bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of carrier selection on immunogenicity of protein conjugate vaccines against Plasmodium falciparum circumsporozoites
Authors: Que J U , Cryz S J , Ballou R , Fürer E , Gross M , Young J , Wasserman G F , Loomis L A , Sadoff J C
Published: January 2020

Conjugate vaccines against the sporozoite stage of Plasmodium falciparum were synthesized by covalently coupling the recombinant protein R32 [with the one-letter amino acid code of MDP-[(NANP)15NVDP]2LR] to tetanus toxoid, cholera toxin, choleragenoid, and Pseudomonas aeruginosa toxin A. Conjugates were produced by using adipic acid dihydrazide as a spacer molecule and carbodiimide as a coupling agent. The molar ratio of R32 to carrier protein ranged from 2.5:1 to 8.4:1. These conjugates were found to be stable, nontoxic, and nonpyrogenic. When adsorbed onto Al(OH)3, all conjugates were capable of inducing anti-R32 antibody. Conjugates made with either cholera toxin or Pseudomonas aeruginosa toxin A were significantly more immunogenic than those constructed with tetanus toxoid or choleragenoid. However, the magnitude of the immune response to the R32 moiety was not governed by the antibody response to the carrier protein.
Comparative analysis of immunological responses to oral (Ty21a) and parenteral (TAB) typhoid vaccines
Authors: D'Amelio R , Tagliabue A , Nencioni L , Di Addario A , Villa L , Manganaro M , Boraschi D , Le Moli S , Nisini R , Matricardi P M
Published: January 2020

The clinical and immunological responses to typhoid vaccination with parenteral (TAB) and oral (Ty21a) vaccines in two groups of 30 adult male subjects were studied. Parameters monitored included specific anti-Salmonella typhi cell-mediated immunity and total and specific antilipopolysaccharide fecal immunoglobulin A (IgA) titers in Ty21a-vaccinated subjects. Peripheral blood lymphocytes antibacterial activity was significantly increased only in Ty21a-vaccinated subjects. Serum arming activity and results of human F(ab')2 anti-IgG and -IgA inhibition tests suggest antibody-dependent cellular cytotoxicity mediated by IgA in those vaccinated with Ty21a. Interestingly enough, the cells of TAB-vaccinated subjects were able to mediate IgG-dependent cellular cytotoxicity, as was observable from the results of blocking experiments. Moreover, total and specific antilipopolysaccharide fecal IgA levels were observed to be significantly increased with Ty21a, up to 8 months post-vaccination schedule. An early-onset, transitory increase in serum IgM rheumatoid factor was also found, exclusively in subjects treated with TAB, and was no longer detectable on day 240. Ty21a was well tolerated and free of side effects, whereas 65% of subjects administered TAB reported fever, headache, malaise, and local tenderness at the injection site. Our data show that the two typhoid vaccines induce different cell-mediated specific immune responses. The role of these responses in protection against Salmonella infection, however, requires further investigation.
Mediation of adherence of streptococci to human endothelial cells by complement S protein (vitronectin)
Authors: Valentin-Weigand P , Grulich-Henn J , Chhatwal G S , Müller-Berghaus G , Blobel H , Preissner K T
Published: January 2020

The role of S protein in the adherence of group A and G streptococci to human umbilical vein endothelial cells cultivated in 96-well microdilution plates was studied by utilizing fluorescein-labeled streptococci. The assay proved suitable for quantitative determination of bacterial adherence to cultured endothelial cells for all tested strains of streptococci. Only bacterial strains with significant S protein binding but weak fibronectin binding were included in these studies. Fibronectin-mediated adherence to endothelial cells of these streptococci was less than 25% of total and could be blocked by antifibronectin immunoglobulin G. Further treatment of endothelial cell monolayers with anti-S protein immunoglobulin G at concentrations up to 1 mg per well led to an almost complete inhibition of adherence for all tested streptococcal cultures, indicating significant contribution of S protein in the streptococcus-endothelial cell interaction. Blocking of S-protein-binding sites on streptococci by preincubation with exogeneous S protein at a concentration of 10 micrograms per 4 x 10(7) streptococci led to about 75% reduction of S-protein-mediated adherence to endothelial cells. Trypsin pretreatment of group G streptococci and pronase pretreatment of group A and G streptococci, modifications known to destroy the bacterial binding sites of S protein, also inhibited the capacity of the streptococci for S-protein-mediated adherence to endothelial cells by 75 to 80%. These results indicate that S protein plays a mediatory role in adherence of streptococci to endothelial cells and that S-protein-specific binding sites on streptococci are involved in this interaction.
N-acetylneuraminyllactose-binding fibrillar hemagglutinin of Campylobacter pylori: a putative colonization factor antigen
Authors: Evans D G , Evans D J , Moulds J J , Graham D Y
Published: January 2020

