bluuebunny/crossref_metadata_2025 huggingface dataset:
Fractalkine receptor polymorphisms V249I and T280M as genetic risk factors for restenosis
Authors: Marculescu Rodrig , Kvakan Heda , Haschemi Arvand , Endler Georg , Weyand Cornelia M. , Maurer Gerald , Mannhalter Christine , Wojta Johann , Wagner Oswald , Huber Kurt , Niessner Alexander
Published: November 2005

The chemokine fractalkine (FKN) recruits leukocytes into lesions of the arterial wall, which may lead to restenosis after stenting. FKN also regulates proliferation of smooth muscle cells, another mechanism pivotal to neointimal thickening. We assessed the hypothesis that functionally important polymorphisms of the FKN receptor CX3CR1 influence restenosis after coronary stenting. Three hundred and sixty-five patients undergoing coronary stenting were genotyped for the CX3CR1 polymorphisms V249I and T280M. Restenosis occurred in 25% of patients, and recurrent (> 1) restenosis at the target lesion in 8%.The allele I249 was associated with an increased risk of restenosis (adjusted odds ratio 2.4, 95% confidence interval: 1.3–4.2,P = 0.003) and recurrent restenosis (odds ratio 2.7,95% confidence interval:1.3–5.9,P = 0.011).Particularly, patients with I249 lacking the allele M280 were at an elevated risk of restenosis (P = 0.006) and, accordingly, the haplotype containing I249 but not M280 was more frequent in patients with restenosis (P = 0.001). In conclusion, the CX3CR1 I249 allele is associated with an increased risk of restenosis while the CX3CR1 M280 allele might counteract the harmful influence of I249.These findings show the importance of the chemokine FKN and genetic variations of its receptor for restenosis after coronary stenting. Recognition of these inherited risk modifiers may help to individualize treatment of coronary stenosis.
The influence of the haematocrit on primary haemostasis in vitro
Authors: Eugster Marco , Reinhart Walter H.
Published: November 2005

Primary haemostasis consists of platelet adhesion to subendothelial collagen, their activation and aggregation and finally the formation of a platelet plug. Erythrocytes are involved in this process because they flow in the center of the vessel and push platelets towards the site of action on the vessel wall and enhance shear forces, which activate platelets. In the platelet function analyzer PFA-100® (Dade Behring, Düdingen, Switzerland), the in vivo situation is simulated in vitro with blood being aspirated at high shear rates (5000s-1) through a capillary into a membrane pore with a diameter of 150 μm coated with type I collagen and either epinephrine or adenosine diphosphate. Aggregating platelets plug the pore and stop the flow, which is measured as the closure time. We analysed the influence of erythrocytes on platelet function analyzer measurements by systematic variation of the haematocrit (20,30,40,and 50%) at constant platelet counts of 289±61 ×103/μl plasma, or 152±30 ×103/μl blood, 96±9 ×103/μl blood and 54±5 ×103/μl blood, respectively. An inverse correlation was found between haematocrit and closure time under all circumstances. A decrease of the platelet count by 50 ×103 /μl could be compensated for by a 10% increase in haematocrit. The haematocrit must, therefore, be taken into consideration for the correct interpretation of PFA-100® measurements. Our data also provide a pathophysiological rationale to reduce the risk of bleeding in patients with thrombocytopenia and anaemia by normalizing the haematocrit with erythrocyte transfusions.
Molecular analysis of the heavy chain variable region genes of human hybridoma clones specific for coagulation factor VIII
Authors: Gharagozlou Soheila , Kardar Gholam Ali , Rabbani Hodjattallah , Shokri Fazel
Published: November 2005

Hemophilia A is a X-linked hematologic disorder characterized by undetectable or low amounts of functional coagulation factor VIII (FVIII). Replacement therapy induces FVIII neutralizing antibody (Ab) (inhibitor) in a proportion of patients which makes further treatment of these patients ineffective and costly. To envisage mechanisms underlying inhibitor development, seven hybridoma clones specific for FVIII were generated from two hemophiliaA patients with high titer of inhibitor. Specificity and isotype of the monoclonal antibodies (mAbs) were determined by ELISA. Immunoglobulin (Ig) variable region heavy (VH) chain gene family usage was identified by RT-PCR usingVH1–6 specific primers. Nucleotide sequences of the VH gene of FVIII specific clones were determined and aligned to the most homologous germ line genes in the GenBank. Analysis of the expressed VH genes by RT-PCR revealed that the hybridomas utilized either the VH1 (71%) or the VH3 (29%) gene family. Three VH domains were encoded by V1–69 (DP-10),V1–2 (DP-8),andV1–8 (DP-15) genes and two by V1–18 (DP-14) gene, all from the VH1 gene family. Of the VH3-gene family expressing clones, one belonged to V3–66 (DP-86) and the other one toV3–21 (DP-77) germline genes. The CDR3 length was found to be highly different amongst these clones ranging from 11 to 22 amino acid residues. These data suggest that FVIII-specific Abs preferentially use VH gene segments derived from VH1 gene family. Diversity of the expressed VH genes and their CDR3 length implies that different epitopes are recognized by these mAbs.
Molecular mechanisms of antithrombin deficiency in two Chinese families
Authors: Zhou Rong-Fu , Fu Qi-Hua , Wang Wen-Bin , Xie Shuang , Dai Jin , Ding Qiu-Lan , Wang Xue-Feng , Wang Zhen-Yi , Wang Hong-Li
Published: November 2005

