bluuebunny/crossref_metadata_2025 huggingface dataset:
Epidemiology of hepatitis B infection in Liberian infants
Authors: Prince A M , White T , Pollock N , Riddle J , Brotman B , Richardson L
Published: January 2020

To provide background for a hepatitis B vaccine efficacy trial, sera were collected from 0- to 4-year-old Liberian infants and their mothers, on two occasions an average of 14.75 months apart, and tested for serological markers of hepatitis B virus infection. The prevalence of the hepatitis B surface antigen (HBsAg) was 2.9% in the 0- to 6-month age group and 23% in infants 3 to 4 years of age. HBsAg persisted for the 14.75-month average follow-up period in 80.8% of the infants tested. The annual incidence of development of HBsAg was 18.9% for infants less than 1 year of age and 13.6% in infants 3 to 4 years of age. Infants born to HBsAg carrier mothers had significantly higher age-specific prevalence and incidence of hepatitis B virus infection. However, it was estimated that only a minor proportion of hepatitis B infections in Liberia are derived by vertical transmission from carrier mothers.
Evaluation of A/Alaska/6/77 (H3N2) cold-adapted recombinant viruses derived from A/Ann Arbor/6/60 cold-adapted donor virus in adult seronegative volunteers
Authors: Murphy B R , Chanock R M , Clements M L , Anthony W C , Sear A J , Cisneros L A , Rennels M B , Miller E H , Black R E , Levine M M , Betts R F , Douglas R G , Maassab H F , Cox N J , Kendal A P
Published: January 2020

The influenza A/Ann Arbor/6/60 (H2N2) cold-adapted (ca) virus was evaluated as a donor of attenuating genes to new variants of influenza A virus. This ca donor virus was mated with the A/Alaska/6/77 (H3N2) wild-type virus, and three A/Alaska/6/77 (H3N2) ca recombinant viruses were produced. The parental origin of the genes in the three ca recombinants had been determined previously (2), and their virulence for adult seronegative volunteers was assessed in the present study to identify the genes present in the ca donor virus that confer attenuation. Each of the recombinants received the hemagglutinin and neuraminidase genes from the A/Alaska/6/77 (H3N2) wild-type parent. One ca recombinant (CR-29) received all six transferable genes from the ca parent and was found to be satisfactorily attenuated in the volunteers. The two other ca recombinants received five of the six transferable genes with a wild-type gene at the M or NS locus. The pattern of infection in humans with these latter two ca recombinants was similar to the CR-29 ca recombinant. These findings demonstrate that inheritance of a gene in ca recombinants at the M or NS locus segregates independently of attenuation and suggest that the M and NS genes present in the ca donor virus are not the major determinants of attenuation conferred by this virus.
Simple adult rabbit model for Vibrio cholerae and enterotoxigenic Escherichia coli diarrhea
Authors: Spira W M , Sack R B , Froehlich J L
Published: January 2020

We developed an adult rabbit model for enteric infection by Vibrio cholerae and enterotoxigenic Escherichia coli. The cecum of each animal was first ligated to prevent it from retaining fluid secreted by the small intestine. A temporary reversible obstruction (a slip knot tie) of the small bowel was introduced at the time of challenge and removed 2 h later. With this modification, we were able to elicit a massive and usually fatal cholera-like diarrhea in adult (3.5- to 6-lb [1.6- to 2.7-kb]) animals challenged with V. cholerae. Animals challenged with enterotoxigenic E. coli also developed diarrhea which was severe and watery but less explosive and less rapidly fatal than that produced by V. cholerae. The susceptibility of animals in this model to infection by V. cholerae was similar to the susceptibility of infant rabbits challenged intraintestinally. The death rate was almost 25% when 10(3) Vibrio cells were given and 90% or more when the dose was greater than or equal to 10(6) cells per animal. We designated this procedure the RITARD (for removable intestinal tie-adult rabbit diarrhea) model.
Effect of formalin toxoiding on Pseudomonas aeruginosa toxin A: biological, chemical, and immunochemical studies
Authors: Cryz S J , Friedman R L , Pavlovskis O R , Iglewski B H
Published: January 2020

We investigated the effect of Formalin toxoiding on the biological, chemical, and immunological activities of Pseudomonas aeruginosa toxin A. Formalin treatment alone resulted in a 1,000-fold decrease in toxin-induced cell cytotoxicity and altered the antigenicity of the toxin A molecule without adversely affecting enzymatic activity. Competitive blocking experiments indicated that Formalin-mediated detoxification proceeded via alterations in a region of the toxin molecule other than the active site of the enzyme. The addition of lysine to Formalin-toxin mixtures not only increased the rate and extent of detoxification and antigenic alteration, but also completely destroyed enzymatic activity. The immunogenicities of different toxoids varied substantially. Upon dialysis and storage, Formalin-derived toxoids underwent partial toxic reversion, whereas a Formalin-lysine-derived toxoid did not. The sodium dodecyl sulfate-polyacrylamide gel patterns of Formalin- and Formalin-lysine-treated toxins indicated that these treatments caused the formation of a heterogenous group of high-molecular-weight species and produced substantial changes in the electrophoretic mobility of toxin A.
Binding Specificity of Piliated Strains of
Escherichia coli
and
Salmonella typhimurium
to Epithelial Cells,
Saccharomyces cerevisiae
Cells, and Erythrocytes
Authors: Korhonen Timo K. , Leffler Hakon , Edén Catharina Svanborg
Published: January 2020

