bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of alternative buffers for use with a new live oral cholera vaccine, Peru-15, in outpatient volunteers
Authors: Sack D A , Shimko J , Sack R B , Gomes J G , MacLeod K , O'Sullivan D , Spriggs D
Published: January 2020

During development of Peru-15, a new live oral vaccine for cholera, the role of buffer needed to be evaluated. Generally, oral bacterial vaccines are acid labile and need to be administered by using a formulation which protects them from gastric acid. We compared three different buffers for use with Peru-15, including a standard bicarbonate-ascorbic acid buffer, Alka-Seltzer, and a new electrolyte-rice buffer, CeraVacx. Saline served as the control. Thirty-nine healthy adult volunteers received Peru-15 (10(8) CFU) with one of the three buffers or saline in a double-masked study. The volunteers were monitored for symptoms for 7 days after the dose, serum was tested for antibody responses by vibriocidal antibody and immunoglobulin G antitoxin enzyme-linked immunosorbent assays, and stool samples were tested for excretion of the vaccine strain. Side effects were minimal in all groups. All 30 volunteers who took Peru-15 with a buffer showed significant rises in vibriocidal antibody titer. The magnitude of the rises was higher in the CeraVacx group than in the other two buffer groups. Four of nine volunteers who took the vaccine with saline also showed increased titers, but they were lower than those in any of the three buffer groups. Excretion of the vaccine strain was similar in the buffer groups, but excretion was not associated with the magnitude of the vibriocidal responses. Excretion of Peru-15 was not detected in the saline group. We conclude that buffer does amplify the serological response to Peru-15 and that CeraVacx may provide benefits not provided by other buffers.
Vaccine- and antigen-dependent type 1 and type 2 cytokine induction after primary vaccination of infants with whole-cell or acellular pertussis vaccines
Authors: Ausiello C M , Urbani F , la Sala A , Lande R , Cassone A
Published: January 2020

Cytokine profiles were examined 1 month after primary vaccination of infants with a whole-cell pertussis vaccine (wP) (Connaught) or either of two acellular pertussis vaccines, aP-Chiron Biocine (aP-CB) or aP-SmithKline Beecham (aP-SB), each combined with diphtheria-tetanus toxoids (DT), in Bordetella pertussis antigen-stimulated or unstimulated peripheral blood mononuclear cells (PBMC). Pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (PRN) were used as antigens, and the children were defined as responsive when their PBMC proliferated in response to these antigens. The controls were either children who received only DT or children who received pertussis vaccine but whose PBMC did not proliferate upon stimulation with B. pertussis antigens (unresponsive children). Antigen-stimulated PBMC of responsive wP recipients were characterized by an elevated production of T-helper-cell type 1 cytokines gamma interferon (IFN-gamma) and interleukin 2 (IL-2), low to minimal production of IL-5, and no production of IL-4. The PBMC of aP vaccine-responsive recipients showed, in addition to the elevated IFN-gamma production, a consistent, antigen-dependent production of type 2 cytokines (IL-4 and IL-5), with PRN being the most and PT being the least effective antigen. Type 2 cytokine induction was more pronounced in aP-SB than in aP-CB recipients, as shown by the presence of IL-4 mRNA transcripts and higher IL-5 production in the former (161.6 +/- 36 and 47.9 +/- 44 pg/ml [mean +/- standard error for five subjects each], respectively, after PRN stimulation). Appreciable, antigen-unstimulated (constitutive) IFN-gamma production was also detected in PBMC cultures of all vaccinees. However, this spontaneous IFN-gamma production was, in most vaccinees, significantly lower than the antigen-driven cytokine production. In contrast, no constitutive type 2 cytokine production was ever observed in any vaccine group. PBMC from the two control groups (either DT or pertussis vaccine recipients) did not show any type 2 cytokine production, while IFN-gamma production was comparable in both antigen-stimulated and unstimulated conditions. Absence of type 2 cytokines and low levels of constitutive IFN-gamma production were also seen in prevaccination children. Thus, pertussis vaccines induce in infants a basically type 1 cytokine profile, which is, however, accompanied by some production of type 2 cytokines. The latter are more expressed by aP-SB than by aP-CB recipients, and with PRN than with other antigens, and they are minimally expressed in wP recipients and with PT as antigen. Our data also highlight a constitutive IFN-gamma production in infancy, which might reflect natural immunization and/or the burden of concomitant vaccinations and which may have an impact on T-helper-cell cytokine pattern polarization consequent to pertussis vaccination.
Evidence of genetic susceptibility to Chlamydia trachomatis-induced pelvic inflammatory disease in the pig-tailed macaque
Authors: Lichtenwalner A B , Patton D L , Cosgrove Sweeney Y T , Gaur L K , Stamm W E
Published: January 2020

