bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of the alternative C3 convertase and classical C5 convertase of complement by group A streptococcal M protein
Authors: Hong K , Kinoshita T , Takeda J , Kozono H , Pramoonjago P , Kim Y U , Inoue K
Published: January 2020

When Streptococcus pyogenes group A type 3 strain C203 (M+) and its M-protein-lacking derivative, strain C203S (M-), were treated with normal human serum in the presence of magnesium-EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], virulent M+ bacteria bound only 10 to 30% as much C3 and factors B and P as did avirulent M- bacteria. After treatment of M+ bacteria with trypsin, which inactivates M protein, their binding of these substances was similar to that of M- bacteria. Pretreatment of M+ bacteria with the Fab fragment of rabbit immunoglobulin G anti-M antibody also increased their binding of C3 in the absence of Ca2+. Therefore, M protein inhibits the alternative C3 convertase. In contrast, in the presence of Ca2+ and Mg2+, M+ bacteria bound 75% as much C3 as M- bacteria. This binding was mostly mediated by classical pathway activation, because M+ bacteria bound much smaller amounts of factors B and P than did M- bacteria but consumed amounts of C4 and C2 comparable to those consumed by M- bacteria. On the other hand, the amount of C5 bound to M+ bacteria was much less than that bound to M- bacteria, and the consumption of C5 and C8 by M+ bacteria was also much less than that by M- bacteria. Therefore, M protein does not inhibit the classical C3 convertase but does inhibit the classical C5 convertase. When M+ and M- streptococci were incubated with normal human serum containing radiolabeled C3 in the presence of Ca2+ and Mg2+, more than 85% of the C3 bound to either type of streptococcus was extractable by sodium dodecyl sulfate and alkali treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the C3 extracted from both strains showed that it was mostly C3b and iC3b. The proportions of C3b and iC3b, respectively, were 7.5 and 71.9% on M+ bacteria and 18.9 and 58.4% on M- bacteria. These results support and extend previous findings that the antiphagocytic activity of streptococcal M protein may be due to complement inhibition mediated by the binding of factor H.
Erythrocyte gangliosides act as receptors for Neisseria subflava: identification of the Sia-1 adhesin
Authors: Nyberg G , Strömberg N , Jonsson A , Karlsson K A , Normark S
Published: January 2020

Neisseria gonorrhoeae was recently shown to bind to a subset of lactose-containing glycolipids (N. Strömberg, C. Deal, G. Nyberg, S. Normark, M. So, and K.-A. Karlsson, Proc. Natl. Acad. Sci. USA 85:4902-4906, 1988). A number of commensal Neisseria strains were also shown to be lactose binders. In addition, Neisseria subflava bound to immobilized gangliosides, such as hematoside and sialosyl paragloboside, carrying the NeuAc alpha 2-3Gal beta 1-4Glc sequence. To a lesser extent, N. gonorrhoeae also bound to this receptor in vitro. In N. subflava GN01, this binding property mediated agglutination of human erythrocytes in a neuraminidase-sensitive fashion. Nitrosoguanidine-induced nonhemagglutinative mutants of N. subflava GN01 had lost the ability to bind hematoside and sialosylparagloboside but remained able to bind lactosylceramide and gangliotetraosylceramide. These mutants fell into three classes with respect to their outer membrane protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Class 1 mutants were identical to the parent strain save for the loss of a 27-kilodalton (kDa) protein. Class 2 mutants showed an outer membrane protein profile identical to that of the wild type, whereas mutants belonging to class 3 showed a number of changes, including the apparent absence of the 27-kDa protein. The 27-kDa protein from N. subflava GN01 was purified from the supernatant. A polyclonal antiserum to the purified Sia-1 protein as well as a Sia-1-specific monoclonal antibody inhibited hemagglutination by strain GN01. The purified Sia-1 protein in the presence of diluted anti-Sia-1 antiserum mediated a neuraminidase-sensitive hemagglutination. The purified Sia protein from a class 2 mutant was not able to hemagglutinate when cross-linked with antibodies, suggesting that it is a mutant form of Sia-1 affected in the receptor-binding site. Immunoelectron microscopy with a Sia-1-specific monoclonal antibody revealed that the adhesin was nonfimbrial in nature, with aggregates of the adhesin extended out from the cells in a patchy fashion.
Partial characterization of an interleukin-1-like factor in human gingival crevicular fluid from patients with chronic inflammatory periodontal disease
Authors: Kabashima H , Maeda K , Iwamoto Y , Hirofuji T , Yoneda M , Yamashita K , Aono M
Published: January 2020

