bluuebunny/crossref_metadata_2025 huggingface dataset:
Intestinal Innate Immunity toCampylobacter jejuniResults in Induction of Bactericidal Human Beta-Defensins 2 and 3
Authors: Zilbauer Matthias , Dorrell Nick , Boughan Parjeet K. , Harris Andrew , Wren Brendan W. , Klein Nigel J. , Bajaj-Elliott Mona
Published: October 2005

Campylobacter jejuniis the most prevalent cause of bacterial diarrhea worldwide. Despite the serious health problems caused by this bacterium, human innate immune responses toC. jejuniinfection remain poorly defined. Human β-defensins, a family of epithelial antimicrobial peptides, are a major component of host innate defense at the gastrointestinal mucosal surface. In this study, the effect of two differentC. jejuniwild-type strains on human intestinal epithelial innate responses was investigated. Up-regulation of β-defensin gene and peptide expression during infection was observed and recombinant β-defensins were shown to have a direct bactericidal effect againstC. jejunithrough disruption of cell wall integrity. Further studies using an isogenic capsule-deficient mutant showed that, surprisingly, the absence of the bacterial polysaccharide capsule did not change the innate immune responses induced byC. jejunior the ability ofC. jejunito survive exposure to recombinant β-defensins. This study suggests a major role for this family of antimicrobial peptides in the innate immune defense against this human pathogen.
Effects of Forskolin on Kupffer Cell Production of Interleukin-10 and Tumor Necrosis Factor Alpha Differ from Those of Endogenous Adenylyl Cyclase Activators: Possible Role for Adenylyl Cyclase 9
Authors: Dahle Maria K. , Myhre Anders E. , Aasen Ansgar O. , Wang Jacob E.
Published: October 2005

Proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) that are released from Kupffer cells may trigger liver inflammation and damage. Hence, endogenous mechanisms for limiting TNF-α expression are crucial for avoiding the development of sepsis. Such mechanisms include the anti-inflammatory actions of interleukin-10 (IL-10) as well as signaling induced by the intracellular second messenger cyclic AMP (cAMP). Kupffer cells express several receptors that activate cAMP synthesis, including E-prostanoid receptors and β-adrenergic receptors. The expression and role of specific adenylyl cyclases in the inhibition of Kupffer cell activation have so far not been subject to study. Pretreatment of rat Kupffer cell cultures with cAMP analogues [8-(4-chlorophenyl)-thio-cAMP], adenylyl cyclase activator (forskolin), or ligands for G-coupled receptors (isoproterenol or prostaglandin E2) 30 min before the addition of lipopolysaccharide (LPS) (1 μg/ml) caused attenuated TNF-α levels in culture medium (forskolin/isoproterenol,P≤ 0.05; prostaglandin E2,P≤ 0.01). Forskolin also reduced IL-10 mRNA and protein (P≤ 0.05), which was not observed with the other cAMP-inducing agents. Furthermore, we found that rat Kupffer cells express high levels of the forskolin-insensitive adenylyl cyclase 9 compared to whole liver and that this expression is down-regulated by LPS (P≤ 0.05). We conclude that regulation of TNF-α and IL-10 in Kupffer cells depends on the mechanism by which cAMP is elevated. Forskolin and prostaglandin E2differ in their effects, which suggests a possible role of forskolin-insensitive adenylyl cyclases like adenylyl cyclase 9.
Role of Complement Receptor Type 2 and Endogenous Complement in the Humoral Immune Response to Conjugates of Complement C3d and Pneumococcal Serotype 14 Capsular Polysaccharide
Authors: Mitsuyoshi Joyce K. , Hu Yong , Test Samuel T.
Published: October 2005

Conjugation of the complement fragment C3d to both T-cell-dependent (TD) protein and T-cell-independent type 2 (TI-2) polysaccharide antigens enhances the humoral immune response in mice immunized with either type of antigen. However, the ability of C3d-protein conjugates to enhance the antibody response in mice deficient in complement receptor types 1 and 2 (CR1 and CR2) has raised questions about the role of C3d-CR2 interactions in the adjuvant effect of C3d. In this study, we examined the role of CR2 binding and endogenous complement activation in the antibody response to conjugates of C3d and serotype 14 pneumococcal capsular polysaccharide (PPS14). To block binding of PPS14-C3d conjugates to CR2, mice were immunized with a mixture of vaccine and (CR2)2-immunoglobulin G1 (IgG1). Mice receiving (CR2)2-IgG1 at the time of primary immunization had a marked reduction in the primary anti-PPS14 antibody response but an enhanced secondary anti-PPS14 response, suggesting that C3d-CR2 interactions are required for the primary response but can have negative effects on the memory response. Further, compared with mice receiving PPS14-C3d having a high C3d/PPS14 ratio, mice immunized with PPS14-C3d with low C3d/PPS14 ratios had an enhanced secondary antibody response. Treatment of mice with cobra venom factor to deplete complement had insignificant effects on the antibody response to PPS14-C3d. Experiments with CBA/Nxidmice confirmed that PPS14-C3d conjugates retain the characteristics of TI-2 rather than TD antigens. Thus, the adjuvant effect of C3d conjugated to PPS14 requires C3d-CR2 interactions, does not require activation of endogenous complement, and is not mediated by TD carrier effects.
Influence of CR3 (CD11b/CD18) Expression on Phagocytosis ofBordetella pertussisby Human Neutrophils
Authors: Mobberley-Schuman Paula S. , Weiss Alison A.
Published: October 2005

