bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduction of Astrogliosis by Early Treatment of Pneumococcal Meningitis Measured by Simultaneous Imaging, In Vivo, of the Pathogen and Host Response
Authors: Kadurugamuwa Jagath L. , Modi Kshitij , Coquoz Olivier , Rice Brad , Smith Steven , Contag Pamela R. , Purchio Tony
Published: November 2005

We developed a method for simultaneous in vivo biophotonic monitoring of pneumococcal meningitis and the accompanying neuronal injury in live transgenic mice.
Streptococcus pneumoniae
engineered for bioluminescence (
lux
) was used for direct visualization of disease progression and antibiotic treatment in a mouse model of meningitis. The host response was monitored in transgenic mice containing an inducible firefly luciferase (
luc
) reporter gene under transcriptional control of the mouse glial fibrillary acidic protein (GFAP) promoter. Based on the different spectra of light emission and substrate requirements for
lux
and
luc
, we were able to separately monitor the two reporters using a highly sensitive in vivo imaging system. The level of neuronal damage and recovery following antibiotic treatment was dependent on the time of treatment. This model has potential for simultaneous multiparameter monitoring and testing of therapies that target the pathogen or host response to prevent neuronal injury and recovery.
Decreased In Situ Expression of Interleukin-10 Receptor Is Correlated with the Exacerbated Inflammatory and Cytotoxic Responses Observed in Mucosal Leishmaniasis
Authors: Faria Daniela R. , Gollob Kenneth J. , Barbosa José , Schriefer Albert , Machado Paulo R. L. , Lessa Hélio , Carvalho Lucas P. , Romano-Silva Marco Aurélio , de Jesus Amélia R. , Carvalho Edgar M. , Dutra Walderez O.
Published: November 2005

Human infection withLeishmania braziliensiscan lead to cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). We hypothesize that the intense tissue destruction observed in ML is a consequence of an uncontrolled exacerbated inflammatory immune response, with cytotoxic activity. For the first time, this work identifies the cellular sources of inflammatory and antiinflammatory cytokines, the expression of effector molecules, and the expression of interleukin-10 (IL-10) receptor in ML and CL lesions by using confocal microscopy. ML lesions displayed a higher number of gamma interferon (IFN-γ)-producing cells than did CL lesions. In both ML and CL, CD4+cells represented the majority of IFN-γ-producing cells, followed by CD8+cells and CD4−CD8−cells. The numbers of tumor necrosis factor alpha-positive cells, as well as those of IL-10-producing cells, were similar in ML and CL lesions. The effector molecule granzyme A showed greater expression in ML than in CL lesions, while inducible nitric oxide synthase did not. Finally, the expression of IL-10 receptor was lower in ML than in CL lesions. Thus, our data identified distinct cytokine and cell population profiles for CL versus ML patients and provide a possible mechanism for the development of ML disease through the demonstration that low expression of IL-10 receptor is present in conjunction with a cytotoxic and inflammatory profile in ML.
Active and Passive Intranasal Immunizations with Streptococcal Surface Protein C5a Peptidase Prevent Infection of Murine Nasal Mucosa-Associated Lymphoid Tissue, a Functional Homologue of Human Tonsils
Authors: Park Hae-Sun , Cleary P. Patrick
Published: November 2005

C5a peptidase, also called SCPA (surface-bound C5a peptidase), is a surface-bound protein on group A streptococci (GAS), etiologic agents for a variety of human diseases including pharyngitis, impetigo, toxic shock, and necrotizing fasciitis, as well as the postinfection sequelae rheumatic fever and rheumatic heart disease. This protein is highly conserved among different serotypes and is also expressed in human isolates of group B, C, and G streptococci. Human tonsils are the primary reservoirs for GAS, maintaining endemic disease across the globe. We recently reported that GAS preferentially target nasal mucosa-associated lymphoid tissue (NALT) in mice, a tissue functionally analogous to human tonsils. Experiments using a C5a peptidase loss-of-function mutant and an intranasal infection model showed that this protease is required for efficient colonization of NALT. An effective vaccine should prevent infection of this secondary lymphoid tissue; therefore, the potential of anti-SCPA antibodies to protect against streptococcal infection of NALT was investigated. Experiments showed that GAS colonization of NALT was significantly reduced following intranasal immunization of mice with recombinant SCPA protein administered alone or with cholera toxin, whereas a high degree of GAS colonization of NALT was observed in control mice immunized with phosphate-buffered saline only. Moreover, administration of anti-SCPA serum by the intranasal route protected mice against streptococcal infection. These results suggest that intranasal immunization with SCPA would prevent colonization and infection of human tonsils, thereby eliminating potential reservoirs that maintain endemic disease.
Conjugates of Group A and W135 Capsular Polysaccharides ofNeisseria meningitidisBound to RecombinantStaphylococcus aureusEnterotoxin C1: Preparation, Physicochemical Characterization, and Immunological Properties in Mice
Authors: Jin Zhigang , Bohach Gregory A. , Shiloach Joseph , Norris Scott E. , Freedberg Darón I. , Deobald Claudia , Coxon Bruce , Robbins John B. , Schneerson Rachel
Published: November 2005

