bluuebunny/crossref_metadata_2025 huggingface dataset:
Susceptibility and Serologic Response of Healthy Adults to Reinfection with
Cryptosporidium parvum
Authors: Okhuysen Pablo C. , Chappell Cynthia L. , Sterling Charles R. , Jakubowski Walter , DuPont Herbert L.
Published: December 2019

Healthy adults are susceptible to infection with small numbers of
Cryptosporidium parvum
oocysts, resulting in self-limited infection. We investigated if infection of humans with
C. parvum
is protective 1 year after primary exposure. At 1 year after a primary challenge with 30 to 10
6
oocysts, 19 healthy immunocompetent adults were rechallenged with 500 oocysts and monitored for the development of infection and/or illness. Oocyst excretion was quantitated by direct immunofluorescence with a
C. parvum
-specific monoclonal antibody, and anti-
C. parvum
antibodies in serum were detected by an enzyme-linked immunosorbent assay. Fewer subjects shed oocysts after the second exposure (3 of 19; 16%) than after the first exposure (12 of 19; 63%) (
P
< 0.005). Although the rates of diarrhea were comparable after each of the two exposures, the clinical severity as determined by the mean number of unformed stools passed was lower after reexposure (11.25 versus 8.62;
P
< 0.05). The number of anti-
Cryptosporidium
immunoglobulin G and A seroconversions increased after secondary exposure. However, the
C. parvum
serum antibody response did not correlate with the presence or absence of infection.
Antibodies and Antibody-Secreting Cells in the Female Genital Tract after Vaginal or Intranasal Immunization with Cholera Toxin B Subunit or Conjugates
Authors: Johansson Eva-Liz , Rask Carola , Fredriksson Margareta , Eriksson Kristina , Czerkinsky Cecil , Holmgren Jan
Published: December 2019

We studied the antibody response including antibody-secreting cells (ASC) in the female genital tract of mice after mucosal immunizations with the recombinant B subunit of cholera toxin (rCTB) perorally, intraperitoneally, vaginally, and intranasally (i.n.). The strongest genital antibody responses as measured with a novel perfusion-extraction method were induced after vaginal and i.n. immunizations, and these routes also gave rise to specific immunoglobulin A (IgA) and IgG ASC in the genital mucosa. Specific ASC in the iliac lymph nodes, which drain the female genital tract, were seen only after vaginal immunization. Progesterone treatment increased the ASC response in the genital tissue after all mucosal immunizations but most markedly after vaginal immunization. We also tested rCTB as a carrier for human gamma globulin (HGG) and the effect of adding cholera toxin (CT) as an adjuvant for the induction of systemic and genital antibody responses to HGG after vaginal and i.n. immunizations. Vaginal immunizations with HGG conjugated to rCTB resulted in high levels of genital anti-HGG antibodies whether or not CT was added, while after i.n. immunization the strongest antibody response was seen with the conjugate together with CT. In summary, vaginal and i.n. immunization give rise to a specific mucosal immune response including ASC in the genital tissue, and vaginal immunization also elicits ASC in the iliac lymph nodes. We have also shown that rCTB can act as an efficient carrier for a conjugated antigen for induction of a specific antibody response in the genital tract of mice after vaginal or i.n. immunization.
Mapping of a Protective Epitope of the CopB Outer Membrane Protein ofMoraxella catarrhalis
Authors: Aebi Christoph , Cope Leslie D. , Latimer Jo L. , Thomas Sharon E. , Slaughter Clive A. , McCracken George H. , Hansen Eric J.
Published: December 2019

A monoclonal antibody (MAb) (MAb 10F3) directed against the CopB outer membrane protein ofMoraxella catarrhalispreviously was found to enhance pulmonary clearance ofM. catarrhalisin an animal model (M. Helminen, I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 61:2003–2010, 1993). In the present study, this same MAb was shown to exert complement-dependent bactericidal activity against this pathogen in vitro. Nucleotide sequence analysis of thecopBgene from two MAb 10F3-reactive and two MAb 10F3-unreactive strains ofM. catarrhalisrevealed that the deduced amino acid sequences of these four CopB proteins were at least 90% identical. Comparison of the amino acid sequences of these proteins allowed localization of possible MAb 10F3 binding sites to five relatively small regions of the CopB protein fromM. catarrhalisO35E. When five synthetic peptides representing these regions were tested for their ability to bind MAb 10F3 in a direct enzyme-linked immunosorbent assay system, an oligopeptide containing 26 amino acids was shown to bind this MAb. The actual binding region for MAb 10F3 was localized further through the use of overlapping decapeptides that spanned this 26-mer. A fusion protein containing the same 26-mer readily bound MAb 10F3 and was used to immunize mice. The resultant antiserum contained antibodies that reacted with the CopB protein of the homologousM. catarrhalisstrain in Western blot analysis and bound to the surface of both homologous and heterologous strains ofM. catarrhalis.
The Role of Sialic Acid in Opsonin-Dependent and Opsonin-Independent Adhesion ofListeria monocytogenesto Murine Peritoneal Macrophages
Authors: Maganti Srinivas , Pierce Marcia M. , Hoffmaster Alex , Rodgers Frank G.
Published: December 2019

