bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation, purification, and partial characterization of an enterotoxin from extracts of Entamoeba histolytica trophozoites
Authors: Feingold C , Bracha R , Wexler A , Mirelman D
Published: January 2020

Soluble cell-free extracts of pathogenic Entamoeba histolytica, as well as serum-free minimal media in which trophozoites are incubated, contain substances that cause the rapid rounding up and detachment of tissue-cultured monolayers of mammalian cells (cytopathic activity) and induce fluid secretion in ligated intestinal loops of indomethacin-pretreated rats (enterotoxic activity). A semiquantitative assay for the determination of the cytopathic activity based on the rate of detachment of tissue-cultured baby hamster kidney cells was developed. Two peaks containing cytopathic activity were obtained upon gel filtration of the soluble extracts: peak I, with over 60% of the activity, emerged in the 30,000 to 50,000 molecular weight region, and peak II, containing the remaining activity, was in the 15,000 to 25,000 molecular weight region. The activity of peak I was found to be heat labile and inhibited by sialoglycoproteins such as fetuin and mucin (5 mg/ml), as well as by sialic acid. Protease inhibitors such as antitrypsin, pepstatin, phenylmethylsulfonyl fluoride, metaloprotease inhibitors, and bacitracin had no effect on the cytopathic activity. Marked inhibition of cytopathic activity was observed, however, with iodoacetamide and p-chloromercuribenzoate, which affect sulfhydryl groups. The toxic material in peak II was found to have ionophoric activity and was not inhibited by sialic acid-containing compounds. The materials from both peaks had enterotoxic activity in intestinal ligated loops. The active substance from peak I was further purified (200X) on an agarose-fetuin affinity column, yielding one major protein band with an apparent molecular weight of ca. 30,000 on sodium dodecyl sulfate. Amino acid analysis revealed that the protein was very poor in sulfur amino acids. The sialic acid-sensitive toxic activity was higher in known virulent strains such as HM-1:IMSS and could be markedly augmented after preincubation of the trophozoites with certain Escherichia coli strains.
Adoptive transfer of murine host protection to salmonellosis with T-cell growth factor-dependent, Salmonella-specific T-cell lines
Authors: Paul C , Shalala K , Warren R , Smith R
Published: January 2020

A spent medium antigen was prepared from the avirulent RIA strain of Salmonella typhimurium. Lymph node cells isolated from female BALB/c mice injected subcutaneously with the spent medium antigen exhibited antigen-specific proliferation. By using these cells and T-cell growth factor, continuous spent medium antigen-specific, Thy 1.2-sensitive lines were generated. These cells exhibited antigen-specific proliferation in vitro and were effective in inducing significant (P less than 0.01) host protection when adoptively transferred to naive syngeneic mice.
Identification and characterization of Vibrio cholerae surface proteins by radioiodination
Authors: Richardson K , Parker C D
Published: January 2020

Whole cells and isolated outer membrane from Vibrio cholerae (Classical, Inaba) were radiolabeled with Iodogen or Iodo-beads as catalyst. Radiolabeling of whole cells was shown to be surface specific by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of whole cells and cell fractions. Surface-labeled whole cells regularly showed 16 distinguishable protein species, of which nine were found in radiolabeled outer membrane preparations obtained by a lithium chloride-lithium acetate procedure. Eight of these proteins were found in outer membranes prepared by sucrose density gradient centrifugation and Triton X-100 extraction of radiolabeled whole cells. The mobility of several proteins was shown to be affected by temperature, and the major protein species exposed on the cell surface was shown to consist of at least two different peptides.
Effects of first-order Cryptococcus-specific T-suppressor cells on induction of cells responsible for delayed-type hypersensitivity
Authors: Murphy J W
Published: January 2020

Cell-mediated immunity is an important aspect of host resistance against Cryptococcus neoformans. Using a CBA/J murine model, we demonstrated that injection of cryptococcal antigen (CneF) at dosages sufficient to stimulate the antigenemia observed in cryptococcosis patients induces specific T-cell-mediated suppression of the cryptococcal delayed-type hypersensitivity response. The purpose of this study was to establish whether Lyt 1+, first-order T-suppressor (Ts1) cells block the induction of T cells responsible for delayed-type hypersensitivity (TDH cells) or whether they function by inducing Lyt 2+, efferent suppressor (Ts2) cells. In one set of experiments, suppression was observed when Ts1 cells were adoptively transferred to recipient animals the day before, the day of, or the day after immunization; however, when Ts1 cells were transferred after TDH cells were present, no suppression occurred. In other experiments, putative TDH cells from lymph nodes (LN) or spleens were adoptively transferred from mice after immunization or after a suppressive dose of CneF or adoptive transfer of Ts1 cells and immunization. Delayed-type hypersensitivity could not be transferred with LN or spleen cells from mice receiving the suppressive dose of CneF or the Ts1 cells, even when the LN or spleen cells were treated with anti-Lyt 2.1 antibody and complement to remove any Ts2 cells. Delayed-type hypersensitivity was readily transferred with LN or spleen cells from immunized mice whether the cells were or were not treated with anti-Lyt 2 and complement. Furthermore, the cells in the tolerized LN cell pools responsible for suppression of TDH cell induction were Lyt 1+ 2-, I-J+ cells, which is the phenotype of the Ts1 cells. Taken together, these data indicate that Ts1 cells inhibit the induction of TDH cells. This finding, coupled with the previous demonstration that Ts1 cells or a Ts1 cell-derived soluble factor (TsF1) induces Ts2 cells, establishes that the cryptococcal Ts1 cells are bifunctional in the suppressive pathway.
Uptake and deacylation of bacterial lipopolysaccharides by macrophages from normal and endotoxin-hyporesponsive mice
Authors: Munford R S , Hall C L
Published: January 2020

