bluuebunny/crossref_metadata_2025 huggingface dataset:
A Spontaneous Translational Fusion ofBacillus cereusPlcR and PapR Activates Transcription of PlcR-Dependent Genes inBacillus anthracisvia Binding with a Specific Palindromic Sequence
Authors: Pomerantsev Andrei P. , Pomerantseva Olga M. , Leppla Stephen H.
Published: September 2004

Transformation ofBacillus anthraciswith plasmid pUTE29-plcR-papRcarrying the nativeBacillus cereus plcR-papRgene cluster did not activate expression ofB. anthracishemolysin genes, even though these are expected to be responsive to activation by the global regulator PlcR. To further characterize the action of PlcR, we examined approximately 3,000B. anthracistransformants containing pUTE29-plcR-papRand found a single hemolytic colony. The hemolytic strain contained a plasmid having a spontaneousplcR-papRintergenic region deletion. Transformation of the resulting plasmid pFP12, encoding a fused PlcR-PapR protein, into the nonhemolyticB. anthracisparental strain produced strong activation ofB. anthracishemolysins, including phosphatidylcholine-specific phospholipase C and sphingomyelinase. The fused PlcR-PapR protein present in a lysate ofB. anthraciscontaining pFP12 bound strongly and specifically to the double-stranded palindrome 5′-TATGCATTATTTCATA-3′ that matches the consensus PlcR-binding site. In contrast, native PlcR protein in a lysate from aB. anthracisstrain expressing large amounts of this protein did not demonstrate binding with the palindrome. The results suggest that the activation of PlcR by binding of a PapR pentapeptide as normally occurs inBacillus thuringiensisandB. cereuscan be mimicked by tethering the peptide to PlcR in a translational fusion, thereby obviating the need for PapR secretion, extracellular processing, retrieval into the bacterium, and binding with PlcR.
The B Subunit ofEscherichia coliHeat-Labile Enterotoxin Induces Both Caspase-Dependent and -Independent Cell Death Pathways in CD8+T Cells
Authors: Salmond Robert J. , Williams Rachel , Hirst Timothy R. , Williams Neil A.
Published: September 2004

The nontoxic B subunit ofEscherichia coliheat-labile enterotoxin (EtxB) is a potent immunomodulatory molecule that acts both as an adjuvant and to stimulate immune deviation processes, resulting in the suppression of Th1-associated inflammatory responses. The ability of EtxB to alter immune reactivity is dependent on its ability to modulate immune cell function through binding to cell surface molecules, the principal receptor of which is the ubiquitous GM1-ganglioside. EtxB activates B cells and antigen-presenting cells and induces the selective apoptosis of murine CD8+T cells. We postulated that these effects are mediated by the induction of intracellular signaling pathways following EtxB-receptor interaction. We have previously shown that CD8+T-cell apoptosis induced by EtxB results from the activation of the transcription factor NF-κB and caspases. Here we report that while caspase activity is required for apoptosis, additional features of cell death are caspase independent. EtxB induces a rapid loss of mitochondrial membrane potential and cell viability that are unaffected by caspase inhibitors. In addition, our data suggest that these processes are independent of the activity of Bax and Bcl-2 but are mediated by nitric oxide synthase.
Dysregulated Inflammatory Response toCandida albicansin a C5-Deficient Mouse Strain
Authors: Mullick Alaka , Elias Miria , Picard Serge , Bourget Lucie , Jovcevski Orce , Gauthier Susan , Tuite Ashleigh , Harakidas Penelope , Bihun Craig , Massie Bernard , Gros Philippe
Published: September 2004

Experimental infection of inbred mouse strains withCandida albicansprovides a good model system to identify host genetic determinants that regulate onset of, response to, and ultimate outcome of disseminated candidiasis. The A/J mouse strain is exquisitely sensitive to infection withC. albicans, while the C57BL/6J strain is relatively resistant, as measured by survival following intravenous injection ofCandidablastospores. This differential susceptibility is caused by an A/J-specific loss-of-function mutation in the C5 component of the complement pathway. C5 plays several critical roles in host response to infection, including target lysis and phagocyte recruitment. Therefore, to determine which of its functions were required for host resistance to candidiasis, a detailed comparative analysis of pathophysiology and host response to acuteC. albicansinfection was conducted in A/J and C57BL/6J mice. C5-sufficient C57BL/6J mice were found to succumb late in infection due to severe kidney pathology, typified by fungal replication and robust neutrophil-based inflammatory response associated with extensive tissue damage. In contrast, A/J mice were moribund within 24 h postinfection but displayed little if any kidney damage despite an inability to mobilize granulocytes and a high fungal load in the kidney. Rather, C5 deficiency in A/J mice was associated with higher levels of circulating cytokines tumor necrosis factor alpha, interleukin-6, monocyte chemotactic protein 1 (MCP-1), MCP-5, and eotaxin in response toC. albicans. Transfer of the C5-defective allele from A/J onto a C57BL/6J genetic background in recombinant congenic strain BcA17 recapitulated the phenotypic aspects of the susceptibility of A/J mice toC. albicans, confirming the causative role of C5 deficiency in the dysregulated cytokine response.
A New Cruzipain-Mediated Pathway of Human Cell Invasion by
Trypanosoma cruzi
Requires Trypomastigote Membranes
Authors: Aparicio Isabela M. , Scharfstein Julio , Lima Ana Paula C. A.
Published: September 2004

