bluuebunny/crossref_metadata_2025 huggingface dataset:
Transfection ofTrypanosoma cruziwith Host CD40 Ligand Results in Improved Control of Parasite Infection
Authors: Chamekh Mustapha , Vercruysse Vincent , Habib Mohammed , Lorent Maxime , Goldman Michel , Allaoui Abdelmounaïm , Vray Bernard
Published: September 2005

We have previously shown that infection byTrypanosoma cruzi, a parasitic protozoan, is reduced by injection of CD40 ligand (CD40L)-transfected 3T3 fibroblasts (D. Chaussabel, F. Jacobs, J. de Jonge, M. de Veerman, Y. Carlier, K. Thielemans, M. Goldman, and B. Vray, Infect. Immun.67:1929-1934, 1999). This prompted us to transfectT. cruziwith the murine CD40L gene and to study the consequences of this transfection on the course of infection. For this, epimastigotes (Y strain) were electroporated with the pTEX vector alone or the pTEX-CD40L construct, and transfected cells were selected for their resistance to Geneticin G418. Then strain Y-, pTEX-, and pTEX-CD40L-transfected epimastigotes were transformed by metacyclogenesis into mammalian infective forms called Y, YpTEX, and YpTEX-CD40L trypomastigotes. Transfection of the CD40L gene and expression of the CD40L protein were assessed by reverse transcription-PCR and Western blot analysis. The three strains of parasites were infective in vitro for mouse peritoneal macrophages. When organisms were inoculated into mice, a very low level of parasitemia and no mortality were seen with the YpTEX-CD40L strain compared to the Y and YpTEX strains. Furthermore, the proliferative capacity and the secretion of gamma interferon were both preserved in spleen cells (SCs) from YpTEX-CD40L-infected mice but not with SCs from Y- and YpTEX-infected mice. These results suggest that the CD40L produced by transfectedT. cruziis involved in the modulation of an antiparasite immune response. Moreover, mice surviving YpTEX-CD40L infection resisted a challenge infection with the wild-type strain. Taken together, our data demonstrate the feasibility of generating aT. cruzistrain expressing a bioactive host costimulatory molecule that counteracts the immunodeficiency induced by the parasite during infection and enhances protective immunity against a challenge infection.
Role of NADase in Virulence in Experimental Invasive Group A Streptococcal Infection
Authors: Bricker Angela L. , Carey Vincent J. , Wessels Michael R.
Published: September 2005

Group A streptococci (GAS) produce several exoproteins that are thought to contribute to the pathogenesis of human infection. Two such proteins, streptolysin O (SLO) and NAD
+
-glycohydrolase (NADase), have been shown to interact functionally as a compound signaling toxin. When GAS are bound to the surface of epithelial cells in vitro, SLO forms pores in the cell membrane and delivers NADase to the epithelial cell cytoplasm. In vitro, intoxication of keratinocytes with NADase is associated with cytotoxic effects and induction of apoptosis; however, the importance of NADase during infection of an animal host has not been established. We employed isogenic GAS mutants to assess the contribution of NADase activity to GAS virulence in vivo using mouse models of invasive soft-tissue infection and septicemia. In both models, mutant GAS that lacked NADase activity were significantly attenuated for virulence compared with the isogenic wild-type parent, confirming an important role for NADase in the infection of a host animal. A double mutant lacking SLO and NADase activity had an intermediate virulence phenotype, consistent with the hypothesis that SLO evokes a protective innate immune response. We conclude that NADase and SLO together enhance GAS virulence in vivo.
Markers for Host-Induced Gene Expression inTrichophytonDermatophytosis
Authors: Kaufman Gil , Berdicevsky Israela , Woodfolk Judith A. , Horwitz Benjamin A.
Published: September 2005

Dermatophytes are adapted to infect keratinized tissues by their ability to utilize keratin as a nutrient source. Although there have been numerous reports that dermatophytes likeTrichophytonsp. secrete proteolytic enzymes, virtually nothing is known about the patterns of gene expression in the host or even when the organisms are cultured on protein substrates in the absence of a host. We characterized the expression of an aminopeptidase gene, theTrichophyton mentagrophyteshomolog of theTrichophyton rubrumTri r 4 gene. TheT. rubrumgene was originally isolated based on the ability of the protein encoded by it to induce immediate and delayed-type hypersensitivity in skin tests.T. mentagrophytesTri m 4 is closely related to Tri r 4 (almost 94% identity at the protein level). Tri m 4 resembles other protease-encoding genes thought to be virulence factors (for example, DPP V ofAspergillus fumigatus). The Tri m 4 protein was detected immunochemically both in fungal extracts and in the culture medium. Expression of the Tri m 4 gene was induced severalfold whenT. mentagrophyteswas grown on keratin and elastin. Ex vivo, strong induction was observed after culture on blood plasma, but the use of homogenized skin did not result in a significant increase in Tri m 4 transcript levels. In order to identify additional genes encoding putative virulence factors, differential cDNA screening was performed. By this method, a fungal thioredoxin and a cellulase homolog were identified, and both genes were found to be strongly induced by skin extracellular matrix proteins. Induction by superficial (keratin) and deep (elastin) skin elements suggests that the products of these genes may be important in both superficial and deep dermatophytosis, and models for their function are proposed. Upregulation of several newly identifiedT. mentagrophytesgenes on protein substrates suggests that these genes encode proteins which are relevant to the dermatophyte-skin interaction.
Identification of Immunogenic and Serum Binding Proteins of
Staphylococcus epidermidis
Authors: Sellman Bret R. , Howell Alan P. , Kelly-Boyd Cari , Baker Steve M.
Published: September 2005

