bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of stimulation of T cells by Streptococcus pyogenes: isolation of a major mitogenic factor, cytoplasmic membrane-associated protein
Authors: Itoh T , Satoh H , Isono N , Rikiishi H , Kumagai K
Published: January 2020

Our previous studies established that heat-killed Streptococcus pyogenes, as well as other gram-positive cocci, when incubated with human peripheral blood leukocytes (PBLs) in culture, induced polyclonal activation of T lymphocytes. The activated T lymphocytes included CD4+ CD8- helper T cells and CD3+ and CD4- CD8- double-negative T cells with gamma delta T-cell receptors. In the present study, we isolated a major factor with this unique mitogenic activity against human T lymphocytes from S. pyogenes. This active fraction was found in the cytoplasmic membrane (CM) of the heat-killed organisms but not in other cellular fractions such as cell walls, peptidoglycan, lipoteichoic acids, or cytoplasmic soluble fractions. An active molecule(s) was further isolated from the CM by cholic acid extraction followed by Sephacryl S-200 chromatography. The molecule was protease labile but highly resistant to heat, had a pI of greater than or equal to 9.3 and a molecular weight of 10,000 to 15,000 according to gel filtration experiments, and was termed CM-associated protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein purified by anion-exchange chromatography showed a single band with a molecular weight of 15,000, corresponding to mitogenically active regions. Purified CM-associated protein induced activation of T lymphocytes, which consisted of CD4+ CD8- T cells and CD4- CD8- double-negative T-cell receptor gamma/delta + T-cell populations, as did the whole cells of S. pyogenes.
Monoclonal antibodies specific for elongation factor Tu and complete nucleotide sequence of the tuf gene in Mycobacterium tuberculosis
Authors: Carlin N I , Löfdahl S , Magnusson M
Published: January 2020

Monoclonal antibodies against mycobacterial antigens were produced by immunizing LOU/C rats with live Mycobacterium bovis BCG. The antibodies were characterized by an enzyme-linked immunosorbent assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting (immunoblotting). One antibody, MAMB 2, reactive with a 47-kDa protein was used to screen a lambda gt11 M. tuberculosis gene library (R. A. Young, B. R. Bloom, C. M. Grosskinsky, J. Ivanji, D. Thomas, and R. W. Davis, Proc. Natl. Acad. Sci. USA 82:2583-2587, 1985). Three recombinant phages reactive with MAMB 2 in plaque lysates were isolated, and part of the insert was sequenced. The mycobacterial inserts were all expressed as proteins fused with beta-galactosidase when the phages were induced as lysogens in Escherichia coli. The entire M. tuberculosis tuf gene was obtained by screening the lambda gt11 library with a DNA probe specific for the primary clones. A phage isolated from this screening was able to express the native protein in E. coli when introduced as a lysogen. A comparison of the entire gene sequence and the deduced protein sequence with the EMBL DNA and Swiss-Prot protein data libraries revealed strong homologies with elongation factors of bacteria, yeast mitochondria, and a plant chloroplast.
Pasteurella haemolytica leukotoxin enhances production of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by bovine polymorphonuclear leukocytes
Authors: Henricks P A , Binkhorst G J , Drijver A A , Nijkamp F P
Published: January 2020

The influence of the leukotoxin of Pasteurella haemolytica on the generation of arachidonic acid metabolites by bovine polymorphonuclear leukocytes (PMNs) was investigated. PMNs released 5-, 12-, and 15-hydroxyeicosatetraenoic acids (5-, 12-, and 15-HETE) and leukotriene B4 (LTB4) upon stimulation with arachidonic acid. The leukotoxin preparations dose dependently enhanced the release of the 5-lipoxygenase products 5-HETE and LTB4 in arachidonic acid-stimulated PMNs, whereas the release of 12- and 15-HETE was not affected. The enhanced release of LTB4 and 5-HETE was not due to a decreased cellular retention of the 5-lipoxygenase products. In addition, leukotoxin preparations by themselves were also able to induce LTB4 and 5-HETE production in the absence of exogenous arachidonic acid. Generation of 5-lipoxygenase products by PMNs stimulated by leukotoxin may represent an important cellular event that occurs during infections with P. haemolytica.
Interaction of capsulate Haemophilus influenzae with human airway mucosa in vitro
Authors: Read R C , Rutman A A , Jeffery P K , Lund V J , Brain A P , Moxon E R , Cole P J , Wilson R
Published: January 2020

Two pairs of isogenic capsulate and noncapsulate and one pair of capsulate fimbriate and nonfimbriate strains of Haemophilus influenzae type b were studied in an organ culture of human respiratory mucosa. Over 24 h, the numbers of recovered bacteria increased from the original inoculum size of 10(5) to 10(8) CFU/ml. Transmission electron microscopy and scanning electron microscopy showed that noncapsulate organisms caused significant epithelial damage, whereas capsulate strains did not. Association of noncapsulate bacteria with damaged epithelial cells was observed by 14 h of incubation. In contrast, capsulate organisms were associated with a dense, thick, gel-like matrix which was observed above the epithelial surface. These capsulate organisms were not seen to associate with the epithelial surface (by transmission electron microscopy), though they were occasionally seen adhering to cells by scanning electron microscopy. Fimbriate capsulate H. influenzae showed increased adherence to buccal cells compared with nonfimbriate capsulate organisms. There was also association of fimbriate capsulate bacteria with damaged organ culture epithelium in one of four experiments. It is concluded that both capsule and fimbriae affect the interaction of H. influenzae with human airway mucosa in vitro by influencing adherence to and damage of the epithelium.
Characterization of antibody-mediated inhibition of Pseudomonas aeruginosa adhesion to epithelial cells
Authors: Sexton M , Reen D J
Published: January 2020

