bluuebunny/crossref_metadata_2025 huggingface dataset:
The spliceosomal autoantigen heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) is a major T cell autoantigen in patients with systemic lupus erythematosus
Authors: Fritsch-Stork Ruth , Müllegger Daniela , Skriner Karl , Jahn-Schmid Beatrice , Smolen Josef S , Steiner Günter
Published: July 2006

A hallmark of systemic lupus erythematosus (SLE) is the appearance of autoantibodies to nuclear antigens, including autoantibodies directed to the heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2), which occur in 20% to 30% of SLE patients as well as in animal models of this disease. To investigate the underlying cellular reactivity and to gain further insight into the nature and potential pathogenic role of this autoimmune response we characterized the T cell reactivity against hnRNP-A2 in patients with SLE in comparison to healthy controls. Cellular proliferation of peripheral blood T cells to hnRNP-A2 was determined by [3H]thymidine incorporation and T cell clones (TCCs) specific for hnRNP-A2 were grown by limiting dilution cloning; IFNγ, IL-4 and IL-10 in culture supernatants were measured by ELISA. Bioactivity of culture supernatants was determined by incubation of anti-CD3/anti-CD28 stimulated peripheral blood CD4+ T cells with supernatants of TCCs. Stimulation assays performed with peripheral blood mononuclear cells of 35 SLE patients and 21 healthy controls revealed pronounced proliferative responses in 66% of SLE patients and in 24% of the controls, which were significantly higher in SLE patients (p < 0.00002). Furthermore, hnRNP-A2 specific TCCs generated from SLE patients (n = 22) contained a relatively high proportion of CD8+ clones and mostly lacked CD28 expression, in contrast to TCCs derived from healthy controls (n = 12). All CD4+ TCCs of patients and all control TCCs secreted IFNγ and no IL-4. In contrast, CD8+ TCCs of patients secreted very little IFNγ, while production of IL-10 did not significantly differ from other T cell subsets. Interestingly, all CD8+ clones producing IL-10 in large excess over IFNγ lacked expression of CD28. Functional assays showed a stimulatory effect of the supernatants derived from these CD8+CD28- hnRNP-A2 specific TCCs that was similar to that of CD4+CD28+ clones. Taken together, the pronounced peripheral T cell reactivity to hnRNP-A2 observed in the majority of SLE patients and the distinct phenotype of patient-derived CD8+ TCCs suggest a role for these T cells in the pathogenesis of SLE.
Diagnostic value of antibodies against a modified citrullinated vimentin in rheumatoid arthritis
Authors: Dejaco Christian , Klotz Werner , Larcher Heike , Duftner Christina , Schirmer Michael , Herold Manfred
Published: July 2006

Antibodies directed against citrullinated vimentin are members of the family of autoantibodies reactive with citrullinated proteins and are among the most specific serological markers for the diagnosis of rheumatoid arthritis (RA). This study was performed to test the diagnostic value of a newly developed enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against a genetically modified citrullinated vimentin (anti-MCV) in comparison with a second-generation anti-cyclic citrullinated peptides (anti-CCP2) ELISA test system. Blinded sera from 631 patients (409 consecutive out-patients and 222 randomly selected stored sera) with RA (n = 164) and non-RA (osteoarthritis [n = 120], polymyalgia rheumatica/giant cell arteritis [n = 80], spondyloarthritis [n = 36], and other inflammatory rheumatic or non-inflammatory disease [n = 67]) were tested for the presence of anti-MCV and anti-CCP2 antibodies according to the manufacturers' instructions. The diagnostic performance of the anti-MCV was comparable with the anti-CCP2 assay for the diagnosis of RA according to the calculated area under the curve (0.824; 95% confidence interval (CI) 0.778–0.870 versus 0.818; 95% CI 0.767–0.869) as analysed by receiving operating characteristic curve. When categorised with a cutoff value of 20.0 U/ml (as recommended by the manufacturer), sensitivity and specificity of the anti-MCV ELISA were 69.5% (95% CI 61.9%–76.5%) and 90.8% (86.9%–93.8%), respectively, compared with 70.1% (62.5%–77.0%) and 98.7% (96.7%–99.6%) of the anti-CCP2 assay. Using the cutoff values of 19.0 U/ml and 81.5 U/ml for the anti-MCV test to obtain a sensitivity and specificity identical to the anti-CCP2 assay, showed a reduced specificity (89.8%; 85.8%–92.9%) and sensitivity (53.7%; 45.7%–61.5%), respectively, of the anti-MCV ELISA compared with the anti-CCP2 test. In conclusion, the serum ELISA testing for anti-MCV antibodies as well as the anti-CCP-2 assay perform comparably well in the diagnosis of RA. In the high-specificity range, however, the anti-CCP2 assay appears to be superior to the anti-MCV test.
Synovial fluid leukocyte apoptosis is inhibited in patients with very early rheumatoid arthritis
Authors: Raza Karim , Scheel-Toellner Dagmar , Lee Chi-Yeung , Pilling Darrell , Curnow S John , Falciani Francesco , Trevino Victor , Kumar Kanta , Assi Lakhvir K , Lord Janet M , Gordon Caroline , Buckley Christopher D , Salmon Mike
Published: July 2006

