bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical and immunological characterization of cell surface proteins of Pasteurella multocida strains causing atrophic rhinitis in swine
Authors: Lugtenberg B , van Boxtel R , Evenberg D , de Jong M , Storm P , Frik J
Published: January 2020

In a previous paper (B. Lugtenberg, R. van Boxtel, and M. de Jong, Infect. Immun., 46:48-54, 1984) we showed that among 34 isolates from swine the membrane protein and lipopolysaccharide (LPS) patterns, as analyzed by sodium dodecyl sulfate-gel electrophoresis, could be classified into three and six patterns, respectively. In all cases a certain LPS pattern was correlated with a certain protein pattern. Certain combinations of types of cell surface proteins and LPSs were correlated with pathogenicity, the latter property being judged by the guinea pig skin test. In the present paper the immunological and biochemical properties of cell surface constituents were analyzed. The reaction between electrophoretically separated cell surface constituents with guinea pig and sow antisera showed that LPS as well as several proteins were immunogenic. Among these is protein H, whose electrophoretic mobility is the main criterium for typing of cell envelope protein patterns. Protein H was the most heavily labeled component when whole cells were iodinated by the Iodo-Gen procedure showing its accessibility at the cell surface. These properties of protein H make it an attractive vaccine candidate. Further biochemical analyses revealed that protein H shares many properties with pore proteins of members of the family Enterobacteriaceae. One of these properties, association between pore proteins and peptidoglycan, was used as the basis for a simple procedure developed to partially purify protein H.
Serotype-related HEp-2 cell interaction of Yersinia enterocolitica
Authors: Lassen J , Kapperud G
Published: January 2020

The ability of human clinical isolates of Yersinia enterocolitica to interact with HEp-2 cells was found to vary considerably between the serotypes O:3, O:8, and O:9. Although all three serotypes adhered initially to the cell surface, regardless of incubation temperature of the bacterial inoculum or presence of the 40- to 48-megadalton virulence plasmid, the ability to localize intracellularly was not uniformly expressed. By using a combined light optical method based on differential interference contrast and UV incident light microscopy, we found that in serotype O:3, resistance to internalization was dependent upon prior growth at 37 degrees C and carriage of the virulence plasmid; in serotype O:9, this property was plasmid dependent but not temperature dependent; in serotype O:8, it was constitutive. The ability of serotype O:3 to resist internalization was correlated with the expression of plasmid-associated fibrillae on the bacterial surface. No relationship between fibrillation and HEp-2 cell interaction was apparent for serotype O:8 or O:9. Serotypes O:8 and O:9, unlike the O:3 strains studied, associated with HEp-2 cells in greater number after cultivation at 22 degrees C than after cultivation at 37 degrees C. We failed to establish a correlation between the expression of surface fibrillae and the ability to evoke guinea pig conjunctivitis.
Development of antibodies to protective antigen and lethal factor components of anthrax toxin in humans and guinea pigs and their relevance to protective immunity
Authors: Turnbull P C , Broster M G , Carman J A , Manchee R J , Melling J
Published: January 2020

A competitive inhibition enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies in serum to the protective antigen (PA) and lethal factor (LF) components of anthrax toxin. Current human vaccination schedules with an acellular vaccine induce predictable and lasting antibody titers to PA and, when present in the vaccine, to LF. Live spore vaccine administered to guinea pigs in a single dose conferred significantly better protection than the human vaccines (P less than 0.001), although they elicited significantly lower (P less than 0.0005) anti-PA and anti-LF titers at time of challenge with virulent Bacillus anthracis. Substantial anti-PA and anti-LF titers may not, therefore, indicate solid protective immunity against anthrax infection. The ELISA system was also shown to be capable of detecting anti-PA and anti-LF antibodies in the sera of individuals with histories of clinical anthrax. The advantage of ELISA over the Ouchterlony gel diffusion test and indirect microhemagglutination assay are demonstrated. There was a highly significant degree of correlation between ELISA and the indirect microhemagglutination assay (P less than 0.0005); but ELISA was markedly superior in terms of reproducibility, reliability, specificity, and simplicity in performance and stability of the bound antigen.
Isolation and characterization of protective T cells induced by Listeria monocytogenes
Authors: Chen-Woan M , McGregor D D , Noonan S K
Published: January 2020

Rats convalescing from a recent infection with Listeria monocytogenes generate T cells which can protect recipient rats against a challenge infection with that organism. Using monoclonal antibodies that react with some but not all rat peripheral T cells, the T-cell mediators of acquired resistance to infection (TCRI) were isolated by panning and characterized by using a fluorescence-activated cell sorter. Many L. monocytogenes-immune TCRI were relatively large cells as judged by their light-scattering properties. That finding accords with previously reported cytokinetic data and velocity sedimentation analyses which revealed that the majority of L. monocytogenes-immune TCRI are lymphoblasts. In the current investigation, the surface antigenic profile of these mediator T cells was revealed as W3/25+ OX8+ OX4+ RT6.1-. That phenotype is identical to that of L. monocytogenes-induced prekiller cells which are formed as part of the animal's cell-mediated response to infection. Like prekiller cells and their differentiated counterparts, L. monocytogenes-immune TCRI adhere preferentially to monolayers of syngeneic fibroblasts. The results indicate that L. monocytogenes-immune TCRI belong to a minor subset of peripheral T cells which also contains T cells that have the cytotoxic potential by which L. monocytogenes-induced prekiller cells have been defined.
Ureaplasma infection of cell cultures
Authors: Kotani H , McGarrity G J
Published: January 2020