Staphylococcus epidermidis
is a commensal of human skin and a leading cause of nosocomial bloodstream infections. Limited information is available about
S. epidermidis
proteins that are expressed upon transition to the bloodstream or those involved in host-pathogen interactions. A cell surface fraction from
S. epidermidis
0-47 grown in rabbit serum to mimic environmental signals encountered during a bloodstream infection was separated by two-dimensional (2D) gel electrophoresis. Following 2D separation, the proteins were transferred to nitrocellulose and detected with either pooled sera generated in rabbits immunized with live
S. epidermidis
0-47 or with biotin-labeled serum proteins eluted from the surface of bacteria grown in rabbit serum. Twenty-nine immunoreactive or serum binding proteins of
S. epidermidis
were identified by mass spectrometry. Twenty-seven of the corresponding genes were expressed in
Escherichia coli
, and the purified recombinant proteins were used to immunize mice. In a preliminary screen, 12 of the 27 recombinant proteins induced a response that reduced the number of bacteria recovered from the spleen or bloodstream of infected mice. In subsequent vaccination studies, 5 of the 12 proteins resulted in a statistically significant reduction in the number of bacteria. The identification of five candidate vaccine antigens from the initial screen of only 29 proteins demonstrates the utility of this approach.
Human Antibody Response to Outer Membrane Protein G1a, a Lipoprotein of
Moraxella catarrhalis
Authors: Adlowitz Diana G. , Sethi Sanjay , Cullen Paul , Adler Ben , Murphy Timothy F.
Published: September 2005

Moraxella catarrhalis
is an important cause of respiratory infections in adults with chronic obstructive pulmonary disease (COPD) and of otitis media in children. Outer membrane protein (OMP) G1a is an ∼29-kDa surface lipoprotein and is a potential vaccine candidate. The gene that encodes OMP G1a was expressed and purified using a novel plasmid vector. [
3
H]palmitic acid labeling demonstrated that both native and recombinant OMP G1a contain covalently bound palmitic acid. To assess the expression of OMP G1a during human infection, paired sera and sputum supernatants from adults with COPD followed prospectively were studied by enzyme-linked immunosorbent assays with recombinant lipidated OMP G1a to detect antibodies made specifically during carriage of
M. catarrhalis
. Overall, 23% of patients developed either a serum immunoglobulin G (IgG) response (9%) or sputum IgA response (21%) to OMP G1a, following 100 episodes of acquisition and clearance of
M. catarrhalis
. Patients developed antibody responses at similar rates following episodes of clinical exacerbation compared to asymptomatic colonization. Serum IgG antibodies following natural infection were directed predominantly at OMP G1a epitopes that are not exposed on the bacterial surface. These data show that OMP G1a is expressed during infection of the human respiratory tract and is a target of systemic and mucosal antibodies. These observations indicate that OMP G1a, a highly conserved surface protein, should be evaluated further as a vaccine candidate.
Protection against Hemorrhagic Colitis in an Animal Model by Oral Immunization with Isogeneic Rabbit Enteropathogenic
Escherichia coli
Attenuated by Truncating Intimin
Authors: Agin Tonia S. , Zhu Chengru , Johnson Laura A. , Thate Timothy E. , Yang Zhuolu , Boedeker Edgar C.
Published: September 2005

Strains of Shiga toxin (Stx)-producing
Escherichia coli
, also called enterohemorrhagic
E. coli
(EHEC), are important food-borne pathogens for humans. Most EHEC strains intimately adhere to the intestinal mucosa in a characteristic attaching and effacing (A/E) pattern, which is mediated by the bacterial adhesin intimin. Subsequent release of Stx1 and/or Stx2 leads to the frequent development of hemorrhagic colitis and, less commonly, to hemolytic-uremic syndrome. The aim of the present study was to develop an attenuated A/E
E. coli
strain for use as a vaccine against EHEC infection encoding a truncated intimin lacking adhesive capacity, but which would still express somatic antigens, other products of the locus of enterocyte effacement pathogenicity island, and an immunogenic remnant of the intimin molecule. A single-nucleotide deletion was generated in the
eae
gene in the prototype rabbit A/E
E. coli
strain RDEC-1 (O15:H−), which resulted in truncation of intimin by 81 C-terminal residues (860 to 939 amino acids) containing a disulfide loop. Inoculation of rabbits with large doses of the truncated intimin mutant (RDEC-1Δeae
860-939
) was well tolerated, as observed by the absence of clinical signs of disease or evidence of intestinal A/E lesions. The efficacy of RDEC-1Δeae
860-939
as a vaccine was evaluated by orogastric inoculation of rabbits with RDEC-1Δeae
860-939
followed by challenge with the virulent strain RDEC-H19A, an Stx1-producing derivative of wild-type RDEC-1 capable of inducing hemorrhagic colitis in rabbits. Following RDEC-H19A challenge, nonimmunized control rabbits exhibited characteristic weight loss with watery to bloody diarrhea and demonstrated intimate bacterial attachment, effacement of microvilli, submucosal edema, mucosal heterophile infiltrates, and Shiga toxin-induced vascular lesions. In contrast, the RDEC-1Δeae
860-939
-immunized rabbits showed no clinical signs of disease, maintained normal weight gain, had reduced fecal shedding of challenge organisms, and showed an absence of gross or microscopic lesions in the intestinal mucosa. Serum antibodies specific to intimin were detected among rabbits immunized with RDEC-1Δeae
860-939
, indicating that truncation of the intimin functional domain not only attenuated bacterial virulence, but also retained at least some of the immunogenicity of native intimin. Although it is not possible to gauge the exact contribution of residual intimin immunity to protection, this attenuation strategy for A/E
E. coli
strains shows promise for the development of effective vaccines to prevent EHEC infection in humans and animals.
Expression of the
Rickettsia prowazekii pld
or
tlyC
Gene in
Salmonella enterica
Serovar Typhimurium Mediates Phagosomal Escape
Authors: Whitworth Ted , Popov Vsevolod L. , Yu Xue-Jie , Walker David H. , Bouyer Donald H.
Published: September 2005

