Early prognostic factors in septic shock cancer patients: a prospective study with a proteomic approach.

BACKGROUND: Organ failures are the main prognostic factors in septic shock. The aim was to assess classical clinico-biological parameters evaluating organ dysfunctions at intensive care unit admission, combined with proteomics, on day-30 mortality in critically ill onco-hematology patients admitted to the intensive care unit for septic shock. METHODS: This was a prospective monocenter cohort study. Clinico-biological parameters were collected at admission. Plasma proteomics analyses were performed, including protein profiling using isobaric Tag for Relative and Absolute Quantification (iTRAQ) and subsequent validation by ELISA. RESULTS: Sixty consecutive patients were included. Day-30 mortality was 47%. All required vasopressors, 32% mechanical ventilation, 33% non-invasive ventilation and 13% renal-replacement therapy. iTRAQ-based proteomics identified von Willebrand factor as a protein of interest. Multivariate analysis identified four factors independently associated with day-30 mortality: positive fluid balance in the first 24 h (odds ratio = 1.06, 95% CI = 1.01-1.12, P = 0.02), severe acute respiratory failure (odds ratio = 6.14, 95% CI = 1.04-36.15, P = 0.04), von Willebrand factor plasma level > 439 ng/ml (odds ratio = 9.7, 95% CI = 1.52-61.98, P = 0.02), and bacteremia (odds ratio = 6.98, 95% CI = 1.17-41.6, P = 0.03). CONCLUSION: Endothelial dysfunction, revealed by proteomics, appears as an independent prognostic factor on day-30 mortality, as well as hydric balance, acute respiratory failure and bacteremia, in critically ill cancer patients admitted to the intensive care unit. Endothelial failure is underestimated in clinical practice and represents an innovative therapeutic target.

Antifibroblast antibodies from systemic sclerosis patients bind to {alpha}-enolase and are associated with interstitial lung disease.

OBJECTIVE: To identify target antigens of antifibroblast antibodies (AFA) in systemic sclerosis (SSc) patients. PATIENTS AND METHODS: In the first part, sera from 24 SSc patients (12 with pulmonary arterial hypertension (PAH) and 12 without) and 36 idiopathic PAH patients, tested in pooled sera for groups of three, were compared with a sera pool from 14 healthy controls (HC). Serum IgG reactivity was analysed by the use of a two-dimensional electrophoresis and immunoblotting technique with normal human fibroblasts antigens. In the second part, serum IgG reactivity for two groups: 158 SSc, 67 idiopathic PAH and 100 HC; and 35 SSc and 50 HC was tested against alpha-enolase from Saccharomyces cerevisiae and recombinant human (rHu) alpha-enolase, respectively, on ELISA. RESULTS: In the first part, alpha-enolase was identified as a main target antigen of AFA from SSc patients. In the second part, 37/158 (23%) SSc patients, 6/67 (9%) idiopathic PAH patients and 4/100 (4%) HC (p<0.001) had anti-S cerevisiae alpha-enolase antibodies; 12/35 (34%) SSc patients and 3/50 (6%) HC had anti-rHu alpha-enolase antibodies (p = 0.001). In SSc, the presence of anti-S cerevisiae alpha-enolase antibodies was associated with interstitial lung disease (ILD), decreased total lung capacity (73.2% vs 89.7%; p<0.001) and diffusion capacity for carbon monoxide (47.4% vs 62.3%; p<0.001), and antitopoisomerase 1 antibodies (46% vs 21%; p = 0.005) but not anticentromere antibodies (11% vs 34%; p = 0.006). Results were similar with rHu alpha-enolase testing. CONCLUSION: In SSc, AFA recognise alpha-enolase and are associated with ILD and antitopoisomerase antibodies.

SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations.

The SLX4 tumor suppressor is a scaffold that plays a pivotal role in several aspects of genome protection, including homologous recombination, interstrand DNA crosslink repair and the maintenance of common fragile sites and telomeres. Here, we unravel an unexpected direct interaction between SLX4 and the DNA helicase RTEL1, which, until now, were viewed as having independent and antagonistic functions. We identify cancer and Hoyeraal-Hreidarsson syndrome-associated mutations in SLX4 and RTEL1, respectively, that abolish SLX4-RTEL1 complex formation. We show that both proteins are recruited to nascent DNA, tightly co-localize with active RNA pol II, and that SLX4, in complex with RTEL1, promotes FANCD2/RNA pol II co-localization. Importantly, disrupting the SLX4-RTEL1 interaction leads to DNA replication defects in unstressed cells, which are rescued by inhibiting transcription. Our data demonstrate that SLX4 and RTEL1 interact to prevent replication-transcription conflicts and provide evidence that this is independent of the nuclease scaffold function of SLX4.

