bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunization of guinea pigs with recombinant TmpB antigen induces protection against challenge infection with Treponema pallidum Nichols
Authors: Wicher K , Schouls L M , Wicher V , Van Embden J D , Nakeeb S S
Published: January 2020

Treponema pallidum-susceptible guinea pigs of strain C4D were immunized with recombinant T. pallidum antigens TmpA, TmpB, TmpC, and TmpA plus TmpB plus TmpC; with Escherichia coli membranes; or with adjuvant alone. Animals in groups of five received six immunizing injections, each of 100 micrograms of antigen incorporated in RIBI adjuvant. After the sixth immunization, all experimental and nonimmunized controls were intradermally challenged with 3 x 10(6) T. pallidum Nichols freshly extracted from infected rabbit testes. Although high titers of antitreponemal antibodies in the fluorescent-treponemal-antibody test or an enzyme-linked immunosorbent assay were evoked in all animals immunized with recombinant antigens, only guinea pigs receiving TmpB antigen demonstrated protection expressed by the development of significantly (P less than 0.01) smaller, atypical lesions of significantly (P less than 0.01) shorter duration and devoid of or containing fewer T. pallidum organisms than lesions in the remaining immunized and control animals.
Antibody responses in the lungs of mice following oral immunization with Salmonella typhimurium aroA and invasive Escherichia coli strains expressing the filamentous hemagglutinin of Bordetella pertussis
Authors: Guzmán C A , Brownlie R M , Kadurugamuwa J , Walker M J , Timmis K N
Published: January 2020

The filamentous hemagglutinin (FHA) of Bordetella pertussis was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, and in a strain of Escherichia coli harboring Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness for epithelial cells. Expression of FHA in these strains did not interfere with their ability to invade Henle cells. Immunoglobulins A and G specific for FHA were detected in lung washes of mice following oral immunization with the live recombinant organisms; antibody levels were significantly higher than those in mice immunized with killed bacteria administered orally or intraperitoneally. Live oral vaccines carrying protective antigens of B. pertussis may be an important alternative to new-generation component vaccines against whooping cough.
Mobilization of F-actin and clathrin during redistribution of Chlamydia trachomatis to an intracellular site in eucaryotic cells
Authors: Majeed M , Kihlström E
Published: January 2020

Immunofluorescence was used to examine the distribution of Chlamydia trachomatis serovars L2 and E, F-actin, and clathrin in infected McCoy and HeLa cells. After incubation at 4 degrees C, C. trachomatis serovar L2 was randomly distributed on the McCoy cell surface. After a temperature shift to 37 degrees C, chlamydiae redistributed, within 30 min, to one local aggregate in the central or perinuclear region of individual cells. About 90% of these aggregated chlamydiae were intracellularly localized, but some remained randomly distributed on the cell surface. Similar results were obtained with HeLa cells and C. trachomatis serovar E, except that the redistribution was slower in HeLa cells than in McCoy cells and fewer cells infected with serovar E exhibited a local aggregate than those infected with serovar L2. Cytochalasin D inhibited more than 90% of this local aggregation. Instead, in cytochalasin D-treated cells, the entry of chlamydiae was inhibited and the organisms became localized on the cell surface in a peripheral local aggregate that distributed in a manner similar to that of phalloidin-stained actin. In a double immunofluorescence assay, F-actin and clathrin aggregated correspondingly in time and position with central or perinuclear aggregation of chlamydiae. These results indicate that polymerized actin and clathrin participate in a rapid redistribution of chlamydiae to an intracellular aggregate.
Heat-killed Listeria monocytogenes and L. monocytogenes soluble antigen induce clonable CD4+ T lymphocytes with protective and chemotactic activities in vivo
Authors: Brocke S , Hahn H
Published: January 2020

In the present study we attempted to analyze the possibility to induce in mice a T-cell response using killed Listeria monocytogenes in adjuvant. Clearly, nonviable antigen is capable of inducing protective and granuloma-forming T cells in C57BL/6 mice when emulsified in complete Freund's adjuvant. These T cells were cloned in vitro by using antigen and irradiated splenocytes, as antigen-presenting cells, and the clones were characterized in vivo. Listeria-specific T-cell clones showed protective and chemotactic activity upon adoptive transfer, although some degree of functional heterogeneity among different clones was observed. The heterogeneous in vivo functions could not be correlated with the ability of the clones to produce gamma interferon or T-cell growth factor (interleukin-2 and/or interleukin-4). It was demonstrated that an in vivo relevant fraction of listeria-specific T lymphocytes can be induced by nonviable antigen in complete Freund's adjuvant. These results show that the low immunogenicity of heat-killed bacteria is not due to the expression of specific protective T-cell epitopes only by live bacteria.
Role of a cell surface-associated protein in adherence and dental caries
Authors: Bowen W H , Schilling K , Giertsen E , Pearson S , Lee S F , Bleiweis A , Beeman D
Published: January 2020