An enzyme-linked immunosorbent assay system was developed and used to study adhesion of Pseudomonas aeruginosa to human epithelial cells and the abilities of specific antibodies to inhibit this process. Human buccal epithelial cells coated onto microtiter plates were incubated with P. aeruginosa suspensions, and adherent bacteria were detected by using anti-P. aeruginosa serum and a horseradish peroxidase-conjugated secondary antiserum. Adhesion, quantitated as an increase in A405, varied linearly with increasing numbers of bacterial CFU added per well in the range of 10(5) to 10(8) CFU per well. Adhesion of P. aeruginosa increased following trypsinization of buccal epithelial cells. Preincubation of bacteria with monoclonal antibodies directed against P. aeruginosa outer membrane protein H2 inhibited adhesion with all eight of the isolates tested. Preincubation of P. aeruginosa with sera from infected cystic fibrosis patients also resulted in inhibition of adhesion in the enzyme-linked immunosorbent assay system. This inhibitory activity was shown to be due to two factors: P. aeruginosa-specific immunoglobulin G and a non-immunoglobulin G serum component. These data support the hypothesis that bacterial components other than pili are involved in adhesion and suggest that anti-P. aeruginosa antibodies may be of use in preventing adhesion and subsequent colonization with P. aeruginosa.
Growth characteristics and proteins of plaque-purified strains of Rickettsia tsutsugamushi
Authors: Moree M F , Hanson B
Published: January 2020

Six plaque-purified strains of Rickettsia tsutsugamushi (Karp, Gilliam, Kato, JC472B, TA716, and TA763) that fall into three categories of virulence for mice were compared by several parameters. Five of the six strains formed plaques of identical size in mouse cells, but each of three strains tested (representing three mouse virulence types) had a different doubling time in mouse cell cultures. Neither of these properties correlated strictly with virulence in mice, although the avirulent TA716 strain replicated much more slowly than the more virulent Karp and Gilliam strains. R. tsutsugamushi strain heterogeneity was also manifested at the polypeptide level by migration rates in sodium dodecyl sulfate-polyacrylamide gels of three of the major scrub typhus antigens (Sta110, Sta56, and Sta47), with those of Sta110 differing most widely. As expected, immunoblotting with polyclonal mouse sera showed substantial cross-reactivity among the major antigens of the six strains. Similar tests with Karp-induced monoclonal antibodies (MAb) demonstrated that some epitopes on Sta110 and Sta56 were shared by fewer than the six strains, but they identified no epitope unique to Karp. In contrast to the ready demonstration of antigenic heterogeneity in Sta110 and Sta56, four of the five Sta47-specific MAb reacted well with Sta47 from each of the six strains; the remaining MAb bound Sta47 from Karp and the Karp-like JC472B strain more strongly than Sta47 from the other four strains. The MAb also were useful in indicating the possible occurrence of Sta47 as dimers and trimers, the presence of Sta110 (as well as Sta56 and Sta47) in the rickettsial membrane, and the apparent interaction of the putative heat shock protein Sta58 with Sta47 or Sta47-Sta56 complexes.
An enhanced murine model for studies of Serpulina (Treponema) hyodysenteriae pathogenesis
Authors: Nibbelink S K , Wannemuehler M J
Published: January 2020

A defined diet was used to increase the susceptibility of mice to Serpulina hyodysenteriae. BALB/cByJ, C3H/HeN, and C3H/HeJ mice, when fed the defined diet 7 to 14 days prior to and throughout the challenge period, consistently showed higher incidences of disease than mice maintained on normal rodent chow. The use of this defined diet will increase the consistency of in vivo studies following infection with S. hyodysenteriae in the mouse model.
Elevated body temperature restricts growth of Haemophilus influenzae type b during experimental meningitis
Authors: O'Reilly T , Zak O
Published: January 2020

Elevation of the environmental temperature appeared to counteract the temperature-depressing effects of urethane anesthetic and allowed rabbits intracisternally infected with Haemophilus influenzae type b to mimic the development of a fever following infection. Elevated core body temperature (greater than 39 degrees C) was associated with an inhibition of the growth of H. influenzae in cerebrospinal fluid (CSF) during the first 12 h postinfection, whereas bacterial growth was essentially unrestricted in rabbits with reduced (approximately 37 degrees C) body temperature. Bacterial densities 24 h postinfection were different, hyperthermic animals having log 6.0 +/- 0.4 CFU/ml of CSF and hypothermic rabbits having log 8.2 +/- 0.8 CFU/ml of CSF (P less than 0.05, Wilcoxon rank sum test). However, the growth of this bacterium in vitro, in either pooled rabbit CSF or brain heart infusion broth, was not inhibited at 39 degrees C. These results suggest that elevated body temperature associated with the development of fever during meningitis may be associated with restriction of the growth of H. influenzae in vivo but that this effect is apparently not due to an innate inability of the bacterium to grow at elevated temperatures.
L-arginine-dependent killing of intracellular Ehrlichia risticii by macrophages treated with gamma interferon
Authors: Park J , Rikihisa Y
Published: January 2020

