bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of subunits of pertussis toxin in Tn5-induced Bordetella mutants deficient in toxin biological activity
Authors: Marchitto K S , Munoz J J , Keith J M
Published: January 2020

Monoclonal antibodies with specificity for pertussis toxin subunits S1, S2, and S4 were used in Western blots to show that the subunits were not secreted into culture medium from Tn5 insertion mutants. The mutants are deficient in toxin biological activities due to an insertion in the S3 subunit structural gene. The Western blots demonstrated that each of the respective subunits was exported in a wild-type strain. Anti-S1 and anti-S2 monoclonal antibodies were capable of detecting subunits in solubilized whole-cell material from a wild-type strain and from the Tn5 mutants lacking only in biologically active toxin (Tox-). Another Tn5 insertion mutant, lacking all known B. pertussis virulence factors (Vir-), did not produce any of the subunits either in whole cellular extracts or in culture supernatants. The data demonstrate that Tn5 Tox- insertion mutants, though defective in toxin activity, synthesize some toxin subunits. The presence of the S3 subunit is most likely a necessity for transport of the toxin from cells. Alternatively, a nonstructural gene coding for a protein involved in transport of the toxin across the membrane may be affected by the Tn5 mutation.
Isolation of Mycoplasma pulmonis membranes and identification of surface antigens
Authors: Horowitz S A , Garrett B , Davis J K , Cassell G H
Published: January 2020

Labeling of Mycoplasma pulmonis cells by iodination with the Bolton-Hunter reagent was shown to efficiently label membrane-associated proteins without significant loss of viability. Labeled proteins proven to be surface exposed by differential proteolytic digestion were analyzed by autoradiography of two-dimensional polyacrylamide gel electrophoresis (PAGE), and seven labeled major polypeptides were identified. To identify all the membrane-associated antigens, pure membranes were isolated by using the surface-labeled proteins as markers. Analysis of the isolated membranes by autoradiography of both sodium dodecyl sulfate-PAGE and the two-dimensional PAGE indicated that the labeled surface proteins were retained in the pure membranes; by immunoblotting with sera from naturally infected animals, these surface proteins were shown to be the predominant antigens recognized by the host during natural infection.
Macrophage activation as an immune correlate to protective immunity against schistosomiasis in mice immunized with an irradiated, cryopreserved live vaccine
Authors: Lewis F A , Winestock J , James S L
Published: January 2020

Immune responses against Schistosoma mansoni were evaluated in C57BL/6 mice injected with one of two populations of irradiated schistosomules, the larval preparations differing only in the degree of freezing-induced damage sustained upon cryopreservation. Mice injected with larvae which successfully withstood cryopreservation showed a significant reduction in worm burden following cercarial challenge. No protection was achieved in mice which received larvae damaged by a suboptimal thawing rate. Parallel comparison of several humoral and cellular responses in mice which received either inoculum revealed that induction of activated macrophages and production of macrophage-activating lymphokine activity were the strongest correlates to development of protective immunity. Protected mice also showed marginal 30-min skin test reactivity and weak but transient 24-h delayed-type hypersensitivity to a soluble adult worm preparation. In contrast, indistinguishable levels of circulating antibodies to soluble and tegumental antigens developed in the two immunization groups, and antigen-stimulated lymphocyte blastogenic responses were strong and essentially equivalent in magnitude. These studies strongly suggested that in this new model for investigating anti-schistosome effector mechanisms, responses contributing to the development of activated macrophages may be essential for induction of protective immunity.
Screening of a recombinant mycobacterial DNA library with polyclonal antiserum and molecular weight analysis of expressed antigens
Authors: Young D B , Kent L , Young R A
Published: January 2020

A lambda gt11 expression library containing recombinant DNA from Mycobacterium tuberculosis was screened using hyperimmune anti-M. tuberculosis rabbit serum. The majority (22 of 29) of the recombinant clones selected by using polyclonal serum expressed three antigens that were previously identified by using mouse monoclonal antibodies, thus indicating the immunodominance of these proteins. Western blot analysis of the recombinant clones demonstrated that expression of these antigens is frequently independent of the formation of beta-galactosidase fusion proteins. The molecular weight of each expressed antigen can vary between clones and is not necessarily identical to that found in mycobacterial extracts.
Anti-infectious activity of liposomal muramyl dipeptides in immunodeficient CBA/N mice
Authors: Phillips N C , Chedid L
Published: January 2020

Two muramyl dipeptides, N-acetylmuramyl-L-alanyl-D-isoglutamine and its adjuvant-inactive isomer N-acetylmuramyl-D-alanyl-D-isoglutamine, were examined for their ability to protect mice carrying the CBA/N immune deficiency gene (xid) against lethal bacterial challenge. Prophylactic treatment with N-acetylmuramyl-L-alanyl-d-isoglutamine gave significant protection against Streptococcus pneumoniae, Salmonella typhimurium, and Salmonella enteritidis infection. N-Acetylmuramyl-D-alanyl-D-isoglutamine was unable to confer protection. Incorporation of the lipophilic glycerol dipalmitate derivatives of the two muramyl dipeptides within liposomal carriers resulted in a significant enhancement of anti-infectious activity, both with respect to number of survivors and length of survival. Liposomal muramyl dipeptides were 10- to 15-fold more potent than free muramyl dipeptide; enhanced potency was most evident with N-acetylmuramyl-D-alanyl-D-isoglutamine. Prophylactic treatment with liposomes containing the lipophilic muramyl dipeptides resulted in enhanced clearance of bacteria from the blood (greater than 3-fold increase in rate) when compared with that of hydrosoluble N-acetylmuramyl-L-alanyl-D-isoglutamine, indicating a correlation between reticuloendothelial stimulation and anti-infectious activity.
Temperature-dependent transcriptional regulation of expression of fimbriae in an Escherichia coli strain isolated from a child with severe enteritis
Authors: Williams P H , Hinson G
Published: January 2020

