bluuebunny/crossref_metadata_2025 huggingface dataset:
Ovine pulmonary surfactant induces killing of Pasteurella haemolytica, Escherichia coli, and Klebsiella pneumoniae by normal serum
Authors: Brogden K A
Published: January 2020

Pulmonary surfactant has been shown to play an increasingly important role in bacterial clearance at the alveolar surface in the lung. This study describes a bactericidal mechanism in which ovine pulmonary surfactant induces killing of Pasteurella haemolytica by normal serum. To demonstrate killing, six bacterial species were incubated first with pulmonary surfactant for 60 min at 37 degrees C and then with serum for an additional 60 min at 37 degrees C. P. haemolytica type A1 strains 82-25 and L101, a P. haemolytica type 2 strain, Escherichia coli, and Klebsiella pneumoniae were susceptible and Pasteurella multocida, Serratia marcescens, and Pseudomonas aeruginosa were not susceptible to killing by ovine pulmonary surfactant and normal serum. No bacteria incubated with bovine pulmonary surfactant were killed by normal serum. Although the species origin of pulmonary surfactant was selective, the species origin of serum was not. P. haemolytica incubated with ovine pulmonary surfactant was killed by fetal calf serum, gnotobiotic calf serum, pooled normal sheep serum, pooled normal rabbit serum, and pooled guinea pig serum. Ultrastructurally, killed P. haemolytica suspensions contained dead cells and cells distorted with vacuoles between the cytoplasmic membrane and the cytoplasm. The mechanism of killing did not correlate with concentrations of complement or lysozyme or titers of residual antibody in either the pulmonary surfactant or the serum, and killing was reduced by preincubation of surfactant with P. haemolytica lipopolysaccharide. Preliminary characterization of both surfactant and serum implicate a low-molecular-weight proteinaceous component in the surfactant and serum albumin in the serum. This mechanism may help clear certain gram-negative bacteria from the lungs of sheep as a part of the pulmonary innate defense system.
Human colonic epithelial cells, HT29/C1, treated with crude Bacteroides fragilis enterotoxin dramatically alter their morphology
Authors: Weikel C S , Grieco F D , Reuben J , Myers L L , Sack R B
Published: January 2020

Bacteroides fragilis has been associated with causation of diarrheal disease in livestock and humans. To date, conventional tissue culture and animal assays used to detect the biologic activity of bacterial enterotoxins have failed with enterotoxigenic B. fragilis. Although enterotoxigenic B. fragilis stimulates intestinal secretion in lamb and calf ligated intestinal loops, infant rabbits, and adult rabbits with ligated ceca, these animal systems are costly and complicated, which limits their usefulness for identification of enterotoxigenic B. fragilis strains. Using the cloned human colonic-epithelial-cell line HT29/C1, we have developed an in vitro assay that is 89% sensitive and 100% specific in detecting enterotoxigenic B. fragilis strains as defined by the lamb ligated-intestinal-loop assay. Subconfluent HT29/C1 cells treated with concentrated bacterium-free culture supernatants of enterotoxigenic B. fragilis strains develop specific and striking morphologic changes including loss of cell-to-cell attachments, rounding, swelling, and, in some cases, pyknosis. These morphologic changes are initially visible at 1 h after treatment and progress over at least the first 24 h. This tissue culture assay should prove useful in epidemiologic studies of enterotoxigenic B. fragilis and may facilitate basic studies to identify the B. fragilis toxin(s) and its mechanism of action.
Human platelet aggregation by Yersinia pseudotuberculosis is mediated by invasin
Authors: Simonet M , Triadou P , Frehel C , Morel-Kopp M C , Kaplan C , Berche P
Published: January 2020

Plasmid-free strains of Yersinia pseudotuberculosis induce aggregation of human platelets in vitro. It appears that this phenomenon is mediated by invasin (Inv), a 103-kDa outer membrane protein that permits bacteria to penetrate mammalian cells, since (i) an isogenic inv-deficient mutant failed to aggregate platelets compared with the parental strain; (ii) a monoclonal antibody directed against invasin inhibited platelet aggregation; (iii) Inv+ Escherichia coli HB101 promoted platelet aggregation. Platelet receptors for invasin were identified by using a panel of anti-platelet glycoprotein monoclonal antibodies in a bacterial adhesion assay. We found that bacteria bind to platelet membrane glycoproteins Ic and IIa. Electron microscopic study of bacterium-platelet interactions also revealed that bacteria expressing invasin attach to and are phagocytized by thrombocytes, in contrast to inv-deficient bacteria, indicating that these anucleated cells are able to internalize bacteria in vitro after specific interaction with invasin.
Identification of a genetic locus essential for capsule sialylation in type III group B streptococci
Authors: Wessels M R , Haft R F , Heggen L M , Rubens C E
Published: January 2020

