bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of NAD
+
-Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics
Authors: Korycka-Machala Malgorzata , Rychta Ewelina , Brzostek Anna , Sayer Heather R. , Rumijowska-Galewicz Anna , Bowater Richard P. , Dziadek Jarosław
Published: June 2007

Mycobacteria contain genes for several DNA ligases, including
ligA
, which encodes a NAD
+
-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type
ligA
cannot be deleted from the chromosome of
Mycobacterium smegmatis
. Deletions of native
ligA
in
M. smegmatis
could be obtained only after the integration of an extra copy of
M. smegmatis
or
Mycobacterium tuberculosis ligA
into the
attB
site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the
M. smegmatis
chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from
M. smegmatis
could be substituted with the NAD
+
-dependent DNA ligase of
Escherichia coli
or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of
M. smegmatis
. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of
M. tuberculosis
. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD
+
-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD
+
-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.
Fluoroquinolone Resistance in Atypical Pneumococci and Oral Streptococci: Evidence of Horizontal Gene Transfer of Fluoroquinolone Resistance Determinants from
Streptococcus pneumoniae
Authors: Ip Margaret , Chau Shirley S. L. , Chi Fang , Tang Julian , Chan Paul K.
Published: June 2007

Atypical strains, presumed to be pneumococcus, with ciprofloxacin MICs of ≥4.0 μg/ml and unique sequence variations within the quinolone resistance-determining regions (QRDRs) of the gyrase and topoisomerase genes in comparison with the
Streptococcus pneumoniae
R6 strain, were examined. These strains were reidentified using phenotypic methods, including detection of optochin susceptibility, bile solubility, and agglutination by serotype-specific antisera, and genotypic methods, including detection of pneumolysin and autolysin genes by PCR, 16S rRNA sequencing, and multilocus sequence typing (MLST). The analysis based on concatenated sequences of the six MLST loci distinguished the “atypical” strains from pneumococci, and these strains clustered closely with
S. mitis
. However, all these strains and five of nine strains from the viridans streptococcal group possessed one to three
gyrA
,
gyrB
,
parC
, and
parE
genes whose QRDR sequences clustered with those of
S. pneumoniae
, providing evidence of horizontal transfer of the QRDRs of the gyrase and topoisomerase genes from pneumococci into viridans streptococci. These genes also conferred fluoroquinolone resistance to viridans streptococci. In addition, the fluoroquinolone resistance determinants of 32 well-characterized
Streptococcus mitis
and
Streptococcus oralis
strains from bacteremic patients were also compared. These strains have unique amino acid substitutions in GyrA and ParC that were distinguishable from those in fluoroquinolone-resistant pneumococci and the “atypical” isolates. Both recombinational events and de novo mutations play an important role in the development of fluoroquinolone resistance.
Activity against Human Immunodeficiency Virus Type 1, Intracellular Metabolism, and Effects on Human DNA Polymerases of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine
Authors: Nakata Hirotomo , Amano Masayuki , Koh Yasuhiro , Kodama Eiichi , Yang Guangwei , Bailey Christopher M. , Kohgo Satoru , Hayakawa Hiroyuki , Matsuoka Masao , Anderson Karen S. , Cheng Yung-Chi , Mitsuya Hiroaki
Published: June 2007

We examined the intracytoplasmic anabolism and kinetics of antiviral activity against human immunodeficiency virus type 1 (HIV-1) of a nucleoside reverse transcriptase inhibitor, 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), which has potent activity against wild-type and multidrug-resistant HIV-1 strains. When CEM cells were exposed to 0.1 μM [
3
H]EFdA or [
3
H]3′-azido-2′,3′-dideoxythymidine (AZT) for 6 h, the intracellular EFdA-triphosphate (TP) level was 91.6 pmol/10
9
cells, while that of AZT was 396.5 pmol/10
9
cells. When CEM cells were exposed to 10 μM [
3
H]EFdA, the amount of EFdA-TP increased by 22-fold (2,090 pmol/10
9
cells), while the amount of [
3
H]AZT-TP increased only moderately by 2.4-fold (970 pmol/10
9
cells). The intracellular half-life values of EFdA-TP and AZT-TP were ∼17 and ∼3 h, respectively. When MT-4 cells were cultured with 0.01 μM EFdA for 24 h, thoroughly washed to remove EFdA, further cultured without EFdA for various periods of time, exposed to HIV-1
NL4-3
, and cultured for an additional 5 days, the protection values were 75 and 47%, respectively, after 24 and 48 h with no drug incubation, while those with 1 μM AZT were 55 and 9.2%, respectively. The 50% inhibitory concentration values of EFdA-TP against human polymerases α, β, and γ were >100 μM, >100 μM, and 10 μM, respectively, while those of ddA-TP were >100 μM, 0.2 μM, and 0.2 μM, respectively. These data warrant further development of EFdA as a potential therapeutic agent for those patients who harbor wild-type HIV-1 and/or multidrug-resistant variants.
New Plasmid-Mediated Fluoroquinolone Efflux Pump, QepA, Found in an
Escherichia coli
Clinical Isolate
Authors: Yamane Kunikazu , Wachino Jun-ichi , Suzuki Satowa , Kimura Kouji , Shibata Naohiro , Kato Haru , Shibayama Keigo , Konda Toshifumi , Arakawa Yoshichika
Published: June 2007

