bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and sequencing of Haemophilus influenzae outer membrane protein P6
Authors: Nelson M B , Apicella M A , Murphy T F , Vankeulen H , Spotila L D , Rekosh D
Published: January 2020

P6, a 16,600-dalton protein present in the outer membranes of both typeable and nontypeable strains, may be an important antigen in immunity to Haemophilus influenzae. The gene encoding P6 of a nontypeable strain of H. influenzae was cloned by using bacteriophage lambda gt11. Four recombinant phages were detected by screening plaques with monoclonal antibodies and a polyclonal antiserum. One recombinant phage, clone O, produced a full-length gene product which was expressed at a high yield. The DNA insert contained within this phage was cloned into the plasmid vector pUC18 to create the recombinant plasmid pBUD1. An Escherichia coli transformant containing this plasmid produced a protein which had an apparent molecular weight identical to that of H. influenzae P6, as determined by Western blot (immunoblot) analyses. Expression of the P6 polypeptide by both clone 0 and the transformant was independent of induction of the lac operon by isopropyl-beta-D-thiogalactopyranoside, suggesting that transcription was from the promoter of the P6 gene. Immunoelectron microscopy using a monoclonal antibody with specificity for a P6 surface epitope detected the presence of P6 on the surface of the transformant. The insert in pBUD1 was cut down in size to approximately 800 base pairs. The resultant plasmid, pBUD5, also coded for a full-length gene product. DNA sequence analysis revealed that the P6 gene contains transcriptional and translational sequences resembling those recognized in E. coli and a signal sequence characteristic of procaryotic membrane proteins. In addition, the carboxy terminus of this signal sequence shares homology with a common sequence found in bacterial lipoproteins, suggesting that P6 is a lipoprotein. Posttranslational proteolytic cleavage of the signal sequence would result in a protein composed of 134 amino acids.
Neutralization of toxic shock syndrome toxin-1 by monoclonal antibodies in vitro and in vivo
Authors: Bonventre P F , Thompson M R , Adinolfi L E , Gillis Z A , Parsonnet J
Published: January 2020

Sixteen monoclonal antibodies (MAbs) directed against toxic shock syndrome toxin-1 (TSST-1) were generated by immunization of mice with purified TSST-1 and subsequent fusion of spleen cells with myeloma cells. Antibody-producing clones, identified by an enzyme-linked immunosorbent assay, were maintained as ascites tumors, and MAbs were purified by protein A chromatography. High-titered clones were further characterized and tested for the ability to neutralize several biological activities of TSST-1. The MAbs, which are of several immunoglobulin subtypes, reacted specifically with purified TSST-1 and TSST-1 present in Staphylococcus aureus culture supernatants. Three MAbs neutralized TSST-1-induced mitogenesis in a dose-dependent manner. Three of eight MAbs tested were able to neutralize induction by TSST-1 of interleukin-1 production by human monocytes. One neutralizing MAb, 8-5-7, was tested for the ability to protect rabbits from a constant infusion of TSST-1. Rabbits given the MAb had an attenuated clinical illness and were protected from the hypocalcemia, lipemia, and hepatic and renal insufficiency seen in control rabbits. Six of seven control rabbits died, compared with only one of seven rabbits treated with MAb 8-5-7. These experiments suggest that MAb 8-5-7 is directed against an antigenic determinant critical to the toxicity of TSST-1 and that the MAbs should be useful as probes in structure-function analyses of the TSST-1 molecule.
T-cell modulation of the murine antibody response to Neisseria meningitidis group A capsular polysaccharide
Authors: Muller E , Apicella M A
Published: January 2020

T-cell modulation of the antibody response of BALB/c mice to group A meningococcal capsular polysaccharide (PS) was examined by using an enzyme-linked immunosorbent assay. An optimal dose (5 micrograms) of antigen induced an immunoglobulin M (IgM) response of short duration; no IgG or IgA antibody could be detected. The capacity to produce serum antibody begins at about 3 weeks of age. Concanavalin A (ConA) inhibited the magnitude of the response by 40 to 60% when given at the time of immunization; it enhanced the response two- to eightfold when given 2 days after PS. T-cell-mediated suppression could be transferred to naive mice by injection of spleen cells from low-dose-primed mice. A secondary antibody response could be induced by immunization with live meningococci. Here, the IgM response was 8- to 10-fold greater than that of mice given an optimal dose of PS; IgG antibody against group A PS increased 1 week after immunization to levels that were 100- to 1,000-fold greater than those of mice immunized with PS. The antibody response could not be augmented by multiple injections of PS; suppression occurred after low-dose priming or hyperimmunization with PS. These studies indicate that the antibody response to PS is not completely T-cell independent; rather, it is inhibited and amplified by T cells.
Impact of proteases on iron uptake of Pseudomonas aeruginosa pyoverdin from transferrin and lactoferrin
Authors: Döring G , Pfestorf M , Botzenhart K , Abdallah M A
Published: January 2020

The Pseudomonas aeruginosa siderophore pyoverdin was unable to acquire iron from human transferrin or lactoferrin at physiological pH. However, in the presence of P. aeruginosa elastase, rapid iron release and pyoverdin iron uptake from transferrin but not from lactoferrin were detected. Neither P. aeruginosa alkaline protease nor elastase from polymorphonuclear leukocytes revealed this effect.
Attachment of Escherichia coli via mannose- or Gal alpha 1----4Gal beta-containing receptors to human colonic epithelial cells
Authors: Wold A E , Thorssén M , Hull S , Edén C S
Published: January 2020

