bluuebunny/crossref_metadata_2025 huggingface dataset:
Headache Profile in Patients With Idiopathic Hypertrophic Cranial Pachymeningitis
Authors: Wang Yuh‐Jen , Fuh Jong‐Ling , Lirng Jiing‐Feng , Lu Shiang‐Ru , Wang Shuu‐Jiun
Published: September 2004

Background.—Idiopathic hypertrophic cranial pachymeningitis (IHCP) is an uncommon disorder due to localized or diffuse thickening of the dura mater. While headache is the most common manifestation, the clinical characteristics of the headache in IHCP have not been well characterized.

Methods.—From 1996 to 2002, 6 consecutive patients with IHCP presenting with headache were reviewed (3 women, 3 men; mean age: 49 years). Diagnosis was based on characteristic neuroimaging findings and the exclusion of secondary causes of cranial pachymeningitis. This study reported the headache characteristics, neuroimaging features, and longitudinal follow‐up.

Results.—Chronic daily headache, especially chronic migraine, was the most common headache pattern observed (4/6 patients). Lateralization of headache location appeared to correlate with the distribution of the hypertrophied dural lesions. Diagnostic delay was due to failure to evaluate with gadolinium‐enhanced MRI. After treatment, headache (5/6, 83%) and neurologic deficits (2/3, 67%) improved in most patients; however, follow‐up MRIs (n = 5) showed deterioration in 3 patients.

Conclusion.—The headache of IHCP is typically a chronic daily headache, often resembling chronic migraine. Correspondingly, IHCP should be considered in the differential diagnosis of refractory chronic daily headache, with or without associated cranial neuropathy or other associated neurologic deficits. The typical imaging finding on gadolinium‐enhanced MRI is localized or diffuse pachymeningitis and failure to order a gadolinium‐enhanced MRI is the primary reason for delayed diagnosis. Despite symptomatic improvement on longitudinal follow‐up, the MRI abnormalities may not improve in parallel with the clinical symptoms.
The societal unit cost of allogenic red blood cells and red blood cell transfusion in Canada
Authors: Amin Mo , Fergusson Dean , Wilson Kumanan , Tinmouth Alan , Aziz Ashique , Coyle Doug , Hébert Paul
Published: September 2004

BACKGROUND:  There is a dearth of information about the cost of allogenic red blood cells (RBCs) and RBC transfusion in Canada in the aftermath of the Canadian blood system reorganization and the introduction of various safety measures. The unit cost of allogenic RBCs and RBC transfusion in Canada in 1994 was estimated at US$152.17. The objective of this study was to determine the unit cost of allogenic RBC transfusion in Canada from a societal perspective.

STUDY DESIGN AND METHODS:  A cost‐structure analysis using the cost information from 2001 through 2002 was used. Costs of blood collection, production, distribution, delivery (hospital transfusion service processing and patient administration), transfusion reaction management, and opportunity cost of donor's time were included in the analysis. Canadian Blood Services and Héma‐Québec supplied the data for collection, production, and distribution stages. Delivery and transfusion reaction costs were collected from eight hospitals across six Canadian provinces. In‐patient costs were assessed for the intensive care unit, emergency, general medicine ward, and operating room.

RESULTS:  The aggregate mean societal unit cost of RBCs transfused on an inpatient basis in 2002 was US$264.81 (95% confidence interval [CI], $256.29‐$275.65). The mean cost of blood collection, production, and distribution was US$202.74 (95% CI, $199.63‐$204.31), the mean opportunity cost of donor time was US$18.21 (95% CI, $17.11‐$21.63), the mean cost of hospital transfusion service processing was US$16.65 (95% CI, $13.50‐$19.79), of RBC transfusion was US$26.92 (95% CI, $25.33‐$28.52), and of transfusion reaction management was US$0.29 (95% CI, $0.22‐$0.36). There were substantial variations in hospital transfusion service processing and RBC transfusion costs across hospitals.

CONCLUSION:  The societal unit cost of RBC transfusion has doubled since 1994 to 1995. Further increases in unit costs would be expected as additional safety measures are introduced. This will have important financial implications for treating patient populations that require a high level of RBC transfusions.
Red cell requirements for intensive care units adhering to evidence‐based transfusion guidelines
Authors: Walsh Timothy S. , Garrioch Magnus , Maciver Caroline , Lee Robert J. , MacKirdy Fiona , McClelland D. Brian , Kinsella John , Wallis Charles ,   Audit of Transfusion in Intensive Care in Scotland (ATICS) Study Group
Published: September 2004

BACKGROUND:  Anemia commonly complicates critical illness. Restrictive transfusion triggers are appropriate in this setting, but no large studies have measured red cell (RBC) requirements for intensive care patients when evidence‐based transfusion guidelines are followed consistently.

STUDY DESIGN AND METHODS:  Data were recorded daily for 1023 of 1042 sequential admissions to 10 intensive care units (ICUs) over 100 days. The sample comprised 44 percent of all ICU admissions in Scotland during this period. RBC transfusions and the occurrence of clinically significant hemorrhage were recorded for every ICU day. Transfusion episodes were classified as either associated with or not associated with hemorrhage. Measures of RBC use were derived for the cohort and for Scotland with national audit data.