Plasmid-mediated Qnr and AAC(6′)-Ib-cr have been recognized as new molecular mechanisms affecting fluoroquinolone (FQ) resistance. C316, an
Escherichia coli
strain demonstrating resistance to various FQs, was isolated in Japan. Resistance to FQs was augmented in an
E. coli
CSH2 transconjugant, but PCR failed to detect
qnr
genes, suggesting the presence of novel plasmid-mediated FQ resistance mechanisms. Susceptibility tests, DNA manipulation, and analyses of the gene and its product were performed to characterize the genetic determinant. A novel FQ-resistant gene,
qepA
, was identified in a plasmid, pHPA, of
E. coli
C316, and both
qepA
and
rmtB
genes were mediated by a probable transposable element flanked by two copies of IS
26
. Levels of resistance to norfloxacin, ciprofloxacin, and enrofloxacin were significantly elevated in
E. coli
transformants harboring
qepA
under AcrB-TolC-deficient conditions. QepA showed considerable similarities to transporters belonging to the 14-transmembrane-segment family of environmental actinomycetes. The effect of carbonyl cyanide
m
-chlorophenylhydrazone (CCCP) on accumulation of norfloxacin was assayed in a
qepA
-harboring
E. coli
transformant. The intracellular accumulation of norfloxacin was decreased in a
qepA
-expressing
E. coli
transformant, but this phenomenon was canceled by CCCP. The augmented FQ resistance level acquired by the probable intergeneric transfer of a gene encoding a major facilitator superfamily-type efflux pump from some environmental microbes to
E. coli
was first identified. Surveillance of the
qepA
-harboring clinical isolates should be encouraged to minimize further dissemination of the kind of plasmid-dependent FQ resistance determinants among pathogenic microbes.
Cross-Resistance to Medical and Agricultural Azole Drugs in Yeasts from the Oropharynx of Human Immunodeficiency Virus Patients and from Environmental Bavarian Vine Grapes
Authors: Müller Frank-Michael C. , Staudigel Andrea , Salvenmoser Stefanie , Tredup Antje , Miltenberger Rudolf , Herrmann Josef V.
Published: June 2007

Cross-resistance among
Candida albicans
isolates from the oropharynges of human immunodeficiency virus-infected patients (
n
= 16) and environmental yeast strains of various species (
n
= 54) to medical and agricultural azole drugs was observed. Precautions against the unnecessary widespread use of azoles in the environment and human medicine are strongly recommended to prevent patients from acquiring azole-resistant yeasts.
Synergistic Activity of Dispersin B and Cefamandole Nafate in Inhibition of Staphylococcal Biofilm Growth on Polyurethanes
Authors: Donelli G. , Francolini I. , Romoli D. , Guaglianone E. , Piozzi A. , Ragunath C. , Kaplan J. B.
Published: June 2007

Antibiotic therapies to eradicate medical device-associated infections often fail because of the ability of sessile bacteria, encased in their exopolysaccharide matrix, to be more drug resistant than planktonic organisms. In the last two decades, several strategies to prevent microbial adhesion and biofilm formation on the surfaces of medical devices, based mainly on the use of antiadhesive, antiseptic, and antibiotic coatings on polymer surfaces, have been developed. More recent alternative approaches are based on molecules able to interfere with quorum-sensing phenomena or to dissolve biofilms. Interestingly, a newly purified β-
N
-acetylglucosaminidase, dispersin B, produced by the gram-negative periodontal pathogen
Actinobacillus actinomycetemcomitans
, is able to dissolve mature biofilms produced by
Staphylococcus epidermidis
as well as some other bacterial species. Therefore, in this study, we developed new polymeric matrices able to bind dispersin B either alone or in combination with an antibiotic molecule, cefamandole nafate (CEF). We showed that our functionalized polyurethanes could adsorb a significant amount of dispersin B, which was able to exert its hydrolytic activity against the exopolysaccharide matrix produced by staphylococcal strains. When microbial biofilms were exposed to both dispersin B and CEF, a synergistic action became evident, thus characterizing these polymer-dispersin B-antibiotic systems as promising, highly effective tools for preventing bacterial colonization of medical devices.
Disease Burden Due to Enterotoxigenic
Escherichia coli
in the First 2 Years of Life in an Urban Community in Bangladesh
Authors: Qadri Firdausi , Saha Amit , Ahmed Tanvir , Al Tarique Abdullah , Begum Yasmin Ara , Svennerholm Ann-Mari
Published: June 2007

A cohort of 321 children was followed from birth up to 2 years of age to determine the incidence of enterotoxigenic
Escherichia coli
(ETEC) in Bangladesh. The average number of diarrheal days and incidence rates were 6.6 and 2.3/child/year, respectively. ETEC was the most common pathogen and was isolated in 19.5% cases, with an incidence of 0.5 episode/child/year. The prevalence of rotavirus diarrhea was lower (10%). ETEC expressing the heat-stable enterotoxin (ST) was predominant. Strains isolated from diarrheal cases were positive for colonization factors (CFs) in higher frequency (66%) than from healthy children (33%) (
P
< 0.001). The heat-labile toxin (LT)-positive strains from healthy children were more often CF negative (92%) than those isolated from children with diarrhea (73%) (
P
< 0.001). In children with symptomatic or asymptomatic infections by CFA/I, CS1 plus CS3, CS2 plus CS3, or CS5 plus CS6 strains, a repeat episode of diarrhea or infection by the homologous CF type was uncommon. Repeat symptomatic infections were noted mostly for LT- and ST-expressing ETEC. ETEC diarrhea was more prevalent in children in the A and AB groups than in those in the O blood group (
P
= 0.032 to 0.023). Children with ETEC diarrhea were underweight and growth stunted at the 2-year follow-up period, showing the importance of strategies to prevent and decrease ETEC diarrheal morbidity in children.
Inactivation of
traP
Has No Effect on the Agr Quorum-Sensing System or Virulence of
Staphylococcus aureus
Authors: Shaw Lindsey N. , Jonsson Ing-Marie , Singh Vineet K. , Tarkowski Andrej , Stewart George C.
Published: June 2007

