bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of immune response to oral administration of Streptococcus sobrinus ribosomal preparations in liposomes
Authors: Gregory R L , Michalek S M , Richardson G , Harmon C , Hilton T , McGhee J R
Published: January 2020

Gnotobiotic rats gastrically intubated with a total of 12.5 micrograms of Streptococcus sobrinus ribosomal protein incorporated into cholesterol-based liposomes had significantly (P less than or equal to 0.01) fewer carious lesions on their molar surfaces than did nonimmunized infected controls after challenge with a virulent organism. The immunized animals had significantly (P less than or equal to 0.01) lower numbers of molar-adherent S. sobrinus cells and higher levels of salivary immunoglobulin A antibodies to S. sobrinus whole cells and ribosomes than did the control group. Dose-response studies indicated that 12.5 micrograms of S. sobrinus ribosomal protein in liposomes induced slightly higher immune responses than did 62.5, 125.0, and 250.0 micrograms of ribosomal protein incorporated into liposomes. Intubation of rats with up to 250.0 micrograms of S. sobrinus ribosomal protein alone was no more effective in reducing the numbers of molar-adherent S. sobrinus cells than were nonimmunized infected controls, establishing that insertion of ribosomes into liposomes was required for inducing an effective immune response. These results indicate that oral administration of as little as 12.5 micrograms of S. sobrinus ribosomal protein incorporated into liposomes can protect rats from caries formation after challenge with the virulent organism by inducing specific salivary immunoglobulin A antibodies which can inhibit colonization by the challenged S. sobrinus.
Secretory immunoglobulin A response following peroral priming and challenge with Shigella flexneri lacking the 140-megadalton virulence plasmid
Authors: Keren D F , McDonald R A , Formal S B
Published: January 2020

This study evaluates the ability of noninvasive Shigella spp., lacking the 140-megadalton virulence plasmid, to elicit a mucosal immunoglobulin A immune response in the intestine. For these studies, we used Shigella flexneri M4243A1 (which lacks the plasmid and is Sereny test negative) to prime and challenge three groups of rabbits perorally. Both primary and immunoglobulin A memory responses were detectable in these secretions. These findings indicate that a mucosal memory response can be primed by nonpathogenic strains of Shigella which lack the virulence plasmid.
Bordetella pertussis adenylate cyclase: isolation and purification by calmodulin-sepharose 4B chromatography
Authors: Friedman R L
Published: January 2020

Purified preparations of adenylate cyclase were obtained from crude urea extracts of Bordetella pertussis by a one-step calmodulin affinity chromatography technique. Diluted extract was loaded onto the column and washed, and adenylate cyclase was eluted with 10mM EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. A 104-fold purification was accomplished in one step. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the affinity-purified adenylate cyclase was dissociated into one major protein band with an apparent molecular weight of 60,000 and a minor band at 200,000. The affinity-purified adenylate cyclase was observed to have adenylate cyclase enzymatic activity which was activated by calmodulin, to bind 125I-calmodulin, and to be free of pertussis toxin as determined by in vivo and in vitro assays.
Chemical characterization of binding properties of opacity-associated protein II from Neisseria gonorrhoeae
Authors: Bessen D , Gotschlich E C
Published: January 2020

Binding of an opacity-associated protein II (PIIop) from Neisseria gonorrhoeae to eucaryotic macromolecules was studied. HeLa cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose, and purified PIIop bound to approximately 50 distinct molecular species. The binding of PIIop to HeLa cell components was stable in high salt and nonionic detergent and was not inhibited by a variety of monosaccharides and polyionic substances. PIIop binding behavior was compared with that of two model carbohydrate-binding proteins, wheat germ agglutinin (WGA) and concanavalin A (ConA). Model glycoproteins (ovomucoid, fetuin, mucin, ovalbumin) inhibited binding by PIIop, WGA, and ConA to various degrees. HeLa cell glycopeptides, generated by pronase digestion of chloroform-methanol-extracted cells, were tested for their ability to inhibit binding by PIIop to Western blots of HeLa cell macromolecules. HeLa cell extracts inhibited PIIop binding before pronase treatment, but inhibitory activity was lost as a result of pronase digestion. Direct binding to defined glycosylated and nonglycosylated proteins revealed that ConA and WGA bound only glycoproteins, whereas PIIop bound to proteins lacking carbohydrate as well. PIIop binding to human and bovine serum albumins was of high affinity and required partial unfolding of albumin; native albumin was not bound by PIIop; however, both the denatured, reduced form of albumin and the compact, nonreduced form of carboxymethylated albumin were bound strongly by PIIop. Albumin-PIIop interaction did not involve covalent bond formation through sulfhydryl groups. The predominant binding interactions of PIIop found in this study were with protein rather than carbohydrate, and the chemical nature of the interactions is more complex than involvement of purely ionic or hydrophobic forces.
A membrane-associated neuraminidase in Entamoeba histolytica trophozoites
Authors: Udezulu I A , Leitch G J
Published: January 2020

