bluuebunny/crossref_metadata_2025 huggingface dataset:
Intercellular spread of Shigella flexneri through a monolayer mediated by membranous protrusions and associated with reorganization of the cytoskeletal protein vinculin
Authors: Kadurugamuwa J L , Rohde M , Wehland J , Timmis K N
Published: January 2020

The spread of Shigella flexneri in a monolayer of infected Henle and HeLa cells was studied by using immunofluorescence and electron microscopy. Infected cells produced numerous bacterium-containing membranous protrusions up to 18 microns in length that penetrated adjacent cells and were subsequently phagocytosed. Fluorescence staining of actin and vinculin in infected cells with phalloidin and monoclonal antibody to vinculin, respectively, demonstrated that the protrusions containing the bacteria consisted of these cytoskeletal proteins. Actin accumulated predominantly at the poles of bacteria distal to the tip of protrusions and appeared as trails extending back towards the host cell cytoplasm. Vinculin, however, was distributed uniformly around the bacteria and throughout the protrusion. A profound rearrangement of vinculin occurred in Henle and HeLa cells following infection with shigellae: whereas in uninfected cells it was distributed mainly around the cell periphery, in infected cells it concentrated mainly around clusters of bacteria in the cytoplasm. This suggests a possible involvement of the vinculin cytoskeletal protein in the intercellular spread of shigellae during an infection.
Cryptococcus neoformans serotype A glucuronoxylomannan-protein conjugate vaccines: synthesis, characterization, and immunogenicity
Authors: Devi S J , Schneerson R , Egan W , Ulrich T J , Bryla D , Robbins J B , Bennett J E
Published: January 2020

We synthesized Cryptococcus neoformans serotype A glucuronoxylomannan (GXM) conjugate vaccines under conditions suitable for human use to prevent disseminated cryptococcosis. The purified, sonicated GXM was derivatized with adipic acid dihydrazide through either hydroxyl or carboxyl groups and then covalently bound to tetanus toxoid (TT) or Pseudomonas aeruginosa exoprotein A (rEPA). The immunogenicity of these conjugates was evaluated in BALB/c and general purpose mice by subcutaneous injection in saline. The conjugates elicited higher GXM antibody responses than GXM alone. Booster immunoglobulin G (IgG) and IgM responses were elicited by all conjugates in BALB/c mice. The conjugates prepared through hydroxyl activation (GXM-TT2 and GXM-rEPA) were more immunogenic than the one prepared through carboxyl activation (GXM-TT1). GXM antibody response was enhanced by the administration of monophosphoryl lipid A 2 days following the injection of GXM-TT2 (P less than 0.03). The conjugates also elicited IgG antibodies to the carrier proteins. Gel diffusion tests using conjugate-induced hyperimmune sera and chemically modified GXMs suggested that the specificity of GXM-TT1-induced antibodies was conferred by the O-acetyl groups. Hyperimmune sera generated by GXM-TT2 precipitated with the chemically unmodified and the de-O-acetylated GXMs but not with the carboxyl-reduced and de-O-acetylated GXM. GXM-TT2-induced hyperimmune serum also precipitated with the capsular polysaccharides of C. neoformans serotypes D, B, and C. The conjugate vaccines prepared through hydroxyl activation of the GXM are sufficiently immunogenic and appear to be suitable for clinical evaluation.
Extraction, purification, and characterization of major outer membrane proteins from Wolinella recta ATCC 33238
Authors: Kennell W L , Holt S C
Published: January 2020

The outer membrane of Wolinella recta ATCC 33238 was isolated by French pressure cell disruption and differential centrifugation. Outer membrane proteins (OMPs) were solubilized by Zwittergent 3.14 extraction and separated by DEAE-Sephacel ion-exchange chromatography. The major OMPs that were found in W. recta ATCC 33238 and in several other Wolinella spp. consisted of proteins with apparent molecular masses of 51, 45, and 43 kDa. These three conserved proteins were purified to essential homogeneity by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and characterized chemically. Heating at between 75 and 100 degrees C revealed both the 43- and 51-kDa proteins to be heat modified from apparent molecular masses of 32 and 38 kDa, respectively. The 45-kDa protein was unmodified at all temperatures tested. Two-dimensional isoelectric focusing-SDS-PAGE revealed the 51-kDa protein to be composed of multiple pIs between a pH of 5.0 and greater than 8.0 while the 43- and 45-kDa proteins had a pI of approximately 6.0. N'-terminal amino acid sequence analysis of the first 30 to 40 amino acids and search of the Protein Identification Resource data base for similar proteins only revealed the 43-kDa protein to be similar to the P.69 OMP of Bordetella pertussis; however, the homology was weak (33%). Amino acid analysis revealed the 43-kDa protein to be noncharged and the 45- and 51-kDa proteins to be hydrophilic, containing between 38 to 42% polar residues but no cysteine. This study reports the purification and partial characterization of three conserved proteins in W. recta ATCC 33238.
Efficacy of a Proteus mirabilis outer membrane protein vaccine in preventing experimental Proteus pyelonephritis in a BALB/c mouse model
Authors: Moayeri N , Collins C M , O'Hanley P
Published: January 2020

