bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of the Treponema denticola prtA gene coding for chymotrypsinlike protease activity and detection of a closely linked gene encoding PZ-PLGPA-hydrolyzing activity
Authors: Que X C , Kuramitsu H K
Published: January 2020

The chymotrypsinlike protease gene (prtA) from Treponema denticola ATCC 35405 was isolated from a lambda gt11 clone bank as one of several clones expressing protease activity. The DNA from one positive clone capable of hydrolyzing type IV collagen was subcloned into plasmid vector pUC119 for further analysis. Deletion analysis of subclone pXQ27.2 revealed the approximate location of the prtA gene on the DNA insert. DEAE-Sephadex chromatography of crude cell extracts of the subclone revealed two distinct T. denticola enzymes, one hydrolyzing SAAPNA (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide [chymotrypsin substrate]) and the other hydrolyzing PZ-PLGPA (phenylazobenzyl-oxycarbonyl-L-leucylglycyl-L-prolyl-D -arginine [collagenase substrate]). Each activity was purified to near homogeneity and exhibited by sodium dodecyl sulfate-polyacrylamide gel electrophoresis estimated molecular sizes of 67 and 36 kDa, respectively. Western blot (immunoblot) analysis demonstrated that only the 67-kDa SAAPNA-hydrolyzing enzyme reacted with antibody against the T. denticola chymotrypsinlike protease. The purified SAAPNA-hydrolyzing enzyme degraded type IV collagen, laminin, and fibronectin, but not type I collagen. These results indicate that the prtA gene coding for the chymotrypsinlike protease from T. denticola has been isolated. Another distinct gene encoding an enzyme hydrolyzing PZ-PLGPA appears to be adjacent to the prtA gene.
Roles of different coli surface antigens of colonization factor antigen II in colonization by and protective immunogenicity of enterotoxigenic Escherichia coli in rabbits
Authors: Svennerholm A M , Wennerås C , Holmgren J , McConnell M M , Rowe B
Published: January 2020

The roles of the subcomponents of colonization factor antigen II, the coli surface antigens CS1, CS2, and CS3, as colonization factors and protective antigens was studied in a nonligated rabbit intestine model (RITARD). Infection with enterotoxigenic Escherichia coli (ETEC) carrying CS3 alone or CS1 plus CS3 induced diarrhea in most (80%) of the rabbits, whereas nonenterotoxigenic strains expressing CS1 or CS2 rarely induced diarrhea. Strains carrying CS1, CS2, or CS3 alone were all shed in stools for a significantly longer period than normal fecal flora-type E. coli. Initial infection with ETEC positive for CS1 plus CS3 induced significant protection against disease caused by reinfection with a highly diarrheagenic dose of the homologous strain; rabbits previously infected with serotype-heterologous, nontoxigenic bacteria carrying CS1 only were also protected against this challenge, whereas no such protection was induced by serogroup-homologous E. coli carrying CS2 only. Animals previously infected with CS1-, CS3-, or CS1-plus-CS3-positive bacteria excreted the CS1-plus-CS3 challenge strain for a significantly shorter period than did "nonimmunized" rabbits, whereas initial infection with bacteria carrying CS2 only did not result in such reduced shedding. Monoclonal antibodies against CS1, CS2, or CS3 all protected against experimental infection with ETEC carrying the corresponding CS factor. These results suggest that all the subcomponents of colonization factor antigen II are colonization factors and may induce anticolonization immunity.
Suppression of bactericidal activity of human polymorphonuclear leukocytes by Bacteroides gingivalis
Authors: Yoneda M , Maeda K , Aono M
Published: January 2020

The direct effects of the culture supernatant of oral microorganisms on the bactericidal activity of human polymorphonuclear leukocytes (PMNs) were investigated. The bactericidal activity of PMNs, which were preincubated with the supernatant of Bacteroides gingivalis, Bacteroides intermedius, Bacteroides melaninogenicus or phosphate-buffered saline, was examined by counting the surviving bacteria. B. gingivalis-treated PMNs were found to have a diminished ability for killing bacteria in the presence or absence of serum. The chemiluminescence response of PMNs, which were preincubated with the culture supernatant of B. gingivalis, was strikingly reduced compared with that of PMNs preincubated with phosphate-buffered saline or other bacterial culture supernatants. The production of superoxide anion (O2-) by PMNs stimulated with either formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate was reduced in both cases after the PMNs were preincubated with the culture supernatant of B. gingivalis. However, it was observed that there was more reduction in superoxide anion (O2-) production stimulated with formyl-methionyl-leucyl-phenylalanine compared with that stimulated with phorbol myristate acetate. These results suggest that B. gingivalis releases a factor which interferes with the bactericidal activity of PMNs by modulating the generation of reactive oxygen species. These suppressive effects on bactericidal activity may be important in the pathogenesis of this microorganism.
Antibody response to Brucella ovis outer membrane proteins in ovine brucellosis
Authors: Riezu-Boj J I , Moriyón I , Blasco J M , Gamazo C , Díaz R
Published: January 2020