The binding to mammalian cells of piliated enteric bacteria and the inhibition of the binding by antibodies to purified pili were studied. The target cells were epithelial cells from human bucca and human and rat urinary tracts, erythrocytes from various species, and
Saccharomyces cerevisiae
cells. The strains were selected to represent the two main agglutination patterns of enteric bacteria: mannose-resistant agglutination of human and other erythrocytes and mannose-sensitive agglutination of guinea pig and other erythrocytes.
Escherichia coli
3669 caused only mannose-resistant agglutination,
E. coli
6013 caused only mannose-sensitive agglutination, and
E. coli
3048 caused both types of agglutination simultaneously.
Salmonella typhimurium
SH6749 exhibited only mannose-sensitive hemagglutination and was included to allow comparison of its pili with those of
E. coli
strains. The range of epithelial cells to which the bacteria adhered was related to their agglutination patterns. All four strains attached to human buccal cells. Only
E. coli
strains 3669 and 3048, which caused mannose-resistant agglutination, adhered to human urinary tract epithelial cells, and only those strains that caused mannose-sensitive agglutination adhered to rat urinary tract epithelial cells. The binding of
S. typhimurium
SH6749, but not of the other strains with mannose-sensitive agglutination, was significantly inhibited by
d
-mannose. Globotetraosylceramide, a glycolipid present in the human urinary tract epithelium, inhibited attachment to human uroepithelial cells of the two strains with mannose-resistant hemagglutination. As tested by the enzyme-linked immunosorbent assay, cross-reactions between type 1 pili of the
E. coli
strains were strong, but those between
S. typhimurium
and
E. coli
mannose-sensitive pili were weak. The two pili that induced mannose-resistant hemagglutination on
E. coli
did not cross-react. Significant inhibition of adhesion of all four strains was obtained with the homologous anti-pilus antiserum. The binding of bacteria to mammalian cells may thus be mediated by several types of bacterial pili reacting with different receptors on mammalian cells.
Attachment Factors Among Enterotoxigenic
Escherichia coli
from Patients with Acute Diarrhea from Diverse Geographic Areas
Authors: Bergman Mark J. , Updike Wanda S. , Wood Sheila J. , Brown Stuart E. , Guerrant Richard L.
Published: January 2020

To cause diarrhea, enterotoxigenic
Escherichia coli
(ETEC) must initially colonize the small bowel. Different surface structures have been implicated in this initial attachment. Recognized attachment factors include colonization factor antigens I and II (CFA/I and CFA/II) and type I pili. Several methods of detection for each of these factors have been reported. In this study, we screened for the presence of these attachment factors among enterotoxigenic
E. coli
isolated from 40 patients with acute diarrhea and 40 asymptomatic control individuals and examined their ability to attach to ATCC 407 human intestinal cells in vitro. Of 40 patients with diarrhea, 16 (40%) had enterotoxigenic
E. coli
isolates which exhibited an attachment trait. Fourteen (35%) of these isolates demonstrated the ability to attach to ATCC 407 cells, whereas only four isolates from asymptomatic controls attached (
P
< 0.02). A total of 20% of the patient isolates and 7.5% of the control isolates possessed CFA/I. Only one patient isolate demonstrated CFA/II. Evidence for type I pili was found on 10% of the patient isolates and 12.5% of the control isolates. Attachment to ATCC 407 cells allowed the detection of 87.5% (14 of 16) of enterotoxigenic
E. coli
isolates with any type of attachment trait. Of the 14 cases demonstrating attachment ability to ATCC 407 cells, 7 did not attach in the presence of mannose. Three of these showed evidence for both CFA/I and type I pili, one showed only CFA/I, and one showed only type I pili. Two of those mannose-sensitive attaching isolates showed no other demonstrable trait. Seven patient isolates showed mannose-resistant attachment. Of these, two were classified as possessing CFA/I, and one was classified as possessing CFA/II. The four remaining isolates could not be classified into any recognized attachment factor category, suggesting that other attachment factors remain to be identified.
Replication of vesicular stomatitis virus in mouse spleen cells
Authors: Hecht T T , Paul W E
Published: January 2020

Mouse spleen cells which normally cannot support the in vivo replication of vesicular stomatitis virus (VSV) became susceptible to VSV infection after the intraperitoneal growth of certain syngeneic and allogeneic tumors. After 3 days' growth of P815 tumor cells in syngeneic DBA/2 mice, the viral-permissive state for VSV replication had been established. By 7 days after tumor in inoculation, up to 18% of the spleen cells were producing virus yielding greater than 10(8) plaque-forming units per spleen. Similarly, P815 cells induced the viral-permissive state in allogeneic C3H/HeN mice. Tumors other than P815 were also effective in permitting VSV growth in the spleen. The presence of tumor cells themselves was not sufficient for VSV growth, yet cell-free ascitic fluid from mice bearing syngeneic tumors inoculated 3 h before infection allowed for VSV replication. Cell-free supernatant from a T-cell hybridoma synthesizing interleukin-2 was also effective in permitting virus growth when inoculated 3 h before infection. The virus-permissive cell has been characterized as a nylon wool-adherent and plastic dish-nonadherent spleen cell.
Immunization against bacteria- and endotoxin-induced hypotension
Authors: Abdelnoor A M , Johnson A G , Anderson-Imbert A , Nowotny A
Published: January 2020