A BALB/c mouse model of nonobstructive, ascending Proteus mirabilis pyelonephritis was characterized bacteriologically, histologically, and serologically from 3 to 28 days. Intravesicular administration of 2 X 10(8) P. mirabilis K7 resulted in the septic death of 9 (16%) of 57 mice by day 15. Among the survivors, K7 colonized the kidneys in great numbers until day 21. Histological examination of the kidneys revealed acute inflammation which was characterized by neutrophil infiltration by day 3, renal necrosis by day 7, and fibroblastic infiltration by day 14 which persisted at least until day 28. The immunoglobulin G response to the outer membrane proteins (OMP) was assessed by enzyme-linked immunosorbent assay and Western blotting (immunoblotting). Anti-OMP immunoglobulin G antibodies were detected as early as day 7, and the reciprocals of their titers rose progressively up to day 28 (i.e., greater than or equal to 500). This model was also used to assess the efficacy of OMP and lipopolysaccharide (LPS) immunization in preventing renal infection. K7 OMP or LPS (100 micrograms) preparations were administered intramuscularly in Freund's complete adjuvant. After 2 weeks, mice were intravesicularly challenged with 2 X 10(8) bacteria of the homologous K7 strain or one of four heterologous strains. Compared with the saline-immunized control group and K7 LPS-immunized mice, K7 OMP recipients were protected from death when challenged by homologous or heterologous strains. In addition, K7 OMP recipients were protected (P less than 0.003) from subsequent renal infection when challenged by the K7 strain and had more rapid bacterial renal clearance when challenged by three of four heterologous strains. OMP recipients produced antibodies which bound major OMP moieties (viz., 36- to 39-kDa cell wall constituents) as assessed by Western blotting. These results support the concept that immunization with selected bacterial protein surface coat constituents can prevent uromucosal infection by interfering with colonization or renal injury.
Scanning and transmission electron microscopic study of adherence of Escherichia coli O103 enteropathogenic and/or enterohemorrhagic strain GV in enteric infection in rabbits
Authors: Licois D , Reynaud A , Federighi M , Gaillard-Martinie B , Guillot J F , Joly B
Published: January 2020

The GV strain (serotype O103:H2:K-), originally isolated from a diarrheic rabbit, is an enteropathogenic Escherichia coli strain that produces diarrhea without synthesizing the classical enterotoxins and that is not invasive. This strain is characterized by a 117-kb plasmid (pREC-1). Histological study of the gut by scanning electron microscopy and transmission electron microscopy was performed on the GV strain, on a derivative strain cured of pREC-1, and on transconjugants obtained by transfer of pREC-1 to nonpathogenic strains E. coli K-12 and 6100, not belonging to the O103 serogroup. The GV strain adhered to the epithelial cells of the ileum and large intestine, whereas the cured GV strain did not. Transfer of plasmid pREC-1 to E. coli K-12 or 6100 allowed the bacteria to attach to the intestinal mucosa in the same manner as that of the wild-type GV strain. Thus, pREC-1 seems to play an important role in attachment to and colonization of the intestinal tract of rabbits by E. coli serogroup O103. Scanning electron microscopy showed numerous bacteria attached together and closely associated with intestinal villi. Transmission electron microscopy revealed effacing lesions characteristic of enteropathogenic E. coli strains: effacing of microvilli and cuplike projections (pedestal formations) associated with an acute inflammatory and hemorrhagic response. In contrast with the results reported for rabbit pathogenic O15 strains, it appeared that the Peyer's patches were not involved in the early stages of infection with the O103 GV strain. This strain may represent a model for the study of the virulence and pathogenic effects of enteropathogenic and enterohemorrhagic E. coli strains.
Persistent cryptosporidiosis in horses with severe combined immunodeficiency
Authors: Bjorneby J M , Leach D R , Perryman L E
Published: January 2020

Cryptosporidial infections were established in five young foals with severe combined immunodeficiency following oral administration of 10(8) Cryptosporidium parvum oocysts. All foals shed oocysts (average of 8 x 10(6) to 2 x 10(8)/g of feces) until death. Inflammation and C. parvum organisms were observed in the common bile duct, duodenum, jejunum, and ileum. Since foals with severe combined immunodeficiency lack functional T and B lymphocytes and are incapable of antigen-specific immune responses, they are well suited for evaluating the pathogenesis and treatment of persistent cryptosporidiosis.
Comparison of pertussis toxin (PT)-neutralizing activities and mouse-protective activities of anti-PT mouse monoclonal antibodies
Authors: Sato H , Sato Y , Ohishi I
Published: January 2020

An assay of neutralizing activity against CHO cell-clustering activity, one of the most common assays for anti-pertussis toxin antibodies, did not correlate well with an assay of the protective activities of monoclonal antibodies. A better correlation between neutralization of leukocytosis-promoting or islet-activating activity and mouse protection against aerosol challenge was seen.
Prevention of murine cerebral malaria by a stable prostacyclin analog
Authors: Sliwa K , Grundmann H J , Neifer S , Chaves M F , Sahlmüller G , Blitstein-Willinger E , Bienzle U , Kremsner P G
Published: January 2020

Iloprost, a synthetic prostacyclin analog, successfully prevents the development of cerebral malaria in mice. Malaria antigen-induced tumor necrosis factor (TNF) production could be inhibited by iloprost in vitro and in vivo. Northern analysis of TNF mRNA revealed that malaria antigen-induced TNF expression was suppressed at the transcription level.
Isolation and molecular characterization of spontaneously occurring cytolysin-negative mutants of Actinobacillus pleuropneumoniae serotype 7
Authors: Anderson C , Potter A A , Gerlach G F
Published: January 2020

