bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Cultural Sensitivity Training on Health Care Provider Attitudes and Patient Outcomes
Authors: Majumdar Basanti , Browne Gina , Roberts Jacqueline , Carpio Barbara
Published: May 2004

Purpose:

To determine the effectiveness of cultural sensitivity training on the knowledge and attitudes of health care providers, and to assess the satisfaction and health outcomes of patients from different minority groups with health care providers who received training.

Design:

In this randomised controlled trial, 114 health care providers (nurses and homecare workers) and 133 patients (from two community agencies and one hospital) were randomly assigned to experimental (training) and control groups, and were followed for 18 months.

Methods:

Providers completed the Cultural Awareness Questionnaire and the Dogmatism Scale. Patients completed the Off‐Axis‐Ratio (OAR) Multidimensional Measure of Functional Capacity, the Client Satisfaction Questionnaire, the Physical and Mental Health Assessment Questionnaire, and the Health and Social Services Utilization Questionnaire. A qualitative analysis was conducted to identify and analyse themes from personal journals kept by participating nurses.

Findings:

Cultural sensitivity training resulted in increased open‐mindedness and cultural awareness, improved understanding of multiculturalism, and ability to communicate with minority people. After 1 year patients of mostly European and British origin, who received care from trained providers, showed improvement in utilizing social resources and overall functional capacity without an increase in health care expenditures.

Conclusions:

The results of this study indicate that a cultural sensitivity training program not only improved knowledge and attitudes among health care providers, but it also yielded positive health outcomes for their patients.
Incentives for Children in Research
Authors: Rice Marti , Broome Marion E.
Published: May 2004

Purpose:

To describe the challenges of using incentives for children in research.

Organizing Framework:

A discussion of incentives in the context of recruitment for research, including a review of the literature, a description of the types of incentives used with children and their families, factors influencing choice of incentives and ethical considerations. Recommendations to guide decisions about incentives are proposed.

Conclusions:

Since the 1997 mandate from the U.S. National Institutes of Health that investigators provide justification for excluding children, researchers have become more willing to include children. Whether all parts of the research process pertain to children without careful consideration and adaptation is unclear. One area lacking clarity is the use of incentives to encourage participation in research. The issue of incentives for children or parents should be considered early in the design of the research proposal, with attention to developmental age, ethical considerations, purpose of the research, and burden to the child and family.
Disulfide-Dependent Multimeric Assembly of Resistin Family Hormones
Authors: Patel Saurabh D. , Rajala Michael W. , Rossetti Luciano , Scherer Philipp E. , Shapiro Lawrence
Published: May 2004

Resistin, founding member of the resistin-like molecule (RELM) hormone family, is secreted selectively from adipocytes and induces liver-specific antagonism of insulin action, thus providing a potential molecular link between obesity and diabetes. Crystal structures of resistin and RELMβ reveal an unusual multimeric structure. Each protomer comprises a carboxy-terminal disulfide-rich β-sandwich “head” domain and an amino-terminal α-helical “tail” segment. The α-helical segments associate to form three-stranded coiled coils, and surface-exposed interchain disulfide linkages mediate the formation of tail-to-tail hexamers. Analysis of serum samples shows that resistin circulates in two distinct assembly states, likely corresponding to hexamers and trimers. Infusion of a resistin mutant, lacking the intertrimer disulfide bonds, in pancreatic-insulin clamp studies reveals substantially more potent effects on hepatic insulin sensitivity than those observed with wild-type resistin. This result suggests that processing of the intertrimer disulfide bonds may reflect an obligatory step toward activation.
Autoimmune Disease and Impaired Uptake of Apoptotic Cells in MFG-E8-Deficient Mice
Authors: Hanayama Rikinari , Tanaka Masato , Miyasaka Kay , Aozasa Katsuyuki , Koike Masato , Uchiyama Yasuo , Nagata Shigekazu
Published: May 2004

Apoptotic cells expose phosphatidylserine and are swiftly engulfed by macrophages. Milk fat globule epidermal growth factor (EGF) factor 8 (MFG-E8) is a protein that binds to apoptotic cells by recognizing phosphatidylserine and that enhances the engulfment of apoptotic cells by macrophages. We report that tingible body macrophages in the germinal centers of the spleen and lymph nodes strongly express MFG-E8. Many apoptotic lymphocytes were found on the
MFG-E8
–/–
tingible body macrophages, but they were not efficiently engulfed. The
MFG-E8
–/–
mice developed splenomegaly, with the formation of numerous germinal centers, and suffered from glomerulonephritis as a result of autoantibody production. These data demonstrate that MFG-E8 has a critical role in removing apoptotic B cells in the germinal centers and that its failure can lead to autoimmune diseases.
Rapid Rise of Sea Level 19,000 Years Ago and Its Global Implications
Authors: Clark Peter U. , McCabe A. Marshall , Mix Alan C. , Weaver Andrew J.
Published: May 2004

Evidence from the Irish Sea basin supports the existence of an abrupt rise in sea level (meltwater pulse) at 19,000 years before the present (B.P.). Climate records indicate a large reduction in the strength of North Atlantic Deep Water formation and attendant cooling of the North Atlantic at this time, indicating a source of the meltwater pulse from one or more Northern Hemisphere ice sheets.Warming of the tropical Atlantic and Pacific oceans and the Southern Hemisphere also began at 19,000 years B.P. These responses identify mechanisms responsible for the propagation of deglacial climate signals to the Southern Hemisphere and tropics while maintaining a cold climate in the Northern Hemisphere.
Definition of a Bacterial Type IV Secretion Pathway for a DNA Substrate
Authors: Cascales Eric , Christie Peter J.
Published: May 2004