Neisseria meningitidisgroups A (GAM) and W135 capsular polysaccharides (CPs) were bound to recombinantStaphylococcus aureusenterotoxin C1 (rSEC). The CPs were activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate and then bound to adipic acid dihydrazide derivatives ofrSEC. Syntheses were conducted with native GAM CP (GAMP), W135 CP (W135P), and ultrasonicated or hydrazine-treated W135P at various concentrations of reactants, pHs, and ionic strengths. The conjugates were characterized by compositional and serologic analyses, high-performance size-exclusion chromatography with multi-angle laser light scattering detection, and immunogenicity in 5- to 6-week-old mice. Conjugates injected subcutaneously in phosphate-buffered saline elicited immunoglobulin G (IgG) responses against their respective CPs andrSEC, whereas GAMP and W135P alone did not induce detectable CP antibodies. The O-acetyl content of W135P was low, and its removal had no adverse effect upon the conjugate's immunogenicity. Reduction of the molecular size of W135P by treatment with hydrazine improved the immunogenicity of W135P-rSEC. IgG anti-CP elicited by the conjugates showed complement-dependent bactericidal activity against their respective organisms, and IgG anti-rSEC neutralized the T-cell proliferative activity of native SEC. A bivalent formulation of GAMP-rSEC and W135P-rSEC elicited IgG anti-CP at comparable levels to those induced by the conjugates administered separately.
Identification of Core and Variable Components of the
Salmonella enterica
Subspecies I Genome by Microarray
Authors: Anjum Muna F. , Marooney Chris , Fookes Maria , Baker Stephen , Dougan Gordon , Ivens Al , Woodward Martin J.
Published: November 2005

We have performed microarray hybridization studies on 40 clinical isolates from 12 common serovars within
Salmonella enterica
subspecies I to identify the conserved chromosomal gene pool. We were able to separate the core invariant portion of the genome by a novel mathematical approach using a decision tree based on genes ranked by increasing variance. All genes within the core component were confirmed using available sequence and microarray information for
S. enterica
subspecies I strains. The majority of genes within the core component had conserved homologues in
Escherichia coli
K-12 strain MG1655. However, many genes present in the conserved set which were absent or highly divergent in K-12 had close homologues in pathogenic bacteria such as
Shigella flexneri
and
Pseudomonas aeruginosa
. Genes within previously established virulence determinants such as SPI1 to SPI5 were conserved. In addition several genes within SPI6, all of SPI9, and three fimbrial operons (
fim
,
bcf
, and
stb
) were conserved within all
S. enterica
strains included in this study. Although many phage and insertion sequence elements were missing from the core component, approximately half the pseudogenes present in
S. enterica
serovar Typhi were conserved. Furthermore, approximately half the genes conserved in the core set encoded hypothetical proteins. Separation of the core and variant gene sets within
S.enterica
subspecies I has offered fundamental biological insight into the genetic basis of phenotypic similarity and diversity across
S. enterica
subspecies I and shown how the core genome of these pathogens differs from the closely related
E. coli
K-12 laboratory strain.
Syndecan 1 Shedding Contributes to
Pseudomonas aeruginosa
Sepsis
Authors: Haynes Allan , Ruda Frank , Oliver Jeffrey , Hamood Abdul N. , Griswold John A. , Park Pyong Woo , Rumbaugh Kendra P.
Published: November 2005

The innate immune system is comprised of many components that function coordinately to prevent bacterial sepsis. However, thermal injury suppresses many of these factors, and the opportunistic pathogen
Pseudomonas aeruginosa
takes advantage of this condition, making it one of the leading causes of morbidity and mortality in the setting of thermal injury.
P. aeruginosa
is extremely efficient at colonizing burn wounds, spreading systemically, and causing sepsis, which often results in a systemic inflammatory response, multiple-organ failure, and death. The pathogenicity of
P. aeruginosa
is due to the arsenal of virulence factors produced by the pathogen and the immunocompromised state of the host. Syndecan 1 is a major heparan sulfate proteoglycan present on many host cells involved in thermal injury. Syndecan 1 anchored to the cell surface can be cleaved in a process termed ectodomain shedding. Syndecan 1 shedding results in the release of intact, soluble proteoglycan ectodomains that have diverse roles in innate immunity. Here we show for the first time that thermal injury results in shedding of syndecan 1 from host tissue. Our data show that syndecan 1 null mice are significantly less susceptible to
P. aeruginosa
infection than their wild-type counterparts, as demonstrated by (i) significantly lower mortality; (ii) absence of systemic spread of
P. aeruginosa
; and (iii) significant reductions in some proinflammatory cytokines. These results suggest that shed syndecan 1 plays an important role in the pathogenesis of
P. aeruginosa
infection of thermal injury and that syndecan 1-neutralizing agents may be effective supplements to current
P. aeruginosa
treatments.
Tissue Destruction Induced by
Porphyromonas gingivalis
Infection in a Mouse Chamber Model Is Associated with Host Tumor Necrosis Factor Generation
Authors: Lin Yuh-Yih , Huang Jan-Hung , Lai Yo-Yin , Huang Han-Ching , Hu Suh-Woan
Published: November 2005

Intrachamber challenge with
Porphyromonas gingivalis
strain 381 in a mouse subcutaneous chamber model results in a local infection that progresses to exfoliation of the chambers within 15 days. This study was designed to elucidate the contribution of host reactions to tissue destruction manifested by chamber exfoliation in animals infected with
P. gingivalis
. Chamber fluids showed increasing levels of prostaglandin E
2
with infection, and the levels of tumor necrosis factor (TNF) in chamber fluids peaked just before chamber exfoliation. Intraperitoneal injection of a TNF inhibitor, thalidomide (TH), reduced the number of exfoliated chambers, while indomethacin had no effect. Exogenous TNF in chambers without bacterial infection did not cause chamber exfoliation but induced neutrophil infiltration. In a dual-chamber model, two chambers were implanted in the same mouse. One chamber was infected with
P. gingivalis
, and 9 days later exogenous TNF was added to the other chamber. Altogether, 66.67% of uninfected chambers were exfoliated between day 11 and day 16, although no bacteria were recovered from uninfected chambers. TH treatment alleviated both infected and uninfected chamber exfoliation. In this study, tissue destruction caused by
P. gingivalis
381 infection was due to the elevation of the TNF levels and not due to local bacterial activities. Our results further indicate that local infection by
P. gingivalis
381, a nondisseminating strain, actually has systemic effects on the host pathological outcome.
Increased Sensitivity toSalmonella entericaSerovar Typhimurium Infection in Mice Undergoing Withdrawal from Morphine Is Associated with Suppression of Interleukin-12
Authors: Feng Pu , Wilson Qiana M. , Meissler Joseph J. , Adler Martin W. , Eisenstein Toby K.
Published: November 2005

