bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression and purification of a fimbria-associated adhesin of Streptococcus parasanguis
Authors: Oligino L , Fives-Taylor P
Published: January 2020

A Streptococcus parasanguis adhesin that blocks the attachment of S. parasanguis FW213 to saliva-coated hydroxyapatite (SCHA) has been purified. Previous work demonstrated that the attachment of FW213 to SCHA is mediated by fimbriae and that one component associated with fimbriae is a 36-kDa protein (FimA) that reacts with antifimbria serum in Western blots (immunoblots) and is not present in afimbriated mutants. To obtain amounts of FimA sufficient for adhesion blocking assays, we cloned the gene coding for FimA into an Escherichia coli T7 overexpression system. The resulting strain produced large amounts of FimA, as much as 50% of the total cell protein. FimA was purified by elution from sodium dodecyl sulfate-polyacrylamide gels, and its native conformation was reestablished by sodium dodecyl sulfate removal, resolubilization in guanidine hydrochloride, and 50-fold dilution. Some refolded FimA aggregated into dimers and trimers. Preincubation of SCHA with 100 micrograms of purified, renatured FimA per ml blocked 85% of the binding of FW213. The FimA-SCHA complex was quite stable and could be washed continuously for at least 2 h with only a slight loss of FimA blocking activity. When FimA was added to preformed bacterium-SCHA complexes, it displaced 40% of the bacteria already bound to SCHA. The results suggest that FimA is an adhesin with a high substrate affinity and may prove useful in the development of a therapeutic agent for the prevention of plaque formation and endocarditis initiated by the sanguis streptococci.
Induction of bradyzoite-specific Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages
Authors: Bohne W , Heesemann J , Gross U
Published: January 2020

By using stage-specific monoclonal antibodies, an in vitro model has been developed to analyze the kinetics of expression of stage-specific antigens during the conversion process between tachyzoites and bradyzoites of Toxoplasma gondii. Following infection of murine macrophages with bradyzoites, the expression of bradyzoite-specific antigens declined, whereas the expression of tachyzoite-specific antigens increased during the first 72 h postinfection. Conversely, in gamma interferon-treated murine macrophages infected with tachyzoites, the inhibitory effect of gamma interferon on replication of parasites was accompanied by the induction of bradyzoite-specific antigens.
C1q binding and activation of the complement classical pathway by Klebsiella pneumoniae outer membrane proteins
Authors: Albertí S , Marqués G , Camprubí S , Merino S , Tomás J M , Vivanco F , Benedí V J
Published: January 2020

The mechanisms of killing of Klebsiella pneumoniae serum-sensitive strains in nonimmune serum by the complement classical pathway have been studied. The bacterial cell surface components that bind C1q more efficiently were identified as two major outer membrane proteins, presumably the porins of this bacterial species. These two outer membrane proteins were isolated from a representative serum-sensitive strain. We have demonstrated that in their purified form, they bind C1q and activate the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted in factor D). Binding of C1q to other components of the bacterial outer membrane, in particular the rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the two outer membrane proteins. The antibody-independent binding of C1q to serum-sensitive strains was independent of the presence of capsular polysaccharide, while strains possessing lipopolysaccharide O antigen bind less C1q and are resistant to complement-mediated killing.
Phenotypic characterization of xpr, a global regulator of extracellular virulence factors in Staphylococcus aureus
Authors: Smeltzer M S , Hart M E , Iandolo J J
Published: January 2020

We recently described a Tn551 insertion in the chromosome of Staphylococcus aureus S6C that resulted in drastically reduced expression of extracellular lipase (M. S. Smeltzer, S. R. Gill, and J. J. Iandolo, J. Bacteriol. 174:4000-4006, 1992). The insertion was localized to a chromosomal site (designated omega 1058) distinct from the lipase structural gene (geh) and the accessory gene regulator (agr), both of which were structurally intact in the lipase-negative (Lip-) mutants. In this report, we describe a phenotypic comparison between strains S6C, a hyperproducer of enterotoxin B; KSI9051, a derivative of S6C carrying the Tn551 insertion at omega 1058; ISP546, an 8325-4 strain that carries a Tn551 insertion in the agr locus; and ISP479C, the parent strain of ISP546 cured of the Tn551 delivery plasmid pI258repA36. Compared with their respective parent strains, ISP546 and KSI9051 produced greatly reduced amounts of lipase, alpha-toxin, delta-toxin, protease, and nuclease. KSI9051 also produced reduced amounts of staphylococcal enterotoxin B. Coagulase production was increased in ISP546 but not in KSI9051. Using a mouse model, we also demonstrated that ISP546 and KSI9051 were far less virulent than ISP479C and S6C. We have designated the genetic element defined by the Tn551 insertion at omega 1058 xpr to denote its role as a regulator of extracellular protein synthesis. We conclude that xpr and agr are similar and possibly interactive regulatory genes that play an important role in pathogenesis of staphylococcal disease.
Plasmodium falciparum-specific immunoglobulin G (IgG), IgM, and IgE antibodies in paired maternal-cord sera from east Sepik Province, Papua New Guinea
Authors: Desowitz R S , Elm J , Alpers M P
Published: January 2020

Enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot) serological analyses for immunoglobulin G (IgG), IgM, and IgE antibodies to Plasmodium falciparum were made from 46 maternal-cord serum pairs obtained from parturient East Sepik (Papua New Guinea) women and their newborn. Concurrent study of these women had shown that placental parasitemia rates were related to parity with the highest rate (41%) in the primiparous group and the lowest rate (3%) in the women who had given birth more than three times (> 3 parity group). Overall ELISA positivity rates for antimalarial IgG, IgM, and IgE antibodies in the maternal sera were 54.3, 28.2, and 8.3%, respectively, while those for the cord sera were 36.9, 0, and 16.6% respectively. Seropositivity rates were not related to maternal parity group, except for maternal IgE, in which there was a higher rate, of borderline significance, in the > 3 parity group than in the primiparous group. Cord IgE positivity was largely independent of maternal positivity and vice versa. Cord and maternal IgG immunoblot pairs showed near homology. IgG antibodies to the P. falciparum antigens of sizes < 36 kDa were either weak or absent in parity group 1 and 2 maternal-cord serum pairs. Neither ELISA or immunoblot revealed IgM antibody in the cord serum samples. Maternal IgM antibodies showed a heterogeneity of responses both between paired IgG immunoblots and between different serum samples. The IgE immunoblots exhibited a similar diversity, albeit of less complexity. The presence of P. falciparum-specific IgE in the cord sera would indicate that prenatal immune hypersensitization of the fetus to malaria had occurred.
LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression
Authors: Gambello M J , Kaye S , Iglewski B H
Published: January 2020

The lasR gene of Pseudomonas aeruginosa is required for transcription of the genes for elastase (lasB) and LasA protease (lasA), two proteases associated with virulence. We report here that the alkaline protease gene (apr) also requires the lasR gene for transcription. Alkaline protease mRNA was absent in the lasR mutant PAO-R1 and present when an intact lasR gene was supplied in trans as determined by Northern (RNA) analysis. The lasR gene also enhances exotoxin A production. Exotoxin A activity in supernatants of PAO-R1 were 30% less than in supernatants of the parental strain, PAO-SR. Multiple copies of lasR in trans in PAO-R1 in increased toxin A activity to twice the parental levels. Analysis of PAO-R1 containing the toxA promoter fused to beta-galactosidase suggests that LasR acts at the toxA promoter or at upstream toxA mRNA sequences. beta-Galactosidase activity was approximately 40% lower in PAO-R1 than in the parental strain, PAO-SR. Furthermore, the effect of LasR on the toxA promoter is not due to the stimulation of transcription of regA, a transcriptional activator of toxA. No difference in chloramphenicol acetyltransferase (CAT) activity was noted between PAO-SR and PAO-R1 containing transcriptional regA promoter-CAT gene fusions. These results broaden the regulatory dominion of lasR and suggest that the lasR gene plays a global role in P. aeruginosa pathogenesis.
Immunochemistry of capsular type polysaccharide and virulence properties of type VI Streptococcus agalactiae (group B streptococci)
Authors: von Hunolstein C , D'Ascenzi S , Wagner B , Jelínková J , Alfarone G , Recchia S , Wagner M , Orefici G
Published: January 2020

The immunochemistry of capsular type polysaccharide and virulence characteristics of group B streptococci (GBS), type VI, were studied. By high-pressure anion-exchange chromatography and pulsed amperometric detection, as well as by 13C nuclear magnetic resonance analysis, both extracellular and cell-bound polysaccharides were found to contain glucose, galactose, and N-acetylneuraminic acid in the molar ratio of 2:2:1, respectively. At variance with all other GBS serotypes described to date (Ia, Ib, II, III, IV, and V), no N-acetylglucosamine was present, whatever the source of the material (secreted or cell bound; reference or clinical isolate). Sialic acid was probably involved in the immunodeterminant structure of this new serotype since cleavage of this sugar from the polysaccharide gave rise to an antigen which reacted very weakly with type VI antiserum and to a precipitation line in immunodiffusion with no identity with the native type VI polysaccharide. By using type VI antiserum and the protein A-gold technique, a large capsule was observed in the type VI GBS reference strain by electron microscopy. All type VI strains examined were lethal for CD-1 mice, the 50% lethal dose after intraperitoneal challenge ranging from 1.0 (+/- 0.9, standard deviation) x 10(5) to 2.5 (+/- 1.5, standard deviation) x 10(5) CFU per mouse. A rabbit antiserum against capsular type polysaccharide exhibited both protective activity for mice injected intraperitoneally with type VI reference strain or with clinical isolates and opsonic activity in a phagocytosis assay.
Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection
Authors: Kawamoto S , Shibano Y , Fukushima J , Ishii N , Morihara K , Okuda K
Published: January 2020

Both Pseudomonas aeruginosa elastase and Bacillus thermoproteolyticus thermolysin are zinc metalloproteases. On the basis of the high homology of the P. aeruginosa elastase with the Bacillus thermolysin, we hypothesized that Glu-141 and His-223 are the key residues for catalytic activity of the Pseudomonas elastase. To test this possibility, we replaced Glu-141 with Asp, Gln, and Gly and His-223 with Gly, Glu, and Leu by site-directed mutagenesis. These substitutions dramatically diminished the proteolytic activities of the mutant elastases when they were expressed in Escherichia coli cells. Although these mutant elastase precursors (proelastases) were produced, no appreciable processing was observed with these mutants. The possibility that autocatalysis is involved in both the processing and activation of elastase is discussed. Furthermore, by immunizing mice with vaccines made from these mutant elastase, we were able to obtain good protection against an intraperitoneal P. aeruginosa challenge.
Changes in inflammatory mediators in experimental periodontitis in the rhesus monkey
Authors: Smith M A , Braswell L D , Collins J G , Boyd D L , Jeffcoat M K , Reddy M , Li K L , Wilensky S , Vogel R , Alfano M
Published: January 2020