Escherichia coli 469-3 synthesizes fibrillar fimbriae at 37 degrees C, but not at 18 degrees C, which promote its adherence to human colonocytes. RNA homologous with a cloned fragment of the strain 469-3 chromosome necessary for normal expression of fimbriae was virtually undetectable in bacteria grown at 18 degrees C, indicating that environmental temperature affects expression by regulating transcription.
Phenotypic variation in epitope expression of the Neisseria gonorrhoeae lipooligosaccharide
Authors: Apicella M A , Shero M , Jarvis G A , Griffiss J M , Mandrell R E , Schneider H
Published: January 2020

Gonococcal lipooligosaccharides (LOSs) are a series of antigenically complex heteropolymers. To investigate whether all members of clonally selected populations of Neisseria gonorrhoeae express antigenically similar LOS, we studied gonococcal strains 4505 and 220 with monoclonal antibodies 6B4 and 3F11 which have specificity for different oligosaccharide epitopes on the same or comigrating LOS unit(s) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent-antibody and immunoelectron microscopy studies indicated that all members of the clonally selected populations were not homogenous for the epitopes these antibodies recognized. Fluorescence-activated cell sorting studies of 3F11-coated strain 220 indicated that the density of epitope expression was a function of time of growth. The population could be separated into two broad groups corresponding to organisms staining strongly or weakly for the 3F11 epitope, and the epitope density decreased during the late-log and stationary phases of growth. Sequentially staining organisms on Formvar grids with 6B4 and 3F11, followed by staining with either 5- or 15-nm colloidal gold spheres conjugated to goat anti-mouse immunoglobulin M demonstrated the following populations of cells among organisms derived from a single clone: organisms which stained for both 6B4 and 3F11 epitopes and organisms which stained for either 6B4 epitopes alone or 3F11 epitopes alone. Immunofluorescence microscopy studies with rhodamine and fluorescein goat anti-mouse immunoglobulin M conjugates sequentially staining organisms on Formvar grids with 3F11 and 6B4 also demonstrated these three populations. Analysis of LOS preparations made over the last 5 years indicated no change in serotype antigen concentration or in sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration pattern. These studies indicate that while clonally selected strains of Neisseria gonorrhoeae undergo phenotypic variation at the epitope level, the impact of this variation on the total LOS of the population has little overall effect on its antigenic or physicochemical properties.
Small-bowel colonization alone is a cause of diarrhea
Authors: Wanke C A , Guerrant R L
Published: January 2020

Bacterial overgrowth in the small bowel is known to occur in patients with persistent diarrhea, but it has been unclear if colonization alone causes fluid secretion. We studied isogenic, nontoxigenic Escherichia coli strains with colonizing factor antigen II (CFA/II) (1392+) and without CFA/II (1392-) in 18-h rabbit ileal loops. Neither strain produced fluid, but quantitative cultures of rabbit ileal loops with the CFA/II-positive strain had a mean of 10(3) more CFU of bacteria per cm2 than did cultures of rabbit ileal loops with the CFA/II-negative strain or a negative control (P less than 0.05). Of 11 rabbits given strain 1392+ via the small bowel in the reversible ileal tie model, 7 became colonized (mean, 10(10.4) +/- 1.6 CFU/cm2) over 72 h, and all 7 developed diarrhea and had gross gut fluid accumulation, versus none of 10 rabbits given strain 1392- (mean, 10(3.6) +/- 1.9 CFU/cm2) (P less than 0.05). Both strains grew equivalently in vitro. The mechanism by which this E. coli strain which colonizes small-bowel mucosa but produces no recognized toxins causes fluid accumulation and diarrhea in a rabbit model at 72 h remains to be elucidated.
Nucleotide sequence of the leukotoxin genes of Pasteurella haemolytica A1
Authors: Lo R Y , Strathdee C A , Shewen P E
Published: January 2020

A 4.4-kilobase-pair DNA fragment coding for the leukotoxin of Pasteurella haemolytica A1 has been isolated, and its nucleotide sequence has been determined. Two open reading frames, designated lktC and lktA, coding for proteins of 19.8 and 101.9 kilodaltons, respectively, were identified. Expression of the two genes in minicell-labeling experiments resulted in the production of the predicted proteins LKTC and LKTA. By using an antiserum against the soluble antigens of P. haemolytica A1 in Western blot (immunoblot) analysis of total cellular proteins from the Escherichia coli clones, LKTA was identified as an additional antigenic protein. Results from subcloning of the DNA fragment suggested that expression from both lktC and lktA is required for leukotoxin activity, indicating that the leukotoxin of P. haemolytica A1 is encoded by two genes. A comparison of the organization and the DNA sequence of the leukotoxin genes with those of the E. coli alpha-hemolysin genes showed a significant degree of homology between the two loci. This analysis suggested that the leukotoxin genes of P. haemolytica A1 and the E. coli alpha-hemolysin genes may have evolved from a common ancestor and that the two toxins may share similar activities or functional domains or both.
Granulocyte-macrophage colony-stimulating factor enhances the production of eosinophil chemotactic lymphokine by egg-associated granulomas of Schistosoma japonicum-infected mice
Authors: Owhashi M , Maruyama H , Nawa Y
Published: January 2020

Granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by splenic lymphocytes obtained from Schistosoma japonicum-infected mice was partially purified by a combination of DEAE anion-exchange chromatography, concanavalin A-Sepharose affinity chromatography, and high-pressure liquid chromatography. When this partially purified GM-CSF was added to the culture of isolated intact granulomas, eosinophil chemotactic factor (ECF) lymphokine production by granulomas was significantly enhanced. The partially purified GM-CSF also enhanced ECF lymphokine production by granuloma T cells cocultured with syngeneic macrophages and specific antigen. The partially purified GM-CSF itself had neither ECF activity nor a synergistic effect with ECF lymphokine. When normal splenic macrophages were preincubated with the partially purified GM-CSF, they potentiated the ECF production by granuloma T cells under the presence of specific antigen. Augmentation of ECF lymphokine production by partially purified GM-CSF was further confirmed by using T-cell clones that were established from granuloma T cells. These results suggest that T-cell-derived GM-CSF primarily activate macrophages so that these activated macrophages can cooperate more effectively with T lymphocytes to produce ECF. Such potentiation of macrophage-T-cell interaction by GM-CSF may be important in the mechanisms of granuloma formation during an acute stage of schistosomiasis.
Recombinant murine interleukin-1 alpha enhancement of nonspecific antibacterial resistance
Authors: Czuprynski C J , Brown J F
Published: January 2020

In this study we report that treatment with recombinant murine interleukin-1 alpha (rIL-1 alpha) significantly enhanced the resistance of mice to infection by the facultative intracellular pathogen Listeria monocytogenes. The greatest level of protection was observed at a dose of 1,000 lymphocyte-activating factor units (approximately 0.17 micrograms) of rIL-1 alpha per mouse. Although rIL-1 alpha enhanced antibacterial resistance when administered either intravenously or intraperitoneally, greater protection was observed when the rIL-1 alpha and the L. monocytogenes challenge were given by the same parenteral route. When the intravenous route was used, antibacterial resistance was maximal when the rIL-1 alpha and L. monocytogenes were injected concomitantly. In contrast, intraperitoneal administration of rIL-1 alpha was most effective when given 48 h before an intraperitoneal L. monocytogenes challenge. Based on the following lines of evidence, we concluded that contaminating lipopolysaccharide (LPS) was unlikely to be responsible for the enhanced antibacterial resistance that was observed: (i) LPS was not detectable (less than 0.2 ng/ml by the lysate assay) at the concentration of rIL-1 alpha that was injected; (ii) polymyxin B did not abrogate rIL-1 alpha-enhanced antibacterial resistance; (iii) rIL-1 alpha treatment enhanced the antibacterial resistance of LPS-nonresponsive C3H/HeJ mice; and (iv) injection of up to 10 micrograms of LPS per mouse (calculated to be greater than 50,000 times the concentration of LPS in the rIL-1 alpha administered) failed to duplicate the marked enhancement of antibacterial resistance that was mediated by rIL-1 alpha. These data provide evidence for the beneficial role of IL-1 in nonspecific antibacterial resistance.
Effects of fibronectin and other salivary macromolecules on the adherence of Escherichia coli to buccal epithelial cells
Authors: Hasty D L , Simpson W A
Published: January 2020

The effect of saliva and fibronectin (Fn) on the adherence of a type 1 fimbriated strain of Escherichia coli to human buccal epithelial cells was studied. Saliva pretreatment of epithelial cells led to a dose-dependent increase in adherence that was inhibited by alpha-methyl mannoside, which is typical of a type 1 fimbria-mediated event. The molecules responsible for affecting this increased adherence were nondialyzable and were recovered after lyophilization. E. coli adherence was stimulated by individual saliva samples from each of 11 volunteers. Fn inhibited E. coli adherence to saliva-treated buccal cells by more than 60%. Biotinylated E. coli and Fn were reacted with Western blots of whole saliva to identify the receptors that might explain the phenomenon described above. Both E. coli and Fn bound to 57- and 62-kilodalton (kDa) protein bands in Western blots of sodium dodecyl sulfate gels of whole saliva. The binding of E. coli to these bands was inhibited by pretreatment with unlabeled Fn. To study these salivary components, samples of saliva were electrophoresed on sodium dodecyl sulfate gels, strips corresponding to the appropriate molecular weights were cut out, and the proteins were eluted electrophoretically. Material that eluted from strips at 57 and 62 kDa, but not that from a control strip, stimulated E. coli adherence to buccal cells. Alternatively, saliva was fractionated over 100- and 50-kDa cutoff filters. Of the three fractions obtained, only the fraction passing through the 100-kDa filter and retained by the 50-kDa filter stimulated E. coli adherence to buccal cells. This fraction also increased the binding of Fn to buccal cells. These observations suggest the possibility that one or more salivary components bind to the surface of buccal cells and serve as receptors for type 1 fimbriated E. coli. Fn also binds to this isolated material; and it is apparently by these interactions, at least in part, that saliva stimulates and Fn inhibits E. coli adherence. The way in which these interactions may affect bacterial adherence in vivo remains to be elucidated.
Effects of iron and temperature on Shiga-like toxin I production by Escherichia coli
Authors: Weinstein D L , Holmes R K , O'Brien A D
Published: January 2020