We investigated the molecular mechanisms responsible for type I congenital antithrombin (AT) deficiency in two unrelated Chinese pedigrees manifesting multiple site venous thrombosis. Phenotype analysis showed both probands had almost 50% of normal AT levels. Direct sequencing of amplified DNA revealed 2757C>T in proband 1 and 13328G>A in proband 2, predicting a heterozygous Thr98Ile (T98I) and Ala404Thr (A404T), respectively. No proband had 20210A allele or factorV Leiden mutation. Transient expression of complementary DNA coding for the mutations in COS-7 cells showed impaired secretion of the mutant molecules. Real-time quantitative PCR indicated that the mutant AT mRNA was transcribed at a similar or even higher level as that of wild-type (wt). Pulse-chase labeling studies suggested both AT variants did not accumulate, but degraded intracellularly. Immunohistochemical staining of the transfected cells revealed that CHO cells expressing the AT-I98 mutant were stained diffusely without perinuclear enhancement and cells expressing AT-T404 mutant mainly in the whole cytoplasm with weaker perinuclear enhancement. We conclude that the impaired secretion of the mutant AT molecules, due to intracellular degradation, is the molecular pathogenesis of AT deficiency caused by T98I and A404T mutation for the two families, respectively.
Interleukin-18 and Macrophage Migration Inhibitory Factor Are Associated With Increased Carotid Intima–Media Thickening
Authors: Korshunov Vyacheslav A. , Nikonenko Tatiana A. , Tkachuk Vsevolod A. , Brooks Andrew , Berk Bradford C.
Published: November 2005

Objective—Carotid intima–media thickening (IMT) is a form of vascular remodeling that has a strong genetic component. Recently, we discovered that in response to decreased carotid blood flow SJL mice developed the largest intima among 5 inbred strains. Because the SJL strain is prone to autoimmune diseases, we hypothesized that inflammation contributed to IMT in SJL mice.

Methods and Results—We compared vascular remodeling (induced by 2 weeks of low flow) in 2 strains with small IMT (C3H/HeJ and C3HeB/FeJ) versus 2 strains with large IMT (FVB/NJ and SJL/J). Quantitative immunohistochemistry showed a dramatic increase in inflammatory cells per intima area in SJL compared with other strains. Microarray profiling of inflammatory gene mRNAs from carotids showed significant increases in interleukin (IL)-18 and Mif gene expression in SJL compared with C3HeB/FeJ mice. Increased expression of these genes was confirmed by quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. Furthermore, greater cell proliferation in the intima of SJL accounted for increased intima–media thickening, whereas a higher level of apoptosis and a lower level of proliferation were observed in C3HeB/FeJ mice.

Conclusion—The present study indicates that increased expression of Mif and IL-18 cytokines is associated with intima–media thickening in SJL mice, likely by stimulating inflammation and proliferation.
Endothelial Nitric Oxide Synthase
Authors: Rabelink Ton J. , Luscher Thomas F.
Published: November 2005

This article explores the physiology of superoxide generation by endothelial nitric oxide synthase (eNOS), the so-called “uncoupled” state of the enzyme. The fact that this alternative chemistry of the eNOS enzyme is evolutionary strongly conserved, suggests that it may play a physiological role. It is proposed that this uncoupled state may contribute to defense against infections. As the switch from NO production to reactive oxygen species by eNOS is also the final common pathway in atherogenesis, the uncoupling of eNOS further builds on the hypothesis that atherogenesis is driven by cellular mechanisms that originally serve host defense. The central role of uncoupled eNOS in redox signaling in the endothelium may open up new avenues for therapy to prevent atherosclerosis.
Correlation of Vasa Vasorum Neovascularization and Plaque Progression in Aortas of Apolipoprotein E
−/−
/Low-Density Lipoprotein
−/−
Double Knockout Mice
Authors: Langheinrich Alexander C. , Michniewicz Agata , Sedding Daniel G. , Walker Gerhard , Beighley Patricia E. , Rau Wigbert S. , Bohle Rainer M. , Ritman Erik L.
Published: November 2005

Objective—

We hypothesized that apolipoprotein E (apoE)
−/−
/low-density lipoprotein (LDL)
−/−
double knockout mice might develop vasa vasorum (VV) in association with advanced lesion formation.

Methods and Results—

Aortas from apoE
−/−
/LDL
−/−
mice aged 16, 18, 20, or 80 weeks were infused in situ with Microfil, harvested, and scanned with micro-computed tomography (CT). We characterized plaque volume and CT “density” as well as VV luminal volume along the aorta using Analyze 6.0 software. Results were complemented by a detailed histological plaque classification according to American Heart Association guidelines. From 16 to 80 weeks, plaque volume and VV opacified lumen volume increased with age (
P
<0.001). The 3-dimensional micro-CT images of arterial and venous VV trees allowed perfusion territories to be delineated. The spatial location and magnitude of VV density and adventitial inflammation were strongly correlated in advanced atherosclerotic lesions (
r
=0.91) and identified as an independent correlate to advanced lesions. At age 80 weeks, VV luminal volume was increased 20-fold compared with animals at age 16 weeks (
P
<0.001). Micro-CT showed that adventitial VV communicate with intraplaque microvessels.

Conclusion—

Our results show that apoE
−/−
/LDL
−/−
double knockout mice develop VV and advanced atheromas along the aorta. Lesion volume was closely associated with amount of neovascularization in advanced atheromas.
Aspirin Has A Gender-Dependent Impact on Antiinflammatory 15-Epi-Lipoxin A
4
Formation
Authors: Chiang Nan , Hurwitz Shelley , Ridker Paul M. , Serhan Charles N.
Published: November 2005

Objective—

Aspirin blocks thromboxane production that contributes to its well-appreciated antiplatelet action. Aspirin also initiates the biosynthesis of novel antiinflammatory mediators from arachidonic acid, namely aspirin-triggered 15-epi-lipoxin A
4
. We recently conducted a double-blinded clinical trial with healthy subjects in whom low-dose aspirin (81 mg daily) significantly increased aspirin-triggered 15-epi-lipoxin A
4
and concomitantly inhibited thromboxane. Here, we assessed whether plasma aspirin–triggered 15-epi-lipoxin A
4
was age or gender dependent in subjects taking low-dose aspirin.

