bluuebunny/crossref_metadata_2025 huggingface dataset:
Vitamin A deficiency predisposes to Staphylococcus aureus infection
Authors: Wiedermann U , Tarkowski A , Bremell T , Hanson L A , Kahu H , Dahlgren U I
Published: January 2020

We have investigated the consequences of vitamin A deficiency in a rat model of T-cell-dependent and superantigen-mediated Staphylococcus aureus arthritis. After intravenous inoculation of enterotoxin A-producing staphylococci, the vitamin-A-deficient rats showed a decreased weight gain compared with the paired fed controls despite equal food consumption. The control rats developed arthritis in the first few days after bacterial inoculation, with a peak frequency at day 5, and then gradually recovered; however, the frequency of arthritis 18 days after bacterial inoculation was 86% among the vitamin A-deficient rats and 44% among the control rats. During this period, 3 of 10 deficient rats and 1 of 10 control rats died. Further in vitro analysis revealed that T-cell responses to S. aureus were significantly higher in the vitamin A-deficient rats than in the control animals. In contrast, B-cell reactivity, measured as immunoglobulin levels, autoantibody levels, and specific antibacterial antibody levels in serum, did not differ between the groups. Interestingly, the innate host defense mechanisms against S. aureus were also profoundly affected by vitamin A deficiency. Thus, despite a larger number of circulating phagocytic cells in the vitamin-A-deficient group, the capacity to phagocytize and exert intracellular killing of S. aureus was significantly decreased in comparison with the control rats. Furthermore, serum from the vitamin A-deficient rats inoculated with Staphylococcus aureus displayed decreased complement lysis activity. Our results suggest that the increased susceptibility to S. aureus infection observed in the vitamin-A-deficient rats is due to a concerted action of antigen-specific T-cell hyperactivity, impaired function of the phagocytes, and decreased complement activity.
A recombinant baculovirus 42-kilodalton C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 protects Aotus monkeys against malaria
Authors: Chang S P , Case S E , Gosnell W L , Hashimoto A , Kramer K J , Tam L Q , Hashiro C Q , Nikaido C M , Gibson H L , Lee-Ng C T , Barr P J , Yokota B T , Hut G S
Published: January 2020

The immunogenicity and protective efficacy of baculovirus recombinant polypeptide based on the Plasmodium falciparum merozoite surface protein 1 (MSP-1) has been evaluated in Aotus lemurinus griseimembra monkeys. The MSP-1-based polypeptide, BVp42, corresponds to the 42-kDa C-terminal processing fragment of the precursor molecule. Immunization of Aotus monkeys with BVp42 in complete Freund's adjuvant resulted in high antibody titers against the immunogen as well as parasite MSP-1. Fine specificity studies indicated that major epitopes recognized by these antibodies localize to conserved determinants of the 19-kDa C-terminal fragment derived from cleavage of the 42-kDa processing fragment. Effective priming of MSP-1-specific T cells was also demonstrated in lymphocyte proliferation assays. All three Aotus monkeys immunized with BVp42 in complete Freund's adjuvant showed evidence of protection of protection against blood-stage challenge with P. falciparum. Two animals were completely protected, with only one parasite being detected in thick blood films on a single days after injection. The third animal had a modified course of infection, controlling its parasite infection to levels below detection by thick blood smears for an extended period in comparison with adjuvant control animals. All vaccinated, protected Aotus monkeys produced antibodies which inhibited in vitro parasite growth, indicating that this assay may be a useful correlate of protective immunity and that immunity induced by BVp42 immunization is mediated, at least in part, by a direct effect of antibodies against the MSP-1 C-terminal region. The high level of protection obtained in these studies supports further development of BVp42 as a candidate malaria vaccine.
Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome-lysosome interaction
Authors: Oh Y K , Straubinger R M
Published: January 2020

Mycobacterium avium is a facultative intracellular pathogen that can survive and replicate within macrophages. We tested the hypotheses that survival mechanisms may include alteration of phagosomal pH or inhibition of phagosome-lysosome fusion. M. avium was surface labeled with N-hydroxysuccinimidyl esters of carboxyfluorescein (CF) and rhodamine (Rho) to enable measurement of the pH of individual M. avium-containing phagosomes and the interactions of bacterium-containing phagosomes with labeled secondary lysosomes. CF fluorescence is pH sensitive, whereas Rho is pH insensitive; pH can be calculated from their fluorescence ratios. Surface labeling of M. avium did not affect viability in broth cultures or within J774, a murine macrophage-like cell line. By fluorescence spectroscopy, live M. avium was exposed to an environmental pH of approximately 5.7 at 6 h after phagocytosis, whereas similarly labeled Salmonella typhimurium, zymosan A, or heat-killed M. avium encountered an environmental pH of < 5.0. Video fluorescence and laser scanning confocal microscopy gave consistent pH results and demonstrated the heterogeneity of intracellular fate early in infection. pH became more homogeneous 6 h after infection. M. avium cells were coated with immunoglobulin G (IgG) or opsonized to investigate whether phagocytosis by the corresponding receptors would alter intracellular fate. Opsonized, unopsonized, and IgG-coated M. avium cells entered compartments of similar pH. Finally, the spatial distribution of intracellular bacteria and secondary lysosomes was compared. Only 18% of live fluorescent M. avium cells colocalized with fluorescent lysosomes, while 98% of heat-killed bacteria colocalized. Thus, both inhibition of phagosome-lysosome fusion and alteration of phagosomal pH may contribute to the intracellular survival of M. avium.
Antibody against the capsule of Vibrio cholerae O139 protects against experimental challenge
Authors: Sengupta D K , Boesman-Finkelstein M , Finkelstein R A
Published: January 2020