A significant level of interleukin-1-(IL-1)-like activity was detected in gingival crevicular fluid obtained from sites in patients with chronic inflammatory periodontal disease, confirming a previous report of IL-1-like activity detected in human gingival crevicular fluid from patients with chronic inflammatory periodontitis (J. A. Charon, T. A. Luger, S. E. Mergenhagen, and J. J. Oppenheim, Infect. Immun. 38:1190-1195, 1982). In the present study, we sought to investigate whether this IL-1-like activity belonged to IL-1 alpha or IL-1 beta and to characterize some of the biochemical properties of this factor. Polyclonal antibodies against recombinant human IL-1 alpha or IL-1 beta (rIL-1 alpha or rIL-1 beta) have been used for serological comparison of the IL-1-like factor. IL-1-like activity was completely neutralized by anti-human rIL-1 alpha antiserum, but not by anti-human rIL-1 beta antiserum. On gel filtration with a high-pressure liquid chromatographic Superose 12 column, IL-1-like activity was separated into two peaks, one with a molecular weight of about 43,000 and the other with a molecular weight of less than 17,000. The majority of the IL-1-like factor with a low molecular weight in human gingival crevicular fluid migrated at a molecular weight of about 17,000 under the reducing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specificity of the IL-1-like factor was further confirmed by an immunochemical method (Western blotting [immunoblotting]) by using anti-human rIL-1 alpha monoclonal antibodies. On isoelectric chromatography with a high-pressure liquid chromatographic Mono P column, the pI of this IL-1-like factor was between pH 4.9 and 5.2. These results suggest that the IL-1-like factor in human gingival crevicular fluid from diseased sites in patients with chronic inflammatory periodontitis consists predominantly of IL-1 alpha.
Production of a monoclonal antibody to Vibrio cholerae non-O1 heat-stable enterotoxin (ST) which is cross-reactive with Yersinia enterocolitica ST
Authors: Takeda T , Nair G B , Suzuki K , Shimonishi Y
Published: January 2020

A monoclonal antibody (MAb) against synthetic heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was produced. The MAb, namely, 2F, belonged to the immunoglobulin G1 class. Ascitic fluid drawn from pristane-primed BALB/c mice injected with a 2F-producing clone demonstrated anti-NAG-ST activity which could be detected in enzyme-linked immunosorbent assay even at a dilution of 1:128,000. Fifty-fold-diluted ascitic fluid could completely neutralize the activity of NAG-ST (synthetic and native) and Vibrio mimicus ST (identical to NAG-ST) in suckling mice. In the same assay, 2F could also neutralize Yersinia enterocolitica ST (Y-ST) but did not neutralize Escherichia coli STh and STp. A similar pattern of reactivity occurred in a competitive enzyme-linked immunosorbent assay with homologous and heterologous toxins. Competitive inhibition curves with synthetic peptides representing NAG-ST and its shorter analogs revealed that aspartic acid located at position 2 from the N terminus of NAG-ST was the essential residue of the recognized epitope. Significantly, in Y-ST, to which 2F cross-reacted, aspartic acid is in the corresponding position as that of NAG-ST, thereby confirming our conclusions that the epitope defining this MAb is aspartic acid.
Surface characteristics of Wolinella recta ATCC 33238 and human clinical isolates: correlation of structure with function
Authors: Borinski R , Holt S C
Published: January 2020

Selected characteristics of the surface of Wolinella recta ATCC 33238 and three W. recta clinical isolates (CI) were studied as well as the adherence of these strains to human gingival fibroblasts (HGF). W. recta ATCC 33238 and the CI were examined by electron microscopy, electrophoresis, isoelectric focusing, and adherence to HGF. Electron microscopic examination of CI revealed the presence of a periodic paracrystalline layer external to and associated with the outer membrane. This surface layer (S layer) was not observed on ATCC 33238. Whole cells and outer envelope protein profiles of the CI revealed major bands of 159- to 138-kilodalton proteins which were barely detectable in ATCC 33238. Repeated in vitro subculturing of the CI on solid or liquid medium resulted in both the physical loss of this layer and the loss of the high-molecular-weight proteins. Low-passage-number CI demonstrated 40 to 60% less adherence to HGF than ATCC 33238. These observations suggest that short term in vitro-subcultured W. recta strains possess surface characteristics which are significantly different from those of their long-term in vitro-subcultured counterparts. These differences may have significant effects on host cell interactions.
Selection and characterization of recombinant clones that produce Mycobacterium leprae antigens recognized by antibodies in sera from household contacts of leprosy patients
Authors: Hartskeerl R A , van Rens R M , Stabel L F , de Wit M Y , Klatser P R
Published: January 2020

A Mycobacterium leprae expression library was constructed in the vectors EX1, pEX2, and pEX3 and screened with a pool of 19 well-absorbed sera from household contacts of leprosy patients. Twelve selected recombinants that were further characterized differed clearly from recombinants selected with murine monoclonal antibodies. Whereas the monoclonal antibodies recognized mainly six recombinant antigens, the human sera from contacts reacted with a range of different recombinant antigens. None of the contact recombinant antigens was identical or related to well-characterized antigens from M. leprae or other mycobacteria selected with monoclonal antibodies, including proteins of the heat shock families. Two groups of recombinant antigens could be distinguished: one that was recognized by all sera used in the pool and one that was recognized by only a limited number of sera. These antigens, selected with sera from household contacts of previously untreated lepromatous leprosy patients, may be relevant to the immune responses during the early phase of infection with M. leprae.
Purification and characterization of an extracellular proteinase from Hendersonula toruloidea
Authors: Rojanavanich V , Yoshiike T , Tsuboi R , Takamori K , Ogawa H
Published: January 2020