Methods and Results—

A total of 128 subjects were allocated to: placebo, 81, 325, or 650 mg daily aspirin for an 8-week period. Plasma thromboxane B
2
and aspirin-triggered 15-epi-lipoxin A
4
were assessed from blood collected at baseline and the conclusion of the trial. We then performed a post-trial analysis in the group receiving low-dose aspirin. In female subjects, we found a positive correlation between age and aspirin-triggered 15-epi-lipoxin A
4
(increase of 0.37 ng/mL per decade), and a negative correlation was observed in men (decrease of 0.29 ng/mL per decade). These trends were significantly different from each other (
P
=0.045).

Conclusions—

Low-dose aspirin has a gender-specific impact on aspirin-triggered 15-epi-lipoxin A
4
production, which may contribute to the gender-dependent clinical benefits of aspirin. Also, they may provide a molecular rationale for low-dose aspirin therapies in elderly women to reduce inflammation-related disorders.
Role of cAMP Response Element Binding Protein in Cardiovascular Remodeling
Authors: Ichiki Toshihiro
Published: November 2005

The cAMP response element binding protein (CREB) is a ubiquitously expressed nuclear transcription factor that is activated by various extracellular stimuli. CREB is known to regulate the expression of genes important to cell proliferation, differentiation, adaptation, and survival in many cell types. Loss of CREB function by transgenic overexpression of dominant negative CREB or targeted deletion of the CREB gene revealed that CREB is involved in the differentiation of T lymphocytes, production of growth hormone, and the long-term potentiation of neuronal memory. The role of CREB in cardiovascular system is incompletely characterized and several controversies remain. A growing body of recent evidence, however, has suggested that CREB plays an important role in the cardiovascular remodeling process, including inflammation, cell migration, and apoptosis. Thus, CREB may be a possible target for the treatment of cardiovascular diseases such as atherosclerosis, restenosis, and reperfusion injury.
Protective Effect of the
KCNMB1
E65K Genetic Polymorphism Against Diastolic Hypertension in Aging Women and Its Relevance to Cardiovascular Risk
Authors: Sentí Mariano , Fernández-Fernández José M. , Tomás Marta , Vázquez Esther , Elosua Roberto , Marrugat Jaume , Valverde Miguel A.
Published: November 2005

The E65K polymorphism in the β
1
-subunit of the large-conductance, Ca
2+
-dependent K
+
(BK) channel, a key element in the control of arterial tone, has recently been associated with low prevalence of diastolic hypertension. We now report the modulatory effect of sex and age on the association of the E65K polymorphism with low prevalence of diastolic hypertension and the protective role of E65K polymorphism against cardiovascular disease. We analyzed the genotype frequency of the E65K polymorphism in 3924 participants selected randomly in two cross-sectional studies. A five-year follow-up of the cohort was performed to determine whether cardiovascular events had occurred since inclusion. Estrogen modulation of wild-type and mutant ion channel activity was assessed after heterologous expression and electrophysiological studies. Multivariate regression analyses showed that increasing age upmodulates the protective effect of the K allele against moderate-to-severe diastolic hypertension in the overall group of participants (odds ratio [OR], 0.35;
P
=0.006). The results remained significant when analyses were restricted to women (OR, 0.18;
P
=0.02) but not men (OR, 0.46;
P
=0.09). This effect was independent of the reported acute modulation of BK channels by estrogen. A five-year follow-up study also demonstrated a reduced age- and sex-adjusted hazard ratio of 0.11, 95% CI, 0.01 to 0.79 of K-carriers for “combined cardiovascular disease” (myocardial infarction and stroke) compared with EE homozygotes. Our study provides the first genetic evidence for the different impact of the BK channel in the control of human blood pressure in men and women, with particular relevance in aging women, and highlights the E65K polymorphism as one of the strongest genetic factors associated thus far to protection against myocardial infarction and stroke.
Targeted Deletion of Kv4.2 EliminatesIto,fand Results in Electrical and Molecular Remodeling, With No Evidence of Ventricular Hypertrophy or Myocardial Dysfunction
Authors: Guo Weinong , Jung W. Edward , Marionneau Céline , Aimond Franck , Xu Haodong , Yamada Kathryn A. , Schwarz Thomas L. , Demolombe Sophie , Nerbonne Jeanne M.
Published: November 2005

Previous studies have demonstrated a role for voltage-gated K+(Kv) channel α subunits of the Kv4 subfamily in the generation of rapidly inactivating/recovering cardiac transient outward K+current, Ito,f, channels. Biochemical studies suggest that mouse ventricular Ito,fchannels reflect the heteromeric assembly of Kv4.2 and Kv4.3 with the accessory subunits, KChIP2 and Kvβ1, and that Kv4.2 is the primary determinant of regional differences in (mouse ventricular) Ito,fdensities. Interestingly, the phenotypic consequences of manipulatingIto,fexpression in different mouse models are distinct. In the experiments here, the effects of the targeted deletion of Kv4.2 (Kv4.2−/−) were examined. Unexpectedly, voltage-clamp recordings from Kv4.2−/−ventricular myocytes revealed thatIto,fis eliminated. In addition, the slow transient outward K+current,Ito,s, and the Kv1.4 protein (which encodesIto,s) are upregulated in Kv4.2−/−ventricles. Although Kv4.3 mRNA/protein expression is not measurably affected, KChIP2 expression is markedly reduced in Kv4.2−/−ventricles. Similar to Kv4.3, expression of Kvβ1, as well as Kv1.5 and Kv2.1, is similar in wild-type and Kv4.2−/−ventricles. In addition, and in marked contrast to previous findings in mice expressing a truncated Kv4.2 transgene, the eliminationIto,fin Kv4.2−/−mice does not result in ventricular hypertrophy. Taken together, these findings demonstrate not only an essential role for Kv4.2 in the generation of mouse ventricularIto,fchannels but also that the loss ofIto,fper se does not have overt pathophysiological consequences.
Excitation–Contraction Coupling in Na
+
–Ca
2+
Exchanger Knockout Mice
Authors: Pott Christian , Philipson Kenneth D. , Goldhaber Joshua I.
Published: November 2005