We have shown previously that withdrawal from morphine induces immunosuppression in mice. The present study reports the effects of morphine withdrawal on infection withSalmonella entericaserovar Typhimurium. Mice were made dependent on morphine by the implantation of a slow-release morphine pellet for 96 h. Controls received a placebo pellet. Withdrawal was induced by pellet removal. Mice were inoculated intraperitoneally withSalmonella24 h postwithdrawal. Morphine withdrawal sensitized mice toSalmonellainfection, as evidenced by increased mortality, shortened mean survival time, and increased bacterial load in the blood, spleen, and liver. Examination of the levels of a panel of proinflammatory cytokines in sera of infected, morphine-withdrawn mice showed that morphine withdrawal inhibited the elevation of interleukin-12p70 (IL-12p70). The production of IL-12p40 in morphine withdrawal mice was also suppressed. The administration of exogenous IL-12 significantly decreased the bacterial burden in morphine-withdrawn mice. These studies show a correlation between the suppression of IL-12 production and a heightened susceptibility toSalmonellainfection in mice undergoing withdrawal from morphine.
Type 1 Chemokine Receptor Expression in Chagas' Disease Correlates with Morbidity in Cardiac Patients
Authors: Gomes Juliana A. S. , Bahia-Oliveira Lilian M. G. , Rocha Manoel Otávio C. , Busek Solange C. U. , Teixeira Mauro M. , Silva João Santana , Correa-Oliveira Rodrigo
Published: November 2005

Chemokines and chemokine receptors (CKRs) control the migration of leukocytes during the inflammatory process and are important immunological markers of type 1 (CCR5 and CXCR3) and type 2 (CCR3 and CCR4) responses. The coexpression of CKRs (CCR2, CCR3, CCR5, CXCR3, and CXCR4) and intracellular cytokines (interleukin-10 [IL-10], IL-4, tumor necrosis factor alpha [TNF-α], and gamma interferon [IFN-γ]) on T CD4+and CD8+peripheral cells from individuals with indeterminate (IND) or cardiac (CARD) clinical forms of Chagas' disease after in vitro stimulation withTrypanosoma cruziantigens, were evaluated in this study. The percentage of T CD4+and CD8+cells coexpressing CCR5 and IFN-γ, CXCR3 and IFN-γ, and CXCR3 and TNF-α were higher in CARD than in IND individuals; on the other hand, the percentage of T CD4+or CD8+cells coexpressing CCR3 and IL-10 or coexpressing CCR3 and IL-4 were lower in CARD individuals than in IND individuals. In addition, a significant positive correlation between the expression of CCR5 or CXCR3 and IFN-γ was observed in CARD individuals contrasting with a significant positive correlation between the expression of CCR3 and IL-4 and of CCR3 and IL-10 in IND patients. These results reinforce the hypothesis that aT. cruzi-exacerbated specific type 1 immune response developed by CARD chagasic patients is associated with the development of heart pathology.
Synergistic Effect of Nod1 and Nod2 Agonists with Toll-Like Receptor Agonists on Human Dendritic Cells To Generate Interleukin-12 and T Helper Type 1 Cells
Authors: Tada Hiroyuki , Aiba Setsuya , Shibata Ken-Ichiro , Ohteki Toshiaki , Takada Haruhiko
Published: November 2005

A synthetic Nod2 agonist, muramyldipeptide (MDP), and two Nod1 agonists, FK565 and FK156, mimic the bacterial peptidoglycan moiety and are powerful adjuvants that induce cell-mediated immunity, especially delayed-type hypersensitivity. In this study, we used human dendritic cell (DC) cultures to examine possible T helper type 1 (Th1) responses induced by MDP and FK565/156 in combination with various synthetic Toll-like receptor (TLR) agonists, including synthetic lipid A (TLR4 agonist), the synthetic triacyl lipopeptide Pam3CSSNA (TLR2 agonist), poly(I:C) (TLR3 agonist), and CpG DNA (TLR9 agonist). Immature DCs derived from human monocytes expressed mRNAs for Nod1, Nod2, TLR2, TLR3, TLR4, and TLR9. The stimulation of DCs with MDP and FK565 in combination with lipid A, poly(I:C), and CpG DNA, but not with Pam3CSSNA, synergistically induced interleukin-12 (IL-12) p70 and gamma interferon (IFN-γ), but not IL-18, in culture supernatants and induced IL-15 on the cell surface. In correlation with the cytokine induction, an upregulation of the mRNA expression of these cytokine genes was observed. Notably, IL-12 p35 mRNA expression increased >1,000-fold upon stimulation with lipid A plus either MDP or FK565 compared with stimulation with each stimulant alone. In contrast, for the expression of CD83 and costimulatory molecules such as CD40, CD80, and CD86, no synergistic effects were observed upon stimulation with Nod plus TLR agonists. The culture supernatants of DCs stimulated with lipid A plus either MDP or FK565 activated human T cells to produce high levels of IFN-γ, and the activity was attributable to DC-derived IL-12. These findings suggest that Nod1 and Nod2 agonists in combination with TLR3, TLR4, and TLR9 agonists synergistically induce IL-12 and IFN-γ production in DCs to induce Th1-lineage immune responses.
Deletions of Endocytic Components
VPS28
and
VPS32
Affect Growth at Alkaline pH and Virulence through both
RIM101
-Dependent and
RIM101
-Independent Pathways in
Candida albicans
Authors: Cornet Muriel , Bidard Frédérique , Schwarz Patrick , Da Costa Grégory , Blanchin-Roland Sylvie , Dromer Françoise , Gaillardin Claude
Published: November 2005

