurella haemolytica leukotoxin
Authors: Murphy G L , Whitworth L C , Clinkenbeard K D , Clinkenbeard P A
Published: January 2020

A Pasteurella haemolytica mutant incapable of producing leukotoxin was created by allelic replacement. Concentrated culture supernatants from wild-type P. haemolytica, but not from the mutant, contained the 102-kDa leukotoxin protein and lysed bovine lymphoma cells and sheep erythrocytes. Wild-type P. haemolytica demonstrated the typical beta-hemolytic phenotype on sheep and rabbit blood agar, whereas the mutant did not.
Localization of the promoter for the ptl genes of Bordetella pertussis, which encode proteins essential for secretion of pertussis toxin
Authors: Kotob S I , Hausman S Z , Burns D L
Published: January 2020

The ptl locus of Bordetella pertussis, which encodes proteins necessary for the secretion of pertussis toxin into the extracellular medium, is located directly downstream from the ptx locus, which encodes the structural subunits of the toxin. We have found that the ptx promoter is essential for expression of the ptl genes. A strain of B. pertussis which lacked only the ptx promotor region but which retained all other portions of the ptx-ptl region did not produce PtlF. Moreover, insertion of a functional ptx promoter from B. pertussis at the 5' end of the ptx region of Bordetella bronchiseptica resulted in the production of PtlF in B. bronchiseptica, a species which normally does not produce PtlF. These results suggest that the ptx operon is larger than originally proposed and contains both the ptx and ptl genes.
Functional properties of nonhuman primate antibody to Porphyromonas gingivalis
Authors: Anderson D M , Ebersole J L , Novak M J
Published: January 2020

The nonhuman primate (NHP) serves as a useful model for examining the host-parasite interactions in Porphyromonas gingivalis-associated periodontal disease. This study determined the influence of NHP sera on (i) the direct killing of P. gingivalis, (ii) P. gingivalis-induced superoxide anion (O2-) release from human polymorphonuclear leukocytes (PMNs), and (iii) the ability of PMNs to bind and phagocytize P. gingivalis. Three types of NHP sera were utilized: (i) normal or baseline sera; (ii) sera obtained after ligature-induced periodontitis; and (iii) sera obtained following active immunization with formalinized P. gingivalis. All assays were performed with or without the addition of human complement. Significantly more (P < 0.01) direct killing of P. gingivalis occurred with immunized sera and complement than with any of the other treatments. The sera from ligature-induced periodontitis NHPs had significantly less (P < 0.03) killing capacity than the baseline sera, which contained natural antibody produced to P. gingivalis colonization. Sera from immunized NHPs were used to opsonize P. gingivalis and caused significantly greater (P < 0.01) levels of O2- release from PMNs. Finally, the sera from immunized NHPs significantly enhanced (P < 0.009) the uptake of P. gingivalis by PMNs, although binding of the bacteria to PMNs was similar among all three serum types. Active immunization of NHPs with P. gingivalis elicited a functional antibody that enhanced direct killing, positively influenced the activation of PMNs, and enhanced the ability of PMNs to phagocytize P. gingivalis. Moreover, antibody produced as a sequela of progressing periodontitis appeared to lack these functions. A wide variability in functional capacity of the sera from individual NHPs, which may contribute to an individual's susceptibility to P. gingivalis-induced disease, was noted. This variability suggested that results from functional tests of serum antibody may aid in predicting host susceptibility to disease and response to therapy.
Characterization of an invasive phenotype associated with enteroaggregative Escherichia coli
Authors: Benjamin P , Federman M , Wanke C A
Published: January 2020

