bluuebunny/crossref_metadata_2025 huggingface dataset:
Reassessment of the role of splenic leukocyte oxidative activity and macrophage activation in expression of immunity to malaria
Authors: Cavacini L A , Guidotti M , Parke L A , Melancon-Kaplan J , Weidanz W P
Published: January 2020

The role of splenic leukocyte oxidative activity and macrophage activation in the development of protective immunity was examined during acute Plasmodium chabaudi adami malaria. Splenic leukocyte oxidative activity was compared in infected BALB/c and P/J mice; the latter are known to suffer from defects in macrophage function. Phorbol myristate acetate-stimulated chemiluminescence and superoxide anion production by splenic leukocytes from infected BALB/c mice were found to be increased dramatically, while the response of splenic leukocytes from infected P/J mice was elevated only minimally. Hydrogen peroxide release was slightly increased in splenic leukocytes from infected BALB/c mice but remained essentially unchanged in those from infected P/J mice. Macrophage function was assessed on the basis of measurements of tumoricidal activity. Splenic macrophages from uninfected BALB/c mice displayed significant tumoricidal activity against L929 target cells. As a result of splenomegaly during infection, tumoricidal activity, when calculated on a per-spleen basis, was increased further in infected BALB/c mice. In contrast, the tumoricidal activity of splenic macrophages from P/J mice was minimal, regardless of infection. Despite these differences, both strains of mice developed malarial infections that resolved within 16 days. Thus, while the production of reactive oxygen radicals by splenic leukocytes and the phenomenon of macrophage activation have traditionally been associated with the resolution of malarial infection, this study failed to establish a correlation between these parameters and the development of protective immunity to blood-stage infection with P. chabaudi adami.
Analysis of human antibody epitopes on the 65-kilodalton protein of Mycobacterium leprae by using synthetic peptides
Authors: Meeker H C , Williams D L , Anderson D C , Gillis T P , Schuller-Levis G , Levis W R
Published: January 2020

In order to study antibody reactivity to the Mycobacterium leprae 65-kilodalton (kDa) antigen, peptides representing overlapping sequences of the 65-kDa protein were synthesized, and a recombinant protein expression system for r65-kDa was constructed. Mouse monoclonal antibodies and leprosy patient seroreactivity to peptides and r65-kDa were tested by an enzyme-linked immunosorbent assay. All seven of the monoclonal antibodies used in this study reacted with their previously defined epitopes when tested against peptides. All monoclonal antibodies also reacted with r65-kDa. Leprosy patient seroreactivity to peptides and r65-kDa was seen in about one-third of active multibacillary cases. Specimens from patients positive for antibodies to peptides were seen to recognize different epitopes than did mouse monoclonal antibodies used in this study. It is concluded that substantial differences exist between mouse monoclonal antibodies and human leprosy patient reactivity to the 65-kDa antigen and that human seroreactivity to the 65-kDa antigen is indicative of a highly elevated bacillary load.
Degradation of basement membranes by Pseudomonas aeruginosa elastase
Authors: Bejarano P A , Langeveld J P , Hudson B G , Noelken M E
Published: January 2020

Pseudomonas aeruginosa virulence has been attributed in part to extracellular proteinases. We found that one of these proteinases, elastase, extensively degrades intact basement membranes from bovine anterior-lens capsules, bovine glomeruli, and bovine lung, producing about 9, 14, and 9 fragments, respectively, with Mrs in the range of 15,000 to greater than 200,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (nonreducing conditions). Release of hydroxyproline showed that collagen IV was degraded by elastase. Degradation of the newly discovered alpha 3(IV) collagen chain was shown by immunoblotting of digests with Goodpasture's syndrome serum, which contains antibodies that react with an epitope located in the carboxyl-terminal globular (NCl) domain of alpha 3(IV). Comparison of total protein release with collagen IV release showed that noncollagenous protein components were solubilized to the same extent as collagen IV. The extensive degradation of the basement membranes described here suggests a role for elastase in the pathogenic mechanism at the local level when P. aeruginosa infection is present.
Reaction of staphylococcal alpha-toxin with peptide-induced antibodies
Authors: Harshman S , Alouf J E , Siffert O , Baleux F
Published: January 2020

Two peptides representing separate 13-amino-acid sequences of staphylococcal alpha-toxin have been synthesized and acrylamide gel-purified alpha-toxin monomer and hexamer forms have been prepared and used to produce antisera in rabbits. We report here that each synthetic peptide, P-I and P-II, induces the formation of a specific precipitating antiserum. Moreover, these sera also react with the toxin monomer and sometimes with the hexamer, indicating that each peptide has more than one epitope. The purified toxin monomer can induce antibodies to fragments of toxin but is significantly less potent than the hexamer in inducing antibodies to the toxin monomer and almost not effective in inducing a response to the toxin hexamer. The purified toxin hexamer induces responses that are almost the reciprocals of the monomers, with the antihexamer and -monomer responses dominating and almost no responses to fragments of toxin being induced. These responses are interpreted in terms of the stability of the toxin hexamer to proteolytic degradation, compared with the relative sensitivity of the monomer to proteases. In assays of toxin-neutralization activity, only those sera containing antihexamer antibodies can block toxin hemolytic activity. This is true for both peptide- and toxin-induced antisera. The basis for this apparent association between toxin-neutralizing potency and antihexamer reactivity is being studied. Peptide P-I contains the uniquely reactive tyrosine residue and may be involved in monomer-to-monomer associations required to form hexamers. Peptide P-II is near the carboxyl terminus of alpha-toxin and may be involved in the binding of toxin to membranes. In a study of the ability of each peptide to inhibit the rate of hexamer formation induced by membrane lipoprotein, peptide P-I (as expected) proves to be more efficient than peptide P-II. Finally, one rabbit immunized with the toxin hexamer produces antibodies to peptides P-I and P-II. This finding suggests that the two synthetic peptides selected for study are relevant to the in vivo immunoprocessing of staphylococcal alpha-toxin.
Hypha formation in the white-opaque transition of Candida albicans
Authors: Anderson J , Cundiff L , Schnars B , Gao M X , Mackenzie I , Soll D R
Published: January 2020