The intracellular protozoan
Trypanosoma cruzi
causes Chagas' disease, a chronic illness associated with cardiomyopathy and digestive disorders. This pathogen invades mammalian cells by signaling them through multiple transduction pathways. We previously showed that cruzipain, the main cysteine protease of
T. cruzi
, promotes host cell invasion by activating kinin receptors. Here, we report a cruzipain-mediated invasion route that is not blocked by kinin receptor antagonists. By testing different strains of
T. cruzi
, we observed a correlation between the level of cruzipain secreted by trypomastigotes and the capacity of the pathogen to invade host cells. Consistent with a role for cruzipain, the cysteine protease inhibitor
N
-methylpiperazine-urea-Phe-homophenylalanine-vinylsulfone-benzene impaired the invasion of human smooth muscle cells by strains Dm28c and X10/6 but not by the G isolate. Cruzipain-rich supernatants of Dm28c trypomastigotes enhanced the infectivity of isolate G parasites twofold, an effect which was abolished by the cysteine protease inhibitor
l
-
trans
-epoxysuccinyl-leucylamido-(4-guanidino)butane and by thapsigargin, a drug that induces depletion of the intracellular Ca
2+
stores. The enhancement due to Dm28 supernatants was abolished upon cruzipain immunodepletion, and the activity was restored by purified cruzipain. In contrast, supernatants from isolate G trypomastigotes (with low levels of cruzipain) or supernatants from Dm28c epimastigotes or purified cruzipain alone did not enhance parasite invasion, indicating that the protease is required but not sufficient to engage this invasion pathway. We provide evidence that activation of this pathway requires cruzipain-mediated processing of a trypomastigote molecule associated with parasite-shed membranes. Our results couple cruzipain to host cell invasion through a kinin-independent route and further suggest that high-level cruzipain expression may contribute to parasite infectivity.
Characterization of the Antigenic Lipopolysaccharide O Chain and the Capsular Polysaccharide Produced by
Actinobacillus pleuropneumoniae
Serotype 13
Authors: MacLean Leann L. , Perry Malcolm B. , Vinogradov Evguenii
Published: September 2004

Serotyping
of
Actinobacillus pleuropneumoniae
, the etiologic agent of
porcine pleuropneumonia, is important for epidemiological studies and
for the development of homologous vaccine cell preparations. The
serology is based on the specific chemical structures of capsular
polysaccharides (CPSs) and lipopolysaccharide (LPS) antigenic
O-polysaccharide moieties (O-PSs), and knowledge of these structures is
required for a molecular-level understanding of their serological
specificities. The structures of
A. pleuropneumoniae
serotype
1 to 12 CPSs and O-PSs have been elucidated; however, the structures
associated with three newly proposed serotypes (serotypes 13, 14, and
15) have not been reported. Herein we described the structures of the
antigenic O-PS and CPS of
A. pleuropneumoniae
serotype 13. The
O-PS of the
A. pleuropneumoniae
serotype 13 LPS is a polymer
of branched tetrasaccharide repeating units composed of
l
-rhamnose, 2-acetamido-2-deoxy-
d
-galactose, and
d
-galactose residues (1:1:2). By use of hydrolysis,
methylation, and periodate oxidation chemical methods together with the
application of one- and two-dimensional
1
H and
13
C nuclear magnetic resonance spectroscopy and mass
spectrometry, the structures of the O chain and CPS were
determined. The CPS of
A. pleuropneumoniae
serotype 13 was characterized as a teichoic-acid type polymer. The LPS
O antigen was identical to the O-PS produced by
A.
pleuropneumoniae
serotype 7. The CPS has the unique structure of a
1,3-poly(glycerol phosphate) teichoic acid type I polymer and
constitutes the macromolecule defining the
A. pleuropneumoniae
serotype 13 antigenic
specificity.
Sm14 of
Schistosoma mansoni
in Fusion with Tetanus Toxin Fragment C Induces Immunoprotection against Tetanus and Schistosomiasis in Mice
Authors: Abreu Patrícia A. E. , Miyasato Patrícia A. , Vilar Mônica M. , Dias Waldely O. , Ho Paulo L. , Tendler Míriam , Nascimento Ana L. T. O.
Published: September 2004

We have constructed vectors that permit the expression in
Escherichia coli
of
Schistosoma mansoni
fatty acid-binding protein 14 (Sm14) in fusion with the nontoxic, but highly immunogenic, tetanus toxin fragment C (TTFC). The recombinant six-His-tagged proteins were purified by nickel affinity chromatography and used in immunization and challenge assays. Animals inoculated with TTFC in fusion with or coadministered with Sm14 showed high levels of tetanus toxin antibodies, while animals inoculated with Sm14 in fusion with or coadministered with TTFC showed high levels of Sm14 antibodies. In both cases, there were no changes in the type of immune response (Th2) obtained with the fusion proteins compared to those obtained with the nonfused proteins. Mice immunized with the recombinant proteins (TTFC in fusion with or coadministered with Sm14) survived the challenge with tetanus toxin and did not show any symptoms of the disease. Control animals inoculated with either phosphate-buffered saline (PBS) or Sm14 died with severe symptoms of tetanus after 24 h. Mice immunized with the recombinant proteins (Sm14 in fusion with or coadministered with TTFC) showed a 50% reduction in worm burden when they were challenged with
S. mansoni
cercariae, while control animals inoculated with either PBS or TTFC were not protected. The results show that the expression of other antigens in fusion at the carboxy terminus of TTFC is feasible for the development of a multivalent recombinant vaccine.
Experimental Assessment of the Roles of Linear Plasmids lp25 and lp28-1 of
Borrelia burgdorferi
throughout the Infectious Cycle
Authors: Grimm Dorothee , Eggers Christian H. , Caimano Melissa J. , Tilly Kit , Stewart Philip E. , Elias Abdallah F. , Radolf Justin D. , Rosa Patricia A.
Published: September 2004