Hot saline extracts of Brucella ovis were composed of vesicles with outer membrane proteins (OMPs), lipopolysaccharide, and phospholipid as constituents. Extraction with petroleum ether-chloroform-phenol yielded a protein fraction free of detectable lipopolysaccharide, in which group 3 OMPs (28,500 apparent molecular weight [28.5K], 27.0K, and 25.5K) represented 81% of the total. Group 1 OMPs and 67.0K, 22.5K to 21.5K, and 19.5K to 18.0K proteins were also detected. Adsorption of immune sera with whole bacteria suggested that group 3 OMPs and 67.0K, 22.5K to 21.5K, and 19.5K to 18.0K proteins had antigenic determinants exposed on the surfaces of both B. ovis and rough B. melitensis cells but not on smooth B. melitensis cells. Antibodies to group 3 OMPs and the 67.0K protein in the sera of 93 and 87%, respectively, of B. ovis-infected rams were found by immunoblotting. Antibodies to other proteins were present in 67% of these animals. Compared with B. ovis-infected rams which had not developed lesions, rams with epididymo-orchitis had antibodies to a larger variety of proteins. Although ewes infected with B. melitensis also showed antibodies to OMPs, the immunoblot reactions were less intense.
Phenotypic characterization of Streptococcus sanguis virulence factors associated with bacterial endocarditis
Authors: Herzberg M C , Gong K , MacFarlane G D , Erickson P R , Soberay A H , Krebsbach P H , Manjula G , Schilling K , Bowen W H
Published: January 2020

Certain strains of Streptococcus sanguis adhere (Adh+) selectively to human platelets and, in plasma, induce them to aggregate (Agg+) into in vitro thrombi. In this study, we examined 18 recent endocarditis and dental plaque isolates of microorganisms that were biotyped as S. sanguis for coexpression of platelet interactivity phenotypes with another possible virulence factor in bacterial endocarditis, dextran synthesis. Detectable production of extracellular glucosyltransferase ranged from 0.2 to 66 mU/mg of culture fluid for 10 representative strains tested. Production of extracellular or cell-associated glucosyltransferase, fructosyltransferase, and soluble or insoluble dextrans was not necessarily coexpressed with platelet interactivity phenotypes, since the levels of production of soluble and insoluble dextrans varied among representative Adh+ Agg+ and Adh- Agg- strains. Analysis of a second panel of 38 fresh dental plaque isolates showed that S. sanguis distributes in a reproducible manner into the possible phenotype groups. Strains with different platelet interactivity phenotypes were distinguished with a panel of four murine monoclonal antibodies (MAbs) raised against Adh+ Agg+ strain 133-79 and screened to rule out artifactual reactions with antigenic components in culture media. The MAbs reacted selectively with Adh+ Agg+ strains in a direct-binding, whole-cell, enzyme-linked immunosorbent assay and also inhibited their interactions with platelets. Analysis of minimal tryptic digests of many strains, including variants that failed to bind the MAbs, suggested that some noninteractivity phenotypes possess cryptic surface determinants. Since the ability to adhere to platelets and induce them to aggregate is relatively stable, these traits may be useful in a phenotyping scheme for these Lancefield nontypeable streptococci.
Inhibition of Streptococcus mutans by the antibiotic streptozotocin: mechanisms of uptake and the selection of carbohydrate-negative mutants
Authors: Jacobson G R , Poy F , Lengeler J W
Published: January 2020

The antibiotic streptozotocin [2-deoxy-2-(3-methyl-3-nitrosoureido)-D-glucopyranoside], an analog of N-acetylglucosamine (NAG), has been shown to be useful for the selection of carbohydrate-negative and auxotrophic bacterial mutants. We have adapted this method for use with the oral pathogen Streptococcus mutans, a gram-positive, aerotolerant anaerobe that uses predominantly carbohydrates as carbon sources for growth. Streptozotocin selectively kills growing cells of S. mutans GS-5, and under appropriate conditions it can reduce the number of viable cells in actively growing cultures by a factor of 10(3) to 10(4). However, unlike in enteric bacteria, which take up this antibiotic by a single NAG-specific transport system, streptozotocin appears to be taken up in S. mutans by both a NAG-specific system and a relatively nonspecific system that is also involved in glucose, fructose, and mannose uptake. Combining streptozotocin selection and a screening procedure involving indicator plates containing triphenyl-tetrazolium chloride, we developed a general method for the isolation of carbohydrate-negative and auxotrophic mutants of S. mutans. A preliminary characterization of both pleiotropic and specific carbohydrate-negative mutants isolated by using this procedure is presented.
Human natural killer cells do not inhibit growth of Cryptococcus neoformans in the absence of antibody
Authors: Miller M F , Mitchell T G , Storkus W J , Dawson J R
Published: January 2020

The interaction between human natural killer (NK) cells and yeast cells of Cryptococcus neoformans was investigated because experiments in mice indicated that NK cells inhibited the growth of C. neoformans. Strains of C. neoformans serotype A that differed in both resistance to alveolar macrophages and the size and composition of their capsules were evaluated. Human NK cells, which were isolated from normal peripheral blood, were activated by preincubation with interleukin-2 and alpha interferon to generate lymphokine-activated killer (LAK) cells. Yeast cells of C. neoformans were incubated with effector cells (NK or LAK cells); and inhibition of yeast cell growth was measured at 4, 8, and 24 h by comparing quantitative plate counts with controls consisting of yeasts in the absence of effector cells. The cytolytic activity of effector cells against target cells was confirmed by the release of radiolabel from 51Cr-labeled K-562 tumor cells. Neither NK nor LAK cells inhibited the growth of 13 strains of C. neoformans at effector to target cell ratios of as high as 500:1. Monocytes, which were isolated from the same populations of leukocytes as the NK cells, inhibited the growth of two strains of C. neoformans at effector to target cell ratios of 100:1 (92 and 46% inhibition), 50:1 (87 and 17%), and 1:1 (49 and 0%). NK cells could inhibit the growth of C. neoformans by an antibody-dependent cellular cytotoxicity mechanism in the presence of rabbit anticryptococcal antiserum at dilutions up to 1:4,000. Purified capsular polysaccharide of C. neoformans had no effect on the viability or tumoricidal activity of NK or LAK cells. These data suggest that human NK and LAK cells are not impaired by C. neoformans, and in the absence of antibody, which is rarely detectable in patients, they afford much less protection against C. neoformans than monocytes do.
Expression of systemic protection and delayed-type hypersensitivity to Listeria monocytogenes is mediated by different T-cell subsets
Authors: Baldridge J R , Barry R A , Hinrichs D J
Published: January 2020