RESULTS:  A total of 39.5 percent (95% confidence interval [CI], 36.5%‐42.5%) of admissions received transfusions. Eighteen percent of admissions received at least one transfusion associated with hemorrhage and 26 percent received at least one transfusion not associated with hemorrhage. The median (interquartile range) transfusion trigger in the absence of hemorrhage was 78 (73‐78) g/L. The overall mean RBC use was 1.87 (95% CI, 1.79‐1.96) units per admission or 0.34 (95% CI, 0.33‐0.36) units per ICU‐day. Forty‐seven percent of RBCs administered were not associated with clinically significant hemorrhage. Mean RBC requirements for intensive care in Scotland were estimated to be 3950 (95% CI, 3780‐4140) per million‐adult‐population per year. This represented 7 to 8 percent of the Scottish blood supply.

CONCLUSIONS:  Despite evidence‐based transfusion practice, 40 percent of ICU patients receive transfusions, which account for 7 to 8 percent of the national blood supply.
Full siblings impersonating parent/child prove most difficult to discredit with DNA profiling alone
Authors: Fung Wing K. , Wong Dart‐Man , Hu Yue‐Qing
Published: September 2004

DNA profiling is currently the most widely used method for parentage verification, although many forms of it have limitations of some sort. In this paper, a general formula is derived to depict a simple relationship between the probability that a random man and the probably that a male relative of the child, other than the child's father, is excluded from paternity, when the phenotype of the child's mother is unavailable. With this, the possible limitations of a finite set of STR loci in excluding close relatives of the child from paternity are illustrated. Genetically, among the commonly encountered biologic relationships, to exclude a full sibling of the child from paternity if they pose themselves as father and child remains the most difficult.
How regular blood donors explain their behavior
Authors: Belda Suárez Isabel Maria , Fernández‐Montoya Antorio , Rodríguez Fernández Andrés , López‐Berrio Antorio , Cillero‐Peñuela Manuel
Published: September 2004

BACKGROUND:  To understand repeated donation, it is crucial to know its meaning. This issue was explored by asking a sample of regular donors to explain why they maintain this behavior.

STUDY DESIGN AND METHODS:  Discourse analysis was conducted in five groups of regular donors, selected according to the setting and number of their donations (from 1990 to 2001), on their motivation, attitudes, commitment to donation, personality, and self‐concept.

RESULTS:  Three discursive positions were distinguished in relation to the different cultural meanings attributed to blood donation. Blood donation was understood in a rational and evaluative manner by the majority but in an emotional, personalized, and stereotyped manner by a minority. Continual donation as a form of help was rationalized as a function of internal and external factors, notably personal convenience in comparison to other helping behaviors and ease of access to collection points.

CONCLUSIONS:  The discourse referents offered to the donor greatly influence the meanings they attribute to their behavior. Some promotional materials should be revised because they may not connect with the reasons for donating that people find most comprehensible.
Photochemical treatment of platelet concentrates with amotosalen and long‐wavelength ultraviolet light inactivates a broad spectrum of pathogenic bacteria
Authors: Lin Lily , Dikeman Roberta , Molini Barbara , Lukehart Sheila A. , Lane Robert , Dupuis Kent , Metzel Peyton , Corash Laurence
Published: September 2004

BACKGROUND: Bacterial contamination of platelet (PLT) concentrates can result in transfusion‐transmitted sepsis. A photochemical treatment (PCT) process with amotosalen HCl and long‐wavelength ultraviolet light (UVA), which cross‐links nucleic acids, was developed to inactivate bacteria and other pathogens in PLT concentrates.

STUDY DESIGN AND METHODS: High titers of pathogenic aerobic and anaerobic Gram‐positive bacteria (10 species), aerobic Gram‐negative bacteria (7 species), and spirochetes (2 species) were added to single‐donor PLT concentrates containing 3.0 × 1011to 6.0 × 1011PLTs in approximately 300 mL of 35 percent plasma and 65 percent PLT additive solution (InterSol, Baxter Healthcare) or saline. After PCT with 150 µmol per L amotosalen and 3 J per cm2UVA, residual bacterial levels were detected by sensitive microbiologic methods.

RESULTS: The level of inactivation of viable bacteria was expressed as log reduction. Log reduction of Gram‐positive bacteria forStaphylococcus epidermidiswas > 6.6; forStaphylococcus aureus,6.6; forStreptococcus pyogenes,> 6.8; forListeria monocytogenes,> 6.3; forCorynebacterium minutissimum,> 6.3; forBacillus cereus(vegetative), > 5.5; forLactobacillussp., > 6.4; forBifidobacterium adolescentis,> 6.0; forPropionibacterium acnes,> 6.2; and forClostridium perfringens,> 6.5. Log reduction of Gram‐negative bacteria forEscherichia coliwas > 6.4; forSerratia marcescens,> 6.7; forKlebsiella pneumoniae,> 5.6; forPseudomonas aeruginosa,4.5; forSalmonella choleraesuis,> 6.2; forYersinia enterocolitica,> 5.9; and forEnterobacter cloacae,5.9. Log reduction of spirochetes forTreponema pallidumwas 6.8 to 7.0, and forBorrelia burgdorferi,> 6.9.

CONCLUSION: PCT inactivates high levels of a broad spectrum of pathogenic bacteria. The inactivation of bacteria in PLT concentrates offers the potential to prospectively prevent PLT‐transfusion‐associated bacteremia.
In vitro testing of fresh and lyophilized reconstituted human and baboon platelets
Authors: Valeri C.R. , Macgregor H. , Barnard M.R. , Summaria L. , Michelson A.D. , Ragno G.
Published: September 2004

BACKGROUND:  Studies have been performed on human fresh, liquid‐preserved, and cryopreserved platelets (PLTs) to assess PLT‐adhesive surface receptors, PLT membrane procoagulant activity, PLT aggregation, and thromboxane production. Lyophilization has been developed as a method to preserve PLTs. This study was performed to evaluate these measurements on human and baboon fresh and lyophilized reconstituted PLTs.