Borrelia burgdorferi
, which causes Lyme disease in humans, has an unusual genome composed of a linear chromosome and up to 21 extrachromosomal elements. Experimental data suggest that two of these elements, linear plasmids lp25 and lp28-1, play essential roles for infectivity in mice. In this study, we prove the essential natures of these two plasmids by selectively displacing lp25 or lp28-1 in an infectious wild-type clone with incompatible shuttle vectors derived from the native plasmids, rendering the respective transformants noninfectious to mice. Conversely, restoration of plasmid lp25 or lp28-1 in noninfectious clones that naturally lack the corresponding plasmid reestablished infectivity in mice. This approach establishes the ability to manipulate the plasmid content of strains by eliminating or introducing entire plasmids in
B. burgdorferi
and will be valuable in assessing the roles of plasmids even in unsequenced
B. burgdorferi
strains.
Distribution of Surface Protein Variants among Hyperinvasive Meningococci: Implications for Vaccine Design
Authors: Urwin Rachel , Russell Joanne E. , Thompson Emily A. L. , Holmes Edward C. , Feavers Ian M. , Maiden Martin C. J.
Published: September 2004

The bacterium
Neisseria meningitidis
is a major cause of meningitis and septicemia worldwide. Outer membrane proteins (OMPs) are candidates in the search for comprehensive meningococcal vaccines; however, the formulation of OMP vaccines is complicated by antigenic diversity, which is generated by high levels of genetic reassortment and strong positive selection in the meningococcal antigen genes. The genetic and antigenic diversity of three OMPs (FetA, PorA, and PorB) among a global collection of meningococcal isolates representative of the major hyperinvasive clonal complexes was determined. There was evidence for antigenic structuring among the three OMPs that could not be explained purely by descent. These observations violated the predictions of the clonal and epidemic clonal models of population structure but were in concordance with models of strain structure which propose that host immunity selects for nonoverlapping antigen combinations. The patterns of antigenic variant combinations suggested that an OMP-based vaccine with as few as six PorA and five FetA variant sequences could generate homologous immune responses against all 78 isolates examined.
Magnetic Resonance Imaging of Pulmonary Lesions in Guinea Pigs Infected withMycobacterium tuberculosis
Authors: Kraft Susan L. , Dailey Deanna , Kovach Matthew , Stasiak Karen L. , Bennett Jamie , McFarland Christine T. , McMurray David N. , Izzo Angelo A. , Orme Ian M. , Basaraba Randall J.
Published: September 2004

We utilized magnetic resonance imaging to visualize lesions in the lungs of guinea pigs infected by low-dose aerosol exposure toMycobacterium tuberculosis. Lesions were prominent in such images, and colorized three-dimensional reconstructions of images revealed a very uniform distribution in the lungs. Lesion numbers after 1 month were approximately similar to the aerosol exposure algorithm, suggesting that each was established by a single bacterium. Numbers of lesions in unprotected and vaccinated animals were similar over the first month but increased thereafter in the control animals, indicating secondary lesion development. Whereas lesion sizes increased progressively in control guinea pigs, lesions remained small in BCG-vaccinated animals. A prominent feature of the disease pathology in unprotected animals was rapid and severe lymphadenopathy of the mediastinal lymph node cluster, which is paradoxical given the strong state of cellular immunity at this time. Further development of this technical approach could be very useful in tracking lesion size, number, and progression in the search for new tuberculosis vaccines.
Legionella pneumophilaDotU and IcmF Are Required for Stability of the Dot/Icm Complex
Authors: Sexton Jessica A. , Miller Jennifer L. , Yoneda Aki , Kehl-Fie Thomas E. , Vogel Joseph P.
Published: September 2004

Legionella pneumophilautilizes a type IV secretion system (T4SS) encoded by 26dot/icmgenes to replicate inside host cells and cause disease. In contrast to all otherL. pneumophila dot/icmgenes,dotUandicmFhave homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead,dotUandicmForthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here thatdotUis partially required forL. pneumophilaintracellular growth, similar to the known requirement foricmF. In addition, we show thatdotUandicmFare necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at whichL. pneumophilabecomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ΔdotUΔicmFstrain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ΔdotUΔicmFmutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize theL. pneumophilaT4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.
The Pathogenicity Island-Associated K15 Capsule Determinant Exhibits a Novel Genetic Structure and Correlates with Virulence in Uropathogenic
Escherichia coli
Strain 536
Authors: Schneider György , Dobrindt Ulrich , Brüggemann Holger , Nagy Gábor , Janke Britta , Blum-Oehler Gabriele , Buchrieser Carmen , Gottschalk Gerhard , Emödy Levente , Hacker Jörg
Published: September 2004