Insertional inactivation of the Streptococcus mutans spaP gene was used to construct an isogenic mutant (834) of strain NG8 (serotype c) which lacked the major cell surface-associated protein referred to as P1 (15). Results of several studies suggest that P1 is involved in the adherence of S. mutans to saliva-coated apatite surfaces. With an in vitro model system of hydroxyapatite (HA) beads coated with parotid saliva (PS) and additional HA surfaces coated with PS and in situ-formed glucan, it was observed that mutant 834 adhered poorly to the PS/HA surfaces. In contrast, both parent and mutant strains bound to the PS-glucan/HA surface. Groups of intact and desalivated rats were infected with each strain to determine relative capacities to induce dental caries. Rats were fed a highly cariogenic diet containing 56% sucrose for 3 to 5 weeks. Each strain colonized the rodent model and caused similar levels of smooth-surface caries under these dietary conditions. It was concluded that P1 influences the ability of organisms to adhere to saliva-coated surfaces and possibly affects primary colonization of the oral cavity in the absence of a glucan surface but has no effect on glucan-mediated adherence in vitro or in vivo.
Transformation of Actinobacillus actinomycetemcomitans by electroporation, utilizing constructed shuttle plasmids
Authors: Sreenivasan P K , LeBlanc D J , Lee L N , Fives-Taylor P
Published: January 2020

Actinobacillus actinomycetemcomitans, a periodontal pathogen, has been strongly implicated in human periodontal disease. Advances in the molecular analysis of A. actinomycetemcomitans virulence factors have been limited due to the unavailability of systems for genetic transfer, transposon mutagenesis, and gene complementation. Slow progress can be traced almost exclusively to the lack of gene vector systems and methods for the introduction of DNA into A. actinomycetemcomitans. An electrotransformation system that allowed at least five strains of A. actinomycetemcomitans to be transformed with stable shuttle plasmids which efficiently replicated in both Escherichia coli and A. actinomycetemcomitans was developed. One plasmid, a potential shuttle vector designated pDL282, is 5.7 kb in size, has several unique restriction enzyme sites, and codes for resistance to spectinomycin and ampicillin. E. coli and A. actinomycetemcomitans were transformed with equal efficiencies of approximately 10(5) transformants per micrograms of DNA. Similar transformation efficiencies were obtained whether the plasmid DNA was isolated from A. actinomycetemcomitans or E. coli. In addition, frozen competent cells of A. actinomycetemcomitans yielded comparable efficiencies of transformation. Restriction enzyme analysis of pDL282 isolated after transformation confirmed the presence of intact donor plasmids. A plasmid isolated from A. pleuropneumoniae was also capable of transforming some isolates of A. actinomycetemcomitans, although generally at a lower frequency. The availability of these shuttle plasmids and an efficient transformation procedure should significantly facilitate the molecular analysis of virulence factors of A. actinomycetemcomitans.
Immunopathology of experimental African sleeping sickness: detection of cytokine mRNA in the brains of Trypanosoma brucei brucei-infected mice
Authors: Hunter C A , Gow J W , Kennedy P G , Jennings F W , Murray M
Published: January 2020

The immunopathology of Trypanosoma brucei brucei in the central nervous system was studied by using an experimental model of chronic meningoencephalitis in outbred CD-1 mice. Mice infected with T. b. brucei were treated with a subcurative dose of the trypanocidal compound diaminazine aceturate. These mice relapsed and were again drug treated. The brains were examined histologically and by immunocytochemistry to identify activated astrocytes. The polymerase chain reaction was used to detect cytokine RNA transcripts. The infected and treated animals developed severe chronic meningoencephalitis characterized by large numbers of inflammatory cells and widespread astrocyte proliferation. In uninfected controls, only interleukin 1 and beta-actin RNA transcripts were detected while transcripts for beta-actin, tumor necrosis factor alpha, macrophage inflammatory protein 1 and interleukins 1 and 4 were demonstrated in the brains of infected animals. Gamma interferon and interleukin 6 were also detected in a few of the infected animals, but interleukin 2 was not found in any of these animals.
Effects of macrophage colony-stimulating factor on reduction of viable bacteria and survival of mice during Listeria monocytogenes infection: characteristics of monocyte subpopulations
Authors: Kayashima S , Tsuru S , Shinomiya N , Katsura Y , Motoyoshi K , Rokutanda M , Nagata N
Published: January 2020

Mice could well tolerate infection with a lethal dose of Listeria monocytogenes after intraperitoneal preinjections with 250 micrograms of macrophage colony-stimulating factor (M-CSF) per kg of body weight for 5 days. The characteristic changes in the surface markers (Mac-1, LFA-1, and F4/80) of peripheral monocytes were also investigated in order to analyze the mechanism of protection by M-CSF. This investigation shows the excellent effect of intraperitoneal preinjections of M-CSF on the reduction of viable Listeria organisms and the improvement of survival after an intravenous Listeria infection.
Gamma interferon-activated human macrophages and Toxoplasma gondii, Chlamydia psittaci, and Leishmania donovani: antimicrobial role of limiting intracellular iron
Authors: Murray H W , Granger A M , Teitelbaum R F
Published: January 2020

Iron-saturated transferrin did not reverse the intracellular killing or inhibition of Toxoplasma gondii, Chlamydia psittaci, or Leishmania donovani by gamma interferon-activated human macrophages. Deferoxamine, an iron chelator, also did not impair replication within unstimulated macrophages. Limiting the availability of intracellular iron is an unlikely mechanism in human macrophage activity against these three diverse pathogens.
A plasmid-encoded surface protein found in chronic-disease isolates of Coxiella burnetti
Authors: Minnick M F , Heinzen R A , Reschke D K , Frazier M E , Mallavia L P
Published: January 2020