The characteristics of hypotension induced in rabbits by intravenous injection of viable Salmonella typhosa 0901 organisms were studied and found to be a function of the number and age of the bacterial cells. Effective neutralization of the blood pressure lowering was achieved by immunization with homologous organisms as well as heterologous endotoxins and a detoxified derivative. In addition, native endotoxins derived from a number of different genera of gram-negative bacilli, as well as lipopolysaccharides deficient in either the polysaccharide or lipid components, were tested for their ability to induce hypotension in rabbits and tolerance to the lowering of blood pressure. Hypotension was elicited by intravenous injection of all native endotoxins as well as polysaccharide-deficient endotoxins, but was absent in preparations from which the lipid was removed. On the other hand, protection against the hypotension effect could be induced after injection of either the lipid- or polysaccharide-deficient derivatives.
Regulation of extracellular slime production by Actinomyces viscosus
Authors: Ooshima T , Kuramitsu H K
Published: January 2020

Extracellular slime polysaccharides produced two Actinomyces viscosus strains, T14V and T14AV, were compared. In various media containing glucose, T14Av produced abundant extracellular viscous slime polysaccharide, whereas T14V produced lower levels. Furthermore, fractionation of these polysaccharides showed that the two extracellular polysaccharides differed in molecular size and net charge. Since there was a significant difference in the relative abilities of chemically defined medium and chemically defined tissue culture medium to support slime production by T14Av, the nutritional factors influencing the production of extracellular slime were examined. Sodium bicarbonate was demonstrated to stimulate both cellular growth and the production of extracellular slime. In chemically defined medium with and without sodium bicarbonate, strain T14Av produced large quantities of viscous slime in glucose and sucrose media. In contrast, relatively low levels of slime were produced in fructose, lactose, raffinose, and inositol media, even though sodium bicarbonate stimulated the growth of T14Av in these latter media.
Protection of mice against mouse hepatitis virus by Corynebacterium parvum
Authors: Schindler L , Streissle G , Kirchner H
Published: January 2020

C57BL/6 mice that are highly susceptible to infection with mouse hepatitis virus type 3 were protected against intraperitoneal viral infection by simultaneous intraperitoneal injection of Corynebacterium parvum. No protection was observed when C. parvum was given intravenously or when it was injected intraperitoneally 3 days before viral infection. Protective effects were, however, consistently found when C. parvum was given 2 h before or 2 h after viral infection. Activity was seen only against 10 50% lethal doses and not against 100 50% lethal doses. C. parvum also caused a significant decrease of virus type 3. These data suggest a direct effect of C. parvum on virus-susceptible cells. Injection of C. parvum in mice caused activation of natural killer (NK) cells and of interferon production. However, these two effects were equally demonstrable at high and low doses of C. parvum, whereas protection against mouse hepatitis virus type 3 was not demonstrable at low doses of C. parvum. Thus, antiviral protection may be dissociated from activation of NK cells and induction of interferon.
Zinc and regulation of growth and phenotype in the infectious yeast Candida albicans
Authors: Soll D R , Bedell G W , Brummel M
Published: January 2020

When Candida albicans is grown at 25 degrees C in suspension in defined medium, cells accumulate at stationary phase as singlets in G1 of the deoxyribonucleic acid replication cycle and acquire the capacity to form mycelia. When cells were removed from a stationary-phase culture and a low concentration of fresh cells was inoculated into the cell-free, stationary-phase medium, the fresh cells grew to approximately the same cell density as the original culture. We demonstrated that in the accompanying decrease in pH, nor due to a depletion of O2, an accumulation of CO2, a physical crowding effect, or accumulation of the putative autoinhibitors tryptophol and 2-phenylethyl alcohol. Rather, cells stop multiplying at stationary phase due to the depletion of zinc from the culture medium. The manipulation of cultures with glassware to remove stationary-phase cells and to add fresh cells led to the addition of zinc to the medium and hence a new round of culture growth. The same manipulations with plasticware did not result in zinc supplementation and hence in now new round of culture growth. When cells enter stationary phase in excess zinc, they do not accumulate as singlets; rather, they accumulate as budded cells. When these cells were induced to form mycelia, they did so in half the time it took zinc-starved cells. The usefulness of employing zinc starvation as a method for obtaining a uniform stationary-phase phenotype and for synchronizing induced mycelium or bud formation is discussed.
Circulating antitoxin in rabbits after ingestion of diphtheria toxoid
Authors: Peri B A , Rothberg R M
Published: January 2020

Immune responses following antigen ingestion vary from stimulation to suppression depending on animal species, antigen, and experimental protocol. Young adult rabbits were given either 0.02% diphtheria toxoid or 0.1% bovine serum albumin in drinking water for 10-day periods, a protocol previously found to immunize human infants fed bovine serum albumin. Specific serum antibody was detected by radioimmunoassay in 10 of 13 rabbits fed diphtheria toxoid for 10 days and 11 of 13 rabbits fed bovine serum albumin. Response to oral diphtheria toxoid in some animals was equal to that after an injection of alum-precipitated diphtheria and tetanus toxoids. All rabbits fed a second course of either antigen showed an enhanced, rapid rise in antibody concentration. Avidity of the antibody also increased markedly, suggesting that priming and memory had been established during the first feeding. Similar anamnestic responses occurred in animals given a parenteral challenge after ingesting antigen. One month after administration of diphtheria toxoid, no significant difference in serum antibody levels was present between unfed animals challenged with diphtheria-tetanus toxoids and although avidity of the antitoxin was higher in fed animals. Antibody activity could be detected only in the immunoglobulin G serum fractions with either antigen. These responses suggest that oral immunization protocols using protein antigens can be adapted for use against toxins causing disease.
Chemiluminescence by polymorphonuclear leukocytes adhering to surfaces
Authors: Yanai M , Quie P G
Published: January 2020