Macrophages are thought to play a central role in the responses of animals to gram-negative bacterial lipopolysaccharides (LPS). Since nothing is known about the metabolism of LPS by these cells, we studied the uptake and deacylation of radiolabeled LPS by thioglycolate-elicited peritoneal macrophages from normal (C3H/HeN) and LPS-hyporesponsive (C3H/HeJ) mice. Macrophages from both kinds of mice took up and deacylated LPS that were added to the culture medium. Opsonization of the LPS with anti-LPS immunoglobulin G antibodies greatly increased LPS uptake; the opsonized LPS also underwent deacylation at rates that were directly related to the amount of cell-associated LPS. An analysis of the fatty acid composition of the cell-associated LPS indicated that the cells have one or more acyloxyacyl hydrolases that remove the non-hydroxylated fatty acids that are normally substituted to the hydroxyl groups of (glucosamine-linked) 3-hydroxytetradecanoate residues in lipid A; we also found evidence for deacylation of 3-hydroxytetradecanoate from the glucosamine backbone. LPS deacylation by macrophages from C3H/HeN and C3H/HeJ mice was qualitatively and quantitatively similar. Nonopsonized LPS are able to stimulate LPS-responsive cells; in these studies we established that animal cells can deacylate nonopsonized LPS, thus raising the possibility that LPS metabolism may play a role in modulating cellular stimulation.
Antibody response to phenolic glycolipid I in inbred mice immunized with Mycobacterium leprae
Authors: Teuscher C , Yanagihara D , Brennan P J , Koster F T , Tung K S
Published: January 2020

The level of circulating antibody to phenolic glycolipid I of Mycobacterium leprae was determined in 18 inbred strains of mice after immunization with M. leprae organisms. By using a solid-phase radioimmunoassay with phenolic glycolipid I as test antigen, a continuous distribution of antibody levels ranging from high to low was observed. The level was found to be controlled by multiple genes, including both H-2 complex- and Igh allotype complex-linked genes. Low antibody response to phenolic glycolipid I was shown to be inherited as a dominant trait in three combinations of high X low responder F1 progeny.
Human ciliated epithelial cells from nasal polyps as an experimental model for Mycoplasma pneumoniae infection
Authors: Almagor M , Kahane I , Wiesel J M , Yatziv S
Published: January 2020

Human ciliated epithelial cells derived from nasal polyps and cultured in a monolayer were studied as an experimental model for Mycoplasma pneumoniae infection. Scanning electron microscopy revealed two types of cultured epithelial cells: one which was covered by microvilli only and another which had microvilli and actively beating cilia. M. pneumoniae adhered to both types of cells, and the adherence followed saturation kinetics as a function of time. Infection of the cells for 20 h resulted in 75% inhibition of their intracellular catalase activity and a 3.5-fold increase in their malonyldialdehyde levels compared with noninfected controls. This indicates the presence of cellular oxidative damage due to M. pneumoniae infection. It is suggested that human nasal ciliated epithelial cells may serve as a representative model for studying M. pneumoniae in relation to its natural host.
Effect of doxycycline on immune response in mice
Authors: Bellahsene A , Forsgren A
Published: January 2020

The effect of doxycycline on immune response has been studied in mice, cell-mediated immunity being evaluated with the split heart allograft technique. Survival duration of heart transplants in animals treated with 2.5 mg of doxycycline per kg per day from the day of transplantation until rejection was slightly but significantly longer than in untreated animals, 18.8 days (P less than 0.05) as compared with 14.5 days. In doxycycline-treated animals, both agglutinating and hemolytic antibody response to sheep erythrocytes was slightly but significantly decreased, though there was no inhibition of splenic production of antibodies to sheep erythrocytes (as measured by the number of plaques of hemolysis detected). The results show the immune response in mice to be only moderately inhibited by doxycycline. The relevance of experiments in mice is also discussed.
Antibodies that bind to fimbriae block adhesion of Streptococcus sanguis to saliva-coated hydroxyapatite
Authors: Fachon-Kalweit S , Elder B L , Fives-Taylor P
Published: January 2020

Antibodies raised against a fimbriated, adhesive strain of Streptococcus sanguis (FW213) were found to block the adhesion of this organism to saliva-coated hydroxyapatite. Antibodies were made specific for adhesion antigens by adsorption with isogenic, nonadhesive mutants (for rabbit polyclonal adsorbed antibody) or selection based on nonreactivity with two nonadhesive mutants (for monoclonal antibody). Rabbit antibody raised against isogenic, nonfimbriated nonadhesive mutants served as a control for antibodies present, but not related to fimbriation. Adsorbed antibody and monoclonal antibody were shown to be specific for fimbriae (antigen 1), since both antibodies could be seen by immune electron microscopy to bind 3.6-nm fimbriae, reacted only with the fimbriated parent and not the mutants in a whole bacterial cell enzyme-linked immunosorbent assay, and could immunoprecipitate fimbriae from fimbrial extracts of FW213. Antibodies isolated from preimmune and mutant sera did not react with fimbriae in any of the above assays. Only adsorbed antibody and monoclonal antibody were capable of blocking the adhesion of FW213 to saliva-coated hydroxyapatite. Adsorbed antibody, purified to immunoglobulin G (IgG), was an effective inhibitor of adhesion without causing interfering cellular aggregation. Monoclonal IgG, papain-cleaved to Fab fragments to prohibit cell-to-cell cross-linking, was also a potent inhibitor of S. sanguis FW213 adhesion. Both IgG from mutant sera and Fab fragments from normal mouse IgG could not be shown to block adhesion. These data further support the hypothesis that S. sanguis fimbriae are involved in adhesion.
Influence of pH and fluoride on properties of an oral strain of Lactobacillus casei grown in continuous culture
Authors: Hamilton I R , Boyar R M , Bowden G H
Published: January 2020