Campylobacter pylori is the causative agent of gastritis and possibly of peptic and duodenal ulcers in adults. Histological observations show C. pylori attached to gastric epithelium as well as in the mucus layer of the stomach. We found that clinical isolates of C. pylori possess a cell-bound hemagglutinin detectable with human erythrocytes (all phenotypes tested) and those of a variety of animal species. The C. pylori hemagglutinin is antigenic, heat sensitive, and destroyed by pronase and papain but resistant to pepsin and trypsin. The hemagglutinin has fibrillar morphology; C. pylori-erythrocyte interaction displays very intimate contact, which is typical of fibrillae-mediated attachment. Fibrillae were removed from C. pylori by solubilization with N-octylglucose. After partial purification and removal of N-octylglucose by dialysis, the protein reaggregated, with the assembly of fibrillar structures. Hemagglutination inhibition was observed with the sialoproteins fetuin, alpha 2-macroglobulin, and glycophorin A but not with asialofetuin or asialoglycophorin A. The erythrocyte receptor was more sensitive to destruction by a neuraminidase specific for the N-acetylneuraminyl-alpha(2-3)-galactopyranosyl [NeuAc(2-3)Gal] sequence than one specific for NeuAc(2-6)Gal. Hemagglutination-inhibition assays with N-acetylneuraminyl-alpha(2-3)-lactose [NeuAc(2-3)-lactose] and NeuAc(2-6)-lactose confirmed that the C. pylori hemagglutinin preferentially binds to the NeuAc(2-3)Gal isomer of NeuAc-lactose. Based upon the above-described properties of the C. pylori fibrillar hemagglutinin, we conclude that this antigen should be designated as a putative colonization factor antigen.
Pseudomonas aeruginosa alkaline protease degrades human gamma interferon and inhibits its bioactivity
Authors: Horvat R T , Parmely M J
Published: January 2020

This study was performed to determine the effect of Pseudomonas aeruginosa on gamma interferon (IFN-gamma) production by antigen-stimulated human T-cell clones. Crude bacterial filtrates prepared from certain strains of P. aeruginosa inhibited IFN-gamma production by T cells and reduced the antiviral activity of preformed IFN-gamma. Bacterial filtrates prepared from mutant strains that did not produce the exoenzyme alkaline protease (AP) did not inhibit IFN-gamma activity. The inhibitory activity of bacterial filtrates was heat and trypsin sensitive and was neutralized by an antiserum to AP. Crystalline AP mimicked the effects of the bacterial filtrates, and an inactive filtrate from a protease-deficient mutant strain was reconstituted by the addition of AP. AP-treated recombinant IFN-gamma showed altered migration on Western blots (immunoblots) of polyacrylamide gels, and this modification correlated with a dose-dependent loss of antiviral activity. The ability of recombinant IFN-gamma to elevate the expression of Fc receptors on cells of the U-937 histiocytic cell line was also diminished by AP treatment. These results indicate that the Pseudomonas protease AP can inhibit the antiviral and immunomodulatory activities of IFN-gamma.
Gastric colonization by Campylobacter pylori subsp. mustelae in ferrets
Authors: Fox J G , Cabot E B , Taylor N S , Laraway R
Published: January 2020

Campylobacter pylori subsp. mustelae was cultured from both normal and inflamed gastric mucosa of ferrets. Examination of neonatal, juvenile, and adult ferrets established that the gastric mucosa in the majority of preweanling (age, less than 6 weeks) ferrets sampled were not colonized with C. pylori subsp. mustelae, whereas the gastric mucosa of 100% of adult ferrets were colonized with this gastric organism. C. pylori subsp. mustelae was isolated from the gastric mucosa on a sequential basis from nine ferrets during a several-month period, inferring either persistent colonization or frequent reinfection with C. pylori subsp. mustelae.
Cell walls from avirulent variants of Histoplasma capsulatum lack alpha-(1,3)-glucan
Authors: Klimpel K R , Goldman W E
Published: January 2020

Cell wall composition of isogenic virulent-avirulent strain pairs of Histoplasma capsulatum varied markedly with respect to alpha-(1,3)-glucan content. When yeast cell walls were fractionated by standard techniques, the avirulent strains contained up to 1,000-fold less alpha-(1,3)-glucan than did their virulent parents. No alpha-(1,3)-glucan could be detected on the surface of the avirulent strain yeast cells if we used a mouse monoclonal antibody that recognized this polymer. A similar relationship between virulence and alpha-(1,3)-glucan has been described for Paracoccidioides brasiliensis. alpha-(1,3)-Glucan is also found in several other pathogenic fungi and may thus be an important common virulence determinant.
Quantitative analysis and partial characterization of cytotoxin production by Salmonella strains
Authors: Ashkenazi S , Cleary T G , Murray B E , Wanger A , Pickering L K
Published: January 2020

The pathogenesis of the wide-spectrum human disease caused by Salmonella species is poorly understood. Cytotoxin production by other enteric pathogens has been increasingly investigated recently, and data are accumulating regarding the role of cytotoxins in enteric infections and hemolytic uremic syndrome. We studied the cytotoxic activity of 131 Salmonella strains of the major serotypes, including 94 strains of Salmonella enteritidis, 12 strains of Salmonella typhi, and 25 strains of Salmonella choleraesuis. Cytotoxicity was quantitatively determined in sonic extracts by a [3H]thymidine-labeled HeLa cell assay. All Salmonella strains examined showed some degree of cytotoxic activity. The geometric means +/- standard deviations of the amounts of cytotoxin produced (50% cytotoxic dose per milligram of bacterial protein) were 27 +/- 2 for S. typhi, 65 +/- 2 for S. enteritidis, and 117 +/- 2 for S. choleraesuis. Analysis of variance showed that the differences in cytotoxin production by the three species were significant (P less than 0.001). No significant differences were found between stool isolates and invasive strains of the same species. Neutralization studies showed that the cytotoxins produced by all Salmonella strains were immunologically distinct from Shiga toxin and the closely related Shiga-like toxins produced by Escherichia coli. DNA hybridization studies with DNA probes for Shiga-like toxins of types I and II showed no hybridization. In each species the cytotoxin was heat labile and sensitive to trypsin treatment, which indicated that its active component was probably protein in nature. Upon ultrafiltration with Amicon membranes and gel filtration chromatography, cytotoxic activity was found in the molecular weight range of 56,000 to 78,000. Our findings indicate that salmonellae produce cytotoxin(s) that may play a role in the manifestations of the various species.
Augmentation of host resistance to microbial infections by recombinant human interleukin-1 alpha
Authors: Minami A , Fujimoto K , Ozaki Y , Nakamura S
Published: January 2020

