bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of Lyme disease spirochetes with cultured eucaryotic cells
Authors: Thomas D D , Comstock L E
Published: January 2020

The association of the Lyme disease spirochete, Borrelia burgdorferi, with cultured human endothelial cells was investigated. Attachment was time and temperature dependent, with optimal adherence occurring after 4 h of incubation at 37 degrees C. Pretreatment of borreliae with heat, immune human serum, or monoclonal antibodies directed against outer surface protein B (OspB) reduced the attachment of organisms to host cell monolayers. These results suggest that the adherence of B. burgdorferi may be mediated, at least in part, by borrelial surface proteins.
Role of C1q in phagocytosis of Salmonella minnesota by pulmonary endothelial cells
Authors: Ryan U S , Schultz D R , Goodwin J D , Vann J M , Selvaraj M P , Hart M A
Published: January 2020

The Re mutant of Salmonella minnesota adheres in much greater numbers than the wild type to endothelial cells derived from the bovine pulmonary artery. Since the Re mutant is distinguished from wild-type S. minnesota by its ability to bind C1q and since endothelial cells possess receptors for C1q, we examined the role of C1q in the phagocytosis of the S. minnesota Re mutant. First, preincubating endothelial cells with C1q-enriched medium resulted in increased adherence of the Re mutant (17.9 x 10(4) versus 6.6 x 10(4]. Second, preincubating the Re mutant with C1q-enriched medium resulted in increased numbers of adherent bacteria (62.1 x 10(4) versus 6.6 x 10(4]. Preincubation of both endothelial cells and bacteria with C1q-enriched medium resulted in increased adherence above control levels but less adherence than when either cells or bacteria were preincubated separately in C1q-enriched medium. If serum depleted of C1q was used for preincubation of endothelial cells or bacteria, adherence was reduced below control levels. Thus, C1q plays an important role in the initial steps (recognition, binding, and ingestion) of phagocytosis. Next, the role of C1q was investigated in the respiratory burst response. Levels of superoxide anion released from endothelial cells 15 min after phagocytosis of the Re mutant (100 bacteria per endothelial cell) were assayed by measurement of the superoxide dismutase-inhibitable reduction of ferricytochrome c. Superoxide anion release was increased during phagocytosis of the Re mutant (35 nmol of O2- per 3 x 10(6) endothelial cells) and was also elevated above control values by incubation with soluble C1q (10 nmol of O2- per 3 x 10(6) endothelial cells). These results indicate a role for C1q in both the ingestion and the response of endothelial cells to the S. minnesota Re mutant.
Gamma interferon controls Eimeria vermiformis primary infection in BALB/c mice
Authors: Rose M E , Wakelin D , Hesketh P
Published: January 2020

Neutralization of endogenous gamma interferon by treatment with a rat monoclonal antibody caused enhancement of infection with the protozoon Eimeria vermiformis in naive BALB/c mice. The effect was dose dependent and was apparent when a monoclonal antibody was given at 2 h before infection or up to 7 days postinfection, but it decreased with increasing time postinfection between days 4 and 7. The titers of parasite-specific antibodies in the serum were not significantly affected by the injection of monoclonal antibodies. Treatment during priming did not prevent the development of resistance to challenge, and treatment at the time of challenge did not abrogate established immunity. The results indicate that gamma interferon is involved in the control of primary infection with E. vermiformis in BALB/c mice but not in the expression of immunity to challenge.
Penetration of endothelial cell monolayers by Borrelia burgdorferi
Authors: Comstock L E , Thomas D D
Published: January 2020

The ability of Borrelia burgdorferi, the agent of Lyme disease, to penetrate cultured human umbilical vein endothelial cell monolayers was investigated. After 4 h of coincubation, approximately 7.7% of added bacteria passed through the host cell monolayers. Electron microscopy revealed that the borreliae entered the endothelial cells and suggested that the organisms penetrated the host monolayers primarily by passing through them.
Complement activation and C3 binding by serum-sensitive and serum-resistant strains of Pseudomonas aeruginosa
Authors: Schiller N L , Hatch R A , Joiner K A
Published: January 2020

The relationship among complement consumption, C3 deposition, and C3 fragmentation pattern was compared for serum-sensitive (Sers) and serum-resistant (Serr) strains of Pseudomonas aeruginosa. The Sers strains, which were mucoid strains derived from patients with cystic fibrosis, had lipopolysaccharide deficient in O-antigen side chains. These organisms generally activated much less complement per organism than their Serr counterparts, characterized by the presence of lipopolysaccharide with long lipopolysaccharide O side chains. Surprisingly, however, although the Serr strains consumed more total hemolytic complement, less C3 was deposited onto the surface of these strains than onto that of the Sers strains. Maximal C3 binding required the participation of both the classical and alternative complement pathways, although classical complement pathway involvement was more important for Serr strains. Finally, while more than half of the C3 deposited on most Sers strains was in the form of C3b, most of the C3 on the Serr strains was in the form of iC3b, indicating a more rapid and extensive conversion of C3b to iC3b on the surface of these strains. Limited complement activation by Sers mucoid strains of P. aeruginosa may confer a selective survival advantage to these organisms in colonizing the airways of patients with cystic fibrosis.
Cloning and expression of the streptococcal C5a peptidase gene in Escherichia coli: linkage to the type 12 M protein gene
Authors: Chen C C , Cleary P P
Published: January 2020