The role of bacterial adhesion for the maintenance of the large-intestinal microflora has not been established. In this study, colonic cells from the adenocarcinoma cell line HT-29 or from surgical specimens were tested for the ability to bind Escherichia coli. The E. coli strains were manipulated by transformation or by mutagenesis to express either mannose-specific type 1 fimbriae (strains 506 MS and HU742) or Gal alpha 1----4Gal beta-specific P fimbriae (506 MR and HU824). Binding to HT-29 cells was seen with strains of either receptor specificity and was inhibited by alpha-methyl mannoside or globotetraosylceramide (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc-ceramide), respectively. The Gal alpha 1----4Gal beta-specific strains interacted with a loosely surface-associated substance, which was sensitive to mechanical treatment and incubation at 37 degrees C, while the mannose-specific strains bound both directly to the cell and to the loosely associated substance. Isolated colonic epithelial cells bound the mannose-specific bacteria in high numbers, while the attachment of the Gal alpha 1----4Gal beta-specific strains depended on the elution method. Cells eluted sequentially with magnetic stirring were unable to bind the Gal alpha 1----4Gal beta-specific bacteria, while elution by a more gentle method resulted in binding of these strains to material loosely associated with the epithelial cells. Thus, the binding pattern of isolated colonic epithelial cells paralleled that of the HT-29 cell line. Conceivably, binding to mannose- and Gal alpha 1----4Gal beta-containing receptors could contribute to the maintenance of E. coli in the human large intestine.
Role of competition for nutrients in suppression of Clostridium difficile by the colonic microflora
Authors: Wilson K H , Perini F
Published: January 2020

The cecal flora of mice is able to eliminate Clostridium difficile from the mouse cecum even when C. difficile is the first organism established. We used a continuous-flow (CF) culture model of the cecal flora to investigate the possibility that competition for nutrients is one mechanism for this antagonism. The medium for the CF cultures consisted of homogenates of fecal pellets from germfree mice. Carbohydrate analysis showed that mouse flora depleted 74 to 99.8% of the various carbohydrates from this environment-simulating medium. When inoculated into filtrates made from CF cultures of mouse flora, C. difficile multiplied slower than the dilution rate of the CF cultures unless glucose, N-acetylglucosamine, or N-acetylneuraminic acid was added. C. difficile did not synthesize hydrolytic enzymes able to cleave these monosaccharides from oligosaccharide side chains. As found previously, veal infusion broth did not support the growth of a microflora that could be transferred to gnotobiotic mice and fully suppress C. difficile. When mucin or monosaccharides found in mucin were added to veal infusion broth, the flora functioned normally in this regard. These data suggest that as yet unidentified organisms compete more efficiently than C. difficile for monomeric glucose, N-acetylglucosamine, and sialic acids found in colonic contents.
Human bactericidal antibody response to outer membrane protein P2 of nontypeable Haemophilus influenzae
Authors: Murphy T F , Bartos L C
Published: January 2020

The human bactericidal antibody response to the major outer membrane protein, P2, of nontypeable Haemophilus influenzae was studied. P2 was isolated from two strains of nontypeable H. influenzae and coupled to affinity columns. Pooled normal human serum was subjected to affinity chromatography with the P2 columns and a control column. Reducing the titer of antibody to P2 resulted in reduced bactericidal activity of that serum for the organism. Immunopurified antibody to P2 from human serum was bactericidal for the homologous strain. The extent to which these bactericidal determinants on P2 are conserved among strains was investigated. Immunopurified antibodies to P2 of two epidemiologically unrelated isolates were bactericidal for four of six strains tested. We conclude that P2 is a target for human bactericidal antibody and that some of these determinants that are recognized by human bactericidal antibody are conserved among strains of nontypeable H. influenzae.
Cloning and expression of genes encoding Haemophilus somnus antigens
Authors: Corbeil L B , Chikami G , Yarnall M , Smith J , Guiney D G
Published: January 2020

A genomic library of Haemophilus somnus 2336, a virulent isolate from a calf with pneumonia (later used to reproduce H. somnus experimental pneumonia), was constructed in the cosmid vector pHC79. The gene bank in Escherichia coli DH1 was screened by filter immunoassay with convalescent-phase serum, which reacted with several outer membrane antigens of H. somnus. On Western blotting (immunoblotting) of immunoreactive colonies, five clones were found to express proteins which comigrated with H. somnus surface antigens. Three clones (DH1 pHS1, pHS3, and pHS4) expressed both a 120-kilodalton (kDa) antigen and a 76-kDa antigen, one clone (DH1 pHS2) expressed only the 76-kDa antigen, and the fifth clone (DH1 pHS5) expressed a 60-kDa antigen. The 120-kDa and 76-kDa antigens were found internally, whereas the 60-kDa protein was detected in the DH1 pHS5 culture supernatant as membrane blebs or insoluble protein. Both the H. somnus 120-kDa antigen and the recombinant 120-kDa antigen had immunoglobulin Fc-binding activity. Restriction endonuclease mapping demonstrated that the genomic DNA inserts of clones expressing the 76-kDa antigen shared a common 28.4-kilobase-pair region, and the three clones also expressing the 120-kDa antigen shared an additional 7.0-kilobase-pair region. The restriction endonuclease map of pHS5, which expressed the 60-kDa antigen, was not similar to the maps of the other four plasmids. Since these three H. somnus antigens reacted with protective convalescent-phase serum, the recombinants which express these proteins should be useful in further studies of protective immunity in bovine H. somnus disease.
Strain variation in phagocytosis of Cryptococcus neoformans: dissociation of susceptibility to phagocytosis from activation and binding of opsonic fragments of C3
Authors: Kozel T R , Pfrommer G S , Guerlain A S , Highison B A , Highison G J
Published: January 2020