The macaque model of chlamydial pelvic inflammatory disease (PID) demonstrates individual variability in the time of onset of intrapelvic adhesions. Some animals develop adhesions rapidly, within 2 weeks after a single tubal inoculation with Chlamydia trachomatis, while in others, adhesions are not observed until 2 weeks after a second tubal inoculation. To test whether this variability correlates with major histocompatibility complex (MHC) class I haplotype, we used macaque alloantisera and mouse anti-HLA monoclonal antibodies to determine the MHC class I haplotypes of 44 C. trachomatis-infected macaques (Macaca nemestrina). Macaques developing gross tubal adhesions after the first chlamydial inoculation were classified as susceptible (n = 29), while those not developing adhesions until after the second chlamydial inoculation were classified as relatively resistant (n = 15), to adhesion formation. Three antibody specificities correlated with susceptibility (odds ratio [OR] 5.2, P < 0.01; OR 6.1 and 4.3, P < 0.05), and two correlated with relative resistance to adhesions (OR 0.1, P < 0.05; OR 0.2, P < 0.01). Because several of these antibodies are cross-reactive, as many as five different MHC class I alleles (three increasing and two decreasing ORs) or as few as two different MHC class I alleles (one increasing and one decreasing OR) could be correlated with risk of adhesion formation. We conclude that in macaques, susceptibility or relative resistance to rapid formation of tubal adhesions is correlated with expression of MHC class I alleles, consistent with reports of MHC class I restriction of chlamydial immunopathology in humans.
Saturable CD14-dependent binding of fluorescein-labeled lipopolysaccharide to human monocytes
Authors: Troelstra A , Antal-Szalmas P , de Graaf-Miltenburg L A , Weersink A J , Verhoef J , Van Kessel K P , Van Strijp J A
Published: January 2020

We used rough lipopolysaccharide (ReLPS) to construct a fluorescein-labeled LPS (FITC-LPS) with a very high labeling efficiency that bound to isolated human monocytes in a CD14-dependent fashion and that in this respect behaved indistinctively from native LPS. The CD14-dependent binding could be inhibited either by a 1,000-fold excess of unlabeled LPS or by polymyxin B, bactericidal/permeability-increasing protein, cationic protein 18, or soluble CD14. Although this FITC-LPS preparation no longer possessed the ability to prime neutrophils for the production of reactive oxygen species or to stimulate human monocytes to produce tumor necrosis factor, activation of the Limulus amoebocyte lysate cascade was comparable to activation by native LPS. Binding to monocytes was enhanced by human pooled serum (HPS) or LPS-binding protein (LBP) for LPS concentrations up to 100 ng/ml and was completely CD14 dependent. For LPS concentrations exceeding 100 ng/ml, binding was still partially CD14 dependent, but not HPS or LBP dependent. CD14-dependent association of LPS with monocytes was shown to be totally saturable. In conclusion, we found an HPS- or LBP-dependent binding of FITC-LPS to monocytes that was CD14 dependent at up to 100 ng of LPS per ml, and saturation of binding was shown.
The decrease in nonsplenic interleukin-6 (IL-6) production after splenectomy indicates the existence of a positive feedback loop of IL-6 production during endotoxemia in dogs
Authors: Moeniralam H S , Bemelman W A , Endert E , Koopmans R , Sauerwein H P , Romijn J A
Published: January 2020

The spleen is involved in endotoxin-induced interleukin-6 (IL-6) production. To quantitate the relative contribution of the spleen to endotoxin-induced IL-6 production, we studied the effect of endotoxin (1.0 microg/kg of body weight) in control dogs (n = 7) and splenectomized dogs (n = 7). Blood for analysis of tumor necrosis factor (TNF) and IL-6 was sampled from the femoral artery and the portal, hepatic, and splenic (only in controls) veins. Arterial plasma endotoxin and cortisol levels were also measured. Whole-body IL-6 production was calculated by a deconvolution technique. Splenic IL-6 production in control dogs was measured from splenic blood flow and arteriovenous concentration differences. Endotoxin levels were higher in splenectomized dogs (P < 0.05) because of a decreased distribution volume (P < 0.05) and decreased clearance of endotoxin (P < 0.05). Endotoxin-induced plasma IL-6 levels were decreased by approximately 75% in splenectomized dogs (P < 0.01), and whole-body IL-6 production rates were severalfold lower (median of 8.7 mg/4 h and range of 3.9 to 11.4 mg/4 h versus a median of 32.3 mg/4 h and a range of 22.7 to 70.2 mg/4 h) (P < 0.05). However, in control dogs splenic IL-6 production (0.6 +/- 0.2 mg/4 h) was only approximately 2% of whole-body IL-6 production. Plasma TNF levels increased in both groups (P < 0.01) but were not different between the groups. Plasma cortisol levels were slightly higher in splenectomized dogs than in control dogs (P < 0.05). In conclusion, splenectomy decreases the distribution volume and clearance rate of endotoxin. Splenectomy results in decreased endotoxin-induced IL-6 production, which is caused not by the absence of splenic IL-6 production, but by a decrease in nonsplenic IL-6 production. Therefore, the spleen is an important mediator in the complete activation of nonsplenic IL-6 production by endotoxin.
Role of alphabeta and gammadelta T cells in the host response to Salmonella infection as demonstrated in T-cell-receptor-deficient mice of defined Ity genotypes
Authors: Weintraub B C , Eckmann L , Okamoto S , Hense M , Hedrick S M , Fierer J
Published: January 2020