A genomic library of Streptococcus pyogenes CS24 DNA was constructed by cloning streptococcal DNA partially digested with Sau3A into the lambda replacement vector EMBL3. The expression of streptococcal C5a peptidase (SCP) was analyzed by radioimmunoassay with hyperimmune rabbit serum. Two clones, lambda 4.1 and lambda 4.2, were found to express the desired antigen, and various DNA fragments from the hybrid bacteriophage lambda 4.1 were subcloned into the plasmid vector pUC9 in Escherichia coli. One of the recombinant plasmids, designated pTT1, contained a 5.8-kilobase (kb) streptococcal DNA insert. Analysis of total cellular protein from this E. coli clone by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blotting identified a 140,000-Mr protein, similar in size to the native protein purified from S. pyogenes. Cloned SCP was functionally active, as shown by its ability to inhibit C5a-mediated chemotaxis. By deletion analysis with both restriction endonucleases and BAL 31 nuclease, the SCP gene was localized to a 4.3-kb segment of DNA. Southern hybridization experiments showed that the type 12 M protein-coding sequence is also present in the hybrid phage lambda 4.1, at approximately 2 kb upstream of the SCP structural gene. Western blot analysis indicated that the cloned streptococcal DNA in lambda 4.1 directed the expression of both SCP and M12 protein.
Isolation of Rickettsia prowazekii with reduced sensitivity to gamma interferon
Authors: Turco J , Winkler H H
Published: January 2020

The growth of Rickettsia prowazekii Madrid E was monitored in mouse L929 cells subcultured for several weeks in the presence of gamma interferon (IFN-gamma) to determine whether the rickettsiae would be eliminated from or would persist in these cultures. R. prowazekii exhibited two distinct patterns in these IFN-gamma-treated cultures. In some cases, IFN-gamma-induced inhibition of rickettsial growth led to elimination of the rickettsiae from the L929 cell cultures; in other cases, the initial inhibition of rickettsial growth was followed by establishment of a persistent rickettsial infection in the IFN-gamma-treated L929 cells. During the first 3 days after infection, the growth rate of the L929 cells was significantly lower and higher percentages of the cells were killed in the IFN-gamma-treated, R. prowazekii-infected cultures than in the untreated, R. prowazekii-infected cultures or the mock-infected cultures, whether treated or untreated. This suppression of cell growth occurred in the infected, IFN-gamma-treated cultures that eventually exhibited the elimination pattern as well as the IFN-gamma-treated cultures that became persistently infected. It was not possible to predict the outcome of a particular infection from the early growth pattern of the culture. It was determined that the L929 cells in the persistently infected, IFN-gamma-treated cultures had not lost the ability to respond to IFN-gamma. These cells, after treatment with an antibiotic to eliminate the persistent rickettsiae, retained the ability to inhibit both the replication of vesicular stomatitis virus and the growth of R. prowazekii Madrid E after treatment with IFN-gamma. In contrast, rickettsiae isolated from two persistently infected, IFN-gamma-treated cultures were less sensitive than R. prowazekii Madrid E to the antirickettsial effects of IFN-gamma in standard L929 cells. The maintenance of the phenotype of these altered rickettsiae during plaque purification and passage in the absence of IFN-gamma suggests an alteration at the genetic level rather than phenotypic adaptation.
Selective proliferation of natural killer cells among monocyte-depleted peripheral blood mononuclear cells as a result of stimulation with staphylococcal enterotoxin B
Authors: Garcia-Peñarrubia P , Lennon M P , Koster F T , Kelley R O , Bankhurst A D
Published: January 2020

In vitro stimulation of monocyte-depleted peripheral blood mononuclear cells with staphylococcal enterotoxin B (SEB) resulted in selective proliferation of cells which express the phenotypic and functional characteristics of natural killer (NK) cells. This culture system provides an easy method for obtaining highly purified NK cells, by sequential incubation of monocyte-depleted cells with SEB and then with interleukin-2 (IL-2). After culture for 4 to 5 days in the presence of SEB, 98 to 100% of the cells expressed the CD16 (Leu11) and HNK-1 (Leu19) antigens. This purification occurred through the death of lymphocytes lacking NK cell markers and marked proliferation of NK cells themselves, which leads to an enrichment of the NK cell population. Activation of NK cells was detected by the appearance of the gamma interferon receptor and IL-2 receptor antigens. This homogeneous population showed the morphology of large granular lymphocytes, were potent effectors of cell-mediated cytotoxicity against K562 and Daudi tumor cell lines, and were able to kill gram-positive and gram-negative bacteria. IL-2 was necessary to maintain the activation and proliferation after SEB stimulation for 4 days. Moreover, the maximum frequency of binding to K562 cells (60.6%) was similar to that recently found (58 +/- 3%) (P. Garcia-Peñarrubia, F. T. Koster, and A. D. Bankhurst, J. Immunol. Methods 118:199-208, 1989) with fresh and highly purified NK cells. This method can be used as a source of highly purified NK cells to study their functional properties and applications to the treatment of cancer.
Characterization of cell surface carbohydrate receptors for Entamoeba histolytica adherence lectin
Authors: Ravdin J I , Stanley P , Murphy C F , Petri W A
Published: January 2020