Cardiac-specific Na
+
–Ca
2+
exchanger (NCX) knockout (KO) mice surprisingly survive into adulthood without compensatory changes in protein expression levels. To determine how cardiac function is maintained in the absence of NCX, we investigated membrane currents, intracellular Ca
2+
, and action potentials (APs) in whole cell patch-clamped myocytes from wild-type (WT) and NCX knockout mice. There was no difference in resting Ca
2+
or sarcoplasmic reticular Ca
2+
load between KO and WT. During prolonged caffeine exposure, the decrease of the Ca
2+
transient was drastically slowed in KO versus WT myocytes, indicating that no alternative Ca
2+
-extrusion mechanism is upregulated to compensate for the absence of NCX. Peak L-type Ca
2+
current (
I
Ca
) was reduced by 62% in KO myocytes compared with WT. Nevertheless, the corresponding Ca
2+
transients were similar, implying an increase in the gain of excitation–contraction coupling in KO cells. APs recorded from KO cells repolarized more rapidly than in WT. In WT myocytes, applying a KO AP waveform voltage clamp reduced Ca
2+
influx via
I
Ca
by 59% compared with WT AP waveform clamps. Again, the corresponding Ca
2+
transients remained similar. Our findings indicate that NCX KO myocytes limit Ca
2+
influx to &20% of that in WT by reducing
I
Ca
and by abbreviating the AP. Contractility is maintained by an increase in the gain of excitation–contraction coupling resulting from both a more rapid repolarization of the AP and an as yet unidentified AP-independent mechanism.
Association of
WNK1
Gene Polymorphisms and Haplotypes With Ambulatory Blood Pressure in the General Population
Authors: Tobin Martin D. , Raleigh Stuart M. , Newhouse Stephen , Braund Peter , Bodycote Clare , Ogleby Jenny , Cross Deborah , Gracey Jay , Hayes Saija , Smith Terry , Ridge Cathy , Caulfield Mark , Sheehan Nuala A. , Munroe Patricia B. , Burton Paul R. , Samani Nilesh J.
Published: November 2005

Background—

Blood pressure (BP) is a heritable trait of major public health concern. The
WNK1
and
WNK4
genes, which encode proteins in the WNK family of serine-threonine kinases, are involved in renal electrolyte homeostasis. Mutations in the
WNK1
and
WNK4
genes cause a rare monogenic hypertensive syndrome, pseudohypoaldosteronism type II. We investigated whether polymorphisms in these
WNK
genes influence BP in the general population.

Methods and Results—

Associations between 9 single-nucleotide polymorphisms (SNPs) in
WNK1
and 1 in
WNK4
with ambulatory BP were studied in a population-based sample of 996 subjects from 250 white European families. The heritability estimates of mean 24-hour systolic BP (SBP) and diastolic BP (DBP) were 63.4% and 67.9%, respectively. We found statistically significant (
P
<0.05) associations of several common SNPs and haplotypes in
WNK1
with mean 24-hour SBP and/or DBP. The minor allele (
C
) of rs880054, with a frequency of 44%, reduced mean 24-hour SBP and DBP by 1.37 (95% confidence interval, −2.45 to −0.23) and 1.14 (95% confidence interval, −1.93 to −0.38) mm Hg, respectively, per copy of the allele.

Conclusions—

Common variants in
WNK1
contribute to BP variation in the general population. This study shows that a gene causing a rare monogenic form of hypertension also plays a significant role in BP regulation in the general population. The findings provide a basis to identify functional variants of
WNK1
, elucidate any interactions of these variants with dietary intake or with response to antihypertensive drugs, and determine their impact on cardiovascular morbidity and mortality.
Deficiency of Neuronal Nitric Oxide Synthase Increases Mortality and Cardiac Remodeling After Myocardial Infarction
Authors: Saraiva Roberto M. , Minhas Khalid M. , Raju Shubha V.Y. , Barouch Lili A. , Pitz Eleanor , Schuleri Karl H. , Vandegaer Koenraad , Li Dechun , Hare Joshua M.
Published: November 2005

Background—

Neuronal nitric oxide synthase (NOS1) plays key cardiac physiological roles, regulating excitation-contraction coupling and exerting an antioxidant effect that maintains tissue NO-redox equilibrium. After myocardial infarction (MI), NOS1 translocates from the sarcoplasmic reticulum to the cell membrane, where it inhibits β-adrenergic contractility, an effect previously predicted to have adverse consequences. Counter to this idea, we tested the hypothesis that NOS1 has a protective effect after MI.