Actinobacillus pleuropneumoniae serotype 7 strains are shown to spontaneously lose cytolytic activity with a frequency of approximately 10(-4). The phenotypic change is associated with the loss of approximately 8.5 kbp of chromosomal DNA. A genomic fragment encoding the cytolysin and its flanking sequences was cloned and characterized. Also, the corresponding truncated fragment was cloned from a spontaneous mutant. Comparison of the two clones allowed the definition of the excision site. The ends of the excised fragment are composed of 1,201 bp long direct identical repeats, possibly facilitating the genotypic change by homologous recombination. In accordance with this hypothesis, one repeat is conserved in the spontaneous mutant. Each repeat contains one open reading frame preceded by a Shine-Dalgarno consensus sequence, and the ends of each repeat contain 26-bp complementary sequences with four mismatches.
Expression of adhesion molecules in leprosy lesions
Authors: Sullivan L , Sano S , Pirmez C , Salgame P , Mueller C , Hofman F , Uyemura K , Rea T H , Bloom B R , Modlin R L
Published: January 2020

Leprosy presents as a clinical spectrum that is precisely paralleled by a spectrum of immunological reactivity. The disease provides a useful and accessible model, in this case in the skin, in which to study the dynamics of cellular immune responses to an infectious pathogen, including the role of adhesion molecules in those responses. In lesions characterized by strong delayed-type hypersensitivity against Mycobacterium leprae (tuberculoid, reversal reaction, and Mitsuda reaction), the overlying epidermis exhibited pronounced keratinocyte intracellular adhesion molecule 1 (ICAM-1) expression and contained lymphocytes expressing the ICAM-1 ligand, LFA-1. Conversely, in lesions in which delayed-type hypersensitivity was lacking (lepromatous), keratinocyte ICAM-1 expression was low and LFA-1+ lymphocytes were rare. Expression of these adhesion molecules on the cells within the dermal granulomas was equivalent throughout the spectrum of leprosy. The percentage of lymphocytes in these granulomas containing mRNA coding for gamma interferon and tumor necrosis factor alpha, synergistic regulators of ICAM-1 expression, paralleled epidermal ICAM-1 expression. In lesions of erythema nodosum leprosum, a reactional state of lepromatous leprosy thought to be due to immune complex deposition, keratinocyte ICAM-1 expression and gamma interferon mRNA+ cells were both prominent. Antibodies to LFA-1 and ICAM-1 blocked the response of both alpha beta and gamma delta T-cell clones in vitro to mycobacteria. Overall, the expression of adhesion molecules by immunocompetent epidermal cells, as well as the cytokines which regulate such expression, correlates with the outcome of the host response to infection.
Extracellular release of reactive oxygen species from human neutrophils upon interaction with Escherichia coli strains causing renal scarring
Authors: Mundi H , Björkstén B , Svanborg C , Ohman L , Dahlgren C
Published: January 2020

The production of reactive oxygen metabolites by neutrophils plays a key role in the host defense against invading microorganisms and in tissue damage resulting from infection. In the present study we measured the ability of different uropathogenic Escherichia coli strains to induce generation of oxygen metabolites upon interaction with human neutrophils. The strains were selected to represent two groups of patients with recurrent episodes of acute pyelonephritis: one with renal scars (12 strains) and one without renal scarring (11 strains). The majority of strains (from both groups) induced a pronounced neutrophil respiratory burst activity. When the intracellular and extracellular oxidative responses were measured separately, it was found that the response induced by nonscarring strains was primarily of intracellular (intraphagosomal) origin, whereas a proportionally larger fraction of the response induced by the scarring strains was extracellular. Since reactive oxygen products are toxic to the renal tissue, this release can be of importance in the development of renal scars.
Release of leukotriene B4 from human neutrophils after interaction with nontypeable Haemophilus influenzae
Authors: Garofalo R , Faden H , Sharma S , Ogra P L
Published: January 2020

Opsonization of nontypeable Haemophilus influenzae with antibody is critical for the interaction between the organism and human polymorphonuclear leukocytes (PMNs). Nontypeable H. influenzae opsonized in fresh antibody-positive serum induced the release of 42.5 +/- 17.9 ng of leukotriene B4 per ml from PMNs after 20 min of incubation at 37 degrees C. On the other hand, opsonization of the organisms in fresh antibody-negative serum stimulated the release of significantly smaller amounts of leukotriene B4 by the PMNs. Simultaneous determinations of phagocytosis demonstrated similar patterns of response. A small amount (26.7 +/- 7.6%) of unopsonized nontypeable H. influenzae was phagocytosed by PMNs during 20 min of incubation at 37 degrees C. In contrast, 89.3 +/- 2.0% of nontypeable H. influenzae opsonized in fresh antibody-positive serum was phagocytosed during the same incubation period (P less than 0.001). Removal of complement through heat inactivation at 56 degrees C for 30 min did not significantly affect phagocytosis. These data suggest that the humoral immune response to nontypeable H. influenzae plays an important role in the inflammatory process and may contribute to the production of middle ear effusions in otitis media.
Inhibition of chemotaxis of neutrophil leukocytes to interleukin-8 by endotoxins of various bacteria
Authors: Bignold L P , Rogers S D , Siaw T M , Bahnisch J
Published: January 2020

