Characterizing the pathotype of neonatal meningitis causing Escherichia coli (NMEC).

BACKGROUND: Neonatal meningitis-causing Escherichia coli (NMEC) is the predominant Gram-negative bacterial pathogen associated with meningitis in newborn infants. High levels of heterogeneity and diversity have been observed in the repertoire of virulence traits and other characteristics among strains of NMEC making it difficult to define the NMEC pathotype. The objective of the present study was to identify genotypic and phenotypic characteristics of NMEC that can be used to distinguish them from commensal E. coli. METHODS: A total of 53 isolates of NMEC obtained from neonates with meningitis and 48 isolates of fecal E. coli obtained from healthy individuals (HFEC) were comparatively evaluated using five phenotypic (serotyping, serum bactericidal assay, biofilm assay, antimicorbial susceptibility testing, and in vitro cell invasion assay) and three genotypic (phylogrouping, virulence genotyping, and pulsed-field gel electrophoresis) methods. RESULTS: A majority (67.92%) of NMEC belonged to B2 phylogenetic group whereas 59% of HFEC belonged to groups A and D. Serotyping revealed that the most common O and H types present in NMEC tested were O1 (15%), O8 (11.3%), O18 (13.2%), and H7 (25.3%). In contrast, none of the HFEC tested belonged to O1 or O18 serogroups. The most common serogroup identified in HFEC was O8 (6.25%). The virulence genotyping reflected that more than 70% of NMEC carried kpsII, K1, neuC, iucC, sitA, and vat genes with only less than 27% of HFEC possessing these genes. All NMEC and 79% of HFEC tested were able to invade human cerebral microvascular endothelial cells. No statistically significant difference was observed in the serum resistance phenotype between NMEC and HFEC. The NMEC strains demonstrated a greater ability to form biofilms in Luria Bertani broth medium than did HFEC (79.2% vs 39.9%). CONCLUSION: The results of our study demonstrated that virulence genotyping and phylogrouping may assist in defining the potential NMEC pathotype.

Protective effect of the multitarget compound DPH-4 on human SSAO/VAP-1-expressing hCMEC/D3 cells under oxygen-glucose deprivation conditions: an in vitro experimental model of cerebral ischaemia.

BACKGROUND AND PURPOSE: Stroke and Alzheimer's disease (AD) are related pathologies in which the cerebrovascular system is involved. Plasma levels of semicarbazide-sensitive amine oxidase/vascular adhesion protein 1 (SSAO/VAP-1, also known as Primary Amine Oxidase -PrAO) are increased in both stroke and AD patients and contribute to the vascular damage. During inflammation, its enzymatic activity mediates leukocyte recruitment to the injured tissue, inducing damage in the blood-brain barrier (BBB) and neuronal tissue. We hypothesized that by altering cerebrovascular function, SSAO/VAP-1 might play a role in the stroke-AD transition. Therefore, we evaluated the protective effect of the novel multitarget-directed ligand DPH-4, initially designed for AD therapy, on the BBB. EXPERIMENTAL APPROACH: A human microvascular brain endothelial cell line expressing human SSAO/VAP-1 was generated, as the expression of SSAO/VAP-1 is lost in cultured cells. To simulate ischaemic damage, these cells were subjected to oxygen and glucose deprivation (OGD) and re-oxygenation conditions. The protective role of DPH-4 was then evaluated in the presence of methylamine, an SSAO substrate, and/or ß-amyloid (Aß). KEY RESULTS: Under our conditions, DPH-4 protected brain endothelial cells from OGD and re-oxygenation-induced damage, and also decreased SSAO-dependent leukocyte adhesion. DPH-4 was also effective at preventing the damage induced by OGD and re-oxygenation in the presence of Aß as a model of AD pathology. CONCLUSIONS AND IMPLICATIONS: From these results, we concluded that the multitarget compound DPH-4 might be of therapeutic benefit to delay the onset and/or progression of the neurological pathologies associated with stroke and AD, which appear to be linked.

BBB on chip: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function.

