bluuebunny/crossref_metadata_2025 huggingface dataset:
Bactericidal Activity of Mammalian Cathelicidin-Derived Peptides
Authors: Travis Sue M. , Anderson Norma N. , Forsyth William R. , Espiritu Cesar , Conway Barbara D. , Greenberg E. P. , McCray Paul B. , Lehrer Robert I. , Welsh Michael J. , Tack Brian F.
Published: July 2002

Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against
Pseudomonas aeruginosa
,
Escherichia coli
,
Staphylococcus aureus
, and methicillin-resistant
Staphylococcus aureus
. Each peptide manifested activity against
P. aeruginosa
irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both low- and high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive
S. aureus
, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an α-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis.
In Situ Production of Gamma Interferon, Interleukin-4, and Tumor Necrosis Factor Alpha mRNA in Human Lung Tuberculous Granulomas
Authors: Fenhalls Gael , Wong Anthony , Bezuidenhout Juanita , van Helden Paul , Bardin Philip , Lukey Pauline T.
Published: July 2002

Human tuberculous granulomas from five adults undergoing surgery for hemoptysis were analyzed by nonradioactive in situ hybridization for tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin-4 (IL-4) gene expression. All of the patients produced TNF-α mRNA. Three patients stained positive for both IFN-γ and IL-4 mRNA; the other two stained positive for IFN-γ but not IL-4 mRNA. Heterogeneity between the granulomas was observed in those patients staining positive for both IFN-γ and IL-4 mRNA; these patients exhibited granulomas having IFN-γ and not IL-4 mRNA as well as granulomas positive for both cytokine mRNAs. There was no evidence of caseation in these granulomas, and the cytokine patterns may represent events in the evolution of the granuloma. However, in those granulomas exhibiting caseous necrosis, very little IFN-γ or IL-4 mRNA was observed, implying that progression of the granuloma is accompanied by a down regulation of T-cell responses. TNF-α mRNA expression was highest in patients with both IFN-γ and IL-4 mRNA. Populations of CD68 positive macrophage-like cells within the granulomas produce mRNA for TNF-α, IFN-γ, and IL-4. This implies that macrophages within the tuberculous granuloma may not be dependent on T-cell cytokines for modulation of their function but may be able to regulate their own activation state and that of the surrounding T cells. These findings have implications on the delivery of immunotherapies to patients with tuberculosis.
Synthesis of Polymerized Melanin byCryptococcus neoformansin Infected Rodents
Authors: Rosas Ángel L. , Nosanchuk Joshua D. , Feldmesser Marta , Cox Gary M. , McDade Henry C. , Casadevall Arturo
Published: July 2002

The ability ofCryptococcus neoformansto synthesize polymerized melanin in vitro has been associated with virulence, but it is unclear whether this fungus synthesizes polymerized melanin during infection. To study this question, we used two approaches: one involved the generation of monoclonal antibodies (MAbs) to melanin for use in immunohistochemical studies ofC. neoformans-infected rodents, and the other sought to isolate fungal melanin from infected tissues. Digestion of in vitro-melanizedC. neoformanscells with proteases, denaturant, and hot concentrated acid yields melanin particles that retain the shape of fungal cells and are therefore called melanin ghosts. BALB/c mice were immunized with melanin ghosts, and two immunoglobulin M MAbs to melanin were generated from the spleen of one mouse. Immunofluorescence analyses of lung and brain tissues of rodents infected with wild-type melanin-producing (Mel+)C. neoformansstrains demonstrated binding of the MAbs to the fungal cell wall. No binding was observed when infections were performed with mutant albino (Mel−)C. neoformansstrains. Particles with striking similarity to melanin ghosts were recovered after digestion of lung and brain tissues from Mel+C. neoformans-infected rodents and were reactive with the MAbs to melanin. No particles were recovered from tissues infected with Mel−C. neoformans. A Mel+C. neoformansstrain grown on lung or brain homogenate agar became lightly pigmented and also yielded particles similar to melanin ghosts upon digestion, providing additional evidence that lung and brain tissues contain substrate forC. neoformansmelanization. These results demonstrate thatC. neoformanssynthesizes polymerized melanin during infection, which has important implications for pathogenesis and antifungal drug development.
Receptor Structure for F1C Fimbriae of Uropathogenic
Escherichia coli
Authors: Khan A. Salam , Kniep Bernhard , Oelschlaeger Tobias A. , Van Die Irma , Korhonen Timo , Hacker Jörg
Published: July 2002