The effects of endotoxins from various bacteria (Escherichia coli, Klebsiella pneumoniae, Vibrio cholerae, Shigella flexneri, Salmonella typhosa, and Pseudomonas aeruginosa) on chemotaxis of neutrophil leukocytes to formyl peptide and interleukin-8 were tested in an improved chemotaxis assay involving a "sparse-pore" polycarbonate (Nuclepore) membrane in a Boyden-type chamber. The possible chemotactic activity of the endotoxins themselves were tested by the same technique. In addition, the effects of these substances on random motility of neutrophils were tested with a corresponding assay involving similar chambers fitted with membranes of standard pore density. Possible activation of the complement system of serum by each endotoxin was tested with sheep erythrocyte assays and the maximum endotoxin concentration (100 micrograms/ml) used in the chemotaxis and motility assays. All endotoxins inhibited chemotaxis of neutrophils to interleukin-8. No endotoxin affected chemotaxis to formyl peptide or was itself chemotactic for neutrophils. Endotoxin of S. flexneri inhibited random motility of neutrophils, while the others had no such effect. Endotoxins of K. pneumoniae and of P. aeruginosa produced moderate and marked inhibition, respectively, of total complement, as measured by hemolysis of sheep erythrocytes, without affecting the levels of C3c and C4 in these assays. Endotoxins of the other bacteria had no demonstrable effect in any of these assays of complement activation. These results suggest that chemotaxis to interleukin-8 may be mediated by cellular mechanisms different from those involved in chemotaxis to formyl peptide. Furthermore, the presence of these endotoxins could be significant for the suppression of neutrophil accumulation in inflammatory lesions mediated by interleukin-8.
Preparation, characterization, and immunogenicity of conjugates composed of the O-specific polysaccharide of Shigella dysenteriae type 1 (Shiga's bacillus) bound to tetanus toxoid
Authors: Chu C Y , Liu B K , Watson D , Szu S S , Bryla D , Shiloach J , Schneerson R , Robbins J B
Published: January 2020

The background for developing conjugate vaccines for shigellosis composed of the O-specific polysaccharide (O-SP) bound to a protein is described elsewhere (C. Y. Chu, R. Schneerson, and J. B. Robbins, submitted for publication). Briefly, there is direct evidence for type (lipopolysaccharide [LPS])-specific protection after infection with the wild type or with attenuated strains of shigellae. Prospective studies of Israeli armed forces recruits show a correlation between preexisting serum immunoglobulin G (IgG) LPS antibodies and resistance to shigellosis (D. Cohen, M. S. Green, C. Block, R. Slephon, and I. Ofek, J. Clin. Microbiol. 29:386-389, 1991). In order to elicit IgG LPS-specific antibodies to Shigella dysenteriae type 1, the O-SP of this pathogen was purified and bound to tetanus toxoid (TT) by three schemes. The most immunogenic used a modification of a published method (C. Y. Chu, R. Schneerson, J. B. Robbins, and S. C. Rastogi, Infect. Immun. 40:245-256, 1983). The resultant O-SP-TT conjugates were stable and elicited high levels of IgG O-SP antibodies and booster responses in young mice when injected subcutaneously in saline at 1/10 the proposed human dose. Adsorption onto alum or concurrent administration with monophosphoryl lipid A enhanced both the IgG and IgM antibody responses to the O-SP of the conjugate; both the nonadsorbed and adsorbed conjugates elicited higher rises of IgG than of IgM antibodies. Clinical evaluations of S. dysenteriae type 1 O-SP-TT conjugates are planned.
Cytolysins of Actinobacillus pleuropneumoniae serotype 9
Authors: Smits M A , Briaire J , Jansen R , Smith H E , Kamp E M , Gielkens A L
Published: January 2020

Cytolysin I (ClyI) and cytolysin II (ClyII), which are present in the culture supernatant of Actinobacillus pleuropneumoniae serotype 9, are thought to play an important role in the pathogenesis of pig pleuropneumonia. The purpose of this study was to clone and characterize the genetic determinants of these cytolysins. Cloning was accomplished by the screening of DNA libraries for the presence of cytolytic activity and for the presence of DNA sequences homologous to leukotoxin DNA of Pasteurella haemolytica. Both genetic determinants were found to be members of the RTX cytotoxin family. The ClyII determinant was characterized in more detail. It appeared that ClyII more closely resembled the leukotoxin of P. haemolytica than the alpha-hemolysin of Escherichia coli. The ClyII amino acid sequence was identical to a hemolysin gene sequence of A. pleuropneumoniae serotype 5; this finding indicates that the latter gene also codes for ClyII and not for ClyI, as has previously been suggested. The genetic organization of the ClyII determinant differed from the genetic organization of other RTX determinants. Genes responsible for secretion of ClyII were not contiguous with the toxin gene. Instead, secretion genes were present elsewhere in the genome. These secretion genes, however, belong to the ClyI operon. This indicates that the secretion genes of the ClyI operon are responsible for secretion of ClyI and ClyII.
Effects of gamma interferon, tumor necrosis factor alpha, and interleukin-2 on infection and proliferation of Theileria parva-infected bovine lymphoblasts and production of interferon by parasitized cells
Authors: DeMartini J C , Baldwin C L
Published: January 2020