The type III capsular polysaccharide of group B streptococci (GBS) consists of a linear backbone with short side chains ending in residues of N-acetylneuraminic acid, or sialic acid. The presence of sialic acid on the surface of the organism inhibits activation of the alternative pathway of complement and is thought to be an important element in the virulence function of the capsule. We showed previously that a mutant strain of GBS that expressed a sialic acid-deficient, or asialo, form of the type III polysaccharide was avirulent, supporting a virulence function for capsular sialic acid. We now report the derivation of an asialo capsule mutant from a highly encapsulated wild-type strain of type III GBS, strain COH1, by insertional mutagenesis with transposon Tn916 delta E. In contrast to the wild-type strain, the asialo mutant strain COH1-11 was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat model of GBS infection. The asialo mutant accumulated free intracellular sialic acid, suggesting a defect subsequent to sialic acid synthesis in the biosynthetic pathway leading to capsule sialylation. The specific biosynthetic defect in mutant strain COH1-11 was found to be in the activation of free sialic acid to CMP-sialic acid: CMP-sialic acid synthetase activity was present in the wild-type strain COH1 but was not detected in the asialo mutant strain COH1-11. One of the two transposon insertions in the asialo mutant COH1-11 mapped to the same chromosomal location as one of the two Tn916 insertions in the previously reported asialo mutant COH31-21, identifying this site as a genetic locus necessary for expression of CMP-sialic acid synthetase activity. These studies demonstrate that the enzymatic synthesis of CMP-sialic acid by GBS is an essential step in sialylation of the type III capsular polysaccharide.
Haemophilus ducreyi, a cytotoxin-producing bacterium
Authors: Purvén M , Lagergård T
Published: January 2020

An extra- and an intracellular product from the bacterium Haemophilus ducreyi were shown to have a cytotoxic effect on cell lines of epithelial origin, e.g., HEp-2 and HeLa cells. The cytotoxic effect appeared on cells within 24 h and resulted in cell death and morphologically changed cells. The cytotoxic activity was heat and pronase sensitive. The activity could be removed by incubation with the target cells, suggesting attachment of the agent to the cells. The cytotoxic products were secreted into the environment during exponential growth of bacteria and produced by most of the H. ducreyi strains tested. The activity was neutralized by homologous rabbit immune serum but not by the corresponding preimmune serum. The results indicate that strains of H. ducreyi produce a cytotoxin which may be of importance to the pathogenesis of chancroid.
The variable C-terminal region of the Mycobacterium leprae 70-kilodalton heat shock protein is the target for humoral immune responses
Authors: Davenport M P , McKenzie K R , Basten A , Britton W J
Published: January 2020

The 70-kDa heat shock protein of Mycobacterium leprae has a high degree of homology with the human hsp70 protein, yet it still elicits T-lymphocyte responses in subjects infected with M. leprae or vaccinated with the related Mycobacterium bovis BCG. We examined the serological responses to this protein by using recombinant protein fragments expressed from mutants with deletions of the M. leprae p70 gene. Monoclonal antibodies raised against either M. bovis or M. leprae p70 reacted with the C-terminal fragments but not the N-terminal fragments in a solid-phase enzyme-linked immunosorbent assay and an immunoblot assay. Inhibition enzyme-linked immunosorbent assays confirmed that two separate epitopes were defined by these monoclonal antibodies. Murine polyclonal sera also showed stronger binding to the C-terminal fragments. Sera from 33 and 48% of lepromatous leprosy patients reacted with M. leprae and M. bovis p70. This reactivity was mycobacterium specific, since few sera from control subjects in the same leprosy-endemic region were seropositive. The levels of anti-mycobacterial hsp70 antibodies were higher in patients with lepromatous leprosy than in those with tuberculoid leprosy or tuberculosis. The reactivity of sera from patients with leprosy was maximal with the C-terminal fragments. Therefore the C-terminal portion of M. leprae hsp70, which includes the region of maximum divergence from human hsp70, is the major target for the humoral immune response to the protein.
Adherence, coaggregation, and hydrophobicity of Streptococcus gordonii associated with expression of cell surface lipoproteins
Authors: Jenkinson H F
Published: January 2020

Streptococcus gordonii Challis incorporated exogenous [3H]palmitate into 13 polypeptides extractable from intact cells with sodium dodecyl sulfate. A 76-kDa surface-exposed polypeptide, implicated previously as a cell aggregation determinant, was shown to be one of these lipid-modified polypeptides. Differences in sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of lipopolypeptides were detected with mutants of S. gordonii that were altered in adherence, aggregation, coaggregation, or hydrophobicity. Lipid-modified polypeptides, tightly associated with the cell membrane, may be involved in the expression of cell surface properties of S. gordonii important for colonization of the human oral cavity.
Defective production of interleukin-6 in very small premature infants in response to bacterial pathogens
Authors: Yachie A , Takano N , Ohta K , Uehara T , Fujita S , Miyawaki T , Taniguchi N
Published: January 2020

In the present study, a whole-blood culture was employed to examine the ability of preterm and term newborn infants to produce interleukin-6 (IL-6) in response to major bacterial pathogens such as group B streptococci, Escherichia coli, Listeria monocytogenes, and Streptococcus pneumoniae. Similarly, in response to stimulation with lipopolysaccharide, a potent stimulant for monocyte cytokine production, appreciable levels of IL-6 activity in the stimulated whole blood from term newborns as well as adults was effectively induced by all of these pathogens. In contrast to that of term infants, the bacteria-induced IL-6 production of preterm infants, especially those born before 30 weeks of gestation, was somewhat decreased (P less than 0.01 for each pathogen). It was also demonstrated that IL-6 responses to lipopolysaccharide stimulation were reduced in preterm newborns (for term versus preterm newborns less than 30 weeks of gestation, P was less than 0.01). These findings imply some inherent abnormality of monocytes in preterm babies. The diminished IL-6 production may be partly responsible for the susceptibility of preterm newborn infants to bacterial infections.
Long-term protection of mice from Lyme disease by vaccination with OspA
Authors: Fikrig E , Barthold S W , Kantor F S , Flavell R A
Published: January 2020