Thioglycolate-induced murine peritoneal macrophages infected with Ehrlichia risticii and treated in vitro with gamma interferon (IFN-gamma) developed antiehrlichial activity that eliminated the intracellular bacteria. This antiehrlichial activity was suppressed by NG-monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthesis from L-arginine, but not by L-tryptophan. Increased levels of nitrite, an oxidative product of nitric oxide, were measured in cultures of infected macrophages treated with IFN-gamma. Sodium nitroprusside, which spontaneously releases nitric oxide, also showed the antiehrlichial activity. The antiehrlichial activity by reactive nitrogen intermediates was not mediated by elevation of the cellular concentration of cyclic GMP since the addition of 8-bromo-cyclic GMP itself had no influence on ehrlichial infection of macrophages. Addition of the intracellular iron chelator deferoxamine also inhibited E. risticii infection in vitro. These results suggest that intracellular E. risticii survival is iron dependent and that production of reactive nitrogen intermediates triggers iron loss from critical target enzymes of E. risticii, leading to lethal metabolic inhibition. However, addition of excess FeSO4, ferric citrate, or iron-saturated transferrin did not counteract the antiehrlichial effect induced by IFN-gamma.
Synthesis of a conjugate vaccine composed of pneumococcus type 14 capsular polysaccharide bound to pertussis toxin
Authors: Schneerson R , Levi L , Robbins J B , Bryla D M , Schiffman G , Lagergard T
Published: January 2020

Type 14 is one of the common types isolated from patients of all ages with infections caused by Streptococcus pneumoniae. Its capsular polysaccharide (Pn14) is composed of a neutrally charged tetrasaccharide repeat unit. Pn14 does not elicit protective levels of antibodies in infants and children and is a less than optimal immunogen of the 23-valent vaccine for adults. Pertussis toxin (PT) is both a virulence factor and protective antigen of Bordetella pertussis: it is not soluble at neutral pH and forms insoluble complexes with acidic polysaccharides. Both Pn14 and PT are potential components of vaccines for infants and children. Accordingly, a synthetic scheme was devised to prepare a conjugate of Pn14 and PT. An adipic acid hydrazide derivative of Pn14 was bound to PT at pH 3.9 by carbodiimide-mediated condensation. The conjugation procedure inactivated the PT as assayed by CHO cell and histamine-sensitizing activity. The Pn14-PT conjugate elicited antibodies in mice to Pn14 at levels estimated to be protective in humans and elicited neutralizing antibodies to PT. We plan to evaluate Pn14-PT clinically.
Effects of gamma interferon on release of tumor necrosis factor alpha from lipopolysaccharide-tolerant human monocyte-derived macrophages
Authors: Matic M , Simon S R
Published: January 2020

After an initial stimulation of human monocyte-derived macrophages with bacterial lipopolysaccharide (LPS), which produces substantial release of tumor necrosis factor-alpha (TNF-alpha), a subsequent exposure to LPS results in about an order-of-magnitude reduction in the levels of TNF-alpha released. We have shown that macrophages which have been stimulated with LPS and then maintained in culture without LPS for as long as 2 weeks do not regain their original capacity to secrete TNF-alpha upon a second LPS challenge. After 2 to 4 days in adherent culture, monocyte-derived macrophages which were not pretreated with LPS also experience a measurable decline in their capacity to release TNF-alpha in response to an initial LPS stimulation. When compared with these previously nonstimulated cells, however, the levels of TNF-alpha released by LPS-pretreated cells in response to a second LPS challenge decline by over 90% after 8 to 9 days in culture. Unstimulated cells spontaneously release barely detectable levels of TNF-alpha. In contrast to the release of TNF-alpha, unstimulated cells release significant levels of prostaglandin E2 continuously over time, and these levels are variably increased by no more than a factor of two in response to a single LPS stimulation. Prostaglandin E2 levels released by LPS-pretreated cells in response to a second LPS stimulation are much closer to the levels released by unstimulated cells. We have also demonstrated that gamma interferon (IFN-gamma) enhances TNF-alpha release from LPS-stimulated macrophages but not from phorbol myristate acetate-stimulated cells. Addition of IFN-gamma to macrophages either during the initial stimulation or during a second stimulation with LPS enhances levels of TNF-alpha released after the second LPS challenge. The greatest enhancement is observed when IFN-gamma is added during both exposures to LPS, but addition of IFN-gamma during only the initial LPS stimulation still results in marked enhancement of TNF-alpha release in response to a second stimulation with LPS 24 h later. If an interval of 2 days of culture in medium alone separates the first and second 24-h LPS stimulations, IFN-gamma enhances TNF-alpha release only when it is included during the second LPS exposure, indicating that, unlike the persistence of endotoxin tolerance, enhancement of TNF-alpha release by IFN-gamma is transient.
Inhibition of bactericidal activity of anticapsular antibody by nonspecific antibodies reactive with surface-exposed antigenic determinants on Actinobacillus pleuropneumoniae
Authors: Udeze F A , Kadis S
Published: January 2020