Studies were performed to characterize the effects of ureaplasmas in HeLa, 3T6, and CV-1 cell cultures. The ureaplasmas studied were human Ureaplasma urealyticum T960 (serotype VIII), bovine U. diversum T95, simian strain T167-2, ovine strain 1202, canine strain D1M-C, and feline strains 382 and FT2-B. FT2-B was the only ureaplasma to grow in the cell free culture medium, Dulbecco modified Eagle-Earle medium containing 10% fetal bovine serum. The growth pattern of the ureaplasmas varied in the different cell cultures, but each strain grew in at least two of the cell cultures, suggesting a requirement for a product of the cell culture and for low concentrations of urea. When growth occurred, organisms grew to concentrations that approached, but did not equal, those observed in 10B broth. Most, but not all, ureaplasmas grew quickly, reaching peak titers 2 days after infection. Canine strain D1M-C did not grow in 3T6, but showed rapid growth in HeLa and CV-1 cells, killing both cultures, In some systems, e.g., U. urealyticum T960 and simian strain T167-2, the infection persisted, and ureaplasmas could be recovered from cell cultures four passages after infection, when studies were terminated. The cell culture ureaplasmas grew on T agar, but not on mycoplasma agar medium.
Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig
Authors: Little S F , Knudson G B
Published: January 2020

Several strains of Bacillus anthracis have been reported previously to cause fatal infection in immunized guinea pigs. In this study, guinea pigs were immunized with either a protective antigen vaccine or a live Sterne strain spore vaccine, then challenged with virulent B. anthracis strains isolated from various host species from the United States and foreign sources. Confirmation of previously reported studies (which used only protective antigen vaccines) was made with the identification of 9 of the 27 challenge isolates as being vaccine resistant. However, guinea pigs immunized with the live Sterne strain spore vaccine were fully protected against these nine isolates. In experiments designed to determine the basis of vaccine resistance, guinea pigs which were immunized with individual toxin components and which demonstrated enzyme-linked immunosorbent assay antibody titers comparable to those induced by Sterne strain vaccine were not protected when challenged with a vaccine-resistant isolate. We concluded that antibodies to toxin components may not be sufficient to provide protection against all strains of B. anthracis and that other antigens may play a role in active immunity. As a practical matter, it follows that the efficacy of anthrax vaccines must be tested by using vaccine-resistant isolates if protection against all possible challenge strains is to be assured.
Quantitative and qualitative analyses of serum antibodies elicited in adults by Haemophilus influenzae type b and pneumococcus type 6A capsular polysaccharide-tetanus toxoid conjugates
Authors: Schneerson R , Robbins J B , Parke J C , Bell C , Schlesselman J J , Sutton A , Wang Z , Schiffman G , Karpas A , Shiloach J
Published: January 2020

Covalent binding to immunogenic proteins increases the immunogenicity of the capsular polysaccharides of Haemophilus influenzae type b (Hib) and pneumococcus type 6A (Pn6A). Conjugates composed of Hib, Pn6A, or the cross-reacting Escherichia coli K100 covalently bound to tetanus toxoid (TT) were injected into young adult volunteers. Local reactions were common and were probably due to Arthus reactivity mediated by the preexisting antibodies reacting with the TT component of the conjugates. Fever occurred in about 10% of the volunteers after the first injection; no volunteers had fever after the second injection. Similar levels of Hib or Pn6A antibodies were elicited by either 50- or 100-micrograms doses or by concurrent injection of two different conjugates (Hib-TT and Pn6A-TT or Hib-TT and K100-TT). The Hib-TT elicited about a 180-fold increase in Hib antibodies, and the Pn6A-TT conjugate elicited about an 8-fold increase in Pn6A antibodies after one injection. Booster reactions were not elicited in adults; similar levels of antibodies in the five experimental groups suggested that the responses elicited by the conjugates were maximal. A one-way cross-reaction was noted as Pn6A conjugates elicited rises of Hib antibodies in 13 of 20 volunteers; only 4 of 59 volunteers immunized with Hib-TT had increases in Pn6A antibodies. The preimmunization Hib antibodies were composed of immunoglobulin M (IgM), IgA, and IgG. The postimmunization sera showed an increase in all three isotypes; the elevation of the IgG was the highest of the three isotypes. Conjugate-induced antibodies to both the polysaccharide and TT exerted biological activities that have been correlated with immunity. Adsorption of the Hib-TT onto aluminium hydroxide resulted in higher levels and an earlier Hib antibody response in infant rhesus. These results encourage the evaluation of Hib and Pn6A conjugates in human children and infants.
A Borrelia-specific monoclonal antibody binds to a flagellar epitope
Authors: Barbour A G , Hayes S F , Heiland R A , Schrumpf M E , Tessier S L
Published: January 2020

In immunofluorescence assays monoclonal antibody H9724 recognized eight species of the spirochetal genus Borrelia but not representatives of the genera Treponema, Leptospira, and Spirochaeta. We examined the reactivity of H9724 against subcellular components of Borrelia hermsii, an agent of relapsing fever, and B. burgdorferi, the cause of Lyme disease. H9724 bound to isolated periplasmic flagella of the two borreliae. In Western blots the antibody reacted with the predominant protein in flagellar preparations from B. hermsii and B. burgdorferi; the apparent molecular weights of these flagellins were 39,000 and 41,000, respectively.
Strains of Streptococcus mutans and Streptococcus sobrinus attach to different pellicle receptors
Authors: Gibbons R J , Cohen L , Hay D I
Published: January 2020