Mice vaccinated with recombinant outer surface protein A (OspA) have been shown to be protected from infection with Borrelia burgdorferi, the agent of Lyme disease, when sacrificed 14 days after challenge with an intradermal inoculum of the spirochete. To determine whether infection was not merely delayed and that protection was long-lasting, we sacrificed vaccinated mice 60, 120, and 180 days after challenge; and to determine whether vaccinated mice retained their immune state over long periods, we challenged mice with B. burgdorferi 60, 90, 120, and 150 days after vaccination. The results of both groups of experiments show that the mice remained free from infection and disease and extend the usefulness of OspA as a vaccine candidate for Lyme borreliosis.
Activation of macrophages for destruction of Francisella tularensis: identification of cytokines, effector cells, and effector molecules
Authors: Fortier A H , Polsinelli T , Green S J , Nacy C A
Published: January 2020

Francisella tularensis live vaccine strain (LVS) was grown in culture with nonadherent resident, starch-elicited, or Proteose Peptone-elicited peritoneal cells. Numbers of bacteria increased 4 logs over the input inoculum in 48 to 72 h. Growth rates were faster in inflammatory cells than in resident cells: generation times for the bacterium were 3 h in inflammatory cells and 6 h in resident macrophages. LVS-infected macrophage cultures treated with lymphokines did not support growth of the bacterium, although lymphokines alone had no inhibitory effects on replication of LVS in culture medium devoid of cells. Removal of gamma interferon (IFN-gamma) by immunoaffinity precipitation rendered lymphokines ineffective for induction of macrophage anti-LVS activity, and recombinant IFN-gamma stimulated both resident and inflammatory macrophage populations to inhibit LVS growth in vitro. Inflammatory macrophages were more sensitive to effects of IFN-gamma: half-maximal activity was achieved at 5 U/ml for inflammatory macrophages and 20 U/ml for resident macrophages. IFN-gamma-induced anti-LVS activity correlated with the production of nitrite (NO2-), an oxidative end product of L-arginine-derived nitric oxide (NO). Anti-LVS activity and nitrite production were both completely inhibited by the addition of either the L-arginine analog NG-monomethyl-L-arginine or anti-tumor necrosis factor antibodies to activated macrophage cultures. Thus, macrophages can be activated by IFN-gamma to suppress the growth of F. tularensis by generation of toxic levels of NO, and inflammatory macrophages are substantially more sensitive to activation activities of IFN-gamma for this effector reaction than are more differentiated resident cells.
A synthetic glycoconjugate representing the genus-specific epitope of chlamydial lipopolysaccharide exhibits the same specificity as its natural counterpart
Authors: Fu Y , Baumann M , Kosma P , Brade L , Brade H
Published: January 2020

The tetrasaccharide 3-deoxy-alpha-D-manno-2-octulosonic acid (alpha-KDO) (2----8)-alpha-KDO(2----4)-alpha-KDO(2----6)-beta GlcNAc, a partial structure of chlamydial lipopolysaccharide (LPS) representing a genus-specific epitope, was synthesized and covalently linked to bovine serum albumin, resulting in an artificial glycoconjugate antigen. Mice were immunized with the glycoconjugate to prepare chlamydia-specific monoclonal antibodies. They were selected with chlamydia-specific LPS antigens and the structurally and antigenically related Re-type LPS of a Salmonella minnesota rough mutant. Characterization of the selected antibodies was by (i) hemagglutination of sheep erythrocytes coated with recombinant chlamydia-specific LPS, (ii) inhibition by synthetic polyacrylamide derivatives containing the genus-specific epitope or partial structures thereof, (iii) enzyme immunoassay with recombinant LPS and synthetic bovine serum albumin glycoconjugates as solid-phase antigens, (iv) immunofluorescence of L929 monolayers infected with Chlamydia psittaci or C. trachomatis, and (v) Western immunoblots with glycoconjugates and LPS as the antigen. Two groups of monoclonal antibodies were obtained; the monoclonal antibodies in one group cross-reacted with chlamydial and Re-type LPS, but those of the other group were chlamydia specific. Among the latter, KDO trisaccharide-specific antibodies that had the same epitope specificity as antibodies obtained after immunization with chlamydial elementary bodies were identified; however, they exhibited a more than 100-fold higher affinity. In addition, antibodies that bound preferentially to the 2.8-linked KDO disaccharide were detected, although with lower affinity. The data show that the artificial glycoconjugate antigen is similar to its natural counterpart.
Purification and characterization of an enterotoxin from Bacteroides fragilis
Authors: Van Tassell R L , Lyerly D M , Wilkins T D
Published: January 2020