In an attempt to understand the mechanism of serum resistance in Actinobacillus pleuropneumoniae, in the present study we examined various interactions among the bacterial surface constituents, serum antibodies, and complement. Analysis of swine sera revealed the presence of anticapsular antibodies in convalescent-phase sera but not in preimmune sera. Both types of sera contained antibodies which reacted with each of 14 polypeptides present in saline extracts of the bacteria. Absorption of the preimmune sera with intact bacteria depleted antibodies to two of the polypeptides (27 and 32 kDa) and high-molecular-weight (greater than 97.4,000) components which did not stain with Coomassie blue. Data derived from complement consumption and C3-binding experiments indicated that the organism was capable of initiating complement activation and binding C3 during incubation in preimmune and immune sera. Experiments designed to evaluate the bactericidal effectiveness of anticapsular antibody revealed that the purified antibody was bactericidal only when preimmune sera absorbed with intact bacteria were used as a source of complement. The bactericidal effects of anticapsular antibody and absorbed preimmune sera were inhibited in a dose-dependent manner by heat-inactivated preimmune sera and immunoglobulin G derived from the sera. The inhibitory activity of the preimmune sera was neutralized by preincubating the sera with column fractions of the saline extract which contained either the 27- or the 32-kDa polypeptide. These results indicate that serum resistance in A. pleuropneumoniae 4074 could be related to inhibition of the bactericidal action of anticapsular antibody by nonspecific antibodies which recognize surface-exposed epitopes on the polypeptides.
Influence of saliva on aggregation and adherence of Streptococcus gordonii HG 222
Authors: Ligtenberg A J , Walgreen-Weterings E , Veerman E C , de Soet J J , de Graaff J , Amerongen A V
Published: January 2020

The influence of saliva on the aggregation and adherence of Streptococcus gordonii HG 222 was studied. The aggregation was measured spectrophotometrically, and the adherence of S. gordonii to microtiter plate wells was measured in an enzyme-linked immunosorbent assay system. The aggregation of HG 222 was induced primarily by mucous saliva, whereas the adherence of HG 222 to microtiter plates was mediated by both mucous and serous saliva. Fractions of submandibular saliva, obtained by gel filtration and containing low-molecular-weight mucins (MG-2), induced both bacterial aggregation and adherence. Purified MG-2 induced aggregation and promoted adherence, whereas high-molecular-weight mucins (MG-1) did not. After incubating clarified human whole saliva with HG 222, only MG-2, and not MG-1, was bound by the bacteria. Proline-rich proteins (PRPs) and proline-rich glycoprotein (PRG) promoted the adherence of HG 222. These proteins in solution bound to HG 222 but did not induce aggregation of the bacterial cells. PRPs and PRG in solution were not able to inhibit adherence to microtiter plate wells coated with the same components. Purified alpha-amylase hardly promoted adherence to microtiter plates but, in the soluble state, readily bound to HG 222. In conclusion, these results indicate that the aggregation of S. gordonii HG 222 is mediated primarily by MG-2. These mucins also promote adherence. Several other salivary components, such as PRPs and PRG, are also involved in the adherence of HG 222.
Identification of an acute-phase reactant in murine infections with Trypanosoma brucei
Authors: Shapiro S Z , Black S J
Published: January 2020

A 42-kDa protein appeared at a much higher concentration in plasma from Trypanosoma brucei-resistant (C57BL/6) mice after infection than in plasma from trypanosome-susceptible (C3H/He) mice. This protein was purified by sequential steps of gel filtration, protein A-Sepharose affinity chromatography, isoelectric focusing, and ammonium sulfate precipitation. The purified protein was identified as a subunit of the acute-phase reactant haptoglobin. Causes of elevated plasma haptoglobin and its implications for resistance to trypanosomiasis are discussed.
Complete nucleotide sequence of the Actinomyces viscosus T14V sialidase gene: presence of a conserved repeating sequence among strains of Actinomyces spp
Authors: Yeung M K
Published: January 2020

The nucleotide sequence of the Actinomyces viscosus T14V sialidase gene (nanH) and flanking regions was determined. An open reading frame of 2,703 nucleotides that encodes a predominately hydrophobic protein of 901 amino acids (M(r), 92,871) was identified. The amino acid sequence at the amino terminus of the predicted protein exhibited properties characteristic of a typical leader peptide. Five 12-amino-acid units that shared between 33 and 67% sequence identity were noted within the central domain of the protein. Each unit contained the sequence Ser-X-Asp-X-Gly-X-Thr-Trp, which is conserved among other bacterial and trypanosoma sp. sialidases. Thus, the A. viscosus T14V nanH gene and the other prokaryotic and eukaryotic sialidase genes evolved from a common ancestor. Southern hybridization analyses under conditions of high stringency revealed the existence of DNA sequences homologous to A. viscosus T14V nanH in the genomes of 18 strains of five Actinomyces species that expressed various levels of sialidase activity. The data demonstrate that the sialidase genes from divergent groups of Actinomyces spp. are highly conserved.
Analysis of B-cell epitopes in the variable C-terminal region of the Mycobacterium leprae 70-kilodalton heat shock protein
Authors: Peake P W , Britton W J , Davenport M P , Roche P W , McKenzie K R
Published: January 2020