Salmonella spp. are facultative intracellular bacteria which enter the body through the intestinal tract. We studied the roles of T cells expressing either the alpha and beta chains or the gamma and delta chains of the T-cell receptor (alphabeta T cells or gammadelta T cells, respectively) in the host defense against Salmonella using mice genetically deficient in either alphabeta T cells, gammadelta T cells, or both T-cell subsets. These mutant strains of mice were infected orally or intraperitoneally with Salmonella dublin, and the progression of the disease was monitored by determining bacterial numbers in the feces, gut wall, Peyer's patches, mesenteric lymph nodes, spleen, and liver. Since susceptibility to Salmonella infection in mice is strongly affected by the alleles at the Ity locus, T-cell-mutant mice with either the Ity-sensitive or Ity-resistant phenotype were tested for resistance to S. dublin infection. We found that even though large numbers of intraepithelial and mucosal alphabeta and gammadelta T cells populate the normal intestine, they have no role in controlling the invasion of S. dublin into the intestine or the subsequent bacterial replication in the Peyer's patches or gut wall. Furthermore, systemic infections were equally severe for the first 6 days in normal, alphabeta T-cell-deficient, and gammadelta T-cell-deficient mice, and alphabeta but not gammadelta T cells were required for clearance of S. dublin, regardless of the Ity phenotype. However, mice that lacked both T-cell subsets had higher bacterial counts in their livers 15 to 18 days after infection than did alphabeta T-cell-deficient mice, suggesting that gammadelta T cells can contribute to acquired immunity to S. dublin.
Characterization of the canine type C enterotoxin produced by Staphylococcus intermedius pyoderma isolates
Authors: Edwards V M , Deringer J R , Callantine S D , Deobald C F , Berger P H , Kapur V , Stauffacher C V , Bohach G A
Published: January 2020

The type C staphylococcal enterotoxins (SECs) are a group of highly conserved proteins with substantial antigenic cross-reactivity. Although Staphylococcus intermedius and coagulase-positive species of staphylococci are reported to produce SEC and other SEs, toxins produced by species other than Staphylococcus aureus have not been previously characterized. In this study we report the molecular, biological, and immunological properties of the canine SEC (SECcanine) expressed by pathogenic isolates of S. intermedius. The mature form of SECcanine has 239 amino acid residues and a pI of 7.0. Typical of the SEs, purified SECcanine induces an emetic response in monkeys and the proliferation of T cells in a Vbeta-dependent manner. Although SECcanine has >95% sequence identity to previously described SEC variants, its sequence is most related to SEC2 and SEC3. In contrast to the sequence similarity, the Vbeta profile induced by SECcanine is typical of that induced by SEC1. This result is likely explained by the conservation of a cysteine residue at position 26 in SECcanine; residues at this position have been previously shown to determine subtype-dependent differences in T-cell receptor interactions of other SEs. Overall, these results show that superantigen toxins produced by the multiple members of the genus Staphylococcus are highly conserved in respect to biological and structural properties. Further, the frequent association of SECcanine with pyoderma in dogs supports the notion that the toxins are important for staphylococcal survival and pathogenesis.
Broad-spectrum antimicrobial activity of human intestinal defensin 5
Authors: Porter E M , van Dam E , Valore E V , Ganz T
Published: January 2020

Defensins are antibiotic peptides expressed in human and animal myeloid and epithelial cells. Due to the limited availability of natural peptides, the properties of human epithelial defensins have not been studied. We assayed the microbicidal activity of recombinant human intestinal defensin 5 (rHD-5) in the presence of salt (O to 150 mM NaCl) with varied pH (pH 5.5 to pH 8.5) and trypsin (25 and 250 microg/ml). rHD-5 exhibits microbicidal activity against Listeria monocytogenes, Escherichia coli, and Candida albicans. In contrast to cryptdins, the mouse intestinal defensins, rHD-5 is active against both mouse-virulent wild-type Salmonella typhimurium and its isogenic, mouse-avirulent phoP mutant. In the presence of salt, rHD-5 activity was reduced, and at 100 mM NaCl, activity against S. typhimurium was abolished. However, at all salt concentrations tested, rHD-5 remained bactericidal to L. monocytogenes. Activity against L. monocytogenes was not pH dependent but was diminished at pH 5.5 against wild-type S. typhimurium. This acid-induced resistance may have been mediated by the virulence gene regulator phoP, since the phoP mutant was equally sensitive at pH 5.5 and pH 7.4. In the presence of trypsin, rHD-5 was partially cleaved, but even then, rHD-5 at 100 microg/ml decreased the number of CFU of wild-type S. typhimurium by more than 99%. The persistence of microbicidal activity of rHD-5 under these conditions supports the notion that naturally occurring human intestinal defensin is an effective arm of mucosal host defense.
Interleukin-6 production by human monocytes stimulated with Cryptococcus neoformans components
Authors: Delfino D , Cianci L , Lupis E , Celeste A , Petrelli M L , Curró F , Cusumano V , Teti G
Published: January 2020

In order to ascertain if Cryptococcus neoformans components can induce interleukin-6 (IL-6) production, we stimulated human whole blood with purified capsular products. Their potencies in stimulating IL-6 release were mannoproteins > galactoxylomannan = glucuronoxylomannan > alpha(1-3)glucan. IL-6 production was tumor necrosis factor alpha independent and required the presence of monocytes and plasma. Since IL-6 can stimulate replication of the human immunodeficiency virus in monocytic cells, these findings may be clinically relevant.
Modulation of neutrophil superoxide response and intracellular diacylglyceride levels by the bacterial pigment pyocyanin
Authors: Muller M , Sorrell T C
Published: January 2020

Low concentrations of pyocyanin are reported to enhance superoxide production by human neutrophils exposed to various stimuli, yet the mechanism remains unknown. Using lucigenin-enhanced chemiluminescence, we examined the kinetics of the neutrophil superoxide response in the presence of pyocyanin. At all concentrations (12.5 to 200 microM), pyocyanin decreased the peak superoxide response while prolonging the duration of the response. The prolonged response may be associated with an observed increase in intracellular diacylglyceride levels due to pyocyanin exposure.
Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis
Authors: Merriam J J , Mathur R , Maxfield-Boumil R , Isberg R R
Published: January 2020

