bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution, persistence, and recall of serum and salivary antibody responses to peroral immunization with protein antigen I/II of Streptococcus mutans coupled to the cholera toxin B subunit
Authors: Russell M W , Wu H Y
Published: January 2020

After peroral immunization of mice with surface protein antigen (Ag) I/II of Streptococcus mutans conjugated to the cholera toxin B (CTB) subunit, cells actively secreting immunoglobulin A (IgA) antibodies specific for Ag I/II, but not for CT, were induced in the salivary glands; salivary IgA anti-Ag I/II antibodies and total salivary IgA were also elevated. The development of large numbers of IgA and IgG antibody-secreting cells in the mesenteric lymph nodes and spleen and high levels of serum IgA and IgG antibodies to Ag I/II and CT demonstrated that a response to both antigens occurred. At least two to three intragastric doses of 15 micrograms or more of Ag I/II-CTB conjugate, plus free CT as an adjuvant, were needed to induce the salivary IgA anti-Ag I/II response, which peaked at about 35 days and persisted at lower levels for 5 to 6 months. A single booster intragastric immunization did not induce enhanced salivary IgA anti-Ag I/II antibodies relative to the primary response, but serum IgA and IgG antibodies to both Ag I/II and CT showed evidence of marked anamnestic responses. The results indicated that relatively long-term mucosal IgA antibody responses could be induced by peroral immunization with small quantities of a CTB-conjugated protein. However, additional factors governed the distribution of cells secreting antibodies of different specificities, or capable of mounting anamnestic responses, between different compartments of the mucosal and circulatory immune systems.
Small-animal model to measure efficacy and immunogenicity of Shigella vaccine strains
Authors: Hartman A B , Powell C J , Schultz C L , Oaks E V , Eckels K H
Published: January 2020

The development of a small-animal model to test the protective efficacy and immunogenicity of a vaccine strain against shigellosis would greatly facilitate the evaluation of potential vaccine candidates. In guinea pigs, the ability of shigellae to invade and multiply within the corneal epithelium, causing keratoconjunctivitis, closely mimics the invasion process in the intestinal epithelium (B. Sereny, Acta Microbiol. Acad. Sci. Hung. 4:367-376, 1957). The serum response of animals recovering from a Shigella keratoconjunctival infection was determined and found to be consistent with that shown by convalescent humans and primates. This model was used to test the efficacy of two vaccine candidates, and the immune response of the guinea pigs to the vaccine strains was examined. Both vaccine strains demonstrated significant protection against challenge by homologous virulent Shigella strains, and the results were comparable with results obtained in trials with monkeys. The guinea pig model also provides a rapid and inexpensive means of evaluating different immunization regimens as well as of testing other variables such as length of protection against disease.
Identification of Mycobacterium leprae antigens from a cosmid library: characterization of a 15-kilodalton antigen that is recognized by both the humoral and cellular immune systems in leprosy patients
Authors: Sela S , Thole J E , Ottenhoff T H , Clark-Curtiss J E
Published: January 2020

Screening of the Mycobacterium leprae cosmid library with pooled sera from lepromatous leprosy (LL) patients by a colony immunoblot technique resulted in the identification of about 100 colonies that produced immunologically reactive proteins. Twenty-four of these clones were purified, analyzed, and found to comprise two groups according to the reactivity of the recombinant proteins with LL sera and to the DNA restriction patterns of the recombinant plasmids and cosmids. Proteins specified by clones from group I reacted strongly with LL patients' sera on a Western blot (immunoblot), demonstrating a 15-kDa protein band designated A15. The A15 antigen also reacted with pooled sera from patients with tuberculoid leprosy from the United States and Brazil. Clones from group II did not show any reactive protein band on a Western blot, when reacted with patients' sera. DNAs from cosmids of group II all contain a 10-kb PstI fragment that hybridized to the unique repetitive M. leprae DNA. Sequence analysis of a 1.2-kb fragment containing the entire coding sequence of A15 revealed three open reading frames (ORFs), only one of which (ORF II) contains sufficient genetic information to encode for A15. Part of the A15 gene was found to exist also in a group of lambda gt11:M. leprae clones previously isolated in our laboratory by immunological screening with LL patients' sera. One of the lambda gt11 clones (L8) expresses a beta-galactosidase fusion protein with 89 amino acids from the C terminus of A15. An important result was that the fusion protein was clearly recognized by T cells from leprosy patients. Interestingly, Mycobacterium tuberculosis-stimulated T cells from M. leprae nonresponder (LL as well as borderline tuberculoid) patients were able to respond to the isolated recombinant M. leprae antigen, indicating that nonresponsiveness to M. leprae antigens can be reversible. The sequence of the M. leprae DNA fused to the beta-galactosidase gene of lambda gt11 clone L8 was identical to that of a lambda gt11:M. leprae clone isolated recently that expresses an immunologically reactive fusion protein (S. Laal, Y. D. Sharma, H. K. Prasad, A. Murtaza, S. Singh, S. Tangri, R. Misra, and I. Nath, Proc. Natl. Acad. Sci. USA 88:1054-1058, 1991). Besides the complete sequence of the A15 gene, sequencing data of two flanking ORFs are presented. Downstream from ORF II (A15), ORF III has a high degree of similarity to the genes for tomato ATP-dependent proteases that are members of a larger class of highly conserved proteases ubiquitous among prokaryotes and eukaryotes.(ABSTRACT TRUNCATED AT 400 WORDS)
Characterization of a proteinaceous adhesin of Staphylococcus epidermidis which mediates attachment to polystyrene
Authors: Timmerman C P , Fleer A , Besnier J M , De Graaf L , Cremers F , Verhoef J
Published: January 2020