Cells of Candida albicans strain WO-1 and related strains switch frequently and reversibly between a white-colony-forming unit (white phase) and a gray-colony-forming unit (opaque phase). Cells in the budding white phase exhibit the usual smooth round phenotype observed in other C. albicans strains, but cells in the budding opaque phase exhibit a unique elongate shape with surface pimples or protrusions. In this study, it was demonstrated that opaque cells formed hyphae at low to negligible levels in suspension cultures but could be induced to form hyphae at high levels when anchored to the chamber wall of a perfusion chamber or to a monolayer of human skin epithelial cells. Variability in the proportion of hyphae formed between experiments appeared to be due to variability between individual opaque clones. The hyphae formed by opaque cells were morphologically identical to hyphae formed by white cells (i.e., they were devoid of pimples or protrusions and exhibited the same shape and septal locations). They also did not stain with an opaque-specific antiserum which differentially stained opaque budding cells in a punctate fashion. However, when stimulated to form buds, opaque hyphae formed opaque-shaped daughter buds, demonstrating that although they are morphologically similar to hyphae formed by white cells, they are genetically opaque.
Association of microneme antigens of Plasmodium brasilianum merozoites with knobs and other parasite-induced structures in host erythrocytes
Authors: Torii M , Matsumoto Y , Kamboj K K , Maracic M , Guo S Q , Nussenzweig R S , Aikawa M , Cochrane A H
Published: January 2020

The localization of Plasmodium brasilianum antigens, common to merozoite micronemes and parasite-induced structures in the host erythrocyte, was determined by means of immunogold electron microscopy and monoclonal antibodies directed against blood stages of this parasite. All monoclonal antibodies reacted with micronemes. In addition, some reacted with either knob protrusions or caveolae of the host erythrocyte membrane; one reacted with a parasite-derived antigen present in the erythrocyte cytoplasm. Gold particles appeared over the membranes of ring-infected cells before the appearance of knobs and caveolae. We hypothesize that at least some knob- and caveolae-associated antigens of P. brasilianum are inserted into the erythrocyte membrane at the time of merozoite invasion.
Antibodies raised against synthetic peptides from the Arg-Gly-Asp-containing region of the Leishmania surface protein gp63 cross-react with human C3 and interfere with gp63-mediated binding to macrophages
Authors: Russell D G , Talamas-Rohana P , Zelechowski J
Published: January 2020

The Leishmania surface glycoprotein gp63 binds to complement receptor type 3 on the macrophage surface. Antibody raised against a synthetic peptide containing the Arg-Gly-Asp region of the amino acid sequence of gp63 recognizes both gp63 and the alpha-chain of human C3. Monovalent Fab fragments from this antibody block gp63-mediated binding to macrophages.
Primary structure of an oligomeric antigen of Treponema pallidum
Authors: Walfield A M , Roche E S , Zounes M C , Kirkpatrick H , Wild M A , Textor G , Tsai P K , Richardson C
Published: January 2020

The properties and sequence of an oligomeric antigen of Treponema pallidum are presented. Antigen C1-5 assembles into oligomers of 140,000 and greater. The nucleotide sequence predicts an open reading frame for a protein monomer of 19,400, confirmed by amino-terminal sequencing of the recombinant antigen.
Role of M cells in initial antigen uptake and in ulcer formation in the rabbit intestinal loop model of shigellosis
Authors: Wassef J S , Keren D F , Mailloux J L
Published: January 2020

Strains of Shigella flexneri with different invasive and pathogenic potentials were inoculated into the intestinal lumen of acutely ligated loops in nonimmune rabbits. After 90 min, tissues processed for ultrastructural as well as light microscopy showed that the bacilli were phagocytosed by M cells over lymphoid follicles of Peyer's patches and carried in vacuoles into the epithelium. Nonpathogenic as well as pathogenic strains were readily taken up regardless of the presence of the 140-megadalton virulence plasmid. More virulent than avirulent shigellae were found in M cells at 90 min, reflecting replication or preferential uptake of the virulent strains. Heat-killed shigellae of the virulent strain were taken up by M cells to the same degree as the avirulent strains. Incubation of the bacteria for 18 h resulted in surface ulceration which was limited to epithelium overlying lymphoid follicles (M cell areas) in acute loops exposed to the virulent shigellae. Villus epithelium adjacent to the ulcerated follicular domes was intact, although there was mucus depletion. In the present study, we found that pathogenic shigellae appear to replicate in the M cells, escape from the phagocytic vesicles, and thereby initiate the ulcerations in this experimental model of dysentery. While initial antigen processing in the gut for a mucosal immune response may require uptake of luminal microorganisms by M cells, this may pose a threat under some circumstances.
Experimental cecitis in gnotobiotic quails monoassociated with Clostridium butyricum strains isolated from patients with neonatal necrotizing enterocolitis and from healthy newborns
Authors: Bousseboua H , Le Coz Y , Dabard J , Szylit O , Raibaud P , Popoff M R , Ravisse P
Published: January 2020