Phagocytosis of Cryptococcus neoformans is markedly influenced by the presence of a polysaccharide capsule. We examined activation and binding of C3 fragments to eight isolates of C. neoformans. All isolates were shown to have capsules by light and electron microscopy. These strains differed in susceptibility to phagocytosis by neutrophils. Yeast cells were opsonized by incubation in normal human serum. Five strains were resistant to ingestion, two strains showed intermediate levels of resistance to ingestion, and one strain was quite sensitive to phagocytosis. Yeast cells opsonized with heat-inactivated serum (56 degrees C for 30 min) neither attached nor were ingested by neutrophils. A quantitative estimate of the amount of C3 bound to the yeast cells was determined by use of normal human serum containing 125I-labeled C3. The results showed approximately 5 X 10(6) to 10 X 10(6) C3 molecules per yeast cell regardless of whether the yeast cells were sensitive or resistant to phagocytosis. Bound C3 was eluted from the yeast cells by treatment with 0.1 M NH2OH (pH 10), and the eluted fragments were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Results of this analysis showed that little of the C3 was in the form of C3b, and there was substantial decay to iC3b, the inactive decay product of C3b. This pattern of decay was similar with all strains. Immunoelectron microscopy was used to assess the ultrastructural location of the C3 fragments bound to the yeast cells. C3 fragments were bound to the perimeter of the capsule regardless of whether the isolate was sensitive or resistant to phagocytosis. Thus, phagocytosis-sensitive and phagocytosis-resistant isolates were similar with regard to the amount, molecular form, and ultrastructural location of C3 fragments bound to the cryptococcal capsule. These results further indicate that activation of the complement cascade is necessary but not sufficient for phagocytosis of the yeast cell.
Production of an interleukin-1 inhibitor by cell line P388D1 murine macrophages stimulated with Haemophilus actinomycetemcomitans lipopolysaccharide
Authors: Nishihara T , Koga T , Hamada S
Published: January 2020

Murine macrophages of the P388D1 cell line stimulated with lipopolysaccharide (LPS) from Haemophilus actinomycetemcomitans Y4 released an interleukin-1 (IL-1) inhibitor, as well as IL-1. Maximal IL-1 activity in culture supernatants was detected after 24 h of culture. On the other hand, IL-1 inhibitor activity reached a maximum level after 72 h of culture. An IL-1 inhibitor was partially purified from the culture supernatant of P388D1 cells stimulated with Y4 LPS for 72 h by ammonium sulfate precipitation, followed by Sephacryl S-200 gel chromatography. A 160-kilodalton peak inhibitory to IL-1 and a 14-kilodalton peak showing IL-1 activity were separated by Sephacryl S-200 column chromatography. The partially purified IL-1 inhibitor significantly suppressed the proliferation of C3H/HeJ murine thymocytes that had been induced with murine and human IL-1 in the presence of a submitogenic dose of concanavalin A. The IL-1 inhibitor more strongly suppressed human recombinant IL-1 beta than human recombinant IL-1 alpha. This inhibitory activity of the partially purified preparation was unaffected by the presence of trypsin inhibitor and the protease inhibitor aprotinin. The IL-1 inhibitor did not exhibit either IL-2 or IL-2 inhibitor activity. The inhibitor suppressed C3H/HeJ thymocyte proliferation induced by IL-1 in the presence of a saturated concentration of IL-2 instead of a suboptimal concentration of concanavalin A. These results indicate that prolonged culture of Y4 LPS-stimulated murine macrophages releases a specific inhibitor of IL-1.
Acid phosphatase activity of virulent and avirulent clones of Leishmania donovani promastigotes
Authors: Katakura K , Kobayashi A
Published: January 2020

Virulent and avirulent clones of Leishmania donovani promastigotes were examined for their acid phosphatase activity. The acid phosphatase activity of whole-cell lysates of virulent clones was 1.5 to 2.0 times higher than that of avirulent clones. Pellet fractions (260,000 x g, 30 min) from sonicated promastigotes of a virulent clone and an avirulent clone contained 60 and 40% of the total enzyme activity, respectively. Membrane-bound acid phosphatase was extracted with Triton X-100 from the pellet. This membrane-bound phosphatase activity was 2.4-fold higher in virulent organisms than in avirulent organisms. The membrane acid phosphatase exhibited two distinct bands on polyacrylamide gels stained for enzyme activity. One diffuse, faster-migrating band showed identical electrophoretic mobility in both virulent and avirulent clones, although a higher enzymatic activity was observed with the extract from virulent cells. In contrast, a slower-migrating band was different between the two clones in the mobility. These results suggest that membrane-bound acid phosphatase was quantitatively and qualitatively different between virulent and avirulent promastigotes of L. donovani. In addition, virulent cells produced a relatively high level of acid phosphatase throughout the growth in culture.
Effect of Mycobacterium tuberculosis-derived sulfolipid I on human phagocytic cells
Authors: Zhang L , Goren M B , Holzer T J , Andersen B R
Published: January 2020

