bluuebunny/crossref_metadata_2025 huggingface dataset:
Staphylococcal exopolysaccharides inhibit lymphocyte proliferative responses by activation of monocyte prostaglandin production
Authors: Stout R D , Ferguson K P , Li Y N , Lambe D W
Published: January 2020

The glycocalyx (exopolysaccharides) of Staphylococcus epidermidis has been reported to inhibit a variety of host defense mechanisms. We have examined the inhibitory effects of glycocalyx on the proliferation of human peripheral blood mononuclear cells (PBMC) and the mechanism of this inhibition. Glycocalyx isolated and partially purified under endotoxin-free conditions from defined liquid medium cultures of S. epidermidis and Staphylococcus lugdunensis inhibited the proliferative response of PBMC when added to cultures at 10 to 100 micrograms/ml. Glycocalyx-mediated inhibition of phytohemagglutinin-stimulated proliferation of PBMC required the presence of plastic-adherent peripheral blood monocytes. Culture supernatants of monocytes stimulated with glycocalyx contained a soluble factor that inhibited the proliferation of monocyte-depleted PBMC. This soluble inhibitory factor was not produced in the absence of glycocalyx or in the presence of both glycocalyx and indomethacin. Analysis of the supernatants of cultures of adherent monocytes revealed that glycocalyx from S. epidermidis and from S. lugdunensis could activate monocyte production of prostaglandin E2 (PGE2), human interleukin-1, and tumor necrosis factor alpha. The addition of purified PGE2, at the same levels of PGE2 (greater than or equal to 10(-9) M) generated in the monocyte cultures, to PBMC cultures resulted in a similar inhibition of proliferative responses. It is concluded that, contrary to previous suggestions, the bacterial glycocalyx does not have a direct inhibitory effect on T lymphocytes. However, it does appear that glycocalyx from coagulase-negative staphylococci can activate monocyte PGE2 production and that it is this activity that in turn contributes to the inhibition of T-cell proliferation.
Plasmodium falciparum-infected erythrocytes do not adhere well to C32 melanoma cells or CD36 unless rosettes with uninfected erythrocytes are first disrupted
Authors: Handunnetti S M , Hasler T H , Howard R J
Published: January 2020

Plasmodium falciparum malaria parasites modify the human erythrocytes in which they grow so that some parasitized erythrocytes (PE) can cytoadhere (C+) to host vascular endothelial cells or adhere in rosettes (R+) to uninfected erythrocytes. These C+ and R+ adherence properties of PE appear to mediate much of the pathogenesis of severe malaria infections, in part by blocking blood flow in microvessels. From one parasite strain, PE were selected in vitro for C+ R+ or C+ R- adherence properties and examined in model adherence assays. The C+ R+ PE cytoadhered poorly to C32 melanoma cells or to immobilized CD36 in a settled-cell assay when uninfected human erythrocytes were present and formed rosettes with PE. C+ R- PE adhered well in the same assays. However, C+ R+ PE adhered very well, even better than C+ R- PE, when the rosettes were disrupted and the C+ R+ PE were purified. Adding back rabbit erythrocytes, which do not form rosettes with C+ R+ PE, had simply a dilutional effect. The ability of rosettes to interfere with the detection of adherence must be dealt with in all future assays of malarial PE adherence. Individual PE were observed attached simultaneously to C32 cells and to a few erythrocytes, suggesting that C+ and R+ adherence properties are coexpressed on the same PE. Coexpression of these adherence properties on the same PE may have pathological importance in vivo, where passage of rosettes through capillaries may shear uninfected erythrocytes from rosetted PE and allow direct PE attachment to postcapillary venule walls before rosettes reform.
Level of neutrophil chemotactic factor CINC/gro, a member of the interleukin-8 family, associated with lipopolysaccharide-induced inflammation in rats
Authors: Iida M , Watanabe K , Tsurufuji M , Takaishi K , Iizuka Y , Tsurufuji S
Published: January 2020

Rat cytokine-induced neutrophil chemoattractant (CINC), which is a counterpart of human gro and belongs to the interleukin-8 family, has been quantified by a new sandwich enzyme-linked immunosorbent assay. Administration of lipopolysaccharide (LPS) into an air pouch performed by subcutaneous injection of air caused inflammation and severe neutrophil infiltration. After the LPS injection, changes in the concentration of CINC/gro, chemotactic activity, and the number of neutrophils in the air pouch exudate were determined. The chemotactic activity of neutrophils was augmented before practical neutrophil infiltration. More than half of the chemotactic activity was neutralized by the antisera. The time kinetics of the level of CINC/gro coincided with the changes in chemotactic activity. The maximal level of rat CINC/gro was 85 ng/ml, which is sufficient to cause neutrophil migration in vitro and in vivo as described previously. These data suggest that rat CINC/gro is a functional chemoattractant for neutrophils in LPS-induced inflammation in rats.
Susceptibility of porcine intestine to pilus-mediated adhesion by some isolates of piliated enterotoxigenic Escherichia coli increases with age
Authors: Nagy B , Casey T A , Whipp S C , Moon H W
Published: January 2020