The blood-brain barrier (BBB) is a unique feature of the human body, preserving brain homeostasis and preventing toxic substances to enter the brain. However, in various neurodegenerative diseases, the function of the BBB is disturbed. Mechanisms of the breakdown of the BBB are incompletely understood and therefore a realistic model of the BBB is essential. We present here the smallest model of the BBB yet, using a microfluidic chip, and the immortalized human brain endothelial cell line hCMEC/D3. Barrier function is modulated both mechanically, by exposure to fluid shear stress, and biochemically, by stimulation with tumor necrosis factor alpha (TNF-α), in one single device. The device has integrated electrodes to analyze barrier tightness by measuring the transendothelial electrical resistance (TEER). We demonstrate that hCMEC/D3 cells could be cultured in the microfluidic device up to 7 days, and that these cultures showed comparable TEER values with the well-established Transwell assay, with an average (± SEM) of 36.9 Ω.cm(2) (± 0.9 Ω.cm(2)) and 28.2 Ω.cm(2) (± 1.3 Ω.cm(2)) respectively. Moreover, hCMEC/D3 cells on chip expressed the tight junction protein Zonula Occludens-1 (ZO-1) at day 4. Furthermore, shear stress positively influenced barrier tightness and increased TEER values with a factor 3, up to 120 Ω.cm(2). Subsequent addition of TNF-α decreased the TEER with a factor of 10, down to 12 Ω.cm(2). This realistic microfluidic platform of the BBB is very well suited to study barrier function in detail and evaluate drug passage to finally gain more insight into the treatment of neurodegenerative diseases.

The association of statins plus LDL receptor-targeted liposome-encapsulated doxorubicin increases in vitro drug delivery across blood-brain barrier cells.

BACKGROUND AND PURPOSE: The passage of drugs across the blood-brain barrier (BBB) limits the efficacy of chemotherapy in brain tumours. For instance, the anticancer drug doxorubicin, which is effective against glioblastoma in vitro, has poor efficacy in vivo, because it is extruded by P-glycoprotein (Pgp/ABCB1), multidrug resistance-related proteins and breast cancer resistance protein (BCRP/ABCG2) in BBB cells. The aim of this study was to convert poorly permeant drugs like doxorubicin into drugs able to cross the BBB. EXPERIMENTAL APPROACH: Experiments were performed on primary human cerebral microvascular endothelial hCMEC/D3 cells, alone and co-cultured with human brain and epithelial tumour cells. KEY RESULTS: Statins reduced the efflux activity of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells by increasing the synthesis of NO, which elicits the nitration of critical tyrosine residues on these transporters. Statins also increased the number of low-density lipoprotein (LDL) receptors exposed on the surface of BBB cells, as well as on tumour cells like human glioblastoma. We showed that the association of statins plus drug-loaded nanoparticles engineered as LDLs was effective as a vehicle for non-permeant drugs like doxorubicin to cross the BBB, allowing its delivery into primary and metastatic brain tumour cells and to achieve significant anti-tumour cytotoxicity. CONCLUSIONS AND IMPLICATIONS: We suggest that our 'Trojan horse' approach, based on the administration of statins plus a LDL receptor-targeted liposomal drug, might have potential applications in the pharmacological therapy of different brain diseases for which the BBB represents an obstacle.

Robot-assisted minimally invasive esophagectomy is equivalent to thoracoscopic minimally invasive esophagectomy.

The use of the surgical robot has been increasing in thoracic surgery. Its three-dimensional view and instruments with surgical wrists may provide advantages over traditional thoracoscopic techniques. Our initial experience with thoracoscopic robot-assisted minimally invasive esophagectomy (RAMIE) for esophageal cancer was compared with our traditional thoracoscopic minimally invasive esophagectomy (MIE) approach for esophageal cancer. A retrospective review of a prospective database was performed. From July 2008 to October 2009, 43 patients underwent MIE resection. Patients who had benign disease and intrathoracic anastomosis were excluded. Results are presented as mean ± SD. Significance was set as P < 0.05. Eleven patients who underwent RAMIE and 26 who underwent MIE were included in the cohort. No differences in age, sex, race, body mass index, or preoperative radiotherapy or chemotherapy between the groups were observed. No significant differences in operative time, blood loss, number of resected lymph nodes, postoperative complications, days of mechanical ventilation, length of intensive care unit stay, or length of hospital stay were also observed. In this short-term study, RAMIE was found to be equivalent to thoracoscopic MIE and did not offer clear advantages.

Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid-beta variants in cells composing the cerebral vessel walls.

Cerebral amyloid angiopathy (CAA) is an age-associated condition and a common finding in Alzheimer's disease in which amyloid-beta (Abeta) vascular deposits are featured in >80% of the cases. Familial Abeta variants bearing substitutions at positions 21-23 are primarily associated with CAA, although they manifest with strikingly different clinical phenotypes: cerebral hemorrhage or dementia. The recently reported Piedmont L34V Abeta mutant, located outside the hot spot 21-23, shows a similar hemorrhagic phenotype, albeit less aggressive than the widely studied Dutch E22Q variant. We monitored the apoptotic events occurring after stimulation of human brain microvascular endothelial and smooth muscle cells with nonfibrillar structures of both variants and wild-type Abeta40. Induction of analogous caspase-mediated mitochondrial pathways was elicited by all peptides, although within different time frames and intensity. Activated pathways were susceptible to pharmacological modulation either through direct inhibition of mitochondrial cytochrome c release or by the action of pan- and pathway-specific caspase inhibitors, giving a clear indication of the independent or synergistic engagement of both extrinsic and intrinsic mechanisms. Structural analyses of the Abeta peptides showed that apoptosis preceded fibril formation, correlating with the presence of oligomers and/or protofibrils. The data support the notion that rare genetic mutations constitute unique paradigms to understand the molecular pathogenesis of CAA.

Molecular cytogenetic characterization of the human cerebral microvessel endothelial cell line hCMEC/D3.

The immortalized human cerebral microvessel endothelial cell line hCMEC/D3 has been repeatedly used as a model of human blood-brain barrier (BBB). hCMEC/D3 cells between passage 25 and 35 are most often applied in research, remained phenotypically nontransformed, and cells maintained many characteristics of human brain endothelial cells. Also hCMEC/D3 was thought to have conserved a normal diploid karyotype over all these passages. Here we characterized the cell line using high-resolution multicolor fluorescence in situ hybridization (FISH) approaches and revealed a complex karyotype in the 30th passage. Clonal cryptic unbalanced structural rearrangements and numerical aberrations were discovered and described as follows: 45 approximately 48,XX, -X,del(5)(q11)[2],del(9)(q11)[3],+9[3],del(11)(q13 approximately 14)[2], der(14)t(14;21)(q32.33;q22.3)[28],der(15)t(9;15)(p11;p11)[13], dup(15)(p11q11)[5],der(21)t(17;21)(p12;q22)[9],-22[6][cp28]. In summary, a complex karyotype with clonal unbalanced chromosomal rearrangements is present in hCMEC/D3. Thus, we solicit to include molecular cytogenetics in the testing of all cell lines prior to application of their use in complex studies.

Review article: the pathophysiology of thrombocytopenia in hepatitis C virus infection and chronic liver disease.

BACKGROUND: The pathophystology of thrombocytopenia in patients with chronic liver disease resulting from hepatitis C virus (HCV) infection is complex and involves several complementary mechanisms that likely act in concert. AIM: To summarize the available data on the etiology of thrombocytopenia in patients with chronic liver disease. RESULTS: In patients with untreated hepatitis C, both prevalence and severity of thrombocytopenia increase in parallel with the extent of disease, usually becoming clinically relevant when patients develop extensive fibrosis and/or cirrhosis. Pathogenetic mechanisms include hypersptenism secondary to portal hypertension, bone marrow suppression resulting from either HCV itself or interferon treatment, aberrations of the immune system resulting in the formation of anti-platelet antibodies and/or immune-complexes that bind to platelets and facilitate their premature clearance, development of immunologically-mediated extrahepatic manifestations including mixed cryoglobulinemia with or without associated joint, renal, or cutaneous involvement, and thrombopoietin (TPO) deficiency secondary to liver dysfunction. In chronic liver disease, the natural inverse relationship between TPO and platelet levels is not maintained; therefore, blood TPO levels fail to have clinical relevance or predictive value in assessing the thrombocytopenic status of a given patient. CONCLUSIONS: The development of thrombocytopenisa in patients with chronic liver disease is complex and multifactorial.