Publisher Correction: SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: SLX4 interacts with RTEL1 to prevent transcription-mediated DNA replication perturbations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The effect of stromelysin-1 (MMP-3) on non-collagenous extracellular matrix proteins of demineralized dentin and the adhesive properties of restorative resins.

Dentin non-collagenous matrix components (NCPs) are structural proteins involved in the formation, the architecture and the mineralization of the extracellular matrix (ECM). We investigated here how recombinant metalloproteinase stromelysin-1, also termed MMP-3, initiates the release of ECM molecules from artificially demineralized human dentin. Analysis of the supernatants by Western blotting reveals that MMP-3 extracts PGs (decorin, biglycan), and also a series of phosphorylated proteins: dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP) and MEPE, but neither dentin matrix protein-1 (DMP1), another member of the SIBLING family, nor osteocalcin (OC), a non-phosphorylated matrix molecule. After treatment of dentin surfaces by MMP-3, scanning electron microscope (SEM) examination of resin replica shows an increased penetration of the resin into the dentin tubules when compared to surfaces only treated by demineralizing solutions. This preclinical investigation suggests that MMP-3 may be used to improve the adhesive properties of restorative materials.

[Update on thrombotic thrombocytopenic purpura]. / Le purpura thrombotique thrombocytopénique acquis de l'adulte : actualités.

PURPOSE: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder and early treatment is vital. Here, we review the recent advances in the understanding of the pathophysiology of TTP and its treatment. CURRENT KNOWLEDGE AND KEY POINTS: Recent advances have shown that TTP is caused by deficiency of the (ADAMTS-13) metalloprotease that cleaves von Willebrand factor multimers. Acquired TTP is associated to inhibitory antibodies directed against ADAMTS-13. This has led to assess new therapeutic approaches in refractory and relapsing forms of TTP and the use of rituximab has shown very encouraging results. FUTURE PROSPECTS AND PROJECTS: A better characterization of TTP amongst the other thrombotic microangiopathies has allowed the use of new therapeutic approaches with the use of rituximab. The encouraging results reported with rituximab in some forms of TTP challenge the classic treatment based on plasma exchanges.

In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant.

Microparticles (MPs) resulting from vesiculation of platelets and other blood cells have been extensively documented in vitro and have been found in increased numbers in several vascular diseases, but little is known about MPs of endothelial origin. The aim of this study was to analyze morphological, immunological, and functional characteristics of MPs derived from human umbilical vein endothelial cells (HUVECs) stimulated by TNF, and to investigate whether these MPs are detectable in healthy individuals and in patients with a prothrombotic coagulation abnormality. Electron microscopy evidenced bleb formation on the membrane of TNF-stimulated HUVECs, leading to increased numbers of MPs released in the supernatant. These endothelial microparticles (EMPs) expressed the same antigenic determinants as the corresponding cell surface, both in resting and activated conditions. MPs derived from TNF-stimulated cells induced coagulation in vitro, via a tissue factor/factor VII-dependent pathway. The expression of E-selectin, ICAM-1, alphavbeta3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with alphavbeta3 was selected to discriminate EMPs from those of other origins. We provide evidence that endothelial-derived MPs are detectable in normal human blood and are increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant. Thus, MPs can be induced by TNF in vitro, and may participate in vivo in the dissemination of proadhesive and procoagulant activities in thrombotic disorders.

Antigenic profile, prevalence, and clinical significance of antiphospholipid antibodies in women referred for in vitro fertilization.

The aim of this prospective study was to assess the prevalence of antiphospholipid antibodies (aPL) in women who had undergone in vitro fertilization (IVF) and the relationship between aPL and IVF outcome. A total of 101 infertile women with at least three unsuccessful IVF attempts were consecutively included in this study. Samples were collected in the follicular phase of a spontaneous ovarian cycle 2 months after the last ovulation induction treatment. Age-matched healthy fertile women (n = 160) were included as controls. All were evaluated for the presence of lupus anticoagulant (LA), antibodies (IgG, IgA, IgM) to cardiolipin (aCL), beta2-glycoprotein I (abeta2GPI), and phosphatidylethanolamine (aPE). Out of the 101 infertile women, 40 were persistently positive for aPL, showing a prevalence significantly higher than in controls (39.6% versus 5%, P < 0.0001). Among aPL, aPE were found with a significantly higher prevalence compared with LA, aCL, and aP2GPI (67.5% versus 0%, 15%, and 40%, respectively). Interestingly, aPE were found in 70% of the cases in the absence of the other aPL. The predominant isotype of aPL was IgA, in particular for abeta2GPI. Finally, no significant association was found between the presence of aPL and IVF outcome. This prospective study shows aPE as the most prevalent aPL in infertile women and IgA as more common than IgG and IgM. However, our results do not support an association between aPL and IVF outcome.