F1C fimbriae are correlated with uropathogenic
Escherichia coli
strains. Although F1C fimbriae mediate binding to kidney tubular cells, their receptor is not known. In this paper, we demonstrate for the first time specific carbohydrate residues as receptor structure for F1C-fimbria-expressing
E. coli
. The binding of the F1C fimbriated recombinant
E. coli
strain HB101(pPIL110-54) and purified F1C fimbriae to reference glycolipids of different carbohydrate compositions was evaluated by using thin-layer chromatography (TLC) overlay and solid-phase binding assays. TLC fimbrial overlay analysis revealed the binding ability of purified F1C fimbriae only to glucosylceramide (GlcCer), β1-linked galactosylceramide 2 (GalCer2) with nonhydroxy fatty acids, lactosylceramide, globotriaosylceramide, paragloboside (nLc
4
Cer), lactotriaosylceramide, gangliotriaosylceramide (asialo-GM
2
[GgO
3
Cer]) and gangliotetraosylceramide (asialo-GM
1
[GgO
4
Cer]). The binding of purified F1C fimbriae as well as F1C fimbriated recombinant
E. coli
strain HB101(pPIL110-54) was optimal to microtiter plates coated with asialo-GM
2
(GgO
3
Cer). The bacterial interaction with asialo-GM
1
(GgO
4
Cer) and asialo-GM
2
(GgO
3
Cer) was strongly inhibited only by disaccharide GalNAcβ1-4Galβ linked to bovine serum albumin. We observed no binding to globotetraosylceramide or Forssman antigen (Gb
5
Cer) glycosphingolipids or to sialic-acid-containing gangliosides. It was demonstrated that the presence of a GalCer or GlcCer residue alone is not sufficient for optimal binding, and additional carbohydrate residues are required for high-affinity adherence. Indeed, the binding efficiency of F1C fimbriated recombinant bacteria increased by 19-fold when disaccharide sequence GalNAcβ1-4Galβ is linked to glucosylceramide as in asialo-GM
2
(GgO
3
Cer). Thus, it is suggested that the disaccharide sequence GalNAcβ1-4Galβ of asialo-GM
2
(GgO
3
Cer) which is positioned internally in asialo-GM
1
(GgO
4
Cer) is the high-affinity binding epitope for the F1C fimbriae of uropathogenic
E. coli
.
Gram-Positive Bacteria Are Potent Inducers of Monocytic Interleukin-12 (IL-12) while Gram-Negative Bacteria Preferentially Stimulate IL-10 Production
Authors: Hessle Christina , Andersson Bengt , Wold Agnes E.
Published: July 2002

Interleukin-10 (IL-10) and IL-12 are two cytokines secreted by monocytes/macrophages in response to bacterial products which have largely opposite effects on the immune system. IL-12 activates cytotoxicity and gamma interferon (IFN-γ) secretion by T cells and NK cells, whereas IL-10 inhibits these functions. In the present study, the capacities of gram-positive and gram-negative bacteria to induce IL-10 and IL-12 were compared. Monocytes from blood donors were stimulated with UV-killed bacteria from each of seven gram-positive and seven gram-negative bacterial species representing both aerobic and anaerobic commensals and pathogens. Gram-positive bacteria induced much more IL-12 than did gram-negative bacteria (median, 3,500 versus 120 pg/ml at an optimal dose of 25 bacteria/cell;P< 0.001), whereas gram-negative bacteria preferentially stimulated secretion of IL-10 (650 versus 200 pg/ml;P< 0.001). Gram-positive species also induced stronger major histocompatibility complex class II-restricted IFN-γ production in unfractionated blood mononuclear cells than did gram-negative species (12,000 versus 3,600 pg/ml;P< 0.001). The poor IL-12-inducing capacity of gram-negative bacteria was not remediated by addition of blocking anti-IL-10 antibodies to the cultures. No isolated bacterial component could be identified that mimicked the potent induction of IL-12 by whole gram-positive bacteria, whereas purified LPS induced IL-10. The results suggest that gram-positive bacteria induce a cytokine pattern that promotes Th1 effector functions.
Pseudomonas aeruginosa
Cell-to-Cell Signaling Is Required for Virulence in a Model of Acute Pulmonary Infection
Authors: Pearson James P. , Feldman Matthew , Iglewski Barbara H. , Prince Alice
Published: July 2002

Cell-to-cell signaling controls many virulence genes in
Pseudomonas aeruginosa
. We tested the virulence of
las
and
rhl
quorum-sensing mutants in neonatal mice. A
lasI rhlI
double mutant was nearly avirulent, and the respective single mutant strains were reduced in virulence compared with the wild-type strain. Quorum sensing plays a role in
P. aeruginosa
pneumonia in neonatal mice.
Interleukin-10 Gene Therapy-Mediated Amelioration of Bacterial Pneumonia
Authors: Morrison Daniel F. , Foss Dennis L. , Murtaugh Michael P.
Published: July 2002

Respiratory infection byActinobacillus pleuropneumoniaecauses a highly pathogenic necrotizing pleuropneumonia with severe edema, hemorrhage and fever. Acute infection is characterized by expression of inflammatory cytokines, including interleukin-1 (IL-1), IL-6 and IL-8. To determine if high level production of inflammatory cytokines contributed to disease pathogenesis, we investigated if inhibiting macrophage activation with adenovirus type 5-expressed IL-10 (Ad-5/IL-10) reduced the severity of acute disease. Porcine tracheal epithelial cells infected with Ad-5/IL-10 produced bioactive human IL-10. When pigs were intratracheally infected withA. pleuropneumoniae, pigs pretreated with Ad-5/IL-10 showed a significant reduction in the amount of lung damage when compared to adenovirus type 5-expressing β-galactosidase (Ad-5/β-Gal)-treated and untreated pigs. In addition, serum zinc levels were unchanged, the lung weight/body weight ratio (an indicator of vascular leakage) was significantly reduced, and lung pathology scores were reduced. Myeloperoxidase activity in lung lavage fluid samples, an indicator of neutrophil invasion, was decreased to levels similar to that seen in pigs not infected withA. pleuropneumoniae. Reduction in inflammatory cytokine levels in lung lavage fluid samples correlated with the clinical observations in that pigs pretreated with Ad-5/IL-10 showed a corresponding reduction of IL-1 and tumor necrosis factor (TNF) compared with untreated and Ad-5/β-Gal-treated pigs. IL-6 levels were unaffected by pretreatment with Ad-5/IL-10, consistent with observations that IL-6 was not derived from alveolar macrophages. Since inflammatory cytokines are expressed at high levels in acute bacterial pleuropneumonia, these results indicate that macrophage activation, involving overproduction of IL-1 and TNF, is a prime factor in infection-related cases of massive lung injury.
MaternalTrypanosoma cruziInfection Upregulates Capacity of Uninfected Neonate Cells To Produce Pro- and Anti-Inflammatory Cytokines
Authors: Vekemans Johan , Truyens Carine , Torrico Faustino , Solano Marco , Torrico Mary-Cruz , Rodriguez Patricia , Alonso-Vega Cristina , Carlier Yves
Published: July 2002