Coagulase-negative staphylococci (CoNS) have evolved into important agents of foreign body-related infections. Adhesion of causative bacteria to biomaterials is considered to be an essential step in these infections. We and others have shown that adhesion of CoNS to biomaterials may be mediated by protease-sensitive surface constituents. In the present study we expanded on these investigations by characterizing a biomaterial adhesin of Staphylococcus epidermidis 354 by using a strain-specific monoclonal antibody (MAb 36.4). MAb 36.4 was strongly and exclusively reactive with strain 354 in an enzyme-linked immunosorbent assay in which whole bacteria were used as antigens. Immunoblotting of cell wall polypeptides of strain 354 revealed strong reactivity with a 200- to 220-kDa band and a weaker reaction in the 100- to 110-kDa range. Preincubation of strain 354 with MAb 36.4 resulted in a 54 to 91% (mean +/- standard deviation, 74% +/- 14%; n = 10) inhibition of adhesion to polystyrene spheres. Fab fragments prepared from MAb 36.4 also inhibited adhesion effectively, indicating specific blocking of an adhesion antigen rather than aspecific inhibition. Immunogold electron microscopy with MAb 36.4 revealed deposition of gold particles on the cell surface and possibly also on fimbrialike surface projections. It is concluded that a surface-located protein antigen of S. epidermidis 354 recognized by MAb 36.4 acts as an adhesin mediating attachment to uncoated foreign material. It is speculated that this type of adhesion to biomaterials may play an important role in the pathogenesis of foreign body-related infections caused by CoNS.
Characterization of murine monoclonal antibodies that recognize defined epitopes of pertussis toxin and neutralize its toxic effect on Chinese hamster ovary cells
Authors: Walker M J , Wehland J , Timmis K N , Raupach B , Schmidt M A
Published: January 2020

Three murine monoclonal antibodies (MAb), E19, E205, and E251, raised against pertussis toxin reacted in Western blots (immunoblots) with the S1, S4, and S2-S3 subunits, respectively, and neutralized the Chinese hamster ovary cell-clustering activity of pertussis toxin. MAb E251 recognized a linear synthetic peptide corresponding to amino acids 107 to 120 of the S2 subunit, suggesting a role for this region in receptor binding.
Listeriolysin as a virulence factor in Listeria monocytogenes infection of neonatal mice and murine decidual tissue
Authors: McKay D B , Lu C Y
Published: January 2020

Listeriolysin is a 60-kDa protein which allows the growth of Listeria monocytogenes in macrophages and other cells and has been shown to be a virulence factor in Listeria infections of adult mice. However, the neonate and fetoplacental unit are major populations susceptible to listeriosis. Recent data indicate that macrophage and T-cell functions are markedly inhibited in these young mice, and the virulence of listeriolysin-negative (HLY-) and listeriolysin-positive (HLY+) Listeria cells in the setting of such inhibited macrophage and T-cell functions has not previously been examined. We now compare CNL 85/162, a transposon-induced, HLY- Listeria strain, and CNL 85/163, a spontaneous HLY+ revertant. We found that all 18 neonates injected with CNL 85/163 (HLY+) died within 12 days after an injection of 10(4) Listeria cells per mouse. In contrast, all 16 neonates injected with 1,000 times more CNL 85/162 (HLY-) cells survived more than 14 days. Three days after injection, growth of CNL 85/163 (HLY+) in the internal organs was more than 5 log greater than that of CNL 85/162 (HLY-). We also found that CNL 85/162 (HLY-) did not proliferate well in decidual tissue, which is a major component of the placental region. Our studies indicate that HLY- bacteria are not virulent in the neonate and the fetoplacental unit despite the inhibited immune functions at these sites.
Adhesion-dependent activation of mucosal interleukin-6 production
Authors: Linder H , Engberg I , Hoschützky H , Mattsby-Baltzer I , Svanborg C
Published: January 2020

Mucosal exposure to Escherichia coli elicits an inflammatory response in the urinary tract. Interleukin-6 (IL-6) is secreted into the urine, and polymorphonuclear leukocytes (PMNLs) are recruited to the site of infection. This study analyzed the ability of mucosally administered bacterial components to activate IL-6 and PMNL responses. P, S, and type 1 fimbrial preparations with adhesins specific for Gal alpha 1-4Gal beta, NeuAc alpha 2-3Gal, and mannose, respectively, were inoculated intravesically into lipopolysaccharide (LPS)-responder (C3H/HeN) and LPS-nonresponder (C3H/HeJ) mice. The role of the fimbrial adhesin was examined by comparing P and S fimbriae with (Adh+) and without (Adh-) the receptor-binding domain. Isolated lipid A was used in parallel. The urinary IL-6 levels were elevated after challenge with Adh+ P fimbriae, but not after challenge with the Adh- P fimbriae, Adh+ or Adh- S fimbriae, or type 1 fimbriae. The activation was not a function of contaminating LPS, since it occurred in both LPS-responder and -nonresponder mice and since isolated lipid A was a poor activator of the IL-6 response. In contrast, lipid A was a potent inducer of the PMNL response. The results suggested that the IL-6 and PMNL responses were activated via different pathways; the IL-6 response was activated mainly by an adhesion-dependent interaction with the mucosa, and the PMNLs were activated mainly by lipid A. The results emphasize the active role of the mucosal barrier in the production of mediators in response to diverse bacterial stimulants.
Identification of monoclonal antibody-binding domains within antigen P1 of Streptococcus mutans and cross-reactivity with related surface antigens of oral streptococci
Authors: Brady L J , Piacentini D A , Crowley P J , Bleiweis A S
Published: January 2020