Ambient pH signaling involves a cascade of conserved Rim or Pal products in ascomycetous yeasts or filamentous fungi, respectively. Recent evidences in the fungi
Aspergillus nidulans
,
Saccharomyces cerevisiae
,
Yarrowia lipolytica
, and
Candida albicans
suggested that components of endosomal sorting complexes required for transport (ESCRT) involved in endocytic trafficking were needed for signal transduction along the Rim pathway. In this study, we confirm these findings with
C. albicans
and show that Vps28p (ESCRT-I) and Vps32p/Snf7p (ESCRT-III) are required for the transcriptional regulation of known targets of the Rim pathway, such as the
PHR1
and
PHR2
genes encoding cell surface proteins, which are expressed at alkaline and acidic pH, respectively. We additionally show that deletion of these two
VPS
genes, particularly
VPS32
, has a more drastic effect than a
RIM101
deletion on growth at alkaline pH and that this effect is only partially suppressed by expression of a constitutively active form of Rim101p. Finally, in an in vivo mouse model, both
vps
null mutants were significantly less virulent than a
rim101
mutant, suggesting that
VPS28
and
VPS32
gene products affect virulence both through Rim-dependent and Rim-independent pathways.
Chronic
Leishmania donovani
Infection Promotes Bystander CD8
+
-T-Cell Expansion and Heterologous Immunity
Authors: Polley Rosalind , Sanos Stephanie L. , Prickett Sara , Haque Ashraful , Kaye Paul M.
Published: November 2005

It has been proposed that long-lived memory T cells generated by vaccination or infection reside within a memory compartment that has a finite size. Consequently, in a variety of acute infection models interclonal competition has been shown to lead to attrition of preexisting memory CD8
+
T cells. Contrary to expectations, therefore, we found that chronic
Leishmania donovani
infection of
Listeria
-immune mice results in heightened protection against subsequent
Listeria
challenge. This protection was associated with bystander expansion of
Listeria
-specific CD8
+
T cells and a bias in these cells toward a central memory T-cell phenotype with an enhanced capacity for gamma interferon production. We propose that splenomegaly, which is characteristic of visceral leishmaniasis and other tropical infections, may help promote heterologous immunity by resetting the size of the memory compartment during chronic infection.
H2-M3 Major Histocompatibility Complex Class Ib-Restricted CD8 T Cells Induced bySalmonella entericaSerovar Typhimurium Infection Recognize Proteins Released bySalmonellaSerovar Typhimurium
Authors: Ugrinovic S. , Brooks C. G. , Robson J. , Blacklaws B. A. , Hormaeche C. E. , Robinson J. H.
Published: November 2005

Salmonella entericaserovar Typhimurium causes a typhoid-like disease in mice which has been studied extensively as a model for typhoid fever in humans. CD8 T cells contribute to protection againstS. entericaserovar Typhimurium in mice, but little is known about the specificity and major histocompatibility complex (MHC) restriction of the response. We report here that CD8 T-cell lines derived fromS. entericaserovar Typhimurium-infected BALB/c mice lysed bone marrow macrophages infected withS. entericaserovar Typhimurium or pulsed with proteins fromS. entericaserovar Typhimurium culture supernatants. Cytoxicity was beta-2-microglobulin dependent and largely TAP dependent, although not MHC class Ia restricted, as target cells of several different MHC haplotypes were lysed. The data suggested the participation of class Ib MHC molecules although no evidence for the presence of Qa1-restricted T cells could be found, unlike in previous reports. Instead, the T-cell lines lysed H2-M3-transfected fibroblasts infected withS. entericaserovar Typhimurium SL3261 or treated withSalmonellaculture supernatants. Thus, this report increases the number of MHC class Ib antigen-presenting molecules known forSalmonellaantigens to three: Qa-1, HLA-E, and now H2-M3. It also expands the range of pathogens that induce H2-M3-restricted CD8 T cells to include an example of gram-negative bacteria.
Essential Role for Humoral Immunity duringEhrlichiaInfection in Immunocompetent Mice
Authors: Yager Eric , Bitsaktsis Constantine , Nandi Bisweswar , McBride Jere W. , Winslow Gary
Published: November 2005

Although cellular immunity is essential for host defense during intracellular bacterial infections, humoral immunity can also play a significant role in host defense during infection by some intracellular bacteria, including the ehrlichiae. Antibodies can protect susceptible SCID mice from fatalEhrlichia chaffeensisinfection, an observation that has been hypothesized to involve the opsonization of bacteria released from host cells. To determine whether humoral immunity plays an essential role during ehrlichia infection in immunocompetent mice, we utilized a murine model of fatal monocytotropic ehrlichiosis caused byIxodes ovatusehrlichia. Mice lacking either B cells or FcγRI were unable to resolve a low-dose (sublethal)I. ovatusehrlichia infection, which suggested that humoral immunity is essential for resistance. Polyclonal sera generated inI. ovatusehrlichia-infected mice recognized a conserved ehrlichia outer membrane protein and, when administered to infected mice, caused a significant decrease in bacterial infection. Mice experimentally depleted of complement, or deficient for complement receptors 1 and 2, were also susceptible to sublethalI. ovatusehrlichia infection, as were mice that lacked the phox91 subunit of NADPH oxidase. The data are consistent with a mechanism whereby bacteria released from infected cells are lysed directly by complement or undergo antibody-mediated FcγR-dependent phagocytosis and subsequent exposure to reactive oxygen intermediates. The findings suggest mechanisms whereby antibodies contribute to immunity against intracellular bacteria in immunocompetent mice.
Sensitization of Human Aortic Endothelial Cells to Lipopolysaccharide via Regulation of Toll-Like Receptor 4 by Bacterial Fimbria-Dependent Invasion
Authors: Yumoto Hiromichi , Chou Hsin-Hua , Takahashi Yusuke , Davey Michael , Gibson Frank C. , Genco Caroline A.
Published: November 2005