Iron is known to depress Shiga toxin production by Shigella dysenteriae 1, and temperature has been shown to regulate several genes required for Shigella invasiveness. In this study, the influence of iron and temperature on regulation of a highly related toxin, Shiga-like toxin I (SLT-I) of enterohemorrhagic Escherichia coli, was examined in strains lysogenic for the toxin-converting coliphage 933J and in strains carrying the cloned slt-I genes on a high-copy-number plasmid vector. For comparison, S. dysenteriae 1 was included in these studies. As expected, iron suppressed Shiga toxin synthesis, and reduced growth temperature was also found to decrease Shiga toxin production. Iron also suppressed SLT-I synthesis in E. coli lysogenized with phage 933J but did not demonstrably repress toxin synthesis in E. coli strains carrying the cloned slt-I genes. Temperature had no effect on SLT-I synthesis. Mini-Mu lac operon fusions were then isolated in the cloned slt-I genes and used to test for regulation of beta-galactosidase by iron. Iron did not decrease beta-galactosidase production in strains that harbored these operon fusion plasmids. Taken together, these results indicate that iron but not temperature represses SLT-I synthesis when the slt-I genes are phage associated but this suppression is not easily demonstrated when the slt-I genes are cloned on a high-copy-number plasmid.
Analysis of cross-protection between serotypes and passively transferred immune globulin in experimental nutritionally variant streptococcal endocarditis
Authors: van de Rijn I
Published: January 2020

Nutritionally variant streptococci (NVS), which account for 5 to 10% of all cases of streptococcal endocarditis, were recently subdivided into three serotypes. In the past, using a rabbit endocarditis model, I demonstrated that by immunization of rabbits with NVS and by challenge with a strain from the homologous serotype a level of 90 to 100% protection was elicited. In the present study, the level of cross-protection between strains from different serotypes was measured. No cross-protection was demonstrated between serotype I and II or III strains. However, significant cross-protection was observed when serotype II and III strains were analyzed in the model. Since high levels of immunoglobulin G were demonstrated against the surface of the NVS after immunization, passive transfer experiments were initiated. Even at comparable levels of surface immunoglobulin G, none of the rabbits given immune globulin were protected against challenge with a dose of live NVS that equaled 20 times the 50% infective dose. Therefore, it appears that components from the immune system, in addition to humoral components, are required for active protection against NVS endocarditis.
Immunosuppressive properties of Actinobacillus actinomycetemcomitans leukotoxin
Authors: Rabie G , Lally E T , Shenker B J
Published: January 2020

Actinobacillus actinomycetemcomitans produces a leukotoxin that kills human polymorphonuclear cells (PMNs) and monocytes but not lymphocytes. In this study, we examined A. actinomycetemcomitans leukotoxin for its ability to alter human peripheral blood lymphocyte (HPBL) responsiveness. After a 90-min exposure to the leukotoxin, all monocytes were killed and HPBL responsiveness to mitogens and antigens was significantly inhibited. The ability of the leukotoxin to inhibit HPBL responses was not surprising, since monocytes and macrophages are required for many lymphocyte functions. However, we were unable to totally restore HPBL responsiveness when adherent autologous monocytes were added back to cultures of leukotoxin-treated lymphocytes. These studies demonstrate that A. actinomycetemcomitans leukotoxin may also exert nonlethal effects directly on lymphocytes. Furthermore, impaired lymphocyte function did not appear to be the result of indirect effects of products released by dying monocytes. Although it is not clear how A. actinomycetemcomitans acts to cause disease, several investigators have proposed that impaired host defenses may play a pivotal role. Several studies have demonstrated defects in PMN, monocyte, and lymphocyte function in patients with periodontal disease. These findings, along with the data presented in this paper, support the hypothesis that patients who harbor A. actinomycetemcomitans could suffer from local or systemic immune suppression. The effects of this suppression may be to enhance the pathogenicity of A. actinomycetemcomitans itself or that of some other opportunistic organism.
Importance of fatty acid substituents of chemically synthesized lipid A-subunit analogs in the expression of immunopharmacological activity
Authors: Kumazawa Y , Nakatsuka M , Takimoto H , Furuya T , Nagumo T , Yamamoto A , Homma Y , Inada K , Yoshida M , Kiso M
Published: January 2020

The immunopharmacological activities of chemically synthesized lipid A-subunit analogs, 4-O-phosphono-D-glucosamine derivatives carrying different N- and 3-O-linked acyl groups, were investigated. None of the synthetic compounds tested exhibited any detectable pyrogenicity at a dose of 10 micrograms/kg. Weaker lethal toxicity in galactosamine-sensitized mice was detected at 1 microgram per mouse for all the synthetic compounds except GLA-58. Among (RS) stereoisomers of 4-O-phosphono-D-glucosamine derivatives carrying a 3-O-tetradecanoyl (C14) group with different N-linked acyloxyacyl groups, i.e., 3-dodecanoyloxytetradecanoyl [C14-O-(C12)], 3-tetradecanoyloxytetradecanoyl [C14-O-(C14)], and 3-hexadecanoyloxytetradecanoyl [C14-O-(C16)] groups (termed GLA-57, GLA-27, and GLA-58, respectively), GLA-27 exhibited significant colony-stimulating factor-inducing and tumor necrosis factor-inducing activities, mitogenicity, polyclonal B-cell activation activity, macrophage activation, and adjuvanticity. The activities of GLA-57, which had an N-linked C14-O-(C12) group, were equivalent to or somewhat weaker than those of GLA-27 with a C14-O-(C14) group. Significant immunopharmacological activities were not observed for GLA-58, carrying a C14-O-(C16) group bound to the amino group. GLA-59, carrying 3-O-linked 3-hydroxytetradecanoyl (C14OH) and N-linked C14-O-(C14) groups, showed much higher activities than GLA-27, GLA-60, a compound which possesses the same fatty acid substituents as GLA-59 but with reversed binding sites, showed the strongest B-cell activation and adjuvant activities among the synthetic compounds. Among stereoisomers of GLA-59 and GLA-60 composed of fatty acid substituents with the (RR) and (SS) configuration, compounds with the (RR) configuration elicited stronger activities than the (SS) stereoisomers. The importance of fatty acid substituents, including stereospecificity for the expression of immunopharmacological activities of 4-O-phosphono-D-glucosamine derivatives, was demonstrated.
Volunteer studies of deletion mutants of Vibrio cholerae O1 prepared by recombinant techniques
Authors: Levine M M , Kaper J B , Herrington D , Losonsky G , Morris J G , Clements M L , Black R E , Tall B , Hall R
Published: January 2020