Bacteria use conjugation systems, a subfamily of the type IV secretion systems, to transfer DNA to recipient cells. Despite 50 years of research, the architecture and mechanism of action of the channel mediating DNA transfer across the bacterial cell envelope remains obscure. By use of a sensitive, quantifiable assay termed transfer DNA immunoprecipitation (TrIP), we identify contacts between a DNA substrate (T-DNA) and 6 of 12 components of the VirB/D4 conjugation system of the phytopathogen
Agrobacterium tumefaciens
. Our results define the translocation pathway for a DNA substrate through a bacterial conjugation machine, specifying the contributions of each subunit of the secretory apparatus to substrate passage.
Mutational Analysis of the Tyrosine Phosphatome in Colorectal Cancers
Authors: Wang Zhenghe , Shen Dong , Parsons D. Williams , Bardelli Alberto , Sager Jason , Szabo Steve , Ptak Janine , Silliman Natalie , Peters Brock A. , van der Heijden Michiel S. , Parmigiani Giovanni , Yan Hai , Wang Tian-Li , Riggins Greg , Powell Steven M. , Willson James K. V. , Markowitz Sanford , Kinzler Kenneth W. , Vogelstein Bert , Velculescu Victor E.
Published: May 2004

Tyrosine phosphorylation, regulated by protein tyrosine phosphatases (PTPs) and kinases (PTKs), is important in signaling pathways underlying tumorigenesis. A mutational analysis of the tyrosine phosphatase gene superfamily in human cancers identified 83 somatic mutations in six PTPs (
PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, PTPN14
), affecting 26% of colorectal cancers and a smaller fraction of lung, breast, and gastric cancers. Fifteen mutations were nonsense, frameshift, or splice-site alterations predicted to result in truncated proteins lacking phosphatase activity. Five missense mutations in the most commonly altered PTP (
PTPRT
) were biochemically examined and found to reduce phosphatase activity. Expression of wild-type but not a mutant PTPRT in human cancer cells inhibited cell growth. These observations suggest that the mutated tyrosine phosphatases are tumor suppressor genes, regulating cellular pathways that may be amenable to therapeutic intervention.
Optical Signatures of the Aharonov-Bohm Phase in Single-Walled Carbon Nanotubes
Authors: Zaric Sasa , Ostojic Gordana N. , Kono Junichiro , Shaver Jonah , Moore Valerie C. , Strano Michael S. , Hauge Robert H. , Smalley Richard E. , Wei Xing
Published: May 2004

We report interband magneto-optical spectra for single-walled carbon nanotubes in high magnetic fields up to 45 tesla, confirming theoretical predictions that the band structure of a single-walled carbon nanotube is dependent on the magnetic flux ϕ threading the tube. We have observed field-induced optical anisotropy as well as red shifts and splittings of absorption and photoluminescence peaks. The amounts of shifts and splittings depend on the value of ϕ/ϕ
0
and are quantitatively consistent with theories based on the Aharonov-Bohm effect. These results represent evidence of the influence of the Aharonov-Bohm phase on the band gap of a solid.
h/e
Magnetic Flux Modulation of the Energy Gap in Nanotube Quantum Dots
Authors: Coskun Ulas C. , Wei Tzu-Chieh , Vishveshwara Smitha , Goldbart Paul M. , Bezryadin Alexey
Published: May 2004

We report experiments on quantum dot single-electron-tunneling (SET) transistors made from short multiwall nanotubes and threaded by magnetic flux. Such systems allow us to probe the electronic energy spectrum of the nanotube and its dependence on the magnetic field. Evidence is provided for the interconversion between gapped (semiconducting) and ungapped (metallic) states. Our tubes exhibit
h
/
e
-period magnetic flux dependence, in agreement with simple tight-binding calculations.
Discovery and Directed Evolution of a Glyphosate Tolerance Gene
Authors: Castle Linda A. , Siehl Daniel L. , Gorton Rebecca , Patten Phillip A. , Chen Yong Hong , Bertain Sean , Cho Hyeon-Je , Duck Nicholas , Wong James , Liu Donglong , Lassner Michael W.
Published: May 2004

The herbicide glyphosate is effectively detoxified by
N
-acetylation. We screened a collection of microbial isolates and discovered enzymes exhibiting glyphosate
N
-acetyltransferase (GAT) activity. Kinetic properties of the discovered enzymes were insufficient to confer glyphosate tolerance to transgenic organisms. Eleven iterations of DNA shuffling improved enzyme efficiency by nearly four orders of magnitude from 0.87 mM
–1
min
–1
to 8320 mM
–1
min
–1
. From the fifth iteration and beyond, GAT enzymes conferred increasing glyphosate tolerance to
Escherichia coli, Arabidopsis
, tobacco, and maize. Glyphosate acetylation provides an alternative strategy for supporting glyphosate use on crops.
Food for the Gods
Authors: Ronald Pamela
Published: May 2004

The Art of Rice
Spirit and Sustenance in Asia. by Roy W. Hamilton [with 26 Contributors]. UCLA Fowler Museum of Cultural History, Los Angeles, 2004. 562 pp. Paper, $60. ISBN 0-930741-98-6.

The contributors to this profusely illustrated catalog explore beliefs and practices relating to rice as they are represented in the arts and cultures of south, southeast, and east Asia.
Genetic Structure of the Purebred Domestic Dog
Authors: Parker Heidi G. , Kim Lisa V. , Sutter Nathan B. , Carlson Scott , Lorentzen Travis D. , Malek Tiffany B. , Johnson Gary S. , DeFrance Hawkins B. , Ostrander Elaine A. , Kruglyak Leonid
Published: May 2004

We used molecular markers to study genetic relationships in a diverse collection of 85 domestic dog breeds. Differences among breeds accounted for ∼30% of genetic variation. Microsatellite genotypes were used to correctly assign 99% of individual dogs to breeds. Phylogenetic analysis separated several breeds with ancient origins from the remaining breeds with modern European origins. We identified four genetic clusters, which predominantly contained breeds with similar geographic origin, morphology, or role in human activities. These results provide a genetic classification of dog breeds and will aid studies of the genetics of phenotypic breed differences.
An Ancient Lineage Worth Saving
Authors: Garland Mark S.
Published: May 2004

The American Horseshoe Crab
. by Carl N. Shuster Jr., Robert B. Barlow, and H. Jane Brockmann, Eds. Harvard University Press, Cambridge, MA, 2003. 439 pp. $95, £61.95, €87.50. ISBN 0-674-01159-7.