Eleven monoclonal antibodies (MAbs) specific for P1, the major protein surface antigen of Streptococcus mutans serotype c, were characterized by Western blot (immunoblot) analysis and by radioimmunoassay using whole bacterial cells. The approximate binding domains of the MAbs were determined by using full-length and truncated P1 polypeptides. The accessibility of these binding sites on the surfaces of intact bacteria was determined by radioimmunoassay. The ability of each MAb to cross-react with related proteins from strains of S. mutans serotypes e and f, S. sanguis, and S. sobrinus serotype g is also reported.
Sugar metabolism by fusobacteria: regulation of transport, phosphorylation, and polymer formation by Fusobacterium mortiferum ATCC 25557
Authors: Robrish S A , Oliver C , Thompson J
Published: January 2020

Strains of eight Fusobacterium species differed in the ability to use sugars as energy sources for growth. For Fusobacterium russii ATCC 25533, F. gonidiaformans ATCC 25563, and F. nucleatum ATCC 10953 (except for fructose), growth was marginal to poor on all of the sugars tested. Other species displayed reasonable growth on glucose, fructose, mannose, and galactose, and two strains of F. mortiferum (ATCC 25557 and ATCC 9817) grew well on six of the sugars tested, including sucrose and maltose. Glucose transport by resting cells of most of the species was dependent upon (or markedly stimulated by) the presence of a fermentable amino acid. By contrast, F. mortiferum cells rapidly accumulated glucose and other sugars in the absence of amino acids. Although these cells were constitutive for glucose uptake, accumulation of other sugars was specifically induced by growth of F. mortiferum on the appropriate sugar. Spectrophotometric analyses and in situ staining of anionic polyacrylamide gels showed that glucose and fructose (mannose) are phosphorylated by separate ATP-dependent kinases. Fructokinase was stable in air at 4 degrees C, but under these conditions, greater than 70% of the glucokinase activity was lost. After overnight dialysis of the extract, no glucokinase activity was detectable; however, 65% of the initial enzyme activity was retained by inclusion of 1 mM dithiothreitol in the dialysis buffer. Thin-section electron microscopy showed that cells of F. mortiferum produced various amounts of intracellular glycogen during growth on the following sugars (in decreasing order of formation): galactose greater than sucrose greater than glucose greater than mannose greater than fructose. Mechanisms for sugar transport regulation, phosphorylation, and polymer synthesis by F. mortiferum cells are proposed.
Comparison of immune repertoires of Chinese and Philippine patients infected with Schistosoma japonicum
Authors: Kresina T F , Guan X H , Posner M , Ramirez B , Olds G R
Published: January 2020

Chinese and Philippine patients exhibited detectable titers of serum antibodies to Schistosoma japonicum worm and egg antigens. Western blot (immunoblot) data revealed differing antigenic profiles. Antigen inhibition studies showed low and high levels of cross-reactivity with anti-worm and anti-egg antibodies, respectively, derived from both Chinese and Philippine patients. Thus, anti-egg antibodies and egg antigens are more conserved than anti-worm antibodies and worm antigens.
Enhanced resistance against Listeria monocytogenes achieved by pretreatment with granulocyte colony-stimulating factor
Authors: Shinomiya N , Tsuru S , Katsura Y , Kayashima S , Nomoto K
Published: January 2020

Phagocytosis, H2O2 production, Mac-1 expression, and in vivo elimination of Listeria monocytogenes were enhanced in granulocyte colony-stimulating factor (G-CSF)-treated mice. Transfer of polymorphonuclear leukocytes prolonged survival of mice infected with a lethal dose of L. monocytogenes. G-CSF augments the functions of polymorphonuclear leukocytes and thus plays a role in protection.
A monoclonal antibody to gamma interferon blocks augmentation of natural killer cell activity induced during systemic cryptococcosis
Authors: Salkowski C A , Balish E
Published: January 2020

These studies demonstrate that the cytotoxic activity of splenic natural killer (NK) cells is augmented in both nu/nu and nu/+ mice during systemic cryptococcosis. Both the kinetics and the regulation of NK cell activity differed in Cryptococcus neoformans-infected nu/nu and nu/+ mice. Greater augmentation was observed following challenge with 10(5) cells than with smaller inocula, and augmented NK cell activity was not always associated with enhanced control of systemic cryptococcosis. Infection with a nonencapsulated strain of C. neoformans induced an early but transient increase in splenic NK cell activity in nu/nu and nu/+ mice. Injection of capsular polysaccharide induced a transient augmentation of splenic NK cell activity in nu/+ mice but caused a persistent increase in splenic NK cell activity in nu/nu mice. In vivo treatment with monoclonal antibody to gamma interferon abrogated the augmentation of splenic NK cell activity induced during cryptococcal infections in both nu/nu and nu/+ mice and enhanced the susceptibility of nu/+ mice to C. neoformans to a greater extent than it did that of nu/nu mice. These results suggest that gamma interferon is an important mediator of resistance to C. neoformans.
Immunoreactive epitopes on an expressed recombinant flagellar protein of Borrelia burgdorferi
Authors: Collins C , Peltz G
Published: January 2020

