bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of anti-core glycolipid monoclonal antibodies with chemically defined lipopolysaccharides
Authors: de Jongh-Leuvenink J , Schellekens J , Verhoef J
Published: January 2020

Five anti-core glycolipid monoclonal antibodies (MAb) (four against Escherichia coli J5 lipopolysaccharide [LPS] and one against the Re core glycolipid of Salmonella typhimurium) were characterized using LPS from several rough and smooth strains and derivatives of E. coli J5 LPS, obtained by N acetylation and hydrolysis. The MAb against E. coli J5 were not only weakly cross-reactive with clinical isolates, whereas the anti-Re MAb was highly cross-reactive. The MAb differed in their reaction pattern with E. coli J5 LPS. MAb 4-7B5 (immunoglobulin M) and MAb 4-6A1 (immunoglobulin G1) cross-reacted with LPS of Salmonella minnesota R5 and S. typhimurium Ra and Rc and little with Re and lipid A. The dominant binding site of these MAb was located in the glucose-heptose-heptose region and was independent of phosphate substitution. The MAb 4-9A1 reacted with the terminal part of the core region (glucose-heptose) and was dependent on phosphate substitution of the LPS. The MAb BA7 (immunoglobulin G3) was E. coli J5 LPS specific and reacted with the glucosaminyl-heptose disaccharide. Antibody 8-2C1 was directed against the common parts of LPS, 3-deoxy-D-manno-octulosonic acid, and lipid A, which are not (or only weakly) recognized by the four anti-J5 LPS MAb. Thus, MAb that are not cross-reactive can be directed against at least three different antigenic determinants present on the core oligosaccharide of E. coli J5 LPS.
Induction of circulating tumor necrosis factor cannot be demonstrated during septicemic salmonellosis in calves
Authors: Peel J E , Voirol M J , Kolly C , Gobet D , Martinod S
Published: January 2020

The concentration of tumor necrosis factor in the circulation of calves, which were infected with Salmonella typhimurium and exhibited septicemia as indicated by clinical signs and blood culture, was measured with a radioimmunoassay. These levels were compared with those in calves before infection and in other calves that had received an intravenous dose of gram-negative endotoxin. The tumor necrosis factor levels measured in samples taken during septicemia were not different from those in samples from infected nonsepticemic calves or samples from calves before infection. In contrast, the levels of tumor necrosis factor rose rapidly in calves after treatment with endotoxin by intravenous injection.
Molecular characterization of the Clostridium difficile toxin A gene
Authors: Dove C H , Wang S Z , Price S B , Phelps C J , Lyerly D M , Wilkins T D , Johnson J L
Published: January 2020

The gene encoding the toxin A protein of Clostridium difficile (strain VPI 10463) was cloned and sequenced. The coding region of 8,133 base pairs had a mol% G + C of 26.9 and encodes 2,710 amino acids. The deduced polypeptide has a molecular mass of ca. 308 kilodaltons. Nearly a third of the gene, at the 3' end, consists of 38 repeating sequences. The repeating units were grouped into two classes, I and II, on the basis of length and the low levels of DNA sequence similarities between them. There were seven class I repeating units, each containing 90 nucleotides, and 31 class II units, which, with two exceptions, were either 60 or 63 nucleotides in length. On the basis of DNA sequence similarities, the class II repeating units were further segregated into subclasses: 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID. The dipeptide tyrosine-phenylalanine was found in all 38 repeating units, and other amino acid sequences were unique to a specific class or subclass. This region of the protein has epitopes for the monoclonal antibody PCG-4 and includes the binding region for the Gal alpha 1-3Gal beta 1-4GlcNAc carbohydrate receptor. Located 1,350 base pairs upstream from the toxin A translation start site is the 3' end of the toxin B gene. Between the two toxin genes is a small open reading frame, which encodes a deduced polypeptide of ca. 16 or 19 kilodaltons. The role of this open reading frame is unknown.
Transmembrane diffusion channels in Mycoplasma gallisepticum induced by tetanolysin
Authors: Rottem S , Groover K , Habig W H , Barile M F , Hardegree M C
Published: January 2020

The permeability properties of Mycoplasma gallisepticum cells treated with a purified preparation of tetanolysin were investigated by determining the initial swelling rates of cells suspended in an isoosmotic solution of electrolytes or nonelectrolytes. The swelling, initiated by the tetanolysin, depended on the tetanolysin concentration and was markedly affected by the molecular size of the various osmotic stabilizers utilized. Thus, the initial swelling rates in an isoosmotic solution of monosaccharides were much higher than those in isoosmotic solutions of di-, tri-, or tetrasaccharides. Cell swelling induced by tetanolysin was much lower with energy-depleted M. gallisepticum cells, with arsenate-treated cells, or when the membrane potential (delta psi) was collapsed by valinomycin (10 microM) plus KCl (100 mM). Swelling was not affected by the proton-conducting ionophore carbonyl cyanide-m-chlorophenylhydrazone (1 to 10 microM) or by nigericin (5 microM). These results support the concept that the damage induced by tetanolysin is due to the formation of water-filled pores within the membranes of energized M. gallisepticum cells. Such pores allow the diffusion of hydrophilic molecules into the cells and may vary in size, depending on the tetanolysin concentration utilized.
Comparison of the glycolipid receptor specificities of Shiga-like toxin type II and Shiga-like toxin type II variants
Authors: Samuel J E , Perera L P , Ward S , O'Brien A D , Ginsburg V , Krivan H C
Published: January 2020

