bluuebunny/crossref_metadata_2025 huggingface dataset:
Serological, electrophoretic, and biological properties of Cryptococcus neoformans antigens
Authors: Murphy J W , Mosley R L , Cherniak R , Reyes G H , Kozel T R , Reiss E
Published: January 2020

We compared a cryptococcal culture filtrate antigen referred to as CneF with chemically defined cryptococcal antigen fractions isolated by Cherniak and co-workers by using double immunodiffusion gels, polyacrylamide gel electrophoresis, immunoblots, and footpad reactivity of immunized mice. The three previously described components of cryptococcal culture filtrates are a high-molecular-weight glucuronoxylo-mannan (GXM), which is the major constituent, a galactoxylomannan (GaIXM), and a mannoprotein (MP). In this study we demonstrated that CneF contained components which were serologically and electrophoretically similar to the three previously described cryptococcal culture filtrate fractions. The MP fraction elicited significantly stronger delayed-type hypersensitivity responses than did the GXM or GaIXM fraction when used in mice immunized either with the CneF in complete Freund adjuvant or whole heat-killed Cryptococcus neoformans yeast cells. These findings were confirmed when the footpads of immunized mice were challenged with GaIXM and MP preparations from a culture filtrate of a C. neoformans acapsular mutant that does not produce GXM. Thus, we concluded that the MP was the primary component recognized by the anticryptococcal cell-mediated immune response in mice.
Human salivary acidic proline-rich proteins and statherin promote the attachment of Actinomyces viscosus LY7 to apatitic surfaces
Authors: Gibbons R J , Hay D I
Published: January 2020

Actinomyces viscosus LY7 cells adsorbed in high numbers to experimental pellicles formed on hydroxyapatite (HA) from human parotid or submandibular saliva but not to pellicles prepared from human plasma or serum. To determine the nature of the salivary components responsible for promoting adhesion, pellicles were prepared from fractions of submandibular and parotid saliva obtained by chromatography on Trisacryl GF 2000 columns. Adsorption of LY7 cells was promoted by two groups of fractions. Each group was rechromatographed on DEAE-agarose. Fractions which promoted adsorption of LY7 cells were found by polyacrylamide gel electrophoresis to contain the acidic proline-rich proteins (PRPs) and statherin. Pellicles prepared from 12-micrograms/ml solutions of pure PRP-1, PRP-2, or parotid isoelectric focusing (PIF-slow) variant promoted maximal adsorption of A. viscosus LY7 cells. Somewhat higher concentrations of PRP-3 and PRP-4 were required for maximal adsorption, indicating that the 44-residue carboxy-terminal segment of PRP-1, PRP-2, and PIF-slow enhances LY7 binding but is not essential. Much higher concentrations of statherin were required to promote LY7 adsorption. Adsorption of LY7 cells to pellicles prepared from PRP-1 was not affected over the range of pH 5 to 8. Adsorption was also not inhibited by 50 mM lactose, which is consistent with the notion that type 1 fimbriae, rather than type 2 fimbriae, were responsible. A. viscosus T14, Actinomyces odontolyticus ATCC 17982, and Actinomyces israelii 12597 also adsorbed to PRP-1 pellicles, whereas Actinomyces naeslundii ATCC 12104 did not. Although A. viscosus cells bind strongly to adsorbed PRP-1, the presence of PRP-1 or PRP-3 in solution did not inhibit adhesion. Similarly, [3H]PRP-1 did not bind to LY7 cells, nor was it degraded when incubated with the organism. However, LY7 cells adsorbed to [3H]PRP-1 pellicles. These data suggest that hidden molecular segments of PRP become exposed when the protein adsorbs to HA; these segments then react with adhesins of LY7 cells. The apparent ability of A. viscosus cells to recognize segments of PRPs which are exposed only in surface-adsorbed molecules provides a novel mechanism which enables the organism to attach to teeth when suspended in salivary secretions.
Recombinant interleukin-2 limits the replication of Mycobacterium lepraemurium and Mycobacterium bovis BCG in mice
Authors: Jeevan A , Asherson G L
Published: January 2020

BALB/c mice were infected with Mycobacterium lepraemurium in the footpad or with Mycobacterium bovis BCG intravenously with 5 x 10(7) bacilli. Recombinant interleukin-2 (IL-2) was injected intraperitoneally as a single dose (20,000 U), as a single course of five injections (400 U each), or as a 6-month course starting 3 days after the M. lepraemurium infection. BCG-infected mice received a single dose (1,000 U) or five daily injections of 100 or 1,000 U each. IL-2 significantly reduced the total bacterial counts in the footpad, lymph nodes, and liver of M. lepraemurium-infected mice (50 to 85%) by 6 months and viable counts in the spleen (30 to 50%) by 60 days after BCG infection. The courses of IL-2 started at 60 days were more effective than those started at 3 days after M. lepraemurium infection (P less than 0.05 to 0.001), and for BCG, 100 U of IL-2 was better than 1,000 U (P less than 0.05 to 0.01). These results indicate that IL-2 limits mycobacterial infections in mice and raise the question of its possible use in humans.
Kinetics of proteolysis of hog gastric mucin by human neutrophil elastase and by Pseudomonas aeruginosa elastase
Authors: Poncz L , Jentoft N , Ho M C , Dearborn D G
Published: January 2020