An enterotoxin produced by Bacteroides fragilis was purified to homogeneity and characterized as to its biological activity and basic molecular properties. Toxin preparations were prepared by growing B. fragilis VPI 13784 in brain heart infusion broth to early stationary phase, immediately precipitating the culture supernatant fluid with 70% ammonium sulfate, and stabilizing the precipitate with the protease inhibitor TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone). The toxin was sequentially purified by anion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on phenyl-agarose, and high-resolution ion-exchange chromatography on Mono Q. The toxin appeared homogeneous as judged by polyacrylamide gel electrophoresis. The estimated molecular weight of the highly purified toxin as determined by gel filtration chromatography on Superose-12 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 19,000. It has an isoelectric point of approximately 4.5 and is stable at pHs 5 to 10. The purified toxin is stable at -20 and 4 degrees C and upon freeze-drying, but it is unstable at temperatures above 55 degrees C. It is sensitive to proteinase K and Streptomyces protease but is resistant to trypsin and chymotrypsin. The activity of the purified toxin is neutralized by antiserum to a toxigenic strain of B. fragilis but not by antiserum to nontoxigenic strains. N-terminal amino acid analysis reveal an unambiguous sequence of Ala-Val-Pro-Ser-Glu-Pro-Lys-Thr-Val-Tyr-Val-Ile-Xxx-Leu-Arg-Glu-Asn-Gly- Ser-Thr . The highly purified toxin induced a strong fluid accumulation response in the lamb ileal-loop assay as well as a cytotoxic response (cell rounding) on HT-29 colon carcinoma cells. Thus, the purified toxin can cause both enterotoxic and cytotoxic activities.
Role of the Staphylococcus epidermidis slime layer in experimental tunnel tract infections
Authors: Patrick C C , Plaunt M R , Hetherington S V , May S M
Published: January 2020

An experimental animal model was used to assess the slime layer of Staphylococcus epidermidis as a pathogenic factor in tunnel tract infections. Mice were inoculated with high-slime-producing or non-slime-producing strains of S. epidermidis, either along the length of a subcutaneous catheter or in the area where a catheter had been placed and immediately removed (controls). Among the catheter-bearing mice, the phenotypically distinct staphylococci produced similar, high frequencies of abscess formation (72% [44 of 61] versus 81% [31 of 38]; P = 0.29). In controls, the non-slime-producing organisms were significantly more pathogenic (87% [40 of 46] versus 57% [25 of 44] abscess formation; P = 0.001). No consistent difference was detected between blood isolates obtained from patients with central venous catheter bacteremia and those from neonates with bacteremia in the absence of a prosthetic medical device. Quantitative culture of removed catheters showed greater adherence by the slime-producing isolates (P = 0.014). In this mouse model, slime production by S. epidermidis did not increase the risk of catheter tunnel tract infection, despite the greater catheter adherence of the slime-producing organisms. These findings suggest that traumatized tissue may be a sufficient condition for the development of S. epidermidis catheter-associated infections.
Tumor necrosis factor production in patients with leprosy
Authors: Barnes P F , Chatterjee D , Brennan P J , Rea T H , Modlin R L
Published: January 2020

The spectrum of host responses to Mycobacterium leprae provides a model for investigating the role of cytokines in the pathogenesis of mycobacterial disease. Of particular interest is tumor necrosis factor (TNF), a cytokine which may have both antimycobacterial and immunopathologic effects. To evaluate the potential role of TNF in leprosy, we measured TNF production in response to M. leprae and its defined constituents by peripheral blood mononuclear cells from patients across the spectrum of disease. The levels of TNF induced through the stimulation of cells with M. leprae or its dominant "lipopolysaccharide," lipoarabinomannan, were higher in patients with the tuberculoid form of the disease than in those with the lepromatous form. In patients with erythema nodosum leprosum (ENL), a reactional state of lepromatous leprosy, the levels of TNF release by peripheral blood mononuclear cells were higher than in any other form of the disease. Treatment of ENL patients with thalidomide reduced TNF secretion by more than 90%. The mycolylarabinogalactan-peptidoglycan complex of Mycobacterium species, the protein-peptidoglycan complex, and muramyl dipeptide all elicited significant TNF release. Therefore, TNF release appears to be triggered by at least two major cell wall structural constituents of M. leprae, lipoarabinomannan and segments of the cell wall skeleton. The prominent TNF release in patients with the paucibacillary tuberculoid form of the disease compared with that in patients with the multibacillary lepromatous form suggests that this cytokine contributes to a resistant immune response to mycobacterial infection. However, the marked TNF release in patients with ENL indicates that TNF may also mediate immunopathologic effects, such as fever and tissue damage.
Improved rat model of Pneumocystis carinii pneumonia: induced laboratory infections in Pneumocystis-free animals
Authors: Boylan C J , Current W L
Published: January 2020

An immunosuppressed rat model of Pneumocystis carinii pneumonia is described that utilizes simple, noninvasive intratracheal (i.t.) inoculation of cryopreserved parasites and results in development of severe P. carinii pneumonia within 5 weeks. This is an improvement over the most commonly used models of P. carinii pneumonia that rely on immune suppression to activate latent P. carinii infections and that often require 8 to 12 weeks to produce heavy infections of P. carinii. It is also less labor intensive than more recent models requiring surgical instillation of parasites. Our report describes a series of preliminary studies to select an appropriate strain of rat; to determine suitable methods for inducing uniform immunosuppression, P. carinii inoculation, and laboratory maintenance of P. carinii; and to determine effective animal husbandry methods for maintaining animals free from serious secondary infections. Results of our more detailed studies demonstrate that animals receiving two or three i.t. inoculations of approximately 10(6) cryopreserved P. carinii organisms have a predictable course of disease progression which includes moderate P. carinii infections within 3 weeks, severe P. carinii pneumonia in 5 weeks, and a high percentage of mortality due to P. carinii pneumonia in 6 weeks. Parasites were distributed evenly between the right and left lungs, regardless of the number of P. carinii inoculations administered. Non-P. carinii-inoculated immunosuppressed control rats maintained in microisolator cages remained free of P. carinii, thus providing an important control that is missing from many P. carinii pneumonia models. Most non-P. carinii-inoculated control animals and P. carinii-inoculated rats treated with trimethoprim-sulfamethoxazole that were housed in open caging in the same room containing heavily infected animals had no detectable infections after 5 to 6 weeks of immunosuppression; however, some had a small number of P. carinii in their lungs. Because heavy, reproducible infections are achieved 5 weeks after i.t. inoculation, because few animals are lost to secondary infections, and because animals can be maintained as noninfected contemporaneous controls, this animal model is useful for the maintenance of P. carinii strains, for studies of the transmission and natural history of P. carinii, for the production of large numbers of organisms for laboratory studies, and for the evaluation of potential anti-P. carinii drugs.
Murine monoclonal antibodies to type Ib polysaccharide of group B streptococci bind to human milk oligosaccharides
Authors: Pritchard D G , Gray B M , Egan M L
Published: January 2020

