bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence of the hemolysin I gene from Actinobacillus pleuropneumoniae
Authors: Frey J , Meier R , Gygi D , Nicolet J
Published: January 2020

The DNA sequence of the gene encoding the structural protein of hemolysin I (HlyI) of Actinobacillus pleuropneumoniae serotype 1 strain 4074 was analyzed. The nucleotide sequence shows a 3,072-bp reading frame encoding a protein of 1,023 amino acids with a calculated molecular size of 110.1 kDa. This corresponds to the HlyI protein, which has an apparent molecular size on sodium dodecyl sulfate gels of 105 kDa. The structure of the protein derived from the DNA sequence shows three hydrophobic regions in the N-terminal part of the protein, 13 glycine-rich domains in the second half of the protein, and a hydrophilic C-terminal area, all of which are typical of the cytotoxins of the RTX (repeats in the structural toxin) toxin family. The derived amino acid sequence of HlyI shows 42% homology with the hemolysin of A. pleuropneumoniae serotype 5, 41% homology with the leukotoxin of Pasteurella haemolytica, and 56% homology with the Escherichia coli alpha-hemolysin. The 13 glycine-rich repeats and three hydrophobic areas of the HlyI sequence show more similarity to the E. coli alpha-hemolysin than to either the A. pleuropneumoniae serotype 5 hemolysin or the leukotoxin (while the last two are more similar to each other). Two types of RTX hemolysins therefore seem to be present in A. pleuropneumoniae, one (HlyI) resembling the alpha-hemolysin and a second more closely related to the leukotoxin. Ca(2+)-binding experiments using HlyI and recombinant A. pleuropneumoniae prohemolysin (HlyIA) that was produced in E. coli shows that HlyI binds 45Ca2+, probably because of the 13 glycine-rich repeated domains. Activation of the prohemolysin is not required for Ca2+ binding.
Regulation of leukotriene B4 generation from human polymorphonuclear granulocytes after stimulation with formyl-methionyl-leucyl phenylalanine: effects of pertussis and cholera toxins
Authors: Hensler T , Köller M , König W
Published: January 2020

The effects of holotoxins and toxin subunits from Bordetella pertussis and Vibrio cholerae strains on intact and digitonin-permeabilized human polymorphonuclear neutrophils were studied. Our data clearly demonstrate that formyl-methionyl-leucyl-phenylalanine (fMLP)-induced generation of chemotactic active leukotriene B4 was inhibited by both holotoxins as well as by their isolated enzymatic A protomers. In contrast, the respective binding components (B oligomers) did not affect leukotriene formation. Priming of digitonin-permeabilized neutrophils with either guanylylimidodiphosphate or inositol trisphosphate increased subsequent stimulation with fMLP. In contrast, diacylglycerol decreased fMLP-induced leukotriene B4 formation, but inositol trisphosphate and diacylglycerol had no effect on inhibition mediated by the toxins. In addition, pertussis and cholera toxins reduced the specific binding of [3H]fMLP. Scatchard plot analysis revealed that the observed decrease of peptide binding was due to a reduced number of receptor sites. The fMLP-stimulated [3H]guanylylimidodiphosphate binding and GTPase activity used as parameters for the activation of G proteins were decreased in parallel. These results suggest altered chemotactic receptor numbers and G-protein functions responsible for the toxin-dependent suppression of fMLP-mediated response for neutrophils.
Cloning and sequencing of the genes coding for the 10- and 60-kDa heat shock proteins from Pseudomonas aeruginosa and mapping of a species-specific epitope
Authors: Sipos A , Klocke M , Frosch M
Published: January 2020

A genomic library of Pseudomonas aeruginosa DNA was screened with a monoclonal antibody (MAb 2528) specific for the P. aeruginosa 60-kDa heat shock protein. A positive clone, pAS-1, was isolated. The gene coding for P. aeruginosa chaperonin (hsp60) was localized to a 2-kb EcoRI fragment subcloned in pAS-2. A sequence analysis of pAS-2 and parts of pAS-1 identified two open reading frames that encoded proteins with calculated molecular masses of 10 and 57 kDa. In amino acid sequence comparison studies the sequences of these proteins, which were designated GroES and GroEL, exhibited up to 78% homology with known prokaryotic sequences of 10- and 60-kDa heat shock proteins (hsp10 and hsp60). In order to map the epitope recognized by MAb 2528, a series of GroEL nested carboxy-terminal deletion clones were tested with MAb 2528. We identified the clone with the shortest insertion that was still recognized by MAb 2528 and the clone with the largest insertion that was not recognized by MAb 2528. The 3' ends of the insertions were determined by sequencing and were found to delimit a region that encoded 25 amino acid residues. Synthetic oligonucleotides that coded for peptides possibly resembling the epitope within this region were ligated into expression vector pGEX-3X, and fusion proteins expressed by these clones were tested for reactivity with MAb 2528. By using this method we determined that the decapeptide QADIEARVLQ (positions 339 to 348 on GroEL) was responsible for the binding of P. aeruginosa-specific MAb 2528.
Blood proteins do not promote adherence of coagulase-negative staphylococci to biomaterials
Authors: Muller E , Takeda S , Goldmann D A , Pier G B
Published: January 2020

We studied the effects of in vitro and in vivo coating of catheters with human blood proteins on binding of coagulase-negative staphylococci. Coating resulted in no enhancement of binding. Catheters coated in vitro bound fewer organisms than uncoated catheters. Host proteins do not enhance adherence of coagulase-negative staphylococci to biomaterials.
Successive synovial Mycoplasma hominis isolates exhibit apparent antigenic variation
Authors: Olson L D , Renshaw C A , Shane S W , Barile M F
Published: January 2020