Two porcine isolates of enterotoxigenic Escherichia coli (ETEC) (serogroup O157 and O141) derived from fatal cases of postweaning diarrhea and lacking K88, K99, F41, and 987P pili (4P- ETEC) were tested for adhesiveness to small-intestinal epithelia of pigs of different ages. Neither strain adhered to isolated intestinal brush borders of newborn (1-day-old) pigs in the presence of mannose. However, mannose-resistant adhesion occurred when brush borders from 10-day- and 3- and 6-week-old pigs were used. Electron microscopy revealed that both strains produced fine (3.5-nm) and type 1 pili at 37 degrees C but only type 1 pili at 18 degrees C. Mannose-resistant in vitro adhesion to brush borders of older pigs correlated with the presence of fine pili. These strains produced predominantly fine pili in ligated intestinal loops of both older and newborn pigs, but adherence was greater in loops in older pigs. Immunoelectron microscopic studies, using antiserum raised against piliated bacteria and absorbed with nonpiliated bacteria, of samples from brush border adherence studies revealed labelled appendages between adherent bacteria and intestinal microvilli. Orogastric inoculation of pigs weaned at 10 and 21 days of age indicated significantly (P less than 0.001) higher levels of adhesion by the ETEC to the ileal epithelia of older pigs than to that of younger ones. We suggest that small-intestinal adhesion and colonization by these ETEC isolates is dependent on receptors that develop progressively with age during the first 3 weeks after birth. Furthermore, our data are consistent with the hypothesis that the fine pili described mediate intestinal adhesion by the 4P- ETEC strains studied.
Phagocytic killing of encapsulated and microencapsulated Staphylococcus aureus by human polymorphonuclear leukocytes
Authors: Xu S , Arbeit R D , Lee J C
Published: January 2020

Phagocytosis by human polymorphonuclear leukocytes (PMNs) is an important host defense against infections caused by Staphylococcus aureus. Using an in vitro assay, we compared the opsonic requirements for phagocytic killing of prototype strains of encapsulated (type 1) and microencapsulated (type 5 and type 8) S. aureus by human PMNs. More than 85% of broth-grown, logarithmic-phase type 5 and 8 S. aureus organisms were killed by PMNs incubated with fresh normal human, rabbit, or guinea pig serum with complement activity. Under similar conditions, the highly encapsulated type 1 strain was not killed. Both encapsulated and microencapsulated strains were opsonized for phagocytosis by heat-inactivated serum raised in rabbits to killed bacteria. Opsonization by homologous serum was required for phagocytosis of the type 1 strain. In contrast, microencapsulated type 5 and 8 S. aureus organisms were killed by heat-inactivated rabbit serum raised to type 5, type 8, or nonencapsulated isolates; this result suggested that antibodies to the capsule or to cell wall components other than the capsule could opsonize these organisms for phagocytosis. The specificity of the assay was confirmed with capsule type 5-specific monoclonal antibodies, which were opsonic only for the type 5 S. aureus isolate. These studies indicate that, unlike the highly encapsulated type 1 strain, broth-grown microencapsulated S. aureus strains do not resist opsonophagocytic killing in vitro by normal serum.
Identification of the mucin-binding adhesin of Pseudomonas cepacia isolated from patients with cystic fibrosis
Authors: Sajjan S U , Forstner J F
Published: January 2020

In previous experiments, we have shown that isolates of Pseudomonas cepacia from sputa of patients with cystic fibrosis (CF), particularly those with severe lung infection, exhibited specific binding to purified respiratory or intestinal mucins (U. Sajjan, M. Corey, M. Karmali, and J. Forstner, J. Clin. Invest. 89:648-656, 1992). The present report describes the identification of the adhesin as a protein located on fimbriae of mucin-binding P. cepacia. From a total of 53 isolates available (from 22 patients with CF), we used three mucin-binding and three non-mucin-binding isolates for our experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude P. cepacia homogenates was performed, the separated proteins were blotted onto nitrocellulose and overlaid with purified mucin, and mucin-binding components were detected with an antimucin antibody and then a second-antibody-alkaline phosphatase conjugate system. Only mucin-binding isolates exhibited a positively stained band at an Mr of 22,000. The 22-kDa protein was purified, and a polyclonal antibody specific for it was developed in rabbits. By electron microscopy and immunogold labelling, both the antibody and mucin (separately) were localized to pili present over the entire surface of the bacterial cells. Non-mucin-binding isolates did not have (or had very few) pili and did not stain with either mucin or the antibody to the 22-kDa protein. The purified 22-kDa protein and its antibody were each able to inhibit piliated P. cepacia binding to mucin. The amino acid composition of the 22-kDa protein was dissimilar to those of the major pilin proteins of Escherichia coli (type 1 pilus) and P. aeruginosa (PAK and PAO1 strains). Both the pili of P. aeruginosa PAK and PAO1 and antibodies to these pili failed to inhibit P. cepacia binding to mucin. Thus, P. cepacia adhesion to mucin is mediated by a pilin-associated 22-kDa protein which differs from epithelial-cell-binding pilin proteins of P. aeruginosa. We postulate that the 22-kDa adhesin may play a role in the virulence of P. cepacia lung infections of patients with CF.
Plasmodium falciparum malaria: evidence for an isotype imbalance which may be responsible for delayed acquisition of protective immunity
Authors: Bouharoun-Tayoun H , Druilhe P
Published: January 2020

