bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of plasmid-borne afa-3 gene clusters encoding afimbrial adhesins expressed by Escherichia coli strains associated with intestinal or urinary tract infections
Authors: Le Bouguenec C , Garcia M I , Ouin V , Desperrier J M , Gounon P , Labigne A
Published: January 2020

The afa gene clusters encode afimbrial adhesins (AFA) that are expressed by uropathogenic and diarrhea-associated Escherichia coli strains and belong to a family of hemagglutinins recognizing the Dr blood group antigen as a receptor. This family so far includes AFA-I and AFA-III as well as the Dr and F1845 adhesins (B. Nowicki, A. Labigne, S. Moseley, R. Hull, S. Hull, and J. Moulds, Infect. Immun. 58:279-281, 1990). Reported in this work is the genetic organization of the afa-3 gene cluster cloned from a uropathogenic E. coli strain (A30) which expressed a subtype of AFA designated AFA-III. The amino acid sequence of AFA-III was deduced from the nucleotide sequence of the afaE3 gene and was found to be highly homologous to that of the Dr adhesin (98.1% identity). A polymerase chain reaction assay was developed to detect the presence of afa-3 gene clusters in E. coli strains. Study of the genetic support of the afa-3 gene clusters in the strains which showed positive amplification revealed that they were always located on large, 100-kb plasmids whether the strains originated from patients with cystitis or with diarrhea. Moreover, the cloned afa-3 gene clusters from A30 and from the diarrhea-associated strain AL845 appeared to be carried by 9-kb plasmid regions which displayed a similar genetic organization. Chloramphenicol was reported to be a potent inhibitor of receptor binding by the Dr adhesin (Nowicki et al., Infect. Immun. 58:279-281, 1990). AFA-III expressed by strains AL845 and AL847 appeared to mediate, like the Dr adhesin, chloramphenicol-sensitive hemagglutination, whereas AFA-III produced by A30 conferred chloramphenicol-resistant adherence. A comparison of the sequences of these four proteins indicated that the amino acid at position 52 of the processed AFA could be part of the receptor-binding domain.
Gamma interferon-dependent temporary resistance to acute Toxoplasma gondii infection independent of CD4+ or CD8+ lymphocytes
Authors: Johnson L L , VanderVegt F P , Havell E A
Published: January 2020

It is well established that resistance to acute primary Toxoplasma gondii infection is mediated by a gamma interferon (IFN-gamma)-dependent mechanism. The present in vivo experiments were undertaken to investigate the cellular basis for this resistance. We show here that immunocompetent T. gondii-infected C57BL/6 (B6) mice treated with anti-IFN-gamma or with anti-Thy-1 or anti-asialo-GM1 antibodies die sooner than infected mice treated with antibodies that deplete both CD4+ and CD8+ T lymphocytes. Thy-1+ CD4- CD8- cells accumulated in the peritoneal cavities of B6 mice during the early stages of an intraperitoneal infection but did not accumulate in sham-infected control mice, and substantial numbers of Thy-1+ CD4- CD8- cells were recovered from the peritoneal cavities of infected B6 mice treated with antibodies that depleted CD4+ and CD8+ lymphocytes. Depletion of Thy-1+ cells reduced IFN-gamma to undetectable levels, whereas depletion of CD4+ and CD8+ cells did not reduce IFN-gamma levels. Thus T. gondii infection in immunocompetent B6 mice elicits Thy-1+ CD4- CD8- cells which either produce protective IFN-gamma themselves or control its production by other cells. It is likely that the function of these Thy-1+ CD4- CD8- cells is to control T. gondii tachyzoites during the early stages of primary infection before specific CD4(+)- and/or CD8(+)-dependent immunity develops.
A new fimbrial putative colonization factor, PCFO20, in human enterotoxigenic Escherichia coli
Authors: Viboud G I , Binsztein N , Svennerholm A M
Published: January 2020

The ability to colonize the small intestine is essential for enterotoxigenic Escherichia coli (ETEC) to cause diarrhea. Several colonization factor antigens (CFAs) and putative colonization factors (PCFs) have been described for ETEC. However, there are still many ETEC strains isolated from patients with diarrhea which do not possess any of these antigens. To identify CFAs in ETEC lacking the above-mentioned antigens, we exploited the ability of ETEC to adhere to tissue-cultured cells from an enterocyte-like cell line, Caco-2. An ETEC strain producing heat-labile toxin and heat-stable toxin of serotype O20:K27:H- (ARG-2) that was isolated from a child with diarrhea in Argentina and bound to Caco-2 cells was studied in further detail. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of this strain revealed a band of 25 kDa when bacteria were grown at 37 degrees C that was missing when the same strain was cultured at 20 degrees C. Furthermore, electron microscopy examination revealed the presence of fimbriae on the surfaces of cells of this strain when cells were grown at 37 degrees C but not at 20 degrees C. Rabbit antiserum raised against purified fimbriae reacted with the 25-kDa protein in immunoblotting and bound specifically to the fimbriae, as shown by immunoelectron microscopy. The presence of fimbriae, adhesion to Caco-2 cells, and the 25-kDa band seen in the SDS-PAGE were all simultaneously lost by single-insertion mutations. The N-terminal amino acid sequence of the protein subunit of the fimbriae showed no relation with those of the known colonization factors of ETEC. Furthermore, the fimbriae of the ARG-2 strain did not cross-react immunologically with any of the previously described adhesive factors in human ETEC when specific antisera against colonization factor antigens and putative colonization factors were used. Moreover, a specific antiserum raised against the fimbriae in ARG-2 did not react with ETEC carrying known colonization factors. We propose to name these new fimbriae PCFO20.
An avirulent lipophosphoglycan-deficient Leishmania major clone induces CD4+ T cells which protect susceptible BALB/c mice against infection with virulent L. major
Authors: Kimsey P B , Theodos C M , Mitchen T K , Turco S J , Titus R G
Published: January 2020