Recombinant human interleukin-1 alpha augmented resistance of mice to microbial infections caused by Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Salmonella typhimurium, and Candida albicans. The effective doses of interleukin-1 alpha ranged from 0.01 to 10 micrograms per mouse, depending on the infecting organism, route of administration, and challenge dose. Intravenous interleukin-1 alpha was, dose for dose, more effective than intravenous muramyl dipeptide and lentinan against the P. aeruginosa and K. pneumoniae infections. Augmentation by interleukin-1 alpha of resistance to infection was also observed in P. aeruginosa-infected mice in a state of cyclophosphamide-induced leucopenia. Interleukin-1 alpha may be useful for controlling obstinate infections not curable by antimicrobial agents alone.
Comparison of type 2 and type 6 fimbriae of Bordetella pertussis by using agglutinating monoclonal antibodies
Authors: Li Z M , Brennan M J , David J L , Carter P H , Cowell J L , Manclark C R
Published: January 2020

Two types of fimbriae have been identified on the pathogenic gram-negative organism Bordetella pertussis. Monoclonal antibodies to these fimbriae were produced to better understand the role of fimbriae as serotype-specific agglutinogens and to investigate the antigenic relationship between these fimbriae. Three monoclonal antibodies were identified that specifically agglutinated B. pertussis cells containing the U.S. Reference Factor 2 agglutinogen, and six monoclonal antibodies were produced that agglutinated only those strains containing the U.S. Reference Factor 6 agglutinogen. Indirect immunofluorescence studies and immunogold electron microscopy demonstrated that these monoclonal antibodies bind to an outer membrane component on serotype-specific strains of B. pertussis. All of the monoclonal antibodies reacted with native or partially assembled type-specific fimbriae but not with monomeric fimbrial subunits as indicated by Western blot (immunoblot) analysis. The fimbrial agglutinogens recognized by the monoclonal antibodies were also uniquely reactive with either U.S. Reference Factor 2 or 6 antiserum (Eldering agglutinogen 2 or 6 polyclonal antiserum) in an indirect ELISA. No cross-reactivity of the monoclonal antibodies with the unrelated fimbriae was observed in any of the comparative immunological studies. Some of the monoclonal antibodies agglutinated certain strains of B. bronchiseptica, suggesting that this closely related species can contain antigenically similar fimbriae. These monoclonal antibodies should prove useful for further structural and functional analysis of Bordetella fimbriae and for studies on the role that these antigens play in prevention of infection and disease.
Specific and nonspecific inhibition of adhesion of oral actinomyces and streptococci to erythrocytes and polystyrene by caseinoglycopeptide derivatives
Authors: Neeser J R , Chambaz A , Del Vedovo S , Prigent M J , Guggenheim B
Published: January 2020

Various caseinoglycopeptide derivatives prepared from mammalian milk were evaluated as inhibitors of hemagglutinations mediated by Actinomyces viscosus Ny1, Streptococcus sanguis OMZ9, and, for comparative purposes, plant lectins from Arachis hypogaea and Bauhinia purpurea. It was found that recognition of the beta-D-galactose-(1----3)-2-acetamido-2-deoxy-D-galactose carbohydrate chain by Actinomyces viscosus Ny1 organisms and Arachis hypogaea and B. purpurea agglutinins had similar structural requirements; in all cases, the desialylated bovine caseinoglycomacropeptide, on which several units of the above mentioned disaccharide are clustered, behaved as the most potent hemagglutination inhibitor. By contrast, none of the preparations tested inhibited erythrocyte agglutination by S. sanguis OMZ9. Thus, the desialylated bovine caseinoglycomacropeptide acts as a potent and specific inhibitor of oral Actinomyces adhesion to cell membranes (a soft surface) and could be used as a probe for the study of recognition mechanisms mediated by Actinomyces galactose-binding lectins. During the present study, both native and desialylated variants of the same bovine glycomacropeptide also totally prevented the adhesion of Actinomyces viscosus Ny1, S. sanguis OMZ9, and S. mutans OMZ176 to polystyrene surfaces. Comparative evaluations of various structurally different compounds gave the following results. Neither mono- nor disaccharides related to caseinoglycopeptide carbohydrates prevented adhesion; highly positively or negatively charged polypeptides and polysaccharides were either not or only moderately active. Besides these glycomacropeptides, an inhibitory activity was also exhibited by other mucin-type glycoproteins carrying short O-linked carbohydrate chains (including bovine submaxillary mucin), polyethylene glycol, and bovine serum albumin. Consequently, caseinoglycopeptide prevention of oral bacterial adhesion to polystyrene tubes (a hard surface) takes place with no species specificity and can be compared to nonspecific inhibition exhibited by various polymers with very different structural characteristics.
Expression of the histidine-rich protein PfHRP1 by knob-positive Plasmodium falciparum is not sufficient for cytoadherence of infected erythrocytes
Authors: Rock E P , Saul A J , Taylor D W , Leech J H , Sherwood J A , Howard R J
Published: January 2020