In view of the recent demonstration that antibodies that are protective against Plasmodium falciparum malaria may act in collaboration with blood monocytes, we investigated the isotype content of sera from individuals with defined clinical states of resistance or susceptibility to malaria. Profound differences in the distribution of each immunoglobulin subclass were found. Immunoglobulin G1 (IgG1) and IgG3, two cytophilic isotypes, predominated in protected subjects. In nonprotected subjects, i.e., children and adults that have sustained a primary malarial attack, four different situations were encountered: (i) an imbalance in which IgG2, a noncytophilic class, predominated (mostly seen in primary attacks), (ii) an imbalance also concerning IgG2 but only of a given antigenic specificity, (iii) an imbalance in which mostly IgM antibodies predominated (a frequent event in children), and, less frequently, (iv) an overall low level of antimalarial antibodies. Of 33 nonimmune subjects studied, all but one had one of the above defects. The function of total immunoglobulin presenting such an isotype imbalance was studied in vitro in antibody-dependent cellular inhibition assays. IgG from protected subjects cooperated efficiently with blood monocytes, whereas IgG from nonprotected groups did not. Also, the latter could inhibit the in vitro effect of the former: in competition assays whole IgG from primary-attack cases with increased IgG2 content and IgG or IgM from children from endemic areas competed with IgG from immune adults. This led us to formulate the hypothesis that nonprotected subjects have antibodies to epitopes critical for protection, but that these antibodies are nonfunctional. These results bring some clues to the very long delay required to reach protection against malaria and clearly stress the need to investigate immune responses in both quantitative and qualitative terms.
Identification of Candida albicans antigens reactive with immunoglobulin E antibody of human sera
Authors: Ishiguro A , Homma M , Torii S , Tanaka K
Published: January 2020

Candida albicans antigens which reacted with immunoglobulin E (IgE) antibodies of 57 allergic patients were detected by immunoblotting. Of the various antigens, the 175-, 125-, 46-, 43-, and 37-kDa antigenic components reacted most frequently with the patient sera. To purify the major antigens, C. albicans cells were fractionated. The 46-, 43-, and 37-kDa antigens were recovered in cytoplasmic fractions, but the 175- and 125-kDa antigens were not recovered in any fraction. The 46-, 43-, and 37-kDa antigens were purified from cytoplasmic fractions by DEAE and P11 ion-exchange chromatography. Antigens were isolated by cutting bands out of sodium dodecyl sulfate-polyacrylamide gels. The purified components confirmed by immunoblotting were next processed for amino acid sequencing. Parts of the sequences of the 46-, 43-, and 37-kDa antigens had significant levels of homology with Saccharomyces cerevisiae glycolytic enzyme enolase, phosphoglycerate kinase, and aldolase, respectively. Rabbit IgG antibodies prepared against the 46- and 43-kDa antigens strongly cross-reacted with the homologous proteins of S. cerevisiae. However, S. cerevisiae enolase and phosphoglycerate kinase did not cross-react with IgE of patient sera. This result suggests that IgE antibodies against only small parts of their epitopes are elevated in the allergic patients. Since enolase is reported to be a major antigen for systemic candidiasis, this enzyme may be the immunodominant protein in both allergies and fungal infections.
Analysis of the N-terminal region of the 47-kilodalton integral membrane lipoprotein of Treponema pallidum
Authors: Weigel L M , Brandt M E , Norgard M V
Published: January 2020

The 47-kDa lipoprotein is an abundant integral membrane protein and dominant immunogen of Treponema pallidum subsp. pallidum. Previous DNA sequencing of the 47-kDa-lipoprotein gene did not reveal consensus features representative of other bacterial lipoprotein genes; this prompted further analyses with emphasis on elucidation of the N terminus of the molecule. To assist in localizing start signals for the protein, the transcription initiation site for the 47-kDa-antigen gene was determined. RNA isolated from both T. pallidum and recombinant Escherichia coli expressing the 47-kDa antigen was used as a template in reverse transcriptase primer extension. Upon analysis of cDNA products, transcription initiation was localized to one nucleotide in T. pallidum and to two adjacent nucleotides in E. coli. When various primers were used in DNA sequencing reactions for these analyses, a previously undetected nucleotide (G) was found in the purported 5' untranslated region; this altered the upstream reading frame to reveal plausible sites for ribosome binding (GGAGG), translation initiation (GTG start codon), and signal peptidase II processing (Val-Val-Gly-Cys). Discounting acylation, the molecular weight of the mature polypeptide is 45,756 (approximately 46,600 with acylation). To derive nonacylated 47-kDa antigen for further structure-function studies, the 47-kDa-antigen gene was subcloned without acylation signals as a genetic construct encoding a glutathione S-transferase fusion protein; following cleavage with thrombin, the nonacylated 47-kDa protein was hydrophilic rather than amphiphilic but retained its antigenicity when tested against 116 human serum samples from patients with various stages of syphilis.
Biochemical characterization of rat colonic mucins secreted in response to Entamoeba histolytica
Authors: Tse S K , Chadee K
Published: January 2020

Invasion of the colonic mucosa by Entamoeba histolytica trophozoites is preceded by colonic mucus depletion. The aim of our studies was to determine whether E. histolytica caused a differential secretion of mucin species in a rat colonic loop model. Mucus secretion in response to amoebae was followed by release of acid-precipitable 3H-glucosamine metabolically labelled glycoproteins and in vitro labelling of glycoprotein secretion with NaB3H4. The secretory response consisted of high-Mr goblet cell mucins and an increase in the secretion of low-Mr nonmucin glycoproteins as determined by Sepharose 4B column chromatography. High-Mr mucins subfractionated by Cellex-E (ECTEOLA) ion-exchange chromatography demonstrated a minor neutral and a major acidic mucin (greater than 98%) species. Marked differences between the neutral and acidic mucin species were indicated by immunogenicity and amino acid compositions. Thin-section histochemistry of rat colons confirmed secretion of neutral and acidic mucins in response to E. histolytica and demonstrated secretory activity from goblet cells from both the crypts and interglandular epithelium. E. histolytica mucus secretagogue activity was generalized and may function to deplete the host's protective mucus layer, facilitating invasion by the parasites.
Synthetic peptides analogous to the fimbrillin sequence inhibit adherence of Porphyromonas gingivalis
Authors: Lee J Y , Sojar H T , Bedi G S , Genco R J
Published: January 2020