Human neutrophil and Pseudomonas aeruginosa elastases were compared for their ability to degrade hog gastric mucin, which was used as a model substrate. P. aeruginosa elastase was more active than neutrophil elastase, and 2 to 10 peptide bonds were hydrolyzed within 5 min. The results demonstrate that both elastases degrade mucins actively at concentrations comparable to physiological levels of neutrophil elastase, which raises the possibility that proteolysis of mucins may be one mechanism of damage during chronic infection and inflammation of the respiratory tract.
Effect of protein malnutrition on salmonellosis and fever
Authors: Bradley S F , Kauffman C A
Published: January 2020

Protein-malnourished rats had greater numbers of bacteria in their livers and spleens compared with numbers in control rats following a sublethal intraperitoneal dose of Salmonella typhimurium. However, the febrile responses of both groups of rats to infection were similar. The ability to mount a normal febrile response does not necessarily reflect the ability of the host to contain an infection.
Quantitative relationship of Treponema denticola to severity of periodontal disease
Authors: Simonson L G , Goodman C H , Bial J J , Morton H E
Published: January 2020

The Treponema denticola content of plaque was quantitatively estimated for samples taken from periodontitis patients as well as periodontally healthy subjects among two separate human populations. The populations studied included military volunteers and civilians at a university dental clinic. The plaque samples from each population were grouped according to pocket depth measurements at the collection site. A biotin-avidin enzyme-linked immunosorbent assay procedure was developed with a monoclonal antibody specific for a serovariety of T. denticola. T. denticola was present at significantly elevated levels in plaque samples collected from deep-pocket sites of patients with severe periodontitis relative to the healthy controls and a group with moderate disease. The ratio of T. denticola content per milligram of plaque in the deep pocket groups to that of the other two groups was about 2:1 for both populations. This is the first quantitative evidence of a positive relationship between a specific spirochete species and severe periodontitis.
Steric hindrance of antibody binding to surface proteins of Coxiella burnetti by phase I lipopolysaccharide
Authors: Hackstadt T
Published: January 2020

The exposure of surface protein antigens on virulent phase I Coxiella burnetti was compared with that on avirulent phase II cells. Although anti-phase II antibodies did not bind to the surfaces of native intact phase I cells, they bound to phase I proteins if the proteins were solubilized for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by immunoblotting. In addition, removal of the phase I lipopolysaccharide (LPS) by trichloroacetic acid exposed surface proteins for reactivity with anti-phase II antibodies, as shown by immunofluorescence assays, direct antibody binding, and immunoelectron microscopy using protein A-colloidal gold conjugates. Based on these observations, a simple model of phase variation is proposed to explain the apparently conflicting notions of the identity of the phase II antigen(s). The model suggests that the phase I LPS sterically hinders access of anti-phase II antibodies to a multitude of shared protein antigens, any one of which may confer phase II specificity. Exposure of these shared protein antigens through the appearance of a more truncated LPS (phase II) or extraction of the smooth-type phase I LPS allows antibody accessibility and therefore confers apparent phase II serospecificity.
Trichinella pseudospiralis larvae express natural killer (NK) cell-associated asialo-GM1 antigen and stimulate pulmonary NK activity
Authors: Niederkorn J Y , Stewart G L , Ghazizadeh S , Mayhew E , Ross J , Fischer B
Published: January 2020

Natural killer (NK) cell function was evaluated in mice infected with either Trichinella pseudospiralis or T. spiralis larvae. T. pseudospiralis-infected mice consistently demonstrated augmented pulmonary NK cell-mediated clearance of YAC-1 tumor cells in vivo but failed to display enhanced splenic NK cell-mediated lysis of the same tumor cells in vitro. Attempts to alter NK cell function in vivo by the injection of anti-asialo-GM1 antibody resulted in anaphylaxis and death of the hosts infected with T. pseudospiralis but not of mice infected with T. spiralis. Immunofluorescence studies demonstrated that larvae of both species bound large quantities of anti-asialo-GM1 antibody but did not react with another NK-associated antibody, anti-NK-1.1. Even though anti-asialo-GM1 antibody reacted with larvae of both species, intravenous injection of antibody elicited anaphylaxis only in hosts that were infected with T. pseudospiralis.
Variation in the expression of cell wall proteins of Staphylococcus aureus grown on solid and liquid media
Authors: Cheung A L , Fischetti V A
Published: January 2020

To evaluate the variation in the expression of cell wall antigens between Staphylococcus aureus grown in liquid medium and solid support, bacteria were harvested from liquid chemically defined medium and chemically defined medium in a 1% agar base. Cell wall proteins were then extracted by lysostaphin in a protoplast-stabilizing medium (30% raffinose). After separation of the cell wall antigens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots, they were probed with chicken antiserum to an S. aureus strain grown on a solid support. For each of the 15 clinical strains analyzed, high-molecular-size bands (molecular size range, 120 to 220 kilodaltons) were either enhanced or distinctly present when compared with those from the cell wall extract of the same strain grown in liquid medium. Results of enzymatic treatment of whole staphylococci grown on solid medium suggested the proteinaceous nature and the surface location of these antigens. Limited passage studies demonstrated the ability of the staphylococci to alter these surface proteins when passaged alternately on liquid and solid media. These observations suggested the importance of the microenvironment to the expression of cell wall proteins in S. aureus. Correlations with observations in vivo may help identify the determinants of microbial pathogenicity in S. aureus.
Heterologous expression of the 65-kilodalton antigen of Mycobacterium leprae and murine T-cell responses to the gene product
Authors: Lamb F I , Kingston A E , Estrada I , Colston M J
Published: January 2020