Stimulation of the plasma membranes of granulocytes results in an oxidative metabolic response. This response can be measured by measuring the reduction of oxidizable substrates, such as Nitro Blue Tetrazolium, as well as by measuring the energy released as light (chemiluminescence). While investigating the oxidative response of human granulocytes, we observed a marked variation in the chemiluminescence response when leukocytes were suspended in a balanced salt solution without gelatin or any other protein. We performed systematic study to investigate the role of protein in suspensions of human polymorphonuclear leukocytes. Final results were identical with human serum, albumin, fetal calf serum, and gelatin; gelatin was used as the protein source in most experiments. Polymorphonuclear leukocytes suspended in Hanks balanced salt solution without gelatin decreased in numbers during incubation at room temperature (approximately 50 percent after 60 min). Cell structures were observed on the walls of the tubes containing leukocyte suspensions without gelatin. Numbers of polymorphonuclear leukocytes were stable in suspensions containing gelatin. A chemiluminescence response which peaked at approximately 10 min and was sustained for at least 30 min was observed in suspensions of polymorphonuclear leukocytes without gelatin. This surface attachment-stimulated chemiluminescence occurred in the absence of either soluble or particulate stimuli. Chemiluminescence was inhibited by either superoxide dismutase or sodium azide and did not occur with suspensions of granulocytes from patients with chronic granulomatous disease. We postulate that both superoxide- and myeloperoxidase-dependent oxidative metabolic reactions are induced during the adherence of polymorphonuclear leukocytes to surfaces. Gelatin or other proteins in leukocyte suspending media are necessary when assays are performed to evaluate the metabolic responses of these cells to particulate or soluble stimuli.
Membrane-damaging action of alveolysin from Bacillus alvei
Authors: Thelestam M , Alouf J E , Geoffroy C , Möllby R
Published: January 2020

We investigated membrane damage to human diploid, embryonic lung fibroblasts caused by highly purified alveolysin by measuring leakage of cytoplasmic markers and uptake of various metabolites, and we observed a leakage pattern typical of SH-activated cytolysins. However, the mode of membrane interaction resembled the mode of membrane interaction of theta-toxin from Clostridium perfringens rather than that of streptolysin O in the following respects: the activity on fibroblast membranes was high compared with the activity on sheep erythrocytes; the toxin did not bind irreversibly to fibroblast cytoplasmic membranes; considerable membrane damage was caused at 0 degrees C; and inhibition of amino acid uptake occurred in the absence of significant structural membrane damage. These findings imply that data on membrane effects caused by streptolysin O cannot be related indiscriminately to other SH-activated cytolysins. With regard to the mode of membrane interaction, two apparently different groups of SH-activated cytolysins exist.
Tumor-associated bacteria capable of producing a human choriogonadotropin-like substance
Authors: Backus B T , Affronti L F
Published: January 2020

Aerobic microorganisms were isolated and identified from 9 of 10 malignant tissues aseptically obtained from surgical patients. The organisms isolated are species commonly associated with the flora of the human body. When these cancer-associated organisms were grown in Trypticase soy broth (BBL Microbiology Systems), a protein substance was isolated from the culture filtrates by acetone precipitation. The acetone precipitates of 12 of 14 organisms tested were positive when assayed by radioimmunoassay for the beta subunit of human chorionic gonadotropin (hCG). All but one of the bacterial isolates from the malignancies were capable of producing the hCG-like substance, but in varying quantities. Control organisms (not isolated from a malignancy) and uninoculated Trypticase soy broth were either completely negative in the radioimmunoassay for beta hCG or had levels of beta hCG near the limit of the sensitivity of the method. These results suggest the possibility that bacteria-tumor relationships do exist and are in agreement with the findings of other workers. Investigation of these relationships may have important and provocative implications in the study of neoplastic diseases.
Interaction of Leishmania donovani promastigotes with human monocyte-derived macrophages: parasite entry, intracellular survival, and multiplication
Authors: Pearson R D , Romito R , Symes P H , Harcus J L
Published: January 2020

Leishmania donovani promastigotes were incubated with human monocyte-derived macrophages in vitro to assess the role of macrophages in the early stage of visceral leishmaniasis. Adherent mononuclear cells, obtained from nonimmune human donors, were cultivated on glass cover slips for 5 days and then incubated with axenically grown promastigotes in the presence of heat-inactivated autologous serum. Promastigotes attached to macrophages with either their flagellar or aflagellar ends, and macrophage pseudopodia formed around them. Intracellular parasites were identified within phagocytic vacuoles by electron microscopy, and the parasites assumed a form similar to that of amastigotes obtained from infected hamster spleens. Initially, 67 +/- 5% of the macrophages were infected with a mean of 4.2 +/- 0.7 parasites per infected cell. After 6 days of incubation, 79 +/- 7% of the macrophages were infected with 15.9 +/- 3.2 parasites per infected cell. The total number of parasites per monolayer increased from 4.8 +/- 0.8 x 10(5) to 1.8 +/- 0.4 x 10(6) (P less than 0.05). Dividing parasites were identified in macrophage vacuoles by electron microscopy. Human monocyte-derived macrophage vacuoles by electron microscopy. Human monocyte-derived macrophages can phagocytize promastigotes, allow the conversion of promastigotes to an amastigote-like state, and support intracellular multiplication.
Bacteriolysis of Veillonella alcalescens by lysozyme and inorganic anions present in saliva
Authors: Tortosa M , Cho M I , Wilkens T J , Iacono V J , Pollock J J
Published: January 2020