The success of
Staphylococcus aureus
as a pathogen can largely be attributed to the plethora of genetic regulators encoded within its genome that temporally regulate its arsenal of virulence determinants throughout its virulence lifestyle. Arguably the most important of these is the two-component, quorum-sensing system
agr
. Over the last decade, the controversial presence of a second quorum-sensing system (the TRAP system) has been proposed, and it has been mooted to function as the master regulator of virulence in
S. aureus
by modulating
agr
. Mutants defective in TRAP are reported to be devoid of
agr
expression, lacking in hemolytic activity, essentially deficient in the secretion of virulence determinants, and avirulent in infection models. A number of research groups have questioned the validity of the TRAP findings in recent years; however, a thorough and independent analysis of its role in
S. aureus
physiology and pathogenesis has not been forthcoming. Therefore, we have undertaken such an analysis of the TRAP locus of
S. aureus
. We found that a
traP
mutant was equally hemolytic as the wild-type strain. Furthermore, transcriptional profiling found no alterations in the
traP
mutant in expression levels of
agr
or in expression levels of multiple
agr
-regulated genes (
hla
,
sspA
, and
spa
). Analysis of secreted and surface proteins of the
traP
mutant revealed no deviation in comparison to the parent. Finally, analysis conducted using a murine model of
S. aureus
septic arthritis revealed that, in contrast to an
agr
mutant, the
traP
mutant was just as virulent as the wild-type strain.
Forget me not: palliative care for people with dementia
Authors: Harris Dylan
Published: June 2007

Dementia is a progressive life limiting condition with increasing prevalence and complex needs. Palliative care needs of patients with dementia are often poorly addressed; symptoms such as pain are under treated while these patients are over subjected to burdensome interventions. Research into palliative care in dementia remains limited but recent developments together with national guidelines and policies set foundations for improving the delivery of palliative care to this group of the population.
Acute rhinosinusitis in adults: an update on current management
Authors: Masood Ajmal , Moumoulidis Ioannis , Panesar Jaan
Published: June 2007

Acute rhinosinusitis is a common disease with worldwide prevalence. It is a significant burden on the health services. It is most commonly caused by viruses and is self-limiting in nature. The diagnosis of acute rhinosinusitis is clinical and sinus radiography is not indicated routinely. Most cases of acute rhinosinusitis are treated symptomatically. However, symptoms may persist beyond 10 days when secondary bacterial infection prevails. Antibiotics are reserved for moderate or severe cases or when there is development of complications of acute rhinosinusitis. This paper provides an update on the current management of acute rhinosinusitis.
Sir Henry Halford, president of the Royal College of Physicians, with a note on his involvement in the exhumation of King Charles I
Authors: Morris John S
Published: June 2007

Henry Halford (formerly Vaughan) (1766–1844) was president of the Royal College of Physicians for an unprecedented 24 years. A successful physician, he had to resign his post at the Middlesex Hospital because of his growing private practice. He was physician to four reigning monarchs and had many famous patients including Geogiana Duchess of Devonshire in whom he correctly diagnosed a liver abscess when other physicians had failed. He was also involved in the exhumation of King Charles I, and the fourth cervical vertebra, through which the King had been executed, came into his possession.
Larval therapy from antiquity to the present day: mechanisms of action, clinical applications and future potential
Authors: Whitaker Iain S , Twine Christopher , Whitaker Michael J , Welck Mathew , Brown Charles S , Shandall Ahmed
Published: June 2007

When modern medicine fails, it is often useful to draw ideas from ancient treatments. The therapeutic use of fly larvae to debride necrotic tissue, also known as larval therapy, maggot debridement therapy or biosurgery, dates back to the beginnings of civilisation. Despite repeatedly falling out of favour largely because of patient intolerance to the treatment, the practice of larval therapy is increasing around the world because of its efficacy, safety and simplicity. Clinical indications for larval treatment are varied, but, in particular, are wounds infected with multidrug-resistant bacteria and the presence of significant co-morbidities precluding surgical intervention. The flies most often used in larval therapy are the facultative calliphorids, with the greenbottle blowfly (Lucilia sericata) being the most widely used species. This review summarises the fascinating and turbulent history of larval therapy from its origin to the present day, including mechanisms of action and evidence for its clinical applications. It also explores future research directions.
Long term prognosis of women with gestational diabetes in a multiethnic population
Authors: Oldfield Matthew D , Donley Penelope , Walwyn Linda , Scudamore Ian , Gregory Robert
Published: June 2007

Aim

To assess the glucose tolerance of South Asian and Caucasian women with previous gestational diabetes mellitus (GDM).

Method

A retrospective follow-up study of 189 women diagnosed with GDM between 1995 and 2001. Glucose tolerance was reassessed by oral glucose tolerance test at a mean duration since pregnancy of 4.38 years.