Vibrio cholerae O1 A-B- vaccine strain JBK 70 and A-B+ CVD 101 prepared by recombinant DNA techniques from pathogenic EI Tor Inaba N16961 and classical Ogawa 395, respectively, were fed to 38 volunteers in single doses of 10(4) to 10(10). Although severe diarrhea did not occur in any vaccine, more than one-half developed mild diarrhea. These attenuated strains colonized well and elicited prominent vibriocidal and antitoxic (CVD 101) antibody responses. Recipients of a single dose of JBK 70 were significantly protected when challenged with 10(6) wild-type N16961. Diarrhea occurred in 7 of 8 controls but in only 1 of 10 vaccines (P less than 0.003, 89% vaccine efficacy), demonstrating the potency of immune mechanisms that do not involve cholera antitoxin. Further derivatives were prepared to explore the pathogenesis of the residual diarrhea, considering that either intestinal colonization by the vaccine itself or accessory toxins might be responsible. CVD 102, an auxotrophic mutant of CVD 101, did not cause diarrhea but colonized poorly and elicited feeble immune responses. Derivatives of JBK 70 and CVD 101 (CVD 104 and 105) deleted of genes encoding the EI Tor hemolysin still caused mild diarrhea. Genetically engineered strains can be colonizing, highly immunogenic, and protective single-dose oral vaccines, but they must be further attenuated before they can be considered for use as public health tools.
Adherence of Pasteurella multocida or Bordetella bronchiseptica to the swine nasal epithelial cell in vitro
Authors: Nakai T , Kume K , Yoshikawa H , Oyamada T , Yoshikawa T
Published: January 2020

The interaction of Bordetella bronchiseptica or Pasteurella multocida with swine nasal epithelial cells was studied in vitro. The mean number of B. bronchiseptica organisms adhered per cell was about three times as high as that of P. multocida (P less than 0.01), and the adherence was specifically inhibited by the homologous antiserum prepared with the whole-cell antigen of each bacterium. The poor affinity of P. multocida to the swine nasal mucosa as compared with that of B. bronchiseptica was also demonstrated in the cultured fragments of the nasal mucosa. When observed with a scanning electron microscope, B. bronchiseptica organisms colonized the fragments, whereas few P. multocida organisms adhered. Morphologically, the P. multocida-infected fragments had an essentially normal structure, whereas marked degeneration and marked desquamation of the epithelial cells and severe inflammatory reactions were observed in many areas of the B. bronchiseptica-infected fragments. These morphological observations were consistent with those for the nasal mucosa of P. multocida- or B. bronchiseptica-infected neonatal pigs (T. Nakai, K. Kume, H. Yoshikawa, T. Oyamada, and T. Yoshikawa, Jpn. J. Vet. Sci. 48:693-701, 1986; T. Oyamada, T. Yoshikawa, H. Yoshikawa, M. Shimizu, T. Nakai, and K. Kume, Jpn. J. Vet. Sci. 48:377-387, 1986). Cultured swine nasal fragments, however, were equally injured when they were incubated in a medium containing purified dermonecrotic toxin (DNT) preparations of B. bronchiseptica or P. multocida. Therefore, these DNT preparations can induce morphological damage closely resembling that induced in vivo. Hence, colonization of B. bronchiseptica and production of its DNT on the swine nasal mucosa appear to result in the production of mucosal damage. On the other hand, P. multocida seems to lack the ability to colonize normal swine nasal mucosa, thus resulting in no production or the slight production of DNT to such an extent as to produce mucosal damage. The present data support our previous hypothesis (Nakai et al.; Oyamada et al.) that B. bronchiseptica induces swine atrophic rhinitis, whereas P. multocida does not.
Partial characterization of the enzymatic activity associated with the binary toxin (type C2) produced by Clostridium botulinum
Authors: Simpson L L , Zepeda H , Ohishi I
Published: January 2020

Clostridium botulinum produces a binary toxin that possesses a heavy chain (approximately 100,000 daltons) and a light chain (approximately 50,000 daltons). The heavy chain is a binding component that directs the toxin to vulnerable cells, and the light chain is an enzyme that has mono(ADP-ribosyl)ating activity. A number of experiments have been done to help characterize the enzymatic activity of the toxin. The data reveal that the enzyme has a pH optimum within the range of 7.0 to 8.0. It is not inhibited or stimulated by physiological concentrations of sodium, potassium, calcium, or magnesium. The enzyme is inhibited by high concentrations of salt, however, as well as high concentrations of nicotinamide, thymidine, theophylline, and histamine; and it is stimulated by histone and lysolecithin. Boiling irreversibly denatures the light chain of the toxin, but denaturation caused by guanidine and urea is substantially reversible. Enzymatic activity is not altered by short exposure to lysosomal proteases, including cathepsin B, cathepsin H, dipeptidyl aminopeptidase, and catheptic carboxypeptidase B.
Identification of C3d receptors on Candida albicans
Authors: Calderone R A , Linehan L , Wadsworth E , Sandberg A L
Published: January 2020