A freshly isolated oral strain, Lactobacillus casei RB1014, was grown in continuous culture to compare the effects of pH and fluoride on growth and metabolism. The cells were grown at pH 7.0 to 3.2 in the absence of fluoride and from pH 7.0 to 5.4 with 20 mM NaF. Cell numbers varied from 3 X 10(9) to 30 X 10(9)/ml on blood agar during alterations in the growth pH from 7.0 to 4.27. Only when the culture was stressed by lowering the pH to 3.2 were cell numbers drastically reduced. Cells growing at pH 7.0 without fluoride were unable to grow when plated on fluoride agar (10.5 mM) at pH 5.5; however, when the growth pH was allowed to decrease to 4.94, cells grew on the fluoride plates in numbers equal to those growing on blood agar. This fluoride tolerance trait appeared rapidly once pH control was removed and was lost when the culture was returned to pH 7.0. The addition of 20 mM NaF to the culture medium did not adversely affect growth, provided that the pH was maintained at 6.0 or above; cells tolerant to 10.5 and 16 mM NaF appeared on pH 5.5 plates during this phase. In cells removed from the chemostat throughout the experiment and incubated at the pH of growth in a pH stat, glycolytic activity was optimum at pH 5.5 in the absence of NaF. Fluoride stimulated glycolytic activity by cells incubated at pH 7.0 and by cells growing with 20 mM NaF, provided that the pH of growth remained at or above 6.0. A more detailed examination of the adaptation to fluoride tolerance during shifts to acidic pH values revealed that cells capable of growth on acidic fluoride agar plates appeared within 2 h of the start of the fall in pH of the chemostat culture. Estimation of the intracellular pH during the period of the initial pH fall revealed that the intracellular pH was identical to the extracellular pH (i.e., no pH gradient [delta pH]), indicating that fluoride would not be transported into the cells to inhibit metabolism. However, once the pH of the medium was stabilized, delta pHs were generated, with the delta pH increasing as the pH declined. The inhibition of glycolysis by fluoride increased in proportion to the delta pH. Cells grown at pH 5.5 generated larger delta pHs than did cells grown at pH 7.0, although the values were normally small (approximately 0.9 U). The data suggest that the inherent fluoride tolerance of L. casei RB1014 was associated with relatively small delta pHs.
Ontogeny of macrophage-mediated protection against Listeria monocytogenes
Authors: Ohara R , Mitsuyama M , Miyata M , Nomoto K
Published: January 2020

We investigated the ontogenic development of macrophage functions which are important in the expression of host defense against infection by Listeria monocytogenes. Macrophage functions, including accumulation in response to local stimuli, chemotaxis in vitro, and intracellular killing, as well as number of macrophages, were examined by using mice 1, 2, 3, 4, and 8 weeks old. The number of peritoneal macrophages was extremely low in younger mice even when their body weights were taken into consideration. Macrophage accumulation in response to infectious stimulus with viable listeria was poor in younger mice and showed an age-dependent development. In younger groups, chemotaxis in vitro was as immature as chemotaxis in vivo. In 1- and 2-week-old mice, macrophages did not show any intracellular killing activity against L. monocytogenes, but killing was observed in mice over 3 weeks of age. These functions developed in an age-dependent manner and reached the 8-week-old adult level after the mice were 4 weeks of age. In adult mice, these macrophage functions were shown to be enhanced after immunization with viable listeria; however, such an immunization-induced enhancement was very poor in the younger groups of mice. Protection judged by mortality and in vivo bacterial growth was weaker in the younger groups against both primary and secondary challenges. In vivo protection against L. monocytogenes seemed to develop in the same age-dependent manner as the development of macrophage functions. These results indicate that age-dependent immaturity of macrophage functions mainly comprises the age-dependent immaturity of protection against L. monocytogenes.
Characterization of antibodies mediating protection and cure of Trypanosoma musculi infection in mice
Authors: Wechsler D S , Kongshavn P A
Published: January 2020

Plasma samples were collected from mice infected with Trypanosoma musculi at different times postinfection and administered to naive recipient mice either before or during T. musculi infection. The protective and curative activities of these plasma samples were shown to increase as the time of collection postinfection increased; plasma collected at 14 days postinfection was partially protective and partially curative, whereas that collected at 28 days postinfection was completely protective and curative. The curative activity was labile to heat treatment (30 min at 56 degrees C), whereas the protective activity was heat stable. Additional kinetic parameters relating to the efficacy of protection were investigated. Evidence is presented that both activities are immunoglobulin in nature. Protein A-Sepharose chromatography indicated that the activities are associated with the immunoglobulin G2a or immunoglobulin G3 subclasses of immunoglobulin G. The curative antibody appears to be intrinsically heat labile, since heat treatment of a purified immunoglobulin preparation abolished the ability to cure. Studies on the mechanism of parasite elimination from blood suggest that the process not only requires antibody but is also complement dependent.
Escherichia coli contains plasmids coding for heat-stable b, other enterotoxins, and antibiotic resistance
Authors: Echeverria P , Seriwatana J , Taylor D N , Tirapat C , Rowe B
Published: January 2020