The contributors offer a detailed and comprehensive review of the horseshoe crab's biology.
Predominant Outer Membrane Antigens of
Bartonella henselae
Authors: Chenoweth Matthew R. , Greene Craig E. , Krause Duncan C. , Gherardini Frank C.
Published: May 2004

A hallmark of
Bartonella henselae
is persistent bacteremia in cats despite the presence of a vigorous host immune response. To understand better the long-term survival of
B. henselae
in cats, we examined the feline humoral immune response to
B. henselae
outer membrane (OM) proteins in naturally and experimentally infected cats. Initially, a panel of sera (
n
= 42) collected throughout North America from naturally infected cats was used to probe
B. henselae
total membranes to detect commonly recognized antigens. Twelve antigens reacted with sera from at least 85% of cats, and five were recognized by sera from all cats. To localize these antigens further, OMs were purified on discontinuous sucrose density step gradients. Each membrane fraction (OM, hybrid or inner membrane [IM]) contained less than 1% of the total malate dehydrogenase activity (soluble marker), indicating very little contamination by cytoplasmic proteins. FtsI, an integral IM cell division protein, was used to identify the low-density fraction (ρ = 1.13 g/cm
3
) as putative IM (<5% of the total FtsI localized to the high-density fraction) while lipopolysaccharide (LPS) and Pap31, a homolog of the
Bartonella quintana
heme-binding protein A (HbpA), defined the high-density fraction (ρ = 1.20 g/cm
3
) as putative OM. Additionally, little evidence of cross-contamination between the IM and OM was evident by two-dimensional gel electrophoresis. When purified OMs were probed with feline sera, antigenic proteins profiles were very similar to those observed with total membranes, indicating that many, but not all, of the immunoreactive proteins detected in the initial immunoblots were OM components. Interestingly, two-dimensional immunoblots indicated that
B. henselae
LPS and members of the Hbp family of proteins did not appear to stimulate an humoral response in any infected cats. Seven proteins were recognized by at least 70% of sera tested, but only three were recognized by all sera. Nanospray-tandem mass spectrometry was used to identify OM components, including the immunodominant OM proteins. Recognition of the nonimmunogenic nature of the major OM components, such as LPS, and identification of the predominant immunogens should elucidate the mechanisms by which
B. henselae
establishes persistent bacteremic infections within cats. Additionally, the common antigens may serve as potential feline vaccine candidates to eliminate the pathogen from its animal reservoir.
Disassembly of F-Actin Cytoskeleton after Interaction of
Bacillus cereus
with Fully Differentiated Human Intestinal Caco-2 Cells
Authors: Minnaard Jessica , Lievin-Le Moal Vanessa , Coconnier Marie-Helene , Servin Alain L. , Pérez Pablo F.
Published: May 2004

In the present study, the role of direct procaryote-eucaryote interactions in the virulence of
Bacillus cereus
was investigated. As a model of human enterocytes, differentiated Caco-2 cells were used. Infection of fully differentiated Caco-2 cells with
B. cereus
in the exponential phase of growth, in order to minimize the concentration of spores or sporulating microorganisms, shows that a strain-dependent cytopathic effect develops. Interestingly, addition of 3-h-old cultures of some strains resulted in complete detachment of the cultured cells after a 3-h infection whereas no such effect was found after a 3-h infection with 16-h-old cultures. Infection of enterocyte-like cells with
B. cereus
leads to disruption of the F-actin network and necrosis. Even though the effect of secreted factors cannot be ruled out, direct eucaryote-procaryote interaction seems to be necessary. In addition, we observed that some
B. cereus
strains were able to be internalized in Caco-2 cells. Our findings add a new insight into the mechanisms of virulence of
B. cereus
in the context of intestinal infection.
Experimental Pneumococcal Meningitis: Impaired Clearance of Bacteria from the Blood Due to Increased Apoptosis in the Spleen in Bcl-2-Deficient Mice
Authors: Wellmer Andreas , von Mering Matthias , Spreer Annette , Diem Ricarda , Eiffert Helmut , Noeske Christiane , Bunkowski Stefanie , Gold Ralf , Nau Roland
Published: May 2004

Necrotic and apoptotic neuronal cell death can be found in pneumococcal meningitis. We investigated the role of Bcl-2 as an antiapoptotic gene product in pneumococcal meningitis using Bcl-2 knockout (Bcl-2−/−) mice. By using a model of pneumococcal meningitis induced by intracerebral infection, Bcl-2-deficient mice and control littermates were assessed by clinical score and a tight rope test at 0, 12, 24, 32, and 36 h after infection. Then mice were sacrificed, the bacterial titers in blood, spleen, and cerebellar homogenates were determined, and the brain and spleen were evaluated histologically. The Bcl-2-deficient mice developed more severe clinical illness, and there were significant differences in the clinical score at 24, 32, and 36 h and in the tight rope test at 12 and 32 h. The bacterial titers in the blood were greater in Bcl-2-deficient mice than in the controls (7.46 ± 1.93 log CFU/ml versus 5.16 ± 0.96 log CFU/ml [mean ± standard deviation];P< 0.01). Neuronal damage was most prominent in the hippocampal formation, but there were no significant differences between groups. In situ tailing revealed only a few apoptotic neurons in the brain. In the spleen, however, there were significantly more apoptotic leukocytes in Bcl-2-deficient mice than in controls (5,148 ± 3,406 leukocytes/mm2versus 1,070 ± 395 leukocytes/mm2;P< 0.005). Bcl-2 appears to counteract sepsis-induced apoptosis of splenic lymphocytes, thereby enhancing clearance of bacteria from the blood.
Interleukin-10 Limits Local and Body Cavity Inflammation during Infection with Muscle-StageTrichinella spiralis
Authors: Beiting Daniel P. , Bliss Susan K. , Schlafer Donald H. , Roberts Victoria L. , Appleton Judith A.
Published: May 2004