Synovial leukocyte apoptosis is inhibited in established rheumatoid arthritis (RA). In contrast, high levels of leukocyte apoptosis are seen in self-limiting crystal arthritis. The phase in the development of RA at which the inhibition of leukocyte apoptosis is first apparent, and the relationship between leukocyte apoptosis in early RA and other early arthritides, has not been defined. We measured synovial fluid leukocyte apoptosis in very early arthritis and related this to clinical outcome. Synovial fluid was obtained at presentation from 81 patients with synovitis of ≤ 3 months duration. The percentages of apoptotic neutrophils and lymphocytes were assessed on cytospin preparations. Patients were assigned to diagnostic groups after 18 months follow-up. The relationship between leukocyte apoptosis and patient outcome was assessed. Patients with early RA had significantly lower levels of neutrophil apoptosis than patients who developed non-RA persistent arthritis and those with a resolving disease course. Similarly, lymphocyte apoptosis was absent in patients with early RA whereas it was seen in patients with other early arthritides. The inhibition of synovial fluid leukocyte apoptosis in the earliest clinically apparent phase of RA distinguishes this from other early arthritides. The mechanisms for this inhibition may relate to the high levels of anti-apoptotic cytokines found in the early rheumatoid joint (e.g. IL-2, IL-4, IL-15 GMCSF, GCSF). It is likely that this process contributes to an accumulation of leukocytes in the early rheumatoid lesion and is involved in the development of the microenvironment required for persistent RA.
Psychological pain treatment in fibromyalgia syndrome: efficacy of operant behavioural and cognitive behavioural treatments
Authors: Thieme Kati , Flor Herta , Turk Dennis C
Published: July 2006

The present study focused on the evaluation of the effects of operant behavioural (OBT) and cognitive behavioural (CBT) treatments for fibromyalgia syndrome (FMS). One hundred and twenty-five patients who fulfilled the American College of Rheumatology criteria for FMS were randomly assigned to OBT (n = 43), CBT (n = 42), or an attention-placebo (AP) treatment (n = 40) that consisted of discussions of FMS-related problems. Assessments of physical functioning, pain, affective distress, and cognitive and behavioural variables were performed pre-treatment and post-treatment as well as 6 and 12 months post-treatment. Patients receiving the OBT or CBT reported a significant reduction in pain intensity post-treatment (all Fs > 3.89, all Ps < 0.01). In addition, the CBT group reported statistically significant improvements in cognitive (all Fs > 7.95, all P < 0.01) and affective variables (all Fs > 2.99, all Ps < 0.02), and the OBT group demonstrated statistically significant improvements in physical functioning and behavioural variables (all Fs > 5.99, all Ps < 0.001) compared with AP. The AP group reported no significant improvement but actually deterioration in the outcome variables. The post-treatment effects for the OBT and CBT groups were maintained at both the 6- and 12-month follow-ups. These results suggest that both OBT and CBT are effective in treating patients with FMS with some differences in the outcome measures specifically targeted by the individual treatments compared with an unstructured discussion group. The AP group showed that unstructured discussion of FMS-related problems may be detrimental.
Independent associations of anti-cyclic citrullinated peptide antibodies and rheumatoid factor with radiographic severity of rheumatoid arthritis
Authors: Mewar Devesh , Coote Annabel , Moore David J , Marinou Ioanna , Keyworth Jodie , Dickson Marion C , Montgomery Doug S , Binks Michael H , Wilson Anthony G
Published: July 2006

Several recent publications have established a strong association between anti-cyclic citrullinated peptide antibody (anti-CCP)-positive rheumatoid arthritis (RA) and carriage of shared epitope (SE) alleles. Although anti-CCP have also been associated with more severe RA, the issue of whether this is independent of rheumatoid factor (RF) has not been addressed. To identify associations between RF, anti-CCP, SE status and radiological damage, we studied a large cross-sectional cohort with longstanding RA. Individuals (n = 872) enrolled in the study all fulfilled the American College of Rheumatology criteria for RA, had a minimum disease duration of 3 years, and at least one definite radiographic erosion was present in hands or feet. Radiographs were scored blind at study entry by a single musculoskeletal radiologist using a modified Larsen's score. Anti-CCP and RF levels were determined using enzyme-linked immunosorbent assay, and DRB1 typing was performed using polymerase chain reaction based methodology. Both anti-CCP and RF levels were strongly associated with radiographic severity (P < 0.0001). In subgroups stratified for both anti-CCP and RF status, evidence of independent associations of both antibodies with radiographic outcome was found (P < 0.0001). An association of SE alleles with radiographic severity was present only in RF-negative individuals. Anti-CCP positivity was associated with SE status with evidence of a gene-dose effect, most markedly in RF-negative individuals (P < 0.01). Anti-CCP and RF status are independent severity factors for RA, with SE alleles playing at most a secondary role. Our data support the view that previously described associations between SE and radiological severity, especially in RF-negative patients, may be indirect and due to an association with anti-CCP.
Statin-induced expression of CD59 on vascular endothelium in hypoxia: a potential mechanism for the anti-inflammatory actions of statins in rheumatoid arthritis
Authors: Kinderlerer Anne R , Steinberg Rivka , Johns Michael , Harten Sarah K , Lidington Elaine A , Haskard Dorian O , Maxwell Patrick H , Mason Justin C
Published: July 2006

Hypoxia, which leads to dysfunctional cell metabolism, and complement activation both play central roles in the pathogenesis of rheumatoid arthritis (RA). Recent studies have reported that mice deficient for the complement-inhibitory protein CD59 show enhanced susceptibility to antigen-induced arthritis and reported that statins have anti-inflammatory effects in RA. We hypothesized that the anti-inflammatory effect of statins in RA relates in part to their ability to increase CD59 expression in hypoxic conditions and therefore to reduce complement activation.