The chemical structures of the repeating units of the type Ib polysaccharide of group B streptococci and of the desialylated form of this antigen are almost identical to those of some oligosaccharides in human milk and certain fetal antigens. The structural similarities suggested that the molecules may be immunologically cross-reactive. Mouse monoclonal antibodies to the sialylated and nonsialylated forms of the type Ib polysaccharide were produced and tested for their ability to bind to immobilized human milk oligosaccharides. One antibody, SMB19, reacted specifically with the sialylated form of the type Ib polysaccharide and was also bound by an affinity column containing immobilized sialyllacto-N-tetraose a. The antibody was eluted from the affinity column with EDTA, since its binding to the antigen was calcium dependent. A second monoclonal antibody. SIbD2, bound specifically to the nonsialylated form of the type Ib polysaccharide and also to immobilized lacto-N-tetraose. The antibody was eluted from the affinity column at an acidic pH and retained immunologic activity. These results further extend our previous observations that certain antibodies raised against group B streptococci can also react with normal human glycoconjugates.
Anti-infective effect of poly-beta 1-6-glucotriosyl-beta 1-3-glucopyranose glucan in vivo
Authors: Onderdonk A B , Cisneros R L , Hinkson P , Ostroff G
Published: January 2020

Mice challenged with Escherichia coli or Staphylococcus aureus were protected against lethal peritonitis by the intravenous administration of 10 micrograms of poly-beta 1-6-glucotriosyl-beta 1-3-glucopyranose (PGG) glucan per animal 4 to 6 h prior to bacterial challenge. Subsequent studies with the rat model for intra-abdominal sepsis indicated that intramuscular doses of 10 to 100 micrograms per animal 24 and 4 h prior to surgical implantation of the bacterial inoculum reduced the early mortality associated with the peritonitis phase of this experimental disease process. Quantitative cultures of blood obtained from challenged rats showed that significantly fewer organisms were present in the blood of PGG glucan-treated animals than in that of untreated animals. Quantitative studies of leukocytes of rats and mice following a single injection of PGG glucan showed a modest transient increase in the total leukocyte count. The possible mechanisms by which protection occurs in the animal model system are discussed.
Modulation of a surface antigen of Entamoeba histolytica in response to bacteria
Authors: Bhattacharya A , Ghildyal R , Prasad J , Bhattacharya S , Diamond L S
Published: January 2020

Changes in the cell surface of Entamoeba histolytica, a human intestinal parasite and the causative agent of amebic dysentery, were examined with a monoclonal antibody, 2D7.10, which selectively recognizes carbohydrate epitopes in some axenic amebic strains. While high-level expression of this epitope was observed in axenic amebae, it was either absent or present only in small amounts in xenic amebae. Furthermore, reassociation of the axenic amebae with intestinal flora resulted in loss of the 2D7.10 epitope. Our data suggest that surface antigens of E. histolytica can be modulated in response to bacteria and may provide an explanation for the observed influence of bacteria on amebic virulence.
Low biological activity of Helicobacter pylori lipopolysaccharide
Authors: Muotiala A , Helander I M , Pyhälä L , Kosunen T U , Moran A P
Published: January 2020

Lipopolysaccharide from the gastroduodenal pathogen Helicobacter pylori was tested for its ability to induce mitogenicity in mouse spleen cells, pyrogenicity in rabbits, and toxic lethality in galactosamine-sensitized mice. Compared with those for enterobacterial lipopolysaccharide, mitogenicity and pyrogenicity were a thousand-fold lower and lethal toxicity was 500-fold lower. We suggest that the phosphorylation pattern and acylation in lipid A are responsible for the low biological activity.
Characterization of rickettsial attachment to host cells by flow cytometry
Authors: Li H , Walker D H
Published: January 2020