Binding and cytolysis of Chinese hamster ovary (CHO) cells by Entamoeba histolytica trophozoites is inhibitable by galactose (Gal) or N-acetyl-D-galactosamine (GalNAc). To better define the carbohydrate receptor for E. histolytica, we compared the binding and cytolytic target properties of 10 CHO glycosylation mutants. Each mutant expresses a uniquely altered array of N- and/or O-linked cell surface carbohydrates. Amebic adherence was reduced when lactosamine-containing N-linked carbohydrates were essentially absent (Lec1 mutant), almost undetectable when Gal and GalNAc residues were absent on both N- and O-linked carbohydrates (ldlD.Lec1 mutant), and enhanced for mutants with increased terminal Gal residues (Lec2 and Lec3). Parental CHO cells treated with neuraminidase to expose Gal residues behaved like Lec2 mutants. Binding of purified Gal or GalNAc lectin to parental, Lec1, ldlD.Lec1, and Lec2 mutant CHO cells corroborated the adherence results. The suitability of CHO cell mutants as targets for amebic cytolysis correlated with their glycosylation phenotype: the Lec1 mutants were less susceptible than parental CHO cells, the ldlD.Lec1 mutants were highly resistant, and the Lec2 mutants required higher concentrations of Gal for inhibition. The E. histolytica Gal or GalNAc adherence lectin bound preferentially to beta 1-6-branched, N-linked carbohydrates lacking terminal sialic acid or fucose residues. However, amebic lectin binding to either N- or O-linked cell surface carbohydrates was sufficient to initiate parasite cytolytic activity.
Reduced adherence to traumatized rat heart valves by a low-fibronectin-binding mutant of Staphylococcus aureus
Authors: Kuypers J M , Proctor R A
Published: January 2020

Isogenic strains of Staphylococcus aureus, differing in fibronectin binding, were constructed for studies of the contribution of fibronectin binding to the in vivo pathogenesis of staphylococcal disease. Mutagenesis of S. aureus 879R4S was accomplished by mating with Enterococcus faecalis FA378 that carried the transposon Tn918. Four low-fibronectin-binding mutants were identified that bound 24- to 35-fold less fibronectin than the parent strain did. A spectinomycin-resistant strain, R4SSp, was transduced by a bacteriophage (80 alpha) lysate propagated on a low-binding mutant of 879R4S to produce R4SSp/1536, which bound less fibronectin, contained a single copy of the transposon, and grew on spectinomycin-containing medium. Using a rat model of endocarditis, we determined the distribution of S. aureus R4SSp and its transductant in normal and cardiac catheterized rats. Cultures of heart tissue showed that catheterized rats challenged with the fibronectin-binding parent strain had over 250-fold more organisms in the left heart than did rats challenged with the low-binding transductant. The ability to bind fibronectin had no effect on the number of S. aureus cells cultured from other tissues. These data suggest that the ability of S. aureus to bind fibronectin is an important factor in establishing adherence to damaged heart valves in vivo.
Cellular immune responses of leprosy contacts to fractionated Mycobacterium leprae antigens
Authors: Lee S P , Stoker N G , Grant K A , Handzel Z T , Hussain R , McAdam K P , Dockrell H M
Published: January 2020

Antigens of armadillo-derived Mycobacterium leprae sonic extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane, and the unstained blot was converted into 20 fractions of antigen-bearing particles. These were tested in cellular proliferation assays, and reproducible results were obtained between batches of fractions. Peripheral blood mononuclear cells from healthy contacts of leprosy patients (presumed to have protective immunity) were tested with the fractions to investigate which antigens they recognized. A small group of tuberculoid leprosy patients were also tested. Both groups showed a wide range of responses. Almost every fraction stimulated proliferation with at least one donor, yet none was clearly immunodominant or inhibitory in either group. Thus, protective immunity did not appear to be associated with proliferation caused by any single fraction.
Roles of alpha-toxin and beta-toxin in virulence of Staphylococcus aureus for the mouse mammary gland
Authors: Bramley A J , Patel A H , O'Reilly M , Foster R , Foster T J
Published: January 2020

Mutants of Staphylococcus aureus which fail to express alpha-toxin (Hly), beta-toxin (Hlb), or both have been constructed by site-specific mutagenesis. The virulence of the mutants was compared with that of wild-type toxigenic strains by intramammary inoculation of lactating mice. A bovine strain, M60, and a laboratory strain, 8325-4, caused acute mastitis and death within 48 h for 60% of the mice inoculated. Animals inoculated with Hly mutants also developed acute mastitis, but no deaths occurred. Comparisons of Hly- or Hlb-positive strains with the double mutation Hly Hlb showed that both toxins led to a significantly higher recovery of S. aureus from the gland 48 h postinfection. Histopathological examination of mammary glands showed that phagocytosis of bacteria occurred irrespective of toxigenicity, but toxigenic strains, particularly those which were Hly+, continued to multiply, invaded the interalveolar tissues, and produced severe lesions. Stimulation of an inflammatory response by inoculation of the mammary gland with endotoxin prior to challenge with S. aureus reduced recovery of the bacteria 10- to 100-fold and, under these conditions, neither alpha-toxin nor beta-toxin contributed significantly to growth and survival.
Loss of catalase activity in Tn1545-induced mutants does not reduce growth of Listeria monocytogenes in vivo
Authors: Leblond-Francillard M , Gaillard J L , Berche P
Published: January 2020

Two catalase-negative mutants of Listeria monocytogenes were obtained by chromosomal insertions of the conjugative transposon Tn1545. The loss of catalase activity did not reduce the level of virulence of these mutants in mice. Indeed, both mutants were capable of growing in host tissues at the same rate as the parental catalase-positive strain. These results favor the view that catalase does not play a critical role in the resistance of L. monocytogenes to macrophage killing.
Chlamydia trachomatis-induced production of interleukin-1 by human monocytes
Authors: Rothermel C D , Schachter J , Lavrich P , Lipsitz E C , Francus T
Published: January 2020