The gene encoding the immunodominant 65-kilodalton antigen of Mycobacterium leprae was subcloned from a lambda gt11 clone into the high-copy-number plasmid pUC8. Escherichia coli containing these recombinants produced large amounts of the antigen, which was purified by polyacrylamide gel electrophoresis in the presence of urea. The ability of E. coli to recognize the mycobacterial promoter was confirmed by constructing additional clones in which the gene is flanked by transcriptional terminators from phage fd. A similar approach was used to demonstrate the expression of this gene in Streptomyces lividans. Mice immunized with killed M. leprae showed cell-mediated immune reactivity to the purified 65-kilodalton protein which stimulated both in vitro lymphoproliferative and in vivo delayed-type hypersensitivity responses.
The cytolytic toxin aerolysin must aggregate to disrupt erythrocytes, and aggregation is stimulated by human glycophorin
Authors: Garland W J , Buckley J T
Published: January 2020

The hole-forming toxin aerolysin was shown to aggregate after binding to erythrocytes at 37 degrees C. Although the protein also bound and aggregated at 4 degrees C, hole formation was not observed, indicating that aggregation preceded penetration of the lipid bilayer. Aggregation, but not binding, could be blocked by pretreatment of the toxin with diethyl pyrocarbonate, a histidine-reactive reagent. This resulted in inactivation of the toxin. Incubation of aerolysin with glycophorin purified from human erythrocytes caused aggregation and complete inactivation. Erythrocytes which lacked glycophorin were less sensitive to the toxin. Proaerolysin, the inactive precursor of aerolysin, also bound to erythrocytes; however, it did not aggregate, nor did it aggregate when preincubated with glycophorin. The protoxin could be activated by treatment with trypsin even after it had bound to erythrocytes. Activation could also be achieved by reaction of proaerolysin with a variety of other proteases, each of which brought about a similar reduction in protein molecular weight. The activated protein was resistant to further proteolysis. These results indicate that aggregation is a necessary step in hole formation and that the sites on aerolysin required for binding and for aggregation and hole formation are separate.
Alveolar macrophage function is selectively altered after endotoxemia in rats
Authors: Christman J W , Petras S F , Hacker M , Absher P M , Davis G S
Published: January 2020

The alveolar macrophage (AM) is exquisitely sensitive to activation by gram-negative bacterial endotoxin, an agent associated with adult respiratory distress syndrome. We tested the hypothesis that specific functions of the AM are activated selectively by in vivo endotoxin while others remain unaffected. AMs were recovered from the airspaces of control and endotoxin-treated (5.0 mg/kg) rats, and functional assays were performed. We measured macrophage adherence, viability, and survival; chemotactic movement; hydrogen peroxide production; phagocytic function; and the secretion of representative biological response modifiers. Endotoxemia enhanced AM adherence during a 15-h incubation period, while not affecting cell number or viability. There was a 60% reduction in AM chemotactic movement and a 65% augmentation of hydrogen peroxide production, but no effect on AM phagocytosis of Staphylococcus aureus. Endotoxemia enhanced AM production of macrophage-derived chemotactic activity for neutrophils by 70% and interleukin-1 activity by 100%, but did not affect the production of macrophage-derived growth factor activity for fibroblasts. We conclude that endotoxemia alters the functions of the AM in a selective manner; certain functions are enhanced, while others are inhibited or not affected. We believe that this selective effect on AM functional capacity may be an important mechanism explaining certain aspects of the course, duration, or outcome of adult respiratory distress syndrome associated with gram-negative sepsis.
Identification of a galactose-binding lectin on Fusobacterium nucleatum FN-2
Authors: Murray P A , Kern D G , Winkler J R
Published: January 2020

A previous study has suggested that Fusobacterium nucleatum FN-2 contains a galactose-binding protein (lectin) on the cell surface (P. A. Murray, V. Matarese, C. I. Hoover, and J. R. Winkler, FEMS Microbiol. Lett. 40:123-127, 1987). In the present study, the molecular specificity and size of this lectin were investigated by several techniques. Whole-cell affinity chromatography with asialofetuin covalently coupled to Sepharose 6MB demonstrated that 81% of 3H-labeled F. nucleatum were specifically eluted by 0.5 M galactose. Specific binding was calcium dependent and did not occur in the presence of calcium chelators. Binding was inhibited by preincubation with galactose. Agglutination of human parotid saliva by F. nucleatum was also inhibited by galactose and its structural analogs. Inhibition by lactose was 2 times that of galactose, inhibition by p-aminophenyl galactosides was 4 times that of galactose, and inhibition by asialoglycopeptides was 100 times that of galactose. Similar inhibition results were obtained for hemagglutination of neuraminidase-treated erythrocytes. These findings suggest that the binding specificity of F. nucleatum FN-2 is more complex than simply the recognition of the monosaccharide galactose. This is consistent with the concept that lectins considered identical in terms of monosaccharide specificity can recognize fine differences in more complex structures. To identify the specific bacterial component(s) involved in galactose recognition, proteins of F. nucleatum FN-2 were separated on a 4 to 11% gradient sodium dodecyl sulfate slab gel, transferred to nitrocellulose paper to renature bacterial binding sites, and then incubated with 125I-labeled asialofetuin. Autoradiographs of the nitrocellulose revealed a band at a range of Mr 300,000 to 330,000 which was not present when the blots were preincubated with galactose. These data support the concept that F. nucleatum FN-2 possesses a lectin that recognizes galactose and galactose-containing substrates.
Resistance to infection with Eimeria vermiformis in mouse radiation chimeras is determined by donor bone-marrow cells
Authors: Joysey H S , Wakelin D , Rose M E
Published: January 2020