The K15 capsule determinant of uropathogenic
Escherichia coli
strain 536 (O6:K15:H31) is part of a novel 79.6-kb pathogenicity island (PAI) designated PAI V
536
that is absent from the genome of nonpathogenic
E. coli
K-12 strain MG1655. PAI V
536
shows typical characteristics of a composite PAI that is associated with the
pheV
tRNA gene and contains the
pix
fimbriae determinant as well as genes coding for a putative phosphoglycerate transport system, an autotransporter protein, and hypothetical open reading frames. A gene cluster coding for a putative general secretion pathway system, together with a
kps
K15
determinant, is localized downstream of a truncated
pheV
gene (
′pheV
) also present in this chromosomal region. The distribution of genes present on PAI V
536
was studied by PCR in different pathogenic and nonpathogenic
E. coli
isolates of various sources. Analysis of the 20-kb
kps
locus revealed a so far unknown genetic organization. Generally, the
kps
K15
gene cluster resembles that of group 2 and 3 capsules, where two conserved regions (regions 1 and 3) are located up- or downstream of a highly variable serotype-specific region (region 2). Interestingly, recombination of a group 2 and 3 determinant may have been involved in the evolution of the K15 capsule-encoding gene cluster. Expression of the K15 capsule is important for virulence in a murine model of ascending urinary tract infection but not for serum resistance of
E. coli
strain 536.
Enhanced Lung Injury and Delayed Clearance ofPneumocystis cariniiin Surfactant Protein A-Deficient Mice: Attenuation of Cytokine Responses and Reactive Oxygen-Nitrogen Species
Authors: Atochina Elena N. , Beck James M. , Preston Angela M. , Haczku Angela , Tomer Yaniv , Scanlon Seth T. , Fusaro Trevor , Casey John , Hawgood Samuel , Gow Andrew J. , Beers Michael F.
Published: September 2004

Surfactant protein A (SP-A), a member of the collectin family, selectively binds toPneumocystis cariniiand mediates interactions between pathogen and host alveolar macrophages in vitro. To test the hypothesis that mice lacking SP-A have delayed clearance ofPneumocystisorganisms and enhanced lung injury, wild-type C57BL/6 (WT) and SP-A-deficient mice (SP-A−/−) with or without selective CD4+-T-cell depletion were intratracheally inoculated withPneumocystisorganisms. Four weeks later, CD4-depleted SP-A-deficient mice had developed a more severePneumocystisinfection than CD4-depleted WT (P. cariniipneumonia [PCP] scores of 3 versus 2, respectively). Whereas all non-CD4-depleted WT mice were free of PCP, intact SP-A−/−mice also had evidence of increased organism burden.Pneumocystisinfection in SP-A-deficient mice was associated histologically with enhanced peribronchial and/or perivascular cellularity (score of 4 versus 2, SP-A−/−versus C57BL/6 mice, respectively) and a corresponding increase in bronchoalveolar lavage (BAL) cell counts. Increases in SP-D content, gamma interferon, interleukin-4, interleukin-5, and tumor necrosis factor alpha in BAL fluid occurred but were attenuated in PCP-infected SP-A−/−mice compared to WT mice. There were increases in total BAL NO levels in both infected groups, but nitrite levels were higher in SP-A−/−mice, indicating a reduction in production of higher oxides of nitrogen that was also reflected in lower levels of 3-nitrotyrosine staining in the SP-A−/−group. We conclude that despite increases in inflammatory cells, SP-A-deficient mice infected withP. cariniiexhibit an enhanced susceptibility to the organism and attenuated production of proinflammatory cytokines and reactive oxygen-nitrogen species. These data support the concept that SP-A is a local effector molecule in the lung host defense againstP. cariniiin vivo.
A
Vibrio cholerae
Classical TcpA Amino Acid Sequence Induces Protective Antibody That Binds an Area Hypothesized To Be Important for Toxin-Coregulated Pilus Structure
Authors: Taylor Ronald K. , Kirn Thomas J. , Meeks Michael D. , Wade Terri K. , Wade William F.
Published: September 2004

Vibrio cholerae
is a gram-negative bacterium that has been associated with cholera pandemics since the early 1800s. Whole-cell, killed, and live-attenuated oral cholera vaccines are in use. We and others have focused on the development of a subunit cholera vaccine that features standardized epitopes from various
V. cholerae
macromolecules that are known to induce protective antibody responses. TcpA protein is assembled into toxin-coregulated pilus (TCP), a type IVb pilus required for
V. cholerae
colonization, and thus is a strong candidate for a cholera subunit vaccine. Polypeptides (24 to 26 amino acids) in TcpA that can induce protective antibody responses have been reported, but further characterization of their amino acid targets relative to tertiary or quaternary TCP structures has not been done. We report a refinement of the TcpA sequences that can induce protective antibody. One sequence, TcpA 15 (residues 170 to 183), induces antibodies that bind linear TcpA in a Western blot as well as weakly bind soluble TcpA in solution. These antibodies bind assembled pili at high density and provide 80 to 100% protection in the infant mouse protection assay. This is in sharp contrast to other anti-TcpA peptide sera (TcpA 11, TcpA 13, and TcpA 17) that bind very strongly in Western blot and solution assays yet do not provide protection or effectively bind TCP, as evidenced by immunoelectron microscopy. The sequences of TcpA 15 that induce protective antibody were localized on a model of assembled TCP. These sequences are centered on a site that is predicted to be important for TCP structure.
Cryptosporidium parvumat Different Developmental Stages Modulates Host Cell Apoptosis In Vitro
Authors: Mele Raffaella , Gomez Morales Maria Angeles , Tosini Fabio , Pozio Edoardo
Published: September 2004