Fimbriae are important in the adherence of many bacterial species to the surfaces they eventually colonize. Porphyromonas (Bacteroides) gingivalis fimbriae appear to mediate adherence to oral epithelial cells and the pellicle-coated tooth surface. The role and contribution of fimbriae in the binding of P. gingivalis to hydroxyapatite (HAP) coated with saliva as a model for the pellicle-coated tooth surface were investigated. 3H-labeled P. gingivalis or the radioiodinated purified fimbriae were incubated with 2 mg of HAP beads coated with whole human saliva (sHAP) and layered on 100% Percoll to separate unbound from sHAP-bound components. The radioactivity of the washed beads was a measure of the bound components. The binding of P. gingivalis 2561 (381) cells and that of purified fimbriae were concentration dependent and saturable at approximately 10(8) cells and 40 micrograms of fimbriae added, respectively. The addition of fimbriae inhibited binding of P. gingivalis to sHAP beads by 65%, while the 75-kDa protein, which is another major surface component of P. gingivalis 2561, did not show significant inhibition, suggesting that the fimbriae are important in adherence. Encapsulated and sparsely fimbriated P. gingivalis W50 did not bind to sHAP beads. On the basis of the predicted sequence of the fimbrillin, a structural subunit of fimbriae, a series of peptides were synthesized and used to localize the active fimbrillin domains involved in P. gingivalis adherence to sHAP beads. Peptides from the carboxyl-terminal one-third of the fimbrillin strongly inhibited P. gingivalis binding to sHAP beads. Active residues within the sequence of inhibitory peptide 226-245 (peptide containing residues 226 to 245) and peptide 293-306 were identified by using smaller fragments prepared either by trypsin cleavage of the peptide 226-245 or by synthesis of smaller segments of peptide 293-306. Hemagglutinin activity, lectinlike binding, and ionic interaction did not seem to be involved in this binding since lysine, arginine, carbohydrates, and calcium ions failed to affect the binding of P. gingivalis. The observation that poly-L-lysine, bovine serum albumin, and defatted bovine serum albumin, even at high concentrations, only partially blocked the binding of P. gingivalis indicates that hydrophobic interactions are not the major forces involved in P. gingivalis binding to sHAP beads. Protease inhibitors such as EDTA, leupeptin, pepstatin, 1,10-phenanthroline, and phenylmethylsulfonyl fluoride did not interfere with the binding of P. gingivalis. However, the binding of P. gingivalis to trypsin- or chymotrypsin-pretreated sHAP beads was reduced.(ABSTRACT TRUNCATED AT 400 WORDS)
Monoclonal secretory immunoglobulin A protects mice against oral challenge with the invasive pathogen Salmonella typhimurium
Authors: Michetti P , Mahan M J , Slauch J M , Mekalanos J J , Neutra M R
Published: January 2020

Hybridomas producing monoclonal immunoglobulin A (IgA) antibodies against Salmonella typhimurium were generated by mucosal immunization of BALB/c mice with attenuated strains of S. typhimurium and subsequent fusion of Peyer's patch lymphoblasts with myeloma cells. To test the role of secretory IgA (sIgA) in protection against Salmonella sp., we analyzed in detail the protective capacity of a monoclonal IgA, Sal4, produced in polymeric as well as monomeric forms, that is directed against a carbohydrate epitope exposed on the surface of S. typhimurium. BALB/c mice bearing subcutaneous Sal4 hybridoma tumors and secreting monoclonal sIgA into their gastrointestinal tracts were protected against oral challenge with S. typhimurium. This protection was directly dependent on specific recognition by the monoclonal IgA, since mice secreting Sal4 IgA from hybridoma tumors were not protected against a fully virulent mutant that lacks the Sal4 epitope. Although monoclonal Sal4 IgA was present in the bloodstreams and tissues of tumor-bearing mice, it did not protect against intraperitoneal challenge and did not possess complement-fixing or bacteriocidal activity in vitro. Taken together, these results indicate that secretion of sIgA alone can prevent infection by an invasive enteric pathogen, presumably by immune exclusion at the mucosal surface.
Synthetic trimer and tetramer of 3-beta-D-ribose-(1-1)-D-ribitol-5-phosphate conjugated to protein induce antibody responses to Haemophilus influenzae type b capsular polysaccharide in mice and monkeys
Authors: Peeters C C , Evenberg D , Hoogerhout P , Käyhty H , Saarinen L , van Boeckel C A , van der Marel G A , van Boom J H , Poolman J T
Published: January 2020

Synthetic oligosaccharides derived from the capsular polysaccharide (PRP) of Haemophilus influenzae type b were conjugated to carrier proteins via a thioether linkage. Conjugates were made of trimeric and tetrameric ribose-ribitol-phosphate and tetanus toxoid or diphtheria toxin. All conjugates elicited anti-PRP antibody responses with an increasing immunoglobulin G/immunoglobulin M ratio in adult mice and monkeys. Trimer conjugates elicited lower anti-PRP antibody responses compared with tetramer conjugates. Adult monkeys responded equally well to the tetrameric oligosaccharide-tetanus toxoid conjugate as to the oligosaccharide-CRM197 conjugate (HbOC), which elicits protective levels of serum antibodies in human infants after two or three injections.
Delivery and expression of a heterologous antigen on the surface of streptococci
Authors: Pozzi G , Contorni M , Oggioni M R , Manganelli R , Tommasino M , Cavalieri F , Fischetti V A
Published: January 2020