An extracellular proteinase from a fast-growing strain of Hendersonula toruloidea was demonstrated when it was cultivated in liquid medium containing bovine serum albumin as the sole nitrogen source. Purification to homogeneity of the proteinase was performed by carboxymethyl cellulose, CM Sephadex G-50, and Sephacryl S-200 column chromatographies. The purified enzyme was a chymotrypsinlike serine proteinase, as indicated by the strong inhibitory activities of diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, L-1-tosylamido-2-phenylethylchloromethyl ketone, and chymostatin and good kinetic constants for a synthetic substrate, Suc-Ala-Ala-Pro-Phe-MCA. The enzyme had a pI of 8.4, a pH optimum of 9.0, and a molecular weight of 34,000. Skin constituents such as stratum corneum and nail, but not hair, were easily digested by this enzyme. Thus, this extracellular proteinase may play a role in the invasion of thickly keratinized skin and nail by this organism.
Inactivation of suppressor T cell activity by the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides
Authors: Baker P J , Taylor C E , Stashak P W , Fauntleroy M B , Hasløv K , Qureshi N , Takayama K
Published: January 2020

Antibody responses of mice immunized with type III pneumococcal polysaccharide were examined with and without treatment with nontoxic lipopolysaccharide from Rhodopseudomonas sphaeroides (Rs-LPS). The results obtained were similar to those described previously for mice treated with monophosphoryl lipid A (MPL) except that lower amounts of Rs-LPS were needed. Both were without effect when given at the time of immunization with type III pneumococcal polysaccharide but elicited significant enhancement when given 2 to 3 days later. Such enhancement was T cell dependent and not due to polyclonal activation of immunoglobulin M synthesis by B cells. Treatment with either Rs-LPS or MPL abolished the expression but not induction of low-dose paralysis, a form of immunological unresponsiveness known to be mediated by suppressor T cells (Ts). The in vitro treatment of cell suspensions containing Ts with extremely small amounts of Rs-LPS or MPI completely eliminated the capacity of such cells to transfer suppression to other mice. These findings indicate that the immunomodulatory effects of both MPL and Rs-LPS are mainly the result of eliminating the inhibitors effects of Ts; this permits the positive effects of amplifier T cells to be more fully expressed, thereby resulting in an increased antibody response. The significance of these and other findings to the use of Rs-LPS as a pharmacotherapeutic agent for gram-negative bacterial sepsis is discussed.
Expression of Neisseria meningitidis iron-regulated outer membrane proteins, including a 70-kilodalton transferrin receptor, and their potential for use as vaccines
Authors: Banerjee-Bhatnagar N , Frasch C E
Published: January 2020

The iron-regulated proteins (IRPs) of five group B meningococcal strains expressing class 2 outer membrane proteins were compared with those of five strains expressing class 3 proteins. Three to four high-molecular-weight IRPs were expressed by each strain, but their molecular sizes varied between strains and were not related to class 2 or 3 protein expression. Transferrin and hemoglobin could be used as a sole iron source. By using anti-human transferrin antibodies, it was shown that meningococcal cells and purified outer membranes bound transferrin. Growth under conditions of iron limitation caused a several-fold increase in the amount of transferrin bound to the cell surface. The transferrin-binding protein was detergent solubilized from outer membranes and partially purified. The isolated protein bound human transferrin and had an apparent molecular mass of 70 kilodaltons. To evaluate the potential of vaccines containing IRPs, we prepared outer membrane vaccines from strains M986-NCV-1 (M986) (--:2a: P1.2) and 44/76-M25 (44/76) (--:15:P1.15) grown to fully express their IRPs. Both vaccines induced significant anti-IRP antibodies as measured by enzyme immunoassay and by Western immunoblot with both M986 and 44/76 outer membranes. By Western blot analysis, the M986 vaccine induced antibodies to two different IRPs, one of which was shared with 44/76. Since the IRPs are major in vivo-expressed outer membrane proteins and are required for survival in vivo, these proteins should be evaluated for their usefulness in a group B meningococcal vaccine.
Cloning, expression, and occurrence of the Brucella Cu-Zn superoxide dismutase
Authors: Bricker B J , Tabatabai L B , Judge B A , Deyoe B L , Mayfield J E
Published: January 2020

Recently, the complete amino acid sequence of a protein expressed in Escherichia coli from cloned Brucella abortus DNA was reported. On the basis of amino acid homology, this protein was identified as a copper-zinc superoxide dismutase (Cu-Zn SOD) (B. L. Beck, L. B. Tabatabai, and J. E. Mayfield, Biochemistry 29:372-376, 1990). We demonstrate in this paper that the sequenced protein is the same as the previously studied salt-extractable protein BCSP20. The plasmid-encoded protein expressed from recombinant E. coli is identical to the Brucella-derived BCSP20 in molecular mass, N-terminal amino acid sequence, and cross-reactivity with homologous and heterologous rabbit sera against either the recombinant gene product or the Brucella-derived protein. A survey of the expression of the Cu-Zn SOD protein in Brucella biovars representing all species was done by Western blotting (immunoblotting) using antisera raised against the recombinant E. coli-derived protein. With the exception of B. neotomae and B. suis biovar 2, the Cu-Zn SOD protein was detectable in all Brucella species and biovars tested, including eight biovars of B. abortus.
Immunotherapeutic efficacy of bovine colostral immunoglobulins from a hyperimmunized cow against cryptosporidiosis in neonatal mice
Authors: Fayer R , Guidry A , Blagburn B L
Published: January 2020