We compared the levels of adsorption of Streptococcus mutans JBP and Streptococcus sobrinus 6715 to experimental pellicles formed from unsupplemented and glucosyltransferase (GTF)-supplemented saliva. Pellicles formed on hydroxyapatite beads from GTF or from saliva-GTF mixtures possessed detectable GTF activity. Low levels of GTF activity were also detected in clarified whole human saliva, but not in samples of submandibular saliva. The adsorptive behavior of S. mutans JBP to pellicles formed from saliva or saliva-GTF mixtures was strikingly different from that of S. sobrinus 6715. S. mutans JBP adsorbed in higher numbers to pellicles formed from whole or submandibular saliva than to buffer-treated hydroxyapatite under the assay conditions used, in which blocking with albumin was used. In contrast, S. sobrinus 6715 attached in lower numbers and did not show enhanced adsorption to pellicles prepared from saliva. Pellicles prepared from the high-molecular-weight mucin fraction of submandibular saliva effectively promoted adsorption of S. mutans JBP, but none of the saliva fractions tested enhanced the attachment of S. sobrinus 6715 above the levels of buffer controls. Exposure of pellicles which contained GTF to sucrose to permit in situ synthesis of glucan markedly enhanced attachment of S. sobrinus 6715 but not attachment of S. mutans JBP. Also, the presence of sucrose throughout the adsorption period did not enhance attachment of S. mutans JBP. Both organisms possessed cell-associated GTF, and GTF preparations derived from S. sobrinus 6715 and Streptococcus sanguis FC-1 behaved like GTF derived from S. mutans JBP. S. sobrinus 6715 attached in high numbers to dextran-treated hydroxyapatite, whereas S. mutans JBP did not. These observations suggest that S. mutans JBP cells possess an adhesin which binds to salivary components in the pellicles. In contrast, S. sobrinus 6715 cells appear to possess an adhesin which binds to glucan in the pellicles. Four additional strains of S. mutans and four additional strains of S. sobrinus behaved qualitatively like strains JBP and 6715, respectively, and thus the differences observed appear to be representative of these species. Collectively, our data indicate that S. mutans and S. sobrinus attach to different receptors in experimental pellicles.
Cellular and humoral immune responses of jirds resistant to Dipetalonema viteae infection
Authors: Abraham D , Weiner D J , Farrell J P
Published: January 2020

Jirds with prepatent Dipetalonema viteae infections develop an acquired immunity to challenge infections. The objective of the present study was to observe parasite-specific and nonspecific cellular and humoral immune responses in immune jirds. Splenic hyperplasia was observed in infected jirds during the first 5 weeks of infection. Antigen-reactive spleen cells were observed in the lymphocyte transformation assay at 3 weeks postinfection. A depressed response to concanavalin A (ConA) was seen at 1 week postinfection through week 5. Mitomycin C-treated cells from infected jirds were capable of suppressing the response of normal cells to ConA. Sephadex G-10-nonadherent spleen cells from infected jirds showed elevated responses to D. viteae antigen at 1, 3, and 5 weeks and elevated responses to ConA at 3 and 5 weeks. Filaria-specific antibodies were seen at 1 week postinfection, and titers rose through week 5. Plaque-forming cell production to sheep erythrocytes was not depressed in infected jirds. It was concluded that jirds react immunologically with both cellular and humoral responses during the prepatent period of D. viteae infection. A concurrent immune depression was seen. Its effect on resistance and tolerance remains to be determined.
A 160-kilodalton epithelial cell surface glycoprotein recognized by plant lectins that inhibit the adherence of Actinomyces naeslundii
Authors: Brennan M J , Cisar J O , Sandberg A L
Published: January 2020

The adherence of Actinomyces naeslundii to human epithelial (KB) cells is mediated by the interaction of a fimbrial lectin on this oral bacterium with epithelial cell receptors exposed by sialidase. The D-galactose- and N-acetyl-D-galactosamine-reactive plant lectins from peanut and from Bauhinia purpurea inhibit this interaction. This report describes the partial purification and characterization of a 160-kilodalton (kDa) cell surface glycoprotein which is the principal receptor for these lectins. Radioiodinated lectins detected a band of 160 kDa on sialidase-treated Western blots of epithelial cell extracts but did not detect bands on nontreated filters. However, wheat germ agglutinin was reactive with the 160-kDa band on filters that were not treated with sialidase, suggesting that this lectin recognizes the sialic acid residues of this molecule. The 160-kDa component was partially purified from n-octylglucoside extracts of the epithelial cells by wheat germ agglutinin affinity chromatography. This molecule was metabolically labeled with D-[14C]glucosamine and labeled at the cell surface by lactoperoxidase-catalyzed iodination or periodate oxidation followed by sodium borotritide reduction. Incubation of epithelial cells with sialidase before extraction resulted in the loss of the 160-kDa band and the appearance of a band at 200 kDa which was directly reactive with 125I-labeled peanut agglutinin. These results indicate that the 160-kDa glycoprotein on the surface of the epithelial cell serves as a receptor for the agglutinins from the peanut and B. purpurea and presumably the fimbrial lectin of actinomyces.
Pulmonary microvascular injury induced by Pseudomonas aeruginosa cytotoxin in isolated rabbit lungs
Authors: Seeger W , Walmrath D , Neuhof H , Lutz F
Published: January 2020