Plasmid DNAs obtained from 18 Escherichia coli isolates that hybridized with the heat-stable b (ST-b) enterotoxin gene probe were examined by Southern blot analysis for genes coding for heat-labile, ST-a, and ST-b enterotoxins with specific radiolabeled DNA probes. Four E. coli isolates contained plasmids coding for both heat-labile and ST-b enterotoxins, and one isolate contained a plasmid coding for ST-a and ST-b. Five of 11 isolates of antibiotic-resistant enterotoxigenic E. coli isolates containing ST-b-coding DNA transferred a plasmid coding for both antibiotic resistance and ST-b to E. coli K-12, suggesting that the widespread use of antibiotics could increase the distribution of genes coding for ST-b.
Effects of splenectomy on antibody-independent immunity to Plasmodium chabaudi adami malaria
Authors: Grun J L , Long C A , Weidanz W P
Published: January 2020

Splenectomy of B-cell-deficient mice and immunologically intact mice before infection with Plasmodium chabaudi adami led to the development of significant parasitemias which eventually resolved in the latter mice. Whereas both eusplenic B-cell-deficient mice and immunologically intact mice resolved their acute P. chabaudi adami infection, only B-cell-deficient mice subsequently developed chronic low-grade malaria. Splenectomy of B-cell-deficient mice with chronic malaria led to recrudescing infections, suggesting that the expression of antibody-independent immunity to reinfection was spleen dependent. When dispersed spleen cells were injected into splenectomized mice before challenge with P. chabaudi adami, the kinetics of the resulting infection resembled that seen in splenectomized mice which had not been grafted with normal spleen cells. This finding indicates that immunity to P. chabaudi adami requires the presence of an architecturally intact spleen.
Human monoclonal antibodies against Mycobacterium leprae
Authors: Atlaw T , Kozbor D , Roder J C
Published: January 2020

Human hybridomas were constructed which produce antibodies against three different extracts of Mycobacterium leprae. A thioguanine-resistant (Thgr), ouabain-resistant (Ouar), human lymphoblastoid cell line, KR-4, was hybridized with Epstein-Barr virus-transformed cell lines from lepromatous leprosy patients with fusion frequencies of greater than 10(-5). Non-Epstein-Barr virus-transformed donor cells fused at much lower rates (less than 2 X 10(-7]. Hybrids were selected in medium containing hypoxanthine aminopterin thymidine and 10(-5) M ouabain. An enzyme-linked immunosorbent assay was used to screen for antibodies against three crude extracts of armadillo-derived M. leprae, including (i) a soluble sonic extract preparation, (ii) sodium dodecyl sulfate extract of insoluble sonicated M. leprae, and (iii) a purified phenolic glycolipid antigen. Of a total of 2,200 final clones screened, 359 were found to secrete antibody which bound to soluble sonic extracts and the sodium dodecyl sulfate extract (6.7 and 9.6%, respectively), whereas 12.5% (21 out of 168) showed positivity to the glycolipid antigen. Four selected hybridomas also reacted with the deacylated derivative of M. leprae phenolic-glycolipid antigen. The specificity of these monoclonal antibodies was partially determined by screening on a panel of crude extracts from four other mycobacteria. Nine clones of 122 showed reactivity to M. leprae only. The predominant immunoglobulin was immunoglobulin M, and quantities up to 10 micrograms/ml were produced. Antibody production by hybrid clones was stable in more than 75% of the clones grown in continuous culture. By comparison, 10,000 Epstein-Barr virus-transformed lymphocyte clones from lepromatous leprosy patients were screened for anti-M. leprae antibody production, and all of the 42 clones that were initially positive in the enzyme-linked immunosorbent assay lost their antibody-producing capabilities within 6 weeks in culture. These results suggest that a combination of Epstein-Barr virus transformation and hybridization may be an optimal method in producing human monoclonal antibodies from leprosy patients.
Chemical and immunochemical analyses of Bacteroides fragilis lipopolysaccharides
Authors: Weintraub A , Larsson B E , Lindberg A A
Published: January 2020

Lipopolysaccharides (LPSs) from 17 different Bacteroides fragilis strains were extracted in a two-step procedure. The first step was a hot phenol-water extraction of whole bacteria, resulting in a crude aqueous phase, which after lyophilization in a second step was extracted with a phenol-chloroform-light petroleum mixture. The resulting LPSs, which were essentially free from contaminating nucleic acid, proteins, and capsular polysaccharide, were investigated for their qualitative and quantitative sugar and fatty acid composition, immunochemical specificity by enzyme-linked immunosorbent inhibition assays, and particle weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the 17 strains, 13 had LPSs which all contained L-rhamnose, D-glucose, D-galactose, and D-glucosamine in approximately the same ratio. Three of these LPSs also contained D-galactosamine. The fatty acid composition was also similar in that the same fatty acids, although in slightly varying proportions, were found in all LPSs. The 13 strains also showed the same specificity in inhibition studies by enzyme immunoassay with rabbit anti-LPS antisera and LPS antigen. The LPS particle weights were also very similar, in the range of what is found for LPSs from rough mutant strains of enterobacteria. Our results suggest that most strains of B. fragilis have similar, if not identical, LPSs with relatively short polysaccharide chains.
Correlation of binding of rabbit granulocyte peptides to Candida albicans with candidacidal activity
Authors: Lehrer R I , Szklarek D , Ganz T , Selsted M E
Published: January 2020