The relationship between acquired cellular resistance and delayed-type hypersensitivity (DTH) during the immune response to Listeria monocytogenes was investigated. Treatment of concanavalin A-stimulated Listeria-immune spleen cells with anti-CD8 antibody plus complement abrogated the adoptive transfer of systemic antilisterial immunity but had no effect on the transfer of DTH. In contrast, in vitro depletion of the CD4+ T-cell subset eliminated the ability of culture-activated cells to transfer DTH reactivity but did not interfere with the adoptive transfer of protection. In vivo, the infusion of anti-CD8 antibody inhibited the expression of both actively and adoptively transferred protection but did not influence the development of DTH skin test reactivity to L. monocytogenes antigens. In vivo depletion of the CD4+ T-cell subset eradicated the DTH response, with only minor influence of the protective anti-Listeria response. The apparent functional dissociation of the CD4+ (DTH) and CD8+ (protection) T-cell populations was further emphasized by our findings that the adoptive transfer of protection was dependent on a cyclophosphamide-sensitive cell population, whereas DTH reactivity was mediated by a cyclophosphamide-resistant population.
Purification and characterization of the Dr hemagglutinins expressed by two uropathogenic Escherichia coli strains
Authors: Kist M L , Salit I E , Hofmann T
Published: January 2020

The fibrillar Dr hemagglutinins expressed by two uropathogenic Escherichia coli isolates were mechanically sheared from whole cells and subsequently purified by using anion-exchange high-pressure liquid chromatography. The isolated hemagglutinins were proteins with apparent subunit molecular masses of 14,500 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric points of 5.4 in denaturing isoelectric focusing gels. The two proteins were serologically related to each other but distinct from P fimbriae, as assessed by bacterial agglutination and immunoblotting. The amino acid compositions of the two hemagglutinins were highly similar both to each other and to other Dr hemagglutinins. N-terminal amino acid sequencing of the major hemagglutinin subunit proteins demonstrated homology with afimbrial E. coli adhesins.
Monoclonal antibodies that inhibit ADP-ribosyltransferase but not NAD-glycohydrolase activity of pertussis toxin
Authors: Kaslow H R , Schlotterbeck J D , Kenimer J G
Published: January 2020

Kenimer et al. (J. G. Kenimer, J. Kim, P. G. Probst, C. R. Manclark, D. G. Burstyn, and J. L. Lowell, Hybridoma 8:37-51, 1989) identified three classes of monoclonal antibodies, termed A, B, and C, that recognize the S1 subunit of pertussis toxin. This report presents data demonstrating that class A monoclonal antibodies (3CX4, 6D11C, and 3C4D), which block the ADP-ribosyltransferase activity and recognize the predominant neutralizing epitope on the S1 subunit of the toxin, do not inhibit the NAD-glycohydrolase activity of the toxin. In addition, alkylation of cysteine 41 of the S1 subunit, which may interact with NAD, inactivates the toxin but does not prevent binding by class A antibodies. Taken together, these results support the conclusion that proper alterations of amino acids that interact with NAD should allow for inactivation of the toxin without destruction of the predominant neutralizing epitope. The class A antibodies recognized control but not heat-treated pertussis toxin spotted onto nitrocellulose, indicating that class A antibodies do not recognize denatured S1 subunit. In contrast, a nonneutralizing class C antibody (X2X5) failed to bind to control toxin or S1 subunit in solution and recognized heat-treated pertussis toxin better than control toxin when spotted onto nitrocellulose. Thus, this type of analysis presents a heterogeneous mixture of fully or partially denatured and native S1 proteins and fails to distinguish between neutralizing and nonneutralizing antibodies.
Characterization of binding of Escherichia coli strains which are enteropathogens to small-bowel mucin
Authors: Wanke C A , Cronan S , Goss C , Chadee K , Guerrant R L
Published: January 2020

Before an enteropathogen binds to the small bowel, it must interact with the small-bowel mucus (SBM) layer. To determine whether this interaction involves specific binding of diarrheagenic Escherichia coli, we used a quantitative assay with labeled, purified rabbit SBM. Binding of SBM from an adult rabbit was significantly greater to strain 162, an agglutinating E. coli strain, than it was to RDEC-1, a rabbit pathogen, and was significantly greater to strain 2348/PMAR, an enteropathogenic E. coli strain, than it was to strains 1392+ and 1392-, which are enterotoxigenic E. coli strains with and without colonizing fimbriae, respectively. Binding of strains RDEC-1, 2348/PMAR, and 162-4 was significantly greater to SBM than to bovine serum albumin. Binding of all strains increased in a linear fashion with increasing amounts of SBM and was reproducible (r = 0.85). Binding was significantly greater at pH 5.7 than at pH 7.4 or 8.0 for all five strains. Temperature did not alter the binding of any strain. Strains 162-4 and RDEC-1 bound significantly more to proximal SBM than to rabbit distal SBM, while strains 1392+ and 1392- bound significantly more to distal SBM. Oxidation of sugars from SBM significantly decreased the binding of all strains. Each pathogenic E. coli strain bound distinctively to SBM; the SBM sugars appeared to mediate this binding for all E. coli strains. Binding was also dependent on mucin characteristics, as binding varied by region of the gut (increased for proximal SBM for strains 162-4 and RDEC-1 and for distal SBM for strains 1392+ and 1392-). The developmental age of the gut significantly affected binding only of the rabbit pathogen RDEC-1.
Causative agent of spotted fever group rickettsiosis in Japan
Authors: Okada T , Tange Y , Kobayashi Y
Published: January 2020

