bluuebunny/crossref_metadata_2025 huggingface dataset:
Cryptosporidium muris in adult mice: adoptive transfer of immunity and protective roles of CD4 versus CD8 cells
Authors: McDonald V , Robinson H A , Kelly J P , Bancroft G J
Published: January 2020

The aim of this study was to investigate the role of the CD4 and CD8 T cells in immunity to cryptosporidia by using Cryptosporidium muris and a mouse model of infection. Two approaches were used, each involving the use of rat anti-T-cell surface marker monoclonal antibodies (MAbs). In the first, the adoptive transfer of immunity was studied by using the CB.17 SCID mouse (which lacks T and B cells) as the host; in the second, the effect on susceptibility of BALB/c mice to infection was examined following depletion of T cells or subsets of T cells. In adoptive immunity experiments, the conditions which differentiated between resistance associated with reconstitution of SCID mice with naive BALB/c lymphocytes and the transfer of immunity with primed lymphocytes from infected animals were determined. Primed spleen or mesenteric lymph node cells conferred better protection to recipients than naive cells when obtained from donors which had developed resistance to infection. Adoptive immunity was abrogated when Thy.1 cells or CD4 cells were depleted from primed cells, while depletion of CD8 cells could reduce the level of protection. In the study of C. muris in BALB/c mice, treatment with either anti-Thy.1 plus anti-Lyt.1 or anti-CD4 MAbs increased susceptibility to a primary infection as determined by the size and duration of oocyst production, but an anti-CD8 MAb produced an increase only in oocyst shedding. Thus, both CD4 and, to a lesser extent, CD8 cells appeared to be involved in resistance to primary and secondary C. muris infection.
Identification, localization, and distribution of the PilT protein in Neisseria gonorrhoeae
Authors: Brossay L , Paradis G , Fox R , Koomey M , Hébert J
Published: January 2020

A monoclonal antibody (MAb) directed against a highly conserved protein of Neisseria gonorrhoeae with a molecular size of 40 kDa was isolated and characterized. The protein antigen detected by this MAb was detected by enzyme-linked immunosorbent assay and immunoblotting in all strains of N. gonorrhoeae tested across a wide range of serovars. The 40-kDa protein was found to be expressed at relatively low levels and localized to both the cytosolic and cytoplasmic membrane fractions. Screening of a lambda gt11 expression library derived from gonococcal genomic DNA with the anti-40-kDa MAb and DNA sequence analysis suggested that the 40-kDa protein and the product of the gonococcal pilT gene were identical. Immunoblotting analysis of gonococcal mutants carrying defined mutations in the pilT gene confirmed that the 40-kDa protein was indeed PilT. The N-terminal sequence derived by microsequencing of the protein purified from gonococci led to the correction of the previously published pilT gene sequence. Sequencing of the pilT gene from three different strains revealed an extremely high degree of conservation at both the amino acid and DNA levels.
Staphylococcal iron requirements, siderophore production, and iron-regulated protein expression
Authors: Lindsay J A , Riley T V
Published: January 2020

Despite the ability of staphylococci to grow in iron-restricted conditions in vivo, their iron requirements and the mechanisms possessed by them for the uptake of iron are poorly understood. Many bacteria are known to produce siderophores. By using the chrome azurol S universal method for the detection of siderophores, all 14 isolates of Staphylococcus aureus tested grew well under conditions of iron restriction and produced iron-regulated siderophore in large quantities, while all 19 isolates of coagulase-negative staphylococci (CoNS) grew poorly under conditions of iron restriction and produced low levels of iron chelator. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of S. aureus isolates revealed altered protein patterns due to iron restriction, while altered profiles were not seen in the CoNS group. The ability to grow in iron-restricted conditions, possibly with the assistance of siderophore-mediated iron uptake, may contribute to the increased pathogenicity of S. aureus when compared with that of the CoNS.
Treatment with anti-interleukin-10 monoclonal antibody enhances early resistance to but impairs complete clearance of Listeria monocytogenes infection in mice
Authors: Wagner R D , Maroushek N M , Brown J F , Czuprynski C J
Published: January 2020

Mice that received an anti-interleukin-10 (anti-IL-10) neutralizing monoclonal antibody (MAb) (SXC-1) prior to infection with Listeria monocytogenes initially demonstrated resistance to the infection, as indicated by reduced recovery of L. monocytogenes from their spleens and livers during the first 5 days after challenge. Anti-IL-10 MAb-treated mice then demonstrated reduced resistance during the later stage of infection, as indicated by persistent infection with L. monocytogenes in their livers 11 days after challenge. Aspartate aminotransferase (AST) levels (a measure of liver damage) in the sera of control mice increased between 1 and 5 days after challenge, while anti-IL-10 MAb-treated mice maintained lower AST levels. At 7 days after challenge, AST levels in the sera of control mice decreased as the numbers of organisms declined. In contrast, AST levels increased as the infections persisted in anti-IL-10 MAb-treated mice. The AST levels in serum reflected liver histopathology as anti-IL-10 MAb-treated mice exhibited fewer granulomatous lesions and less necrosis of liver tissue than the control mice during the first 5 days after challenge. Anti-IL-10 MAb treatment altered the expression of inflammatory cytokine mRNAs during L. monocytogenes infection. Control MAb-treated mice exhibited increased expression of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor mRNA in their lives during L. monocytogenes infection, but this increase did not occur in anti-IL-10 MAb-treated mice. Gamma interferon mRNA expression in the livers of the control MAb-treated mice was increased between 1 and 5 days after L. monocytogenes challenge and then decreased at 7 days after challenge. In contrast, gamma interferon mRNA expression in the livers of anti-IL-10 MAb-treated mice was not decreased until 7 days after challenge. These results indicate that endogenous IL-10 has both beneficial and detrimental effects on the host response to L. monocytogenes infection in mice.
Inhibition of intracellular Histoplasma capsulatum replication by murine macrophages that produce human defensin
Authors: Couto M A , Liu L , Lehrer R I , Ganz T
Published: January 2020