Veillonella alcalescens subsp. dispar was grown in a synthetic medium containing either radiolabeled thymidine or uridine to monitor cell lysis by assay of the release of deoxyribonucleic acid or ribonucleic acid (RNA), respectively. Biochemical analyses demonstrated that, although human or hen egg white lysozymes alone did not release deoxyribonucleic acid or RNA, the nucleic acids were liberated in equal amounts from lysozyme-treated cells by the addition of low concentrations of the sodium salts of HCO-3, SCN-, Cl-, and F-, RNA release was dependent on enzyme and anion concentration. Human lysozyme was more potent than hen egg white lysozyme, and bicarbonate was the most effective anion in promoting bacteriolysis. Surprisingly, ultrastructural analyses differed from biochemical results. Lysozyme alone caused lysis in approximately 40% of the cell population. Detailed ultrastructural examination revealed aggregated cytoplasmic components which appeared as small clumps, explaining why nucleic acids were not measurable in the biochemical assays. In reaction mixtures containing lysozyme plus inorganic salts, electron microscopy results were compatible with biochemical data. Ultrastructural studies demonstrated that the addition of inorganic salts to lysozyme-treated cells resulted in the solubilization of the protoplasmic aggregates of lysed cells, presumably freeing the complexed RNA, and in the rapid lysis of the remaining cells (approximately 60%). These data suggest that electron microscopy must be used in conjunction with biochemical assays to assess lytic damage of bacterial cells.
Specificities of Albumin Receptors and Albumin Antibodies
Authors: Thung Swan N. , Gerber Michael A.
Published: January 2020

Receptor sites for polymerized human serum albumin (pHSA) on the envelope of hepatitis B virus (hepatitis B surface antigen) and antibodies to pHSA in sera of patients with liver disease were differentiated by an indirect fluorescent antibody method. Analysis of the specificities of pHSA receptors and antibodies to pHSA by hemagglutination inhibition suggested that pHSA receptors reacted with a species-specific site on pHSA, whereas “autoantibodies” to pHSA cross-reacted with pHSA from different species. These human antibodies inhibited the binding of hepatitis B surface antigen-associated receptors to pHSA.
Antigen-specific serotyping of Neisseria gonorrhoeae: characterization based upon principal outer membrane protein
Authors: Buchanan T M , Hildebrandt J F
Published: January 2020

Principal outer membrane protein (protein I) of Neisseria gonorrhoeae was prepared nearly free of lipopolysaccharide (LPS) and substantially purified from other membrane proteins by chromatography of partially purified gonococcal outer membranes over Sepharose 6B in the presence of deoxycholate at pH 9.0. This protein I of nine separate antigenic types was coated to polystyrene tubes and used in the enzyme-linked immunosorbent assay (ELISA) to measure antibody to protein I or in inhibition tests to quantitate protein I antigen. No significant inhibition of the ELISA test was produced by purified LPS from the strain used to prepare each of the protein I types or by whole gonococci bearing the same LPS but different protein I antigens as the strain used to produce a given protein I antigen. Of 125 strains of gonococci used as whole organisms to inhibit the protein I ELISA, 124 (99%) typed with one or more of the nine protein I types, and 35% of these typed with a single protein I serotype. Sixty-one of 65 (94%) strains from Seattle and Atlanta patients with disseminated gonococcal infection contained protein I serotype 1, and 16 of 24 (64%) strains from Seattle patients with salpingitis bore one or both of protein I serotypes 1 and 2.
Interaction of rat platelets with Listeria monocytogenes
Authors: Czuprynski C J , Balish E
Published: January 2020

Listeria monocytogenes induced aggregation of rat platelets in vitro and stimulated the nonlytic release of [3H]serotonin. Listeria-induced platelet aggregation and serotonin release required the presence of intact Listeria, was maximal at a 1:1 Listeria/platelet ratio, required a plasma cofactor, and was not inhibited by indomethacin, acetylsalicylic acid, or apyrase. Aggregation either of platelets in platelet-rich plasma with adenosine diphosphate or of washed platelets with thrombin resulted in the release of a listericidin from the platelets; however, direct interaction of L. monocytogenes with platelet-rich plasma did not kill Listeria. The ability of rats to clear an intravenous challenge of L. monocytogenes (0.005 50% lethal dose), as determined by the recovery of viable L. monocytogenes from the spleen and liver, was unaffected by prior treatment with antiplatelet serum.
Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate
Authors: Ohman D E , Chakrabarty A M
Published: January 2020