The C-terminal region of the Mycobacterium leprae 70-kDa heat-shock protein is the major target for the humoral immune response to this protein and contains M. leprae-specific sequences. To examine B-cell responses to this region more closely, we constructed and expressed a recombinant fragment of the M. leprae P70 gene that encodes the C-terminal 142 residues (C-142) and synthesized a series of 10 overlapping peptides to encompass this region. The affinities of three monoclonal antibodies (MAbs) reactive with this region of P70 were measured, and the binding site of the highest-affinity MAb was determined to lie between residues 498 and 515. This reactivity was confirmed by a fluid-phase inhibition enzyme-linked immunosorbent assay. By contrast, sera from leprosy patients which were strongly reactive with the C-142 fragment failed to bind directly to the conjugated or unconjugated peptides. To determine whether the M. leprae-specific C-terminal 70 residues could stimulate B-cell responses, the reactivity of hyperimmune anti-M. leprae P70 antisera with the peptides was examined. Rabbit polyclonal anti-M. leprae P70 antisera recognized epitopes between residues 498 and 515 and in the M. leprae-specific region between residues 567 and 591. The latter, in turn, when coupled to ovalbumin, was able to generate a strong anti-P70 response specific for mycobacterial, but not human, HSP70. Three strains of mice immunized with either C-142 or P70 recognized epitopes in the region between residues 487 and 532, but the response varied with the strain and immunogen. These data demonstrate that two regions in the C-terminal portion of M. leprae P70 contain linear B-cell epitopes recognized by MAbs or hyperimmune serum. Sera from leprosy patients, however, react predominantly with conformational determinants in the immunodominant C-terminal part of the protein.
Nucleotide sequence of the variable domains within the major outer membrane protein gene from serovariants of Chlamydia trachomatis
Authors: Lampe M F , Suchland R J , Stamm W E
Published: January 2020

During studies in which we serotyped large numbers of Chlamydia trachomatis clinical isolates by using monoclonal antibodies, three novel serological variants (D-, D*, and I-) were identified. To determine the molecular basis for the altered monoclonal antibody reactions of these strains and other previously identified variants (Da, Ia, and L2a), we determined the nucleotide sequences of the variable domains in their major outer membrane protein genes. Da, D-, and D* variants differed by a single nucleotide and also an amino acid in the carboxy terminus of variable domain IV (VDIV) from the D serovar. The L2a variant also differed from L2 by a single amino acid but in VDII. Ia variants differed in VDI, III, and IV and I- variants differed in all four VDs from the I serovar. These studies demonstrate the potential for using major outer membrane protein VD sequencing as a highly sensitive typing method and further identify immunologically reactive major outer membrane protein epitopes.
Relative avidity of serum immunoglobulin G antibody for the fimbria antigen of Actinobacillus actinomycetemcomitans in patients with adult periodontitis
Authors: Saito A , Hosaka Y , Nakagawa T , Yamada S , Okuda K
Published: January 2020

The relative avidity of serum immunoglobulin G antibody for the fimbria antigen of Actinobacillus actinomycetemcomitans was assessed by diethylamine dissociation enzyme-linked immunosorbent assay in patients with adult periodontitis. High-titer sera from patients not harboring A. actinomycetemcomitans had significantly higher avidities for the fimbria antigen than did high-titer sera from patients with A. actinomycetemcomitans in their periodontal pockets. The elicited antibodies against the fimbria antigen may afford protection against A. actinomycetemcomitans infection.
Binding of pertussis toxin to lipid vesicles containing glycolipids
Authors: Hausman S Z , Burns D L
Published: January 2020

The binding of pertussis toxin and its B oligomer to lipid vesicles containing glycosphingolipids was studied. Both pertussis toxin and the B oligomer bound to lipid vesicles containing ganglioside GD1a. Binding of pertussis toxin to these vesicles decreased upon treatment of the vesicles with neuraminidase, suggesting that sialic acid residues are important for efficient binding of the toxin to GD1a.
Adherence of Helicobacter pylori to primary human gastrointestinal cells
Authors: Clyne M , Drumm B
Published: January 2020

Helicobacter pylori adheres only to gastric cells in vivo. However, the organism adheres to a wide variety of nongastric cells in vitro. In this study, we have used flow cytometry to assess the adherence of H. pylori to primary epithelial cells isolated from gastric, duodenal, and colonic biopsy specimens by collagenase digestion. After incubation of bacteria and cells together and subsequent staining with a two-stage fluorescein isothiocyanate-labelled H. pylori antibody method, cells with adherent bacteria could be easily distinguished from cells without bacteria. Binding to Kato III cells (a gastric adenocarcinoma cell line) was saturable when bacteria and cells were mixed at a ratio of 250:1. Adherence to cells isolated from gastric biopsy specimens was significantly better than adherence to cells isolated from duodenal or colonic biopsy specimens. Almost 70% of gastric cells had bacteria bound, in contrast to 30% of duodenal cells and 32% of colonic cells (P < 0.0001). There was no correlation between expression of hemagglutinins by the bacteria and ability to bind to either Kato III cells or primary epithelial cells isolated from gastric biopsy specimens. In view of the strict tropism that the organism exhibits in vivo for gastric cells, the results of this study indicate that primary cells are ideal for assessing the factors that might play a role in the pathogenesis of disease caused by the organism.
Characterization of novel type C staphylococcal enterotoxins: biological and evolutionary implications
Authors: Marr J C , Lyon J D , Roberson J R , Lupher M , Davis W C , Bohach G A
Published: January 2020