Theileria parva is a protozoan parasite that infects bovine B cells and alpha beta and gamma delta T cells and transforms them into continually proliferating cells. CD4+ T. parva-antigen-specific immune T cells have been shown to produce cytokines in response to stimulation with parasitized cells, and T. parva-infected lymphocytes produce and consume T-cell growth factors and interleukin-2 (IL-2). To ascertain the role of T-cell cytokines on T. parva infections, we evaluated recombinant gamma interferon (rIFN-gamma), rIL-2, and tumor necrosis factor alpha (rTNF-alpha) for their effects on establishment, proliferation, and survival of parasitized cells. The results indicate that neither rIFN-gamma nor rTNF-alpha had an enhancing or inhibitory effect on the growth of established T. parva-infected T-cell clones, whereas bovine rIL-2 increased the proliferation of infected B-cell and alpha beta T-cell clones but not that of gamma delta T-cell clones. To evaluate the effects of the cytokines on establishment of parasitized cell lines, peripheral blood mononuclear cells were cultured in their presence immediately following infection with T. parva sporozoites. Neither rIFN-gamma nor rIL-2 altered the proportion of cells initially developing schizonts, but both enhanced establishment of infected cell lines by about twofold. In contrast, rTNF-alpha resulted in about a 33% decrease in the proportion of schizont-infected cells. Inhibitory effects on establishment of parasitized cell lines by rTNF-alpha were no longer apparent by 12 days following infection. Tests conducted during this study indicated that T. parva-infected lymphocytes also spontaneously produce IFN that is neutralized by acidic pH treatment. In conclusion, we speculate that none of these T-cell cytokines are likely to have a profound inhibitory effect in vivo on T. parva infections. Instead, IFN-gamma and IL-2 may facilitate the establishment of infection by T. parva.
Binding of a Streptococcus mutans cationic protein to kidney in vitro
Authors: Choi S H , Stinson M W
Published: January 2020

An 8-kDa protein, with binding activity for heparin and heparan sulfate of basal laminae of animal tissues, was isolated from Streptococcus mutans MT703 and purified to homogeneity. Binding of radioiodinated 8-kDa protein to rabbit kidney tissue in vitro showed a high degree of specificity, as indicated by saturation kinetics, time dependence, and competitive inhibition by unlabeled protein. Binding activity for kidney tissue was competitively inhibited by selected glycosaminoglycans and polyanions in the following order: heparin greater than dextran sulfate greater than heparan sulfate greater than chondroitin sulfate greater than lipoteichoic acid greater than keratan sulfate greater than hyaluronic acid. Binding of the streptococcal protein to rabbit kidney tissue was also strongly inhibited by protamine sulfate, polylysine, and a random copolymer of lysine and alanine. Among the monosaccharides tested at 50 mM, glucosamine 2,3- or 2,6-disulfate, glucuronic acid, glucose 6-phosphate, and glucose 6-sulfate inhibited 50% or more of the binding activity, whereas N-acetylglucosamine 3-sulfate, glucosamine 6-sulfate, N-acetyl-glucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and a selection of neutral sugars were not inhibitory. The heparin-binding protein was detected on the cell wall of S. mutans and in the culture medium following growth. Several other species of streptococci produce an immunologically related protein of similar size.
Evidence for peptidoglycan absorption in rats with experimental small bowel bacterial overgrowth
Authors: Lichtman S N , Keku J , Schwab J H , Sartor R B
Published: January 2020

Surgical creation of jejunal self-filling blind loops (SFBL) causes small bowel bacterial overgrowth which is associated with hepatobiliary inflammation in the susceptible Lewis and Wistar rat strains. Since hepatic injury occurs when small bowel anaerobic bacterial concentrations are increased 4 to 6 log10 units per ml and hepatic bacterial cultures are negative, we postulate that the inflammation is caused by absorption of phlogistic cell wall polymers originating from bacteria within the loop. To demonstrate absorption of bacterial cell wall polymers, we measured plasma and hepatic levels of immunoreactive peptidoglycan-polysaccharide (PG-PS) following intraluminal injection as well as anti-PG antibodies as an indirect measure of absorption and/or accumulation of endogenous PG. PG-PS purified from group A streptococci was detected in plasma by enzyme-linked immunosorbent assay after intraluminal injection; rats with SFBL showed significantly more uptake into plasma and the liver than sham-operated rats or SFBL rats which were treated with metronidazole (P less than 0.025). Total plasma immunoglobulin A (IgA), IgG, and IgM levels did not differ among sham-operated rats and those with self-emptying blind loops or SFBL, but plasma anti-PG IgA (P less than 0.05), IgG, and IgM (P less than 0.01) levels were increased in rats with SFBL. Metronidazole and tetracycline prevented the elevation of anti-PG antibody, but gentamicin and polymyxin B did not. Anti-lipid A, anti-soy protein, and anti-chow antibodies in plasma were not consistently increased in rats with SFBL indicating the lack of a generalized antibody response to luminal antigens. These data suggest that PG from normal flora bacteria is absorbed from the intestinal lumen and that mucosal injury and/or increased luminal concentrations of PG, such as those induced by small bowel bacterial overgrowth, lead to enhanced absorption of potentially inflammatory bacterial polymers.
Blood stage-induced Plasmodium brasilianum infection in the squirrel monkey induces antibodies which react with the circumsporozoite protein
Authors: Cochrane A H , Maracic M
Published: January 2020

A blood stage-induced P. brasilianum infection in a naive squirrel monkey induced antibodies which reacted with the circumsporozoite protein of the parasite. Titers increased with duration of infection and persisted for 3 months after cure. In an immunoblot, these antibodies detected two polypeptides with molecular weights identical to those of the circumsporozoite protein and its precursor.
Sequence diversity of the 60-kilodalton protein and of a putative 15-kilodalton protein between the trachoma and lymphogranuloma venereum biovars of Chlamydia trachomatis
Authors: de la Maza L M , Fielder T J , Carlson E J , Markoff B A , Peterson E M
Published: January 2020