The expression of surface proteins by 14 successive Mycoplasma hominis isolates obtained from the synovial fluid of a chronically infected septic arthritis patient was examined. Marked differences in the expression of surface proteins, as determined by monoclonal antibodies raised against the first isolate, were observed. However, identical restriction patterns and virtually identical hybridization patterns with probes containing the conserved genes of the Mycoplasma capricolum rRNA operon and the Escherichia coli elongation factor Tu suggest that the protein differences might reflect antigenic variation by M. hominis during infection.
Occurrence and Persistence of “Australia” Antigen Determinants
Authors: Schmidt Nathalie J. , Roberto Ronald R. , Lennette Edwin H.
Published: January 2020

“Australia” (Au) antigens from various groups of individuals were examined for the presence of
d
and
y
determinants. Antigens from all of the 214 individuals examined were found to possess either the
d
or
y
determinant, in addition to the
a
determinant. With the
ay
antiserum employed, antibody absorption was found to be a more effective means than demonstration of spur formation for detection of the
y
determinant. Antigens with
ad
specificity predominated in a collection of sera from non-ill Tongan children and adults, but no significant differences were noted in the specificities of antigens from individuals from four different regions. Almost all of the antigens from various groups of individuals in California, including inmates of a state hospital, a group of heroin users, and hepatitis patients from San Francisco General Hospital, were of the
ay
specificity. With one exception, antigenic specificities were found to persist for 3 years in a group of Tongan school children. Specificities also persisted in chronic carriers from California and in hepatitis patients over the course of antigenemia. Of 15 human and 4 animal antisera examined, antibodies to the
y
determinant were demonstrable only in a single (human) antiserum, and antibodies to the
d
determinant were demonstrable in one guinea pig antiserum and one horse antiserum.
Tests for Antibody- and Cell-Mediated Hypersensitivity to Trypanosome Antigens in Rabbits Infected with
Trypanosoma congolense
Authors: Mansfield John M. , Kreier Julius P.
Published: January 2020

Rabbits infected with
Trypanosoma congolense
were tested for immediate and delayed-type hypersensitivity responses to parasite antigens. Positive skin-test reactions were exclusively of the Arthus type, as shown by sequential histological analyses which revealed that more than 90% of the infiltrating cells at 24 hr postinjection were polymorphonuclear neutrophils. Skin reactions could be elicited in normal rabbits by intradermal injection of trypanosome antigen-antibody complexes. The absence of cell-mediated hypersensitivity to trypanosome antigens in infected rabbits was demonstrated by skin tests and by migration inhibitory factor and skin reactive factor tests. The role of the immediate-type skin reaction to trypanosome antigens in the pathology of infection and the possibility of its utilization for diagnosis are discussed.
Organ Cultures of Respiratory Epithelium Infected with Rhinovirus or Parainfluenza Virus Studied in a Scanning Electron Microscope
Authors: Reed Sylvia E. , Boyde A.
Published: January 2020

Organ cultures of bovine tracheal epithelium were infected with a rhinovirus or a strain of parainfluenza type 3 virus, and the epithelial surfaces were studied by scanning electron microscopy. When washed free from mucus, normal control cultures showed a thick carpet of normal cilia, whereas the two viruses each produced specific morphological abnormalities. In rhinovirus-infected cultures, degenerating ciliated and nonciliated cells with finely granular surfaces were rapidly extruded from the epithelium. The denuded epithelial surface was relatively smooth, and showed some evidence of squamous metaplasia. By contrast, in cultures infected with parainfluenza type 3 virus, damage developed more slowly and the epithelial surface was ultimately covered with a profuse array of short microvillous projections. In thin sections, some of these were shown to be the sites of viral maturation.
Parameters Affecting the Association of Vibrios with the Intestinal Surface in Experimental Cholera
Authors: Freter R.
Published: January 2020

Intestinal antibody (coproantibody) significantly reduced the adsorption of heat-killed
Vibrio cholerae
to the mucosa of in vivo isolated ileal loops of adult rabbits. This suggests a direct effect of coproantibody on adsorption, which appears to function in addition to the antibacterial mechanism described earlier. When antivibrio serum was administered passively into intestinal loops, it showed a predilection for the intestinal mucosa. In vivo adsorption of vibrios appeared to parallel their viability, i.e., vibrios killed by heat or in the presence of neomycin adsorbed significantly less than live vibrios. In contrast, in vivo adsorption was only slightly affected in the presence of bacteriostatic concentrations of tetracycline. Adsorption of
Salmonella senftenberg
and
V. cholerae
to slices of rabbit ileum in Krebs-Ringer solution appeared to involve different mechanisms, in that the former was strongly removed from the intestinal tissues by sodium lauryl sulfate, whereas vibrios were not affected by this agent. Triton X-100 increased the adsorption of vibrios, whereas rabbit bile and changes in
p
H had no effect.
Isolation of Protective Somatic Antigen from
Vibrio cholerae
(Ogawa) Ribosomal Preparations
Authors: Jensen Ronald , Gregory Barry , Naylor James , Actor Paul
Published: January 2020