The type C staphylococcal enterotoxins (SEC) are a group of highly conserved proteins with significant immunological cross-reactivity. Although three antigenically distinct SEC subtypes (SEC1, SEC2, and SEC3) have been reported in the literature, we observed that the isoelectric points of SEC from several Staphylococcus aureus isolates are different from those of any of these three subtypes. This observation led us to propose that additional SEC molecular variants exist. For assessment of this possibility, the sec genes from representative human, animal, and food isolates were cloned and sequenced. The toxins encoded by the 18 isolates used in this study included five unique SEC proteins in addition to SEC1, SEC2, and SEC3. Six of the SEC proteins (including SEC1, SEC2, and SEC3) were produced by human and food isolates. Analysis of seven bovine and ovine isolates showed that isolates from each animal species produced a unique host-specific SEC. All of the SEC caused lymphocyte proliferation, although some of the toxins differed in their ability to stimulate cells from several animal species. An explanation for these results, which is supported by our phenotypic analysis of Sec+ staphylococcal isolates, is that toxin heterogeneity is due to selection for modified SEC sequences that facilitate the survival of S. aureus isolates in their respective hosts.
The gene encoding phosphoribosylaminoimidazole carboxylase (ADE2) is essential for growth of Cryptococcus neoformans in cerebrospinal fluid
Authors: Perfect J R , Toffaletti D L , Rude T H
Published: January 2020

A cryptococcal meningitis model in corticosteroid-treated rabbits was used to assess the requirement for the phosphoribosylaminoimidazole gene (ADE2) for virulence of Cryptococcus neoformans. A wild-type strain (H99), an ade2 auxotroph of H99 (M001), and a randomly selected prototrophic transformant of M001 (M001.1c) which had received the cloned ADE2 cDNA copy were inoculated intrathecally into immunosuppressed rabbits. While M001 was avirulent in the central nervous system model, virulence was completely restored to wild-type pathogenicity in the prototrophic transformant. This study identifies the pathogenic importance of an endogenous adenine pathway in this yeast and confirms that purine biosynthesis is a potential target for antifungal therapy. It also demonstrates that the virulence of C. neoformans can be molecularly changed and detected within a clinically relevant animal model.
Three-dimensional reconstruction of Coxiella burnetii-infected L929 cells by high-voltage electron microscopy
Authors: Hechemy K E , McKee M , Marko M , Samsonoff W A , Roman M , Baca O
Published: January 2020

Previous examination of thin sections of L929 cells heavily infected with the Q fever Priscilla isolate by conventional transmission electron microscopy indicated that the rickettsiae resided within multiple vacuoles. The present study using high-voltage electron microscopy and three-dimensional reconstruction revealed that, in heavily infected cells, the rickettsiae, in fact, reside in one multilobed vacuole. As a result of asymmetric cell division, the multilobed vacuole containing the rickettsiae apparently segregates into one daughter cell, while the companion daughter cell emerges parasite free. This likely explains the appearance of naive uninfected cells in long-term-infected (i.e., ca. 2 years) cell populations that had not been supplemented with uninfected L929 host cells.
The 39-kilodalton protein of Borrelia burgdorferi: a target for bactericidal human monoclonal antibodies
Authors: Scriba M , Ebrahim J S , Schlott T , Eiffert H
Published: January 2020

Three human monoclonal immunoglobulin M antibodies against Borrelia burgdorferi, obtained from in vitro-stimulated peripheral blood lymphocytes, reacted in Western blots (immunoblots) with a prominent 39-kDa peptide and a faint band of approximately 66 kDa. Two of these antibodies showed bactericidal activity without addition of complement. All three antibodies were reactive in an enzyme immunoassay with cloned P39 (W.J. Simpson, M.E. Schrumpf, and T.G. Schwan, J. Clin. Microbiol. 28:1329-1337, 1990), suggesting that the target molecule of these antibodies is identical to the P39 protein. In addition, the majority of supernatants from human lymphocytes stimulated in vitro with crude B. burgdorferi antigen reacted in this assay, demonstrating that P39, although a minor component of B. burgdorferi, is an immunodominant antigen in these spirochetes. A fourth monoclonal antibody, reacting with OspA, also exhibited bactericidal activity.
Novel antigens expressed by Aeromonas salmonicida grown in vivo
Authors: Thornton J C , Garduño R A , Carlos S J , Kay W W
Published: January 2020