The cbbE' gene codes for the E' protein of Coxiella burnetii and was detected in genomic DNA from all known human isolates of the biotzere strain but not in DNA from the other five strains of C. burnetti. The biotzere strain is strictly associated with chronic disease in humans. Extrinsic iodination of biotzere strain cells radiolabeled a 55-kDa protein which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the E' protein synthesized in vitro from recombinants containing the cbbE' gene. The 125I-labeled 55-kDa protein was immunoprecipitated with polyclonal anti-E' antiserum, confirming its identity as E'. Predicted secondary structure of the E' polypeptide shows six regions of beta-sheet structure and an alpha-helix near the C terminus with adequate lengths to span a membrane. The predicted hydropathy profile of E' is similar to profiles of known outer membrane proteins and corroborates the biochemical data, indicating that the protein is located in the outer membrane of C. burnetii.
Contribution of a 28-kilodalton membrane protein to the virulence of Haemophilus influenzae
Authors: Chanyangam M , Smith A L , Moseley S L , Kuehn M , Jenny P
Published: January 2020

A Haemophilus influenzae b (Hib) membrane protein with a molecular mass of 28 kDa bound polyclonal antisera raised against a highly purified Hib fimbrial subunit. We cloned the gene encoding this protein and found that the gene was expressed in Escherichia coli. DNA sequence analysis identified an 843-bp open reading frame which predicted a 26.78-kDa protein with an amino-terminal signal sequence and a mature protein with 70% similarity to the 28-kDa lipoprotein of E. coli (F. Yu, S. Inouye, and M. Inouye, J. Biol. Chem. 261:2284, 1986). Colony blot hybridization analysis with an intergenic probe of the cloned gene demonstrated that 29 of 32 H. influenzae strains hybridize with this gene. Insertion of a chloramphenicol acetyltransferase gene into the open reading frame inactivated expression of the 28-kDa protein in E. coli. Isogenic Hib strains were derived by marker exchange mutagenesis to generate mutants which no longer expressed the 28-kDa protein as recognized with Western immunoblot analysis. There was no difference in the rate of nasopharyngeal colonization of infant rats or monkeys by the isogenic mutants which lacked the 28-kDa protein compared with colonization by the wild-type strain. In contrast, the frequency of invasion and density of bacteremia in infant rats caused by the isogenic mutants were reduced relative to those caused by the wild-type Hib strain. We conclude that this 28-kDa outer membrane protein aids transepithelial invasion of type b strains but is not essential.
Effect of lysozyme on glucose fermentation, cytoplasmic pH, and intracellular potassium concentrations in Streptococcus mutans 10449
Authors: Wang Y B , Germaine G R
Published: January 2020

Several previous findings have suggested that the cationic nature of lysozyme is a major factor in its bactericidal activity. Since a number of cationic proteins or peptides have been reported to cause membrane damage in bacteria, we investigated the effect of lysozyme on glucose fermentation and intracellular pH and K+ in Streptococcus mutans under conditions in which lysis does not occur. Results showed that lysozyme and poly-D-lysine (PDL) cause inhibition of glucose fermentation at pH 5.5 in a dose-dependent manner. Human placental lysozyme and hen egg-white lysozyme exhibited similar inhibitory potency on glucose fermentation. Both lysozyme and PDL caused a marked acidification of the cytoplasm of S. mutans. However, when cytoplasmic pH was examined as a function of fermentation rate, the relationship was similar regardless of the presence or absence of lysozyme or PDL. Therefore, acidification of the cytoplasm appeared to not depend specifically on lysozyme or PDL. In contrast, the same relationship between the profound loss of intracellular K+, when fermenting cells were exposed to either lysozyme or PDL, and the fermentation rate was not exhibited in the controls. These results indicate that lysozyme and PDL specifically affected the ability of the cells to maintain intracellular K+. We concluded that lysozyme and PDL indeed perturb membrane function, perhaps in a selective manner. Furthermore, the similarity in action of lysozyme and the cationic homopolypeptide PDL supports the notion that the cationic property of lysozyme indeed plays a significant role in its antibacterial activity.
Mycobacterium leprae renders Schwann cells and mononuclear phagocytes susceptible or resistant to killer cells
Authors: Steinhoff U , Wand-Württenberger A , Bremerich A , Kaufmann S H
Published: January 2020

Acquired resistance to Mycobacterium leprae, the etiologic agent of leprosy, crucially depends on cellular immune mechanisms. In addition to interleukin-mediated helper functions, killer mechanisms seem to be involved. This study addresses the question of how M. leprae render mononuclear phagocytes and Schwann cells, its natural targets, susceptible or resistant to killer cells. Killer activities were stimulated in peripheral blood mononuclear cells from healthy individuals by incubation with mycobacteria plus interleukin-2. These cells lysed Schwann cells and mononuclear phagocytes which had been pulsed with dead M. leprae, while unpulsed targets remained virtually unaffected. Importantly, targets infected with viable M. leprae were not lysed; furthermore, infection with viable M. leprae as well as gamma interferon stimulation or heat shock caused resistance in otherwise susceptible targets which had been pulsed with dead M. leprae. Thus, M. leprae markedly influenced the effect of killer cells on Schwann cells and mononuclear phagocytes.
Expression and deletion analysis of the Trypanosoma brucei rhodesiense cysteine protease in Escherichia coli
Authors: Pamer E G , Davis C E , So M
Published: January 2020