Antiserum to the capsular polysaccharide of an opaque variant of Vibrio cholerae O139 strain MDO-12 recognizes capsular antigen in three different colonial variants of the strain, although the amount of recognition varies with the extent of opacity. The anti-capsular-polysaccharide serum, at subagglutinating doses, protected suckling mice against challenge with both the most opaque variant and the most translucent variant. Further studies indicated that the protection was associated with inhibition of intestinal colonization by the vibrios. These results thus highlight the potential importance of the capsule in immunoprophylaxis against cholera caused by V. cholerae O139.
Protective immunity to Shiga-like toxin I following oral immunization with Shiga-like toxin I B-subunit-producing Vibrio cholerae CVD 103-HgR
Authors: Acheson D W , Levine M M , Kaper J B , Keusch G T
Published: January 2020

This study addresses a mechanism for inducing systemic immunity to Shiga-like toxins by oral administration of a Shiga-like toxin I B-subunit-expressing Vibrio cholerae vaccine strain [CVD 103-HgR(pDA60)]. Two sets of three rabbits were given either CVD 103-HgR or CVD 103-HgR(pDA60) orally. All rabbits immunized with CVD 103-HgR(pDA60) developed neutralizing serum antibodies to Shiga-like toxin I. None of the controls developed such antibodies.
Contribution of specific Pseudomonas aeruginosa virulence factors to pathogenesis of pneumonia in a neonatal mouse model of infection
Authors: Tang H B , DiMango E , Bryan R , Gambello M , Iglewski B H , Goldberg J B , Prince A
Published: January 2020

We sought to identify which Pseudomonas aeruginosa products are involved initiating respiratory tract infection. Defined mutants derived from strain PAO i.e., PAOR1 (lasR),PAO-pmm (algC) (an LPS mutant), and AK1152 (which is Fla- and lacks functional pili), were significantly less virulent than PAO1 in a BALBc/ByJ neonatal mouse model of infection as measured by their abilities to cause acute pneumonia, bacteremia, and death. All three mutants were also less adherent to epithelial cells in an in vitro binding assay. PAOR1 and AK1152 were less able to elicit epithelial production of interleukin-8 than PAO1. LasR was found to be required for the optimal expression of neuraminidase under conditions of increased osmolarity, as might be present in certain pathological conditions. PAO-exsA::omega,, which lacks exoenzyme S expression, was fully virulent, causing at least as much pathology as PAO1. The expression of several P. aeruginosa virulence factors appears to be required to establish pulmonary infection in the neonatal mouse.
Effect of nitric oxide on staphylococcal killing and interactive effect with superoxide
Authors: Kaplan S S , Lancaster J R , Basford R E , Simmons R L
Published: January 2020

The role of reactive nitrogen intermediates (RNI) such as nitric oxide (.NO) in host defense against pyogenic microorganisms is unclear, and the actual interactive effect of RNI and reactive oxidative intermediates (ROI) for microbial killing has not been determined. Since, in nature, ROI and RNI might be generated together within any local infection, we evaluated the separate and interactive effects of .NO and O2- on staphylococcal survival by using a simplified system devoid of eukaryotic cells. These studies showed that prolonged exposure of staphylococci to .NO does not result in early loss of viability but instead is associated with a dose-related delayed loss of viability. This effect is abrogated by the presence of hemoglobin, providing further evidence that the effect is RNI associated. Superoxide-mediated killing also is dose related, but in contrast to RNI-mediated killing, it is rapid and occurs within 2 h of exposure. We further show that the interaction of .NO and O(2)- results in decreased O(2)--mediated staphylococcal killing at early time points. .NO, however, appears to enhance or stabilize microbial killing over prolonged periods of incubation. This study did not produce evidence of early synergism of ROI and RNI, but it does suggest that .NO may contribute to host defense, especially when ROI-mediated killing is compromised.
Susceptibility to Pneumocystis carinii infection: host responses of neonatal mice from immune or naive mothers and of immune or naive adults
Authors: Garvy B A , Harmsen A G
Published: January 2020

Mice from either naive or immunized dams were given intranasal inoculations of Pneumocystis carinii as neonates (24 to 48 h old). Lung P. carinii burdens increased through day 13 postinoculation in all pups and declined to nearly undetectable numbers by day 23 in pups from immune mothers. However, P. carinii numbers in pups from naive mothers did not begin to decline significantly until after day 33, and P. carinii organisms were still detectable in low numbers through day 45. In contrast, the lungs of naive or immunized adult mice contained detectable numbers of P. carinii organisms only up to 9 or 3 days, respectively, after inoculation. The onset of clearance of P. carinii organisms from the lungs of neonatal mice and naive adults was coincident with infiltration of neutrophils and CD4+ CD45RBlo cells into the alveolar spaces and increased titers of P. carinii-specific antibody in sera. Immunized dams had high levels of P. carinii-specific antibody in both their sera and milk, and pups from these dams had higher titers of P. carinii-specific antibody than did pups from naive dams. These data indicate that P. carinii survives for a much longer period in neonates than in adult mice, which is the result of a delay in the onset of the immune response in neonates. Furthermore, immunized mothers contributed to an early clearance of P. carinii organisms by their offspring presumably because of the transfer of P. carinii-specific antibody. However, the passively acquired antibody did not seem to have an effect until the neonates began to mount their own responses.
Purification and characterization of novel hemagglutinins from Vibrio mimicus: a 39-kilodalton major outer membrane protein and lipopolysaccharide
Authors: Alam M , Miyoshi S I , Tomochika K I , Shinoda S
Published: January 2020