STUDY DESIGN AND METHODS:  In both human and baboon fresh and lyophilized PLTs, aggregation response and PLT production of thromboxane A2 were measured after stimulation, and PLT surface markers P‐selectin, glycoprotein (GP) Ib, GPIIb‐IIIa, and factor (F) V were measured before and after stimulation.

RESULTS:  Fresh PLTs responded to the dual agonists arachidonic acid and adenosine diphosphate (ADP) to aggregate and produce thromboxane A2, and in both the PLT surface markers P‐selectin and GPIIb‐IIIa increased and GPIb decreased after stimulation. Neither human nor baboon lyophilized reconstituted PLTs aggregated to dual agonists, and neither produced thromboxane A2, increased PLT surface markers P‐selectin or GPIIb‐IIIa, or decreased PLT GPIb after stimulation. Nevertheless, after recalcification the lyophilized reconstituted PLTs accumulated FV to a significantly greater degree than fresh PLTs.

CONCLUSIONS:  Lyophilized reconstituted PLTs exhibited modification of the PLT membrane that interfered with aggregation and thromboxane production, prevented increases in PLT P‐selectin and GPIIb‐IIIa and decreases in GPIb after stimulation, and increased FV accumulation after recalcification. The in vitro data suggest that lyophilized PLTs may have reduced in vivo survival. In vivo studies are needed to determine the survival and function of lyophilized PLTs.
The independent association of massive blood loss with mortality in cardiac surgery
Authors: Karkouti Keyvan , Wijeysundera Duminda N. , Yau Terrence M. , Beattie W. Scott , Abdelnaem Esamelden , McCluskey Stuart A. , Ghannam Mohammed , Yeo Eric , Djaiani George , Karski Jacek
Published: September 2004

BACKGROUND:  Although the association between massive perioperative blood loss (MBL) and adverse outcomes is well recognized, it is unclear whether MBL is an independent risk factor or, instead, simply a marker for other adverse events or severity of illness. The objective of this cohort study was to quantify the independent association of MBL in cardiac surgery with all‐cause in‐hospital mortality.

STUDY DESIGN AND METHODS:  Data were prospectively collected on consecutive patients who underwent cardiac surgery with cardiopulmonary bypass at a quaternary‐care academic center from 1999 to 2003. The number of red blood cell (RBC) units transfused within 1 day of surgery was used as a surrogate measure of perioperative blood loss. Receiver‐operating characteristic curve analyses were employed to identify the most appropriate cutoff for defining MBL. The independent association of MBL with mortality was determined with multivariable logistic regression analyses. Bootstrapping and sensitivity analyses were used to confirm the validity of the results.

RESULTS:  MBL was defined as receiving at least 5 units of RBCs within 1 day of surgery. Of 9215 patients analyzed, 1.8 percent (n = 169) died and 9.7 percent (n = 890) had MBL. After adjusting for multiple potential confounders (including perioperative adverse events), MBL was associated with an 8.1‐fold (95% confidence interval, 3.9‐17.0) increase in the odds of death. This risk estimate was stable across different modeling conditions as well as in bootstrap sampling.

CONCLUSION:  MBL after cardiac surgery has a strong, independent association with in‐hospital mortality.
Quantifying losses to the donated blood supply due to donor deferral and miscollection
Authors: Custer Brian , Johnson Eric S. , Sullivan Sean D. , Hazlet Tom K. , Ramsey Scott D. , Hirschler Nora V. , Murphy Edward L. , Busch Michael P.
Published: September 2004

BACKGROUND:  Donors are deferred for multiple reasons. Losses related to disease marker rates are well established. Donor and donation losses for other reasons, however, have not been extensively quantified.

STUDY DESIGN AND METHODS:  To quantify these losses, three data sets from the Blood Centers of the Pacific were combined, permitting detailed analysis of year 2000 allogeneic whole‐blood donations.

RESULTS:  During 2000, 13.6 percent of 116,165 persons who presented for donation were deferred at presentation. Short‐term deferral accounted for 68.5 percent (hematocrit was most common at 60%); long‐term deferral accounted for 21 percent (travel to a malarial area and tattoo or other nonintravenous drug use needle exposure were most common at 59 and 29%, respectively); and multiple‐year or permanent deferral accounted for 10.5 percent (UK travel [variant Creutzfeldt‐Jakob disease] risk and emigration from a malarial area were most common at 38 and 11%, respectively). Disease‐marker‐reactive donations represented 0.9 percent of donor outcomes. The prevalence of deferral and also miscollection (under‐ and overweight units) varied by age, sex, and first‐time versus repeat donor status. Overall, miscollection led to a loss of 3.8 percent of 100,141 collections, ranging from 1.9 percent in repeat male donors 40 to 54 years of age to 10.7 percent in first‐time female donors 16 to 24 years of age.