Trophozoites of the parasitic amoeba Entamoeba histolytica HM-1:IMSS possess a surface neuraminidase capable of liberating N-acetylneuraminic acid (NANA) from N-acetylneuramin-lactose (alpha 2----3 or alpha 2----6) or mucin in their medium. The neuraminidase was found to be membrane associated, with more than 50% of the yield being recovered in the plasma membrane fraction. The neuraminidase specific activity of the plasma membrane fraction was six times that of internal membrane fraction enzyme. The optimum pH and temperature for this enzyme were 6.7 and 37 degrees C, respectively. Neuraminidase activity was inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and the optimum Ca2+ concentration was 2 mM. The microfilament disruptor cytochalasin D (30 micrograms/ml) inhibited motility and neuraminidase activity of intact Entamoeba trophozoites. The cytochalasin D-induced loss of surface neuraminidase activity was explained in part by a redistribution of enzyme with a loss of plasma membrane enzyme and an increase in intracellular membrane enzyme. A qualitatively similar cytochalasin D effect was observed with two other membrane-associated enzymes, calcium-regulated ATPase and acid phosphatase. Membrane-associated enzyme was minimally affected by Triton X-100 and saponin. An N-acetylneuraminic acid aldolase, optimum pH, 7.4, was found in trophozoite homogenate supernatant fractions. NANA and NANA-containing compounds stimulated trophozoite-directed motility. This motility stimulation by NANA-containing compounds did not apparently require prior release of free NANA by the trophozoite surface neuraminidase. Entamoeba neuraminidase is one of a series of enzymes that may modify the mucus blanket and target cell surface and thereby play a role in the pathogenesis of amebiasis.
Role of natural killer cells in infection with the mouse pneumonitis agent (murine Chlamydia trachomatis)
Authors: Williams D M , Schachter J , Grubbs B
Published: January 2020

Natural killer (NK) activity is increased in both spleen and lung early in pulmonary infection by murine Chlamydia trachomatis in both susceptible nude and resistant heterozygous (nu/+) mice. Ablation of the rise in NK activity by giving the mice antiasialo GM-1 antibody or stimulation of NK activity by immunomodulators did not affect quantitative tissue counts of the mouse pneumonitis biovar of C. trachomatis or significantly affect survival. Studies are needed to further define the role of NK cells in host defense, immunoregulation, and immunopathology during chlamydial infection.
Functional capacity of marginated and bone marrow reserve granulocytes
Authors: Steele R W , Steele C R , Pilkington N S , Charlton R K
Published: January 2020

Marginated and bone marrow reserve granulocytes were obtained from young healthy volunteers after subcutaneous administration of aqueous epinephrine (0.4 ml/m2) or intravenous administration of hydrocortisone sodium succinate (250 mg), respectively. These leukocytes were compared with circulating granulocytes for the ability to adhere to surfaces, migrate in a random fashion, respond to chemoattractants, interact with autologous serum opsonins, and phagocytize and kill five common bacterial pathogens. As contrasted with circulating neutrophils, marginated cells had enhanced phagocytic and killing capacity for some pathogens, whereas adherence, random migration, chemotaxis, and chemiluminescence for the two cell populations were equivalent. Bone marrow reserve cells demonstrated increased activity for three functional mechanisms; chemotaxis for these cells averaged 21% higher than that for circulating cells, and phagocytosis was 32% higher, with 6 to 17% greater killing of the five bacterial species studied. All of these differences were statistically significant (P less than 0.05). Random migration and interaction with serum opsonins were unchanged in bone marrow granulocytes. These enhanced functional properties of neutrophils which are outside of the circulating pool may represent important host defense mechanisms during episodes of bacterial infection.
Cloning and characterization of Streptococcus mutans LM7 plasmid pAM7
Authors: Noji S , Date S , Abiko Y , Takiguchi H , Taniguchi S
Published: January 2020

The 5.6-kilobase-pair cryptic plasmid, pAM7, of Streptococcus mutans LM7 was cloned into Escherichia coli plasmids or a shuttle plasmid to examine whether the plasmid encodes bacteriocin. Plasmid pAM7 encoded proteins with molecular weights of 30,000, 22,000, and 12,000, but none of them appeared to be bacteriocin.
Restoration of normal febrile response to endotoxin in pyrogen-tolerant rabbits by injection with human beta interferon
Authors: Kawasaki H , Moriyama M , Ohtani Y , Naitoh M , Nariuchi H , Tanaka A
Published: January 2020

Early-phase pyrogen tolerance was induced in rabbits by two consecutive daily injections of 125 ng of endotoxin per kg of body weight. The second injection of the same dose of endotoxin evoked only a monophasic fever with a peak response 1.5 h after the injection; no second peak was observed. The rabbits were released from the tolerance to develop a typical biphasic fever by an injection of 125 ng of endotoxin along with human beta interferon (HuIFN-beta), although the tolerance-inducing amount of endotoxin alone could not. The profile of the febrile response of tolerant rabbits injected with both endotoxin and HuIFN-beta could not be distinguished from that of normal rabbits. There was no essential difference between natural and recombinant HuIFN-beta in breaking tolerance. Heat-stable (70 degrees C, 30 min) endogenous pyrogen or tumor necrosis factor was increased significantly in concentration in the serum of tolerance-broken rabbits. These results suggest that HuIFN-beta stimulates the production of tumor necrosis factor in tolerant rabbits to elicit the second peak of febrile response.
Effect of vaccination on feline Chlamydia psittaci infection
Authors: Wills J M , Gruffydd-Jones T J , Richmond S J , Gaskell R M , Bourne F J
Published: January 2020