Ligature-induced periodontitis was monitored for 6 months in eight Macaca mulatta monkeys to examine clinical status, radiographic bone level, and crevicular fluid (CF) levels of prostaglandin E2 (PGE2), thromboxane B2 (TxB2), interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, and leukotriene B4 (LTB4). A split-mouth design was used, with eight ligated teeth and eight contralateral nonligated teeth which develop soft-chow-promoted (spontaneous) disease. Ligated sites experienced an average attachment loss of 0.94 mm per site and linear bone loss of 0.88 mm per site, with spontaneous-periodontitis sites experiencing approximately half the loss of ligated sites. The CF mediator levels showed increased levels of PGE2 and TxB2 at the ligated sites, as compared with the spontaneous sites, with no significant contralateral differences in the IL-1 beta or LTB4 responses. The concentrations of LTB4 in CF reached an early threefold peak over the baseline level at 1 month. By 2 months there was a statistically significant threefold elevation in CF-PGE2 in the ligated sites and a twofold elevation in the spontaneous sites as compared to the baseline level (P = 0.041 and 0.008, respectively). The monocyte product IL-1 beta increased sharply at 2 months and returned to the baseline level by 6 months at both ligated and nonligated sites. Tumor necrosis factor alpha in CF was below the limit of detection at all sites throughout the experiment (i.e., < 2 ng/ml). The selective elevation of both PGE2 and TxB2 in ligated sites, compared with levels in spontaneous sites, in the presence of similar levels of LTB4 and IL-1 beta provides further evidence that these molecules regulate the magnitude of the tissue-destructive response in progressive periodontitis.
Complement component C5 modulates the systemic tumor necrosis factor response in murine endotoxic shock
Authors: Barton P A , Warren J S
Published: January 2020

Patients with disseminated Neisseria meningitidis infections (meningococcemia) suffer from a fulminant shock syndrome that is accompanied by extraordinarily high concentrations in serum of tumor necrosis factor (TNF). People with homozygous deficiencies of late complement components (C5, C6, C7, and C8) experience a high incidence of disseminated neisserial infections yet suffer from an attenuated form of the disease. The mechanisms that account for this disparity in host response are unclear, but they may in part be related to differences in the systemic TNF response that are modulated by terminal complement components (C5 to C9). The role of C5 in the modulation of the systemic endotoxin-induced TNF response was studied with matched strains of C5-deficient (B10 D2/Osn) and complement-sufficient (B10 D2/Nsn) mice. Following lipopolysaccharide (LPS) administration, complement-sufficient mice exhibited more rapid increases in pulmonary and hepatic vascular permeabilities than did C5-deficient controls. Complement-sufficient mice developed acute passive hepatic congestion, they appeared more ill than C5-deficient mice, and they exhibited a twofold greater rise in serum TNF activity compared with that by C5-deficient mice. C5-deficient mice reconstituted with normal serum before an LPS injection exhibited pulmonary and hepatic vascular permeability increases and serum TNF levels approaching those observed in complement-sufficient mice. Alveolar and peritoneal macrophages isolated from complement-sufficient and C5-deficient mice and incubated in heat-inactivated serum did not exhibit differences in TNF mRNA expression or secreted TNF activity following stimulation with LPS. However, incubation of macrophages in complement-sufficient mouse serum (before LPS stimulation) resulted in increased TNF mRNA expression and TNF activity compared with those in cells incubated in C5-deficient serum. In vitro studies employing human complement components and peripheral blood monocytes revealed that recombinant C5a, in the presence or absence of LPS, can induce increased concentrations of TNF and that C5b to C9 had no additional modulatory effect on the TNF response. These data suggest that C5 modulates the endotoxin-triggered TNF response. The role of complement components distal to C5 (i.e., C5b to C9) in the endotoxin-triggered TNF response remains unclear.
Analysis of a DNA polymorphic region in the gtfB and gtfC genes of Streptococcus mutans
Authors: Chia J S , Lin S W , Hsu T Y , Chen J Y , Kwan H W , Yang C S
Published: January 2020

We have previously demonstrated the existence of DNA polymorphisms at the 5' coding regions of the gtfB and gtfC genes specifying the streptococcal glucosyltransferases (J.S. Chia, T.Y. Hsu, L.J. Teng, J.Y. Chen, L.J. Hahn, and C.S. Yang, Infect. Immun. 59:1656-1660, 1991). DNA sequence analysis by polymerase chain reaction and direct sequencing revealed that while several nucleotide changes were identified, accounting for the polymorphisms, the amino acids which they code for remain unchanged. The polymorphic region is located in a highly conserved amino terminus of the glucosyltransferases. A peptide of 19 amino acids from this region reversed the inhibiting activity of an antiserum raised against the proteins coded for by the gtfB and gtfC genes. The results suggest that the polymorphic region, varying in DNA but not in amino acid sequences, might specify some biological function.
Neutralizing antibodies to Haemophilus ducreyi cytotoxin
Authors: Lagergård T , Purvén M
Published: January 2020