NP-1, a candidacidal peptide purified from rabbit granulocytes, bound extensively and with biphasic kinetics to Candida albicans. The primary phase of binding was temperature independent and occurred even at 0 degrees C. This primary binding was relatively specific, reversible, saturable, and of high capacity. It was inhibited by increased salt concentrations in the incubation medium, but was relatively unaffected by increasing the calcium ion concentration or by lowering the incubation temperature to 0 degrees C. The secondary phase of binding was only noted under conditions that supported candidacidal activity. Secondary binding was inhibited by millimolar concentrations of calcium, but not magnesium, ions and did not occur at 0 degrees C or when subtoxic concentrations of NP-1 were tested. NP-2 and NP-3a, other potent candidacidal peptides from rabbit granulocytes, also bound directly and extensively to C. albicans and competed for binding with NP-1. NP-4 and NP-5, less candidacidally active homologs of the aforementioned peptides, showed relatively little direct binding activity and competed poorly for binding with NP-1 or NP-2. NP-3b, another less candidacidal homolog, bound extensively to C. albicans, but did not compete effectively with NP-1 or NP-2. By comparing candidacidal and binding activity of the peptides, we conclude that the candidacidal activity of NP-1 involves primary binding to C. albicans followed by postbinding events that are temperature dependent and inhibitable by calcium ions.
Granuloma formation by muramyl dipeptide associated with branched fatty acids, a structure probably essential for tubercle formation by Mycobacterium tuberculosis
Authors: Emori K , Nagao S , Shigematsu N , Kotani S , Tsujimoto M , Shiba T , Kusumoto S , Tanaka A
Published: January 2020

Muramyl dipeptide, which does not induce epithelioid granuloma when injected alone dissolved in phosphate-buffered saline, could induce extensive granulomas in guinea pigs when chemically conjugated with branched, but not linear, fatty acids. Peptidoglycan fragments of Staphylococcus epidermidis could evoke epithelioid granulomas when incorporated in a water-in-oil emulsion. These findings suggest the importance of a lipid bound to muramyl dipeptide for granuloma formation. In view of the fact that mycobacteria uniquely contain large amounts of branched fatty acids, it was proposed that the complex of muramyl dipeptide and branched fatty acids, mostly mycolic acids, is a structure in tubercle bacilli responsible for tubercle formation.
Purification and characterization of an extracellular cytolysin produced by Vibrio damsela
Authors: Kothary M H , Kreger A S
Published: January 2020

Large amounts of an extremely potent extracellular cytolysin produced by the halophilic bacterium Vibrio damsela were obtained free of detectable contamination with medium constituents and other bacterial products by sequential ammonium sulfate precipitation, gel filtration with Sephadex G-100, and hydrophobic interaction chromatography with phenyl-Sepharose CL-4B. The cytolysin is heat labile and protease sensitive and has a molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of ca. 69,000 and an isoelectric point of ca. 5.6. The first 10 amino-terminal amino acid residues of the cytolysin are Phe-Thr-Gln-Trp-Gly-Gly-Ser-Gly-Leu-Thr. The cytolysin was very active against erythrocytes from 4 of the 18 animal species examined (mice, rats, rabbits, damselfish) and against Chinese hamster ovary cells and was lethal for mice (ca. 1 microgram/kg, intraperitoneal median lethal dose). Lysis of mouse erythrocytes by the cytolysin is a multi-hit, at least two-step process consisting of a temperature-independent, toxin-binding step followed by a temperature-dependent, membrane-perturbation step(s).
Influence of protein synthesis inhibitors on regulation of extent of O-acetylation of gonococcal peptidoglycan
Authors: Rosenthal R S , Gfell M A , Folkening W J
Published: January 2020

The effects of protein synthesis inhibitors on the extent of O-acetylation of Neisseria gonorrhoeae peptidoglycan (PG) and on the resistance of PG to degradation by human PG hydrolases were examined. Addition of chloramphenicol, tetracycline, and streptomycin (in amounts equal to approximately twice their respective MICs) rapidly increased the level of O-acetylation of [3H]glucosamine-labeled N. gonorrhoeae FA19 PG from 46% to about 70% and simultaneously enhanced the resistance of the PG to degradation by human polymorphonuclear leukocyte lysozyme. Entry into the stationary phase also enhanced O-acetylation of FA19 PG, but neither protein synthesis inhibitors nor the stationary phase had a detectable effect on the O-acetyl-deficient, lysozyme-sensitive PG of N. gonorrhoeae RD5. Mild alkali treatment of PG derived from chloramphenicol-treated FA19 specifically removed O-acetyl groups and simultaneously reduced the extents of O-acetylation and polymorphonuclear leukocyte lysozyme resistance to the level of RD5 PG, suggesting that the O-acetyl substituents were solely responsible for the increased PG hydrolase resistance of PG from chloramphenicol-treated FA19. Pulse-chase experiments indicated that the drug-mediated enhancement of O-acetylation was limited to newly assembled PG. In summary, conditions favoring unbalanced macromolecular synthesis and bacteriostasis increased the level of O-acetylation and the PG hydrolase resistance of gonococcal PG. Similar conditions encountered by gonococci in vivo might potentiate the pathobiological consequences of PG-host interactions.
Marked enhancement in vivo of adjuvant activity of muramyl dipeptide to protein antigens and to synthetic weak immunogens with monoclonal anti-muramyl dipeptide antibodies
Authors: Bahr G M , Tello D S , Chedid L A
Published: January 2020