Infection with Cryptosporidium parvum, a ubiquitous protozoan parasite of virtually all mammals, can cause mild to severe diarrhea in immunocompetent hosts and life-threatening diarrhea in immunocompromised hosts. Passive immunotherapy of experimentally infected animals and naturally infected humans with hyperimmune bovine colostrum has been reported to be efficacious, whereas chemotherapy has not. In this study, the efficacy of specific immunoglobulin isotypes purified from bovine colostrum from a cow hyperimmunized with Cryptosporidium parvum was assessed in neonatal BALB/c mice. Mice were orally infected with oocysts and treated with whole whey immunoglobulin G1 (IgG1), IgG2, IgA, or IgM at six intervals from 22 to 66 h postinfection. In histologic sections of intestine examined at 72 h postinfection, the reduction in number of intestinal stages in treated mice versus untreated controls was very highly significant (P less than 0.0001). The greatest reduction in parasite number was found in mice treated with IgG1, IgA, or whey.
Evidence for diversity of Plasmodium falciparum sporozoite surface antigens derived from analysis of antibodies elicited in humans
Authors: Galey B , Druilhe P , Ploton I , Desgranges C , Asavanich A , Harinasuta T , Marchand C , Brahimi K , Charoenvit Y , Paul C
Published: January 2020

We have compared the reactivities of antibodies developed by individuals frequently exposed to Plasmodium falciparum infections with the epitopes contained within the repeats of the circumsporozoite (CS) protein and their reactivities with the epitopes of a native molecule(s) accessible on the sporozoite surface. Results of direct-binding assays and competition assays between artificial and native molecules or between human antibodies and anti-CS monoclonal antibodies suggest that humans respond preferentially to epitopes not contained within the repeats of the CS protein and probably not contained in the whole CS protein. Human monoclonal antibodies reactive with P. falciparum sporozoite surface antigens were produced by Epstein-Barr virus transformation of human lymphocytes. Their pattern of reactivity with sporozoites from a number of different isolates indicates the existence of several distinct epitopes on the parasite surface. Differences between isolates and between sporozoites within a given sample were observed. No single human monoclonal antibody capable of detecting an epitope expressed in all the parasites studied was found.
Epitopes of Escherichia coli alpha-hemolysin: identification of monoclonal antibodies that prevent hemolysis
Authors: Ji G E , O'Hanley P
Published: January 2020

The antigenic regions of Escherichia coli alpha-hemolysin were determined by antibody binding to cyanogen bromide (CnBr) fragments of this protein under denatured conditions. Alpha-hemolysin was isolated from filtered culture supernatants of a recombinant strain by a combination of trichloroacetic acid precipitation and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Alpha-hemolysin was used to (i) produce polyclonal rabbit antisera and murine monoclonal immunoglobulin G (IgG) antibodies and (ii) generate CnBr fragments. Rabbit IgG and 13 murine IgG monoclonal antibodies (MAbs) were elicited to alpha-hemolysin as determined by enzyme-linked immunosorbent and immunoprecipitation assays. Antibodies bound to three specific CnBr fragments of alpha-hemolysin in Western blots (immuno-blots) from sodium dodecyl sulfate-polyacrylamide gels: CnBrII (encompassing residues [R] 2 to 160), CnBrV (R 425 to 892), and CnBrVI (R 893 to 1023). Five MAbs bound to CnBrII, seven MAbs bound to CnBrV, and one MAb bound to CnBrVI. These specific CnBr fragments are predicted to be hydrophilic and charged. There was no antibody binding to the highly hydrophobic CnBrIII (R 161 to 416). Similar binding patterns were observed when rabbit polyclonal anti-alpha-hemolysin IgG was used. Polyclonal antibodies to alpha-hemolysin readily inhibited hemolysis and its neutralization capacity was 4- to 64-fold more potent than neutralizing MAbs. The five MAbs that bind to CnBrII possessed hemolytic neutralizing activity to various degrees. In contrast, only three of seven MAbs that bind to CnBrV fragment exhibited neutralization capacity to various degrees; the MAb to CnBrVI did not exhibit this capacity. Based on these data, we predict that denatured alpha-hemolysin and its CnBrII and CnBrV fragments might be worthwhile immunoprophylactic candidates for the prevention of hemolysin-mediated E. coli tissue injury.
Conservation of Salmonella typhimurium virulence plasmid maintenance regions among Salmonella serovars as a basis for plasmid curing
Authors: Tinge S A , Curtiss R
Published: January 2020

The association of large plasmids with virulence in invasive Salmonella serovars has led to a number of studies designed to uncover the role of these plasmids in virulence. This study addresses two aspects of virulence-associated plasmids. The first is the distribution of the replication and maintenance regions among the plasmids of different Salmonella serovars, and the second is the use of the conserved virulence plasmid par region to provide a rapid method for eliminating the virulence plasmids specifically. Colony blots revealed that the par and repB regions of the S. typhimurium virulence plasmid hybridized with 80% of the isolates of S. choleraesuis, S. dublin, S. enteritidis, S. gallinarum, S. pullorum, and S. typhimurium, while the repC region was not detected in any of the isolates of S. dublin, S. gallinarum, or S. pullorum. None of these maintenance regions was found in any of the 30 additional serovars tested. The large plasmids of those serovars that hybridized with par were labeled with a Kmr insert within parA via P22HTint or P1L4 transduction, which destabilized the plasmids and allowed the rapid isolation of plasmid-free derivatives for all of the serovars, except for S. dublin, which exhibited weak homology with par.
Role of the P plasmid in attenuation of Vibrio cholerae O1
Authors: Bartowsky E J , Attridge S R , Thomas C J , Mayrhofer G , Manning P A
Published: January 2020