Neutralizing antibodies against cytotoxin produced by Haemophilus ducreyi bacteria were studied in rabbits by an assay employing HEp-2 cells and diluted crude cytotoxin preparations from the organism. Antisera to 12 different H. ducreyi strains were prepared by immunization of rabbits with bacterial sonicates combined with Freund's adjuvant. The antibody response during infection with H. ducreyi was studied in two groups of rabbits which were infected with five live strains by either single or multiple intradermal injections. Neutralizing antibodies in hyperimmune sera to sonicates from 12 H. ducreyi strains tested against their homologous cytotoxin preparations were present with titers ranging from 1:80 to 1:640. Similar antibody titers against heterologous cytotoxin preparations were recorded, indicating immunological similarity or identity between cytotoxins from the various H. ducreyi strains. Three strains did not produce cytotoxin, and these strains did not induce toxin-neutralizing antibodies. Hyperimmune sera to other gram-negative bacteria had no detectable neutralizing capacity, indicating species specificity of the H. ducreyi cytotoxin. Cytotoxin-neutralizing antibodies were not detectable in rabbit sera before infection with H. ducreyi. Repeated single injections with live bacteria resulted in development of low levels of neutralizing antibodies. Multiple primary injection of live bacteria of the cytotoxin-producing strain CCUG 7470 resulted in a low immune response to the cytotoxin preparation from the same strain. A booster infection resulted in the development of neutralizing antibodies in all rabbits infected with cytotoxin-producing strains. The antibody titers determined against the homologous cytotoxin preparation were similar to those recorded for two heterologous cytotoxin preparations.
Evidence for absence of the MPB64 gene in some substrains of Mycobacterium bovis BCG
Authors: Li H , Ulstrup J C , Jonassen T O , Melby K , Nagai S , Harboe M
Published: January 2020

Substrains of Mycobacterium bovis BCG have been divided in two major groups, high producers and low producers of the secreted proteins MPB64 and MPB70. Of these, Mycobacterium tuberculosis secretes only the analog MPT64 during growth on Sauton medium. It has been confirmed that high-producer and low-producer substrains of BCG as well as M. tuberculosis contain the gene for the MPB/MPT70 protein. By contrast, polymerase chain reaction and hybridization experiments are reported here which indicate that the MPB64 gene is absent in the BCG substrains Copenhagen, Pasteur, Glaxo, and Tice, in which previous methods did not permit distinction between secretion of small amounts or absence of the protein in culture fluids.
Purification of a mycobacterial adhesin for fibronectin
Authors: Ratliff T L , McCarthy R , Telle W B , Brown E J
Published: January 2020

Previous studies have demonstrated that mycobacteria attach to fibronectin (FN). The attachment of mycobacteria to FN is considered to be biologically important in Mycobacterium bovis BCG therapy for superficial bladder cancer, initiation of delayed hypersensitivity to mycobacterial antigens, and the phagocytosis of mycobacteria by epithelial cells. Therefore, we purified the mycobacterial receptor for FN. Culture supernatants from 3-week cultures of Mycobacterium vaccae, which contained proteins that bound FN and inhibited the attachment of both M. vaccae and BCG to FN, were used as a source of receptor. Lyophilized M. vaccae supernatants were reconstituted in 0.02 M bis-Tris (pH 6.0) and applied sequentially to an ACA 54 gel filtration column and a DEAE-Sephacel anion-exchange column. A purified inhibitory protein of 55 kDa (p55) was obtained. The purified p55 protein was observed to bind to FN and to inhibit 125I-FN binding to viable BCG in a dose-dependent manner. Polyclonal and monoclonal antibodies to the protein were generated. The resulting polyclonal antiserum blotted a single protein band at 55 kDa in crude M. vaccae supernatants, cross-reacted with a 55-kDa BCG protein by Western blot (immunoblot), and recognized a 55-kDa band that was associated with the BCG cell wall, which is consistent with its function as a FN receptor. A monoclonal immunoglobulin M(lambda) was isolated from mice immunized with purified M. vaccae p55 protein that was not functional in Western blots but inhibited the attachment of viable BCG to FN. These studies demonstrate that a protein or antigenically related proteins with M(r)s of 55,000 function as FN receptors for at least two distinct mycobacteria.
Directional actin polymerization associated with spotted fever group Rickettsia infection of Vero cells
Authors: Heinzen R A , Hayes S F , Peacock M G , Hackstadt T
Published: January 2020

Members of the spotted fever group (SFG) of rickettsiae spread rapidly from cell to cell by an unknown mechanism(s). Staining of Rickettsia rickettsii-infected Vero cells with rhodamine phalloidin demonstrated unique actin filaments associated with one pole of intracellular rickettsiae. F-actin tails greater than 70 microns in length were seen extending from rickettsiae. Treatment of infected cells with chloramphenicol eliminated rickettsia-associated F-actin tails, suggesting that de novo protein synthesis of one or more rickettsial proteins is required for tail formation. Rickettsiae were coated with F-actin as early as 15 min postinfection, and tail formation was detected by 30 min. A survey of virulent and avirulent species within the SFG rickettsiae demonstrated that all formed actin tails. Typhus group rickettsiae, which do not spread directly from cell to cell, lacked F-actin tails entirely or exhibited only very short tails. Transmission electron microscopy demonstrated fibrillar material in close association with R. rickettsii but not Rickettsia prowazekii. Biochemical evidence that actin polymerization plays a role in movement was provided by showing that transit of R. rickettsii from infected cells into the cell culture medium was inhibited by treatment of host cells with cytochalasin D. These data suggest that the cell-to-cell transmission of SFG rickettsiae may be aided by induction of actin polymerization in a fashion similar to that described for Shigella flexneri and Listeria monocytogenes.
Borrelia burgdorferi infection and immunity in mice deficient in the fifth component of complement
Authors: Bockenstedt L K , Barthold S , Deponte K , Marcantonio N , Kantor F S
Published: January 2020