An avirulent clone of Leishmania major was used to immunize susceptible BALB/c mice against challenge with virulent L. major. By using the immunized animals as a source of cells, CD4+ parasite-specific T-cell lines could be generated in vitro which, when adoptively transferred to naive BALB/c recipients, conferred marked protection against challenge with virulent L. major. Compared with CD4+ parasite-specific T-cell lines generated from nonimmunized BALB/c mice infected with L. major, the protective T-cell lines generated from immunized mice produced substantially less interleukin-4 and substantially more tumor necrosis factor and interleukin-2. Interestingly, the protective CD4+ T cells did not mediate L. major-specific delayed-type hypersensitivity in vivo and proliferated in vitro only in response to living L. major and not to frozen-and-thawed antigen preparations of the parasite. Finally, the avirulent clone of L. major was found to express the major surface glycolipid of L. major, lipophosphoglycan, at a level that was sixfold less than expression of this molecule by virulent L. major. In addition, lipophosphoglycan of the avirulent parasite failed to mature into the larger, or metacyclic, form of the molecule.
Expression of monocyte chemoattractant protein 1 (MCP-1) in adult periodontal disease: increased monocyte chemotactic activity in crevicular fluids and induction of MCP-1 expression in gingival tissues
Authors: Hanazawa S , Kawata Y , Takeshita A , Kumada H , Okithu M , Tanaka S , Yamamoto Y , Masuda T , Umemoto T , Kitano S
Published: January 2020

The present study shows that monocyte chemotactic activity in crevicular fluids increases with severity of the disease and that a monocyte chemoattractant, monocyte chemoattractant protein 1 (MCP-1), is expressed as the predominant cytokine of gingival tissues and their fibroblasts treated with Porphyromonas (Bacteroides) gingivalis lipopolysaccharide (P-LPS). High monocyte chemotactic activity in the crevicular fluids was neutralized significantly by antiserum specific for the JE/MCP-1 protein. Marked expression of the MCP-1 gene was observed in the gingival tissues of all adult periodontal patients tested, but not in those of healthy subjects. Monocyte chemotactic activity was observed in culture supernatants of human normal gingival tissues treated with P-LPS, and the chemotactic activity increased in a dose-related manner. Expression of MCP-1 in P-LPS-treated human gingival fibroblasts was further examined. P-LPS induced the MCP-1 gene expression in a dose- and treatment time-dependent manner. The MCP-1 gene product in the culture supernatant was detected as two forms with molecular masses of 11,000 and 15,000 Da by immunoprecipitation with the specific antiserum. The MCP-1 gene expression was induced in the fibroblasts treated with interleukin-1 beta and tumor necrosis factor alpha, but not with interleukin-6. These results suggest that gingival fibroblasts can participate in monocyte recruitment in gingival tissues of adult periodontal patients via the MCP-1 gene product and that MCP-1 plays an important role in the inflammatory reaction in the disease.
Toxicity of staphylococcal enterotoxins potentiated by lipopolysaccharide: major histocompatibility complex class II molecule dependency and cytokine release
Authors: Stiles B G , Bavari S , Krakauer T , Ulrich R G
Published: January 2020

The biological effects of staphylococcal enterotoxins (SE), potentiated by bacterial lipopolysaccharide (LPS), were studied with mice. Control animals survived the maximum dose of either SE or LPS, while mice receiving both agents died. SEA was 43-fold more potent than SEB and 20-fold more potent than SEC1. The mechanism of toxicity was further examined with transgenic mice deficient in major histocompatibility complex class I or II expression. Class II-deficient mice were resistant to SEA or SEB. However, class I-deficient animals were less susceptible to SEA (30% lethality) than wild-type mice (93% lethality). In vitro stimulation of T cells from the three mouse phenotypes by SEA correlated well with toxicity. T cells from transgenic or wild-type mice were similarly responsive to SEA when presented by irradiated, wild-type mononuclear cells. These data confirmed that the toxicity of SE was mainly exerted through a mechanism dependent on the expression of major histocompatibility complex class II molecules. Toxicity was also linked to stimulated cytokine release. Levels in serum of tumor necrosis factor alpha, interleukin-6, and gamma interferon peaked 2 to 4 h after the potentiating dose of LPS but returned to normal within 10 h. Concentrations of interleukin-1 alpha were also maximal after 2 h but remained above the background for up to 22 h. Relative to the levels in mice given only SEA or LPS, the levels in serum of tumor necrosis factor alpha, interleukin-6, and gamma interferon increased 5-, 10-, and 15-fold, respectively, after injections of SEA plus LPS. There was only an additive effect of SEA and LPS on interleukin-1 alpha concentrations.
Identification of Legionella pneumophila genes required for growth within and killing of human macrophages
Authors: Sadosky A B , Wiater L A , Shuman H A
Published: January 2020