The conjugative plasmid P of Vibrio cholerae has been shown to have a suppressive effect on the virulence of hypertoxigenic strains like 569B. In this study, we have sought to analyze this phenomenon. Utilizing the infant mouse cholera model, we have demonstrated that the presence of P increases the 50% lethal dose of V. cholerae classical Inaba 569B by more than 300-fold. No effect of P on cholera toxin (CT) production, whether measured by GM1 enzyme-linked immunosorbent assay, by CT activity in ligated rabbit ileal loops, or by transcription from the CT promoter, could be discerned. Colonization of the intestine by P+ derivatives was dramatically reduced although only a minor effect could also be demonstrated on in vitro attachment to intestinal strips. Electron microscopic examination suggested that the P plasmid was affecting the production of the TCP pilus. Another conjugative plasmid, V, has also been examined, but it had no effect on virulence.
Nucleotide sequence of htpB, the Legionella pneumophila gene encoding the 58-kilodalton (kDa) common antigen, formerly designated the 60-kDa common antigen
Authors: Sampson J S , O'Connor S P , Holloway B P , Plikaytis B B , Carlone G M , Mayer L W
Published: January 2020

Gene htpB, which encodes the 58-kilodalton protein of Legionella pneumophila, was cloned in Escherichia coli and its complete nucleotide sequence was determined. Analysis of this sequence revealed an open reading frame of 1,644 nucleotides encoding a protein with a predicted molecular mass of 57,952 daltons. Data obtained by amino-terminal sequencing of the purified 58-kilodalton protein agreed, except for one amino acid residue, with the predicted amino acid sequence, identifying this open reading frame as htpB. A comparison of the primary structure of this protein to other proteins of similar molecular weights from E. coli, Mycobacterium leprae, M. tuberculosis, and Coxiella burnetii revealed significant regions of sequence similarity, which are discussed.
Localization of protective epitopes within the pilin subunit of the Vibrio cholerae toxin-coregulated pilus
Authors: Sun D X , Seyer J M , Kovari I , Sumrada R A , Taylor R K
Published: January 2020

From a collection of monoclonal antibodies (MAbs) that recognize the native structure of the toxin-coregulated pilus of Vibrio cholerae, two protective MAbs (16.1 and 169.1) were used to localize the corresponding epitopes on the pilus. These MAbs were shown to specifically recognize the carboxyl half of the TcpA pilin subunit, as determined by their recognition of proteolytic fragments and hybrid pilin proteins. The positions of the epitopes were precisely determined through the use of overlapping synthetic peptides corresponding to this region of the pilin. The MAbs were found to recognize adjacent peptides, delineating a region between residues 157 and 199. Since the protective nature is specific for these two antibodies, the findings suggest that this region defines a domain that participates in toxin-coregulated pilus-mediated colonization and therefore represents a target for studies of its potential as an immunogen for incorporation into a component cholera vaccine.
Murine macrophage interleukin-1 release by capsularlike serotype-specific polysaccharide antigens of Actinobacillus actinomycetemcomitans
Authors: Takahashi T , Nishihara T , Ishihara Y , Amano K , Shibuya N , Moro I , Koga T
Published: January 2020

Serotype-specific polysaccharide antigens (SPAs) were extracted from whole cells of Actinobacillus actinomycetemcomitans ATCC 29523 (serotype a), Y4 (serotype b), and NCTC 9710 (serotype c) by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300 columns. Y4 SPA induced interleukin-1 (IL-1) release by P388D1 murine macrophages. Polymyxin B had virtually no effect on the release of IL-1. Rabbit anti-murine IL-1 serum strongly suppressed the proliferation of C3H/HeJ mouse thymocytes induced with the culture supernatants of Y4 SPA-stimulated P388D1 cells and a submitogenic dose of concanavalin A. Gel filtration of the culture supernatants of Y4 SPA-stimulated macrophages on Sephacryl S-200 showed that an IL-1 peak at a point corresponding to approximately 16.5 kDa was eluted. The ability of SPAs from strains ATCC 29523 and NCTC 9710 to induce the release of IL-1 was lower than that of Y4 SPA. The IL-1-releasing ability of serotype a and c antigens was enhanced by deacetylation of both polysaccharides, suggesting that acetyl groups of these antigens might hinder the interaction between the antigens and macrophages.
Role of bacterial association with Kupffer cells in occurrence of endogenous systemic bacteremia
Authors: Hirakata Y , Tomono K , Tateda K , Matsumoto T , Furuya N , Shimoguchi K , Kaku M , Yamaguchi K
Published: January 2020