A recombinant Borrelia burgdorferi flagellin protein expressed in Escherichia coli is bound by a murine monoclonal antiflagellin antibody (H9724) and by antibodies in the sera of patients with Lyme disease. Immunoreactive epitopes on the flagellar protein were identified by immunoblot analysis of antibody binding to expressed truncated flagellar proteins. The epitope recognized by the murine monoclonal antibody is within the central heterologous region of the flagellar protein (amino acids 90 to 266). However, antiflagellin antibodies in the sera of patients with Lyme arthritis bound an epitope entirely within, or whose conformation was partly formed by, the 90 NH2-terminal amino acids of the flagellar protein. The binding of antibodies in the sera of patients with Lyme arthritis to the NH2-terminal region of the flagellar protein, a region with sequence homology to the flagellar proteins of other bacterial species, suggests the possibility that antigenic mimicry contributes to the immunopathogenesis of Lyme disease. The fact that human antibodies bind to a highly conserved and hence shared portion of the flagellin reduces the specificity of serological assays for the diagnosis of Lyme disease which use the flagellar protein as antigen.
Comparison of albumin receptors expressed on bovine and human group G streptococci
Authors: Raeder R , Otten R A , Boyle M D
Published: January 2020

The albumin receptor expressed by bovine group G streptococci was extracted and affinity purified. The protein was characterized for species reactivity, and monospecific antibodies were prepared to the purified receptor. The bovine group G albumin receptor was compared functionally, antigenically, and for DNA homology with the albumin-binding protein expressed by human group G streptococci. In agreement with previous reports, the albumin-binding activity of human strains was mediated by a unique domain of the type III immunoglobulin G-Fc-binding molecule, protein G. The albumin receptor expressed by bovine group G strains was found to lack any immunoglobulin G-binding potential but displayed a wider profile of species albumin reactivity than protein G. Both albumin receptors could inhibit the binding of the other to immobilized human serum albumin, and each displayed similar binding properties. Antigenic comparison of the two albumin receptors demonstrated a low level of cross-reactivity; however comparison at the DNA level, using an oligonucleotide probe specific for the albumin-binding region of protein G, demonstrated that the two albumin receptors expressed by human and bovine group G streptococcal strains do not display significant homology.
Characterization of cell envelope proteins of Staphylococcus epidermidis cultured in human peritoneal dialysate
Authors: Smith D G , Wilcox M H , Williams P , Finch R G , Denyer S P
Published: January 2020

The cell envelope protein profiles of Staphylococcus epidermidis cultured in used human peritoneal dialysate (HPD) differed markedly from those of cells cultured in nutrient broth. Compared with broth-grown cells, many cell wall proteins were repressed in HPD, although three proteins of 42, 48, and 54 kDa predominated and an iron-repressible 130-kDa protein was induced. Growth in HPD also resulted in expression of two cell membrane proteins of 32 and 36 kDa which were iron repressible. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis using monospecific polyclonal antisera raised against the 32- and 36-kDa proteins revealed considerable antigenic and molecular mass homology among 12 S. epidermidis isolates from patients with continuous ambulatory peritoneal dialysis-related peritonitis. The 32-kDa antiserum also cross-reacted with a 32-kDa S. aureus cell membrane protein. Immunoblots of S. epidermidis cell walls and membranes were also probed with normal human serum and serum and HPD from continuous ambulatory peritoneal dialysis patients. While the cell wall proteins of S. epidermidis appeared to be relatively poorly immunogenic, the 32- and 36-kDa membrane proteins reacted strongly with antibodies present in each of the body fluids evaluated. These results suggest that the highly conserved 32- and 36-kDa iron-repressible proteins are expressed during growth in vivo and may be involved in iron transport, since all 12 S. epidermidis strains examined also produced iron chelators.
Monoclonal antibody characterization of a leukoagglutinin produced by Renibacterium salmoninarum
Authors: Wiens G D , Kaattari S L
Published: January 2020

Renibacterium salmoninarum causes a chronic disease of salmonid fish known as bacterial kidney disease. High concentrations of bacterially produced extracellular protein (ECP) are present in plasma, kidney, and spleen tissue of naturally and experimentally infected fish. ECP agglutinated salmonid leukocytes in vitro at concentrations which correspond to levels found in highly infected fish. Association of biological activity with the structure of the major protein constituent of ECP, p57, was accomplished by monoclonal antibody (MAb) analysis. Location of the antigenic binding sites recognized by the MAbs was determined by two-dimensional electrophoresis and Western immunoblotting of the proteolytic breakdown fragments of p57. Eight MAbs have been classified into three groups on the basis of their differential recognition of these proteolytic breakdown products. Group I MAbs bound a region proximal to the amino terminus of the protein. Two of these MAbs were also able to block leukoagglutinating activity. Group III MAbs bound to a region associated with the bacterial cell surface, while group II MAbs bound a region between group I and group III. These analyses have allowed the identification of potential structural and functional regions of p57.
Enhancement of lipopolysaccharide-induced tumor necrosis factor production in mice by carrageenan pretreatment
Authors: Ogata M , Yoshida S , Kamochi M , Shigematsu A , Mizuguchi Y
Published: January 2020