Flow-cytometric analysis showed that CD59 expression on endothelial cells (EC) was unaffected by atorvastatin in normoxia (21% O2), whereas in hypoxic conditions (1% O2) an up to threefold dose-dependent increase in CD59 expression was seen. This effect of hypoxia was confirmed by treatment of EC with chemical mimetics of hypoxia. The upregulation of CD59 protein expression in hypoxia was associated with an increase in steady-state mRNA. L-Mevalonate and geranylgeraniol reversed the response, confirming a role for inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase and geranylgeranylation. Likewise, inhibition by NG-monomethyl-L-arginine and NG-nitro-L-arginine methyl ester confirmed that CD59 upregulation in hypoxia was nitric oxide dependent. The expression of another complement-inhibitory protein, decay-accelerating factor (DAF), is known to be increased by atorvastatin in normoxia; this response was also significantly enhanced under hypoxic conditions. The upregulation of CD59 and DAF by atorvastatin in hypoxia prevented the deposition of C3, C9 and cell lysis that follows exposure of reoxygenated EC to serum. This cytoprotective effect was abrogated by inhibitory anti-CD59 and anti-DAF mAbs. The modulation of EC CD59 and DAF by statins under hypoxic conditions therefore inhibits both early and late complement activation and may contribute to the anti-inflammatory effects of statins in RA.
β3Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells
Authors: Galliher Amy J , Schiemann William P
Published: July 2006

Introduction

Transforming growth factor (TGF)-β suppresses breast cancer formation by preventing cell cycle progression in mammary epithelial cells (MECs). During the course of mammary tumorigenesis, genetic and epigenetic changes negate the cytostatic actions of TGF-β, thus enabling TGF-β to promote the acquisition and development of metastatic phenotypes. The molecular mechanisms underlying this conversion of TGF-β function remain poorly understood but may involve signaling inputs from integrins.

Methods

β3 Integrin expression or function in MECs was manipulated by retroviral transduction of active or inactive β3 integrins, or by transient transfection of small interfering RNA (siRNA) against β3 integrin. Altered proliferation, invasion, and epithelial-mesenchymal transition (EMT) stimulated by TGF-β in control and β3 integrin manipulated MECs was determined. Src involvement in β3 integrin mediated alterations in TGF-β signaling was assessed by performing Src protein kinase assays, and by interdicting Src function pharmacologically and genetically.

Results

TGF-β stimulation induced αvβ3 integrin expression in a manner that coincided with EMT in MECs. Introduction of siRNA against β3 integrin blocked its induction by TGF-β and prevented TGF-β stimulation of EMT in MECs. β3 integrin interacted physically with the TGF-β receptor (TβR) type II, thereby enhancing TGF-β stimulation of mitogen-activated protein kinases (MAPKs), and of Smad2/3-mediated gene transcription in MECs. Formation of β3 integrin:TβR-II complexes blocked TGF-β mediated growth arrest and increased TGF-β mediated invasion and EMT. Dual β3 integrin:TβR-II activation induced tyrosine phosphorylation of TβR-II, a phosphotransferase reaction mediated by Src in vitro. Inhibiting Src activity in MECs prevented the ability of β3 integrin to induce TβR-II tyrosine phosphorylation, MAPK activation, and EMT stimulated by TGF-β. Lastly, wild-type and D119A β3 integrin expression enhanced and abolished, respectively, TGF-β stimulation of invasion in human breast cancer cells.

Conclusion

We show that β3 integrin alters TGF-β signaling in MECs via Src-mediated TβR-II tyrosine phosphorylation, which significantly enhanced the ability of TGF-β to induce EMT and invasion. Our findings suggest that β3 integrin interdiction strategies may represent an innovative approach to re-establishing TGF-β mediated tumor suppression in progressing human breast cancers.
The mTOR inhibitor rapamycin down-regulates the expression of the ubiquitin ligase subunit Skp2 in breast cancer cells
Authors: Shapira Ma'anit , Kakiashvili Eli , Rosenberg Tzur , Hershko Dan D
Published: July 2006

Introduction

Loss of the cyclin-dependent kinase inhibitor p27 is associated with poor prognosis in breast cancer. The decrease in p27 levels is mainly the result of enhanced proteasome-dependent degradation mediated by its specific ubiquitin ligase subunit S phase kinase protein 2 (Skp2). The mammalian target of rapamycin (mTOR) is a downstream mediator in the phosphoinositol 3' kinase (PI3K)/Akt pathway that down-regulates p27 levels in breast cancer. Rapamycin was found to stabilize p27 levels in breast cancer, but whether this effect is mediated through changes in Skp2 expression is unknown.

Methods

The expression of Skp2 mRNA and protein levels were examined in rapamycin-treated breast cancer cell lines. The effect of rapamycin on the degradation rate of Skp2 expression was examined in cycloheximide-treated cells and in relationship to the anaphase promoting complex/Cdh1 (APC\C) inhibitor Emi1.

Results

Rapamycin significantly decreased Skp2 mRNA and protein levels in a dose and time-dependent fashion, depending on the sensitivity of the cell line to rapamycin. The decrease in Skp2 levels in the different cell lines was followed by cell growth arrest at G1. In addition, rapamycin enhanced the degradation rate of Skp2 and down-regulated the expression of the APC\C inhibitor Emi1.

Conclusion

These results suggest that Skp2, an important oncogene in the development and progression of breast cancer, may be a novel target for rapamycin treatment.
Pharmacokinetics of recombinant activated factor VII in trauma patients with severe bleeding
Authors: Klitgaard Thomas , y Palacios Rene Tabanera , Boffard Kenneth D , Iau Philip TC , Warren Brian , Rizoli Sandro , Rossaint Rolf , Kluger Yoram , Riou Bruno ,   the NovoSeven® Trauma Study Group
Published: July 2006

Introduction

Recombinant activated factor VII (rFVIIa) has been used as adjunctive therapy in trauma patients with severe bleeding. However, its pharmacokinetics profile remains unknown.