The mechanisms of rickettsial attachment have been studied by measuring quantitative changes in rickettsial binding to host cells by flow cytometry after different treatments of the rickettsiae and host cells. Time-dependent binding of Rickettsia conorii to host cells was demonstrated by the increasing intensity of host cell surface fluorescence of rickettsia-host cell combinations when examined with a rickettsia-specific monoclonal antibody. More than 70% of host cells had intensity of fluorescence above the threshold value after 10 min of incubation, owing to rickettsiae bound to the cell surface, and the greatest fluorescence intensity indicative of binding occurred at 20 min. The binding kinetics was rickettsial dose dependent. The binding of rickettsiae to host cells was greatly decreased when host cells or rickettsiae were treated with 1% paraformaldehyde for 30 min or 0.25% trypsin for 5 or 15 min, respectively. Rickettsiae that were heated at 56 degrees C for 15 min lost more than 80% of their ability to attach to host cells. R. rickettsii, an organism closely related to R. conorii, competitively inhibited the attachment of R. conorii (51% inhibition when mixed in equal numbers). These results indicate that the rickettsial binding structures are trypsin and heat sensitive and likely to be surface proteins.
Demonstration of chlamydial RNA and DNA during a culture-negative state
Authors: Holland S M , Hudson A P , Bobo L , Whittum-Hudson J A , Viscidi R P , Quinn T C , Taylor H R
Published: January 2020

Trachoma is a common blinding disease of humans caused by ocular infections with Chlamydia trachomatis. The cynomolgus monkey is a valuable primate model for the detection, pathobiology, and treatment of this infection. We have used this model system to compare the relative ability of tissue culture, direct fluorescence cytology, a modified polymerase chain reaction, and RNA blotting to detect C. trachomatis following primary infection and reinfection over 34 weeks. Six cynomolgus monkeys were given a primary ocular chlamydia infection, and 20 weeks later they were reinoculated with the same organism. All animals showed brisk inflammatory responses to the primary infection and milder inflammatory reactions to reinfection. All four diagnostic techniques detected chlamydia at 1 week after primary infection, but both nucleic acid detection methods suggested that organisms were present longer after primary infection than did either tissue culture or direct fluorescence cytology (16 weeks for RNA blotting versus 12 weeks for tissue culture). Following reinoculation at 20 weeks, the period of C. trachomatis detection by tissue culture or direct fluorescence cytology (4 weeks) was much shorter than after primary infection. In contrast, nucleic acid detection was positive for up to 5 weeks longer than tissue culture or direct fluorescence cytology. Both polymerase chain reaction and RNA blotting, which involved no amplification step, indicated the presence of organisms during the culture-negative period. These data suggest that live chlamydiae may remain at a site of infection and produce inflammation beyond the time at which standard microbiological techniques are able to detect them.
Parallel induction by glucose of adherence and a polysaccharide antigen specific for plastic-adherent Staphylococcus epidermidis: evidence for functional relation to intercellular adhesion
Authors: Mack D , Siemssen N , Laufs R
Published: January 2020

The initial attachment and the accumulation of Staphylococcus epidermidis on polymer surfaces in multilayered cell clusters embedded in amorphous slime, which together lead to the plastic-adherent phenotype detected by the adherence assay used in this study, have been proposed to be major virulence factors of these bacteria. An antigen specific for plastic-adherent S. epidermidis strains was detected by an indirect immunofluorescence test using absorbed antiserum raised against the strongly plastic-adherent S. epidermidis 1457. A coagglutination assay was established, which allowed the quantitation of the antigen in bacterial extracts under different physiologic growth conditions. Expression of the antigen and of plastic adherence depended significantly on the presence of glucose in the growth medium. Parallel to increased plastic adherence, a 32- to 64-fold increase in the amount of the antigen was detected in bacterial extracts of cells grown in tryptone soya broth (TSB) compared with that in extracts of cells grown in TSB lacking glucose. A parallel time-dependent increase of plastic adherence and expression of the antigen was observed after stimulation by glucose of stationary-phase cultures of plastic-adherent S. epidermidis strains grown in TSB lacking glucose. The antigen consisted most probably of polysaccharide, because its immunologic reactivity was completely abolished by periodate oxidation but was resistant to protease digestion. A significant proportion of cells of plastic-adherent as compared with nonadherent S. epidermidis strains grown in TSB were located in large cell clusters exceeding 50 cells, which completely disintegrated after periodate oxidation of the cell preparations. Periodate oxidation of adherent bacterial films in situ led to release of the adherent cells from the plastic surface. These results strongly indicate a functional relation of the antigen to adherence of S. epidermidis to polymer surfaces, most probably by mediating intercellular adhesion of cells leading to accumulation in multilayered cell clusters.
Inhibition of expression of major histocompatibility complex class II molecules in macrophages infected with Leishmania donovani occurs at the level of gene transcription via a cyclic AMP-independent mechanism
Authors: Kwan W C , McMaster W R , Wong N , Reiner N E
Published: January 2020

Among the important pleiotropic responses to gamma interferon (IFN-gamma) during the activation of macrophages (M phi) is the increased expression of major histocompatibility complex class II genes. In the present study, infection with Leishmania donovani was shown to inhibit in parallel the induction by IFN-gamma of H-2 A beta gene transcription, class II mRNA accumulation, and H-2 Ad protein expression in cells of the murine macrophage cell line P388D1. Treatment of P388D1 cells with either the adenylate cyclase activator cholera toxin or the protein kinase A activator N6-2'-O-dibutyryl cyclic AMP (dibutyryl cAMP) similarly inhibited the induction by IFN-gamma of class II protein expression, and in parallel with Leishmania infection, cholera toxin inhibited the induction of mRNA for the H-2 A alpha and H-2 A beta proteins. Concentrations of intracellular cAMP were significantly increased in cholera toxin-treated cells but not in leishmania-infected cells. These findings indicate that at least one mechanism by which Leishmania infection attenuates the activation of M phi by IFN-gamma involves selective, transcriptional inhibition of major histocompatibility complex class II genes via a cAMP-independent mechanism.
Human T-lymphocyte proliferation, lymphokine production, and amebicidal activity elicited by the galactose-inhibitable adherence protein of Entamoeba histolytica
Authors: Schain D C , Salata R A , Ravdin J I
Published: January 2020