Tumor necrosis factor (TNF) is a cytokine which mediates endotoxin shock and causes multiple organ damage. It is thought that macrophage (MP) activation is necessary to increase lipopolysaccharide (LPS)-induced TNF production and lethality. Carrageenan (CAR) is sulfated polygalactose which destroys MP; it is used as a MP blocker. We found that CAR pretreatment can increase both endotoxin-induced TNF production and the mortality rate in mice. The ddY mice (7 to 8 weeks old) were injected intraperitoneally with CAR (5-mg dose) and challenged intravenously with LPS 24 h later. Without CAR pretreatment, LPS doses of less than 10 micrograms did not induce TNF in sera. After pretreatment, however, about 3 x 10(3) to 4 x 10(4) U of TNF per ml was produced after LPS injection at doses of 0.1 to 10 micrograms, respectively. TNF production was significantly increased by CAR pretreatment at LPS doses of more than 10 micrograms. CAR pretreatment rendered the mice more sensitive to the lethal effect of LPS; 50% lethal doses of LPS in CAR-pretreated mice and nonpretreated mice were 26.9 and 227 micrograms, respectively. The mortality of the two groups was significantly different at doses of 50, 100, and 200 micrograms of LPS. CAR increased LPS-induced TNF production and mortality within 2 h, much earlier than MP activators, which needed at least 4 days. Our results made clear that TNF production is enhanced not only by a MP activator but also by a MP blocker.
Sequence analysis of the gene encoding the Chlamydia pneumoniae DnaK protein homolog
Authors: Kornak J M , Kuo C C , Campbell L A
Published: January 2020

The antigen-coding region of a 4.2-kb PstI fragment of Chlamydia pneumoniae (pLC3), which encodes a 75-kDa immunoreactive protein recognized during human C. pneumoniae infection, was localized to a 2.0-kb EcoRI fragment. This subclone expressed an immunoreactive fusion protein of ca. 82 kDa. Nucleotide sequence analysis of the C. pneumoniae gene revealed that it consisted of a 1,980-base open reading frame with an inferred 71,550-Da protein of 660 amino acids. Putative Escherichia coli-like promoters and a ribosomal binding site were located in the 5' upstream region, and an 11-base dyad forming a stable stem-loop structure following two in-frame stop codons was identified. The C. pneumoniae 75-kDa protein is a member of the hsp70 family of heat shock proteins and has 87% amino acid similarity with the Chlamydia trachomatis protein.
Use of Peyer's patch and lymph node fragment cultures to compare local immune responses to Morganella morganii
Authors: Logan A C , Chow K P , George A , Weinstein P D , Cebra J J
Published: January 2020

Lymphoid tissue fragment cultures were established to analyze the differentiative processes among B cells in Peyer's patches (PP) and peripheral lymph nodes (PLN), especially those in germinal centers. PP cultures from both conventionally reared mice and formerly germ-free mice colonized with Morganella morganii could be maintained for greater than 12 days with continued B-cell division, especially among cells binding high levels of peanut agglutinin, a characteristic of germinal center cells. PLN cultures from conventionally reared mice injected with a heat-killed vaccine of M. morganii could be maintained for the same amount of time. Over this period, PP cultures continued to secrete immunoglobulin A (IgA) as well as smaller amounts of IgM. PP cultures from formerly germ-free mice colonized with M. morganii showed net increases of IgA antiphosphocholine (anti-PC) antibodies with avidities as high as those of the prototypic T15 monoclonal antibody. Similar PLN fragment cultures from conventionally reared mice given footpad injections of M. morganii showed net increases of IgM and IgG anti-PC antibodies in the culture fluid. Thus, although M. morganii stimulated lymphoid tissues in vivo to produce an anti-PC response in vitro when given by either the oral or the parenteral route, the antibody isotypes differed between PP and PLN fragment cultures. Fragment culturing may offer a complementary and simpler way to detect a local secretory IgA response than does either measuring IgA antibody in secretions or detecting IgA antibody in the cytoplasm of plasma cells in the lamina propria of gastrointestinal or respiratory tissue.
Identification and characterization of proteinase K-resistant proteins in members of the class Mollicutes
Authors: Butler G H , Kotani H , Kong L , Frick M , Evancho S , Stanbridge E J , McGarrity G J
Published: January 2020

Proteins resistant to proteinase K are rare because of the potency, wide pH optimum, and low peptide bond specificity of this enzyme. Previously, only the prion proteins associated with transmissible spongiform encephalopathies, possibly related proteins in the mollicute Spiroplasma mirum, and proteinase K itself have been reported. We identified a new proteinase K-resistant protein, p40-pr, in two strains of Mycoplasma hyorhinis and in extracts of these organisms. p40-pr's are similar to prion proteins in their resistance to high doses of proteinase K and in the reversal of this resistance by strong denaturing conditions. However, p40-pr's were distinct immunologically, in relative molecular mass, and in their method of extraction. Two immunologically related forms of p40-pr were identified on sodium dodecyl sulfate (SDS) gels and Western immunoblots, a 40-kDa species in boiled samples and a 120-kDa species dissociable by boiling in SDS. Reduction with 2-mercaptoethanol did not affect the mass of p40-pr's or the 120-kDa forms. The development of proteinase K resistance of p40-pr correlated to age-dependent increases in organism protein-lipid ratios. p40-pr-like proteinase K-resistant proteins of 46 to 50 kDa were identified in four of eight additional species of the class Mollicutes but not in S. mirum. However, these mycoplasmal proteins did not react with antibody to the denatured 40-kDa form of M. hyorhinis p40-pr purified by electroelution. The chromatographically purified 46-kDa proteinase K-resistant protein of Mycoplasma orale was an arginine deiminase.
Further analysis of the sequence of the S1 subunit of pertussis toxin
Authors: Pizza M , Bugnoli M , Pucci P , Siciliano R , Marino G , Rappuoli R
Published: January 2020