Pseudohyphal but not yeast forms of Candida albicans possess both iC3b and C3d receptors, as determined by rosetting with erythrocytes carrying iC3b (EAC3bi) or C3d (EAC3d). Rosetting with EAC3d was markedly reduced when pseudohyphae were heat killed or treated with trypsin or pronase but was not inhibited by several saccharides or aminosaccharides, including alpha-methyl-D-mannoside, or by pretreatment of pseudohyphae with concanavalin A. However, mannoproteins obtained by concanavalin A affinity chromatography of whole pseudohyphal extracts inhibited the attachment of EAC3d to C. albicans, whereas soluble (nonmannosylated) proteins were less active. Thus, although the C3d receptors appeared to be glycosylated, the oligosaccharide component of the receptor was apparently not involved in the recognition of C3d. To isolate these receptors, whole-cell extracts were separated by DEAE-Trisacryl chromatography. Fractions that inhibited rosetting were pooled and affinity purified by C3d-Thiol-Sepharose chromatography. The eluate from this affinity column inhibited attachment of C. albicans to EAC3d. Monoclonal antibodies to C. albicans were prepared, and three of these antibodies blocked rosetting. Western blotting (immunoblotting) with these antibodies indicated the presence of 62- and 70-kilodalton receptors for C3d in the extracts purified by C3d affinity chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Fungal-strain-dependent alterations in the time course and mortality of chronic murine pulmonary blastomycosis
Authors: Moser S A , Koker P J , Williams J E
Published: January 2020

Intratracheal injection of 10(4) conidia of Blastomyces dermatitidis strain M1-A into mice was shown in previous work to induce chronic pulmonary and disseminated infection with histopathologic features of chronic human blastomycosis. Furthermore, 10-fold variations in inoculum density produced marked changes in mean survival times (MSTs), i.e., 32 days at 10(6), 36 days at 10(5), 97 days at 10(4), and 172 days at 10(3). A second strain (M1-B) failed to induce death in this model. To assess fungal-strain-dependent virulence, we extended these previous studies to 11 additional human isolates. Groups of male BALB/cByJ mice (6 to 8 weeks old) were injected intratracheally with 10(4) conidia from each of the 13 strains; the mice were weighed weekly and monitored for mortality, and their lungs were examined histopathologically. Infection rate and mortality were 100% in all groups except for the M1-B inoculated mice. For strains M1-B and M1-A, MST and mortality curves were not significantly different from those observed in our previously reported experiments. Four different survival patterns were noted in infected mice. The shortest MSTs were produced by strains M2-E, M2-B, M2-K, M2-H, and M2-A (24, 26, 26, 27, and 31 days, respectively), and the longest MST was seen in animal groups inoculated with strains M2-J and M2-G (130 and 134 days, respectively). Strains M2-I, M2-F, and M2-D produced intermediate MSTs of 65, 79, and 80 days, respectively. The 107-day MST induced by M1-A did not differ from strain M2-C-induced MST but differed significantly from the MST produced by the other strains. Pulmonary histopathology was similar in all animals dying with blastomycosis. The wide spectrum in survival times was not related to differences in clinical status of the patient from whom the isolate had been obtained, to fungal inoculum viability, or to individual mouse weight at inoculation. Strain-dependent virulence factors present in these fungal isolates alter the disease course in inbred mice in a fashion similar to that induced by 10-fold inoculum variation of strain M1-A conidia. These 13 isolates of B. dermatitidis, 1 avirulent and 12 with differing levels of virulence, provide tools for future studies into the nature of fungal virulence determinants in chronic blastomycosis.
Protective monoclonal antibodies recognize heat-labile epitopes on surface proteins of spotted fever group rickettsiae
Authors: Li H , Lenz B , Walker D H
Published: January 2020

Thirty-eight monoclonal antibodies that have not been reported previously were developed from mice immunized with Rickettsia rickettsii, R. conorii, and R. sibirica. Western immunoblotting showed that these monoclonal antibodies are directed against heat-sensitive epitopes which are located on two major surface polypeptides with molecular sizes ranging from 115 to 150 kilodaltons. The detection of the two bands did not depend on the presence of 2-mercaptoethanol. Both bands were destroyed by treatment with proteinase K. Monoclonal antibodies examined by immunofluorescence assay reacted with epitopes that are species specific, group reactive, or shared among a smaller subset of species of spotted fever group rickettsiae. Nine of the monoclonal antibodies were evaluated for their ability to neutralize rickettsial infection and thus protect animals against disease caused by homologous species of rickettsiae. Treatment of rickettsiae with monoclonal antibodies F3-12, F3-14, and F3-36 completely protected guinea pigs against illness caused by the homologous organism R. rickettsii. Monoclonal antibodies F9-5G11 and F15-5B12, derived from mice immunized with R. sibirica, conferred partial protection by delaying the onset and shortening the duration of fever in guinea pigs inoculated with R. sibirica. Monoclonal antibodies F2-15, F2-31, F2-53, and F3-12 protected mice from a lethal infection with R. conorii. Heat-labile epitopes of spotted fever group rickettsial surface proteins are important candidate antigens for development of vaccines to confer protective immunity.
Binding characteristics of Escherichia coli adhesins in human urinary bladder
Authors: Virkola R , Westerlund B , Holthöfer H , Parkkinen J , Kekomäki M , Korhonen T K
Published: January 2020