The possibility of maternal in utero modulation of the innate and/or adaptive immune responses of uninfected newborns fromTrypanosoma cruzi-infected mothers was investigated by studying the capacity of their whole blood cells to produce cytokines in response toT. cruzilysate or lipopolysaccharide-plus-phytohemagglutinin (LPS-PHA) stimulation. Cells of such newborns occasionally released gamma interferon (IFN-γ) and no interleukin-2 (IL-2) and IL-4 upon specific stimulation, while their mothers responded by the production of IFN-γ, IL-2, and IL-4. Infection in mothers was also associated with a hyperactivation of maternal cells and also, strikingly, of cells of their uninfected neonates, since their release of proinflammatory (IL-1β, IL-6, and tumor necrosis factor alpha [TNF-α]) as well as of anti-inflammatory (IL-10 and soluble TNF receptor) cytokines or factors was upregulated in the presence of LPS-PHA and/or parasite lysate. These results show thatT. cruziinfection in mothers induces profound perturbations in the cytokine response of their uninfected neonates. Such maternal influence on neonatal innate immunity might contribute to limit the occurrence and severity of congenital infection.
Activation of Vα14+Natural Killer T Cells by α-Galactosylceramide Results in Development of Th1 Response and Local Host Resistance in Mice Infected withCryptococcus neoformans
Authors: Kawakami Kazuyoshi , Kinjo Yuki , Yara Satomi , Koguchi Yoshinobu , Uezu Kaori , Nakayama Toshinori , Taniguchi Masaru , Saito Atsushi
Published: July 2002

We examined the effect of α-galactosylceramide (α-GalCer) on the synthesis of gamma interferon (IFN-γ) and local resistance in mice infected intravenously withCryptococcus neoformans. The level of IFN-γ in serum increased on day 3, reached a peak level on day 7, and decreased to the basal level on day 14 postinfection in mice treated with α-GalCer, while in vehicle-treated mice, no increase was detected at any time points except for a small increase on day 7. Such effects were not observed in NKT-KO mice. In CD4KO mice, minor synthesis of IFN-γ was detected on day 3 in sera but was completely abolished by day 7. The α-GalCer-induced IFN-γ production on day 3 was partially reduced in mice depleted of NK cells by treatment with anti-asialo-GM1antibody (Ab). Spleen cells obtained from infected and α-GalCer-treated mice on day 7 produced a large amount of IFN-γ upon restimulation with live organisms, while only a marginal level of production was detected in splenocytes from infected and vehicle-treated mice. Such effects were abolished in CD4KO and NKT-KO mice. Finally, the fungal loads in the lungs and spleen on days 7 and 14 were significantly reduced in α-GalCer-treated mice compared to those in control mice. In NKT-KO mice, local resistance elicited by α-GalCer was completely abolished, although no obvious exacerbation of infection was detected. Furthermore, treatment with anti-IFN-γ monoclonal Ab mostly abrogated the protective effect of this agent. Thus, our results indicated that activation of Vα14+NKT cells resulted in an increased Th1 response and local resistance toC. neoformansthrough production of IFN-γ.
Ferric Dicitrate Transport System (Fec) of
Shigella flexneri
2a YSH6000 Is Encoded on a Novel Pathogenicity Island Carrying Multiple Antibiotic Resistance Genes
Authors: Luck Shelley N. , Turner Sally A. , Rajakumar Kumar , Sakellaris Harry , Adler Ben
Published: July 2002

Iron uptake systems which are critical for bacterial survival and which may play important roles in bacterial virulence are often carried on mobile elements, such as plasmids and pathogenicity islands (PAIs). In the present study, we identified and characterized a ferric dicitrate uptake system (Fec) in
Shigella flexneri
serotype 2a that is encoded by a novel PAI termed the
Shigella
resistance locus (SRL) PAI. The
fec
genes are transcribed in
S. flexneri
, and complementation of a
fec
deletion in
Escherichia coli
demonstrated that they are functional. However, insertional inactivation of
fecI
, leading to a loss in
fec
gene expression, did not impair the growth of the parent strain of
S. flexneri
in iron-limited culture media, suggesting that
S. flexneri
carries additional iron uptake systems capable of compensating for the loss of Fec-mediated iron uptake. DNA sequence analysis showed that the
fec
genes are linked to a cluster of multiple antibiotic resistance determinants, designated the SRL, on the chromosome of
S. flexneri
2a. Both the SRL and
fec
loci are carried on the 66,257-bp SRL PAI, which has integrated into the
serX
tRNA gene and which carries at least 22 prophage-related open reading frames, including one for a P4-like integrase. This is the first example of a PAI that carries genes encoding antibiotic resistance and the first report of a ferric dicitrate uptake system in
Shigella
.
Motility and Chemotaxis in Tissue Penetration of Oral Epithelial Cell Layers byTreponema denticola
Authors: Lux Renate , Miller James N. , Park No-Hee , Shi Wenyuan
Published: July 2002