Results

South Asian women comprised 65% of the GDM population. Diabetes developed in 36.9% of the population, affecting more South Asian (48.6%) than Caucasian women (25.0%). Women developing diabetes were older at follow-up (mean (SD) 38.8 (5.7) vs 35.9 (5.6) years; p<0.05) and had been heavier (body mass index 31.4 (6.3) vs 27.7 (6.7) kg/m2; p<0.05), more hyperglycaemic (Gl0 6.5 (1.7) vs 5.2 (1.1) mmol/l; p<0.01: G120 11.4 (3.3) vs 9.6 (1.8) mmol/l; p<0.01: HbA1c 6.4 (1.0) vs 5.6 (0.7); p<0.01) and more likely to require insulin during pregnancy (88.1% vs 34.0%; p<0.01). Future diabetes was associated with and predicted by HbA1c taken at GDM diagnosis in both South Asian (odds ratio 4.09, 95% confidence interval 1.35 to 12.40; p<0.05) and Caucasian women (OR 9.15, 95% CI 1.91 to 43.87; p<0.01) as well as by previously reported risk factors of increasing age at follow-up, pregnancy weight, increasing hyperglycaemia and insulin requirement during pregnancy.

Conclusion

GDM represents a significant risk factor for future DM development regardless of ethnicity. Glycated haemoglobin values at GDM diagnosis have value in predicting future diabetes mellitus.
Matrix metalloproteinase-1 therapy improves muscle healing
Authors: Bedair Hany , Liu T. Thomas , Kaar Joel L. , Badlani Shawn , Russell Alan J. , Li Yong , Huard Johnny
Published: June 2007

Muscle undergoes time-dependent phases of healing after injury, which ultimately results in residual fibrosis in the injured area. The use of exogenous matrix metalloproteinases (MMPs) may improve recovery after muscle injury by promoting the digestion of existing fibrous tissue and releasing local growth factors. In the current experiment, bilateral gastrocnemius (GM) lacerations were created in severe combined immunodeficient mice. Twenty-five days after injury (peak posttraumatic fibrosis), C2C12 cells (myoblasts) transduced with the LacZ reporter gene were injected with exogenous MMP-1 into the right GMs at the site of injury; the cells were also injected along with PBS (control) at the site of injury in the left GMs. The muscle tissues were examined histologically via X-gal, hemotoxylin and eosin, and Masson's trichrome staining. The MMP-treated limbs contained more regenerating myofibers than did the control limbs (MMP 170 ± 96 fibers, control 62 ± 51 fibers; P < 0.001). Less fibrous tissue was observed within MMP-treated muscles (MMP: 24 ± 11%, control: 35 ± 15%; P < 0.01). These results suggest that the direct injection of MMP-1 into the zone of injury during fibrosis can enhance muscle regeneration by increasing the number of myofibers and decreasing the amount of fibrous tissue.
Cerebral haemodynamics during hypo- and hypercapnia
Authors: Terborg C. , Hautzel H. , Herzog H. , Witte O. W. , Mueller H.-W. , Krause B. J. , Poeppel T. D.
Published: June 2007

Aim: Transcranial Doppler sonography (TCD) is increasingly used in cerebrovascular disease for monitoring brain perfusion. It allows estimation of cerebral blood flow (CBF) by the measurement of cerebral blood flow velocity (CBFV). The CBFV as well as CBF are intimately associated with the intravascular CO2-concentration. Thus, hyper- or hypocapnia can be used to induce a defined range of blood flows. The aim of our study was the comparison of vasomotor reactivity assessed with simultaneous TCD and quantitative regional CBFmeasurements (rCBF) by PET (serving as the reference method for in-vivo quantification of rCBF). Patients, methods: Six healthy young volunteers participated in this study. CBF was measured using 15O-butanol PET. A flow and dispersion- model was fitted to the measured time activity curves using arterial input curves. Each subject underwent five scans at five different end-tidal CO2 levels (EtCO2): 25, 32, 40, 48, and 55 mmHg. CBFV was assessed by continuous bilateral TCD of the middle cerebral artery (MCA). Volumes of interest for rCBF determination were placed in grey matter of the prefrontal cortex (PFC) as determined from individual MRIs. Comparisons between the rCBF, EtCO2 and CBFV were carried out with regression and correlation analysis and paired t-tests. Results: Strong positive linear correlations of rCBF and CBFV with the CO2-concentration and linear relationships between rCBF and CBFV were found in each individual. Normalised CO2-reactivities measured by TCD and PET were closely correlated. Conclusions: TCD-measurements of vascular reactivity in healthy volunteers show a high correlation to those acquired with PET that serves as the reference method of quantitative rCBF-measurement. The results of the MCA insonation are a close approximation of the rCBF changes induced by variations of EtCO2.
Proteomic discovery of 21 proteins expressed in human plasma-derived but not platelet-derived microparticles
Authors: Smalley David M. , Root Karen E. , Cho HyungJun , Ross Mark M. , Ley Klaus
Published: June 2007