The antigenically distinct Shiga-like toxins (SLTs) SLT-1 and SLT-II are cytotoxic for both Vero and HeLa cells and use Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb3) molecules as functional receptors. SLT-II-related variants SLT-IIvp and SLT-IIvh, produced by a porcine isolate and a human isolate, respectively, are cytotoxic for Vero but not HeLa cells. To investigate the basis for these differences in cytotoxic specificity among SLTs, the nature of the receptor for the SLT-II variants was examined. First, the patterns of binding of SLT-II and the SLT-II variants to Gb3 receptor analogs Gal alpha 1-4Gal-bovine serum albumin and Gal alpha 1-4Gal beta 1-4Glc-bovine serum albumin were compared. SLT-IIvp bound the trisaccharide neoglycoprotein preferentially, while SLT-IIvh bound both analogs equally but with less affinity than did SLT-II. Next, the glycolipids to which the SLT-II variants bound in Vero and HeLa cells were identified by thin-layer chromatography. SLT-IIvp bound to Gb3, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb4), and Gal beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer (Gb5) in Vero cells but only Gb3 in HeLa cells. However, SLT-IIvh bound to Gal alpha 1-4Gal beta 1-1Cer (Gb2) and Gb3 in HeLa cells but only Gb3 in Vero cells. In addition, hybrid toxins (SLT-IIvp subunit A with SLT-II subunit B or SLT-II subunit A with SLT-IIvp subunit B) were used to show that the receptor specificities of the SLTs was B subunit specific. These differences in receptor specificities are important in vivo, as evidenced by a 400-fold difference in the 50% lethal doses of purified SLT-IIvp and SLT-II (200 versus 0.5 ng, respectively) for mice. These data indicate that SLT-II-cytotoxic variants can occur as a consequence of differences in receptor specificity and affinity.
Sequence analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingbritt
Authors: Banas J A , Russell R R , Ferretti J J
Published: January 2020

The nucleotide sequence of the gbp gene, which encodes the glucan-binding protein (GBP) of Streptococcus mutans, was determined. The reading frame for gbp was 1,689 bases. A ribosome-binding site and putative promoter preceded the start codon, and potential stem-loop structures were identified downstream from the termination codon. The deduced amino acid sequence of the GBP revealed the presence of a signal peptide of 35 amino acids. The molecular weight of the processed protein was calculated to be 59,039. Two series of repeats spanned three-quarters of the carboxy-terminal end of the protein. The repeats were 32 to 34 and 17 to 20 amino acids in length and shared partial identity within each series. The repeats were found to be homologous to sequences hypothesized to be involved in glucan binding in the GTF-I of S. downei and to sequences within the protein products encoded by gtfB and gtfC of S. mutans. The repeated sequences may represent peptide segments that are important to glucan binding and may be distributed among GBPs from other bacterial inhabitants of plaque or the oral cavity.
Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress
Authors: Woods M L , Bonfiglioli R , McGee Z A , Georgopoulos C
Published: January 2020

We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis.
Enrichment of suppressor T cells by means of binding to monophosphoryl lipid A
Authors: Baker P J , Haslov K R , Fauntleroy M B , Stashak P W , Myers K , Ulrich J T
Published: January 2020

The binding and elution of spleen cells from plastic dishes coated with monophosphoryl lipid A (MPL) resulted in a greater than 1,000-fold enrichment of antigen-specific suppressor T-cell (TS) activity when spleen cells from mice 18 to 24 h after exposure to a low dose of type III pneumonococcal polysaccharide (SSS-III) were used. The removal of MPL-adherent TS cells resulted in an increase in the degree of amplifier T-cell (TA) activity present in the remaining MPL-nonadherent cell fraction; however, both TS and TA activities were found in the MPL-adherent cell fraction when spleen cells from mice 4 days after immunization with an optimal dose of SSS-III were examined. These findings, as well as others, suggest that both TS and TA, once activated, acquire a cell surface receptor that enables them to bind to MPL. Because of differences in the kinetics for the activation of TS and TA during the course of the antibody response and the fact that TS, but not TA, activity appears as early as 18 to 24 h after exposure to SSS-III, it is possible to use this experimental approach to obtain cell suspensions greatly enriched in TS activity.
Demonstration and immunochemical characterization of natural, autologous anti-idiotypic antibodies throughout the course of experimental syphilis
Authors: Baughn R E
Published: January 2020

Immunoglobulin Gs (IgGs) from serial bleeds obtained 1 week to 5 months after infection of rabbits with Treponema pallidum were examined for autologous anti-idiotypic (auto-anti-Id) antibodies. The capacities of IgGs to bind 125I-IgG F(ab')2 anti-T. pallidum prepared from earlier bleeds were determined by using radioimmunoassays. Results obtained in these assays suggested that auto-anti-Id IgGs were demonstrable between 60 and 180 days postinfection. Following reinfection, 90 days after the initial infection, auto-anti-Id IgGs peaked 6 to 8 weeks later, even though they were first noted as early as 2 weeks postreinfection. In the reinfection model the auto-anti-Id continued to persist throughout the remaining 3 months of the study. As auto-anti-Id antibodies to anti-fibronectin antibodies were demonstrable also in reinfected animals, our findings raise questions concerning the possible role of the anti-Id in modulating the Id expression of both T. pallidum-specific and autoimmune humoral responses.
The 987P gene cluster in enterotoxigenic Escherichia coli contains an STpa transposon that activates 987P expression
Authors: Klaasen P , Woodward M J , van Zijderveld F G , de Graaf F K
Published: January 2020