Legionella pneumophila was mutagenized with Tn903dIIlacZ, and a collection of mutants was screened for defects in macrophage killing (Mak-). Of 4,564 independently derived mutants, 55 (1.2%) showed a reduced or complete lack in the ability to kill HL-60-derived human macrophages. Forty-nine of the Mak- mutants could be assigned to one of 16 DNA hybridization groups. Only one group (9 of the 10 members) could be complemented for macrophage killing by a DNA fragment containing icm and dot, two recently described L. pneumophila loci that are required for macrophage killing. Phenotypic analysis showed that none of the mutants were any more sensitive than the wild type to human serum, oxidants, iron chelators, or lipophilic reagents nor did they require additional nutrients for growth. The only obvious difference between the Mak-mutants and wild-type L. pneumophila was that almost all of the Mak- mutants were resistant to NaCl. The effects of LiCl paralleled the effects of NaCl but were less pronounced. Resistance to salt and the inability to kill human macrophages are linked since both phenotypes appeared when Tn903dIIlacZ mutations from two Mak- strains were transferred to wild-type backgrounds. However, salt sensitivity is not a requisite for killing macrophages since a group of Mak- mutants containing a plasmid that restored macrophage killing remained resistant to NaCl. Mak- mutants from groups I through IX associated with HL-60 cells similarly to wild-type L. pneumophila. However, like the intracellular-multiplication-defective (icm) mutant 25D, the Mak- mutants were unable to multiply within macrophages. Thus, the ability of L. pneumophila to kill macrophages seems to be determined by many genetic loci, almost all of which are associated with sensitivity to NaCl.
CD4+ T-cell receptor V beta usage during the immune response to Schistosoma mansoni
Authors: Vella A T , Pearce E J
Published: January 2020

T-cell receptor V beta usage by CD4+ cells during the immune response of C57BL/6J mice to Schistosoma mansoni was examined. During acute infection, the distribution of splenic CD4+ cells utilizing V beta s 3, 5, 6, 7, 8.1/8.2, 9, and 11 was not significantly different from that in uninfected mice. Preferential V beta usage or deletion was not detected when either the primary or the memory immune response to parasite eggs was examined.
Class II major histocompatibility complex molecule expression on murine eosinophils activated in vivo by Brugia malayi
Authors: Mawhorter S D , Pearlman E , Kazura J W , Boom W H
Published: January 2020

The Th-2 response to microfilariae of Brugia malayi in BALB/c mice is associated with peritoneal eosinophilia. These eosinophils had an activated hypodense phenotype in vivo and expressed high levels of the class II major histocompatibility complex (MHC). Interleukin 4 up-regulated the class II MHC, whereas gamma interferon inhibited class II MHC expression on eosinophils.
Cell-mediated immunity to Bordetella pertussis: role of Th1 cells in bacterial clearance in a murine respiratory infection model
Authors: Mills K H , Barnard A , Watkins J , Redhead K
Published: January 2020

A murine respiratory infection model was used to study the mechanism of protective immunity to Bordetella pertussis. We found that nude mice, which are deficient in T cells, developed a persistent infection and failed to clear the bacteria after aerosol inoculation. In contrast, normal adult nonimmune mice cleared a respiratory infection approximately 35 days after challenge. Before bacterial clearance, antipertussis antibody levels in serum were low or undetectable, whereas consistent antigen-specific T-cell responses were demonstrated throughout the course of infection. The in vitro responses detected in immune spleen cells were mediated by a population of CD4+ major histocompatibility complex class II-restricted Th1-like cells that secreted interleukin-2 and gamma interferon but not interleukin-4. Adoptive transfer of immune spleen cells into nude or sublethally irradiated immunosuppressed mice before challenge resulted in bacterial clearance within 14 to 21 days. In contrast, injection of serum from convalescent mice before challenge only marginally reduced the bacterial load early in the course of infection. Furthermore, transfer of enriched T cells or purified CD4+ T cells but not CD8+ T cells from immune mice conferred a high level of protection. Recipients of CD4+ T cells cleared the bacteria from the lungs within 20 days of challenge, at which time B. pertussis-specific antibodies in the serum were undetectable. Although we do not rule out a contribution of mucosal immunoglobulin A, our findings suggest that cellular responses mediated by CD4+ Th1 cells play an important role in protective immunity to B. pertussis.
Comparison of the alpha-toxin genes of Clostridium perfringens type A and C strains: evidence for extragenic regulation of transcription
Authors: Katayama S , Matsushita O , Minami J , Mizobuchi S , Okabe A
Published: January 2020

The Clostridium perfringens plc gene encoding phospholipase C (alpha-toxin) was cloned from type C NCIB 10662, a strain which produces low levels of phospholipase C activity. The nucleotide sequence of a cloned 3.1-kb HindIII fragment was determined. The same fragment was also cloned from type A NCTC 8237, a phospholipase C-overproducing strain. In this case, an open reading frame (ORF2) truncated in the previously cloned 2-kb fragment was also sequenced. Comparison of the nucleotide sequence between the 3.1-kb fragments of the two type strains shows some differences both in the plc gene and in ORF2. However, when the 3.1-kb fragment was cloned into plasmid pUC19 and expressed in Escherichia coli, the plc genes from both type strains were similarly expressed and the toxins produced showed similar levels of activity. Northern blot analysis revealed that the type A strain produced 16 to 23 times more plc mRNA than the type C strain. These results indicate that in C. perfringens the two plc genes are transcribed at different rates, probably because of a difference in a locus lying outside of the cloned fragments. Gel retardation analysis showed that the type A strain possessed two different proteins that bound different regions of the plc gene. However, one of these proteins, which binds within the plc coding region, was not found in the type C strain, suggesting that it plays a role in the regulation of the plc gene expression.
Cysteine-dependent zinc binding by membrane proteins of Giardia lamblia
Authors: Zhang Y Y , Aley S B , Stanley S L , Gillin F D
Published: January 2020