Experimental ocular infection of specific-pathogen-free cats with the feline pneumonitis strain of Chlamydia psittaci produced an acute, severe conjunctivitis characterized by blepharospasm, conjunctival hyperemia, chemosis, and ocular discharge. Organisms were recovered from the conjunctiva for several weeks, and persistent genital and gastrointestinal infection also resulted from the ocular infection in some cats. Subcutaneous vaccination with live feline pneumonitis C. psittaci 4 weeks before ocular challenge significantly reduced the severity of the conjunctivitis. However, there was no effect on shedding of organisms from the eye or on the transmission of infection to the gastrointestinal and genital tracts. It is suggested that the acute stage of this ocular disease is caused largely by release of pathogenic antigen(s) from chlamydia-infected conjunctival cells, rather than by a direct cytopathic effect of chlamydial replication. Thus, vaccination with whole live organisms reduced the acute disease in experimentally infected cats but did not prevent shedding of the organism. The implications of these findings are discussed.
Enhanced epitopic response to a synthetic human malarial peptide by preimmunization with tetanus toxoid carrier
Authors: Lise L D , Mazier D , Jolivet M , Audibert F , Chedid L , Schlesinger D
Published: January 2020

Successful human vaccination by synthetic malarial sporozoite peptides may depend on the choice of an appropriate carrier. Tetanus toxoid (TT) has been proposed because of its safe and widespread use in humans. Paradoxically, however, prior exposure to this toxoid vaccine could produce specific epitopic suppression against synthetic malarial peptides conjugated to this same protein as carrier. Indeed, we have previously reported that such a phenomenon can occur in the case of a synthetic vaccine made with a streptococcal peptide conjugated to TT. Our present study shows that similar results can be observed in mice preimmunized with TT 1 month before the administration of a conjugate containing TT and a Plasmodium knowlesi peptide. Analysis of the isotypic pattern of the antipeptide response showed that the immunoglobulin G1 (IgG1) subclass and especially the IgG2a and IgG2b subclasses were suppressed. In contrast, when a sporozoite peptide from Plasmodium falciparum was coupled to TT, the total antipeptide antibodies and particularly the IgG1 subclass were enhanced by preimmunization by TT. This increase of antipeptide antibodies was correlated with a greater ability of the sera to neutralize sporozoite infectivity. These results indicate that prior exposure to TT does not systematically impair the antibody response against a peptide administered as a peptide-TT conjugate.
Attachment of oral bacteria to a basement-membrane-like matrix and to purified matrix proteins
Authors: Winkler J R , John S R , Kramer R H , Hoover C I , Murray P A
Published: January 2020

The purpose of this study was to investigate the adherence of oral bacteria to an in vitro basement-membrane-like matrix and to selected individual macromolecular constituents of this matrix. Radiolabeled bacteria were incubated with basement-membrane-like matrices isolated from PF HR-9 cells. Bacteroides gingivalis 33277, Fusobacterium nucleatum FN-2, and Actinobacillus actinomycetemcomitans GA3(A) bound to the matrix in the range of 44 to 70%, considerably higher than the ranges of A. actinomycetemcomitans GA3(NA) and SUNY AB67 (range, 20 to 25%). The attachment of selected strains of gram-positive bacteria such as Streptococcus and Actinomyces spp. was much less frequent (range, 6 to 25%). Competitive inhibition studies demonstrated that preincubating the bacteria with fibronectin significantly decreased the binding of B. gingivalis by 51% but increased the binding of other gram-negative and gram-positive organisms tested. Similarly, preincubating the matrices with antifibronectin antibodies decreased the binding of B. gingivalis by 31%, whereas the other bacteria tested were either unaffected or binding was increased. The adherence of bacteria to purified basement membrane proteins was also investigated. Strain and species differences were seen in binding, but no clear relationship emerged between binding to an intact matrix and binding to isolated matrix proteins. The results of this study suggest that some gram-negative oral bacteria commonly associated with periodontal disease, such as B. gingivalis, A. actinomycetemcomitans, and F. nucleatum, bound in high numbers to basement-membrane-like matrices in vitro. On the other hand, the gram-positive strains tested bound in much fewer numbers. The results suggest that further studies with this in vitro model may aid in understanding the mechanisms by which oral bacteria adhere to basement membranes.
Cytoadherence of Plasmodium falciparum-infected erythrocytes to human melanoma cell lines correlates with surface OKM5 antigen
Authors: Panton L J , Leech J H , Miller L H , Howard R J
Published: January 2020

OKM5 antigen and thrombospondin are currently under investigation as potential receptors on the surface of human monocytes, endothelial cells, and melanomas responsible for the cytoadherence of Plasmodium falciparum-infected erythrocytes. We have studies the binding capacity of six human melanoma cell lines and related this property to the cytoplasmic and surface expression of the OKM5 antigen and thrombospondin by using indirect immunofluorescence assays on methanol-fixed and nonfixed melanomas. The presence of OKM5 antigen was detectable only in the melanoma lines which bound P. falciparum-infected erythrocytes. Thrombospondin was present in the cytoplasm of all the melanoma lines but was not detectable on the surface of any cells. Our work demonstrates a direct correlation between surface OKM5 antigen and cytoadherence in vitro. While our results do not exclude thrombospondin as a mediator of cytoadherence to endothelial cells in vivo, they showed no correlation between the presence of thrombospondin and the ability of melanoma cell lines to cytoadhere in vitro.
Establishment of gastric Campylobacter pylori infection in the neonatal gnotobiotic piglet
Authors: Krakowka S , Morgan D R , Kraft W G , Leunk R D
Published: January 2020