Mucoid Pseudomonas aeruginosa strain FRD, a sputum isolate from a cystic fibrosis patient, was used to develop a genetic system. The mucoid appearance is due to the biosynthesis of the exopolysaccharide alginate and is a potential virulence factor of the organism. The sex factor plasmid FP2 was used for uninterrupted genetic exchange to investigate the nature of spontaneous mutations which produce frequent alginate-negative (Alg-) derivatives. Crosses between Alg+ donors and Alg- recipients demonstrated linkage between alginate genes and chromosomal markers. Crosses between an Alg- donor and Alg- recipients produced Alg+ recombinants at frequencies that varied, depending on the recipient strains used. This indicated that more than one genetic locus was associated with spontaneous mutation leading to loss of the mucoid character. Three classes of Alg- mutants were identified. Genetic exchange experiments showed that the loci of the alginate (alg) mutations of the three mutant classes are in the same region of the chromosome. The sex factor plasmid R68.45 was used for nonpolarized chromosome transfer and demonstrated close linkage between chromosomal markers (his-1, met-1) and alg markers. This was consistent with the data obtained in FP2-mediated crosses. Thus, the evidence obtained indicated that the alg genes which undergo frequent mutation are chromosomal, that several loci are involved, and that these alg loci are apparently clustered on the chromosome.
Macrophages as primary target cells and mediators of immune dysfunction in African trypanosomiasis
Authors: Grosskinsky C M , Askonas B A
Published: January 2020

African trypanosomiasis is accompanied by profound general immunosuppression. The experiments described here were designed to characterize the contribution of macrophages to the immune pathology of this disease. We used peptone-stimulated, uninfected mice and injected them intraperitoneally with lethally irradiated and 35S-labeled Trypanosoma brucei and parasite-specific antisera. Peritoneal macrophages were thus induced to take up in vivo a defined number of trypanosomes. After the phagocytosis of parasites, macrophages were transferred into uninfected syngeneic mice, where they mimicked some of the important immunological changes normally associated with active trypanosome infection: (i) splenic background plaque-forming cells increased nonspecifically and (ii) the specific immune response to sheep erythrocytes was either enhanced or suppressed, depending on the timing of the antigen challenge: priming simultaneously with the transfer of trypanosome-containing macrophages enhanced immune responsiveness; in contrast, if parasite-containing macrophages were transferred and recipient mice were primed 4 days later, the immune response was suppressed. A contribution of suppressor T cells was ruled out by the treatment of peritoneal exudate cells with anti-Thy 1.2 and complement before transfer into recipient mice. The results indicate that macrophages are key cells in the mediation of parasite-induced immune dysfunction.
Inhibition of nonspecific tumoricidal activity by activated macrophages with antiserum against a soluble cytotoxic factor
Authors: Männel D N , Falk W , Meltzer M S
Published: January 2020

Mouse peritoneal macrophages activated for tumor cytotoxicity by any of several in vivo or in vitro treatments released a soluble cytotoxin into culture fluids only after exposure to small amounts of bacterial lipopolysaccharides. This cytotoxic factor was physicochemically similar to the cytotoxic factor (tumor necrosis factor) in sera of BCG-infected mice injected with lipopolysaccharide. A rabbit antiserum against partially purified serum cytotoxic factor also inhibited the activity of macrophage-derived cytotoxic factor. Of special interest was the observation that rabbit anti-cytotoxic factor inhibited the cytotoxic activity of macrophages both in the presence and in the absence of exogenously added lipopolysaccharide. Inhibition was not complete but was consistent in all experiments. Thus, cytotoxic factor was implicated as a possible effector molecule in the nonspecific tumoricidal activity of activated macrophages.
Comparison of the biological actions of three purified heat-stable enterotoxins: effects on ion transport and guanylate cyclase activity in rabbit ileum in vitro
Authors: Rao M C , Orellana S A , Field M , Robertson D C , Giannella R A
Published: January 2020

The biological activities of three purified preparations of heat-stable enterotoxin (ST), elaborated by different strains of Escherichia coli and known to differ in their amino acid composition and molecular size, were compared in the rabbit ileum. The mechanisms of action and potencies of all three purified STs were similar and resembled those previously demonstrated for partially purified ST. They all increased electrical potential difference and short-circuit current, inhibited active Cl- absorption, increased cyclic guanosine 3',5'-monophosphate production, and stimulated particulate guanylate cyclase activity in ileal mucosa. Their molar potencies were also similar, the concentrations of toxin required for half-maximal response differing less than fourfold in short-circuit current response and twofold in guanylate cyclase activity. However, there were 10-fold differences in potency when activity was expressed in mouse units per milliliter. Thus, heterogeneity in the size of these three ST molecules is not reflected in a difference in their mechanisms of action or potencies.
Development of a radioimmunoassay for Escherichia coli heat-stable enterotoxin: comparison with the suckling mouse bioassay
Authors: Giannella R A , Drake K W , Luttrell M
Published: January 2020

Escherichia coli strains which produce heat-stable enterotoxin (ST) are usually identified by demonstrating the production of ST. At present, ST can be detected only by bioassay methods. Recently, we purified E. coli ST, which enabled us to develop a radioimmunoassay for ST. Radioiodination of ST was performed by the lactoperoxidase method, which resulted in a high specific activity and retained the biological activity of St. Anti-ST antisera were raised in goats by injecting the goats with pure ST coupled to bovine immunoglobin G. Antibody titers ranged from 1:8,000 to 1:40,000. Using these reagents, we examined assay conditions thoroughly and found that a 14- to 18-h incubation at 4 degrees C in sodium acetate buffer with an ionic strength of 120 mM (pH 6.2) gave maximal sensitivity and reproducibility. Free ST was separated from antibody-bound ST by dextran-coated charcoal. This radioimmunoassay accurately and reproducibly measured ST in the range from 50 to 500 pg of ST per tube and could quantitate ST accurately in complex bacteriological media. This assay was specific for STa, measured human and porcine STa equally well, and did not cross-react with STb, with several other enterotoxins, or with various gastrointestinal peptides. Intact disulfide bridges in the ST molecule were required for immunoreactive activity.
Immunological properties of Escherichia coli heat-stable enterotoxins: development of a radioimmunoassay specific for heat-stable enterotoxins with suckling mouse activity
Authors: Frantz J C , Robertson D C
Published: January 2020