Members of the genus
Rickettsia
possess the ability to invade host cells and promptly escape from phagosomal vacuoles into the host cell cytosol, thereby avoiding destruction within the endosomal pathway. The mechanism underlying rickettsial phagosomal escape remains unknown, although the genomic sequences of several rickettsial species have allowed for the identification of four genes with potential membranolytic activities (
tlyA
,
tlyC
,
pat1
, and
pld
). This study was undertaken to determine which of the selected genes of
Rickettsia prowazekii
mediate the escape process. Quantitative ultrastructural analyses indicated that the period of active phagosomal escape was between 30 and 50 min postinfection. Reverse transcriptase PCR analyses determined that
tlyC
and
pld
were transcribed during the period of active phagosomal escape but that
tlyA
and
pat1
were not. The functionality of both
tlyC
and
pld
was determined by complementation studies of
Salmonella
, which replicates within endosomes. Complementation of
Salmonella
organisms with either
tlyC
or
pld
resulted in the escape of transformants from endosomal vacuoles into the host cell cytosol demonstrated by quantitative ultrastructural analyses. These data suggest a role for
tlyC
and
pld
in the process of phagosomal escape by
R. prowazekii
.
Hyperinfectivity of Human-Passaged
Vibrio cholerae
Can Be Modeled by Growth in the Infant Mouse
Authors: Alam Ashfaqul , LaRocque Regina C. , Harris Jason B. , Vanderspurt Cecily , Ryan Edward T. , Qadri Firdausi , Calderwood Stephen B.
Published: September 2005

It has previously been shown that passage of
Vibrio cholerae
through the human intestine imparts a transient hyperinfectious phenotype that may contribute to the epidemic spread of cholera. The mechanism underlying this human-passaged hyperinfectivity is incompletely understood, in part due to inherent difficulties in recovering and studying organisms that are freshly passed in human stool. Here, we demonstrate that passage of
V. cholerae
through the infant mouse intestine leads to an equivalent degree of hyperinfectivity as passage through the human host. We have used this infant mouse model of host-passaged hyperinfectivity to characterize the timing and the anatomic location of the competitive advantage of mouse-passaged
V. cholerae
as well as the contribution of three type IV pili to the phenotype.
Identification of a Novel Sialic Acid Transporter in
Haemophilus ducreyi
Authors: Post Deborah M. B. , Mungur Rachna , Gibson Bradford W. , Munson Robert S.
Published: September 2005

Haemophilus ducreyi
, the causative agent of chancroid, produces a lipooligosaccharide (LOS) which terminates in
N
-acetyllactosamine. This glycoform can be further extended by the addition of a single sialic acid residue to the terminal galactose moiety.
H. ducreyi
does not synthesize sialic acid, which must be acquired from the host during infection or from the culture medium when the bacteria are grown in vitro. However,
H. ducreyi
does not have genes that are highly homologous to the genes encoding known bacterial sialic acid transporters. In this study, we identified the sialic acid transporter by screening strains in a library of random transposon mutants for those mutants that were unable to add sialic acid to
N
-acetyllactosamine-containing LOS. Mutants that reacted with the monoclonal antibody 3F11, which recognizes the terminal lactosamine structure, and lacked reactivity with the lectin
Maackia amurensis
agglutinin, which recognizes α2,3-linked sialic acid, were further characterized to demonstrate that they produced a
N
-acetyllactosamine-containing LOS by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analyses. The genes interrupted in these mutants were mapped to a four-gene cluster with similarity to genes encoding bacterial ABC transporters. Uptake assays using radiolabeled sialic acid confirmed that the mutants were unable to transport sialic acid. This study is the first report of bacteria using an ABC transporter for sialic acid uptake.
NK Cells in Gamma-Interferon-Deficient Mice Suppress Lung Innate Immunity againstMycoplasmaspp
Authors: Woolard Matthew D. , Hudig Dorothy , Tabor Leslie , Ivey James A. , Simecka Jerry W.
Published: September 2005

The purpose of this study was to examine the 100-fold difference in mycoplasma levels in lungs of gamma interferon knockout (IFN-γ−/−) mice compared to those seen with wild-type BALB/c mice at 3 days postinfection. NK cells secreted IFN-γ; however, their cytotoxic granule extracts failed to kill mycoplasma. We found a conundrum: the clearance of organisms was as effective in NK-depleted IFN-γ−/−animals as in wild-type mice (with both IFN-γ and NK cells). NK+IFN-γ−/−animals had high mycoplasma burdens, but, after NK-like cell depletion, mycoplasma numbers were controlled. Essentially, IFN-γ was important in animals with NK-like cells and unimportant in animals without NK cells, suggesting that IFN-γ counters deleterious effects of NK-like cells. Impairment of innate immunity in IFN-γ−/−mice was not due to NK-like cell killing of macrophages. The increased levels of inflammatory cytokines and neutrophils in lung fluids of NK+IFN-γ−/−mice were reduced after NK cell depletion. In summary, in the murine model that resembles chronic human disease, innate immunity to mycoplasma requires IFN-γ when there are NK-like cells and the positive effects of IFN-γ counteract negative effects of NK-like cells. When imbalanced, NK-like cells promote disease. Thus, it was not the lack of IFN-γ but the presence of a previously unrecognized NK-like cell-suppressive activity that contributed to the higher mycoplasma numbers. It appears that pulmonary NK cells may contribute to the immunosuppressive environment of the lung, but when needed, these dampening effects can be counterbalanced by IFN-γ. Furthermore, there may be instances where perturbation of this regulatory balance contributes to the susceptibility to and severity of disease.
Comparative Opsonic and Protective Activities of
Staphylococcus aureus
Conjugate Vaccines Containing Native or Deacetylated Staphylococcal Poly-
N
-Acetyl-β-(1-6)-Glucosamine
Authors: Maira-Litrán Tomás , Kropec Andrea , Goldmann Donald A. , Pier Gerald B.
Published: September 2005

