bluuebunny/crossref_metadata_2025 huggingface dataset:
An in vitro system to study listericidal capacity of macrophages from separate mice: resident macrophages exhibit different activation patterns
Authors: Ottendorfer D , Bitter-Suermann D , Hadding U
Published: January 2020

An in vitro system with macrophages from individual mice was established to study their listericidal capacity. Because no antibiotics were used, bacterial killing was really due to macrophages in short-term culture. To restrict the extracellular growth of bacteria, cell culture medium was changed at 1-h intervals. We demonstrated that intracellular growth of listeria in macrophage pools from untreated animals varies considerably. Obviously, preactivated macrophages are constantly present, so that the common procedure of using macrophage pools from several animals is no longer acceptable. In addition, we demonstrated that in vitro mixtures of listeria-immune macrophages of one animal with cells from untreated animals at different ratios exhibit enhanced bacterial killing above a mere additive effect. Consequently, by using macrophages from individual untreated mice, we found that cells of different animals exhibited various activation stages, although unstimulated, inbred specific-pathogen-free mice of the same age, weight, and sex were used. When equal numbers of macrophages from untreated separate animals were mixed in vitro, intracellular growth of listeria was only moderate; that is, the number of preactivated macrophages of the individual animals determined listerial growth in the pooled preparation. Furthermore, we showed that identical doses of phorbol myristate acetate exerted different effects on the listericidal activities of macrophages as a function of their preactivation states. These experiments clearly demonstrate the advantage of using macrophages from individual mice for in vitro studies of macrophage activation.
Opsonic requirements for phagocytosis of Legionella micdadei by polymorphonuclear leukocytes
Authors: Steffensen D O , Weinbaum D L , Dowling J N
Published: January 2020

The roles of the classical and alternative pathways of complement activation and of antibody in the phagocytosis of Legionella micdadei by polymorphonuclear leukocytes were studied. Normal serum was treated with the appropriate chelators or with heat to inactivate the classical, alternate, or both pathways of complement activation. Normal and complement-depleted sera with or without antibody were employed as opsonins for L. micdadei in phagocytosis assays. There was no difference in the phagocytosis of L. micdadei promoted by normal serum and either C4-deficient serum or serum in which the classical pathway had been inactivated. Both normal and classical pathway-deficient sera promoted significantly greater phagocytosis than did sera in which the alternate pathway or both the alternate and classical pathways had been inactivated. Thus, polymorphonuclear leukocyte phagocytosis of L. micdadei in the absence of antibody required an intact alternate pathway. Specific antibody partially restored opsonization to sera deficient in the alternate or both complement pathways, but phagocytosis was still significantly less than that with the alternate pathway intact.
Virulence of Staphylococcus aureus in a mouse mastitis model: studies of alpha hemolysin, coagulase, and protein A as possible virulence determinants with protoplast fusion and gene cloning
Authors: Jonsson P , Lindberg M , Haraldsson I , Wadström T
Published: January 2020

Mutants of a genetically well-characterized strain of Staphylococcus aureus [SA113(83A)] were isolated after mutagenization. Alpha-hemolysin- (hla), coagulase- (coa), and protein A- (spa) negative mutants were characterized by more than 90 biochemical tests for production of extracellular proteins and biochemical profile to exclude pleiotropy. Protoplast fusion was then used to isolate double-defective (hla and coa) recombinants and recombinants with regained properties, i.e., production of alpha-hemolysin and coagulase. Studies of such mutants and recombinants in the mouse mastitis model showed that one alpha-hemolysin [SA113(83A) hla-5] and one coagulase-negative [SA113(83A) coa-147] mutant were lower in virulence compared with the wild-type strain SA113(83A). The double-negative mutant SA113(83A) hla-5 coa-147 showed a drastic decline in virulence and only induced very mild changes, as determined by microscopic examinations of infected mammary gland tissue. The recombinant with regained properties, however, was as virulent as the wild-type strain. This suggests that alpha-hemolysin and coagulase are virulence determinants of S. aureus. A high-level protein A-producing mutant (U300) showed the same virulence as the parent strain SA113(83A) in this model. One low virulence protein A-negative mutant (U320) did not markedly increase in virulence when a plasmid containing the cloned gene for protein A (pSPA15) was introduced into this mutant. By these and earlier observations, it seems likely that protein A is not an important virulence determinant in mastitis of mice. The reduced virulence of the protein A-negative mutant U320 compared with the wild-type SA113(83A) may be due to pleiotropic loss of some other unknown virulence determinant(s). Our data confirm earlier findings that pleiotropic changes are common in protein A-negative mutants.
Carbohydrate Metabolism and Survival of Endotoxin-Poisoned Mice Given Tryptophan
Authors: Moon Robert J.
Published: January 2020

Severe hypoglycemia and increased deaths were observed among two strains of endotoxin-poisoned mice within 3 to 6 hr after tryptophan injection. Sensitivity to tryptophan could be demonstrated in Rockland Farms mice by 4 hr after endotoxin and in Carworth Farms (CF-1) mice by 10 hr after endotoxin. If allopurinol was given to CF-1 mice concurrently with endotoxin, severe hypoglycemia and increased deaths were observed when tryptophan was given only 4 hr after the bacterial poison. Cyproheptadine, an antiserotonin drug, decreased the number of deaths as well as the depletion of blood glucose in both strains of endotoxin-poisoned mice given a delayed injection of tryptophan. In most instances, liver glycogen was depleted by 8 to 10 hr after endotoxin. Correlation of liver glycogen levels with sensitivity to tryptophan was not as consistent as the correlation between blood glucose levels and hyperreactivity to the amino acid. The data show that severe hypoglycemia is a significant factor which must be considered in resolving the basis for increased deaths among endotoxin-poisoned mice given tryptophan.
Biological Activity of a Partially Purified Inhibitor of Vaccinia Virus Hemagglutinin
Authors: Lau Stephen C. W. , Cassel W. A.
Published: January 2020