The course of infection with Eimeria vermiformis was determined in BALB/b, BALB/c, and C57BL/10ScSn (B10) mice and in radiation chimeras prepared from the H-2-compatible BALB/b and B10 mice. The BALB strains, irrespective of H-2 haplotype, were resistant, the B10 mice were susceptible, and in the chimeras infection was characterized by the genotype of the donated bone-marrow cells and not by the phenotype of the recipient. Thus, the genetic control of relative resistance or susceptibility to infection with this parasite is expressed through bone-marrow-derived cells.
Isolation and nucleotide sequence of the F17-A gene encoding the structural protein of the F17 fimbriae in bovine enterotoxigenic Escherichia coli
Authors: Lintermans P , Pohl P , Deboeck F , Bertels A , Schlicker C , Vandekerckhove J , Van Damme J , Van Montagu M , De Greve H
Published: January 2020

The genetic determinant for production of the fimbrial F17 adhesive antigen was isolated from a bovine enterotoxigenic Escherichia coli strain. The F17-A gene, coding for the structural component of the F17 fimbrial adhesin, was cloned and sequenced. An open reading frame of 540 base pairs encoding a polypeptide of 180 amino acids, of which the NH2-terminal 21 residues are characteristic of a signal sequence, has been characterized. The mature protein lacks histidine, methionine, and tryptophan. A possible promoter and ribosome binding site as well as a possible site for termination of transcription are proposed. An important homology of the F17-A protein with fimA and papA fimbrial proteins was found. The N-terminal sequence of the mature F17-A pilin is extremely similar to the N-terminal sequence of the G fimbriae identified on human pyelonephritogenic E. coli strains.
Synthetic peptides of Shiga toxin B subunit induce antibodies which neutralize its biological activity
Authors: Harari I , Donohue-Rolfe A , Keusch G , Arnon R
Published: January 2020

Shiga toxin B chain, the binding subunit of Shiga toxin, was recently purified; and the amino acid sequence of this 7,716-dalton polypeptide was determined (N.G. Seidah, A. Donohue-Rolfe, C. Lazure, F. Auclair, G. T. Keusch, and M. Chretien, J. Biol. Chem. 261:13928-13931, 1986). In the present study, synthetic peptides corresponding to three overlapping sequences from the N-terminal region of this subunit were prepared. The peptides synthesized consisted of residues 5 to 18, 13 to 26, and 7 to 26. This region coincides with the major peak of hydrophilicity and surface area residues predicted from a computer analysis. For the purpose of immunization, the peptides either were conjugated with a protein or synthetic carrier or were polymerized with glutaraldehyde. Antisera against these peptide derivatives raised in rabbits reacted not only with the respective homologous peptide but also to a comparable extent with the intact Shiga toxin. The anti-peptide antisera effectively neutralized the various biological activities of the Shiga toxin, namely, cytotoxicity to HeLa cells, enterotoxic activity (the fluid secretion into ligated ileal loops in rats), and neurotoxicity in mice. Furthermore, active immunization with the peptide conjugates was found to protect mice against the lethal effect of Shiga toxin.
Novel 17-kilodalton Leishmania antigen revealed by immunochemical studies of a purified glycoprotein fraction recognized by murine T lymphocytes
Authors: Rodrigues M M , Xavier M T , Mendonça-Previato L , Barcinski M A
Published: January 2020

Recently, a glycoprotein fraction, designated gp10/20, purified from Leishmania mexicana amazonensis was shown to induce a cellular immune response mediated by murine L3T4+ T lymphocytes. This fact led us to pursue further the characterization of this fraction. The present study demonstrated that gp10/20 is a degradation product of a 17-kilodalton antigen present in promastigotes and amastigotes of L. mexicana amazonensis. This antigen was easily detected in promastigotes of L. mexicana mexicana, L. donovani, L. chagasi, L. major, and L. tropica. However, culture forms of L. braziliensis complex expressed either low amounts of the 17-kilodalton antigen or an antigenically unrelated antigen. The recognition of gp10/20 by several serum samples of patients with kala-azar was also shown.
Alteration of sleep in rabbits by Staphylococcus aureus infection
Authors: Toth L A , Krueger J M
Published: January 2020