Methods

In two placebo-controlled studies in patients with blunt and penetrating trauma, the pharmacokinetics of rFVIIa given at an initial dose of 200 μg.kg-1 after transfusion of eight red blood cell units, followed by additional doses of 100 μg.kg-1, one and three hours later, have been studied, based on the FVII coagulant activity assay. Both non-compartment and population pharmacokinetic analyses were performed. A two-compartment, population pharmacokinetic model was used to estimate a population profile for the rFVIIa dosing regimen. Data are population means (percent coefficient of variation (CV)).

Results

Based on the two-compartment population model, the estimated pharmacokinetic parameters were: clearance 40 (30% CV) ml.kg-1.h-1; central volume of distribution 89 (32% CV) ml.kg-1; inter-compartmental clearance 24 ml.kg-1.h-1; and peripheral compartment volume 31 ml.kg-1. Baseline FVII coagulant activity was estimated at 0.29 (39% CV) U.ml-1, initial half-life was 0.6 (34% CV) hours, and terminal half-life 2.4 (50% CV) hours. High intra- and inter-patient variability was noted in volume of distribution and clearance, which was in part correlated with the transfusion requirements as the single significant covariate. The non-compartmental analysis led to almost identical estimates of key parameters.

Conclusion

A high intra- and inter-patient variability was noted in the volume of distribution and clearance of rFVIIa in trauma patients with severe bleeding, mainly related with the transfusion requirements and thus blood loss and/or bleeding rate.
Open lung biopsy in early-stage acute respiratory distress syndrome
Authors: Kao Kuo-Chin , Tsai Ying-Huang , Wu Yao-Kuang , Chen Ning-Hung , Hsieh Meng-Jer , Huang Shiu-Feng , Huang Chung-Chi
Published: July 2006

Introduction

Acute respiratory distress syndrome (ARDS) has heterogeneous etiologies, rapid progressive change and a high mortality rate. To improve the outcome of ARDS, accurate diagnosis is essential to the application of effective early treatment. The present study investigated the clinical effects and safety of open lung biopsy (OLB) in patients with early-stage ARDS of suspected non-infectious origin.

Methods

We undertook a retrospective study of 41 patients with early-stage ARDS (defined as one week or less after intubation) who underwent OLB in two medical intensive care units of a tertiary care hospital from 1999 to 2005. Data analyzed included baseline characteristics, complication rate, pathological diagnoses, treatment alterations, and hospital survival.

Results

The age of patients was 55 ± 17 years (mean ± SD). The average ratio of arterial partial pressure of oxygen (PaO2) to fraction of inspired oxygen (FiO2) was 116 ± 43 mmHg (mean ± SD) at biopsy. Seventeen patients (41%) were immunocompromised. Postoperative complications occurred in 20% of patients (8/41). All biopsies provided a pathological diagnosis with a diagnostic yield of 100%. Specific pathological diagnoses were made for 44% of patients (18/41). Biopsy findings led to an alteration of treatment modality in 73% of patients (30/41). The treatment alteration rate was higher in patients with nonspecific diagnoses than in patients with specific diagnoses (p = 0.0024). Overall mortality was 50% (21/41) and was not influenced by age, gender, pre-OLB oxygenation, complication rate, pathological results, and alteration of treatment. There was no surgery-related mortality. The survival rate for immunocompromised patients was better than that for immunocompetent patients (71% versus 33%; p = 0.0187) in this study.

Conclusion

Our retrospective study suggests that OLB was a useful and acceptably safe diagnostic procedure in some selected patients with early-stage ARDS.
Dehydroepiandrosterone administration modulates endothelial and neutrophil adhesion molecule expression in vitro
Authors: Barkhausen Tanja , Westphal Britt-Mailin , Pütz Claudia , Krettek Christian , van Griensven Martijn
Published: July 2006

Introduction

The steroid hormone dehydroepiandrosterone (DHEA) exerts protecting effects in the treatment of traumatic and septic complications in several animal models. This effect goes along with reduced amounts of infiltrating immune cells in organs such as lung and liver. However, the underlying mechanisms of DHEA action are still not known. Adhesion molecules are important for the extravasation of neutrophils into organs where they may exhibit detrimental effects. Therefore, we investigated the in vitro effect of DHEA on the expression pattern of adhesion molecules of human endothelial cells and neutrophils.

Methods

Endothelial cells derived from human umbilical cord were subjected to an lipopolysaccharide (LPS) challenge. DHEA was administered in two different concentrations, 10-5 M and 10-8 M, as a single stimulus or in combination with LPS challenge. After two, four and 24 hours, fluorescence activated cell sorter (FACS) analysis for vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and E-selectin was performed. Neutrophils were freshly isolated from blood of 10 male healthy volunteers, stimulated the same way as endothelial cells and analyzed for surface expression of L-selectin, CD11b and CD18.

Results

In the present study, we were able to demonstrate effects of DHEA on the expression of every adhesion molecule investigated. DHEA exhibits opposite effects to those seen upon LPS exposure. Furthermore, these effects are both time and concentration dependent as most DHEA specific effects could be detected in the physiological concentration of 10-8 M.