When immunocompetent mice are inoculated with Borrelia burgdorferi, they develop acute arthritis and carditis that undergo spontaneous regression despite the persistence of infection. Specific T- and/or B-cell immunity appears to be necessary for resolution of disease manifestations. Humoral immune responses to B. burgdorferi are also important in prevention of B. burgdorferi infection, in that passive transfer of immune sera or protective monoclonal antibodies prevents the spirochete from establishing infection. It has previously been suggested that complement is necessary for effective antibody-mediated host responses against B. burgdorferi. To investigate the role of complement in the pathogenesis and prevention of Lyme disease, we compared the responses to B. burgdorferi challenge inoculation of mice genetically deficient in the fifth component of complement (C5) with those of C5-sufficient mice. All C5-deficient strains tested were susceptible to B. burgdorferi infection, and disease manifestations underwent regression in a similar time-course to those of complement-sufficient mice. Moreover, passive immunization of C5-deficient mice with either immune rabbit sera or neutralizing monoclonal antibody protected them from challenge infection. These results demonstrate that the expression of Lyme disease is not altered in mice deficient in C5 and that C5-mediated complement activation is not necessary for antibody-mediated protection from infection.
Dietary vitamin D affects cell-mediated hypersensitivity but not resistance to experimental pulmonary tuberculosis in guinea pigs
Authors: Hernandez-Frontera E , McMurray D N
Published: January 2020

Outbred, Hartley strain guinea pigs were fed purified diets varying only in their levels of vitamin D. The amounts of vitamin D in the diets were adjusted to represent 0, 25, 50, 100, or 200% of the recommended level (1,180 IU/kg of body weight) for guinea pigs. In some experiments, half of the animals in each diet group were vaccinated with Mycobacterium bovis BCG vaccine at the time the diets were introduced. Six weeks later, all guinea pigs were infected by the respiratory route with a low dose of virulent M. tuberculosis H37Rv. Vitamin D-deficient animals exhibited marked reductions in levels of the major vitamin D metabolite, 25-hydroxyvitamin D3, in plasma. Altered vitamin D intake was accompanied by changes in antigen (purified protein derivative)-induced, cell-mediated immune responses both in vivo (tuberculin hypersensitivity) and in vitro (lymphoproliferation). Dermal tuberculin reactivity developed more slowly in vitamin D-deficient guinea pigs but eventually achieved normal levels. The proliferation of splenocytes cultured with purified protein derivative was suppressed by both deficiency and excess of dietary vitamin D. Vitamin D status did not affect the abilities of naive guinea pigs to control primary, pulmonary tuberculosis, nor did it influence the protective efficacy of BCG vaccination. We conclude that changes in dietary vitamin D are associated with alterations in some cellular immune functions but may not be an important determinant of disease outcome in pulmonary tuberculosis, as has been suggested previously.
Immune response of the female rat genital tract after oral and local immunization with keyhole limpet hemocyanin conjugated to the cholera toxin B subunit
Authors: Menge A C , Michalek S M , Russell M W , Mestecky J
Published: January 2020

The immune response of the female rat genital tract was evaluated with Lewis rats given primary and secondary immunizations with keyhole limpet hemocyanin (KLH) alone or coupled to the cholera toxin (CT) B subunit (CTB) by the oral or intravaginal-uterine route or a combination of routes. CT (2 to 5 micrograms) was administered as an adjuvant with the KLH-CTB conjugate. While a significant mucosal immunoglobulin A (IgA) response was induced by KLH, there were no significant differences among the immunized groups in the levels of IgA antibodies in salivary gland, gut, vaginal, and uterine secretions, with the exception that rats immunized only orally with the KLH-CTB conjugate lacked a detectable vaginal response. Levels of IgA antibodies to CT, however, were significantly increased in genital tract secretions of rats immunized locally versus orally with the KLH-CTB conjugate. Antibody activity of the IgG isotype against both KLH and CT was significantly elevated in genital tract secretions of rats immunized with KLH-CTB by the oral or intravaginal-uterine route and given genital tract boosters, in comparison with the results for the other groups. IgM antibody titers were generally negligible in the different secretions. An enzyme-linked spot-forming assay revealed IgA and IgG antibody-secreting cells in salivary gland and uterine tissues. A highly significant correlation between the numbers of antibody-secreting cells and antibody titers existed for uterine IgG but not IgA responses to KLH among the different groups of rats. In conclusion, a vigorous local immune response was induced after immunization of the female rat reproductive tract alone or in combination with peroral challenge with the KLH-CTB conjugate.
Protective anti-idiotype antibodies in the primate model of pyelonephritis
Authors: Kaack M B , Martin L N , Svenson S B , Baskin G , Steele R H , Roberts J A
Published: January 2020