An inhibitor acting against vaccinia virus hemagglutinin, and producing a hemagglutinin-negative virus, was separated from tumorous ascitic plasma. The finding that the partially purified inhibitor was biologically active strengthens the evidence for a specific role of the inhibitor in altering the virus.
Location of Feline Leukemia-Sarcoma Group-Specific Antigen in Infected Human Tissue Culture Cells
Authors: Ubertini Tito R.
Published: January 2020

Feline leukemia-sarcoma group-specific antigens were located in human embryo cells infected with feline leukemia and feline sarcoma viruses. This was done by using the fluorescent-antibody and enzyme-labeled antibody techniques at both light and electron microscopic levels. The antigens were found to be exclusively intracytoplasmic, diffuse, and located in discrete punctate foci.
In Vitro Phagocytosis and Intracellular Fate of Variously Encapsulated Strains of
Cryptococcus neoformans
Authors: Mitchell Thomas G. , Friedman Lorraine
Published: January 2020

Five isolates of
Cryptococcus neoformans
type A with stable capsular thicknesses were used. Three of the isolates had capsules of medium size, one had a minimal capsule, and the other, a large capsule. Peritoneal exudate cells from Lewis rats were cultured on cover slips in Leighton tubes containing medium 199 and 20% fresh, isologous normal rat serum. Yeast cells were added to the Leighton tube cultures, and, 2 hr later, the extracellular yeasts were rinsed out. Cover slips were removed from some tubes for Wright staining and measurement of both phagocytosis and loss of macrophages. The remaining tubes were reincubated and sampled at 24 or 48 hr. To determine fate of yeast cells after ingestion, washed cover slips were inverted onto agar slide cultures, and specific macrophages were observed in situ for subsequent multiplication of their intracellular yeasts. More than half of the macrophages survived 24 to 48 hr of exposure to different strains of
C. neoformans
, with small, medium, or large capsules. Phagocytic activity was dependent upon a heat-labile factor in normal rat serum. The number of yeast ingested by macrophages was inversely proportional to the capsular size. Although most of the ingested yeasts were resistant to intracellular killing, the agar culture technique clearly demonstrated that many were unable to multiply, presumably dead. Three of the isolates were more susceptible than the other two, and the fate of these yeasts after engulfment was not correlated with their capsular size.
Association of
Escherichia coli
with the Small Intestinal Epithelium I. Comparison of Enteropathogenic and Nonenteropathogenic Porcine Strains in Pigs
Authors: Bertschinger Hans U. , Moon Harley W. , Whipp Shannon C.
Published: January 2020

Two enteropathogenic strains of
Escherichia coli
(EEC) differed from a nonenteropathogenic strain of
E. coli
(NEEC) in their association with porcine small intestinal epithelium. The EEC characteristically were found along villi from tip to base and contiguous to the brush border. They were not in crypts. In contrast, the NEEC characteristically remained in the central lumen near the tips of villi and was only occasionally contiguous to the brush border. No organisms were detected within epithelial cells. The difference in distribution between EEC and NEEC was apparent in ligated jejunal loops 45 min postexposure. The association between host and bacterial cells was most consistently demonstrated on frozen sections of intestine, as other histological techniques removed many bacteria. However, cellular details of the association were best demonstrated in chemically fixed tissues.
Plasmid-Associated Enterotoxin Production in a Strain of
Escherichia coli
Isolated from Humans
Authors: Skerman F. J. , Formal S. B. , Falkow Stanley
Published: January 2020

A strain of
Escherichia coli
isolated from an epidemic of “nonspecific” diarrhea has been shown to harbor a plasmid which specifies enterotoxin production.
Experimental Pneumococcal Infection in Embryonated Eggs Previously Infected with Influenza C Virus
Authors: Blouse Louis E. , Buddingh G. John
Published: January 2020

Cumulative mortality rates for embryonated eggs, compiled for a variety of pneumococcal strains, indicated a uniformly high degree of virulence. Based on median lethal dose values for combined infection with influenza C virus and single infection with pneumococcus, it is difficult to conclude that pneumococcal virulence is enhanced in combined infection. However, there was a significant increase in mortality during the first 24 hr following combined infection. A significantly greater in vitro growth rate for pneumococcus was shown in amniotic fluid from virus-infected embryonated eggs. Microscopy study of selected embryos revealed the absence of a discernible inflammatory reaction during the first 24 hr. From 48 through 96 hr after inoculation, inflammatory reactions were restricted to the upper and lower respiratory tract and consisted of a polymorphonuclear leukocyte infiltration in response to the localization of pneumococci in the paranasal sinuses, bronchi, alveolar ducts, and alveolar spaces. Extensive, severe inflammatory reactions were more frequently encountered in the combined infection. The various phases from early exudation to eventual resolution, which characterize pneumococcal pneumonia in the human host, were in clear evidence following both the single and combined infection of the chick embryo.
Sex Difference in Antibody Response of CFW Mice to
Candida albicans
Authors: Rifkind David , Frey Janet A.
Published: January 2020