Since 1984, it has been known that spotted fever group rickettsiosis exists in Japan. We isolated three strains of the causative rickettsiae, designated Katayama, Misaka, and Abe, from patients with the disease and studied the characteristics of the isolates. Nude mice and cyclophosphamide-treated mice died after infection with the isolates. However, infected normal mice recovered and acquired immunity. Infected adult male guinea pigs had fever, a scrotal reaction, and seroconversion. The isolates propagated well in tissue-cultured Vero cells. Analysis by the cross-immunofluorescence antibody method showed that these isolates were closely related serologically. To reveal their immunological properties in detail, we produced 21 anti-Katayama monoclonal antibodies. Seven of these antibodies reacted with all representative strains of spotted fever group rickettsiae used in this study, and five others reacted only with the homologous strain, revealing that the Katayama strain has a strain-specific antigen(s) different from those of other spotted fever group rickettsiae. Moreover, these strain-specific antibodies also reacted with the Misaka and Abe strains. These results demonstrate that the causative agent of spotted fever group rickettsiosis in Japan is a new serotype of spotted fever group rickettsiae.
Secretion of toxin A from Pseudomonas aeruginosa PAO1, PAK, and PA103 by Escherichia coli
Authors: Hamood A N , Wick M J , Iglewski B H
Published: January 2020

The exotoxin A gene (toxA) from Pseudomonas aeruginosa PAO1 was expressed from the lac promoter in Escherichia coli, and the localization of the toxin A protein was determined. Throughout the growth cycle, the ADP-ribosyltransferase activity of toxin A was gradually reduced in the periplasm of E. coli, with no apparent degradation of the toxin A protein. This suggests the presence of an E. coli periplasmic factor that interferes with the ADP-ribosyltransferase activity in toxin A. Such an inactivating factor was found in the periplasmic extract from control E. coli cells. The processing of toxin A in E. coli was examined by pulse-chase immunoprecipitation experiments. Mature toxin was detected in both the periplasm and cytoplasm, whereas the membranes contained both mature and precursor forms. Toxin A precursor appears to be processed in both the cytoplasm and the periplasm of E. coli. Toxin A proteins from P. aeruginosa PAO1, PA103, and PAK were compared for their secretion in E. coli. Despite the differences in the amino acid sequences of their leader peptides, toxin A proteins from strains PAO1, PA103, and PAK were processed and secreted to the periplasm of E. coli.
Correlation between the presence of sequences homologous to the vir region of Salmonella dublin plasmid pSDL2 and the virulence of twenty-two Salmonella serotypes in mice
Authors: Roudier C , Krause M , Fierer J , Guiney D G
Published: January 2020

Large plasmids encoding important virulence properties have been found in several Salmonella serotypes. We have studied the relationship between the presence of a highly conserved 4-kilobase (kb) EcoRI fragment from the plasmid virulence region and pathogenicity for mice of 53 isolates representing 22 serotypes of Salmonella. Only strains possessing the homologous 4-kb region were virulent for mice. In addition, we transferred the virulence plasmid from S. dublin into nine different serotypes, including S. typhi and S. paratyphi A, that lack a native virulence plasmid. Only S. heidelberg and S. newport were rendered mouse virulent by the introduction of the S. dublin plasmid. This study demonstrates that plasmid-mediated virulence sequences are required for Salmonella virulence in mice, but many strains, including the agents of human typhoid fever, also lack chromosomal genes necessary to produce lethal systemic disease in mice. Since all the major Salmonella strains that are host-adapted to animals carry virulence plasmids, it appears that these plasmids are important in mediating systemic infection in animals and may contribute to septicemic, nontyphoid salmonellosis in humans.
Antibody response to epitopes of chlamydial major outer membrane proteins on infectious elementary bodies and of the reduced polyacrylamide gel electrophoresis-separated form
Authors: Baghian A , Shaffer L , Storz J
Published: January 2020

Approximately 60% of the outer membrane of chlamydial elementary bodies (EBs) consists of the major outer membrane protein (MOMP) that has structural and metabolic functions. The antigenic properties of MOMPs from mammalian strains of serovars 1 and 2 and an avian strain of Chlamydia psittaci were analyzed. Polyclonal-monospecific antisera (PMAs), one monoclonal antibody (MAb), and polyclonal antisera (PAs) were produced against reduced polyacrylamide gel electrophoresis-separated MOMPs and against infectious EBs. Three PMAs and the MAb, which were induced by reduced polyacrylamide gel electrophoresis-separated MOMPs, reacted strongly in Western blot (immunoblot) assays with MOMPs of serovar 1 and 2 strains as well as with that of the avian strain 6BC, and two of these PMAs reacted weakly (dilution, 1:20) with the MOMP of strain LGV-2. The third PMA and the MAb against the MOMP of the serovar 2 strain did not react with the MOMP of LGV-2. Four PAs were produced against infectious EBs of the serovar 1 strain. One of these PAs reacted with the homologous MOMP and that of the avian strain 6BC but did not recognize MOMPs of other chlamydial strains. Three of the PAs reacted with MOMPs of homologous strains only and failed to recognize MOMPs of avian, serovar 2, and LGV-2 strains. Five PAs induced against infectious EBs of the serovar strain 2 reacted only with the MOMPs of the homologous strains and failed to recognize MOMPs of other strains of chlamydiae. Consequently, MOMPs of C. psittaci strains possess genus-, species-, and serovar-specific epitopes whereby the immune response to serovar-specific epitopes of MOMP predominate when infectious EBs are used for immunization.
Escherichia coli F-18 makes a streptomycin-treated mouse large intestine colonization factor when grown in nutrient broth containing glucose
Authors: Cohen P S , Kjelleberg S , Laux D C , Conway P L
Published: January 2020