The ability to penetrate tissue is an important virulence factor for pathogenic spirochetes. Previous studies have recognized the role of motility in allowing pathogenic spirochetes to invade tissues and migrate to sites favorable for bacterial proliferation. However, the nature of the movements, whether they are random or controlled by chemotaxis systems, has yet to be established. In this study, we addressed the role of motility and chemotaxis in tissue penetration by the periodontal disease-associated oral spirocheteTreponema denticolausing an oral epithelial cell line-based experimental approach. Wild-typeT. denticolaATCC 35405 was found to penetrate the tissue layers effectively, whereas a nonmotile mutant was unable to overcome the tissue barrier. Interestingly, the chemotaxis mutants also showed impaired tissue penetration. AcheAmutant that is motile but lacks the central kinase of the chemotaxis pathway showed only about 2 to 3% of the wild-type penetration rate. The two known chemoreceptors ofT. denticola, DmcA and DmcB, also appear to be involved in the invasion process. Thedmcmutants were actively motile but exhibited reduced tissue penetration of about 30 and 10% of the wild-type behavior, respectively. These data suggest that not only motility but also chemotaxis is involved in the tissue penetration byT. denticola.
Characterization of Saa, a Novel Autoagglutinating Adhesin Produced by Locus of Enterocyte Effacement-Negative Shiga-Toxigenic
Escherichia coli
Strains That Are Virulent for Humans
Authors: Paton Adrienne W. , Srimanote Potjanee , Woodrow Matthew C. , Paton James C.
Published: July 2002

The capacity of Shiga toxigenic
Escherichia coli
(STEC) to adhere to the intestinal mucosa undoubtedly contributes to pathogenesis of human disease. The majority of STEC strains isolated from severe cases produce attaching and effacing lesions on the intestinal mucosa, a property mediated by the locus of enterocyte effacement (LEE) pathogenicity island. This element is not essential for pathogenesis, as some cases of severe disease, including hemolytic uremic syndrome (HUS), are caused by LEE-negative STEC strains, but the mechanism whereby these adhere to the intestinal mucosa is not understood. We have isolated a gene from the megaplasmid of a LEE-negative O113:H21 STEC strain (98NK2) responsible for an outbreak of HUS, which encodes an auto-agglutinating adhesin designated Saa (STEC autoagglutinating adhesin). Introduction of
saa
cloned in pBC results in a 9.7-fold increase in adherence of
E. coli
JM109 to HEp-2 cells and a semilocalized adherence pattern. Mutagenesis of
saa
in 98NK2, or curing the wild-type strain of its megaplasmid, resulted in a significant reduction in adherence. Homologues of
saa
were found in several unrelated LEE-negative STEC serotypes, including O48:H21 (strain 94CR) and O91:H21 (strain B2F1), which were also isolated from patients with HUS. Saa exhibits a low degree of similarity (25% amino acid [aa] identity) with YadA of
Yersinia enterocolitica
and Eib, a recently described phage-encoded immunoglobulin binding protein from
E. coli
. Saa produced by 98NK2 is 516 aa long and includes four copies of a 37-aa direct repeat sequence. Interestingly, Saa produced by other STEC strains ranges in size from 460 to 534 aa as a consequence of variation in the number of repeats and/or other insertions or deletions immediately proximal to the repeat domain.
Relative Importance of NF-κB p50 in Mycobacterial Infection
Authors: Yamada Hiroyuki , Mizuno Satoru , Reza-Gholizadeh Mohammad , Sugawara Isamu
Published: July 2002

To understand the role of NF-κB in the development of murine tuberculosis in vivo, NF-κB p50 knockout mice were infected withMycobacterium tuberculosisby placing them in the exposure chamber of an airborne-infection apparatus. These mice developed multifocal necrotic pulmonary lesions or lobar pneumonia. Compared with the levels in wild-type mice, pulmonary inducible nitric oxide synthase, interleukin-2 (IL-2), gamma interferon, and tumor necrosis factor alpha mRNA levels were significantly low but expression of IL-10 and transforming growth factor β mRNAs were within the normal ranges. The pulmonary IL-6 mRNA expression level was higher. Therefore, NF-κB and its interaction with host cells play an important role in the pathogenesis of tuberculosis.
Intra- and Interspecies Regulation of Gene Expression byActinobacillus actinomycetemcomitansLuxS
Authors: Fong Karen P. , Chung Whasun O. , Lamont Richard J. , Demuth Donald R.
Published: July 2002

The cell density-dependent control of gene expression is employed by many bacteria for regulating a variety of physiological functions, including the generation of bioluminescence, sporulation, formation of biofilms, and the expression of virulence factors. Although periodontal organisms do not appear to secrete acyl-homoserine lactone signals, several species, e.g.,Porphyromonas gingivalis,Prevotella intermedia, andFusobacterium nucleatum, have recently been shown to secrete a signal related to the autoinducer II (AI-2) of the signal system 2 pathway inVibrio harveyi. Here, we report that the periodontal pathogenActinobacillus actinomycetemcomitansexpresses a homolog ofV. harveyi luxSand secretes an AI-2-like signal. Cell-free conditioned medium fromA. actinomycetemcomitansor from a recombinantEscherichia colistrain (E. coliAIS) expressingA. actinomycetemcomitans luxSinduced luminescence inV. harveyiBB170 >200-fold over controls. AI-2 levels peaked in mid-exponential-phase cultures ofA. actinomycetemcomitansand were significantly reduced in late-log- and stationary-phase cultures. Incubation of early-log-phaseA. actinomycetemcomitanscells with conditioned medium fromA. actinomycetemcomitansor fromE. coliAIS resulted in a threefold induction of leukotoxic activity and a concomitant increase in leukotoxin polypeptide. In contrast, no increase in leukotoxin expression occurred when cells were exposed to sterile medium or to conditioned broth fromE. coliAIS−, a recombinant strain in whichluxSwas insertionally inactivated.A. actinomycetemcomitansAI-2 also induced expression ofafuA, encoding a periplasmic iron transport protein, approximately eightfold, suggesting that LuxS-dependent signaling may play a role in the regulation of iron acquisition byA. actinomycetemcomitans. Finally,A. actinomycetemcomitansAI-2 added intranscomplemented aluxSknockout mutation inP. gingivalisby modulating the expression of theluxS-regulated genesuvrBandhasFin this organism. Together, these results suggest that LuxS-dependent signaling may modulate aspects of virulence and the uptake of iron byA. actinomycetemcomitansand induce responses in other periodontal organisms in mixed-species oral biofilm.
Immunological Studies of Chronic Ocular Toxoplasmosis: Up-Regulation of Major Histocompatibility Complex Class I and Transforming Growth Factor β and a Protective Role for Interleukin-6
Authors: Lyons R. E. , Anthony J. P. , Ferguson D. J. P. , Byrne N. , Alexander J. , Roberts F. , Roberts C. W.
Published: July 2002