Six culture-adapted knob-positive Plasmodium falciparum parasites, four of which were nonbinding in an in vitro cytoadherence assay, were tested for the presence of the knob-associated histidine-rich protein PfHRP1. Two knob-positive, strongly cytoadherence-positive P. falciparum strains from Aotus monkeys were also examined. All parasites expressed PfHRP1, suggesting that it plays a structural or functional role related to knobs but is not by itself sufficient for cytoadherence of P. falciparum-infected erythrocytes.
Frequency of fimbriation of nontypable Haemophilus influenzae and its ability to adhere to chinchilla and human respiratory epithelium
Authors: Bakaletz L O , Tallan B M , Hoepf T , DeMaria T F , Birck H G , Lim D J
Published: January 2020

To date, we have examined nearly 60 clinical isolates of nontypable Haemophilus influenzae (26 nasopharyngeal, 33 from middle ear effusions) and have found that 100% were fimbriated. The percentage of cells bearing fimbriae within each isolate varied from less than 10 to 100%, with fimbriae being either peritrichous or bipolar in distribution. Fimbriae were approximately 2.4 to 3.6 nm in width; however, there was a high degree of variability in both length and number of fimbriae per individual bacterial cell among these isolates. All isolates tested adhered to both human oropharyngeal cells and chinchilla tracheal epithelium regardless of the degree to which the particular isolate was fimbriate. The level or degree of fimbriation did not correlate with either site of isolation, biotype, strength of hemagglutination reaction, or type of effusion present in the ear. These appendages appear to be quite different from those described for type b H. influenzae in which the ability to adhere and strength of ability to hemagglutinate correlated strongly with degree of fimbriation.
Bovine T cells, B cells, and null cells are transformed by the protozoan parasite Theileria parva
Authors: Baldwin C L , Black S J , Brown W C , Conrad P A , Goddeeris B M , Kinuthia S W , Lalor P A , MacHugh N D , Morrison W I , Morzaria S P
Published: January 2020

The target cells for infection and transformation by Theileria parva were investigated. Peripheral blood mononuclear cells were reacted with monoclonal antibodies specific for bovine leukocyte differentiation antigens, sorted into subpopulations with a fluorescence-activated cell sorter, and infected in vitro with T. parva sporozoites. Infected cells were cultured at limiting dilution, and transformed clones were screened with monoclonal antibodies. The results indicated that B cells, T cells (including BoT4+ and BoT8+ cells), and null cells but not monocytes or neutrophils were transformed in vitro after infection with T. parva. After transformation, peripheral blood T cells and T-cell clones retained expression of most or all of the T-cell differentiation antigens including the mature T-cell marker recognized by monoclonal antibody IL-A27, BoT2, and BoT4 or BoT8, and some cells acquired a low level of expression of BoT4, BoT8, or the null cell marker recognized by monoclonal antibody IL-A29. T. parva-transformed null cells retained expression of the IL-A29 determinant and acquired expression of BoT2 and BoT8 but not the IL-A27 determinant or BoT4. T. parva-transformed B cells in most instances lost expression of surface immunoglobulin and never acquired expression of the IL-A27 determinant, BoT2, BoT4, or BoT8, although some cells acquired a low level of expression of the null cell marker recognized by monoclonal antibody IL-A29. Further studies on cell lines and clones grown in vitro from populations isolated from T. parva-infected cattle suggested that the majority of the in vivo T. parva-transformed cells were of T-cell origin.
Modulation of macrophage lysosomal pH by Mycobacterium tuberculosis-derived proteins
Authors: Chicurel M , García E , Goodsaid F
Published: January 2020

Macrophage lysosomal pH was significantly (greater than 1 pH unit) increased in a reversible, concentration-dependent manner characterized by a saturable and cyclic kinetics after exposure to culture filtrate protein extract derived from Mycobacterium tuberculosis. Lysosomal alkalinization peaked 30 min after administration of culture filtrate protein extract to cells of the macrophagelike cell line J774A.1. The alkalinization was reversible, and a second peak was observed approximately 60 min after incubation. Maximum lysosomal alkalinization increased as a function of culture filtrate protein extract concentration, reaching an apparent saturation level around 700 to 1,000 micrograms/ml, although the time course for this process was not significantly dependent on antigen concentration. The alkalinizing agent(s) was heat labile and produced a similar effect in cells which had a different lysosomal enzyme composition. Our observations are consistent with the presence of one or more mycobacterial antigens which have a pH-dependent affinity for lysosomal structures essential for lysosomal acidification and which are able to inhibit this lysosomal acidification.
Lacteal immunity to enteric cryptosporidiosis in mice: immune dams do not protect their suckling pups
Authors: Moon H W , Woodmansee D B , Harp J A , Abel S , Ungar B L
Published: January 2020

The susceptibilities of passively immunized principal and nonimmunized control suckling mice to orogastric challenge with Cryptosporidium parvum oocysts were compared. Principals were suckled by dams that had recovered from C. parvum infection. Controls were suckled by dams reared free of C. parvum infection. Principals and controls were equally susceptible to challenge. Principals were susceptible even when their dams were hyperimmunized by oral and parenteral booster inoculations with C. parvum oocysts. Immune dams produced serum antibody against C. parvum, while nonimmune dams did not. Anti-cryptosporidia immunoglobulin G (IgG) and IgA were demonstrated in whey extracted from the stomachs of principals that had suckled immune dams but not in whey extracted from the stomachs of controls. It was concluded that passive lacteal immunity is not an efficient means of protection against cryptosporidiosis in mice. As in other coccidian infections, protective immunity against cryptosporidiosis may depend more on immune cells than on antibody.
Agglutinating monoclonal antibodies that specifically recognize lipooligosaccharide A of Bordetella pertussis
Authors: Li Z M , Cowell J L , Brennan M J , Burns D L , Manclark C R
Published: January 2020