The two published sequences of the pertussis toxin operon differ in 3 bp in the S1 subunit gene. In this report, we provide evidence that Bordetella pertussis strains are able to produce active pertussis toxin only when they contain one of the two possible nucleotide sequences.
Serological responses to the B subunit of Shiga-like toxin 1 and its peptide fragments indicate that the B subunit is a vaccine candidate to counter action of the toxin
Authors: Boyd B , Richardson S , Gariepy J
Published: January 2020

The B subunit of Shiga toxin and Shiga-like toxin (SLT-1) and its fragments are potentially immunogenic and may generate protective humoral responses against the action of these toxins. We have analyzed the antibody response of rabbits immunized with pure B subunit of SLT-1 or synthetic fragments of the subunit. The immune response to the native B subunit was found to be largely directed at conformational epitopes. More importantly, rabbits immunized with the B subunit were protected from a lethal challenge with SLT-1, indicating that the B subunit represents an excellent vaccine candidate to counter the effects of Shiga toxin and SLT-1 in humans. Polyclonal antibodies against a synthetic peptide corresponding to residues 28 to 40 of the B subunit neutralized the cytotoxicity of SLT-1 towards Vero cells. This region is thus exposed in the native state of the B subunit. The sequence specificity of other antipeptide antisera also provides clues to the state of folding and assembly of the B subunit. Antisera to synthetic peptides representing the N- and C-terminal regions of the SLT-1 B subunit did not cross-react with native B subunit but strongly recognized denatured forms of the protein. Finally, the monoclonal antibody 13C4 was shown to bind to a discontinuous epitope expressed only on the native form of the protein. These immunological reagents can be used to probe the conformational state of the B subunit and the holotoxin as it relates to their functional properties.
Resolution of chlamydial genital infection with antigen-specific T-lymphocyte lines
Authors: Ramsey K H , Rank R G
Published: January 2020

To determine cell-mediated immune mechanisms involved in the resolution of chlamydial genital infection of mice, we utilized an established murine model in which it has been demonstrated that resolution of infection occurs independently of the antibody response. Splenic T lymphocytes were obtained from mice that had previously been immunized with viable elementary bodies of the mouse pneumonitis agent (MoPn), a Chlamydia trachomatis biovar. Antigen-reactive T lymphocytes were maintained and expanded in vitro by frequent restimulation with UV light-inactivated MoPn in the presence of antigen-presenting cells and recombinant interleukin-2 (rIL-2). Flow cytometry indicated that this cell line was at least 92% positive for the pan-specific T-cell marker Thy1.2. Stimulation of the cells in the presence of syngeneic antigen-presenting cells plus MoPn antigen and in the absence of exogenous IL-2 induced the cells to produce IL-2 activity in culture supernatants. Following adoptive transfer, this T-lymphocyte line was effective in inducing resolution of an ongoing MoPn genital infection in congenitally athymic nude mice which otherwise maintain chronic unresolved infections. The line was less efficient in resolving the infection after longer periods in culture. An additional T-lymphocyte line was derived from the spleens of athymic mice that had received the first line and had resolved the infection. These T cells were also capable of inducing resolution of the infection. Lastly, this cell line was treated with specific antibody and complement to delete either CD4+ or CD8+ T lymphocytes in an attempt to enrich for T-cell subpopulations prior to transfer into infected athymic mice. The anti-CD4-treated line was essentially depleted of CD4 cells, while the anti-CD8-treated line was only partially enriched for CD4 cells, with a large proportion of CD8 cells still present. Nude mice that received either of the treated T-cell lines or the parental cell line were capable of resolving the infection, although the line with increased numbers of CD4 cells was more efficient than either the parental line or the CD8 line.
New model for analysis of mucosal immunity: intestinal secretion of specific monoclonal immunoglobulin A from hybridoma tumors protects against Vibrio cholerae infection
Authors: Winner L , Mack J , Weltzin R , Mekalanos J J , Kraehenbuhl J P , Neutra M R
Published: January 2020

Secretory immunoglobulin A (sIgA) plays a role in defense against Vibrio cholerae and other microorganisms that infect mucosal surfaces, but it is not established whether sIgA alone can prevent disease. We report here a strategy for identifying the antigen specificities of monoclonal sIgA antibodies that are capable of providing such protection. IgA hybridomas were generated from Peyer's patch lymphocytes after oral immunization with V. cholerae Ogawa 395. A clone was selected that produced dimeric monoclonal IgA antibodies directed against an Ogawa-specific lipopolysaccharide carbohydrate antigen exposed on the bacterial surface. Hybridoma cells were used to produce subcutaneous "backpack" tumors in syngeneic mice, resulting in secretion of monoclonal sIgA onto mucosal surfaces. Neonatal mice bearing anti-lipopolysaccharide hybridoma backpack tumors were specifically protected against oral challenge with 100 50% lethal doses of virulent Ogawa 395 organisms. Thus, the IgA hybridoma backpack tumor method identifies protective epitopes in the mucosal system and demonstrates that a single monoclonal sIgA can be sufficient to protect against intestinal disease.
Adherence of cell surface mutants of Candida albicans to buccal epithelial cells and analyses of the cell surface proteins of the mutants
Authors: Fukayama M , Calderone R A
Published: January 2020