The abundant, highly variable surface proteins (VSPs) which cover the surface of Giardia lamblia trophozoites compose a group of extremely cysteine (C)-rich proteins in which more than half of the cysteines are in the motif CXXC. Because of the constancy of these features among the known VSPs and the prominence of cysteine and particularly CXXC in proteins that bind zinc and other metals, we asked whether G. lamblia VSPs bind zinc in vitro. VSPs are the major protein component of Triton X-114 detergent-phase extracts of G. lamblia trophozoites and can be readily identified by surface iodination of intact cells. The partitioning of 65Zn binding into the Triton X-114 detergent phase and the correspondence between surface iodination and zinc binding patterns of four G. lamblia strains or sublines with different VSPs support the idea that VSPs bind zinc. The requirement for renaturation of blots with a reducing agent indicates that Zn2+ is coordinated by cysteine residues, rather than by other amino acids. Binding did not appear to be specific to zinc since it was inhibited by competition with other divalent metal ions. The abundance of the VSPs and the prevalence of metal binding motifs among all known variants suggest that they may play an important role in trophozoite survival and colonization in the host.
Shiga-like toxin II-related cytotoxins in Citrobacter freundii strains from humans and beef samples
Authors: Schmidt H , Montag M , Bockemühl J , Heesemann J , Karch H
Published: January 2020

By hybridizing colonies grown from 928 individual stool samples of patients suffering from diarrhea with oligonucleotide probes 772 and 849 complementary to Shiga-like toxin I (SLT-I) and SLT-II gene sequences, respectively, we identified two strains that hybridized with probe 849, which biochemical identification revealed as Citrobacter freundii. An additional five slt-II probe-positive isolates were screened from 81 beef samples. Polymerase chain reaction analysis and restriction of amplified products provided evidence for slt-II-related genes in all seven strains. From C. freundii LM 76, the genes encoding the A and B subunits were cloned in pUC 18 vectors and sequenced. The gene encoding the A subunit differed from that of Escherichia coli slt-IIvhc in 4 bases, resulting in two amino acid residue differences. In 11, 13, and 11 nucleotides, differentiation of slt-IIA, slt-IIcA, and vtx2haA, respectively, was found. These differences affected the predicted amino acid sequence as follows: there were six amino acid differences with SLT-IIA, five with SLT-IIcA, and four with VTx2haA. The nucleotide sequence of the gene encoding the B subunit is identical to slt-IIvhcB and differed from slt-IIcB and vtx2haB by only a single nucleotide base, but this resulted in a predicted amino acid sequence identical to that reported for these toxins. We therefore termed the toxin genes C. freundii slt-IIcA and slt-IIcB. Culture filtrates inoculated with material from the colonies from primary cultures were cytotoxic to Vero cells. Neutralization assays with antisera to E. coli SLT-I, SLT-II, and SLT-IIvhc revealed that antibodies against SLT-IIvhc reduced the C. freundii cytotoxic activity specifically and to the same degree as with the E. coli SLT-IIvhc control strain. In five of the seven strains tested, subcultivation on both a liquid or solid medium resulted in loss of cytotoxic activity. With polymerase chain reaction, we demonstrated that loss of cytotoxic activity ran parallel with the loss of slt genes. These data demonstrate the intergeneric occurrence of SLT-II-related toxins, which may well be a new marker of enteropathogenicity in C. freundii. Our findings that the toxin genes belong to the slt-II family plus their evident instability in the majority of strains should help pave the way to a better understanding of their role in diarrhea or food poisoning.
Cloning and characterization of the S fimbrial adhesin II complex of an Escherichia coli O18:K1 meningitis isolate
Authors: Hacker J , Kestler H , Hoschützky H , Jann K , Lottspeich F , Korhonen T K
Published: January 2020

S fimbrial adhesins (Sfa), which are able to recognize sialic acid-containing receptors on eukaryotic cells, are produced by Escherichia coli strains causing urinary tract infections or newborn meningitis. We recently described the cloning and molecular characterization of a determinant, termed sfaI, from the chromosome of an E. coli urinary tract infection strain. Here we present data concerning a S fimbria-specific gene cluster, designated sfaII, of an E. coli newborn meningitis strain. Like the SfaI complex, SfaII consists of the major subunit protein SfaA (16 kDa) and the minor subunit proteins SfaG (17 kDa), SfaS (15 kDa), and SfaH (29 kDa). The genes encoding the subunit proteins of SfaII were identified and sequenced. Their protein sequences were calculated from the DNA sequences and compared with those of the SfaI complex subunits. Although the sequences of the two major SfaA subunits differed markedly, the sequences of the minor subunits showed only a few amino acid exchanges (SfaG, SfaH) or were completely identical (SfaS). The introduction of a site-specific mutation into the gene sfaSII and subsequent analysis of an SfaS-negative clone indicated that sfaSII codes for the sialic acid-specific adhesin of the meninigitis isolate. These data were confirmed by the isolation and characterization of the SfaSII protein and the determination of its N-terminal amino acid sequence. The identity between the sialic acid-specific adhesins of SfaI and SfaII revealed that differences between the two Sfa complexes with respect to their capacities to agglutinate erythrocytes must result from sequence alterations of subunit proteins other than SfaS.
Identification by two-dimensional gel electrophoresis of a 58-kilodalton tumor necrosis factor-inducing protein of Mycobacterium tuberculosis
Authors: Wallis R S , Paranjape R , Phillips M
Published: January 2020