Bilaterally gonadectomized and control unilaterally gonadectomized male and female CFW mice were given a course of
Candida albicans
vaccine, and the agglutinating antibody response was measured. The mean titers in the control females were higher than those in the control males. Bilateral gonadectomy did not affect the mean titer in the females but in the males resulted in a significant increase in the antibody. Similar groups of mice were inoculated intraperitoneally with viable
C. albicans
, and 10 weeks later antibody was measured and the kidneys were cultured. There was no apparent relationship between the degree of the agglutinin response and either gonadectomy or renal candidiasis at autopsy.
Ultrastructural Study of the Behavior of Macrophages Toward Parasitic Mycobacteria
Authors: Hart P. D'Arcy , Armstrong J. A. , Brown Carolyn A. , Draper P.
Published: January 2020

Mycobacterium lepraemurium
and
M. microti
(causal agent of vole tuberculosis) were isolated from tissues of experimentally infected mice and used to infect normal mouse peritoneal macrophage cultures. The cellular response to these bacteria up to 4 days after infection was studied quantitatively by electron microscopy. Prelabeling with ferritin was used to facilitate observation of fusion between secondary lysosomes in the cells and phagosomes containing the bacteria. All bacteria were intraphagosomal, and a high proportion of them was morphologically “intact.” Nearly all phagosomes containing morphologically damaged (presumed nonviable) bacteria also contained ferritin, having fused with secondary lysosomes. Fusion of lysosomes had also occurred with most phagosomes containing intact
M. lepraemurium
but was infrequent with phagosomes containing intact
M. microti.
This tendency of multiplying mycobacteria of the tubercle type to avoid contact with lysosomal contents has already been reported for
M. tuberculosis
strain H37Rv. The different intracellular circumstances of the parasites may reflect different means of intracellular survival.
Study of the One-Step Growth Curve of Equine Infectious Anemia Virus by Immunofluorescence
Authors: Ushimi Chuzo , Henson James B. , Gorham John R.
Published: January 2020

Primary horse leukocyte cultures were inoculated with 2 or 10 50% tissue culture infective doses (TCID
50
) of equine infectious anemia (EIA) virus per cell, and the titer of cell-associated and fluid-phase virus was determined from 1 to 72 hr postinoculation (PI). Cover slips were collected from 4 to 72 hr PI and stained for EIA viral antigen by the indirect immunofluorescent (FA) technique. Viral replication was detected after a latent period of approximately 18 to 24 hr and reached peak titers of approximately 10
4.5
to 10
6
TCID
50
/0.5 ml from 48 to 72 hr PI. The fluid phase contained 10
1
to 10
2
TCID
50
/0.5 ml more virus than the cells. Viral antigen was first detected by FA from 18 to 24 hr PI. Approximately 75% of the cells contained antigen in their cytoplasm 72 hr PI. The FA technique is a sensitive method for detecting EIA virus in horse leukocyte cultures.
Lymphokine-induced inhibition of growth of Eimeria bovis and Eimeria papillata (Apicomplexa) in cultured bovine monocytes
Authors: Speer C A , Reduker D W , Burgess D E , Whitmire W M , Splitter G A
Published: January 2020

Sporozoites of Eimeria bovis penetrated and developed normally to first-generation meronts in bovine monocytes (BM) and Madin-Darby bovine kidney (MDBK) cells that had been pretreated with culture medium (CM) or supernatant (NS) from nonstimulated bovine T cells. At 240 h after sporozoite inoculation (ASI), the mean percent development (meronts/[sporozoites + meronts]) in CM- and NS-pretreated BM was 52 and 28%, respectively; values for MDBK cells were 36 and 35%, respectively. Pretreatment of BM and MDBK cells with supernatant (ConAS) from concanavalin A-stimulated bovine T cells had no effect on the ability of sporozoites to penetrate cells; however, at 240 h ASI, only 1% of the sporozoites in ConAS-pretreated BM cultures had developed to meronts. In contrast, ConAS had no adverse effect on the ability of E. bovis sporozoites to develop to first-generation meronts in MDBK cells. At 240 h ASI, E. bovis meronts in ConAS-pretreated BM were abnormal in appearance and retarded in development, whereas sporozoites appeared structurally normal by light microscopy. Pretreatment of BM with ConAS had no effect on the ability of sporozoites of Eimeria papillata (Apicomplexa) to penetrate cells. Sporozoites of E. papillata did not develop to meronts in ConAS-pretreated BM and, in contrast to E. bovis, most sporozoites were destroyed intracellularly.
Specific nature of Trichomonas vaginalis parasitism of host cell surfaces
Authors: Alderete J F , Garza G E
Published: January 2020