Strains of Candida albicans, selected on the basis of their reduced agglutination with a polyclonal anti-Candida antiserum, were tested for their adherence to human buccal epithelial cells (BEC). Of four strains, one (A9V2) had reduced binding to BEC in vitro. Adherence of wild type (wt) yeast cells (A9), as measured by the percentage of BEC with adhering Candida cells, was 73.4% +/- 3.8% compared with 49.3% +/- 3.1% for A9V2 (P less than 0.01). From yeast cells of A9 and A9V2, whole-cell extracts and dithiothreitol-, Zymolyase-, or beta-mercaptoethanol-solubilized cell extracts were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting). From dithiothreitol-solubilized cell extracts, proteins with molecular masses of 55 to 60, 80 to 84, 115, and 165 kDa were observed from wt (A9) cells but were highly reduced in amount or absent from A9V2 cells. Western blot profiles of Zymolyase-solubilized extracts from both A9 and A9V2 were similar in appearance, while 55-, 80- to 84-, 115-, and 165-kDa proteins were observed only in A9 cells extracted with beta-mercaptoethanol. Strain A9V4, also selected by reduced agglutination but which adhered as well as strain A9, lacked the 80- to 84-kDa and 115-kDa proteins but otherwise was similar to strain A9. These results indicate that the 55- to 60- and 165-kDa proteins may be related to an adhesin function in C. albicans. The differences observed in the protein profiles of the wt adhering strain and its derived nonadhering mutant are similar to those described for another matched pair of C. albicans strains.
Binding interactions of murine natural killer cells with the fungal target Cryptococcus neoformans
Authors: Murphy J W , Hidore M R , Nabavi N
Published: January 2020

Murine natural killer (NK) cells have been shown to inhibit the growth of the yeastlike organism Cryptococcus neoformans both in vivo and in vitro. An essential first step in NK cell-mediated damage of cryptococcal cells is the binding of the NK cell to the cryptococcal cell. The studies presented here focused on the binding event. Electron photomicrographs and three-dimensional reconstructions of NK cell-C. neoformans conjugates show that NK cells bind to cryptococci through many microvilli. This is in contrast to the broad membrane-membrane interactions which form the binding site of NK cell-YAC-1 tumor cell conjugates. NK cell binding to cryptococci is much slower than NK cell binding to YAC-1 targets. Maximal conjugate formation with cryptococcal targets is reached after 2 h, whereas maximal conjugate formation with YAC-1 targets is obtained after 20 min. Once maximum NK cell-C, neoformans conjugate formation is obtained, another 4 h is required before damage to the cryptococcal cells can be detected with the CFU assay. These data indicate that the binding and action of NK cells on C. neoformans cells requires considerably more time than is necessary for similar events to occur in the NK cell-tumor cell model. NK cell membrane integrity is necessary for NK cells to bind to tumor targets, since some disruption of membrane integrity with 0.1 M dimethyl sulfoxide reduces conjugate formation and tumor cell lysis. In contrast, 0.1 M dimethyl sulfoxide did not diminish NK cell binding to cryptococcal targets; however, it significantly reduced cryptococcal growth inhibition. Although we have observed several differences in NK cell binding to the cryptococcal target compared with NK cell binding to tumor cell targets, there are some similarities in binding interactions of NK cells with the two different targets. Disulfide bonding appears to play a role in the binding of NK cells to both targets, since 5 mM 2-mercaptoethanol, a reagent that reduces disulfide bonds, prevented NK cells from binding to the tumor targets as well as the cryptococcal targets. Actin filaments, components of the cytoskeletal network, must be intact for NK cells to bind to YAC-1 cells or cryptococci. Taken together, our data confirm that binding of NK cells to the cryptococcal target is prerequisite to the stages that result in damage to the cryptococcal cell and that there are similarities and differences in NK cell-binding interactions with structurally different target cells.
Morphological and immunohistochemical studies of the lungs and bronchus-associated lymphoid tissue in a rat model of chronic pulmonary infection with Pseudomonas aeruginosa
Authors: Iwata M , Sato A
Published: January 2020

Pseudomonas aeruginosa is one of the most frequently encountered bacterial pathogens in patients with chronic pulmonary infections, including cystic fibrosis and diffuse panbronchiolitis. Bronchus-associated lymphoid tissue (BALT), noted frequently in patients with cystic fibrosis and diffuse panbronchiolitis, is considered to play an important role in the local immunologic defense mechanisms in the respiratory tract. To investigate the role of BALT in chronic pulmonary infections, we developed an animal model for chronic pulmonary infection and studied the morphological and immunohistochemical characteristics of BALT. Experimental pneumonia was produced in rats by intratracheal inoculation of P. aeruginosa enmeshed in agar beads. The histological changes corresponded to those occurring in chronic bronchiolitis. Immunohistochemically, surface immunoglobulin M-positive (sIgM+) cells and sIgA+ cells were recognized in the inflamed bronchial walls from day 4, and sIgG+ cells were recognized from day 14, W3/25+ cells exceeded OX8+ cells in number until day 14. In the BALT, there was a massive accumulation of lymphocytes in the lymphatics and high endothelial venules. The development of germinal centers was accompanied by increased numbers of sIgM+ and sIgA+ cells. W3/25+ cells exceeded OX8+ cells in number in the BALT until day 14. On the other hand, OX8+ cells were predominant in comparison with W3/25+ cells at day 21, and then both sIgM+ and sIgA+ cells and inflammatory changes in the lung decreased at day 28. These findings suggest that BALT regulates the local immune responses against chronic pulmonary infection due to P. aeruginosa.
Activation of particulate guanylate cyclase by Escherichia coli heat-stable enterotoxin is regulated by adenine nucleotides
Authors: Gazzano H , Wu H I , Waldman S A
Published: January 2020