Escherichia coli F-18 FimA-, a type 1 fimbria-less derivative of a normal human fecal isolate, E. coli F-18, has previously been shown to be as good a colonizer of streptomycin-treated mouse large intestine as its parent, suggesting that type 1 fimbriae are not necessary in this process. In this study it was found that when E. coli F-18 FimA- was grown standing overnight at 37 degrees C in nutrient broth, it remained uniformly suspended; however, when grown in nutrient broth containing 1% (wt/wt) D-glucose, it settled to the bottom of culture tubes. Settling was associated with the formation of clumps (microcolonies) of more than 10 cells each. The effect of glucose could be partially reversed by growing E. coli F-18 FimA- in nutrient broth containing 1% D-glucose supplemented with cyclic AMP (greater than or equal to 1 mM). A reduced-settling mutant of E. coli F-18 FimA-, E. coli F-18 FimA- Set-, selected after Tn5 mutagenesis, was found to be a poor colonizer of streptomycin-treated mouse large intestine when fed to mice simultaneously with the parent strain. These results suggest that glucose-induced settling is, at least in part, regulated in a way related to catabolite repression and that the ability of E. coli F-18 FimA- to form microcolonies plays an important role in its ability to colonize streptomycin-treated mouse large intestine.
Functional analysis of Histoplasma capsulatum-reactive T-cell hybridomas
Authors: Deepe G S , Brunner G D
Published: January 2020

Activation of CD4+ T cells is a crucial step in the elimination of Histoplasma capsulatum yeast cells from tissues. However, only a limited amount of information exists concerning the immunobiology of H. capsulatum-reactive T cells that are CD4+. To facilitate the analysis of the functional activities of this T-cell subpopulation, we developed a panel of 10 murine T-cell hybridomas from splenocytes of immune C57BL/6 mice. All hybridomas reacted with monoclonal anti-CD4+ antibody and released interleukin-2 after stimulation with histoplasmin. Within 3 weeks, the reactivity of hybridomas to histoplasmin declined dramatically, yet the cells responded vigorously to yeast-phase preparations that were enriched for cytosol, cell wall, or cell membrane. Of 10 hybridomas studied, only one recognized heterologous fungal antigens. Responsiveness to yeast-phase antigens was restricted by I-Ab. We mapped determinants in cytosol and cell wall or cell membrane by the technique of one-dimensional T-cell immunoblotting. The patterns of responses of hybridomas to cytosol were nearly uniform. All hybridomas responded to two immunodominant regions in cytosol with masses ranging from less than or equal to 18 to 26 kilodaltons (kDa) and 35 to 39 kDa. All hybridomas tested responded to determinants in the cell wall or cell membrane preparation with masses of 35 to 39 kDa. These hybridomas provide a useful tool for defining yeast-phase antigens that trigger T-cell activation.
Strain differences in sensitivity of rats to Mycoplasma arthritidis ISR 1 infection are under multiple gene control
Authors: Binder A , Gärtner K , Hedrich H J , Hermanns W , Kirchhoff H , Wonigeit K
Published: January 2020

At least 5 female rats from each of 24 inbred (ACI, AS, BDIX, BH, BN, BS, BUF, DA, LE, LEW, MWF, OM, SPRD-Cu3, W-Krypt, and WKY), RT1 congenic [BH.1L(LEW), LEW.1A(AVN), LEW.1C(WIST), LEW.1LV3(BH), LEW.1K(SHR), and LEW.1N(BN)], and F1 hybrid [(LEW x BN)F1, (LEW.1W x LEW.1A)F1, and (LEW x LEW.1W)F1] strains, representing eight independent major histocompatibility complex (MHC) haplotypes (a, b, c, dv1, k, l, n, and u) and five related RT1 haplotypes (av1, lv1, lv3, uv2, and uv3), were inoculated intravenously with Mycoplasma arthritidis, and the severity of the polyarthritis that developed was determined by estimating arthritis scores and weight reductions. The 24 inbred, congenic, and F1 hybrid rat strains differed considerably in their sensitivity to infection with M. arthritidis and in the severity of the polyarthritis that they developed. Statistical evaluation showed that in the acute phase (days 1 to 42 after infection) as well as in the chronic phase (days 39 to 121 after infection) of the disease, the means of the arthritis scores for the strains form a continuous variation without significant interruptions, with the very sensitive LEW rats, the RT1 congenic rats on LEW background, the F1 hybrids with LEW, and the MWF, BS, BH, and DA rats on one end and the resistant WKY, BUF, W-Krypt, LE, and OM rats on the other end. A continuous variation was also observed for the means of the growth rates. There were, however, no significant differences between the sensitive and the resistant rat strains in the antibody titers determined by complement fixation test and enzyme immunoassay. Heritabilities of arthritis scores were calculated for all strains (h2 = 0.39 to 0.62), for the RT1 congenic strains (h2 = 0.04 to 0.14), and for several strains with identical MHC genes (h2 = 0.61 to 0.93). The results show that non-MHC genes are probably responsible for the sensitivity of rats to infection with M. arthritidis.
Immunogenic properties of Pseudomonas aeruginosa mucoid exopolysaccharide
Authors: Garner C V , DesJardins D , Pier G B
Published: January 2020