The adherence of Trichomonas vaginalis NYH 286 to host cells was evaluated by using monolayer cultures of HeLa and HEp-2 epithelial cells and human fibroblast cell lines. Saturation of sites on HeLa cells was achieved, yielding a maximal T. vaginalis NYH 286-to-cell ratio of two. The ability of radiolabeled NYH 286 to compete with unlabeled trichomonads for attachment and the time, temperature, and pH-dependent nature of host cell parasitism reinforced the idea of specific parasite-cell associations. Other trichomonal isolates (JH31A, RU375, and JHHR) were also found to adhere to cell monolayers, albeit to different degrees, and all isolates produced maximal contact-dependent HeLa cell cytotoxicity. The avirulent trichomonad, Trichomonas tenax, did not adhere to cell monolayers and did not cause host cell damage. Interestingly, parasite cytadherence was greater with HeLa and HEp-2 epithelial cells than with fibroblast cells. In addition, cytotoxicity with fibroblast cells never exceeded 20% of the level of cell killing observed for epithelial cells. Elucidation of properties of the pathogenic human trichomonads that allowed for host cell surface parasitism was also attempted. Treatment of motile T. vaginalis NYH 286 with trypsin diminished cell parasitism. Incubation of trypsinized organisms in growth medium allowed for regeneration of trichomonal adherence, and cycloheximide inhibited the regeneration of attachment. Organisms poisoned with metronidazole or iodoacetate failed to attach to host cells, and adherent trichomonads exposed to metronidazole or iodoacetate were readily released from parasitized cells. Coincubation experiments with polycationic proteins and sugars and pretreatment of parasites or cells with neuraminidase or periodate had no effect on host cell parasitism. Colchicine and cytochalasin B, however, did produce some inhibition of adherence to HeLa cells. The data suggest that metabolizing T. vaginalis adheres to host cells via parasite surface proteins in a specific receptor-ligand fashion. Furthermore, parasitism of epithelial cells appears to render this cell type more susceptible than fibroblast cell types to contact-dependent cytotoxicity.
Inhibition of plaque and caries formation by a glucan produced by Streptococcus mutans mutant UAB108
Authors: Takada K , Shiota T , Curtiss R , Michalek S M
Published: January 2020

A mutant (UAB108) derived from Streptococcus mutans UAB66, a spectinomycin-resistant (Spcr) isolate of strain 6715, inhibited plaque formation when grown with strain 6715 in a sucrose medium and also inhibited caries formation in gnotobiotic rats infected with both strain UAB108 and 6715. A substance obtained from UAB108 culture supernatant fluid after ethanol precipitation and DEAE-cellulose treatment, designated glucan 108, inhibited S. mutans 6715 virulence and was shown to be a water-soluble glucan. In the presence of sucrose and increasing concentrations of glucan 108, the activity of a glucosyltransferase (GTase) preparation from S. mutans 6715 to synthesize adhesive water-insoluble glucan (ad-WIG) was inhibited, and the activity to synthesize non-ad-WIG was stimulated. Glucan 108 similarly inhibited sucrose-dependent adherence of heat-treated cells, was a poor inducer of cell aggregation, and inhibited S. mutans 6715-induced dental caries in gnotobiotic rats. In the presence of GTase, glucan 108, and sucrose, the glucose moiety of sucrose was found to be incorporated into glucan 108, and most of this glucose-incorporated glucan 108 was found in the non-ad-WIG fraction. The mode of inhibition of plaque formation by S. mutans 6715 appears to involve a shift from ad-WIG to non-ad-WIG formation. The water-soluble glucan 108 was found to have an approximate molecular weight of 2 X 10(6) and was hydrolyzed by fungal dextranase to yield glucans with an average molecular weight of about 1.2 X 10(4). This glucan (designated glucan 12k) was further hydrolyzed by bacterial dextranase to yield smaller glucans and oligosaccharides, but was refractile to alpha (1----3) glucanase. These results suggest that glucan 108 is a branched alpha (1----6) glucan, and it is proposed that UAB108 is defective in its ability to polymerize glucan 12k with alpha (1----3)-linked glucosyl residues.
Effect of thiol-activated toxins (streptolysin O, alveolysin, and theta toxin) on the generation of leukotrienes and leukotriene-inducing and -metabolizing enzymes from human polymorphonuclear granulocytes
Authors: Bremm K D , König W , Pfeiffer P , Rauschen I , Theobald K , Thelestam M , Alouf J E
Published: January 2020

The generation of leukotrienes (LTC4, LTD4, LTE4, and LTB4; 12-epi-LTB4 isomer) from human granulocytes by thiol-activated toxins (streptolysin O, alveolysin from Bacillus alvei, and theta toxin from Clostridium perfringens) is described. The release occurs under noncytolytic conditions. Although LTB4 is the major component after calcium ionophore stimulation, more LTC4 as compared with LTB4 is released with the toxins. The 5-lipoxygenase pathway of toxin-mediated activation can effectively be inhibited by caffeic acid, a lipoxygenase inhibitor. The toxins also induce the release of leukotriene-metabolizing enzymes such as gamma-glutamyltranspeptidase, which transfers LTC4 into LTD4, and dipeptidase, which metabolizes LTD4, into LTE4. Dipeptidase activity is more pronounced than the gamma-glutamyltranspeptidase activity but still does not reach the levels obtained when cells were triggered with opsonized zymosan.
Trehalose dimycolate from various mycobacterial species induces differing anti-infectious activities in combination with muramyl dipeptide
Authors: Masihi K N , Brehmer W , Lange W , Werner H , Ribi E
Published: January 2020

Significant resistance against influenza virus and Mycobacterium tuberculosis infections was induced when trehalose dimycolate from M. tuberculosis or M. bovis but not M. avium was combined with muramyl dipeptide. Trehalose dimycolate from M. tuberculosis, in contrast to that from M. avium, could confer resistance against Toxoplasma gondii infections.
New fimbrial antigen F165 from Escherichia coli serogroup O115 strains isolated from piglets with diarrhea
Authors: Fairbrother J M , Larivière S , Lallier R
Published: January 2020