The genetic determinant for the production of 987P fimbriae has been cloned into pBR322. Analysis of frequently occurring deletions in the resultant recombinant plasmid, pPK180, revealed that the 987P gene cluster contains a transposon that encodes the synthesis of heat-stable enterotoxin STpa and is flanked by inverted repeats of IS1. Hybridization experiments with STpa- and 987P-specific probes demonstrated that a variety of STpa+ 987P+ wild-type Escherichia coli strains contained contiguous STpa-987P DNA, most likely on their chromosome. Transcription of the 987P gene cluster appeared to be activated by the adjacent IS1 element.
Cloning and expression of functional fragment C of tetanus toxin
Authors: Halpern J L , Habig W H , Neale E A , Stibitz S
Published: January 2020

A segment of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by using the polymerase chain reaction. This fragment was cloned into expression vector pTTQ8, under the control of the tac promoter. Expression of this plasmid in Escherichia coli resulted in the production of a protein consisting of 8 amino acids of the vector fused to the C-terminal 460 amino acids of tetanus toxin. This protein (rFragment C) was recognized by an antipeptide antibody specific for fragment C in an enzyme-linked immunosorbent assay and on immunoblots. rFragment C could be purified significantly in one step by immunoaffinity chromatography. Immunization of mice with rFragment C resulted in the production of antibodies that were able to protect the mice against a challenge with tetanus toxin. rFragment C bound to ganglioside GT1b and to neuronal cells in a manner indistinguishable from that of fragment C obtained by papain cleavage of tetanus toxin. For many applications, rFragment C appears to be a suitable alternative to tetanus toxin or toxin-derived fragment C.
Construction of stable cloning vectors that do not segregate from a human fecal Escherichia coli strain in the streptomycin-treated mouse large intestine
Authors: Burghoff R L , Laux D C , Cohen P S
Published: January 2020

Escherichia coli F-18 Col- was previously shown to be a poor colonizer of the streptomycin-treated mouse large intestine, relative to its parent, E. coli F-18. Prior to attempting to clone genes responsible for the colonization phenotype of E. coli F-18 into E. coli F-18 Col-, a suitable cloning vector had to be found. In this investigation, we report that the commonly used cloning vectors pBR322, pHC79, and pBR329 all segregate from E. coli F-18 Col- both when grown in L broth under conditions of nonselection (i.e., in vitro) and when fed to streptomycin-treated mice (i.e., in vivo). Insertion of the cer region (which promotes resolution of replicating plasmids into monomeric forms) into pHC79 stabilized this plasmid in E. coli F-18 Col- in vitro and in vivo. In contrast, two independent cer insertions into pBR329 did not stabilize the plasmid completely in E. coli F-18 Col- in vitro, and feeding the strain to streptomycin-treated mice resulted in rapid segregation of the plasmids in vivo. Also, stability of all three plasmids in E. coli F-18 Col- in vitro was achieved by insertion of the parB region of plasmid R1, which encodes a cell-killing protein, Hok, that is active only postsegregationally. However, as with cer, complete in vitro and in vivo stabilization was achieved only in parB constructs of pBR322 and pHC79.
Characterization of a phosphomonoesterase from Brucella abortus
Authors: Saha A K , Mukhopadhyay N K , Dowling J N , Ficht T A , Adams L G , Glew R H
Published: January 2020

Brucellae are facultative intracellular bacterial pathogens that reside primarily in cells of the reticuloendothelial system. The high-speed supernatant obtained after centrifuging a suspension of Brucella abortus that had been frozen-thawed and sonicated contained abundant phosphomonoesterase activity, determined by using 4-methylumbelliferylphosphate as the substrate; this enzyme was purified 2,900-fold (yield, 570%) by chromatography on DE-52 cellulose and hydroxylapatite columns and high-performance liquid chromatography-gel filtration. The native enzyme had a molecular mass of 120,000 daltons (+/- 10,000 daltons), as determined by gel filtration chromatography, and resolved into two bands (60,000 and 66,000 daltons) when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The B. abortus phosphomonoesterase had the following properties: pH optimum, 6.0 to 6.5; isoelectric point, 3.0; substrate specificity, 5'-AMP greater than 3'-AMP greater than 3'-GMP greater than 5'-GDP greater than 5'-CDP greater than 5'-CTP greater than 5'-UPT greater than phosphotyrosine greater than phosphoserine greater than phosphothreonine. The Km for 5'-AMP was 0.37 mM. Phosphatidylinositol 4,5-bisphosphate and myo-inositol 1,3,4-trisphosphate were poor substrates for the B. abortus enzyme. The phosphomonoesterase did not inhibit superoxide anion production by human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine. The phosphomonoesterase may be one of the bacterial enzymes in the pathway leading to the production of adenine, which is secreted by B. abortus and blocks the activation of neutrophils.
Phagocytosis of opsonized Treponema pallidum subsp. pallidum proceeds slowly
Authors: Alder J D , Friess L , Tengowski M , Schell R F
Published: January 2020