Human diseases caused by the intracellular bacterium Chlamydia trachomatis include genital tract infections and blinding trachoma. Chlamydial infections are characterized by chronic inflammation and scarring, and development of such complications is thought to be immunologically mediated. In this study, we show that coculture of C. trachomatis (serovar L2) with human blood monocytes induced the production of interleukin-1 (IL-1), an important mediator of inflammation, tissue remodeling, and scarring. IL-1 was produced in response to UV-inactivated elementary bodies containing from 0.1 to 50 micrograms of protein per ml, with a maximal response at 5 to 10 micrograms/ml. IL-1 activity was detected by 6 h of incubation and was maximal by 24 h. Peak levels were maintained throughout 96 h of incubation. Rabbit antibody to human IL-1(alpha + beta) effectively neutralized the thymocyte-stimulating activity of the supernatants. The apparent molecular weight of chlamydia-induced IL-1 was 16,000, as determined by gel filtration on a Bio-Gel P-60 column. Isoelectric focusing yielded two peaks of activity, with pIs of 5.5 and 6.9. Neutralization studies with antisera against human IL-1 alpha and IL-1 beta showed that the acidic and neutral peaks corresponded to IL-1 alpha and IL-1 beta, respectively, with IL-1 beta predominating. Heat-killed chlamydiae, which are not internalized by monocytes, were effective IL-1 inducers, indicating that phagocytosis was not required for IL-1 induction. Purified C. trachomatis lipopolysaccharide was also an effective IL-1 inducer, suggesting that the response to intact organisms may be largely a response to chlamydial lipopolysaccharide. Finally, purified chlamydial major outer membrane protein induced low but detectable IL-1 activity.
Fibronectin as an enhancer of nonopsonic phagocytosis of Pseudomonas aeruginosa by macrophages
Authors: Kluftinger J L , Kelly N M , Jost B H , Hancock R E
Published: January 2020

Fibronectin is capable of enhancing uptake by macrophages of Pseudomonas aeruginosa grown in vivo in rats or mice or in vitro on nutrient agar plates. It was demonstrated that concentrations as low as 27 nM fibronectin produced significant enhancement of macrophage phagocytosis. Washing of fibronectin-treated macrophages did not prevent phagocytosis enhancement, but washing of fibronectin-treated bacteria did. The tetrapeptide arginine-glycine-aspartic acid-serine, which comprises the eucaryotic cell-binding domain of fibronectin, was also capable of promoting bacterial uptake, whereas the control tetrapeptide tetraglycine was not. Fibronectin caused depolarization of the mouse macrophage cell line P388D1, plasma membrane, as demonstrated by using a polarization-sensitive fluorescent probe. These data indicate that promotion by fibronectin of nonopsonic phagocytosis is mediated by the action of fibronectin on the macrophages.
Purification and characterization of a protease produced by Vibrio cholerae non-O1 and comparison with a protease of V. cholerae O1
Authors: Honda T , Lertpocasombat K , Hata A , Miwatani T , Finkelstein R A
Published: January 2020

A protease produced by a clinical isolate of Vibrio cholerae non-O1 was purified to apparent homogeneity by ammonium sulfate fractionation and successive column chromatography on DEAE-Sephadex A25, Sephadex G100, Mono Q, and Phenyl Superose. Like the hemagglutinin-protease of V. cholerae O1, the purified protease had both hemagglutinating and proteolytic activities. The protease was heat labile, and in contrast to crude preparations, no Arrhenius effect was observed with the purified protein. Immunological analyses indicated that the proteases (or hemagglutinins) derived from V. cholerae O1 and non-O1 are identical.
Monoclonal anti-idiotypic antibodies regulate the expression of virus-induced murine myocarditis
Authors: Paque R E , Miller R
Published: January 2020

A monoclonal anti-idiotypic antibody (anti-Id), produced by electrofusion and designated anti-Id88, was able to modulate expression of murine autoimmune myocarditis mediated by coxsackievirus B3 (CVB3). The anti-Id was characterized as an immunoglobulin G2b species possessing kappa light chains and was able to reduce expression of inflammatory myocarditis in anti-Id-pretreated mice challenged with CVB3. Anti-Id88 was able to stimulate specific cell-mediated immunity against anti-Id88, as well as CVB3, and exerted a suppressive effect on the proliferation of mixed spleen cell populations from virus-exposed mice. Anti-Id stimulated an anti-anti-Id antibody 3 population able to bind antibody 2 F(ab')2 fragments or virus antigen in an indirect enzyme-linked immunosorbent assay. Western blot (immunoblot) analysis of anti-Id88 exhibited binding of syngeneic anti-Id antibody to idiotypes present on immunoglobulin G molecules from virus-immunized mice.
Composition, serologic reactivity, and immunolocalization of a 120-kilodalton tube precipitin antigen of Coccidioides immitis
Authors: Cole G T , Kruse D , Zhu S W , Seshan K R , Wheat R W
Published: January 2020