Conclusion

Thus, we conclude that one mechanism by which DHEA may exert its protection in animal models is via the differential regulation of adhesion molecule expression.
Serum procalcitonin level and leukocyte antisedimentation rate as early predictors of respiratory dysfunction after oesophageal tumour resection
Authors: Bogar Lajos , Molnar Zsolt , Tarsoly Piroska , Kenyeres Peter , Marton Sandor
Published: July 2006

Introduction

Postoperative care after oesophageal tumour resection holds a high risk of respiratory complications. We therefore aimed to determine the value of systemic inflammatory markers in predicting arterial hypoxaemia as the earliest sign of developing lung injury after oesophageal tumour resection.

Methods

In a prospective observational study, 33 consecutive patients were observed for three days (T1–T3) after admission (T0) to an intensive care unit following oesophageal tumour resection. The daily highest values of the heart rate, axillary temperature, leukocyte count and PaCO2 were recorded. Serum C-reactive protein and procalcitonin concentrations and the leukocyte antisedimentation rate (LAR) were determined at T1 and T2. Respiratory function was monitored 6-hourly measurement of the PaO2/FIO2 ratio, and the lowest value was recorded at T3. Patients were categorised as normoxaemic or hypoxaemic using the cutoff value of 300 mmHg for PaO2/FIO2.

Results

Seventeen out of 33 patients were classified as hypoxaemic and 16 patients as normoxaemic at T3. Increases of temperature at T0 and of the procalcitonin and LAR values at T2 were predictive of hypoxaemia at T3 (P < 0.05, P < 0.01 and P < 0.001, respectively). The area under the receiver-operating characteristic curve was 0.65 for the temperature at T0, which was significantly lower than that for the procalcitonin level at T2 (0.83; 95% confidence interval, 0.69–0.97; P < 0.01) and that for LAR at T2 (0.89; 95% confidence interval, 0.77–1.00; P < 0.001).

Conclusion

These results suggest that an elevated LAR (>15%) and an elevated procalcitonin concentration (>2.5 ng/ml) measured on the second postoperative day can predict next-day arterial hypoxaemia (PaO2/FIO2 < 300 mmHg) after oesophageal tumour resection.
Timing of adequate antibiotic therapy is a greater determinant of outcome than are TNF and IL-10 polymorphisms in patients with sepsis
Authors: Garnacho-Montero Jose , Aldabo-Pallas Teresa , Garnacho-Montero Carmen , Cayuela Aurelio , Jiménez Rocio , Barroso Sonia , Ortiz-Leyba Carlos
Published: July 2006

Introduction

Genetic variations may influence clinical outcomes in patients with sepsis. The present study was conducted to evaluate the impact on mortality of three polymorphisms after adjusting for confounding variables, and to assess the factors involved in progression of the inflammatory response in septic patients.

Method

The inception cohort study included all Caucasian adults admitted to the hospital with sepsis. Sepsis severity, microbiological information and clinical variables were recorded. Three polymorphisms were identified in all patients by PCR: the tumour necrosis factor (TNF)-α 308 promoter polymorphism; the polymorphism in the first intron of the TNF-β gene; and the IL-10-1082 promoter polymorphism. Patients included in the study were followed up for 90 days after hospital admission.

Results

A group of 224 patients was enrolled in the present study. We did not find a significant association among any of the three polymorphisms and mortality or worsening inflammatory response. By multivariate logistic regression analysis, only two factors were independently associated with mortality, namely Acute Physiology and Chronic Health Evaluation (APACHE) II score and delayed initiation of adequate antibiotic therapy. In septic shock patients (n = 114), the delay in initiation of adequate antibiotic therapy was the only independent predictor of mortality. Risk factors for impairment in inflammatory response were APACHE II score, positive blood culture and delayed initiation of adequate antibiotic therapy.

Conclusion

This study emphasizes that prompt and adequate antibiotic therapy is the cornerstone of therapy in sepsis. The three polymorphisms evaluated in the present study appear not to influence the outcome of patients admitted to the hospital with sepsis.
Design principles of molecular networks revealed by global comparisons and composite motifs
Authors: Yu Haiyuan , Xia Yu , Trifonov Valery , Gerstein Mark
Published: July 2006

Background

Molecular networks are of current interest, particularly with the publication of many large-scale datasets. Previous analyses have focused on topologic structures of individual networks.

Results

Here, we present a global comparison of four basic molecular networks: regulatory, co-expression, interaction, and metabolic. In terms of overall topologic correlation - whether nearby proteins in one network are close in another - we find that the four are quite similar. However, focusing on the occurrence of local features, we introduce the concept of composite hubs, namely hubs shared by more than one network. We find that the three 'action' networks (metabolic, co-expression, and interaction) share the same scaffolding of hubs, whereas the regulatory network uses distinctly different regulator hubs. Finally, we examine the inter-relationship between the regulatory network and the three action networks, focusing on three composite motifs - triangles, trusses, and bridges - involving different degrees of regulation of gene pairs. Our analysis shows that interaction and co-expression networks have short-range relationships, with directly interacting and co-expressed proteins sharing regulators. However, the metabolic network contains many long-distance relationships: far-away enzymes in a pathway often have time-delayed expression relationships, which are well coordinated by bridges connecting their regulators.