Toll-like receptors (TLRs) are differentially up-regulated in response to microbial infection and chronic inflammatory diseases such as atherosclerosis. Epidemiological data support the idea that periodontal disease may be a risk factor for acceleration of atherosclerosis.Porphyromonas gingivalis, the etiological agent of periodontal disease, invades endothelium, has been detected in human atheromatous tissue, and accelerates atheroma formation in apolipoprotein E−/−mice with concurrent induction of TLRs in the aorta. As endothelial cells can present antigen via TLRs and play an important role in the development of atherosclerosis, we examined TLR expression in human aortic endothelial cells (HAEC) cultured with wild-typeP. gingivalis, a fimbria-deficient mutant, and purified antigens. We observed increased TLR expression in HAEC infected with wild-typeP. gingivalisby fluorescence-activated cell sorter, but not with noninvasive, fimbria-deficient mutant or purifiedP. gingivalisantigens. Following a wild-typeP. gingivalischallenge, functional TLR2 and TLR4 activation was assessed by subsequent stimulation with TLR agonistsStaphylococcus aureuslipoteichoic acid (SLTA; TLR2 ligand) andEscherichia colilipopolysaccharide (LPS; TLR4 ligand). Unchallenged HAEC failed to elicit monocyte chemoattractant protein 1 (MCP-1) in response to LPS or SLTA but did so when cultured with wild-typeP. gingivalis. P. gingivalis-induced TLR2 and -4 expression on HAEC functionally reacted to SLTA andE. coliLPS as measured by a further increase in MCP-1 production. Furthermore, MCP-1 expression elicited byE. coliLPS was inhibitable with TLR4-specific antibody and polymyxin B. These results indicate that invasiveP. gingivalisstimulates TLR expression on the surface of endothelium and these primed cells respond to defined TLR-specific ligands.
Toxoplasma gondii-Specific Immunoglobulin M Limits Parasite Dissemination by Preventing Host Cell Invasion
Authors: Couper Kevin N. , Roberts Craig W. , Brombacher Frank , Alexander James , Johnson Lawrence L.
Published: November 2005

An important role for immunoglobulin M (IgM) during early acute virulentToxoplasma gondiiinfection was identified using IgM−/−mice that lack surface and secretory IgM but maintain normal B-cell functionality and isotype class switching. Following intraperitoneal inoculation with the virulent RH strain, IgM−/−mice displayed significantly fewer peritoneal parasites than wild-type (WT) mice, which correlated with increased tachyzoite dissemination to the liver, lung, and spleen in IgM−/−mice compared with WT mice. Early splenic T-cell activation, as measured by CD69 expression, was augmented in IgM−/−mice, and serum and peritoneal cavity gamma interferon levels were also elevated in IgM−/−mice compared with WT controls. Consequently, the difference in parasite dissemination was not attributable to an impaired proinflammatory immune response in the IgM−/−mice. Specific IgM was found to bind to tachyzoites in vivo in WT mice, and this correlated with an increased ability of antiserum collected from WT mice at day 6 postinfection to block tachyzoite cell invasion, compared with comparable serum collected from IgM−/−mice at the same time point. Tachyzoite invasion of host cells was similar if parasites were incubated with WT or IgM−/−nonimmune serum, suggesting that natural IgM does not function to limit parasite dissemination during earlyT. gondiiinfection. Our results highlight an important role for parasite-specific IgM in limiting systemic dissemination of tachyzoites during early acuteT. gondiiinfection.
Vaccination with the RecombinantBrucellaOuter Membrane Protein 31 or a Derived 27-Amino-Acid Synthetic Peptide Elicits a CD4+T Helper 1 Response That Protects againstBrucella melitensisInfection
Authors: Cassataro Juliana , Estein Silvia M. , Pasquevich Karina A. , Velikovsky Carlos A. , de la Barrera Silvia , Bowden Raúl , Fossati Carlos A. , Giambartolomei Guillermo H.
Published: November 2005

The immunogenicity and protective efficacy of the recombinant 31-kDa outer membrane protein fromBrucella melitensis(rOmp31), administered with incomplete Freund's adjuvant, were evaluated in mice. Immunization of BALB/c mice with rOmp31 conferred protection againstB. ovisandB. melitensisinfection. rOmp31 induced a vigorous immunoglobulin G (IgG) response, with higher IgG1 than IgG2 titers. In addition, spleen cells from rOmp31-immunized mice produced interleukin 2 (IL-2) and gamma interferon, but not IL-10 or IL-4, after in vitro stimulation with rOmp31, suggesting the induction of a T helper 1 (Th1) response. Splenocytes from rOmp31-vaccinated animals also induced a specific cytotoxic-T-lymphocyte activity, which led to the in vitro lysis ofBrucella-infected macrophages. In vitro T-cell subset depletion indicated that rOmp31 immunization elicited specific CD4+T cells that secrete IL-2 and gamma interferon, while CD8+T cells induced cytotoxic-T-lymphocyte activity. In vivo depletion of T-cell subsets showed that the rOmp31-elicited protection againstB. melitensisinfection is mediated by CD4+T cells while the contribution of CD8+T cells may be limited. We then evaluated the immunogenicity and protective efficacy of a known exposed region from Omp31 on theBrucellamembrane, a peptide that contains amino acids 48 to 74 of Omp31. Immunization with the synthetic peptide in adjuvant did not elicit a specific humoral response but elicited a Th1 response mediated by CD4+T cells. The peptide in adjuvant induced levels of protection similar to those induced by rOmp31 againstB. melitensisbut less protection than was induced by rOmp31 againstB. ovis. Our results indicate that rOmp31 could be a useful candidate for the development of subunit vaccines againstB. melitensisandB. ovis.
Identification of Novel Antigenic Proteins in a ComplexAnaplasma marginaleOuter Membrane Immunogen by Mass Spectrometry and Genomic Mapping
Authors: Lopez Job E. , Siems William F. , Palmer Guy H. , Brayton Kelly A. , McGuire Travis C. , Norimine Junzo , Brown Wendy C.
Published: November 2005