DNA from Chlamydia trachomatis serovars L3, C, and E corresponding to the open reading frames of the 60-kDa protein and of a putative 15-kDa protein was sequenced. The open reading frames coding for the 60-kDa protein had 1,641 bp in the three serovars. Compared with the L3 serovar, there were 9 and 11 amino acid changes in the C and E serovars, respectively. The open reading frames corresponding to the putative 15-kDa protein had 450, 456, and 453 bp for the L3, C, and E serovars, respectively. When compared with the L3 serovar, the C and E serovars had 14 and 16 amino acid differences, respectively.
Purification and characterization of major antigens from a Mycobacterium bovis culture filtrate
Authors: Fifis T , Costopoulos C , Radford A J , Bacic A , Wood P R
Published: January 2020

Ten major antigens from Mycobacterium bovis culture filtrate of 39, 32, 30, 25, 24, 22 (a and b forms), 19, 15, and 12 kDa have been purified and characterized by classical physicochemical methods. With monoclonal antibodies and/or N-terminal amino acid sequencing data, it was found that the antigens of 32, 30, 24, 22 (a), 19, and 12 kDa are related to M. bovis or M. tuberculosis antigens P32, MPB59, MPB64, MPB70, 19 kDa, and 12 kDa, respectively. The 39-, 25-, 22 (b)-, and 19-kDa antigens showed concanavalin A-binding properties and were positive in a glycan detection test, suggesting that they are glycoproteins. The 25- and 22 (b)-kDa proteins were found to be glycosylated forms of MPB70.
Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II
Authors: Perera L P , Samuel J E , Holmes R K , O'Brien A D
Published: January 2020

Shiga-like toxin type II (SLT-II) is one of two antigenically distinct cytotoxins produced by enterohemorrhagic Escherichia coli that are believed to play a central role in the pathogenesis of enterohemorrhagic E. coli-induced disease. SLT-II is a bipartite toxin with an enzymatically active A subunit that inhibits protein synthesis and an oligomeric B subunit that binds to the glycolipid globotriaosylceramide on eukaryotic cells. In this study, functional boundaries of the slt-II operon were mapped. Mutant proteins lacking the last four amino acids from the carboxy terminus of the 70-amino-acid mature SLT-II B polypeptide had no cytotoxic activity. However, when only two amino acids were removed from the carboxy terminus of the B subunit, the cytotoxic activity of the holotoxin was not altered drastically. Furthermore, a 21-amino-acid extension to the carboxy terminus of the SLT-II B polypeptide was tolerated with a minimum reduction in cytotoxic activity of the holotoxin. Deletion of the region coding for amino acids 3 through 18 of the 296-amino-acid mature SLT-II A polypeptide resulted in complete ablation of the cytotoxic activity of the holotoxin as well as abolition of the enzymatic activity of the A subunit. Thus, it appears that both 5'- and 3'-terminal coding sequences are essential for function of the slt-II operon.
Splenic and granuloma T-lymphocyte responses to fractionated soluble egg antigens of Schistosoma mansoni-infected mice
Authors: Lukacs N W , Boros D L
Published: January 2020

Soluble egg antigens (SEA) secreted by the eggs of Schistosoma mansoni worms induce a T-cell-mediated granulomatous response that is principally responsible for the pathology of the disease. In the present study sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated SEA proteins were divided into nine fractions (less than 21, 25 to 30, 32 to 38, 40 to 46, 50 to 56, 60 to 66, 70 to 90, 93 to 125, and greater than 200 kDa), electroeluted, and utilized in in vitro lymphoproliferation assays. T-cell-enriched spleen cells from acutely infected mice responded to all nine fractions, while those from chronically infected mice responded to only the 50- to 56- and the 60- to 66-kDa fractions. Depletion of the CD4+ T-cell subset among acute and chronic-infection spleen cells abrogated the response. Depletion of the CD8+ T-cell population resulted in increased proliferation in response to fractions by acute-infection T cells and facilitated responsiveness to hitherto-inactive SEA fractions in chronic-infection T cells. Acute-infection CD4+ granuloma T cells responded to the 40- to 46-, 50- to 56-, 70- to 90-, 93- to 125-, and greater than 200-kDa fractions, while the chronic-infection granuloma T cells responded only to the greater than 200-kDa fraction of SEA. Selective depletion of the CD4+ T-cell subset when acute-infection granuloma lymphocytes were tested abrogated proliferation, whereas subset depletions when chronic-infection granuloma cells were tested indicated that both CD4+ and CD8+ T cells respond to the greater than 200-kDa fraction. The present study reveals differences between acute- and chronic-infection splenic and granuloma T cells in the pattern of T-cell blastogenic responses to fractionated SEA.
Molecular characterization of the Enterococcus faecalis cytolysin activator
Authors: Segarra R A , Booth M C , Morales D A , Huycke M M , Gilmore M S
Published: January 2020

The gene encoding component A (cylA), the activator protein of the Enterococcus faecalis cytolysin, has been localized on pAD1, and the nucleotide sequence was determined. cylA consists of a 1,236-bp open reading frame encoding a 412-amino-acid polypeptide. A search of the National Biomedical Research Foundation data base revealed significant homology between the inferred amino acid sequence of component A and subtilisin BPN'. Component A activation of the cytolysin precursor (component L) was observed to be inhibited by the serine protease inhibitor diisopropylfluorophosphate. Mature component A exhibits a molecular weight of approximately 30,000 and an isoelectric point of 4.5. Differences between the size of the primary translation product (45,625 daltons) and the mature enzyme suggest that, as for subtilisin, component A is secreted as a proenzyme. These results provide the basis for a model of component A activation of component L and a role for component A in protecting the cytolysin-producing cell from lysis.
Physicochemical and biological properties of purified Escherichia coli Shiga-like toxin II variant
Authors: MacLeod D L , Gyles C L , Valdivieso-Garcia A , Clarke R C
Published: January 2020