Virulent and avirulent Aeromonas salmonicida strains grown inside intraperitoneal implants in Rainbow trout (Oncorhynchus mykiss) were examined for unique antigen expression. Western blots (immunoblots), performed with immune rabbit serum raised against in vivo-grown cells, revealed several unique antigens. With the exception of lipopolysaccharide (LPS), these novel antigens were destroyed after proteinase K treatment. The majority of these antigens were not induced in vitro in response to either iron limitation or anaerobiosis. In addition, electron microscopy demonstrated the presence of a putative capsule on in vivo-grown cells. Purification and fractionation of this carbohydrate material from cells grown in carbon-rich synthetic media resulted in the isolation and separation of an antigenically distinct LPS not seen with cells grown in standard media. Antiserum raised against in vivo-grown cells recognized both this LPS and the typical LPS of A. salmonicida apparent in in vitro-grown cells. Antiserum raised against in vitro-grown cells recognized only the LPS expressed in vitro. Antiserum directed against in vivo-grown cells was approximately 10 times more sensitive than serum directed against in vitro-grown cells in detecting A. salmonicida in infected fish kidney tissue.
Characterization of neuraminidases produced by various serotypes of Pasteurella haemolytica
Authors: Straus D C , Jolley W L , Purdy C W
Published: January 2020

Neuraminidases produced by 16 strains of Pasteurella haemolytica (serotypes 1 to 16) were characterized by molecular weight, antigenic identity, and substrate specificity. After growth in a chemically defined medium, stage I (lyophilized) culture supernatants were assayed for activity with N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Neuraminidase produced by P. haemolytica serotype A1 (Ph A1) was purified by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. Purified Ph A1 neuraminidase was used to immunize rabbits, and the resultant antiserum reduced the activity of Ph A1 neuraminidase by 46%. This antiserum also reduced the activity of neuraminidase produced by the other serotypes by between 15 and 66%. Molecular weight estimates of the neuraminidases produced by the various serotypes were obtained by gel filtration chromatography on Sephadex G-200. Fifteen of the 16 serotypes examined produced a neuraminidase with a molecular weight of approximately 150,000 to 200,000. One serotype (serotype 11) produced no material with neuraminidase activity. In addition, all 15 high-molecular-weight neuraminidases showed similar substrate specificities. That is, they were all most active against N-acetylneuramin lactose and least active against bovine submaxillary mucin. On the basis of these results, it appears that the high-molecular-weight neuraminidases produced by the different P. haemolytica serotypes are quite similar.
Stimulation of protective antibodies against type Ia and Ib group B streptococci by a type Ia polysaccharide-tetanus toxoid conjugate vaccine
Authors: Wessels M R , Paoletti L C , Rodewald A K , Michon F , DiFabio J , Jennings H J , Kasper D L
Published: January 2020

Antisera elicited by type Ia group B streptococci (GBS) contain antibodies that react with both type Ia and type Ib strains. Previous studies suggested that antibodies elicited by type Ia organisms recognized a carbohydrate antigen or epitope common to Ia and Ib strains. We now report the synthesis and immunogenicity testing of a type Ia polysaccharide-tetanus toxoid (Ia-TT) conjugate vaccine. Ia-TT elicited type Ia polysaccharide-specific immunoglobulin G antibodies in all three of the rabbits inoculated. In competitive enzyme-linked immunosorbent assay, these antibodies reacted with high affinity to type Ia polysaccharide and with lower affinity to the structurally related GBS type Ib polysaccharide. Despite the lower binding affinity of the Ia-TT-induced antibodies for the type Ib polysaccharide, Ia-TT antiserum opsonized not only type Ia GBS but also type Ib GBS for killing by human blood leukocytes. Ia-TT antiserum was also evaluated in a mouse model designed to test the efficacy of maternal antibodies in protecting neonates against GBS infection. Pups born to dams that had received Ia-TT antiserum were protected against lethal challenge with either type Ia or Ib GBS. These studies using a polysaccharide-protein conjugate as an immunogen support the view that the carbohydrate immunodeterminant recognized on Ib strains by Ia antisera is a common epitope contained within the structurally related Ia and Ib capsular polysaccharides. Although antibodies elicited by Ia-TT had protective activity against both Ia and Ib strains, these antibodies reacted with lower affinity to Ib than to Ia polysaccharide.
Correlation between vacuolating cytotoxin production by Helicobacter pylori isolates in vitro and in vivo
Authors: Cover T L , Cao P , Lind C D , Tham K T , Blaser M J
Published: January 2020

Approximately 50 to 60% of Helicobacter pylori isolates produce a vacuolating cytotoxin in vitro. To assess cytotoxin production in vivo, we sought to determine whether infection with a Tox+ H. pylori strain is associated with the presence of serum antitoxin antibodies. H. pylori isolates and serum samples were obtained from 30 patients, and serum samples were obtained from 20 uninfected patients as controls. Sera were tested by enzyme-linked immunosorbent assay for reactivity with the purified 87-kDa vacuolating cytotoxin, and the 30 H. pylori isolates were tested for vacuolating cytotoxin production. Supernatants from 14 (47%) of the 30 H. pylori isolates induced vacuolation of HeLa cells. Sera from the 30 H. pylori-infected patients reacted with the purified 87-kDa cytotoxin to a greater extent than sera from the uninfected controls for both immunoglobulin G (IgG) and IgA classes (P = 0.0004 and P < 0.0001, respectively). Serum IgG and IgA responses to the purified 87-kDa cytotoxin were higher among the 14 patients infected with Tox+ strains than among the 16 patients infected with Tox- strains (mean optical densities +/- standard errors of the means of 0.603 +/- 0.11 versus 0.234 +/- 0.07 [P = 0.005] and 0.644 +/- 0.12 versus 0.341 +/- 0.08 [P = 0.04] for IgG and IgA, respectively). Infection with a Tox+ strain compared with a Tox- strain was associated with increased antral polymorphonuclear leukocyte inflammation scores (P = 0.04). These data indicate that cytotoxin production by H. pylori isolates in vitro correlates with cytotoxin production in vivo and that infection with Tox+ H. pylori isolates may be associated with increased antral mucosal polymorphonuclear leukocyte infiltration.
Immunomodulation of human peripheral blood mononuclear cell functions by defined lipid fractions of Mycobacterium avium
Authors: Barrow W W , de Sousa J P , Davis T L , Wright E L , Bachelet M , Rastogi N
Published: January 2020