Although purified defensins are effective microbicides in vitro, their operation within intact phagocytes has not been established. To address this question, we inserted cDNA encoding human defensin HNP-1 into a pBabe/neo retroviral vector and transduced it into RAW 264.7 cells, a murine macrophage line that lacks endogenous defensins. We isolated five independent clones of HNP-1-transduced cells, all of which secreted prodefensin and contained small amounts of fully processed HNP-1. The two clones that produced the largest amounts of defensin (clones 5 and 14), together with wild-type RAW cells and pBabe/neo-transduced RAW cells (control), were used for the present study. All cells were grown in Dulbecco's modified Eagle's medium-F12 medium that contained 10% heat-inactivated fetal bovine serum and gentamicin. The medium used for the transduced cells contained aminoglycoside G418 in lieu of gentamicin. Both wild-type and transduced cells were placed in antibiotic-free medium 96 h prior to challenge with a yeast-phase strain of Histoplasma capsulatum. Phagocytosis of yeast cells was allowed to proceed for 90 min and was followed by washing and further incubation for 18.5 h. Whereas the phagocytic index did not differ significantly among the four cell populations under study, the mean level of intracellular growth of H. capsulatum in the defensin-transduced RAW cells was significantly lower than those observed for any other cell types (P < 0.05). These findings constitute the first instance of xenogeneic expression of an antimicrobial peptide by phagocytes and suggest that macrophages can be armed with defensins to enhance their ability to restrict certain intracellular pathogens.
Pathogenicity of porcine intestinal spirochetes in gnotobiotic pigs
Authors: Neef N A , Lysons R J , Trott D J , Hampson D J , Jones P W , Morgan J H
Published: January 2020

Twelve intestinal spirochete strains of porcine origin were characterized on the basis of their phenotypic properties, by multilocus enzyme electrophoresis, and by pathogenicity testing in gnotobiotic pigs. The spirochetes used included two strains of Serpulina hyodysenteriae (B204 and P18A), two strains of Serpulina innocens (B256 and 4/71), one strain from the proposed new genus and species "Anguillina coli" (P43/6/78), and seven non-S. hyodysenteriae strains recently isolated from United Kingdom pig herds with a history of nonspecific diarrhea and typhlocolitis. By multilocus enzyme electrophoresis, five of these were identified as S. innocens, one was identified as an unspecified Serpulina sp., and one was identified as "A. coli." S. hyodysenteriae B204 and P18A, "A. coli" P43/6/78 and 2/7, and three (22/7, P280/1, and 14/5) of the five S. innocens field isolates induced mucoid feces and typhlocolitis in gnotobiotic pigs. None of the other spirochetes produced clinical signs or large intestinal pathology in this model. The "A. coli" strains induced a more watery diarrhea, with lesions present more proximally in the large intestine, than did the other pathogenic spirochetes. S. innocens 22/7 was also tested for pathogenicity in hysterotomy-derived pigs that had previously been artificially colonized with a spirochete-free intestinal flora and shown to be susceptible to swine dysentery. Despite effective colonization, strain 22/7 did not produce any disease, nor was there any exacerbation of large intestinal pathology or clinical signs when pigs with an experimentally induced existing colitis caused by Yersinia pseudotuberculosis were superinfected with strain 22/7. Certain non-S. hyodysenteriae spirochetes are therefore capable of inducing disease in gnotobiotic pigs, but their role as primary or opportunistic pathogens in conventional pigs remains equivocal.
B cells are required for the switch from Th1- to Th2-regulated immune responses to Plasmodium chabaudi chabaudi infection
Authors: Taylor-Robinson A W , Phillips R S
Published: January 2020

The induction of T-helper cell subsets during the course of blood stage Plasmodium chabaudi chabaudi infection was compared in immunologically intact NIH mice and mice that were depleted of B cells from birth by treatment with anti-mu antibodies. For intact mice, in which the acute primary parasitemia peaked 10 days following infection, purified splenic CD4+ T cells recovered during the ascending parasitemia produced high levels in vitro of interleukin 2 (IL-2) (peak levels on day 10) and gamma interferon (IFN-gamma) (peak levels on day 7). Sera collected from these mice at around this time contained relatively high levels of P. c. chabaudi-specific immunoglobulin 2a (peak levels on day 12), and serum nitric oxide activity was significantly elevated at peak parasitemia. During the descending primary parasitemia, production of IFN-gamma and IL-2 decreased, while levels of IL-4 and IL-10 produced by splenic CD4+ T cells were significantly raised from the time at which subpatency was recorded (day 17) and persisted for at least 50 days. This was concomitant with a significant increase in levels of parasite-specific immunoglobulin G1, which peaked at around the time of recrudescence. Thus, in normal mice, sequential appearance of Th1 and Th2 responses was observed. In contrast, in B-cell-depleted mice, recovery from acute primary parasitemia was followed by a persistent patent infection which did not drop below 0.1% for at least 75 days after initiation of infection. These mice were unable to mount a significant Th2 response, manifest as an enduring inability of splenic CD4+ T cells to produce significant levels of IL-4 and IL-10. IL-2 and IFN-gamma levels remained significantly elevated throughout the 50-day observation period, and there was sustained production of nitric oxide. These data show that immune responses mediated by CD4+ T cells of the Th1 subset are capable of limiting infection beyond the initial acute phase, but that they do not eliminate parasitemia. Furthermore, as the progression from a Th1-regulated to a Th2-regulated immune response fails to occur in B-cell-depleted mice, the data suggest that B cells are required for the downregulation of Th1-mediated and/or the generation of Th2-mediated protective immunity to P. c. chabaudi.
Cloning and characterization of bys1, a temperature-dependent cDNA specific to the yeast phase of the pathogenic dimorphic fungus Blastomyces dermatitidis
Authors: Burg E F , Smith L H
Published: January 2020