Microparticles (MPs) are small membrane vesicles generated by essentially all cell types. In the plasma, most MPs are derived from platelets, but those from other sources, particularly leukocytes (macrophages, lymphocytes, and neutrophils), endothelial cells, and even smooth muscle cells can be detected and appear to play an important role in normal physiology and various diseases. In previous work we analyzed the proteome of MPs generated from isolated platelets (platelet MPs). Here, we report on a comparative analysis of microparticles isolated from plasma (plasma MPs) versus platelet MP using two complementary methods of comparative analysis. The first method, spectral count analysis, yielded 21 proteins detected in plasma MPs (with a total spectral count of 10 or greater) that were essentially absent in platelet MPs (with a total spectral count of 1 or 0). An additional two proteins (von Willebrand Factor, albumin) were present in both types of MPs but enriched in the plasma MPs. The second method, isotope-coded affinity tag (ICAT) labeling of proteins, supported the spectral count results for the more abundant proteins and provided better relative quantitation of differentially expressed proteins. Proteins present only in the plasma MPs include several associated with apoptosis (CD5-like antigen, galectin 3 binding protein, several complement components), iron transport (transferrin, transferrin receptor, haptoglobin), immune response (complement components, immunoglobulin J and kappa chains), and the coagulation process (protein S, coagulation factor VIII).
Relatively increased von Willebrand factor activity after off-pump coronary artery bypass graft surgery
Authors: Nierich Arno P. , Kalkman Cor J. , Fijnheer Rob , Lo Bernard
Published: June 2007

High shear forces can induce structural changes in the shape of the von Willebrand factor (VWF) molecule, making it more sensitive for ADAMTS-13, a specific VWF protease. This leads to proteolysis of the highest-molecular-weight multimers, which are the most effective in platelet-mediated hemostasis under conditions of high shear stress. The use of cardiopulmonary bypass (CPB) is accompanied with high shear forces and could therefore lead to diminished VWF activity. Therefore, we studied VWF activity in patients undergoing myocardial revascularization. We enrolled 60 patients undergoing coronary artery bypass graft (CABG) surgery with and without CPB (30 in each group). ADAMTS-13 activity, VWF antigen (VWF:Ag) and propeptide levels were measured directly before and after the procedure. VWF activity was determined using both the ristocetin cofactor activity (VWF:Rcof) and collagen binding (VWF:CB) assays. VWF:Rcof and VWF:CB, both corrected for VWF:Ag, were significantly increased after the procedure in the off-pump group (p<0.001 and p=0.05, respectively), but not in the CPB group. Postoperative VWF:Ag and VWF-propeptide levels significantly increased in both groups. ADAMTS-13 activity increased after both types of surgery after correction for hemodilution. Postoperative VWF: Rcof levels correlated with postoperative D-dimer levels (p=0.025) and were associated with early cognitive decline in the off-pump group (p=0.025). Our data indicate that VWF activity is increased after off-pump CABG, but not after CABG with CPB. Since release of VWF is comparable in both groups, shear stress-induced proteolysis due to the use of CPB may be responsible for the decreased VWF activity.
The role of fibrin monomers in optimizing the diagnostic work-up of deep vein thrombosis
Authors: Haas Fred , Agterof Mariette , Vos Marike , Biesma Douwe , Schutgens Roger
Published: June 2007

Despite the use of a clinical score and D-dimers to exclude deep vein thrombosis (DVT), the majority of patients still need repeated ultrasound (US).The aim of the study was to investigate whether fibrin monomers (FMs), as markers of thrombin generation, have additional value in the diagnosis of DVT. This is a posthoc analysis of 464 outpatients, participants in a management study using D-dimers (Tina-Quant® ) and a clinical score in the exclusion of DVT. Two new FM assays (Auto LIA-FM® and IATRO SF®, Japan) were performed. Overall sensitivity, negative predictive value (NPV) and specificity of the D-dimer test were 98%, 98% and 42%.The optimal cut-off point for the Auto LIAFM test was ≤ 3 µ g/ml with values of 88%, 88% and 59%, respectively. The IATRO SF test had an optimal cut-off point of ≤ 2 µ g/ ml with values of 92%, 81 and 22%, respectively.The NPV of a non-high clinical score and a normal D-dimer (n=97) was 100%. In patients with a high clinical score (n=160), the NPV of the D-dimer was 88%. In these patients, a single US combined with a normal D-dimer or FM test had an equal NPV as serial US (100 versus 98%, respectively) and lead to a reduction in the need for US by 36–53%, respectively. In patients with abnormal D-dimer concentrations (n=343), a normal US combined with a normal Auto LIA-FM test had a NPV of 97%,which was also true for serial US.This could lead to a reduction in the need for US by 45%. The present studied FMs are inferior to theTina-Quant D-dimer test when used as primary screening tool to exclude DVT.Adding these FMs to patients with a normal Tina-Quant D-dimer has no benefit. In patients with a high pretest clinical probability score, a single US in combination with a normal D-dimer or FM test might be as safe as serial US. In patients with abnormal D-dimer concentrations and a normal US, a normal FM test might be able to replace the second US.
Impaired pericyte recruitment and abnormal retinal angiogenesis as a result of angiopoietin-2 overexpression
Authors: Feng Yuxi , vom Hagen Franziska , Pfister Frederick , Djokic Snezana , Hoffmann Sigrid , Back Walter , Wagner Patrick , Lin Jihong , Deutsch Urban , Hammes Hans-Peter
Published: June 2007