Blood-brain barrier-specific properties of a human adult brain endothelial cell line.

Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.

Rituximab for autoimmune haemophilia: a proposed treatment algorithm.

We previously reported durable complete responses following brief courses of rituximab and prednisone with or without cyclophosphamide in four patients with autoimmune haemophilia and inhibitor titres of 5-60 BU. We report here responses to this monoclonal anti-CD20 antibody in four additional patients, including two patients with inhibitor titres >200 BU. Factor VIII levels became normal 2-35weeks after 4 or 8 weekly doses of rituximab, brief courses of prednisone and in one patient immunoglobulin. Complete responses are ongoing at 10 months in two patients. Two patients relapsed: a patient whose initial inhibitor titre was 525 BU relapsed at 3.5 months and a long-term prednisone-dependent patient at 8.5 months. Both responded to second courses of rituximab and prednisone and are in remission. Our experience suggests that rituximab is a safe and effective addition to immunosuppression with prednisone and cyclophosphamide to treat autoimmune haemophilia, and may permit early discontinuation or even avoidance of these potentially toxic agents. High-titre inhibitor patients, however, may require multiple courses of rituximab or the addition of cyclophosphamide. Pending randomized studies, we propose an algorithm based on our experience and other reports for incorporating rituximab in the treatment of this rare disorder.

Anti-endothelial cell antibodies (AECA) in systemic sclerosis--increased sensitivity using different endothelial cell substrates and association with other autoantibodies.

OBJECTIVE: One of the main features of systemic sclerosis (SSc) is vascular damage, the mechanism of which is not understood. In the present study we examined whether screening of SSc patients for different anti-endothelial cells antibodies (AECA) of various origins increase the sensitivity of AECA detection in SSc patients. Secondary aim was an attempt to correlate AECA with other common autoantibodies. MATERIALS & METHODS: 478 SSc patients were studied for the presence AECA, anti-cardiolipin (aCL), anti-dsDNA, anti-heparin (AHA), anti-pyruvate dehydrogenase (PDH) and anti-PDC-E2 autoantibodies. AECA levels were detemined using human umbilical vein EC (HUVEC), bone marrow EC (BMEC), EC hybridoma (EA.hy 926) and Kaposi sarcoma EC (KS). RESULTS: Positive AECA were found in 49.5% of SSc patients (27.1% HUVEC; 34.3% BMEC; 26.3% EaHy 926 and 22.7% KS). The highest percent reactivity of AECA was obtained using microvascular BMEC. When combining BMEC and either other cell lines the reactivity ranged from 41.4% to 46%. A significant association between AECA on the one hand and AHA (p<0.001)) and anti-PDH (p<0.05) on the other was secn. Cross-reactivity with anti-PDC-E2 was excluded by inhibition tests, but AHA and anti-PDH may be part of the spectrum of AECA. CONCLUSIONS: Since false-negative AECA may result from lack of expression of various antigens on a specific EC, analysis of AECA in SSc patients requires using several EC types, including microvascular EC.

Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor signals through multiple pathways in endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) encodes a chemokine-like G protein-coupled receptor (KSHV-GPCR) that is implicated in the pathogenesis of Kaposi's sarcoma (KS). Since endothelial cells appear to be targets for the virus, we developed an in vitro mouse lung endothelial cell model in which KSHV-GPCR is stably expressed and KSHV-GPCR signaling was studied. In mouse lung endothelial cells: 1) KSHV-GPCR does not exhibit basal signaling through the phosphoinositide-specific phospholipase C pathway but inositol phosphate production is stimulated by growth-related oncogene alpha (Gro-alpha) via a pertussis toxin (PTX)-insensitive pathway; 2) KSHV-GPCR signals basally through a PTX-sensitive pathway leading to a lowering of intracellular cAMP level that can be lowered further by Gro alpha and increased by interferon gamma-inducible protein 10; 3) KSHV-GPCR stimulates phosphatidylinositol 3-kinase via a PTX-insensitive mechanism; and 4) KSHV-GPCR activates nuclear factor-kappa B (NF-kappa B) by a PTX-sensitive G beta gamma subunit-mediated pathway. These data show that KSHV-GPCR couples to at least two G proteins and initiates signaling via at least three cascades in endothelial cells thereby increasing the complexity of regulation of endothelial cell function by KSHV-GPCR that may occur during viral infection.

[Arachidonic acid and prostaglandins, inflammation and oncology]. / Acide arachidonique et prostaglandines, inflammation et oncologie.

CYCLOOXYGENASE PATHWAY: Among the 3 metabolic pathways leading to oxidation of arachidonic acid, the cyclooxygenase (COX) pathway produces prostaglandin G2 that is rapidly transformed into prostaglandin H2. PROSTAGLANDINS: Prostaglandins are inflammation mediators that are strongly implicated in tumorgenesis. They participate in tumor initiation, promotion and growth. INFLAMMATION AND EPITHELIAL CANCER: Chronic inflammation is a risk factor for epithelial cancer. It induces prostaglandin synthesis via activation of COX-2. There is a cause and effect relationship between chronic inflammation and carcinogeneis via COX-2 expression. It has been demonstrated that COX-2 favors tumor invasion and inhibits apoptosis. Tumor growth is favored by PGE2-induced reduction in immunity; COX-2 inhibitors reinforce the immune response. Finally COX-2 is expressed in tumor neovessels and plays a role in angiogenesis.

[Cyclooxygenases]. / Les cyclooxygénases.

TWO ISOFORMS: There are two isoforms of cyclooxygenase (COX) with similar structure and metabolic activity. COX-1 is a constitutional enzyme. COX-2 is an inductible form. CYCLOOXYGENASE-1: COX-1 is present in most tissues, particularly in the kidney, the digestive tract mucosa, platelets, brain, liver and spleen. CYCLOOXYGENASE-2: COX-2 expression can be induced by an inflammatory process. Implicated in the development of certain cancers, COX-2 is expressed in numerous tumor cell lines and in most epithelial carcinomas. COX-2 favors tumor invasion and inhibits apoptosis. When it is absent, tumor growth is slower or stopped.

[Cyclooxygenase inhibitors]. / Les inhibiteurs des cyclooxygénases.

DISTINCT EFFECTS: Since the discovery of the two isoforms of COX, the therapeutic effects of nonsteroidal antiinflammatory drugs (NSAID) can be distinguished from their adverse effects linked to the inhibition of the constitutional form via selective inhibition of the inducible form. Non-selective NSAID that inhibit both COX isoforms are difficult to use for long-term regimens. NSAID AND CANCER PREVENTION: Epidemiology studies and animal and in vitro studies have demonstrated that regular use of NSAID reduced the incidence of colorectal cancer and certain precancer lesions. PROMISING PERSPECTIVES: COX-2 specific inhibitors, for example cerecoxib or rofecoxib, are potentially interesting for human therapy for chemoprevention of epithelial cancer or as medical treatment, alone or in combination with other anticancer treatments.

Structure and function of a vimentin-associated matrix adhesion in endothelial cells.

The alpha4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the alpha4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by alphav and beta3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the alpha4 laminin subunit G domain in an alphavbeta3-integrin-dependent manner. The alphavbeta3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the alphavbeta3 integrin-positive focal contacts. We have investigated the function of alpha4-laminin and alphavbeta3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.

Effects of tobacco smoke and benzo[a]pyrene on human endothelial cell and monocyte stress responses.