Ribosomal preparations from
Vibrio cholerae
Ogawa and Inaba are protective immunogens for mice challenged with either serotype. Column chromatography of ribosomal fractions separated protective antigen from the ribosomes. The antigen is a heterogeneous colloid which contains protein, lipid, and carbohydrate in the ratio 3:1:1. Amino acid composition is that of a slightly acidic protein with no unusual complement of amino acids. The lipid component consists of several longchain fatty acids and of phosphatidyl ethanolamine. The major sugars identified were glucose and galactose. The colloid can be dissolved by treatment with chelating agents and sodium dodecyl sulfate. Purification of the solubilized material or of the colloid was unsuccessful.
Effect of Parainfluenza-3 Neuraminidase on Bovine Nasal Secretion
Authors: Morein Bror , Bergman Rune
Published: January 2020

Three samples of bovine nasal secretion were each separated into a sol phase and a surface gel phase. In all samples, the gel phase contained an approximately four times greater amount of bound
N
-acetylneuraminic acid (NANA) than the sol phase. From the gel phase, bound NANA could be released by exposure to parainfluenza-3 virus neuraminidase. The surface gel appears to be a natural substrate for this enzyme.
Concentration and Purification of Hepatitis B Antigen with Polyethylene Glycol and Polyelectrolyte 60, a Cross-Linked Copolymer of Isobutylene Maleic Anhydride
Authors: de Rizzo Edda , Pandey Ramayan , Wallis Craig , Melnick Joseph L.
Published: January 2020

A simple two-step procedure is described for the concentration and purification of hepatitis B antigen. The antigen is precipitated by polyethylene glycol and then adsorbed on and eluted from an insoluble polyelectrolyte, polyelectrolyte 60, which is a cross-linked copolymer of isobutylene maleic anhydride.
Detection of Delayed Hypersensitivity in Mice Injected with Ribonucleic Acid-Protein Fractions of
Salmonella typhimurium
Authors: Smith Randall A. , Bigley Nancy J.
Published: January 2020

Manifestations of delayed hypersensitivity (cell-mediated immunity) were found to be present in mice immunized with virulent
Salmonella typhimurium
SR-11 subfractions which contained appreciable concentrations of bacterial protein and in mice immunized with the living, attenuated RIA strain of
S. typhimurium.
Delayed hypersensitive responses were measured by footpad sensitivity in the immunized mice and in vitro by inhibition of spleen cell migration and by increase in
3
H-thymidine uptake by lymphoid cell populations when exposed to bacterial fractions rich in protein content.
Murine Resistance to Inhaled
Neisseria meningitidis
After Infection with an Encephalomyocarditis Virus
Authors: Goldstein E. , Buhles W. C. , Akers T. G. , Vedros N.
Published: January 2020

A reduction in pulmonary anti-bacterial activity due to a preceding viral illness has been suggested as the mechanism responsible for some meningococcal infections of the lung. We investigated this proposed pathogenesis by infecting mice with airborne encephalomyocarditis virus (EMC) and then challenging them 1, 4, and 7 days later with aerosols of
Neisseria meningitidis.
Meningococcal clearance was assessed by comparing the numbers of bacteria present immediately after inhaling the aerosols with the numbers present 3 hr later. To insure that EMC virus adequately depressed murine defense mechanisms, we also determined staphylococcal killing rates at 4 hr by using radiophosphorus-labeled staphylococcal aerosols. Viral infection depressed murine pulmonary antimeningococcal activity at 1 and 4 days (
P
< 0.01) but not at 7 days. Intrapulmonary staphylococcal killing was impaired on day 4 (
P
< 0.01) but not on days 1 or 7. Pulmonary viral titers decreased rapidly from 10
7
to 10
3
plaque-forming units/ml of lung during the experimental period. According to these data viral disease transiently depresses resistance to meningococcal infection. This impairment in host resistance is present while the viral titer is decreasing and follows a relatively similar pattern to the transient decrease noted for staphylococci.
Response of Cultured Mammalian Cells to Diphtheria Toxin IV. Isolation of KB Cells Resistant to Diphtheria Toxin
Authors: Moehring T. J. , Moehring J. M.
Published: January 2020

KB cell strains which are resistant to the lethal effects of diphtheria toxin were isolated from the toxin-sensitive KB cell line. For one such strain, KB-R2, the concentration of diphtheria toxin required to cause a 50% reduction of incorporation of amino acids into protein was more than 2,000 times that required in the sensitive KB-S cell. The KB-R2 strain was cloned by a single-cell isolation method to yield uniform strains with an even greater resistance to toxin. Resistance is a stable, inheritable property, providing a good genetic marker, and appears to be related to some alteration in the cell membrane. Preliminary studies showed that these cell strains are resistant to poliovirus and vesicular stomatitis virus as well as to toxin.
Model Approach to Immunological Rejuvenation of the Aged
Authors: Perkins E. H. , Makinodan T. , Seibert Charlene
Published: January 2020

Reconstitution of a damaged or exhausted immune system by injection of genetically compatible immunocompetent cells (immunologic rejuvenation) is a promising approach for restoration of immune activity. By using this model, spleen cells from young-adult mice, previously immunized with
Salmonella typhimurium
, were transferred to either young-adult or old, syngeneic recipients before or after storage at −196 C. The susceptibility of recipient mice was then determined by challenging them at increasing time intervals after reconstitution with lethal doses of the virulent organisms. The findings, although preliminary in nature, demonstrate that (i) immunological rejuvenation of mice is possible with immunocompetent cells from specifically immunized donors; (ii) prolonged “takes” of these cells can occur even in nonirradiated recipient mice, and (iii) storage at −196 C does not impair their protective capacity.
Biochemical Basis of the Immunogenicity of the Common Enterobacterial Antigen
Authors: Mayer H. , Schmidt G. , Whang H. Y. , Neter E.
Published: January 2020