Bacteremia in immunocompromised hosts often arises from their endogenous intestinal flora. We produced experimental endogenous bacteremia by administering cyclophosphamide and ampicillin to conventional and specific-pathogen-free mice. The frequencies of bacteremia and mortality in the conventional mice were significantly higher than for the specific-pathogen-free mice. Pseudomonas aeruginosa was the major pathogen causing systemic bacteremia in conventional mice and was associated with a high mortality rate. Morganella morganii caused systemic bacteremia in both conventional and specific-pathogen-free mice. In contrast, Escherichia coli, enterococci, or other species most often caused portal bacteremia only. To determine the mechanism of occurrence of systemic bacteremia, we investigated bacterial blood clearance in mice and association with murine Kupffer cells, using several bacterial strains isolated from mice with bacteremia. Blood clearance rates and the abilities of isolated Kupffer cells to associate with bacteria were significantly greater for the organisms causing portal bacteremia than for those causing systemic bacteremia. There were no significant differences between the blood clearance rates in carrageenan-treated mice and that in normal mice. Moreover, association at 4 degrees C was not different from that at 37 degrees C. The results suggest that blood clearance of bacteria reflects bacterial adherence to Kupffer cells and that the resistance of bacteria to association with Kupffer cells plays an important role in the occurrence of overwhelming systemic bacteremia in this animal model.
Identification and characterization of an immunodominant 58-kilodalton antigen of Aspergillus fumigatus recognized by sera of patients with invasive aspergillosis
Authors: Fratamico P M , Buckley H R
Published: January 2020

Sera from 38 patients with invasive aspergillosis were tested by Western immunoblotting for the presence of antibodies to antigens of Aspergillus fumigatus present in a mycelial extract of the organism. All of the sera contained antibodies to an antigen of molecular weight 58,000, which was which was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was the only antigen recognized in approximately 90% of the sera tested. The 58-kDa antigen is an abundant component of mycelial extracts composing approximately 50% of the Coomassie blue-stained protein. The antigen also contains carbohydrate, since it is stained by the carbohydrate stain periodic acid-Schiff and it binds to the lectin concanavalin A. It was purified by immunoaffinity chromatography employing a monoclonal antibody directed against an epitope on the 58-kDa antigen. Analysis of the purified antigen by gas-liquid chromatography revealed the presence of mannose, galactose, and glucose residues in a 2:1:2 ratio. The ratio of protein to carbohydrate is 1.16:1. The protein is slightly acidic, containing relatively high quantities of glutamic and aspartic acids, glycine, alanine, serine, and threonine. The 58-kDa antigen also contains phosphate groups as part of its structure. Serological activity was totally destroyed after treatment with sodium metaperiodate and was partially destroyed after treatment with pronase. The 58-kDa antigen was not able to hydrolyze protein.
Pulsed-field gel electrophoretic analysis of leptospiral DNA
Authors: Taylor K A , Barbour A G , Thomas D D
Published: January 2020

The genomic structures of spirochete species are not well characterized, and genetic studies on these organisms have been hampered by lack of a genetic exchange mechanism in these bacteria. In view of these observations, pulsed-field gel electrophoresis was used to examine the genomes of Leptospira species. Live cells, prepared in agarose plugs, were lysed in situ, and the DNA was analyzed under different electrophoretic conditions. Pulsed-field gel electrophoresis of DNA digested with infrequently cutting restriction enzymes showed that the genome of Leptospira interrogans serovar canicola is approximately 3.1 Mb, while that of the saprophytic L. biflexa serovar patoc I is 3.5 Mb. DNA forms of approximately 2,000 and 350 kb which were present in samples from L. interrogans serovars were not readily detected in nonpathogenic serovars. Three distinct populations, designated type alpha, beta, and gamma, of L. interrogans DNA molecules were further analyzed with two-dimensional gel electrophoresis. Evidence suggested that two of these DNA forms, type alpha and gamma, were linear structures. Pulsed-field gel electrophoresis has proven to be a valuable tool with which to size bacterial genomes and to take the first steps toward characterization of a form of leptospiral DNA which behaves as a linear molecule and which may be related to the virulence of L. interrogans.
Prostaglandin E release from human monocytes treated with lipopolysaccharides isolated from Bacteroides intermedius and Salmonella typhimurium: potentiation by gamma interferon
Authors: Nichols F C , Peluso J F , Tempro P J , Garrison S W , Payne J B
Published: January 2020

The purpose of this investigation was to examine gamma interferon potentiation of lipopolysaccharide (LPS) responses in human monocytes by using phenol-water-extracted (unfractionated) and highly purified LPS preparations isolated from Bacteroides intermedius and Salmonella typhimurium. Phenol-water-extracted LPS preparations from these bacteria were further purified by chromatography over Sepharose-CL-4B. LPS enrichment in pooled column fractions was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantitation of hydroxy-fatty acid and 2-keto-3-deoxyoctulosonic acid content, protein contamination, and anthrone-reactive material. Monocyte stimulation by LPS, measured as prostaglandin E (PGE) release, was assessed with and without gamma interferon treatment. Cells were either treated simultaneously with gamma interferon and LPS or pretreated with gamma interferon prior to LPS stimulation. PGE release from counterflow-isolated monocytes was quantitated during the 0- to 24-h and 24- to 48-h culture intervals. Contrary to previous results from this laboratory, phenol-water-extracted LPS preparations from B. intermedius and S. typhimurium were similar in their capacities to stimulate PGE release from monocytes. Molecular sieve chromatography was found to remove substantial amounts of high-molecular-weight polysaccharide contaminants only from the B. intermedius LPS but did not significantly alter the potency of either B. intermedius or S. typhimurium LPS. Gamma interferon cotreatment did not potentiate the release of PGE with any of the LPS preparations tested. However, 24-h pretreatment of monocytes with gamma interferon followed by a 24-h exposure to LPS resulted in significant potentiation of PGE release over LPS alone. In addition, B. intermedium preparations were approximately threefold more potent than similarly prepared LPS isolates from S. typhimurium following gamma interferon pretreatment. These results indicate that gamma interferon can selectively potentiate the effects of B. intermedius LPS in human monocyte isolates.
Toxin A of Clostridium difficile binds to the human carbohydrate antigens I, X, and Y
Authors: Tucker K D , Wilkins T D
Published: January 2020