The adherence of P-fimbriated Escherichia coli to a receptor containing alpha-D-Gal-(1-4)-beta-D-Gal (Gal-Gal) on urothelial cells is an important pathogenic mechanism in the development of pyelonephritis. Antibodies (Ab1) that had been produced by immunization with Gal-Gal conjugated with bovine serum albumin were specifically purified and used to stimulate the production of anti-idiotypic antibodies (Ab2) in cynomolgus monkeys (Macaca fascicularis). While sera from all of the Ab2-producing monkeys contained antibodies reactive with Ab1 and P-fimbriae, not all of the sera inhibited P-fimbrial binding to the Gal-Gal receptor. On the basis of the inhibition of binding, Ab2-producing monkeys were divided into two groups, termed reactive and nonreactive. The reactive and nonreactive Ab2-producing monkeys, together with a group of control monkeys, were challenged with a renal inoculation with P-fimbriated Escherichia coli. Hematologic, immunologic, microbiologic, and pathologic data were compared among the three groups. The reactive monkeys, whose Ab2 in serum inhibited binding between P-fimbriae and the Gal-Gal receptor, were protected against renal damage compared with the control group. The nonreactive group shared some parameters with the reactive group but overall developed renal damage comparable to that of the controls.
Immunogenicity of overlapping synthetic peptides covering the entire sequence of Haemophilus influenzae type b outer membrane protein P2
Authors: Chong P , Yang Y P , Fahim R , McVerry P , Sia C , Klein M
Published: January 2020

Haemophilus influenzae type b is a major cause of bacterial meningitis in young children. Antibodies against the outer membrane protein P2 are protective in the infant rat model of bacteremia. To identify conserved, surface-exposed, and protective epitopes of P2, 17 overlapping peptides covering the entire sequence of the protein were synthesized. Antisera from mice, guinea pigs, and rabbits raised against chromatographically purified P2 were tested for their reactivities to the peptides by enzyme-linked immunosorbent assays (ELISA). Three major linear immunodominant B-cell epitopes were mapped to residues 53 to 81, 241 to 265, and 314 to 341 of mature P2. Human convalescent-phase antisera also reacted strongly with these three epitopes. Rabbit antisera against all peptide-keyhole limpet hemocyanin conjugates except two peptides containing residues 8 to 19 and 302 to 319 recognized the corresponding peptides in ELISA and reacted with P2 on immunoblots. Immunization with all unconjugated peptides, except the 19 N-terminal residues, induced very strong peptide-specific antibody responses, and these antisera reacted with P2 on immunoblots. Rabbit antisera raised against peptides corresponding to residues 1 to 14, 125 to 150, 193 to 219, and 241 to 319 also recognized P2 purified from H. influenzae nontypeable isolates. Identification of these immunodominant B-cell epitopes and conserved regions is a first step toward the rational design of a universal H. influenzae vaccine.
A bacterial protease perturbs the paracellular barrier function of transporting epithelial monolayers in culture
Authors: Azghani A O , Gray L D , Johnson A R
Published: January 2020

Tight junctions between cells and adhesion to the substratum maintain the barrier function of epithelia throughout the body. Damage to the epithelial barrier by microbial products allows penetration of bacteria and promotion of infection. We studied the effects of Pseudomonas elastase (PE) on the barrier function of epithelia by using Madin-Darby canine kidney (MDCK) epithelial cells; these cells form tight junctions (zonula occludens [ZO]) in vitro. PE decreased electrical resistance across the monolayers in a concentration- and time-dependent manner. Immunostaining of selected proteins of the ZO and zonula adherens was used to explore the effects of PE on junctional proteins. PE-treated monolayers of MDCK cells had markedly decreased immunostaining of ZO-1, a protein of the ZO, but light microscopy of PE-treated cells revealed no obvious morphologic changes. A chromium release assay indicated that, even with marked changes in transmonolayer electrical resistance, the permeability defect was not due to membrane disruption. Fluorescence staining of F-actin indicated diminution of cellular microfilaments in PE-treated cells, but E cadherin (uvomorulin), a protein of the zonula adherens, was unaffected by the enzyme. Elastases from porcine pancreas and human leukocytes with similar enzymatic activity (6 U/ml) did not decrease transmonolayer electrical resistance or degrade ZO-1. These results suggest that PE disturbs the barrier function of epithelial monolayers, in part, by changing the cell architecture and altering at least one protein of the ZO.
Association of treatment-resistant chronic Lyme arthritis with HLA-DR4 and antibody reactivity to OspA and OspB of Borrelia burgdorferi
Authors: Kalish R A , Leong J M , Steere A C
Published: January 2020

Chronic Lyme arthritis that is unresponsive to antibiotic therapy is associated with an increased frequency of the HLA-DR4 specificity. To determine whether the immune response to a particular polypeptide of Borrelia burgdorferi may be associated with treatment-resistant chronic Lyme arthritis, we correlated the clinical courses and HLA-DR specificities of 128 patients with Lyme disease with their antibody responses to spirochetal polypeptides. Antibody reactivity was determined by Western blotting (immunoblotting) with sonicated whole B. burgdorferi and recombinant forms of its outer surface proteins, OspA and OspB, as the antigen preparations. Of 15 patients monitored for 4 to 12 years, 11 (73%) developed strong immunoglobulin G responses to both OspA and OspB near the beginning of prolonged episodes of arthritis, from 5 months to 7 years after disease onset. When single serum samples from 80 patients with Lyme arthritis, were tested, 57 (71%) showed antibody reactivity to recombinant Osp proteins; in contrast, none of 43 patients who had erythema migrans or Lyme meningitis (P < 0.00001) and 1 of 5 patients who had chronic neuroborreliosis but who never had arthritis (P = 0.03) showed antibody reactivity to these proteins. Among the 60 antibiotic-treated patients with Lyme arthritis, those with the HLA-DR4 specificity and Osp reactivity had arthritis for a significantly longer time after treatment than those who lacked Osp reactivity (median duration, 9.5 versus 4 months; P = 0.009); a similar trend was found for the HLA-DR2 specificity. For other HLA-DR specificities, arthritis resolved within a median duration of 2 months in both Osp-reactive and nonreactive patients. We conclude that the combination of the HLA-DR4 specificity and OspA or OspB reactivity is associated with chronic arthritis and the lack of a response to antibiotic therapy.
Investigation of the influences of puberty, genetics, and environment on the composition of subgingival periodontal floras
Authors: Moore W E , Burmeister J A , Brooks C N , Ranney R R , Hinkelmann K H , Schieken R M , Moore L V
Published: January 2020