Sixteen strains of Escherichia coli serogroup O115 isolated from piglets with diarrhea were examined for mannose-sensitive or mannose-resistant hemagglutination (MSHA or MRHA, respectively) for the presence of fimbriae by electron microscopy and for enterotoxigenicity by the ligated gut loop technique in 10-day-old piglets. Four strains demonstrated MRHA of sheep, goat, pig, dog, cat, chicken, and human erythrocytes but no MRHA of calf, horse, guinea pig, and rabbit erythrocytes. They were divided into pattern I (MSHA negative) and pattern II (MSHA positive). The remaining 12 strains were classified as pattern III (MRHA negative, MSHA positive) and pattern IV (hemagglutination negative). An antiserum produced against the MRHA-positive, MSHA-negative strain 4787 and absorbed by the same strain grown at 15 degrees C agglutinated all of the MRHA-positive strains but none of the MRHA-negative strains and completely inhibited the MRHA of these strains. The surface antigen against which this absorbed antiserum was directed was designated "F165." Fimbriae (pili) purified from strain 4787 hemagglutinated erythrocytes in the same mannose-resistant pattern as the strain itself and reacted with the anti-F165 antiserum in an enzyme-linked immunosorbent assay, thus demonstrating the fimbrial nature of the hemagglutinating F165 antigen. The F165 antigen showed no serological relationship with the fimbrial antigens F4, F5, F6, and "F41". A positive correlation between the presence of F165 and the lack of enterotoxigenicity was demonstrated. Thus, we found a new mannose-resistant, hemagglutinating fimbrial antigen, F165, which is produced only by nonenterotoxigenic strains of E. coli serogroup O115. The possible role of F165 as a virulence attribute of E. coli strains causing extraintestinal disease is discussed.
Immunogenicity and antigenicity of synthetic Escherichia coli lipid A
Authors: Brade L , Rietschel E T , Kusumoto S , Shiba T , Brade H
Published: January 2020

The immunogenicity and antigenicity of synthetic Escherichia coli lipid A (compound 506) and its 1- and 4'-monophosphorylated derivatives (compounds 505 and 504, respectively) and nonphosphorylated derivative (compound 503) were compared with those of bis- and 4'-monophosphorylated natural free lipid A from E. coli. The synthetic compounds under study were either coated onto sheep erythrocytes (except for the water-insoluble preparation 503) or incorporated into liposomes and used for the immunization of rabbits. Both types of immunogens (the latter representing fully synthetic immunogens) resulted in high-titered polyclonal antisera which were characterized before or after absorption in a passive hemolysis assay as well as in a passive hemolysis inhibition assay with the synthetic compounds as test antigens. All antisera were found to react with their corresponding homologous antigens coated onto sheep erythrocytes, with titers of up to 2,048, and were comparable to those antisera obtained after immunization with natural lipid A exposed on the bacterial surface after acid hydrolysis. Antisera against bisphosphorylated compound 506 were highly specific for the homologous antigens, showing no interaction with compounds 504 and 505 in the passive hemolysis test. The same held true for the absorption experiments in which glutaraldehyde-fixed sheep erythrocytes were sensitized with the respective antigens. Antisera against monophosphorylated compounds 504 and 505 exhibited, besides their expected homologous reactivity, complete cross-reactivity with compound 506, but they did not cross-react with each other. Thus, anti-504 and anti-505 antibodies recognized distinct antigenic determinants, being related to the ester linked 4'-phosphate or the glycosidically linked 1-phosphate, respectively. Both antigenic determinants were also expressed by bisphosphorylated compound 506 used as an antigen; however, upon immunization, only antibodies against compound 506 were elicited.
Specific cleavage of human type III and IV collagens by Pseudomonas aeruginosa elastase
Authors: Heck L W , Morihara K , McRae W B , Miller E J
Published: January 2020

Purified Pseudomonas aeruginosa elastase cleaved human type III and IV collagens with the formation of specific cleavage products. Furthermore, type I collagen appeared to be slowly cleaved by both P. aeruginosa elastase and alkaline protease. These cleavage fragments from type III and IV collagens were separated from the intact collagen chains by SDS polyacrylamide gradient gel electrophoresis run under reducing conditions, and they were detected by their characteristic Coomassie blue staining pattern. The results of these studies suggest that the pathogenesis of tissue invasion and hemorrhagic tissue necrosis observed in P. aeruginosa infections may be related to the degradation of these collagen types by bacterial extracellular proteases.
Expression of the pilin gene from Bacteroides nodosus in Escherichia coli
Authors: Elleman T C , Hoyne P A , Emery D L , Stewart D J , Clark B L
Published: January 2020

Bacterial plasmids that direct the expression in Escherichia coli of the pilin of Bacteroides nodosus were constructed. The quantity of pilin produced was greater than that of the pilin synthesized by B. nodosus, but no surface structural pili were present; pilin was found associated with the inner membrane of E. coli. Vaccination of sheep with E. coli containing pilin elicited increases in agglutinating and enzyme-linked immunosorbent assay antibody titers, which in turn were lower than the titers in sheep immunized with pilin from B. nodosus. The E. coli-produced pilin vaccine initially appeared to delay the progression of infection in immunized sheep after a challenge with virulent homologous B. nodosus, but at a later time the severity of foot rot was similar to that in sheep vaccinated with a placebo.
Immunogenic proteins of Trichomonas vaginalis as demonstrated by the immunoblot technique
Authors: Garber G E , Proctor E M , Bowie W R
Published: January 2020