Conclusion

We demonstrate how basic molecular networks are distinct yet connected and well coordinated. Many of our conclusions can be mapped onto structured social networks, providing intuitive comparisons. In particular, the long-distance regulation in metabolic networks agrees with its counterpart in social networks (namely, assembly lines). Conversely, the segregation of regulator hubs from other hubs diverges from social intuitions (as managers often are centers of interactions).
Shuffling of cis-regulatory elements is a pervasive feature of the vertebrate lineage
Authors: Sanges Remo , Kalmar Eva , Claudiani Pamela , D'Amato Maria , Muller Ferenc , Stupka Elia
Published: July 2006

Background

All vertebrates share a remarkable degree of similarity in their development as well as in the basic functions of their cells. Despite this, attempts at unearthing genome-wide regulatory elements conserved throughout the vertebrate lineage using BLAST-like approaches have thus far detected noncoding conservation in only a few hundred genes, mostly associated with regulation of transcription and development.

Results

We used a unique combination of tools to obtain regional global-local alignments of orthologous loci. This approach takes into account shuffling of regulatory regions that are likely to occur over evolutionary distances greater than those separating mammalian genomes. This approach revealed one order of magnitude more vertebrate conserved elements than was previously reported in over 2,000 genes, including a high number of genes found in the membrane and extracellular regions. Our analysis revealed that 72% of the elements identified have undergone shuffling. We tested the ability of the elements identified to enhance transcription in zebrafish embryos and compared their activity with a set of control fragments. We found that more than 80% of the elements tested were able to enhance transcription significantly, prevalently in a tissue-restricted manner corresponding to the expression domain of the neighboring gene.

Conclusion

Our work elucidates the importance of shuffling in the detection of cis-regulatory elements. It also elucidates how similarities across the vertebrate lineage, which go well beyond development, can be explained not only within the realm of coding genes but also in that of the sequences that ultimately govern their expression.
What makes species unique? The contribution of proteins with obscure features
Authors: Gollery Martin , Harper Jeff , Cushman John , Mittler Taliah , Girke Thomas , Zhu Jian-Kang , Bailey-Serres Julia , Mittler Ron
Published: July 2006

Background

Proteins with obscure features (POFs), which lack currently defined motifs or domains, represent between 18% and 38% of a typical eukaryotic proteome. To evaluate the contribution of this class of proteins to the diversity of eukaryotes, we performed a comparative analysis of the predicted proteomes derived from 10 different sequenced genomes, including budding and fission yeast, worm, fly, mosquito, Arabidopsis, rice, mouse, rat, and human.

Results

Only 1,650 protein groups were found to be conserved among these proteomes (BLAST E-value threshold of 10-6). Of these, only three were designated as POFs. Surprisingly, we found that, on average, 60% of the POFs identified in these 10 proteomes (44,236 in total) were species specific. In contrast, only 7.5% of the proteins with defined features (PDFs) were species specific (17,554 in total). As a group, POFs appear similar to PDFs in their relative contribution to biological functions, as indicated by their expression, participation in protein-protein interactions and association with mutant phenotypes. However, POF have more predicted disordered structure than PDFs, implying that they may exhibit preferential involvement in species-specific regulatory and signaling networks.

Conclusion

Because the majority of eukaryotic POFs are not well conserved, and by definition do not have defined domains or motifs upon which to formulate a functional working hypothesis, understanding their biochemical and biological functions will require species-specific investigations.
yrGATE: a web-based gene-structure annotation tool for the identification and dissemination of eukaryotic genes
Authors: Wilkerson Matthew D , Schlueter Shannon D , Brendel Volker
Published: July 2006

Your Gene structure Annotation Tool for Eukaryotes (yrGATE) provides an Annotation Tool and Community Utilities for worldwide web-based community genome and gene annotation. Annotators can evaluate gene structure evidence derived from multiple sources to create gene structure annotations. Administrators regulate the acceptance of annotations into published gene sets. yrGATE is designed to facilitate rapid and accurate annotation of emerging genomes as well as to confirm, refine, or correct currently published annotations. yrGATE is highly portable and supports different standard input and output formats. The yrGATE software and usage cases are available at http://www.plantgdb.org/prj/yrGATE.
The prokaryotic antecedents of the ubiquitin-signaling system and the early evolution of ubiquitin-like β-grasp domains
Authors: Iyer Lakshminarayan M , Burroughs A Maxwell , Aravind L
Published: July 2006

Background

Ubiquitin (Ub)-mediated signaling is one of the hallmarks of all eukaryotes. Prokaryotic homologs of Ub (ThiS and MoaD) and E1 ligases have been studied in relation to sulfur incorporation reactions in thiamine and molybdenum/tungsten cofactor biosynthesis. However, there is no evidence for entire protein modification systems with Ub-like proteins and deconjugation by deubiquitinating enzymes in prokaryotes. Hence, the evolutionary assembly of the eukaryotic Ub-signaling apparatus remains unclear.

Results

We systematically analyzed prokaryotic Ub-related β-grasp fold proteins using sensitive sequence profile searches and structural analysis. Consequently, we identified novel Ub-related proteins beyond the characterized ThiS, MoaD, TGS, and YukD domains. To understand their functional associations, we sought and recovered several conserved gene neighborhoods and domain architectures. These included novel associations involving diverse sulfur metabolism proteins, siderophore biosynthesis and the gene encoding the transfer mRNA binding protein SmpB, as well as domain fusions between Ub-like domains and PIN-domain related RNAses. Most strikingly, we found conserved gene neighborhoods in phylogenetically diverse bacteria combining genes for JAB domains (the primary de-ubiquitinating isopeptidases of the proteasomal complex), along with E1-like adenylating enzymes and different Ub-related proteins. Further sequence analysis of other conserved genes in these neighborhoods revealed several Ub-conjugating enzyme/E2-ligase related proteins. Genes for an Ub-like protein and a JAB domain peptidase were also found in the tail assembly gene cluster of certain caudate bacteriophages.