CR3 (CD11b/CD18) is expressed on neutrophils, and the engagement of CR3 can promote phagocytosis. CR3 serves as the receptor for theBordetella pertussisadhesin filamentous hemagglutinin (FHA) and for the adenylate cyclase toxin (ACT), which blocks neutrophil function. The influence of CR3, FHA, and ACT on the phagocytosis ofB. pertussisby human neutrophils was examined. The surface expression and function of CR3 are regulated. Tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) increased CR3 surface expression, but only TNF-α increased the ability of neutrophils to phagocytoseB. pertussis, suggesting that elevated CR3 expression alone is not sufficient to promote phagocytosis. Purified FHA and pertussis toxin also increased the surface expression of CR3 on neutrophils, while ACT and the B subunit of pertussis toxin did not affect CR3 expression. FHA-mediated attachment to CR3 can lead to phagocytosis, especially in the absence of ACT. FHA mutants failed to attach and were not phagocytosed by neutrophils. Similarly, an antibody to CR3 blocked both attachment and phagocytosis. The addition of exogenous FHA enhanced the attachment and phagocytosis of wild-typeB. pertussisand FHA mutants. Mutants lacking the SphB1 protease, which cleaves FHA and allows the release of FHA from the bacterial surface, were phagocytosed more efficiently than wild-type bacteria. ACT mutants were efficiently phagocytosed, but wild-typeB. pertussisor ACT mutants plus exogenous ACT resisted phagocytosis. These studies suggest that the activation and surface expression of CR3, FHA expression, and the efficiency of ACT internalization all influence whetherB. pertussiswill be phagocytosed and ultimately killed by neutrophils.
Intraspecies and Temperature-Dependent Variations in Susceptibility of
Yersinia pestis
to the Bactericidal Action of Serum and to Polymyxin B
Authors: Anisimov Andrey P. , Dentovskaya Svetlana V. , Titareva Galina M. , Bakhteeva Irina V. , Shaikhutdinova Rima Z. , Balakhonov Sergey V. , Lindner Buko , Kocharova Nina A. , Senchenkova Sof'ya N. , Holst Otto , Pier Gerald B. , Knirel Yuriy A.
Published: October 2005

Lipopolysaccharide (LPS) structure impacts the bactericidal action of cationic peptides, such as polymyxin B (PMB), and sensitivity to killing by normal human serum (NHS). Cultivation of different subspecies strains of
Yersinia pestis
isolated from unrelated geographic origins at various temperatures (mammals, 37°C; fleas, 25°C; or winter hibernation, 6°C) affects LPS composition and structure. We tested the susceptibilities of various strains of
Y. pestis
grown at these different temperatures to PMB and serum bactericidal killing. Both properties varied significantly in response to temperature changes. In
Y. pestis
subsp.
pestis
(the main subspecies causing human plague), high levels of resistance to PMB and NHS were detected at 25°C. However, at the same temperature,
Y. pestis
subsp.
caucasica
was highly sensitive to PMB. At both of the extreme temperatures, all strains were highly susceptible to PMB. At 25°C and 37°C,
Y. pestis
subsp.
caucasica
strain 1146 was highly susceptible to the bactericidal activity of 80% NHS. All
Y. pestis
strains studied were able to grow in heat-inactivated human serum or in 80% normal mouse serum. At 6°C, all strains were highly sensitive to NHS. Variations in the PMB resistance of different bacterial cultures related to both the content of cationic components (4-amino-4-deoxyarabinose in lipid A and glycine in the core) and a proper combination of terminal monosaccharides in the LPS. The NHS resistance correlated with an elevated content of
N
-acetylglucosamine in the LPS. Structural variation in the LPS of
Y. pestis
correlates with the organism's ability to resist innate immunity in both fleas and mammals.
Burkholderia pseudomallei-Induced Expression of Suppressor of Cytokine Signaling 3 and Cytokine-Inducible Src Homology 2-Containing Protein in Mouse Macrophages: a Possible Mechanism for Suppression of the Response to Gamma Interferon Stimulation
Authors: Ekchariyawat P. , Pudla S. , Limposuwan K. , Arjcharoen S. , Sirisinha S. , Utaisincharoen P.
Published: October 2005

Burkholderia pseudomallei, the causative agent of melioidosis, is a facultative intracellular gram-negative bacterium that is able to survive and multiply in macrophages. Previously, we reported thatB. pseudomalleiwas able to escape macrophage killing by interfering with the expression of inducible nitric oxide synthase (iNOS). In the present study, we extended this finding and demonstrated thatB. pseudomalleiwas able to activate the expression of suppressor of cytokine signaling 3 (SOCS3) and cytokine-inducible Src homology 2-containing protein (CIS) but not SOCS1 in a mouse macrophage cell line (RAW 264.7). The expression of SOCS3 and CIS inB. pseudomallei-infected macrophages directly correlated with a decreased gamma interferon (IFN-γ) signaling response, as indicated by a reduction in Y701-STAT-1 phosphorylation (pY701-STAT-1). Moreover, a reduction in the expression of IFN-γ-induced proteins, such as interferon regulatory factor 1 (IRF-1), was observed inB. pseudomallei-infected macrophages that were treated with IFN-γ. Since pY701-STAT-1 and IRF-1 are essential transcription factors for regulating iNOS expression, the failure to activate these factors could also result in depression of iNOS expression and a loss of macrophage killing capacity. Taken together, the data indicate that the activation of SOCS3 and CIS expression inB. pseudomallei-infected macrophages interfered with IFN-γ signaling, thus allowing the bacteria to escape killing by these phagocytic cells.
Characterization of Two Novel Cryptococcal Mannoproteins Recognized by Immune Sera
Authors: Biondo Carmelo , Messina Luciano , Bombaci Mauro , Mancuso Giuseppe , Midiri Angelina , Beninati Concetta , Cusumano Valentina , Gerace Elisabetta , Papasergi Salvatore , Teti Giuseppe
Published: October 2005