[Auto-immune hepatitis and antiphospholipids]. / Etude de l'association hépatite auto-immune et antiphospholipides.

INTRODUCTION: Only few series have reported the association of autoimmune hepatitis with antiphospholipid antibodies. The aim of our study is to investigate the frequency of these antibodies in a series of autoimmune hepatitis and to search for a correlation with clinical, biological or histological characteristics. MATERIAL AND METHODS: Antiphospholipid were investigated in 24 patients with well defined autoimmune hepatitis. Characteristics were compared between antiphopholipids positive and negative patients. Characteristics of our patients were also compared toward cases collected in a literature review. RESULTS: The frequency of antiphospholipid antibodies is of 70.8% in our series. Four patients had a well defined antiphospholid syndrome. Seven patients had a systemic lupus erythematosus in the antiphospholipid group whereas none in the antiphospholipid negative group. The frequency of the different antiphopholipid antibodies was: IgG ACL (52.9%), IgM APE (52.9%), ACC (43.7%), IgG Abeta2GP1 (41.2%). We found no correlation between hypergammaglobulinemia and the presence or the isotype of antiphospholipid antibodies. Clinical presentation and outcome as biological and histological parameters were similar in both groups. CONCLUSION: Our study report a high frequency of antiphospholipids antibodies in autoimmune hepatitis patients. However we found no clinical, biological or histological correlation with the presence of antiphospholipids. Further longitudinal studies on larger cohorts should clarify the association between antiphospholipid antibodies and autoimmune hepatitis and potential therapeutic issues.

[The MYH9 syndrome: report of a new case with a new mutation of the MYH9 gene]. / Le syndrome MYH9: à propos d'une nouvelle observation et de la mise en évidence d'une nouvelle mutation du gène MYH9.

INTRODUCTION: Familial macrothrombocytopenias are a group of rare autosomal dominant platelet disorders including many syndromes in particular the May-Hegglin anomaly. They are characterized by thrombocytopenia with giant platelets and in some cases neutrophilic inclusions in peripheral blood granulocytes. Recently these different clinical entities have been demonstrated to be linked to mutations in the same gene, MYH9. CASE REPORT: We report in a young African woman presenting as a May-Hegglin anomaly a new mutation of the MYH9 gene. In regard of this case we present a brief review of the MYH9 syndrome. CONCLUSION: The MYH9 syndrome includes now several clinical entities who share some common clinical and biological characteristics such as a thrombocytopenia with giant platelets, presence or absence of other manifestations including Dohle like bodies, nephritis, sensorineural hearing loss, cataract. We report a new case in which a new mutation of the MYH9 gene was evidenced.

The iodocyanopindolol and SM-11044 binding protein belongs to the TM9SF multispanning membrane protein superfamily.

SM-11044 is the only beta-adrenergic agonist that inhibits guinea pig eosinophil chemotaxis and induces relaxation of depolarized rat colon tonus. We have previously reported the purification of a 34 kDa photoaffinity-labeled SM-11044 binding protein (SMBP) from rat colon that may mediate the biological effects of the ligand and that differs from all known monoamine receptors (Sugasawa et al., J. Biol. Chem. 272 (1997) 21244). The present report describes partial amino acid sequence of rat SMBP and molecular cloning of corresponding human SMBP (hSMBP) cDNA. This cDNA encodes a 588 amino acid residue polypeptide comprising a signal peptide, a long hydrophilic amino-terminal region, and a highly hydrophobic C-terminal portion organized into nine putative transmembrane domains. The sequence and structure of hSMBP shows homology to members of a new transmembrane protein 9 superfamily (TM9SF). Comparison of hSMBP with related protein sequences from yeast, plant and human revealed two subgroups within TM9SF. The members of these groups differ in length and have characteristic amino acid sequence motifs in their amino-terminal portion. Northern blot analysis revealed two major SMBP mRNAs, at 3.4 and 3.8 kb, that were present in all the human tissues examined. Western blot experiments detected SMBP as a 70 kDa protein that may be further cleaved into an active 34 kDa N-terminal polypeptide. Stable Chinese Hamster Ovary cell transfectants expressing hSMBP cDNA displayed specific binding of [(125)I]iodocyanopindolol that was displaced by SM-11044 in a dose-dependent manner. Thus, SMBP is the first member of TM9SF with functional ligand binding properties, suggesting that some of these integral membrane proteins may function as channels, small molecule transporters or receptors.