Immunization with purifiedAnaplasma marginaleouter membranes induces complete protection against infection that is associated with CD4+T-lymphocyte-mediated gamma interferon secretion and immunoglobulin G2 (IgG2) antibody titers. However, knowledge of the composition of the outer membrane immunogen is limited. Recent sequencing and annotation of theA. marginalegenome predicts at least 62 outer membrane proteins (OMP), enabling a proteomic and genomic approach for identification of novel OMP by use of IgG serum antibody from outer membrane vaccinates. Outer membrane proteins were separated by two-dimensional electrophoresis, and proteins recognized by total IgG and IgG2 in immune sera of outer membrane-vaccinated cattle were detected by immunoblotting. Immunoreactive protein spots were excised and subjected to liquid chromatography-tandem mass spectrometry. A database search of theA. marginalegenome identified 24 antigenic proteins that were predicted to be outer membrane, inner membrane, or membrane-associated proteins. These included the previously characterized surface-exposed outer membrane proteins MSP2, operon associated gene 2 (OpAG2), MSP3, and MSP5 as well as recently identified appendage-associated proteins. Among the 21 newly described antigenic proteins, 14 are annotated in theA. marginalegenome and include type IV secretion system proteins, elongation factor Tu, and members of the MSP2 superfamily. The identification of these novel antigenic proteins markedly expands current understanding of the composition of the protective immunogen and provides new candidates for vaccine development.
Regulation of Cellular Caveolin-1 Protein Expression in Murine Macrophages by Microbial Products
Authors: Lei Mei G. , Tan Xiaoyu , Qureshi Nilofer , Morrison David C.
Published: November 2005

Previously, we reported that expression of caveolin-1 in elicited peritoneal mouse macrophages was up-regulated by remarkably low (1.0-pg/ml) concentrations ofEscherichia coliO111 lipopolysaccharide (LPS). Here we report that increases in caveolin-1 expression are manifested by different types of LPS, LPS-mimetic taxol, and heat-killedE. coliand to a much lesser extent by zymosan, polysaccharide-peptidoglycan, and heat-killedStaphylococcus aureus. Rhodobacter sphaeroideslipid A (RsDPLA) could not induce caveolin-1 expression in macrophages. Interestingly, polymyxin B (5 μg/ml) and RsDPLA show only a limited capacity to inhibit LPS-induced caveolin-1 expression. These findings suggest that expression of caveolin-1 in response to LPS may only partially be dependent upon lipid A. Recombinant tumor necrosis factor alpha marginally induces caveolin-1, suggesting that the ability of LPS to regulate caveolin-1 is not mediated primarily through an autocrine/paracrine mechanism involving this cytokine. Under conditions in which cellular levels of caveolin-1 are profoundly induced, no significant changes in TLR4 expression are observed. Of interest, caveolin-1 appears to localize to two cellular compartments, one associated with lipid rafts and a second associated with TLR4. Gamma interferon treatment inhibits the induction of caveolin-1 by LPS in macrophages. Inhibition of the p38 kinase-dependent pathway, but not the extracellular signal-regulated kinase pathway, effectively reduced the ability of LPS to mediate caveolin-1 up-regulation. Lactacystin, a potent inhibitor of the proteasome pathway, significantly modulates LPS-independent caveolin-1 expression, and lactacystin inhibits LPS-triggered caveolin-1 responses. These studies suggest that caveolin-1 up-regulation in response to LPS is likely to be proteasome dependent and triggered through the p38 kinase pathway.
Comparative Toll-Like Receptor 4-Mediated Innate Host Defense toBordetellaInfection
Authors: Mann Paul B. , Wolfe Daniel , Latz Eicke , Golenbock Douglas , Preston Andrew , Harvill Eric T.
Published: November 2005

Bordetella pertussis,B. parapertussis, andB. bronchisepticaare closely related species associated with respiratory disease in humans and other mammals. WhileB. bronchisepticahas a wide host range,B. pertussisandB. parapertussisevolved separately from aB. bronchiseptica-like progenitor to naturally infect only humans. Despite very different doubling times in vitro, all three establish similar levels of infection in the mouse lung within 72 h. Recent work has revealed separate roles for Toll-like receptor 4 (TLR4) in immunity toB. pertussisandB. bronchiseptica, while no role for TLR4 duringB. parapertussisinfection has been described. Here we compared the requirement for TLR4 in innate host defense to these organisms using the same mouse infection model. WhileB. bronchisepticacauses lethal disease in TLR4-deficient mice,B. pertussisandB. parapertussisdo not. Correspondingly, TLR4 is critical in limitingB. bronchisepticabut notB. pertussisorB. parapertussisbacterial numbers during the first 72 h. Interestingly,B. bronchisepticainduces a TLR4-dependent cytokine response that is considerably larger than that induced byB. pertussisorB. parapertussis. Analysis of their endotoxins using RAW cells suggests thatB. bronchisepticalipopolysaccharide (LPS) is 10- and 100-fold more stimulatory thanB. pertussisorB. parapertussisLPS, respectively. The difference in LPS stimulus is more pronounced when using HEK293 cells expressing human TLR4. Thus, it appears that in adapting to infect humans,B. pertussisandB. parapertussisindependently modified their LPS to reduce TLR4-mediated responses, which may compensate for slower growth rates and facilitate host colonization.
Antigenic Specificity of the Mucosal Antibody Response to
Moraxella catarrhalis
in Chronic Obstructive Pulmonary Disease
Authors: Murphy Timothy F. , Brauer Aimee L. , Aebi Christoph , Sethi Sanjay
Published: November 2005