Using a PCR-based strategy and degenerate oligonucleotides, we isolated a Legionella pneumophila gene that showed high sequence similarity to members of the fliI gene family. An insertion mutation that disrupted the fliI open reading frame was recombined onto the L. pneumophila chromosome and analyzed for its effects on production of flagella and intracellular growth. The mutation resulted in loss of surface-localized flagellin protein but had no effect on the ability of the bacteria to grow within cultured cells. Therefore, in spite of the fact that some aflagellar mutations render L. pneumophila unable to grow within macrophages, the isolation of this defined mutant confirms that production of flagella is not required for intracellular growth.
The tumor necrosis factor alpha-stimulating region of galactose-inhibitable lectin of Entamoeba histolytica activates gamma interferon-primed macrophages for amebicidal activity mediated by nitric oxide
Authors: Séguin R , Mann B J , Keller K , Chadee K
Published: January 2020

Entamoeba histolytica adheres via galactose-lectin (Gal-lectin) to human colonic mucins and intestinal epithelial cells as a prerequisite to amebic invasion. Native Gal-lectin is a protective antigen in the gerbil model of amebiasis. Amino acids 596 to 1082 of Gal-lectin mediate E. histolytica adherence to target cells and stimulate tumor necrosis factor alpha (TNF-alpha) production by naive murine bone marrow macrophages (BMM). Resistance to amebiasis requires an effective cell-mediated immune response against E. histolytica trophozoites mediated by nitric oxide (NO) released from activated macrophages. Herein, we determine whether the TNF-alpha-stimulating region of Gal-lectin can activate gamma interferon (IFN-gamma)-primed BMM for NO production and amebicidal activity. Native Gal-lectin (100 to 500 ng/ml) stimulated TNF-alpha and inducible nitric oxide synthase (iNOS) mRNA expression in IFN-gamma-primed BMM as did lipopolysaccharide (100 ng/ml). Primed BMM produced TNF-alpha and NO in response to Gal-lectin in a dose-dependent manner. Antilectin monoclonal antibody IG7, which recognizes a domain (amino acids 596 to 818) of the TNF-alpha mRNA-stimulating region of Gal-lectin, specifically inhibited TNF-alpha and iNOS mRNA induction and TNF-alpha and NO production by primed BMM in response to Gal-lectin (100 ng/ml). Simultaneous treatment of BMM with IFN-gamma and Gal-lectin (100 ng/ml) activated the cells to kill E. histolytica trophozoites, whereas IFN-gamma treatment alone had no effect. In the presence of monoclonal antibody 1G7 or aminoguanidine (an iNOS inhibitor), NO production and amebicidal activity were inhibited >80%. These results suggest that the TNF-alpha-stimulating region of native Gal-lectin is a potent stimulus of IFN-gamma-primed BMM for NO production, which is essential for host defense against amebiasis.
Predominant recognition of the ESAT-6 protein in the first phase of interferon with Mycobacterium bovis in cattle
Authors: Pollock J M , Andersen P
Published: January 2020

Tuberculosis continues to be a worldwide health problem for both humans and animals. The development of improved vaccines and diagnostic tests requires detailed understanding of the immune responses generated and the antigens recognized during the disease. This study examined the T-cell response which develops in cattle experimentally infected with Mycobacterium bovis. The first significant T-cell response was found 3 weeks after the onset of infection and was characterized by a pronounced gamma interferon (IFN-gamma) response from peripheral blood mononuclear cells directed to antigens in culture filtrates. Short-term culture filtrate (ST-CF) was separated into molecular mass fractions and screened for recognition by T cells from experimentally infected and field cases of bovine tuberculosis. Cattle in the early stages of experimental infection were characterized by strong IFN-gamma responses directed predominantly toward the lowest-mass (<10-kDa) fraction of ST-CF, but cattle in later stages of experimental infection (16 weeks postinfection) exhibited a broader recognition of antigens of various molecular masses. Field cases of bovine tuberculosis, in comparison, preferentially recognized low-mass antigens, characteristic of animals in the early stages of infection. The major T-cell target for this dominant IFN-gamma response was found to be the secreted antigen ESAT-6. This antigen was recognized strongly by the majority of field cases of bovine tuberculosis tested. As ESAT-6 is unique to pathogenic mycobacterial species, our study suggests that ESAT-6 is an antigen with major potential for vaccination against and specific diagnosis of bovine tuberculosis.
Extracellular proteins of Cryptococcus neoformans and host antibody response
Authors: Chen L C , Pirofski L A , Casadevall A
Published: January 2020

Proteins secreted by the fungal pathogen Cryptococcus neoformans may be involved in invasion and could be useful in vaccine design. Despite the medical importance of this fungus, little is known about its extracellular proteins or the immune response to these antigens. To study C. neoformans extracellular proteins, 12 strains were metabolically radiolabeled and protein supernatants were analyzed. Both strain- and growth condition-dependent differences were observed. Enzymatic assays of filtered culture supernatants revealed butyrate esterase and caprylate esterase lipase activity for 11 of 12 strains, as well as acid phosphatase, naphthol-AS-BI-phosphohydrolase, and beta-glucosidase activities in some strains. Serum from infected rodents immunoprecipitated several secreted proteins, consistent with in vivo expression and development of an antibody response. For strain 24067, two immunodominant species, of approximately 75 and 30 kDa, were recognized. The relative intensity of the autoradiographic bands depended on the route of infection for both rats and mice. In summary, our results indicate that (i) there are multiple proteins in C. neoformans culture supernatants, (ii) there are strain differences in supernatant protein profiles, (iii) there are differences in supernatant protein profile depending on the growth conditions, (iv) there are several new extracellular and/or cell-associated enzymatic activities, and (v) antibodies to several supernatant proteins are made in the course of infection.
Enteropathogenic Escherichia coli protein secretion is induced in response to conditions similar to those in the gastrointestinal tract
Authors: Kenny B , Abe A , Stein M , Finlay B B
Published: January 2020