Using axenic quails fed a diet containing lactose, we have investigated the potentially pathogenic roles of six Clostridium butyricum strains of human origin. Three strains (CB155-3, CB1002, and CB203-1) isolated from neonatal necrotizing enterocolitis patients and two of three strains (CB19-1 and CB25-2) isolated from healthy newborns led to cecal or crop lesions or both similar to those observed in human neonatal necrotizing enterocolitis: thickening of the cecal wall with gas cysts, hemorrhagic ulcerations, and necrotic areas. The lactose-negative strain (CB46-1) did not develop any lesions. The neuraminidase-producing strain (CB155-3) caused lesions in all monoassociated quails, whereas the other strains caused lesions in 28 to 85% of animals. Removal of dietary lactose suppressed all pathological incidence. These results show that lactose fermentation is a prerequisite in these pathological changes and stress the roles played by both the strain and the host in the expression of C. butyricum enteropathogenicity.
Biological activities of monoclonal antibodies to Mycoplasma pneumoniae membrane glycolipids
Authors: Chandler D K , Olson L D , Kenimer J G , Probst P G , Rottem S , Grabowski M W , Barile M F
Published: January 2020

A purified preparation of membranes was obtained by using a unique method of treating Mycoplasma pneumoniae with the ATPase inhibitor, diethylstilbestrol. This method was shown to yield highly purified membranes with little or no cytoplasmic contamination. These membranes were used to immunize mice for subsequent productions of monoclonal antibodies (MAbs). Hybridoma culture supernatants were screened by enzyme-linked immunosorbent assay with whole-cell M. pneumoniae and lipid extract antigens. Four stable MAbs were obtained and characterized. MAb CP3-46F5 reacted with a protein of a molecular weight of approximately 52,000 as determined by Western blot (immunoblot). MAbs CP3-50C2, CP3-53C5, and CP3-53C8 did not react with any antigens on Western blots but did bind to at least 10 distinct glycolipid bands as determined by orcinol staining on thin-layer chromatograms of M. pneumoniae lipid extracts. The MAbs did not react with similarly prepared lipid extracts from Mycoplasma genitalium, Mycoplasma neurolyticum, and Mycoplasma gallisepticum. These MAbs did not inhibit M. pneumoniae metabolism or attachment to WiDr cell cultures. The anti-glycolipid MAbs recognize determinants specific to M. pneumoniae, unlike polyclonal hyperimmune sera against M. pneumoniae, which cross-react with lipid extracts of M. genitalium.
Diphosphoryl lipid A from Rhodopseudomonas sphaeroides ATCC 17023 blocks induction of cachectin in macrophages by lipopolysaccharide
Authors: Takayama K , Qureshi N , Beutler B , Kirkland T N
Published: January 2020

Purified diphosphoryl lipid A (DPLA) obtained from the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 was shown to block the induction of cachectin (tumor necrosis factor) in the RAW 264.7 macrophage cell line by toxic deep-rough-chemotype lipopolysaccharide (ReLPS) of Escherichia coli in a concentration-dependent manner. The ReLPS-to-DPLA mass ratios of 1:10 and 1:100 (when 1.0 ng of ReLPS per ml was used) gave 55 and 95% inhibitions, respectively, of the induction of cachectin. Since the structure of the DPLA from R. sphaeroides is so similar to that of the lipid A moiety of the toxic ReLPS from E. coli, we suggest that this inhibition could have been due to competitive binding by DPLA to the active sites on the macrophage. This DPLA could become a useful reagent to study the nature of lipopolysaccharide/lipid A binding in macrophages and perhaps other responding cells.
Effect of immunoglobulin M from normal human serum on Leishmania donovani promastigote agglutination, complement-mediated killing, and phagocytosis by human monocytes
Authors: Navin T R , Krug E C , Pearson R D
Published: January 2020

Serum from healthy, nonimmune humans contained immunoglobulin M (IgM) antibodies that agglutinated Leishmania donovani promastigotes, activated complement, and enhanced promastigote ingestion by human monocytes. The findings indicate that IgM antibodies have the capacity to affect the initial interaction of L. donovani promastigotes with human host defenses.
Vaccination with Mycobacterium bovis BCG affects the distribution of Fc receptor-bearing T lymphocytes in experimental pulmonary tuberculosis
Authors: Bartow R A , McMurray D N
Published: January 2020

Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated and challenged 6 weeks later by the respiratory route with virulent Mycobacterium tuberculosis. By using a double rosette assay with isotype-specific antibody-coated ox and uncoated rabbit erythrocytes, the proportions of T lymphocytes bearing Fc receptors for immunoglobulin G (IgG) (T gamma cells) or IgM (T mu cells) were quantified in tissues taken from animals that were killed within 4 weeks postchallenge. Tuberculin reactivity in vivo and in vitro and antimycobacterial resistance were also measured. BCG vaccination protected the guinea pigs and resulted in significantly enhanced proportions of T mu cells in the blood during the first 3 weeks and in the spleen during weeks 2 and 3 postchallenge. Levels of T gamma cells declined in all tissues during the first 3 weeks of infection and were unaffected by prior vaccination with BCG. Increased proportions of T mu cells in the blood were accompanied by dramatic tuberculin skin reactions and purified protein derivative-induced lymphoproliferation in BCG-vaccinated guinea pigs during the first 2 weeks following virulent pulmonary challenge. Peak levels of T mu cells in the spleens of vaccinated animals at 2 weeks coincided with the first appearance of virulent mycobacteria in that organ. BCG vaccination appears to influence immunoregulatory events in pulmonary tuberculosis through effects on the distribution of IgM Fc receptor-bearing (T mu cell) T lymphocytes.
Comparative analyses of proteins extracted by hot saline or released spontaneously into outer membrane blebs from field strains of Brucella ovis and Brucella melitensis
Authors: Gamazo C , Winter A J , Moriyón I , Riezu-Boj J I , Blasco J M , Díaz R
Published: January 2020