The adhesion of listeriae to host cells employs mechanisms which are complex and not well understood.Listeria monocytogenesis a facultative intracellular pathogen responsible for meningoencephalitis, septicemia, and abortion in susceptible and immunocompromised individuals. Subsequent to colonization and penetration of the gut epithelium, the organism attaches to resident macrophages and replicates intracellularly, thus evading the humoral immune system of the infected host. The focus of these studies was to investigate the attachment of the organism to murine peritoneal macrophages in an opsonin-dependent and opsonin-independent fashion. Assessment of competitive binding experiments by immunofluorescence and enzyme-linked immunosorbent assays showed that adhesion of the organism to macrophages in the presence or absence of opsonins was inhibited (90%) byN-acetylneuraminic acid (NAcNeu). In addition, the lectin fromMaackia amurensis, with affinity for NAcNeu-α(2,3)galactose, blocked binding ofL. monocytogenesto host cells. Oxidation of the surface carbohydrates on the organism by using sodium metaperiodate resulted in a dose-dependent reduction (up to 98%) in adherence to macrophages. Monoclonal antibody to complement receptor 3 did not prevent listeriae from binding to mouse macrophages or from replicating within the infected cells whether or not normal mouse serum was present. Based on our results, we propose the involvement of NAcNeu, a member of the sialic acid group, in the attachment ofL. monocytogenesto permissive murine macrophages.
Therapeutic Effects of Nitric Oxide Inhibition during Experimental Fecal Peritonitis: Role of Interleukin-10 and Monocyte Chemoattractant Protein 1
Authors: Hogaboam Cory M. , Steinhauser Matthew L. , Schock Harold , Lukacs Nicholas , Strieter Robert M. , Standiford Theodore , Kunkel Steven L.
Published: December 2019

This study demonstrates that the therapeutic effect of a nitric oxide inhibitor in a murine model of fecal peritonitis is mediated in part by increased levels of interleukin-10 (IL-10) and monocyte chemoattractant protein 1 (MCP-1). Female CD1 mice were subjected to cecal ligation and puncture (CLP) with a 21-gauge needle and, immediately following surgery, were injected intraperitoneally with saline,NG-nitro-l-arginine methyl ester (l-NAME; 8 mg/kg), orNG-nitro-d-arginine methyl ester (d-NAME; 8 mg/kg). At 96 h after surgery and drug treatment, 20% of mice that receivedd-NAME had survived whereas 60% of mice that receivedl-NAME were alive. To elucidate the effect ofl-NAME treatment on chemokine and cytokine production during fecal peritonitis, the levels of macrophage inflammatory protein 2 (MIP-2), IL-10, and MCP-1 were measured in peritoneal washings from additional groups of mice 24 h after the CLP surgery. Peritoneal fluids froml-NAME-treated mice contained significantly higher levels of IL-10 and MCP-1 than did those fromd-NAME-treated mice. To elucidate the effect of nitric oxide inhibition on potential cellular sources of IL-10 and MCP-1 in the CLP model, cultured alveolar and peritoneal macrophages were activated with bacterial lipopolysaccharide in the presence ofl-NAME; these macrophages produced significantly more MCP-1 than did similarly activated macrophages in the presence ofd-NAME. In the CLP surgery model, immunoneutralization of IL-10 alone or IL-10 and MCP-1 together with polyclonal antibodies prior to surgery significantly reduced the survival rates inl-NAME-treated groups compared withl-NAME-treated groups that received preimmune serum. Taken together, these data demonstrate that the inhibition of nitric oxide following experimental CLP fecal peritonitis is therapeutic, in part through the modulatory effect of this treatment on the synthesis of IL-10 and MCP-1.
Comprehensive Study of the Intestinal Stage of Listeriosis in a Rat Ligated Ileal Loop System
Authors: Pron Benedicte , Boumaila Claire , Jaubert Francis , Sarnacki Sabine , Monnet Jean-Paul , Berche Patrick , Gaillard Jean-Louis
Published: December 2019

The intestinal stage of listeriosis was studied in a rat ligated ileal loop system.
Listeria monocytogenes
translocated to deep organs with similar efficiencies after inoculation of loops with or without Peyer’s patches. Bacterial seeding of deep organs was demonstrated as early as 15 min after inoculation. It was dose dependent and nonspecific, as the
ΔinlAB
, the
Δhly
, and the
ΔactA L. monocytogenes
mutants and the nonpathogenic species,
Listeria innocua
, translocated similarly to wild-type
L. monocytogenes
strains. The levels of uptake of listeriae by Peyer’s patches and villous intestine were similar and low, 50 to 250 CFU per cm
2
of tissue. No listeria cells crossing the epithelial sheet of Peyer’s patches and villous intestine were observed by transmission electron microscopy. The lack of significant interaction of listeriae and the follicle-associated epithelium of Peyer’s patches was confirmed by scanning electron microscopy. The follicular tissue of Peyer’s patches was a preferential site of
Listeria
replication. With all doses tested, the rate of bacterial growth was 10 to 20 times higher in Peyer’s patches than in villous intestine. At early stages of Peyer’s patch infection, listeriae were observed inside mononuclear cells of the dome area. Listeriae then disseminated throughout the follicular tissue except for the germinal center. The virulence determinants
hly
and, to a lesser extent,
actA
, but not
inlAB
, were required for the completion of this process. This study suggests that Peyer’s patches are preferential sites for replication rather than for entry of
L. monocytogenes
, due to the presence of highly permissive mononuclear cells whose nature remains to be defined.
Protection againstMycobacterium tuberculosisInfection by CD8+T Cells Requires the Production of Gamma Interferon
Authors: Tascon Ricardo E. , Stavropoulos Evangelos , Lukacs Katalin V. , Colston M. Joseph
Published: December 2019