Previous studies have shown that antibodies to Pseudomonas aeruginosa mucoid exopolysaccharide (MEP) can be divided into two types on the basis of their functional activity. One type is able to mediate opsonic killing in conjunction with leukocytes and complement, and the other type is not. We investigated, in mice, the properties of this antigen associated with elicitation of opsonic killing antibody. We found that smaller-sized material (Kav = 0.26), which has been tested as a human vaccine, elicited opsonic killing antibody in mice at low doses (1 to 10 micrograms) and at doses of greater than or equal to 40 micrograms, only nonopsonic killing antibody was produced. A similar dose effect was seen with heat-killed mucoid P. aeruginosa cells. After immunization with high doses of this MEP or the heat-killed cells, the mice were refractory to induction of opsonic killing antibody no matter what dose of MEP was used as a booster. In contrast, a larger-molecular-weight preparation (Kav = 0.05) elicited opsonic killing antibody over a wide dose range (1 to 400 micrograms). Additionally, 50 micrograms of the larger-sized preparation could overcome the suppression induced by 50 micrograms of the smaller material. Suppression elicited by 50 micrograms of the smaller material could be adoptively transferred to nonimmune mice with the T-cell fraction of spleen cells. These results indicate that both molecular size and dose are critical determinants for eliciting opsonic killing antibody to mucoid P. aeruginosa after immunizing with MEP.
Diarrhea and intestinal invasiveness of Aeromonas strains in the removable intestinal tie rabbit model
Authors: Pazzaglia G , Sack R B , Bourgeois A L , Froehlich J , Eckstein J
Published: January 2020

Twelve Aeromonas strains were tested for virulence by using the removable intestinal tie adult rabbit diarrhea model. Mortality was 50% or greater for 7 of 12 strains; 23 of 37 rabbits that died developed diarrhea before death, and 11 of 27 surviving rabbits developed diarrhea. Aeromonas bacteremia was detected in 36 of 37 (97%) animals that died, but only in 2 of 27 (7%) survivors. Death, diarrhea, and bacteremia were all strongly strain dependent. Gastrointestinal lesions varied from moderate focal enteritis to severe multifocal necrosis and hemorrhage of the ileal mucosa, often accompanied by hepatic and splenic lesions. Intestinal colonization assays performed after infection indicated that the ileum was the most heavily colonized portion of the gut and the probable site of invasion. The application of the removable intestinal tie adult rabbit diarrhea model for intestinal challenge with Aeromonas strains has shown that some isolates are capable of invading the mucosa of rabbits, causing diarrhea and bacteremia. These data suggest that such strains may be important in causing human invasive diarrhea.
Anaerobiosis, type 1 fimbriae, and growth phase are factors that affect invasion of HEp-2 cells by Salmonella typhimurium
Authors: Ernst R K , Dombroski D M , Merrick J M
Published: January 2020

The invasion of HEp-2 cells by Salmonella typhimurium was studied under various conditions. Anaerobiosis was shown to markedly affect the internalization of bacterial cells by HEp-2 cells. Anaerobically grown bacteria incubated with HEp-2 cells under anaerobic conditions markedly stimulated the rate of invasion. Anaerobiosis may therefore be a controlling factor in the invasion process. Cells obtained during the logarithmic phase of growth invaded at much higher rates than cells obtained during the stationary phase of growth. The presence of mannose-sensitive type 1 fimbriae on the bacterial surface also promoted invasion, and these fimbriae appear to play a role as an accessory virulence factor.
Primary antibody responses to thymus-independent antigens in the lungs and hilar lymph nodes of mice
Authors: Goud S N , Kaplan A M , Subbarao B
Published: January 2020

B lymphocytes from the pulmonary lymphoid tissues were stimulated with a variety of thymus-independent (TI) antigens by intratracheal (i.t.) immunization. Immune responses in the lungs and hilar lymph nodes (HLN), which are part of the localized lymphoid tissue, as well as in the spleen, the systemic lymphoid organ, were studied. Thus, primary i.t. immunization of mice with the TI-1 antigen trinitrophenyl-lipopolysaccharide (TNP-LPS) elicited both antigen-specific and polyclonal plaque-forming cell responses from HLN, lung, and splenic B lymphocytes. These responses appeared as early as 3 days after immunization and declined by day 7. Similar immunization with another TI-1 antigen, TNP-Brucella abortus, resulted in anti-TNP responses in both pulmonary and systemic lymphoid tissues, although the kinetics of the antibody response were different than those to TNP-LPS. Interestingly an i.t. immunization with a TI-2 antigen, TNP-Ficoll, failed to induce an anti-TNP PFC response from HLN and lung B cells, although there was good antibody formation from splenic B cells. Antibody response to TNP-Ficoll was restored in pulmonary tissues when mice were immunized with TNP-Ficoll mixed with unconjugated B. abortus. In conclusion, our results indicate that TI-1 and TI-2 antigens differ in their ability to induce antibody responses in the pulmonary lymphoid tissues. The inability of TNP-Ficoll to elicit an antibody response in pulmonary lymphoid tissues has significance in the development of vaccines containing bacterial polysaccharides.
Oral immunization of mice with attenuated Salmonella typhimurium containing a recombinant plasmid which codes for production of a 31-kilodalton protein of Brucella abortus
Authors: Stabel T J , Mayfield J E , Tabatabai L B , Wannemuehler M J
Published: January 2020