Sheep infected with Brucella ovis produce antibody responses to the rough lipopolysaccharide and to proteins present in hot saline (HS) extracts of B. ovis (J. I. Riezu-Boj, I. Moriyón, J. M. Blasco, C. M. Marín, and R. Díaz, J. Clin. Microbiol. 23:938-942, 1986). The distribution and antigenic relatedness of proteins in HS extracts and in outer membrane blebs were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting for 41 strains of B. ovis and 26 strains of Brucella melitensis of diverse geographic origin. Five major groups of proteins were identified in HS extracts of B. ovis that had been freed of rough lipopolysaccharide: proteins of 43 kilodaltons (kDa), group A (25.5 to 32.0 kDa), group B (21.5 to 22.5 kDa); group C (18.0 to 19.5 kDa), and group D (13.0 to 15.5 kDa). Group A, B, C, and D proteins were also present in blebs. The profiles of proteins in HS extracts or blebs from strains of both Brucella species were very similar. Cross-reactions were demonstrated among HS extracts and blebs of all strains tested in immunoblots performed with an antiserum against the HS extract of a reference strain of B. ovis. Evidence was also provided of an antigenic relationship between group 3 proteins of the outer membrane and some of the proteins in groups A, B, and C. The conservation of these antigens and their immunogenicity in infected animals provide promise that they may serve as components of an effective subcellular vaccine for ovine brucellosis.
Antipneumococcal effects of C-reactive protein and monoclonal antibodies to pneumococcal cell wall and capsular antigens
Authors: Briles D E , Forman C , Horowitz J C , Volanakis J E , Benjamin W H , McDaniel L S , Eldridge J , Brooks J
Published: January 2020

Antibodies to pneumococcal capsular polysaccharides are well known for their ability to protect against pneumococcal infection. Recent studies indicate that antibodies to cell wall antigens, including pneumococcal surface protein A and the phosphocholine (PC) determinant of teichoic acids as well as human C-reactive protein (which also binds to PC), can protect mice against pneumococcal infection. In the present study we compared the protective effects of these agents as measured by mouse protection, the blood bactericidal assay, and clearance of pneumococci from the blood and peritoneal cavity. Our findings extend previous results indicating that human C-reactive protein and antibodies to noncapsular antigens are generally less protective than anticapsular antibodies. The new results obtained indicate the following: (i) mouse protection studies with intraperitoneal and intravenous infections provide very similar results; (ii) monoclonal immunoglobulin G2a (IgG2a) antibodies to PC, like IgG1, IgG2b, and IgG3 antibodies to PC, are highly protective against pneumococcal infection in mice; (iii) human antibody to PC is able to protect against pneumococcal infection in mice; (iv) antibodies to PspA are effective at mediating blood and peritoneal clearance of pneumococci; (v) complement is required for the in vivo protective effects of both IgG and IgM antibodies to PC; (vi) IgG1, IgG2b, and IgG3 anti-PC antibodies all mediate complement-dependent lysis of PC-conjugated erythrocytes; and (vii) antibodies and human C-reactive proteins that are reactive with capsular antigens but not cell wall antigens are able to mediate significant antibacterial activity in the blood bactericidal assay.
lcrH, a gene necessary for virulence of Yersinia pestis and for the normal response of Y. pestis to ATP and calcium
Authors: Price S B , Straley S C
Published: January 2020

We are investigating the functions of the three proteins encoded by the V operon (lcrGVH) of the low-calcium response virulence plasmid pCD1 of Yersinia pestis KIM5. The purpose of this study was to define the role of the 18-kilodalton protein encoded by lcrH, the third gene of the V operon. Using marker exchange mutagenesis, we constructed a Y. pestis mutant that failed to express the LcrH protein. This LcrH- mutant was "ATP blind" in that it failed to show altered growth and V-antigen expression at 37 degrees C when 18 mM ATP was present. It also showed only a partial response to 2.5 mM Ca2+. The parental Y. pestis strain showed full growth yield at 37 degrees C and depressed expression of V antigen and of yop (yersinial pCD1-encoded outer membrane protein) genes in response to ATP or Ca2+. In contrast, the LcrH- mutant failed to grow at 37 degrees C in the presence of ATP and showed only limited growth when Ca2+ was present. V-antigen expression in the mutant was not depressed by ATP and only partially depressed by Ca2+. These findings show that LcrH is necessary for the normal response of Y. pestis to ATP and that LcrH contributes to Ca2+ responsiveness. The mutant also showed abnormal yopJ expression, indicating that LcrH also is necessary for normal yop regulation. The LcrH- mutant was avirulent in mice, probably because of its compromised growth at 37 degrees C. These findings indicate that the responses of Y. pestis to ATP and Ca2+ are distinct and that lcrH encodes a protein that is an important mediator of Ca2+ and ATP regulation of pCD1-encoded virulence determinant(s) in Y. pestis.
Tumor necrosis factor alpha and the anemia associated with murine malaria
Authors: Miller K L , Silverman P H , Kullgren B , Mahlmann L J
Published: January 2020