Of the numerous members of the family
Enterobacteriaceae
only a few strains, notably
Escherichia coli
O14 and R mutants of the
E. coli
R1-core type, engender antibodies against the common enterobacterial antigen (CA) following immunization of rabbits with heated suspensions or culture supernatants; other members produce nonimmunogenic CA of identical serological specificity. The biochemical basis of the immunogenic properties of CA of the former strains was investigated by determining the relationship between the CA determinant and the lipopolysaccharide molecule. Lipopolysaccharides extracted from R mutants of the
E. coli
R1-core type or of
E. coli
O14 by the phenol-chloroform-petrol ether method contain the CA determinant, in contrast to extracts of other CA-producing R mutants. This is evident from the observation that only the former absorb CA antibodies, utilizing erythrocytes coated with alkali-treated lipopolysaccharide preparations. Based on the findings that CA of R mutants of
E. coli
R1-core type follows lipopolysaccharide during all purification steps and that alkali treatment increases its affinity for erythrocytes parallel to that of the lipopolysaccharide, it is concluded that the CA determinant either is part of the lipopolysaccharide molecule or is strongly complexed with it. It is suggested that this association between CA and the lipopolysaccharide of
E. coli
R1-core type and
E. coli
O14 accounts for the heat stability of the immunogenicity of CA of these unusual strains.
Cytotoxic Antibody Response to Tumors Induced in Adult and Newborn Rabbits by Fibroma Virus
Authors: Tompkins W. A. F. , Schultz R. M.
Published: January 2020

Cells cultured from tumors induced in rabbits by inoculation of fibroma virus possessed virus-specific cell surface and cytoplasmic antigens. Tumor cell cultures were capable of a limited number of cell divisions before degenerating. Employing the
51
Cr-release test and the microcytotoxicity test, it was demonstrated that sera from rabbits with regressed fibroma tumors contained antibodies cytotoxic for cells infected in culture with fibroma virus. These sera were only weakly cytotoxic for cultured fibroma tumor cells. In addition, newborn rabbits bearing progressively growing tumors had serum antibodies cytotoxic for cells infected with fibroma virus in culture but not for fibroma tumor cells. Immunofluorescence studies also showed that the reaction of immune serum with the surface antigens of fibroma tumor cells was markedly weaker than with the surface antigens of cells infected with virus in culture. Furthermore, membrane cytotoxicity and immunofluorescence reaction were significantly reduced when cells were tested after prolonged incubation or cell division, or both, following infection with fibroma virus. The failure of tumors to regress in newborn rabbits in spite of the presence of cytotoxic antibodies is discussed in respect to a possible reduction or alteration of virus-specific surface antigens on proliferating tumor cells.
Production of Staphylococcal Alpha Toxin II. Glucose Repression of Toxin Formation
Authors: Duncan James L. , Cho Gloria J.
Published: January 2020

The effect of glucose on alpha toxin production was studied in the Wood 46 strain of
Staphylococcus aureus.
Optimal toxin production occurred when 0.2% glucose was present in the medium. Omission of glucose gave lower yields of toxin, and concentrations of 0.5% and higher severely depressed toxin formation. Glucose affected the initiation of alpha toxin synthesis in growing cultures. As the glucose concentration increased, the time lag prior to the onset of toxin production also increased, and maximal rates of synthesis were not obtained until essentially all the glucose had been exhausted from the medium. The addition of glucose to toxin-producing cultures caused a temporary, almost complete repression of toxin formation which was not due to
p
H changes in the culture. The synthesis of most extracellular proteins was not inhibited during the period of repression. After recovery, toxin was produced at rates equal to those of untreated control cultures. The kinetics of toxin repression and the observation that the glucose analogues, 2-deoxy-
d
-glucose and α-methyl-glucoside, as well as other carbon sources, inhibit toxin production suggest that transient repression is responsible for the inhibition of toxin formation. No evidence for a regulatory role of adenosine 3′, 5′-cyclc monophosphate in alpha toxin production was obtained.
Detection and Localization of Viruses in Human Fetal Intestinal Organ Cultures by Immunofluorescence
Authors: Dolin Raphael , Blacklow Neil R. , Wyatt Richard G. , Sereno Mitzi M.
Published: January 2020

Viral antigens from viruses belonging to four different viral groups were detected directly in human fetal intestinal organ cultures by the application of immunofluorescent techniques. The time of appearance and the cellular localization of fluorescent-stainable antigen varied with the type of virus under investigation. After infection with adenovirus or with adeno-associated virus, fluorescent-stainable antigen was seen in the epithelial cells of the explants, though no light microscopic changes could be observed. In infection with herpes simplex virus and echovirus, fluorescence was noted in both the epithelium and the lamina propria, along with histological changes throughout the organ culture. These techniques offer promise for the investigation of possible viral agents implicated in gastrointestinal disease.
Effects of alginase on the natural history and antibiotic therapy of experimental endocarditis caused by mucoid Pseudomonas aeruginosa
Authors: Bayer A S , Park S , Ramos M C , Nast C C , Eftekhar F , Schiller N L
Published: January 2020