Two hemagglutinins (HAs) mediating the agglutinability to rabbit erythrocytes were isolated from 32-h culture supernatant of enterotoxigenic strain E-33 of Vibrio mimicus by ultrafiltration followed by gel filtration and anion-exchange column chromatography. The HAs were designated R-HA and C-HA on the basis of specific hemagglutinating activity towards rabbit erythrocytes only (R-HA) and towards chicken and rabbit erythrocytes (C-HA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent staining with Coomassie brilliant blue revealed no detectable protein band and a single band of Mr 39,000 in the case of R-HA and C-HA, respectively. However, silver staining of the gel containing R-HA revealed the appearance of low-molecular-weight material. These two HAs differed from each other and from previously reported HA/protease in receptor specificity, molecular composition, and biochemical and immunochemical properties. No simple sugar other than glycoproteins, including mucin, inhibited hemagglutinating activities of both C-HA and R-HA. Rabbit antibody against R-HA or C-HA could agglutinate E-33 whole cells, implying a possible cell surface origin of the two HAs. The isolated E-33 lipopolysaccharide (LPS) or its polysaccharide moiety conferred biochemical and immunochemical properties identical to those of R-HA, confirming that the R-HA represents polysaccharide of LPS. The LPS preparations from heterologous strains of Vibrio mimicus and Vibrio cholerae non-O1 confirmed that the hemagglutinating ability is a common function of LPS. On the other hand, the antibody against C-HA specifically recognized a major outer membrane protein (OMP) with an Mr of around 39,000 in both homologous and heterologous strains of V. mimicus, suggesting an OMP origin of C-HA. Furthermore, the antibody recognized a major OMP with an Mr of around 37,000 in V. cholerae. Although the immunogenicity of LPS and OMP is well documented for important intestinal pathogens, the hemagglutinating properties of such attractive cell surface components are hitherto unrecognized and will definitely contribute towards understanding their role in bacterial adherence.
Human immunoglobulin E responses to a recombinant 22.6-kilodalton antigen from Schistosoma mansoni adult worms are associated with low intensities of reinfection after treatment
Authors: Webster M , Fulford A J , Braun G , Ouma J H , Kariuki H C , Havercroft J C , Gachuhi K , Sturrock R F , Butterworth A E , Dunne D W
Published: January 2020

Schistosoma mansoni-infected individuals who have low intensities of reinfection following treatment produce immunoglobulin E (IgE) antibodies against a range of S. mansoni adult-worm antigens. One of the targets of the IgE response is an adult-worm sodium dodecyl sulfate-polyacrylamide gel electrophoresis band of 22 kDa (Sm22), which contains an antigen(s) located within the tegument and gut lining of adult worms and relatively late schistosomula life cycle stages only. A significant negative correlation between the level of anti-Sm22 IgE and the intensity of reinfection following treatment suggests that IgE responses against this antigen(s) are characteristic of individuals who are resistant to reinfection. To identify the antigen(s) in the Sm22 band that are associated with these IgE responses, we have cloned and characterized a recombinant 22-kDa protein (rSm22) that cross-reacts immunologically with Sm22. There was a high correlation between native and recombinant Sm22 isotype responses, indicating that the correct antigen had been cloned and that responses against rSm22 made up the majority of the responses against Sm22. By analyzing human isotype responses to rSm22 with human sera from a longitudinal treatment and reinfection study and correlating the anti-rSm22 isotype responses, retrospectively, with the intensity of reinfection following treatment for each individual, we observed a negative correlation between the IgE response to rSm22 and the intensity of reinfection. This relationship remained significant after allowing for age and other isotype responses to rSm22, in particular IgG4.
Expression of sialyltransferase is not required for interaction of Neisseria gonorrhoeae with human epithelial cells and human neutrophils
Authors: McGee D J , Chen G C , Rest R F
Published: January 2020

Sialyltransferase (Stase) in Neisseria gonorrhoeae organisms (gonococci [GC]) transfers sialic acid (N-acetylneuraminic acid [NANA]) from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) mainly to the terminal galactose (Gal) residue in the Gal beta-1,4 N-acetylglucosamine (Gal-GlcNAc)-R lipooligosaccharide (LOS) structure. Sialylated GC resist killing by normal human serum, sometimes show reduced invasion of epithelial cells, and have reduced adhesion to and stimulation of human neutrophils. We questioned whether Stase itself modulates the interactions of GC with human epithelial cells and neutrophils in the absence of exogenous CMP-NANA. To that end, we treated strain F62 with ethyl methanesulfonate and grew approximately 175,000 colonies on CMP-NANA plates, and screened them with monoclonal antibody 1B2-1B7 (MAb 1B2). MAb 1B2 is specific for Gal-GlcNAc and reacts only with asialylated GC. We isolated 13 MAb 1B2-reactive mutants, including five null mutants, that had Stase activities ranging from barely detectable to fivefold less than that of wild-type (WT) F62. The LOS phenotype of Stase null mutants was identical to that of WT F62, yet the mutants could not sialylate their LOS when grown with CMP-NANA. The Stase null phenotype was rescuable to Stase+ by transformation with chromosomal DNA from WT F62. Stase null mutants remained serum sensitive even when grown with CMP-NANA. One Stase null mutant, ST94A, adhered to and invaded the human cervical epithelial cell line ME-180 at levels indistinguishable from that of WT F62 in the absence of CMP-NANA. In human neutrophil studies, ST94A stimulated the oxidative burst in and adhered to human neutrophils at levels similar to those of WT F62. ST94A and WT F62 were also phagocytically killed by neutrophils at similar levels. These results indicate that expression of Stase activity is not required for interaction of GC with human cells.
Binding and internalization of the Helicobacter pylori vacuolating cytotoxin by epithelial cells
Authors: Garner J A , Cover T L
Published: January 2020