Amino acid sequence and three-dimensional structure of the Tn-specific isolectin B4 from Vicia villosa.

The partial amino acid sequence of the tetrameric isolectin B4 from Vicia villosa seeds has been determined by peptide analysis, and its three-dimensional structure solved by molecular replacement techniques and refined at 2.9 A resolution to a crystallographic R-factor of 21%. Each subunit displays the thirteen-stranded beta-barrel topology characteristic of legume lectins. The amino acid residues involved in metal- and sugar-binding are similar to those of other GalNAc-specific lectins, indicating that residues outside the carbohydrate-binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.

The formation of an intrachain disulfide bond in the leptin protein is necessary for efficient leptin secretion.

Leptin is a cytokine secreted by the adipose tissue that is involved in the control of body weight. We previously showed that a point mutation (R105W) in leptin results in leptin deficiency, marked obesity and hypogonadism in humans adults. Expression in COS1 cells showed impaired secretion and intracellular accumulation of the mutated protein. However, impaired secretion of the mutant leptin had not been demonstrated in adipose cells. In this work, we demonstrate that secretion of R105W mutant is impaired in rat and human adipocytes. We also show that R105W mutant expressed in COS1 cells and in PAZ6 adipocytes forms large molecular aggregates that cannot cross a filtration membrane with a cut-off of 100 kDa. Moreover, we have engineered, by site directed mutagenesis, the cDNAs coding for leptin in which either Cys 117, Cys 167, or both, were replaced by a serine. When expressed in COS1 cells or PAZ6 adipocytes, cysteine mutants also show impaired secretion and formation of large molecular aggregates. Therefore, our work indicates that the formation of an intramolecular disulfide bridge is necessary for normal processing and secretion of leptin. Moreover, the similarity of the behavior of R105W mutant and cystein mutants suggests that the lack of secretion observed with the naturally occurring mutant could result from impaired disulfide bond formation.

Evidence for the expression of urokinase-type plasminogen activator by human venous endothelial cells in vivo.

Endothelial cells (ECs) in culture synthesize and secrete urokinase-type plasminogen activator (u-PA), but the normal vascular endothelium is believed to synthesize only tissue plasminogen activator (t-PA), which is thought to be responsible for intravascular fibrinolysis. More recently, animal studies have shown that the biological role of u-PA in fibrinolysis has been underestimated, prompting a re-examination of its synthesis by the endothelium. In this study, we investigated whether u-PA was synthesized by non-atherosclerotic endothelial cells in vivo by testing ECs dislodged by venipuncture from 12 normal volunteers and 17 patients admitted for plasmapheresis. The ECs were isolated with an anti-endothelial monoclonal antibody coupled to immunomagnetic beads and characterized by morphology and by labelling for vWF, CD31, and UEA-1 binding. U-PA antigen was found in 50% of the ECs from the normal subjects and in 60% of those from patients. U-PA enzymatic activity on zymograms was detected in 50% of the normal samples and 60% of the patient samples, with the latter being more frequently and more strongly positive. U-PA mRNA was found in all the normal and patient samples tested. The results indicate that u-PA is synthesized by the venous endothelium in vivo but that its expression is highly variable.

Chemokines control fat accumulation and leptin secretion by cultured human adipocytes.

In addition to their role in inflammation, cytokines like TNFalpha have been reported to regulate the adipose tissue function suggesting a role for these soluble mediators in metabolism. However, it is not known whether adipocytes have the capacity to secrete chemokines, a group of low molecular weight inflammatory mediators that control leukocyte migration into tissues. Here we show that primary cultures of human preadipocytes constitutively produce three chemokines, interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1), while their level of expression is low in mature adipocytes. Upon TNFalpha treatment, the expression of all the three chemokines is upregulated in adipocytes differentiated in vitro. In addition, we describe the presence of seven different chemokine receptors, mainly in mature adipocytes, both in vitro and in human fat tissue sections. Prolonged stimulation of cultured human adipocytes with exogenous chemokines leads to a decrease in lipid content in association with the downregulation of PPARgamma mRNA expression. Moreover, chemokines positively control the secretion of leptin, a hormone that regulates appetite, by a post-transcriptional mechanism. These findings reveal a new role for chemokines in the regulation of adipose tissue and suggest a novel therapeutic basis for the treatment of obesity, diabetes and cachexia.

Serum leptin activity in obese and lean patients.