A murine model was used to characterize the local immune and inflammatory response during ocular toxoplasmosis. Major histocompatibility complex (MHC) class I, normally expressed at low levels in immune-privileged sites such as the eye, was up-regulated during infection as determined by competitive reverse transcriptase (RT)-PCR and immunocytochemistry for both β2-microglobulin and the MHC class I heavy chain. However, the eyes of chronically infected mice also had increased levels of mRNA transcripts for transforming growth factor β, a cytokine associated with immune privilege and constitutively expressed in normal eyes. Transcripts for a number of inflammatory mediators, including interleukin-6 (IL-6), were increased during chronic infection. The role of IL-6 was further investigated by comparing disease progression and the development of the local immune response in wild-type (WT) and IL-6-deficient mice (IL-6−/−mice). Following infection, IL-6−/−mice developed more severe inflammation in the retina and vitreous humor compared with WT mice. This increased severity of disease was associated with reduced ocular IL-1α and increased tumor necrosis factor α mRNA production compared with WT mice. Moreover, the increased severity of disease in IL-6−/−mice correlated with increased eye parasite burden as determined by RT-PCR for theToxoplasma gondiibradyzoite-specificLDH2gene. These results demonstrate alterations to components of immune privilege as a result of ocular toxoplasmosis and a role for IL-6 in controlling parasite numbers and inflammation in the eye.
Passive Immunization with Melanin-Binding Monoclonal Antibodies Prolongs Survival of Mice with Lethal
Cryptococcus neoformans
Infection
Authors: Rosas Ángel L. , Nosanchuk Joshua D. , Casadevall Arturo
Published: July 2002

Passive immunization with monoclonal antibodies (MAbs) to melanin prolonged the survival of and reduced the fungal burden in
Cryptococcus neoformans
-infected mice in comparison to controls. MAbs to melanin reduced the growth rate of in vitro-melanized
C. neoformans
cells, suggesting a new mechanism of antibody-mediated protection.
Molecular Basis for Variable Expression of Merozoite Surface Antigen gp45 among American Isolates ofBabesia bigemina
Authors: Fisher Tina G. , McElwain Terry F. , Palmer Guy H.
Published: July 2002

Immunization with the merozoite surface glycoprotein gp45 induces protection against challenge using the homologousBabesia bigeminastrain. However, gp45 B-cell epitopes are highly polymorphic amongB. bigeminastrains isolated from different geographical locations within North and South America. The molecular basis for this polymorphism was investigated using the JG-29 biological clone of a Mexico strain ofB. bigeminaand comparison with the Puerto Rico, St. Croix, and Texcoco strains. The molecular size and antibody reactivity of gp45 expressed by the JG-29 clone were identical to those of the parental Mexico strain.gp45cDNA and the genomic locus encompassinggp45were cloned and sequenced from JG-29. The locus sequence and Southern blot data were consistent with a singlegp45copy in the JG-29 genome. The JG-29 cDNA expressed the full-length protein recognized by the gp45-specific monoclonal antibody 14/1.3.2. The genomes of the Puerto Rico and St. Croix strains ofB. bigeminawere shown to lack a closely relatedgp45-like gene by PCR using multiple primer sets and by Southern blots using both full-length and region-specificgp45probes. This genomic difference was confirmed using unpassaged isolates from a 1999 disease outbreak in Puerto Rico. In contrast, the Texcoco strain retains agp45gene, encoding an open reading frame identical to that of JG-29. However, the Texcocogp45gene is not transcribed. These two mechanisms, lack of a closely relatedgp45-like gene and failure to transcribegp45, result in generation of antigenic polymorphism amongB. bigeminastrains, and the latter mechanism is unique compared to prior mechanisms of antigenic polymorphism identified in babesial parasites.
Induction of Autoimmune Valvular Heart Disease by Recombinant Streptococcal M Protein
Authors: Quinn Anthony , Kosanke Stanley , Fischetti Vincent A. , Factor Stephen M. , Cunningham Madeleine W.
Published: July 2002