Conclusion

These observations imply that members of the Ub family had already formed strong functional associations with E1-like proteins, UBC/E2-related proteins, and JAB peptidases in the bacteria. Several of these Ub-like proteins and the associated protein families are likely to function together in signaling systems just as in eukaryotes.
A method for high-throughput gene expression signature analysis
Authors: Peck David , Crawford Emily D , Ross Kenneth N , Stegmaier Kimberly , Golub Todd R , Lamb Justin
Published: July 2006

Genome-wide transcriptional profiling has shown that different biologic states (for instance, disease and response to pharmacologic manipulation) can be recognized by the expression pattern of relatively small numbers of genes. However, the lack of a practical and cost-effective technology for detection of these gene expression 'signatures' in large numbers of samples has severely limited their exploitation in important medical and pharmaceutical discovery applications. Here, we describe a solution based on the combination of ligation-mediated amplification with an optically addressed microsphere and flow cytometric detection system.
A strategy for extracting and analyzing large-scale quantitative epistatic interaction data
Authors: Collins Sean R , Schuldiner Maya , Krogan Nevan J , Weissman Jonathan S
Published: July 2006

Recently, approaches have been developed for high-throughput identification of synthetic sick/lethal gene pairs. However, these are only a specific example of the broader phenomenon of epistasis, wherein the presence of one mutation modulates the phenotype of another. We present analysis techniques for generating high-confidence quantitative epistasis scores from measurements made using synthetic genetic array and epistatic miniarray profile (E-MAP) technology, as well as several tools for higher-level analysis of the resulting data that are greatly enhanced by the quantitative score and detection of alleviating interactions.
Critical role for serum opsonins and complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in phagocytosis of Francisella tularensis by human dendritic cells (DC): uptake of Francisella leads to activation of immature DC and intracellular survival of the bacteria
Authors: Ben Nasr Abdelhakim , Haithcoat Judith , Masterson Joseph E , Gunn John S , Eaves-Pyles Tonyia , Klimpel Gary R
Published: July 2006

Francisella tularensis is one of the most infectious human pathogens known. Although much has been learned about the immune response of mice using an attenuated live vaccine strain (LVS) derived from F. tularensis subspecies holarctica (Type B), little is known about the responses of human monocyte-derived immature dendritic cells (DC). Here, we show that optimal phagocytosis of LVS by DC is dependent on serum opsonization. We demonstrate that complement factor C3-derived opsonins and the major complement receptors expressed by DC, the integrins CR3 (CD11b/CD18) and CR4 (CD11c/CD18), play a critical role in this adhesion-mediated phagocytosis. LVS induced proinflammatory cytokine production and up-regulation of costimulatory surface proteins (CD40, CD86, and MHC Class II) on DC but resisted killing. Once taken up, LVS grew intracellularly, resulting in DC death. DC maturation and cytokine production were induced by direct contact/phagocytosis of LVS or interaction with soluble products of the bacteria, and enhanced activation was seen when LVS was pretreated with serum. Sonicated LVS and supernatants from LVS cultures were potent activators of DC, but LVS LPS failed to activate DC maturation or cytokine production. Serum-treated LVS rapidly induced (within 6 h) a number of cytokines including IL-10, a potent suppressor of macrophage functions and down-regulator of Th1-like responses and the Th1 response inducer IL-12. These results suggest that the simultaneous production of an activating (IL-12, IL-1β, and TNF-α) and a suppressing (IL-10) cytokine profile could contribute to the immunopathogenesis of tularemia.
Suppression of Prolactin-Induced Signal Transducer and Activator of Transcription 5b Signaling and Induction of Suppressors of Cytokine Signaling Messenger Ribonucleic Acid in the Hypothalamic Arcuate Nucleus of the Rat during Late Pregnancy and Lactation
Authors: Anderson Greg M. , Beijer Paulien , Bang Angela S. , Fenwick Mark A. , Bunn Stephen J. , Grattan David R.
Published: July 2006

During late pregnancy and lactation, the tuberoinfundibular dopamine (TIDA) neurons that regulate prolactin secretion by negative feedback become less able to produce dopamine in response to prolactin, leading to hyperprolactinemia. Because prolactin-induced activation of dopamine synthesis in these neurons requires the Janus kinase/signal transducer and activator of transcription 5b (STAT5b) signaling pathway, we investigated whether prolactin-induced STAT5b signaling is reduced during lactation and whether induction of suppressors of cytokine signaling (SOCS) mRNAs occur at this time and in late pregnancy. During lactation, the ability of exogenous prolactin to induce STAT5 phosphorylation and STAT5b nuclear translocation was markedly reduced when compared with diestrous rats. In nonpregnant female rats, acute treatment with ovine prolactin markedly increased levels of SOCS-1 and -3 and cytokine-inducible SH2-containing protein mRNA in arcuate nucleus micropunches. On gestation d 22, SOCS-1 and SOCS-3 mRNA levels were 10-fold that on G20. SOCS-1 and -3 and cytokine-inducible SH2-containing protein mRNA levels were also elevated on lactation d 7. At these times, dopaminergic activity was decreased and the rats were hyperprolactinemic. The high levels of SOCS mRNA were prevented by bromocriptine pretreatment (gestation d 22) or pup removal (lactation d 7), which suppressed circulating prolactin to basal levels. These results demonstrate that around the end of pregnancy, prolactin loses the ability to activate STAT5b, associated with an increase in SOCS mRNAs. The loss of this stimulating pathway may underlie the reduced tuberoinfundibular dopamine neuron dopamine output and hyperprolactinemia that characterizes late pregnancy and lactation. The high maternal levels of SOCS mRNAs appear to be dependent on prolactin, presumably acting through an alternative signaling pathway to STAT5b.
Calpain System Regulates the Differentiation of Adult Primitive Mesenchymal ST-13 Adipocytes
Authors: Yajima Yukiko , Sato Mayumi , Sorimachi Hiroyuki , Inomata Mitsushi , Maki Masatoshi , Kawashima Seiichi
Published: July 2006