Fifteen clinical isolates of Trichomonas vaginalis were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with transfer to nitrocellulose and by immunoblots probed with human sera. All T. vaginalis isolates showed similar banding patterns by Coomassie brilliant blue and silver staining of the electrophoresis gels and by amido black staining of the nitrocellulose. However, by the immunoblot technique, differences in banding patterns were noted, particularly in the high-molecular-weight zone (greater than 1.5 X 10(5], which were consistent in numerous experiments. A common immunogenic band was noted at a molecular weight of approximately 100,000 in all T. vaginalis isolates probed with six sera reactive in an enzyme-linked immunosorbent assay but was not seen or was only faintly visible when isolates were probed with sera considered to be nonreactive by the assay. Many other bands were identified, some of which appeared common to all isolates, but were not recognized by all sera tested. These studies demonstrate the antigenic heterogeneity of T. vaginalis and show that different individuals appear to respond immunologically to different T. vaginalis antigens.
Clonal analysis of Escherichia coli O2:K1 isolated from diseased humans and animals
Authors: Achtman M , Heuzenroeder M , Kusecek B , Ochman H , Caugant D , Selander R K , Väisanen-Rhen V , Korhonen T K , Stuart S , Orskov F
Published: January 2020

Forty-six Escherichia coli isolates of serotype O2:K1 from human urinary tract infections, chicken sepsis, and bovine mastitis were obtained from laboratories in England, Denmark, Sweden, and Finland. The bacteria were compared for outer membrane protein (OMP) pattern, lipopolysaccharide pattern, electrophoretic mobilities of enzymes, and flagellar serotype and were tested for fimbriation, biotype, hydroxamate production, hemolysin production, antibiotic resistance, plasmid content, colicin production, and virulence in neonatal rats. Isolates from humans were assigned to two clonal groups; poultry isolates belonged to one of these clonal groups, whereas bovine isolates belonged to the other. Poultry and human isolates of the same clonal group could be distinguished only by their plasmid content. Strains within this group were heterogeneous with respect to biotype, fimbriation, virulence, and flagellar serotype. Human and bovine isolates of the second clonal group were distinguished by a minor change in OMP pattern and by their plasmid content. It is concluded that meaningful clonal groupings are best recognized by the combination of OMP and electrophoretic enzyme patterns. The O:K serotype can aid in the recognition of important subclones, whereas the other microbiological properties tested can vary widely within clonal groupings. Furthermore, we conclude that certain O:K serotypes can contain very different clonal groupings having little genetic relatedness.
Intestinal receptor for heat-stable enterotoxin of Escherichia coli is tightly coupled to a novel form of particulate guanylate cyclase
Authors: Waldman S A , Kuno T , Kamisaki Y , Chang L Y , Gariepy J , O'Hanley P , Schoolnik G , Murad F
Published: January 2020

A novel form of particulate guanylate cyclase tightly coupled by cytoskeletal components to receptors for heat-stable enterotoxin (ST) produced by Escherichia coli can be found in membranes from rat intestinal mucosa. Intestinal particulate guanylate cyclase was resistant to solubilization with detergent alone, with only 30% of the total enzyme activity being extracted with Lubrol-PX. Under similar conditions, 70% of this enzyme was solubilized from rat lung membranes. The addition of high concentrations of sodium chloride to the extraction buffer resulted in greater solubilization of particulate guanylate cyclase from intestinal membranes. Although extraction of intestinal membranes with detergent and salt resulted in greater solubilization of guanylate cyclase, a small fraction of the enzyme activity remained associated with the particulate fraction. This activity was completely resistant to solubilization with a variety of detergents and chaotropes. Particulate guanylate cyclase and the ST receptor solubilized by detergent retained their abilities to produce cyclic GMP and bind ST, respectively. However, ST failed to activate particulate guanylate cyclase in detergent extracts. In contrast, guanylate cyclase resistant to solubilization remained functional and coupled to the ST receptor since enzyme activation by ST was unaffected by various extraction procedures. The possibility that the ST receptor and particulate guanylate cyclase were the same molecule was explored. ST binding and cyclic GMP production were separated by affinity chromatography on GTP-agarose. Similarly, guanylate cyclase migrated as a 300,000-dalton protein, while the ST receptor migrated as a 240,000-dalton protein on gel filtration chromatography. Also, thiol-reactive agents such as cystamine and N-ethylmaleimide inhibited guanylate cyclase activation by ST, with no effect on receptor binding of ST. These data suggest that guanylate cyclase and the ST receptor are independent proteins coupled by cytoskeletal components in membranes of intestinal mucosa.
Activation of the alternative complement pathway by Sporothrix schenckii
Authors: Scott E N , Muchmore H G , Fine D P
Published: January 2020

We studied the activation of complement by Sporothrix schenckii yeast cells. Total complement activity, and the effect of various activators on this activity, were assayed on aliquots of fresh nonimmune human serum with and without prior treatment with chelators. Both total hemolytic complement and C3 were consumed (activated) in serum chelated with magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, which blocks the classical pathway but leaves the alternative pathway intact. Further, C3 was consumed, but C4 (exclusively a component of the classical pathway) was preserved, in nonchelated serum after challenge by S. schenckii yeast cells. Absorption of serum with S. schenckii yeast cells to deplete antibodies did not alter these results. Furthermore, immunofluorescence studies demonstrated that C3, but not immunoglobulin G, was deposited on yeast cells during incubation with nonimmune serum. These data indicate that S. schenckii yeast cells activate the alternative complement pathway in vitro independently of antibody. These data do not define a role for the alternative pathway in in vivo host defenses against infection with this organism but provide a foundation for studies to evaluate such a role.
Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins
Authors: Straley S C , Bowmer W S
Published: January 2020