The pathogenic dimorphic fungal organism Blastomyces dermatitidis exists as a budding yeast at 37 degrees C and as a mycelium at 25 degrees C. While the conversion of one morphological phase of B. dermatitidis to another has long been known to be a thermally dependent process, little of the accompanying biochemical or genetic events controlling the phase transition has been elucidated. Using differential cDNA library screening, we have identified one transcript, bys1, in B. dermatitidis that is expressed at very high levels in the yeast phase but whose levels diminish rapidly when yeast cells are transferred to 25 degrees C to promote conversion to the mycelial phase. Although the 0.95-kb bys1 transcript is absent in B. dermatitidis mycelia maintained at 25 degrees C, transfer of mycelial cultures to 37 degrees C results in the reappearance of bys1 within 12 h. bys1 codes for a protein of 18.6 kDa that contains multiple putative phosphorylation sites, a hydrophobic N terminus, and two 34-amino-acid domains with similarly spaced nine-amino-acid degenerative repeating motifs. Although the nature of the thermal dependency of bys1 expression and the function of the bys1 protein are unknown, the strong expression of this transcript specifically in the yeast phase of B. dermatitidis may prove to be very useful in the development of more specific and sensitive diagnostic methods for blastomycosis.
Expansion of gamma interferon-producing CD8+ T cells following secondary infection of mice immune to Leishmania major
Authors: Müller I , Kropf P , Louis J A , Milon G
Published: January 2020

Reinfection of immune mice with Leishmania major elicits a secondary gamma interferon (IFN-gamma) response to which specific CD8+ T cells are essential. We have shown previously that specific CD8+ T cells from reinfected immune mice release substantially higher levels of IFN-gamma, a cytokine essential for the efficient activation of parasitized macrophages to kill intracellular L. major. By using an ELISPOT assay, which allows the detection of IFN-gamma production by individual cells, it is shown here that this elevated IFN-gamma response is the result of an increase of up to 50-fold in the frequency of parasite-specific CD8+ T lymphocytes in the spleens and draining lymph nodes of both immune reinfected CBA and BALB/c mice. This observation is additional evidence of the role that CD8+ T cells play in immunity to reinfection with L. major.
Infection of human endothelial cells by Rickettsia rickettsii causes a significant reduction in the levels of key enzymes involved in protection against oxidative injury
Authors: Devamanoharan P S , Santucci L A , Hong J E , Tian X , Silverman D J
Published: January 2020

The activities of glucose-6-phosphate dehydrogenase, catalase, and glutathione peroxidase were significantly decreased in human endothelial cells infected with the obligate intracellular bacterium Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. This observation lends additional support to our hypothesis implicating oxidative damage in endothelial cell injury caused by this microorganism.
Diversity of the P2 protein among nontypeable Haemophilus influenzae isolates
Authors: Bell J , Grass S , Jeanteur D , Munson R S
Published: January 2020

The genes for outer membrane protein P2 of four nontypeable Haemophilus influenzae strains were cloned and sequenced. The derived amino acid sequences were compared with the outer membrane protein P2 sequence from H. influenzae type b MinnA and the sequences of P2 from three additional nontypeable H. influenzae strains. The sequences were 76 to 94% identical. The sequences had regions with considerable variability separated by regions which were highly conserved. The variable regions mapped to putative surface-exposed loops of the protein.
Genetic, enzymatic, and pathogenic studies of the iron superoxide dismutase of Campylobacter jejuni
Authors: Pesci E C , Cottle D L , Pickett C L
Published: January 2020

Campylobacter jejuni is a microaerobic bacterium that produces an acute, self-limiting, watery or bloody diarrhea in humans. Little is known about how C. jejuni causes disease or even what specific capabilities it requires for survival in vivo. The enzyme, superoxide dismutase (SOD), which catalyzes the breakdown of superoxide radicals to hydrogen peroxide and dioxygen is one of the bacterial cell's major defense mechanisms against oxidative damage. A PCR-based search for sod genes in C. jejuni 81-176 revealed that this bacterium contained at least one sod gene. We cloned and sequenced a sod gene from 81-176 and determined that its predicted protein product was most similar to that of FeSODs (sodB genes). Transcriptional analysis indicated that this gene is monocistronic and may be transcribed from a sigma 70-like promoter. Nondenaturing polyacrylamide gels stained to reveal SOD activities, accompanied by inhibition studies, demonstrated that C. jejuni produces five electrophoretically distinct bands of SOD activity, all of which appeared to be FeSODs. Analysis of an 81-176 sodB strain revealed that all of these FeSOD activities may be products of the one sodB gene that we cloned. The expression and enzymatic activity of the respective sodB and FeSOD produced by both C. jejuni and Helicobacter pylori were examined in Escherichia coli. Both genes were expressed in E. coli, and the proteins produced were enzymatically active. Finally, the ability of the 81-176 sodB strain to survive INT407 cell invasion was found to be significantly decreased (12-fold) compared with that of the parent, suggesting a potential role for SodB in C. jejuni intracellular survival.
Listeria monocytogenes infection enhances transcription factor NF-kappa B in P388D1 macrophage-like cells
Authors: Hauf N , Goebel W , Serfling E , Kuhn M
Published: January 2020