The effects of Pseudomonas aeruginosa cytotoxin on the pulmonary microvasculature were studied in blood-free, perfused, isolated rabbit lungs. Cytotoxin was administered to the recirculating Krebs Henseleit albumin (1%) buffer during two consecutive 30-min-perfusion phases (phases 1 and 2) at a concentration of 13 micrograms/ml, followed by a third perfusion phase (phase 3) without toxin. After perfusion phases 2 and 3, the capillary filtration coefficient (Kf,c) and vascular compliance were determined gravimetrically from two-step microvascular pressure increments under zero-flow conditions. Cytotoxin caused a continuous release of K+ and lactate dehydrogenase, which started within the first 5 min and amounted to about 50% of the total lung cellular K+ and 5 to 7% of the total lactate dehydrogenase by the end of the experiment. The toxin caused the continuous generation of prostaglandin I2, which was detectable in the perfusates of all perfusion phases at maximum values five times above the control values and which was measured in the bronchoalveolar lavage fluid at the end of the experiment. Thromboxane generation in toxin-treated lungs did not significantly exceed that of control lungs or of lungs with mechanically induced edema. Cytotoxin caused a gradual increase in pulmonary vascular resistance, to maximum values 2.5 times above the control, starting within 1 min; the increase was partially reversible after washout of the toxin. After a lag period of 20 to 30 min, the lungs gained weight, amounting to a mean gain of 9.1 g at the end of the experiments. After perfusion phases 2 and 3, an almost fourfold increase in Kf,c, which was not reversible after washout of the toxin, was measured, whereas the values of vascular compliance were not altered. We conclude that pseudomonal cytotoxin may be an important factor in the pathogenesis of prolonged microvascular injury, encountered in states of P. aeruginosa sepsis or acute lung failure with secondarily acquired P. aeruginosa pneumonia.
Primary murine immunoglobulin M responses to certain pneumococcal capsular polysaccharides consist primarily of anti-pneumococcal cell wall carbohydrate antibodies
Authors: Fairchild R L , Sterner K E , Braley-Mullen H
Published: January 2020

The antibody induced in mice immunized with a vaccine preparation of type 6 (Danish type 6A) pneumococcal capsular polysaccharide (S6) reacted with several chemically disparate pneumococcal capsular polysaccharides. Equivalent numbers of plaque-forming cells were observed when sheep erythrocytes coated with either S6, type 19 pneumococcal capsular polysaccharide (S19), or the pneumococcal cell wall carbohydrate (PnC) were used to detect the response to S6 or to S19. The addition of exogenous PnC to the plaquing mixtures of spleen cells from S6-, S19-, or PnC-immunized mice inhibited the appearance of most (greater than or equal to 85%) of the plaque-forming cells. Furthermore, the addition of monoclonal antibody specific for the dominant (TEPC 15) idiotype of anti-phosphorylcholine (a component of PnC) antibodies also inhibited the appearance of most of the plaque-forming cells. A suppressed S19 response was induced by priming mice with a low dose of S19 or PnC 3 days before immunization with an optimal dose of S19 (low-dose paralysis). These results demonstrated that most, if not all, of the antibody stimulated by these preparations of S6 and S19 was actually induced by and was specific for PnC.
Production of mouse monoclonal antibodies which inhibit in vitro adherence of Entamoeba histolytica trophozoites
Authors: Ravdin J I , Petri W A , Murphy C F , Smith R D
Published: January 2020

Adherence by axenic Entamoeba histolytica trophozoites to mammalian cells is mediated by an N-acetylgalactosamine (GalNAc)-inhibitable adhesin on the surface of the parasite. We isolated 35 hybridoma cell lines producing antibodies to E. histolytica as indicated by ELISA with sonicated amebic protein or by immunofluorescence assay with fixed whole trophozoites. Tissue culture supernatants were further screened for subcloning by the ability to bind to Chinese hamster ovary (CHO) cells which were first exposed to a partially purified soluble preparation of the amebic GalNAc-inhibitable lectin. Eight tissue culture supernatants were positive in this assay. Antibodies from four subcloned cell lines (D3-14, H8-5, I12-2, and I1-21) inhibited amebic adherence to CHO cells (P less than 0.01). Of the original 35 tissue culture supernatants, 3 also inhibited amebic adherence (P less than 0.01; F1, F14, and J10); monoclonal antibodies in these supernatants did not bind to lectin-exposed CHO cells. Three purified monoclonal antibodies (H8-5, I12-2, and I1-21) inhibited amebic adherence at greater than or equal to 2 micrograms/10(4) amebae (P less than 0.05). None of these inhibitory monoclonal antibodies immunoprecipitated with a soluble amebic protein preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions. Monoclonal antibodies which inhibit in vitro adherence by E. histolytica will be useful in purification of the GalNAc-inhibitable lectin.
Quantification of specific antibody response to Cryptosporidium antigens by laser densitometry
Authors: Ungar B L , Nash T E
Published: January 2020