The role of CD8 T cells in controllingMycobacterium tuberculosisinfections in mice was confirmed by comparing the levels of growth of the organism in control, major histocompatibility complex class II knockout, and athymic mice and by transferring T-cell populations into athymic mice. By using donor mice which were incapable of making gamma interferon (IFN-γ), it was shown that IFN-γ production was essential for CD8 cell mediation of protective immunity againstM. tuberculosis.
Enterococcus faecalis
Localization in Experimental Endophthalmitis: Role of Plasmid-Encoded Aggregation Substance
Authors: Jett Bradley D. , Atkuri Rajeshwari V. , Gilmore Michael S.
Published: December 2019

Enterococci have emerged as leading agents of nosocomial infection, yet relatively little is known about the pathogenesis of enterococcal disease. In previous studies, we developed an
Enterococcus faecalis
endophthalmitis infection model which provides unique opportunities to study the evolution of enterococcal disease by direct observation, as well as through sensitive electrophysiologic measures of organ function. The present study was designed to determine whether
E. faecalis
possesses traits that permit its attachment to mammalian tissues during infection. It was also of interest to determine whether a plasmid-encoded adhesin, aggregation substance, contributes to enterococcal localization or otherwise mediates adherence to alternate sites. These studies found that, in this model, enterococci attach to membranous structures occurring within the vitreous but that this attachment or the course or severity of disease is unaffected by the aggregation substance phenotype.
Cell-Specific Proteins Synthesized bySalmonella typhimurium
Authors: Burns-Keliher Lisa , Nickerson Cheryl A. , Morrow Brian J. , Curtiss Roy
Published: December 2019

Studies of the proteins synthesized bySalmonella typhimuriumduring growth within tissue culture cells have previously focused on a single cell type. In the present study we examine the different protein patterns exhibited byS. typhimuriumduring growth within three different cell types relevant to those it would encounter throughout the course of a natural infection, including intestinal epithelial cells (Intestine-407), macrophages (J774.A, rat bone marrow-derived macrophages, and mouse bone marrow-derived macrophages), and liver cells (NMuLi). Side-by-side comparisons reveal thatS. typhimuriumresponds to these different cellular environments with specific patterns of protein synthesis unique to each cell type. The numbers of proteins detected in each cell line are as follows: 142 proteins in Intestine-407, of which 58 appear to be unique to growth within this cell line; 413 proteins in J774.A, of which 157 appear to be unique; 260 proteins in rat bone marrow-derived macrophages, of which 40 appear to be unique; 336 proteins in mouse bone marrow-derived macrophages, of which 113 appear to be unique; and 183 proteins in NMuLi, of which 91 appear to be unique.
Expression of β-Defensin Genes in Bovine Alveolar Macrophages
Authors: Ryan Lisa K. , Rhodes Janice , Bhat Meenakshi , Diamond Gill
Published: December 2019

Bovine alveolar macrophages (BAM) were examined for the expression of β-defensins and to determine whether their expression could be upregulated by bacterial lipopolysaccharide (LPS), as observed with β-defensins expressed in bovine tracheal epithelial cells. Four β-defensins were expressed constitutively in BAM, with bovine neutrophil β-defensin (BNBD)-4 and BNBD-5 being the most predominant. This is the first evidence of β-defensin gene expression in a mature myeloid cell. LPS had no effect on β-defensin expression in BAM, even though tumor necrosis factor alpha (TNF-α) production was induced. Nonbacterial inflammatory particles had little effect on β-defensin gene expression or TNF-α production in BAM. We hypothesize that constitutively expressed β-defensins of alveolar macrophages may have a role in lung host defense.
Down Regulation of Macrophage Activation inBrugia pahangi-Infected Jirds (Meriones unguiculatus)
Authors: Nasarre C. , Krahenbuhl J. L. , Klei T. R.
Published: December 2019

The macrophage is a major component of the inflammatory response induced by lymphatic tissue-dwelling filariae. Intraperitoneal (i.p.) infections withBrugia pahangiin Mongolian gerbils, or jirds (Meriones unguiculatus), induce a peritoneal inflammatory response characterized by accumulation of numerous macrophages and fewer eosinophils. This inflammatory response is associated with the release of microfilariae by female worms. The aim of this study was to investigate the activation state of the peritoneal macrophages during the course of i.p. infections with either male or female worms. Activation was determined by a toxoplasmacidal assay and assays which measured the production of tumor necrosis factor (TNF)-like activity and nitric oxide (NO) production. The development of these assays with jirds was initially conducted in parallel with the mouse system, which served as a positive control. Jird macrophages became activated to killToxoplasma gondiiby in vivo immunization withMycobacterium bovisBCG in a pattern similar to that of mouse macrophages. However, unlike the mouse system, supernatants from purified protein derivative- or concanavalin A-stimulated jird splenocytes plus lipopolysaccharide failed to activate jird macrophages in vitro or induce NO production. These results indicate that factors involved in jird macrophage activation may differ from those demonstrated in the mouse system and other systems. i.p. infections of 15 days in duration with either male or female worms induced macrophage activation as measured byToxoplasmakilling and TNF production. These responses decreased as the infection progressed to the chronic period on a time course that parallels the down regulation of experimentalB. pahangigranulomas. There was no evidence of NO production by activated jird macrophages. These data indicate that macrophage function is down modulated during filarial infection and suggest that mechanisms involved in macrophage deactivation are related to those that induce down modulation of the systemic granulomatous inflammatory response in the jird. This response is not dependent on the microfilarial stage of the parasite and is also independent of mechanisms which induce peritoneal accumulations of macrophages.
Isolation and Characterization of the Outer Membrane of
Borrelia hermsii
Authors: Shang Ellen S. , Skare Jonathan T. , Exner Maurice M. , Blanco David R. , Kagan Bruce L. , Miller James N. , Lovett Michael A.
Published: December 2019