Staphylococcus aureus
and
Staphylococcus epidermidis
both synthesize the surface polysaccharide poly-
N
-acetyl-β-(1-6)-glucosamine (PNAG), which is produced in vitro with a high level (>90%) of the amino groups substituted by acetate. Here, we examined the role of the acetate substituents of PNAG in generating opsonic and protective antibodies. PNAG and a deacetylated form of the antigen (dPNAG; 15% acetylation) were conjugated to the carrier protein diphtheria toxoid (DT) and used to immunize animals. Mice responded in a dose-dependent fashion to both conjugate vaccines, with maximum antibody titers observed at the highest dose and 4 weeks after the last of three weekly immunizations. PNAG-DT and dPNAG-DT vaccines were also very immunogenic in rabbits. Antibodies raised to the conjugate vaccines in rabbits mediated the opsonic killing of various staphylococcal strains, but the specificity of the opsonic killing was primarily to dPNAG, as this antigen inhibited the killing of
S. aureus
strains by both PNAG- and dPNAG-specific antibodies. Passive immunization of mice with anti-dPNAG-DT rabbit sera showed significant levels of clearance of
S. aureus
from the blood (54 to 91%) compared to control mice immunized with normal rabbit sera, whereas PNAG-specific antibodies were ineffective at clearing
S. aureus
. Passive immunization of mice with a goat antiserum raised to the dPNAG-DT vaccine protected against a lethal dose of three different
S. aureus
strains. Overall, these data show that immunization of animals with a conjugate vaccine of dPNAG elicit antibodies that mediated opsonic killing and protected against
S. aureus
infection, including capsular polysaccharide types 5 and 8 and an untypable strain.
Successful Selection of Cross-Protective Vaccine Candidates for
Ornithobacterium rhinotracheale
Infection
Authors: Schuijffel D. F. , van Empel P. C. M. , Pennings A. M. M. A. , van Putten J. P. M. , Nuijten P. J. M.
Published: September 2005

Ornithobacterium rhinotracheale
is a bacterial pathogen known for causing respiratory disease in poultry. In this study, we demonstrate for the first time that cross-protective immunity against different
O. rhinotracheale
serotypes can be induced by live vaccination. Sera from these live-vaccinated and cross-protected birds were used to identify new vaccine targets by screening an
O. rhinotracheale
expression library. Out of 20,000 screened plaques, a total of 30 cross-reactive clones were selected for further analysis. Western blot analysis and DNA sequencing identified eight different open reading frames. The genes encoding the eight cross-reactive antigens were amplified, cloned in an expression vector, and expressed in
Escherichia coli
. Purified recombinant proteins with a molecular mass ranging from 35.9 kDa to 62.9 kDa were mixed and tested as a subunit vaccine for (cross-)protection against challenge with homologous and heterologous
O. rhinotracheale
serotypes in chickens. Subunit vaccination resulted in the production of antibodies reactive to the recombinant proteins on Western blot, and this eight-valent vaccine conferred both homologous and heterologous protection against
O. rhinotracheale
challenge in chickens.
Influence of Cystic Fibrosis Transmembrane Conductance Regulator on Gene Expression in Response to
Pseudomonas aeruginosa
Infection of Human Bronchial Epithelial Cells
Authors: Reiniger Nina , Ichikawa Jeffrey K. , Pier Gerald B.
Published: September 2005

Chronic lung infection by
Pseudomonas aeruginosa
causes significant morbidity in cystic fibrosis patients initiated by the failure of innate immune responses. We used microarray analysis and real-time PCR to detect transcriptional changes associated with cytokine production in isogenic bronchial epithelial cell lines with either wild-type (WT) or mutant cystic fibrosis transmembrane conductance regulator (CFTR) in response to
P. aeruginosa
infection. The transcription of four NF-κB-regulated cytokine genes was maximal in the presence of WT CFTR: the interleukin-8 (IL-8), IL-6, CXCL1, and intracellular adhesion molecule 1 (ICAM-1) genes. Analysis of protein expression in two cell lines paired for wild-type and mutant CFTR with three
P. aeruginosa
strains showed IL-6 and IL-8 expressions were consistently enhanced by the presence of WT CFTR in both cell lines with all three strains of
P. aeruginosa
, although some strains gave small IL-8 increases in cells with mutant CFTR. CXCL1 production showed consistent enhancement in cells with WT CFTR using all three bacterial strains in one cell line, whereas in the other cell line, CXCL1 showed a significant increase in cells with either WT or mutant CFTR. ICAM-1 was unchanged at the protein level in one of the cell lines but did show mild enhancement with WT CFTR in the other cell pair. Inhibitions of NF-κB prior to infection indicated differing degrees of dependence on NF-κB for production of the cytokines, contingent on the cell line. Cytokine effectors of innate immunity to
P. aeruginosa
were found to be positively influenced by the presence of WT CFTR, indicating a role in resistance to
P. aeruginosa
infection.
Evaluation of Recombinant Lipidated P2086 Protein as a Vaccine Candidate for Group B
Neisseria meningitidis
in a Murine Nasal Challenge Model
Authors: Zhu Duzhang , Zhang Ying , Barniak Vicki , Bernfield Liesel , Howell Alan , Zlotnick Gary
Published: September 2005