Host defenses against the encapsulated yeast
Cryptococcus neoformans
involve both humoral and cell-mediated immunity. Mannoproteins (MPs) are a heterogeneous class of immunodominant glycoproteins which have been only incompletely characterized. In this study, we report on the molecular features of two novel MPs that are recognized by serum antibodies during cryptococcosis. After fractionation of extracellular cryptococcal products, MPs reacted more strongly than other components with sera from
C. neoformans
-infected AIDS patients. Further fractionation and Western blot analysis of MPs evidenced the presence of highly reactive bands with molecular masses of 250, 125, 115, and 84 kDa. The 115- and 84-kDa bands contained significant amounts of N-linked oligosaccharides, as shown by decreased molecular mass after peptide-
N
-glycosidase F treatment. N-terminal amino acid sequences of the two bands were used to search
C. neoformans
nucleotide databases. Homologous genomic sequences were used to synthesize DNA probes and isolate cDNA clones containing the full-length genes, which were designated
MP84
and
MP115.
Both genes showed the presence of a serine/threonine-rich region, a potential site for heavy glycosylation.
MP84
and
MP115
showed homology with, respectively, polysaccharide deacetylases and carboxylesterases from other organisms. Recombinant, deglycosylated proteins expressed in
Escherichia coli
still reacted with sera from patients, albeit more weakly than natural MPs, indicating that at least some of the reactive epitopes were retained in the recombinant forms. In conclusion, we identified two novel MPs that are important targets of antibody responses during cryptococcosis. These data may be useful to devise alternative immunity-based strategies to control the disease.
Immune Responses against a Single CD8+-T-Cell Epitope Induced by Virus Vector Vaccination Can Successfully ControlTrypanosoma cruziInfection
Authors: Miyahira Yasushi , Takashima Yasuhiro , Kobayashi Seiki , Matsumoto Yasunobu , Takeuchi Tsutomu , Ohyanagi-Hara Mutsuko , Yoshida Ayako , Ohwada Akihiko , Akiba Hisaya , Yagita Hideo , Okumura Ko , Ogawa Hideoki
Published: October 2005

In order to develop CD8+-T-cell-mediated immunotherapy against intracellular infectious agents, vaccination using recombinant virus vectors has become a promising strategy. In this study, we generated recombinant adenoviral and vaccinia virus vectors expressing a single CD8+-T-cell epitope, ANYNFTLV, which is derived from aTrypanosoma cruziantigen. Immunogenicity of these two recombinant virus vectors was confirmed by the detection of ANYNFTLV-specific CD8+T cells in the spleens of immunized mice. Priming/boosting immunization using combinations of these two recombinant virus vectors revealed that the adenovirus vector was efficient for priming and the vaccinia virus vector was effective for boosting the CD8+-T-cell responses. Moreover, we also demonstrated that the ANYNFTLV-specific CD8+-T-cell responses were further augmented by coadministration of recombinant vaccinia virus vector expressing the receptor activator of NFκB (RANK) ligand as an adjuvant. By priming with the adenovirus vector expressing ANYNFTLV and boosting with the vaccinia virus vectors expressing ANYNFTLV and RANK ligand, the immunized mice were efficiently protected from subsequent challenge with lethal doses ofT. cruzi. These results indicated, for the first time, that the induction of immune responses against a single CD8+-T-cell epitope derived from an intrinsicT. cruziantigen was sufficient to control lethalT. cruziinfection.
Effects of Ploidy and Mating Type on Virulence of
Candida albicans
Authors: Ibrahim Ashraf S. , Magee B. B. , Sheppard D. C. , Yang Molly , Kauffman Sarah , Becker Jeff , Edwards John E. , Magee P. T.
Published: October 2005

Candida albicans
is the most common fungal pathogen of humans. The recent discovery of sexuality in this organism has led to the demonstration of a mating type locus which is usually heterozygous, although some isolates are homozygous. Tetraploids can be formed between homozygotes of the opposite mating type. However, the role of the mating process and tetraploid formation in virulence has not been investigated. We describe here experiments using a murine model of disseminated candidiasis which demonstrate that in three strains, including CAI-4, the most commonly used strain background, tetraploids are less virulent than diploids and can undergo changes in ploidy during infection. In contrast to reports with other strains, we find that
MTL
homozygotes are almost as virulent as the heterozygotes. These results show that the level of ploidy in
Candida albicans
can affect virulence, but the mating type configuration does not necessarily do so.
Nasal Immunization with a Malaria Transmission-Blocking Vaccine Candidate, Pfs25, Induces Complete Protective Immunity in Mice against Field Isolates of
Plasmodium falciparum
Authors: Arakawa Takeshi , Komesu Ai , Otsuki Hitoshi , Sattabongkot Jetsumon , Udomsangpetch Rachanee , Matsumoto Yasunobu , Tsuji Naotoshi , Wu Yimin , Torii Motomi , Tsuboi Takafumi
Published: October 2005