Antiserum was raised against the purified heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli strain 431, a class II porcine enteropathogen. The antiserum was used to examine the antigenic determinants of STs produced by enterotoxigenic strains of different host origins and develop a sensitive radioimmunoassay specific for ST having biological activity in suckling mice and piglets (STA). The antiserum neutralized one effective dose of toxin at a dilution of 1:5,000 and neutralized approximately 40 microgram of toxin per ml of serum. In the radioimmunoassay, protein A-bearing staphylococci was used as the primary solid-phase adsorbent. The purified STs produced by a class I enteropathogen (strain 667) and by a bovine enterotoxigenic strain (B-41) exhibited patterns of competitive inhibition identical to those of homologous unlabeled strain 431 ST in the radioimmunoassay when specific antibody to strain 431 ST was used. The levels of ST in culture supernatants determined by the suckling mouse assay correlated with the concentrations of toxin measured by the radioimmunoassay. The antiserum was specific for STA produced by enterotoxigenic E. coli of porcine, bovine, and human origins and did not react with heat-labile enterotoxin or with ST that had biological activity in piglets but not in suckling mice (STB). These results suggest that STA molecules having different host origins share at least one antigenic determinant.
Host defenses in murine malaria: nonspecific resistance to Plasmodium berghei generated in response to Mycobacterium bovis infection or Corynebacterium parvum stimulation
Authors: Murphy J R
Published: January 2020

Infection with Mycobacterium bovis (BCG) or injection of killed Corynebacterium parvum protected some strain B6D2 F1 (C57BL/6xDBA/2) mice but did not protect strain ICR or A mice from lethal challenge with Plasmodium berghei strain NYU-2. B6D2 mice were not protected against challenges delivered immediately after intravenous injection of these materials, but rather protection developed by day 7 and persisted through at least day 84. Infections in protected mice progressed to about 10% parasitemia in parallel with infections initiated with the same inoculum in untreated controls. However, infections in most of the protected mice were cleared subsequently, whereas infections in untreated controls were uniformly fatal. A small number of treated mice developed protracted high-level erythrocytic infections, which led to markedly delayed death. BCG-infected mice which survived P. berghei infections had a factor in their sera which protected passively immunized recipients from P. berghei. BCG-infected mice passively immunized with protective serum controlled P. berghei infections better than normal mice given the same amount of the same serum and challenged with the same P. berghei inoculum. The capacity of BCG-infected B6D2 mice to resist P. berghei infection was not directly related to the pattern of growth of BCG, to the degree of splenomegaly, or to the level of activation of macrophages (measured as microbicidal capacity) caused by BCG infection. Therefore, I concluded that (i) BCG infection or injection of killed C. parvum altered the immunological potential of B6D2 mice in such a way as to allow the production of measurable levels of a protective humoral factor in response to infection with P. berghei; (ii) BCG infection caused the generation of a capacity which, when expressed in the presence of immune serum, provided an anti-P. berghei capacity which was superior to that provided by BCG infection alone or immune serum in the absence of BCG infection; and (iii) not all strains of mice could be protected from P. berghei by BCG or C. parvum injection.
Role of neutrophil degranulation in streptococcal leukotoxicity
Authors: Sullivan G W , Mandell G L
Published: January 2020

Cinemicrography and electron microscopy suggested that leukotoxic Streptococcus pyogenes killed polymorphonuclear neutrophils (PMN) by inducing intracytoplasmic rupture of the PMN granules. To further study the relationship between granule rupture and the mode of action of the streptococcal leukotoxin, PMN degranulation was experimentally altered. Exocytosis of PMN granule contents was blocked with 80 mM tetraethylammonium chloride, 2 mM dibutyryl cyclic adenosine monophosphate, or 2 mM magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetate in calcium-free medium. This treatment did not prevent the granules from firing into the cytoplasm of the PMN, nor did it significantly diminish leukotoxicity. Degranulating the PMN before exposure to the leukotoxic streptococci did partially block leukotoxicity if both the specific and the primary granules were released with either 5 microM calcium ionophore A23187 or 10% zymosan-activated serum plus 5 micrograms of cytochalasin B per ml. Leukotoxic streptococci stimulated intracytoplasmic rupture of granules, and this granule lysis contributed significantly to the ability of these streptococci to kill PMN.
Purified Clostridium difficile cytotoxin stimulates guanylate cyclase activity and inhibits adenylate cyclase activity
Authors: Vesely D L , Straub K D , Nolan C M , Rolfe R D , Finegold S M , Monson T P
Published: January 2020