Mycobacterial fractions, some of which are associated with the cell envelope of Mycobacterium avium serovar 4, were assessed for their ability to affect various immunological functions of human peripheral blood mononuclear cells (PBM). Treatment of PBM with a total lipid fraction derived from M. avium serovar 4 resulted in a significant suppression of lymphoproliferative responsiveness to phytohemagglutinin stimulation at concentrations not affecting cell viability. Although a similar suppression was not observed when PBM were treated with purified serovar 4-specific glycopeptidolipids (GPL), treatment with the beta-lipid fragment derived from the GPL did result in a significant suppression of phytohemagglutinin responsiveness. Further studies revealed that the total lipid fraction and the beta-lipid fragment were effective at significantly reducing the ability of human macrophages to restrict the intracellular growth of mycobacteria and at stimulating PBM to secrete prostaglandin E2. These same effects were not observed when purified GPL or the reduced oligosaccharide fragment of the GPL was used. Other studies revealed that the total lipid and purified GPL fractions were effective at stimulating tumor necrosis factor alpha release from human PBM, whereas the beta-lipid fragment was not. These results indicate that mycobacterial lipids have various immunomodulatory capabilities, depending upon their chemical nature and ability to interact with certain host cells.
Regulation of inflammation-primed activation of macrophages by two serum factors, vitamin D3-binding protein and albumin
Authors: Yamamoto N , Kumashiro R , Yamamoto M , Willett N P , Lindsay D D
Published: January 2020

A very small amount (0.0005 to 0.001%) of an ammonium sulfate [50% saturated (NH4)2SO4]-precipitable protein fraction of alpha 2-globulin efficiently supported inflammation-primed activation of macrophages. This fraction contains vitamin D3-binding protein essential for macrophage activation. Comparative macrophage activation studies with fetal calf serum, alpha 2-globulin fraction, 50% (NH4)2SO4 precipitate, and purified bovine vitamin D3-binding protein revealed that fetal calf serum and alpha 2-globulin fraction appear to contain an inhibitor for macrophage activation while ammonium sulfate precipitate contains no inhibitor. This inhibitor was found to be serum albumin. When bovine serum albumin (25 micrograms/ml) was added to a medium supplemented with 0.0005 to 0.05% (NH4)2SO4 precipitate or 1 to 10 ng of vitamin D3-binding protein per ml, activation of macrophages was inhibited.
Purification and characterization of pili of a Vibrio cholerae non-O1 strain
Authors: Yamashiro T , Nakasone N , Iwanaga M
Published: January 2020

Pili of the Vibrio cholerae non-O1 strain V10 were purified and characterized. The V10 pili were physicochemically and immunologically different from those of the previously reported V. cholerae non-O1 strain S7, although the pili of the two strains had homologous N-terminal amino acid sequences. V10 plus antigen was detected only in V. cholerae non-O1 strains.
Circulating tumor necrosis factor alpha (TNF), soluble TNF receptors, and interleukin-6 in human subacute bacterial endocarditis
Authors: Kern W V , Engel A , Schieffer S , Prümmer O , Kern P
Published: January 2020

Cell surface components of viridans streptococci and enterococci have been shown to stimulate the release of tumor necrosis factor alpha (TNF) and interleukin-6 from monocytes/macrophages. In the sera from 10 patients with subacute enterococcal or streptococcal endocarditis, however, the levels of both cytokines were low or undetectable, with elevated TNF levels on admission in 3 patients with complicated disease. Soluble TNF receptor levels were significantly elevated compared with those of healthy controls. When patients with malaria were used as a control group of acute intravascular infection with high circulating TNF values, the ratio between soluble TNF receptors and TNF on admission was significantly greater in the patients with subacute bacterial endocarditis. Besides different amounts of circulating TNF, enhanced TNF receptor shedding may have an important role in the pathogenesis of subacute versus acute clinical disease following human intravascular infection.
Mutations in the promoter spacer region and early transcribed region increase expression of staphylococcal enterotoxin A
Authors: Borst D W , Betley M J
Published: January 2020

The mechanism leading to increased production of staphylococcal enterotoxin type A (SEA) in mutant Staphylococcus aureus FRI722 compared within its wild-type parent strain, FRI100, was examined. Sequence analysis revealed two mutations in the upstream promoter region of FRI722 at nucleotides -28 and +3 with respect to the transcriptional initiation site at An sea translational fusion of the upstream region of FRI722 to the structural gene from FRI100 showed an increase in sea expression by Northern (RNA) analysis and in SEA production by Western (immunoblot) analysis. To independently evaluate the effect of each mutation, site-directed mutagenesis was done and revealed that each mutation was responsible for an increase in SEA production.