Cryptosporidium spp. is a protozoan parasite with worldwide distribution associated with diarrhea in immunocompromised patients (particularly those with acquired immunodeficiency syndrome [AIDS]) and in immunocompetent humans. Immunoglobulin M (IgM) and IgG antibody responses are readily detected by an enzyme-linked immunosorbent assay. To determine which Cryptosporidium antigens invoke antibody responses in humans, we performed polyacrylamide gel electrophoresis using purified oocysts, followed by Western blots with human sera from various populations. Of 40 sera from persons with cryptosporidiosis (24 AIDS and 16 non-AIDS patients), in 37 (93%) a 23,000-dalton antigen measured quantitatively by laser densitometry was recognized. Of 63 sera from IgM- or IgG-positive individuals, as determined by enzyme-linked immunosorbent assay, in 58 (92%) this same antigen was recognized. Up to three additional bands between 125,000 and 175,000 daltons were identified by some of these sera. These results suggest that most persons infected with Cryptosporidium spp. produce antibodies which recognize at least one common low-molecular-weight antigen. Isolation of this antigen will be useful in development of diagnostic tests and may be important in the study of immunity.
Biochemical and immunological characterization of the variant surface coat glycoprotein shed by African trypanosomes
Authors: Diffley P , Straus D C
Published: January 2020

As the variant surface coat glycoprotein (VSG) was shed from Trypanosoma brucei rhodesiense into the blood of infected rats, it was biochemically characterized and compared with VSG that had been purified from trypanosomal homogenates. To determine if VSG was in association with lipid, membranes and lipoproteins in plasma of infected rats (IRP), VSG isolated from plasma (PVSG), and VSG isolated from trypanosomal homogenates (HVSG) were all concentrated by ultracentrifugation and assayed for the presence of VSG by radial immunodiffusion (minimum level of detection, 25 micrograms/ml) and by immunoelectroblots (minimum level of detection, 1 microgram/ml). Crimson red was used to detect lipid (minimum level of detection, 10 micrograms per sample) in electrophoresed samples. The VSG was neither concentrated with membrane or lipoprotein fractions nor stained by lipid crimson. Lipids from normal rat plasma, IRP, trypanosomal homogenates, HVSG, and PVSG were also extracted and separated by thin-layer chromatography (minimum level of detection, 20 micrograms of trypanosomal phospholipid per sample). The trypanosomal homogenates had five bands as detected by iodine vapors, of which three were phospholipids as detected by molybdenum blue. Both normal rat plasma and IRP had identical patterns of bands with a single phospholipid. The PVSG had one neutral lipid contaminant that apparently was not physically associated with the shed surface coat. The HVSG contained no lipids at all. Therefore, no evidence was obtained to implicate an association between membranes and VSG, once the latter had been shed into the blood of infected hosts. From immunoelectroblots of denatured material, it was determined that both HVSG and PVSG had the same reduced molecular weight. From molecular sieve column chromatography, however, it was determined that VSG released during the homogenization of trypanosomes is a noncovalently linked dimer, whereas that shed in the blood is apparently a trimer. This difference in native structure made no difference in immunological effect. Administered in a regimen that mimicked what the host encounters during a first peak of parasitemia, both HVSG and PVSG induced nonspecific proliferation of splenic lymphocytes and production of unelicited antibodies without the generation of nonspecific immunosuppression. This polyclonal activation of lymphocytes was not the result of contamination by exogenous pyrogen, because the activity was lost if VSG was immunologically absorbed from plasma.(ABSTRACT TRUNCATED AT 400 WORDS)
Analysis of Trypanosoma cruzi antigens bound by specific antibodies and by antibodies to related trypanosomatids
Authors: Araujo F G
Published: January 2020

Antigens of the epimastigote stage of Trypanosoma cruzi were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and examined for their ability to bind antibodies in sera from humans infected with this organism or infected with one or both of the related protozoa Leishmania braziliensis and Leishmania donovani by protein blot analysis and enzyme-linked immunosorbent assay. Most of the antigens were bound by antibodies against each one of the organisms. A group of antigens with Mrs between 31,000 and 21,000 were bound by antibodies against T. cruzi only. These antigens were isolated and used in an enzyme-linked immunosorbent assay for the differential diagnosis of Chagas' disease, with excellent results. All sera from individuals proven to be infected with T. cruzi reacted with the antigens, whereas none of the sera from individuals proven to be infected with L. braziliensis or L. donovani reacted with the antigens, even when tested at a low dilution. Biochemical characterization of the isolated antigens revealed the presence of protein and carbohydrate. The reactivity of the isolated antigens with antibodies was completely abolished by pronase and partially abolished by sodium periodate. Protein blot analysis of sera from mice immunized with the antigens revealed a major large band with an Mr between 31,000 and 21,000 and a minor band with an Mr of 45,000, suggesting sharing of epitopes between antigens of different Mrs. These sera did not agglutinate or lyse live epimastigotes. Indirect immunofluorescent antibody tests with live and dead epimastigotes revealed that antibodies in the sera only bound to Formalin-killed organisms.
Application of multilocus enzyme gel electrophoresis to Haemophilus influenzae
Authors: Porras O , Caugant D A , Lagergård T , Svanborg-Edén C
Published: January 2020