Clostridium difficile causes pseudomembranous colitis in humans. The enterotoxin (i.e., toxin A) from this organism is believed to be responsible for the initial intestinal pathology associated with this disease. Previous work shows that this toxin binds to carbohydrates that contain Gal alpha 1-3Gal beta 1-4GlcNAc. However, this carbohydrate is not present on normal human cells. Therefore, this study was undertaken to identify potential receptors for toxin A that do exist on human intestinal epithelium. Using enzyme-linked immunosorbent assay, affinity chromatography, and altered migration in an electric field, we assayed the binding of toxin A to purified carbohydrates and glycoproteins. We found that toxin A bound to the carbohydrate antigens designated I, X, and Y. Each of these carbohydrates exist on the intestinal epithelium of humans.
Effects of immunization with Porphyromonas gingivalis and Prevotella intermedia on progression of ligature-induced periodontitis in the nonhuman primate Macaca fascicularis
Authors: Ebersole J L , Brunsvold M , Steffensen B , Wood R , Holt S C
Published: January 2020

The nonhuman primate (Nhp) has proven to be a useful model of human periodontitis. This study describes the immunological characteristics of this model and the ability of active immunization to interfere with ecological changes in the microbiota and its associated disease symptoms. Nhps were parenterally immunized with whole-cell antigens of Porphyromonas gingivalis and Prevotella intermedia. The immunization elicited an approximate 2-log increase in serum immunoglobulin G (IgG), IgM, and IgA isotype antibody that was highly specific for these immunogens. Postimmunization and postligation, there was minimal change in the levels of specific antibody. P. gingivalis immunization significantly inhibited the emergence of this species during disease progression. In contrast, induction of anti-P. intermedia antibody had a minimal effect on this species within the subgingival plaque. Plaque indices showed few changes that could be attributed to active immunization. Both bleeding on probing and loss of attachment were higher in ligated sites of immunized animals than in the placebo-treated group. A significant increase in bone density loss was observed in the ligated teeth from immunized versus control animals. These findings indicate that active immunization of Nhps can elicit a substantial systemic immune response; however, while this response may effect the emergence of an individual microorganism, it appears that other ecological considerations are critical in disease progression. It is also possible that the induction of a broad-based immune response to multiple bacterial antigens can result in increased disease, potentially associated with hypersensitivity reactions to the bacteria in the subgingival plaque.
Specific T-cell response to a Pneumocystis carinii surface glycoprotein (gp120) after immunization and natural infection
Authors: Fisher D J , Gigliotti F , Zauderer M , Harmsen A G
Published: January 2020

T cells have been shown to be important in recovery from Pneumocystis carinii pneumonitis, although no specific antigen of P. carinii has been defined as containing T-cell epitopes. P. carinii has an abundant mannosylated surface glycoprotein of approximately 120 kDa (gp120) which induces a prominent host antibody response in experimental animals after exposure to P. carinii in the environment or after recovery from P. carinii pneumonitis. P. carinii gp120 was purified from infected lungs by lectin affinity chromatography. Standard in vitro lymphocyte stimulation assays using purified gp120 and control normal lung preparations were performed on isolated T cells obtained from BALB/c mice after immunization with P. carinii-infected crude lung homogenates or lectin-purified gp120. Lymphocytes from reconstituted severe combined immunodeficient mice which had recovered from naturally acquired P. carinii pneumonitis were also tested. A specific T-cell response was elicited by gp120 after immunization with P. carinii gp120 and after recovery from P. carinii pneumonitis. In addition, the mice developed a strong antibody response to gp120 as ascertained by Western blot (immunoblot). These data suggest that gp120 may be important in the recognition of P. carinii by T cells.
Endogenous gamma interferon-independent host resistance against Listeria monocytogenes infection in CD4+ T cell- and asialo GM1+ cell-depleted mice
Authors: Nakane A , Numata A , Chen Y , Minagawa T
Published: January 2020

The effects of in vivo administration of antibodies against T-cell subsets and asialo GM1 (ASGM1)-bearing cells on endogenous gamma interferon (IFN-gamma) production and host defense in Listeria monocytogenes-infected mice were investigated. Endogenous IFN-gamma titers in the bloodstreams and spleen extracts of mice on day 2 of infection were partially suppressed by administration of rabbit anti-ASGM1 antibody, but not by anti-CD4 monoclonal antibody (MAb) or anti-CD8 MAb. Of the different combinations of these three antibodies, the most suppressive effect on IFN-gamma production was observed after administration of anti-CD4 Mab and anti-ASGM1 antibody, although anti-CD8 MAb combined with anti-CD4 MAb partially inhibited IFN-gamma production. In contrast, antilisterial resistance was suppressed by the administration of anti-CD8 MAb but not by anti-CD4 MAb or anti-ASGM1 antibody. Antilisterial resistance in mice in which both CD4+ cells and ASGM1+ cells had been depleted was performed as efficiently as in normal mice in spite of the fact that endogenous IFN-gamma production was markedly suppressed. Furthermore, these mice also eliminated L. monocytogenes cells efficiently from the spleens even when they were pretreated with anti-mouse IFN-gamma MAb. These results indicate that CD4+ T cells, CD8+ T cells, and ASGM1+ cells are all responsible for endogenous IFN-gamma production and that antilisterial resistance and endogenous IFN-gamma production are not absolutely correlated.
Ability of recombinant or native proteins to protect monkeys against heterologous challenge with Plasmodium falciparum
Authors: Etlinger H M , Caspers P , Matile H , Schoenfeld H J , Stueber D , Takacs B
Published: January 2020