We have developed a system in which a foreign antigen replaces nearly all of the surface-exposed region of the fibrillar M protein from Streptococcus pyogenes and is fused to the C-terminal attachment motif of the M molecule. The fusion protein is thus expressed on the surface of Streptococcus gordonii, a commensal organism of the oral cavity. The antigen chosen to be expressed within the context of the M6 molecule was the E7 protein (98 amino acids) of human papillomavirus type 16. Stable recombinant streptococci were obtained by integrating genetic constructs into the chromosome, exploiting in vivo homologous recombination. The M6-E7 fusion protein expressed on the S. gordonii surface was shown to be immunogenic in mice. This is the first step in the construction of recombinant live vaccines in which nonpathogenic streptococci as well as other gram-positive bacteria may be used as vectors to deliver heterologous antigens to the immune system.
Interactions between conidia of Aspergillus fumigatus and human complement component C3
Authors: Sturtevant J E , Latgé J P
Published: January 2020

Activation and deposition of C3 on Aspergillus fumigatus conidia have been previously demonstrated. This study investigated in further detail the interactions between complement component C3 and the conidia of A. fumigatus. Immunoblotting and 125I-C3 binding studies showed that C3 deposition was rapid (less than 15 min) and parallel to the formation of iC3b. Immunoblotting experiments identified a 54- to 58-kDa conidial protein which binds human complement component C3 and/or a C3 fragment(s). 125I labeling of the outer layer of the conidia demonstrated that this protein doublet was present on the surface of the spore. The further degradation of C3 to low-molecular-mass fragments (40, 37, and 30 kDa), in the absence of plasma, by intact living conidia and a preparation of the outer conidial wall layer indicated the ability of fungal components to cleave C3. These data suggest that interactions between conidia and C3 are not limited only to deposition via activation of the alternative complement pathway; they also include degradation of bound C3.
Identification and characterization of B-cell epitopes of IpaC, an invasion-associated protein of Shigella flexneri
Authors: Phalipon A , Arondel J , Nato F , Rouyre S , Mazie J C , Sansonetti P J
Published: January 2020

Invasion plasmid antigen C (IpaC) is a 43-kDa plasmid-encoded protein associated with the ability of shigellae to invade epithelial cells. This protein is consistently strongly recognized by sera from convalescent patients and monkeys experimentally infected with shigellae. The strong immunogenicity of IpaC in the course of natural infection makes it a good candidate as a potentially protective antigen. To map the B-cell epitopes of this protein, the gene encoding IpaC was cloned and expressed at a high level in Escherichia coli. The partially purified recombinant protein was used to raise rabbit polyclonal antisera and murine monoclonal antibodies. A lambda gt11 ipaC gene library was screened with the antisera and antibodies. Recombinant DNA clones producing specific antigenic determinants were isolated, and the sequence of their DNA inserts was determined. The amino acid sequence of each determinant was deduced from the minimal overlap of DNA inserts of multiple antibody-positive DNA clones. Two distinct epitopes, located between amino acid residues 25 and 33 and 90 and 97, were identified. Two additional B-cell epitopes which were located between residues 297 and 349, near the carboxy-terminal end of the protein, were characterized. Each of these epitopes was also recognized by sera from convalescent humans and monkeys. Therefore, it seems likely that these epitopes are relevant to the humoral response against IpaC during natural infection.
Aspergillus antigen-induced eosinophil differentiation in a murine model
Authors: Murali P S , Dai G , Kumar A , Fink J N , Kurup V P
Published: January 2020

Eosinophilia is a prominent feature of the cellular response in allergic and parasitic diseases. Allergic bronchopulmonary aspergillosis due to colonization of the lungs of some asthmatics with Aspergillus fumigatus is characterized by high levels of serum immunoglobulin E and peripheral blood (PB) and lung eosinophilia. This study investigates the role of eosinophils in the pathogenesis of allergic bronchopulmonary aspergillosis by using a mouse model. BALB/c mice were immunized intranasally and intraperitoneally with A. fumigatus antigens (Ag), and the eosinophils in PB and bone marrow (BM) were enumerated. Eosinophilopoiesis in BM cultures was studied in the presence of murine recombinant interleukin-5 (mrIL-5) and supernatants from pokeweed mitogen-stimulated spleen cells as the source of eosinophil differentiation factors. Eosinophils were quantitated by direct counting and by estimating eosinophil peroxidase activity. The results indicate that the percentage of eosinophils in the PB (5.77 +/- 1.17) and the BM (11.19 +/- 4.31) of mice exposed to A. fumigatus Ag was higher than in controls (PB, 2.42 +/- 0.76; BM, 5.12 +/- 2.79; P less than 0.01 for both). Similarly, a significant increase in eosinophils was observed in the BM population from mice exposed to A. fumigatus Ag compared with that in controls when cultured with murine recombinant interleukin-5 (23.13 +/- 7.14 versus 13.77 +/- 5.79, P less than 0.01), indicating that the mice exposed to A. fumigatus Ag had significantly greater numbers of eosinophil precursors in their BM. This study demonstrates that A. fumigatus Ag may be involved in the in vivo commitment of stem cells in the eosinophil differentiation pathway.
Indirect measurement of intestinal immune responses to an orally administered attenuated bacterial vaccine
Authors: Forrest B D
Published: January 2020