Multilocus enzyme electrophoresis was adapted to the study of Haemophilus influenzae. Protein extracts from sonicated whole bacteria were subjected to starch gel electrophoresis. After staining with substrates, the position of each isoenzyme (electromorph) was registered. Each isolate was assigned an electrophoretic type (ET) by the combination of electromorphs for the enzymes stained. Twenty-seven enzymes were tested; 12 were expressed in H. influenzae. Six enzymes were selected for subsequent study: malate dehydrogenase (MDH), phenylalanylleucine peptidase (PE2), 6-phosphogluconate dehydrogenase (6PG), adenylate kinase (AK), glucose 6-phosphate dehydrogenase (G6P), and phosphoglucose isomerase (PGI). They were polymorphic and occurred in all isolates. Six electromorphs were found for PE2, G6P, and PGI, five for MDH, four for 6PG, and three for AK. PE2, G6P, and PGI contributed most of the ET resolution (48 of 49 ETs). Multilocus enzyme electrophoresis showed several advantages over previous typing techniques. An ET could be assigned to both typable and nontypable (NT) isolates. The technique was powerful in resolving differences among isolates. The 94 isolates comprised 49 ETs, five biotypes, and six capsular types and NT isolates. Strains known to be related expressed the same ET, e.g., RAB b+ and b-, ET12; Ma a+ and a-, ET1. ET variability among type b isolates was low; 26 of 28 clinical isolates expressed ET14; 2 of 28 expressed ET13 and ET15, differing from ET14 by one electromorph each. In contrast, the 47 NT isolates comprised 38 different ETs. No ETs were shared between non-type b capsulated strains and type b or NT strains. Interestingly, five NT isolates expressed the same ET as type b strains. (iv) Strains of the same capsular type but different biotypes expressed different ETs. ET determinations will thus be useful in studying the epidemiology and evolution of H. influenzae.
Variation in antigenic determinants specific to the infective stage of Trypanosoma cruzi
Authors: Wrightsman R A , Leon W , Manning J E
Published: January 2020

Monoclonal antibodies reactive with the surface antigens of the Peru strain of Trypanosoma cruzi were analyzed by Western blots and immunofluorescence assays to determine their reactivity with three life cycle stages and five strain isolates of T. cruzi. One monoclonal antibody, 7.6, recognized a 68-kilodalton (kDa) polypeptide in Western blots of Peru strain trypomastigotes, epimastigotes, and amastigotes. A 68-kDa polypeptide was also detected by monoclonal antibody 7.6 in trypomastigotes of the CL and Y strains and in the clonal isolates Esmeraldo clone 3 and Silvio X10 clone 1. Positive immunofluorescence results were obtained for all life cycle stages of the five strains that were reacted with monoclonal antibody 7.6, thus indicating that the antigen recognized by monoclonal antibody 7.6 is universally present in all T. cruzi strains tested. In contrast, monoclonal antibody 4.2 reacted with a polypeptide doublet of 90 and 105 kDa in Western blots of Peru strain trypomastigotes, but it did not detect these antigens in epimastigotes or amastigotes. The same polypeptide doublet of 90 and 105 kDa was also detected in Western blots of Y strain trypomastigotes; however, no bands were detected in blots of strain CL or isolate Silvio X10 clone 1 trypomastigotes. In blots of Esmeraldo clone 3 trypomastigotes, a single band of 130 kDa was detected by monoclonal antibody 4.2. In immunofluorescence assays of monoclonal antibody 4.2, positive reactions were obtained only with trypomastigotes of Peru, Y, and Esmeraldo clone 3 strains. Thus, monoclonal antibody 4.2 recognizes a trypomastigote-specific antigen which is not universally present on all strains of T. cruzi.
Isolation and characteristics of collagenolytic enzyme produced by Candida albicans
Authors: Kaminishi H , Hagihara Y , Hayashi S , Cho T
Published: January 2020

In media containing collagen as the nitrogen source, the pathogenic yeast Candida albicans secreted a collagenolytic enzyme. Purification of the enzyme from a culture filtrate was achieved by DEAE-Sephacel chromatography at pH 6.7. The molecular weight was found to be 46,000 by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was at pH 4.2. The pH optimum lay between 3.5 and 4.0, and above pH 6.0, the enzyme underwent alkaline denaturation. The enzyme was heat labile, and a decrease in activity was found above 55 degrees C. The enzyme activity was inhibited by the addition of urea, cysteine, and pepstatin. No other inhibitor among those tested had any effect. The C. albicans enzyme degraded both the native acid-soluble collagen and the insoluble dentinal collagen.
Pathogenesis of Campylobacter spp. in athymic and euthymic germfree mice
Authors: Yrios J W , Balish E
Published: January 2020

Adult athymic (nu/nu) and euthymic (+/nu) germfree BALB/c mice were orally challenged with pure cultures of Campylobacter jejuni (human clinical fecal strains) and a human blood isolate of Campylobacter fetus subsp. fetus. After a period of adaptation to the mouse intestinal tract, all three C. jejuni strains caused disease in gnotobiotic mice. Mouse-adapted, weakly cytotoxic C. jejuni 45100 consistently induced disease symptoms (transient diarrhea, cecal shrinkage, and acute inflammatory changes with eosinophilia in the lower intestinal mucosa) in nu/nu mice 7 to 9 days after oral challenges. Conversely, no overt disease or histopathology was evident in +/nu mice challenged with the same strain (45100). After periods of adaptation in the murine alimentary tract, the two C. jejuni strains, 24 and INN 73-83, with greater cytotoxin-producing capacities, decreased cecal size and caused minor mucosal inflammatory changes in both nu/nu and +/nu BALB/c mice 1 to 2 weeks after intestinal colonization. A transient splenomegaly was also evident at 1 to 2 weeks after germfree nu/nu mice were colonized with each of the three C. jejuni strains used in this study. Occult blood was observed in a small percentage (approximately 11%) of nu/nu and +/nu BALB/c mice that were colonized with C. jejuni strains 45100 and INN 73-83. C. fetus subsp. fetus 255 colonized the alimentary tract of gnotobiotic mice, but neither morbidity nor mortality was evident. The disease we observed in the gnotobiotic mice, along with the histological changes in the intestinal tract after oral challenges, resembles symptoms of campylobacteriosis in humans. The gnotobiotic BALB/c mouse model of Campylobacter disease provides a unique opportunity to detail basic aspects of the acute and chronic pathogenesis of and immunity to this recently recognized disease.
Hormonal factors in vaginal candidiasis in rats
Authors: Kinsman O S , Collard A E
Published: January 2020