The outer membrane of
Borrelia hermsii
has been shown by freeze-fracture analysis to contain a low density of membrane-spanning outer membrane proteins which have not yet been isolated or identified. In this study, we report the purification of outer membrane vesicles (OMV) from
B. hermsii
HS-1 and the subsequent identification of their constituent outer membrane proteins. The
B. hermsii
outer membranes were released by vigorous vortexing of whole organisms in low-pH, hypotonic citrate buffer and isolated by isopycnic sucrose gradient centrifugation. The isolated OMV exhibited porin activities ranging from 0.2 to 7.2 nS, consistent with their outer membrane origin. Purified OMV were shown to be relatively free of inner membrane contamination by the absence of measurable β-NADH oxidase activity and the absence of protoplasmic cylinder-associated proteins observed by Coomassie blue staining. Approximately 60 protein spots (some of which are putative isoelectric isomers) with 25 distinct molecular weights were identified as constituents of the OMV enrichment. The majority of these proteins were also shown to be antigenic with sera from
B. hermsii
-infected mice. Seven of these antigenic proteins were labeled with [
3
H]palmitate, including the surface-exposed glycerophosphodiester phosphodiesterase, the variable major proteins 7 and 33, and proteins of 15, 17, 38, 42, and 67 kDa, indicating that they are lipoprotein constituents of the outer membrane. In addition, immunoblot analysis of the OMV probed with antiserum to the
Borrelia garinii
surface-exposed p66/Oms66 porin protein demonstrated the presence of a p66 (Oms66) outer membrane homolog. Treatment of intact
B. hermsii
with proteinase K resulted in the partial proteolysis of the Oms66/p66 homolog, indicating that it is surface exposed. This identification and characterization of the OMV proteins should aid in further studies of pathogenesis and immunity of tick-borne relapsing fever.
Yersinia enterocolitica
-Induced Interleukin-8 Secretion by Human Intestinal Epithelial Cells Depends on Cell Differentiation
Authors: Schulte Ralf , Autenrieth Ingo B.
Published: December 2019

In response to bacterial entry epithelial cells up-regulate expression and secretion of various proinflammatory cytokines, including interleukin-8 (IL-8). We studied
Yersinia enterocolitica
O:8-induced IL-8 secretion by intestinal epithelial cells as a function of cell differentiation. For this purpose, human T84 intestinal epithelial cells were grown on permeable supports, which led to the formation of tight monolayers of polarized intestinal epithelial cells. To analyze IL-8 secretion as a function of cell differentiation, T84 monolayers were infected from the apical or basolateral side at different stages of differentiation. Both virulent (plasmid-carrying) and nonvirulent (plasmid-cured)
Y. enterocolitica
strains invaded nondifferentiated T84 cells from the apical side.
Yersinia
invasion into T84 cells was followed by secretion of IL-8. After polarized differentiation of T84 cells
Y. enterocolitica
was no longer able to invade from the apical side or to induce IL-8 secretion by T84 cells. However,
Y. enterocolitica
invaded and induced IL-8 secretion by polarized T84 cells after infection from the basolateral side. Basolateral invasion required the presence of the
Yersinia
invasion locus,
inv
, suggesting β
1
integrin-mediated cell invasion. After basolateral infection,
Yersinia
-induced IL-8 secretion was not strictly dependent on cell invasion. Thus, although the plasmid-carrying
Y. enterocolitica
strain did not significantly invade T84 cells, it induced significant IL-8 secretion. Taken together, these data show that
Yersinia
-triggered IL-8 secretion by intestinal epithelial cells depends on cell differentiation and might be induced by invasion as well as by basolateral adhesion, suggesting that invasion is not essential for triggering IL-8 production. Whether IL-8 secretion is involved in the pathogenesis of
Yersinia
-induced abscess formation in Peyer’s patch tissue remains to be shown.
d
-Lactate Production and [
14
C]Succinic Acid Uptake by Adherent and Nonadherent
Escherichia coli
Authors: McCabe K. , Mann M. D. , Bowie M. D.
Published: December 2019

Escherichia coli
isolates of different adherence phenotypes produced different amounts of
d
-lactate. Alterations of culture conditions did not influence the amount of
d
-lactate produced. The observed pH decreases in tissue culture medium corresponded with increases in
d
-lactate concentration. Very little [
14
C]succinic acid was incorporated into cells during the in vitro incubation of adherent and nonadherent
E. coli
with HeLa cells, but the amounts of tracer removed from the culture medium by adherent and nonadherent strains differed. The results are further evidence of a difference in the metabolic behavior of adherent and nonadherent
E. coli.
Structural Properties of Lipopolysaccharides fromRickettsia typhiandRickettsia prowazekiiand Their Chemical Similarity to the Lipopolysaccharide fromProteus vulgarisOX19 Used in the Weil-Felix Test
Authors: Amano Ken-ichi , Williams Jim C. , Dasch Gregory A.
Published: December 2019

The lipopolysaccharides (LPSs) isolated from typhus group (TG) rickettsiaeRickettsia typhiandRickettsia prowazekiiwere characterized by chemical analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. LPSs from two species of TG rickettsiae contained glucose, 3-deoxy-d-manno-octulosonic acid, glucosamine, quinovosamine, phosphate, and fatty acids (β-hydroxylmyristic acid and heneicosanoic acid) but not heptose. The O-polysaccharides of these LPSs were composed of glucose, glucosamine, quinovosamine, and phosphorylated hexosamine. Resolution of these LPSs by their apparent molecular masses by SDS-PAGE showed that they have a common ladder-like pattern. Based on the results of chemical composition and SDS-PAGE pattern, we suggest that these LPSs act as group-specific antigens. Furthermore, glucosamine, quinovosamine, and phosphorylated hexosamine were also found in the O-polysaccharide of the LPS fromProteus vulgarisOX19 used in the Weil-Felix test, suggesting that they may represent the antigens common to LPSs from TG rickettsiae andP. vulgarisOX19.
Identification of Linked
Legionella pneumophila
Genes Essential for Intracellular Growth and Evasion of the Endocytic Pathway
Authors: Andrews Helene L. , Vogel Joseph P. , Isberg Ralph R.
Published: December 2019