Angiopoietin-2 (Ang2) is among the relevant growth factors induced by hypoxia and plays an important role in the initiation of retinal neovascularizations. Ang2 is also involved in incipient diabetic retinopathy, as it may cause pericyte loss. To investigate the impact of Ang2 on developmental and hypoxia-induced angiogenesis, we used a transgenic mouse line overexpressing human Ang2 in the mouse retina. Transgenic mice displayed a reduced coverage of capillaries with pericytes (-14 %; p<0.01) and a 46% increase of vascular density of the capillary network at postnatal day 10 compared to wild type mice. In the model of oxygen-induced retinopathy (OIR), Ang2 overexpression resulted in enhanced preretinal (+103%) and intraretinal neovascularization (+29%). Newly formed intraretinal vessels in OIR were also pericyte-deficient (-26 %; p<0.01). The total expression of Ang2 in transgenic mice was seven-fold, compared with wild type controls. Ang2 modulated expression of genes encoding VEGF (+65%) and Ang1 (+79%) in transgenic animals. These data suggest that Ang2 is involved in pericyte recruitment, and modulates intraretinal, and preretinal vessel formation in the eye under physiological and pathological conditions.
High post-treatment platelet reactivity is associated with a high incidence of myonecrosis after stenting for non-ST elevation acute coronary syndromes
Authors: Frere Corinne , Quilici Jacques , Morange Pierre-Emmanuel , Nait-Saidi Lyassine , Mielot Christopher , Bali Laurent , Lambert Marc , Alessi Marie-Christine , Bonnet Jean-Louis , Cuisset Thomas
Published: June 2007

High post-treatment platelet reactivity (HPPR= adenosine diphosphate [ADP] 10 µM-induced platelet aggregation > 70%) identifies low responders to dual antiplatelet therapy with increased risk of recurrent cardiovascular (CV) events after stenting for non-ST elevation acute coronary syndromes (NSTEACS). This study was designed to compare the incidence of periprocedural myocardial infarction (MI) after stenting for NSTEACS patients between non-responders to dual antiplatelet therapy defined by HPPR and normo-responders. One hundred ninety NSTE-ACS consecutive patients undergoing coronary stenting were included in this prospective study. They received 250 mg aspirin and a 600 mg loading dose of clopidogrel at least 12 hours (h) before percutaneous coronary intervention (PCI). A single post-treatment blood sample was obtained before PCI to analyze maximal intensity of ADP-induced platelet aggregation, and troponin levels were analyzed before PCI, and 12 and 24 h after PCI. Troponin I was considered elevated if > 0.4 ng/ ml. HPPR was present in 22% of patients (n=42). Periprocedural MI occurred significantly more frequently in patients with HPPR than in the normo-responders (43% vs. 24%, p=0.014). After being correlated with recurrent ischemic events after stenting for NSTE-ACS, the HPPR seems to be also a marker of increased risk of periprocedural MI for NSTE-ACS patients.
Recombinant annexin-2 inhibits the progress of diabetic nephropathy in a diabetic mouse model via recovery of hypercoagulability
Authors: Ishii Hideto , Hiraoka Megumi , Tanaka Akira , Shimokado Kentaro , Yoshida Masayuki
Published: June 2007

Diabetic nephropathy, a major complication of diabetes mellitus that leads to mortality, has been shown to involve a dysregulation of the coagulation system. Annexin-2, a co-receptor for plasminogen and tissue plasminogen activator on endothelial cells, is one of the molecules required to maintain the antithrombogenic properties of endothelial cells. Previously, we showed that recombinant annexin-2 protein (rAN II) modulated impaired fibrinolytic activity in the carotid arteries of rats. In the present study, to investigate its protective effects against diabetic nephropathy, rAN II was administered to KK-Ay mice, a murine model of type 2 diabetes, for eight weeks, and albuminuria, kidney size, and histological glomerular lesions were investigated. The mean weight of kidneys from KK-Ay mice treated with rAN II was significantly less than that of those treated with PBS (control) (p<0.02). Furthermore, the level of albuminuria observed in rAN II-treated KK-Ay mice was significantly less than that of the control group (rAN II, 0.90+/-0.12 µg/day; PBS, 1.55+/-0.31 µg/day; p<0.01); also, the area of diffuse glomerular lesions was significantly smaller (rAN II, 41.51+/-4.54%; PBS, 81.81+/-8.10%; p<0.01). Bleeding time, prothrombin time (PT), and active partial thromboplastin time (APTT) did not significantly differ between the two groups. Our results suggest that rAN II may inhibit the progression of diabetic nephropathy in KK-Ay mice without influencing the coagulation system, indicating that annexin-2 may be considered as a possible new therapeutic tool for patients with diabetic nephropathy.
Genetic variation in estrogen receptor, C-reactive protein and fibrinogen does not predict the plasma levels of inflammation markers after longterm hormone replacement therapy
Authors: Madsen Jonna , Langdahl Bente , Bladbjerg Else , Tofteng Charlotte , Abrahamsen Bo , Rejnmark Lars , Brixen Kim , Christensen Kaare , Jespersen Jørgen , Kristensen Søren , de Maat Moniek
Published: June 2007

Markers of inflammation, such as C-reactive protein (CRP) and fibrinogen, are associated with the risk of atherothrombosis. Plasma levels of these markers of inflammation are affected by hormone replacement therapy (HRT) and modulated by smoking. We studied whether genetic variation in the estrogen receptor- 1 (ESR1), CRP and fibrinogen-β genes influences the plasma levels of inflammation markers after HRT. Plasma CRP and fibrinogen were measured after five years follow-up in healthy postmenopausal women (per-protocol group) who were randomised to hormone therapy (n=187) or no treatment (n=249). The effect of HRT, smoking and genetic variations in ESR1 (PvuII and XbaI), CRP (1444C/T) and fibrinogen-β ( FGB, –455G/A) were determined. The plasma concentration of CRP was higher in the HRT group than in the control group (2.03 mg/l and 1.41 mg/l, respectively; p < 0.001), while the concentration of fibrinogen was lower in the HRT group than in the control group (3.02 g/l and 3.20 g/l, respectively; p < 0.001), indicating that it is unlikely that inflammation is the common underlying pathway. There was a significant interaction between smoking and HRT on the ibrinogen (p=0.02), but not on the CRP concentration (n.s.). Genetic polymorphisms in ESR1, CRP and fibrinogen were not associated with an effect of HRT on the CRP and fibrinogen plasma levels, and no significant interaction with smoking was observed. In conclusion, higher plasma levels of CRP and lower plasma levels of fibrinogen were observed in women using HRT; however, genetic polymorphisms in ESR1, CRP and FGB were not associated with these effects of HRT.
Sequential adhesion of platelets and leukocytes from flowing whole blood onto a collagen-coated surface: Requirement for a GpVI-binding site in collagen
Authors: Butler Lynn , Metson-Scott Tom , Felix Jo , Abhyankar Anita , Rainger G. , Farndale Richard , Watson Stephen , Nash Gerard
Published: June 2007