Priming of mice with complexes of antigen coupled to muramyl dipeptide and monoclonal anti-muramyl dipeptide antibodies enhanced the adjuvant activity of muramyl dipeptide on the humoral response to the antigen. The enhancement did not occur with free (uncoupled) muramyl dipeptide and required the presence of an adjuvant-active hapten within the complex as well as the Fc fragment of the monoclonal antibody. This system proved highly effective in eliciting antibodies to synthetic weak immunogens whereas muramyl dipeptide, on its own, exerts very little or no adjuvant activity. The effect was not due to a general polyclonal activation and was restricted to the antigen coupled to the synthetic adjuvant. Possible pathways involved in this phenomenon are discussed.
Activation of neonatal and adult human macrophages by alpha, beta, and gamma interferons
Authors: Wilson C B , Westall J
Published: January 2020

Increasing evidence suggests that gamma interferon (IFN-gamma) is the major or sole factor in human lymphokines which activates blood monocyte-derived macrophages (M phi) to inhibit or kill Toxoplasma gondii and certain other intracellular pathogens. In the current studies, we found that IFN-gamma effectively activated tissue M phi from adults (peritoneal M phi) and from newborns (placental M phi) as well as blood-derived M phi from adults and from newborns to kill or to inhibit the replication of T. gondii. Results with purified and recombinant IFN-gamma and with adult and newborn M phi were similar. IFN-gamma-treated M phi were equally or more active against T. gondii than were freshly isolated monocytes and M phi. Recombinant IFN-alpha A and IFN-beta were less effective than IFN-gamma. IFN-gamma also inhibited survival and replication of T. gondii in WISH cells more effectively than did IFN-alpha and IFN-beta. These findings are consistent with an important role for IFN-gamma in the control of Toxoplasma infection and indicate that the anti-Toxoplasma activity of resting and IFN-gamma-activated adult and neonatal M phi is similar. The increased susceptibility of neonates to T. gondii is not due to a defect in M phi effector function.
Bacteroides endodontalis and other black-pigmented Bacteroides species in odontogenic abscesses
Authors: van Winkelhoff A J , Carlee A W , de Graaff J
Published: January 2020

Twenty-eight odontogenic abscesses were examined for the presence of black-pigmented Bacteroides spp. Of the 28 samples, 26 were found to contain one or more species of black-pigmented Bacteroides. Abscesses were divided into three categories according to the tissue of origin: endodontal, periodontal, and pericoronal. Four abscesses which developed after extraction were also examined. It was found that Bacteroides endodontalis, a newly described species of asaccharolytic black-pigmented Bacteroides, was isolated almost exclusively from periapical abscesses of endodontal origin. B. intermedius proved to be the most frequently isolated species in all of the samples. B. gingivalis was present in all of the periodontal abscesses studied, as well as in two endodontal abscesses. B. melaninogenicus was recovered once from a pericoronal abscess. Precautions for the isolation of B. endodontalis are discussed.
Opsonized streptococcal cell walls cross-link human leukocytes and erythrocytes by complement receptors
Authors: Pryzwansky K B , Lambris J D , MacRae E K , Schwab J H
Published: January 2020

Serum-opsonized group A streptococcal cell walls, consisting of peptidoglycan-polysaccharide polymers (PG-APS), induced monolayers of human neutrophils, monocytes, and eosinophils to aggregate. When erythrocytes were present in the incubation medium, they also were associated with the leukocyte aggregates. By immunofluorescence staining, PG-APS was localized at the site of cell-to-cell contact. By scanning electron microscopy the cells appeared to adhere to one another by surface contact; filopodia often acted as connectors, particularly in leukocyte-erythrocyte interaction. Cellular binding of PG-APS and aggregation were dependent upon C3 fixation. No aggregation was observed when heat-inactivated serum was used as an opsonin. In contrast to peptidoglycan, an activator of the alternative complement pathway, the group-specific polysaccharide moiety of PG-APS induced no cellular aggregation. Rosette formation was observed in suspensions when neutrophils were incubated with erythrocytes coated with C3b-opsonized PG-APS. Cell monolayers bound serum-opsonized PG-APS, but aggregation was observed only when serum was present in the incubation medium. Similar results were obtained with C5-deficient serum. No aggregation was observed with heat-inactivated serum or bovine serum albumin. A heat-labile serum component(s) appears to be required to elicit leukocyte aggregation. It is suggested that C3 fixed to PG-APS acts as a bridge to link cells together in clusters as a result of common recognition of C3 by leukocyte and erythrocyte complement receptors.
Genetic Control of Host Responses to Endotoxin
Authors: Sultzer Barnet M.
Published: January 2020