Campylobacter pylori, a gram-negative microaerophilic bacterium, has been implicated in the genesis of human gastritis, dyspepsia, and gastroduodenal ulceration. Previous attempts to reproduce the diseases in conventional laboratory animal species have been unsuccessful. To determine if neonatal gnotobiotic piglets were susceptible to C. pylori, we orally challenged two litters (n = 17) with 10(9) CFU after pretreating them with cimetidine. Controls housed in separate units received nothing or peptone water alone. Piglets were examined 1, 2, 3, and 4 weeks after challenge. Colonization by the bacterium and inflammation of the gastric mucosa persisted throughout the study period. Organisms were revealed by Warthin-Starry silver stain to reside between the mucus layer and the gastric epithelium. Culturing of samples from sites along the gastrointestinal tract revealed that the bacterium colonized essentially only the gastric and proximal duodenal mucosae. Gross pathological changes were restricted to the stomachs of infected piglets and consisted of submucosal edema, increased gastric mucus production, and progressive development of mucosal lymphoid follicles. Microscopic lesions consisted of transient neutrophilic infiltrates followed by diffuse and follicular infiltrations of mononuclear leukocytes into the mucosa and submucosa. Alcian blue-periodic acid-Schiff stains suggested that the infection resulted in the depletion of mucopolysaccharide production by deep gastric glands. These data indicate that gnotobiotic piglets reproduce many of the features of diseases associated with C. pylori in humans.
Fungicidal activation of murine macrophages by recombinant gamma interferon
Authors: Beaman L
Published: January 2020

Alveolar macrophages from BALB/c mice readily phagocytized endospores (2 to 5 micron) and arthroconidia of Coccidioides immitis in vitro. Within 24 to 30 h at 37 degrees C, the phagocytized endospores started developing into spherules, and the arthroconidia formed germ tubes and hyphae. However, these processes did not occur if the macrophages were incubated with murine recombinant gamma interferon (rIFN-gamma) during infection with C. immitis. Treatment with rIFN-gamma activated the fungicidal capabilities of the alveolar macrophages, as evidenced by the 50% reduction in the CFU which could be recovered from macrophages infected in the presence of gamma interferon compared with alveolar macrophages infected without gamma interferon (P less than 0.05). Similar results were seen with peritoneal macrophages incubated with rIFN-gamma and infected with C. immitis. As little as 10 U of rIFN-gamma per ml reduced by half the number of C. immitis CFU which could be recovered from the phagocytes 8 h after infection with arthroconidia, although interferon alone did not affect the viability of the fungi.
Bacterial-lipopolysaccharide-induced release of lactoferrin from human polymorphonuclear leukocytes: role of monocyte-derived tumor necrosis factor alpha
Authors: Koivuranta-Vaara P , Banda D , Goldstein I M
Published: January 2020

We have examined the role played by human peripheral blood monocytes in mediating responses of human polymorphonuclear leukocytes (PMN) to bacterial lipopolysaccharide (LPS) in vitro. When incubated with Salmonella typhimurium LPS at 37 degrees C, human PMN suspended in serum-free buffer released the specific granule constituent lactoferrin into the surrounding medium. Release of lactoferrin from PMN varied with the concentration of LPS (1 to 1,000 ng/ml) as well as with the duration of incubation (2 to 60 min) and was not accompanied by significant release of the cytoplasmic enzyme lactate dehydrogenase. LPS-induced release of lactoferrin from PMN was augmented significantly when cell suspensions were supplemented with additional monocytes and lymphocytes. Only monocytes, however, secreted significant amounts of lactoferrin-releasing activity (in a time- and concentration-dependent manner) when incubated separately with LPS. Lactoferrin-releasing activity was heat (80 degrees C for 15 min) labile, eluted after chromatography on Sephadex G-100 with an apparent molecular weight of approximately 60,000, and was inhibited by antibodies to tumor necrosis factor alpha. Thus, LPS-induced noncytotoxic release of lactoferrin from human PMN suspended in serum-free buffer is mediated, at least in part, by tumor necrosis factor alpha derived from contaminating monocytes.
Adherence of Salmonella typhimurium to small-intestinal enterocytes of the rat
Authors: Lindquist B L , Lebenthal E , Lee P C , Stinson M W , Merrick J M
Published: January 2020

The adherence of radiolabeled Salmonella typhimurium to freshly isolated enterocytes of rats was studied. The results established that type 1 fimbriated strains adhered in significantly higher numbers than did related nonfimbriated strains. Adherence was inhibited by D-mannose and methyl alpha-D-mannoside. Results of kinetic studies indicated that adherence was biphasic; the number of bacteria that adhered per enterocyte remained constant for approximately 20 min and then increased rapidly under the assay conditions. The second phase was associated with structural damage to the enterocytes. The addition of chloramphenicol did not prevent the initial attachment of bacteria to enterocytes but did prevent the second phase. Viable and nonviable bacterial cells adhered to enterocytes, but only viable bacteria were destructive. Freshly isolated enterocytes (trypan blue impermeable) and enterocytes stored overnight (trypan blue permeable) were infected by viable S. typhimurium in a similar manner, suggesting that metabolic activity of the host cell was of less consequence than metabolic activity of the bacterial cells. A model for the role of mannose-sensitive fimbriae as a virulence factor is proposed.
Induction of murine gamma interferon production by lipopolysaccharide and interleukin-2 in Propionibacterium acnes-induced peritoneal exudate cells
Authors: Okamura H , Wada M , Nagata K , Tamura T , Shoji K
Published: January 2020