Trypanosoma brucei, the cause of African sleeping sickness, differentiates in the mammalian bloodstream from a long, slender trypanosome into a short, stumpy trypanosome. This event is necessary for infection of the tsetse fly and maintenance of the life cycle. We have previously shown that the stumpy form contains 10- to 15-fold-greater cysteine protease activity than either the slender form or the insect midgut procyclic, and we have isolated a cDNA encoding the protease. In order to determine whether the cDNA encodes the developmentally regulated cysteine protease, we have purified the protease from trypanosomes and have made a polyclonal antiserum against it. The trypanosomal protease gene was then expressed in Escherichia coli with three different methionines within the pre- and propeptides acting as initiation sites. In each case, a protein was synthesized that was recognized by an antiserum specific for the developmentally regulated trypanosomal cysteine protease. The protein synthesized from the more upstream initiation site within the propeptide was proteolytically active. The recombinant protease and the trypanosomal enzyme were identical with respect to peptide substrates and protease inhibitors. The protein remained active when synthesized in a truncated form lacking the nine consecutive prolines and carboxy-terminus extension, indicating that the terminal 108 amino acids are not necessary for proteolytic activity.
The receptor repertoire defines the host range for attaching Escherichia coli strains that recognize globo-A
Authors: Lindstedt R , Larson G , Falk P , Jodal U , Leffler H , Svanborg C
Published: January 2020

Escherichia coli strains which colonize the human urinary tract express lectins specific for different members of the globoseries of glycolipids, e.g., globotetraosylceramide and globo-A. This study investigated the importance of globo-A expression for attachment to human uroepithelial cells, colonization of the urinary tract, and severity of urinary tract infection. The expression of receptor-active glycolipids by erythrocytes and epithelial cells was analyzed by thin-layer chromatography and bacterial overlay as well as by bacterial binding to those cells. The epithelial expression of the globo-A receptor was restricted to individuals of blood group A with a positive secretor state. Consequently, globo-A binding E. coli strains attached only to epithelial cells from these individuals. In contrast, globoside-recognizing strains attached in similar numbers to uroepithelial cells regardless of the ABH blood group and secretor state of the donor. The role of host receptor expression for infection with globo-A-specific E. coli was analyzed in 1,473 children with urinary tract infections. All those infected with strains exclusively expressing globo-A-specific adhesins were found to be of blood group A, compared with 45% in the population at large (P less than 0.006). The inflammatory response (fever, C-reactive protein, erythrocyte sedimentation rate) of individuals infected with these strains was lower than that in individuals with infections caused by globoside binding strains. The results demonstrate the importance of fitness between host receptors and bacterial adhesins for infection and suggest that minor receptor epitope differences have profound effects on the disease process.
Protection against experimental pseudomembranous colitis in gnotobiotic mice by use of monoclonal antibodies against Clostridium difficile toxin A
Authors: Corthier G , Muller M C , Wilkins T D , Lyerly D , L'Haridon R
Published: January 2020

The pathogenicity of Clostridium difficile is due to the production of two toxins (toxins A and B). We prepared monoclonal antibodies against toxin A and determined whether axenic mice passively immunized with the monoclonal antibodies were protected against C. difficile disease. The mice were kept in an isolator and were given ascites fluid intravenously prior to challenge with a toxinogenic strain of C. difficile. Control mice and mice receiving ascites fluid devoid of toxin antibody died within 2 days and had high levels of toxins A and B in their feces. Mice that received ascites fluid containing high amounts of toxin A monoclonal antibodies directed against the repeating units of the toxin survived. In protected mice, toxin B levels were similar to those in dying mice, but toxin A levels were greatly reduced. These data show that passive immunity induced by monoclonal antibodies against toxin A was effective against pseudomembranous cecitis.
Rapid elimination of a synthetic adjuvant peptide from the circulation after systemic administration and absence of detectable natural muramyl peptides in normal serum at current analytical limits
Authors: Fox A , Fox K
Published: January 2020

Although it is clear that muramyl peptides are involved in sleep associated with bacterial infection, their role in normal physiological sleep is less certain. It has been speculated that "natural" muramyl peptides, derived from degraded gut flora, may pass into the bloodstream, where they play a role in normal sleep (M. Karnovsky, Fed. Proc. 45:2556-2560, 1986). Muramic acid serves as a chemical marker for muramyl peptides, since it is not synthesized by mammals. After injection of synthetic muramyl dipeptide in rabbits, muramic acid was readily detected (after release by acid hydrolysis) in the circulation; however, levels rapidly decreased. This was an important positive control in assessing circulating levels of natural muramyl peptides. Muramic acid was not found in normal serum (detection limit, approximately 500 pmol/ml), demonstrating the absence of appreciable amounts of circulating natural muramyl peptides. At this time we are unable to provide supportive evidence for Karnovsky's hypothesis.
Resistance to acute Trypanosoma cruzi infection resulting from immunization of mice with a 90-kilodalton antigen from metacyclic trypomastigotes
Authors: González J , Araguth M F , Yoshida N
Published: January 2020