Experiments were performed to determine the effects of Mycobacterium tuberculosis-derived sulfolipid I on phagocytic cells. Sulfolipid I was taken up in significant amounts by human neutrophils and in lesser amounts by monocytes and lymphocytes. Superoxide (O2-) production by neutrophils was significantly increased by sulfolipid I, but the rate of production was slower than that reported previously for other stimuli. The optimal concentration of sulfolipid I for stimulation of O2- production was 27 micrograms/ml, while higher concentrations produced less. At substimulatory levels sulfolipid I caused enhancement of O2- release from neutrophils when it was subsequently stimulated by other agents. Nonadherent monocytes from most normal donors failed to produce O2- when treated with sulfolipid I; however, adherent monocytes pretreated with gamma interferon did produce O2- with sulfolipid I stimulation. Priming for an enhanced oxidative response of activated monocytes was also observed. These sulfolipid I-induced changes in phagocytic cell function may be important in altering the ability of phagocytes to respond effectively to M. tuberculosis and may also cause exaggerated inflammatory responses.
Role of yeast cell growth temperature on Candida albicans virulence in mice
Authors: Antley P P , Hazen K C
Published: January 2020

Previous studies have suggested that yeast cell growth temperature may influence the relative virulence of the opportunistic dimorphic fungus Candida albicans. To test this possibility, mice were challenged with C. albicans yeast cells which were grown at either room temperature or 37 degrees C, and their survival was monitored daily. Mice which received room temperature-grown cells died faster. The interaction of glycogen-elicited polymorphonucleated neutrophils (PMNs) with C. albicans yeast cells grown at the two temperatures was examined, because PMNs have been shown to have a critical role in preventing development of candidiasis in normal individuals. In the absence of serum (i.e., nonopsonic conditions), more PMNs conjugated and engulfed C. albicans cells grown at room temperature than those grown at 37 degrees C. However, PMNs were less able to kill cells grown at room temperature than cells grown at 37 degrees C. Cells grown at room temperature also produced abundant germ tubes after engulfment and were thus more likely to escape killing by phagocytes. These results suggest that cells grown at room temperature are more virulent because they are less likely to be killed by phagocytes and are more likely to disseminate. The possibility that expression of cell surface hydrophobicity is involved in these events is discussed.
Iron-regulated hemolysin production and utilization of heme and hemoglobin by Vibrio cholerae
Authors: Stoebner J A , Payne S M
Published: January 2020

El Tor and non-O1 strains of Vibrio cholerae were analyzed to determine whether synthesis of secreted hemolysin was influenced by the concentration of iron in the medium. Synthesis of hemolysin was found to be iron regulated in both El Tor and non-O1 isolates. Increased levels of hemolytic activity were detected in supernatants of iron-starved cells. Spontaneous hemolysin-deficient mutants of one non-O1 strain were found to occur at high frequency. These variants also failed to synthesize vibriobactin, the iron transport compound utilized by V. cholerae. Another non-O1 strain was found to synthesize both hemolysin and vibriobactin constitutively. When the cloned Escherichia coli fur gene, encoded on the plasmid pABN203, was introduced into this constitutive strain, normal iron regulation of both hemolysin and vibriobactin was reestablished. The ability of V. cholerae to utilize mammalian iron compounds was determined, and it was found that both hemin and hemoglobin could serve as sole sources of iron.
Cloning and characterization of a new type of fimbria (S/F1C-related fimbria) expressed by an Escherichia coli O75:K1:H7 blood culture isolate
Authors: Pawelzik M , Heesemann J , Hacker J , Opferkuch W
Published: January 2020

The Escherichia coli blood culture isolate BK658 (O75:K1:H7) expresses F1A and F1B fimbriae as well as a third fimbrial type which reacts with anti-S-fimbrial antiserum but fails to show S-specific binding properties (i.e., agglutination of bovine erythrocytes). To characterize these fimbriae, we cloned the respective genetic determinant in E. coli K-12. The resulting recombinant clone HB101(pMMP658-6) expresses fimbriae of 1.2-micron length and a diameter of approximately 7 nm. The determinant codes for the fimbrillin subunit, a protein of 17 kilodaltons in size, and for at least five other proteins of 87, 31, 23, 14.3, and 13.8 kilodaltons. By restriction analysis and by DNA-DNA hybridization, it could be shown that the cloned fimbrial determinant of strain BK658 exhibits a high degree of sequence homology to the gene clusters coding for S fimbrial adhesins (sfa) and F1C fimbriae (foc). By using the Western blot (immunoblot) technique and a quantitative enzyme-linked immunosorbent assay, it could be further demonstrated that the cloned fimbriae of BK658, S fimbriae, and F1C fimbriae share cross-reactive epitopes as well as antigenic determinants specific for each fimbrial type. No antigenic cross-reactivity with F1C fimbriae could be detected. The results indicate a genetical and serological relatedness of the cloned fimbriae to S fimbriae and F1C fimbriae. Therefore, this new type of fimbriae is preliminarily termed S/F1C-related fimbriae (Sfr).
Chemical and structural studies of serotype polysaccharide antigens of Streptococcus sobrinus 6715
Authors: Takada K , Shiota T , Ikeda T
Published: January 2020