In the present study, we investigated the effect of Listeria monocytogenes infection on the cellular level of the transcription factors NF-kappa B, AP-1, and NF-IL6 in the macrophage-like cell line P388D1 by using electrophoretic mobility shift assays. Infection with L. monocytogenes enhanced the formation of two NF-kappa B-like DNA-protein complexes, C1 and C2, whereas the concentration of AP-1 and NF-IL6 complexes remained unaffected. In supershift assays using NF-kappa B-specific antibodies, complex C2 was identified to be a p50 homodimer (KBF1) and complex C1 was identified as a p50/p65 heterodimer. Both complexes were formed within 10 min after addition of the bacteria. Since the synthesis of tumor necrosis factor alpha and interleukin-1 occurs at later times, these cytokines cannot be the mediators of enhanced NF-kappa B formation. Infection experiments with different nonhemolytic mutants of L. monocytogenes and the use of the phagocytosis inhibitor cytochalasin B suggest that events prior to invasion and escape of the bacteria from the phagosome into the cytoplasm enhance the nuclear transport of p50/p65 NF-kappa B components.
Induction of tumor necrosis factor alpha by the group- and type-specific polysaccharides from type III group B streptococci
Authors: Mancuso G , Tomasello F , von Hunolstein C , Orefici G , Teti G
Published: January 2020

Previous studies suggested that circulating tumor necrosis factor alpha (TNF-alpha) may have a pathophysiologic role in experimental neonatal sepsis induced by group B streptococci (GBS). This study was undertaken to investigate the ability of the type III and group-specific polysaccharides of GBS to induce TNF-alpha production and TNF-alpha-dependent lethality in neonatal rats. The cytokine was detected in plasma samples by the L929 cytotoxicity assay. Intracardiac injections of either polysaccharide induced dose-dependent, transient elevations in plasma TNF-alpha levels that returned to baseline values after 5 h. The group-specific antigen induced significantly higher mean peak TNF-alpha levels than the type III antigen (125 +/- 47 versus 44 +/- 15 U/ml with 70 mg/kg of body weight). Glycogen (70 mg/kg), used as a negative control, did not induce TNF-alpha. The lipopolysaccharide-neutralizing agent polymyxin B did not decrease TNF-alpha levels induced by either polysaccharide, ruling out contamination with endotoxin as a possible cause of TNF-alpha induction. Fifty percent lethal doses of the type III and group-specific antigens given as intracardiac injections were 105 and 16 mg/kg, respectively. Salmonella endotoxin, used as a positive control, had a 50% lethal dose of 0.1 mg/kg. The lethal activities of GBS polysaccharides, as well as endotoxin, were completely prevented by pretreatment of neonatal rats with the respective specific antibodies or anti-murine TNF-alpha serum. To assess the relative importance of the type-specific substance in TNF-alpha induction by whole bacteria, two unrelated GBS transposon mutants devoid of only the type-specific capsular polysaccharide (COH1-13 and COH31-15) were employed. Each of the heat-killed unencapsulated mutants was able to produce plasma TNF-alpha level elevations or TNF-alpha-dependent lethality but was significantly less efficient in these activities than the corresponding encapsulated wild-type strain. These data suggest that the presence of type-specific material on GBS is not necessary for the stimulation of TNF-alpha production. Type III capsular polysaccharide, however, can significantly increase the ability of GBS to induce TNF-alpha. Further studies will be needed to assess the importance of TNF-alpha induction by the group- and type-specific antigens in the pathophysiology of GBS disease.
Presence of a capsule in Erysipelothrix rhusiopathiae and its relationship to virulence for mice
Authors: Shimoji Y , Yokomizo Y , Sekizaki T , Mori Y , Kubo M
Published: January 2020

Three avirulent insertional mutants of Erysipelothrix rhusiopathiae were obtained by the technique of transposon mutagenesis with the self-conjugative transposon Tn916. The interactions between murine polymorphonuclear leukocytes and parent and mutant strains were studied in vitro. In the presence of normal serum, the virulent parent strain was resistant to phagocytosis, whereas the avirulent mutant strains were efficiently phagocytosed. In the presence of immune serum, the parent and the mutant strains were both efficiently phagocytosed. Electron microscopic examination of the parent strain demonstrated the presence of a structure resembling a capsule which was absent on the mutant strains, suggesting that a capsule may be involved in virulence. This was confirmed in studies in which an avirulent mutant strain reverted to virulence following acquisition of a capsule when the transposon was lost by spontaneous excision. These results strongly suggest that virulence of E. rhusiopathiae is associated, at least in part, with resistance to phagocytosis by polymorphonuclear leukocytes and that this antiphagocytic ability of the bacterium results from its possession of a capsule.
Actin accumulation associated with clustered and localized adherence in Escherichia coli isolated from patients with diarrhea
Authors: Yamamoto T , Kaneko M , Changchawalit S , Serichantalergs O , Ijuin S , Echeverria P
Published: January 2020