The stage-specific 90-kDa surface antigen of Trypanosoma cruzi metacyclic trypomastigotes was affinity purified and used for immunization studies. Mice immunized with the 90-kDa antigen plus alum as an adjuvant were resistant to challenge with Tulahuen strain metacyclic forms, displaying either reduced or subpatent parasitemias. Mice immunized with the purified antigen without adjuvant or with antigen plus Freund adjuvant or glucan developed high parasitemias, comparable to those of nonimmunized controls, upon metacyclic challenge. Sera of mice immunized with the 90-kDa antigen plus alum showed complement-dependent trypanolytic activity and also greatly inhibited the invasion of Vero cells by metacyclic forms of G and Tulahuen strains in vitro. These sera were found by a competition binding assay to contain antibodies that recognized the same or topographically related sites as the monoclonal antibody 1G7, an antibody previously shown to reduce the infectivity of metacyclic forms in vivo and in vitro. In contrast, 1G7-related antibodies were poorly represented in sera with negative or low trypanolytic activity, such as those from mice immunized with purified antigen without adjuvant.
Clinical correlates of in vitro Plasmodium falciparum cytoadherence
Authors: Ho M , Singh B , Looareesuwan S , Davis T M , Bunnag D , White N J
Published: January 2020

To determine whether isolates of Plasmodium falciparum have intrinsically different cytoadherent properties and whether these differences contribute to the clinical severity of human falciparum malaria, we studied the cytoadherence to C32 melanoma cells in vitro of 59 parasite isolates from patients with naturally acquired infections in Thailand. Parasitized erythrocytes adhere to these melanoma cells principally via the glycoprotein CD36, which is also expressed on most vascular endothelium. In vitro cytoadherence was significantly greater for isolates from patients with biochemical evidence of severe malaria. The cytoadherent properties of P. falciparum parasites may thus be a virulence factor in human falciparum malaria. However, there was no correlation between the degree of in vitro cytoadherence and cerebral symptoms, which suggests that other receptors and/or host factors may be important in the adherence of malaria parasites to cerebral vascular endothelium. The cytokines tumor necrosis factor, interleukin-1, and gamma interferon, which have been implicated in the pathogenesis of cerebral malaria and are known to promote intercellular adhesion in other systems, did not enhance the cytoadherence of P. falciparum isolates to C32 melanoma cells.
Effect of growth rate and pH on intracellular levels and activities of the components of the phosphoenolpyruvate: sugar phosphotransferase system in Streptococcus mutans Ingbritt
Authors: Vadeboncoeur C , St Martin S , Brochu D , Hamilton I R
Published: January 2020

The growth of Streptococcus mutants Ingbritt in continuous culture at low pH or high growth rates repressed the biosynthesis of the components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The cellular concentrations of the soluble components HPr, enzyme I (EI), and EIII for mannose (IIIman) and EII activity for glucose, mannose, 2-deoxyglucose (2DG), and fructose were determined in membrane preparations from cells grown at pHs from 8.0 to 5.0 and at dilution (D) or growth rates from 0.1 to 1.0 h-1. The cellular levels of HPr and EI varied less than threefold under all of the growth conditions tested. On the other hand, EII activity in membranes from cells grown at D = 0.1 h-1 was repressed by growth at pHs below 8.0, with cells grown at pH 5.0 completely devoid of EII activity. In addition, cells grown at D = 0.5 and 1.0 h-1 exhibited little PTS activity for glucose, mannose, and 2DG and twofold-lower activity for fructose. These activities were stimulated by the addition of a membrane-free cytoplasmic fraction, and this activating activity was shown to be due to the presence of IIIman. Estimation of the cellular content of IIIman indicated that the synthesis of this factor was repressed by growth above and below pH 7.0 and was particularly sensitive to growth at high rates. These results indicate that with S. mutans Ingbritt, both pH and growth rate regulate the genes for the synthesis of EIIs involved in the phosphorylation of glucose, mannose, 2DG, and fructose and the gene for the formation of IIIman.
Modifications of plasma fibronectin in cystic fibrosis patients
Authors: Stanislawski L , Sorin M
Published: January 2020

Recurrent pulmonary infections with Staphylococcus aureus and Pseudomonas aeruginosa are a major problem in cystic fibrosis (CF), leading to severe and progressive deterioration of the respiratory tract. Fibronectin (FN) has a binding site(s) for S. aureus and Streptococcus pyogenes. This bacterial bindings site(s) was studied by using FN from patients with CF and comparing them with FN purified from the plasma of bronchitis and normal subjects by an enzyme-linked immunosorbent assay technique. We found a decreased binding of the CF FN to these bacteria in all seven CF patients. Other differences between the CF FN and normal FN were a marked modification in the pattern of peptide migration in polyacrylamide gel electrophoresis after hydrolysis with various proteinases and an increase or decrease of the total sugar content of the FN.
Antibody-producing cells in peripheral blood and salivary glands after oral cholera vaccination of humans
Authors: Czerkinsky C , Svennerholm A M , Quiding M , Jonsson R , Holmgren J
Published: January 2020

We examined whether immunization with a newly developed oral cholera vaccine would elicit gut-derived antibody-producing cells in the blood and in distant mucosal tissues, such as the minor salivary glands, in 30 adult Swedish volunteers. The results of this study demonstrated that this vaccine indeed induced production of specific antibody-producing cells against the cholera toxin B subunit in both peripheral blood and salivary glands. The response in blood, which after primary and booster immunizations comprised both immunoglobulin A (IgA) and IgG antibody-forming cells, was highly transient and preceded the response in salivary glands; the latter response was restricted to the IgA isotype. The results provide further evidence of the existence of a common mucosal immune system in humans. Furthermore, these findings support previous observations that in animals, the cholera toxin B subunit may be a useful carrier protein for preparing enteric vaccines against pathogens encountered at intestinal and extraintestinal mucosal sites.
Cloning and expression of a species-specific early immunogenic 36-kilodalton protein of Mycoplasma hyopneumoniae in Escherichia coli
Authors: Strasser M , Frey J , Bestetti G , Kobisch M , Nicolet J
Published: January 2020