CONCLUSION:  Loss of units from both first‐time and repeat donors due to temporary deferral and loss of units from miscollection are more common events than losses due to disease marker testing. Some of these losses may be avoidable and could increase the blood supply without having to recruit new donors.
A Framework for Facilitating Sourcing and Allocation Decisions for Make‐to‐Order Items
Authors: Murthy Nagesh N. , Soni Samit , Ghosh Soumen
Published: September 2004

This paper provides a fundamental building block to facilitate sourcing and allocation decisions for make‐to‐order items. We specifically address the buyer's vendor selection problem for make‐to‐order items where the goal is to minimize sourcing and purchasing costs in the presence of fixed costs, shared capacity constraints, and volume‐based discounts for bundles of items. The potential suppliers for make‐to‐order items provide quotes in the form of single sealed bids or participate in a dynamic auction involving open bids. A solution to our problem can be used to determine winning bids amongst the single sealed bids or winners at each stage of a dynamic auction. Due to the computational complexity of this problem, we develop a heuristic procedure based on Lagrangian relaxation technique to solve the problem. The computational results show that the procedure is effective under a variety of scenarios. The average gap across 2,250 problem instances is 4.65%.
A Bayesian Network Estimation of the Service‐Profit Chain for Transport Service Satisfaction
Authors: Anderson Ronald D. , Mackoy Robert D. , Thompson Vincent B. , Harrell Gilbert
Published: September 2004

Bayesian network methodology is used to model key linkages of the service‐profit chain within the context of transportation service satisfaction. Bayesian networks offer some advantages for implementing managerially focused models over other statistical techniques designed primarily for evaluating theoretical models. These advantages are (1) providing a causal explanation using observable variables within a single multivariate model, (2) analysis of nonlinear relationships contained in ordinal measurements, (3) accommodation of branching patterns that occur in data collection, and (4) the ability to conduct probabilistic inference for prediction and diagnostics with an output metric that can be understood by managers and academics. Sample data from 1,101 recent transport service customers are utilized to select and validate a Bayesian network and conduct probabilistic inference.
The Impact of Organizational Culture on Time‐Based Manufacturing and Performance
Authors: Nahm Abraham Y. , Vonderembse Mark A. , Koufteros Xenophon A.
Published: September 2004

Firms are utilizing an array of manufacturing practices in their quest for survival and success in the marketplace. The implementation of those practices has not always resulted in success stories as the focus had been mostly on technical issues, with little concern for “soft issues.” For example, the enabling role of organizational culture has often been ignored. Using Schein's conceptualization of culture as underlying assumptions, espoused values, and artifacts, we examine a framework that relates culture and manufacturing practices to performance. The underlying assumption of customer orientation is posited to affect espoused values such as beliefs on investing in facilities and equipment to leverage intellectual work and to promote creativity, beliefs on working with others, beliefs on making decisions that are global, beliefs on management control, and beliefs on integrating with suppliers. The espoused values are hypothesized to affect visible attributes of culture (behaviors) such as time‐based manufacturing practices, which firms are employing for competitive advantage. A sample of 224 firms is used for developing research instruments and testing the hypothesized relationships advanced. Results indicate that high levels of customer orientation lead to a set of managerial beliefs that are collaborative and integrative. In turn, certain espoused values support a high level of time‐based manufacturing practice, which leads to high performance.
Quantity Flexibility Contracts: Optimal Decisions with Information Updates*
Authors: Sethi Suresh P. , Yan Houmin , Zhang Hanqin
Published: September 2004

We study single and multiperiod quantity flexibility contracts involving one demand forecast update in each period and a spot market. We obtain the optimal order quantity at the beginning of a period and order quantities on contract and from the spot market at the then prevailing price after the forecast revision and before the demand materialization. The amount that can be purchased on contract is bounded by a given flexibility limit. We discuss the impact of the forecast quality and the level of flexibility on the optimal decisions and managerial insights behind the results.
The
vimE
Gene Downstream of
vimA
Is Independently Expressed and Is Involved in Modulating Proteolytic Activity in
Porphyromonas gingivalis
W83
Authors: Vanterpool Elaine , Roy Francis , Fletcher Hansel M.
Published: September 2004

Regulation/activation of the
Porphyromonas gingivalis
gingipains is poorly understood. A unique 1.3-kb open reading frame downstream of the
bcp-recA-vimA
transcriptional unit was cloned, insertionally inactivated with the
ermF-ermAM
antibiotic resistance cassette, and used to create a defective mutant by allelic exchange. In contrast to the wild-type W83 strain, the growth rate of the mutant strain (designated FLL93) was reduced, and when plated on
Brucella
blood agar it was nonpigmented and nonhemolytic. Arginine- and lysine-specific gingipain activities were reduced by approximately 90 and 85%, respectively, relative to activities of the parent strain. These activities were unaffected by the culture's growth phase, in contrast to the
vimA
-defective mutant
P. gingivalis
FLL92, which has increased proteolytic activity in stationary phase. Expression of the
rgpA
,
rgpB
, and
kgp
gingipain genes was unaltered in
P. gingivalis
FLL93 compared to that of the wild-type strain. Further, in extracellular protein fractions a 64-kDa band was identified that was immunoreactive with the RgpB-specific proenzyme antibodies. Active-site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone or immunoblot analysis showed no detectable protein band representing the gingipain catalytic domain. In vitro protease activity could be slightly induced by a urea denaturation-renaturation cycle in an extracellular protein fraction, in contrast to the
vimA
-defective mutant
P. gingivalis
FLL92. Expression of flanking genes, including
recA
,
vimA
, and
Pg079
2, was unaltered by the mutation. Taken together, these results suggest that the
vimA
downstream gene, designated
vimE
(for virulence-modulating gene E), is involved in the regulation of protease activity in
P. gingivalis
.
A Bicistronic DNA Vaccine Containing Apical Membrane Antigen 1 and Merozoite Surface Protein 4/5 Can Prime Humoral and Cellular Immune Responses and Partially Protect Mice against VirulentPlasmodium chabaudi adamiDS Malaria
Authors: Rainczuk A. , Scorza T. , Spithill T. W. , Smooker P. M.
Published: September 2004

The ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. In order to efficiently deliver multiple antigens with use of DNA vaccine technology, new antigen delivery systems must be assessed. This study utilized a bicistronic vector construct, containing an internal ribosome entry site, expressing a combination of malarial candidate antigens: merozoite surface protein 4/5 (MSP4/5) (fused to a monocyte chemotactic protein 3 chemoattractant sequence) and apical membrane antigen 1 (AMA-1) (fused to a tissue plasminogen activator secretion signal). Transfection of COS 7 cells with bicistronic plasmids resulted in production and secretion of both AMA-1 and MSP4/5 in vitro. Vaccination of BALB/c mice via intraepidermal gene gun and intramuscular routes against AMA-1 and MSP4/5 resulted in antibody production and significant in vitro proliferation of splenocytes stimulated by both AMA-1 and MSP4/5. Survival of BALB/c mice vaccinated with bicistronic constructs after lethalPlasmodium chabaudi adamiDS erythrocytic-stage challenge was variable, although significant increases in survival and reductions in peak parasitemia were observed in several challenge trials when the vaccine was delivered by the intramuscular route. This study using a murine model demonstrates that the delivery of malarial antigens via bicistronic vectors is feasible. Further experimentation with bicistronic delivery systems is required for the optimization and refinement of DNA vaccines to effectively prime protective immune responses against malaria.
Microarray Analysis of Pneumococcal Gene Expression during Invasive Disease
Authors: Orihuela Carlos J. , Radin Jana N. , Sublett Jack E. , Gao Geli , Kaushal Deepak , Tuomanen Elaine I.
Published: September 2004

Streptococcus pneumoniae
is a leading cause of invasive bacterial disease. This is the first study to examine the expression of
S. pneumoniae
genes in vivo by using whole-genome microarrays available from The Institute for Genomic Research. Total RNA was collected from pneumococci isolated from infected blood, infected cerebrospinal fluid, and bacteria attached to a pharyngeal epithelial cell line in vitro. Microarray analysis of pneumococcal genes expressed in these models identified body site-specific patterns of expression for virulence factors, transporters, transcription factors, translation-associated proteins, metabolism, and genes with unknown function. Contributions to virulence predicted for several unknown genes with enhanced expression in vivo were confirmed by insertion duplication mutagenesis and challenge of mice with the mutants. Finally, we cross-referenced our results with previous studies that used signature-tagged mutagenesis and differential fluorescence induction to identify genes that are potentially required by a broad range of pneumococcal strains for invasive disease.
Humoral Immunity through Immunoglobulin M Protects Mice from an Experimental Actinomycetoma Infection byNocardia brasiliensis
Authors: Salinas-Carmona Mario C. , Pérez-Rivera Isabel
Published: September 2004

An experimental model of infection withNocardia brasiliensis, used as an example of a facultative intracellular pathogen, was tested.N. brasiliensiswas injected into the rear foot pads of BALB/c mice to establish an infection. Within 30 days, infected animals developed a chronic actinomycetoma infection. Batch cultures ofN. brasiliensiswere used to purify P61, P38, and P24 antigens; P61 is a catalase, and P38 is a protease with strong caseinolytic activity. Active and passive immunizations of BALB/c mice with these three purified soluble antigens were studied. Protection was demonstrated for actively immunized mice. However, immunity lasted only 30 days. Other groups of immunized mice were bled at different times, and their sera were passively transferred to naive recipients that were then infected withN. brasiliensis. Sera collected 5, 6, and 7 days after donor immunization conferred complete, long-lasting protection. The protective effect of passive immunity decreased when sera were collected 2 weeks after donor immunization. However, neither the early sera (1-, 2-, and 3-day sera) nor the later sera (30- or 45-day sera) prevented the infection. Hyperimmune sera with the highest levels of immunoglobulin G (IgG) toN. brasiliensisantigens did not protect at all. The antigens tested induced two IgM peaks. The first peak was present 3 days after immunization but was not antigen specific and did not transfer protection. The second peak was evident 7 days after immunization, was an IgM response, was antigen specific, and conferred protection. This results clearly demonstrate that IgM antibodies protect the host against a facultative intracellular bacterium.
Structural Organization of the pFra Virulence-Associated Plasmid of Rhamnose-Positive
Yersinia pestis
Authors: Golubov Andrey , Neubauer Heinrich , Nölting Christina , Heesemann Jürgen , Rakin Alexander
Published: September 2004

The
137,036-bp plasmid pG8786 from rhamnose-positive
Yersinia
pestis
G8786 isolated from the high mountainous Caucasian plague
focus in Georgia is an enlarged form of the pFra virulence-associated
plasmid containing genes for synthesis of the antigen fraction 1 and
phospholipase D. In addition to the completely conserved genes of the
pFra backbone, pG8786 contains two large regions consisting of 4,642
and 32,617 bp, designated regions 1 and 2, respectively. Region 1
retains a larger part of
Salmonella enterica
serovar Typhi
plasmid pHCM2 resembling the backbone of pFra replicons, while region 2
contains 25 open reading frames with high levels of similarity to the
transfer genes of the F-like plasmids. Surprisingly, region 1 is also
present in the pFra plasmid of avirulent
Y. pestis
strain
91001 isolated in Inner Mongolia, People's Republic of China.
Despite the fact that some genes typically involved in conjugative
transfer of the F-like replicons are missing in pG8786, we cannot
exclude the possibility that pG8786 might be transmissive under certain
conditions. pG8786 seems to be an ancient form of the pFra group of
plasmids that were conserved due to the strict geographical isolation
of rhamnose-positive
Y. pestis
strains in the high mountainous
Caucasian plague
locus.
Pseudomonas aeruginosa relA
Contributes to Virulence in
Drosophila melanogaster
Authors: Erickson David L. , Lines J. Louise , Pesci Everett C. , Venturi Vittorio , Storey Douglas G.
Published: September 2004