We studied apoptosis in a human ileocecal adenocarcinoma tumor cell line (HCT-8) infected withCryptosporidium parvum, from 2 to 72 h postinfection (h.p.i.). At 2 h.p.i., the percentage of annexin V-positive cells in the cell culture had increased to 10% compared to 2.5% in noninfected control culture; sorted infected cells expressed mRNA of FasL, the active form of caspase 3, and high caspase 3 activity, whereas the noninfected neighboring cells sorted from the same culture showed no signs of apoptosis. At 24 h.p.i., the percentages of early (annexin V positive) and late (DNA fragment) apoptotic cells were 13 and 2%, respectively, in the entire cell culture, and these percentages were not statistically significant in comparison with those from noninfected control cultures. At this time, sorted infected cells expressed the inactive form of caspase 3, a low caspase 3 activity, and the antiapoptotic protein Bcl-2. Noninfected cells sorted from the same culture showed expression of the active form of caspase 3, a moderate caspase 3 activity, and no Bcl-2 expression. At 48 h.p.i., the percentages of early and late apoptotic cells and caspase 3 activity had increased in the total cell culture, and both sorted infected and noninfected cells showed the active form of caspase 3. These results show thatC. parvum, depending on its developmental stage, can inhibit (at the trophozoite stage) or promote (at the sporozoite and merozoite stages) host cell apoptosis, suggesting that it is able to interact with and regulate the host-cell gene expression.
Expression Profiling Reveals Novel Innate and Inflammatory Responses in the Jejunal Epithelial Compartment during Infection withTrichinella spiralis
Authors: Knight Pamela A. , Pemberton Alan D. , Robertson Kevin A. , Roy Douglas J. , Wright Steven H. , Miller Hugh R. P.
Published: September 2004

Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematodeTrichinella spiralisin jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected andT. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule β (RELMβ) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmβ and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmβ).
Correct Promoter Control Is Needed for Trafficking of the Ring-Infected Erythrocyte Surface Antigen to the Host Cytosol in Transfected Malaria Parasites
Authors: Rug Melanie , Wickham Mark E. , Foley Michael , Cowman Alan F. , Tilley Leann
Published: September 2004

Following invasion of human erythrocytes, the malaria parasite,
Plasmodium falciparum
, exports proteins beyond the confines of its own plasma membrane to modify the properties of the host red cell membrane. These modifications are critical to the pathogenesis of malaria. Analysis of the
P. falciparum
genome sequence has identified a large number of molecules with putative atypical signal sequences. The signals remain poorly characterized; however, a number of molecules with these motifs localize to the host erythrocyte. To examine the role of these atypical signal sequences in the export of parasite proteins, we have generated transfected parasites expressing a chimeric protein comprising the N-terminal region of the
P. falciparum
ring-infected erythrocyte surface antigen (RESA) appended to green fluorescent protein (GFP). This N-terminal region contains a hydrophobic stretch of amino acids that is presumed to act as a noncanonical secretory signal sequence. Modulation of the timing of transgene expression demonstrates that trafficking of malaria proteins into the host erythrocyte is dependant on both the presence of an appropriate transport signal and the timing of expression. Transgene expression under the control of a trophozoite-specific promoter mistargets the chimeric molecule to the parasitophorous vacuole surrounding the parasite. However, expression of RESA-GFP in schizont stages, under the control of the
RESA
promoter, enables correct trafficking of a population of the chimeric protein to the host erythrocyte.
Sigma Factor B and RsbU Are Required for Virulence in
Staphylococcus aureus
-Induced Arthritis and Sepsis
Authors: Jonsson Ing-Marie , Arvidson Staffan , Foster Simon , Tarkowski Andrzej
Published: September 2004

The prototype
Staphylococcus aureus
strain 8325-4 produces high levels of hemolysins and proteases. Recently it has been shown that this property depends on a deficiency of sigma factor B (SigB) activity controlling the activation of regulatory genes such as
agr
and
sarA
. SigB deficiency is in turn due to a mutation in the
rsbU
gene, which is required for posttranslational activation of SigB. The
rsbU
defect of strain 8325-4 has recently been repaired, and we used this strain (SH1000), along with its isogenic
sigB
-negative mutant, to investigate the contributions of RsbU and SigB in a murine model of septic arthritis. Intravenous inoculation with the
rsbU
-repaired isogenic strain SH1000 resulted in significantly more severe arthritis, weight decrease, and mortality compared to those of the parental strain 8325-4 (
rsbU
-negative) or the isogenic
sigB
-negative mutant (MJH502). SH1000 also persisted more in kidneys and joints of infected mice. Our data strongly suggest that RsbU and SigB regulate important virulence factors, thereby contributing significantly to the outcome of staphylococcal infection.
Nucleic Acid Vaccination withSchistosoma mansoniAntioxidant Enzyme Cytosolic Superoxide Dismutase and the Structural Protein Filamin Confers Protection against the Adult Worm Stage
Authors: Cook Rosemary M. , Carvalho-Queiroz Claudia , Wilding Gregory , LoVerde Philip T.
Published: September 2004