Yersinia pestis, the causative agent of plague, has a virulence determinant called the low-Ca2+ response (Lcr+ phenotype) that confers on the bacterium Ca2+ dependence for growth at 37 degrees C and expression of V antigen. This virulence determinant is common to the three species of Yersinia and is mediated by Lcr plasmids (called pCD in Y. pestis). In this study, we generated insertions of Mu dI1(Ap lac) in pCD1 of Y. pestis KIM, screened for cells showing transcriptional regulation by Ca2+, and obtained inserts that define at least four pCD1 genes. Their patterns of transcription under different growth conditions closely paralleled the pattern of expression of the V antigen. We tested for expression of Lcr-specific yersinial outer membrane proteins (Yops) by the pCD1::Mu dI1(Ap lac) plasmids. Four of the inserts each eliminated expression of a different Yop; one of these Yops was unique to Y. pestis. Two of the insertions affecting Yops caused avirulence, and one caused strongly decreased virulence of Y. pestis in mice. These data indicate that Yops, like the V antigen, are virulence attributes regulated in the low-Ca2+ response.
Antigenic heterogeneity in high- and low-virulence strains of Rickettsia rickettsii revealed by monoclonal antibodies
Authors: Anacker R L , List R H , Mann R E , Wiedbrauk D L
Published: January 2020

Previously it has been reported that strains of Rickettsia rickettsii that differ greatly in their ability to cause disease in guinea pigs are similar by serological and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. In this study, we used monoclonal antibodies to the virulent R and the relatively avirulent HLP strains to investigate strain differences which might account for the disparate behavior of the strains in guinea pigs. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the R and HLP strains were nearly identical for polypeptides with apparent molecular weights greater than 32 kilodaltons (kDa). All of the monoclonal antibodies to a lipopolysaccharide-like antigen reacted equally well with antigen from both strains by immunoblotting. None of the antibodies to the lipopolysaccharide-like antigen protected mice against challenge with viable rickettsiae. Some antibodies reacted with both 120- and 155-kDa polypeptides of both strains in radioimmune precipitation and immunoblotting tests, and other antibodies reacted only with the homologous strain. The monoclonal antibodies cross-reacted with the heterologous strain in the enzyme-linked immunosorbent assay essentially either completely or not at all. The ability of the monoclonal antibodies to the 120- and 155-kDa polypeptides to protect mice against the two strains was correlated with the ability of the antibodies to react with the antigens in the enzyme-linked immunosorbent assay and radioimmune precipitation or immunoblotting tests. These results demonstrate that R and HLP antigens which appear identical in molecular weight differ in their compositions of antigenic determinants.
Evidence for macrophage-mediated protection against lethal Candida albicans infection
Authors: Bistoni F , Vecchiarelli A , Cenci E , Puccetti P , Marconi P , Cassone A
Published: January 2020

Systemic infection of mice with a Candida albicans strain (PCA-2) incapable of yeast-mycelial conversion conferred protection against a subsequent intravenous challenge with the pathogenic strain of the parent organism, strain CA-6. Protection was nonspecific since it was also detected upon challenge of mice with Staphylococcus aureus. Moreover, the PCA-2 organisms had to be viable, their effects being most evident when they were given intravenously at a dose of 10(6) cells 7 to 14 days prior to microbial challenge. Thus, all mice pretreated with PCA-2 and challenged 14 days later with viable CA-6 cells lived through a 60-day observation period, whereas all control mice not treated with PCA-2 died within 3 days. In an attempt to correlate the immunostimulatory effects observed in vivo with possible modifications in in vitro functions, it was found that administration of PCA-2 was accompanied by an increase in the number of peripheral blood polymorphonuclear cells and by the activation in the spleen of cells with highly candidacidal activity in vitro. Moreover, the adoptive transfer of plastic-adherent cells from PCA-2-infected mice into histocompatible recipients conferred considerable protection against subsequent CA-6 challenge.
In vivo identification of sialic acid as the ocular receptor for Pseudomonas aeruginosa
Authors: Hazlett L D , Moon M , Berk R S
Published: January 2020

In vivo bacterial adherence of Pseudomonas aeruginosa to the immature ocular epithelium was mediated by a sialic (N-acetylneuraminic) acid (NANA) receptor. Saturation of binding sites on the bacterial surface by NANA prevented attachment of the organism to the epithelial cell membrane receptor. Additionally, a significant number of animals receiving NANA-treated organisms were protected from septicemia and death. In vivo protection studies showed excellent correlation with scanning electron microscopy, in that the number of adherent organisms at the corneal surface decreased dramatically in the presence of NANA. These studies exhibit a strong correlation with clinical cases of human infant ocular infection.
Purification and characterization of an extracellular protease of Legionella pneumophila
Authors: Dreyfus L A , Iglewski B H
Published: January 2020