We have previously identified proteins in fractions of culture filtrate of Mycobacterium tuberculosis with the capacity to induce cytokine production in monocytes, by using a technique we defined as "monocyte Western blotting" (immunoblotting). In this series of experiments, we have extended this technique to two-dimensional gel electrophoresis and have identified a novel 58-kDa protein of M. tuberculosis which induces production of tumor necrosis factor by human monocytes. Nitrocellulose particles bearing this protein were used to develop murine monoclonal antibodies by the technique of intrasplenic immunization. The protein was purified by preparative isoelectric focusing and gel electrophoresis and subjected to N-terminal amino acid sequence analysis. As tumor necrosis factor is a mediator of both pulmonary necrosis and macrophage activation for intracellular killing, this 58-kDa protein may play an important role both in the immunopathogenesis of tuberculosis and in mycobacterial immunity.
Light emission from a Mudlux transcriptional fusion in Salmonella typhimurium is stimulated by hydrogen peroxide and by interaction with the mouse macrophage cell line J774.2
Authors: Francis K P , Gallagher M P
Published: January 2020

Hydrogen peroxide is known to induce a multigenic response in Salmonella typhimurium cells. We have used a Mudlux transcriptional reporter system to identify and isolate fusions in the virulent strain SL1344 which respond to hydrogen peroxide in vitro by light production, and one of these fusions, MPG203, has been further characterized. Transient light production was observed from MPG203 at levels of hydrogen peroxide as low as 10 microM. However, high levels of this toxic oxidizing agent resulted in light suppression, particularly at low bacterial densities. This fusion was also shown to produce light following adhesion to cells of the mouse macrophage cell line J774.2. Furthermore, the response was greatly reduced in the presence of catalase, directly implicating hydrogen peroxide as the eliciting agent and suggesting the involvement of the hydrogen peroxide-induced bacterial stress response in the infection process. Chemiluminescence studies also indicated that inhibition of the respiratory burst may occur as the infection ratio is increased. In addition, the level of light produced from bacteria within individual macrophage cells was shown to vary.
Role of lipooligosaccharide in virulence of the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius for infant rats
Authors: Rubin L G , St Geme J W
Published: January 2020

Clonally related strains of Haemophilus influenzae biogroup aegyptius have recently been associated with Brazilian purpuric fever (BPF), a fulminant, systemic disease in children. Using an infant rat bacteremia model for BPF, we found that a rat blood-passaged BPF isolate of H. influenzae biogroup aegyptius was more virulent than the original strain was. When compared with the original strain, the animal-passaged variant was found to display an altered lipooligosaccharide (LOS) phenotype and to lack pili. To examine the role of LOS phenotype and pili in virulence, we isolated isogenic variants differing in LOS phenotype or expression of pili. The virulence of variants was compared by examining the results of blood cultures obtained 24 h after intraperitoneal inoculation with 10(5) CFU. Our results indicate that the LOS phenotype is a critical determinant of BPF clone virulence for infant rats. To a lesser extent, the absence of piliation and an undefined additional factor(s) contribute to virulence.
An antagonist of platelet-activating factor suppresses endotoxin-induced tumor necrosis factor and mortality in mice pretreated with carrageenan
Authors: Ogata M , Matsumoto T , Koga K , Takenaka I , Kamochi M , Sata T , Yoshida S , Shigematsu A
Published: January 2020

We found that carrageenan (CAR), that is, sulfated polygalactose, can enhance both lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production and the rate of lethality in mice (M. Ogata, S. Yoshida, M. Kamochi, A. Shigematsu, and Y. Mizuguchi, Infect. Immun. 59:679-683, 1991). It has been reported that platelet-activating factor (PAF) antagonists reduce the rate of mortality from endotoxin shock. However, there are few reports regarding the effect of PAF antagonists on TNF production. The aim of the present study is to examine the effect of TCV-309, a new PAF antagonist, on LPS-induced TNF production and mortality in mice pretreated with CAR. ddY mice (6 to 7 weeks old) were injected intraperitoneally with CAR (5 mg per mouse) and were then divided into two groups: mice treated with a PAF antagonist (TCV-309; Takeda Pharmaceutical Co.) and control mice. The mice treated with PAF antagonist received indicated doses of TCV-309 subcutaneously (s.c.) at 30 min before LPS injection, while the control mice received 1 ml of saline s.c. at the same time. All mice were stimulated by intravenous injection of LPS (50 micrograms per mouse) at 24 h after pretreatment with CAR. At intervals after injection of LPS, serum samples were obtained for a TNF assay in which cytotoxicity to L929 cells was measured. TCV-309 both significantly suppressed LPS-induced TNF production and reduced mortality in a dose-dependent manner. When TCV-309 was administered at 30 min before injection of LPS, the effect of TCV-309 on the suppression of TNF activity was at its peak. Treatment with TCV-309 (990 micrograms per mouse) s.c. significantly improved the survival rate after challenge with LPS compared with the survival rate of control mice. Although the 50% lethal dose of LPS was 15 micrograms per mouse for control mice, it increased to 102 micrograms per mouse for mice that were treated s.c. with TCV-309 (990 micrograms per mouse). Even in vitro, TCV-309 also inhibited LPS-induced TNF production in thioglycolate-elicited macrophages. It was concluded that PAF plays an important role in endotoxin-induced TNF production and mortality.
Secondary Vibrio cholerae-specific cellular antibody responses following wild-type homologous challenge in people vaccinated with CVD 103-HgR live oral cholera vaccine: changes with time and lack of correlation with protection
Authors: Losonsky G A , Tacket C O , Wasserman S S , Kaper J B , Levine M M
Published: January 2020