Neisseria meningitidis
is a major causative agent of bacterial meningitis in human beings, especially among young children (≤2 years of age). Prevention of group B meningococcal disease represents a particularly difficult challenge in vaccine development, due to the inadequate immune response elicited against type B capsular polysaccharide. We have established an adult mouse intranasal challenge model for group B
N. meningitidis
to evaluate potential vaccine candidates through active immunization. Swiss Webster mice were inoculated intranasally with meningococci, and bacteria were recovered from the noses for at least 3 days postchallenge. Iron dextran was required in the bacterial inoculum to ensure sufficient meningococcal recovery from nasal tissue postchallenge. This model has been utilized to evaluate the potential of a recombinant lipidated group B meningococcal outer membrane protein P2086 (rLP2086) as a vaccine candidate. In this study, mice were immunized subcutaneously with purified rLP2086 formulated with or without an attenuated cholera toxin as an adjuvant. The mice were then challenged intranasally with
N. meningitidis
strain H355 or M982, and the colonization of nasal tissue was determined by quantitative culture 24 h postchallenge. We demonstrated that immunization with rLP2086 significantly reduced nasal colonization of mice challenged with the two different strains of group B
N. meningitidis
. Mice immunized with rLP2086 produced a strong systemic immunoglobulin G response, and the serum antibodies were cross-reactive with heterologous strains of group B
N. meningitidis
. The antibodies have functional activity against heterologous
N. meningitidis
strain, as demonstrated via bactericidal and infant rat protection assays. These results suggest that rLP2086 is a potential vaccine candidate for group B
N. meningitidis
.
Enterotoxin-Based Mucosal Adjuvants Alter Antigen Trafficking and Induce Inflammatory Responses in the Nasal Tract
Authors: van Ginkel Frederik W. , Jackson Raymond J. , Yoshino Naoto , Hagiwara Yukari , Metzger Daniel J. , Connell Terry D. , Vu Hong L. , Martin Michael , Fujihashi Kohtaro , McGhee Jerry R.
Published: September 2005

The safety of nasal vaccines containing enterotoxin-based mucosal adjuvants has not been studied in detail. Previous studies have indicated that native cholera toxin (nCT) can alter antigen trafficking when applied nasally. In this study, we determined the enterotoxin-based variables that alter antigen trafficking. To measure the influence of enterotoxin-based mucosal adjuvants on antigen trafficking in the nasal tract, native and mutant enterotoxins were coadministered with radiolabeled tetanus toxoid (TT). The nCT and heat-labile enterotoxin type 1 (LTh-1) redirected TT into the olfactory neuroepithelium (ON/E). Antigen redirection occurred mainly across the nasal epithelium without subsequent transport along olfactory neurons into the olfactory bulbs (OB). Thus, no significant accumulation of the vaccine antigen TT was observed in the OB when coadministered with nCT. In contrast, neither mutant CT nor mutant LTh-1, which lack ADP-ribosyltransferase activity, redirected TT antigen into the ON/E. Thus, ADP-ribosyltransferase activity was essential for antigen trafficking across the olfactory epithelium. Accumulation of TT in the ON/E was also due to B-subunit binding to GM1 gangliosides, as was demonstrated (i) by redirection of TT by LTh-1 in a dose-dependent manner, (ii) by ganglioside inhibition of the antigen redirection by LTh-1 and nCT, and (iii) by the use of LT-IIb, a toxin that binds to gangliosides other than GM1. Redirection of TT into the ON/E coincided with elevated production of interleukin 6 (IL-6) but not IL-1β or tumor necrosis factor alpha in the nasal mucosa. Thus, redirection of TT is dependent on ADP-ribosyltransferase activity and GM1 binding and is associated with production of the inflammatory cytokine IL-6.
Role of HtrA in Surface Protein Expression and Biofilm Formation by
Streptococcus mutans
Authors: Biswas Saswati , Biswas Indranil
Published: September 2005

The HtrA surface protease in gram-positive bacteria is involved in the processing and maturation of extracellular proteins and degradation of abnormal or misfolded proteins. Inactivation of
htrA
has been shown to affect the tolerance to thermal and environmental stress and to reduce virulence. We found that inactivation of
Streptococcus mutans htrA
by gene-replacement also resulted in a reduced ability to withstand exposure to low and high temperatures, low pH, and oxidative and DNA damaging agents. The
htrA
mutation affected surface expression of several extracellular proteins including glucan-binding protein B (GbpB), glucosyltransferases, and fructosyltransferase. In addition,
htrA
mutation also altered the surface expression of enolase and glyceraldehyde-3-phosphate dehydrogenease, two glycolytic enzymes that are known to be present on the streptococcal cell surface. As expected, microscopic analysis of in vitro grown biofilm structure revealed that the
htrA
deficient biofilms adopted a much more granular patchy appearance, rather than the relatively smooth confluent layer normally seen in the wild type. These results suggest that HtrA plays an important role in the biogenesis of extracellular proteins including surface associated glycolytic enzymes and in biofilm formation of
S. mutans
.
Binding Hot Spot for Invasion Inhibitory Molecules on
Plasmodium falciparum
Apical Membrane Antigen 1
Authors: Harris Karen S. , Casey Joanne L. , Coley Andrew M. , Masciantonio Rosella , Sabo Jennifer K. , Keizer David W. , Lee Erinna F. , McMahon Andrew , Norton Raymond S. , Anders Robin F. , Foley Michael
Published: September 2005