The hormonal status of rats affected vaginal infection with Candida albicans. Four hours after infection viable counts were higher and germ tubes were longer in those animals in estrous than in other animals. However, the infection was not maintained with the change in epithelial cell type which occurred as part of the estrous cycle. Estrogen dosing following ovariectomy predisposed toward infection, while progesterone dosing did not. In rats injected with progesterone, germ tube clumping was seen, leukocytes were present, and elimination occurred before hyphal growth was evident. In rats injected with estrogen, however, infection was maintained, with hyphal growth extending throughout the cornified epithelial layer. Vaginal washings from rats dosed with estrogen promoted elongation of germ tubes in vitro to a greater extent than washings from other rats. Preincubation of blastospores in progesterone and subsequent infection of rats in pseudoestrous promoted clumping of germ tubes in the vagina. Strains of C. albicans varied in their virulence, which correlated with their ability to produce germ tubes in vitro. Loss of virulence occurred on subculture of a clinical isolate.
Biliary lipids support serum-free growth of Giardia lamblia
Authors: Gillin F D , Gault M J , Hofmann A F , Gurantz D , Sauch J F
Published: January 2020

Giardia lamblia has been grown in vitro only in media containing serum or serum fractions. How this pathogen can grow in the human small intestinal lumen without serum is not known. We found that samples of human hepatic or gall bladder bile maintained G. lamblia survival for 24 to 48 h in medium without serum but did not support growth. By contrast, an artificial biliary lipid dispersion containing six bile salts, phosphatidylcholine (PC), and cholesterol, in the ratios characteristic of human bile, supported parasite growth in medium without serum or serum fractions. To define the requirements, we showed that 1-palmitoyl-2-linoleoyl-PC or 1-palmitoyl-2-oleoyl-PC (which predominate in human bile) satisfied the requirement for PC. Moreover, either glycocholate or glycodeoxycholate could be substituted for the bile salt mixture. The finding that biliary lipids can support serum-free growth of G. lamblia may help explain why this parasite colonizes the upper small intestine.
Induction of early-phase endotoxin tolerance in athymic (nude) mice, B-cell-deficient (xid) mice, and splenectomized mice
Authors: Madonna G S , Vogel S N
Published: January 2020

Early-phase endotoxin tolerance was inducible in mice which were T cell deficient (nude), B cell deficient (xid), or asplenic, which suggests that these lymphoid cell subsets and the spleen do not contribute significantly to the induction of acquired lipopolysaccharide hyporesponsiveness. C3H/HeJ mice did not exhibit the hematopoietic changes observed in mice made endotoxin tolerant, which suggests that multiple mechanisms may underlie lipopolysaccharide hyporesponsiveness.
Experimental evidence for the role of lipids in adherence of Candida spp. to human buccal epithelial cells
Authors: Ghannoum M A , Burns G R , Elteen K A , Radwan S S
Published: January 2020

Lipids extracted from Candida albicans and C. tropicalis, but not from the weakly adherent C. pseudotropicalis, significantly blocked in vitro adherence of the respective yeast cells to buccal epithelial cells. The percentage of reduction from control values ranged between 16.4 and 42.1%, depending on the species, the strain, and the solvent used for lipid extraction. The constituent lipid classes of both the acetone and chloroform-methanol extracts of C. albicans ATCC 10231 were qualitatively and quantitatively analyzed. The individual classes were isolated by preparative thin-layer chromatography and then tested for their effects on the adherence of this strain to buccal epithelial cells. Individual phospholipids, sterols, and steryl esters blocked adherence significantly (between 15.5 and 55.7% reduction). Triacylglycerols and free fatty acids showed no effect whatsoever. The same results were obtained when standard lipid samples were investigated.
Characterization of toxins A and B of Clostridium difficile with monoclonal antibodies
Authors: Lyerly D M , Phelps C J , Toth J , Wilkins T D
Published: January 2020

Two monoclonal antibodies (MAbs) were used to learn more about the structures of Clostridium difficile toxins A and B. One of the antibodies, the PCG-4 MAb, reacted specifically with toxin A. This MAb precipitated toxin A and neutralized the enterotoxic but not the cytotoxic activity of the toxin. The site to which the antibody bound was resistant to denaturation with sodium dodecyl sulfate; however, it was destroyed by N-bromosuccinimide. Immunoblot analysis with the PCG-4 MAb revealed the presence of a large number of bands in preparations of denatured toxin A, suggesting that toxin A exists as an aggregate of smaller components. The antibody was covalently coupled to Affi-Gel 10, and the gel was used to purify toxin A from the culture filtrate of a highly toxigenic strain of C. difficile by immunoaffinity chromatography. The second antibody, the G-2 MAb, cross-reacted with toxins A and B. The cross-reaction was confirmed by immunoblot analysis. These results show that toxins A and B share an epitope and suggest that they have a common subunit. The G-2 MAb did not neutralize or precipitate either toxin. The site to which the G-2 MAb bound was partially destroyed by sodium dodecyl sulfate and was resistant to oxidation with N-bromosuccinimide.
Immunization against fatal experimental Klebsiella pneumoniae pneumonia
Authors: Cryz S J , Fürer E , Germanier R
Published: January 2020