Schistosomiasis remains a worldwide endemic cause of chronic and debilitating illness. There are two paradigms that exist in schistosome immunology. The first is that the schistosomule stages are the most susceptible to immune killing, and the second is that the adult stage, through evolution of defense mechanisms, can survive in the hostile host environment. One mechanism that seems to aid the adult worm in evading immune killing is the expression of antioxidant enzymes to neutralize the effects of reactive oxygen and nitrogen species. Here, we challenge one paradigm by targeting adultSchistosoma mansoniworms for immune elimination in an experimental mouse model using twoS. mansoniantioxidants, cytosolic superoxide dismutase (SmCT-SOD) and glutathione peroxidase (SmGPX), and a partial coding sequence for a structural protein, filamin, as DNA vaccine candidates. DNA vaccination with SmCT-SOD induced a mean of 39% protection, filamin induced a mean of 50% protection, and SmGPX induced no protection compared to controls following challenge with adult worms by surgical transfer. B- and T-cell responses were analyzed in an attempt to define the protective immune mechanism(s) involved in adult worm killing. SmCT-SOD-immunized mice presented with a T1 response, and filamin-immunized mice showed a mixed T1-T2 response. We provide evidence for natural boosting after vaccination. Our results demonstrate that adult worms can be targeted for immune elimination through vaccination. This represents an advance in schistosome vaccinology and allows for the development of a therapeutic as well as a prophylactic vaccine.
Host Cell Tropism Underlies Species Restriction of Human and Bovine
Cryptosporidium parvum
Genotypes
Authors: Hashim Amna , Clyne Marguerite , Mulcahy Grace , Akiyoshi Donna , Chalmers Rachel , Bourke Billy
Published: September 2004

It has been recognized recently that human cryptosporidiosis is usually caused by
Cryptosporidium parvum
genotype I (“human”
C. parvum
), which is not found in animals. Compared to
C. parvum
genotype II, little is known of the biology of invasion of the human-restricted
C. parvum
genotype I. The aims of the present study were (i) to explore and compare with genotype II the pathogenesis of
C. parvum
genotype I infection by using an established in vitro model of infection and (ii) to examine the possibility that host-specific cell tropism determines species restriction among
C. parvum
genotypes by using a novel ex vivo small intestinal primary cell model of infection. Oocysts of
C. parvum
genotypes I and II were used to infect HCT-8 cells and primary intestinal epithelial cells in vitro. Primary cells were harvested from human endoscopic small-bowel biopsies and from bovine duodenum postmortem.
C. parvum
genotype I infected HCT-8 cells with lower efficiency than
C. parvum
genotype II. Actin colocalization at the host parasite interface and reduction in levels of invasion after treatment with microfilament inhibitors (cytochalasin B and cytochalasin D) were observed for both genotypes.
C. parvum
genotype II invaded primary intestinal epithelial cells, regardless of the species of origin. In contrast,
C. parvum
genotype I invaded only human small-bowel cells. The pathogenesis of
C. parvum
genotype I differs from
C. parvum
genotype II.
C parvum
genotype I does not enter primary bovine intestinal cells, suggesting that the species restriction of this genotype is due to host tissue tropism of the infecting isolate.
A Calcium-Calmodulin Antagonist Blocks Experimental
Vibrio vulnificus
Cytolysin-Induced Lethality in an Experimental Mouse Model
Authors: Lee Young-Rae , Park Kwang-Hyun , Lin Zhao-Zhen , Kho Young-Jong , Park Jin-Woo , Rho Hye-Won , Koo Bon-Sun , Kim Hyung-Rho , Song Eun-Kyung , Yu Hong-Nu , Han Myung-Kwan , Lee Seung-Ok , Jhee Eun-Chung , Kim Jong-Suk
Published: September 2004

We demonstrated that trifluoperazine, a calcium-calmodulin antagonist, blocked the hyperpermeability induced by
Vibrio vulnificus
cytolysin in in vitro-modeled endothelium and prevented the deaths of mice. Furthermore, compared to tetracycline alone, tetracycline combined with trifluoperazine enhanced the survival rate of
V. vulnificus
-infected mice, indicating the role of the cytolysin as an important factor in pathogenesis.
The Extracellular Adherence Protein fromStaphylococcus aureusInhibits Neutrophil Binding to Endothelial Cells
Authors: Haggar Axana , Ehrnfelt Cecilia , Holgersson Jan , Flock Jan-Ingmar
Published: September 2004

Extracellular adherence protein (Eap) fromStaphylococcus aureusinhibits the adherence of neutrophils to nonstimulated and tumor necrosis factor alpha-stimulated endothelial cells in both static adhesion assays and flow adhesion assays. Consequently, Eap also impaired their transendothelial migration. During anS. aureusinfection, Eap may thus serve to reduce inflammation by inhibiting neutrophil adhesion and extravasation.
Limited Role for Interleukin-18 in the Host Protection Response to Pulmonary Infection with
Pseudomonas aeruginosa
in Mice
Authors: Nakasone Chikara , Kawakami Kazuyoshi , Hoshino Tomoaki , Kawase Yusuke , Yokota Koichi , Yoshino Kohichiro , Takeda Kiyoshi , Akira Shizuo , Saito Atsushi
Published: September 2004

We report that clearance of
Pseudomonas aeruginosa
, accumulation of neutrophils, and synthesis of tumor necrosis factor alpha and macrophage inflammatory protein 2 in the infected lung were not largely different in interleukin-18 (IL-18) knockout or transgenic mice compared with control mice. Our results suggest a limited role for IL-18 in the host defense against
P. aeruginosa
.
The Fine Specificity, but Not the Invasion Inhibitory Activity, of 19-Kilodalton Merozoite Surface Protein 1-Specific Antibodies Is Associated with Resistance to Malarial Parasitemia in a Cross-Sectional Survey in The Gambia
Authors: Corran Patrick H. , O'Donnell Rebecca A. , Todd Jim , Uthaipibull Chairat , Holder Anthony A. , Crabb Brendan S. , Riley Eleanor M.
Published: September 2004