Apical membrane antigen 1 (AMA1) is expressed in schizont-stage malaria parasites and sporozoites and is thought to be involved in the invasion of host red blood cells. AMA1 is an important vaccine candidate, as immunization with this antigen induces a protective immune response in rodent and monkey models of human malaria. Additionally, anti-AMA1 polyclonal and monoclonal antibodies inhibit parasite invasion in vitro. We have isolated a 20-residue peptide (R1) from a random peptide library that binds to native AMA1 as expressed by
Plasmodium falciparum
parasites. Binding of R1 peptide is dependent on AMA1 having the proper conformation, is strain specific, and results in the inhibition of merozoite invasion of host erythrocytes. The solution structure of R1, as determined by nuclear magnetic resonance spectroscopy, contains two structured regions, both involving turns, but the first region, encompassing residues 5 to 10, is hydrophobic and the second, at residues 13 to 17, is more polar. Several lines of evidence reveal that R1 targets a “hot spot” on the AMA1 surface that is also recognized by other peptides and monoclonal antibodies that have previously been shown to inhibit merozoite invasion. The functional consequence of binding to this region by a variety of molecules is the inhibition of merozoite invasion into host erythrocytes. The interaction between these peptides and AMA1 may further our understanding of the molecular mechanisms of invasion by identifying critical functional regions of AMA1 and aid in the development of novel antimalarial strategies.
Characterization of the Antibody Response to the Receptor Binding Domain of Botulinum Neurotoxin Serotypes A and E
Authors: Baldwin Michael R. , Tepp William H. , Pier Christina L. , Bradshaw Marite , Ho Mengfei , Wilson Brenda A. , Fritz Robert B. , Johnson Eric A. , Barbieri Joseph T.
Published: September 2005

Clostridium botulinum
neurotoxins (BoNTs) are the most toxic proteins for humans. The current clostridial-derived vaccines against BoNT intoxication have limitations including production and accessibility. Conditions were established to express the soluble receptor binding domain (heavy-chain receptor [HCR]) of BoNT serotypes A and E in
Escherichia coli
. Sera isolated from mice and rabbits immunized with recombinant HCR/A1 (rHCR/A1) from the classical type A-Hall strain (ATCC 3502) (BoNT/A1) and rHCR/E from BoNT serotype E Beluga (BoNT/E
B
) neutralized the homologous serotype of BoNT but displayed differences in cross-recognition and cross-protection. Enzyme-linked immunosorbent assay and Western blotting showed that α-rHCR/A1 recognized epitopes within the C terminus of the HCR/A and HCR/E, while α-rHCR/E recognized epitopes within the N terminus or interface between the N and C termini of the HCR proteins. α-rHCR/E
B
sera possessed detectable neutralizing capacity for BoNT/A1, while α-rHCR/A1 did not neutralize BoNT/E. rHCR/A was an effective immunogen against BoNT/A1 and the Kyoto F infant strain (BoNT/A2), but not BoNT serotype E Alaska (BoNT/E
A
), while rHCR/E
B
neutralized BoNT/E
A
, and under hyperimmunization conditions protected against BoNT/A1 and BoNT/A2. The protection elicited by rHCR/A1 to BoNT/A1 and BoNT/A2 and by rHCR/E
B
to BoNT/E
A
indicate that immunization with receptor binding domains elicit protection within sub-serotypes of BoNT. The protection elicited by hyperimmunization with rHCR/E against BoNT/A suggests the presence of common neutralizing epitopes between the serotypes E and A. These results show that a receptor binding domain subunit vaccine protects against serotype variants of BoNTs.
Cloning Vectors and Fluorescent Proteins Can Significantly Inhibit
Salmonella enterica
Virulence in Both Epithelial Cells and Macrophages: Implications for Bacterial Pathogenesis Studies
Authors: Knodler Leigh A. , Bestor Aaron , Ma Caixia , Hansen-Wester Imke , Hensel Michael , Vallance Bruce A. , Steele-Mortimer Olivia
Published: September 2005

Plasmid vectors and fluorescent protein reporter systems are commonly used in the study of bacterial pathogenesis. Here we show that they can impair the ability of
Salmonella enterica
serovar Typhimurium to productively infect either cultured mammalian cells or mice. This has significant implications for studies that rely on these systems.
Identification of
Actinobacillus suis
Genes Essential for the Colonization of the Upper Respiratory Tract of Swine
Authors: Ojha Shivani , Sirois Marc , MacInnes Janet I.
Published: September 2005

Actinobacillus suis
has emerged as an important opportunistic pathogen of high-health-status swine. A colonization challenge method was developed, and using PCR-based signature-tagged transposon mutagenesis, 13 genes belonging to 9 different functional classes were identified that were necessary for
A. suis
colonization of the upper respiratory tract of swine.
HIGH ENERGY NEUTRINOS FROM A SLOW JET MODEL OF CORE COLLAPSE SUPERNOVAE
Authors: RAZZAQUE SOEBUR , MÉSZÁROS PETER , WAXMAN ELI
Published: September 2005

It has been hypothesized recently that core collapse supernovae are triggered by mildly relativistic jets following observations of radio properties of these explosions. Association of a jet, similar to a gamma-ray burst jet but only slower, allows shock acceleration of particles to high energy and non-thermal neutrino emission from a supernova. Detection of these high energy neutrinos in upcoming kilometer scale Cherenkov detectors may be the only direct way to probe inside these astrophysical phenomena as electromagnetic radiation is thermal and contains little information. Calculation of high energy neutrino signal from a simple and slow jet model buried inside the pre-supernova star is reviewed here. The detection prospect of these neutrinos in water or ice detector is also discussed in this brief review. Jetted core collapse supernovae in nearby galaxies may provide the strongest high energy neutrino signal from point sources.
MODELING NETWORK TRAFFIC USING CAUCHY CORRELATION MODEL WITH LONG-RANGE DEPENDENCE
Authors: LI MING , LIM S. C.
Published: September 2005