Salmonella typhimurium chi 4064, an attenuated delta cya delta crp mutant of S. typhimurium SR-11, was used as a carrier for the plasmid pBA31-R7. This plasmid codes for the expression of a 31-kilodalton (kDa) protein from Brucella abortus (BCSP31). Recombinant S. typhimurium chi 4064(pBA31-R7) expressed BCSP31 in vitro as shown by Western blot (immunoblot) analysis. The plasmid was stable in vitro and in vivo and did not affect the ability of the mutant to invade and colonize the small intestine, mesenteric lymph nodes, liver, or spleen of BALB/cByJ mice. Animals orally immunized with S. typhimurium chi 4064(pBA31-R7) developed serum and intestinal antibody responses to the B. abortus 31-kDa protein and to salmonella endotoxin as measured by enzyme-linked immunosorbent assay. Mice orally immunized with S. typhimurium chi 4064pBA31-R7 did not develop a delayed-type hypersensitivity following a footpad injection with recombinant BCSP31. Antigen-specific blastogenic data also support these in vivo results. All data indicate that this route of antigen delivery is effective for stimulating antibody-mediated immunity but that the B. abortus 31-kDa protein is a poor immunogen for inducing a cell-mediated immune response in BALB/cByJ mice.
Opsonic activity of cerebrospinal fluid in experimental cryptococcal meningitis
Authors: Hobbs M M , Perfect J R , Granger D L , Durack D T
Published: January 2020

The role of antibody in protection against infection with Cryptococcus neoformans is undefined. In this paper we describe the development of opsonic activity in the cerebrospinal fluid (CSF) of rabbits in response to cryptococcal meningitis. The opsonin appeared to be immunoglobulin G (IgG); the activity was heat stable, copurified with the IgG fraction during protein A separation, and could be absorbed by encapsulated cryptococci. Immunosuppression with cyclosporine could be administered to prevent or allow in vivo deposition of IgG on the polysaccharide capsule of yeast in the CSF. Both early and late cyclosporine regimens resulted in prolonged, severe meningeal infections corresponding to the complete absence of in vitro opsonic activity in the CSF. While the production of opsonic antibody is part of the successful host response against C. neoformans in the central nervous system of rabbits, the presence of specific immunoglobulin by itself is insufficient for complete protection.
Glycosphingolipid receptors for Pseudomonas aeruginosa
Authors: Baker N , Hansson G C , Leffler H , Riise G , Svanborg-Edén C
Published: January 2020

The binding of Pseudomonas aeruginosa to glycosphingolipids and to buccal and bronchial epithelial cells was analyzed. Three independently expressed specificities were found by bacterial binding to glycosphingolipids separated by thin-layer chromatography. All strains bound gangliotria- and gangliotetrasylceramide. All but one of the strains bound sialic acid-containing glycosphingolipids and lactosylceramide. The latter two specificities could be separated in that the lactosylceramide binding was retained and the sialic acid binding was suppressed when bovine serum albumin was used as a blocking agent in the thin-layer chromatography assay. The attachment to buccal epithelial cells, like the binding to sialylated compounds and lactosylceramide, was abolished by Formalin treatment of the bacteria, suggesting the importance of these specificities for cell adherence. In contrast, the binding to gangliotria- and gangliotetraosylceramide was retained by nonattaching Formalin-treated bacteria.
Synthesis and immunologic properties in mice of vaccines composed of Staphylococcus aureus type 5 and type 8 capsular polysaccharides conjugated to Pseudomonas aeruginosa exotoxin A
Authors: Fattom A , Schneerson R , Szu S C , Vann W F , Shiloach J , Karakawa W W , Robbins J B
Published: January 2020

Epidemiological, serological and in vitro phagocytosis experiments provide evidence that the newly discovered type 5 and type 8 capsular polysaccharides (CPs) are both virulence factors and protective antigens for bacteremia caused by Staphylococcus aureus. Neither type 5 nor type 8 CP elicited serum antibodies when injected into mice. These two CPs were bound to Pseudomonas aeruginosa exotoxin A (ETA) to form conjugates by using the synthetic scheme devised for the CP (Vi) of Salmonella typhi and of pneumococcus type 12F (A. Fattom, W. F. Vann, S. C. Szu, A. Sutton, X. Li, D. Bryla, G. Schiffman, J. B. Robbins, and R. Schneerson, Infect. Immun. 56:2292-2298, 1988; S. C. Szu, A. L. Stone, J. D. Robbins, R. Schneerson, and J. B. Robbins, J. Exp. Med. 166:1510-1524, 1987). Both S. aureus CP-ETA conjugates elicited a rise in CP antibodies. As components of conjugates, both S. aureus CPs acquired T-cell-dependent properties, as shown by their ability to respond to carrier priming and to stimulate booster responses. The conjugate-induced antibodies facilitated type-specific opsonization of S. aureus by human polymorphonuclear leukocytes. The conjugates also induced ETA antibodies which neutralized the native toxin in vitro. Clinical studies of these two conjugates for active or passive immunization of patients at risk for S. aureus bacteremia are planned.
Cytokine response by monocytes and macrophages to free and lipoprotein-bound lipopolysaccharide
Authors: Cavaillon J M , Fitting C , Haeffner-Cavaillon N , Kirsch S J , Warren H S
Published: January 2020