Abundant evidence suggests that sleep might be altered during infectious disease, although the relationship between sleep and infectious disease has never been examined systematically. To address this issue, we determined the effects of Staphylococcus aureus infection on rabbit sleep. Rabbits inoculated intravenously with S. aureus demonstrated the expected physiological changes consistent with a state of infectious disease (e.g., lymphopenia, neutrophilia, and fever), as well as time-dependent changes in sleep patterns. The sleep changes were characterized initially by increases in (i) the time spent in slow-wave sleep, (ii) the electroencephalographic slow-wave amplitudes during slow-wave sleep, and (iii) the duration of individual bouts of slow-wave sleep. At 20 to 36 h after inoculation, sleep responses fell to levels below corresponding control values for 6 to 12 h. At 6 to 10 h after inoculation, rapid-eye-movement sleep was suppressed and remained at low levels throughout the remainder of the 48-h recording period. These effects of bacterial infection on sleep were attenuated by antibiotic (cephalothin) therapy. Inoculation with killed bacteria produced similar changes in sleep and other physiological parameters, although significantly higher numbers of organisms were required to produce equivalent responses. We postulate that changes in sleep may represent an adaptive response of the host to infectious disease.
Boosting of transmission-blocking immunity during natural Plasmodium vivax infections in humans depends upon frequent reinfection
Authors: Ranawaka M B , Munesinghe Y D , de Silva D M , Carter R , Mendis K N
Published: January 2020

The infectivity to mosquitoes of 31 acute Plasmodium vivax patients was measured by permitting mosquitoes to feed directly on the patients. The infectivity of these patients correlated closely with titers of antibodies in their serum as measured by indirect immunofluorescence against air-dried female gametes of P. vivax. Infectivity by direct feeding was also closely parallel to the transmission-blocking activity of the sera of patients as measured by the suppression of infectivity of parasitized blood by autologous serum relative to normal (nonmalarial) human serum when fed to mosquitoes through a membrane. These results are consistent with serum antibodies in human P. vivax infections as major factors determining the infectivity of an infected individual to mosquitoes. It was further noted that individuals having a second attack of P. vivax within less than 4 months were considerably less infectious to mosquitoes than first-attack patients were. This "boosting" of transmission-blocking immunity was much less if longer intervals intervened between attacks. We discuss the immunological implications and possible epidemiological significance of this short-term boosting of transmission-blocking immunity by successive P. vivax infections.
Role of alveolar macrophages in lipopolysaccharide-induced neutrophil accumulation
Authors: Harmsen A G
Published: January 2020

B6D2F1/TRU mice were exposed to a lipopolysaccharide (LPS) aerosol that resulted in a 1-h-postexposure lung burden of about 290 ng of LPS. This exposure caused an accumulation of neutrophils in the lung that peaked between 6 and 12 h after exposure. To determine the potential role of alveolar macrophages (AM) in the induction of neutrophil accumulation by LPS, 10(6) AM from normal or LPS-exposed mice were transferred to the lungs of groups of naive recipient mice. A third group of mice was instilled intratracheally with vehicle only. After 5 h, the lungs of the mice were lavaged and the numbers of neutrophils in the lavage fluids were determined. The instillation of AM from unexposed mice did not cause significantly more neutrophils to accumulate than did the instillation of vehicle only, whereas the instillation of AM from LPS-exposed mice caused nearly a threefold increase in the numbers of neutrophils in lavage fluids. Transfer of AM from LPS-exposed mice into cutaneous air pouches of naive mice also caused greater local neutrophil accumulation (10-fold) than did the transfer of AM from normal mice. Repeated freeze-thawing of the suspensions of AM before transfer to recipients significantly reduced the ability of the suspensions to induce neutrophil accumulation. This indicated that AM viability is necessary to cause a maximal neutrophil infiltration upon transfer of the AM. To determine the extent to which LPS-induced neutrophil accumulation depends on the presence of AM, the ability of LPS to elicit neutrophil accumulation when injected alone or together with AM into air pouches was determined. The injection of either AM or LPS alone caused few neutrophils to accumulate, whereas the injection of LPS and AM together caused a large number of neutrophils to accumulate. The results of this study indicate that LPS deposition in the lung can stimulate AM to induce neutrophil accumulation and that this may be the major mechanism by which LPS causes neutrophil accumulation.
Susceptibility of germfree or antibiotic-treated adult mice to Cryptosporidium parvum
Authors: Harp J A , Wannemuehler M W , Woodmansee D B , Moon H W
Published: January 2020

Adult mice are more resistant than neonatal mice to intestinal colonization with the protozoan parasite Cryptosporidium parvum. Development of a mature intestinal flora may play a role in this resistance. We compared susceptibilities to colonization with C. parvum in adult conventional mice, adult germfree mice, and adult conventional mice treated with oral antibiotics to deplete the intestinal flora. Germfree mice of both CD1 and BALB/c strains were colonized at day 7 following inoculation with C. parvum oocysts isolated from the feces of an infected, diarrheic calf. Age-matched conventional mice of the same strains were comparatively resistant to colonization. Conventional mice treated with antibiotics remained resistant to colonization. These results suggest that the microflora in the intestine was not the sole determinant of resistance or susceptibility to colonization. The germfree adult mouse as an experimental model of cryptosporidiosis is discussed.
Induction of immunity with avirulent Listeria monocytogenes 19113 depends on bacterial replication
Authors: Baldridge J R , Thomashow M F , Hinrichs D J
Published: January 2020