We studied human T-lymphocyte responses to the purified Entamoeba histolytica galactose-inhibitable adherence protein. Individuals having serum anti-adherence protein antibodies possess peripheral blood lymphocytes which demonstrate antigen-specific responses to the purified adherence protein (10 micrograms/ml) and whole soluble amebic antigen (100 micrograms/ml). This was determined by incorporation of [3H]thymidine (53,080 and 73,114 dpm, respectively) and by increased production of interleukin-2 and gamma interferon (42.0 and 67.5 U/ml, respectively) (P less than 0.05 for each in comparison with values for control lymphocyte responses). Lymphocytes from antiamebic antibody-positive subjects develop in vitro amebicidal activity only when incubated for 5 days with the purified adherence protein (P = 0.02). In conclusion, the E. histolytica galactose-inhibitable adherence protein elicits an in vitro amebicidal cell-mediated immune response, further supporting the potential for the use of this protein in a subunit amebiasis vaccine.
Rat monoclonal antibodies against Aspergillus galactomannan
Authors: Stynen D , Sarfati J , Goris A , Prévost M C , Lesourd M , Kamphuis H , Darras V , Latgé J P
Published: January 2020

Monoclonal antibodies (MAbs) against Aspergillus fumigatus galactomannan were produced in rats. Seven of them, EB-A1 through EB-A7, were characterized in more detail. They were all immunoglobulin M antibodies, reacting in an indirect enzyme-linked immunosorbent assay with purified A. fumigatus galactomannan, with avidity constants of between 2 x 10(9) and 5 x 10(9)/M. Enzyme-linked immunosorbent assay inhibition experiments with modified galactomannan and synthetic oligomers of beta (1----5)galactofuranose demonstrated that the MAbs bound to an epitope located on the beta(1----5)galactofuranose-containing side chains of the galactomannan molecule. An identical or similar epitope also seemed to be present in other fungi. Immunofluorescence and immunoelectron microscopy experiments with EB-A2 revealed the presence of the antigen in the fungal wall and inside the cell. Immunoblotting experiments demonstrated that the epitope recognized by the MAbs was a common oligosaccharide moiety of a wide range of intracellular and extracellular glycoproteins in A. fumigatus. The characteristics of the MAbs justify their use in the diagnosis of invasive aspergillosis by antigen detection.
Characterization of the heat shock response in Brucella abortus and isolation of the genes encoding the GroE heat shock proteins
Authors: Lin J , Adams L G , Ficht T A
Published: January 2020

In an effort to define the heat shock response in the bovine intracellular pathogen Brucella abortus, a rough variant lacking extensive lipopolysaccharide was pulse-labeled with [35S]methionine following exposure to elevated temperatures. The major heat shock proteins observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography migrate at 70, 62, 18, and 10 kDa. The maximum response was observed between 42 and 46 degrees C and within 2 to 3 h of the shif in temperature and varied slightly for the different proteins. Accumulation of the 62-kDa heat shock protein (62-kDa Hsp) was observed to continue for up to 5 h following the shift in temperature. In an effort to better define the heat shock response and its potential relationship with protective immunity, genes encoding the major heat shock proteins were isolated from recombinant libraries constructed from B. abortus S19 and S2308 and sequenced. The 62-kDa Hsp shares more than 60% amino acid homology with members of the GroEL family and is immunoprecipitated with polyclonal antibodies to Escherichia coli GroEL and monoclonal antibodies to mycobacterial Hsp 65. Western blot (immunoblot) analysis with pooled sera from vaccinated and infected cattle revealed that the 62-kDa Hsp is a predominantly recognized antigen. The roles of these gene products during environmental stress and in protective immunity against brucellosis are under investigation.
Adjuvant effects of liposomes containing lipid A: enhancement of liposomal antigen presentation and recruitment of macrophages
Authors: Verma J N , Rao M , Amselem S , Krzych U , Alving C R , Green S J , Wassef N M
Published: January 2020

Liposomes containing lipid A induced potent humoral immune responses in mice against an encapsulated malaria antigen (R32NS1) containing NANP epitopes. The immune response was not enhanced by lipid A alone or by empty liposomes containing lipid A. Experiments to investigate the adjuvant mechanisms of liposomes and lipid A revealed that liposome-encapsulated R32NS1 was actively presented by bone marrow-derived macrophages to NANP-specific cloned T cells. The degree of presentation was related to the amount of liposomal antigen added per macrophage in the culture medium. At high cell densities, poor presentation occurred when liposomes lacked lipid A but excellent presentation occurred when the liposomes contained lipid A. Liposomes containing lipid A and encapsulated antigen also activated gamma interferon-treated macrophages to produce nitric oxide. Macrophage activation and antigen presentation occurred with liposomes that could not be detected by the Limulus amebocyte lysis assay. Intraperitoneal injection of liposomal lipid A caused a marked increase in the recruitment of immature (peroxidase-positive) macrophages to the peritoneum. On the basis of these experiments, we propose that the mechanism of the adjuvant action of liposomal lipid A is partly due to increased antigen presentation by macrophages and partly due to recruitment of an increased number of macrophages serving as antigen-presenting cells.
Contribution of the pAD1-encoded cytolysin to the severity of experimental Enterococcus faecalis endophthalmitis
Authors: Jett B D , Jensen H G , Nordquist R E , Gilmore M S
Published: January 2020