Monoclonal antibodies that specifically agglutinate strains of Bordetella pertussis having serotype 1 agglutinogen were uniquely reactive with the electrophoretically slow-migrating A form of lipooligosaccharide. These monoclonal antibodies should be useful for the structural analysis of B. pertussis lipooligosaccharide and for the establishment of a better-defined serogroup for Bordetella species.
Antiheart antibody-dependent cytotoxicity in the sera of mice chronically infected with Trypanosoma cruzi
Authors: Laguens R P , Meckert P C , Chambó J G
Published: January 2020

Sera of mice chronically infected with Trypanosoma cruzi contain antibodies that bind to the surface of living adult syngeneic heart muscle cells. In a syngeneic system, with nonadherent spleen mononuclear cells as effector cells and cardiocytes as targets, antibody-dependent cytotoxicity (ADCC), revealed by the liberation of creatine phosphokinase from damaged cardiocytes, was observed after incorporation of serum samples from infected mice. Target damage was decreased after absorption with syngeneic myocardium, but absorption with T. cruzi epimastigotes or trypomastigotes or with syngeneic skeletal muscle had no effect on ADCC. No complement-dependent lysis against heart muscle cells was detected in the same serum samples. These observations indicate that serum from chronically chagasic mice contain antibodies that bind to the surface of living adult syngeneic cardiocytes and are able to exert ADCC, suggesting that they could play a role in the pathogenesis of the heart damage that occurs in Chagas' disease.
X-linked immunodeficient mice as a model for testing the protective efficacy of monoclonal antibodies against Pseudomonas aeruginosa
Authors: Zweerink H J , Gammon M C , Hutchison C F , Jackson J J , Pier G B , Puckett J M , Sewell T J , Sigal N H
Published: January 2020

(DBA/N[female] X CBA/2[male])F1 males have been reported to be deficient in producing antibodies against a number of antigens, including carbohydrates (I. Scher, Adv. Immunol. 35:1-71, 1982). We show that F1 male mice, in contrast to females, made less lipopolysaccharide (LPS)-specific antibodies after immunization with heat-inactivated Pseudomonas aeruginosa and had significantly less naturally occurring LPS-specific antibodies. Furthermore, neutropenic males were 50 to 1,000 times more sensitive to challenge with representative isolates belonging to the seven Fisher immunotypes. Administration to neutropenic F1 males of a human monoclonal antibody specific for the O carbohydrates of P. aeruginosa immunotype 2 LPS or administration of serum from rabbits immunized with heat-inactivated P. aeruginosa immunotype 1 raised the level of resistance to bacterial challenge close to that of females. The results show that the X-linked immunodeficient mouse is an excellent model with which to test the protective efficacy of P. aeruginosa-specific monoclonal antibodies.
Resolution of chlamydial genital infection in B-cell-deficient mice and immunity to reinfection
Authors: Ramsey K H , Soderberg L S , Rank R G
Published: January 2020

The purpose of this investigation was to determine the relative roles of the humoral and cell-mediated immune responses in the resolution of chlamydial genital infection of mice and resistance to reinfection. To this end, female BALB/c mice were rendered B cell deficient by treatment with heterologous anti-immunoglobulin M (IgM) serum from birth. Controls were similarly treated with either normal serum or phosphate-buffered saline. Before inclusion in each experiment, anti-IgM-treated mice were screened for the absence of IgM in serum and for the presence of cell-mediated immune responses. In addition, spleen cells from anti-IgM-treated mice responded to concanavalin A and phytohemagglutinin but not to lipopolysaccharide. By these criteria, mice were designated B cell deficient. B-cell-deficient mice and controls were inoculated intravaginally with a suspension of mouse pneumonitis agent (MoPn), a Chlamydia trachomatis biovar. All B-cell-deficient mice resolved the infection. Additionally, no significant difference was seen in the course of the infection in B-cell-deficient mice when compared with controls. In contrast to control mice, B-cell-deficient mice displayed no detectable antibody responses to MoPn in serum or in genital secretions. However, both B-cell-deficient mice and controls developed delayed-type hypersensitivity and T-cell proliferative responses to MoPn. When challenged 53 days after primary infection, no significant difference was seen in the resistance of B-cell-deficient mice to reinfection when compared with that of the controls. These data indicate that cell-mediated immune mechanisms play an important role in the resolution of and resistance to chlamydial genital infection in this model.
Identification of a human erythrocyte receptor for colonization factor antigen I pili expressed by H10407 enterotoxigenic Escherichia coli
Authors: Pieroni P , Worobec E A , Paranchych W , Armstrong G D
Published: January 2020