Many Helicobacter pylori strains produce a cytotoxin (VacA) that induces vacuolation in epithelial cells. In this study, binding and internalization of the cytotoxin by HeLa or AGS (human gastric adenocarcinoma) cells were characterized by indirect fluorescence microscopy. Cells incubated with the cytotoxin at 4 degrees C displayed a uniform fluorescent plasma membrane signal. Preincubation of the cytotoxin with either rabbit antiserum to approximately 90-kDa H. pylori VacA or sera from H. pylori-infected persons inhibited its binding to cells and blocked its capacity to induce cytoplasmic vacuolation. Recombinant VacA fragments (approximately 34 and approximately 58 kDa), corresponding to two proteolytic cleavage products of approximately 90-kDa VacA, each bound to the plasma membrane of HeLa cells. Antiserum reactive with the approximately 58-kDa VacA fragment inhibited the binding of native H. pylori cytotoxin to cells and inhibited cytotoxin activity, whereas antiserum to the approximately 34-kDa fragment had no effect. When incubated with cells at 37 degrees C for > or = 3 h, the H. pylori cytotoxin localized intracellularly in a perinuclear location but did not localize within cytotoxin-induced vacuoles. When cells with previously bound cytotoxin were incubated with anticytotoxin serum at 4 degrees C and then shifted to 37 degrees C, vacuolation was completely inhibited. Bound cytotoxin became inaccessible to the neutralizing effects of antiserum after 60 to 120 min of incubation with cells at 37 degrees C. These data suggest a model in which (i) VacA binds to cells primarily via amino acid sequences in its 58-kDa fragment, (ii) VacA internalization occurs slowly in a temperature-dependent process, and (iii) VacA interacts with an intracellular target.
Gastrointestinal colonization by methicillin-resistant Staphylococcus aureus in immunosuppressed mice
Authors: Kato-Matsunaga N , Okonogi K
Published: January 2020

ICR mice were inoculated intranasally with methicillin-resistant Staphylococcus aureus (MRSA) N133, and the inoculated MRSA was quantitatively recovered from the ceca and feces. The viable counts of the MRSA recovered from ceca correlated well with those from feces. Some mice eliminated MRSA from the cecum by 14 days after inoculation. Intraperitoneal administration of cyclophosphamide at a dose of 200 mg/kg 3 days before inoculation inhibited the elimination of the MRSA from both ceca and feces. All mice treated with cyclophosphamide excreted more than 10(4) CFU of the MRSA per g of feces for at least 70 days, indicating persistent colonization of the MRSA in the gastrointestinal tract. Some beta-lactam antibiotics decreased the colonization level, but others did not. The colonization level was suggested to depend on the antibacterial activity of the antibiotic against the MRSA and the degree of disturbance of intestinal flora by the antibiotic.
Effects of Clostridium difficile toxin A and toxin B on phospholipase D activation in human promyelocytic leukemic HL60 cells
Authors: Ohguchi K , Banno Y , Nakashima S , Kato N , Watanabe K , Lyerly D M , Nozawa Y
Published: January 2020

The possible involvement of Rho family GTP-binding proteins in the regulation of phospholipase D (PLD) activity has recently been demonstrated. In the present study, to further examine the role of Rho family proteins in PLD activation of human promyelocytic leukemic HL60 cells, we used toxin A and toxin B from the anaerobic bacterium Clostridium difficile, which was shown to glucosylate Rho family proteins and inhibit their interaction with effectors. Pretreatment of [3H]oleic acid-labeled HL60 cell lysates with either one of the toxins resulted in a remarkable inhibition of membrane PLD activity stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The magnitude of inhibition of PLD activity was correlated well with the extent of toxin A- or B-induced glucosylation of 22-kDa RhoA in HL60 cells, toxin B being more effective than toxin A. GTPgammaS-stimulated PLD activation measured with the exogenous substrate containing phosphatidylinositol 4,5-bisphosphate was also inhibited by toxin B. Toxin B had no effect on GTP-gammaS-induced translocation of RhoA from cytosol to membranes. Furthermore, the toxin B pretreatment also suppressed PLD activation induced by 4beta-phorbol 12-myristate 13-acetate in HL60 cell lysates. Thus, it was indicated that Rho family proteins play a key role in GTPgammaS- and 40-phorbol 12-myristate 13-acetate-induced PLD activity in HL60 cells. In addition, the results obtained here indicate that C. difficile toxins are a useful tool for researching the regulation of the Rho family protein-mediated PLD activation and also provide a clue toward understanding the pathogenic background of pseudomembranous colitis from the viewpoint of signal transduction.
Isolation, antimicrobial activities, and primary structures of hamster neutrophil defensins
Authors: Mak P , Wójcik K , Thogersen I B , Dubin A
Published: January 2020