The adhesion of leukocytes to immobilised platelets may contribute to inflammatory and thrombotic responses in damaged tissue. To investigate the conditions under which platelets and leukocytes might be deposited together in vessels, we perfused fluorescently-labelled whole blood through glass capillaries coated with various collagen preparations. Video-microscopic observations of the surface showed that platelets formed numerous, individual, rolling and stationary attachments to surfaces coated with acid-soluble, monomeric collagen. However, leukocyte interactions with the deposited platelets were rare. If the blood was washed out, the adherent platelets became more activated, and many rolling adherent leukocytes were observed if a second bolus of blood was perfused over them. This suggested that platelet activation had initially been inadequate to support leukocyte capture. Next, fibrillar collagen was adsorbed to the capillaries to present an ordered array of peptide motifs to platelet receptor glycoprotein (Gp)VI and transduce an activating signal. In this case, platelets were deposited in discrete, stable aggregates and the bound platelets captured many flowing leukocytes.Alternatively, acid-soluble collagen was seeded with collagen-related peptide (CRP) known to contain a GpVI-binding motif.Again, platelet adhesion became stable, and numerous flowing leukocytes were captured.Addition of antibody against GpVI or against P-selectin greatly reduced leukocyte adhesion to the platelets. Thus, in whole blood, platelets binding to exposed collagen need to be activated through GpVI in order to expose sufficient P-selectin to allow efficient capture of flowing leukocytes to take place.
Factor XIII activation peptide is released into plasma upon cleavage by thrombin and shows a different structure compared to its bound form
Authors: Schroeder Verena , Vuissoz Jean-Marc , Caflisch Amedeo , Kohler Hans
Published: June 2007

The first step of coagulation factor XIII (FXIII) activation involves cleavage of the FXIII activation peptide (FXIII-AP) by thrombin. However, it is not known whether the FXIII-AP is released into plasma upon cleavage or remains attached to activated FXIII. The aim of the present work was to study the structure of free FXIII-AP, develop an assay for FXIII-AP determination in human plasma, and to answer the question whether FXIII-AP is released into plasma. We used ab-initio modeling and molecular dynamics simulations to study the structure of free FXIII-AP. We raised monoclonal and polyclonal antibodies against FXIII-AP and developed a highly sensitive and specific ELISA method for direct detection of FXIII-AP in human plasma. Structural analysis showed a putative different conformation of the free FXIII-AP compared to FXIII-AP bound to the FXIII protein. We concluded that it might be feasible to develop specific antibodies against the free FXIII-AP. Using our new FXIII-AP ELISA, we found high levels of FXIII-AP in in-vitro activated plasma samples and serum. We showed for the first time that FXIIIAP is detached from activated FXIII and is released into plasma, where it can be directly measured. Our findings may be of major clinical interest in regard to a possible new marker in thrombotic disease.
Sex-specific, independent associations of insulin resistance with erythrocyte sedimentation rate in apparently healthy subjects
Authors: Hernández-Aguado Ildefonso , Jarrín Vera Inmaculada , Recasens Mónica , Esteve Eduardo , Casamitjana Roser , Ricart Wifredo , Fernández-Real José , López-Bermejo Abel
Published: June 2007

Insulin resistance and erythrocyte sedimentation rate (ESR, a non-specific marker of inflammation) are known risk factors for cardiovascular disease. Although obesity is associated with increased ESR, it is unclear whether insulin resistance is associated with ESR in humans. The relationship between insulin resistance and ESR was studied in a cross-sectional, health-area based study of 140 (89 men and 51 women) apparently healthy Caucasians subjects. ESR, additional inflammatory markers [soluble tumor necrosis alpha receptors 1 and 2 (sTNFR1 and sTNFR2); C-reactive protein (CRP)], and insulin sensitivity ( S I, frequently sampled intravenous glucose tolerance test with minimal model analysis) were assessed in all subjects. An interaction with sex was documented in the relationship between ESR and both insulin resistance and obesity (p < 0.05), as log ESR correlated with log S I in men (r= –0.29, p = 0.009), but not in women (r= –0.14, p = 0.33), and correlated with body mass index (BMI) in women (r=0.49, p= < 0.0001), but not in men (r=0.15, p = 0.16). On multivariate analyses, these associations proved to be independent of known covariates, such as age, hematocrit, smoking and additional inflammatory markers in both men and women. In a replication study, variables independently associated with ESR were also insulin resistance (homeostasis model assessment) in men and obesity markers (either BMI or fat mass) in women. In conclusion, ESR is independently associated with either insulin resistance or obesity in a sex-specific manner. These findings contribute to explain the known relationship of this inflammatory marker with cardiovascular disease.
Fibrinogen gene haplotypes in relation to risk of coronary events and coronary and extracoronary atherosclerosis: The Rotterdam Study
Authors: Kardys Isabella , Uitterlinden André , Hofman Albert , Witteman Jacqueline , de Maat Moniek
Published: June 2007