The g antigen of Streptococcus sobrinus 6715 was previously shown to consist of polysaccharides of various molecular weights. In this study, two such polysaccharides, LII and LIII, were purified by gel filtration and affinity chromatography procedures. By a double immunodiffusion analysis, fraction LII was found to contain a region in the serotype-specific g site not present in the serospecific g site of fraction LIII. This region was designated x. In addition to the serotype-specific g site, the cross-reactive sites, g-a, g-d, and g-(a-d), were all present on a single molecule of fractions LII and LIII. Polysaccharides LII and LIII were composed of galactose, glucose, and rhamnose. Analysis of inhibition of the precipitin reaction suggested that the serotype g site of fraction LII (the putative form of g antigen) may consist of two immunodominant regions, one a galactose-containing region (region x) and the other a glucose-containing region, while the serotype g site of fraction LIII appeared to have one immunodominant region containing a glucose determinant. The methylation and 13C nuclear magnetic resonance analyses of LII and LIII fractions provided information on the linkage and the anomeric structures of the sugar components of the polysaccharides.
A pneumococcal surface protein (PspB) that exhibits the same protease sensitivity as streptococcal R antigen
Authors: McDaniel L S , Briles D E
Published: January 2020

A monoclonal antibody against Streptococcus pneumoniae, designated T4A49, detected a 64,000-molecular-weight protein that was immunologically unrelated to the previously described surface protein PspA. This new protein, designated PspB, was pepsin sensitive and trypsin resistant, as is the R antigen of type 28 group A streptococci.
Role in vivo for gamma interferon in control of pneumonia caused by Chlamydia trachomatis in mice
Authors: Williams D M , Byrne G I , Grubbs B , Marshal T J , Schachter J
Published: January 2020

In a model of pneumonia caused by murine Chlamydia trachomatis, depletion experiments with monoclonal antibody to gamma interferon (IFN-gamma) made mice more susceptible. Repletion experiments giving exogenous recombinant murine IFN-gamma were not consistently protective. IFN-gamma may be necessary but not sufficient in host defense against the organism.
Isolation and characterization of a recombinant lambda gt11 bacteriophage which expresses an immunoreactive Mycobacterium intracellulare protein in Escherichia coli
Authors: Morris S L , Rouse D A , Hussong D , Chaparas S D
Published: January 2020

Disseminated Mycobacterium avium-Mycobacterium intracellulare (M. avium complex) disease is a prevalent opportunistic infection in patients with acquired immune deficiency syndrome. Because of the increasing importance of this disease, an M. avium complex lambda gt11 expression library was prepared. We screened the library with an absorbed anti-M. intracellulare serum and identified a recombinant phage which expressed a 190-kilodalton beta-galactosidase-M. intracellulare fusion protein. Lysates containing the 190-kilodalton fusion protein evoked strong humoral and cell-mediated responses. The immunoreactivity of the M. intracellulare recombinant protein suggests that antigens isolated from the expression library may be useful as skin test, serodiagnostic, or immunoprophylactic reagents for M. avium complex disease.
Serum antibody and ocular responses to murine corneal infection caused by Pseudomonas aeruginosa
Authors: Berk R S , Montgomery I N , Hazlett L D
Published: January 2020

The serum antibody response and differential corneal response to primary and secondary infections by Pseudomonas aeruginosa were investigated in DBA/2J (resistant) and C57BL/6J (susceptible) mice, since they respond differently to intracorneal challenge. Using an enzyme-linked immunosorbent assay, we found that naturally resistant DBA/2J mice mounted a significant immunoglobulin M (IgM) and IgG response to P. aeruginosa within 7 days postinfection of one eye; this was subsequently followed by a drop in the IgM response. Of 31 mice, 30 were able to restore corneal clarity within 3 to 4 weeks. However, when C57BL/6J mice were infected intracorneally, their levels of serum antibody developed more slowly than did those of the DBA/2J mice, and they were unable to restore corneal clarity within 8 to 12 weeks. None of the mice from either test strain mounted a detectable serum IgA response to P. aeruginosa over a 90-day holding period. However, infection of the contralateral, normal cornea of mice of both test strains resulted in a heightened IgG response to P. aeruginosa within 30 days after the secondary infection. Many (50%) of the susceptible C57BL/6J mice recovered or exhibited less severe corneal damage within the 30-day holding period. If the C57BL/6J mice were reinfected 60 days after the primary infection instead of after 30 days, most (89%) of the mice had restored corneal clarity within 3 to 6 days. Passive transfer of immune serum from either recovered DBA/2J or C57BL/6J mice to naive C57BL/6J mice resulted in the restoration of corneal clarity in many of the recipients following infection.
Identification of leishmanial antigens in the sera of patients with American visceral leishmaniasis
Authors: Evans T G , Pearson R D
Published: January 2020

Circulating immune complexes are present in the sera of patients with visceral leishmaniasis caused by Leishmania donovani chagasi. In order to determine whether these complexes contain parasite antigens, sera were collected from Brazilian patients with visceral leishmaniasis and from hospitalized control subjects with other diagnoses. High-molecular-weight complexes were precipitated from pooled sera with 2.5% polyethylene glycol. Approximately 140-fold-more protein was precipitated from patient sera than from control sera; 12% of the total patient serum protein was precipitated. Patient serum precipitates contained immunoglobulins G (525 mg/dl), M (27 mg/dl), and A (8 mg/dl). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the patient serum precipitates revealed multiple bands, including a prominent band at 70 kilodaltons, that were not seen in precipitates of control sera. The 70-kilodalton band was recognized by human and hamster sera with antileishmanial antibodies, but not by control sera. Finally, immunization of BALB/c mice with the high-molecular-weight precipitates from patients elicited antileishmanial antibodies against L. donovani chagasi antigens as detected by enzyme-linked immunosorbent assay and Western blot (immunoblot) assay. In summary, sera of patients with American visceral leishmaniasis were found to have high-molecular-weight complexes that contained one or more parasite antigens. These complexes may play a role in the immunology of the disease, and detection of circulating parasite antigens has potential diagnostic importance.
Plasmid-containing strains of Streptococcus mutans cluster within family and racial cohorts: implications for natural transmission
Authors: Caufield P W , Ratanapridakul K , Allen D N , Cutter G R
Published: January 2020