Enteroaggregative Escherichia coli (EAggEc) strains are associated with persistent diarrhea in children in the developing world and exhibit a classic aggregative phenotype. We have demonstrated that EAggEc strains isolated from children with persistent diarrhea in Brazil, Bangladesh, and Pakistan also have the potential to be internalized by HeLa cells in the gentamicin protection assay. We have confirmed this phenomenon with transmission electron micrographs of bacteria engulfed by HeLa cells. We examined the mechanisms by which this process occurs. Staurosporine inhibited internalization of EAggEc strain 162 by 50% at a concentration of 0.1 microM. Genistein inhibited internalization of this same organism by 50% at a concentration of 50 microM. Cytochalasin D inhibited internalization by 50% at a concentration of 1 microgram/ml. Staurosporine, genistein, and cytochalasin D inhibited the internalization of EAggEc strain 162 by HeLa cells in a dose-dependent manner. These data suggest that active cell processes such as signal transduction by protein kinase and/or tyrosine kinase may be involved in the internalization of EAggEc strain 162 by HeLa cells and that actin filaments and cytoskeletal structure may be important for this process.
Deposition of complement components on Streptococcus agalactiae in bovine milk in the absence of inflammation
Authors: Rainard P , Poutrel B
Published: January 2020

Bovine milk is generally considered to be almost devoid of complement, on the basis of undetectable hemolytic activity, unless inflammation recruits plasma components. This study examines the deposition of complement components on a mastitis-causing isolate of Streptococcus agalactiae by enzyme-linked immunosorbent assay (ELISA). Neat milk from mid-lactating, uninflamed mammary glands (normal milk) effected marked C3 deposition on bacteria. Kinetic studies showed a protracted lag period (30 to 45 min) preceding C3 deposition, which required about 2 h to reach a maximum. Experiments with diluted serum suggested that this slow C3 deposition resulted mainly from the low concentration of certain components of complement in milk. Bacteria incubated in neat milk readily bound bovine conglutinin, indicating the presence of iC3b. Elution of covalently bound C3 fragments with hydroxylamine confirmed the deposition of C3b and iC3b on bacteria. Deposition of C4 on bacteria was not detected in neat milk, suggesting that C3 deposition did not result from the activation of the classical pathway. This was not the result of a lack of antibodies. However, C4 deposition could be obtained by adding purified bovine C1q to normal milk, and C3 deposition was accelerated, suggesting the participation of the classical pathway. The deposition of C1q on antibody-sensitized bacteria was impeded by milk compared with that of C1q diluted in phosphate-buffered saline. Concentrations of C1q in normal milk were very low, ranging from 150 to 250 ng/ml. Overall, these findings indicate that C1q was a limiting factor of the classical pathway in normal milk. The capacity of milk to deposit C3 fragments on mastitis-causing S. agalactiae prompts further studies to investigate its role in opsonophagocytosis.
The role of the eaeA gene in diarrhea and neurological complications in a gnotobiotic piglet model of enterohemorrhagic Escherichia coli infection
Authors: Tzipori S , Gunzer F , Donnenberg M S , de Montigny L , Kaper J B , Donohue-Rolfe A
Published: January 2020

We reported previously that mutation of the chromosomal gene eaeA from enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 prevented bacterial attachment in vivo. Attachment was restored when the EHEC or enteropathogenic E. coli (EPEC) eaeA gene was introduced into the mutant on a plasmid. In this communication we have compared in gnotobiotic piglets the pathogenicities of wild-type O157:H7 strain 86-24 and its eaeA mutant UMD619 with those of the two plasmid-complemented strains expressing IntiminO157 (EHEC) and IntiminO127 (EPEC). 86-24 colonized the surface and glandular epithelium of the large intestine and induced diarrhea, while UMD619 did not colonize any intestinal site and induced little or no diarrhea. Surprisingly, strain UMD619 expressing IntiminO127 behaved in pigs more like EPEC than EHEC strains; it colonized the distal half of the small intestine and the surface of the large intestine, inducing serious diarrhea. In contrast, strain UMD619 expressing IntiminO157 colonized the colon extremely poorly, inducing little or no diarrhea. While only the two strains causing extensive attachment--86-24 and UMD619 expressing IntiminO127--induced diarrhea, neurological symptoms attributed to Shiga-like toxin II occurred equally in all four groups of animals. The intimate bacterial attachment and mucosal damage were not a prerequisite for Shiga-like toxin II translocation from the gut lumen into the circulation. IntiminO127 appears not only to facilitate intimate attachment to cells but also to influence the site of intestinal colonization and other characteristics of EPEC infection.