Moraxella catarrhalis
is an important human mucosal pathogen causing otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Little is known about the mucosal antibody response to
M. catarrhalis
in adults with COPD. In this study, 10 pairs of well-characterized sputum supernatant samples from adults with COPD who had acquired and subsequently cleared
M. catarrhalis
from their respiratory tracts were studied in detail in an effort to begin to elucidate potentially protective immune responses. Flow cytometry analysis was used to study the distribution of immunoglobulin isotypes in paired preacquisition and postclearance sputum samples. The results showed that immunoglobulin A (IgA) is the predominant
M. catarrhalis
-specific immunoglobulin isotype and that the sputum IgA contains a secretory component, indicating that it is locally produced at the mucosal site. Most patients made new sputum IgA responses to the adhesins UspA1 and Hag, along with the surface protein UspA2. A smaller proportion of patients made new sputum IgA responses to the iron-regulated proteins TbpB and CopB and to lipooligosaccharide. These results have important implications in understanding the mucosal immune response to
M. catarrhalis
in the setting of COPD and in elucidating the elements of a protective immune response.
Iron and Fur Regulation in
Vibrio cholerae
and the Role of Fur in Virulence
Authors: Mey Alexandra R. , Wyckoff Elizabeth E. , Kanukurthy Vanamala , Fisher Carolyn R. , Payne Shelley M.
Published: November 2005

Regulation of iron uptake and utilization is critical for bacterial growth and for prevention of iron toxicity. In many bacterial species, this regulation depends on the iron-responsive master regulator Fur. In this study we report the effects of iron and Fur on gene expression in
Vibrio cholerae
. We show that Fur has both positive and negative regulatory functions, and we demonstrate Fur-independent regulation of gene expression by iron. Nearly all of the known iron acquisition genes were repressed by Fur under iron-replete conditions. In addition, genes for two newly identified iron transport systems, Feo and Fbp, were found to be negatively regulated by iron and Fur. Other genes identified in this study as being induced in low iron and in the
fur
mutant include those encoding superoxide dismutase (
sodA
), fumarate dehydratase (
fumC
), bacterioferritin (
bfr
), bacterioferritin-associated ferredoxin (
bfd
), and multiple genes of unknown function. Several genes encoding iron-containing proteins were repressed in low iron and in the
fur
mutant, possibly reflecting the need to reserve available iron for the most critical functions. Also repressed in the
fur
mutant, but independently of iron, were genes located in the
V. cholerae
pathogenicity island, encoding the toxin-coregulated pilus (TCP), and genes within the
V. cholerae
mega-integron. The
fur
mutant exhibited very weak autoagglutination, indicating a possible defect in expression or assembly of the TCP, a major virulence factor of
V. cholerae
. Consistent with this observation, the
fur
mutant competed poorly with its wild-type parental strain for colonization of the infant mouse gut.
Novel AttenuatedSalmonella entericaSerovar Choleraesuis Strains as Live Vaccine Candidates Generated by Signature-Tagged Mutagenesis
Authors: Ku Yu-We , McDonough Sean P. , Palaniappan Raghavan U. M. , Chang Chao-Fu , Chang Yung-Fu
Published: November 2005

Salmonella entericaserovar Choleraesuis is a host-adapted pathogen that causes swine paratyphoid. Signature-tagged mutagenesis (STM) was used to understand the pathogenicity ofS. entericaserovar Choleraesuis in its natural host and also to develop novel attenuated live vaccine candidates against this disease. A library of 960 signature-tagged mutants ofS. entericaserovar Choleraesuis was constructed and screened for attenuation in pigs. Thirty-three mutants were identified by the STM screening, and these mutants were further screened for attenuation by in vivo and in vitro competitive growth. Of these, 20 mutants targeting the outer membrane, type III secretion, transporter, lipopolysaccharide biosynthesis, and other unknown proteins were confirmed for attenuation. Five highly attenuated mutants (SC2D2 [ssaV], SC4A9 [gifsy-1], SC6F9 [dgoT], SC12B12 [ssaJ], and SC10B1[spiA]) were selected and evaluated for safety and protective efficacy in pigs by comparison with a commercially available vaccine strain. STM-attenuated live vaccine strains SC4A9 (gifsy-1) and SC2D2 (ssaV) were superior to commercially available live vaccine because they provided both safety and a protective immune response against challenge in pigs.
Giardia lamblia
-Induced Changes in Gene Expression in Differentiated Caco-2 Human Intestinal Epithelial Cells
Authors: Roxström-Lindquist Katarina , Ringqvist Emma , Palm Daniel , Svärd Staffan
Published: November 2005