The exopolysaccharide (alginate) of mucoid strains of Pseudomonas aeruginosa is believed to be an important virulence factor. The ability of an alginate-deploymerizing enzyme (alginase) to modify the polymorphonuclear leukocyte (PMN)-directed and antibiotic-mediated phagocytosis and killing of mucoid P. aeruginosa was studied both in vitro and in vivo. In vitro, pretreatment of a mucoid P. aeruginosa strain (144MR) resulted in a significant enhancement of PMN phagocytosis and killing of the organism (P less than 0.05), to levels similar to that observed with its nonmucoid mate, strain 144NM. Moreover, alginase treatment of the mucoid strain 144MR caused a substantial removal of bacterial cell surface alginate as assessed by immunofluorescence staining with a murine monoclonal antialginate antibody. The experimental endocarditis model was used to evaluate the in vivo effect of alginase in modifying the course of a deep-seated pseudomonal infection caused by mucoid strain 144MR. In right-sided endocarditis, in which PMNs normally mediate spontaneous clearance of the organism from cardiac vegetations (A. S. Bayer, J. Yih, C. Y. Chiu, and C. C. Nast, Chemotherapy 35:278-288, 1989), the presence of the alginate exopolysaccharide on strain 144MR was associated with an inability to reduce intravegetation pseudomonal counts over a 13-day postinfection period; in contrast, right-sided vegetations infected with the nonmucoid strain 144NM underwent significant reductions in bacterial densities over this same time (P less than 0.05). Administration of alginase intravenously (i.v.) (750 enzyme units per day for 7 days) to animals with right-sided endocarditis caused by the mucoid strain 144MR was associated with a significant reduction in intravegetation pseudomonal counts (P less than 0.05), to levels similar to that seen with endocarditis caused by the nonmucoid strain. In left-sided endocarditis caused by mucoid strain 144MR, animals received either no therapy, amikacin (20 or 40 mg/kg twice a day for 7 or 14 days), or amikacin plus alginase (750 U/day [i.v.]). The coadministration of alginase for 14 days with the higher-dose amikacin regimen rendered more left-sided vegetations culture negative than those in animals receiving the antibiotic alone for 7 or 14 days (P = 0.001 and 0.056, respectively). These salutary effects of alginase in vivo were paralleled by the ability of the enzyme to remove the exopolysaccharide from the surface of mucoid pseudomonal cells within cardiac vegetations, as assessed by transmission electron microscopy.(ABSTRACT TRUNCATED AT 400 WORDS)
WI-1, a novel 120-kilodalton surface protein on Blastomyces dermatitidis yeast cells, is a target antigen of cell-mediated immunity in human blastomycosis
Authors: Klein B S , Sondel P M , Jones J M
Published: January 2020

A large body of experimental data has demonstrated the central role of T cells in acquired resistance to the dimorphic fungus Blastomyces dermatitidis. We examined the human T-cell response to WI-1, a 120-kDa B. dermatitidis yeast cell surface protein recently shown to be an immunodominant antigen of the B-cell response in infected humans. Peripheral blood lymphocytes from 10 blastomycosis patients studied proliferated in response to WI-1 (mean, 19,431 cpm) and to the standard, crude cell wall antigen, Blastomyces alkali- and water-soluble antigen (B-ASWS) (mean, 19,131 cpm); lymphocytes from 10 histoplasmosis patients and 10 normal control subjects did not respond to WI-1. WI-1 stimulation of patient lymphocytes and rechallenge with WI-1 or B-ASWS showed that the antigens share immunodominant epitopes. Of 100 WI-1-responsive T-cell clones derived from peripheral blood, 10 were studied in detail to assess the phenotype, function, and ligands recognized. The clones exhibit the CD3+ CD4+ phenotype of helper T cells; 2 of 10 clones (and 21% of antigen-stimulated peripheral blood lymphocytes) use the V beta 8 T-cell receptor gene element to respond to WI-1. All the clones proliferate in response to both WI-1 and B-ASWS but not other fungal antigens, and some mediate potent cytolytic effects on WI-1- and B-ASWS-labeled targets. WI-1 recognition requires antigen processing and presentation of epitopes in association with HLA-DR (to noncytolytic clones) and HLA-DP (to cytolytic clones). From these findings, we conclude that CD4+ T cells with regulatory and cytolytic properties are involved in the development of acquired resistance of B. dermatitidis, that the cells are directed against WI-1, and that the manner of display of WI-1 peptide epitopes in conjunction with major histocompatibility complex class II may influence the profile of the immune response.
Effect of growth temperature on outer membrane components and virulence of Aeromonas hydrophila strains of serotype O:34
Authors: Merino S , Camprubí S , Tomás J M
Published: January 2020

Growth of Aeromonas hydrophila strains from serotype O:34 at 20 and 37 degrees C in tryptic soy broth resulted in changes in the lipids, lipopolysaccharide (LPS), and virulence of the strains tested. Cells grown at 20 degrees C contained, relative to those cultured at 37 degrees C, increased levels of the phospholipid fatty acids hexadecanoate and octadecanoate and reduced levels of the corresponding saturated fatty acids. Furthermore, the lipid A fatty acids also showed thermoadaptation. In addition, LPS extracted from cells cultivated at 20 degrees C was smooth, while the LPS extracted from the same cells cultivated at 37 degrees C was rough. Finally, the strains were more virulent for fish and mice when they were grown at 20 degrees C than when they were grown at 37 degrees C and also showed increased different extracellular activities when they were grown at 20 degrees C.
von Willebrand factor release and thrombomodulin and tissue factor expression in Rickettsia conorii-infected endothelial cells
Authors: Teysseire N , Arnoux D , George F , Sampol J , Raoult D
Published: January 2020