Escherichia coli D2 (serotype 07:H-) that was isolated from a child with diarrhea hybridized with an F1845 DNA probe used to detect diffuse adherence. Strain D2 adhered to tissue culture cells (HeLa and HEp-2 cells) in a clustered pattern but did not autoagglutinate on the cell surface and induced the elongation of microvilli after 3 h of incubation. After 6 h of incubation, the infected cells were positive for fluorescent-actin staining at the site of clustered adherence. When analyzed with a confocal laser scanning microscope, each D2 cell was surrounded by accumulated actin in a capsule-like formation. Capsule-like, accumulated actin was also observed with enteropathogenic E. coli (EPEC), although in this case, actin accumulation was associated with EPEC microcolonies in a localized pattern. Four other strains of F1845 DNA probe-positive, diffusely adhering E. coli were negative for actin accumulation. Strain D2 did not hybridize with EPEC attaching and effacing DNA or EPEC adherence factor DNA probes. In addition, clustered D2 cells were found inside tissue culture cells. The data suggest a novel infectious mechanism as well as genetic heterogeneity of F1845 DNA probe-positive E. coli. Capsule-like, accumulated actin may protect the bacteria from host defense mechanisms.
Immunological variants of the aerobactin-cloacin DF13 outer membrane protein receptor IutA among enteric bacteria
Authors: Bouchet A , Valvano M A , Dho-Moulin M , Le Roy D , Andremont A
Published: January 2020

Mouse monoclonal antibodies (MAbs) were generated against a 76-kDa IutA receptor of pathogenic avian Escherichia coli 15972. Six of the eight IutA-specific MAbs isolated (AB1 to AB6) were shown to be directed toward membrane-exposed conformational epitopes, although they did not interfere with the uptake of ferric aerobactin and cloacin DF13 as assessed by competition experiments with purified ligands. The two remaining IutA MAbs (AB9 and AB10) recognized linear epitopes buried in the IutA molecule. The panel of IutA MAbs was used to characterize IutA variants occurring in strains of E. coli, Klebsiella pneumoniae, Enterobacter spp., and Shigella spp., resulting in the identification of four immunological groups of IutAs. MAb AB9 defined an epitope conserved in all IutA variants. In addition, the panel of IutA MAbs served to identify the presence of IutA in wild-type bacteria grown in the presence of diphenylamine to reduce the expression of O-specific polysaccharide.
Degradation of endogenous plasma membrane fibronectin concomitant with Treponema denticola 35405 adhesion to gingival fibroblasts
Authors: Ellen R P , Song M , McCulloch C A
Published: January 2020

Treponema denticola adhesion and degradation of fibronectin (Fn) on human gingival fibroblasts (HGF) were studied by immunofluorescence and enzyme-linked immunosorbent assays. The number of adherent bacteria increased and the amount of immunoreactive Fn decreased as a function of increasing T. denticola concentration. The distribution of cell-bound Fn was punctate in micrographs. Anti-human Fn impaired bacterial adhesion to HGF. Phenylmethylsulfonyl fluoride inhibited Fn degradation but not adhesion. Sonicated extracts and diluted spent growth medium degraded HGF Fn but, unlike intact T. denticola cells, they hardly stimulated F-actin rearrangements.
Molecular characterization of clustered variants of genes encoding major surface antigens of human Pneumocystis carinii
Authors: Garbe T R , Stringer J R
Published: January 2020

A 13-kb genomic fragment from human Pneumocystis carinii was cloned as repetitive DNA. The fragment contains a cluster of three related genes, each 3 kb in size, and the 5' end of a fourth gene. The predicted polypeptide of the first gene in the cluster comprises 1,030 amino acid residues with a total molecular mass of 116 kDa. The gene's predicted amino acid sequence bears 32% identity to predicted sequences of recently described gene fragments of ferret P. carinii, which encode an immunodominant surface glycoprotein (gpA) (P. J. Haidaris, T. W. Wright, F. Gigliotti, and C. G. Haidaris, J. Infect. Dis. 166:1113-1123, 1992), and 36% identity to the predicted sequence of a rat P. carinii major surface glycoprotein gene (msg) (J. A. Kovacs, F. Powell, J. C. Edman, B. Lundgren, A. Martinez, B. Drew, and C. W. Angus, J. Biol. Chem. 268:6034-6040). DNA hybridization showed that sequences related to the cloned msg genes reside on at least 12 chromosomes of human P. carinii at various degrees of multiplicity and/or homology. Affinity-purified antibodies with specificity to a fusion protein made from the human P. carinii msgI gene recognized two bands on a Western immunoblot containing total human P. carinii protein; they also recognized fusion proteins derived from the other two genes of the cluster. Monoclonal antibodies with reactivity to Msg of human P. carinii recognized fusion proteins produced from two msg genes. Fusion proteins were also recognized by sera from healthy humans and from patients. The msg genes are candidates for the development of immunotherapy and subunit vaccines for the treatment and prevention of P. carinii pneumonia.
Internalization of Proteus mirabilis by human renal epithelial cells
Authors: Chippendale G R , Warren J W , Trifillis A L , Mobley H L
Published: January 2020