The classical twin model was utilized in this study in an attempt to determine the importance of host genetics to the composition of the subgingival flora. Simultaneously, the effect of puberty on the flora composition was assessed. The compositions of the floras were significantly different at ages 11 and 14 in the same people, indicating that transition to an adult flora composition may be initiated during puberty. However, the numbers of subjects who had prepubertal and postpubertal testosterone levels in this study were too small to demonstrate significant differences based solely on testosterone level (P = 0.053 and 0.11 for tests of unrelated members, i.e., all twins "a," the first twin of each pair, and all twins "b," the second twin of each pair). Sixteen unrelated 11-year-old subjects had prepubertal levels of less than 30 ng of testosterone per dl of serum, and only six of these unrelated subjects had levels above 300 ng/dl by age 14. Of their twin siblings, who formed the second group of unrelated individuals, 15 had prepubertal levels and only 5 reached postpubertal levels. Unpaired t tests indicated that Veillonella atypica, Prevotella denticola, and Prevotella melaninogenica were among the species that contributed most to changes in flora composition during puberty. The compositions of subgingival floras of 11-year-old monozygous and dizygous male twins were significantly more similar than those of unrelated subjects in the study (P = 0.004 and 0.009, respectively). At 12.5 years of age, the floras of monozygous twins remained more similar than those of unrelated subjects (P = 0.001), but the dizygous-twin floras were not significantly more similar than those of unrelated people. This difference corresponded with moderate and varied testosterone levels within dizygous-twin pairs at age 12.5. By age 14 both monozygous and dizygous twins again had floras with compositions more similar than those of unrelated people (P = 0.008 and 0.002, respectively). Estimates of the genetic contributions to the increased similarity of the floras of twins as compared with floras of unrelated people indicated that the concentrations of several species in the flora may be influenced by host genetic factors. The prevalence of certain other species appeared to be controlled primarily by environment.
Purification and characterization of alpha-toxin produced by Clostridium novyi type A
Authors: Ball D W , Van Tassell R L , Roberts M D , Hahn P E , Lyerly D M , Wilkins T D
Published: January 2020

Our study describes the production, purification, and properties of alpha-toxin from Clostridium novyi type A 19402. The bacterium produced maximal amounts of alpha-toxin when grown at 37 degrees C for 72 h in dialysis flask cultures containing brain heart infusion supplemented with 0.75% Tween 80 and 2% glycerol. The alpha-toxin was purified by precipitation with polyethylene glycol 6000, followed by chromatography on Q-Sepharose, phenyl-agarose, and Mono-Q. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the toxin exhibited a single band with an M(r) of 200,000. The toxin also exhibited a single immunoprecipitin arc by crossed immunoelectrophoresis with antiserum against crude toxin. It was stable when stored at 4 degrees C and also following exposure to buffers with pHs in the range of 4 to 7. The toxin had a minimum lethal dose in mice of 5 to 10 ng, caused rounding of a variety of cells in tissue culture, and was negative in the rabbit ileal loop assay. The cytotoxic activity was inhibited by agents that affect receptor-mediated processes, and the toxin was less active on a CHO mutant cell line that is defective in endosomal acidification. The analysis of the amino acid composition revealed an unusually high proline content. The N-terminal sequence is Met-Leu-Ile-Thr-Arg-Glu-Gln-Leu-Met-Lys.
A Campylobacter jejuni homolog of the LcrD/FlbF family of proteins is necessary for flagellar biogenesis
Authors: Miller S , Pesci E C , Pickett C L
Published: January 2020

A Campylobacter jejuni homolog of the lcrD/flbF family of genes was cloned and sequenced. The nucleotide sequence of the gene, called flbA, predicted a protein of 78,864 Da, with significant homology to a group of related proteins including the Yersinia pestis LcrD, Salmonella typhimurium InvA, and Caulobacter crescentus FlbF proteins. The greatest homology was seen with the C. crescentus FlbF protein, with an overall amino acid sequence homology of 57%. An insertion mutation in the C. jejuni 81-176 flbA gene was constructed. The resultant strain did not synthesize flagellin and was nonmotile.
Immune responses associated with susceptibility of C57BL/10 mice to Leishmania amazonensis
Authors: Afonso L C , Scott P
Published: January 2020