Methods and Results—

We studied mortality, cardiac remodeling, and upregulation of oxidative stress pathways after MI in NOS1-deficient (NOS1
−/−
) and wild-type C57BL6 (WT) mice. Compared with WT, NOS1
−/−
mice had greater mortality (hazard ratio, 2.06;
P
=0.036), worse left ventricular (LV) fractional shortening (19.7±1.5% versus 27.2±1.5%,
P
<0.05), higher LV diastolic diameter (5.5±0.2 versus 4.9±0.1 mm,
P
<0.05), greater residual cellular width (14.9±0.5 versus 12.8±0.5 μm,
P
<0.01), and equivalent β-adrenergic hyporesponsiveness despite similar MI size. Superoxide production increased after MI in both NOS1
−/−
and WT animals, although NO increased only in WT. NADPH oxidase (
P
<0.05) activity increased transiently in both groups after MI, but NOS1
−/−
mice had persistent basal and post-MI elevations in xanthine oxidoreductase activity.

Conclusions—

Together these findings support a protective role for intact NOS1 activity in the heart after MI, despite a potential contribution to LV dysfunction through β-adrenergic hyporesponsiveness. NOS1 deficiency contributes to an imbalance between oxidative stress and tissue NO signaling, providing a plausible mechanism for adverse consequences of NOS1 deficiency in states of myocardial injury.
Optimization of Transplant Regimens for Patients with Myelodysplastic Syndrome (MDS)
Authors: Deeg H. Joachim
Published: November 2005

Myelodysplastic syndrome (MDS) is a hemopoietic stem cell disorder that is potentially curable by transplantation of normal hemopoietic stem cells. The optimum timing, however, and the best conditioning strategy have remained controversial. Both conventional and reduced-intensity/nonmyeloablative regimens have been used successfully. Among selected patients with less advanced/low-risk MDS (< 5% marrow myeloblasts), 3-year survivals of 65% to 75% are achievable with HLA-matched related and unrelated donors. Among patients with more advanced/ high-risk disease (≥ 5% marrow blasts; high International Prognostic Scoring System [IPSS] scores), the probability of post-transplant relapse ranges from 10% to 40%, and, as a result, relapse-free survival is inferior. In addition to disease stage, co-morbid conditions, pre-transplant chemotherapy, conditioning regimen, source of stem cells, and post-transplant immunosuppression affect transplant outcome. Reduced-intensity conditioning regimens are associated with a decrease in non-relapse mortality and allow for successful hemopoietic stem cell transplants even in patients 60–70 years of age. Graft-versus-host disease, both acute and chronic, and post-transplant relapse remain challenging problems.
Specialization, kinetics, and repertoire of type 1 interferon responses by human plasmacytoid predendritic cells
Authors: Ito Tomoki , Kanzler Holger , Duramad Omar , Cao Wei , Liu Yong-Jun
Published: November 2005

Recent studies suggest plasmacytoid predendritic cells (pDCs) and myeloid dendritic cells (mDCs) have the functional plasticity to produce similar amounts of type 1 interferons (IFNs) and interleukin-12 (IL-12), challenging the concept and existence of DC subsets with distinct function. In this study, we demonstrate that previous studies showed human pDCs produce large amounts of IL-12 because of contaminating mDCs. Using highly purified human DC subsets, we found that although pDCs make 300 times more IFN-α than mDCs and mDCs make 13 times more IL-12 p70 than pDCs in response to all the toll-like receptor ligands and CD40 ligands, pDCs rapidly make large amounts of IFN-α within the first 12 hours of activation and become refractory to further stimulation. pDCs preferentially expressed the transcriptional factors critical for type 1 IFN, but not for IL-12 transcription, and they dedicated 60% of new transcriptional activity to make 19 type 1 IFN subtypes. This study provides formal proof that the plasticity of DC subsets is limited and that different DC subsets evolve to perform distinct functions in linking innate and adaptive immunity. (Blood. 2006;107:2423-2431)
GILZ expression in human dendritic cells redirects their maturation and prevents antigen-specific T lymphocyte response
Authors: Cohen Nicolas , Mouly Enguerran , Hamdi Haifa , Maillot Marie-Christine , Pallardy Marc , Godot Véronique , Capel Francis , Balian Axel , Naveau Sylvie , Galanaud Pierre , Lemoine François M. , Emilie Dominique
Published: November 2005

Interleukin (IL)-10 and glucocorticoids (GCs) inhibit the ability of antigen-presenting dendritic cells (DCs) to stimulate T lymphocytes. We show that induction of GILZ (GC-induced leucine zipper) is involved in this phenomenon. IL-10, dexamethasone (DEX), and transforming growth factor (TGF)β stimulate GILZ production in human immature DCs derived from monocytes and from CD34+ cells. GILZ is necessary and sufficient for DEX, IL-10, and TGFβ modulation of CD80, CD83, CD86, immunoglobulin-like transcript (ILT)-3, and B7-H1 expression by DCs, and alteration of DC functions. GILZ stimulates the production of IL-10 by immature DCs and prevents the production of inflammatory chemokines by CD40L-activated DCs. In contrast, GILZ does not prevent CD40 ligand-mediated inhibition of phagocytosis, indicating that it affects some but not all aspects of DC maturation. GILZ prevents DCs from activating antigen-specific T lymphocyte responses. Administration of GCs to patients stimulates GILZ expression in their circulating antigen-presenting cells, and this contributes to the weak lymphocyte responses of GC-treated patients. Thus, regulation of GILZ expression is an important factor determining the decision of DCs whether or not to stimulate T lymphocytes, and IL-10, GCs, and TGFβ share this mechanism for influencing DC functions and the balance between immune response and tolerance.
Identification of glucocorticoid-response genes in children with acute lymphoblastic leukemia
Authors: Schmidt Stefan , Rainer Johannes , Riml Stefan , Ploner Christian , Jesacher Simone , Achmüller Clemens , Presul Elisabeth , Skvortsov Sergej , Crazzolara Roman , Fiegl Michael , Raivio Taneli , Jänne Olli A. , Geley Stephan , Meister Bernhard , Kofler Reinhard
Published: November 2005