The aim of this study was to characterize cellular responses to muscle-stageTrichinella spiralis. From its intracellular habitat in muscle,T. spiralissecretes potent glycoprotein antigens that elicit a strong systemic host immune response. Despite the magnitude and prolonged nature of this response, nurse cells are rarely destroyed by infiltrating cells. We tested the hypothesis that the anti-inflammatory cytokine interleukin-10 (IL-10) moderates cellular responses to muscle-stage parasites.Trichinellalarvae colonize the diaphragm in large numbers, prompting us to evaluate regional responses in body cavities in addition to local responses in muscle. Mice deficient in IL-10 demonstrated an exaggerated inflammatory response around nurse cells and in the pleural cavity. The effect of IL-10 was most evident 20 days following muscle infection. The increased intensity of the response in IL-10-deficient mice did not affect parasite establishment or survival. Between 20 and 50 days postinfection, the inflammatory response was diminished in both wild-type and IL-10-deficient mice. Muscle infection also elicited an antibody response, characterized initially by mixed isotypes directed at somatic larval antigens and changing to an immunoglobulin G1-dominated response directed at tyvelose-bearing excreted or secreted antigens. We conclude that IL-10 limits local and regional inflammation during the early stages of muscle infection but that chronic inflammation is controlled by an IL-10-independent mechanism that is coincident with a Th2 response.
Borrelia burgdorferi
Binds to, Invades, and Colonizes Native Type I Collagen Lattices
Authors: Zambrano Maria C. , Beklemisheva Anastasia A. , Bryksin Anton V. , Newman Stuart A. , Cabello Felipe C.
Published: May 2004

Borrelia burgdorferi
binds strongly to the extracellular matrix and cells of the connective tissue, a binding apparently mediated by specific proteins and proteoglycans. We investigated the interactions between
B. burgdorferi
cells and intact type I collagen using hydrated lattices that reproduce features of in vivo collagen matrices.
B. burgdorferi
cells of several strains adhered avidly to these acellular matrices by a mechanism that was not mediated by decorin or other proteoglycans. Moreover, following adhesion to these matrices,
B. burgdorferi
grew and formed microcolonies. The collagen used in these studies was confirmed to lack decorin by immunoblot analysis;
B. burgdorferi
cells lacking the decorin adhesin bound readily to intact collagen matrices.
B. burgdorferi
also bound to collagen lattices that incorporated enzymes that degraded glycosaminoglycan chains in any residual proteoglycans. Binding of the bacteria to intact collagen was nonetheless specific, as bacteria did not bind agar and showed only minimal binding to bovine serum albumin, gelatin, pepsinized type I collagen, and intact collagen that had been misassembled under nonphysiological pH and ionic-strength conditions. Proteinase K treatment of
B. burgdorferi
cells decreased the binding, as did a lack of flagella, suggesting that surface-exposed proteins and motility may be involved in the ability of
B. burgdorferi
to interact with intact collagen matrices. The high efficiency of binding of
B. burgdorferi
strains to intact collagen matrices permits replacement of the commonly used isotopic binding assay with visual fluorescent microscopic assays and will facilitate future studies of these interactions.
Comparative Analysis of B- and T-Cell Epitopes ofMycobacterium lepraeandMycobacterium tuberculosisCulture Filtrate Protein 10
Authors: Spencer John S. , Kim Hee Jin , Marques Angela M. , Gonzalez-Juarerro Mercedes , Lima Monica C. B. S. , Vissa Varalakshmi D. , Truman Richard W. , Gennaro Maria Laura , Cho Sang-Nae , Cole Stewart T. , Brennan Patrick J.
Published: May 2004

Culture filtrate protein 10 (CFP-10) fromMycobacterium tuberculosisis a well-characterized immunodominant 10-kDa protein antigen known to elicit a very potent early gamma interferon response in T cells fromM. tuberculosis-infected mice and humans. The sequence of theMycobacterium lepraehomologue of CFP-10 shows only 40% identity (60% homology) at the protein level withM. tuberculosisCFP-10 and thus has the potential for development as a T- or B-cell reactive antigen for specific diagnosis of leprosy. Antisera raised in mice or rabbits against recombinantM. lepraeandM. tuberculosisCFP-10 proteins reacted only with homologous peptides from arrays of overlapping synthetic peptides, indicating that there was no detectable cross-reactivity at the antibody level. Sera from leprosy and tuberculosis patients were also specific for the homologous protein or peptides and showed distinct patterns of recognition for eitherM. lepraeorM. tuberculosisCFP-10 peptides. At the cellular level, only 2 of 45 mouse T-cell hybridomas raised against eitherM. lepraeorM. tuberculosisCFP-10 displayed a cross-reactive response against the N-terminal heterologous CFP-10 peptide, the region that exhibits the highest level of identity in the two proteins; however, the majority of peptide epitopes recognized by mouse T-cell hybridomas specific for each protein did not cross-react with heterologous peptides. Coupled with the human serology data, these results raise the possibility that peptides that could be used to differentiate infections caused by these two related microorganisms could be developed. Immunohistochemical staining of sections ofM. leprae-infected nude mouse footpads resulted in strongly positive staining in macrophages and dendritic cells, as well as weaker staining in extracellular areas, suggesting thatM. lepraeCFP-10, like its homologue inM. tuberculosis, is a secreted protein.
Identification of Acyloxyacyl Hydrolase, a Lipopolysaccharide- Detoxifying Enzyme, in the Murine Urinary Tract
Authors: Feulner J. Amelia , Lu Mingfang , Shelton JohnM. , Zhang Mei , Richardson James A. , Munford Robert S.
Published: May 2004