Lipopolysaccharide (LPS) induces high levels of gamma interferon (IFN-gamma) in the circulation of mice pretreated with heat-killed Propionibacterium acnes. The following results were obtained in the present study. LPS, as well as interleukin-2 (IL-2), was also able to induce IFN-gamma in vitro in peritoneal exudate cells (PEC) from such mice. Splenocytes and lymph node cells from these mice or resident peritoneal cells from control mice produced trace or undetectable amount of IFN-gamma upon exposure to LPS. A synergistic effect on IFN-gamma induction was observed when LPS was added to a culture of PEC together with IL-2. Indomethacin augmented the induction of IFN-gamma by LPS or IL-2, and prostaglandin E2 reversed its effect. Deprivation of plastic-adherent or nylon wool-adherent cells abolished the induction by LPS or IL-2, whereas it did not affect that by concanavalin A. Culture supernatant of plastic-adherent cells incubated with LPS stimulated the nylon wool-nonadherent cells to produce IFN-gamma in the presence of IL-2, but interleukin-1 or phorbol myristic acetate did not replace the LPS-stimulated supernatant. The ability of PEC to produce IFN-gamma measured as a function of time after P. acnes injection increased in proportion to their natural killer (NK)-like activity against YAC-1 cells. Moreover, treatment of PEC with monoclonal anti-Thy-1 antibody or with anti-asialo GM1 antiserum plus complement eliminated the production of IFN-gamma and the NK-like activity simultaneously, whereas treatment with monoclonal anti-Lyt-2 antibody plus complement did not. These results suggest that IL-2 and some unidentified factor released from plastic-adherent cells by LPS stimulation cooperatively induce IFN-gamma production in activated, Thy-1- and asialo GM1-positive NK-like cells appearing in inflammatory reactions and that prostaglandin E2 regulates IFN-gamma production in these cells.
Inhibition of malaria parasite invasion of human erythrocytes by a lymphocyte membrane polypeptide
Authors: Benzaquen-Geffin R , Milner Y , Ginsburg H
Published: January 2020

Extraction by boiling of the buffy coat of human blood yields a protein solution which inhibits the propagation of the human malaria parasite Plasmodium falciparum in culture with a 50% inhibitory dose of 105 micrograms of protein per ml. The inhibitory activity is associated exclusively with the lymphocytes and affects solely the invasion of erythrocytes by free merozoites. Boiled extracts of isolated lymphocytes had a 50% inhibitory dose of 22 micrograms/ml. Fractionation of surface-labeled or pronase-treated lymphocytes revealed that the antimalarial lymphocyte factor is associated with the intracellular aspect of the membrane fraction and is probably not involved in the host defense system against malaria. Further purification by salt extraction, ion-exchange chromatography, molecular gel filtration, and electroelution from lithium dodecyl sulfate-polyacrylamide gels resulted in 300- to 550-fold purification, i.e., a 50% inhibitory dose of 40 to 70 ng/ml. All inhibitory fractions contained a 48-kilodalton polypeptide which eluted from a gel filtration column as a 400-kilodalton species, implying multimeric association. Some 6,000 molecules of the 48-kilodalton polypeptide bind with high affinity to one merozoite, the free form of the parasite. The Kd of 0.1 to 0.5 nM for the binding of the 48-kilodalton polypeptide correlated well with the 50% inhibitory dose of 0.3 to 0.4 nM obtained with purified active antimalarial lymphocyte factor. We therefore suggest that the 48-kilodalton polypeptide partially purified from lymphocyte membranes is the antimalarial lymphocyte factor and that it exerts its inhibitory activity by binding to merozoites, thereby preventing their invasion into erythrocytes. The antimalarial lymphocyte factor or a polypeptide sequence thereof could serve for further probing of invasion at the molecular level.
Protection of Babesia bigemina-immune animals against subsequent challenge with virulent Babesia bovis
Authors: Wright I G , Goodger B V , Leatch G , Aylward J H , Rode-Bramanis K , Waltisbuhl D J
Published: January 2020

Two groups of cattle, one previously exposed to Babesia bigemina and one not, were challenged with Babesia bovis. The group previously infected with Babesia bigemina was only mildly affected upon challenge with B. bovis, whereas four of five of the other group were severely affected. Immunoblotting studies performed in both homologous and heterologous systems showed that there were polypeptides of similar molecular weight in both species, but species-specific polypeptides were demonstrated only in B. bovis by the homologous B. bovis reaction. B. bovis antisera reacted avidly with B. bigemina-infected erythrocytes in fluorescent-antibody assays. In contrast, B. bigemina antisera did not cross-react with B. bovis-infected erythrocytes. Two groups of splenectomized calves were immunized with an enriched antigen fraction of B. bigemina. A third group was immunized by infection with B. bigemina and treatment with a drug. One of the groups of calves immunized with the antigenic fraction of B. bigemina, the group immunized by infection with B. bigemina, and a control group were challenged with B. bovis. All control calves died, whereas 50% of the calves immunized by infection with B. bigemina and 75% of the animals immunized with the B. bigemina antigen survived. The second group immunized with the B. bigemina antigen and a control group were challenged with B. bigemina. All control animals died by day 6, whereas 50% of the vaccinates survived, the deaths occurring on days 8 and 11. The nature of the probable protective mechanism is discussed.
"Haemophilus somnus," a facultative intracellular pathogen of bovine mononuclear phagocytes
Authors: Lederer J A , Brown J F , Czuprynski C J
Published: January 2020