The ability of glucocorticoids (GCs) to kill lymphoid cells led to their inclusion in essentially all chemotherapy protocols for lymphoid malignancies, particularly childhood acute lymphoblastic leukemia (ALL). GCs mediate apoptosis via their cognate receptor and subsequent alterations in gene expression. Previous investigations, including expression profiling studies with subgenome microarrays in model systems, have led to a number of attractive, but conflicting, hypotheses that have never been tested in a clinical setting. Here, we present a comparative whole-genome expression profiling approach using lymphoblasts (purified at 3 time points) from 13 GC-sensitive children undergoing therapy for ALL. For comparisons, expression profiles were generated from an adult patient with ALL, peripheral blood lymphocytes from GC-exposed healthy donors, GC-sensitive and -resistant ALL cell lines, and mouse thymocytes treated with GCs in vivo and in vitro. This generated an essentially complete list of GC-regulated candidate genes in clinical settings and experimental systems, allowing immediate analysis of any gene for its potential significance to GC-induced apoptosis. Our analysis argued against most of the model-based hypotheses and instead identified a small number of novel candidate genes, including PFKFB2, a key regulator of glucose metabolism; ZBTB16, a putative transcription factor; and SNF1LK, a protein kinase implicated in cell-cycle regulation.
CD38 orchestrates migration, survival, and Th1 immune response of human mature dendritic cells
Authors: Frasca Loredana , Fedele Giorgio , Deaglio Silvia , Capuano Cristina , Palazzo Raffaella , Vaisitti Tiziana , Malavasi Fabio , Ausiello Clara Maria
Published: November 2005

CD38, an ectoenzyme and a signaling receptor, is a novel marker of human mature monocyte-derived dendritic cells (MDDCs). The working hypothesis is that CD38 is not only a marker but also contributes to functions specifically gained by MDDCs with maturation. This was tested by assessing the role(s) of CD38 after signaling with agonistic anti-CD38 monoclonal antibodies or by blocking the interactions taking place between CD38 and CD31, its counterreceptor. The results indicate the following: (1) CD38 engagement in MDDCs ensures efficient chemotaxis and transendothelial migration driven by CC chemokine ligand 21 (CCL21); (2) CD38 is laterally associated with the CCL21-specific CC chemokine receptor 7 and with CD83 and CD11b; (3) CD38 localizes in membrane lipid domains; (4) CD38 signaling contributes to support longevity of lipopolysaccharide (LPS)–matured MDDCs after growth factor withdrawal; and (5) IFN-γ is produced by cocultured T lymphocytes, thus affecting T-helper 1 (Th1) polarization. These data suggest that the localization of CD38 in lipid rafts and its multiple interactions with signaling receptors rule innate and adaptive immune responses by tuning DC migration, survival, and Th1-polarization ability. These findings may lay out the basis to assess the functional role(s) of human CD38 in infections, autoimmune diseases, and neoplastic disorders.
High frequency of alternative first exons in erythroid genes suggests a critical role in regulating gene function
Authors: Tan Jeff S. , Mohandas Narla , Conboy John G.
Published: November 2005

The human genome uses alternative pre-mRNA splicing as an important mechanism to encode a complex proteome from a relatively small number of genes. An unknown number of these genes also possess multiple transcriptional promoters and alternative first exons that contribute another layer of complexity to gene expression mechanisms. Using a collection of more than 100 erythroid-expressed genes as a test group, we used genome browser tools and genetic databases to assess the frequency of alternative first exons in the genome. Remarkably, 35% of these erythroid genes show evidence of alternative first exons. The majority of the candidate first exons are situated upstream of the coding exons, whereas a few are located internally within the gene. Computational analyses predict transcriptional promoters closely associated with many of the candidate first exons, supporting their authenticity. Importantly, the frequent presence of consensus translation initiation sites among the alternative first exons suggests that many proteins have alternative N-terminal structures whose expression can be coupled to promoter choice. These findings indicate that alternative promoters and first exons are more widespread in the human genome than previously appreciated and that they may play a major role in regulating expression of selected protein isoforms in a tissue-specific manner. (Blood. 2006;107: 2557-2561)
Size regulation of von Willebrand factor–mediated platelet thrombi by ADAMTS13 in flowing blood
Authors: Donadelli Roberta , Orje Jennifer N. , Capoferri Cristina , Remuzzi Giuseppe , Ruggeri Zaverio M.
Published: November 2005

The metalloproteinase ADAMTS13 regulates the size of released von Willebrand factor (VWF) multimers bound to endothelial cells, but it is unknown whether it can cleave plasma VWF during thrombogenesis. To address this issue, we perfused blood over immobilized VWF and used videomicroscopy to visualize an activation-independent platelet aggregation process mediated by soluble VWF at shear rates greater than 10 000 s-1. At normal Ca2+ concentration, platelets formed rolling as well as surface-attached clusters that grew larger during the first 5 minutes but then lost more than 70% of their mass by 10 minutes. In contrast, platelet clusters were stable in size when metal ions were chelated, anti-ADAMTS13 IgG were added, or washed blood cells were perfused with purified VWF but no plasma. In the latter case, addition of recombinant ADAMTS13 reduced platelet cluster size by more than 70%. Incubating ADAMTS13 with VWF before perfusion did not prevent the initial platelet clustering, indicating that the enzyme may act on platelet-bound VWF under shear stress. At the concentrations tested, ADAMTS13 had no effect on platelet aggregates formed upon blood perfusion over collagen fibrils. ADAMTS13, therefore, may regulate thrombus size preferentially when the cohesion between platelets depends on VWF binding induced by pathologically elevated shear stress.
Patterns of expression, membrane localization, and effects of ectopic expression suggest a function for MS4a4B, a CD20 homolog in Th1 T cells
Authors: Xu Hui , Williams Mark S. , Spain Lisa M.
Published: November 2005