Proteus mirabilis, a common agent of bacteriuria in humans, causes acute pyelonephritis and bacteremia. Renal epithelium provides a barrier between luminal organisms and the renal interstitium. We have hypothesized that P. mirabilis may be internalized into renal epithelium. To test this hypothesis, we added suspensions of three P. mirabilis strains (10(8) CFU) to confluent monolayers of primary cultures of human renal proximal tubular epithelial cells (HRPTEC) and, after 3 h, found the bacteria internalized within membrane-bound vacuoles by light and electron microscopy. Internalization of bacteria by HRPTEC was corroborated by using the gentamicin protection assay. Cytolysis of HRPTEC by the HpmA hemolysin, however, was a confounding factor in this assay, and therefore a hemolysin-negative hpmA mutant was used in subsequent experiments. The nonhemolytic mutant WPM111 did not disrupt the monolayer and was recovered in numbers that were 10- to 100-fold higher than those of the hemolytic parent BA6163. Cytochalasin D (20 micrograms/ml) inhibited internalization of Salmonella typhimurium but not that of P. mirabilis, suggesting that the latter species enters HRPTEC by a mechanism that is not dependent on actin polymerization. We suggest that HpmA hemolysin-mediated cytotoxicity and internalization of bacteria by HRPTEC may play a role in the development of Proteus pyelonephritis.
Candida albicans stimulates arachidonic acid liberation from alveolar macrophages through alpha-mannan and beta-glucan cell wall components
Authors: Castro M , Ralston N V , Morgenthaler T I , Rohrbach M S , Limper A H
Published: January 2020

Candida albicans is an increasingly important fungal pathogen. Alveolar macrophages respond to fungal components such as zymosan by releasing arachidonic acid (AA) and AA metabolites. However, few studies hypothesized that macrophages respond to C. albicans by releasing AA and generating AA metabolites as a consequence of interaction of mannose and beta-glucan receptors with fungal cell wall components. [14C]AA-labeled rabbit alveolar macrophages released AA following stimulation with either live or heat-killed C. albicans. High-pressure liquid chromatography analysis revealed that 55% of the AA released was metabolized via cyclooxygenase and lipoxygenase pathways. The metabolites consisted of prostaglandin E2, prostaglandin F2 alpha, 6-ketoprostaglandin F1 alpha, thromboxane B2, and leukotrienes B4 and D4. We further examined the roles of alpha-mannan and beta-glucan components of C. albicans in mediating these alterations of eicosanoid metabolism. Prior work in our laboratory has shown that soluble alpha-mannan and beta-glucan inhibit macrophage mannose and beta-glucan receptors, respectively. Incubation of alveolar macrophages with soluble alpha-mannan derived from C. albicans (1 mg/ml) resulted in 49.8% +/- 2.6% inhibition of macrophage AA release during stimulation with intact C. albicans (P = 0.0001 versus control). Macrophage AA release in response to C. albicans was also inhibited to a significant but lesser degree by soluble beta-glucan (36.2% +/- 1.3%; P = 0.008 versus control). These results indicate that C. albicans stimulates macrophage AA metabolism and that these effects are partly mediated by alpha-mannan and beta-glucan constituents of the fungus.
Inhibition of Legionella pneumophila growth by gamma interferon in permissive A/J mouse macrophages: role of reactive oxygen species, nitric oxide, tryptophan, and iron(III)
Authors: Gebran S J , Yamamoto Y , Newton C , Klein T W , Friedman H
Published: January 2020

A/J mouse macrophages infected with Legionella pneumophila and treated with gamma interferon (IFN-gamma) in vitro developed potent antimicrobial activity. This antilegionella activity was independent of the macrophage capacity to generate reactive oxygen intermediates, since the oxygen radical scavengers catalase, superoxide dismutase, mannitol, and thiourea had no effect on the antilegionella activity of IFN-gamma-activated macrophages. Likewise, whereas the ability of IFN-gamma-activated macrophages to synthesize reactive nitrogen intermediates was markedly inhibited by the L-arginine (Arg) analogs, NG-monomethyl-L-arginine and L-aminoguanidine, as well as by incubation in L-Arg-free medium, their ability to inhibit the intracellular growth of L. pneumophila remained intact. The intracellular growth of L. pneumophila in A/J macrophages was inhibited by the iron(III) chelator desferrioxamine and reversed by Fe-transferrin as well as by ferric salts. Additionally, IFN-gamma-activated macrophages incorporated 28% less 59Fe(III) compared with nonactivated cells. Nonetheless, only partial blocking of growth restriction was observed when IFN-gamma-stimulated macrophages were saturated with iron(III). Indole-propionic acid, which appears to inhibit the biosynthesis of L-tryptophan (L-Trp), was an L-Trp-reversible growth inhibitor of L. pneumophila in macrophages, implying that the intracellular replication of this pathogen is also L-Trp dependent. However, an excess of exogenous L-Trp did not reverse the growth inhibition due to IFN-gamma, though a small synergistic effect was observed when the culture medium was supplemented with both iron(III) and L-Trp. We conclude that IFN-gamma-activated macrophages inhibit the intracellular proliferation of L. pneumophila by reactive oxygen intermediate- and reactive nitrogen intermediate-independent mechanisms and just partially by nutritionally dependent mechanisms. We also suggest that additional mechanisms, still unclear, may be involved, since complete reversion was never obtained and since at higher concentrations of IFN-gamma, iron(III) did not induce any significant reversion in the L. pneumophila growth inhibition.
Peptidoglycan fragments decrease food intake and body weight gain in rats
Authors: Biberstine K J , Rosenthal R S
Published: January 2020