Mycoplasma hyopneumoniae, the etiologic agent of porcine enzootic pneumonia, synthesizes a 36-kDa protein which is an early and strong immunogenic factor in experimentally and naturally infected swine. The gene encoding this protein was cloned by screening a gene library of M. hyopneumoniae DNA with rabbit hyperimmune serum made against whole M. hyopneumoniae cells and convalescent-phase swine serum. Analysis of the recombinant protein expressed in Escherichia coli by immunoblot techniques showed that the protein is expressed in E. coli in its full length and does not cross-react with proteins from M. flocculare or M. hyorhinis. Genetic analysis showed that the gene was expressed from the lac promoter of the vector and seems to be translationally initiated from its own ribosome binding site. Subcloning in a transcriptional fusion vector to optimize expression resulted in production of the 36-kDa protein in E. coli at levels up to 30% of total protein.
Distribution of protein D, an immunoglobulin D-binding protein, in Haemophilus strains
Authors: Akkoyunlu M , Ruan M , Forsgren A
Published: January 2020

Protein D, a novel surface protein of the bacterial species Haemophilus influenzae with specific affinity for human immunoglobulin (Ig) D was detected in all 127 H. influenzae strains studied. All strains representing different serotypes of encapsulated strains and different biotypes of nonencapsulated strains bound 125I-labeled IgD to a high degree (38 to 74%). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis showed that protein D from all H. influenzae strains had the same apparent molecular weight (i.e., 42,000) and reacted with all three different anti-protein D monoclonal antibodies. By Scatchard analysis, the number of protein D residues on a nontypeable H. influenzae strain was estimated to be approximately 2,800 per organism. The equilibrium constant for the reaction between a human IgD myeloma protein and IgD was found to be 5.8 x 10(8) M-1. Also, all strains of H. haemolyticus and H. aegypticus strains tested bound IgD, 21 to 28% and 41 to 48%, respectively. In extracts of those bacteria, a 42,000-molecular-weight protein reactive with IgD and all three anti-protein D monoclonal antibodies was found. In H. parainfluenzae, H. aphrophilus, H. paraphrophilus, and Actinobacillus actinomycetemcomitans, a 42,000-molecular-weight protein that was reactive with one to three of three anti-protein D monoclonal antibodies but not reactive with human IgD was detected with Western blot analysis. Other Haemophilus species (H. ducreyi, H. parasuis, H. parahaemolyticus, H. segnis, and H. haemoglobinophilus) did not react with human monoclonal IgD or anti-protein D antibodies. On the basis of the wide distribution of protein D among H. influenzae strains, we suggest that protein D could be a vaccine candidate.
A novel mouse model to study the virulence of and host response to Porphyromonas (Bacteroides) gingivalis
Authors: Genco C A , Cutler C W , Kapczynski D , Maloney K , Arnold R R
Published: January 2020

We describe here the development of a mouse subcutaneous chamber model that allows for the examination of host-parasite interactions as well as the determination of gross pathology with Porphyromonas (Bacteroides) gingivalis challenge. When inoculated into stainless-steel chambers implanted subcutaneously in female BALB/c mice, P. gingivalis W83, W50, and A7436 (10(8) to 10(10) CFU) caused cachexia, ruffling, general erythema and phlegmonous, ulcerated, necrotic lesions, and death. P. gingivalis W50/BEI, HG405, and 33277 (10(10) CFU) produced localized abscesses in the mouse chamber model with rejection of chambers at the injection site. Analysis of chamber fluid from 33277-, HG405-, and W50/BEI-infected mice by cytocentrifugation revealed inflammatory cell debris, polymorphonuclear leukocytes, and high numbers of dead bacteria. In contrast, fluid from A7436-, W50-, and W83-infected mice revealed infiltration predominantly of polymorphonuclear leukocytes and live bacteria. Bacteria were found primarily associated with polymorphonuclear leukocytes in the fluid from W50-, HG405-, and W83-infected mice but not from A7436-infected mice. Viable isolates were recoverable from the chamber fluid through day 3 for W50/BEI, day 5 for 33277, day 6 for HG405, day 7 for W50, day 14 for W83, and day 26 for A7436. All strains induced a systemic immunoglobulin G response in serum and chamber fluid samples. The use of this model will allow us to examine the virulence of P. gingivalis as defined by the interaction of host response to localized infection with P. gingivalis.
Strain-specific and immunodominant surface epitopes of the P2 porin protein of nontypeable Haemophilus influenzae
Authors: Haase E M , Campagnari A A , Sarwar J , Shero M , Wirth M , Cumming C U , Murphy T F
Published: January 2020