Acyloxyacyl hydrolase (AOAH) is an unusual but highly conserved lipase, previously described only in myeloid cells, that removes secondary fatty acyl chains from bacterial lipopolysaccharides (LPS) and may also act on various glycero(phospho)lipids. Deacylation by AOAH greatly reduces the ability of LPS to stimulate cells via CD14-MD-2-Toll-like receptor 4. We report here that renal cortical tubule cells produce AOAH and secrete it into urine, where it can deacylate LPS. In vitro studies revealed that proximal tubule cells secrete pro-AOAH, which can be taken up by bladder cells and processed to the heterodimeric, more enzymatically active, mature form of AOAH. AOAH can then be used by the recipient cells to deacylate LPS. The enzyme produced by proximal tubule epithelium may thus be shared with downstream cells. In addition, mature AOAH is found in the urine. We suggest that cortical tubule cells may produce and secrete AOAH to limit inflammatory responses to gram-negative bacteria throughout the urinary tract.
Toll-Like Receptor 4 Expression and Cytokine Responses in the Human Urinary Tract Mucosa
Authors: Samuelsson Patrik , Hang Long , Wullt Björn , Irjala Heikki , Svanborg Catharina
Published: May 2004

Mucosal pathogens trigger a local innate host response by activating epithelial cells. Bacterial adherence and Toll-like receptor 4 (TLR4) signaling have been implicated as key events in this process. This study addressed the molecular basis of the epithelial response to gram-negative infection in the human urinary tract. Mucosal biopsies were obtained from kidneys, ureters, and bladders of patients undergoing urinary tract surgery, and epithelial TLR4 and CD14 expression was examined by immunohistochemistry. TLR4 was detected in epithelial cells lining the entire urinary tract and in the renal tubular epithelium. CD14, in contrast, was completely absent from the epithelial tissue. The response of the epithelial cells to infection was studied by in vitro challenge of the biopsies with uropathogenicEscherichia colibacteria. A rapid cytokine response was observed, with production of interleukin-1β (IL-1β), IL-6, and IL-8 but not of IL-4 or gamma interferon. Adhering, P- or type 1-fimbriatedE. coliactivated IL-6 and IL-8 production more efficiently than the nonfimbriated control, as shown by cellular staining and analysis of secreted cytokines. The results demonstrate that human uroepithelial cells possess the molecular machinery needed to respond to uropathogenicE. coli. This includes recognition receptors for fimbriae and TLR4 for transmembrane signaling. We speculate that the lack of membrane-bound CD14 allows the epithelium to regulate its sensitivity to lipopolysaccharide and to discriminate between more-virulent and less-virulent strains.
Bacterial Superantigen-Treated Intestinal Epithelial Cells Upregulate Heat Shock Proteins 25 and 72 and Are Resistant to Oxidant Cytotoxicity
Authors: Musch Mark W. , Petrof Elaine O. , Kojima Keishi , Ren Hongyu , McKay Derek M. , Chang Eugene B.
Published: May 2004

While the pathological events evoked by infection are commonly described, effective host responses to bacteria and their products should primarily be protective. Heat shock protein (Hsp) expression is upregulated by many stimuli and serves to maintain intracellular protein integrity. The ability of the prototypic superantigen,Staphylococcus aureusenterotoxin B (SEB) to induce Hsps was investigated with BALB/c mice and by in vitro addition to the murine small intestinal epithelial cell line MSIE. SEB-treated (5 or 100 μg intraperitoneally) mice revealed increased Hsp25 and Hsp72, but not Hsc73, in jejunal lymphocytes and epithelial cells. A similar Hsp response to SEB occurred in MSIE cells and was preceded by activation of the ERK1/2 and p38 mitogen-activated protein kinases but not the SAPK/JNK pathway; pharmacological inhibition of ERK1/2, but not p38, significantly reduced SEB-induced Hsps. Moreover, SEB-treated MSIE cells were protected against oxidant-induced cytotoxicity (measured by51Cr release) and F-actin depolymerization. Thus, SEB exposure results in a rapid induction of the Hsp25 and Hsp72 in intestinal epithelial cells, both directly and through lymphocyte activation, and we suggest that this event is important in protecting the gut from damage byStaphylococcusinfection or in the reparatory process and may be a generalized response to lumen-derived bacterial toxins.
Comparative Analysis of EspF from Enteropathogenic and Enterohemorrhagic
Escherichia coli
in Alteration of Epithelial Barrier Function
Authors: Viswanathan V. K. , Koutsouris Athanasia , Lukic Sandra , Pilkinton Mark , Simonovic Ivana , Simonovic Miljan , Hecht Gail
Published: May 2004