The ability of serospecific anti-capsular polysaccharide (CPS) antibody to prevent fatal Klebsiella pneumoniae pneumonia was evaluated in a rat lung model. Rats were immunized intramuscularly with 100 micrograms of purified serotype 2 CPS and challenged intrabronchially 14 days later with a serotype 2 strain of K. pneumoniae. Vaccination engendered high levels of serum anti-CPS antibody which afforded significant protection (P less than 0.01) against fatal pneumonia. Immunization promoted clearance of the challenge bacteria from the lungs and prevented bacteremia. Histological examination of lung tissue from infected control animals showed pronounced inflammatory cellular infiltrate in the alveolar spaces, intra- and peribronchial inflammation, and tissue necrosis. In contrast, pathological changes noted in lungs from immunized animals were restricted to infrequent intra- and peribronchial involvement.
A low-Ca2+ response operon encodes the V antigen of Yersinia pestis
Authors: Perry R D , Harmon P A , Bowmer W S , Straley S C
Published: January 2020

Yersinia pestis has a virulence regulon called the low-Ca2+ response that is mediated by the plasmid pCD and manifested as regulation of growth and of expression of several virulence-associated properties by Ca2+ and temperature. We used Mu dI(Ap lac) to obtain a mutation in pCD1 of Y. pestis KIM that rendered the bacteria unable to express one of these properties, the V antigen. This mutant also had lost the Ca2+ requirement for growth at 37 degrees C and was avirulent in mice. Two-dimensional protein gel electrophoresis showed that the Mu dI(Ap lac) insertion had eliminated 13,000- and 18,000-molecular-weight proteins in addition to the V antigen. We mapped the Mu dI(Ap lac) insertion within pCD1, cloned the HindIII fragment spanning the insertion location, prepared two subclones of this fragment, and identified the proteins these clones expressed in Escherichia coli minicells. The data indicated that the V gene lies within an operon containing three genes; lcrG (encoding the 13,000-molecular-weight protein), lcrV (encoding the 38,000-molecular-weight V antigen), and lcrH (encoding the 18,000-molecular-weight protein). Therefore, the V operon contains the structural gene for V antigen, at least one virulence gene, and at least one Ca2+-dependence gene.
Immunochemical analysis of plasmid-encoded proteins released by enteropathogenic Yersinia sp. grown in calcium-deficient media
Authors: Heesemann J , Gross U , Schmidt N , Laufs R
Published: January 2020

Enteropathogenic Yersinia sp. releases plasmid-associated proteins of low molecular mass (26-67 kilodaltons) at 37 degrees C. In this study, the optimum conditions for the release of proteins were assessed and the released proteins (RPs) were analyzed for the manner of release, immunochemical characteristics, and the location of the genes necessary for their synthesis. Protein release was strongly enhanced when growth media were markedly depleted of calcium ions by precipitation with oxalate or chelation with EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. RP yields were greatest when Yersinia spp. were in the exponential growth phase. The RPs appeared to be released from the Yersinia spp. by secretion rather than by pinching off of membrane vesicles, because the RPs did not sediment during high-speed centrifugation nor were they contaminated to any significant degree with lipopolysaccharide. Moreover, immunoblot analysis revealed only traces of protein species related to RPs within the outer membranes of plasmid-positive Yersinia spp. grown at 37 degrees C under calcium-restricted conditions. Immunoblot studies also showed that the RPs of Y. enterocolitica serotypes O:3, O:8, and O:9 and the RP of Y. pseudotuberculosis serotype I are highly cross-reactive. Finally, the immunoprecipitates of the products of minicells which harbor Yersinia plasmids were used to demonstrate that at least three proteins immunochemically related to the released fraction were plasmid encoded. These results suggest that at least three of the RPs may be related to or identical with previously described plasmid-encoded Yersinia outer membrane proteins.
Some galE mutants of Salmonella choleraesuis retain virulence
Authors: Nnalue N A , Stocker B A
Published: January 2020

galE mutants were isolated from three mouse-virulent strains of Salmonella choleraesuis (of group C1, O antigen 6,7) by selection for resistance to 2-deoxygalactose. The galE derivative of strain 381 comprised two components: galactose sensitive, thought to be the original mutant; and galactose resistant, presumably by a second mutation reducing galK or galT function or both. The galactose-sensitive component had an intraperitoneal 50% lethal dose for BALB/c mice of ca. 4 X 10(6) CFU, whereas the galactose-resistant component was about as virulent as its gal+ parent, with a 50% lethal dose of ca. 100 CFU. The galE mutant of strain 110 was somewhat sensitive to galactose, as shown by retardation of growth; its 50% lethal dose, ca. 500 CFU, was not much greater than the ca. 200 CFU value for its parent. The galE mutant of strain 117 showed the same partial sensitivity to galactose as strain 110 galE, but was nonvirulent (50% lethal dose of ca. 10(6) CFU versus ca. 400 CFU for its parent). Growth on galactose-supplemented medium restored the smooth phenotype, as indicated by phage sensitivity to three of the four galE strains, but only partially so for the strain 117 galE mutant. The retention of parental virulence by galE mutants of S. choleraesuis which are galactose resistant or somewhat galactose sensitive contrasts with the greatly reduced virulence of galactose-resistant galE mutants of Salmonella typhimurium and Salmonella typhi; this difference may result from the absence of galactose from the O repeat unit in the lipopolysaccharide of group C1 salmonellae.