Leishmaniae are protozoans which, depending upon both the host and parasite species, can cause either a healing or nonhealing infection. While C57BL/10 mice are able to heal following infection with Leishmania major, they fail to heal following infection with Leishmania amazonensis. In order to address the role of Th1 and Th2 cell responses in the outcome of these infections in C57BL/10 mice, gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production was assessed. While cells from L. major-infected C57BL/10 mice produced high levels of IFN-gamma, cells from L. amazonensis-infected animals produced little or no IFN-gamma. On the other hand, IL-4 was produced only by cells from L. amazonensis-infected C57BL/10 mice, but this production was restricted to the first few weeks of infection. Later in infection, when lesions were evident, no IL-4 was detected. Treatment of BALB/c mice with a monoclonal antibody (11B11) directed against IL-4 induced a dramatic reduction in L. amazonensis lesions. This reduction was associated with a decrease in IL-4 levels and an increase in IFN-gamma production. However, only a slight reduction in lesion sizes and parasite numbers was observed when anti-IL-4-treated C57BL/10 mice were infected with L. amazonensis. These results suggest that IL-4 may have an important role in mediating susceptibility to L. amazonensis in BALB/c mice, as previously demonstrated for L. major. More importantly, however, the data suggest that susceptibility to L. amazonensis in C57BL/10 mice is due to the absence of a Th1 cell response, rather than to the presence of a Th2 cell response.
Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine
Authors: Boman H G , Agerberth B , Boman A
Published: January 2020

Cecropin P1 and PR-39 are two antibacterial peptides isolated from the upper part of the small intestine of the pig. They have been sequenced, and their antibacterial spectra have been investigated (J.-Y. Lee, A. Boman, C. Sun, M. Andersson, H. Jörnvall, V. Mutt, and H. G. Boman, Proc. Natl. Acad. Sci. USA 86:9159-9162, 1989; B. Agerberth, J.-Y. Lee, T. Bergman, M. Carlquist, H. G. Boman, V. Mutt, and H. Jörnvall, Eur. J. Biochem. 202:849-854, 1991). We have now compared these two peptides for their mechanism of action on Escherichia coli K-12 by using three strains with different markers. Our results show that cecropin P1, like other cecropins, kills bacteria by lysis and that this reaction requires more peptide to kill more cells. PR-39 requires a lag period of about 8 min to penetrate the outer membrane of wild-type E. coli; then killing is quite fast. This lag period was absent in the envA1 mutant; in this strain the outer membrane was freely permeable to both peptides. PR-39 killed growing bacteria faster than nongrowing cells; for cecropin P1 there was no such difference. It is suggested from isotope incorporation experiments that PR-39 kills bacteria by a mechanism that stops protein and DNA synthesis and results in degradation of these components.
In utero transmission of Mycoplasma pulmonis in experimentally infected Sprague-Dawley rats
Authors: Steiner D A , Uhl E W , Brown M B
Published: January 2020

Genital mycoplasmosis is important as an animal model for the interaction between infectious agents and the host during pregnancy as well as in its own right as a confounding variable affecting research projects in which the rat is used as a model to study reproductive function and physiology. We report the in utero transmission of Mycoplasma pulmonis and the development of placentitis, amnionitis, and mild fetal bronchopneumonia in Sprague-Dawley rats. A minimum of 10 days prior to breeding, specific-pathogen-free female Sprague-Dawley rats were infected by intravaginal inoculation with 3 x 10(7) CFU of M. pulmonis X1048 or with an equal volume of sterile broth. Rats and fetuses were subjected to necropsy at days 11, 14, and 18 of gestation. M. pulmonis was able to invade the placenta, cross the placental barrier, and establish an amniotic fluid infection by gestational day 14. It was isolated from the oropharynx and lungs of fetuses at gestational day 18. The placenta was more frequently colonized than amniotic fluid, followed by the fetal oropharynx and lungs, supporting an ascending route of infection. Histopathological evidence also support an active infection, with lesions compatible with placentitis, amnionitis, and mild fetal bronchopneumonia. M. pulmonis can traverse the placenta, resulting in infection of the amniotic fluid and in utero transmission of the microorganism to the developing fetus.
Heterogeneity in cytokine profiles of Babesia bovis-specific bovine CD4+ T cells clones activated in vitro
Authors: Brown W C , Woods V M , Dobbelaere D A , Logan K S
Published: January 2020

The central role of T cells in the immune response against hemoprotozoan parasites, both as helper cells for T cell-dependent antibody production and as effector cells acting on intracellular parasites through the elaboration of cytokines, has prompted an investigation of the bovine cellular immune response against Babesia bovis antigens. CD4+ T helper (Th) cell clones generated from four B. bovis-immune cattle by in vitro stimulation with a soluble or membrane-associated merozoite antigen were characterized for reactivity against various forms of antigen and against different geographical isolates of B. bovis and B. bigemina and analyzed for cytokine production following mitogenic stimulation with concanavalin A. Biological assays to measure interleukin-2 (IL-2), IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor alpha or tumor necrosis factor beta and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, IFN-gamma, and tumor necrosis factor alpha revealed differential production of cytokines by the Th cell clones. The majority of clones expressed the Th0 pattern of cytokines: IFN-gamma, IL-4, and IL-2. One clone expressed the Th1 profile (IFN-gamma and IL-2 but not IL-4), whereas none of the clones expressed the Th2 profile. All of the Th cell clones examined expressed the low-molecular-weight isoform of the leukocyte common antigen associated with a memory cell phenotype (CD45RO), and all expressed the lymph node homing receptor (L-selectin). These results extend our previous finding of differential cytokine expression by B. bovis-specific Th cell clones and confirm the identity of the specific cytokines produced, showing that a Th0 response is preferentially induced in a panel of 20 CD4+ T cell clones obtained from immune cattle.