Macrophages were found to phagocytize Treponema pallidum subsp. pallidum attached to polycarbonate filters. This environment simulated the in vivo interaction of surface-adherent treponemes with macrophages. The phagocytosis of T. pallidum subsp. pallidum was found to proceed slowly. Heat-killed T. pallidum subsp. pallidum were susceptible to opsonization with 2% immune serum, whereas live treponemes were resistant to this concentration of antibody. High concentrations of immune serum were found to increase phagocytosis of the spirochetes. Live T. pallidum subsp. pallidum had bound limited quantities of immunoglobulin G in vivo, and only opsonization with 20% immune serum resulted in a detectable increase in surface-bound immunoglobulin in vitro. Kinetic studies suggested a steady rate of phagocytosis that is considerably slower than with other bacteria. Scanning electron microscopy studies of the phagocytizing macrophages showed that the treponemes were detached from the membrane filters and scooped onto the ruffled portion of the macrophage surface. This lengthy physical process, along with the lack of a dramatic increase in ingestion after opsonization, may account for the slow rate of phagocytosis.
Characterization of the immunodeficiency of RIIIS/J mice: immune response to polysaccharide antigens
Authors: Hiernaux J R , Baker P J , McEvoy S J , Stashak P W , Fauntleroy M B , Goidl E A
Published: January 2020

RIIIS/J mice lack an autosomal dominant gene(s) that influences the magnitude of the antibody response to several polysaccharide antigens of bacterial origin. Low responsiveness is demonstrable whether polysaccharide is administered as a T-helper-cell-independent or -dependent antigen conjugated to an immunogenic carrier; however, RIIIS/J mice make good anti-hapten antibody responses to haptenated polysaccharides. The low antibody responses of RIIIS/J mice to type III pneumococcal polysaccharide do not appear to be the results of an imbalance in the activity of regulatory T lymphocytes. Compared with other strains of mice, RIIIS/J mice elicit low antibody responses to lipopolysaccharide (LPS). They do not develop a cyclic primary or secondary antibody response to Escherichia coli O113 LPS; the latter is not due to a lack of mitogenic response to E. coli O113 LPS. They also produce auto-anti-idiotypic antibody after being immunized with trinitrophenyl-Ficoll.
Pyelonephritogenic Escherichia coli and killing of cultured human renal proximal tubular epithelial cells: role of hemolysin in some strains
Authors: Mobley H L , Green D M , Trifillis A L , Johnson D E , Chippendale G R , Lockatell C V , Jones B D , Warren J W
Published: January 2020

Acute pyelonephritis, a complication of Escherichia coli bacteriuria, must represent a bacterial invasion through the kidney epithelium. To study this process, we overlaid bacterial suspensions onto monolayers of cultured human kidney proximal tubular epithelial cells and measured cytotoxicity by release of lactate dehydrogenase (LDH). Thirty-four isolates cultured from patients with acute pyelonephritis were screened for the ability to cause pyelonephritis in CBA mice by transurethral challenge. The eight most virulent strains (greater than or equal to 70% of mice challenged developed greater than or equal to 10(3) CFU/g of kidney after 48 h) were selected for study. Each strain displayed mannose-resistant hemagglutination of human O erythrocytes; three strains were phenotypically and genotypically hemolytic. Pyelonephritogenic strains were significantly more cytotoxic (30.1 +/- 9.5% LDH release after 18 h) than eight fecal control strains (13.5 +/- 11.5% LDH release; P = 0.0068). We selected the most cytotoxic strain, CFT073, for further study. Sterile filtrate from this hemolytic strain was significantly more cytotoxic than was the filtrate of the fecal control strain, FN414. Transposon mutagenesis of CFT073 with TnphoA abolished hemolytic activity and cytotoxicity by both whole cells and sterile filtrate. Southern blot analysis revealed that the Tnphoa insertion mapped to the E. coli chromosomal hly determinant within a 12-kilobase SalI restriction fragment. Transformation of a nonhemolytic strain, CPZ005 with plasmid pSF4000, which carries a cloned hemolysin determinant, resulted in highly elevated cytotoxicity. Light micrographs of proximal tubular epithelial cell cultures demonstrated cell damage by pyelonephritogenic strains that was not induced by a fecal strain or the hemolysin-deficient mutant. Results indicate that pyelonephritogenic E. coli strains are more frequently cytotoxic for a putative target, that is, human renal tubular epithelium, than are fecal isolates. Hemolysin, in some strains, is apparently responsible for this cytotoxicity.
Effect of proteolytic activity on virulence of Candida albicans in burned mice
Authors: Neely A N , Holder I A
Published: January 2020