Rheumatic heart disease is an autoimmune sequela of group A streptococcal infection. Previous studies have established that streptococcal M protein is structurally and immunologically similar to cardiac myosin, a well-known mediator of inflammatory heart disease. In this study, we investigated the hypothesis that streptococcal M protein could produce inflammatory valvular heart lesions similar to those seen in rheumatic fever (RF). Fifty percent (3 of 6) of Lewis rats immunized with recombinant type 6 streptococcal M protein (rM6) developed valvulitis as well as focal lesions of myocarditis. Valvular lesions initiated at the valve surface endothelium spread into the valve. Anitschkow cells and verruca-like lesions were present. T cells from rM6-immunized rats proliferated in the presence of purified cardiac myosin, but not skeletal myosin. A T-cell line produced from rM6-treated rats proliferated in the presence of cardiac myosin and rM6 protein. The study demonstrates that the Lewis rat is a model of valvular heart disease and that streptococcal M protein can induce an autoimmune cell-mediated immune attack on the heart valve in an animal model. The data support the hypothesis that a bacterial antigen can break immune tolerance in vivo, an important concept in autoimmunity.
Arginine-Specific Protease fromPorphyromonas gingivalisActivates Protease-Activated Receptors on Human Oral Epithelial Cells and Induces Interleukin-6 Secretion
Authors: Lourbakos Afrodite , Potempa Jan , Travis James , D'Andrea Michael R. , Andrade-Gordon Patricia , Santulli Rosemary , Mackie Eleanor J. , Pike Robert N.
Published: July 2002

Periodontitis is a chronic inflammatory disease affecting oral tissues. Oral epithelial cells represent the primary barrier against bacteria causing the disease. We examined the responses of such cells to an arginine-specific cysteine proteinase (RgpB) produced by a causative agent of periodontal disease,Porphyromonas gingivalis. This protease caused an intracellular calcium transient in an oral epithelial cell line (KB), which was dependent on its enzymatic activity. Since protease-activated receptors (PARs) might mediate such signaling, reverse transcription-PCR was used to characterize the range of these receptors expressed in the KB cells. The cells were found to express PAR-1, PAR-2, and PAR-3, but not PAR-4. In immunohistochemical studies, human gingival epithelial cells were found to express PAR-1, PAR-2, and PAR-3 on their surface, but not PAR-4, indicating that the cell line was an effective model for the in vivo situation. PAR-1 and PAR-2 expression was confirmed in intracellular calcium mobilization assays by treatment of the cells with the relevant receptor agonist peptides. Desensitization experiments strongly indicated that signaling of the effects of RgpB was occurring through PAR-1 and PAR-2. Studies with cells individually transfected with each of these two receptors confirmed that they were both activated by RgpB. Finally, it was shown that, in the oral epithelial cell line, PAR activation by the bacterial protease-stimulated secretion of interleukin-6. This induction of a powerful proinflammatory cytokine suggests a mechanism whereby cysteine proteases fromP. gingivalismight mediate inflammatory events associated with periodontal disease on first contact with a primary barrier of cells.
CpG Oligodeoxynucleotides Enhance Host Defense during Murine Tuberculosis
Authors: Juffermans Nicole P. , Leemans Jaklien C. , Florquin Sandrine , Verbon Annelies , Kolk Arend H. , Speelman Peter , van Deventer Sander J. H. , van der Poll Tom
Published: July 2002

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate immune cells to produce cytokines. CpG ODNs protect mice against infections with intracellular bacteria by the induction of a T helper 1 (Th1) response. To determine the effect of CpG ODNs in pulmonary tuberculosis, mice were treated with CpG ODNs or control ODNs at the time of intranasal infection. CpG ODNs reduced mycobacterial outgrowth for up to 5 weeks afterMycobacterium tuberculosisinfection and were associated with a decrease in inflammation in lung tissue. CpG treatment was also associated with elevated levels of gamma interferon (IFN-γ) and decreased levels of interleukin 4 in the lungs and an increased capacity of splenocytes to secrete Th1-type cytokines. CpG ODNs given 2 weeks after infection were still able to reduce mycobacterial outgrowth and to enhance a Th1 response 5 weeks postinfection. Administration of CpG ODNs to IFN-γ-gene-deficient mice failed to reduce mycobacterial outgrowth. These data suggest that CpG ODNs improve host defense during pulmonary tuberculosis by an IFN-γ-dependent mechanism.
Role of CsrR, Hyaluronic Acid, and SpeB in the Internalization ofStreptococcus pyogenesM Type 3 Strain by Epithelial Cells
Authors: Jadoun Jeries , Eyal Osnat , Sela Shlomo
Published: July 2002

Internalization of group A streptococcus by human epithelial cells has been extensively studied during the past 6 years. It is now clear that multiple mechanisms are involved in this process. We have previously demonstrated that the CsrR global regulator controls the internalization of an invasive M type 3 strain through regulation of thehas(hyaluronic acid synthesis) operon, as well as another, unknown gene(s). Recently, it was reported that the CsrR-regulated cysteine protease (SpeB) is also involved in bacterial uptake. In this study we have examined the roles of CsrR, hyaluronic acid capsule, and SpeB in streptococcal internalization. We have constructed isogenic mutants of the M3 serotype deficient in thecsrR,hasA, andspeBgenes and tested their ability to be internalized by HEp-2 epithelial cells. Inactivation ofcsrRabolished internalization, while inactivation of eitherhasAorspeBincreased the internalization efficiency. Mutation incsrRderepressedhasAtranscription and lowered the activity of SpeB, while no effect onspeBtranscription was observed. ThespeBmutant expressed smaller amounts of capsule, while thehasAmutant transcribed morecsrRandspeBmRNAs. Thus, it seems that complex interactions between CsrR, SpeB, and capsule are involved in modulation of group A streptococcus internalization.
Identification of Major Outer Surface Proteins of
Streptococcus agalactiae
Authors: Hughes Martin J. G. , Moore Joanne C. , Lane Jonathan D. , Wilson Rebecca , Pribul Philippa K. , Younes Zabin N. , Dobson Richard J. , Everest Paul , Reason Andrew J. , Redfern Joanne M. , Greer Fiona M. , Paxton Thanai , Panico Maria , Morris Howard R. , Feldman Robert G. , Santangelo Joseph D.
Published: July 2002