The membrane-spanning 4A (MS4A) family of proteins includes CD20, FcϵRIβ, and HTm4, whose genes are grouped in a chromosomal location that is associated with increased susceptibility to allergy and atopic asthma. One family member, Chandra/MS4a4B, was reported to be expressed in T helper 1 (Th1) T cells but not Th2 T cells. In the present study, Ms4a4b was isolated in a screen of genes differentially expressed during thymocyte development. MS4a4B was detected in immature CD4-CD8-CD44+CD25- thymocytes, turned off during further stages of thymocyte development and reexpressed in mature single-positive thymocytes. MS4a4B expression was found in naive CD8+ and CD4+ peripheral T cells and natural killer (NK) cells but not in B cells. MS4a4B is expressed at the cell surface with its C-terminus located in the cytoplasm. When expressed in a T-cell hybridoma by retroviral vector, MS4a4B protein constitutively associated with lipid raft microdomains, whereas in primary T cells endogenous MS4a4B protein became enriched in rafts after T-cell activation. Overexpression of MS4a4B in primary CD4+ T-cell blasts enhanced T-cell receptor (TCR)-induced Th1 cytokine production. These results suggest that MS4a4B expression is tightly regulated during T-cell development and that MS4a4B expression promotes Th1 function and/or differentiation. (Blood. 2006;107:2400-2408)
Suicide gene therapy of graft-versus-host disease induced by central memory human T lymphocytes
Authors: Bondanza Attilio , Valtolina Veronica , Magnani Zulma , Ponzoni Maurilio , Fleischhauer Katharina , Bonyhadi Mark , Traversari Catia , Sanvito Francesca , Toma Salvatore , Radrizzani Marina , La Seta-Catamancio Simona , Ciceri Fabio , Bordignon Claudio , Bonini Chiara
Published: November 2005

In allogeneic hematopoietic cell transplantation (allo-HCT), the immune recognition of host antigens by donor T lymphocytes leads to a beneficial graft-versus-leukemia (GvL) effect as well as to life-threatening graft-versus-host disease (GvHD). Genetic modification of T lymphocytes with a retroviral vector (RV) expressing the herpes simplex virus-thymidine kinase (TK) suicide gene confers selective sensitivity to the prodrug ganciclovir (GCV). In patients, the infusion of TK+ lymphocytes and the subsequent administration of GCV resulted in a time-wise modulation of antihost reactivity for a GvL effect, while controlling GvHD. Because activation required for genetic modification with RV may reduce antihost reactivity, we investigated the requirements for maximizing the potency of human TK+ lymphocytes. Whereas T-cell receptor triggering alone led to effector memory (EM) TK+ lymphocytes, the addition of CD28 costimulation through cell-sized beads resulted in the generation of central memory (CM) TK+ lymphocytes. In a quantitative model for GvHD using nonobese diabetic/severely combined immunodeficient mice, CM TK+ lymphocytes were more potent than EM TK+ lymphocytes. GCV administration efficiently controlled GvHD induced by CM TK+ lymphocytes. These results warrant the clinical investigation of CM suicide gene-modified human T lymphocytes for safe and effective allo-HCT.
A MAPK/HNRPK pathway controls BCR/ABL oncogenic potential by regulating MYC mRNA translation
Authors: Notari Mario , Neviani Paolo , Santhanam Ramasamy , Blaser Bradley W. , Chang Ji-Suk , Galietta Annamaria , Willis Anne E. , Roy Denis C. , Caligiuri Michael A. , Marcucci Guido , Perrotti Danilo
Published: November 2005

Altered mRNA translation is one of the effects exerted by the BCR/ABL oncoprotein in the blast crisis phase of chronic myelogenous leukemia (CML). Here, we report that in BCR/ABL+ cell lines and in patient-derived CML blast crisis mononuclear and CD34+ cells, p210BCR/ABL increases expression and activity of the transcriptional-inducer and translational-regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K or HNRPK) in a dose- and kinase-dependent manner through the activation of the MAPKERK1/2 pathway. Furthermore, HNRPK down-regulation and interference with HNRPK translation-but not transcription-regulatory activity impairs cytokine-independent proliferation, clonogenic potential, and in vivo leukemogenic activity of BCR/ABL-expressing myeloid 32Dcl3 and/or primary CD34+ CML-BC patient cells. Mechanistically, we demonstrate that decreased internal ribosome entry site (IRES)-dependent Myc mRNA translation accounts for the phenotypic changes induced by inhibition of the BCR/ABL-ERK-dependent HNRPK translation-regulatory function. Accordingly, MYC protein but not mRNA levels are increased in the CD34+ fraction of patients with CML in accelerated and blastic phase but not in chronic phase CML patients and in the CD34+ fraction of marrow cells from healthy donors. Thus, BCR/ABL-dependent enhancement of HNRPK translation-regulation is important for BCR/ABL leukemogenesis and, perhaps, it might contribute to blast crisis transformation. (Blood. 2006;107:2507-2516)
Siglec-H is an IPC-specific receptor that modulates type I IFN secretion through DAP12
Authors: Blasius Amanda L. , Cella Marina , Maldonado Jorge , Takai Toshiyuki , Colonna Marco
Published: November 2005