Enteropathogenic
Escherichia coli
(EPEC) and enterohemorrhagic
E. coli
(EHEC) are related intestinal pathogens that harbor highly similar pathogenicity islands known as the locus of enterocyte effacement (LEE). Despite their genetic similarity, these two pathogens disrupt epithelial tight junction barrier function with distinct kinetics. EHEC-induced reduction in transepithelial electrical resistance (TER), a measure of barrier function disruption, is significantly slower and more modest in comparison to that induced by EPEC. The variation in bacterial adherence only partially accounted for these differences. The LEE-encoded effector protein EspF has been shown to be critical for EPEC-induced alterations in TER. EspF from both EPEC and EHEC is expressed and secreted upon growth in tissue culture medium. The mutation of EHEC
cesF
suggested that the optimal expression and secretion of EHEC EspF required its chaperone CesF, as has been shown for EPEC. In contrast to EPEC
espF
and
cesF
, mutation of the corresponding EHEC homologs did not dramatically alter the decrease in TER. These differences could possibly be explained by the presence of additional
espF
-like sequences (designated U- and M-
espF
, where the letter designations refer to the specific cryptic prophage sequences on the EHEC chromosome closest to the respective genes) in EHEC. Reverse transcription-PCR analyses revealed coordinate regulation of EHEC U-
espF
and the LEE-encoded
espF
, with enhanced expression in bacteria grown in Dulbecco-Vogt modified Eagle’s medium compared to bacteria grown in Luria broth. Both EHEC
espF
and U-
espF
complemented an EPEC
espF
deletion strain for barrier function alteration. The overexpression of U-
espF
, but not
espF
, in wild-type EHEC potentiated the TER response. These studies reveal further similarities and differences in the pathogenesis of EPEC and EHEC.
Role of Flagellin and the Two-Component CheA/CheY System of
Listeria monocytogenes
in Host Cell Invasion and Virulence
Authors: Dons Lone , Eriksson Emma , Jin Yuxuan , Rottenberg Martin E. , Kristensson Krister , Larsen Charlotte N. , Bresciani José , Olsen John E.
Published: May 2004

The flagellum protein flagellin of
Listeria monocytogenes
is encoded by the
flaA
gene. Immediately downstream of
flaA
, two genes,
cheY
and
cheA
, encoding products with homology to chemotaxis proteins of other bacteria, are located. In this study we constructed deletion mutants with mutations in
flaA. cheY
, and
cheA
to elucidate their role in the biology of infection with
L. monocytogenes
. The Δ
cheY
, Δ
cheA
, and double-mutant Δ
cheYA
mutants, but not Δ
flaA
mutant, were motile in liquid media. However, the Δ
cheA
mutant had impaired swarming and the Δ
cheY
and Δ
cheYA
mutants were unable to swarm on soft agar plates, suggesting that
cheY
and
cheA
genes encode proteins involved in chemotaxis. The Δ
flaA
, Δ
cheY
, Δ
cheA
, and Δ
cheYA
mutants (grown at 24°C) showed reduced association with and invasion of Caco-2 cells compared to the wild-type strain. However, spleens from intragastrically infected BALB/c and C57BL/6 mice showed larger and similar numbers of the Δ
flaA
and Δ
cheYA
mutants, respectively, compared to the wild-type controls. Such a discrepancy could be explained by the fact that tumor necrosis factor receptor p55 deficient mice showed dramatically exacerbated susceptibility to the wild-type but unchanged or only slightly increased levels of the Δ
flaA
or Δ
cheYA
mutant. In summary, we show that listerial
flaA. cheY
, and
cheA
gene products facilitate the initial contact with epithelial cells and contribute to effective invasion but that
flaA
could also be involved in the triggering of immune responses.
Lipopolysaccharide Protects Primary B Lymphocytes from Apoptosis by Preventing Mitochondrial Dysfunction and Bax Translocation to Mitochondria
Authors: Souvannavong Vongthip , Lemaire Christophe , Chaby Richard
Published: May 2004

Mature B lymphocytes undergo apoptosis when they are cultured in the absence of survival factors. Gram-negative bacterial lipopolysaccharide (LPS) prevents this spontaneous apoptosis. This study aimed to better define the signaling pathway(s) involved in the antiapoptotic activity of this endotoxin. We report here that, in addition to its effects on spontaneous apoptosis, LPS protects B cells from apoptosis induced by the broad-spectrum protein kinase inhibitor staurosporine. LPS increased cell viability and concomitantly maintained the mitochondrial transmembrane potential (ΔΨm) and high glutathione levels. Moreover, LPS inhibited cytosolic cytochromecrelease and decreased caspase-9 activation. Unlike staurosporine, LPS induced the retention of Bax, a proapoptotic protein of the Bcl-2 family, in the cytosol by preventing its translocation to mitochondria. These results suggest that Bax relocalization from the cytosol to the mitochondria is an important step of mature B-cell apoptosis and that the antiapoptotic activity of LPS occurs upstream of mitochondrial events.
Enhancement of Anthrax Lethal Toxin Cytotoxicity: a Subset of Monoclonal Antibodies against Protective Antigen Increases Lethal Toxin-Mediated Killing of Murine Macrophages
Authors: Mohamed Nehal , Li Juan , Ferreira Claudia S. , Little Stephen F. , Friedlander Arthur M. , Spitalny George L. , Casey Leslie S.
Published: May 2004

We investigated the ability of using monoclonal antibodies (MAbs) against anthrax protective antigen (PA), an anthrax exotoxin component, to modulate exotoxin cytotoxic activity on target macrophage cell lines. Anthrax PA plays a critical role in the pathogenesis ofBacillus anthracisinfection. PA is the cell-binding component of the two anthrax exotoxins: lethal toxin (LeTx) and edema toxin. Several MAbs that bind the PA component of LeTx are known to neutralize LeTx-mediated killing of target macrophages. Here we describe for the first time an overlooked population of anti-PA MAbs that, in contrast, function to increase the potency of LeTx against murine macrophage cell lines. The results support a possible mechanism of enhancement: binding of MAb to PA on the macrophage cell surface stabilizes the PA by interaction of MAb with macrophage Fcγ receptors. This results in an increase in the amount of PA bound to the cell surface, which in turn leads to an enhancement in cell killing, most likely due to increased internalization of LF. Blocking of PA-receptor binding eliminates enhancement by MAb, demonstrating the importance of this step for the observed enhancement. The additional significance of these results is that, at least in mice, immunization with PA appears to elicit a poly-clonal response that has a significant prevalence of MAbs that enhance LeTx-mediated killing in macrophages.
The LetE Protein Enhances Expression of Multiple LetA/LetS-Dependent Transmission Traits by
Legionella pneumophila
Authors: Bachman Michael A. , Swanson Michele S.
Published: May 2004