Mediterranean spotted fever, a tick-borne rickettsiosis caused by Rickettsia conorii, may lead to small-vessel or deep-vein thrombosis. In order to evaluate the role of endothelial cell alteration in this lesion, we infected human endothelial cells derived from umbilical veins with R. conorii. We report the induction of two previously unreported prothrombotic mechanisms in rickettsial disease: (i) a progressive decline in thrombomodulin antigen and (ii) early expression of tissue factor, and, as described for R. rickettsii infection, later release of von Willebrand factor from Weibel-Palade bodies. Thrombomodulin expression in infected endothelial cells, measured by the thrombin-dependent activation of protein C or flow cytometric analysis, decreased steadily between 4 and 24 h after inoculation with rickettsiae. R. conorii infection induced tissue factor expression, measured by clotting assay and flow cytometric analysis, which was detectable 2 h postinoculation, reached its maximum 4 h postinoculation, and progressively decreased thereafter. Infection resulted in a relatively late release of von Willebrand factor antigen into the culture medium. A double-label immunofluorescence assay for the simultaneous evaluation of von Willebrand factor and R. conorii showed that the depletion of cytoplasmic von Willebrand factor stored in Weibel-Palade bodies was due to a direct effect of the intracellular R. conorii. These disturbances of endothelial function observed with R. conorii-infected cells may provide a paradigm for the elucidation of thrombotic pathobiology with Mediterranean spotted fever.
Some properties of purified Escherichia coli heat-stable enterotoxin II
Authors: Hitotsubashi S , Fujii Y , Yamanaka H , Okamoto K
Published: January 2020

We examined the biological properties of purified Escherichia coli heat-stable enterotoxin II (STII) using mouse intestinal loop assays and compared these properties with those of heat-stable enterotoxin I (STI) and cholera toxin (CT). The action of STII over time differed from those of STI and CT. STII did not alter cyclic GMP or cyclic AMP levels in intestinal mucosal cells. Our results supported the idea that the mechanism of action of STII in inducing fluid secretion is different from the mechanisms of action of STI and CT. This hypothesis was further supported by the fact that an anti-STII neutralizing serum did not neutralize the activities of STI and CT. Subsequently, we examined the involvement of prostaglandins in the action of STII. The level of prostaglandin E2 in the fluid accumulated as a result of the action of STII increased, and the prostaglandin synthesis inhibitors aspirin and indomethacin significantly reduced the response to STII. These results implicate prostaglandin E2 in the mechanism of action of STII.
Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile
Authors: Lyerly D M , Barroso L A , Wilkins T D , Depitre C , Corthier G
Published: January 2020

This study was undertaken to examine toxin production by Clostridium difficile 8864, a naturally occurring isolate that has been reported to produce toxin B in the absence of toxin A. To date, this is the only strain of C. difficile reported to produce only one of the toxins. The results of our initial studies with antibodies against toxins A and B confirmed these observations. Toxin B from strain 8864 and from VPI strain 10463, a strain that produces high levels of both toxin A and toxin B, was purified to homogeneity by sequential anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Ultrogel AcA22, and immunoadsorption chromatography, and their toxic activities were compared. Our results showed that toxin B from strain 8864 and toxin B from C. difficile VPI strain 10463 were comparable in their cytotoxic activities and that the 8864 toxin B was more lethal. In addition, we observed that toxin B from strain 8864 was weakly enterotoxic, which may explain the ability of this strain to cause intestinal disease in hamsters treated with antibiotics. Analysis with specific antibodies showed that the toxin B molecules from these strains were highly related but contained distinct epitopes. The results of hybridization studies with probes specific for the toxin B gene of VPI strain 10463 demonstrated differences between the toxin B genes of the two strains. In addition, probes specific for the toxin A gene of VPI strain 10463 showed that strain 8864 contains a region which shows identity with the 5' end of the toxin A gene but not the region of the gene which encodes a hydrophobic region and the repeating units.
Opsonic antibody activity against Actinobacillus actinomycetemcomitans in patients with rapidly progressive periodontitis
Authors: Sjöström K , Darveau R , Page R , Whitney C , Engel D
Published: January 2020

Actinobacillus actinomycetemcomitans has been closely associated with early-onset, severe periodontitis, and such patients often have serum immunoglobulin G (IgG) antibodies reactive with antigens of this gram-negative pathogen. We examined the functionality and potential importance of these antibodies. The opsonic activity against A. actinomycetemcomitans of sera from 30 patients with rapidly progressive periodontitis (RPP) and from 28 periodontally normal subjects was tested by using polymorphonuclear leukocyte (PMN) chemiluminescence and bactericidal assays. Peak chemiluminescence values correlated strongly with killing observed in the PMN-dependent bactericidal assay (r = 0.88; P < 0.001). Neither the mean IgG titer nor the mean peak chemiluminescence differed significantly between the two groups. However, when the relationship between chemiluminescence and titer was examined, regression analysis showed that antibodies present in low-titer normal sera were significantly more effective at opsonizing A. actinomycetemcomitans than antibodies present in low-titer RPP patient sera (P = 0.04). Thus, periodontally normal individuals may be better able than RPP patients to clear A. actinomycetemcomitans in early stages of colonization, and anti-A. actinomycetemcomitans antibodies in RPP patients may be relatively ineffective in preventing infection by this organism.
Monoclonal antibodies against the native urease of Helicobacter pylori: synergistic inhibition of urease activity by monoclonal antibody combinations
Authors: Nagata K , Mizuta T , Tonokatu Y , Fukuda Y , Okamura H , Hayashi T , Shimoyama T , Tamura T
Published: January 2020