Diagnosis of coccidioidomycosis largely depends on serologic tests. In this investigation, the enzyme-linked immunosorbent assay (ELISA) was used to detect patient immunoglobulin M (IgM) precipitin antibody binding to a 120-kilodalton (kDa) fraction previously isolated from an alkali-soluble, water-soluble extract of the arthroconidial wall and mycelial culture filtrate plus toluene lysate of Coccidioides immitis. Results of the serologic response to this tube precipitin antigen (TP-Ag) in the ELISA correlated well with results of immunodiffusion assays of 30 serum samples from patients. Immunoelectron microscopic examinations of arthroconidia and spherules were performed with patient IgM precipitin antibodies isolated from sera eluted over a solid-phase immunosorbent column containing the purified 120-kDa TP-Ag. The antibody probe located the 120-kDa TP-Ag on the walls of in vitro-grown arthroconidia and spherules. Pronase digestion and heating (100 degrees C, 5 min) had no apparent effect on the activity of the 120-kDa TP-Ag, while periodate oxidation resulted in total loss of its immunodiffusion-TP activity. Analysis of the carbohydrate composition of the TP-Ag revealed xylose, 3-O-methylmannose (3-O-MM), mannose, galactose, and glucose. Competitive inhibition ELISAs were used to demonstrate that 3-O-MM is largely responsible for the reactivity of IgM precipitin antibodies with the 120-kDa TP-Ag. Synthetic 3-O-MM may be a useful probe for detection of anti-Coccidioides precipitin antibodies in the ELISA.
Reduction of wall degradability of clindamycin-treated staphylococci within macrophages
Authors: Wecke J , Johannsen L , Giesbrecht P
Published: January 2020

Clindamycin treatment of Staphylococcus aureus caused a remarkable thickening of the bacterial cell wall and made the bacterial wall much more resistant against lytic enzymes within bone marrow-derived macrophages as revealed by electron microscopy and radiolabeling experiments. This reduced wall degradability resulted from an increased number of O-acetyl groups in the murein. Furthermore, such clindamycin-treated bacteria were ingested by adherent bone marrow-derived macrophages at a higher rate than untreated bacteria. The medical aspects of these results are discussed.
Impaired colonization by and full invasiveness of Escherichia coli K1 bearing a site-directed mutation in the type 1 pilin gene
Authors: Bloch C A , Orndorff P E
Published: January 2020

A type 1 pilus-deficient mutant of a systemically invasive Escherichia coli K1 strain was constructed by directed mutagenesis of pilA, the gene that codes for the major structural subunit of type 1 pili. By comparing this mutant with an isogenic pilA+ strain, we were able to assess the role of type 1 piliation in alimentary tract colonization and bloodstream invasion in neonatal rats. Intestinal colonization was not significantly affected by the pilA mutation; in contrast, loss of type 1 piliation correlated with a dramatic decrease in oropharyngeal colonization. Nevertheless, development of bacteremia after oral administration of E. coli K1 was not diminished by the mutation in pilA. Thus, loss of type 1 piliation correlated with a site-dependent effect on colonization within the alimentary tract while not interfering with bloodstream invasion.
Cholera toxin B-subunit gene fusion: structural and functional analysis of the chimeric protein
Authors: Dertzbaugh M T , Peterson D L , Macrina F L
Published: January 2020

A synthetic peptide, encoding amino acid residues 345 to 359 of the glucosyltransferase B enzyme of Streptococcus mutans GS-5, was genetically fused to the N-terminal end of the B-subunit gene of cholera toxin. The protein was overexpressed in Escherichia coli and retained the antigenicity associated with cholera toxin B subunit (CTB) as well as that associated with glucosyltransferase B. The addition of 15 amino acids to the N-terminal end of CTB did not appear to affect the gross structure of the protein significantly. The chimeric protein monomers assembled into a functional oligomer which exhibited only minor conformational differences from native CTB as measured by circular dichroism. The chimera bound to GM1 ganglioside and thus retained the biological activity of CTB. These results demonstrate that genetic fusion of small peptides to the N terminus of CTB has only a minimal effect on the structure and function of the protein. Furthermore, the chimera was shown to be immunogenic when fed to mice. This work has important implications in the construction of CTB chimeras for use as oral vaccines.
Characterization of the cytochalasin D-resistant (pinocytic) mechanisms of endocytosis utilized by chlamydiae
Authors: Reynolds D J , Pearce J H
Published: January 2020

The cytochalasin D-resistant (pinocytic) portion of the entry of two chlamydia strains (Chlamydia trachomatis L2/434/Bu and Chlamydia psittaci GPIC [guinea pig inclusion conjunctivitis]) was examined. By ultrastructural criteria, few organisms of either strain were observed in association with coated host-cell plasma membrane during entry into McCoy cells; this argues against a coated-pit mechanism of entry. When association with a coated membrane was seen, coat material appeared to pinch off ahead of internalizing chlamydiae. However, entry of both strains was substantially reduced by cytosol acidification, a procedure shown to prevent coated-pit vesiculation (K. Sandvig, S. Olsnes, O. W. Petersen, and B. van Deurs, J. Cell Biol. 105:679-689, 1987). No conclusive evidence of displacement of the fluid-phase marker [3H]sucrose from constitutively forming endocytic vesicles was found. Indeed the entry of strain 434 (but not strain GPIC) was accompanied by the influx of a large volume of fluid, suggesting an inducible mechanism. Additionally, entry of strain 434 (but not strain GPIC) was partially inhibitable by amiloride, yet the drug had no effect on the entry of transferrin, a ligand known to enter solely via coated pits. Our findings endorse the view that chlamydial entry can occur via a pathway involving coated pits. However, the unusual morphology of entry and lack of fluid exclusion are consistent with a process whereby although chlamydiae are not fully enclosed by coat material, their entry is dependent on the vesiculation of coated pits. Furthermore, the data support the proposition that a significant proportion of the entry of strain 434 occurs via an inducible pathway independent of coated-pit uptake.
Protective immunization against experimental Bacteroides (Porphyromonas) gingivalis infection
Authors: Chen P B , Davern L B , Schifferle R , Zambon J J
Published: January 2020