Malaria transmission-blocking vaccines based on antigens expressed in sexual stages of the parasites are considered one promising strategy for malaria control. To investigate the feasibility of developing noninvasive mucosal transmission-blocking vaccines against
Plasmodium falciparum
, intranasal immunization experiments with
Pichia pastoris
-expressed recombinant Pfs25 proteins were conducted. Mice intranasally immunized with the Pfs25 proteins in the presence of a potent mucosal adjuvant cholera toxin induced robust systemic as well as mucosal antibodies. All mouse immunoglobulin G (IgG) subclasses except IgG3 were found in serum at comparable levels, suggesting that the immunization induced mixed Th1 and Th2 responses. Consistent with the expression patterns of the Pfs25 proteins in the parasites, the induced immune sera specifically recognized ookinetes but not gametocytes. In addition, the immune sera recognized Pfs25 proteins with the native conformation but not the denatured forms, indicating that mucosal immunization induced biologically active antibodies capable of recognizing conformational epitopes of native Pfs25 proteins. Feeding
Anopheles dirus
mosquitoes with a mixture of the mouse immune sera and gametocytemic blood derived from patients infected with
P. falciparum
resulted in complete interference with oocyst development in mosquito midguts. The observed transmission-blocking activities were strongly correlated with specific serum antibody titers. Our results demonstrated for the first time that a
P. falciparum
transmission-blocking vaccine candidate is effective against field-isolated parasites and may justify the investigation of noninvasive mucosal vaccination regimens for control of malaria, a prototypical mucosa-unrelated mosquito-borne parasitic disease.
Epitope of the Vaccine-TypeBordetella pertussisStrain 186 Lipooligosaccharide and Antiendotoxin Activity of Antibodies Directed against the Terminal Pentasaccharide-Tetanus Toxoid Conjugate
Authors: Niedziela Tomasz , Letowska Iwona , Lukasiewicz Jolanta , Kaszowska Marta , Czarnecka Anna , Kenne Lennart , Lugowski Czeslaw
Published: October 2005

Lipooligosaccharides (LOS) isolated fromBordetella pertussisstrains 186 and 606 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-resolution magic angle spinning nuclear magnetic resonsnace (NMR). These analyses distinguished between the LOS of strains 186 and 606, suggesting that the structure of LOS inB. pertussisis heterogeneous. The pentasaccharide was selectively cleaved from LOS ofB. pertussisstrain 186, purified, and covalently linked to a monomer fraction of tetanus toxoid. Injection of rabbits with the neoglycoconjugate emulsified in complete Freund's adjuvant yielded immunoglobulin G antibodies that were reactive with the LOS. These antibodies reacted strongly withB. pertussisLOS possessing the complete dodecasaccharide, as determined by an enzyme-linked immunosorbent assay, immunoblotting, and flow cytometry with intact, live bacterial cells. The binding epitope within the pentasaccharide was investigated by saturation transfer difference (STD) NMR spectroscopy. Protons H-1 and H-4 of the terminal α-d-GlcpNAc and proton H-6 and protons of anN-methyl group at H-4 of 3-substituted β-l-FucpNAc4NMe exhibited the largest saturation transfers. STD NMR experiments confirmed that the immunodominant epitope recognized by the antineoglycoconjugate antibodies is located predominantly in the distal trisaccharide ofB. pertussis186 LOS. The antipentasaccharide antibodies induced by the conjugate inhibited the secretion of tumor necrosis factor alpha, interleukin-6, and NO by LOS-stimulated J774A.1 cells.
Enhanced Factor H Binding to Sialylated Gonococci Is Restricted to the Sialylated Lacto-N-Neotetraose Lipooligosaccharide Species: Implications for Serum Resistance and Evidence for a Bifunctional Lipooligosaccharide Sialyltransferase in Gonococci
Authors: Gulati Sunita , Cox Andrew , Lewis Lisa A. , St. Michael Frank , Li Jianjun , Boden Ryan , Ram Sanjay , Rice Peter A.
Published: October 2005

We isolated serologically identical (by serovar determination and porin variable region [VR] typing) strains ofNeisseria gonorrhoeaefrom an infected male and two of his monogamous female sex partners. One strain (termed 398078) expressed the L1 (Galα1 → 3Galβ1 → 4Glcβ1 → 4HepI) lipooligosaccharide (LOS) structure exclusively; the other (termed 398079) expressed the lacto-N-neotetraose (LNT; Galβ1 → 4GlcNAcβ1 → 3Galβ1 → 4Glcβ1 → 4HepI) LOS structure. The strain from the male index case expressed both glycoforms and exhibited both immunotypes. Nuclear magnetic resonance analysis revealed that sialic acid linked to the terminal Gal of L1 LOS via an α2 → 6 linkage and, as expected, to the terminal Gal of LNT LOS via an α2→ 3 linkage. Insertional inactivation of the sialyltransferase gene (known to sialylate LNT LOS) abrogated both L1 LOS sialylation and LNT LOS sialylation, suggesting a bifunctional nature of this enzyme in gonococci. Akin to our previous observations, sialylation of the LNT LOS of strain 398079 enhanced the binding of the complement regulatory molecule, factor H. Rather surprisingly, factor H did not bind to sialylated strain 398078. LOS sialylation conferred the LNT LOS-bearing strain complete (100%) resistance to killing by even 50% nonimmune normal human serum (NHS), whereas sialylation of L1 LOS conferred resistance only to 10% NHS. The ability of gonococcal sialylated LNT to bind factor H confers high-level serum resistance, which is not seen with sialylated L1 LOS. Thus, serum resistance mediated by sialylation of gonococcal L1 and LNT LOS occurs by different mechanisms, and specificity of factor H binding to sialylated gonococci is restricted to the LNT LOS species.
Analysis of a Heme-Dependent Signal Transduction System in
Corynebacterium diphtheriae
: Deletion of the
chrAS
Genes Results in Heme Sensitivity and Diminished Heme-Dependent Activation of the
hmuO
Promoter
Authors: Bibb Lori A. , King Natalie D. , Kunkle Carey A. , Schmitt Michael P.
Published: October 2005