Intestinal fluid, saliva, circulating peripheral blood lymphocytes (PBL), and serum samples obtained from 81 human adult subjects who had been orally vaccinated with either Salmonella typhi Ty21a or one of its recombinant derivatives were examined to determine the value of indirect measurements of an antigen-specific intestinal-immunoglobulin A (IgA) response. Salivary IgA failed to provide consistent or correlative responses, and no evidence of a significant relationship was apparent with the intestinal-IgA responses. No significant correlation between the specific increase in responses in serum IgA and intestinal IgA was evident. While the magnitude of the serum IgG response significantly correlated with the intestinal-IgA response (P = 0.00064), it failed to detect 14.8% of the intestinal-IgA responses. The observation that 16.6% of the subjects had delayed serum IgA responses, with a peak occurring after day 23 compared with days 12 to 14, may have contributed to the inadequacy of the serum IgA response as a correlative indicator. The serum IgG responses in these subjects were also of a diminished magnitude. Specific IgA production by circulating PBL was found to be the most sensitive (92.6% response rate) and correlative (P = 0.00071) indicator of a specific intestinal-IgA immune response. However, its value in predicting protective efficacy is untried. These studies confirm that for the assessment of an enteric bacterial vaccine, determination of in vitro specific IgA production by circulating PBL may offer a single measurement of specific immunity which is as useful as serum and intestinal measurements combined.
Characterization of the major membrane protein of virulent Mycobacterium tuberculosis
Authors: Lee B Y , Hefta S A , Brennan P J
Published: January 2020

A protein with a molecular mass of 19 kDa was isolated and purified from enriched membrane fractions of the virulent Erdman strain of Mycobacterium tuberculosis. The protein is different from another 19-kDa protein, a lipoprotein, that was recently described (D. B. Young and T. R. Garbe, Res. Microbiol. 142:55-65, 1991). The sequencing strategy applied to this major membrane protein employed four different endoproteinases and resulted in sufficient overlapping peptide sequences for assignment of the entire protein sequence. Electron spray ionization mass spectrometry demonstrated a measured mass of 16,100, deviating from the predicted mass by only 2.86 atomic mass units. The sequence of this protein is unique. However, some similarities with other low-molecular-weight heat shock proteins were observed. Immunoblotting indicated that this protein is highly expressed in the virulent strains of M. tuberculosis. Its application to sera from patients with pulmonary tuberculosis showed promise as a serodiagnostic tool.
Fate of conidia of Paracoccidioides brasiliensis after ingestion by resident macrophages or cytokine-treated macrophages
Authors: Cano L E , Brummer E , Stevens D A , Restrepo A
Published: January 2020

Conidia ingested by resident macrophages had an enhanced percentage of transformation to yeast cells compared with those in culture medium without macrophages. The yeast cells subsequently grew intracellularly by budding. Macrophages treated with cytokines from antigen-stimulated spleen cells from immunized mice significantly inhibited transformation of ingested conidia.
Urease-associated heat shock protein of Helicobacter pylori
Authors: Evans D J , Evans D G , Engstrand L , Graham D Y
Published: January 2020

Helicobacter pylori urease is an extracellular, cell-bound enzyme with a molecular weight of approximately 600,000 (600K enzyme) comprising six 66K and six 31K subunits. A 62K protein is closely associated with the H. pylori urease, both in crude preparations and after gel filtration; this protein can be removed from the urease by ion-exchange chromatography without inactivating the enzyme. We purified this urease-associated protein and determined its N-terminal amino acid sequence. The sequence is 80% homologous (identical plus conserved amino acid residues) to the Escherichia coli GroEL heat shock protein (HSP), 75% homologous to the human homolog, and 84% homologous to the HSP homolog found in species of Chlamydia. Thus, the 62K urease-associated protein of H. pylori belongs to the HSP60 family of stress proteins known as chaperonins. Evidently this protein, HSP62, participates in the extracellular assembly and/or protection of the urease against inactivation in the hostile environment of the stomach.
Heat shock treatment of macrophages causes increased release of superoxide anion
Authors: Reddy M V , Gangadharam P R
Published: January 2020

Heat shock treatment of murine macrophages and the J774 cell line resulted in an enhanced capacity to release superoxide anion (O2-) upon stimulation. There was no concomitant increase in hydrogen peroxide production, and the macrophage microbicidal activity against Mycobacterium tuberculosis, Mycobacterium avium complex, and Staphylococcus aureus was not altered.
Altered diacylglycerol level and metabolism in neutrophils from patients with localized juvenile periodontitis
Authors: Tyagi S R , Uhlinger D J , Lambeth J D , Champagne C , Van Dyke T E
Published: January 2020

Diacylglycerol, a physiological activator of protein kinase C, was elevated nearly twofold in unstimulated peripheral blood neutrophils from patients with localized juvenile periodontitis compared with cells from normal individuals. These cells also showed an enhanced and prolonged elevation of diglyceride in response to N-formylmethionylleucylphenylalanine. The metabolism of a cell-permeant diacylglycerol by diglyceride kinase was significantly decreased, because of a fivefold or higher elevation in the apparent Km of cellular diglyceride kinase.
Binding and neutralization of endotoxin by Limulus antilipopolysaccharide factor
Authors: Warren H S , Glennon M L , Wainwright N , Amato S F , Black K M , Kirsch S J , Riveau G R , Whyte R I , Zapol W M , Novitsky T J
Published: January 2020