The anemia associated with malaria is complex, and multiple factors contribute to its severity. An increased destruction and a decreased production of erythrocytes are involved; however, the mechanisms responsible remain unclear. Tumor necrosis factor alpha (TNF-alpha), released by macrophages in response to infection, is thought to play a role through its ability to inhibit erythropoiesis. In these studies we have examined erythropoiesis in mice infected with Plasmodium berghei and in mice infused with recombinant TNF-alpha via implanted osmotic pumps. In both groups of mice there was (i) a reduction of pluripotent stem cells in the bone marrow and a concomitant increase in the spleen, (ii) a reduction of erythroid progenitor cells, and (iii) a reduced incorporation of 59Fe into erythrocytes. When P. berghei-infected mice were given antiserum against recombinant murine TNF, erythropoiesis was partially restored. There was a significant increase in bone marrow stem cells, erythroid progenitor cells, and 59Fe incorporation into erythrocytes in P. berghei-infected mice that had been treated with anti-TNF. How TNF may act, directly or indirectly, to inhibit erythropoiesis is not yet clear. These results demonstrate that TNF mediates, in part, the anemia associated with malaria.
Antilipopolysaccharide factor from horseshoe crab, Tachypleus tridentatus, inhibits lipopolysaccharide activation of cultured human endothelial cells
Authors: Desch C E , O'Hara P , Harlan J M
Published: January 2020

Antilipopolysaccharide (anti-LPS) factor is a basic protein that is purified from the hemocyte lysate of the Japanese and American horseshoe crabs (Tachypleus tridentatus and Limulus polyphemus). Anti-LPS factor has previously been reported to inhibit LPS-mediated activation of limulus factor C, lyse endotoxin-sensitized erythrocytes, and inhibit the growth of some gram-negative bacteria. In this study, we examine the ability of anti-LPS factor purified from T. tridentatus to inhibit the activation of cultured human endothelial cells by LPS. Anti-LPS factor inhibited the stimulation of endothelial adhesiveness for neutrophils by LPS in a dose-dependent manner. Maximum inhibition was achieved when anti-LPS factor was mixed with LPS prior to addition to the endothelial cell monolayers. Anti-LPS factor inhibited endothelial cell activation by LPS derived from Salmonella minnesota Re and Rc mutants as well as from the wild type (smooth), suggesting that it recognizes the lipid A moiety of LPS.
Bacteroides gingivalis vesicles bind to and aggregate Actinomyces viscosus
Authors: Ellen R P , Grove D A
Published: January 2020

Isolated Bacteroides gingivalis 2561 vesicles aggregated suspensions of Actinomyces viscosus and Actinomyces naeslundii of all taxonomy clusters. Vesicles bound near A. viscosus cell walls and among its surface fibrils. Tritiated vesicles bound slightly better to saliva-coated hydroxyapatite (SHA) than to SHA coated with A. viscosus; saturation was approached at the concentrations that were tested. Pretreatment of A. viscosus-coated SHA with vesicles impaired the subsequent adherence of B. gingivalis whole cells.
Metacyclic neutralizing effect of monoclonal antibody 10D8 directed to the 35- and 50-kilodalton surface glycoconjugates of Trypanosoma cruzi
Authors: Yoshida N , Mortara R A , Araguth M F , Gonzalez J C , Russo M
Published: January 2020

It was shown in this work that the infectivity of metacyclic forms of Trypanosoma cruzi was affected upon interaction with the monoclonal antibody (10D8), which reacts with a carbohydrate epitope of the 35- and 50-kilodalton (kDa) surface glycoconjugates. The invasion of Vero cells by metacyclic forms of strains Tulahuen and G was inhibited 50 to 67% in the presence of 10D8 (10 micrograms/ml), whereas a nonrelated monoclonal antibody to Plasmodium berghei had no such effect. In mice that were inoculated with metacyclic forms preincubated with 10D8 or that had passively received 10D8 before challenge with metacyclic forms, a considerable decrease in the parasitemia levels was observed. The 35- and 50-kDa antigens were detectable by the galactose oxidase and sodium boro[3H]hydride procedure but not by surface iodination or metabolic labeling with [35S]methionine, suggesting that they may be of glycolipid nature. The finding that the 35- and 50-kDa antigens are major bands recognized by sera of mice immunized with killed metacyclic forms and protected against acute infection, in addition to the results with 10D8, indicate that these glycoconjugates may play an important role in the metacyclic form-host cell association that initiates T. cruzi infection.
Fixation of Clostridium difficile toxin A and cholera toxin to intestinal brush border membranes from axenic and conventional mice
Authors: Lucas F , Elmer G W , Brot-Laroche E , Corthier G
Published: January 2020

We have tested the in vitro binding of Clostridium difficile toxin A (enterotoxin) and cholera toxin to intestinal brush border membranes prepared from either conventional or axenic mice. Membranes from axenic mice were shown to be saturated at a lower toxin A concentration (at least 2.5 times lower). Because there were no significant differences between membranes from axenic and conventional mice in binding at low toxin A concentrations, the presence of the normal microflora seems to increase the number but not the affinity of brush border membrane receptors on the enterocyte surface. Corroborating the in vitro results, we observed that conventional mice were more sensitive to the pathological effects of toxin A given intragastrically than were axenic mice. In contrast, there was no difference in the binding characteristics of cholera toxin between membranes from conventional and axenic mice. We conclude that the presence of the mouse intestinal bacteria increases the number of C. difficile toxin A intestinal receptors but does not influence cholera toxin receptors.
Subunit structure of the variable V-1 antigen of Mycoplasma pulmonis
Authors: Watson H L , Dybvig K , Blalock D K , Cassell G H
Published: January 2020