Monoclonal antibodies (MAbs) against the native urease of Helicobacter pylori NCTC 11637 were found to clearly inhibit the urease activity. Interestingly, synergistic inhibition by two MAbs recognizing different subunits was also observed. Ten MAbs were produced and classified as two isotypes of the immunoglobulin G (IgG) subclass, IgG1, and IgG2a. Western blot (immunoblot) analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that five MAbs recognized the large subunit and the other five recognized the small subunit of the urease. Among the MAbs, L2 and S2, which recognized the large and the small subunits, respectively, were also able to inhibit the urease activity of clinical isolates from H. pylori-infected patients. The combination of L2 and S2 led to augmented synergistic inhibition. L2, but not S2, could also inhibit the urease activity from Helicobacter mustelae; enzyme-linked immunosorbent assay and Western blot analysis showed that L2 cross-reacted with this urease. These results suggested that the epitope recognized by L2 had a structure common to both Helicobacter species and may be involved in the active site of the urease. In contrast to the MAbs, a polyclonal antibody in sera from mice immunized with H. pylori urease did not have the ability to inhibit H. pylori urease activity. However, the polyclonal antibody retained the ability to abolish the inhibitory action of these MAbs. Moreover, other MAbs which could not inhibit H. pylori urease activity also abolished the inhibitory action.
Feedback suppression of staphylococcal enterotoxin-stimulated T-lymphocyte proliferation by macrophages through inductive nitric oxide synthesis
Authors: Isobe K , Nakashima I
Published: January 2020

Staphylococcal enterotoxin A (SEA)- or SEB-stimulated T-lymphocyte proliferation was suppressed by the addition of high numbers of murine peritoneal macrophages or rat peritoneal or alveolar macrophages, whereas lower numbers of murine peritoneal macrophages enhanced the T-lymphocyte response. Suppression was associated with the increase of accumulation of nitrite, a product of nitric oxide, in the culture supernatants. This macrophage-mediated suppression was totally reversed by the addition of NG-monomethyl-L-arginine, a homolog of L-arginine, indicating that macrophage-mediated suppression of T-lymphocyte proliferation was mediated through the nitric oxide-synthesizing pathway activity. Macrophages in large numbers spontaneously produced nitric oxide in culture supernatant fluids. By the addition of autologous or allogeneic spleen cells but not thymocytes to SEA- or SEB-stimulated macrophage culture, nitric oxide production was greatly increased. When T lymphocytes in spleen cells were killed by antibody before addition to macrophage culture, nitric oxide production was diminished to the basal level. These results suggest that in addition to the action to support the process of T-lymphocyte activation by SEA or SEB, macrophages display a feedback regulatory action on the SEA- or SEB-stimulated T-cell proliferative response by releasing nitric oxide through interaction between macrophages and activated T lymphocytes.
Laminin on Toxoplasma gondii mediates parasite binding to the beta 1 integrin receptor alpha 6 beta 1 on human foreskin fibroblasts and Chinese hamster ovary cells
Authors: Furtado G C , Cao Y , Joiner K A
Published: January 2020

We investigated the role of parasite-bound laminin and the host cell beta 1 integrin receptors for this extracellular matrix protein in Toxoplasma gondii binding to fibroblasts. Laminin but not fibronectin was detected on extracellular tachyzoites by immunofluorescence and immunoblotting. Binding of parasites to CHO cells was inhibited by polyclonal antibodies to laminin and by a monoclonal antibody directed against the globular carboxyl-terminal portion of the long arm of laminin (at or near the suggested ligand-binding sites for alpha 3 beta 1 and alpha 6 beta 1), but not by a monoclonal antibody directed against the lateral short arms of laminin near the cross region of the molecule. Antibodies to the alpha 6 but not the alpha 2, alpha 3, or alpha 5 chains of the beta 1 family of integrins blocked parasite attachment to human foreskin fibroblasts and CHO cells. Attachment of T. gondii to cells via laminin on the parasite surface and laminin receptors on the mammalian cell is consistent with the capacity of the parasite to invade almost all nucleated cells.
Molecular analysis of the P2 porin protein of nontypeable Haemophilus influenzae
Authors: Sikkema D J , Murphy T F
Published: January 2020

The P2 porin protein is the most abundant outer membrane protein (OMP) of nontypeable Haemophilus influenzae (NTHI) and shows extensive antigenic heterogeneity among strains. To study the molecular basis of this heterogeneity, the DNA sequences of the genes encoding the P2 proteins of three unrelated strains of NTHI were determined, and restriction fragment length polymorphisms around the P2 genes of 35 strains were analyzed. The deduced amino acid sequences of the P2 genes from the three strains of NTHI revealed four major (12 to 35 amino acids long) and several smaller (2 to 7 amino acids) hypervariable regions in each protein. The major variations occurred in identical portions of the genes, and these regions showed a high antigenic index and surface exposure probability in computer modeling analysis. Differences in the molecular mass of the P2 protein correlate with differences in the size of the variable region in each strain. Oligonucleotide primers suitable for amplification of the P2 genes by polymerase chain reaction were developed. Restriction fragment length polymorphism analysis showed marked heterogeneity in and around the ompP2 locus of 35 NTHI strains. These results contrast with the high degree of conservation of the P2 genes in H. influenzae type b strains. We conclude that the molecular mass and antigenic heterogeneity of the P2 molecule of NTHI is due to variations in gene sequence that are clustered primarily in four large hypervariable regions of the gene.
Adherence of Legionella pneumophila to guinea pig peritoneal macrophages, J774 mouse macrophages, and undifferentiated U937 human monocytes: role of Fc and complement receptors
Authors: Husmann L K , Johnson W
Published: January 2020

Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen of alveolar macrophages. Although previous studies have demonstrated that specific antibody facilitates uptake of L. pneumophila by phagocytic cells, the role of complement has been unclear. Thus, we have examined the relative contributions of Fc gamma- and complement receptor-mediated adherence to guinea pig peritoneal macrophages, U937 human monocytic cells, and J774 mouse macrophage cells. Opsonization of L. pneumophila (Philadelphia 2) with polyclonal immunoglobulin G promoted maximum adherence to guinea pig macrophages. In contrast, incubation in the presence of 20% fresh nonimmune human serum from a single donor did not promote adherence. The results obtained with U937 and J774 cells paralleled those obtained with guinea pig macrophages. In the absence of specific antibody, opsonization with guinea pig complement did not enhance adherence of the Philadelphia 1, Philadelphia 2, or Knoxville strain. However, when complement was added to heat-inactivated, specific antiserum, a fourfold increase in the number of adherent organisms was observed. Blocking studies utilizing membrane receptor-specific monoclonal antibodies demonstrated that both Fc and complement receptors mediated adherence of organisms treated with complement in the presence of specific antibody. These results suggest that complement augments adherence of L. pneumophila only when acting in concert with specific antibody.
An 87-kilodalton glucan-binding protein of Streptococcus sobrinus B13
Authors: Wu-Yuan C D , Gill R E
Published: January 2020

An 87-kDa glucan-binding protein (GBP) of Streptococcus sobrinus B13 (serotype d) was isolated and purified from extracellular culture supernatant by using affinity chromatography on Sephadex G-50 and elution with a guanidine HCl gradient. Western blot (immunoblot) analysis showed it to be antigenically related, but not completely identical, to the 74-kDa GBP of Streptococcus mutans Ingbritt. The 87-kDa GBP has no glucosyltransferase activity. A possible role for this GBP in the cariogenicity of S. sobrinus B13 is suggested.
Cloning and expression of a Serpula (Treponema) hyodysenteriae hemolysin gene
Authors: Muir S , Koopman M B , Libby S J , Joens L A , Heffron F , Kusters J G
Published: January 2020

Serpula (Treponema) hyodysenteriae, the etiologic agent of swine dysentery, produces a hemolysin which is thought to be an important factor in the pathogenesis of the disease. We report the cloning, sequencing, and expression of a hemolysin gene (tly) from S. hyodysenteriae B204. A pUC19 gene bank of strain B204 was constructed in the Escherichia coli K-12 strain DH5 alpha, and hemolytic recombinants were identified by plating the library on blood agar plates. From the hemolytic recombinants, a 1.5-kb DNA fragment could be isolated that contained information necessary for the production of a hemolysin/cytotoxin in E. coli. Nucleotide sequence determination of this 1.5-kb fragment showed that it contained an open reading frame capable of encoding a 26.9-kDa protein. The recombinant hemolysin was easily released from E. coli by osmotic shock. As with the native hemolysin, the recombinant hemolysin is EDTA insensitive, thermolabile, and cytotoxic for several eukaryotic cell lines. Southern blot hybridization showed that the cloned S. hyodysenteriae hemolysin gene tly is present in all pathogenic strains of S. hyodysenteriae tested and absent in the nonpathogenic, weakly hemolytic spirochete S. innocens.
Comparison of the safety and immunogenicity of delta aroC delta aroD and delta cya delta crp Salmonella typhi strains in adult volunteers
Authors: Tacket C O , Hone D M , Curtiss R , Kelly S M , Losonsky G , Guers L , Harris A M , Edelman R , Levine M M
Published: January 2020

Three attenuated Salmonella typhi strains have been constructed by introducing deletions in aroC and aroD or deletions in cya and crp into one of two wild-type parent strains, Ty2 or ISP1820. These mutant strains were designated CVD 906 (ISP1820 delta aroC delta aroD), CVD 908 (Ty2 delta aroC delta aroD), and chi 3927 (Ty2 delta cya delta crp). Two studies were conducted with 36 healthy adult inpatient volunteers to determine in a double-blind fashion the safety and immunogenicity of approximately 5 x 10(4) and 5 x 10(5) CFU of each of these three vaccine candidates given as a single dose. No statistically significant difference in the incidence of reactions among vaccinees was observed. Fever (oral temperature greater than or equal to 38.2 degrees C) occurred in 2 of 12 volunteers who received CVD 906, in 0 of 12 who received CVD 908, and in 1 of 12 who received chi 3927. Vaccine bacteremia without symptoms occurred in 1 of 12 vaccinees who received CVD 906, in 0 of 12 who received CVD 908, and in 2 of 12 who received chi 3927. Overall, 19 (53%) of 36 vaccinees developed immunoglobulin G antibody to S. typhi lipopolysaccharide after vaccination, with no statistically significant differences in the rate of seroconversion among volunteers in the three groups. We conclude that defined mutations in the aromatic biosynthetic pathway and in the cyclic AMP global regulatory system attenuate S. typhi. Mutant strains CVD 906, CVD 908, and chi 3927 are highly (and approximately equally) immunogenic but possibly differ in their propensity to induce fever. Further studies are needed to document the apparent relative safety of CVD 908 as a typhoid vaccine and as a vaccine carrier of foreign antigens.