Natural interferon (IFN)-producing cells are the primary cell type responsible for production of type I IFN in response to viruses. Herein we report the identification of the first molecular marker of mouse natural interferon-producing cells (IPCs), a novel member of the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family termed Siglec-H. Siglec-H is expressed exclusively on IPCs and is unique among Siglec proteins in that it associates with the adaptor protein DAP12. Moreover, we show that DAP12 modulates the type I IFN response of IPCs to a Toll-like receptor 9 (TLR9) agonist. This observation explains our previous finding that stimulation of IPCs with 440c, a Siglec-H-specific antibody, reduces IPC secretion of type I IFN. Moreover, it supports a model in which engagement of DNAX-activation protein 12 (DAP12)-associated receptors with antibodies or low avidity endogenous ligands interferes with TLR-mediated cellular activation. (Blood. 2006;107:2474-2476)
The structure of the GPIb–filamin A complex
Authors: Nakamura Fumihiko , Pudas Regina , Heikkinen Outi , Permi Perttu , Kilpeläinen Ilkka , Munday Adam D. , Hartwig John H. , Stossel Thomas P. , Ylänne Jari
Published: November 2005

Filamin A (FLNa), a dimeric actin cross-linking and scaffold protein with numerous intracellular binding partners, anchors the platelet adhesion glycoprotein (GP) Ib-IX-V receptor to actin cytoskeleton. We mapped the GPIbα binding site to a single domain of FLNa and resolved the structure of this domain and its interaction complex with the corresponding GPIbα cytoplasmic domain. This is the first atomic structure of this class of membrane glycoprotein–cytoskeleton connection. GPIbα binds in a groove formed between the C and D β strands of FLNa domain 17. The interaction is strikingly similar to that between the β7 integrin tail and a different FLNa domain, potentially defining a conserved motif for FLNa binding. Nevertheless, the structures also reveal specificity of the interfaces, which explains different regulatory mechanisms. To verify the topology of GPIb-FLNa interaction we also purified the native complex from platelets and showed that GPIb interacts with the C-terminus of FLNa, which is in accordance with our biochemical and structural data.
A stepwise framework for the normalization of array CGH data
Authors: Khojasteh Mehrnoush , Lam Wan L , Ward Rabab K , MacAulay Calum
Published: November 2005

Background

In two-channel competitive genomic hybridization microarray experiments, the ratio of the two fluorescent signal intensities at each spot on the microarray is commonly used to infer the relative amounts of the test and reference sample DNA levels. This ratio may be influenced by systematic measurement effects from non-biological sources that can introduce biases in the estimated ratios. These biases should be removed before drawing conclusions about the relative levels of DNA. The performance of existing gene expression microarray normalization strategies has not been evaluated for removing systematic biases encountered in array-based comparative genomic hybridization (CGH), which aims to detect single copy gains and losses typically in samples with heterogeneous cell populations resulting in only slight shifts in signal ratios. The purpose of this work is to establish a framework for correcting the systematic sources of variation in high density CGH array images, while maintaining the true biological variations.

Results

After an investigation of the systematic variations in the data from two array CGH platforms, SMRT (Sub Mega base Resolution Tiling) BAC arrays and cDNA arrays of Pollack et al., we have developed a stepwise normalization framework integrating novel and existing normalization methods in order to reduce intensity, spatial, plate and background biases. We used stringent measures to quantify the performance of this stepwise normalization using data derived from 5 sets of experiments representing self-self hybridizations, replicated experiments, detection of single copy changes, array CGH experiments which mimic cell population heterogeneity, and array CGH experiments simulating different levels of gene amplifications and deletions. Our results demonstrate that the three-step normalization procedure provides significant improvement in the sensitivity of detection of single copy changes compared to conventional single step normalization approaches in both SMRT BAC array and cDNA array platforms.

Conclusion

The proposed stepwise normalization framework preserves the minute copy number changes while removing the observed systematic biases.
An acquisition account of genomic islands based on genome signature comparisons
Authors: van Passel MWJ , Bart A , Thygesen HH , Luyf ACM , van Kampen AHC , van der Ende A
Published: November 2005

Background

Recent analyses of prokaryotic genome sequences have demonstrated the important force horizontal gene transfer constitutes in genome evolution. Horizontally acquired sequences are detectable by, among others, their dinucleotide composition (genome signature) dissimilarity with the host genome. Genomic islands (GIs) comprise important and interesting horizontally transferred sequences, but information about acquisition events or relatedness between GIs is scarce. In Vibrio vulnificus CMCP6, 10 and 11 GIs have previously been identified in the sequenced chromosomes I and II, respectively. We assessed the compositional similarity and putative acquisition account of these GIs using the genome signature. For this analysis we developed a new algorithm, available as a web application.

Results

Of 21 GIs, VvI-1 and VvI-10 of chromosome I have similar genome signatures, and while artificially divided due to a linear annotation, they are adjacent on the circular chromosome and therefore comprise one GI. Similarly, GIs VvI-3 and VvI-4 of chromosome I together with the region between these two islands are compositionally similar, suggesting that they form one GI (making a total of 19 GIs in chromosome I + chromosome II). Cluster analysis assigned the 19 GIs to 11 different branches above our conservative threshold. This suggests a limited number of compositionally similar donors or intragenomic dispersion of ancestral acquisitions. Furthermore, 2 GIs of chromosome II cluster with chromosome I, while none of the 19 GIs group with chromosome II, suggesting an unidirectional dispersal of large anomalous gene clusters from chromosome I to chromosome II.

Conclusion

From the results, we infer 10 compositionally dissimilar donors for 19 GIs in the V. vulnificus CMCP6 genome, including chromosome I donating to chromosome II. This suggests multiple transfer events from individual donor types or from donors with similar genome signatures. Applied to other prokaryotes, this approach may elucidate the acquisition account in their genome sequences, and facilitate donor identification of GIs.