The activity of calpain, a calcium-activated protease, is required during the mitotic clonal expansion phase of 3T3-L1 embryonic preadipocyte differentiation. Here we examined the role of calpain in the adipogenesis of ST-13 preadipocytes established from adult primitive mesenchymal cells, which do not require mitotic clonal expansion. After exposure to the calpain inhibitor, N-benzyloxycarbonyl-l-leucyl-l-leucinal or overexpression of calpastatin, a specific endogenous inhibitor of calpain, ST-13 preadipocytes acquired the adipocyte phenotype. Overexpression of calpastatin in ST-13 adipocytes stimulated the expression of adipocyte-specific CCAAT/enhancer-binding protein-α (C/EBPα), peroxisome proliferator-activated receptor (PPAR)-γ, sterol regulatory element-binding protein 1, and the insulin signaling molecules, insulin receptor α, insulin-receptor substrates, and GLUT4. However, insulin-stimulated glucose uptake was reduced by approximately 52%. The addition of calpain to the nuclear fraction of ST-13 adipocytes resulted in the Ca2+-dependent degradation of PPARγ and C/EBPα but not sterol regulatory element-binding protein 1. Exposing ST-13 adipocytes to A23187 also led to losses of endogenous PPARγ and C/EBPα. Under both conditions, calpain inhibitors almost completely prevented C/EBPα cleavage but partially blocked the decrease of PPARγ. Two ubiquitous forms of calpain, μ- and m-calpain, localized to the cytosol and the nucleus, whereas the activated form of μ- but not m-calpain was found in the nucleus. Finally, stable dominant-negative μ-calpain transfectants showed accelerated adipogenesis and increase in the levels of PPARγ and C/EBPα during adipocyte program. These results support evidence that the calpain system is involved in regulating the differentiation of adult primitive mesenchymal ST-13 preadipocytes.
Insulin-Like Growth Factor-I Is Essential for Embryonic Bone Development
Authors: Wang Yongmei , Nishida Shigeki , Sakata Takeshi , Elalieh Hashem Z. , Chang Wenhan , Halloran Bernard P. , Doty Steven B. , Bikle Daniel D.
Published: July 2006

Although IGF-I has been identified as an important growth factor for the skeleton, the role of IGF-I on embryonic bone development remains unknown. Here we show that, in IGF-I-deficient (IGF-I−/−) mice, skeletal malformations, including short-limbed dwarfism, were evident at days post coitus (dpc) 14.5 to 18.5, accompanied by delays of mineralization in the spinal column, sternum, and fore paws. Reduced chondrocyte proliferation and increased chondrocyte apoptosis were identified in both the spinal ossification center and the growth plate of long bones. Abnormal chondrocyte differentiation and delayed initiation of mineralization was characterized by small size and fewer numbers of type X collagen expressing hypertrophic chondrocytes and lower osteocalcin expression. The Indian hedgehog-PTHrP feedback loop was altered; expression of Indian hedgehog was reduced in IGF-I−/− mice in long bones and in the spine, whereas expression of PTHrP was increased. Our results indicate that IGF-I plays an important role in skeletal development by promoting chondrocyte proliferation and maturation while inhibiting apoptosis to form bones of appropriate size and strength.
Orexin-Induced Apoptosis: The Key Role of the Seven-Transmembrane Domain Orexin Type 2 Receptor
Authors: Voisin Thierry , Firar Aadil El , Avondo Virgile , Laburthe Marc
Published: July 2006

Orexin-A and orexin-B are regulatory peptides involved in the control of feeding, sleep-wakefulness, and exerting various endocrine and metabolic actions. Recently we demonstrated that orexins, acting at OX1 receptor (OX1R), are proapoptotic peptides. The aim of this study was to investigate the role of the receptor subtype OX2R in the control of apoptosis. Orexins caused a caspase-dependent cell death by apoptosis and a drastic cell growth inhibition in Chinese hamster ovary cells transfected with OX2R cDNA. On addition of either orexin (10−6m) for 48 h, apoptosis was demonstrated by DNA fragmentation, chromatin condensation, annexin-V binding, and activation of caspase-3 and caspase-9. Orexins were active on apoptosis and cell growth inhibition in the range of concentrations between 10−10 and 10−5m with an EC50 of 5 × 10−8m peptides. No effect of orexins could be detected in parental Chinese hamster ovary cells. A rat pancreatic acinar cell line, AR42J, which expresses OX2R but not OX1R, also underwent growth suppression and apoptosis on treatment with orexins. Suppression of AR42J cell growth by 10−6m orexin was more than 75% after 24 h. Induction of annexin-V-labeled AR42J cell number was dose dependent, with EC50 of 5.1 × 10−8m orexin-A and 9.8 × 10−8m orexin-B. The OX2R agonist [Ala (11), d-Leu (15)]orexin-B promoted effects on cell growth and apoptosis, which were similar to those elicited by orexins. The OX1R antagonist SB33487 did not alter orexin-induced inhibition of growth or orexin-induced stimulation of apoptosis in AR42J cells. For the first time, we provide functional and pharmacological evidence for a role of the OX2R in orexin-induced apoptosis.