Legionella pneumophila
colonizes freshwater amoebae and can also replicate within alveolar macrophages. When their nutrient supply is exhausted, replicating bacteria become cytotoxic, motile, and infectious, which is thought to promote transmission to a new amoeba. The differentiation of
L. pneumophila
is coordinated by the sigma factors RpoS and FliA and the two-component regulator LetA/LetS and is enhanced by the
letE
locus. Here we demonstrate that
letE
promotes motility by increasing expression of the flagellin gene
flaA
but has little impact on the transcription of
fliA
, the flagellar sigma factor gene. In addition to promoting motility,
letE
induces the characteristic shape, pigment, and heat resistance of stationary-phase
L. pneumophila
. To gain insight into how
letE
promotes the expression of the transmission phenotype, we designed molecular genetic experiments to discriminate between the following three models:
letE
mutations are polar on
milX
;
letE
encodes a small novel protein; or, by analogy to
csrB
,
letE
encodes a regulatory RNA that sequesters CsrA to relieve repression. We report that
letE
encodes an activator protein, as it does not complement an
Escherichia coli csrB
mutant, it directs the synthesis of an ∼12-kDa polypeptide, and a
letE
nonsense mutation eliminates function. A monocistronic
letE
RNA is abundant during the exponential phase, and its decay during the stationary phase requires RpoS and LetA/LetS. We also discuss how the LetE protein may interact with LetA/LetS and CsrA to enhance
L. pneumophila
differentiation to a transmissible form.
Cystathionine β-Lyase Is Important for Virulence ofSalmonella entericaSerovar Typhimurium
Authors: Ejim Linda J. , D'Costa Vanessa M. , Elowe Nadine H. , Loredo-Osti J. Concepción , Malo Danielle , Wright Gerard D.
Published: May 2004

The biosynthesis of methionine in bacteria requires the mobilization of sulfur from Cys by the formation and degradation of cystathionine. Cystathionine β-lyase, encoded bymetCin bacteria andSTR3inSchizosaccharomyces pombe, catalyzes the breakdown of cystathionine to homocysteine, the penultimate step in methionine biosynthesis. This enzyme has been suggested to be the target for pyridinamine antimicrobial agents. We have demonstrated, by using purified enzymes from bacteria and yeast, that cystathionine β-lyase is not the likely target of these agents. Nonetheless, an insertional inactivation ofmetCinSalmonella entericaserovar Typhimurium resulted in the attenuation of virulence in a mouse model of systemic infection. This result confirms a previous chemical validation of the Met biosynthetic pathway as a target for the development of antibacterial agents and demonstrates that cystathionine β-lyase is important for bacterial virulence.
Clearance ofCitrobacter rodentiumRequires B Cells but Not Secretory Immunoglobulin A (IgA) or IgM Antibodies
Authors: Maaser Christian , Housley Michael P. , Iimura Mitsutoshi , Smith Jennifer R. , Vallance Bruce A. , Finlay B. Brett , Schreiber John R. , Varki Nissi M. , Kagnoff Martin F. , Eckmann Lars
Published: May 2004

Citrobacter rodentium, a murine model pathogen for human enteropathogenicEscherichia coli, predominantly colonizes the lumen and mucosal surface of the colon and cecum and causes crypt hyperplasia and mucosal inflammation. Mice infected withC. rodentiumdevelop a secretory immunoglobulin A (IgA) response, but the role of B cells or secretory antibodies in host defense is unknown. To address this question, we conducted oralC. rodentiuminfections in mice lacking B cells, IgA, secreted IgM, polymeric Ig receptor (pIgR), or J chain. Normal mice showed peak bacterial numbers in colon and feces at 1 week and bacterial eradication after 3 to 4 weeks. B-cell-deficient mice were equally susceptible initially but could not control infection subsequently. Tissue responses showed marked differences, as infection of normal mice was accompanied by transient crypt hyperplasia and mucosal inflammation in the colon and cecum at 2 but not 6 weeks, whereas B-cell-deficient mice had few mucosal changes at 2 weeks but severe epithelial hyperplasia with ulcerations and mucosal inflammation at 6 weeks. The functions of B cells were not mediated by secretory antibodies, since mice lacking IgA or secreted IgM or proteins required for their transport into the lumen, pIgR or J chain, clearedC. rodentiumnormally. Nonetheless, systemic administration of immune sera reduced bacterial numbers significantly in normal and pIgR-deficient mice, and depletion of IgG abrogated this effect. These results indicate that host defense againstC. rodentiumdepends on B cells and IgG antibodies but does not require production or transepithelial transport of IgA or secreted IgM.
Age-Specific Immunoglobulin G (IgG) and IgA to Pneumococcal Protein Antigens in a Population in Coastal Kenya
Authors: Laine Catherine , Mwangi Tabitha , Thompson Claudette M. , Obiero Jacktone , Lipsitch Marc , Scott J. Anthony G.
Published: May 2004

Streptococcus pneumoniae
is the primary etiological agent of community-acquired pneumonia and a major cause of meningitis and bacteremia. Three conserved pneumococcal proteins—pneumolysin, pneumococcal surface adhesin A (PsaA), and pneumococcal surface protein A (PspA)—are currently being investigated as vaccine candidates. Such protein-based vaccines, if proven effective, could provide a cheaper alternative to conjugate vaccine formulae. Few data from sub-Saharan Africa exist concerning the development of natural antibody to these antigens, however. To investigate the age-specific development of antiprotein immunoglobulin G (IgG) and IgA antibody responses, the sera of 220 persons 2 weeks to 84 years of age from coastal Kenya were assayed using enzyme-linked immunosorbent assays. IgG and IgA antibody responses to each antigen were observed in all age groups. Serum concentrations of IgG and IgA antibody responses to PspA and PdB (a recombinant toxoid derivative of pneumolysin), but not to PsaA, increased significantly with age (
P
< 0.001). No decline was observed in the sera of the elderly. Anti-protein IgG concentrations were only weakly correlated (0.30 <
r
< 0.56;
P
< 0.0001), as were IgA concentrations (0.24 <
r
< 0.54;
P
< 0.0001).
RecombinantMycobacterium bovisBCG Expressing the Sm14 Antigen ofSchistosoma mansoniProtects Mice from Cercarial Challenge
Authors: Varaldo Paula B. , Leite Luciana C. C. , Dias Waldely O. , Miyaji Eliane N. , Torres Fabio I. G. , Gebara Vera C. , Armôa Geraldo R. G. , Campos Adriano S. , Matos Denise C. S. , Winter Nathalie , Gicquel Brigitte , Vilar Mônica M. , McFadden Johnjoe , Almeida Marilia S. , Tendler Miriam , McIntosh Douglas
Published: May 2004