The transmission of mutans streptococci is thought to occur along familial lines based on investigations which have shown common strains within family units by using phenotypic typing methods, such as bacteriocin production and immunity profiles and serotyping. Difficulties in implementing these typing methods, coupled with conflicting interpretations of results between laboratories, led us to study the conservation of Streptococcus mutans strains within a mother-child cohort by using a genotypic marker, plasmid DNA. Plasmids (all 5.6 kilobases in size) were observed at an overall frequency of 3.3%, with a significantly different frequency in whites (1.5%) compared with blacks (6.6%). Plasmid-containing strains were significantly clustered in mother-child pairs compared with nonrelated individuals (58 versus 3.3%; P less than 0.001). Moreover, the different plasmid groups (I and II) were highly conserved within racial boundaries (P = 0.007). In those instances in which we were able to characterize distinct strains by either biotype or plasmid profile, we found that mothers harbored a more heterogeneous population of mutans streptococci than did their children. This suggested, among other possibilities, that children acquire additional strains as they approach adulthood. Alternatively, we may have been unable to detect more heterogeneity of strains in children because of quantitative differences of strains in saliva. We present collective data which show that strains of S. mutans are highly conserved within not only mothers and their children but also racial groups, suggesting vertical transmission of this organism within human populations. Moreover, we show that levels of S. mutans found in the saliva of the mother correlated with levels found in her child, demonstrating a quantitative relationship within mother-child pairs.
Killing of Brucella abortus by bovine serum
Authors: Corbeil L B , Blau K , Inzana T J , Nielsen K H , Jacobson R H , Corbeil R R , Winter A J
Published: January 2020

Studies of the serum bactericidal system in bovine brucellosis were undertaken to investigate the role of the humoral immune response in protection of cattle against the facultative intracellular parasite Brucella abortus. Fresh sera from normal control cattle, infected cattle, and cattle immunized with B. abortus cell envelopes were collected before treatment and during the course of immunization or infection. Normal fresh bovine serum or fresh agammaglobulinemic serum from colostrum-deprived calves was effective in killing smooth virulent B. abortus 2308, but rough strains RB51 (a rough mutant of strain 2308) and 45/20 were much more sensitive to serum. The difference in susceptibility to serum was shown to be correlated with differences in lipopolysaccharide chemotype, with the more resistant strain 2308 having O polysaccharide and the more susceptible strains 45/20 and RB51 lacking O side chains. By treatment of fresh serum with MgCl2 and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] killing was shown to occur via the classical pathway of complement activation. When antibody to B. abortus was present, killing of strain RB51 increased but killing of smooth strain 2308 decreased. The earliest antibody response in serum from infected animals did not interfere with killing. When affinity-purified bovine immunoglobulins specific for B. abortus smooth lipopolysaccharide were added to fresh normal bovine serum, immunoglobulin G1 (IgG1) and IgG2 isotypes blocked killing but IgM and IgA isotypes did not. Thus, it appears that serum from previously unexposed animals or animals early during infection can kill smooth B. abortus, an appropriate defense mechanism before the organism becomes intracellular. At later stages of infection, blocking antibodies predominate.
Interactions of monospecific antisera with cell surface determinants of Candida albicans
Authors: Chaffin W L , Ringler L , Larsen H S
Published: January 2020

Flow cytometric analysis of indirect immunofluorescence showed that surface determinants recognized by antisera (Candida Check; Iatron Laboratories, Tokyo, Japan) for factors 1, 4, 5, and 6 were expressed to the same extent by all cells of Candida albicans under each growth condition and for each morphology examined. Fluorescence intensity increased with increasing cell size.
Susceptibility of Plasmodium falciparum to a peroxidase-mediated oxygen-dependent microbicidal system
Authors: Malhotra K , Salmon D , Le Bras J , Vilde J L
Published: January 2020

The viability of Plasmodium falciparum in culture was assessed by [3H]hypoxanthine incorporation during 24 h of incubation with lactoperoxidase, glucose-glucose oxidase, hydrogen peroxide, halides, or thiocyanate, alone or in combination. Synergistic inhibition was produced by the following combinations: lactoperoxidase plus hydrogen peroxide, lactoperoxidase plus glucose-glucose oxidase, and lactoperoxidase plus hydrogen peroxide plus halides or thiocyanate. These inhibitory effects were reversed by catalase and glutathione. The presence of plasmodial crisis forms inside the erythrocytes suggests that the oxygen-dependent microbicidal system of phagocytes has a killing effect.
Elastase in the pathogenic free-living amoebae Naegleria and Acanthamoeba spp
Authors: Ferrante A , Bates E J
Published: January 2020

The data showed that pathogenic free-living amoebae contain the proteolytic enzyme elastase. The levels of enzyme were similar in Naegleria fowleri, N. australianis italica, and Acanthamoeba culbertsoni A-1. No difference was found between elastase levels in a highly pathogenic N. fowleri and those in the same organism which had lost pathogenicity as a result of long-term axenic maintenance.
Activation of mouse peritoneal macrophages in vitro or in vivo by recombinant murine gamma interferon inhibits the growth of Chlamydia trachomatis serovar L1
Authors: Zhong G M , de la Maza L M
Published: January 2020