The pathogenicity of enteropathogenic Escherichia coli (EPEC) is associated with the expression and secretion of specific bacterial factors. EspB is one such secreted protein which is required to trigger host signaling pathways resulting in effacement of microvilli and cytoskeletal rearrangements. These events presumably contribute to the ensuing diarrhea associated with EPEC infections. EPEC encounters several environmental changes and stimuli during its passage from the external environment into the host gastrointestinal tract. In this paper we show that the secretion of EspB is subject to environmental regulation, and maximal secretion occurs under conditions reminiscent of those in the gastrointestinal tract. Thus, secretion is maximal at 37 degrees C, pH 7, and physiological osmolarity. In addition, maximal secretion requires the presence of sodium bicarbonate and calcium and is stimulated by millimolar concentrations of Fe(NO3)3. The secretion of the four other EPEC-secreted proteins appears to be modulated in a manner similar to that of EspB. Our results also show that secretion is not dependent on CO2, as originally reported by Haigh et al. (FEMS Microbiol. Lett. 129: 63-67, 1995), but that CO2 more likely acts as a component of the medium buffering system, since CO2 dependence was abolished by the use of alternative buffers.
Analysis of F1F0-ATPase from Helicobacter pylori
Authors: McGowan C C , Cover T L , Blaser M J
Published: January 2020

The adaptive mechanisms that permit Helicobacter species to survive within the gastric mucosa are not well understood. The proton-translocating F1F0-ATPase is an important enzyme for regulating intracellular pH or synthesizing ATP in many other enteric bacteria; therefore, we used degenerate primers derived from conserved bacterial F1F0-ATPase sequences to PCR amplify and clone the gene (atpD) encoding the H. pylori F1F0-ATPase beta subunit. The deduced amino acid sequences of the F1F0-ATPase beta subunits from H. pylori and Wolinella succinogenes were 85% identical (91% similar). To characterize a potential functional role of F1F0-ATPase in H. pylori, H. pylori or Escherichia coli cells were incubated for 60 min in buffered solutions at pH 7, 6, 5, or 4, with or without 100 microM N,N'-dicyclohexylcarbodiimide (DCCD), a specific inhibitor of F1F0-ATPase. At pH 5 and 4, there was no significant decrease in survival of H. pylori in the presence of DCCD compared to its absence, whereas incubation with DCCD at pH 7 and 6 significantly decreased H. pylori survival. E. coli survival was unaffected by DCCD at any pH value tested. We next disrupted the cloned beta-subunit sequence in E. coli by insertion of a kanamycin resistance cassette and sought to construct an isogenic F1F0-ATPase H. pylori mutant by natural transformation and allelic exchange. In multiple transformations of H. pylori cells grown at pH 6 or 7, no kanamycin-resistant F1F0 mutants were isolated, despite consistently successful mutagenesis of other H. pylori genes by using a similar approach and PCR experiments providing evidence for integration of the kanamycin resistance cassette into atpD. The sensitivity of H. pylori to DCCD at pH 7 and 6, and failure to recover F1F0 H. pylori mutants under similar conditions, suggests that the function of this enzyme is required for survival of H. pylori at these pHs.
Nitrite reductase from Pseudomonas aeruginosa induces inflammatory cytokines in cultured respiratory cells
Authors: Oishi K , Sar B , Wada A , Hidaka Y , Matsumoto S , Amano H , Sonoda F , Kobayashi S , Hirayama T , Nagatake T , Matsushima K
Published: January 2020

Persistent infection with Pseudomonas aeruginosa increases interleukin-8 (IL-8) levels and causes dense neutrophil infiltrations in the airway of patients with chronic airway diseases. To investigate the role of P. aeruginosa infection in IL-8 production in the airway of these patients, we examined whether cell lysates of P. aeruginosa could cause IL-8 production from human bronchial epithelial cells. Diluted sonicated supernatants of P. aeruginosa (SSPA) with a mucoid or nonmucoid phenotype stimulated human bronchial epithelial (BET-1A) cells to produce IL-8. In this study, we have purified a 59-kDa heat-stable protein with IL-8-inducing activity from the SSPA by sequential ion-exchange chromatography. The N-terminal sequence of this purified protein completely matched a sequence at the N-terminal part of the mature protein of nitrite reductase from P. aeruginosa. In addition, immunoblotting with a polyclonal immunoglobulin G (IgG) against recombinant Pseudomonas nitrite reductase demonstrated a specific binding to the purified protein. Furthermore, the immunoprecipitates of the SSPA with a polyclonal IgG against recombinant nitrite reductase induced a twofold-higher IL-8 production in the BET-1A cell culture than did the immunoprecipitates of the SSPA with a control IgG. These lines of evidence confirmed that Pseudomonas nitrite reductase was responsible for IL-8 production in the BET-1A cells. The purified nitrite reductase induced maximal expression of IL-8 mRNA in the BET-1A cells at 1 to 3 h after stimulation, and the IL-8 mRNA expression declined by 8 h after stimulation. New protein translation was not required for nitrite reductase-mediated IL-8 mRNA expression in the BET-1A cells. Nitrite reductase stimulated the BET-1A cells, as well as human alveolar macrophages, pulmonary fibroblasts, and neutrophils, to produce IL-8. In contrast, nitrite reductase induced significant levels of tumor necrosis factor alpha and IL-1beta protein only in human alveolar macrophages. These data support the notion that nitrite reductase from P. aeruginosa induces the production of inflammatory cytokines by respiratory cells and may contribute to the pathogenesis of chronic airway diseases and persistent P. aeruginosa infection.
Intranasal immunogenicity and adjuvanticity of site-directed mutant derivatives of cholera toxin
Authors: Douce G , Fontana M , Pizza M , Rappuoli R , Dougan G
Published: January 2020