The parasitic protozoan
Giardia lamblia
is a worldwide cause of diarrhea, but the mechanism of disease remains elusive. The parasite colonizes the small intestinal epithelium, known to be a sensor for the presence of enteric pathogens, without invading or causing severe inflammation. In this study we investigated the epithelial cell response to
G. lamblia
. Differentiated Caco-2 cells were infected with
G. lamblia
isolate WB-A11, and the transcriptome of the intestinal cells was analyzed after 1.5, 6, and 18 h of interaction, using oligonucleotide microarrays. A large number of genes displayed changed expression patterns, showing the complexity of the interaction between
G. lamblia
and intestinal cells. A novel chemokine profile (CCL2, CCL20, CXCL1, CXCL2, and CXCL3) was induced that was different from the response induced by enteric pathogens causing intestinal inflammation. Several genes involved in stress regulation changed their expression. These findings indicate that the intestinal epithelium senses the
G. lamblia
infection, and this is important for induction of innate and adaptive immunity. The induced stress response can be important in the pathogenesis.
Interferon-Inducible Protein 10, but Not Monokine Induced by Gamma Interferon, Promotes Protective Type 1 Immunity in MurineKlebsiella pneumoniaePneumonia
Authors: Zeng Xianying , Moore Thomas A. , Newstead Michael W. , Deng Jane C. , Kunkel Steven L. , Luster Andrew D. , Standiford Theodore J.
Published: November 2005

CXC chemokines that lack the ELR motif, including interferon-inducible protein 10 [IP-10 (CXCL10)] and monokine induced by gamma interferon (IFN-γ) [MIG (CXCL9)], have been shown to mediate the generation of type 1 immune responses. In this study, we found that intrapulmonary administration of the gram-negative bacteriumKlebsiella pneumoniaeresulted in the local and systemic expression of IP-10, followed sequentially by MIG expression. MIG mRNA expression in the lungs ofKlebsiella-infected mice required the endogenous production of IFN-γ, whereas IP-10 was expressed in both an IFN-γ-dependent and an IFN-γ-independent fashion. Antibody-mediated neutralization of IP-10 resulted in reduced bacterial clearance and decreased survival, whereas bacterial clearance was unaltered in mice treated with anti-MIG antibody. Impaired bacterial clearance in anti-IP-10 antibody-treated mice was associated with significant reductions in the number and/or activational status of NK and NK-T cells, CD4+T cells, and γδ T cells, as well as a reduction in the expression of IFN-γ. Conversely, the transient transgenic expression of murine IP-10 using adenovirus-mediated gene transfer resulted in improved bacterial clearance when IP-10 adenovirus was given concomitant with intrapulmonary bacterial challenge. These results indicate that IP-10 is an important component of innate immunity against extracellular bacterial pathogens of the lung and may represent a candidate molecule for immunotherapy in the setting of severe respiratory tract infection.
Cold Shock Response of the UspA1 Outer Membrane Adhesin of
Moraxella catarrhalis
Authors: Heiniger Nadja , Troller Rolf , Meier Patricia Stutzmann , Aebi Christoph
Published: November 2005

Colonization of the human nasopharynx exposes
Moraxella catarrhalis
, a common cause of otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, to sudden downshifts in temperature, occurring when the host breathes cold air. We investigated whether in vitro cold shock influences the expressions of the outer membrane adhesins UspA1 and hemagglutinin, which are considered virulence factors, and of an
M. catarrhalis
homolog of
recA
, a housekeeping gene, which in
Escherichia coli
is induced by cold shock. Quantitative real-time reverse transcriptase PCR was used for measuring mRNA copy number. A screening experiment revealed that a cold shock at 26°C maximally induced the copy number of
uspA1
. In comparison with 37°C conditions, a 1-hour cold shock at 26°C increased copy numbers of
uspA1
and
recA
by 2.5-fold (11.2 ± 1.8 versus 4.5 ± 0.8 copies/CFU) and 2.7-fold (0.30 ± 0.10 versus 0.11 ± 0.06), respectively, but did not induce transcription of
hag
. Exposure to 26°C increased surface expression of UspA1, as assessed by fluorescence-activated cell sorter analysis, and resulted in a significant increase in adherence of strain O35E to Chang human conjunctival cells (97.1% ± 2.0% versus 48.3% ± 9.2% at 37°C;
P
= 0.01). Cold shock induction of
uspA1
and
recA
was detected in strains belonging to either phylogenetic subpopulation of
M. catarrhalis
(16S rRNA types 1 and 2/3) and was most pronounced in type 2/3 strains (4- to 25-fold for
uspA1
), which do not express detectable amounts of UspA1 protein at 37°C. These data indicate that cold shock at a physiologically relevant temperature of 26°C induces the expression of at least one virulence factor (UspA1). To our knowledge, no similar data are available for other nasopharyngeal pathogens.
Plant-Based Vaccine: Mice Immunized with Chloroplast-Derived Anthrax Protective Antigen Survive Anthrax Lethal Toxin Challenge
Authors: Koya Vijay , Moayeri Mahtab , Leppla Stephen H. , Daniell Henry
Published: November 2005

The currently available human vaccine for anthrax, derived from the culture supernatant of
Bacillus anthracis
, contains the protective antigen (PA) and traces of the lethal and edema factors, which may contribute to adverse side effects associated with this vaccine. Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required. In an effort to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants, PA was expressed in transgenic tobacco chloroplasts by inserting the
pagA
gene into the chloroplast genome. Chloroplast integration of the
pagA
gene was confirmed by PCR and Southern analysis. Mature leaves grown under continuous illumination contained PA as up to 14.2% of the total soluble protein. Cytotoxicity measurements in macrophage lysis assays showed that chloroplast-derived PA was equal in potency to PA produced in
B. anthracis
. Subcutaneous immunization of mice with partially purified chloroplast-derived or
B. anthracis
-derived PA with adjuvant yielded immunoglobulin G titers up to 1:320,000, and both groups of mice survived (100%) challenge with lethal doses of toxin. An average yield of about 150 mg of PA per plant should produce 360 million doses of a purified vaccine free of bacterial toxins edema factor and lethal factor from 1 acre of land. Such high expression levels without using fermenters and the immunoprotection offered by the chloroplast-derived PA should facilitate development of a cleaner and safer anthrax vaccine at a lower production cost. These results demonstrate the immunogenic and immunoprotective properties of plant-derived anthrax vaccine antigen.