Legionella pneumophila
replicates within a specialized phagosome in cultured cells, a function necessary for its pathogenicity. The replicative phagosome lacks membrane marker proteins, such as the glycoprotein LAMP-1, that are indicators of the normal endocytic pathway. We describe the isolation of several
Legionella
genes essential for intracellular growth and evasion of the endocytic pathway, using a genetic and cell biological approach. We screened 4,960 ethyl methanesulfonate-mutagenized colonies for defects in intracellular growth and trafficking to the replicative phagosome. Six mutant strains of
L. pneumophila
that had severe intracellular growth defects in mouse bone marrow-derived macrophages were identified. All six mutants were found in phagosomes that colocalized with LAMP-1, indicating defects in intracellular trafficking. The growth defects of two of these strains were complemented by molecular clones from a bank constructed from a wild-type
L. pneumophila
strain. The inserts from these clones are located in a region of the chromosome contiguous with several other genes essential for intracellular growth. Three mutants could be complemented by single open reading frames placed in
trans
, one mutant by a gene termed
dotH
and two additional mutants by a gene termed
dotO
. A deletion mutation was created in a third gene,
dotI
, which is located directly upstream of
dotH
. The Δ
dotI
strain was also defective for intracellular growth in macrophages, and this defect was complemented by a single open reading frame in
trans
. Based on sequence analysis and structural predictions, possible roles of
dotH
,
dotI
, and
dotO
in intracellular growth are discussed.
Phase Variation of Hemoglobin Utilization in
Neisseria gonorrhoeae
Authors: Chen Ching-Ju , Elkins Christopher , Sparling P. Frederick
Published: December 2019

Most
Neisseria gonorrhoeae
isolates are unable to use human hemoglobin as the sole source of iron for growth (Hgb
−
), but a minor population is able to do so (Hgb
+
). This minor population grows luxuriously on hemoglobin, expresses two outer membrane proteins of 42 kDa (HpuA) and 89 kDa (HpuB), and binds hemoglobin under iron-stressed conditions. In addition to the previously reported HpuB, we identified and characterized HpuA, which is encoded by the gene
hpuA
, located immediately upstream of
hpuB
. Expression of both proteins was found to be controlled at the translational level by frameshift mutations in a run of guanine residues within the
hpuA
sequence encoding the mature HpuA protein. The “on-phase” hemoglobin-utilizing variants contained 10 G’s, while the “off-phase” variants contained 9 G’s. Insertional
hpuB
mutants of FA19 Hgb
+
and FA1090 Hgb
+
no longer expressed HpuB but still produced HpuA. A polar insertional mutation of the upstream
hpuA
gene in FA1090 Hgb
+
eliminated production of both HpuA and HpuB, whereas a nonpolar insertional mutant expressed HpuB only. Insertional mutagenesis of either
hpuA
or
hpuB
or both substantially decreased the hemoglobin binding ability of the FA1090 Hgb
+
variant and prevented growth on hemoglobin plates. Therefore, both HpuA and HpuB were required for the utilization of hemoglobin for growth.
Influence of Antibodies in Mother’s Milk on Antigenic Variation ofGiardia lambliain the Murine Mother-Offspring Model of Infection
Authors: Stäger S. , Gottstein B. , Sager H. , Jungi T. W. , Müller N.
Published: December 2019

In the present study, neonatal ZU.ICR mice and their mothers were infected with trophozoites ofGiardia lambliaclone GS/M-83-H7 expressing the variant surface protein (VSP) H7. The infection experiments included a detailed analysis of the specificities of anti-Giardiaimmunoglobulin A (IgA) antibodies in mother’s milk and a determination of the effects of the milk antibodies on both the growth of the parasite during in vitro cultivation and colonization of the parasite within the intestine of suckling offspring. These investigations revealed that transiently emerging milk IgA antibodies against a variant-specific 314-amino-acid N-terminal region of VSP H7 exhibit a strong parasiticidal effect on VSP H7-type trophozoites both in vitro and in vivo. These findings indicated that parasiticidal effects of local IgA antibodies against the N-terminal part of VSP H7 select for new variant types within the intestinal parasite population of suckling mice. The selective influence of such antibodies promotes in vivo antigenic variation ofG. lambliaclone GS/M-83-H7 and modulates the early course of parasite infection in these animals.
Superoxide Dismutase-Dependent, Catalase-Sensitive Peroxides in Human Endothelial Cells Infected byRickettsia rickettsii
Authors: Hong June E. , Santucci Lisa A. , Tian Xiaojiang , Silverman David J.
Published: December 2019