We have identified a receptor for colonization factor antigen I (CFA/I) pili in human erythrocyte membranes. Erythrocyte binding assays, using whole organisms, suggested that the CFA/I receptor was a glycoprotein containing important sialic acid moieties. Subsequently, human erythrocyte membranes were extracted with lithium diiodosalicylate to obtain a soluble glycoprotein fraction from which to isolate receptors. The extracted material caused agglutination of the CFA/I+ but not the CFA/I- organisms at a protein concentration of 0.5 mg/ml. The CFA/I receptor was identified in iodinated extract by an affinity isolation procedure, using whole bacterial cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the washed, extract-coated H10407 CFA/I+ organisms revealed a band with an apparent molecular weight of 26,000 which was present in the original extract but was not observed on extract-coated H10407 CFA/I- bacteria. The addition of purified CFA/I pili reduced binding of the 26,000-molecular-weight receptor to CFA/I+ bacteria. The CFA/I-specific receptor species also bound to wheat germ agglutinin-agarose. This observation supported the suggestion that the CFA/I receptor identified in this report is a sialoglycoprotein.
Gonococci possessing only certain P.II outer membrane proteins interact with human neutrophils
Authors: Fischer S H , Rest R F
Published: January 2020

We investigated the role of the protein II (P.II) family of gonococcal outer membrane proteins in the interaction of seven single P.II variants of Neisseria gonorrhoeae FA1090 with human neutrophils in vitro. The abilities of nonpiliated gonococci to adhere to and be killed by neutrophils and to stimulate luminol-dependent chemiluminescence (CL) depended on the possession of at least one P.II. Gonococci lacking P.II (i.e., P.II-) adhered poorly to and were not killed by neutrophils and induced only minimal CL. Although most P.II-containing (i.e., P.II+) variants adhered to, stimulated, and were readily killed by neutrophils, one variant, containing P.IIa, possessed none of these characteristics; it acted just like a P.II- variant. No correlation was found between the colony opacity phenotype and the interaction of gonococci with neutrophils. Data from CL experiments suggest that the stimulatory effect of P.II was dominant over that of pili; i.e., piliated P.II+ gonococci were much more stimulatory than piliated P.II- gonococci. The results indicate that most but not all P.II proteins mediate, in part or in full, the interaction of N. gonorrhoeae with human neutrophils, including adherence, stimulation of the neutrophil respiratory burst, and phagocytic killing.
Use of recombinant antigens expressed in Escherichia coli K-12 to map B-cell and T-cell epitopes on the immunodominant 65-kilodalton protein of Mycobacterium bovis BCG
Authors: Thole J E , van Schooten W C , Keulen W J , Hermans P W , Janson A A , de Vries R R , Kolk A H , van Embden J D
Published: January 2020

In gene libraries of Mycobacterium bovis BCG, Mycobacterium tuberculosis, and Mycobacterium leprae, recombinants were frequently encountered that expressed an immunodominant 65-kilodalton (kDa) protein antigen that was shown to react with a high proportion of mycobacterium-reactive human and murine T cells and murine monoclonal antibodies. In this study, recombinant antigens were used to map T-cell and B-cell epitopes on the M. bovis BCG 65-kDa protein that was previously designated MbaA. Four different T-cell-epitope-containing regions (amino acid residues 1 through 16, 17 through 61, 85 through 108, and 235 through 279) were defined that were recognized by seven T-cell clones from patients with tuberculoid leprosy. These regions are distinct from two previously described T-cell epitopes recognized by T cells from a tuberculosis patient. As T-cell clones restricted by different class II determinants were shown to be specific for different regions on the 65-kDa protein, the presented data suggested that the products of different human leukocyte antigen class II loci and alleles present different parts of MbaA to the immune system. B-cell epitopes recognized by 20 monoclonal antibodies were assigned to eight different regions of MbaA. Using 15 of these antibodies, we previously showed that MbaA was antigenically related to a common antigen present in many bacterial species. The dispersed localization of the involved epitopes defined here shows that various different parts of MbaA are indeed conserved. These results show that well-defined recombinant antigens are useful tools for the localization of both B- and T-cell-epitope-containing regions of a protein. Peptides synthesized from the sequences of such regions may then exactly define the epitopes relevant for the development of specific diagnostic tests or of vaccines against mycobacteria.
Role of pili in adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells
Authors: Doig P , Todd T , Sastry P A , Lee K K , Hodges R S , Paranchych W , Irvin R T
Published: January 2020

The ability of pili from Pseudomonas aeruginosa K (PAK) to act as an adhesin to human respiratory epithelial cells was examined using an in vitro adhesion assay. Equilibrium analysis of PAK binding to human buccal epithelial cells (BECs) and tracheal epithelial cells (TECs) by means of a Langmuir adsorption isotherm revealed that the maximum numbers of binding sites per epithelial cell (N) were 255 for BECs and 236 for TECs, with apparent association constants (Ka) of 2.8 x 10(-9) and 5.8 x 10(-9) ml/CFU, respectively. Trypsinization of the BECs before the binding assay increased N to 605 and decreased the Ka to 1.7 x 10(-9) ml/CFU. Addition of homologous pili to the binding assay with BECs or TECs or the addition of anti-pilus Fab fragments inhibited PAK adherence. Binding of purified pili to BECs was shown to reach saturation. Purified pili and PAK competed for the same receptor on the BEC surface. Further, by using peptide fragments of PAK pilin (derived from the native pili or produced synthetically) in the binding assay for PAK to BECs, we have presumptively identified the pilus binding domain in the C-terminal region of the pilin and shown that the C-terminal disulfide bridge is important in maintaining the functionality of the binding domain.
Immunogenicity of liposomes and iscoms containing the major outer membrane protein of Neisseria gonorrhoeae: influence of protein content and liposomal bilayer composition
Authors: Kersten G F , van de Put A M , Teerlink T , Beuvery E C , Crommelin D J
Published: January 2020