Recent evidence suggests that bacterial lipopolysaccharide binds to serum lipoproteins in vitro and in vivo and that lipopolysaccharide in the form that is bound to lipoprotein is less biologically active in several experimental models. In order to study the mechanism of this apparent detoxification, we compared the ability of free and lipoprotein-bound lipopolysaccharide from Escherichia coli O18 to stimulate interleukin-1, interleukin-6, and tumor necrosis factor from elicited murine peritoneal macrophages and circulating human monocytes. Lipopolysaccharide bound to lipoprotein was 20- to 1,000-fold less active than the unbound form in inducing the release of each cytokine. We also studied the binding of each form of lipopolysaccharide to the macrophage surface. Lipopolysaccharide complexed to lipoprotein was unable to compete for the binding of radiolabeled heterologous lipopolysaccharide to murine macrophages, and radiolabeled lipopolysaccharide-lipoprotein complexes bound poorly compared with molar equivalents of free lipopolysaccharide. Our experiments suggest that in the process of binding to lipoproteins, lipopolysaccharide may be rendered less toxic through a mechanism of decreased ability to induce monocytes and macrophages to release cytokines, perhaps because of an altered interaction at the cell surface.
Differential cytokine induction by doses of lipopolysaccharide and monophosphoryl lipid A that result in equivalent early endotoxin tolerance
Authors: Henricson B E , Benjamin W R , Vogel S N
Published: January 2020

The phenomenon of early endotoxin tolerance, which is induced by sublethal injection of lipopolysaccharide (LPS), results in a protracted period of hyporesponsiveness that is most profound at 3 to 4 days after injection and is marked by reduced cytokine production after a challenge injection of LPS. Early endotoxin tolerance is also induced by the nontoxic LPS derivative monophosphoryl lipid A (MPL), although much more of the monophosphoryl derivative is required to produce a state of tolerance equivalent to that evoked by LPS. In this study, equivalent tolerance-inducing doses of LPS and MPL were tested, and the levels of cytokines induced by LPS and MPL were compared. Although induced levels of colony-stimulating factor were comparable following doses of LPS and MPL that elicited an equivalent state of early endotoxin tolerance, levels of tumor necrosis factor, interleukin-6, and interferon were significantly lower in MPL-injected animals. These results suggest that the lowered toxicity of MPL may be related to its elicitation of significantly lower levels of potentially toxic intermediaries such as tumor necrosis factor, interferon, and interleukin-6.
Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7
Authors: Wadolkowski E A , Burris J A , O'Brien A D
Published: January 2020

Enterohemorrhagic Escherichia coli O157:H7 isolates produce Shiga-like toxins and carry a 60-megadalton plasmid which encodes an adhesin for Henle 407 intestinal cells. A streptomycin-treated mouse model was used to compare the intestinal colonizing capacity of E. coli O157:H7 strain 933 with that of its 60-megadalton plasmid-cured derivative, strain 933cu. When fed individually to mice, both 933 and 933cu maintained a stable number of organisms per gram of feces, and the greatest numbers of 933 or 933cu were isolated from cecal and proximal colonic epithelial cells. When 933 and 933cu were simultaneously fed to mice, 933cu was unable to maintain a stable level of colonization in about two-thirds of the mice tested. However, in one-third of the mice, the number of 933cu in feces began to increase rapidly until a stable level of co-colonization with 933 was attained. The isolate from these mice, 933cu-rev, was excreted in high numbers when fed alone to mice and was found on epithelial cells throughout the entire large bowel and distal small intestine. Moreover, 933cu-rev grew in mucus from all segments of the intestine and at higher levels than strain 933 or 933cu. Only mice fed strain 933cu-rev died. Histopathological studies confirmed that mice fed 933cu-rev died from bilateral renal cortical tubular necrosis consistent with toxic insult, perhaps due to Shiga-like toxins. The virulence of 933cu-rev may reflect its ability to grow well in mucus and colonize the small as well as large bowel.
Therapeutic efficacy of granulocyte colony-stimulating factor alone and in combination with antibiotics against Pseudomonas aeruginosa infections in mice
Authors: Yasuda H , Ajiki Y , Shimozato T , Kasahara M , Kawada H , Iwata M , Shimizu K
Published: January 2020

The therapeutic efficacy of granulocyte colony-stimulating factor (G-CSF) against an experimental intramuscular infection induced by Pseudomonas aeruginosa in mice was confirmed. Bacterial growth in the infected thigh muscle was suppressed by G-CSF treatment. The change in the number of peripheral blood polymorphonuclear leukocytes (PMN) after bacterial challenge was investigated. The results showed that G-CSF could stimulate stronger defense mechanisms after stimulation by bacterial challenge. In the G-CSF-treated group, more clusters of matured PMN were observed in the infected thigh muscle 6 h after bacterial challenge. Next, the correlation between the number of PMN in the blood at the time of infection and the therapeutic efficacy of antibiotics was investigated. The therapeutic efficacy of ceftazidime, a beta-lactam antibiotic, was affected by the number of blood PMN at the time of infection. In particular, a decrease of peripheral blood PMN at the time of infection resulted in a dramatic decrease in the efficacy of ceftazidime. The reduction in leukopenia by G-CSF remarkably strengthened the therapeutic effect of antibiotics in mice.
Isolation and characterization of an elastinolytic proteinase from Aspergillus flavus
Authors: Rhodes J C , Amlung T W , Miller M S
Published: January 2020

An elastinolytic proteinase of Aspergillus flavus has been isolated to homogeneity, and its physical and biochemical properties have been characterized. Two purification protocols were compared; an initial step of ion-exchange chromatography was found to be equivalent to ammonium sulfate precipitation at neutral pH. A combination of gel filtration and adsorption chromatographies on the resultant crude enzyme produced highly purified elastase with yields of 5 to 10%. The enzyme is a 23-kilodalton protein with a pI of 7.6. The enzyme activity is markedly inhibited by numerous metal ions. Aspergillus elastase appears to be a metalloproteinase EC 3.4.24.X), as determined by its sensitivity to 1,10-phenanthroline.