Events necessary for triggering the cell-mediated response to intracellular parasites are poorly understood. Here we show that extremely high doses of avirulent Listeria monocytogenes 19113 (greater than 10(9] induce a modest and short-lived state of resistance in BALB/c mice. Induction of this protective state could not be achieved with nonviable bacteria and was blocked by inhibiting replication of viable L. monocytogenes 19113 through antibiotic treatment. The immune response was antigen specific and could be adoptively transferred with lymphoid cells. However, unlike the prototypic acquired cellular resistance induced by virulent Listeria strains, the protective response induced by L. monocytogenes 19113 was extremely short-lived, lasting less than 2 weeks, with a precipitous decline in the activity of the immune cells involved. An intervening in vitro culture period with concanavalin A greatly enhanced the activity of the adoptively transferred immune cells.
CS31A, a new K88-related fimbrial antigen on bovine enterotoxigenic and septicemic Escherichia coli strains
Authors: Girardeau J P , Der Vartanian M , Ollier J L , Contrepois M
Published: January 2020

The nature of the common surface antigen of six hemagglutinating and adhesive piliated Escherichia coli strains isolated from diarrheic or septicemic calves was studied. By electron microscopy studies, the E. coli surface antigen designated CS31A was found on bacterial cells and in purified form to consist of thin (2-nm) "fibrillar" fimbriae. E. coli 31A, which was cured of a 105-megadalton plasmid, failed to express CS31A fimbriae, but retained the ability to hemagglutinate and to adhere in vitro on intestinal cells. Conversely, E. coli K-12, harboring the 105-megadalton plasmid originating from strain 31A, produced CS31A fimbriae but was not able to hemagglutinate or adhere on intestinal cells. A single fimbrial subunit of 29 kilodaltons was observed when purified fimbriae from the 105-megadalton plasmid-containing E. coli K-12 strain was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis or eluted by gel filtration after dissociation by 8.5 M guanidium hydrochloride from an S300 Sephacryl column. Western immunoblot analysis and the N-terminal sequence and amino acid composition of CS31A indicate structural and immunological relatedness between CS31A and K88 protein subunits.
Respiratory-mucin inhibition of the opsonophagocytic killing of Pseudomonas aeruginosa
Authors: Vishwanath S , Ramphal R , Guay C M , DesJardins D , Pier G B
Published: January 2020

Pseudomonas aeruginosa is a frequent respiratory tract colonizer in diseases in which mucociliary clearance is defective. The most striking of these is cystic fibrosis. The reasons for this organism's ability to colonize the respiratory tract and to persist there are not fully understood. Earlier studies showed that P. aeruginosa adheres preferentially to tracheobronchial mucin when compared with enterobacteria. We reasoned that if adherence to respiratory mucin protected P. aeruginosa from opsonophagocytic killing, then the ability of this organism to chronically colonize the respiratory tract could be partially explained. Using an opsonophagocytic killing assay with human polymorphonuclear leukocytes, we found that respiratory mucin protected six strains of P. aeruginosa from opsonophagocytic killing but did not protect poorly adhering strains of Escherichia coli, Staphylococcus aureus, or group B streptococci. Incubating P. aeruginosa with the mucin prior to addition to the opsonic assay inhibited phagocytic killing, whereas incubation of polymorphonuclear leukocytes with mucin did not, suggesting that inhibition was not due to an effect of mucin on leukocytes per se but was a consequence of bacterial adherence to mucin. Further studies indicated no decrease in the binding of either antibody or complement component C3 to the bacterial surface in the presence of mucin. This suggests that phagocytic inhibition may be due to a defect in uptake or destruction of mucin-coated bacteria by the leukocytes. Thus, the adherence of P. aeruginosa to respiratory mucin potentially contributes to its persistence in the respiratory tract by interfering with host immune responses.
Protective ability of antibodies against 78- and 40-kilodalton outer membrane antigens of Haemophilus somnus
Authors: Gogolewski R P , Kania S A , Liggitt H D , Corbeil L B
Published: January 2020

The ability of concentrated antibody against the 78- or 40-kilodalton (kDa) outer membrane protein (OMP) of Haemophilus somnus to passively protect calves against H. somnus-induced pneumonia was determined. The 78- and 40-kDa OMPs were evaluated in passive protection experiments, because results of previous studies demonstrated their (i) immunogenicity for cattle, (ii) intense reactivity with convalescent-phase sera which passively protected calves against experimental H. somnus pneumonia, (iii) surface location and accessibility to antibody, and (iv) conservation among a wide range of H. somnus isolates obtained from animals with different diseases and from different geographic locations. The specificity of the two antisera evaluated in this study was verified by (i) immunoblots in which reactivity against the 78- or 40-kDa OMP was present in postimmunization but not preimmunization serum and (ii) immunoblots in which affinity-purified, surface-reactive antibodies in each antisera were used, which demonstrated that essentially only antibody to the 78- or 40-kDa OMP was reactive with the surface of H. somnus. In enzyme-linked immunosorbent assays, the antiserum against the 40-kDa OMP contained immunoglobulin G1 (IgG1), IgG2, and IgM against H. somnus, while the antiserum against the 78-kDa OMP contained IgG1 and IgM but no IgG2 against H. somnus. The antiserum against the 40-kDa OMP contained IgG1 and IgG2 specific for the 40-kDa OMP, as determined by Western blot analysis. Slight reactivity against H. somnus lipopolysaccharide was detected by enzyme-linked immunosorbent assay but not by Western blot analysis. In passive protection experiments, preincubation of bacteria with antibody against the 40-kDa OMP protected calves (P less than 0.025) against H. somnus pneumonia, while antibody against the 78-kDa OMP failed to protect calves against H. somnus pneumonia. Determination of the potential protective capacity of the 78-kDa OMP awaits resolution of the role of anti-78-kDa IgG2 in protection against H. somnus pneumonia. The 40-kDa OMP is, however, a good candidate antigen for evaluation of protective ability against H. somnus pneumonia following active immunization.
Use of lymphokines in treatment of experimental intra-abdominal abscess caused by Bacteroides fragilis
Authors: Gollapudi S V , Gupta A , Thadepalli H , Perez A
Published: January 2020