Much attention has been given to the long-range dependence and fractal properties in network traffic engineering, and these properties are also widely observed in many fields of science and technologies. Traffic time series is conventionally characterized by its fractal dimension D, which is a measure for roughness, and by the Hurst parameter H, which is a measure for long-range dependence, see for examples (Refs. 10–12). Each property has been traditionally modeled and explained by self-affine random functions, such as fractional Gaussian noise (FGN)1,10–13,18,22–28 and fractional Brownian motion (FBM),6,7 where a linear relationship between D and H, say D = 2 - H for one-dimensional series, links the two properties. The limitation of single parameter models (e.g., FGN) in long-range dependent (LRD) traffic modeling has been noticed as can be seen from Refs. 1, 18 and 25. Hence, models which can provide good fitting of LRD traffic for both short-term lags and long-term ones are worth studying due to the importance of accurate models of traffic in network communications.13 This letter utilizes a statistical model called the Cauchy correlation model to model LRD traffic. This model characterizes D and H separately, and it allows any combination of two within the constraint of LRD condition. It is a new power-law correlation model for LRD traffic modeling with its local and global behavior decoupling. Its flexibility in data modeling in comparison with a single parameter model of FGN is briefly discussed, and applications to LRD traffic modeling demonstrated.
ON THE USE OF THE RECIPROCITY-GAP FUNCTIONAL IN INVERSE SCATTERING FROM PLANAR CRACKS
Authors: BEN ABDA AMEL , DELBARY FABRICE , HADDAR HOUSSEM
Published: September 2005

We consider the inverse problem of determining the shape and location of planar screens inside a 3D body through acoustic or electromagnetic imaging. In order to determine the flaws we perform acoustic measurements with prescribed over-determined boundary data. The reciprocity gap concept is exploited to determine sound-hard planar screens of general shapes.
TWO NONCONFORMING QUADRILATERAL ELEMENTS FOR THE REISSNER–MINDLIN PLATE
Authors: MING PINGBING , SHI ZHONG-CI
Published: September 2005

We construct two low order nonconforming quadrilateral elements for the Reissner–Mindlin plate. The first one consists of a modified nonconforming rotated Q1 element for one component of the rotation and the standard four-node isoparametric element for the other component as well as for the the approximation of the transverse displacement, a modified rotated Raviart–Thomas interpolation operator is employed as the shear reduction operator. The second differs from the first only in the approximation of the rotation, which employs the modified rotated Q1 element for both components of the rotation, and a jump term accounting the discontinuity of the rotation approximation is included in the variational formulation. Both elements give optimal error bounds uniform in the plate thickness with respect to the energy norm as well as the L2 norm.
Plant, Synthetic, and Endogenous Cannabinoids in Medicine
Authors: Marzo Vincenzo Di , Petrocellis Luciano De
Published: September 2005

Although used for more than 4000 years for recreational and medicinal purposes, Cannabis and its best-known pharmacologically active constituents, the cannabinoids, became a protagonist in medical research only recently. This revival of interest is explained by the finding in the 1990s of the mechanism of action of the main psychotropic cannabinoid, Δ9-tetrahydrocannabinol (THC), which acts through specific membrane receptors, the cannabinoid receptors. The molecular characterization of these receptors allowed the development of synthetic molecules with cannabinoid and noncannabinoid structure and with higher selectivity, metabolic stability, and efficacy than THC, as well as the development of antagonists that have already found pharmaceutical application. The finding of endogenous agonists at these receptors, the endocannabinoids, opened new therapeutic possibilities through the modulation of the activity of cannabinoid receptors by targeting the biochemical mechanisms controlling endocannabinoid tissue levels.
Albumin Adducts of Naphthalene Metabolites as Biomarkers of Exposure to Polycyclic Aromatic Hydrocarbons
Authors: Waidyanatha Suramya , Zheng Yuxin , Serdar Berrin , Rappaport Stephen M.
Published: September 2005

We investigated the utility of adducts formed by the reaction of the naphthalene metabolites naphthalene-1,2-oxide, 1,2-naphthoquinone (1,2-NPQ), and 1,4-naphthoquinone (1,4-NPQ) with serum albumin (Alb) as biomarkers of exposure to polycyclic aromatic hydrocarbons. Cysteinyl serum Alb adducts of 1,2- and 1,4-NPQ (1,2-NPQ-Alb and 1,4-NPQ-Alb, respectively) but not of naphthalene-1,2-oxide were detected in 28 coke oven workers and 22 controls from the steel industry of northern China. The median level of 1,2-NPQ-Alb in coke oven workers (76.6 pmol/g) was significantly higher than that observed in controls (44.9 pmol/g; P = 0.0027). However, the median level of 1,4-NPQ-Alb in exposed subjects was not significantly different from that of controls (48.6 versus 44.2 pmol/g; P = 0.296). Levels of 1,2-NPQ-Alb were significantly correlated with exposure category (controls, side and bottom workers, and top-of-oven workers) as well as with previously measured levels of urinary naphthalene, 1- and 2-naphthol, and 1-pyrenol in these subjects. Multiple linear regression analysis revealed that 35% of the variation in 1,2-NPQ-Alb could be explained by the work category and age. A negative relationship between 1,2-NPQ-Alb and age was observed, suggesting that cytochrome P450 c metabolism diminished with age at ∼3%/year of life.
Prognostic and Diagnostic Significance of β-Catenin Nuclear Immunostaining in Colorectal Cancer
Authors: Wong Sze Chuen Cesar , Lo Elena Siu Fong , Lee King Chung , Chan John K. C. , Hsiao W. L. Wendy
Published: September 2005