In order to examine the ability of Limulus antilipopolysaccharide factor (LALF) to bind lipopolysaccharide (LPS), we purified LALF to homogeneity from Limulus amoebocyte lysate and coupled it covalently to agarose beads. LALF-coupled beads captured more tritiated LPS from rough and smooth strains of gram-negative bacteria than did control human serum albumin-coupled beads. Unlabeled homologous and heterologous LPS competed for the binding of 3H-LPS to LALF-coupled beads. LALF bound LPS in a dose-dependent manner as assessed by the precipitation of LPS-LALF complexes with 50% saturated ammonium sulfate. We also studied the ability of LALF to neutralize LPS. LPS preincubated with LALF was less mitogenic for murine splenocytes, was less pyrogenic in the rabbit fever assay, was less lethal in mice which had been sensitized to LPS with actinomycin D, and induced less fever, neutropenia, and pulmonary hypertension when infused into sheep. Our findings extend prior studies which suggested that LALF binds to and neutralizes LPS from multiple strains of gram-negative bacteria.
Mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing
Authors: Merino S , Camprubí S , Albertí S , Benedí V J , Tomás J M
Published: January 2020

The different mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing were investigated by using different strains and isogenic mutants previously characterized for their surface components. We found that strains from serotypes whose K antigen masks the lipopolysaccharide (LPS) molecules (such as serotypes K1, K10, and K16) fail to activate complement, while strains with smooth LPS exposed at the cell surface (with or without K antigen) activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b binds far from the cell membrane and that the lytic final complex C5b-9 (membrane attack complex) is not formed. Isogenic rough mutants (K+ or K-) are serum sensitive because they bind C3b close to the cell membrane and the lytic complex (C5b-9) is formed.
Cell envelope proteins of Staphylococcus epidermidis grown in vivo in a peritoneal chamber implant
Authors: Modun B , Williams P , Pike W J , Cockayne A , Arbuthnott J P , Finch R , Denyer S P
Published: January 2020

Staphylococcus epidermidis was grown in vivo in chambers implanted intraperitoneally in rats. The cell wall and cytoplasmic membrane protein profiles of the in vivo-grown organisms were compared with those of S. epidermidis grown in vitro in nutrient broth (NB), in iron-restricted NB, or in pooled human peritoneal dialysate (HPD). Compared with growth in broth and in common with growth in HPD, growth in vivo in chambers resulted in the repression of many S. epidermidis wall proteins, with proteins of 27, 42, 54, and 70 kDa predominating. Growth in vivo also resulted in the induction of two iron-repressible cytoplasmic membrane proteins of 32 and 36 kDa, which were also present in staphylococci grown in HPD and in iron-restricted NB. Immunoblotting experiments revealed that in sera taken 21 days after inoculation of the intraperitoneal chambers, the predominant antibody response to cell envelope proteins was directed against the 32- and 36-kDa iron-repressible membrane proteins.
Alterations in murine macrophage arachidonic acid metabolism following ingestion of nonviable Histoplasma capsulatum
Authors: Wolf J E , Massof S E , Peters S P
Published: January 2020

The effect of ingestion of heat-killed Histoplasma capsulatum yeast cells on the metabolism of arachidonic acid (20:4) to prostenoids and leukotrienes was examined in murine peritoneal macrophages (M phi s). H. capsulatum-containing M phi s exhibited a metabolite profile similar to that of zymosan-challenged phagocytes; however, there were differences with respect to the relative and total amounts of products produced. While proteose peptone-elicited M phi s exposed to H. capsulatum released quantitatively less prostaglandin E2 (PGE2) and leukotriene C4 than zymosan-treated M phi s, they metabolized a greater percentage of total product to prostenoids. In addition, whereas in vitro priming with gamma interferon increased both the PGE2 and leukotriene C4 contents of zymosan-stimulated M phi supernatants, similarly primed M phi s challenged with H. capsulatum selectively increased only PGE2 production. The immunosuppressive effect of a relative excess of prostenoids in H. capsulatum-containing M phi s may contribute to the overall disturbance in cell-mediated immunity characteristic of disseminated histoplasmosis.
Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB
Authors: Hu L T , Foxall P A , Russell R , Mobley H L
Published: January 2020

Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted to the gastric mucosa of humans. Urease, synthesized at up to 6% of the soluble cell protein, hydrolyzes urea, thereby releasing ammonia, which may neutralize acid, allowing survival of the bacterium and initial colonization of the gastric mucosa. The urease protein is encoded by two subunit genes, ureA and ureB; however, accessory genes are necessary for enzyme activity. H. pylori urease genes were isolated from a cosmid gene bank and subcloned on a 5.8-kb Sau3A partial fragment carrying ureCDAB, corresponding to four open reading frames described by A. Labigne, V. Cussac, and P. Courcoux (J. Bacteriol. 173:1920-1931, 1991). Clones were confirmed as ureas gene sequences by polymerase chain reaction amplification. The recombinant enzyme was purified from the soluble protein of French press lysates of Escherichia coli DH5 alpha(pHP402) by chromatography on DEAE-Sepharose, Phenyl-Sepharose, Mono-Q, and Superose 6 resins. Fractions containing a catalytically inactive apoenzyme were identified by an enzyme-linked immunosorbent assay (ELISA) by using antisera to native UreA (29.5 kDa) and UreB (66 kDa). Purified recombinant urease was indistinguishable from native enzyme on a Superose 6 column and on Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels. The protein reacted specifically on Western blots (immunoblots) with anti-UreA and anti-UreB antibodies and was recognized with an intensity equal to that of the native enzyme in an ELISA using human sera. Clones containing only ureA and ureB also produced an assembled but inactive enzyme. Enzyme activity was not restored by in trans complementation with cloned urease accessory gene sequences from Proteus mirabilis or Morganella morganii. H. pylori urease genes (ureCDAB) subcloned into pACYC184 were also not complemented with any of 1,000 cosmid clones containing H. pylori chromosomal sequences. However, larger clones containing 4.5 kb of DNA downstream of ureB synthesized catalytically active urease when grown in minimal medium. These data indicate that the ureA and ureB genes encoding H. pylori urease are transcribed and translated in E. coli and that these genes alone are sufficient for the synthesis and assembly of the native size enzyme. Genes downstream of ureB, however, are necessary for production of a catalytically active urease.
Structural features that influence the ability of lipid A and its analogs to abolish expression of suppressor T cell activity
Authors: Baker P J , Hraba T , Taylor C E , Myers K R , Takayama K , Qureshi N , Stuetz P , Kusumoto S , Hasegawa A
Published: January 2020

Lipid A preparations derived from the lipopolysaccharides of several gram-negative bacteria, as well as chemically defined synthetic lipid A's and their analogs (both glucosamine mono- and disaccharides), were used to establish the chemical structures required for (i) abolishing the expression of suppressor T cell (Ts) function and (ii) inducing polyclonal activation of B cells. Salmonella minnesota R595 lipid A (diphosphoryl lipid A) possesses both of these activities. Decreasing the number of phosphate groups in lipid A from two to one (monophosphoryl lipid A) as well as decreasing the fatty acyl content, primarily by removing the residue at the 3 position, resulted in a progressive reduction in toxicity; however, these structural modifications did not influence its ability to abolish the expression of Ts function. Reducing the fatty acyl content from five to four (lipid A precursor IVA or Ia) eliminated the capacity to influence Ts function but not to induce polyclonal activation of B cells. None of the monosaccharide analogs of lipid A examined influenced the expression of Ts activity, although some were able to activate B cells polyclonally. Thus, in order to be able to abolish the expression of Ts function, lipid A (i) must be a glucosamine disaccharide, (ii) may have either one or two phosphate groups, and (iii) must have at least five fatty acyl groups. Also, the chain length of the nonhydroxylated fatty acid, as well as the location of acyloxyacyl groups (2' versus 3' position), may play an important role. These findings indicate that the chemical structures responsible for the toxicity of lipid A differ from those that influence its capacity to abolish the expression of Ts function and to induce polyclonal activation of B cells.
Construction of a recombinant oral vaccine against Salmonella typhi and Salmonella typhimurium
Authors: Cao Y , Wen Z , Lu D
Published: January 2020

The viaB gene coding for the Vi antigen of Salmonella typhi Ty2 was subcloned into expression vector pYA248. The recombinant plasmid was termed SMM202 and transformed into Salmonella typhimurium chi 4072, an attenuated delta cya delta crp mutant. Recombinant S. typimurium Vi4072 had the ability to produce Vi capsular polysaccharide and also to invade and colonize the small intestine, mesenteric lymph nodes, and spleen of BALB/c mice. Mice orally immunized with Vi4072 developed serum and secretory antibody responses to the Vi antigen, as measured by a passive hemagglutination assay. Mice developed a delayed-type hypersensitivity following a footpad injection with Vi antigen after being sensitized orally with a suitable dose of Vi4072. Immunization of mice with Vi4072 afforded complete protection against fatal infection with virulent S. typhi Ty2. All data indicate that this route of antigen delivery is effective for stimulating antibody-mediated immunity and for inducing a cell-mediated immune response in BALB/c mice. Thus, S. typhimurium Vi4072 may serve as a vaccine for protection against typhoid fever and salmonellosis caused by S. typhimurium.
Humoral and cell-mediated immunity in mice to a 17-kilodalton lipoprotein of Francisella tularensis expressed by Salmonella typhimurium
Authors: Sjöstedt A , Sandström G , Tärnvik A
Published: January 2020

A 17-kDa lipoprotein, TUL4, of the facultative intracellular bacterium Francisella tularensis is one of several membrane proteins that induce an in vitro response in T cells from F. tularensis-primed humans. A DNA fragment of the live vaccine strain F. tularensis LVS encoding TUL4 was cloned into Salmonella typhimurium chi 4072, an attenuated delta cya delta crp mutant. Expression of the protein by the recombinant S. typhimurium chi 4072 (pTUL4-15) was maintained after passage in BALB/cJ mice. When mice were immunized with S. typhimurium chi 4072(pTUL4-15), some animals showed an antibody response and a T-cell response to TUL4. When the immunized mice were challenged with the live vaccine strain F. tularensis LVS, bacterial counts in the liver and spleen were lower than in animals immunized with S. typhimurium chi 4072. Immunization with F. tularensis LVS caused a much stronger protection against the challenge than did immunization with S. typhimurium chi 4072(pTUL4-15). The present study demonstrated that the 17-kDa lipoprotein TUL4 of F. tularensis is involved in a protective immunity to tularemia. Possibly, several T-cell-reactive proteins of the organism have to contribute for optimal protection to be achieved.