The contribution of the pAD1-encoded cytolysin to Enterococcus faecalis virulence in an experimental endophthalmitis model was studied by using isogenic strains differing only in the location of transposon Tn917. The course of experimental endophthalmitis in New Zealand White rabbits was evaluated by postoperative reduction in retinal neuroresponsiveness, thin-section histopathology, and transmission electron microscopy. Infections caused by cytolytic E. faecalis resulted in 99% loss of retinal function at postoperative day 3, while isogenic, noncytolytic strains produced reductions of only 74.2%. Light microscopy revealed near-total destruction of retinal architecture at 24 h postinfection with cytolytic E. faecalis, while noncytolytic strains produced few or no destructive changes. Transmission electron microscopy revealed tissue destruction in retinal layers as early as 6 h postinfection with cytolytic E. faecalis. In vivo and in vitro growth rates of cytolytic and noncytolytic E. faecalis showed similar kinetics. These data demonstrate the contribution of the pAD1-encoded cytolysin to both the course and the severity of experimental E. faecalis endophthalmitis.
Invasion by Salmonella typhimurium is affected by the direction of flagellar rotation
Authors: Jones B D , Lee C A , Falkow S
Published: January 2020

When grown aerobically, Salmonella typhimurium exhibits a low level of entry into tissue culture cells. We have isolated an S. typhimurium Tn10 mutant which, when grown under aerobic conditions, efficiently invades HEp-2 cells. Sequencing of S. typhimurium DNA adjacent to the site of the Tn10 element showed that the insertion disrupted transcription of the aspartate receptor gene, tar. Polar effects of the transposon on downstream genes also eliminated chemotaxis. Isogenic nonchemotactic (Che-), as well as nonmotile (Mot-) and nonflagellated (Fla-), S. typhimurium strains were examined for their ability to invade HEp-2 cells. "Smooth" swimming Che- mutants (cheA, cheW, cheR, and cheY) were found to possess increased invasiveness for cultured mammalian cells. In contrast, a "tumbly" cheB mutant and Mot- (flagellated) strain were found to have decreased levels of tissue culture invasiveness. A Fla- strain was found to be as invasive as the wild-type strain if centrifugation was used to facilitate contact with the monolayer surface. In addition, the observed hyperinvasiveness of the smooth swimming tar::Tn10 mutant was suppressed when the strain was paralyzed by the introduction of a mot or fla mutation. A murine infection model was used to demonstrate that the mutant invasive phenotypes were also observed in vivo. These data are most consistent with the idea that the rotation and physical orientation of flagella around the bacteria affect the ability of salmonellae to enter host cells.
Localization of two epitopes recognized by monoclonal antibody PCG-4 on Clostridium difficile toxin A
Authors: Frey S M , Wilkins T D
Published: January 2020

The toxin A gene of Clostridium difficile contains a 2.5-kb region encoding a series of contiguous repeating units located at the COOH terminus of the molecule. We previously showed that the monoclonal antibody (MAb) PCG-4, which neutralizes the enterotoxic activity of toxin A, binds to epitopes located within these repeating units. In the present study, we subcloned a series of fragments from this portion of the gene. The recombinant peptides expressed from the gene fragments were examined for reactivity with MAb PCG-4 to identify the epitopes involved in binding. Our results showed that MAb PCG-4 recognizes epitopes in amino acid residues 2097 through 2141 and amino acid residues 2355 through 2398.
In vivo administration of recombinant growth hormone or gamma interferon activities macrophages: enhanced resistance to experimental Salmonella typhimurium infection is correlated with generation of reactive oxygen intermediates
Authors: Edwards C K , Ghiasuddin S M , Yunger L M , Lorence R M , Arkins S , Dantzer R , Kelley K W
Published: January 2020

Purified and recombinant forms of growth hormone (GH) as well as of recombinant rat gamma interferon (IFN-gamma) enhance the survival of rats deprived of endogenous pituitary GH secretion by hypophysectomy (HX rats) and infected with virulent Salmonella typhimurium. Macrophages obtained from rats with intact pituitaries (pituitary-intact rats) or HX rats that were treated in vivo with either GH or the closely related hormone prolactin released elevated (P less than 0.05) levels of superoxide anion (O2-) after in vitro opsonized-zymosan stimulation compared with those from placebo-treated animals. These levels of O2- release were similar in magnitude to those of macrophages from rats treated in vivo with IFN-gamma. In time course in vivo macrophage activation studies, both IFN-gamma and GH significantly increased O2- secretion within 24 h, with maximal secretion occurring at day 3. Macrophages obtained from pituitary-intact and HX rats injected in vivo with GH also released elevated (P less than 0.05) levels of hydrogen peroxide (H2O2) and displayed enhanced (P less than 0.01) phagocytic activity toward opsonized Listeria monocytogenes in vitro. The mechanism of action of GH in vivo is likely to be a direct one because resident peritoneal macrophages from rats could be primed in vitro for enhanced secretion of O2- following triggering of these cells with opsonized zymosan. These data show that in vivo administration of two closely related pituitary hormones, GH and prolactin, can effectively prime macrophages, which is consistent with the hypothesis that GH mediates resistance to S. typhimurium by a direct stimulatory action on macrophages.