It was previously shown that multiple structural variants of the V-1 antigen (variable antigen 1) of Mycoplasma pulmonis could be found within a single strain. This antigen is unusual in that it produces a ladder pattern after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The present study showed that some variants of V-1 could be extracted into the aqueous phase of a phenol-H2O system. Analysis with anti-V-1 monoclonal antibodies showed that the phenol-H2O-extracted V-1 had a regular spacing of 3.1 kilodaltons (kDa) between bands and trypsinization of this extracted V-1 resulted in the gradual symmetrical collapse (2.9-kDa increments) of the ladder into a single band, suggesting the presence of multiple identical subunits within the V-1 structure. The upper band from the phenol-H2O-extracted V-1 was isolated and analyzed by SDS-polyacrylamide gel electrophoresis immunoblotting, resulting in the regeneration of the original ladder pattern with 3.1-kDa spacing between bands. When V-1 was boiled for increasing times in the presence of SDS, the staining intensity of the upper band decreased with the concurrent appearance of additional lower-molecular-weight bands. Finally, by using whole cells, it was found that the lower-molecular-weight species of the ladder pattern selectively partitioned into the hydrophobic phase of a Triton X-114 phase partitioning system, and the higher-molecular-weight bands were found in the aqueous phase. These data indicate that the V-1 bands are composed of subunits which may aggregate via hydrophobic interactions and that these aggregates at least partially dissociate when exposed to harsh denaturing conditions, resulting in the characteristic ladder pattern of V-1.
Immunopharmacological activities of 2-keto-3-deoxyoctonic acid-(alpha 2----6)-linked 4-O-phosphono-D-glucosamine derivatives carrying N- and 3-O-acyl substituents
Authors: Kumazawa Y , Matsuura M , Homma J Y , Furuya T , Takimoto H , Inagaki K , Nagumo T , Kiso M , Hasegawa A
Published: January 2020

The immunopharmacological activities of 2-keto-3-deoxyoctonic acid (KDO)-(alpha 2----6)-linked lipid A-subunit analogs, 4-O-phosphono-D-glucosamine derivatives carrying N- and 3-O-acyl substituents, were compared with those of the corresponding analogs without KDO, GLA-27, GLA-47, and GLA-60. Among the analogs tested, GLA-60, a 4-O-phosphono-D-glucosamine carrying N-3-hydroxytetradecanoyl and 3-O-3-tetradecanoyloxytetradecanoyl groups, exhibited the most intensive activities in terms of mitogenicity, adjuvanticity, and mediator (tumor necrosis factor and colony-stimulating factor) induction. Binding (alpha 2----6) of KDO to GLA-60 failed to enhance the activities. Similarly, the activities of GLA-27 and GLA-47 were also decreased by introduction of KDO to the O-6 of the analogs. This indicates that the strengths of the activities of the subunit analogs depend on the kinds of N- and 3-O-linked acyl substituents and not on the presence of the KDO linked to the O-6.
Clearance of Cryptococcus neoformans from immunologically suppressed mice
Authors: Murphy J W
Published: January 2020

To assess the effects of cryptococcal antigen-induced immunosuppression on a Cryptococcus neoformans infection, CBA/J mice were injected intravenously with saline or suppressive doses of cryptococcal antigen (CneF) at weekly intervals and were then infected with viable C. neoformans cells. By the second week after infection, the cryptococcal antigen-injected mice had suppressed anticryptococcal delayed-type hypersensitivity (DTH) responses compared with the responses of the saline-treated, infected control mice. In addition, the immunosuppressed mice had higher numbers of cryptococcal CFU cultured from their lungs, livers, spleens, lymph nodes, and brains than did the control animals. A direct correlation of suppression of the anticryptococcal DTH response and reduced clearance of cryptococci from tissues was also observed after mice were given a single intravenous injection of CneF and infected. To determine whether or not the cryptococcal antigen was specifically reducing the clearance of C. neoformans or had a more generalized effect, mice were injected with saline or suppressive doses of CneF, infected with Listeria monocytogenes, and then followed daily for 7 days for the clearance of L. monocytogenes from spleens and on day 7 for DTH reactivity to Listeria antigen. There were no differences between the saline- and CneF-treated mice with respect to anti-Listeria DTH responses or clearance of L. monocytogenes from spleens, indicating that CneF was not altering natural resistance mechanisms responsible for early clearance of L. monocytogenes, nor was the CneF influencing the induction of the acquired immune response which was responsible for the late clearance of the bacteria. Together, these data indicate that the specific suppression of this cell-mediated immune response induced by cryptococcal antigen reduces the ability of the animals to eliminate the homologous organism (C. neoformans) but not a heterologous infectious agent, such as L. monocytogenes.
Characterization and vaccine potential of a novel recombinant coccidial antigen
Authors: Miller G A , Bhogal B S , McCandliss R , Strausberg R L , Jessee E J , Anderson A C , Fuchs C K , Nagle J , Likel M H , Strasser J M
Published: January 2020