Guanylate cyclase is regulated by adenine nucleotides in membranes of intestinal mucosal cells. Basal guanylate cyclase was activated about twofold by adenine nucleotides. Activation was specific for adenine, as compared with the pyrimidine nucleotides UTP and CTP. In addition, enzyme activation was obtained in the presence of saturating concentrations of GTP, the substrate for guanylate cyclase. The most potent adenine nucleotide was the nonhydrolyzable analog of ATP, adenosine 5'-O-(3-thiotriphosphate). Adenine nucleotide activation was specific for the particulate form of guanylate cyclase, as compared with the soluble form. Also, adenine nucleotides potentiated the activation of guanylate cyclase by the heat-stable enterotoxin produced by Escherichia coli. Indeed, enzyme activation by adenine nucleotides and toxin was greater than the sum of individual activations by these agents. Adenine nucleotides regulate guanylate cyclase by increasing the maximum velocity of the enzyme without altering its affinity for substrate or its cooperativity. In addition to stimulating guanylate cyclase, adenine nucleotides decreased the specific binding of the heat-stable enterotoxin to receptors in intestinal membranes. The coordinated regulation of the toxin-receptor interaction and guanylate cyclase activity by a process utilizing nonhydrolyzable analogs of a purine nucleotide is similar to the mechanisms involved in the hormone regulation of adenylate cyclase by guanine nucleotide-binding proteins. These data suggest that an adenine nucleotide-dependent protein may couple the toxin-receptor interaction to the regulation of particulate guanylate cyclase in intestinal membranes.
Characterization of microneme antigens of Cryptosporidium parvum (Protozoa, Apicomplexa)
Authors: Bonnin A , Dubremetz J F , Camerlynck P
Published: January 2020

Two monoclonal antibodies (MAbs) raised against purified excysted oocysts and sporozoites of cryptosporidium parvum reacted in an immunofluorescence assay with antigens located at the anterior pole of the zoites. On Western blots of purified oocysts, these MAbs reacted with a series of bands between 210 and 40 kDa; several of these bands were recognized by both MAbs; others were specific. One MAb (TOU) did not react after periodic acid treatment and was therefore considered to recognize a carbohydrate epitope; as determined by immunoelectron microscopy, this MAb reacted on micronemes of sporozoites and merozoites and also with the peripheral cytoplasm and the parasitophorous vacuole of trophozoites and macrogametes. The other MAb (HAD) reacted with an epitope that was insensitive to periodate treatment but did not react in the immunoelectron microscopy assay. However, the similar labeling pattern obtained with the immunofluorescence assay with both MAbs and the fact that the two antibodies share common bands on Western immunoblots suggest that both MAbs react with molecules located in Cryptosporidium micronemes, one reacting with a glycannic epitope and the other reacting with a peptidic epitope.
Production of recombinant Bordetella pertussis serotype 2 fimbriae in Bordetella parapertussis and Bordetella bronchiseptica: utility of Escherichia coli gene expression signals
Authors: Walker M J , Guzmán C A , Rohde M , Timmis K N
Published: January 2020

Serotype-specific fimbriae of Bordetella pertussis are considered potential components of new-generation vaccines against whooping cough. Attempts to characterize fimbriae, and indeed other virulence determinants, produced by B. pertussis have been frustrated on one hand by low yields from B. pertussis itself and on the other by an inability to produce native recombinant products in Escherichia coli. In order to try to circumvent this problem, we have examined the expression of B. pertussis serotype 2 fimbriae in Bordetella parapertussis and Bordetella bronchiseptica from native as well as E. coli expression signals. These studies revealed that the fimbrial gene product was expressed from the original B. pertussis promoter and Shine-Dalgarno sequence in both B. parapertussis and B. bronchiseptica. The transcriptional start site of the gene was located 146 nucleotides upstream of its ATG start codon. A recombinant fimbrial subunit gene containing PLAC and the atpE translation initiation region of E. coli was also expressed in B. bronchiseptica. In all cases in which gene expression was detected the gene product was expressed as serotype 2-specific fimbriae as determined by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopic investigation of the bacterial cell surface.
Cutaneous cryptococcosis in athymic and beige-athymic mice
Authors: Salkowski C A , Balish E
Published: January 2020

The dermotropism of Cryptococcus neoformans SLHA in congenitally athymic (nu/nu) and doubly immunodeficient beige-athymic (bg/bg-nu/nu) mice is described. Both bg/bg-nu/nu and nu/nu mice developed cutaneous cryptococcosis within 7 to 12 days following intravenous challenge with 10(4) encapsulated yeast cells. Macroscopically, cutaneous lesions appeared as small subcutaneous nodules without ulceration. Cryptococcal skin lesions were observed primarily on the flank of nu/nu mice, whereas skin lesions in bg/bg-nu/nu mice were distributed over the trunk, abdomen, and face. While bg/bg-nu/nu mice had four times as many macroscopic skin lesions as nu/nu mice on day 14 after intravenous challenge, the skin lesions in nu/nu mice were larger. Histopathology revealed large foci of encapsulated yeasts extending from the basement membrane of the epidermis through the dermis to the underlying musculature. Yeasts in these lesions evoked a minimal inflammatory response that consisted primarily of macrophages. Interestingly, yeast cells appeared to be degrading collagen bundles located in the dermis. The dermotropic strain used in this study produced gelatinase and other proteases in vitro. These results indicate that C. neoformans can be dermotropic in a T-cell-deficient host and that proteases may be a virulence factor(s).