The Sm14 antigen ofSchistosoma mansoniwas cloned and expressed inMycobacterium bovisBCG as a fusion with theMycobacterium fortuitumβ-lactamase protein under the control of its promoter, pBlaF*; the protein was localized in the bacterial cell wall. The rBCG-Sm14 strain was shown to be relatively stable in cultured murine and bovine monocytes in terms of infectivity, bacterial persistence, and plasmid stability. The immunization of mice with rBCG-Sm14 showed no induction of anti-Sm14 antibodies; however, splenocytes of immunized mice released increased levels of gamma interferon upon stimulation with recombinant Sm14 (rSm14), indicating an induction of a Th1-predominant cellular response against Sm14. Mice immunized with one or two doses of rBCG-Sm14 and challenged with liveS. mansonicercaria showed a 48% reduction in worm burden, which was comparable to that obtained by immunization with three doses of rSm14 purified fromEscherichia coli. The data presented here further enhance the status of Sm14 as a promising candidate antigen for the control of schistosomiasis and indicate that a one-dose regimen of rBCG-Sm14 could be considered a convenient means to overcome many of the practical problems associated with the successful implementation of a multiple-dose vaccine schedule in developing countries.
Complement Activation and Complement-Dependent Inflammation byNeisseria meningitidisAre Independent of Lipopolysaccharide
Authors: Sprong Tom , Møller Anne-Sophie W. , Bjerre Anna , Wedege Elisabeth , Kierulf Peter , van der Meer Jos W. M. , Brandtzaeg Petter , van Deuren Marcel , Mollnes T. E.
Published: May 2004

Fulminant meningococcal sepsis has been termed the prototypical lipopolysaccharide (LPS)-mediated gram-negative septic shock. Systemic inflammation by activated complement and cytokines is important in the pathogenesis of this disease. We investigated the involvement of meningococcal LPS in complement activation, complement-dependent inflammatory effects, and cytokine or chemokine production. Whole blood anticoagulated with lepirudin was stimulated with wild-typeNeisseria meningitidisH44/76 (LPS+), LPS-deficientN. meningitidisH44/76lpxA(LPS−), or purified meningococcal LPS (NmLPS) at concentrations that were relevant to meningococcal sepsis. Complement activation products, chemokines, and cytokines were measured by enzyme-linked immunosorbent assays, and granulocyte CR3 (CD11b/CD18) upregulation and oxidative burst were measured by flow cytometry. The LPS+and LPS−N. meningitidisstrains both activated complement effectively and to comparable extents. Purified NmLPS, used at a concentration matched to the amount present in whole bacteria, did not induce any complement activation. Both CR3 upregulation and oxidative burst were also induced, independent of LPS. Interleukin-1β (IL-1β), tumor necrosis factor alpha, and macrophage inflammatory protein 1α production was predominantly dependent on LPS, in contrast to IL-8 production, which was also markedly induced by the LPS−meningococci. In this whole blood model of meningococcal sepsis, complement activation and the immediate complement-dependent inflammatory effects of CR3 upregulation and oxidative burst occurred independent of LPS.
Suppression of Serum Antibody Responses by Pertussis Toxin after Respiratory Tract Colonization byBordetella pertussisand Identification of an Immunodominant Lipoprotein
Authors: Carbonetti Nicholas H. , Artamonova Galina V. , Andreasen Charlotte , Dudley Edward , Mays R. Michael , Worthington Zoe E. V.
Published: May 2004

Pertussis toxin (PT), a virulence factor secreted byBordetella pertussis, contributes to respiratory tract infection and disease caused by this pathogen. By comparing a wild-type (WT)B. pertussisstrain to a mutant strain with an in-frame deletion of theptxgenes encoding PT (ΔPT), we recently found that the lack of PT confers a significant defect in respiratory tract colonization in mice after intranasal inoculation. In this study, we analyzed serum antibody responses in mice infected with the WT or ΔPT strain and found that infection with the ΔPT strain elicited greater responses to severalB. pertussisantigens than did infection with the WT, despite the lower colonization level achieved by the ΔPT strain. The same enhanced antibody response was observed after infection with a strain expressing an enzymatically inactive PT; but this response was not observed after infection withB. pertussismutant strains lacking filamentous hemagglutinin or adenylate cyclase toxin, nor when purified PT was administered with the ΔPT inoculum, indicating a specific role for PT activity in this immunosuppressive effect. In particular, there were consistent strong serum antibody responses to one or more low-molecular-weight antigens after infection with the ΔPT strain. These antigens were Bvg independent, membrane localized, and also expressed by the closely related pathogensBordetella parapertussisandBordetella bronchiseptica. Two-dimensional gel electrophoresis and mass spectrometry were used to identify one of the immunodominant low-molecular-weight antigens as a protein with significant sequence homology to peptidoglycan-associated lipoprotein in several other gram-negative bacterial species. However, a serum antibody response to this protein alone did not protect mice against respiratory tract infection byB. pertussis.