The P2 porin protein is the major outer membrane protein of nontypeable Haemophilus influenzae. Five monoclonal antibodies to P2 of four strains of nontypeable H. influenzae were developed by immunizing mice with whole bacterial cells. All five antibodies recognized epitopes on P2 in immunoblot assays of whole organism lysates, purified outer membrane, and purified P2. Competitive enzyme-linked immunosorbent assays and immunoblot assays of cyanogen bromide-digested P2 showed that two antibodies to the P2 protein of strain 1479 recognized different epitopes on the molecule. Immunofluorescence and immunoelectron microscopy demonstrated that each of the five antibodies recognized epitopes that were abundantly expressed on the bacterial surface. Analysis of 120 H. influenzae strains indicated that three of the five antibodies were reactive exclusively with the homologous strain. The remaining two antibodies were reactive with less than 3% of the strains. These studies indicate that the P2 protein expresses a highly strain-specific and immunodominant epitope on the bacterial surface. The expression of strain-specific and immunodominant epitopes on the bacterial surface may represent a mechanism by which the bacterium induces antibodies that will protect against recurrent infection by the homologous strain but will not protect against infection by heterologous strains.
Antimicrobial activity of rabbit leukocyte defensins against Treponema pallidum subsp. pallidum
Authors: Borenstein L A , Selsted M E , Lehrer R I , Miller J N
Published: January 2020

Defensins, which are peptides with broad antimicrobial activity, are major constituents of rabbit neutrophils and certain macrophages. We tested six rabbit defensins, NP-1, NP-2, NP-3a, NP-3b, NP-4, and NP-5, for activity against Treponema pallidum subsp. pallidum. Mixtures of T. pallidum and defensin in 10% normal rabbit serum (NRS) or heat-inactivated NRS (HI-NRS) were incubated anaerobically for various time periods ranging between 0 and 16 h and then examined by dark-field microscopy for treponemal motility or inoculated intradermally into rabbits to assess treponemal virulence. Immobilization of T. pallidum by NP-1 (400 micrograms/ml) occurred after 4 and 8 h of coincubation in mixtures containing NRS and HI-NRS, respectively. Similarly, neutralization of T. pallidum by NP-1 occurred more rapidly and was complete when incubations were performed in NRS as compared with that in HI-NRS. Endpoint titration confirmed the augmentation of NP-1 antitreponemal activity by heat-labile serum factors; NP-1 showed neutralizing activity at 4 micrograms/ml (about 1 microM) in NRS and at 40 micrograms/ml in HI-NRS. When NP-1 was tested in serum that was deficient in C6, the T. pallidum neutralizing activity of NP-1 was reduced to levels slightly greater than that observed in HI-NRS. NP-1 that had been reduced and alkylated was inactive against T. pallidum. When NP-2, NP-3a, NP-3b, NP-4, and NP-5 were tested at 400 micrograms/ml, all exerted potent treponemicidal activity, manifested by abrogation or delayed development of cutaneous lesions relative to that of controls. These data suggest that defensins may equip certain macrophages and neutrophils to participate in host defense against T. pallidum, that the direct activity of defensins against T. pallidum is enhanced by heat-labile serum factors (presumably complement), and that conformational factors influence the biological activity of the defensin molecule.
Recombinant derivatives of Pasteurella multocida toxin: candidates for a vaccine against progressive atrophic rhinitis
Authors: Petersen S K , Foged N T , Bording A , Nielsen J P , Riemann H K , Frandsen P L
Published: January 2020

Potential vaccine components for protection against atrophic rhinitis in pigs were developed. This was achieved by deletion mutagenesis of the gene encoding the Pasteurella multocida toxin. Four purified toxin derivatives lacking different and widely separated regions in the amino acid sequence were characterized by a lack of toxic activity. One such component was shown to induce efficient protection of vaccinated female mice and their offspring against challenge with purified P. multocida toxin.
Virulence of Campylobacter jejuni for chicken embryos is associated with decreased bloodstream clearance and resistance to phagocytosis
Authors: Field L H , Underwood J L , Payne S M , Berry L J
Published: January 2020

The 11-day-old chicken embryo has been shown to be a useful animal model for comparing the virulence of human isolates of Campylobacter jejuni. Virulence in this system is associated with the ability to invade the chorioallantoic membrane and to survive and proliferate in vivo. In this study, the survival and multiplication of C. jejuni in the embryonic host was investigated. It was possible to enhance the virulence of a relatively avirulent C. jejuni strain by passaging it intravenously through the embryos. The resulting isogenic variants demonstrated enhanced abilities to survive in vivo but were still unable to invade when inoculated onto the chorioallantoic membrane. The bloodstream clearance of C. jejuni was studied, and virulent, but not avirulent, strains persisted and multiplied both in the bloodstream and in embryonic liver. Virulent strains also were cleared significantly more slowly from the bloodstream of adult BALB/c mice after intravenous challenge than were avirulent strains. C. jejuni strains which were cleared slowly in vivo were also ingested slowly in vitro by mouse peritoneal macrophages. Clearance studies in mice pretreated with cobra venom factor demonstrated that opsonization by serum complement was not a prerequisite for clearance of campylobacters from the murine bloodstream.
Effect of adoptive transfer of cloned Actinobacillus actinomycetemcomitans-specific T helper cells on periodontal disease
Authors: Yamashita K , Eastcott J W , Taubman M A , Smith D J , Cox D S
Published: January 2020