A cDNA clone derived from sporulated oocysts of Eimeria tenella and encoding the expression product GX3262 was identified using a monoclonal antibody raised against Eimeria acervulina sporozoites. The cDNA fragment containing the coccidial antigen gene was cloned in bacteriophage lambda gt11, transferred to a plasmid, and introduced into Escherichia coli for analysis of the gene products. The strain carrying the plasmid produced GX3262 as part of a fusion protein consisting of the first 1,006 amino acids of E. coli beta-galactosidase and 112 amino acids of the E. tenella protein of approximately 12 kilodaltons. Partially purified antigen, heat-killed recombinant bacterin, and live E. coli containing the recombinant coccidial antigen were used to immunize 1-week-old or newly hatched broiler chicks. Several immunization protocols were utilized, including boosts with partially purified beta-galactosidase-GX3262, bacterin, or small numbers of live E. tenella oocysts. After challenge with an experimental E. tenella infection, the birds were evaluated by scoring cecal lesions to determine the level of protection. The greatest degree of protection was seen after only a single immunization of 2-day-old birds with a live recombinant E. coli preparation. The results presented here identify GX3262 as a potential candidate coccidial vaccine antigen and provide evidence for the first time that newly hatched chickens can be successfully vaccinated with a recombinant antigen.
Cloning and expression of a cohemolysin, the CAMP factor of Actinobacillus pleuropneumoniae
Authors: Frey J , Perrin J , Nicolet J
Published: January 2020

The genetic determinant of the cohemolysin which is responsible for the CAMP phenomenon, a cohemolysis, of Actinobacillus pleuropneumoniae was cloned in Escherichia coli. Total DNA from the A. pleuropneumoniae serotype 1 type strain 4074 was used to construct a gene library in plasmid pUC18 in E. coli JM83. A total of 10,500 clones containing recombinant plasmids have been screened for hemolysis on blood plates. Fifty-five clones which showed a weak hemolytic response after 24 to 48 h of incubation were screened for the CAMP reaction with Staphylococcus aureus. This led to the identification of one clone which showed a positive CAMP reaction. Immunoblot analysis revealed that the recombinant strain expressed a protein with a molecular mass of 27,000 daltons, similar in size to the CAMP protein of the group B streptococci. Rabbit antibodies against the CAMP+ clone neutralized the CAMP reaction mediated by the E. coli strain containing the cloned CAMP gene as well as that of A. pleuropneumoniae. Antibodies raised against the cloned CAMP cohemolysin cross-reacted with Streptococcus agalactiae protein B. We designate the 27,000-dalton molecule CAMP factor protein and name its corresponding gene cfp.
Modulation of endotoxin lethality in mice by hydrazine sulfate
Authors: Silverstein R , Christoffersen C A , Morrison D C
Published: January 2020

Although the precise mechanism of endotoxin lethality has yet to be defined, it is well recognized that the amount of hepatic phosphoenolpyruvate carboxykinase is reproducibly and significantly reduced after challenge with endotoxin. Hydrazine has been shown to be a specific inhibitor of gluconeogenesis, causing a metabolic crossover at the step catalyzed by phosphoenolpyruvate carboxykinase. More recently, it has also been shown that hydrazine sulfate may be of potential therapeutic value against cancer cachexia. The experiments described in this paper demonstrate that treatment of CF1 mice with hydrazine sulfate 5 h prior to challenge with endotoxin from Salmonella enteritidis significantly improved survival. Furthermore, such treatment counteracted the drop in hepatic phosphoenolpyruvate carboxykinase activity in isolated cytosol otherwise evident at 6 h and 12 h after endotoxin challenge. Despite this, there was no corresponding improvement in the plasma glucose, measured at 6, 12, and 24 h following endotoxin challenge. It is suggested that the endogenous response to the metabolic crossover initiated by hydrazine may contribute to the protection. The response to hydrazine sulfate has yet to be fully elaborated but does include the increase in phosphoenolpyruvate carboxykinase activity. In contrast with the protection seen upon hydrazine sulfate pretreatment, injecting a corresponding dose of hydrazine sulfate after the endotoxin resulted in more fatalities.
Fibrinogen-binding protein/clumping factor from Staphylococcus aureus
Authors: Bodén M K , Flock J I
Published: January 2020

The binding of staphylococcal components to fibrinogen was studied. Fibrinogen-binding material from lysed staphylococcal cells or culture supernatants was affinity purified on fibrinogen-Sepharose and analyzed on Western (immuno-) blots by the use of fibrinogen and antifibrinogen antibodies. Two main bands of 87 and 19 kilodaltons (kDa) and a weaker band of 35 kDa bound specifically to fibrinogen. A monoclonal antibody bound to all three bands, indicating that these were of the same origin. The yield of these components was much higher in the culture supernatant than on washed cells, suggesting that these molecules are essentially extracellular products. In a plasma coagulase test, the 87-kDa band, but not the 19-kDa band, clotted rabbit plasma, demonstrating that the 87-kDa molecule is coagulase. This was further confirmed by the fact that the 87-kDa band binds specifically to prothrombin. It was shown that the 87- and the 19-kDa molecules were present on the cell surface by surface labeling the cells with 125I. In addition, the fact that killed and washed cells could induce plasma clotting demonstrates that staphylococci have coagulase exposed on the surface. It was concluded that cell-bound coagulase has affinity for fibrinogen also in the absence of prothrombin and thus is responsible for the clumping of staphylococci in fibrinogen.
Vimentin-cross-reactive epitope of type 12 streptococcal M protein
Authors: Kraus W , Seyer J M , Beachey E H
Published: January 2020