The influences of Neisseria gonorrhoeae protein IB content and bilayer composition of liposomes and protein content of iscoms on immunogenicity were investigated. Changes in the protein content of liposomes did not influence the immunoglobulin G response, whereas the response was lowered when the amount of protein in iscoms was increased. Bilayer composition only influenced the primary immunoglobulin G response; immunological memory was not affected.
Ultrastructural study of entry of Chlamydia strain TWAR into HeLa cells
Authors: Kuo C C , Chi E Y , Grayston J T
Published: January 2020

Ultrastructural studies of the entry of Chlamydia strain TWAR into HeLa cells showed that the elementary bodies first attach to host cells by the pointed end, secure other binding sites on the host cells by forming cell wall protrusions, enter host cells by invaginating the host cell membrane, and form vacuolated endocytic vesicles. Differences were demonstrated between TWAR and other chlamydiae in the mode of attachment and endocytosis.
Specificity of the glucan-binding lectin of Streptococcus cricetus
Authors: Drake D , Taylor K G , Bleiweis A S , Doyle R J
Published: January 2020

The specificity of the glucan-binding lectin (GBL) of Streptococcus cricetus AHT was determined. Examination of the kinetics of aggregation of cell suspensions with glucans containing various percentages of alpha-1,6, alpha-1,4, alpha-1,3, and alpha-1,2 anomeric linkages revealed that only glucans with at least 80% alpha-1,6 linkages promoted strong aggregation. Moreover, only linear glucans with molecular weights greater than 5 X 10(5) were capable of causing rapid aggregation of the bacteria. The lectin was observed to be present on S. cricetus strains, on Streptococcus sobrinus, and on several Streptococcus mutants strains. Preincubation of suspensions of S. cricetus AHT with glucan T10 (molecular weight of 10,000) before the addition of high-molecular-weight glucan resulted in competitive inhibition in a concentration-dependent manner. Inhibition was achieved also with isomaltopentaose, isomaltohexaose, and isomaltooctaose, but at higher concentrations than glucan T10. In contrast, no inhibition was observed with maltoheptaose, providing additional evidence for the specificity of GBL. Treatment of suspensions of S. cricetus AHT with trypsin before and after aggregation with high-molecular-weight glucan revealed a substantial level of protection of GBL when in a bound state. Collectively, these results indicated that GBL has an absolute affinity for glucans rich in alpha-1,6 linkages and possesses an active site which recognizes internal sequences and accommodates isomaltosaccharides of at least nine residues. This unusual specificity may contribute to the colonization of S. cricetus, S. sobrinus, and S. mutans in glucan-containing plaque in the oral cavity.
Quantitation and purification of the Pasteurella multocida toxin by using monoclonal antibodies
Authors: Foged N T
Published: January 2020

Pasteurella multocida toxin (PMT), derived from a toxigenic strain of P. multocida originally isolated from a pig with clinical atrophic rhinitis, was used to immunize BALB/c mice. Ninety-two hybridomas secreting monoclonal antibodies (MAbs) against PMT were produced by fusion of spleen cells from these mice with P3-X63-Ag8.653 myeloma cells. The specificity for PMT of the MAbs was ascertained by enzyme-linked immunosorbent assay and immunoblotting analysis. The interrelationship of a panel of 10 MAbs and their respective epitopes was characterized by a competitive enzyme-linked immunosorbent assay based on the biotin-avidin system and by an in vitro neutralization assay based on the cytopathic effect of PMT on embryonic bovine lung cells. In vivo neutralization of the lethal effect of PMT in mice was obtained by passive immunization with an anti-PMT MAb 2 days before challenge with PMT. PMT was quantified by a sandwich enzyme-linked immunosorbent assay with a lower detection limit of approximately 50 pg of PMT. Application of supernatant or bacterial extract from cultivation of toxigenic P. multocida to an affinity column containing immobilized MAb resulted in purification of PMT with a yield of 78 to 93% of the PMT applied.
Scanning electron microscopy of epidermal adherence and cavitation in murine candidiasis: a role for Candida acid proteinase
Authors: Ray T L , Payne C D
Published: January 2020

Adherence of blastoconidia to epidermal corneocytes is an early event in Candida colonization and infection of the skin. Pathogenic species adhere more avidly than nonpathogenic species, transform to hyphal growth, and invade the stratum corneum of the skin. Adherence was studied by scanning electron microscopy of experimental murine cutaneous Candida infections, using six species of Candida. Candida albicans and C. stellatoidea blastoconidia, applied to newborn mouse skin, adhered to the stratum corneum in greater numbers than other species tested, acquired fibrils and strands of amorphous mucinlike material ("cohesin") between spores and the corneocyte cell surface, formed cavitations in the corneocyte surface, and invaded the corneocyte envelope by hyphal growth at sites distant to the point of blastoconidia attachment. Other species showed little or no adherence, colonization, or cavitation of the corneocyte surface, except C. tropicalis, which showed intermediate results. Pepstatin, an inhibitor of Candida acid proteinase, did not alter adherence or cohesion formation, but inhibited formation of corneocyte cavitations about adherent blastoconidia, suggesting that this enzyme may facilitate adherence/invasion events on skin. Depletion of surface lipids did not alter the formation of cohesin material or the adherence process. Adherence and invasion of epithelium by pathogenic Candida species include the interaction of blastoconidia with an epithelial surface cohesin material that participates in the adherence process. Candida acid proteinase, a keratinolytic enzyme, may participate in the cavitation process of the corneocyte surface by C. albicans.
Sequence analysis of the Streptococcus mutans scrB gene
Authors: Sato Y , Kuramitsu H K
Published: January 2020