In a cross-sectional survey of 187 Gambian children and adults, we have analyzed prevalence, fine specificity, and 19-kilodalton merozoite surface protein 1 (MSP-119)-specific erythrocyte invasion inhibitory activity of antibodies to MSP-119but find no significant association between any of these parameters and prevalence or density of malarial parasitemia, except that, after correcting for total anti-MSP-119antibody levels, individuals with anti-MSP-119antibodies that compete with an invasion inhibitory monoclonal antibody (12.10) were significantly less likely to have malaria infections with densities of ≥1,000 parasites/μl than were individuals without such antibodies. This association persisted after correction for age and ethnic origin.
ORAL MICROVASCULAR NETWORK GEOMETRY ABNORMALITIES IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS
Authors: PARRINI STEFANO , BIANCIARDI GIORGIO , LATINI GIUSEPPE , DI MAGGIO GIOVANNI , DE FELICE CLAUDIO
Published: September 2004

Infantile hypertrophic pyloric stenosis (IHPS) is a common surgical condition of unknown etiology. The oral mucosal vascular networks of IHPS patients (n=25) and their unaffected parents showed lower blood vessel-free areas, as well as higher box-counting dimensions at two box size scales [D(1–46), D(1–15)], and relative Lempel-Ziv values (P<0.000001), as compared to those of gender- and age-matched controls. These findings may provide a useful phenotypical marker for identifying couples potentially at risk for the birth of an affected infant, while supporting the importance of a genetic component in this condition.
Corticosterone-dependent metabolic and neuroendocrine abnormalities in obese Zucker rats in relation to feeding
Authors: Duclos Martine , Timofeeva Elena , Michel Chantal , Richard Denis
Published: September 2004

The obese Zucker ( fa/fa) rat is characterized by hyperphagia, hyperinsulinemia, an increase in fat deposition, and a hyperactivity in the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis in fa/fa rats is hypersensitive to stressful experimental conditions. Food deprivation even leads to a stress reaction in obese fa/fa rats. The present study was conducted to investigate the role of corticosterone in obese rats on the basal, fasting, and postprandial metabolic rate as well as on the central expression of the thyrotropin-releasing hormone (TRH) in these conditions. In addition, the study was aimed at clarifying whether the high levels of corticosterone in obese rats are responsible for the induction of the stress reaction to food deprivation in these animals. The present results demonstrate that whole body fat oxidation and postprandial metabolic responses in obese Zucker rats were improved by adrenalectomy (ADX). At the level of the central nervous system, ADX reversed a decrease in TRH mRNA expression in the paraventricular hypothalamus (PVH) detected in fasting animals. Considering all feeding conditions, the obese rats demonstrated lower TRH mRNA levels compared with lean animals. ADX resulted in an enhanced postprandial activation of the parvocellular PVH. In contrast, the magnocellular part of the PVH was less responsive to refeeding in ADX animals. Finally, ADX failed to prevent the stress response of obese rats to food deprivation. The present results provide evidence that the removal of adrenals resolve some of the metabolic defects encountered in obese Zucker rats. They also demonstrate that not all the abnormalities of the obese Zucker rats are attributable to the hyperactivity of the HPA axis.
Acute IL-6 treatment increases fatty acid turnover in elderly humans in vivo and in tissue culture in vitro
Authors: Petersen E. W. , Carey A. L. , Sacchetti M. , Steinberg G. R. , Macaulay S. L. , Febbraio M. A. , Pedersen B. K.
Published: September 2004

To determine whether IL-6 increases lipolysis and fat oxidation in patients with type 2 diabetes and/or whether it exerts this effect independently of changes to the hormonal milieu, patients with type 2 diabetes (D) and healthy control subjects (CON) underwent recombinant human (rh)IL-6 infusion for 3 h. Rates of appearance (Ra) and disappearance (Rd) of [U-13C]palmitate and [6,6-2H2]glucose were determined. rhIL-6 infusion increased ( P < 0.05) palmitate Ra and Rd in a similar fashion in both groups. Neither plasma glucose concentration nor glucose Ra/Rd was affected by rhIL-6 infusion in either group, whereas rhIL-6 infusion resulted in a reduction ( P < 0.05) in circulating insulin in D. Plasma growth hormone (GH) was increased ( P < 0.05) by IL-6 in CON, and cortisol increased ( P < 0.05) in response to IL-6 in both groups. To determine whether IL-6 was exerting its effect directly or through activation of these hormones, we performed cell culture experiments. Fully differentiated 3T3-L1 adipocytes were treated with PBS (control) IL-6, or IL-6 plus dexamethasone and GH. IL-6 treatment alone increased ( P < 0.05) lipolysis, but this effect was reduced by the addition of dexamethasone and GH such that IL-6 plus dexamethasone and GH had blunted ( P < 0.05) lipolysis compared with IL-6 alone. To assess whether IL-6 increases fat oxidation, L6 myotubes were treated with PBS (Control), IL-6, or AICAR, a compound known to increase lipid oxidation. Both IL-6 and AICAR markedly increased ( P < 0.05) oxidation of [14C]palmitate compared with Control. Acute IL-6 treatment increased fatty acid turnover in D patients as well as healthy CON subjects. Moreover, IL-6 appears to be activating lipolysis independently of elevations in GH and/or cortisol and appears to be a potent catalyst for fat oxidation in muscle cells.
Bombesin and nutrients independently and additively regulate hormone release from GIP/Ins cells
Authors: Li Lin , Wice Burton M.
Published: September 2004