We studied domains in the human bladder that acted as receptors for Escherichia coli P, S, type 1, type 1C, and O75X fimbriae or adhesin and domains in the human kidneys that were receptors for E. coli type 1C fimbriae. Binding sites in frozen tissue sections were localized by direct staining with fluorochrome-labeled recombinant strains and by indirect immunofluorescence with the purified adhesins. In the bladder, the P and S fimbriae showed closely similar binding to the epithelial and muscular layers, and the S fimbriae also bound to the connective tissue elements. Type 1 fimbriae bound to vascular walls and to muscle cells, whereas the O75X adhesin bound avidly to connective tissue elements and to some extent to epithelial and muscle cells of the bladder. The type 1C fimbriae bound to distal tubules and collecting ducts of the kidney and to vascular endothelial cells in both the kidney and bladder. The binding of all adhesin types was inhibited by specific receptor analogs or Fab fragments. The results reveal a possible mechanism by which the type 1C fimbriae may help invasion of E. coli in the kidneys but do not support a pathogenetic role for type 1 fimbriae. Similar tissue specificity of P and S fimbriae in the human urinary tract indicates that the presence of binding sites on uroepithelia does not fully explain the virulence properties of P fimbriae in human urinary tract infections.
Immunoreactivity of a surface wall fraction produced by spherules of Coccidioides immitis
Authors: Cole G T , Kirkland T N , Franco M , Zhu S , Yuan L , Sun S H , Hearn V M
Published: January 2020

The membranous spherule outer wall (SOW) isolated from liquid cultures of Coccidioides immitis has been shown to elicit reactivity with human anti-Coccidioides antibody by immunofluorescence and the immunodiffusion-tube precipitin assay. The serologically reactive components were extracted from SOW with the nonionic detergent N-octyl-beta-D-glucopyranoside (OG). The OG-soluble fraction of SOW was shown to be reactive with immunoglobulin G in 25 serum samples from coccidioidomycosis patients by an enzyme-linked immunosorbent assay. The isolated SOW and OG-soluble fraction of SOW were also demonstrated to be capable of eliciting lymphocyte blastogenesis. The antigenic and protein compositions of the OG-soluble fraction were examined by two-dimensional immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Two antigens which were extracted from SOW were identified as antigens 2 and CS on the basis of the coccidioidin-anticoccidioidin reference system. The latter was isolated earlier and shown to correspond to a molecular mass (Mr) of 19 X 10(3) by SDS-PAGE under reducing conditions. This same electrophoresis band was shown to be reactive with sera from coccidioidomycosis patients by immunoblot analysis. One other SDS-PAGE component of the OG-soluble fraction of SOW with an Mr of 66 X 10(3) was shown to be reactive with sera from patients by immunoblot analysis. The SOW of C. immitis represents an important reservoir of immunoreactive wall components which has not previously been reported.
T-cell-mediated immunity in persistent Mycobacterium intracellulare infections in mice
Authors: Takashima T , Collins F M
Published: January 2020

Growth of mouse-virulent Mycobacterium intracellulare D673 and TMC 1405 in the lung was affected by T-cell depletion in susceptible C57BL/6 mice. Significant differences also occurred between the growth patterns seen in congenitally athymic (nu/nu) mice and their nu/+ littermates. Treatment of the mice with an immunosuppressive regimen of cyclosporin A (75 mg/kg of body weight per day subcutaneously) provided further evidence of the importance of T cells in controlling growth of M. intracellulare in the normal host. Adoptive transfer experiments indicated the presence of a T-cell-mediated specific protective immunity against a subsequent M. intracellulare challenge when transfer was carried out 3 weeks after immunization of the donor host. At this time, cross-protective immunity was also observed against a virulent M. tuberculosis challenge. There was no difference in the rate of growth by M. intracellulare as challenge in Mycobacterium bovis BCG-activated or normal peritoneal macrophages from C57BL/6 mice tested in vitro during a 7-day period. However, M. tuberculosis growth rates were decreased substantially in the BCG-activated macrophages. These studies suggest that mice infected with M. intracellulare do not eliminate the infection, because this organism can resist the bactericidal activity of the T-cell-activated macrophage better than M. tuberculosis can.
Characterization of the Vibrio cholerae ToxR regulon: identification of novel genes involved in intestinal colonization
Authors: Peterson K M , Mekalanos J J
Published: January 2020

A gene fusion library of Vibrio cholerae classical strain O395 was generated by using a broad host range vector for delivery of the transposon TnphoA. The insertion library was screened for colonies expressing alkaline phosphatase-positive (PhoA+) fusion proteins on LB agar at 30 degrees C in the presence of 0.2% glucose. Over 600 PhoA+ strains were isolated and then tested for regulation of their gene fusions in broth media that permitted high or low expression of cholera toxin. This strategy resulted in the isolation of 60 TnphoA (Tn5 IS50L::phoA) fusions to genes encoding secreted proteins that are apparently coordinately regulated with cholera toxin. Introduction of a toxR null mutation into 10 of these fusion strains confirmed that these TnphoA gene fusions are controlled either directly or indirectly by the cholera toxin transcriptional activator encoded by toxR. A combination of Southern and immunoblot analysis identified 17 distinct ToxR-regulated genes in V. cholerae O395. Many of these insertions were located in one of the two cholera toxin operon copies of strain O395, as well as a large gene cluster involved in the biogenesis of the toxin-coregulated pilus colonization factor. In addition, insertions were identified in genes that had no effect on either cholera toxin or toxin-coregulated pilus expression. Several of these insertions were localized to a cluster of four genes, the disruption of any of which by TnphoA reduced the ability of strain O395 to colonize the intestines of suckling mice. The product encoded by this second gene cluster was named accessory colonization factor to describe its possible role in cholera pathogenesis. These studies reinforce the contribution of ToxR-regulated genes to the virulence properties of V. cholerae. This report also demonstrates a new approach for the identification of bacterial virulence factors, based on the characterization of genes that are regulated by the same environmental signals that control the expression of a known virulence factor.
Roles of CD4- and CD8-bearing T lymphocytes in the immune response to the erythrocytic stages of Plasmodium chabaudi
Authors: Süss G , Eichmann K , Kury E , Linke A , Langhorne J
Published: January 2020