Fibrin network structure has been correlated with coronary disease. Fibrinogen γ and α (FGG and FGA) gene haplotypes (chromosome 4q28) may be associated with fibrin network structure, and thereby with rigidity of the fibrin clot and sensitivity of the fibrin clot to the fibrinolytic system. Through these mechanisms they may influence risk of cardiovascular disease. We set out to investigate the relation between combined fibrinogen FGG and FGA gene haplotypes, representing the common variation of the fibrinogen FGG and FGA genes, coronary events and measures of coronary and extracoronary atherosclerosis. The study was embedded in the Rotterdam Study, a prospective populationbased study among men and women aged ≥ 55 years. Common haplotypes were studied using seven tagging SNPs across a 30-kb region with the FGG and FGA genes. Incident coronary events were registered, and carotid intima-media thickness, carotid plaques, ankle-arm index, aortic calcification and coronary calcification were assessed. Seven haplotypes with frequencies >1% covered 97.5% of the genetic variation. In 5,667 participants without history of coronary heart disease (CHD), 733 CHD cases occurred during a median follow-up time of 11.9 years. Fibrinogen gene haplotypes were not associated with coronary events. Fibrinogen gene haplotypes did not show a consistent association with measures of coronary and extracoronary atherosclerosis. In conclusion, fibrinogen FGG and FGA gene haplotypes are not associated with coronary events, coronary atherosclerosis or extracoronary atherosclerosis. Confirmation of these findings by future population-based studies is warranted.
A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity – a staphylokinase variant
Authors: Stankiewicz Adrian , Rozmyslowicz-Szermńska Wioletta , Mogielnicki Andrzej , Gromotowicz Anna , Buczko Wlodzimierz , Oszajca Katarzyna , Bartkowiak Jacek , Chabielska Ewa , Szemraj Janusz
Published: June 2007

The recombinant protein SAK-RGD-K2-Hir is characterized by its fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. It was previously shown in our in-vitro studies to be a more potent and faster-acting thrombolytic agent compared with standard r-SAK. In order to document the effects of the thrombolytic potential of SAKRGD- K2-Hir we examined this protein in an electrically induced carotid artery thrombosis model and stasis-induced venous model in rats. In the arterial thrombosis model, a bolus injection of SAK-RGD-K2-Hir was less effective than rt-PA and r-SAK. However, the most effective in the improvement and maintenance of carotid patency and in arterial thrombus mass reduction was SAK-RGD-K2. In contrast, all r-SAK derivatives reduced venous thrombus weight significantly in comparison to r-SAK and r-Hir. However, the most observable decrease in thrombus weight was obtained after application of recombinant proteins containing the r-Hir.The bleeding time was significantly prolonged in the animals treated with proteins containing r-Hir at a dose of 1.0 mg/kg.There were no observable changes in plasma fibrinogen concentration.In conclusion,our findings show thrombolytic activity in intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hir in rats.Although this protein compares favourably with r-SAK in rat venous thrombolysis, we were unable to confirm the beneficial effects of SAK-RGDK2- Hir over r-SAK and rt-PA in the carotid artery thrombolysis model. Furthermore, our results also suggest that SAKRGD- K2-Hir bears a risk of bleeding, but this may be true for higher doses.
Lack of association between the 807 C/T polymorphism of glycoprotein Ia gene and post-treatment platelet reactivity after aspirin and clopidogrel in patients with acute coronary syndrome
Authors: Cuisset Thomas , Frere Corinne , Quilici Jacques , Morange Pierre-Emmanuel , Saut Noémie , Romero-Barra Marie , Camoin Laurence , Lambert Marc , Juhan-Vague Irène , Bonnet Jean-Louis , Alessi Marie-Christine
Published: June 2007

Variability in platelet response to antiplatelet therapy and its clinical relevance have been well described. However, the underlying mechanisms remain unclear. It was the aim of the present study to assess whether the response to aspirin and clopidogrel may be influenced by the 807 C/T polymorphism of the glycoprotein Ia (GpIa) gene in patients with non-ST elevation acute coronary syndrome (NSTE ACS). Six hundred one NSTE ACS patients were included in our study and were divided into three groups: CC homozygotes, CT heterozygotes ad TT homozygotes. All patients received loading doses of 600 mg clopidogrel and 250 mg aspirin at least 12 hours before blood samples were
drawn. Post-treatment platelet reactivity was assessed by post treatment ADP 10 µM-induced platelet aggregation (ADP-Ag), VASP phosphorylation (PRI VASP) and P-selectin expression. Non-response to dual antiplatelet therapy was defined by high post-treatment platelet reactivity (HPPR= ADP-Ag>70%). Significant variability in the distribution of platelet parameters was observed in the overall study population. No significant difference in platelet parameters profiles was observed within patients having the same genotype, for ADP-Ag (p=0.33), PRIVASP (p=0.72) and P-selectin expression (p=0.37). The genotype frequencies of the 807 C/T polymorphism of the GpIa gene were similar in responders and non-responders defined by persistent HPPR (p=0.104). In conclusion, our study did not show any influence of 807 C/T polymorphism of GpIa gene on post-treatment platelet reactivity assessed by ADP-Ag, PRI VASP or P-selectin expression in 601 NSTE ACS patients.