Blood levels of the satiety hormone leptin are directly correlated to fat stores in obese and lean people. Therefore, leptin resistance is the logical explanation for the phenomenon of common obesity. However, the important question of whether or not the intrinsic leptin activity could differ between obese and lean people has not been examined before. In the present study, serum leptin activity was measured by an in vitro assay of leptin signaling in a modified culture of HEK-293 cells. The system is based on activation of a luciferase reporter gene through a leptin receptor-dependent activation of the signal transducer and activator of transcription (STAT3). Serum samples from 20 obese and 20 non-obese individuals with leptin levels ranging from 3 to 75 ng/ml, as determined by radioimmunoassay (RIA), were used. A high correlation was observed for each serum sample between leptin RIA values and leptin activity in the bioassay. The results indicate that obesity in the 20 obese patients among the 40 individuals examined cannot be accounted for by alterations in leptin activity in our assay. The assay system provides a tool to screen for possible rare cases exhibiting alteration in leptin activity either due to a change in leptin itself or through interaction with other serum factors.

Purification of the plasmin receptor from human carcinoma cells and comparison to alpha-enolase.

We have characterized a receptor for plasmin (Pli-R) from a human tumor cell line, MCF7MF. The Pli-R was purified from a MCF7 0.1% Triton X-100 solubilisate by affinity chromatography. A protein of 55-60 kDa was obtained, which bound plasminogen and plasmin specifically. Chemical cross-linking of M(r) 90 kDa [125I]-Pli to the surface of MCF7 cells with DSP results in the formation of a labelled complex of M(r) 145 kDa, suggesting a M(r) of 55-60 kDa for the receptor. Comparing Pli-R with alpha-enolase (a candidate for plasminogen receptor in U937 cells) we have found a high homology between both proteins, but not an identity.

Purification and characterization of equine testicular cytochrome P-450 aromatase: comparison with the human enzyme.

Cytochrome P-450 aromatase was purified by five chromatographic steps from adult stallion testis. It was first separated from NADPH-cytochrome P-450 reductase (reductase) on omega-aminohexyl-Sepharose 4B then purified to homogeneity on concanavalin A-Sepharose 4B, hydroxyapatite-Sepharose 4B, DEAE-Sepharose CL-6B and on a second hydroxyapatite-Sepharose 4B. On the other hand, purifications of the equine testicular and rat liver reductases, which allowed the reconstitution of aromatase activity in vitro, were achieved for each species in one chromatographic step on an adenosine 2',5'-diphosphate-agarose affinity column. Analysis on SDS/PAGE indicated single bands with apparent molecular masses of 53, 82, and 80 kDa for purified equine testicular cytochrome P-450 aromatase (eAROM), equine testicular reductase and rat liver reductase respectively. eAROM shows a time- and concentration-dependent activity that was stable for at least 2 months when stored at -78 degrees C. It is a highly hydrophobic protein composed from 505 residues and direct sequencing of its N-terminal part showed good homology when compared with human aromatase. When deglycosylated by N-glycosidase-F the apparent molecular mass of eAROM was decreased from 53 to 51 kDa as revealed by electrophoresis, its activity, however, was not impaired. eAROM exhibits much higher affinity for androgens than for 19-norandrogens, Km values were approximately 3, 16 and 170 nM for androstenedione (A), testosterone (T) and 19-nortestosterone (19-NT) respectively. However, it aromatizes 19-norandrostenedione (19-NA) slightly more efficiently than A, the estrone (E1) formed was 4.27 vs 3.54 pmol min-1 micrograms-1 respectively (P < 0.01). After incubation of eAROM with radiolabelled A and separation of steroids on HPLC, E1, 19-hydroxyandrostenedione (19-OHA) and 19-oxoandrostenedione (19-oxoA) were accumulated in the incubation medium in a time-dependent manner. The presence of 4-hydroxyandrostenedione (4-OHA), a suicide inhibitor of aromatase, cause a time-dependent inactivation of the enzyme. Whereas the activity of eAROM was unchanged in the presence of K+ (up to 250 mM), it was increased in the presence of EDTA (up to 50 mM) and decreased in the presence of DTT or Mg2+ (from 25 mM). We conclude that: (a) eAROM is a glycoprotein, however, deglycosylation by N-glycosidase-F does not appear to impair its activity, (b) eAROM aromatizes really both androgens and 19-norandrogens having a higher affinity for androgens, (c) the intermediary compounds of aromatization 19-OHA and 19-oxoA appear to be synthesized by the same active site that synthesizes E1 as the final product, (d) the inhibition of eAROM by increasing concentrations of Mg2+ and the stimulation of its activity by EDTA, taken together, indicate the importance of negatively charged residues in the polypeptide chain of equine aromatase, which play a role in enzymatic activity.