The
Corynebacterium diphtheriae hmuO
gene encodes a heme oxygenase that is involved in the utilization of heme as an iron source. Transcription of
hmuO
is activated by heme or hemoglobin and repressed by iron and DtxR. Previous studies with
Escherichia coli
showed that heme-dependent transcriptional activation of an
hmuO
promoter-
lacZ
fusion was dependent on the cloned
C. diphtheriae chrA
and
chrS
genes (
chrAS
), which encode the response regulator and sensor kinase, respectively, of a two-component signal transduction system. In this study, nonpolar deletions in the
chrAS
genes were constructed on the chromosome of
C. diphtheriae
. Mutations in
chrAS
resulted in marked reduction in heme-dependent transcription of
hmuO
, which indicates that the ChrA/S system is a key regulator at the
hmuO
promoter. However, low but significant levels of heme-specific transcriptional activity were observed at the
hmuO
promoter in the
chrAS
mutants, suggesting that an additional heme-dependent activator is involved in
hmuO
expression. The
chrAS
mutants were also sensitive to heme, which was observed only in stationary-phase cultures and correlated with reduced cell viability. The heme sensitivity of the mutants was not due to reduced expression of
hmuO
, and these results suggest that additional factors controlled by the ChrA/S system may be involved in protection against heme toxicity. Transcriptional analysis of the
chrAS
operon revealed that it was not autoregulated or affected by iron or heme levels.
Epsilon-Toxin Is Required for Most
Clostridium perfringens
Type D Vegetative Culture Supernatants To Cause Lethality in the Mouse Intravenous Injection Model
Authors: Sayeed Sameera , Fernandez-Miyakawa M. E. , Fisher Derek J. , Adams Vicki , Poon Rachael , Rood Julian I. , Uzal Francisco A. , McClane Bruce A.
Published: October 2005

Clostridium perfringens
type D enterotoxemias have significant economic impact by causing rapid death of several domestic animal species. Consequently, domestic animals are commonly vaccinated, at varying efficacy, with inactivated type D vegetative supernatants. Improved type D vaccines might become possible if the lethal toxins produced by type D isolates were characterized and the contributions of those toxins to supernatant-induced lethality were established. Therefore, the current study evaluated the presence of lethal toxins in supernatants prepared from late-log-phase vegetative cultures of a large collection of genotype D isolates. Under this growth condition, most genotype D isolates produced variable levels of at least three different lethal toxins, including epsilon-toxin (ETX). To model the rapid lethality of type D enterotoxemias, studies were conducted involving intravenous (i.v.) injection of genotype D vegetative supernatants into mice, which were then observed for neurotoxic distress. Those experiments demonstrated a correlation between ETX (but not alpha-toxin or perfringolysin O) levels in late-log-phase genotype D supernatants and lethality. Consistent with the known proteolytic activation requirement for ETX toxicity, trypsin pretreatment was required for, or substantially increased, the lethality of nearly all of the tested genotype D vegetative supernatants. Finally, the lethality of these trypsin-pretreated genotype D supernatants could be completely neutralized by an ETX-specific monoclonal antibody but not by an alpha-toxin-specific monoclonal antibody. Collectively, these results indicate that, under the experimental conditions used in the present study, ETX is necessary for the lethal properties of most genotype D vegetative supernatants in the mouse i.v. injection model.
Toll-Like Receptor 2 Modulates the Proinflammatory Milieu inStaphylococcus aureus-Induced Brain Abscess
Authors: Kielian Tammy , Haney Anessa , Mayes Patrick M. , Garg Sarita , Esen Nilufer
Published: October 2005

Toll-like receptor 2 (TLR2) is a pattern recognition receptor (PRR) that plays an important role in innate immune recognition of conserved structural motifs on a wide array of pathogens, includingStaphylococcus aureus. To ascertain the functional significance of TLR2 in the context of central nervous system (CNS) parenchymal infection, we evaluated the pathogenesis ofS. aureus-induced experimental brain abscess in TLR2 knockout (KO) and wild-type (WT) mice. The expression of several proinflammatory mediators, including inducible nitric oxide synthase, tumor necrosis factor alpha, and macrophage inflammatory protein-2, was significantly attenuated in brain abscesses of TLR2 KO mice compared to WT mice during the acute phase of infection. Conversely, interleukin-17 (IL-17), a cytokine produced by activated and memory T cells, was significantly elevated in lesions of TLR2 KO mice, suggesting an association between innate and adaptive immunity in brain abscess. Despite these differences, brain abscess severity in TLR2 KO and WT animals was similar, with comparable mortality rates, bacterial titers, and blood-brain barrier permeability, implying a role for alternative PRRs. Expression of the phagocytic PRRs macrophage scavenger receptor type AI/AII and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was increased in brain abscesses of both TLR2 KO and WT mice compared to uninfected animals. However, LOX-1 induction in brain abscesses of TLR2 KO mice was significantly attenuated compared to WT animals, revealing that the TLR2-dependent signal(s) influence LOX-1 expression. Collectively, these findings reveal the complex nature of gram-positive bacterial recognition in the CNS which occurs, in part, through engagement of TLR2 and highlight the importance of receptor redundancy forS. aureusdetection in the CNS.
In Vitro Effector Activity ofPneumocystis murina-Specific T-Cytotoxic-1 CD8+T Cells: Role of Granulocyte-Macrophage Colony-Stimulating Factor
Authors: McAllister Florencia , Steele Chad , Zheng Mingquan , Shellito Judd E. , Kolls Jay K.
Published: October 2005