Total circulating proteolytic activity (PA) was determined by measuring the acid-soluble 125I-protein fragments generated per 100 microliters of serum incubated with 125I-protein at 37 degrees C for 15 min. Normal mice had low circulating PA (1.3 +/- 0.2 micrograms/100 microliters), and burned mice had a higher average PA; the actual value depended on the time of measurement postburn. We measured the effect on mortality and on circulating PA of challenging normal and burned mice with high-virulence strain Candida albicans MY 1044 and its less virulent mutant MY 1049. Burned and normal mice challenged with a high dose (10(5)) of MY 1044 had high mortality (greater than 90%) and high circulating PA (greater than 33 micrograms generated per 100 microliters). Burned mice challenged with a lower dose (10(4] of MY 1044 had moderate mortality (63%) and lower PA (27.2 +/- 4.2 micrograms/100 microliters). All other groups of mice, including burned mice challenged with 10(5) MY 1049, had low mortality (less than 10%), and PAs were less than 22 micrograms/100 microliters. Augmentation of burned mice challenged with 10(5) MY 1049 with proteinase significantly increased mortality; with treatment of burned mice challenged with 10(5) MY 1044 with proteinase inhibitor significantly decreased mortality. We conclude that mortality correlated with total circulating PA; that the contribution to this net PA was the background PA level in the normal mice, the PA associated with the burn, and the PA caused by infection with a C. albicans strain with a particular virulence; that most deaths caused by C. albicans occurred past a PA threshold of 25 micrograms/100 microliters in the host; and that the number of burned and infected mice that died of candidiasis could be modulated by the addition of proteinases or proteinase inhibitors to the host. This last finding may lead to some novel treatments for candidiasis in burned hosts.
Interaction of Treponema denticola TD-4, GM-1, and MS25 with human gingival fibroblasts
Authors: Weinberg A , Holt S C
Published: January 2020

The adherence of Treponema denticola GM-1, TD-4, and MS25 to human gingival fibroblasts (HGFs) was studied to serve as an introduction to investigations into the interactions of these oral bacteria with human host cells. Under both aerobic (5% CO2) and anaerobic (85% N2 plus 10% H2 plus 5% CO2) environments, the interactions with the HGFs were such that strains GM-1 and MS25 were consistently more adherent than strain TD-4. Polyclonal antibodies to GM-1 inhibited GM-1 adherence by 70%, while MS25 and TD-4 showed differing degrees of cross-reactive inhibition, indicative of common but not identical epitopes on the surface of the three T. denticola strains. Pretreatment of the three strains with trypsin did not inhibit adherence; proteinase K did, however, inhibit this interaction by 80%. Trypsin pretreatment of the HGFs resulted in increases in adherence of 50 and 86% for GM-1 and MS25, respectively, while a decrease of 41% was noted for TD-4. Exposure of the T. denticola strains to sugars and lectin pretreatment of the HGFs implicated adherence mediation by mannose and galactose residues on the HGF surface. Periodate treatment of HGFs resulted in a 50% drop in adherence for GM-1 and MS25, but did not decrease that of TD-4. Addition of fetal bovine serum inhibited adherence of the three strains to differing degrees, with TD-4 being the most susceptible. Addition of purified fibronectin (100 micrograms/ml) resulted in greater than 50% inhibition in GM-1 and MS25 adherence, while a 25% increase occurred with TD-4. While strain differences were noted in some of the parameters studied, the results indicate two possibilities for T. denticola-HGF adherence: a lectinlike adhesin(s) on the T. denticola surface with affinity for galactose and mannose on the HGF surface, and a serum host factor(s) bridging T. denticola and HGFs.
The HpmA hemolysin is more common than HlyA among Proteus isolates
Authors: Swihart K G , Welch R A
Published: January 2020

Two different hemolysins, HpmA and HlyA, have been reported in Proteus spp. To study the distribution of these hemolysins among Proteus strains, isolates from various infections and normal feces were screened for hemolysin production. All 63 Proteus mirabilis strains and 23 of the 24 Proteus vulgaris strains produced a calcium-independent hemolytic activity detectable in cell-free supernatants. The calcium-independent activity was due to HpmA; this activity correlated with the presence of hpmA sequences and the production of an extracellular 166-kilodalton (kDa) protein that reacted with anti-HpmA antiserum. HpmA- mutants, constructed by deletion of the central portion of the hpmA gene, did not produce the 166-kDa protein and were no longer hemolytic when compared with their respective parent strains. Among the 87 P. mirabilis and P. vulgaris isolates examined, calcium-dependent hemolytic activity was produced by only two P. vulgaris strains. These strains produced a 110-kDa protein which comigrated with the Escherichia coli hemolysin (HlyA) antibodies in immunoblots. These studies show that Proteus spp. produce two distinct extracellular hemolysins, with nearly all strains producing the calcium-independent hemolysin, HpmA, but only an occasional P. vulgaris isolate producing HlyA.
The liver as a major site of immunological elimination of murine trypanosome infection, demonstrated with the liver perfusion model
Authors: Albright J W , Long G W , Albright J F
Published: January 2020

The isolated liver perfusion model has been used to investigate immunological elimination of bacteria and yeasts but not for analysis of mechanisms of immunological destruction of extracellular parasitic protozoa. Extracellular trypanosomes are eliminated primarily through antibody (and complement?)-promoted hepatic (Kupffer cell) uptake and destruction. We studied the suitability of the isolated liver model system for analyzing the mechanism of immune elimination of mouse-specific Trypanosoma musculi and identified several factors which can complicate such analyses: (i) mechanical trapping of trypanosomes that are quite large (for example, reproducing forms or epimastigotes) or are nonviable and, therefore, nondeformable; (ii) variable species and concentrations of cytadhesive molecules; and (iii) the integrity and composition of the trypanosomal surface coat. There was a substantial difference between hepatic retention of infused T. musculi organisms coated with a specific antibody and those devoid of antibody when both were suspended in normal mouse serum. The difference appeared sufficient to allow accurate quantitative studies of immune destruction in the liver. Studies of whole mice indicated that quantitative investigations of immunological elimination of trypanosomes from the bloodstream are likely to be complicated by problems such as cytadherence of parasites to host endothelial cells and mechanical trapping. Uptake by the liver and spleen appeared more reliable. Thus, the isolated liver perfusion model should significantly benefit studies to elucidate the mechanisms of immune elimination of extracellular trypanosomes.
The spermicidal compound nonoxynol-9 increases adhesion of Candida species to human epithelial cells in vitro
Authors: McGroarty J A , Soboh F , Bruce A W , Reid G
Published: January 2020