Hamster (Mesocricetus auratus) neutrophil granules contain at least four microbicidal peptides belonging to the defensin family. These compounds were purified from granule acid extracts by reverse-phase chromatography and termed HaNP-1 to -4 (hamster neutrophil peptide). HaNP-1 and HaNP-3 revealed the most bactericidal activity, with a 50% inhibitory concentration of 0.3 to 0.8 microg/ml for Staphylococcus aureus and Streptococcus pyogenes strains. The HaNP-4 was always isolated in concentrations exceeding about 10 times the concentrations of other hamster peptides, but its antibacterial activity as well as that of HaNP-2 was relatively lower, probably as a result of conserved Arg residue substitutions. Other microorganisms were also tested, and generally, hamster defensins exhibited less potency against gram-negative bacteria. The amino acid sequence of hamster defensins showed a high percentage of identity to the sequence of mouse enteric defensins, reaching about 60% identical residues in the case of HaNP-3 and cryptdin 3.
Evidence for degradation of gastrointestinal mucin by Candida albicans secretory aspartyl proteinase
Authors: Colina A R , Aumont F , Deslauriers N , Belhumeur P , de Repentigny L
Published: January 2020

A zone of extracellular digestion of the mucin layer around Candida albicans blastoconidia was observed by transmission electron microscopy in the jejunum of mice inoculated intragastrically (G. T. Cole, K. R. Seshan, L. M. Pope, and R. J. Yancey, J. Med. Vet. Mycol. 26:173-185, 1988). This observation prompted the hypothesis that a putative mucinolytic enzyme(s) may contribute to the virulence of C. albicans by facilitating penetration of the mucus barrier and subsequent adherence to and invasion of epithelial cells. Mucinolytic activity was observed as zones of clearing around colonies of C. albicans LAM-1 grown on agarose containing yeast nitrogen base, glucose, and hog gastric mucin. In addition, concentrated culture filtrate obtained after growth for 24 h in yeast nitrogen base, supplemented with glucose and mucin as the sole nitrogen source, contained proteolytic activity against biotin-labelled mucin which was inhibited by pepstatin A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the culture filtrate revealed two components of 42 and 45 kDa, with pIs of 4.1 and 5.3, respectively. A zymogram showed that mucin was degraded only by the 42-kDa component, which was also recognized by immunoblotting with an anti-secretory aspartyl proteinase (anti-Sap) 2p monoclonal antibody. The N-terminal sequence of the first 20 amino acids matched that reported for Sap2p. These results demonstrate that Sap2p is responsible for proteolysis of mucin by C. albicans in vitro and may be involved as a virulence factor in the breakdown of mucus and penetration of the mucin barrier by C. albicans.
Immune responses and resistance to brucellosis in mice vaccinated orally with Brucella abortus RB51
Authors: Stevens M G , Olsen S C , Palmer M V , Pugh G W
Published: January 2020

Immune responses and resistance to infection with Brucella abortus 2308 (S2308) were measured in mice following oral or intraperitoneal (i.p.) vaccination with strain RB51 (SRB51). Bacteria persisted in the parotid lymph node for 4 weeks following oral vaccination of mice with 5 x 10(8) or 5 x 10(6) CFU of SRB51. Bacteria did not appear in the spleen during 12 weeks after oral vaccination, whereas they did appear in the spleen for 8 weeks following i.p. vaccination of mice with SRB51 (5 x 10(8) or 5 x 10(6) CFU). Increased resistance to S2308 infection occurred at 12 to 20 weeks in mice vaccinated i.p. with SRB51 (5 x 10(8) or 5 x 10(6) CFU) but occurred at 12 weeks only in mice vaccinated orally with SRB51 (5 x 10(8) CFU). Oral SRB51 vaccination induced lower levels of antibodies to the surface antigens of intact SRB51 bacteria than did i.p. vaccination. However, neither route of vaccination induced anamnestic antibody responses to the surface antigens of intact S2308 bacteria after challenge infection of the vaccinated mice with S2308. Mice vaccinated orally with SRB51 and challenged with S2308 at 12 to 20 weeks had lower and less persistent spleen cell proliferation and production of gamma interferon in response to S2308 and certain immunodominant S2308 proteins (32 to < or = 18 kDa) than did mice vaccinated i.p. with SRB51. However, mice vaccinated orally or i.p. with SRB51 and challenged with S2308 had similar spleen cell tumor necrosis factor alpha production. These results indicate that oral vaccination of mice with SRB51 was effective in inducing protective immunity to S2308 infection, although the immunity was lower and less persistent than that induced by i.p. vaccination. The lower protective immunity induced by oral vaccination may have resulted from lower and less persistent cell-mediated immunity and gamma interferon production in response to S2308 and S2308 proteins.
Safety, immunogenicity, and transmissibility in humans of CVD 1203, a live oral Shigella flexneri 2a vaccine candidate attenuated by deletions in aroA and virG
Authors: Kotloff K L , Noriega F , Losonsky G A , Sztein M B , Wasserman S S , Nataro J P , Levine M M
Published: January 2020