We have reported previously that bovine neutrophils are unable to kill the bovine respiratory pathogen "Haemophilus somnus." In the present study we expanded our efforts and examined the interaction of bovine mononuclear phagocytes with this important veterinary pathogen. Bovine alveolar macrophages and blood monocytes ingested but did not kill opsonized "Haemophilus somnus" in vitro, whereas these same cells ingested and killed opsonized Escherichia coli. Because this suggested that "H. somnus" was a facultative intracellular pathogen, we developed an assay to monitor the intracellular fate of ingested "H. somnus" within bovine monocytes. Our results indicated that ingested "H. somnus" multiplied within bovine monocytes (1- to 2-log10 increase in 4 h); equivalent intracellular growth was noted for both a laboratory strain and a recent field isolate of "H. somnus." Bovine monocytes killed ingested E. coli (1- to 2-log10 decrease in 4 h) under the same assay conditions that were used to follow intracellular growth of "H. somnus," thus indicating that the assay conditions did not induce a generalized defect in monocyte antibacterial activity. Light and electron microscopic examination of "H. somnus"-infected monocytes confirmed that intracellular growth had occurred. We did not observe an obvious correlation between the release of superoxide anion from bovine mononuclear phagocytes that had ingested opsonized "H. somnus" and E. coli and the subsequent intracellular survival of the bacteria. The results of this study suggest that infected mononuclear phagocytes sustain "H. somnus" infections in cattle and thus contribute to the subacute to chronic clinical course that has been reported.
Analgesic effects of N-acetylmuramyl-L-alanyl-D-isoglutamine in decreasing the acetic acid-induced abdominal-writhing response
Authors: Ogawa T , Kotani S
Published: January 2020

A new pharmacological action of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) is described. Pretreatment of male ddY mice with MDP, but not its biologically inactive analogs, significantly decreased the frequency of acetic acid-induced writhing movements more effectively than did acetylsalicylic acid (aspirin). The analgesic effect of MDP, however, was less than that of a narcotic antagonist, pentazocine.
Intracellular fate of Mycobacterium leprae in normal and activated mouse macrophages
Authors: Sibley L D , Franzblau S G , Krahenbuhl J L
Published: January 2020

Mycobacterium leprae replicates within mononuclear phagocytes, reaching enormous numbers in the macrophage-rich granulomas of lepromatous leprosy. To examine the capability of macrophages to digest M. leprae, we studied the intracellular fate of M. leprae organisms in normal and activated mouse macrophages by using the electron-dense secondary lysosome tracer Thoria Sol. Intracellular M. leprae organisms, surrounded by a characteristic electron-transparent zone, were contained within phagosomal vacuoles of macrophages cultured in vitro for 1 to 6 days. In normal macrophages, a majority of phagosomes containing freshly isolated live M. leprae cells resisted fusion with Thoria Sol-labeled lysosomes. The extent of fusion was not significantly affected by pretreatment of M. leprae with human patient serum high in specific immunoglobulin G and M antibodies. In contrast, a majority of phagosomes containing gamma-irradiated M. leprae cells underwent lysosome fusion in normal macrophages. In addition, increased phagolysosome fusion was observed with live M. leprae-containing phagosomes in macrophages activated with gamma interferon. Increased fusion was associated with an increase in the number of fragmented and damaged bacilli, suggesting that increased digestion followed fusion. This study indicates that activated macrophages may have an increased capacity for clearance of normally resistant M. leprae.
Production of monoclonal antibodies that recognize specific and cross-reactive antigens of Fusobacterium nucleatum
Authors: Bird P S , Seymour G J
Published: January 2020

Monoclonal antibodies (MAbs) against the cell surface antigens of Fusobacterium nucleatum 263 were obtained by fusion of murine myeloma cells (P3-NSI/1-Ag4-1) with the splenocytes of BALB/c mice immunized with whole cells of F. nucleatum 263. Screening was performed using an enzyme-linked immunosorbent assay (ELISA) against the immunizing strain, F. nucleatum 263. Further selection was done using a bacterial panel consisting of Bacteroides, Actinomyces, Streptococcus, Fusobacterium, and Escherichia species. Twelve MAbs were selected on the basis of this screening procedure, seven of which reacted specifically with F. nucleatum 263. Two reacted with F. nucleatum 263 and ATCC 25586, and three reacted with F. nucleatum 263, ATCC 25586, and UQD-003 (a clinical isolate) and also cross-reacted with Fusobacterium russii ATCC 25533. The selected MAbs were then further characterized by absorption experiments with suspensions of intact whole bacterial cells, and the residual binding activity of the supernatants was determined in an ELISA. To determine whether the MAbs reacted with the same or different epitopes, pairs of MAbs were reacted together and independently in a checkerboard manner in an ELISA. The additive or nonadditive nature of the reactivity was determined. A competitive inhibition assay was performed using one labeled and selected unlabeled MAbs. The results of these experiments suggested some epitope sharing among the selected MAbs that reacted with a specific antigen on F. nucleatum and also shared cross-reactive antigens with the three strains of F. nucleatum and F. russii.
Distribution of specific DNA sequences among pathogenic and commensal Neisseria species
Authors: Aho E L , Murphy G L , Cannon J G
Published: January 2020

Several traits, including pili and the outer membrane proteins P.II and H.8, have been associated with pathogenic Neisseria species. We examined several Neisseria species for DNA sequence homology to cloned pilin, P.II, and H.8 genes. Strains of Neisseria gonorrhoeae and N. meningitidis showed hybridization to all of these genes. Commensal strains showed little hybridization to any of these genes. Strains of N. lactamica and N. cinerea showed intermediate patterns of hybridization. Generally, organisms that expressed a given trait showed DNA homology to the corresponding cloned gene. However, we observed pili on some commensal strains that did not show hybridization to the cloned gonococcal pilin gene.
Comparative translocation of enteropathogenic Campylobacter spp. and Escherichia coli from the intestinal tract of gnotobiotic mice
Authors: Youssef M , Corthier G , Goossens H , Tancrede C , Henry-Amar M , Andremont A
Published: January 2020