Peritoneal mouse macrophages activated in vitro with recombinant murine gamma interferon (10 ng/ml) or in vivo (10 micrograms per mouse) showed a significant decrease in the growth and yield of Chlamydia trachomatis. The restriction of the growth of C. trachomatis paralleled the expression of Iad on the macrophages. Mice that received macrophages activated in vitro with recombinant murine gamma interferon showed a significant decrease in the yield of chlamydial infection-forming units from their spleens and peritoneal fluids.
Oral immunization of rabbits with enterotoxigenic Escherichia coli protects against intraintestinal challenge
Authors: Sack R B , Kline R L , Spira W M
Published: January 2020

The development of a successful oral vaccine against enterotoxigenic Escherichia coli depends upon the identification of appropriate protective antigens which can be delivered effectively to intestinal mucosa. We have determined in a modified RITARD model the relative protection against intraintestinal challenge afforded by oral immunization with live enterotoxigenic E. coli carrying different candidate antigens. Studies were done with both wild-type strains and genetically manipulated strains of enterotoxigenic E. coli (parent strain E1392/75 2A) which carried plasmids containing intact heat-labile toxin (LT) gene sequences or various mutations of the LT genes. Immunizations were done by orogastric tube inoculation on days 0, 7, and 14; challenges were done on day 33. Protection against diarrhea with a homologous challenge was found to be 84 to 100% (P less than 0.01). Protection against diarrhea with challenges in which specific antigens could be tested included the following: (i) O and H antigens (O6:H16), 87 to 100% protection with different E. coli strains with identical O and H antigens (P less than 0.01) but no protection against a heterologous challenge; (ii) LT or the B subunit of LT only, approximately 50% protection (P less than 0.02). These findings suggest that O antigens are highly protective in this model but afford only serotype-specific protection and that the B subunit (with or without the A subunit) affords less protection but confers cross-protection against heterologous strains producing LT. This model should be useful in further defining appropriate protective antigens for candidate enterotoxigenic E. coli vaccine strains.
Effect of dietary restriction on total and bacterium-specific mucosal secretory immunoglobulin A in bile-diverted intestinal self-filling blind loops
Authors: Lichtman S N , Sherman P M , Forstner G G
Published: January 2020

The effect of starvation on the mucosal secretory immunoglobulin A (sIgA) response to bacterial antigens was studied in bile-free rat self-filling blind loops constructed at the end of a Roux-en-Y branch of jejunum. Rats were fed a 50% restricted diet for 1 to 4 weeks after surgery. sIgA was measured in the mucosa and lumen by an enzyme-linked immunosorbent assay. Dietary restriction caused a final rise of luminal sIgA which was less than 50% of that of normally fed controls Luminal bacteria counts were not different in the two groups. The percentage of total sIgA precipitated with intestinal bacteria was not significantly affected by dietary restriction, and there was no change in the specific binding of sIgA to several bacterial species. Nonprecipitated sIgA exhibited a low but significant specific binding to bacteria in both diet-restricted and fed rats. Diet restriction therefore reduced the total sIgA response to luminal bacteria, but the specific bacterial binding capacity per microgram of sIgA was not altered. In these short-term experiments diet-restricted animals appeared to be capable of secreting sIgA in excess of requirement, since the nonprecipitable luminal fraction contained free sIgA with binding capacity for bacteria. The ability of sIgA to react with specific antigens may therefore be of more significance as an indicator of bacterial susceptibility than the measurement of total sIgA.
Incorporation of the major outer membrane protein of Neisseria gonorrhoeae in saponin-lipid complexes (iscoms): chemical analysis, some structural features, and comparison of their immunogenicity with three other antigen delivery systems
Authors: Kersten G F , Teerlink T , Derks H J , Verkleij A J , van Wezel T L , Crommelin D J , Beuvery E C
Published: January 2020

We incorporated the major outer membrane protein (PI) of Neisseria gonorrhoeae into immunostimulating complexes (iscoms) and examined some analytical, physicochemical, and immunological properties of these structures. The immunogenicity was compared with that of three other PI-containing structures, i.e., liposomes, outer membrane complexes produced by the bacterium, and protein-detergent-adjuvant complexes. AIPO4 and dioctadecyldimethylammonium bromide were used as adjuvants. Our results show that iscoms are much more immunogenic than liposomes and protein-detergent complexes but are also much more toxic. The localization of PI in iscoms was investigated. Therefore, the chymotrypsin susceptibility of PI in iscoms was tested, and the incorporation of fragments of PI was determined. Amphiphilic fragments of PI were incorporated in iscoms, but hydrophilic and hydrophobic fragments were not. Chymotrypsin degradation of PI in iscoms indicated that the protein is exposed to the environment in a similar manner as PI in outer membrane complexes, i.e., with both termini anchored in the iscom.
Haemophilus influenzae serotype a: outer membrane protein classification and correlation with DNA polymorphism at the cap locus
Authors: Allan I , Kroll J S , Dhir A , Moxon E R
Published: January 2020