Antibiotic-associated pseudomembranous colitis has been linked with Clostridium difficile toxin. We examined the effect of toxins from four strains of C. difficile isolated from patients with pseudomembranous colitis on colonic adenylate (EC 4.6.1.1) and guanylate cyclase (EC 4.6.1.2) activities. Partially purified toxins had a cytotoxic effect on hamster fibroblasts in culture at a concentration of 10 ng/ml. Likewise, these toxins enhanced colonic guanylate cyclase activity two- to threefold, with the maximal stimulation being at 10 ng/ml. These toxins also enhanced guanylate cyclase activity in ileum, cecum, and duodenum. Both the cytotoxic activity on hamster fibroblasts and the enhancement of hamster guanylate cyclase activity were inhibited by antiserum to C. difficile toxin. These same toxins inhibited adenylate cyclase activity at a 100-ng/ml concentration, but had no effect at 10 ng/ml. They also had no effect at any concentration on colonic Na+-K+ adenosine triphosphatase. To be sure that the findings were not due to a contaminant, a purified C. difficile cytotoxin was used, and the same findings were found with the pure cytotoxin (at a 100-fold-lower concentration). The data suggest that activation of guanylate cyclase may be a factor in the pathogenesis of antimicrobial-associated pseudomembranous colitis.
Rejection of the intestinal parasite Nippostrongylus brasiliensis by mast cell-deficient W/Wv anemic mice
Authors: Crowle P K , Reed N D
Published: January 2020

The ability of W/Wv anemic mice to accumulate mucosal mast cells and to reject the intestinal parasite Nippostrongylus brasiliensis was examined. W/Wv mice did not accumulate mucosal mast cells in response to infections with N. brasiliensis. They eliminated a primary infection more slowly than did their normal littermate controls but were as refractory as controls to second and third infections. W/Wv mice had higher serum titers of worm-specific immunoglobulin E than did controls. These results indicate that mucosal mast cells are not an absolute requirement for N. brasiliensis rejection.
Intoxication of cultured human lung fibroblasts with Clostridium difficile toxin
Authors: Florin I , Thelestam M
Published: January 2020

The cytopathogenic effect of partially purified toxin from Clostridium difficile on cultured human lung fibroblasts was studied. Conditions for determination of 50% tissue culture dose were standardized. The cytopathogenic effect of the toxin was dependent on toxin concentration, exposure time, and density of the cells. Transfer of the cells to 0 degrees C did not inhibit binding of toxin to the fibroblast surface, but prevented the development of the cytopathogenic effect. Both binding of toxin and some intracellular step(s) were prevented by 2,4-dinitrophenol. These preventative effects were reversible. Before and concomitantly with the appearance of the cytopathogenic effect, the cellular uptake of uridine and of amino acids was markedly stimulated. Protein synthesis was depressed when 100% of the cells showed the cytopathogenic effect, but the synthesis of nucleic acids was inhibited only several hours later. The primary cellular target for the toxin is still unknown.
Lysozyme digestion and chemical characterization of the peptidoglycan of Fusobacterium nucleatum Fev 1
Authors: Vasstrand E N
Published: January 2020

The amino acids in the peptidoglycan of Fusobacterium nucleatum Fev 1 are D-glutamic acid, meso-lanthionine, and D- (42%) and L-alanine (58%). About 70% of the lanthionine residues were not susceptible to dinitrophenylation, evidently because they are involved in cross-linkages. Consequently, lysozyme digestion of the peptidoglycan yielded 20 to 25% uncross-linked disaccharide tri- and tetrapeptides. A chemical analysis of isolated glycopeptides indicated that the structure of the building block of this peptidoglycan is N-acetylgucosamine-N-acetylmuramic acid-L-alanine-D-glutamic acid-meso-lanthionine(-D-alanine). I present evidence which supports the classification of the F. nucleatum Fev 1 peptidoglycan as a new A1 delta, directly cross-linked, meso-lanthionine-containing peptidoglycan.
Killing of Listeria monocytogens by conventional and germfree rat sera
Authors: Czuprynski C J , Balish E
Published: January 2020

Serum from both germfree and conventional rats, but not plasma or plasma serum, killed Listeria monocytogenes in vitro by a calcium-dependent mechanism that was independent of either complement or lysozyme and was not inhibited by the addition of iron. The listericidin was purified by passing either rat serum or platelet lysate through a nitrocellulose filter (0.2 micrometer) and eluting the activity from the filter with 0.02 N HCl. The partially purified listericidin was heat stable (56 degrees C for 30 min), removed by absorption with zymosan or bentonite, sensitive to treatment with trypsin or pronase, and inhibited by the addition of citrate (0.045 M), suggesting that the serum listericidin is a cationic protein. The development of serum listericidal activity, which could be important in the innate resistance of rats to L. monocytogenes, was dependent on both age and microbial status. Although some discrepancies exist between the serum listericidin and previous descriptions of serum beta-lysin, we believe that the rat serum listericidin is a similar cationic protein.
Phagocytosis and killing of salmonella typhimurium by peritoneal exudate cells
Authors: Briles D E , Lehmeyer J , Forman C
Published: January 2020

Normal peritoneal cells from conventional, germfree, or nu/nu mice readily killed opsonized salmonellae, an observation that suggests that this activity in the normal peritoneal cavity may not be dependent on either environmental antigenic stimulation or T-cell mediation. In contrast, peritoneal cells elicited 4 days after injection with thioglycolate medium failed to kill opsonized salmonellae but appeared to be highly phagocytic. Peritoneal cells from thioglycolate-treated mice could be induced to kill opsonized salmonellae by giving the mice a primary footpad injection and a secondary intraperitoneal injection of Corynebacterium parvum. This activation by C. parvum appeared to be thymus dependent, since it did not occur in nu/nu mice.