Glucose-dependent insulinotropic polypeptide (GIP) regulates glucose homeostasis and high-fat diet-induced obesity and insulin resistance. Therefore, elucidating the mechanisms that regulate GIP release is important. GIP is produced by K cells, a specific subtype of small intestinal enteroendocrine (EE) cell. Bombesin-like peptides produced by enteric neurons and luminal nutrients stimulate GIP release in vivo. We previously showed that PMA, bombesin, meat hydrolysate, glyceraldehyde, and methylpyruvate increase hormone release from a GIP-producing EE cell line (GIP/Ins cells). Here we demonstrate that bombesin and nutrients additively stimulate hormone release from GIP/Ins cells. In various cell systems, bombesin and PMA regulate cell physiology by activating PKD signaling in a PKC-dependent fashion, whereas nutrients regulate cell physiology by inhibiting AMPK signaling. Western blot analyses of GIP/Ins cells using antibodies specific for activated and/or phosphorylated forms of PKD and AMPK and one substrate for each kinase revealed that bombesin and PMA, but not nutrients, activated PKC, but not PKD. Conversely, nutrients, but not bombesin or PMA, inhibited AMPK activity. Pharmacological studies showed that PKC inhibition blocked bombesin- and PMA-stimulated hormone release, but AMPK activation failed to suppress nutrient-stimulated hormone secretion. Forced expression of constitutively active vs. dominant negative PKDs or AMPKs failed to perturb bombesin- or nutrient-stimulated hormone release. Thus, in GIP/Ins cells, PKC regulates bombesin-stimulated hormone release, whereas nutrients may control hormone release by regulating the activity of AMPK-related kinases, rather than AMPK itself. These results strongly suggest that K cells in vivo independently respond to neuronal vs. nutritional stimuli via two distinct signaling pathways.
Alterations in lung mechanics in decorin-deficient mice
Authors: Fust Anita , LeBellego Frederique , Iozzo Renato V. , Roughley Peter J. , Ludwig Mara S.
Published: September 2004

Decorin, a small leucine-rich proteoglycan with a widespread tissue distribution, is required for the normal fibrillogenesis of collagen in most tissues. Because collagen is important in determining the elastic behavior of the lung, we hypothesized that lung tissue mechanics would be altered in a mutant mouse in which the single decorin gene was abrogated by targeted deletion ( Dcn−/−). Complex impedance of the respiratory system was measured in C57Bl/6 mice ( Dcn−/− and Dcn+/+) using a small animal ventilator that delivers a volume signal with multiple frequencies to the trachea. A constant-phase model was fit to calculate airway resistance (Raw), tissue damping, and tissue elastance. Compliance of the respiratory system (Crs) was measured from a pressure volume curve during stepwise deflations. Lungs were excised, and parenchymal tissue strips were mounted in an organ bath for in vitro measurement of tissue impedance and quasistatic length-stress curves. In addition, pulmonary tissue was examined by immunohistochemistry and immunoblotting. In vivo, in the Dcn−/− mice, Raw was decreased and Crs was increased. Similarly, in vitro, length-stress curves showed increased compliance of the strips in the Dcn−/− mice. These alterations in lung tissue mechanical behavior in Dcn−/− mice support a critical role for decorin in the formation of the lung collagen network.
Renal epoxyeicosatrienoic acid synthesis during pregnancy
Authors: Zhou Yiqiang , Chang Hsin-Hsin , Du Juan , Wang Cong-Yi , Dong Zheng , Wang Mong-Heng
Published: September 2004

Epoxyeicosatrienoic acids (EETs), which belong to cytochrome P-450 (CYP)-derived eicosanoids, have been implicated to vasodilate renal arterioles, inhibit sodium transport in the nephron, and regulate blood pressure in several animal models. Because pregnancy is associated with changes of blood pressure, the aim of this study was to examine whether renal EET synthesis is altered and whether EETs are involved in blood pressure regulation during pregnancy in rats. Renal microsomal epoxygenase activity increased by 47, 97, and 63% on days 6, 12, and 19 of gestation, respectively. The elevation of epoxygenase activity during pregnancy was associated with an increase in CYP2C11, CYP2C23, and CYP2J2 protein expression on days 6, 12, and 19 of gestation. Moreover, immunohistochemical analysis showed that renal tubular CYP2C11, CYP2C23, and CYP2J2 expression was significantly increased in pregnant rats on days 6, 12, and 19 of gestation. Administration of 6-(2-propargyloxyphenyl)hexanoic acid (PPOH), a selective epoxygenase inhibitor, caused a dose-dependent inhibition of microsomal expoxygenase activity without a significant effect on ω-hydroxylase activity in female rats. Interestingly, administration of PPOH (20 mg·kg−1·day−1for 4 days starting on day 15 of pregnancy) increased blood pressure by 21 mmHg and caused a significant decrease in the body weight of fetal pups (1.3 ± 0.08 g in control vs. 1.1 ± 0.06 g in PPOH). Moreover, PPOH treatment significantly decreased renal microsomal epoxygenase activity and the expression of CYP2C11, CYP2C23, and CYP2J in pregnant rats. This study demonstrates that EET synthesis in the kidney is elevated during pregnancy, and CYP2C11, 2C23, and CYP2J2 are responsible for the change of renal EET synthesis. The inhibition results demonstrate that the downregulation of renal epoxygenase activity by PPOH causes hypertension in pregnant rats. This study suggests that EETs may contribute to the control of blood pressure during pregnancy.