Purified Escherichia coli Shiga-like toxin II variant (SLT-IIv) was characterized with regard to selected physical, chemical, and biological properties. N-terminal amino acid sequencing confirmed the identities of 33,000-, 27,500-, and 7,500-molecular-weight (MW) bands seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified SLT-IIv as the A subunit, A1 fragment, and B subunit, respectively. The arginine-serine bond between amino acids 247 and 248 in the A subunit was determined to be the site for proteolytic cleavage into A1 and A2 fragments. As with other SLTs, gel filtration chromatography of SLT-IIv gave estimates of the MW of holotoxin that were variable and less than predicted for a 1-A-subunit-5-B-subunit configuration. The MWs were estimated to be 40,000 and 43,000 by Sephacryl S-100 and Sephadex G-100 and less than 2,000 by Bio-Sil Sec-250 gel filtration chromatography. The isoelectric point of SLT-IIv holotoxin was 9.0. Cytotoxicity of SLT-IIv was destroyed by heating at 65 degrees C for 30 min and by incubation with 2-mercaptoethanol and dithiothreitol, but it increased 30-fold by incubation with trypsin, chymotrypsin, or pepsin and 2-fold by incubation with thermolysin. SLT-IIv cytotoxic activity was stable at neutral and alkaline pH values but was lost at pHs 3, 4, and 5. SLT-IIv was stable in fluid from the anterior and posterior small intestines of pigs but was not enterotoxic in pig intestinal loops. The smallest doses of SLT-IIv that inhibited protein synthesis in porcine endothelial cells and Vero cells were 0.1 ng and 0.1 fg, respectively.
Susceptibility to reinfection after a primary chlamydial genital infection is associated with a decrease of antigen-specific T cells in the genital tract
Authors: Igietseme J U , Rank R G
Published: January 2020

We tested the hypothesis that the intensity of specific antichlamydial T cell-mediated immunity in the genital tract of female guinea pigs infected intravaginally with the chlamydial agent of guinea pig inclusion conjunctivitis would determine the resistance or susceptibility to reinfection after a primary chlamydial infection. T cell-enriched lymphocytes were isolated by collagenase treatment of genital tract tissues from either infected or control uninfected female guinea pigs at various times after infection. The nylon wool-enriched T lymphocytes were evaluated for expression of antigen-specific T cell-mediated immunity in vitro by using a blast transformation assay. Both uninfected and infected genital tracts contained T cells, as evidenced by reactivity to concanavalin A, although a greater number of T lymphocytes was detected in the genital tracts of infected animals compared with that in controls. Significant antigen-specific T-cell activity could be detected in the genital tract tissue by 7 days after a primary genital tract infection with the chlamydial agent of guinea pig inclusion conjunctivitis. When antigen-specific activity was assessed at different times after infection, the intensity of the response of genital tract-associated T lymphocytes was directly proportional to the degree of resistance of the animals to genital challenge. Thus, susceptibility of animals to reinfection by chlamydiae appears to be associated with the intensity of the local T cell-mediated immune responses in the genital tract of infected animals.
Characterization of a glycosyl-phosphatidylinositol-anchored membrane protein from Trypanosoma cruzi
Authors: Hernández-Munaín C , Fernández M A , Alcina A , Fresno M
Published: January 2020

Four monoclonal antibodies (MAbs) specific for Trypanosoma cruzi were obtained. Flow cytometry analysis showed that these four MAbs stained the membranes of the three main morphological forms of T. cruzi: amastigotes, trypomastigotes, and epimastigotes. The four MAbs seemed to recognize the same 50- to 55-kDa antigen that was revealed by immunoblotting. Competition experiments revealed that they defined at least two different epitopes on the molecule. The antigen was detected on the external surface of the membrane by immunoelectron microscopy. Several experiments indicated that the 50- to 55-kDa antigen recognized by these four MAbs was a glycosyl-phosphatidylinositol-anchored membrane protein. (i) The antigen could be removed from the cell surface by treatment with proteases, NaOH, HNO2, and phosphatidylinositol-specific phospholipase C (PI-PLC). (ii) The phase distribution of the antigen in Triton X-114 solutions changed drastically upon treatment with PI-PLC. The antigen was found mainly in the detergent phase in nontreated samples and in the aqueous phase in PI-PLC-digested samples. (iii) A cross-reacting determinant that was found in other glycosyl-phosphatidylinositol-anchored membrane proteins appeared after PI-PLC treatment.
Expression of Escherichia coli F-18 type 1 fimbriae in the streptomycin-treated mouse large intestine
Authors: Krogfelt K A , McCormick B A , Burghoff R L , Laux D C , Cohen P S
Published: January 2020

Escherichia coli F-18, isolated from the feces of a healthy human, makes type 1 fimbriae and is an excellent colonizer of the streptomycin-treated mouse large intestine. Recently, it was shown that the inability to produce type 1 fimbriae had no effect on the ability of E. coli F-18 to colonize the streptomycin-treated mouse large intestine, suggesting the possibility that E. coli F-18 does not express type 1 fimbriae in vivo. However, we show here that E. coli F-18 does express type 1 fimbriae in mouse cecal mucus in vivo and, in fact, appears to express substantially more type 1 fimbriae in cecal mucus in vivo than in L broth in vitro.
Escherichia coli O128 strains from infants with diarrhea commonly show localized adhesion and positivity in the fluorescent-actin staining test but do not hybridize with an enteropathogenic E. coli adherence factor probe
Authors: Scotland S M , Smith H R , Rowe B
Published: January 2020