To identify the major outer surface proteins of
Streptococcus agalactiae
(group B streptococcus), a proteomic analysis was undertaken. An extract of the outer surface proteins was separated by two-dimensional electrophoresis. The visualized spots were identified through a combination of peptide sequencing and reverse genetic methodologies. Of the 30 major spots identified as
S. agalactiae
specific, 27 have been identified. Six of these proteins, previously unidentified in
S. agalactiae
, were sequenced and cloned. These were ornithine carbamoyltransferase, phosphoglycerate kinase, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, purine nucleoside phosphorylase, enolase, and glucose-6-phosphate isomerase. Using a gram-positive expression system, we have overexpressed two of these proteins in an in vitro system. These recombinant, purified proteins were used to raise antisera. The identification of these proteins as residing on the outer surface was confirmed by the ability of the antisera to react against whole, live bacteria. Further, in a neonatal-animal model system, we demonstrate that some of these sera are protective against lethal doses of bacteria. These studies demonstrate the successful application of proteomics as a technique for identifying vaccine candidates.
Mycobacterial Antigens Exacerbate Disease Manifestations inMycobacterium tuberculosis-Infected Mice
Authors: Moreira Andre L. , Tsenova Liana , Haile Aman Melles , Bekker Linda-Gail , Freeman Sherry , Mangaliso Bande , Schröder Ulf , Jagirdar Jaishree , Rom William N. , Tovey Michael G. , Freedman Victoria H. , Kaplan Gilla
Published: July 2002

To control tuberculosis worldwide, the burden of adult pulmonary disease must be reduced. Although widely used,Mycobacterium bovisBCG vaccination given at birth does not protect against adult pulmonary disease. Therefore, postexposure vaccination of adults with mycobacterial antigens is being considered. We examined the effect of various mycobacterial antigens on mice with priorM. tuberculosisinfection. Subcutaneous administration of live or heat-treated BCG with or without lipid adjuvants to infected mice induced increased antigen-specific T-cell proliferation but did not reduce the bacterial load in the lungs and caused larger lung granulomas. Similarly, additional mycobacterial antigen delivered directly to the lungs by aerosol infection with viableM. tuberculosismixed with heat-killedMycobacterium tuberculosis(1:1) also did not reduce the bacillary load but caused increased expression of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), which was associated with larger granulomas in the lungs. WhenM. tuberculosis-infected mice were treated with recombinant BCG that secreted cytokines shown to reduce disease in a preinfection vaccine model, the BCG secreting TNF-α, and to a lesser extent, IL-2 and gamma interferon (IFN-γ), caused a significant increase in granuloma size in the lungs. Moreover, treatment ofM. tuberculosis-infected mice with recombinant murine TNF-α resulted in increased inflammation in the lungs and accelerated mortality without affecting the bacillary load. Taken together, these studies suggest that administration of mycobacterial antigens to mice with priorM. tuberculosisinfection leads to immune activation that may exacerbate lung pathology via TNF-α-induced inflammation without reducing the bacillary load.
NonviableBurkholderia malleiInduces a Mixed Th1- and Th2-Like Cytokine Response in BALB/c Mice
Authors: Amemiya Kei , Bush Gary V. , DeShazer David , Waag David M.
Published: July 2002

Nonviable cell preparations ofBurkholderia mallei, the causative agent of glanders, were evaluated as potential vaccine candidates in a BALB/c murine model. Three differentB. malleicell preparations plus Alhydrogel were evaluated: a heat-killed preparation, an irradiation-inactivated preparation, and a preparation of a capsule-negative mutant strain which had been irradiation inactivated. BALB/c mice were vaccinated twice with the differentB. malleipreparations, and spleens and sera were collected to determine their cellular and humoral immune responses. All three bacterial cell preparations had essentially the same results in two cellular immune response assays. In a splenocyte proliferation assay, the amount of cell proliferation in response to the homologous immunogen, concanavalin A, or lipopolysaccharide was similar for all the cell preparations. Also, splenocytes from the inoculated mice expressed interleukin 2 (IL-2), gamma interferon, and small amounts of IL-4 and IL-5, and more IL-10 cytokine in the presence of the homologous antigen. When the immunoglobulin subclasses from these mice were examined, they all produced higher levels of IgG1 than IgG2a subclasses. The higher ratio of IgG1 to IgG2a was not due to the amount of the immunogen or the adjuvant (Alhydrogel) used in the BALB/c mice. The cell preparations did not protect the vaccinated mice from a live challenge (>300 50% lethal doses). Our results suggest that in BALB/c mice, a mixed T-helper-cell-like response to nonviableB. malleiis obtained, as demonstrated by a Th1- and Th2-like cytokine response and a Th2-like subclass immunoglobulin response. This may be the reason for the inability of theB. malleicells that were examined as candidate vaccines to protect the mice from a live challenge.
Gamma Interferon Fails To Induce Expression of Indoleamine 2,3-Dioxygenase and Does Not Control the Growth ofChlamydophila abortusin BeWo Trophoblast Cells
Authors: Entrican Gary , Wattegedera Sean , Chui Michelle , Oemar Lyn , Rocchi Mara , McInnes Colin
Published: July 2002