The generation and intracellular accumulation of reactive oxygen species have been shown to be associated with the infection of human umbilical vein endothelial cells (HUVEC) byRickettsia rickettsii. In response to the oxidant superoxide, the activity of the enzyme superoxide dismutase (SOD) increases following infection by this obligate intracellular bacterium. Other oxidants which are capable of oxidizing the fluorescent probe 2′,7′-dichlorofluorescin (DCFH) also accumulate intracellularly within infected cells. In the study reported here, we show that (i) an inhibitor of SOD, diethyldithiocarbamic acid, reduces the observed rise in SOD activity in infected cells by 40 to 60% and at the same time reduces the degree of intracellular oxidation of DCFH; (ii) catalase-sensitive peroxides can be detected in supernatants ofR. rickettsii-infected cells shortly after rickettsial exposure; and (iii) fluorescence-activated cell sorter analysis demonstrates significant intracellular oxidant activity in infected cells within 5 h after exposure toR. rickettsii. The results of these experiments indicate that hydrogen peroxide is a major oxidant associated with infection of HUVEC byR. rickettsiiand that intracellular oxidant activity sensitive to SOD inhibition is detectable early and prior to significant rickettsial multiplication and much earlier than the ultrastructural manifestations of cell injury seen by electron microscopy.
Expression and Bactericidal Activity of Nitric Oxide Synthase inBrucella suis-Infected Murine Macrophages
Authors: Gross Antoine , Spiesser Sandra , Terraza Annie , Rouot Bruno , Caron Emmanuelle , Dornand Jacques
Published: December 2019

We examined the expression and activity of inducible nitric oxide synthase (iNOS) in both gamma interferon (IFN-γ)-treated and untreated murine macrophages infected with the gram-negative bacteriumBrucella suis. The bacteria were opsonized with a mouse serum containing specific antibrucella antibodies (ops-Brucella) or with a control nonimmune serum (c-Brucella). The involvement of the produced NO in the killing of intracellularB. suiswas evaluated.B. suissurvived and replicated within J774A.1 cells. Opsonization with specific antibodies increased the number of phagocytized bacteria but lowered their intramacrophage development. IFN-γ enhanced the antibrucella activity of phagocytes, with this effect being greater inops-Brucellainfection. Expression of iNOS, interleukin-6, and tumor necrosis factor alpha (TNF-α) mRNAs was induced in bothc-Brucella- andops-Brucella-infected cells and was strongly potentiated by IFN-γ. In contrast to that of cytokine mRNAs, iNOS mRNA expression was independent of opsonization. Similar levels of iNOS mRNAs were expressed in IFN-γ-treated cells infected withc-Brucellaorops-Brucella; however, expression of iNOS protein and production of NO were detected only in IFN-γ-treated cells infected withops-Brucella. These discrepencies between iNOS mRNA and protein levels were not due to differences in TNF-α production. The iNOS inhibitorNω-nitro-l-arginine methyl ester increasedB. suismultiplication specifically in IFN-γ-treated cells infected withops-Brucella, demonstrating a microbicidal effect of the NO produced. This observation was in agreement with in vitro experiments showing thatB. suiswas sensitive to NO killing. Together our data indicate that inB. suis-infected murine macrophages, the posttranscriptional regulation of iNOS necessitates an additive signal triggered by macrophage Fcγ receptors. They also support the possibility that in mice, NO favors the elimination ofBrucella, providing that IFN-γ and antibrucella antibodies are present, i.e., following expression of acquired immunity.
Intranasal Administration of a Meningococcal Outer Membrane Vesicle Vaccine Induces Persistent Local Mucosal Antibodies and Serum Antibodies with Strong Bactericidal Activity in Humans
Authors: Haneberg Bjørn , Dalseg Rolf , Wedege Elisabeth , Høiby E. Arne , Haugen Inger Lise , Oftung Fredrik , Andersen Svein Rune , Næss Lisbeth Meyer , Aase Audun , Michaelsen Terje E. , Holst Johan
Published: December 2019

A nasal vaccine, consisting of outer membrane vesicles (OMVs) from group BNeisseria meningitidis, was given to 12 volunteers in the form of nose drops or nasal spray four times at weekly intervals, with a fifth dose 5 months later. Each nasal dose consisted of 250 μg of protein, equivalent to 10 times the intramuscular dose that was administered twice with a 6-week interval to 11 other volunteers. All individuals given the nasal vaccine developed immunoglobulin A (IgA) antibody responses to OMVs in nasal secretions, and eight developed salivary IgA antibodies which persisted for at least 5 months. Intramuscular immunizations did not lead to antibody responses in the secretions. Modest increases in serum IgG antibodies were obtained in 5 volunteers who had been immunized intranasally, while 10 individuals responded strongly to the intramuscular vaccine. Both the serum and secretory antibody responses reached a maximum after two to three doses of the nasal vaccine, with no significant booster effect of the fifth dose. The pattern of serum antibody specificities against the different OMV components after intranasal immunizations was largely similar to that obtained with the intramuscular vaccine. Five and eight vaccinees in the nasal group developed persistent increases in serum bactericidal titers to the homologous meningococcal vaccine strain expressing low and high levels, respectively, of the outer membrane protein Opc. Our results indicate that meningococcal OMVs possess the structures necessary to initiate systemic as well as local mucosal immune responses when presented as a nasal vaccine. Although the serum antibody levels were less conspicuous than those after intramuscular vaccinations, the demonstration of substantial bactericidal activity indicates that a nonproliferating nasal vaccine might induce antibodies of high functional quality.
Molecular Cloning and Sequencing of Three GranulocyticEhrlichiaGenes Encoding High-Molecular-Weight Immunoreactive Proteins
Authors: Storey James R. , Doros-Richert Linda A. , Gingrich-Baker Cindy , Munroe Kenneth , Mather Thomas N. , Coughlin Richard T. , Beltz Gerald A. , Murphy Cheryl I.
Published: December 2019