In the present study, we investigated the prognostic and diagnostic significance of β-catenin nuclear immunostaining in 60 specimens of normal colorectal tissue; 180 specimens of colorectal polyps, adenomas, and carcinomas; and 40 specimens from patients with the simultaneous occurrence of polyps, adenomas, and carcinomas. Additional specimens from 59 patients with colorectal carcinoma and 14 patients with adenoma who subsequently developed carcinoma were examined for possible survival study. Immunohistochemical staining showed that the occurrence of nuclear β-catenin correlated with the sequential stages in colorectal carcinogenesis, in which positive staining was observed in 0% of normal tissues, 8% of polyps, 92% of adenomas, and 100% of carcinomas. High immunohistochemical scores in colorectal carcinoma were significantly associated with lymph node metastasis and poor survival. Adenomas associated with synchronous or metachronous carcinomas showed significantly higher levels of nuclear β-catenin compared with adenomas without associated carcinomas. Nuclear translocation of β-catenin was rare or absent in other types of cytokeratin 20 positive adenocarcinomas examined (99 cases). Thus, it was positive in only 7% of colonic mucinous adenocarcinomas, 3% of pancreatic adenocarcinomas, 8% of ovarian mucinous cystadenocarcinomas, and 0% of gastric adenocarcinomas. However, 100% of primary and metastatic colorectal adenocarcinomas were positive for nuclear staining for β-catenin. Thus, nuclear staining for β-catenin may serve as an additional parameter to help distinguish colorectal adenocarcinomas from adenocarcinomas of other tissue sites. Collectively, the present large-scale study has clearly addressed the clinical significance of β-catenin nuclear translocation with respect to tumor progression, survival, and differential diagnosis.
Effective Intravenous Therapy of Murine Pulmonary Metastases with an Oncolytic Herpes Virus Expressing Interleukin 12
Authors: Wong Richard J. , Chan Mei-Ki , Yu Zhenkun , Kim Teresa H. , Bhargava Amit , Stiles Brendon M. , Horsburgh Brian C. , Shah Jatin P. , Ghossein Ronald A. , Singh Bhuvanesh , Fong Yuman
Published: September 2005

Purpose: There currently is no therapy that enhances the survival of patients with distantly metastatic squamous cell carcinoma (SCC). Engineered herpes oncolytic viruses are effective therapeutic agents when delivered directly to tumors in animal models, but their efficacy in treating disseminated disease is poorly defined.

Experimental Design: We treated disseminated pulmonary SCC in mice with an interleukin (IL)-12-expressing oncolytic herpes virus (NV1042) or with the parent oncolytic virus (NV1023, IL-12 deficient) by i.v. tail vein administration.

Results: Lung IL-12 was 16.1 pg/mg and IFN-γ was 4.3 pg/mg at day 1 after a single dose of NV1042 (5 × 107 plaque-forming units); levels of both were undetectable for NV1023. 5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside histochemistry demonstrated viral infection of disseminated pulmonary tumor nodules by both vectors at day 1, with sparing of adjacent alveolar cells. NV1042-treated lungs showed no surface nodules at day 12, in contrast to NV1023-treated (92 ± 27 surface nodules) and PBS-treated (225 ± 9 surface nodules) lungs. Significantly enhanced survival was observed in NV1042-treated animals compared with NV1023- and PBS-treated animals (log rank < 0.05). In animals with a low tumor burden, 100% of NV1042-treated, 70% of NV1023-treated, and none of the control animals achieved long-term survival. NV1042 efficacy was similar to NV1023 efficacy in animals depleted of CD4/CD8 T lymphocytes, showing that IL-12 expression enhances oncolytic activity through immune effects. Histology showed no cytopathic effects in non-tumor-bearing lung, brain, spleen, liver, and pancreas after completion of viral therapy. No animals demonstrated any visible side effects attributable to viral therapy.

Conclusions: The i.v. delivery of an oncolytic herpes virus may achieve effective infection, oncolysis, and transgene expression at distant tumor sites. This approach to systemic therapy combining oncolysis with IL-12 immune stimulation led to significantly improved survival in animals with disseminated SCC.
Radiosensitization by Antisense Anti-MDM2 Mixed-Backbone Oligonucleotide in
in Vitro
and
in Vivo
Human Cancer Models
Authors: Zhang Zhuo , Wang Hui , Prasad Gautam , Li Mao , Yu Dong , Bonner James A. , Agrawal Sudhir , Zhang Ruiwen
Published: September 2005

Purpose: The MDM2 oncogene, amplified or overexpressed in many human cancers, has been suggested to be a novel target for cancer therapy. We have demonstrated a second-generation antisense antihuman-MDM2 oligonucleotide to have antitumor activity when administered alone or in combination with cancer chemotherapeutic agents. In the present study, we investigated the effect of the antisense oligonucleotide on radiation therapy.

Experimental Design: The in vitro radiosensitization activity was determined in cell lines of human cancers of prostate (LNCaP and PC3), breast (MCF-7 and MDA-MB-468), pancreas (PANC-1), and glioma (U87-MG and A172) and its in vivo radiosensitization activity in xenograft models of LNCaP, PC3, MCF-7, MDA-MB-468, and PANC-1.

Results: In cells containing at least one functional p53 allele (LNCaP, U87-MG, and A172), after specific inhibition of MDM2 expression, p53 and p21 levels were elevated. In LNCaP cells, the Bax level was increased, and Bcl-2 and E2F1 levels were decreased. In PC3 cells that are p53 null, after inhibition of MDM2 expression, Bax and p21 levels were elevated, and E2F1 levels were decreased. On the basis of in vitro clonogenic assay, the antisense oligonucleotide, in a sequence-specific manner, significantly increased radiation-induced antiproliferation effects. It also increased radiation-induced inhibitory effects on tumor growth in SCID or nude mice bearing LNCaP, PC3, MCF-7, MDA-MB-468, and PANC-1 xenografts.

Conclusions: These results suggest that MDM2 has a role in radiation therapy of human cancers, regardless of p53 status, providing a basis for future development of MDM2 inhibitors, such as antisense oligonucleotides, as radiosensitizers.