We hypothesized that peptidoglycan (PG) fragments decrease appetite in rats. Male Lewis rats (150 g; n > or = 7) received intraperitoneal (i.p.) injections of purified soluble PG fragments that had been treated with polymyxin B-agarose to remove residual endotoxin. Food consumption and body weight gain were determined at intervals after injection. Single i.p. injections of macromolecular extensively O-acetylated PG (S-O-PG) and non-O-acetylated PG fragments (24 to 240 micrograms/kg) reduced food intake and body weight gain in a dose-dependent fashion during the first 12 h after injection. Low-molecular-weight disaccharide peptide monomers with nonreducing 1,6-anhydro-N-acetylmuramic acid ends and muramyl dipeptide (MDP; 1.6 mg/kg) were also appetite and weight gain suppressants, albeit at least 10-fold less potent than S-O-PG; however, muramidase-derived monomers and peptide cross-linked dimers with reducing muramic acid ends were inactive. Appetite suppression was not limited to the Lewis rat strain since another strain, F344, exhibited similar decreases in food intake after injection of S-O-PG or MDP. Oral administration of MDP or S-O-PG, at concentrations 3 and 20 times higher, respectively, than those that were active i.p., failed to elicit a hypophagic response. We conclude that soluble PG fragments are potent suppressants of food consumption and body weight gain in rats and that, although macromolecular PG is more potent than low-molecular-weight fragments, neither O-acetylation nor glycosidic linkage of PG fragments is required for activity. We speculate that PG fragments may contribute to loss of appetite during bacterial illness.
Protection against infertility in a BALB/c mouse salpingitis model by intranasal immunization with the mouse pneumonitis biovar of Chlamydia trachomatis
Authors: Pal S , Fielder T J , Peterson E M , de la Maza L M
Published: January 2020

Female BALB/c mice were immunized intranasally with the mouse pneumonitis biovar of Chlamydia trachomatis and subsequently challenged in the ovarian bursa (C. trachomatis immunized, C. trachomatis challenged). Two groups of mice served as controls. One group was sham immunized intranasally with mock-infected HeLa 229 cell extracts and was challenged in the ovarian bursa with C. trachomatis MoPn (sham immunized, C. trachomatis challenged). The second control group was sham immunized and not challenged (sham immunized, nonchallenged). Before challenge, the C. trachomatis-immunized, C. trachomatis-challenged animals mounted a significant humoral response as shown by high immunoglobulin G (IgG), IgM, and IgA levels and high levels of neutralizing antibodies in serum and moderate IgG and IgA titers in vaginal secretions. Reactivity by Western blot (immunoblot) to the lipopolysaccharide, 30-, 40- (major outer membrane protein), and 60-kDa cysteine-rich proteins and 75- and 100-kDa chlamydial components could be demonstrated. However, reactivity to the 60-kDa heat shock protein was only observed 22 days after challenge. In addition, this group of animals mounted a significant immune response to chlamydial antigens, as shown by a lymphocyte proliferation assay, compared with the sham-immunized nonchallenged mice. After intrabursal challenge, there was no C. trachomatis shedding from the vagina in the C. trachomatis-immunized, C. trachomatis-challenged animals, while 63% of the sham-immunized, C. trachomatis-challenged mice had a positive C. trachomatis culture. In addition, histological sections from the genital tract showed, at 2 weeks postchallenge, a marked acute inflammatory reaction in the sham-immunized, C. trachomatis-challenged animals while in the C. trachomatis-immunized, C. trachomatis-challenged mice there was minimal inflammatory reaction. When the animals were mated, only 12% of the mice from the sham-immunized, C. trachomatis-challenged mice were fertile. In contrast, 94 and 80% of the sham-immunized, nonchallenged and C. trachomatis-immunized, C. trachomatis-challenged mice, respectively, were fertile.
Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis
Authors: Amano A , Sojar H T , Lee J Y , Sharma A , Levine M J , Genco R J
Published: January 2020

Fimbriae are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. It has been demonstrated that purified fimbriae bind to whole human saliva adsorbed to hydroxyapatite (HAP) beads, and the binding appears to be mediated by specific protein-protein interactions. Recently, we expressed the recombinant fimbrillin protein (r-Fim) of P. gingivalis corresponding to amino acid residues 10 to 337 of the native fimbrillin (A. Sharma, H.T. Sojar, J.-Y. Lee, and R.J. Genco, Infect. Immun. 61:3570-3573, 1993). We examined the ability of individual salivary components to promote the direct attachment of r-Fim to HAP beads. Purified r-Fim was radiolabeled with 125I and incubated with HAP beads which were coated with saliva or purified individual salivary components. Whole, parotid, and submandibular-sublingual salivas increased the binding of 125I-r-Fim to HAP beads. Submandibular-sublingual saliva was most effective in increasing the binding of 125I-r-Fim to HAP beads (1.8 times greater than that to uncoated HAP beads). The binding of 125I-r-Fim to HAP beads coated with acidic proline-rich protein 1 (PRP1) or statherin was four and two times greater, respectively, than that to uncoated HAP beads. PRP1 and statherin molecules were also found to bind 125I-r-Fim in an overlay assay. The binding of intact P. gingivalis cells to HAP beads coated with PRP1 or statherin was also enhanced, by 5.4 and 4.3 times, respectively, over that to uncoated HAP beads. The interactions of PRP1 and statherin with 125I-r-Fim were not inhibited by the addition of carbohydrates or amino acids. PRP1 and statherin in solution did not show inhibitory activity on 125I-r-Fim binding to HAP beads coated with PRP1 or statherin. These results suggest that P. gingivalis fimbriae bind strongly through protein-protein interactions to acidic proline-rich protein and statherin molecules which coat surfaces.
Cellular responses to a 55-kilodalton recombinant Pneumocystis carinii antigen
Authors: Theus S A , Sullivan D W , Walzer P D , Smulian A G
Published: January 2020