Peripheral blood immunoglobulin A antibody-secreting-cell (ASC) responses are thought to reflect the mucosal immune response to locally presented antigens. We evaluated the ASC response to cholera toxin (CT) and Inaba lipopolysaccharide (LPS) in 26 North American volunteers following immunization with a single oral dose of live attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR and again upon homologous wild-type challenge with V. cholerae classical Inaba 569B. Challenge occurred at either 7, 30, or 180 days after vaccination. The CT and LPS ASC responses of volunteers following vaccination (83 and 55%, respectively) were similar in magnitude and frequency to those of unvaccinated controls following wild-type challenge (80 and 60%, respectively [0.1 < or = P < or = 0.9]). The responses were primarily immunoglobulin A. Vaccinated volunteers challenged within 30 days of vaccination had reduced or nondetectable CT and LPS ASC responses. Challenge at 6 months resulted in a heightened ASC response to LPS, confirming the existence of mucosal memory. ASC responses to CT upon challenge at 6 months were detectable but not different from that seen following primary immunization, suggesting that secondary ASC responses to different antigens from a single vaccine operate independently. In spite of these variable ASC responses, the vaccine efficacy was 100% following challenge for all vaccinees. V. cholerae-specific ASC responses following antigenic reexposure gave information on the presence of mucosal B memory cells but did not correlate with protective immunity. As such, these ASC assays will have limited usefulness for evaluating vaccine responders in vaccine field trials in cholera-endemic areas where prior V. cholerae O1 exposure is unknown.
Leishmania donovani-reactive Th1- and Th2-like T-cell clones from individuals who have recovered from visceral leishmaniasis
Authors: Kemp M , Kurtzhals J A , Bendtzen K , Poulsen L K , Hansen M B , Koech D K , Kharazmi A , Theander T G
Published: January 2020

Infections in humans by Leishmania donovani parasites can result in a fatal disease, visceral leishmaniasis (VL), or in a self-limiting asymptomatic infection. In murine models of the infection employing Leishmania major, the course of the disease can be directed into a VL-like syndrome by interleukin-4 (IL-4)-producing Th2 cells, or cure may result by Th1 cells secreting gamma interferon (IFN-gamma). The present study examined the potential of human T cells to generate Th1 or Th2 responses to L. donovani. The profiles of IFN-gamma, IL-4, and lymphotoxin secretion after antigen stimulation were analyzed in a panel of L. donovani-reactive CD4+ human T-cell clones generated from individuals who had recovered from VL after antimonial treatment. Two of the T-cell clones produced large amounts of IL-4 without production of IFN-gamma, seven clones produced both IFN-gamma and IL-4, and eight produced only IFN-gamma. This is the first report of a Th1- and Th2-type response in human leishmaniasis. These results suggest that in analogy with murine models, there is a dichotomy in the human T-cell response to L. donovani infections. Preferential activation of IL-4-producing Th2-like cells may be involved in the exacerbation of human VL, whereas activation of IFN-gamma-producing Th1 cells may protect the host from severe disease. Identification of leishmanial antigens activating one or the other type of T cells will be important in the development of vaccines against leishmaniasis.
Effects of Clostridium difficile toxin B on human monocytes and macrophages: possible relationship with cytoskeletal rearrangement
Authors: Siffert J C , Baldacini O , Kuhry J G , Wachsmann D , Benabdelmoumene S , Faradji A , Monteil H , Poindron P
Published: January 2020

Toxin B from Clostridium difficile is cytopathic in vitro for various types of cells, including polymorphonuclear cells, lymphocytes, and monocytes. Since intestine lamina propria is rich in macrophages, we studied the effect of toxin B on human monocytes and on human macrophages generated in vitro by long-term culture of purified circulating blood monocytes. Upon addition of toxin B, human monocytes exhibited few modifications whereas macrophages adopted a stellate morphology, with rounding up of the perikaryon. Toxin B made microfilaments of actin disappear and induced an important reorganization of vimentin and a redistribution of tubulin. Membrane area increased by approximately 16%. Toxin B did not affect the viability of human mononuclear phagocytes and did not exert any significant lytic effect. It profoundly altered the phagocytic function of macrophages. When activated by gamma interferon in the presence of toxin B, monocytes were more cytotoxic for U-937 target cells than control monocytes activated in absence of toxin. Finally, the combined treatment of monocytes with gamma interferon and toxin B increased significantly the secretion of tumor necrosis factor alpha, whereas toxin B alone was unable to induce tumor necrosis factor production. These results suggest that morphological and functional alterations induced in human mononuclear phagocytes by toxin B from C. difficile are due to the disorganization of the cytoskeleton and the resulting impairment of the membrane traffic equilibrium.
Molecular cloning, expression, and DNA sequence analysis of the gene that encodes the 16-kilodalton outer membrane lipoprotein of Serpulina hyodysenteriae
Authors: Thomas W , Sellwood R
Published: January 2020

The gene (smpA) that encodes the 16-kDa outer membrane lipoprotein of Serpulina hyodysenteriae was cloned in Escherichia coli, and its primary structure was determined by nucleotide sequencing. The putative open reading frame encodes a prolipoprotein of 16.8 kDa which in its fully acylated and cleaved form is 15.1 kDa. Analysis of the N-terminal amino acid sequence derived from the DNA sequence revealed the presence of a signal sequence and a putative acylation and signal peptidase II cleavage site (Phe-Ala-Val-Ser-Cys). In E. coli, processing of the prolipoprotein was less efficient than that observed in S. hyodysenteriae, and globomycin, an inhibitor of signal peptidase II, inhibited cleavage of the lipoprotein expressed in E. coli but did not inhibit cleavage in S. hyodysenteriae.
Immunolocalization of superoxide dismutase in Dirofilaria immitis adult worms
Authors: Callahan H L , Hazen-Martin D , Crouch R K , James E R
Published: January 2020