The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme. We investigated the role of the
relA
gene in the ability of an extracellular pathogen,
Pseudomonas aeruginosa
, to cause infection. Strains lacking the
relA
gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems. The absence of
relA
abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. We found that strains lacking
relA
exhibited reduced virulence in a
D. melanogaster
feeding assay. In conditions of low magnesium, the
relA
gene enhanced production of the cell-cell signal
N
-[3-oxododecanoyl]-
l
-homoserine lactone, whereas
relA
reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response. In the
relA
mutant, alterations in the
Pseudomonas
quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase. Deletion of
relA
also resulted in reduced levels of the RpoS sigma factor. These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in
P. aeruginosa
adaptation in pathogenic relationships.
The ClpP Protease of
Streptococcus pneumoniae
Modulates Virulence Gene Expression and Protects against Fatal Pneumococcal Challenge
Authors: Kwon Hyog-Young , Ogunniyi A. David , Choi Moo-Hyun , Pyo Suhk-Neung , Rhee Dong-Kwon , Paton James C.
Published: September 2004

Streptococcus pneumoniae
usually colonizes the nasopharynx of humans asymptomatically but occasionally translocates from this niche to the lungs, the brain, and the blood, causing potentially fatal infections. Spread to other host tissues requires a significant morphological change and the expression of virulence factors, such as capsular polysaccharide, and virulence proteins, such as pneumolysin (Ply), PspA, and CbpA. Modulation of the expression of pneumococcal virulence genes by heat shock and by heat shock proteins ClpL and ClpP, as well as the attenuation of virulence of a
clpP
mutant in a murine intraperitoneal infection model, was demonstrated previously. In this study, we further investigated the underlying mechanism of virulence attenuation by the
clpP
mutation. The half-lives of the mRNAs of
ply
and of the first gene of the serotype 2 capsule synthesis locus [
cps(2)A
] in the
clpP
mutant were more than twofold longer than those of the parent after heat shock, suggesting that the mRNA species were regulated posttranscriptionally by ClpP. In addition, the
clpP
mutant was defective in colonization of the nasopharynx and survival in the lungs of mice after intranasal challenge. The mutant was also killed faster than the parent in the murine macrophage RAW264.7 cell line, indicating that ClpP is required for colonization and intracellular survival in the host. Furthermore, fractionation studies demonstrated that ClpP was translocated into the cell wall after heat shock, and immunization of mice with ClpP elicited a protective immune response against fatal systemic challenge with
S. pneumoniae
D39, making ClpP a potential vaccine candidate for pneumococcal disease.
Internalization of
Staphylococcus aureus
by Human Keratinocytes
Authors: Kintarak Sompid , Whawell Simon A. , Speight Paul M. , Packer Samantha , Nair Sean P.
Published: September 2004

Staphylococcus aureus
is among the most important human pathogens and causes various superficial and systemic infections. The ability of
S. aureus
to be internalized by, and survive within, host cells, such as keratinocytes, may contribute to the development of persistent or chronic infections and may finally lead to deeper tissue infections or dissemination. To examine the mechanisms of internalization of
S. aureus
by keratinocytes, isogenic mutants lacking fibronectin-binding proteins (FnBPs), a recombinant protein consisting of the fibronectin-binding domain of
S. aureus
FnBPs, and an anti-α5β1 antibody were used in cocultures with immortalized keratinocytes and primary keratinocytes. We found that internalization of
S. aureus
by immortalized keratinocytes requires bacterial FnBPs and is mediated by the major fibronectin-binding integrin α5β1. In contrast to internalization by immortalized keratinocytes, internalization of
S. aureus
by primary keratinocytes could occur through FnBP-dependent and -independent pathways.
S. aureus
clumping factor B (ClfB), which was recently determined to bind to epithelial cells, was not involved in the uptake of this bacterium by keratinocytes. The identification of an alternate uptake pathway, which is independent of
S. aureus
FnBPs and host cell α5β1, has important implications for the design of therapies targeted to bacterial uptake by host cells.
The Glycan-Rich Outer Layer of the Cell Wall ofMycobacterium tuberculosisActs as an Antiphagocytic Capsule Limiting the Association of the Bacterium with Macrophages
Authors: Stokes Richard W. , Norris-Jones Raymond , Brooks Donald E. , Beveridge Terry J. , Doxsee Dan , Thorson Lisa M.
Published: September 2004

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that infects macrophages and other host cells. We show that sonication ofM. tuberculosisresults in the removal of material from the surface capsule-like layer of the bacteria, resulting in an enhanced propensity of the bacteria to bind to macrophages. This effect is observed with disparate murine and human macrophage populations though, interestingly, not with freshly explanted alveolar macrophages. Enhanced binding to macrophages following sonication is significantly greater within members of theM. tuberculosisfamily (pathogens) than within theMycobacterium aviumcomplex (opportunistic pathogens) or forMycobacterium smegmatis(saprophyte). Sonication does not affect the viability or the surface hydrophobicity ofM. tuberculosisbut does result in changes in surface charge and in the binding of mannose-specific lectins to the bacterial surface. The increased binding of sonicatedM. tuberculosiswas not mediated through complement receptor 3. These results provide evidence that the surface capsule on members of theM. tuberculosisfamily may be an important virulence factor involved in the survival ofM. tuberculosisin the mammalian host. They also question the view thatM. tuberculosisis readily ingested by any macrophage it encounters and support the contention thatM. tuberculosis, like many other microbial pathogens, has an antiphagocytic capsule that limits and controls the interaction of the bacterium with macrophages.
The Down-Regulation of Cathepsin G in THP-1 Monocytes after Infection withMycobacterium tuberculosisIs Associated with Increased Intracellular Survival of Bacilli
Authors: Rivera-Marrero Carlos A. , Stewart Julie , Shafer William M. , Roman Jesse
Published: September 2004