Human immunodeficiency virus (HIV)-related opportunistic infections continue to occur in patients who are newly diagnosed with HIV infection, those in the early course of highly active antiretroviral therapy or nonadherent to HIV care, and other immunosuppressed individuals. One of the most common opportunistic infections in these patients isPneumocystispneumonia. CD8+T cells are recruited to the lung afterP. cariniiinfection and have been associated with both lung injury and host defense. This variability may be due to subpopulations of CD8+T cells recruited to the lung. We have previously shown using adoptive transfer studies that in vivo-generated T-cytotoxic-1 (Tc1) CD8+T cells, defined by the secretion of gamma interferon (IFN-γ), have effector activity against Pneumocystis spp. in vitro as well as in vivo. To better understand the mechanisms of these effects, we generated, expanded, and tested Tc1 and Tc2 CD8+T cells specific forP. murinaex vivo. Tc1-polarized CD8+T cells secreted higher levels of IFN-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) and lower levels of interleukin-4 (IL-4), IL-5, IL-10, and IL-13 than Tc2 CD8+T cells when stimulated withP. murinaantigen. Moreover, Tc1 CD8+T cells demonstrated enhanced effector activity in a macrophage-mediated killing assay which was independent of cell contact. The augmentation in macrophage-mediatedP. murinakilling was significantly abrogated when GM-CSF was neutralized in the Tc1 CD8+T cells. These data support the possibility that antigen-specific GM-CSF secretion is critical for effector activity ofP. murina-specific Tc1 CD8+T cells in vitro.
Immune Response to Pneumococcal Polysaccharides 4 and 14 in Elderly and Young Adults: Analysis of the Variable Heavy Chain Repertoire
Authors: Kolibab Kris , Smithson S. Louise , Rabquer Bradley , Khuder Sadik , Westerink M. A. Julie
Published: October 2005

Streptococcus pneumoniaeis a leading cause of morbidity and mortality in both developed and developing countries. The current pneumococcal polysaccharide (PPS) vaccine is highly effective in young adults; however, vaccine efficacy is dramatically decreased in the elderly population. We hypothesized that the decreased vaccine efficacy in the elderly results from altered variable gene family usage. We have characterized the immunoglobulin G gene usage of the antibody response to PPS4 and PPS14 in 20 young and 20 elderly adults. The variable heavy (VH) gene repertoire of human peripheral B cells was amplified by using PCR. A total of 364 heavy chain sequences with specificity for PPS4 and 305 heavy chain sequences for PPS14 were analyzed from young adults. In addition, a total of 325 sequences for PPS4 and 291 sequences for PPS14 were obtained from elderly adults. Complete sequence identity, somatic mutation frequencies, and VHgene usage was determined in response to PPS4 and PPS14. In all volunteers, the immune response to both polysaccharides consisted predominantly of heavy chains belonging to the VH3 gene family. There were significant differences in the variable gene repertoire between young and elderly adults. Somatic mutation occurred more frequently in sequences derived from young compared to elderly derived sequences. With aging, a loss of oligoclonality was noted in response to PPS4 and PPS14 compared to young adults. The observed differences in VHrepertoire, somatic mutation, and loss of oligoclonality may contribute to decreased vaccine efficacy in the elderly.
Immune Response to Pneumococcal Polysaccharides 4 and 14 in Elderly and Young Adults: Analysis of the Variable Light Chain Repertoire
Authors: Smithson S. Louise , Kolibab Kris , Shriner Anne K. , Srivastava Neeti , Khuder Sadik , Westerink M. A. Julie
Published: October 2005

Streptococcus pneumoniaeis a human bacterial pathogen responsible for serious infections including pneumonia. The currently licensed polysaccharide vaccine provides 60 to 80% protection in young adults, but in the elderly the vaccine efficacy is drastically reduced despite normal antibody levels. We hypothesized that the reduced vaccine efficacy in the elderly results from altered variable gene family usage. We have analyzed the light chain gene usage in 20 young (20 to 30 years of age) and 20 elderly (65 to 86 years of age) adults in response to pneumococcal polysaccharide 4 (PPS4) and PPS14. We generated a variable light chain library using B cells specific for PPS4 and PPS14 from each vaccinated individual. We determined complete sequences and somatic mutation frequencies in all isolated variable light chain fragments. Six gene families, κ1, κ2, κ3, κ4, λ1, and λ3, were identified in response to PPS4 and PPS14 in both age groups. Comparison of young and elderly adults demonstrated significant differences in κ4, λ1, and λ3 gene usage in response to PPS4 and PPS14. With aging, there was a significant increase in κ4 gene usage and a significant decrease in λ1 and λ3 gene usage in response to both PPS4 and PPS14. Although both Vκ1 and Vλ3 gene products demonstrated extensive mutations, there was no age-related difference in mutational frequency per gene family. These findings suggest an age-related change in light chain gene usage in response to PPS4 and PPS14.
Defining the Interaction of theTreponema pallidumAdhesin Tp0751 with Laminin
Authors: Cameron Caroline E. , Brouwer Nathan L. , Tisch Lisa M. , Kuroiwa Janelle M. Y.
Published: October 2005

Various invasive pathogens attach to host tissues via the extracellular matrix component laminin, the major glycoprotein found within basement membranes. Previous investigations identified the laminin-binding adhesin Tp0751 within the spirochete bacteriumTreponema pallidum. In the current study, Tp0751 was shown to attach to a variety of laminin isoforms that are widely distributed throughout the host, including laminins 1, 2, 4, 8, and 10. Such universal attachment is conducive for an adhesin present within a highly invasive pathogen that encounters a variety of tissue sites during the course of infection. Additional studies systematically identified the amino acid residues within Tp0751 that contribute to laminin binding using synthetic peptides designed from the mature protein sequence. The minimum laminin-binding region of the adhesin was localized to 10 amino acids; peptides containing these residues inhibited attachment of Tp0751 andT. pallidumto laminin. Further, Tp0751-specific antibodies inhibited attachment ofT. pallidumto laminin. This study furthers our knowledge of the interaction ofT. pallidumwith laminin, an association that is proposed to facilitate bacterial traversal of basement membranes and subsequent entry into the circulation and tissue invasion. As such, these investigations will reveal new targets for possible prevention of bacterial dissemination and establishment of chronic infection.
The Glucocorticoid-Induced Tumor Necrosis Factor Receptor-Related Gene Modulates the Response toCandida albicansInfection
Authors: Agostini Massimiliano , Cenci Elio , Pericolini Eva , Nocentini Giuseppe , Bistoni Giovanni , Vecchiarelli Anna , Riccardi Carlo
Published: October 2005