Superoxide dismutase (SOD) may not only perform a housekeeping role in filarial worms but also assist in defense against oxidants generated by host immune cells. Both Dirofilaria and Onchocerca adult filariae and microfilariae contain relatively high activities of the antioxidant enzyme SOD; adult Dirofilaria worms also secrete SOD in vitro. In addition, superoxide radicals are relatively impotent against Dirofilaria and Onchocerca microfilariae in vitro. In assessing the role of SOD, we determined the anatomic localization of SOD in D. immitis adult worms by immunolocalization at the light-microscopic level. We found that anti-D. immitis SOD did not stain parasite tissues homogeneously, in support of the hypothesis that SOD does not have only a housekeeping role and that the pattern of staining may suggest another role(s) for SOD.
Characterization of environmental regulators of Bordetella pertussis
Authors: Melton A R , Weiss A A
Published: January 2020

Bordetella pertussis suppresses transcription of its virulence genes in response to specific environmental conditions, a response called modulation. The organism responds to high concentrations of SO4 and CIO4 ions, nicotinic acid, and nicotinic acid analogs in vitro; however, the in vivo modulator has not been identified. We investigated which chemical structures of the nicotinic acid molecule are important for modulation by testing various analogs for their ability to modulate. The ring nitrogen of nicotinic acid was not required, since benzoic acid was a modulator. In contrast, the carboxyl group was required, since derivatives like ethylnicotinate, 3-pyridylcarbinol, 3-acetyl pyridine, and 6-chloronicotinamide with altered carboxyl groups were not modulators. The planar ring structure or resonance in the ring was required for modulation, since nipecotic acid failed to modulate. The most potent modulators were nicotinic acid derivatives with electron-withdrawing substituents in the meta or para position relative to the carboxyl group. Relative hydrophilicity of substituents did not appear to contribute to modulation. Although these modulators elicited a clear biological response, the mechanism of modulation remains unclear, because no binding of the modulator 35SO4 or [14C]4-chlorobenzoic acid to whole B. pertussis was detected. However, modulation appears to involve a charge-charge interaction, since the response was blocked by chlorine ions.
Experimental gastritis induced by Helicobacter pylori in Japanese monkeys
Authors: Shuto R , Fujioka T , Kubota T , Nasu M
Published: January 2020

We used Japanese monkeys (Macaca fuscata) to establish an experimental model in order to clarify the pathogenicity of Helicobacter pylori in gastric and duodenal disorders. A suspension (5 ml; 10(9) CFU/ml) of H. pylori cells isolated from humans was sprayed around the antrum of the stomach of each of 12 of 17 animals with an endoscope. The remaining five animals were not inoculated; they served as a control group. On days 7, 14, and 28 after inoculation, the gastric mucosa samples were examined grossly and were biopsied for microscopic examination with an endoscope. H. pylori was recovered from 7 of the 12 inoculated animals (58%), and infiltration by neutrophils and monocytes was observed histologically. Macroscopic gastritis with erythema and erosions were noted for five of these animals. On day 28 after inoculation, five animals in the infected group were treated with ampicillin. In two infected but untreated animals, the bacteria persisted for more than 6 months. The result of the gastritis scoring of the antral mucosa and the ammonia concentration in the gastric secretion were significantly higher (P < 0.01 to 0.001) for the infected group than for the control group; however, these values decreased to levels comparable to those for the control group after treatment with ampicillin. Urease activity was positive in gastric biopsy specimens from five of the seven animals in the infected group after 7 days and from four of these animals after 14 days but was negative in all specimens from animals in the control group. The level of antibody (immunoglobulin G) in serum for the infected group was elevated but changed very little for the control group. These results suggest that this M. fuscata model can be used to study H. pylori infection and that H. pylori can induce gastritis.
Isolation and characterization of a cloned Porphyromonas gingivalis hemagglutinin from an avirulent strain of Salmonella typhimurium
Authors: Dusek D M , Progulske-Fox A , Whitlock J , Brown T A
Published: January 2020

Identification of surface macromolecules of Porphyromonas gingivalis that act as virulence factors in periodontal disease has important implications for studying host-parasite interactions as well as for potential vaccine development. The objective of this study was to determine whether a cloned, P. gingivalis hemagglutinin gene could be expressed in an intact form in an avirulent Salmonella typhimurium vaccine construct and to characterize the recombinant protein. The recombinant protein was purified from the vaccine strain, characterized, and tested for biological activity as a competitive inhibitor of hemagglutination. Cells of S. typhimurium SL3261/pST7 grown in Luria broth were broken by sonic disruption and fractionated. The purified recombinant protein was found to inhibit hemagglutination of erythrocytes by whole P. gingivalis cells. The same purified protein was analyzed for its N-terminal amino acid sequence and amino acid composition and found to match that predicted from the nucleotide sequence of the cloned gene. These results indicate that a surface macromolecule of P. gingivalis can be expressed in an intact and biologically active form in a Salmonella carrier strain.
Cloning and characterization of the Actinobacillus pleuropneumoniae-RTX-toxin III (ApxIII) gene
Authors: Jansen R , Briaire J , Kamp E M , Gielkens A L , Smits M A
Published: January 2020