We evaluated the safety and immunogenicity of attenuated Shigella flexneri 2a vaccine candidate CVD 1203, which harbors precise deletions in the plasmid gene virG and in the chromosomal gene aroA. CVD 1203 invades epithelial cells but undergoes minimal intracellular proliferation and cell-to-cell spread. Fasting healthy volunteers, aged 18 to 40 years, were randomly allocated (double-blind design) to receive either CVD 1203 vaccine or placebo, along with sodium bicarbonate buffer, on days 0 and 14, as follows. At the time of the first inoculation, 10 subjects received placebo (group 1) and 22 subjects received either 1.5 x 10(8) (group 2; 11 subjects) or 1.5 x 10(9) (group 3; 11 subjects) CFU of CVD 1203. Fourteen days later, subjects from group 1 received 1.2 x 10(6) CFU of CVD 1203 and subjects from groups 2 and 3 received 1.2 x 10(8) vaccine organisms. Clinical tolerance was dose dependent. After a single dose of CVD 1203 at 10(6), 10(8), or 10(9) CFU, self-limited (<48-h duration) objective reactogenicity (fever, diarrhea, or dysentery) developed in 0, 18, and 72% of subjects, respectively, and in no placebo recipients. CVD 1203 induced immunoglobulin G seroconversion to S. flexneri 2a lipopolysaccharide (LPS) in 30, 45, and 36% of subjects from groups 1, 2, and 3, respectively, and stimulated immunoglobulin A-producing anti-LPS antibody-secreting cells in 60, 91, and 100% of subjects, respectively. After vaccination, significant rises in tumor necrosis factor alpha concentration in serum (groups 1, 2, and 3) and stool (group 2) samples were observed. We conclude that engineered deletions in virG and aroA markedly attenuate wild-type S. flexneri but preserve immunogenicity; however, less reactogenic vaccines are needed.
Experimental infection of newly weaned pigs with human and porcine strains of Serpulina pilosicoli
Authors: Trott D J , Huxtable C R , Hampson D J
Published: January 2020

Cultures of Serpulina pilosicoli 95/1000, isolated from a pig with porcine intestinal spirochetosis (PIS), and S. pilosicoli WesB, isolated from an Aboriginal child with diarrhea, were used to infect 5-week-old newly weaned pigs. Four of 12 pigs infected with strain 95/1000 and 2 of 12 pigs infected with strain WesB became colonized and developed watery, mucoid diarrhea within 2 to 11 days postinfection. Affected pigs all had moderate subacute mucosal colitis, with gross and histological changes similar to those previously reported in both natural and experimentally induced cases of PIS. Silver-stained histological sections of the colon and cecum from affected pigs demonstrated spirochetes within dilated intestinal crypts, where they were associated with neutrophilic exocytosis and mucus secretion. Sections from one pig infected with strain 95/1000 showed large numbers of spirochetes attached by one end to the colonic epithelium, a feature consistent with PIS. This study confirms the role of S. pilosicoli in the etiology of PIS and provides evidence that S. pilosicoli strains of human origin have pathogenic potential in an animal model.
Regulation of schistosome egg granuloma formation: host-soluble L-selectin enters tissue-trapped eggs and binds to carbohydrate antigens on surface membranes of miracidia
Authors: El Ridi R , Velupillai P , Harn D A
Published: January 2020

Immunogenic carbohydrate epitopes are prominent in soluble egg antigens (SEA) of Schistosoma mansoni and in vivo are released through ultramicroscopic pores in the eggshell. Previously, one immunogenic carbohydrate was identified as lacto-N-fucopentaose III (LNFPIII), which contains the biologically important trisaccharide Lewis(x) (Le(x)), a weak ligand for E-, L-, and P-selectins. Selectins are involved in various inflammatory reactions, including recruitment of granulocytes. Further, L-selectin molecules are shed from leukocyte cell surfaces upon activation. These independent observations suggest that selectins may play various roles in granuloma formation and/or regulation. We tested in situ for alterations in expression of host E-, L-, or P-selectin in murine liver tissue at various times postinfection with schistosome cercariae. We found that L-selectin was expressed on cells surrounding egg granulomas, but surprisingly we also found mouse L-selectin on the surface membranes of larval miracidia within the S. mansoni ova. In contrast, neither E- nor P-selectin was found within ova. Antibodies to human or rat L-selectin or 15 other distinct mouse leukocyte surface molecules did not bind to the miracidial surface. The anti-mouse L-selectin staining of miracidia could be inhibited by wash buffer containing sulfated carbohydrates such as sulfated Le(x), heparan sulfate, fucoidan, and carrageenan. The elution studies imply that the miracidial surface and, therefore, schistosomes express sulfated glycans. The binding of soluble L-selectin to miracidia was not restricted by the genetic background of the host, as mouse L-selectin was detected on the surface of S. mansoni miracidia in livers from BALB/C, CBA/J, C57BL/6, and outbred Swiss mice. We also detected soluble mouse L-selectin binding to Schistosoma japonicum miracidia, indicating that these observations can be generalized to all schistosome infections. On the basis of these observations we hypothesize that host-soluble L-selectin traverses through pores in the eggshell and binds to target ligands on the surface membranes of miracidia; this complex formation might ultimately impede the release of soluble antigens from the eggs. The intraovum binding of mouse L-selectin to immunogenic carbohydrate antigens is a novel role for selectins, and this model may, in part, explain the down-regulation in granulomatous pathology observed following the acute phase of the disease.
Balance of inflammatory cytokines related to severity and mortality of murine sepsis
Authors: Walley K R , Lukacs N W , Standiford T J , Strieter R M , Kunkel S L
Published: January 2020