The effects of immunization in modulating the pathogenesis of Bacteroides (Porphyromonas) gingivalis infection in a murine model system were examined. BALB/c mice were immunized by intraperitoneal injection with B. gingivalis ATCC 53977 (one injection per week for 3 weeks), or with a lithium diiodosalicylate (LIS) extract (one injection per week for 3 weeks), or with lipopolysaccharide (LPS; one intravenous or intraperitoneal injection) from this same strain. Two weeks after the final immunization, the mice were challenged by subcutaneous injection of B. gingivalis ATCC 53977. Mice immunized with bacteria had no secondary lesions and no septicemia, whereas mice immunized with LIS extract had few secondary lesions and no septicemia. Mice immunized with LPS and nonimmunized mice demonstrated secondary abdominal lesions and septicemia after challenge. Bacterial cells and LIS extract, but not LPS, induced serum antibody and antigen reactive lymphocytes, as measured by enzyme-linked immunosorbent assay, immunoblot, Western immunoblot transfer, and in vitro lymphoproliferative responses. The present study suggests that immunization with a LIS extract or whole cells may induce a protective response against experimental B. gingivalis infection.
Biochemical and histologic findings in experimental pyelonephritis due to Ureaplasma urealyticum
Authors: Pickering W J , Birch D F , Kincaid-Smith P
Published: January 2020

Ureaplasma urealyticum has previously been shown to be capable of persisting in the rat kidney for up to 6 months following a single reflux challenge. We examined kidney tissue from infected animals for evidence of renal damage by using standard cytochemical and immunoenzyme methods. We also monitored changes in renal function during a 6-month study period with standard biochemical assays of plasma and urine. Histologic examination showed tubular atrophy, interstitial fibrosis, and a mononuclear infiltrate in proportion to ureaplasma counts from renal tissue. The most severe damage was accompanied by hyaline cast formation within tubules which gave rise to the typical thyroidlike appearance of chronic pyelonephritis involving conventional urinary pathogens. Macroscopic renal scarring occurred in some animals. Although damage to the renal medulla was moderate to severe, only minor changes were seen in the cortex, and glomeruli were invariably spared. Biochemical tests of renal function showed similar changes in infected and uninfected animals during the study period. Interstitial inflammation was characterized by a mononuclear cell infiltrate in which polymorphonuclear leukocytes were not conspicuous. It is evident that U. urealyticum is capable of producing chronic pyelonephritis in the rat after a single reflux challenge. The results of this study have obvious implications for the pathogenicity of these bacteria in human pyelonephritis.
Effect of type 1 piliation on in vitro killing of Escherichia coli by mouse peritoneal macrophages
Authors: Keith B R , Harris S L , Russell P W , Orndorff P E
Published: January 2020

Escherichia coli K-12 mutants possessing defined lesions affecting type 1 pilus production, receptor binding, or length were examined for their ability to resist killing by mouse peritoneal macrophages in vitro. Mutants were mixed pairwise at known ratios in wells containing macrophages, and after incubation, the ratio of the survivors was assayed. The difference in phagocytic killing between type 1 piliated cells and isogenic nonpiliated cells was significant, the piliated cells being approximately threefold more resistant. Pilus length had little effect upon survival, as the long-piliated mutants were no more resistant to killing than the normal-length parents. Interestingly, the receptor-binding function of type 1 pili was most important in effecting resistance, as mutants lacking the ability to bind receptor were killed as effectively as nonpiliated mutants. These data are consistent with the notion that pili actually impede killing by macrophages rather than serve as passive physical barriers to uptake.
A nonvirulent mutant of Listeria monocytogenes does not move intracellularly but still induces polymerization of actin
Authors: Kuhn M , Prévost M C , Mounier J , Sansonetti P J
Published: January 2020

Listeria monocytogenes has the capacity to penetrate and multiply within professional and nonprofessional phagocytic cells, such as the Caco-2 human enterocytelike cell line. It was shown recently that shortly after listeriae have been phagocytosed, the phagosomal membrane is dissolved, probably by the action of the bacterial cytolysin listeriolysin O. The listeriae, which are then lying obviously free in the cytoplasm, become surrounded by a coat of actin filaments within a few hours. Once formed, this layer of actin filaments is reorganized in an as yet unknown way to form polar tails, which seem to be associated to the generation of listerial movement inside the cytoplasm and in intercellular spread. By using transposon Tn916 mutagenesis, a bank of L. monocytogenes mutants was generated and subsequently screened by the plaque assay system in order to select an intracellular, nonmotile mutant of L. monocytogenes. One such mutant was identified. This mutant, called L. monocytogenes M117 Imt- (for intracellular motility), like the wild type, induced actin polymerization but was not able to rearrange the actin coat to generate movement and as a result remained entrapped within the actin cloud. In a mouse virulence assay, this strain was significantly reduced in virulence. L. monocytogenes M117 is the first example to date of a Listeria mutant which is still hemolytic and invasive but reduced in virulence.
Expression of a repeating phosphorylated disaccharide lipophosphoglycan epitope on the surface of macrophages infected with Leishmania donovani
Authors: Tolson D L , Turco S J , Pearson T W
Published: January 2020