To study the role of Actinobacillus pleuropneumoniae-RTX-toxin III (ApxIII) in the pathogenesis of porcine pleuropneumonia, we cloned and characterized the gene encoding this toxin. For that purpose, we screened an expression library of genomic DNA of serotype 8 with an ApxIII-specific monoclonal antibody and isolated a 425-bp fragment of an immunoreactive clone. Using this fragment as a probe, we identified and cloned an overlapping chromosomal NsiI restriction fragment of 5.0 kbp. Escherichia coli cells that contained this fragment produced a protein similar to ApxIII. Like ApxIII, the protein had a molecular mass of approximately 120 kDa, was recognized by an ApxIII-specific antibody, killed porcine lung macrophages, and was not lytic for sheep erythrocytes. We concluded from these data that the 5.0-kbp NsiI fragment contained the ApxIII-coding gene. Nucleotide sequence analysis of the 5.0-kbp NsiI fragment revealed the presence of two genes, apxIIIC and apxIIIA. These genes coded for proteins ApxIIIC and ApxIIIA, respectively, which were 53 and 50% identical to the prototypic RTX proteins HlyC and HlyA of E. coli. We assumed that the apxIIIA gene coded for the structural RTX toxin and that the apxIIIC gene coded for its activator. In addition, we found that ApxIII could be secreted from E. coli by the heterologous RTX transporter proteins HlyB and HlyD. The deduced amino acid sequence of ApxIIIA was 50% identical to that of ApxIA and 41% identical to that of ApxIIA. We concluded that, beside ApxI and ApxII, ApxIII is the third RTX toxin produced by A. pleuropneumoniae.
Characterization of bovine neutrophil antibacterial polypeptides which bind to Escherichia coli
Authors: Litteri L , Romeo D
Published: January 2020

Bovine neutrophils contain several cationic polypeptides which exert potent microbicidal effects in vitro. To better characterize the repertoire of these polypeptides, we have incubated extracts of bovine neutrophils or neutrophil granules at pH 4 or 7 with either a smooth strain of Escherichia coli or a rough one. Only a few polypeptides interacted with the bacterial surface and were subsequently desorbed with 200 mM MgCl2, as revealed by gel electrophoresis and analysis of Western blots (immunoblots) with appropriate antibodies. Two of the main proteins appearing in Coomassie blue-stained gels have molecular masses of 53 and 15 kDa and correspond to the heavy and light chains of myeloperoxidase. Another prevailing protein band with a molecular mass of 31 kDa was purified and shown to be 87% identical to human azurocidin/CAP37 in its 22-amino-acid N-terminal sequence. Proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose did not react with an antiserum to human bactericidal/permeability-increasing protein. Conversely, immunoglobulin G against Bac7 or Bac5, two members of the antimicrobial proline- and arginine-rich polypeptide family, recognized in Western blots both the inactive precursor molecules, proBac7 and proBac5, and the mature polypeptides.
Elevated interleukin-8 levels in the urine of patients with urinary tract infections
Authors: Ko Y C , Mukaida N , Ishiyama S , Tokue A , Kawai T , Matsushima K , Kasahara T
Published: January 2020

Pyuria is one of the main features of urinary tract infections (UTI). Nevertheless, the mechanism of polymorphonuclear leukocyte (PMN) recruitment into the urine remains to be investigated. We examined whether interleukin-8 (IL-8), a potent neutrophil chemoattractant and activator, was involved in pyuria seen in UTI. Of 113 patients, 112 had elevated levels of IL-8 in their urine (1,078.0 +/- 181.5 pg/ml), regardless of whether they had an upper or lower UTI; this was in contrast to undetectable levels (less than 16 pg/ml) in the urine of all of the 20 normal individuals and 74 control patients without UTI. A concomitant study revealed increases in urine IL-6, but not IL-1 beta, and tumor necrosis factor alpha levels in patients with UTI. In addition to gram-negative bacteria, a wide spectrum of microorganisms was capable of inducing IL-8 production in urine. Local production of IL-8 in the urinary tract was suggested by a urine IL-8 level that was higher than the paired serum IL-8 level. The urine IL-8 level correlated with the number of PMN in the urine, and an average of half of the chemotactic activity in urine from patients with UTI could be abrogated by anti-IL-8 antibody treatment in vitro. Furthermore, urine IL-8 purified from patients was bioactive and showed multiple forms on immunoblotting analysis. This is the first documentation of IL-8 in the urine of patients with UTI, and these results imply that IL-8 is involved in inducing PMN migration into the urinary tract.
Complete development of Cryptosporidium parvum in a human endometrial carcinoma cell line
Authors: Rasmussen K R , Larsen N C , Healey M C
Published: January 2020

Sporozoites of Cryptosporidium parvum, excysted from oocysts isolated from calves, were applied to monolayers of the human endometrial carcinoma cell line RL95-2. Cells were grown as monolayers in 24-well plates at concentrations ranging from 5 x 10(4) to 1 x 10(5) RL95-2 cells per well. At 1 or 7 days postculturing, C. parvum sporozoites (ranging from 1 x 10(5) to 2 x 10(5) were added to the monolayers of RL95-2 cells. The cells were fixed and stained to estimate the extent of parasite colonization. Light microscopy and electron microscopy confirmed the development and replication of C. parvum within the RL95-2 cells. A standardized and reproducible in vitro culture system for C. parvum is necessary to evaluate therapies against cryptosporidiosis.