All 25 strains of Candida spp. tested were able to grow in medium supplemented with 25% nonoxynol-9 in vitro. Adhesion of Candida spp. to HeLa cells was found to increase in the presence of 5% nonoxynol-9 (2.2- to 6.6-fold; P less than 0.001) and, to a lesser extent, in 12.5% nonoxynol-9. Adhesion of Candida strains cultured in medium supplemented with nonoxynol-9 varied, with five of six strains of Candida albicans and the single strain of Candida tropicalis demonstrating increases of 1.4 to 4.0 times control levels (P less than 0.05). The increased adhesion did not correlate with altered yeast cell surface hydrophobicity or germ tube formation.
Variation in virulence among oculogenital serovars of Chlamydia trachomatis in experimental genital tract infection
Authors: Ito J I , Lyons J M , Airo-Brown L P
Published: January 2020

Seven different oculogenital serovars (D, E, F, G, H, I, and K) of Chlamydia trachomatis were inoculated intravaginally into CF-1 mice, and subsequent infection was monitored. The duration of infection was longest with serovars D and E. This may help to explain clinical surveys which demonstrate a high (50%) prevalence of these serovars. Furthermore, a comparison of the invasiveness of strains D and H demonstrated a much higher frequency of uterine horn infection with serovar D.
Resistance of calves to Cryptosporidium parvum: effects of age and previous exposure
Authors: Harp J A , Woodmansee D B , Moon H W
Published: January 2020

Cryptosporidium parvum is a coccidian parasite that causes diarrheal disease in many vertebrate species, including young (less than or equal to 1 month old) calves. Older calves and adult cattle are resistant to infection. In this study, newborn calves were raised in isolation from C. parvum for 1 week to 3 months before experimental challenge with the parasite. Calves orally challenged with C. parvum at 1 week of age shed oocysts in their feces and had diarrhea after challenge exposure. When these calves were rechallenged at 1 and 3 months of age, they neither shed oocysts nor had diarrhea. There was no significant increase in the mean anticryptosporidium enzyme-linked immunosorbent assay serum antibody titer in these calves following any of the challenge exposures. Calves orally inoculated with C. parvum for the first time at 1 month of age shed oocysts, had diarrhea after challenge exposure, and were resistant to rechallenge at 3 months of age. These calves had a twofold increase in serum antibody titer after the first challenge and no increase after the second challenge. Calves orally inoculated with C. parvum for the first time at 3 months of age shed oocysts, and two of seven animals had diarrhea. These calves had a 10-fold increase in serum antibody to C. parvum after exposure. This study demonstrates that calves raised in isolation from C. parvum remain susceptible to challenge until at least 3 months of age. Furthermore, within this time period, initial exposure and recovery renders calves resistant to further challenge with the parasite. The data also suggest that exposure of young calves to C. parvum may inhibit the development of a serum antibody response to the parasite.
Cross-reactivity between the immunodominant determinant of the antigen I component of Streptococcus sobrinus SpaA protein and surface antigens from other members of the Streptococcus mutans group
Authors: Goldschmidt R M , Curtiss R
Published: January 2020

Most members of the Streptococcus mutans group of microorganisms specify a major cell surface-associated protein, SpaA, that is defined by its antigenic properties. The region of the spaA gene from Streptococcus sobrinus 6715 encoding the immunodominant determinant of the major antigenic component (antigen I) of the SpaA protein has recently been characterized. This study examined whether recognition of the immunodominant determinant is independent of the immunized animal host and whether antibodies elicited by the immunodominant determinant cross-react with cell surface proteins from S. mutans of various serotypes. Mouse and rabbit antisera to the undenatured SpaA protein reacted similarly both with the immunodominant determinant and with other antigenic structures of the protein in Western immunoblots with SpaA polypeptides that were specified by spaA gene fragments expressed in recombinant Escherichia coli. This suggests that the antibody responses of inbred and outbred animals were similar. Furthermore, antibodies raised against both the S. sobrinus SpaA immunodominant determinant expressed by recombinant E. coli and the purified protein from S. sobrinus displayed similar strain specificities and protein band profiles towards cells surface proteins from S. mutans of various serotypes in immunodot and Western blot analyses, respectively. This suggests that for S. sobrinus serotype g, the immune response against the SpaA protein is governed by the immunodominant determinant of antigen I. In addition, it indicates that the SpaA protein domain containing the immunodominant determinant overlaps the domain conferring cross-reactivity to cell surface proteins of S. mutans of various serotypes.
Biological evaluation of Mycoplasma pulmonis temperature-sensitive mutants for use as possible rodent vaccines
Authors: Lai W C , Bennett M , Lu Y S , Pakes S P
Published: January 2020