GranulocyticEhrlichiawas isolated from canine blood obtained from animals challenged with field-collectedIxodes scapularisand propagated in HL60 cells. PCR primers specific for the 16S ribosomal DNA (rDNA) of theEhrlichiagenogroup comprisingE. equi,E. phagocytophila, and the agent of human granulocytic ehrlichiosis (HGE) amplified DNA from extracts of these cells. Sequence analysis of this amplified DNA revealed that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocyticEhrlichiaand screened with pooled sera from tick-challenged, granulocyticEhrlichia-infected dogs. Several clones were isolated and sequenced. Three complete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with convalescent-phase sera from dogs and human patients recovering from HGE. This approach will be useful for identifying candidate diagnostic and vaccine antigens for granulocytic ehrlichiosis and aid in the classification of genogroup members.
Characterization of a Strain of
Chlamydia pneumoniae
Isolated from a Coronary Atheroma by Analysis of the
omp1
Gene and Biological Activity in Human Endothelial Cells
Authors: Molestina Robert E. , Dean Deborah , Miller Richard D. , Ramirez Julio A. , Summersgill James T.
Published: December 2019

Chlamydia pneumoniae
is a respiratory pathogen that has been associated with chronic inflammatory diseases such as asthma and atherosclerosis. Recent isolation of
C. pneumoniae
from human carotid and coronary atheromas provides additional support for a role of this organism in atherogenesis. We characterized the coronary strain
C. pneumoniae
A-03 by sequence analysis of the major outer membrane protein gene (
omp1
). In addition, the in vitro activities of A-03 and three respiratory strains of
C. pneumoniae
(BAL-16, TW-183, and T-2634) were examined in infected human umbilical vein endothelial cells (HUVEC) by analysis of the production of interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and soluble intercellular cell adhesion molecule 1 (sICAM-1). Sequence analysis of
omp1
of
C. pneumoniae
A-03, compared to prototype strains TW-183 and AR-39, revealed five nucleotide changes resulting in nonsynonymous codons. Of interest was a nonconservative amino acid substitution (Ser to Pro) in position 61 of variable segment 1. In vitro, the extent of MCP-1, IL-8, and sICAM-1 production was dependent on the
C. pneumoniae
strain examined at low multiplicities of infection following 24 h of incubation. Strain A-03 displayed the lowest stimulatory activity in infected HUVEC, while T-2634 induced the highest levels of MCP-1, IL-8, and sICAM-1 among all strains examined. Heat-inactivated
C. pneumoniae
failed to stimulate production of these proteins by all strains tested. In contrast, only partial inhibition was observed by UV-inactivated organisms. Results from this study demonstrate that unlike prototype respiratory strains of
C. pneumoniae
, the coronary strain A-03 displays divergence in the
omp1
gene. In addition, the stimulation of chemokines and adhesion molecules involved in the recruitment of leukocytes to sites of inflammation by
C. pneumoniae
may be important in the pathogenesis of diseases associated with this organism, including atherosclerosis.
Endotoxin-Neutralizing Protein Protects against Endotoxin-Induced Endothelial Barrier Dysfunction
Authors: Bannerman Douglas D. , Fitzpatrick Michael J. , Anderson Dell Y. , Bhattacharjee Apurba K. , Novitsky Thomas J. , Hasday Jeffrey D. , Cross Alan S. , Goldblum Simeon E.
Published: December 2019

Bacterial lipopolysaccharide induces tyrosine phosphorylation of paxillin, actin reorganization, and opening of the transendothelial paracellular pathway through which macromoles flux. In this study, lipid A was shown to be the bioactive portion of the lipopolysaccharide molecule responsible for changes in endothelial barrier function. We then studied whether endotoxin-neutralizing protein, a recombinant peptide that is derived fromLimulusantilipopolysaccharide factor and targets lipid A, could block the effects of lipopolysaccharide on protein tyrosine phosphorylation, actin organization, and movement of14C-bovine serum albumin across bovine pulmonary artery endothelial cell monolayers. In the presence of serum, a 6-h exposure to lipopolysaccharide (10 ng/ml) increased transendothelial14C-albumin flux compared to the simultaneous media control. Coadministration of endotoxin-neutralizing protein (≥10 ng/ml) with lipopolysaccharide (10 ng/ml) protected against lipopolysaccharide-induced barrier dysfunction. This protection was dose dependent, conferring total protection at endotoxin-neutralizing protein/lipopolysaccharide ratios of ≥10:1. Similarly, endotoxin-neutralizing protein was capable of blocking the lipopolysaccharide-induced endothelial cell responses that are prerequisite to barrier dysfunction, including tyrosine phosphorylation of paxillin and actin depolymerization. Finally, endotoxin-neutralizing protein cross-protected against lipopolysaccharide derived from diverse gram-negative bacteria. Thus, endotoxin-neutralizing protein offers a novel therapeutic intervention for the vascular endothelial dysfunction of gram-negative sepsis and its attendant endotoxemia.
Human Mannose-Binding Protein Inhibits Infection of HeLa Cells byChlamydia trachomatis
Authors: Swanson Albertina F. , Ezekowitz R. Alan B. , Lee Amy , Kuo Cho-chou
Published: December 2019