The role of cell-free soluble factors (lymphokines) derived from mitogen-activated splenic cells of mice previously immunized against Bacteroides fragilis was evaluated in the treatment of B. fragilis intra-abdominal abscess (IAA). Neither clindamycin nor lymphokines alone were effective against an established B. fragilis IAA, but the combination of clindamycin and lymphokines decreased the abscess size and bacterial counts in the majority of animals. This suggests that the synergy of lymphokines with clindamycin effects cure of IAA caused by B. fragilis and that lymphokines might have an application as adjuncts to conventional antimicrobial therapy in this setting.
Heterogeneity of hemolytic efficiency and isoelectric point of streptolysin O
Authors: Suzuki J , Kobayashi S , Kagaya K , Fukazawa Y
Published: January 2020

Using thin-layer agarose gel isoelectric focusing overlaid with thin-layer erythrocyte agar plates, we found that crude streptolysin O (SLO) consisted of a variety of hemolytic components with different isoelectric points (pIs) and that the distribution of pIs in crude SLO was different even in samples which were produced from a single strain of Streptococcus pyogenes under similar conditions. All of the hemolytic components in crude SLO were shown to have the properties of SLO with respect to their susceptibility to oxygen and anti-SLO serum and their molecular weight. The SLO components showed a single molecular weight of 64,000, but they exhibited various pIs ranging from pH 5.4 to 8.3, with major components showing a pI of 6 and/or 7.5. Further examination revealed the slope of the hemolytic titration curve to be dissimilar among the samples of crude SLO. Since the slope of the hemolytic titration curve of a component appears to be based on its hemolytic efficiency, the value of the slope was designated its hemolytic efficiency index. When SLO was purified by isoelectric focusing, the pI of the components was correlated with its hemolytic efficiency index; hemolytic components with lower pIs exhibited a lower hemolytic efficiency index. These results indicate that SLO consists of heterogeneous components with different pIs and suggest that the differences in hemolytic efficiency indices of SLO components are due to the different electrical charges of SLO molecules, which are related to their polymerization and affect hemolytic efficiency.
Recognition of typhus group rickettsia-infected targets by human lymphokine-activated killer cells
Authors: Carl M , Ching W M , Dasch G A
Published: January 2020

Lymphokine-activated killer cells caused significant lysis of target cells infected with typhus group rickettsiae (TGR) but not cells infected with Rickettsia tsutsugamushi. Monoclonal and polyclonal antibodies against TGR or TGR-infected cells failed to bind specifically to infected targets, as determined by flow cytometry or by immunoprecipitation of radiolabeled cell surface proteins. Major unaltered antigenic determinants of TGR therefore cannot be detected on the surface of infected targets.
Characterization of CS4 and CS6 antigenic components of PCF8775, a putative colonization factor complex from enterotoxigenic Escherichia coli E8775
Authors: Wolf M K , Andrews G P , Tall B D , McConnell M M , Levine M M , Boedeker E C
Published: January 2020

PCF8775 is a putative colonization factor complex present on the surface of 10 to 20% of enterotoxigenic Escherichia coli strains and has been reported to be composed of antigen CS6 (morphology undefined) expressed alone or together with either of the rigid fimbrial antigens CS4 and CS5. To better define the individual components of this complex and the determinants of their expression, we prepared antiserum to the PCF8775 complex as it was expressed on prototype strain E8775 and then used the antiserum to identify the subunit structure of the antigens, to study their morphology, and to detect expression of individual components of the complex after transfer of plasmids into laboratory strain HB101. CS4 was purified from strain E8775, confirmed to be fimbrial by electron microscopy, and found to be composed of a 22-kilodalton protein subunit whose N-terminal amino acid sequence (1 to 20) was similar to that of colonization factor antigen I. Transconjugants that express CS6 but not CS4 were obtained by mating prototype strain E8775 with HB101. CS6 expression was mediated by a 61-megadalton plasmid. Expression of CS6 in the transconjugants correlates with expression of a 16-kilodalton cell surface protein. The CS6 antigen was confirmed to be present on the cell surface by immunogold labeling, but its morphology was beyond the limits of resolution by electron microscopy.
Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases
Authors: Simpson L L , Stiles B G , Zepeda H , Wilkins T D
Published: January 2020