We tested the hypothesis that, during sepsis, the balance of pro- and anti-inflammatory cytokines is related to severity and survival. Cecal ligation and puncture (CLP) with a large (18-gauge)-, intermediate (21-gauge)-, or small (26-gauge)-diameter needle, or sham laparotomy, was performed on outbred CD-1 mice. Concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 were measured (by enzyme-linked immunosorbent assay) in serum, peritoneal lavage fluid, and liver and lung samples at 4, 8, 24, 48, and 96 h. As the diameter of the CLP needle decreased, the mortality rate decreased (at 48 h: large, 80%; intermediate, 40%; small, 20%; P < 0.05), the TNF-alpha and IL-6 concentrations decreased, and the time-to-peak TNF-alpha expression increased. In contrast, IL-10 concentration increased compared with baseline (serum at 24 h: large, 2.3-fold +/- 1.6-fold; intermediate, 2.0-fold +/- 0.5-fold; small, 49.9-fold +/- 8.3-fold; P < 0.05). Administration of IL-10 (5 microg, intraperitoneal) prior to CLP decreased mortality (P < 0.001). Administration of polyclonal anti-IL-10 serum prior to CLP (0.5 ml intraperitoneal) had the opposite effect and increased mortality (P < 0.001) and TNF-alpha, IL-6, and TNF-alpha mRNA expression compared with controls. Thus, severe sepsis is associated with a largely unopposed inflammatory response, and a largely unopposed inflammatory response (with anti-IL-10) results in severe sepsis and death. Less severe sepsis is associated with greater anti-inflammatory mediator expression, and greater anti-inflammatory mediator expression (with IL-10) results in less severe sepsis. Thus, the balance of inflammatory mediators is related to the severity and mortality of murine sepsis.
The hemagglutinin genes hagB and hagC of Porphyromonas gingivalis are transcribed in vivo as shown by use of a new expression vector
Authors: Lee S W , Hillman J D , Progulske-Fox A
Published: January 2020

The hemagglutinin genes hagB and hagC of Porphyromonas gingivalis, a putative periodontopathic microorganism, have been cloned, sequenced, and characterized. However, the roles of these putative virulence genes have not yet been determined. In this study, an in vivo expression technology vector termed pPGIVET was constructed and used to determine if hagB and hagC were expressed during an infectious process. We constructed pPGIVET as a conjugative suicide plasmid containing a multiple-cloning site (MCS) upstream of two tandem promoterless reporter genes that encode tetracycline resistance [tetA(Q)2] and galactokinase (galK). The promoter and a portion of the open reading frame (ORF) of hagB were inserted into the MCS in both a positive and a negative orientation relative to the reporter genes. These constructs were conjugated into P. gingivalis 381. Southern blot analysis of different transconjugants indicated that Campbell insertions had occurred at the chromosomal hagB locus and also at the hagC locus, which has high (99%) homology to the ORF of hagB. pPGIVET-labeled clones in which the hag promoters were positively oriented relative to the reporter genes expressed tetracycline resistance and galactokinase activity in vitro and in vivo at significantly higher levels than did the wild-type strain or clones in which the hag promoters were negatively oriented. Expression of tetracycline resistance allowed substantial enrichment of heterodiploids over wild-type cells during a mixed infection in the mouse abscess model. These results indicate that hagB and hagC are transcriptionally active in vivo and suggested that pPGIVET may be used to isolate P. gingivalis genes expressed only during an infectious process.
Murine intranasal challenge model for the study of Campylobacter pathogenesis and immunity
Authors: Baqar S , Bourgeois A L , Applebee L A , Mourad A S , Kleinosky M T , Mohran Z , Murphy J R
Published: January 2020

Campylobacter jejuni infection of mice initiated by intranasal administration was investigated as a potential model for studies of pathogenesis and immunity. By using a standard challenge (5 x 10(9) CFU), C. jejuni 81-176 was more virulent for BALB/c (72% mortality) than for C3H/Hej (50%), CBA/CAJ (30%), or C58/J (0%). Intranasal challenge of BALB/c was used to compare the relative virulence of three reference strains; C.jejuni 81-176 was more virulent (killing 83% of challenged mice) than C. jejuni HC (0%) or C. coli VC-167 (0%). The course of intranasally initiated C. jejuni 81-176 infection in BALB/c was determined. C. jejuni was recovered from the lungs, intestinal tract, liver, and spleen at 4 h after challenge, the first interval evaluated. After this initial interval, three distinct patterns of infection were recognized: (i) a progressive decline in number of C. jejuni CFU (stomach, blood, lungs), (ii) decline followed by a second peak in the number of organisms recovered at 2 or 3 days postchallenge (intestine, liver, mesenteric lymph nodes), and (iii) persistence of approximately the same number of C.jejuni CFU during the course of the experiment (spleen). Intranasally induced infection initiated with a sublethal number of bacteria or intranasal immunization with killed Campylobacter preparations resulted in both the generation of Campylobacter antigen-specific immune responses and an acquired resistance to homologous rechallenge. The model was used to evaluate the relative virulence of nine low-in vitro-passage (no more than five passages) isolates of C. jejuni species from patients with diarrhea. The patient isolates were differentially virulent for mice; one killed all exposed mice, three were avirulent (no deaths) and the remainder showed an intermediate virulence, killing 17 to 33%. Mouse virulence of Campylobacter strains showed a trend toward isolates originating from individuals with watery diarrhea; however, no association was found between mouse virulence and other signs or symptoms. There were no observed relationships between mouse virulence and bacterial Lior serotype or Fla polymorphic group. Intranasal challenge of BALB/c with C. jejuni is a useful model for the study of infection and vaccination-acquired immunity to this agent.
Burkholderia pseudomallei activates complement and is ingested but not killed by polymorphonuclear leukocytes
Authors: Egan A M , Gordon D L
Published: January 2020