The role that collectin (mannose-binding protein) may play in the host’s defense against chlamydial infection was investigated. Recombinant human mannose-binding protein was used in the inhibition of cell culture infection byChlamydia trachomatis(C/TW-3/OT, E/UW-5/Cx, and L2/434/Bu),Chlamydia pneumoniae(AR-39), andChlamydia psittaci(6BC). Mannose-binding protein (MBP) inhibited infection of all chlamydial strains by at least 50% at 0.098 μg/ml for TW-3 and UW-5, and at 6.25 μg/ml for 434, AR-39, and 6BC. The ability of MBP to inhibit infection with strain L2was not affected by supplementation with complement or addition of an L2-specific neutralizing monoclonal antibody. Enzyme-linked immunosorbent assay and dot blot analyses showed MBP bound to the surface of the organism to exert inhibition, which appeared to block the attachment of radiolabeled organisms to HeLa cells. Immunoblotting and affinity chromatography indicated that MBP binds to the 40-kDa glycoprotein (the major outer membrane protein) on the outer surface of the chlamydial elementary body. Hapten inhibition assays with monosaccharides and defined oligosaccharides showed that the inhibitory effects of MBP were abrogated by mannose or high-mannose type oligomannose-oligosaccharide. The latter carbohydrate is the ligand of the 40-kDa glycoprotein ofC. trachomatisL2, which is known to mediate attachment, suggesting that the MBP binds to high mannose moieties on the surface of chlamydial organisms. These results suggest that MBP plays a role in first-line host defense against chlamydial infection in humans.
A Recombinant Live Attenuated Strain of
Vibrio cholerae
Induces Immunity against Tetanus Toxin and
Bordetella pertussis
Tracheal Colonization Factor
Authors: Chen Inês , Finn Theresa M. , Yanqing Liu , Guoming Qi , Rappuoli Rino , Pizza Mariagrazia
Published: December 2019

An attenuated strain of
Vibrio cholerae
was used as a carrier for the expression of heterologous antigens such as fragment C from tetanus toxin (TetC) and tracheal colonization factor from
Bordetella pertussis
(Tcf). In vitro, high levels of protein were obtained when the
Escherichia coli nirB
promoter was used and the bacteria were grown with low aeration. Intranasal immunization of mice with IEM101 expressing TetC elicited serum vibriocidal activity and induced antibodies against tetanus toxin which were protective against lethal challenge with 10 times the 50% lethal dose of tetanus toxin. Bacterial viability was essential for the induction of anti-TetC antibodies. Intranasal administration of IEM101 expressing Tcf induced a significant reduction in bacterial colonization of the tracheas of mice challenged with wild-type
B. pertussis
. These data are in agreement with the putative role of Tcf in
Bordetella
tracheal colonization. In conclusion, we have demonstrated that
V. cholerae
may be used as a live vector to deliver heterologous antigens in vivo and that protection to both systemic and local challenge may be achieved.
Electrotransformation and Expression of Bacterial Genes Encoding Hygromycin Phosphotransferase and β-Galactosidase in the Pathogenic Fungus
Histoplasma capsulatum
Authors: Woods Jon P. , Heinecke Elizabeth L. , Goldman William E.
Published: December 2019

We developed an efficient electrotransformation system for the pathogenic fungus
Histoplasma capsulatum
and used it to examine the effects of features of the transforming DNA on transformation efficiency and fate of the transforming DNA and to demonstrate fungal expression of two recombinant
Escherichia coli
genes,
hph
and
lacZ
. Linearized DNA and plasmids containing
Histoplasma
telomeric sequences showed the greatest transformation efficiencies, while the plasmid vector had no significant effect, nor did the derivation of the selectable
URA5
marker (native
Histoplasma
gene or a heterologous
Podospora anserina
gene). Electrotransformation resulted in more frequent multimerization, other modification, or possibly chromosomal integration of transforming telomeric plasmids when saturating amounts of DNA were used, but this effect was not observed with smaller amounts of transforming DNA. We developed another selection system using a hygromycin B resistance marker from plasmid pAN7-1, consisting of the
E. coli hph
gene flanked by
Aspergillus nidulans
promoter and terminator sequences. Much of the heterologous fungal sequences could be removed without compromising function in
H. capsulatum
, allowing construction of a substantially smaller effective marker fragment. Transformation efficiency increased when nonselective conditions were maintained for a time after electrotransformation before selection with the protein synthesis inhibitor hygromycin B was imposed. Finally, we constructed a readily detectable and quantifiable reporter gene by fusing
Histoplasma URA5
with
E. coli lacZ
, resulting in expression of functional β-galactosidase in
H. capsulatum
. Demonstration of expression of bacterial genes as effective selectable markers and reporters, together with a highly efficient electrotransformation system, provide valuable approaches for molecular genetic analysis and manipulation of
H. capsulatum
, which have proven useful for examination of targeted gene disruption, regulated gene expression, and potential virulence determinants in this fungus.
Bordetella pertussisFilamentous Hemagglutinin Enhances the Immunogenicity of Liposome-Delivered Antigen Administered Intranasally
Authors: Poulain-Godefroy Odile , Mielcarek Nathalie , Ivanoff Nathalie , Remoué Franck , Schacht Anne-Marie , Phillips Nigel , Locht Camille , Capron André , Riveau Gilles
Published: December 2019

In an attempt to increase the immunogenicity of mucosally delivered antigens, we incorporated theBordetella pertussisfilamentous hemagglutinin (FHA) adhesin into liposomes containing the glutathioneS-transferase ofSchistosoma mansoni(Sm28GST) as a model antigen. Outbred mice immunized twice intranasally with liposomes containing a constant suboptimal dose of Sm28GST and increasing doses of FHA produced anti-Sm28GST antibodies in a FHA dose-dependent manner. The addition of 3 μg of FHA to the liposomes induced more than 10-fold-higher anti-Sm28GST antibody titers, compared to those induced by liposomes without FHA. The presence of FHA did not alter the nature of the humoral immune response, and the sera contained anti-Sm28GST immunoglobulin G1 (IgG1), IgG2a, and IgG2b. However, anti-Sm28GST IgA was only detected when at least 3 μg of FHA was added to the preparation. These results show a promising potential for FHA to enhance the immunogenicity of mucosally administered antigens incorporated into liposomes.