Murine peritoneal macrophages were infected with living, virulent Leishmania donovani promastigotes. At intervals after infection, the macrophage surfaces were probed for the expression of lipophosphoglycan (LPG) epitopes by immunofluorescence with anti-LPG monoclonal antibodies. A repeating phosphorylated disaccharide epitope of LPG was detected as early as 5 to 10 min postinfection and was initially localized to the immediate area of internalization of the promastigote into the macrophage. The epitopes were evenly distributed over the entire macrophage surface by 25 min postinfection. Treatments which inhibited macrophage phagolysosomal degradation processes had no effect on epitope expression, whereas reagents that affected macrophage membrane flow and, thus, phagocytosis drastically reduced or abolished expression. Purified LPG or phosphoglycan, the delipidated form of the LPG molecule, was also shown to bind to a variety of different cell types in a temperature-independent manner. Since LPG has been implicated as having an immunoprotective role in leishmaniasis, these results suggest a further mechanism(s) by which Leishmania LPG might be involved in parasite pathogenicity and virulence.
Aspergillus fumigatus complement inhibitor: production, characterization, and purification by hydrophobic interaction and thin-layer chromatography
Authors: Washburn R G , DeHart D J , Agwu D E , Bryant-Varela B J , Julian N C
Published: January 2020

Aspergillus fumigatus has previously been shown to produce a soluble extracellular inhibitor of the alternative complement pathway, called Aspergillus complement inhibitor, or CI. We now report an efficient method for production of CI which relies on the fact that poorly conidiating cultures yielded CI activity with approximately sevenfold-higher potency than CI produced by conidiating cultures. CI from poorly conidiating cultures provided 50% inhibition of alternative pathway-mediated binding of 125I-labeled complement component C3 to cryptococcal blastoconidia at a mean concentration of 60 micrograms/ml. The ability of crude CI to inhibit the alternative complement pathway seemed to be independent of intact protein or polysaccharide structure, as evidenced by resistance of inhibitory activity to digestion by proteases, including subtilisin, alpha-chymotrypsin, papain, and pepsin as well as endoglycosidases F and H. Separation of the active inhibitory component of CI from contaminating materials contained in crude CI preparations was achieved by using Phenylsuperose hydrophobic interaction chromatography in a fast protein liquid chromatography system. The active material proved to be extremely hydrophobic, desorbing from the column only during elution with ethanol; it contained only 15% protein and 5% polysaccharide. Furthermore, results from preparative thin-layer chromatography indicated that lipids which comigrated with phosphatidylserine/phosphatidylinositol and phosphatidylethanolamine possessed significant complement-inhibitory activity. Taken together, these data suggested that phospholipids from A. fumigatus contributed to the functional activity of CI.
Chlamydicidal activity of human alveolar macrophages
Authors: Nakajo M N , Roblin P M , Hammerschlag M R , Smith P , Nowakowski M
Published: January 2020

Pneumonia due to Chlamydia trachomatis is a disease limited mainly to infants under 6 months of age. Rare cases have been reported in immunocompromised adults. One possible reason for the propensity of the pneumonia to occur in the very young may be related to differences in the phagocytic and bactericidal capacity of alveolar macrophages (AMs) in young infants and adults. At birth a function of AMs is clearance of surfactant-related material from the alveolar surface. Studies in animals have suggested that engorgement of AMs with surfactant-related lipids may reduce the microbicidal capacity of these cells. In the present study we determined that AMs obtained from healthy, nonsmoking adults were capable of killing both human biovars of C. trachomatis, with complete killing observed by 48 h after inoculation. Preincubation of AMs from adults with surfactant did not reduce the capacity of the cells to kill C. trachomatis.
Inability of toxin inhibitors to neutralize enhanced toxicity caused by bacteria adherent to tissue culture cells
Authors: Ofek I , Zafriri D , Goldhar J , Eisenstein B I
Published: January 2020

Toxicity to Y-1 adrenal mouse cells caused by heat-labile toxin secreted by an enterotoxigenic strain of Escherichia coli (H-10407-p) was 40-fold enhanced in mixtures containing organisms capable of adhering to the Y-1 cells compared with monolayers exposed to organisms whose adherence was inhibited by mannoside. Severalfold the concentrations of anti-heat-labile toxin antibodies required to neutralize the toxicity of nonadherent bacteria were unable to neutralize the toxicity caused by adherent bacteria. The cytolytic activity toward tissue culture cells and mouse peritoneal macrophages caused by streptolysin S carried by Streptococcus pyogenes was severalfold increased in mixtures containing organisms capable of adhering to the target cells compared with mixtures containing nonadherent bacteria. The ability of trypan blue and RNA core to inhibit the cell-bound streptolysin S was determined in tissue culture cells containing adherent streptococci and mixtures of streptococci randomly colliding with erythrocytes. Both inhibitors were markedly less effective in neutralizing cytolysis than in their ability to neutralize hemolysis. We conclude that compared with toxins produced by nonadherent bacteria, those produced by bacteria adherent to cells are targeted more efficiently and become relatively inaccessible to neutralization by toxin inhibitors.
Cytokine induction by lipopolysaccharide (LPS) corresponds to lethal toxicity and is inhibited by nontoxic Rhodobacter capsulatus LPS
Authors: Loppnow H , Libby P , Freudenberg M , Krauss J H , Weckesser J , Mayer H
Published: January 2020