The complete nucleotide sequence of the Streptococcus mutans GS-5 scrB gene coding for sucrose-6-phosphate hydrolase activity was determined. A potential ribosome-binding site as well as promoter sequences were identified upstream from the gene. The deduced amino acid sequence of the enzyme suggested a molecular weight of 51,750, which is similar to that estimated for the enzyme isolated from strain GS-5. The enzyme is slightly acidic, with a pI of 5.9, and is a relatively hydrophilic protein. The nucleotide and amino acid sequences of the enzyme showed significant homology with those of the sacA protein from Bacillus subtilis. In addition, a region of amino acid homology with the S. mutans fructosyltransferase and B. subtilis levansucrase proteins was also detected.
Intact motility as a Salmonella typhi invasion-related factor
Authors: Liu S L , Ezaki T , Miura H , Matsui K , Yabuuchi E
Published: January 2020

Invasiveness of Salmonella typhi was investigated. At first, we introduced Tn5 into the chromosome of a wild-type S. typhi strain, GIFU 10007, and screened the independent Tn5 insertion mutants for noninvasive (Inv-) strains. During the first half of this work, we obtained 4 Inv- strains from 1,338 independent Tn5 mutants. The four were either nonflagellate (Fla-), nonmotile (Mot-), or nonchemotactic (Che-). We then isolated more Fla-, Mot-, or Che- mutants and examined the invasiveness of these mutants. Sixty-three spontaneous or Tn5 insertion motility mutants, i.e., Fla-, Mot-, or Che-, were independently isolated from the wild-type strain GIFU 10007; all of them were noninvasive. Motile revertants isolated from some of these mutants showed the same invasiveness as the parent strain. P22-mediated transductional crosses were carried out between some of the motility mutants (as the recipients) and the Fla- reference strains of S. typhimurium with known deletion sites on the genome (as the donors). The mutational sites of the S. typhi mutants were assigned almost evenly to the three flagellar gene regions (regions I, II, and III) of S. typhimurium. The invasiveness of the motile recombinants obtained from the transduction assays was examined. The restoration of intact motility resulted in the restoration of invasiveness. Thus, we conclude that intact motility is an invasion-related factor of S. typhi. The relationship of Vi antigen to the invasiveness of S. typhi was also studied. Vi-negative mutants with intact motility remained invasive, whereas all 63 Inv- spontaneous or Tn5 mutants were Vi positive. Therefore, Vi antigen was not related to the invasiveness of S. typhi.
Adherence of Candida albicans germ tubes to plastic: ultrastructural and molecular studies of fibrillar adhesins
Authors: Tronchin G , Bouchara J P , Robert R , Senet J M
Published: January 2020

Germ tubes of Candida albicans produced an additional fibrillar surface layer responsible for enhanced adherence to plastic. The correlation between germination of C. albicans and adherence of germ tubes to a plastic matrix led us to consider the existence of germ tube-specific adhesive components involved in the attachment process. Using concanavalin A-sensitized latex microspheres, we first detected extracellular molecules on the plastic surface after removal of the adherent germ tubes. Electron microscopy confirmed that fibrils of the germ tube involved in cell-substratum interconnections were retained on the plastic surface. Cytochemistry with concanavalin A-gold labeling demonstrated that these fibrillar structures contained mannoproteins. Dithiothreitol and iodoacetamide treatment of washed plastic allowed us to further characterize these fibrillar adhesins. Through analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two components with molecular weights (MWs) of 68,000 and 60,000 were detected on the plastic surface. The 68,000-MW component appeared to be one of the major constituents of the germ tube surface layers. Biosynthetic labeling experiments performed with L-[35S]methionine revealed two additional proteins: a high-MW component (greater than 200,000), and a 200,000-MW component. These four proteins, strongly labeled on the plastic surface and on the germ tube cell wall layers, were in contrast slightly labeled or even nonidentified in the culture supernatant, suggesting their involvement in germ tube adherence.
Antibody responses against Mycobacterium tuberculosis in 11 strains of inbred mice: novel monoclonal antibody specificities generated by fusions, using spleens from BALB.B10 and CBA/J mice
Authors: Ljungqvist L , Worsaae A , Heron I
Published: January 2020

Eleven strains of inbred mice were immunized with a culture filtrate of Mycobacterium tuberculosis H37Rv, and the quality of the antibody responses was determined by immunoblotting. The quantity of mycobacterial antigen used for each immunization ranged from 6 to 750 micrograms per inoculum. The culture filtrate of M. tuberculosis was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose filters. Immunoblotting results were obtained with serum from the following 11 strains of immunized mice: C57BL/6J, BALB/cJ, BALB.B10, C3H.OH, A.CA/Sn, CBA/J, DBA/1J, DBA/2J, C3H/HeJ, B10.A/SgSn, and B10.D2/nSn. Mice were tested individually, and results from each mouse were compared after each immunization. It was found that sera from individual mice within the same strain differed only slightly in their immune response patterns. In contrast, major differences were seen when the reactivities of sera from different strains were compared. Hybridomas were obtained from cell fusions by using spleen cells from BALB.B10 and CBA/J mice. Twelve monoclonal antibodies were raised, which identified epitopes on molecules with different electrophoretic mobilities than those already described by other investigators. The monoclonal antibodies were characterized by immunoblotting with respect to their reactivities with culture filtrates from M. tuberculosis and six other mycobacterial species. One of the monoclonal antibodies (HBT-10) identified an epitope that was present in M. tuberculosis H37Rv but not in Mycobacterium bovis BCG.