The mechanism by which Burkholderia pseudomallei is resistant to lysis by human serum is unknown but may include interference with complement activation, effective opsonization, or complement-mediated lysis. We investigated the interaction of B. pseudomallei with complement in the presence and absence of specific antibody to determine potential mechanisms of serum resistance. We demonstrated rapid activation and consumption of complement by B. pseudomallei which, in the absence of specific antibody, occurred predominantly via the alternative pathway. Complement activation was associated with deposition of the opsonically active C3b and iC3b fragments on the bacterial surface. C5b-9, detected on the bacterial surface after opsonic periods of 1 to 60 min, was susceptible to elution by 1 M NaCl, indicating that resistance to complement-mediated lysis may result from deposition of the membrane attack complex in a nonmicrobicidal location. To define the role of opsonins, we investigated the ability of polymorphonuclear leukocytes (PMNL) to phagocytose B. pseudomallei. Phagocytosis of bacteria by PMNL, and the observed oxidative response, was significantly increased by opsonization of organisms with complement and/or specific antibody. Despite opsonophagocytosis by PMNL and the production of an oxidative response, no significant bacterial killing was observed.
The gene slyA of Salmonella typhimurium is required for destruction of M cells and intracellular survival but not for invasion or colonization of the murine small intestine
Authors: Daniels J J , Autenrieth I B , Ludwig A , Goebel W
Published: January 2020

Recent studies have shown that Salmonella typhimurium invades the M cells of Peyer's patches (PP) of the murine ileum. The slyA gene of S. typhimurium has also recently been reported to affect virulence of this pathogen in mice and survival in macrophages. We therefore compared the effect on PP tissue of four strains of S. typhimurium: a wild-type strain, two slyA insertion mutants, and a recombinant S. typhimurium derivative carrying multiple copies of slyA. Invasion assays performed 2 and 7 days after orogastric infection revealed significantly lower numbers of bacteria of the slyA mutants and of the SlyA-overproducing strain in PP than of the wild type. However, similar numbers of bacteria of all strains were still present in the lumen of the small intestine after these times. Invasion assays of PP tissue after 90-min ileal loop infection yielded comparable numbers of bacteria of all strains in PP. Transmission and scanning electron microscopy of PP tissue after ileal loop infection demonstrated that the two slyA mutants and the SlyA-overproducing strain were able to attach to, induce membrane ruffling of, and invade M cells in a way morphologically and quantitatively similar to that of the wild type. In contrast to the wild type, both slyA mutants and, to a lesser extent, the SlyA-overproducing strain were significantly impaired in their ability to destroy M cells and adjacent enterocytes. Taken together, these data suggest that slyA is involved in intracellular survival and M-cell cytotoxicity but not in the invasion process and that the amount of SlyA needs to be precisely balanced for virulence.
Sera from patients with falciparum malaria induce substance P gene expression in cultured human brain microvascular endothelial cells
Authors: Chiwakata C B , Hort G , Hemmer C J , Dietrich M
Published: January 2020

Substance P is a pluripotent neuropeptide capable of inducing neurogenic inflammation, immunoregulation, and vasodilatation. In an effort to contribute to the understanding of the pathophysiology of cerebral malaria, we have evaluated the effects of sera obtained from patients suffering from severe or mild malaria and from a healthy donor with no previous history of exposure to malaria on the expression of the substance P gene by cultured human brain microvascular endothelial cells (HBMEC) and human umbilical-vein endothelial cells. PCR, Southern blotting, hybridization with an internal probe, and densitometry demonstrated that treatment of HBMEC with sera from patients with severe malaria caused remarkably increased expression of the substance P gene. In HBMEC, substance P was not significantly influenced by serum from a healthy donor. Substance P was expressed at almost undetectable levels in untreated HBMEC. Treatment of cultured human umbilical-vein endothelial cells with the same sera produced no signal. The influence of different sera on the expression of substance P by HBMEC suggests that substance P expression may be involved in events leading to the development of severe malaria.
Changes in the carboxyl-terminal repeat region affect extracellular activity and glucan products of Streptococcus gordonii glucosyltransferase
Authors: Vickerman M M , Sulavik M C , Minick P E , Clewell D B
Published: January 2020

Glucans produced by the glucosyltransferase (GTF) of Streptococcus gordonii confer a hard, cohesive phenotype (Spp+) on colonies grown on sucrose agar plates. S. gordonii strains with specific mutations in the region of gtfG that encodes the GTF carboxyl terminus were characterized. In the parental strain Challis CH1, this region included a series of six direct repeats thought to function in glucan binding. The spontaneous mutant strain CH107 had a 585-bp deletion resulting in the loss of three internal direct repeats. Insertional mutagenesis was used to construct strain CH2RPE, which had the parental repeat region but was missing 14 carboxyl-terminal amino acids. The similarly constructed strain CH4RPE had an in-frame addition of 390 nucleotides encoding two additional direct repeats. Although strains CH1, CH2RPE, and CH4RPE all had similar levels of extracellular GTF activity, strain CH107 had less than 15% of the parental activity; however, Western blots (immunoblots) indicated that the amounts of extracellular GTF protein in all four strains were similar. 13C NMR analyses indicated that partially purified GTFs from the Spp+ strains CH1, CH2RPE, and CH4RPE all produced glucans with similar ratios of alpha1,6 and alpha1,3 glucosidic linkages, whereas the Spp- strain CH107 GTF produced primarily alpha1,6-linked glucans. Transformation of strain CH107 with pAMS57, which carries the gtfG positive regulatory determinant, rgg, increased the amount of GTF activity and GTF antibody-reactive protein ca. fivefold but did not confer a hard colony phenotype on sucrose agar plates, suggesting that the type of glucan product affects the sucrose-promoted colony phenotype.