Temperature-sensitive mutants (TSMs) of Mycoplasma pulmonis were produced by treating the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine. Three TSMs were selected at 38 degrees C, as a restrictive temperature, and at 34 degrees C, as a permissive temperature. Two TSMs, UTCMI and UTCMII, were proven to be nonpathogenic but immunogenic. In addition, they did not induce pneumonia, tracheitis, or tympanitis but did induce mild rhinitis. They were stable after 10 passages in vitro and in vivo. They elicited excellent antibody production and cell-mediated immunity in vaccinated rats. They also were not mitogenic to rat lymphocytes. Rats immunized intranasally with these TSMs were significantly protected against challenge with wild-type organisms. These mutants were morphologically and serologically indistinguishable from the wild-type organisms. The growth characteristics and antibiotic sensitivities were similar to those of wild-type organisms, except that they grew only at 34 degrees C. In contrast to wild-type organisms, they did not bind to or lyse sheep erythrocytes. Thus, these TSMs may qualify as a vaccine to prevent M. pulmonis infection in rats.
Interaction of Clostridium difficile toxin A with cultured cells: cytoskeletal changes and nuclear polarization
Authors: Fiorentini C , Malorni W , Paradisi S , Giuliano M , Mastrantonio P , Donelli G
Published: January 2020

Experiments done on in vitro-cultured cells exposed to toxin A from C. difficile showed a series of cytopathologic changes leading to cell retraction and rounding accompanied by the marginalization of the nucleus, which localized at one pole of the cell. Cytoskeleton appeared to be strongly involved in such modifications. In particular, the microfilament system seemed to be involved in cell retraction, while microtubule network integrity and function seemed to be necessary for the nuclear displacement. The carboxylic ionophore monensin completely blocked the cytopathic effect when added with the toxin. The serine protease inhibitor chymostatin appeared to be protective also upon addition long after the end of the binding step. The Ca2(+)-dependent cytosolic protease inhibitors antipain and leupeptin were uneffective in protecting cells. Thus, our results suggest the involvement of an acidic compartment and the action of a serine protease in toxin A-induced cytopathic effect.
Characterization of Borrelia burgdorferi proteins reactive with antibodies in synovial fluid of a patient with Lyme arthritis
Authors: Mensi N , Webb D R , Turck C W , Peltz G A
Published: January 2020

Four Borrelia burgdorferi proteins reactive with antibodies in the synovial fluid of a patient with Lyme arthritis were characterized. Homology between amino acid sequences of immunoreactive spirochetal proteins and human proteins, including members of the Escherichia coli GroEL protein family, suggests that antigenic mimicry may play a role in the pathogenesis of Lyme arthritis.
Characterization of Candida albicans mannan-induced, mannan-specific delayed hypersensitivity suppressor cells
Authors: Garner R E , Childress A M , Human L G , Domer J E
Published: January 2020

We have shown previously that CBA/J mice immunized with Candida albicans developed delayed hypersensitivity (DH) demonstrable with mannan (MAN) extracted from the same organism and that the intravenous (i.v.) injection of MAN prior to or during the immunization phase resulted in the suppression of the MAN-specific DH response. In this study, we demonstrate that MAN-induced suppression of DH is a T-lymphocyte-mediated phenomenon. Suppressor cells induced in vivo by the i.v. injection of MAN into naive mice 1 to 7 days prior to harvest were passaged through nylon wool, treated with various surface-specific antibodies and complement, and then injected i.v. into immunized syngeneic recipients. Enrichment of splenic T cells by passage over nylon wool and transfer of the nylon-wool-nonadherent populations to immunized recipient mice suppressed DH in a dose-dependent manner. Depletion of Thy+ or Lyt-2+ cells from nylon-wool-nonadherent populations regularly ablated the ability of such suspensions to transfer suppression. Treatment of the same transfer suspensions with anti-Lyt-1 had variable effects, suggesting that the surface density of the Lyt-1 antigen was not as constant from population to population as was the Lyt-2 antigen. In addition, C. albicans MAN-induced suppressor cells were able to suppress DH demonstrable with Candida tropicalis MAN in animals immunized with C. tropicalis. Suppression of DH by MAN in this model, therefore, is mediated by Thy+ Lyt-2+ lymphocytes.
Effect of staphylococcal delta-toxin and bee venom peptide melittin on leukotriene induction and metabolism of human polymorphonuclear granulocytes
Authors: Raulf M , Alouf J E , König W
Published: January 2020

The abilities of delta-toxin from Staphylococcus aureus and melittin to induce and modulate the generation of leukotriene from human polymorphonuclear granulocytes (PMNs) were studied. Stimulation of PMNs with melittin (10 micrograms) induced leukotriene formation, whereas stimulation with delta-toxin did not. Preincubation of the PMNs with delta-toxin modulated the subsequent generation of leukotriene from PMNs induced by Ca ionophore A23187 or opsonized zymosan. The generation of leukotriene B4 (LTB4), induced by the Ca ionophore A23187, was increased when the PMNs were preincubated with delta-toxin for 5 min. When opsonized zymosan was used as a secondary stimulus to activate the delta-toxin-pretreated PMNs, LTB4 generation decreased. In contrast, melittin showed no significant modulatory effect on the generation of leukotriene from PMNs. In addition, preincubation of PMNs with delta-toxin inhibited the conversion of LTB4 to omega-oxidation products. Our data suggest that peptides with similar structures, e.g., delta-toxin and melittin, induce and modify leukotriene generation in different manners.
Neither motility nor chemotaxis plays a role in the ability of Escherichia coli F-18 to colonize the streptomycin-treated mouse large intestine
Authors: McCormick B A , Laux D C , Cohen P S
Published: January 2020