The BeWo trophoblast cell line does not constitutively express the tryptophan degrading enzyme indolamine 2,3-dioxygenase (IDO), nor can IDO expression be induced by gamma interferon. This correlates with the inability of BeWo cells to control the growth ofChlamydophila abortus, in contrast to effects observed in HeLa cells treated with gamma interferon.
Differential Cytokine and Antibody Responses to Adult and Larval Stages ofOnchocerca volvulusConsistent with the Development of Concomitant Immunity
Authors: MacDonald Angus J. , Turaga Prasad S. D. , Harmon-Brown Carolyn , Tierney Tracy J. , Bennett Kristine E. , McCarthy Maggie C. , Simonek Scott C. , Enyong Peter A. , Moukatte Daniel W. , Lustigman Sara
Published: July 2002

The possibility of concomitant immunity and its potential mechanisms inOnchocerca volvulusinfection were examined by analyzing cytokine and antibody responses to infective larval (third-stage larvae [L3] and molting L3 [mL3]), adult female worm (F-OvAg), and skin microfilaria (Smf) antigens in infected individuals in a region of hyperendemicity in Cameroon as a function of age. Peripheral blood mononuclear cell interleukin 5 (IL-5) responses to F-OvAg and Smf declined significantly with age (equivalent to years of exposure toO. volvulus). In contrast, IL-5 secretion in response to L3 and mL3 remained elevated with increasing age. Gamma interferon responses to L3, mL3, and F-OvAg were low or suppressed and unrelated to age, except for responses to Smf in older subjects. IL-10 levels were uniformly elevated, regardless of age, in response to L3, mL3, and F-OvAg but not to Smf, for which levels declined with age. A total of 49 to 60% of subjects had granulocyte-macrophage colony-stimulating factor responses to allO. volvulusantigens unrelated to age. Analysis of levels of stage-specific immunoglobulin G3 (IgG3) and IgE revealed a striking, age-dependent dissociation between antibody responses to larval antigens (L3 and a recombinant L3-specific protein,O. volvulusALT-1) which were significantly increased or maintained with age and antibody responses to F-OvAg, which decreased. Levels of IgG1 to L3 and F-OvAg were elevated regardless of age, and levels of IgG4 increased significantly with age, although not toO. volvulusALT-1, which may have unique L3-specific epitopes. Immunofluorescence staining of whole larvae showed that total anti-L3 immunoglobulin levels also increased with the age of the serum donor. The separate and distinct cytokine and antibody responses to adult and infective larval stages ofO. volvuluswhich are age related are consistent with the acquisition of concomitant immunity in infected individuals.
Analysis of Sequence Diversity at the Highly Polymorphic
Cpgp40
/
15
Locus among
Cryptosporidium
Isolates from Human Immunodeficiency Virus-Infected Children in South Africa
Authors: Leav Brett A. , Mackay Malanie R. , Anyanwu Akudo , O' Connor Roberta M. , Cevallos Ana Maria , Kindra Gurpreet , Rollins Nigel C. , Bennish Michael L. , Nelson Richard G. , Ward Honorine D.
Published: July 2002

Cryptosporidium
sp. is a significant cause of diarrheal disease, particularly in human immunodeficiency virus (HIV)-infected patients in developing countries. We recently cloned and sequenced several alleles of the highly polymorphic single-copy
Cryptosporidium parvum
gene
Cpgp40
/
15.
This gene encodes a precursor protein that is proteolytically cleaved to yield mature cell surface glycoproteins gp40 and gp15, which are implicated in zoite attachment to and invasion of enterocytes. The most-striking feature of the
Cpgp40
/
15
alleles and proteins is their unprecedented degree of sequence polymorphism, which is far greater than that observed for any other gene or protein studied in
C. parvum
to date. In this study we analyzed nucleic acid and amino acid sequence polymorphism at the
Cpgp40
/
15
locus of 20
C. parvum
isolates from HIV-infected South African children. Fifteen isolates exhibited one of four previously identified genotype I alleles at the
Cpgp40
/
15
locus (Ia, Ib, Ic, and Id), while five displayed a novel set of polymorphisms that defined a new
Cpgp40
/
15
genotype I allele, designated genotype Ie. Surprisingly, only 15 of these isolates exhibited concordant type I alleles at the thrombospondin-related adhesive protein of
Cryptosporidium
and
Cryptosporidium
oocyst wall protein loci, while five isolates (all of which displayed
Cpgp40
/
15
genotype Ic alleles) displayed genotype II alleles at these loci. Furthermore, the last five isolates also manifested chimeric genotype Ic/Ib or Ic/II alleles at the
Cpgp40
/
15
locus, raising the possibility of sexual recombination within and between prototypal parasite genotypes. Lastly, children infected with isolates having genotype Ic alleles were significantly older than those infected with isolates displaying other genotype I alleles.
Sequence Diversity of
Pseudomonas aeruginosa
: Impact on Population Structure and Genome Evolution
Authors: Kiewitz Claudia , Tümmler Burkhard
Published: July 2002

Comparative sequencing of
Pseudomonas aeruginosa
genes
oriC
,
citS
,
ampC
,
oprI
,
fliC
, and
pilA
in 19 environmental and clinical isolates revealed the sequence diversity to be about 1 order of magnitude lower than in comparable housekeeping genes of
Salmonella
. In contrast to the low nucleotide substitution rate, the frequency of recombination among different
P. aeruginosa
genotypes was high, leading to the random association of alleles. The
P. aeruginosa
population consists of equivalent genotypes that form a net-like population structure. However, each genotype represents a cluster of closely related strains which retain their sequence signature in the conserved gene pool and carry a set of genotype-specific DNA blocks. The codon adaptation index, a quantitative measure of synonymous codon bias of genes, was found to be consistently high in the
P. aeruginosa
genome irrespective of the metabolic category and the abundance of the encoded gene product. Such uniformly high codon adaptation indices of 0.55 to 0.85 fit the ubiquitous lifestyle of
P. aeruginosa
.