Two pathophysiological effects of endotoxin in mice have been shown to be under the major influence of polygenic inheritance. The first, endotoxemia, was measured by LD
50
assays on hybrids and backcross hybrids of the resistant C
3
H/HeJ and the susceptible A/HJe strains. The LD
50
values of the F1 hybrids and the backcross hybrids were intermediate between the respective parental types. The second effect, the intraperitoneal extravascular leukocyte response to small doses of endotoxin, also was found to follow characteristic patterns of polygenic inheritance. As estimated from an analysis of the variance of mice of the F1 and F2 generations, the degree of genetic determination is approximately 54% for the neutrophil response and 82% for the monuclear response.
Mycoplasmacidal Activity of Peroxidase-H
2
O
2
-Halide Systems
Authors: Jacobs A. A. , Low I. E. , Paul B. B. , Strauss R. R. , Sbarra A. J.
Published: January 2020

A mycoplasmacidal system consisting of myeloperoxidase (MPO)-containing granules, H
2
O
2
, and a halide is described. In all parameters measured, it appears to be identical to the MPO-H
2
O
2
-halide bactericidal system previously reported. It has a
p
H optimum of approximately 5.5 and an optimal MPO:H
2
O
2
ratio of 1:25. The halide requirement can be satisfied by either chloride or iodide. Through the use of taurine or horseradish peroxidase substitution, chloride-mediated killing can be distinguished from iodide-mediated killing. The relationship of this mycoplasmacidal system to other mycoplasmacidal systems and to host surveillance of mycoplasma is discussed.
Production of Potent Anti-Australia Antigen Sera of High Specificity and Sensitivity in Goats
Authors: Dreesman Gordon R. , Hollinger F. Blaine , McCombs Robert M. , Melnick Joseph L.
Published: January 2020

Potent antisera of high specificity and sensitivity were produced, in goats, to purified Australia antigen (Au). The antigen was prepared by one of three methods: (i) pelleting, low
p
H treatment, isopycnic centrifugation two times in CsCl, and rate zonal centrifugation in sucrose; (ii) same as procedure i, with the exception of the low
p
H treatment; or (iii) twice banding in CsCl by using a BXIV batch-type zonal centrifuge rotor with subsequent preparative Pevikon electrophoresis. The goat anti-Au sera contained high levels of precipitating antibody as tested by immunodiffusion in agar gel and discontinuous counterimmunoelectrophoresis (DCIE) as well as specific complement-fixing antibody and could be used for routine screening of sera for Au without prior adsorption with Au-negative normal human serum (NHS). Identification of 66 of 70 positive specimens (94.3%) in a panel of 98 coded sera (49 duplicates) with 100% reproducibility was made by using one of the goat anti-Au sera at a dilution of 1:16 in the DCIE method. No false positives were recorded. Low levels of antibody against NHS components were effectively removed by a single adsorption with glutaraldehyde cross-linked NHS.
Screening of Sera for Antibodies to Foot-and-Mouth Disease Viral Antigens by Radial Immunodiffusion
Authors: Wagner G. G. , Cowan K. M. , McVicar J. W.
Published: January 2020

Radial immunodiffusion procedures have been developed for testing bovine sera for the presence of antibody activity against three foot-and-mouth disease virus types and a virus infection-associated antigen. Reactions with virus antigens were obtained as early as 4 days after infection, and the virus type causing the response was identified. The procedure had the advantages of great sensitivity for the detection of antibody and the ease with which antibodies to a variety of antigens could be detected.
Immunochemical Identification of a Defined Antigen of
Mycobacterium leprae
Authors: Estrada-Parra Sergio
Published: January 2020

An extract from purified
Mycobacterium leprae
precipitated with sera from patients with tuberculosis, mycetoma, and leprosy and gave a band of identity with polysaccharide I from
Nocardia
and
Mycobacteria.
Demonstration of Cytotoxic Antibodies in Rabbits Bearing Tumors Induced by Shope Fibroma Virus
Authors: Singh S. B. , Smith J. W. , Rawls W. E. , Tevethia S. S.
Published: January 2020

The
51
Cr-release test was used to detect cytotoxic antibodies in adult rabbits bearing tumors induced by Shope fibroma virus. The following are the recommended experimental conditions: the infection of RK-13 cells with a multiplicity of 1 to 2 infectious units per cell for 48 hr,
51
Cr labeling of infected cells during the last 12 hr of incubation, sensitization of suspended labeled infected cells for 1 hr with immune serum, and quantitation of cell damage by the amount of
51
Cr released after 6 hr of incubation in the presence of complement. The immune sera reacted only with fibroma virus-infected cells but not with cells infected with vaccinia virus or herpesvirus type 1. Similarly, sera prepared against vaccinia virus and herpesvirus type 1 were not cytotoxic to fibroma virus-infected cells, although they were cytotoxic to cells infected with homologous viruses. The total antibody activity in sera of rabbits infected with Shope fibroma virus was detected first on day 7, gradually rose to its peak by day 23, and persisted at that level for at least 50 days. The 19
S
antibody was detected on day 7, reached peak titers by day 13, and disappeared by day 17. The 7
S
antibody was barely detectable on day 7, reached maximum titers on day 13, and remained high for at least 50 days. The tumors appeared on the 3rd day after virus inoculation, reached maximum size on day 13, and regressed completely by day 23.
Circadian Rhythm of the Resistance of Mice to Acute Pneumococcal Infection
Authors: Wongwiwat Mai , Sukapanit Sirimana , Triyanond Chalermlarp , Sawyer William D.
Published: January 2020