In C3H gnotobiotic mice, no significant difference was observed between the translocation of nine clinical isolates of enteropathogenic Campylobacter spp. and that of seven nonisogeneic Escherichia coli strains with or without various determinants of pathogenicity. In addition, there was no correlation found between the intensity of translocation of Campylobacter strains and the signs of invasiveness in the patients from whom the strains had been isolated.
The Bacteroides by-product succinic acid inhibits neutrophil respiratory burst by reducing intracellular pH
Authors: Rotstein O D , Nasmith P E , Grinstein S
Published: January 2020

Short-chain fatty acids, particularly succinic acid, are major metabolic by-products of Bacteroides species. To determine their role as potential virulence factors in infections containing Bacteroides species, short-chain fatty acids were examined for their effect on the neutrophil respiratory burst. Succinate (30 to 50 mM) irreversibly impaired superoxide and hydrogen peroxide production in response to opsonized zymosan and phorbol myristate acetate when neutrophils were treated at pH 5.5 but not pH 6.0 or greater. Several other short-chain fatty acids tested produced similar inhibition. Reversible inhibition of oxygen consumption was found when neutrophils were incubated in succinate-containing medium (pH 6.0) as well as control medium (pH 6.0 and 5.5). Neutrophil cytoplasmic pH was measured by fluorimetric techniques to determine whether the inhibition was mediated via a reduction in intracellular pH. The intracellular pH of cells in control medium (pH 6.5 or less) was significantly reduced compared with pH 7.4. The addition of succinate (30 mM) to these media caused a further significant reduction in cytoplasmic pH at each pH level. The reduction in intracellular pH was sufficient to account for both the irreversible and reversible impairment of the neutrophil respiratory burst. Thus, short-chain fatty acids appear to exert their inhibition, at least in part, by reducing intracellular pH. These data also demonstrate the potential for interactions between Bacteroides species and their microenvironment to increase the virulence of an infection.
Membrane glycoproteins of human polymorphonuclear leukocytes that act as receptors for mannose-specific Escherichia coli
Authors: Rodriguez-Ortega M , Ofek I , Sharon N
Published: January 2020

Type 1 fimbriated (mannose-specific) Escherichia coli cells bind to mannose residues on human polymorphonuclear leukocytes (PMN); this leads to phagocytosis of the bacteria. To identify the mannose-containing receptors on the PMN, the cells were surface labeled with 125I and lysed in 0.5% Nonidet P-40, and the lysate was fractionated by affinity chromatography on a column of Sepharose-bound fimbriae. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the material eluted from the column with 500 mM methyl-alpha-mannoside revealed two radioactive bands of Mr 70,000 to 80,000 (gp70-80) and 100,000 (gp100). Another weak band of Mr 150,000 (gp150) was observed after prolonged exposure of the gel. Upon blotting of the glycoproteins separated by polyacrylamide gel electrophoresis and overlaying of the blots with concanavalin A, gp150 appeared as the major band. Membrane preparations of the PMN were enriched in gp70-80, gp100, and gp150, in comparison with the cell homogenates, further suggesting that these glycoproteins are surface components. Fractionation of the membrane preparations on the immobilized fimbriae followed by concanavalin A overlay of blots of the methyl-alpha-mannoside-eluted material revealed that gp150 was the major component in this fraction. The eluted fraction, obtained from a cell lysate (4.4 micrograms/ml), inhibited by 70% the agglutination of yeasts by the intact bacteria. Our results suggest that the three surface glycoproteins isolated by us serve as receptors for mannose-specific E. coli on PMN and may be involved in the lectin-mediated phagocytosis of the bacteria.
In vitro and in vivo pathogenicity of Plesiomonas shigelloides
Authors: Herrington D A , Tzipori S , Robins-Browne R M , Tall B D , Levine M M
Published: January 2020

Epidemiologic evidence suggests that Plesiomonas shigelloides is an enteric pathogen. We conducted in vitro, animal, and volunteer studies on P. shigelloides isolates from patients with diarrhea. Five strains gave a negative keratoconjunctivitis reaction in guinea pigs and did not invade HeLa cells. Genetic probes for heat-stable enterotoxins related to those of enterotoxigenic Escherichia coli and for gene sequences common to the invasiveness plasmids of Shigella spp. and enteroinvasive E. coli were negative. Heat-labile enterotoxins were not found when a modified GM1-enzyme-linked immunosorbent assay was used. Rabbits did not develop diarrhea but were transiently colonized when inoculated with up to 10(11) P. shigelloides CFU using the reversible intestinal tie adult rabbit diarrhea model. A very large plasmid (between 118 and 312 megadaltons) was found in all isolates. Strain P012 was cured of its plasmid by novobiocin. This strain, but not its cured derivative, invaded the mucosa of the distal ileum of gnotobiotic piglets given 10(10) CFU. At a lower inoculum (10(9) CFU), strain P012 induced inflammation of the colonic mucosa and diarrhea at day 6. The same isolate was fed to 33 healthy volunteers in doses of 1 X 10(3) to 4 X 10(9) CFU. Thirty-six percent of the volunteers shed the organism, but none became ill. These data are only weakly supportive of a role for P. shigelloides in diarrheal illness and suggest the need for more studies with other strains to better understand its pathogenicity.
Role of myeloperoxidase in the killing of Naegleria fowleri by lymphokine-altered human neutrophils
Authors: Ferrante A , Hill N L , Abell T J , Pruul H
Published: January 2020