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins.
C-reactive protein protects against preerythrocytic stages of malaria
Authors: Pied S , Nussler A , Pontent M , Miltgen F , Matile H , Lambert P H , Mazier D
Published: January 2020

We previously reported that low doses of interleukin-1 strongly inhibited in vitro development of the hepatic stages of Plasmodium falciparum and P. yoelii. Among several hypotheses, we considered the role of C-reactive protein (CRP), a major acute-phase reactant whose concentration increases markedly in infectious disorders. We demonstrated that human hepatocytes cultured in the presence of interleukin-1 released, as early as 30 min after stimulation, an increased amount of CRP. We then established that CRP bound to the P. falciparum and P. yoelii sporozoite surface membranes, probably via a phosphorylcholine binding site. Experiments in which CRP was added to rat hepatocyte monolayers during or after inoculation confirmed that the target of the CRP-mediated inhibition was at the very early phase of infection. These in vitro functional activities were confirmed in an in vivo model; rats with increased levels of CRP in serum following an injection of turpentine oil were found to be largely protected against an inoculation of P. yoelii sporozoites. The same results were observed in animals inoculated with sporozoites previously incubated in purified CRP or in sera of rats pretreated with turpentine oil. The latter effect was inhibited after incubation of serum from turpentine-injected rats with anti-CRP serum.
Structural and antigenic conservation of the P2 porin protein among strains of Haemophilus influenzae type b
Authors: Hansen E J , Pelzel S E , Orth K , Moomaw C R , Radolf J D , Slaughter C A
Published: January 2020

The P2 porin protein is the most abundant protein in the outer membrane of Haemophilus influenzae type b (Hib). Biochemical and immunochemical techniques were used to characterize the P2 proteins from a number of different Hib strains. P2 proteins from Hib outer membrane vesicles were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for in situ tryptic digestion. Solid-phase tryptic digests of P2 from eight Hib strains were resolved by high-pressure liquid chromatography and shown to be similar if not identical. Radioimmunoprecipitation analysis involving Hib cells (containing intrinsically radiolabeled proteins or lipooligosaccharide) and Western blot (immunoblot) analysis were used to identify two P2-specific murine monoclonal antibodies (MAbs). These MAbs were shown to be reactive with 120 Hib strains tested in a colony blot radioimmunoassay. One of these MAbs bound to a surface-exposed P2 epitope that was antibody accessible on all Hib strains tested; the other MAb was directed against a P2 epitope that either was not exposed on the cell surface or was otherwise inaccessible to antibody.
Altered in vivo activity of liposome-incorporated lipopolysaccharide and lipid A
Authors: Dijkstra J , Mellors J W , Ryan J L
Published: January 2020

We compared the abilities of free and liposome-incorporated Salmonella minnesota wild-type lipopolysaccharide (LPS) and lipid A to activate peritoneal macrophages and induce lethal toxicity in mice. Incorporation of lipid A into multilamellar vesicles resulted in a 100-fold-decreased potency to prime macrophages for phorbol myristate acetate-triggered release of H2O2. In addition, liposome incorporation reduced the lethality of LPS and lipid A at least 10-fold in dactinomycin-sensitized mice. Similar results were obtained with multilamellar liposomes delivered intravenously and when small unilamellar vesicles were employed. The observed difference in toxicity was not dependent on dactinomycin treatment, since a similar decrease was obtained with large doses of liposomal LPS in unsensitized mice. Control liposomes, prepared without LPS and lipid A, did not reduce the activities of the free compounds. The administration of a sublethal amount of liposomal LPS induced within 20 days, but not during the first week, tolerance to a subsequently injected lethal dose of free endotoxin. The latter observation suggests that early-phase tolerance is not the mechanism responsible for the reduced toxicity of liposomal LPS. These data show that liposomal LPS and lipid A have reduced endotoxic activity in vivo and are consistent with our hypothesis that a direct interaction of lipid A with appropriate plasma membrane components is necessary to efficiently trigger biologic responses. This interaction, however, is prevented by the stable insertion of LPS into the liposomal membrane.
Binding specificities of wild-type and cloned Escherichia coli strains that recognize globo-A
Authors: Lindstedt R , Baker N , Falk P , Hull R , Hull S , Karr J , Leffler H , Svanborg Edén C , Larson G
Published: January 2020

In this study we compared the specificity for the globoseries of glycolipids of Escherichia coli expressing the O-negative, A-positive (ONAP) adhesin and clones transformed with the pap-like (prs or pap-2) gene cluster. Receptor-active glycolipids were identified by the ability of radiolabeled bacteria to bind to the glycolipids on thin-layer chromatogram plates. The ONAP adhesin and pap-like clones bound with high affinity to the globo-A and Forssman glycolipids. The ONAP strains did not recognize other glycolipids of the globoseries. In contrast, the pap-like clones also showed weak binding to globotriaosylceramide and reacted weakly with Gal alpha 1----4 Gal beta-latex beads. We suggest that the pap-like and ONAP adhesins recognize an epitope shared by the globo-A and Forssman structures, e.g., terminal GalNAc alpha 1----3 bound to Gal alpha 1----4Gal beta-containing glycolipids.