Previously we isolated several Actinobacillus actinomycetemcomitans-specific T-cell clones from the spleens and lymph nodes of immunized Rowett rats. These clones were characterized as W3/13+, W3/25+, OX8-, and OX22-, suggesting a T helper (Th) phenotype. In the current experiments, 10(6) cells from a single A. actinomycetemcomitans-specific clone (A3) were adoptively transferred to a group (AaTh; n = 13) of normal heterozygous rats (rnu/+) at 28 days of age. A second group received no T cells (AaNT; n = 15), and a third group also received no T cells (NAaNT, n = 11). Beginning 1 day after transfer, the first and second groups were infected orally with A. actinomycetemcomitans for 5 consecutive days. The presence of infection was confirmed immediately after challenge and after 5 months, when the experiments were ended. Significantly higher numbers of lymphocytes were recovered from the gingival tissues of the first group than from those of either of the other groups. Also, this group showed significantly elevated (P less than 0.01) serum immunoglobulin G and immunoglobulin M antibody to A. actinomycetemcomitans in an enzyme-linked immunosorbent assay when compared with both other groups. Bone loss was significantly lower (P less than 0.01) in recipients of A. actinomycetemcomitans-specific cloned cells when compared with the other infected group and was approximately equal to the bone loss of the uninfected group. These results are consistent with the hypothesis that T-cell regulation can affect periodontal disease. In this regulation, T helper cells appear to interfere with periodontal bone loss.
Synthetic low-toxicity muramyl dipeptide and monophosphoryl lipid A replace Freund complete adjuvant in inducing growth-inhibitory antibodies to the Plasmodium falciparum major merozoite surface protein, gp195
Authors: Hui G S , Tam L Q , Chang S P , Case S E , Hashiro C , Siddiqui W A , Shiba T , Kusumoto S , Kotani S
Published: January 2020

The Plasmodium falciparum major merozoite surface protein (gp195) is a protective antigen against lethal malaria. However, increasing evidence indicates that the efficacy of a malaria vaccine will require a strong adjuvant that is safe for human use. We compared the efficacies of two low-toxicity synthetic immunomodulators, B30-MDP (a lipophilic muramyl dipeptide derivative) and LA-15-PH (a synthetic equivalent of monophosphoryl lipid A), with that of Freund complete adjuvant (FCA) in eliciting an antibody response to gp195. Rabbits were immunized with native gp195 and B30-MDP, LA-15-PH, or the two in combination, with liposomes as the vehicle. Aluminum hydroxide and FCA were used as reference adjuvants. Results showed that adjuvant formulations based on B30-MDP alone or in combination with LA-15-PH induced high antibody titers to gp195, as compared with FCA. LA-15-PH alone was less effective. Aluminum hydroxide induced significantly lower antibody titers. The functional activity of the rabbit anti-gp195 antibodies induced by different adjuvants was evaluated in an in vitro parasite growth inhibition assay previously shown to correlate with anti-gp195 immunity in the Aotus monkey model. All rabbits immunized with B30-MDP-LA-15-PH and two of three rabbits immunized with B30-MDP alone produced sera that strongly inhibited parasite growth. The degree of growth inhibition was similar to that with FCA. The antibody titers of the rabbits receiving B30-MDP-LA-15-PH strongly correlated with the degree of in vitro growth inhibition. Our findings provided strong evidence that adjuvant formulations based on synthetic B30-MDP and LA-15-PH can replace FCA as adjuvants in stimulating protective immunity specific for gp195.
A 40-kilodalton cell wall protein-coding sequence upstream of the sr gene of Streptococcus mutans OMZ175 (serotype f)
Authors: Ogier J A , Schöller M , Lepoivre Y , Gangloff S , M'Zoughi R , Klein J P
Published: January 2020

Streptococcus mutans surface proteins may be important in immunization against dental caries. We report the existence of an open reading frame of 1,005 bp that lies 1,162 bases upstream of the S. mutans OMZ175 sr gene and that encodes a cell wall-associated protein. This open reading frame codes for 335 amino acid residues. The first 18-amino acid region is predominantly hydrophobic and resembles a signal peptide, and the hydrophobic C-terminal region may function as an anchor to the bacterial cell wall. On the basis of the predicted antigenic determinants of the deduced amino acid sequence, a 16-residue synthetic peptide corresponding to the middle hydrophilic coiled region was synthesized. Antibodies raised against this synthetic peptide reacted with a protein with an apparent Mr of 40,000 that was identified by Western immunoblotting in a cell wall extract from S. mutans OMZ175. The high reactivity in an enzyme-linked immunosorbent assay of the antibodies with whole S. mutans OMZ175 cells showed that this protein was located on the bacterial cell surface. Furthermore, the antipeptide immunoglobulin G recognized an identical determinant on the cell surface of other members of the S. mutans group. However, the function of this protein is not yet known.
Stress fiber-based movement of Shigella flexneri within cells
Authors: Vasselon T , Mounier J , Prevost M C , Hellio R , Sansonetti P J
Published: January 2020

icsA (virG), a gene located on pWR100, the virulence plasmid of Shigella flexneri serotype 5 (M90T), encodes a 120-kDa outer membrane protein. This protein promotes a random intracellular movement of the bacteria and leads to the infection of adjacent cells by the formation of protrusions. This movement, which involves the nucleation, polymerization, and subsequent polarization of actin, is referred to as the Ics phenotype (intra/intercellular spread). Here we present evidence that a second, distinct form of locomotion is also elaborated by S. flexneri in chicken embryo fibroblasts in which the Ics phenotype is not expressed. Using a combination of phase-contrast microcinematography and confocal microscopy, we have demonstrated that bacteria adopt parallel orientations by interacting with stress fibers. This interaction subsequently results in bacterial movement along the stress fibers themselves. This phenomenon occurs independently of the presence of a phagocytic vacuole which is lysed shortly after entry of the bacteria into the cell. It is expressed by M90T and SC560, its icsA mutant. This movement has been termed organelle-like movement (Olm phenotype) and is thought to account for the early accumulation of bacteria seen near the nucleus.