Genetically modified derivatives of cholera toxin (CT), harboring a single amino acid substitution in and around the NAD binding cleft of the A subunit, were isolated following site-directed mutagenesis of the ctxA gene. Two mutants of CT, designated CTS106 (with a proline-to-serine change at position 106) and CTK63 (with a serine-to-lysine change at position 63), were found to have substantially reduced ADP-ribosyltransferase activity and toxicity; CTK63 was completely nontoxic in all assays, whereas CTS106 was 10(4) times less toxic than wild-type CT. The mucosal adjuvanticity and immunogenicity of derivatives of CT were assessed by intranasal immunization of mice, with either ovalbumin or fragment C of tetanus toxin as a bystander antigen. Mice immunized with wild-type CT produced both local (immunoglobulin A in mucosal washes) and systemic immune responses to both CT and bystander antigens. CTS106 showed good local and systemic responses to bystander proteins and to itself. Interestingly, mice immunized with the nontoxic derivative of CT, CTK63, generated weak immune responses to the bystander antigens which were similar to those achieved when CT B subunit was used as an adjuvant. In parallel experiments, an equivalent nontoxic mutant of the Escherichia coli heat-labile enterotoxin, LTK63 (with a serine-to-lysine change at position 63), was tested (9). In contrast to CTK63, LTK63 was found to be more immunogenic and a better intranasal adjuvant than recombinant heat-labile enterotoxin B subunit or CTK63. This information, together with data on immunoglobulin subclass responses, suggests that although highly homologous, CT and heat-labile enterotoxin should not be considered biologically identical in terms of their ability to act as intranasal adjuvants.
Characterization of human T- and B-cell epitopes in the C terminus of Plasmodium falciparum merozoite surface protein 1: evidence for poor T-cell recognition of polypeptides with numerous disulfide bonds
Authors: Egan A , Waterfall M , Pinder M , Holder A , Riley E
Published: January 2020

We have investigated the relationship between cellular and humoral immune responses to defined epitopes of the C terminus of merozoite surface protein 1 (MSP-1) of the human malaria parasite, Plasmodium falciparum, in immune blood donors. Sera from almost all donors contained antibodies to the 33-kDa processing product of the MAD20 allele of MSP-1 (MSP-1(33)), but these antibodies did not cross-react with the equivalent sequence of the Wellcome allele. In contrast, T-cell responses to MSP-1(33) are directed towards epitopes that are conserved between the two allelic families. Only 50% of adult blood donors possessed antibodies which recognized the 19-kDa processing product of MSP-1 (MSP-1(19)). These antibodies predominantly recognized conserved epitopes involving both of the constituent epidermal growth factor-like domains of MSP-1(19). T-cell responses were found in only 26% (for recombinant proteins) or 44% (for synthetic peptides) of donors and were directed mainly at dimorphic sequences of the protein. There was no obvious association, at an individual level, between the presence of antibodies and the detection of T-cell proliferative or gamma interferon responses, suggesting that the T cells identified in this manner are not providing significant levels of help to B cells. T-cell responses to reduced recombinant proteins and linear peptides were more prevalent than responses to disulfide-bonded proteins, suggesting that the complex disulfide-bonded structure of native MSP-1(19) may inhibit antigen processing or presentation.
Effects of Ixodes scapularis and Borrelia burgdorferi on modulation of the host immune response: induction of a TH2 cytokine response in Lyme disease-susceptible (C3H/HeJ) mice but not in disease-resistant (BALB/c) mice
Authors: Zeidner N , Mbow M L , Dolan M , Massung R , Baca E , Piesman J
Published: January 2020

Previous studies have demonstrated that both Ixodes scapularis saliva and Borrelia burgdorferi antigens modulated lymphokines and monokines in vitro. The studies presented here were designed to delineate the role of I. scapularis and B. burgdorferi in modulation of the host immune response in vivo. Infestation of C3H/HeJ mice with infected I. scapularis resulted in an up regulation of IL-4 as early as 8 days after tick infestation, while the levels of T helper cell type 1 (TH1) cytokines, interleukin-2 (IL-2) and gamma interferon (IFN-gamma), were significantly decreased by days 10 to 12. In contrast, the cytokine profile of BALB/c mice exposed to infected nymphal ticks resulted in only transient alterations in IL-4, IL-2, and IFN-gamma production throughout a 12-day period postinfestation. Although the IL-10 level was elevated in both C3H/HeJ and BALB/c mice infested with infected nymphal ticks, no significant difference in the levels of IL-10 was noted between the mouse strains. Flow-cytometric analysis demonstrated increases in the numbers of splenic B-cell and CD4+ lymphocytes in C3H/HeJ but not BALB/c mice exposed to infected ticks. Cell depletion experiments with C3H/HeJ mice demonstrated that CD4+ cells were the sole producers of IFN-gamma and IL-10 while both CD4+ and CD8+ splenocytes contributed to the production of IL-2 and IL-4. These findings suggest that B and CD4+ splenocytes are activated, increase in number, and produce a polarized TH2 response in C3H/HeJ mice exposed to infected I. scapularis. Given that C3H/HeJ mice are susceptible to Lyme disease and the initial TH2 polarization is not evident in BALB/c mice, effective control of this response may have ramifications for spirochete transmission in vivo.
Activation of the interleukin-1beta precursor by Treponema denticola: a potential role in chronic inflammatory periodontal diseases
Authors: Beauséjour A , Deslauriers N , Grenier D
Published: January 2020