An extracellular proteolytic enzyme of Legionella pneumophila was purified by sequential batch separation with DEAE-cellulose, hydrophobic interaction chromatography with octyl-Sepharose, and ion-exchange chromatography with DEAE-Bio-Gel A (Bio-Rad Laboratories, Richmond, Calif.). The resulting protease preparation was determined to be homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Although free of contaminating proteins, the purified protease separated into two antigenically indistinguishable proteins both of which possessed proteolytic activity. The apparent masses of the proteins were 38 and 40 kilodaltons (kDa) as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, whereas gel filtration chromatography revealed a single mass of 34 kDa. Immunoblot analysis indicated that the 38-kDa protein probably originated from the 40-kDa protein during purification. The isoelectric points of the two protease species were 4.20 and 4.42. Enzyme activity, which was optimum between pH 5.5 and 7.5, was inhibited by various metal chelators; however, no effect was observed after treatment with phenylmethylsulfonyl fluoride, chymostatin, trypsin inhibitor, or dithiothreitol. Enzyme activity inhibited by metal chelators was restored upon the addition of various metal ions, including Zn2+, Fe2+, Mn2+, Cu2+, and Fe3+, but was not restored by Mg2+ or Ca2+. Atomic absorption analysis of the purified protease revealed a single gram-atom of zinc per mole of enzyme. Our findings indicate that the L. pneumophila protease resembles neutral zinc-containing metalloproteases similar to those found in other bacterial species.
Collaboration of bovine T lymphocytes and macrophages in T-lymphocyte response to Brucella abortus
Authors: Splitter G A , Everlith K M
Published: January 2020

Brucella abortus-induced bovine macrophage-T-lymphocyte collaboration was studied as a prerequisite for the eventual clearance of this infectious organism. Esterase-positive, peripheral blood monocytes functioned as an adherent antigen-presenting cell population. A dual requirement for expression of bacterial antigens in combination with self major histocompatibility complex class II products was required by adherent cells for the activation of T lymphocytes. Comparison of antigen-presenting cell populations that were either trypsinized or nontrypsinized following B. abortus ingestion substantiated the need for phagocytosis and antigen processing. A monoclonal antibody (H4) directed against major histocompatibility complex class II determinants was able to block or, with complement, to abrogate T-lymphocyte responses. Measurement of both proliferation and interleukin 2 production via [3H]thymidine incorporation confirmed specific activation of an enriched T-lymphocyte population. These results indicate that in vivo-primed T lymphocytes of peripheral blood origin recognize phagocytized bacterial components of the facultative intracellular bacterium B. abortus and may contribute to the removal of the bacteria. Furthermore, bovine peripheral blood-adherent cells function as classic antigen-presenting cells, which suggests that macrophages are capable of processing this bacteria. Therefore, any lymphocyte-mediated dysfunction attributable to B. abortus most likely occurs at some point in the cascade of immune events following initial macrophage-T-lymphocyte collaboration.
Cell surface hydrophobicity of pigmented and nonpigmented clinical Serratia marcescens strains
Authors: Rosenberg M , Blumberger Y , Judes H , Bar-Ness R , Rubinstein E , Mazor Y
Published: January 2020

The cell surface hydrophobicity of 10 pigmented and 4 nonpigmented clinical Serratia marcescens strains was studied, based on the ability of the strains to adhere to hydrocarbons and to polystyrene. The cell surface hydrophobicity depended greatly on growth temperature; all of the strains tested were adherent following growth at 30 degrees C, whereas none was adherent following growth at 38 degrees C. In previous studies, the pigment prodigiosin has been cited as responsible for cell surface hydrophobicity in various Serratia strains. However, the observed ability of the nonpigmented strains to adhere to the test hydrocarbons and to polystyrene indicates that Serratia strains can possess hydrophobic surface properties in the absence of this pigment. Moreover, strain 1785 cells were adherent whether they were grown at 30 or 36.5 degrees C, even though pigment was not synthesized at the higher temperature. In Escherichia coli correlations have been noted between increased cell surface hydrophobicity and the presence of mannose-specific adhesins; no such relationship was found in the S. marcescens strains tested. The expression of cell surface hydrophobicity in clinical S. marcescens strains at 30 degrees C and the loss of hydrophobicity at host temperatures raise the possibility that infective cells from the environment are initially hydrophobic, but lose this property upon subsequent proliferation within a host.
Development of increased serum immunoblot reactivity against a 45,000-dalton polypeptide of Treponema pallidum (Nichols) correlates with establishment of chancre immunity in syphilitic rabbits
Authors: Strugnell R A , Williams W F , Drummond L , Pedersen J S , Toh B H , Faine S
Published: January 2020

Rabbits developed chancre immunity 5.0 to 7.5 weeks after intradermal infection with 10(3) Treponema pallidum (Nichols). The serological response against T. pallidum antigen during this 2.5-week period was examined by Western immunoblotting. Sera from rabbits infected for 5.0 weeks contained antibodies against 7 of 13 major T. pallidum immunogens, with strongest binding detected against a polypeptide of Mr 47,000. By 7.5 weeks of infection, syphilitic rabbit sera recognized 10 of 13 antigens; the most evident increase in serological reactivity was directed against a polypeptide of Mr 45,000, suggesting that the development of a strong serological response against this polypeptide correlated with the onset of chancre immunity.
Aerobactin-mediated iron uptake by Escherichia coli isolates from human extraintestinal infections
Authors: Carbonetti N H , Boonchai S , Parry S H , Väisänen-Rhen V , Korhonen T K , Williams P H
Published: January 2020

A total of 516 strains of Escherichia coli were screened for the presence and expression of the aerobactin iron uptake system. The incidence was markedly higher among clinical isolates from patients with septicemia (68.8%), pyelonephritis (74.6%), and symptomatic (59.8%) and asymptomatic (63.2%) lower urinary tract infections than among normal human fecal isolates (34.3%).