The glucocorticoid-induced tumor necrosis factor (TNF) receptor-related gene (GITR; TNFRSF18) modulates immune response activating coaccessory signals in T cells and is expressed at high levels in CD4+CD25+cells. Its ligand (GITRL) is expressed in antigen-presenting cells, where it is capable of promoting signaling. We investigated the role of GITR/GITRL interaction during disseminated candidiasis in GITR knockout (GITR−/−) mice. GITR−/−mice survived longer and had a significantly decreased yeast load in kidneys and brain compared to GITR+/+mice. Since protective immunity to the fungus is mediated by antigen-specific T helper (Th) 1 cells, we studied in vitro cytokine production following infection. CD4+T cells of GITR−/−mice demonstrated a more efficient Th1 polarization as suggested by a two- to threefold decreased production of interleukin- (IL-)4 and IL-10 and a four- to fivefold increased production of gamma interferon compared to GITR+/+mice. This effect was not due to differences in lymphocyte and dendritic cell (DC) subpopulations in infected mice as demonstrated by flow cytometric studies. To verify whether DC activity was differently modulated, DCs were cocultured with CD4+T cells in the presence of heat-inactivatedCandida albicans. DCs, cocultured with GITR+/+CD4+CD25+cells produced a lower amount of IL-12 than DCs cocultured with GITR−/−CD4+CD25+T cells. These results suggest that GITR regulates susceptibility to systemic candidiasis by negatively modulating IL-12 production and promoting polarization of CD4+T cells towards Th2 by analogy with OX40, another TNF receptor superfamily member.
A
prfA
Transposon Mutant of
Listeria monocytogenes
F2365, a Serotype 4b Strain, Is Able To Survive in the Gastrointestinal Tract but Does Not Cause Systemic Infection of the Spleens and Livers of Intragastrically Inoculated Mice
Authors: Faith N. , Uhlich G. , Luchansky J. B. , Neudeck B. , Czuprynski C.
Published: October 2005

prfA
is a member of the Crp/Fnr family of global regulatory genes in
Listeria monocytogenes
that has been shown previously to regulate several key virulence determinants both in vitro and in parenterally inoculated laboratory rodents. However, the role of
prfA
in the ability of
L. monocytogenes
to cause infection via the gastrointestinal (GI) tract has not been clearly established. In this study, we used a
prfA
transposon mutant of
L. monocytogenes
F2365, a serotype 4b strain, to assess the role of
prfA
in the pathogenesis of gastrointestinal listeriosis in mice. We found that the
prfA
mutant was able to survive in the GI tract (i.e., cecum) of mice, albeit in numbers somewhat less than those of the wild-type parent strain of
L. monocytogenes
. However, mice inoculated with the
prfA
mutant did not exhibit systemic infection of the spleen and liver, as was noted for mice inoculated with the wild-type parent strain. Survival of the
prfA
mutant in synthetic gastric fluid at pH 2.5 or 5 was somewhat reduced compared to that of the wild-type strain, as was its ability to invade and multiply within differentiated human intestinal epithelial cells (Caco-2 cells). Prior infection with the
prfA
mutant gave mice some protection against a subsequent challenge with virulent
L. monocytogenes
, although much less than that gained by prior gastrointestinal infection with the wild-type parent strain. These findings indicate that the global regulatory gene
prfA
is dispensable for colonization of the GI tract in mice but not for systemic infection.
Invariant NKT Cells Are Essential for the Regulation of Hepatic CXCL10 Gene Expression duringLeishmania donovaniInfection
Authors: Svensson Mattias , Zubairi Soombul , Maroof Asher , Kazi Fatima , Taniguchi Masaru , Kaye Paul M.
Published: October 2005

Gamma interferon (IFN-γ)-regulated chemokines of the CXC family have been implicated as key regulators of a variety of T-cell-dependent inflammatory processes. However, the cellular source(s) of IFN-γ that regulates their early expression has rarely been defined. Here, we have directly addressed this question in mice afterLeishmania donovaniinfection. Comparison of CXCL10 mRNA accumulation in normal and IFN-γ-deficient mice confirmed an absolute requirement for IFN-γ for sustained (24 h) expression of CXCL10 mRNA accumulation in this model. In normal mice, IFN-γ was produced by both CD3intNK1.1+NKT cells and CD3−NK1.1+NK cells, as detected by intracellular flow cytometry. Strikingly, B6.Jα281−/−mice lacking NKT cells that express the invariant Vα14Jα18 T-cell-receptor α chain, although retaining a significant population of IFN-γ-producing NK cells and NKT cells, were unable to sustain CXCL10 mRNA accumulation. These data indicate that invariant NKT cells are indispensable for the regulation of hepatic CXCL10 gene expression duringL. donovaniinfection.
Metalloproteinase Inhibitors, Nonantimicrobial Chemically Modified Tetracyclines, and Ilomastat BlockBacillus anthracisLethal Factor Activity in Viable Cells
Authors: Kocer Salih S. , Walker Stephen G. , Zerler Brad , Golub Lorne M. , Simon Sanford R.
Published: October 2005