The resistance of both male and female mice to an acute infection by type I pneumococci varied rhythmically in an approximately 24-hr cycle. Mice inoculated intraperitoneally with 10
5.4±0.1
organisms during the dark period of the daily cycle survived significantly longer than mice similarly inoculated during the light period. The variation in resistance was correlated with the rate of increase of bacteremia, the rate being greatest after inoculation during the light period. Neither the capacity of the blood to support growth of pneumococci in vitro nor clearance of pneumococci from the blood in vivo varied between phases of the daily cycle. Adrenalectomized mice were equally and highly susceptible to infection during both light and dark periods. Stress of mice during the light period, normally a time of relative inactivity of mice, disturbed the rhythm; resistance to infection initiated during the light period increased and equalled that during the dark period.
Cell Wall Composition of Two Strains of
Blastomyces dermatitidis
Exhibiting Differences in Virulence for Mice
Authors: Cox Rebecca A. , Best Gary K.
Published: January 2020

Cell walls isolated from two strains of
Blastomyces dermatitidis
were examined. Whereas strain Ga-1 was practically avirulent for mice, strain KL-1 produced death by 21 days in 50% of the mice inoculated. Analyses of the trypsin-treated cell walls of the two strains revealed a higher chitin and protein content in strain KL-1, whereas a higher polysaccharide content was observed in the cell walls of strain Ga-1. Extraction of the walls with 1
n
NaOH revealed a threefold difference in the amount of alkali-soluble cell wall material present. The alkali-soluble material could be further fractionated into a water-soluble and a water-insoluble fraction. Previous reports have indicated that the water-insoluble fraction of
B. dermatitidis
consists of an alpha-linked glucan; however, we report that in addition a phospholipid moiety is covalently bound to the polysaccharide. Furthermore, on the basis of organic phosphorus content, considerably more phospholipid is associated with the alpha-linked glucan of the more virulent KL-1 strain. These results suggest that this cell wall constituent might be one of the factors related to the virulence of this fungus.
Characterization and Comparison of Mycobacterial Antigens by Two-Dimensional Polyacrylamide Gel Electrophoresis
Authors: Wright George L. , Affronti Lewis F. , Reich Melvin
Published: January 2020

Cell extracts and culture filtrates were prepared from several mycobacterial species and subjected to two-dimensional acrylamide gel electrophoresis. The cell preparations were first electrophoresed in a 7% homogeneous gel followed by a second electrophoresis, at right angles to the first separation, in a 2 to 30% linear gel gradient slab. Because the stained slab resembled a “fingerprint” or “peptide map,” this procedure has been called “electroprotein mapping.” More than 200 protein-staining spots were detected in several of the cell extracts, each map being characteristic of the species examined. The single most characteristic portion of each map was the region between the 16 and 30% gel concentrations. This region consisted of several small-molecular-weight components (many of peptide proportions) which together appeared as the most anodic band in the 7% gel column (first separation). The number and location of these components in the linear gel slab (second separation) were specific for each species tested. By electrophoresis of molecular-weight markers (i.e., albumin, myoglobin, cytochrome
c
) with and across (transverse electrophoresis) the gel gradient, the relative molecular weights of the unknown cell extract components could be estimated. These results suggest that two-dimensional acrylamide gel electrophoresis is a useful tool for the characterization of mycobacterial cell constituents.
Infection at the Subcellular Level I. Localization of Intravenously Injected Brucellae in the Vacuolar Apparatus of Cells of Guinea Pig Liver
Authors: Guerra Humberto , Deter Russell L. , Williams Robert P.
Published: January 2020

The distribution of
Brucella melitensis
in various tissues and in subcellular fractions obtained from liver was investigated to evaluate the initial phases of brucellosis in the guinea pig. Fifty minutes after intravenous infection, brucellae were found principally in the blood and liver, with a substantial number recovered from spleen. Fractionation of liver established that most bacteria were found in the mitochondrial plus lysosomal (M + L) fraction; a significant number, however, sedimented in the nuclear (N) fraction. With time, there was a progressive shift of bacteria from the M + L to the N fraction, accompanied by a similar shift in acid phosphatase activities. Isopycnic centrifugation of mixtures of M + L fractions and brucellae permitted complete separation of acid phosphatase-bearing particles from bacteria. Similar experiments with fractions from infected animals showed that viable bacteria were found in both the acid phosphatase and free brucellae regions of the gradient. At 10 min postinfection, 52% of the recovered organisms were in the acid phosphatase region; at 30 min, 65%; at 60 min, 85%; and at 315 min, 79%. Detergent plus sonic treatment of an M + L fraction from the liver of an animal killed 50 min after infection caused most of the bacteria in the acid phosphatase region to shift to the region where free bacteria were found. These data suggested that brucellae sequestered in the liver were located primarily in the vacuolar apparatus of the cells which phagocytized them.
Effect of Dihydrostreptomycin on Phagocytosis of Mouse-Peritoneal Macrophages In Vitro
Authors: Adam Dieter , Staber Fritz , Belohradsky Bernd H. , Marget Walter
Published: January 2020

The effect of the antibiotic dihydrostreptomycin on the phagocytic and bactericidal ability of peritoneal macrophages obtained from mice has been investigated. In subliminal concentrations which did not influence the bacterial growth, the drug caused macrophages to ingest and kill bacteria (
Escherichia coli
) at a higher rate than did macrophages without antibiotic. The differences for phagocytosis and intracellular killing of
E. coli
with and without a subliminal amount of dihydrostreptomycin were statistically significant. Macrophages pretreated with the antibiotic did not demonstrate any enhancement of phagocytosis.