To circumvent problems associated with polymorphic vaccine candidates for Plasmodium falciparum malaria, we evaluated recombinant proteins representing sequences from relatively high conserved regions of the precursor to the major merozoite surface proteins, gp190, for their ability to protect Saimiri monkeys against malaria challenge. Recombinant proteins represented amino acid residues 147 to 321 (p190-1) or 147 to 321 and 1060 to 1195 (p190-3), and their efficacy was compared with that of native gp190 and its processed products. All antigens were derived from P. falciparum K1, a Thai isolate, while the challenge strain was Palo Alto (from Uganda, Africa), which contains, with the exception of the N-terminal 375 amino acids, which are almost identical to the K1 sequence, essentially the MAD-20 allelic form of gp190. By 12 days following challenge, each control monkey required drug treatment. Three monkeys injected with p190-3 required therapy, while one cleared the parasites without therapy. Two monkeys injected with p190-1 received therapy on day 14, while the remaining two cleared the parasites without therapy. Of four animals injected with native gp190, because of health reasons unrelated to malaria, one was not challenged with parasites and one was removed from the study 8 days after challenge when its parasitemia was 1.1% (parasitemias in control animals ranged from 4.3 to 9%); the remaining two cleared the parasites after maximum parasitemias of 0.45 and 0.53%. The highest levels of antiparasite antibody were produced by animals immunized with native gp190. There was a significant correlation between monkeys which did not require drug treatment and antiparasite antibody. These results may suggest that native gp190 and/or its processed products can provide excellent protection against heterologous challenge and that antibody is important for protection. The challenge for vaccine development is to identify the protective sequence(s).
Depletion of complement and effects on passive transfer of resistance to infection with Borrelia burgdorferi
Authors: Schmitz J L , Lovrich S D , Callister S M , Schell R F
Published: January 2020

When irradiated hamsters are passively immunized with immune serum before challenge with Borrelia burgdorferi, they are completely protected from arthritis and infection. The complement dependency of this protection was addressed by treating hamsters with cobra venom factor. Depletion of complement abrogated the ability of immune serum obtained 1 and 10 weeks after infection to confer complete protection. By contrast, depletion of complement had no effect on the ability of 3-week immune serum to confer protection. These results suggest that complement-dependent, and possibly complement-independent, antibodies are important for preventing the induction of Lyme arthritis.
Recombinant Escherichia coli clones expressing Chlamydia trachomatis gene products attach to human endometrial epithelial cells
Authors: Schmiel D H , Knight S T , Raulston J E , Choong J , Davis C H , Wyrick P B
Published: January 2020

To identify Chlamydia trachomatis genes involved in attachment to host cells, a chlamydial genomic library was screened on the basis of binding characteristics by two methods. In the whole-cell screen, individual recombinant Escherichia coli clones were assayed for adherence to eukaryotic cells. In the membrane-binding screen, each recombinant colony of E. coli was treated with CHCl3 and assayed for binding to purified, 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS)-solubilized, 35S-labeled eukaryotic membrane material. Initial screening with McCoy cells was refined by using HEC-1B cells, a human endometrial epithelial cell line, which discriminate among recombinants adhering to McCoy cells. Some recombinants demonstrate significantly greater adherence to HEC-1B cells than to McCoy cells and appear, by transmission electron microscopy, to associate with electron-dense areas of the epithelial cell plasma membrane, resembling coated pits. Recombinants positive by one or both screening methods were examined by Southern and Western (immunoblot) analyses, which revealed the presence of chlamydial sequences inserted in the plasmids and the expression of novel 18-, 28-, and approximately 82 kDa, and perhaps of 18 Maxicell analysis of selected recombinants confirmed that the proteins of 28 and approximately 82 kDa, and perhaps of 18 kDa, are plasmid encoded. Antiserum generated against the recombinant approximately 82-kDa protein reacted in Western analysis with a similar-sized protein from C. trachomatis serovar E elementary bodies (EB) and reticulate bodies, serovar L2 EB, and C. psittaci EB. E. coli JM109(pPBW58) contains a 6.7-kb plasmid insert which encodes proteins of all three sizes. Under a number of different conditions in the whole-cell attachment assay--i.e., at 4 degrees C, in Ca(2+)- and Mg(2+)-free medium, in the presence of trypsin or dextran sulfate, and with rabbit aortic endothelial cells--the binding specificity of JM109(pPBW58) parallels that of C. trachomatis EB. Finally, the adherence phenotype of E. coli JM109(pPBW58) correlates directly with the presence of the recombinant plasmid; the phenotype is lost concurrently with loss of the recombinant plasmid, and the into E. coli JM109. The role of the 18-, 28-, and approximately 82-kDa proteins in mediating attachment, whether they act in concert as a complex or individually, has yet to be determined.