Previously we have shown that human neutrophils treated with conditioned medium from phytohemagglutinin-stimulated mononuclear leukocytes (sCM) in the presence of antisera have amoebicidal properties for Naegleria fowleri, a pathogenic free-living amoeba. The data now presented show that neutrophils which lack myeloperoxidase (MPO) but have a normal oxygen-dependent respiratory burst could not be altered by sCM to express the amoebicidal activity. Catalase inhibited this amoebicidal activity of sCM-treated neutrophils. Various components and products of the neutrophils were examined for effects on naegleriae. A granule extract was found to have no effect at concentrations up to 100-fold that which killed Salmonella minnesota R595. Hydrogen peroxide appeared to have little effect even at 100 microM. However, in the presence of MPO, H2O2 was amoebicidal at 2.5 microM. The generation of amoebicidal activity required the presence of chloride ions. Azide inhibited the effects of the MPO-H2O2-Cl- system. Arginine, a scavenger of hypochlorite, significantly depressed the ability of sCM-treated neutrophils to kill amoebae and also prevented the amoebicidal properties of the MPO-H2O2-halide system. These results suggest that the MPO-H2O2-halide system is important in the killing of naegleriae by sCM-treated neutrophils and that hypochlorite may be the amoebicidal agent.
Molecular cloning and expression of Rickettsia tsutsugamushi genes for two major protein antigens in Escherichia coli
Authors: Oaks E V , Stover C K , Rice R M
Published: January 2020

Several polypeptide antigens of Rickettsia tsutsugamushi are recognized by human or primate convalescent sera and may be important protective immunogens. Molecular cloning and expression of the genes encoding the 110K (110 kilodalton) and 56K polypeptide antigens of R. tsutsugamushi Karp were accomplished in the lambda gt11 expression vector system. Southern blot analysis with the cloned fragments for the 56K polypeptide antigen (0.7 kilobases) and the 110K polypeptide antigen (5.4 kilobases) confirmed that the insert DNA was rickettsial and not host cell in origin. Expression of a complete 110K polypeptide was shown to be independent of isopropyl-beta-D-thiogalactopyranoside induction, suggesting that an intact rickettsial promoter was operational. Epitopes of the 56K polypeptide were expressed as lac promoter-dependent beta-galactosidase fusion proteins. Polyclonal antibody, affinity purified against the recombinant 110K and 56K polypeptides, reacted with polypeptides of similar size in the Kato and Gilliam strains of R. tsutsugamushi. Group-reactive, but not strain-specific, monoclonal antibodies against the 56K polypeptide reacted with the cloned portion of the 56K polypeptide. Western blot analysis demonstrated that the cloned 56K Karp antigen gene product is recognized by human convalescent serum.
Molecular cloning, characterization, and complete nucleotide sequence of the gene for pneumolysin, the sulfhydryl-activated toxin of Streptococcus pneumoniae
Authors: Walker J A , Allen R L , Falmagne P , Johnson M K , Boulnois G J
Published: January 2020

A recombinant lambda bacteriophage has been isolated that carries DNA from Streptococcus pneumoniae and expresses a potent hemolysin that has been shown to be pneumolysin, the sulfhydryl-activated toxin of the pneumococcus. Hemolytic activity is inhibited by cholesterol and neutralized by serum against streptolysin O. The cloned gene expresses two polypeptides (Mrs, 56,000 and 53,000) in an Escherichia coli in vitro transcription-translation system, and both are precipitated by the addition of anti-alveolysin serum and anti-streptolysin O serum in the presence of Staphylococcus aureus cells. Expression of pneumolysin occurs when the gene is cloned in both possible orientations in pUC8. The DNA sequence of a 5-kilobase ClaI fragment that carries the pneumolysin gene has been determined. An open reading frame was identified that encodes a polypeptide of 471 amino acids that is hydrophobic in character and has an N-terminal amino acid sequence which is identical to that deduced from amino acid sequencing of the purified protein. The predicted amino acid sequence of the polypeptide reveals a single cysteine residue located 44 residues from the C terminus. Putative promoter and ribosome binding sites have been identified 5' to the pneumolysin coding sequence.
Pilus-mediated binding of bovine enterotoxigenic Escherichia coli to calf small intestinal mucins
Authors: Mouricout M A , Julien R A
Published: January 2020

In this study we show that the adhesion to mucus of the enterotoxigenic Escherichia coli strains responsible for diarrhea in calves involves a bacterium-mucin recognition phenomenon in which the bacterial pili and specific mucus receptors carried by the glycoproteins (2,000 to 400 kilodalton) play a major role. An adhesion maximum was observed at a pH of less than 6 (4.75 to 5.25). The sialic acids and galactose appeared to be at least partly responsible for the attachment of K99 pili, whereas F41 pili preferentially recognized desialylated receptors. The attachment of different strains of E. coli characterized by the presence of the three main pili, K99, F41, and FY, known to be responsible for the binding of enterotoxigenic E. coli to the intestinal epithelium of the calf, was studied using Scatchard and Hill analyses. The attachment mechanism of bacteria carrying K99 pili showed positive cooperativity. FY and F41 pili recognized independent receptor sites, the first on sialylated mucus and the second on sialidase-treated mucus. Moreover, F41 pili were found to bind the native mucus according to a negative cooperativity phenomenon. Finally, the recognition sites carried by bacterial pilins may be saturated by some animal glycoprotein glycans which are therefore adhesion inhibitors.