The NH2-terminal amino acid sequence of type 12 M protein was determined by automated Edman degradation of a 38-kilodalton polypeptide fragment purified from a limited pepsin digest of intact type 12 streptococci. The sequence of the first 13 amino acid residues of the polypeptide confirmed that predicted by the nucleotide sequence of the mature type 12 M protein. A chemically synthesized peptide copying the NH2-terminal 25 residues, SM12(1-25)C, evoked opsonic antibodies against type 12 streptococci as well as renal glomerular cross-reactive antibodies. The serum from one of six rabbits reacted in immunofluorescence tests with human glomeruli in a mesangial staining pattern. The cross-reactive antibodies were completely inhibited by the immunizing peptide and absorption with type 12 streptococci. Subpeptides of the 25-residue synthetic peptide were without inhibitory effect, suggesting that the cross-reactive antibodies are directed against a conformational epitope of SM12(1-25)C. Anti-SM12(1-25)C antisera reacted specifically with the intermediate filament protein vimentin extracted from mesangial cells. None of the cross-reactions of anti-SM12(1-25)C were inhibited by a synthetic peptide SM1(1-26)C of type 1 M protein, which was previously shown to share a cross-reactive epitope with vimentin. These results indicate that type 12 M protein contains at least one vimentin cross-reactive epitope that is clearly distinct from the tetrapeptide epitope shared with vimentin by type 1 M protein.
Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells
Authors: Popoff M R , Milward F W , Bancillon B , Boquet P
Published: January 2020

The two components Sa (Mr, 44,000) and Sb (Mr, 92,000) of Clostridium spiroforme toxin were identified and characterized. Serological data permitted the identification of two groups of actin ADP-ribosylating clostridial toxins. The first consists of only C. botulinum C2. The second group includes spiroforme toxin, iota toxin of C. perfringens E, and an enzyme called CDT found in one strain of C. difficile, antibodies against which cross-react with all of the members of both groups. C. spiroforme toxin acted on cells by disrupting microfilaments by ADP-ribosylation of G actin. Toxicity was not blocked by 10 or 20 mM ammonium chloride and was only moderately inhibited by 30 mM NH4Cl. Inhibition of coated-pit formation in HEp-2 cells by potassium depletion strongly protected against the effect of C. spiroforme toxin. Toxicity was not blocked by incubation of HEp-2 cells and spiroforme toxin at 15 degrees C. These results suggest that this new binary toxin enters cells via the coated-pit-coated-vesicle pathway and might reach the cytoplasm at the same time as or before transfer to early endosomes.
Evidence of coordinate regulation of virulence in Salmonella typhimurium involving the rsk element of the 95-kilobase plasmid
Authors: Vandenbosch J L , Kurlandsky D R , Urdangaray R , Jones G W
Published: January 2020

Integration of the Salmonella typhimurium virulence plasmid into the chromosome reduces mouse virulence, serum resistance, and HeLa cell adhesion-invasion while prolonging lag time in minimal medium. The proposed virulence plasmid regulatory element, rsk, partially restores virulence and fully restores the other three phenotypes to wild-type levels. Plasmid curing reduces virulence without affecting the other phenotypes. rsk has no apparent effect on the cured strain.
Invasion of HeLa 229 cells by virulent Bordetella pertussis
Authors: Ewanowich C A , Melton A R , Weiss A A , Sherburne R K , Peppler M S
Published: January 2020

Phase-dependent invasive behavior of Bordetella pertussis was demonstrated by recovery of viable organisms from gentamicin-treated HeLa cell monolayers and by transmission electron microscopy. Several mutants of B. pertussis with Tn5 or Tn5 lac inserted into various vir-regulated genes were evaluated for differences in their invasive abilities. Mutants lacking filamentous hemagglutinin, pertussis toxin, and two as yet uncharacterized vir-regulated products had levels of invasion significantly lower than that of the parent strain BP338. In contrast, invasion by mutants lacking adenylate cyclase toxin was significantly increased compared with that of wild-type B. pertussis. This increase in invasion was eliminated when concentrations of intracellular cyclic 3'-5' AMP were stimulated by treating HeLa cells with cholera toxin or forskolin. Entry of B. pertussis occurred through a microfilament-dependent phagocytic process, as evidenced by the marked reduction in uptake following treatment of HeLa cells with cytochalasin D. Invasion was inhibited with polyclonal anti-B. pertussis and anti-filamentous hemagglutinin antisera. In addition, a monoclonal antibody against lipooligosaccharide A reduced uptake by 65.5%. The preservation of HeLa cell integrity and the limited replication of intracellular bacteria suggest that invasion may represent a means by which B. pertussis evades an active host immune response.
Major integral membrane protein immunogens of Treponema pallidum are proteolipids
Authors: Chamberlain N R , Brandt M E , Erwin A L , Radolf J D , Norgard M V
Published: January 2020

A number of the major pathogen-specific immunogens of Treponema pallidum were characterized recently as amphiphilic, integral membrane proteins by phase partitioning with Triton X-114 (J. D. Radolf, N. R. Chamberlain, A. Clausell, and M. V. Norgard. Infect. Immun. 56:490-498, 1988). In the present study, we demonstrated that the same membrane immunogens (designated as detergent phase proteins [DPPs]) become radiolabeled upon in vitro incubation of T. pallidum with various 3H-labeled fatty acids. Radioimmunoprecipitation with a monoclonal antibody confirmed that the 3H-labeled 47-kilodalton protein corresponded to the well-characterized treponemal antigen with the identical apparent molecular mass. Failure to detect 3H-labeled DPPs following incubation with erythromycin confirmed that protein acylation required de novo protein synthesis by the bacteria. When treponemes were incubated with [3H]myristate, [3H]palmitate, or [3H]oleate, radiolabeled proteins corresponding to the DPPs were detected upon autoradiography. Demonstration that a number of the abundant membrane immunogens of T. pallidum are proteolipids provides information to help clarify their membrane association(s) and may serve to explain their extraordinary immunogenicity.