There are several indications suggesting that interleukin-1beta (IL-1beta) may play an important role in inflammatory periodontal diseases. We hypothesized that periodontal sites would represent a unique combination of both cellular sources of IL-1beta precursor (pro-IL-1beta) and microbial proteases and proposed that Treponema denticola, a suspected periodontal pathogen, would play a critical role in the inflammatory nature of adult chronic periodontitis by activating pro-IL-1beta. The aim of this study was thus to demonstrate the proteolytic cleavage and activation of the inactive precursor pro-IL-1beta by T. denticola. After incubation of bacterial cells with recombinant pro-IL-1beta, proteolytic cleavage was monitored by Western immunoblotting, and the biological activity of the digestion products was tested in a bioassay. We report here that T. denticola can cleave pro-IL-1beta to yield two fragments with molecular masses of 18 and 19 kDa. Cleavage products showed a dose-dependent biological activity in the thymocyte proliferation bioassay, and this activity was inhibited by anti-IL-1beta neutralizing antibodies. These results suggest that T. denticola may have a proinflammatory role in periodontal diseases.
Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium elicit mucosal and systemic neutralizing antibodies in mice
Authors: Nardelli-Haefliger D , Roden R B , Benyacoub J , Sahli R , Kraehenbuhl J P , Schiller J T , Lachat P , Potts A , De Grandi P
Published: January 2020

Attenuated strains of Salmonella are attractive live vaccine candidates for eliciting mucosal as well as systemic immune responses. The ability to induce immune responses in the reproductive tract may be critical for the effectiveness of a prophylactic vaccine against genital human papillomaviruses (HPV), which are important etiologic agents in the development of cervical cancer. To examine the potential of a live Salmonella-based vaccine to prevent genital HPV infection, the L1 major capsid protein from HPV type 16 (HPV16) was constitutively expressed in the PhoPc strain of Salmonella typhimurium. As demonstrated by electron microscopy, the L1 protein expressed in these bacteria assembled into virus-like particles (VLPs) that resemble authentic papillomavirus virions. This is the first demonstration that papillomavirus VLPs can self-assemble in prokaryotes. BALB/c mice were immunized with the HPV16 L1 recombinant PhoPc strain by the oral and nasal routes. Despite a low stability of the L1-expressing plasmid in vivo, a double nasal immunization was effective in inducing L1-specific serum antibodies that recognized mainly native, but not disassembled, VLPs. These antibodies effectively neutralized HPV16 pseudotyped virions in an in vitro infectivity assay. Conformationally dependent anti-VLP immunoglobulin A (IgA) and IgG were also detected in oral and vaginal secretions, indicating that potentially protective antibody responses were elicited at mucosal sites. Recombinant attenuated Salmonella expressing HPV capsids may represent a promising vaccine candidate against genital HPV infection.
Mice lacking the gamma interferon receptor have an impaired granulomatous reaction to Schistosoma mansoni infection
Authors: Rezende S A , Oliveira V R , Silva A M , Alves J B , Goes A M , Reis L F
Published: January 2020

The egg-induced granulomatous reaction in Schistosoma mansoni-infected individuals develops within the portal system of the liver and is the major pathological finding in schistosomiasis. We have infected mice lacking the gamma interferon (IFN-gamma) receptor with S. mansoni larvae and studied the development of hepatic granulomas in these mutant mice in comparison to that in control wild-type mice. In the absence of IFN-gamma activity, a dramatic reduction in the size and architecture of the granuloma was observed. Granulomas from mutant mice were smaller than those from the control group and showed a significant reduction in the number of infiltrating inflammatory cells. Moreover, they appear to prematurely progress to the chronic phase of the reaction at a time when the control group still has acute inflammation. Our data suggests a pivotal role for IFN-gamma in the early events of the granulomatous reaction in vivo.
Characterization of a recombinant fragment that contains a carbohydrate recognition domain of the filamentous hemagglutinin
Authors: Liu D F , Phillips E , Wizemann T M , Siegel M M , Tabei K , Cowell J L , Tuomanen E
Published: January 2020