Escherichia coli F-18, isolated from the feces of a healthy human in 1977, is an excellent colonizer of the streptomycin-treated mouse large intestine and displays normal motility and chemotaxis ability. A chemotaxis-defective derivative of E. coli F-18, E, coli F-18 CheA-, and a nonflagellated derivative, E. coli F-18 Fla-, were constructed. These strains were found to colonize the streptomycin-treated mouse large intestine as well as E. coli F-18 when mice were fed both E. coli F-18 and either the CheA- or Fla- derivative at high levels (10(10) CFU of each strain per mouse) or low levels (10(4) CFU of each strain per mouse). Furthermore, E. coli F-18 lost motility and chemotaxis ability when grown in colonic or cecal mucus in vitro despite retaining the ability to synthesize flagella. Thus, it appears that neither motility nor chemotaxis plays a role in the ability of E. coli F-18 to colonize because this strain becomes functionally nonmotile upon growth in the streptomycin-treated mouse large intestine.
Endogenous carbohydrate-binding proteins in Pneumocystis carinii
Authors: Vierbuchen M , Ortmann M , Uhlenbruck G
Published: January 2020

By using biotinylated neoglycoproteins, the in situ occurrence of endogenous carbohydrate-binding proteins (lectins) in Pneumocystis carinii has been glycohistochemically demonstrated in lung tissue specimens from acquired immunodeficiency syndrome patients with P. carinii pneumonia. While the parasite possessed only a weak to moderate density of receptors for L-fucose and N-acetylated amino sugars, a strong specific binding of beta-D-galactoside and D-mannoside neoglycoproteins was observed on the cyst surface and within intracystic bodies. It is suggested that these endogenous lectins may be involved in the adhesion of P. carinii to the target.
Functional role of mucoid exopolysaccharide (alginate) in antibiotic-induced and polymorphonuclear leukocyte-mediated killing of Pseudomonas aeruginosa
Authors: Bayer A S , Speert D P , Park S , Tu J , Witt M , Nast C C , Norman D C
Published: January 2020

We evaluated in vitro the functional role of mucoid exopolysaccharide (MEP) of Pseudomonas aeruginosa in blocking antibiotic-induced and polymorphonuclear leukocyte (PMN)-mediated pseudomonal killing. The serum-resistant P. aeruginosa isolates used were mucoid strain 144MR and its nonmucoid revertant, strain 144NM. By timed kill curves, early bacterial effects of amikacin against mucoid strain 144MR were substantially less than those observed with nonmucoid strain 144NM; this effect was reversible with enzymatic hydrolysis of MEP of strain 144MR by alginase. Also, early tobramycin uptake (15 to 30 min) by mucoid 144MR cells was less than that seen with nonmucoid strain 144NM; pretreatment of 144MR cells with alginase substantially enhanced early tobramycin uptake compared with untreated 144MR cells (P = 0.08). In strain 144NM (but not in strain 114MR) there was a notable postantibiotic leukocidal enhancement effect manifested by increased nonopsonic killing following brief exposure of these cells to supra-MIC amikacin; pretreatment of strain 144MR with alginase rendered these cells more susceptible to amikacin-induced postantibiotic leukocidal enhancement. Similarly, direct PMN-mediated nonopsonic killing of mucoid strain 144MR was significantly less than that observed with strain 144NM (P less than 0.05); pretreatment of 144MR cells with alginase rendered this strain equal to strain 144NM in susceptibility to nonopsonic killing. In addition, exogenous sodium alginate or extracted MEP of strain 144MR interfered with effective nonopsonic killing of strain 144NM by PMNs. Studies also indicated that mucoid strain 144MR was phagocytosed significantly less well than its nonmucoid mate (P less than 0.00001), an effect reversed by pretreatment of the mucoid cells with alginase. These data confirm that P. aeruginosa MEPs functionally decrease the uptake and early bactericidal effect of aminoglycosides in vitro and interfere with effective PMN-mediated nonopsonic phagocytosis and killing of mucoid strains.
Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis
Authors: Nagai S , Wiker H G , Harboe M , Kinomoto M
Published: January 2020

Five actively secreted proteins (MPT32, MPT45, MPT51, MPT53, and MPT63) and the MPT46 protein were purified to homogeneity from Mycobacterium tuberculosis culture fluid and compared with proteins previously purified by ourselves and other investigators. Antisera were obtained by immunization of rabbits with all of the newly isolated proteins identified to be immunogenic. Two-dimensional electrophoresis of culture fluids obtained each week for 2 to 10 weeks of culturing of M. tuberculosis revealed characteristic changes, permitting identification of two distinct groups of proteins being actively secreted from the mycobacterial cells or appearing later in the culture fluids as a result of the release of soluble proteins from the cytosol after lysis of bacteria. The N-terminal amino acid sequences of five MPTs were shown to be identical to those of proteins previously isolated by other investigators and given different designations, and five new sequences are given. These sequences and the use of the antisera may serve to identify these proteins with mycobacterial constituents isolated by other investigators. The previously identified but not isolated MPT45 protein was shown to correspond to the C component of the antigen 85 complex. The 27-kDa MPT51 protein was demonstrated to cross-react with the three components of the antigen 85 complex, and the N-terminal amino acid sequences of MPT51 and MPT59 showed 60% homology. This finding and the extensive cross-reactivity between the components of the antigen 85 complex may indicate that there is a family of closely related secreted proteins in mycobacteria.