The possible role of CD4- and CD8-bearing T lymphocytes in parasite clearance in vivo was investigated, using Plasmodium chabaudi in C57BL/6 mice as a model. Monoclonal antibodies specific for the CD4 and CD8 molecules were administered in vivo to deplete selectively the appropriate subset of T cells. The efficacy of depletion was ascertained by flow cytometry and functional studies. These mice were then infected with P. chabaudi, and the course of infection was followed. The control groups had maximum parasitemias of approximately 30% 10 days after infection, and the infection was cleared within 27 days. Mice without CD4+ cells had significantly higher parasitemias which they were unable to reduce below 20% for the duration of the experiment. Mice without CD8 cells had slightly higher parasitemias which were cleared after 34 days. Because of the possibility that CD8+ cells alone could not be activated in the absence of growth factors, exogenous interleukin-2 was administered to the mice depleted of CD4 cells. This did not significantly affect parasitemias, and the mice were still unable to clear their infections. The data suggest that CD4+ T cells play a crucial role in the protective immune response to the erythrocytic stages of P. chabaudi.
Toxoid of Pseudomonas aeruginosa exotoxin A generated by deletion of an active-site residue
Authors: Lukac M , Pier G B , Collier R J
Published: January 2020

Glutamic acid-553 of Pseudomonas aeruginosa exotoxin A (ETA), identified previously as an active-site residue, was deleted by oligonucleotide-directed mutagenesis of the cloned toxin gene in Escherichia coli. The purified mutant toxin was stable, fully immunoreactive, and capable of blocking toxin receptors. ADP-ribosyltransferase and cytotoxic activities were at least 10(6)-fold lower than those of wild-type ETA, and injection of mice with 50 micrograms (equivalent to 400 lethal doses of ETA) produced no ill effects. The mutant toxin elicited high levels of neutralizing anti-ETA antibodies in mice, which protected against a challenge with 100 micrograms of authentic ETA (greater than 600 lethal doses). The mutant protein has the attributes of a toxoid and may be useful as a component of vaccines for individuals at risk for infection by P. aeruginosa.
Resistance of mucoid Pseudomonas aeruginosa to nonopsonic phagocytosis by alveolar macrophages in vitro
Authors: Krieg D P , Helmke R J , German V F , Mangos J A
Published: January 2020

A unique, recently described rat alveolar macrophage cell line (NR8383) was used to study the interaction of the pulmonary immune system with a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa (SRM-3), its nonmucoid revertant (SRM-3R), and a non-cystic fibrosis isolate (PAO-1). Strain SRM-3 was cultivated in a chemostat system to allow maintenance of an entirely mucoid population. The alveolar macrophage response to the mucoid and nonmucoid strains of P. aeruginosa was determined by visually quantitating phagocytosis in acridine orange-stained monolayers and measuring the induction of an oxidative burst as indicated by chemiluminescence and H2O2 production. In all experiments, fewer than 2% of the NR8383 cells engulfed the mucoid SRM-3 isolate, while SRM-3R and PAO-1 were phagocytized by 15 and 41%, respectively. Opsonization by normal serum (complement) provided minimal phagocytic enhancement of these strains, whereas specific anti-P. aeruginosa antibody slightly elevated phagocytic responses to strains with nonmucoid phenotypes while providing a sevenfold increase in uptake of SRM-3. Chemiluminescent and H2O2 responses were comparable with the levels of phagocytosis observed, with very little or no response to the mucoid strain SRM-3. The data indicate that the strains with mucoid phenotypes are refractile to ingestion and that studies which describe ingestion of mucoid strains were likely measuring ingestion of revertants. Alginic acid (2 mg/ml) was found to inhibit stimulation of macrophage response to the opsonized and unopsonized nonmucoid strain PAO-1.
Circumsporozoite protein of the human malaria parasite Plasmodium ovale identified with monoclonal antibodies
Authors: Procell P M , Collins W E , Campbell G H
Published: January 2020

Monoclonal antibodies (MAbs) have been produced against Plasmodium ovale sporozoites and used to characterize the circumsporozoite (CS) protein. Six MAbs were produced, and all were species specific. By using Western blot (immunoblot) analysis, three polypeptides were detected: a predominant 51,000-Mr polypeptide and two presumed precursor 57,000- and 67,000-Mr molecules. The presence of a repeating epitope in the CS protein of P. ovale was demonstrated by using one of the MAbs in a single-antibody two-site enzyme immunoassay. Three MAbs recognized epitopes on the surfaces of sporozoites; the presence of at least one other epitope within the CS protein, but not on the surfaces of P. ovale sporozoites, was also demonstrated.