Fifty-four temporally and geographically distinct isolates of Haemophilus influenzae serotype a were characterized by (i) outer membrane protein composition and (ii) DNA polymorphism at the cap locus. In 53 of 54 cases, knowledge of the outer membrane protein type was accurately predictive of the cap locus polymorphism, suggesting that the majority of H. influenzae (type a) isolates have evolved from a limited number of clones.
Susceptibility of HRS/J mice to listeriosis: dynamics of infection
Authors: Archinal W A , Wilder M S
Published: January 2020

Congenitally hairless HRS/J homozygous (hr/hr) mice as well as phenotypically normal littermates (hr/+) were found to exhibit unusual susceptibility to infection with Listeria monocytogenes with 50% of the animals dying within a 10-day period (LD50) at an infecting inoculum approaching 200 microorganisms. In marked contrast to the outbred CD-1 strain as well as other Listeria-susceptible mice, HRS/J hosts are virtually incapable of limiting infection with virulent Listeria. The dynamics of infection reveal early uncontrolled bacterial growth within the peritoneal cavity, followed by a sharp increase of bacterial load in the spleen of both HRS/J homozygotes and heterozygous littermates. Spleen indices obtained for mutant mice indicate substantial splenomegaly which parallels the onset of infection in that organ. Assessment of the exudate population within the peritoneal cavity during infection indicates that HRS/J mice produce an early sustained influx of polymorphonuclear leukocytes while exhibiting a diminished macrophage inflammatory response. Additionally, it was shown that the mutant strain expresses significant increases in the total number of recoverable peritoneal leukocytes in response to other phlogistic stimuli.
Characterization of the susceptibility of Pseudomonas aeruginosa to complement-mediated killing: role of antibodies to the rough lipopolysaccharide on serum-sensitive strains
Authors: Schiller N L
Published: January 2020

The mechanism of complement-mediated killing of seven serum-sensitive Pseudomonas aeruginosa strains was examined. All seven strains were sensitive to the bactericidal activity of 20% pooled normal human serum (PNHS) containing magnesium EGTA, which blocks the classical complement pathway (CCP), or 20% PNHS preheated to 50 degrees C for 20 min, which inactivates the alternative complement pathway, suggesting that either pathway was effective against these strains. However, for four of these strains, optimal killing required the function of both pathways. Preabsorption of PNHS with serum-sensitive strains dramatically reduced the killing activity of serum for the homologous strains when a concentration of 10% serum was used, implying a role for antibody in the activation of complement via the CCP. Affinity purification of antibodies to the rough lipopolysaccharide (LPS) on strain 144M resulted in a pool of antibodies which could restore all of the bactericidal activity and most of the C3 activation-deposition activity of serum which had been lost by preabsorption with 144M. Confirmation that the LPS was the target for these bactericidal antibodies was provided by demonstrating that exogenously added 144M LPS inhibited the killing activity of PNHS. These anti-144M LPS-specific antibodies were also bactericidal for the six other serum-sensitive strains examined, suggesting that all seven strains shared an antigenic determinant recognized by these anti-144M LPS-specific antibodies. Results from cross-absorption studies imply that there are bactericidal antibodies in PNHS directed to additional bacterial targets. These studies suggest that part of the bactericidal activity of PNHS is due to binding of antibodies to the rough LPS on serum-sensitive strains, initiating activation of the CCP, and that all seven strains examined shared this bactericidal antibody-binding site.
Distribution and degree of heterogeneity of the afimbrial-adhesin-encoding operon (afa) among uropathogenic Escherichia coli isolates
Authors: Labigne-Roussel A , Falkow S
Published: January 2020

The afimbrial adhesin (AFA-I) from a pyelonephritic Escherichia coli isolate (KS52) is a mannose-resistant, P-independent, X-binding adhesin, expressed by the afa-1 operon. It is distinct from the E. coli X-binding adhesins with M and S specificity. A total of 138 E. coli isolates belonging to various serotypes, mostly from urinary tract infections, were screened for the presence of DNA sequences related to the afa operon and for the expression of an X-adhesin able to mediate mannose-resistant hemagglutination (MRHA) and adhesion to uroepithelial cells. Fifteen strains were shown to harbor DNA sequences related to the AFA-I-encoding operon, and 13 of them expressed an X-adhesin. Using as probes different DNA segments of the AFA-I-encoding operon in Southern experiments, we demonstrated that only three of these clinical isolates contained genetic determinants closely related to those identified in the original afa prototype strain (KS52): presence of the afaA, afaB, afaC, afaD, and afaE genes associated with the expression of a 16,000-dalton hemagglutinin-adhesin which strongly cross-reacted with AFA-I-specific antibodies. The other E. coli isolates harbored DNA sequences homologous to the afaA, afaB, afaC, and afaD genes, but lacked the sequence corresponding to the adhesin-producing gene afaE; Western blots allowed the detection of polypeptides (15,000, 15,500, or 16,000 daltons) in these strains which cross-reacted with variable intensity with antibodies raised against the denatured AFA-I protein, but did not cross-react with native AFA-I-specific antibodies. Following DNA cloning experiments from chromosomal DNA of two of those strains (A22 and A30), we demonstrated that although the AFA-related operon in A22 and A30 strains lacked the AFA-I adhesin-encoding gene, they synthesized a functional X-adhesin. Thus, strains A22 and A30 encode adhesins designated AFA-II and AFA-III, which were cloned on recombinant plasmids pILL72 and pILL61, respectively. Southern hybridization experiments and Western blot analyses of the 15 AFA-related strains demonstrate the heterogeneity of the genetic sequences encoding the structural adhesin and suggest the bases for the serological diversity of the AFA adhesins.