Cathepsin G (CatG) is a serine protease found in the azurophilic granules of monocytes that is known to have antimicrobial properties, but its role inMycobacterium tuberculosisinfection is unknown. We found thatM. tuberculosisinfection of human THP-1 monocytic cells induced the down-regulation of CatG mRNA expression, as demonstrated by gene array analysis and reverse transcription-PCR. This was associated with a concomitant decrease in CatG protein and enzymatic activity. In contrast, the expression of lysosomal cathepsins B and D was up-regulated in infected cells. This effect was also observed when THP-1 cells were induced to differentiate into adherent macrophages by exposure to bacterial lipopolysaccharide (LPS). In agreement with this, CatG expression was null in adherent macrophages isolated from bronchoalveolar lavages and normal blood. We wanted to determine if the down-regulation of CatG would be relevant toM. tuberculosisinfection. First, we found that addition of CatG to THP-1 cells prior to infection resulted in decreased bacillary viability, presumably due to extracellular killing of bacilli. However, pretreatment of cells with LPS, which decreases intracellular CatG expression, resulted in increased bacillary viability. Second, we found that CatG cationic peptides killedM. tuberculosisbacilli and were five- to sevenfold more bactericidal than full-length CatG. These observations suggest thatM. tuberculosisinfection of human monocytic cells results in a “cathepsin switch” with down-regulation of CatG renderingM. tuberculosisbacilli more viable. Therefore, the down-regulation of CatG in macrophages is advantageous toM. tuberculosisbacilli and possibly is an important mechanism by whichM. tuberculosisis able to evade the host immune defenses.
Neither Neutrophils nor Reactive Oxygen Species Contribute to Tissue Damage duringPneumocystisPneumonia in Mice
Authors: Swain Steve D. , Wright Terry W. , Degel Peter M. , Gigliotti Francis , Harmsen Allen G.
Published: September 2004

Neutrophils are implicated in the damage of lung tissue in many disease states, including infectious diseases and environmental insults. These effects may be due to oxidative or nonoxidative functions of the neutrophil or both. We examined the role of neutrophils in pulmonary damage during infection with the opportunistic fungal pathogenPneumocystissp. in four mouse models of neutrophil dysfunction. These were (i) a knockout of the gp91phoxcomponent of NADPH oxidase, in which reactive oxygen species (ROS) production is greatly reduced; (ii) a double knockout of gp91phoxand inducible nitric oxide synthase, in which ROS and nitric oxide production is greatly decreased; (iii) a knockout of the chemokine receptor CXCR2, in which accumulation of intra-alveolar neutrophils is severely diminished; and (iv) antibody depletion of circulating neutrophils in wild-type mice with the monoclonal antibody RB6. Surprisingly, in each case, indicators of pulmonary damage (respiratory rates, arterial oxygen partial pressures, and intra-alveolar albumin concentrations) were the same in knockout mice and comparable wild-type mice. Therefore, whereas neutrophils are a valid correlative marker of lung damage duringPneumocystisinfection, neither neutrophils nor ROS appear to be the causative agent of tissue damage. We also show that there is no difference inPneumocystisburdens between wild-type and knockout mice, which supports the idea that neutrophils do not have a major role in the clearance of this organism.
NF-κB- and AP-1-Mediated Induction of Human Beta Defensin-2 in Intestinal Epithelial Cells byEscherichia coliNissle 1917: a Novel Effect of a Probiotic Bacterium
Authors: Wehkamp Jan , Harder Jürgen , Wehkamp Kai , Meissner Birte Wehkamp-von , Schlee Miriam , Enders Corinne , Sonnenborn Ulrich , Nuding Sabine , Bengmark Stig , Fellermann Klaus , Schröder Jens Michael , Stange Eduard F.
Published: September 2004

Little is known about the defensive mechanisms induced in epithelial cells by pathogenic versus probiotic bacteria. The aim of our study was to compare probiotic bacterial strains such asEscherichia coliNissle 1917 with nonprobiotic, pathogenic and nonpathogenic bacteria with respect to innate defense mechanisms in the intestinal mucosal cell. Here we report thatE. colistrain Nissle 1917 and a variety of other probiotic bacteria, including lactobacilli—in contrast to more than 40 differentE. colistrains tested—strongly induce the expression of the antimicrobial peptide human beta-defensin-2 (hBD-2) in Caco-2 intestinal epithelial cells in a time- and dose-dependent manner. Induction of hBD-2 throughE. coliNissle 1917 was further confirmed by activation of the hBD-2 promoter and detection of the hBD-2 peptide in the culture supernatants ofE. coliNissle 1917-treated Caco-2 cells. Luciferase gene reporter analyses and site-directed mutagenesis experiments demonstrated that functional binding sites for NF-κB and AP-1 in the hBD-2 promoter are required for induction of hBD-2 throughE. coliNissle 1917. Treatment with the NF-κB inhibitor Helenalin, as well as with SP600125, a selective inhibitor of c-Jun N-terminal kinase, blocked hBD-2 induction byE. coliNissle 1917 in Caco-2 cells. SB 202190, a specific p38 mitogen-activated protein kinase inhibitor, and PD 98059, a selective inhibitor of extracellular signal-regulated kinase 1/2, were ineffective. This report demonstrates that probiotic bacteria may stimulate the intestinal innate defense through the upregulation of inducible antimicrobial peptides such as hBD-2. The induction of hBD-2 may contribute to an enhanced mucosal barrier to the luminal bacteria.
CD28 Costimulation Is Required for the Expression of T-Cell-Dependent Cell-Mediated Immunity against Blood-StagePlasmodium chabaudiMalaria Parasites
Authors: Rummel Thomas , Batchelder Joan , Flaherty Patrick , LaFleur GayeLyn , Nanavati Payal , Burns James M. , Weidanz William P.
Published: September 2004