The host-parasite interaction in Pneumocystis carinii pneumonia is poorly understood. In recent years, two major groups of P. carinii antigens have been identified. One class of antigens is characterized by a broad band of immunoreactivity between 45 and 55 kDa in P. carinii derived from rats. This antigen complex is the P. carinii antigen most commonly found in respiratory tract specimens and most frequently recognized by the host immune response. The availability of a recombinant antigen has permitted studies focusing on the cellular and humoral responses to a single antigen within this class, p55. In this study, we have demonstrated that the p55 antigen elicits a cell-mediated immune response in animals previously exposed to P. carinii. Under conditions of natural exposure, the 5' portion of the molecule, p55(1-200), appears immunologically silent, failing to elicit lymphocyte proliferation or cytokine secretion. Following active immunization, the 5' portion is capable of stimulating lymphocyte proliferation. The 3' portion, p55(268-414), has at least one immunodominant region which contains a 7-amino-acid repeat motif. The cells responding to p55 include a CD4+ T cell which secretes a Th1 cytokine pattern. A detailed understanding of the host-parasite interaction will facilitate the development of immunoprophylaxis and immunotherapy for P. carinii infection.
Inhibition of C3 deposition on Streptococcus equi subsp. equi by M protein: a mechanism for survival in equine blood
Authors: Boschwitz J S , Timoney J F
Published: January 2020

The effect of the M protein of Streptococcus equi subsp. equi on complement deposition, complement degradation, and bacterial survival in equine whole blood was examined in vitro. Preincubation of bacteria with rabbit M protein-specific immunoglobulin G (IgG) inhibited the survival of the M+ strain in whole blood by 20-fold (P < 0.01). In addition, preincubation of bacteria with M protein-specific F(ab')2 fragments inhibited the survival of M+ cells in whole blood by 3.8-fold (P < 0.01). In the absence of specific antibody, an M+ strain (CF32) of S. equi subsp. equi survived 100-fold better in whole blood than an M- isolate (strain 19) (P < 0.01). Complement inactivation by cobra venom factor significantly enhanced the ability of the M- and M+ strains of S. equi subsp. equi to survive in whole blood, the latter in the presence or absence of M protein-specific IgG. The major opsonic forms of C3, C3b and iC3b, were present on both M- and M+ cells after opsonization in nonimmune plasma. However, colloidal gold staining indicated that the M- strain bound four times as much C3 as the M+ strain (P < 0.02) and that preincubation of the M+ strain with M protein-specific IgG or F(ab')2 fragments also enhanced the amount of C3 deposited by a factor of 4 (P < 0.02). Therefore, at least part of the M protein's ability to enhance bacterial survival in equine whole blood may be related to its ability to interfere with the deposition of equine complement on the bacterial surface.
Altered expression of surface protein WI-1 in genetically related strains of Blastomyces dermatitidis that differ in virulence regulates recognition of yeasts by human macrophages
Authors: Klein B S , Chaturvedi S , Hogan L H , Jones J M , Newman S L
Published: January 2020

The molecular basis for pathogenicity and virulence of the dimorphic fungus Blastomyces dermatitidis remains unknown. WI-1 is a major cell wall protein of B. dermatitidis yeasts and is a recognition target of both humoral and cell-mediated immunity. As an initial study to determine if WI-1 might be linked to virulence of B. dermatitidis, we quantified WI-1 expression on three genetically related strains that differ in their virulence for mice: wild-type virulent ATCC strain 26199, mutant ATCC strain 60915 (which is 10,000-fold reduced in virulence), and mutant ATCC strain 60916 (which is avirulent). Two principal alterations in WI-1 expression were observed in the mutants. First, the mutants express more WI-1 on their surface, as quantified by flow cytometry with monoclonal antibody to WI-1 and by radioimmunoassay, but the WI-1 on their cell wall is less extractable than that on the wild-type strain. Second, the mutants shed less WI-1 during culture and demonstrate impaired processing of shed WI-1. Surface alterations in WI-1 were accompanied by significant differences in the binding of the virulent and mutant strains to human monocyte-derived macrophages. Attachment of yeasts to macrophages paralleled and was proportional to the expression of WI-1. Compared with wild-type yeasts, both mutants bound to macrophages more rapidly and in two- to threefold-greater magnitude. Furthermore, about 75% of yeast binding to macrophages was inhibited by a Fab anti-WI-1 monoclonal antibody. These results suggest that altered WI-1 expression on attenuated and avirulent mutant B. dermatitidis yeasts greatly facilitates macrophage recognition and binding of yeasts and, in turn, may contribute to more rapid ingestion and killing in the host.
The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species
Authors: Gouin E , Mengaud J , Cossart P
Published: January 2020

Most known Listeria monocytogenes virulence genes cluster within a 9.6-kb chromosomal region. This region is flanked on one end by two uncharacterized open reading frames (ORF A and ORF B) and ldh, an ORF presumably encoding the L. monocytogenes lactate dehydrogenase (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). We report here that the other end is flanked by prs, and ORF homologous to phosphoribosyl PPi synthetase genes. ORF B and prs were detected in all Listeria species and thus delimit the virulence region. This virulence gene cluster was detected exclusively in hemolytic Listeria species, Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species.