Twenty-nine strains of Escherichia coli O128 isolated from infants with diarrhea that did not produce heat-stable enterotoxin, heat-labile enterotoxins, or Vero cytotoxin showed localized attachment to HEp-2 cells (LA). Only four strains hybridized with the enteropathogenic E. coli adherence factor (EAF) probe. One of the 25 LA+ EAF- strains attached to 72% of cells, while a plasmid-negative variant attached to 0.5% of cells. LA+ EAF- and LA+ EAF+ strains gave a positive fluorescent-actin staining test that correlates with the ability to cause attaching and effacing lesions in the intestine. The use of the EAF probe alone to detect LA+ strains is inadequate for epidemiological studies.
Synthesis of tetanus toxin fragment C in insect cells by use of a baculovirus expression system
Authors: Charles I G , Rodgers B C , Makoff A J , Chatfield S N , Slater D E , Fairweather N F
Published: January 2020

The baculovirus expression vector p36C was used to express in cells of the insect Spodoptera frugiperda fragment C of tetanus toxin under the control of the strong polyhedrin promoter. Fragment C was expressed intracellularly at a high level and was soluble, allowing it to be purified by affinity chromatography with monoclonal antibody TT08. Purified fragment C from baculovirus was used to immunize mice and was shown to successfully prevent the symptoms of tetanus following a toxin challenge. The ganglioside-binding properties of baculovirus-derived fragment C were compared with those of intact tetanus toxin and native fragment C and were found to be dissimilar.
Glucosyltransferase gene polymorphism among Streptococcus mutans strains
Authors: Chia J S , Hsu T Y , Teng L J , Chen J Y , Hahn L J , Yang C S
Published: January 2020

Genetic polymorphisms in genes coding for the glucosyltransferases were detected among Streptococcus mutans serotype c strains by Southern blot analysis with DNA probes located within the gtfB gene (H. Aoki, T. Shiroza, M. Hayakawa, S. Sato, and H. K. Kuramitsu, Infect. Immun. 53:587-594, 1986). Restriction endonucleases were used to examine genomic DNAs isolated from serotype a to h strains. The variations were readily detected among 33 strains of serotype c by EcoRI and PstI restriction enzyme digestions. Serotypes e and f, which are genetically similar to serotype c, also had comparable polymorphism; however, serotypes a, b, d, g, and h did not hybridize to the same DNA probes in parallel experiments. Further analysis of enzymatic activities for glucan synthesis and sucrose-dependent adherence revealed no significant differences among the serotype c strains. Our results suggested that genetic polymorphisms existing in S. mutans serotype c strains may reflect a complexity in genes coding for the glucosyltransferases, which are produced ubiquitously in members of the S. mutans group.
Molecular cloning, expression, and sequence of the pilin gene from nontypeable Haemophilus influenzae M37
Authors: Coleman T , Grass S , Munson R
Published: January 2020

Nontypeable Haemophilus influenzae M37 adheres to human buccal epithelial cells and exhibits mannose-resistant hemagglutination of human erythrocytes. An isogenic variant of this strain which was deficient in hemagglutination was isolated. A protein with an apparent molecular weight of 22,000 was present in the sodium dodecyl sulfate-polyacrylamide gel profile of sarcosyl-insoluble proteins from the hemagglutination-proficient strain but was absent from the profile of the isogenic hemagglutination-deficient variant. A monoclonal antibody which reacts with the hemagglutination-proficient isolate but not with the hemagglutination-deficient isolate has been characterized. This monoclonal antibody was employed in an affinity column for purification of the protein as well as to screen a genomic library for recombinant clones expressing the gene. Several clones which contained overlapping genomic fragments were identified by reaction with the monoclonal antibody. The gene for the 22-kDa protein was subcloned and sequenced. The gene for the type b pilin from H. influenzae type b strain MinnA was also cloned and sequenced. The DNA sequence of the strain MinnA gene was identical to that reported previously for two other type b strains. The DNA sequence of the strain M37 gene is 77% identical to that of the type b pilin gene, and the derived amino acid sequence is 68% identical to that of the type b pilin.
Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic
Authors: Crowle A J , Dahl R , Ross E , May M H
Published: January 2020

Mycobacterium tuberculosis and Mycobacterium avium multiply in cultured human macrophages (MP) within membrane-enclosed vesicles. These vesicles are generally assumed to be acidic. The evidence most frequently cited for this assumption is that pyrazinamide, which requires an acid pH to be effective, is effective and streptomycin, which loses most of its activity at a low pH, is poorly effective against tubercle bacilli. This assumption was tested by using the two weak bases chloroquine and NH4Cl to raise the pH of acidic vesicles in MP experimentally infected with M. tuberculosis or M. avium. An immunocytochemical locator of acidic regions in the MP was used to monitor the association of intracellular bacilli with acidity. MP were infected with M. tuberculosis or M. avium and incubated with various combinations of the drugs and the weak bases. Replication of the bacteria in the MP was measured by culture counts. Intracellular associations of the mycobacteria with acidity were assessed by electron micrographs and by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, which was detected with colloidal gold-labeled antibodies. It was confirmed by immunocytochemistry that both chloroquine and NH4Cl raise the pH of acidic vesicles in the infected MP. However, neither caused any pH-related change in the antimycobacterial activities of pyrazinamide or streptomycin or of the pH-independent drug isoniazid. Immunochemical analyses showed acidity to be associated with killed but not living mycobacteria in the MP. These findings suggest that living M. tuberculosis and M. avium are located in human MP in vesicles which are not acidic.