Mice suppress the parasitemia of acute blood-stagePlasmodium chabaudimalaria by an antibody- or T-cell-dependent cell-mediated mechanism of immunity (AMI and CMI, respectively) or by both mechanisms. To determine whether CD28 costimulation is required for expression of these polar immune responses, we first compared the time courses ofP. chabaudimalaria in CD28-deficient (CD28−/−) and CD28-intact (CD28+/+) mice. Acute infections in both knockout (KO) and control mice followed similar time courses, with the period of descending parasitemia being prolonged ∼2 weeks in KO mice followed by intermittent low-grade chronic parasitemia. Infected CD28−/−mice produced primarily the immunoglobulin M antibody, which upon passive transfer provided partial protection againstP. chabaudichallenge, suggesting that the elimination of blood-stage parasites by CD28−/−mice was achieved by AMI. To determine whether CD28−/−costimulation is required for the expression of CMI against the parasite, we compared the time courses of parasitemia in B-cell-deficient double-KO (JH−/−× CD28−/−) mice and control (JH−/−× CD28+/+) mice. Whereas control mice suppressed parasitemia to subpatent levels within ∼2 weeks postinoculation, double-KO mice developed high levels of parasitemia of long-lasting duration. Although not required for the suppression of acuteP. chabaudiparasitemia by AMI, CD28 costimulation is essential for the elimination of blood-stage parasites by CMI.
Reduced Expression of Gamma Interferon in Serum and Marked Lymphoid Depletion Induced by
Porphyromonas gingivalis
Increase Murine Morbidity and Mortality due to Cytomegalovirus Infection
Authors: Stern Jacob , Shai Ela , Zaks Batia , Halabi Amal , Houri-Haddad Yael , Shapira Lior , Palmon Aaron
Published: September 2004

Porphyromonas gingivalis
, a gram-negative anaerobe, is a major etiological agent of severe forms of periodontal disease. Although periodontal disease is considered a localized disease, accumulating evidence indicates that it may lead to a predisposition to a decline in immunocompetence. Human cytomegalovirus (CMV) commonly infects all human populations without producing significant clinical symptoms. Immunocompromised patients usually develop a primary or reactivated CMV infection, which is associated with high rates of morbidity and mortality. The aim of this study was to determine whether
P. gingivalis
increases animal susceptibility to CMV infection. Mice were inoculated with CMV and infected locally with
P. gingivalis
3 days after the virus inoculation. Mortality rates were monitored, and traces of viral DNA and bacterial infection were detected systemically by using real-time PCR. Local and systemic cytokine secretion was measured, and histological sections were used to assess the pathological state of infected organs.
P. gingivalis
- and CMV-coinfected mice showed dramatically higher mortality rates than mice infected with
P. gingivalis
or CMV only. Although the organs of coinfected mice exhibited decreased viral titers, distinct necrosis and tissue damage were more evident in the livers and spleens of these mice than in those of mice infected with CMV only. Furthermore, systemic gamma interferon levels were decreased in coinfected mice, and marked lymphoid depletion was observed in their necrotic organs. In parallel control
Escherichia coli
-CMV coinfection experiments, the mortality and pathological results were the same as those found in mice infected with CMV only. Our results suggest a specific influence of
P. gingivalis
on the mouse immune response, causing increased susceptibility to CMV infection.
Differential Expression of Toll-Like Receptors 2 and 4 in Tissues of the Human Female Reproductive Tract
Authors: Pioli Patricia A. , Amiel Eyal , Schaefer Todd M. , Connolly John E. , Wira Charles R. , Guyre Paul M.
Published: September 2004

Toll-like receptor (TLR) signal transduction is a central component of the innate immune response to pathogenic challenge. Although recent studies have begun to elucidate differences in acquired immunity in tissues of the human female reproductive tract, there is a relative paucity of work regarding innate defense mechanisms. We investigated TLR mRNA and protein expression in tissues of the human female reproductive tract. Constitutive mRNA expression of TLRs 1 to 6 was observed in fallopian tubes, uterine endometrium, cervix, and ectocervix. Furthermore, transcripts of the signaling adapter MyD88 and the accessory molecule CD14 were also detected in all tissues assayed. Quantitative analysis of TLR2 mRNA levels revealed highest expression of this molecule in fallopian tube and cervical tissues, followed by endometrium and ectocervix. In contrast to TLR2, TLR4 expression declined progressively along the tract, with highest expression in the upper tissues (fallopian tubes and endometrium), followed by cervix and ectocervix. In addition to mRNA, protein expression of TLR2 and TLR4 was also documented in these tissues. These data suggest that TLRs are differentially expressed in distinct compartments of the female reproductive tract and may provide insight regarding the regulation of inflammation and immunity within the tract.