Many pathological effects of gram-negative bacteria are produced by their cell wall-derived lipopolysaccharides (LPSs). Differing pathogenicity of gram-negative LPSs, however, may depend on their capacities to induce cytokines. Thus, we studied the lethal toxicity of four nonenterobacterial LPSs and compared it with their capacity to induce mononuclear cell (MNC)-derived interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF). Unstimulated MNC did not release these cytokines. LPS from the phototrophic strain Rhodobacter capsulatus 37b4 elaborated little toxicity in galactosamine-treated mice (10 micrograms of LPS per mouse was the 100% lethal dose [LD100]) and induced IL-1 and IL-6 release only at high concentrations (10 to 50 micrograms of LPS per ml). R. capsulatus LPS failed to induce TNF activity even at the highest concentration tested (100 micrograms of LPS per ml). In contrast, LPS derived from Pseudomonas diminuta NCTC 8545 or the nodulating species Bradyrhizobium lupini DSM 30140 and Rhizobium meliloti 10406 expressed lethal toxicity (LD100, 1,000, 100, and 10 ng per mouse, respectively) and induced IL-1 or IL-6 (10 to 100, 10, and 1 ng of LPS per ml, respectively) at concentrations 1,000- to 10,000-fold lower than effective levels of R. capsulatus LPS. LPSs from P. diminuta, B. lupini, and R. meliloti also stimulated TNF production and release. MNC accumulated cell-associated IL-1 activities under circumstances in which released activity was readily detected. The cells contained only scant IL-6 activity, indicating release of this mediator rather than intracellular accumulation. Antisera to the respective cytokines inactivated biological activities of the samples selectively. The R. capsulatus LPS inhibited cytokine induction by LPS from P. diminuta, B. lupini, and R. meliloti in coincubation experiments. These results show that the in vivo lethality of the LPSs tested correlates with the induction of monocyte-derived cytokines in vitro. The results of this study suggest that the different lethality of various LPSs from gram-negative bacteria may be due to the differential ability of these LPSs to induce cytokine production.
Isolation of Listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread
Authors: Sun A N , Camilli A , Portnoy D A
Published: January 2020

To dissect the regulatory and structural requirements for Listeria monocytogenes intracellular growth and cell-to-cell spread, we designed a protocol based on transposon mutagenesis and the isolation of mutants which form small plaques in monolayers of mouse L2 cell fibroblasts. Two different transposable elements were used to generate libraries of insertion mutants: Tn916 and a derivative of Tn917-lac, Tn917-LTV3. Ten classes of mutants were isolated and evaluated for growth and cell-to-cell spread in J774 mouse macrophagelike cells, Henle 407 human epithelial cells, and mouse bone marrow-derived macrophages. Mutants were also evaluated for secretion of hemolysin and phospholipase (assayed by egg yolk opacity) and association with F-actin in the cytoplasm of cells, using NBD-phallacidin staining. The ten classes of mutants included (i) mutants showing abortive intracellular and extracellular growth; (ii) mutants showing abortive intracellular growth; (iii) rough mutants; (iv) mutants showing greatly reduced hemolysin and phospholipase secretion but showing normal growth in cells and little or no association with F-actin; (v) mutants with mutations mapping to an open reading frame (ORF) adjacent to hlyA and referred to as ORF U, lacking phospholipase activity, and with 50% normal hemolysin activity; (vi) mutants with reduced secretion of both hemolysin and phospholipase; (vii) nonhemolytic mutants with mutations mapping to the structural gene, hlyA; (viii) mutants with 25% normal hemolysin secretion and absolutely no association with F-actin; (ix) mutants with mutations mapping to ORF U, lacking phospholipase activity, and with normal hemolysin activity; and (x) mutants showing a mixed-plaque morphology but normal for all other parameters.
Effects of Escherichia coli hemolysin on endothelial cell function
Authors: Suttorp N , Flöer B , Schnittler H , Seeger W , Bhakdi S
Published: January 2020

Escherichia coli hemolysin is considered an important virulence factor in extraintestinal E. coli infections. The present study demonstrates that cultured pulmonary artery endothelial cells are susceptible to attack by low concentrations of E. coli hemolysin (greater than or equal to 0.05 hemolytic units/ml; greater than or equal to 5 ng/ml). Sublytic amounts of hemolysin increased the permeability of endothelial cell monolayers in a time- and dose-dependent manner. The hydraulic conductivity increased approximately 30-fold and the reflection coefficient for large molecules dropped from 0.71 to less than 0.05, indicating a toxin-induced loss of endothelial barrier function. The alterations of endothelial monolayer permeability were accompanied by cell retraction and interendothelial gap formation. In addition, E. coli hemolysin stimulated prostacyclin synthesis in endothelial cells. This effect was strictly dependent on the presence of extracellular Ca2+ but not of Mg2+. An enhanced passive influx of 45Ca2+ and 3H-sucrose but not of tritiated inulin and dextran was noted in toxin-treated cells, indicating that small transmembrane pores comparable to those detected in rabbit erythrocytes had been generated in endothelial cell membranes. These pores may act as nonphysiologic Ca2+ gates, thereby initiating different Ca2+-dependent cellular processes. We conclude that endothelial cells are highly susceptible to E. coli hemolysin and that two major endothelial cell functions are altered by very low concentrations of hemolysin.
Binding of viridans group streptococci to human platelets: a quantitative analysis
Authors: Sullam P M , Payan D G , Dazin P F , Valone F H
Published: January 2020

The binding of viridans group streptococci with human platelets was analyzed by two-color flow cytometry. Binding was detected within 15 s of mixing bacteria and platelets. At ratios of bacteria to platelets of 1:1, 10:1, 100:1, and 1,000:1, the percentages of bound streptococci (mean +/- standard deviation) were 93.2% +/- 5.4%, 80.0% +/- 8.6%, 39.8% +/- 11.1%, and 12.5% +/- 2.0%, respectively. Binding of labeled bacteria was reversed by adding a 500-fold excess of unlabeled streptococci. These results demonstrate that streptococcus-platelet binding is rapid, reversible, and saturable, which suggests a specific receptor-ligand interaction.