The filamentous hemagglutinin (FHA) of Bordetella pertussis plays an important role in establishing infection by attaching the bacteria to the ciliated respiratory epithelial cells. Expression of DNA encoding residues 1141 to 1279 of FHA in Escherichia coli yields a protein of 18,000 Da that exhibits some of the carbohydrate recognition properties of FHA (S. M. Prasad, Y. Yin, E. Rodzinski, E. I. Tuomanen, and H. R. Masure, Infect. Immun. 61:2780-2785, 1993). We have constructed an E. coli strain that expresses this protein, designated fragment A, in a soluble form at markedly elevated levels. Fragment A could be purified with high purity and yields and was immunogenic in mice. Both fragment A and anti-fragment A sera inhibited the binding of B. pertussis to asialo-GM2 and to rabbit ciliated cells. These observations demonstrate that this fragment of FHA contains a cellular binding domain capable of eliciting functional antibodies.
Clostridium perfringens enterotoxin lacks superantigenic activity but induces an interleukin-6 response from human peripheral blood mononuclear cells
Authors: Krakauer T , Fleischer B , Stevens D L , McClane B A , Stiles B G
Published: January 2020

We investigated the potential superantigenic properties of Clostridium perfringens enterotoxin (CPE) on human peripheral blood mononuclear cells (PBMC). In contrast to the findings of a previous report (P. Bowness, P. A. H. Moss, H. Tranter, J. I. Bell, and A. J. McMichael, J. Exp. Med. 176:893-896, 1992), two different, biologically active preparations of CPE had no mitogenic effects on PBMC. Furthermore, PBMC incubated with various concentrations of CPE did not elicit interleukin-1, interleukin-2, gamma interferon, or tumor necrosis factor alpha or beta, which are cytokines commonly associated with superantigenic stimulation. However, CPE did cause a dose-related release of interleukin-6 from PBMC cultures.
Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence
Authors: Hube B , Sanglard D , Odds F C , Hess D , Monod M , Schäfer W , Brown A J , Gow N A
Published: January 2020

Secreted aspartyl proteinases (Saps), encoded by a gene family with at least nine members (SAP1 to SAP9), are one of the most discussed virulence factors produced by the human pathogen Candida albicans. In order to study the role of each Sap isoenzyme in pathogenicity, we have constructed strains which harbor mutations at selected SAP genes. SAP1, SAP2, and SAP3, which are regulated differentially in vitro, were mutated by targeted gene disruption. The growth rates of all homozygous null mutants were similar to those of the isogenic wild-type parental strain (SC5314) in complex and defined media. In medium with protein as the sole source of nitrogen, sap1 and sap3 mutants grew with reduced growth rates but reached optical densities similar to those measured for SC5314. In contrast, sap2 null mutants tended to clump, grew poorly in this medium, and produced the lowest proteolytic activity. Addition of ammonium ions reversed such growth defects. These results support the view that Sap2 is the dominant isoenzyme. When sap1, sap2, and sap3 mutants were injected intravenously in guinea pigs and mice, the animals had increased survival rates compared to those of control animals infected with SC5314. However, reduction of proteolytic activity in vitro did not correlate directly with the extent of attenuation of virulence observed for all Sap-deficient mutants. These data suggest that SAP1, SAP2, and SAP3 all contribute to the overall virulence of C. albicans and presumably all play important roles during disseminated infections.
Antigenic heterogeneity and molecular analysis of CopB of Moraxella (Branhamella) catarrhalis
Authors: Sethi S , Surface J M , Murphy T F
Published: January 2020

Outer membrane protein (OMP) CopB, an iron-repressible 81-kDa major OMP of Moraxella (Branhamella) catarrhalis has been a major focus of investigation. To assess CopB as a potential vaccine antigen, we elucidated the degree of antigenic and sequence heterogeneity in this protein among strains of M. catarrhalis. Two monoclonal antibodies, 1F5 and 2.9F, which bind to surface-exposed epitopes on CopB recognized 60 and 70% of the strains, respectively. The degree of sequence heterogeneity in CopB was assessed by cloning and sequencing the CopB gene from two different strains of M. catarrhalis and comparing with the published sequence. There was 92 to 96% homology between the sequences at the nucleotide level and 90 to 95% homology at the amino acid level. The variability in the protein sequence is confined mainly to three moderately variable regions. Restriction fragment length polymorphism (RFLP) analysis of the CopB genes obtained from 20 diverse strains by PCR was performed. Ninety percent of the potential restriction sites in the constant regions and 47% of the potential restriction sites in the variable regions were present in the 20 strains, indicating that the pattern of variable and constant areas in the CopB gene is a general pattern among strains of M. catarrhalis. We conclude that the CopB gene is largely conserved among strains of M. catarrhalis and contains discrete regions which show moderate heterogeneity among strains.
SlyA, a transcriptional regulator of Salmonella typhimurium, is required for resistance to oxidative stress and is expressed in the intracellular environment of macrophages
Authors: Buchmeier N , Bossie S , Chen C Y , Fang F C , Guiney D G , Libby S J
Published: January 2020

Appropriate regulation of genes enables Salmonella typhimurium to adapt to the intracellular environment of the host. The Salmonella slyA gene is in a family of transcriptional regulators that may play an important role in this adaptation. We have previously shown that slyA mutant Salmonella strains are profoundly attenuated for virulence and do not survive in macrophages. In this study, we demonstrate that the expression of multiple Salmonella proteins is regulated by SlyA during stationary phase and during infection of macrophages. Both of these conditions also induced the expression of a slyA::lacZ transcriptional fusion. Expression of the slyA::lacZ transcriptional fusion increased 15-fold in stationary phase and was not dependent on the stationary-phase sigma factor, RpoS. slyA mutant Salmonella strains were sensitive to oxidative products of the respiratory burst, including hydrogen peroxide and the products of the redox cycling compound paraquat, but not to nitric oxide donors. These results suggest that the SlyA regulon is activated during infection of the host and is required for resistance to toxic oxidative products of the reticuloendothelial system.