Lethal toxin, produced by the bacteriumBacillus anthracis, is a major contributor to morbidity and mortality in animals and humans who have contracted anthrax. One component of this toxin, lethal factor (LF), proteolytically inactivates members of the mitogen-activated protein kinase kinase (MAPKK or MEK) family. In this study we show that CMT-300, CMT-308, and Ilomastat, agents initially characterized as matrix metalloproteinase inhibitors which are in early stages of development as pharmaceuticals, effectively inhibit the zinc metalloproteinase activity of LF. All three inhibitors, CMT-300, CMT-308, and Ilomastat, inhibit LF-mediated cleavage of a synthetic peptide substrate based on the N-terminal domain of MEKs. Inhibition of LF-mediated MEK proteolysis by all three agents was also achieved using lysates of the human monocytoid line MonoMac 6 as sources of MAPKKs and visualization of the extent of cleavage after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by detection by Western blotting. Finally, we have demonstrated inhibition of intracellular MEKs in viable human monocytes and MonoMac 6 cells by these agents after incubation of the cells with a reconstituted preparation of recombinant lethal toxin. All three agents are effective inhibitors when incubated with LF prior to exposure to cells, while the CMTs, but not Ilomastat, are also effective when added after LF has already entered the viable cell targets. These results offer promise for strategies to combat effects of the lethal toxin ofB. anthracis.
Moraxella catarrhalisBacterium without Endotoxin, a Potential Vaccine Candidate
Authors: Peng Daxin , Hong Wenzhou , Choudhury Biswa P. , Carlson Russell W. , Gu Xin-Xing
Published: October 2005

Lipooligosaccharide (LOS) is a major surface component ofMoraxella catarrhalisand a possible virulence factor in the pathogenesis of human infections caused by this organism. The presence of LOS on the bacterium is an obstacle to the development of vaccines derived from whole cells or outer membrane components of the bacterium. AnlpxAgene encoding UDP-N-acetylglucosamine acyltransferase responsible for the first step of lipid A biosynthesis was identified by the construction and characterization of an isogenicM. catarrhalis lpxAmutant in strain O35E. The resulting mutant was viable despite the complete loss of LOS. The mutant strain showed significantly decreased toxicity by theLimulusamebocyte lysate assay, reduced resistance to normal human serum, reduced adherence to human epithelial cells, and enhanced clearance in lungs and nasopharynx in a mouse aerosol challenge model. Importantly, the mutant elicited high levels of antibodies with bactericidal activity and provided protection against a challenge with the wild-type strain. These data suggest that the null LOS mutant is attenuated and may be a potential vaccine candidate againstM. catarrhalis.
Dra/AfaE Adhesin of Uropathogenic Dr/Afa
+
Escherichia coli
Mediates Mortality in Pregnant Rats
Authors: Wroblewska-Seniuk K. , Selvarangan R. , Hart A. , Pladzyk R. , Goluszko P. , Jafari A. , du Merle L. , Nowicki S. , Yallampalli C. , Le Bouguénec C. , Nowicki B.
Published: October 2005

Escherichia coli
bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa
+
E. coli
is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using
afaE
and/or
afaD
mutants, we investigated the role of the
afaE
and
afaD
genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the
E. coli afaE
+
afaD
and
afaE afaD
+
mutants. The clinical
E. coli
strain (
afaE
+
afaD
+
) and the
afaE afaD
double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the
afaE
+
afaD
+
strain, followed by the group infected with the
afaE
+
afaD
strain. The mortality was dose dependent. The
afaE afaD
double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the
afaE
+
strains were the most invasive (
afaE
+
afaD
strain >
afaE
+
afaD
+
strain), while the
afaE
mutant strains (
afaE afaD
+
and
afaE afaD
strains) seemed to be noninvasive. This study shows for the first time that the
afaE
gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of
afaE
+
E. coli
strains in vitro.
Peptidoglycan ofStaphylococcus aureusUpregulates Monocyte Expression of CD14, Toll-Like Receptor 2 (TLR2), and TLR4 in Human Blood: Possible Implications for Priming of Lipopolysaccharide Signaling
Authors: Hadley J. S. , Wang J. E. , Foster S. J. , Thiemermann C. , Hinds C. J.
Published: October 2005

Previous studies have indicated that peptidoglycan (PepG) from gram-positive bacteria can exert a priming effect on the innate immune response to lipopolysaccharide (LPS) from gram-negative bacteria. Here, we hypothesized that this priming effect may be preceded by enhanced expression of monocyte CD14, Toll-like receptor 2 (TLR2), and TLR4. In an ex vivo whole human blood model, we observed a substantial synergy between LPS and PepG in the release of tumor necrosis factor alpha and interleukin-1β (IL-1β) over the 24-h experimental period, whereas the effect on IL-8 and IL-10 release was more time dependent. The priming effect of PepG on cytokine release was preceded by a rapid upregulation of CD14, TLR2, and TLR4 expression on monocytes: at 3 hours there was a twofold increase in CD14 expression (P< 0.03), a fivefold increase in TLR2 expression (P< 0.03), and a twofold increase in TLR4 expression (P< 0.03). CD14 and TLR2 remained upregulated throughout the experimental period following exposure to PepG (P< 0.05). Only a transient upregulation of these monocyte receptors was observed following treatment with LPS or LPS plus PepG. In conclusion, the synergistic effect of LPS and PepG on cytokine release is preceded by a reciprocal upregulation of TLR2 and TLR4 by both bacterial cell wall components.