Smoking is an important risk factor for atherosclerosis. We compared tobacco smoke filtrate with benzo[a]pyrene (a prominent xenobiotic component of tobacco smoke) for the capacity to induce stress proteins and cause cell death in human monocytes and vascular endothelial cells, two cell types that are involved in the formation of atherosclerotic lesions. Exposure to freshly prepared filtrates of tobacco smoke induced in both monocytes and endothelial cells expression of the inducible heat shock protein (HSP)70 and heme oxygenase-1 (HO-1) and produced loss of mitochondrial membrane potential. Later, cell death by apoptosis or necrosis occurred depending on the concentration of tobacco smoke. These toxic effects could be prevented by the antioxidant N-acetylcysteine. In contrast, exposure of these cells to benzo[a]pyrene alone evoked neither stress proteins nor mitochondrial damage but did induce cell death by necrosis. Thus our results indicate that tobacco smoke rapidly induces complex oxidant-mediated stress responses in both vascular endothelial cells and circulating monocytes that are independent of the benzo[a]pyrene content of the smoke.

Anti-endothelial cell antibodies from patients with thrombotic thrombocytopenic purpura specifically activate small vessel endothelial cells.

Thrombotic thrombocytopenic purpura (TTP) is an uncommon disease of an unknown etiology, characterized by consumptive thrombocytopenia, microangiopathic hemolytic anemia, fever and acute thrombotic complications, especially within the cerebral circulation. Although anti-endothelial cell antibodies (AECA) have occasionally been shown to be present in TTP, their role in the pathogenesis of the disease has never been ascertained. In the current study we demonstrated the pathogenic activity of affinity-purified anti-endothelial cell F(ab)2 antibodies (AECA/TTP) from four consecutive patients with active TTP. These AECA/TTP bound to and activated only microvascular endothelial cells (EC) and not large vessel EC. The specificity of AECA/TTP binding to microvascular EC was confirmed by competition assay employing membranes derived from small and large vessels EC. Activation included enhanced IL-6 and von Willebrand factor release from the EC followed by increased expression of adhesion molecules P-selectin, E-selectin and vascular cell adhesion molecule-1 on the EC, as evaluated by ELISA. Increased expression of adhesion molecules was followed by an increase in monocyte adhesion to EC. The level of soluble thrombomodulin (TM) also increased in the culture medium of activated microvascular EC upon exposure to AECA/TTP antibodies and was directly correlated to a decrease in cell-associated TM. Our data suggest that AECA/TTP directed against microvascular EC could play a pathogenic role in the development of endothelial injury in TTP that leads to thrombosis.

Genetic hypofibrinolysis in complicated pregnancies.

OBJECTIVE: To assess the hypofibrinolytic 4G/4G mutation of the plasminogen activator inhibitor (PAI-1) gene as a possible factor contributing to severe preeclampsia, abruptio placentae, fetal growth restriction, and stillbirth. METHODS: We compared 94 women from a previous report who had obstetric complications to 95 controls with normal pregnancies matched for ethnic background and age. We collected blood and extracted DNA after delivery. All subjects had been tested for thrombophilic mutations factor V Leiden, C677T mutation in the methylenetetrahydrofolate reductase gene, and the G20210A mutation in the prothrombin gene. In the present study we tested for the hypofibrinolytic 4G/4G mutation in the PAI-1 gene. RESULTS: Women who had obstetric complications were more likely than controls to be 4G/4G homozygotes, 32% (30 of 94) women versus 19% (18 of 95) controls, odds ratio (OR) and 95% confidence intervals (CI) 2.0 (1.02, 3.9). Mutations in the PAI-1 gene were independently associated with obstetric complications (OR 1.56, 95% CI 1.005, 2.43). Heterozygosity for the factor V Leiden mutation was more common in the 30 women who had PAI-1 4G/4G than in the 18 4G/4G controls (33% versus 0%, Fisher P =.008). Seventy-six percent of women had some form of thrombophilia or hypofibrinolysis compared with 37% of controls (Fisher P <.001). CONCLUSIONS: Women with severe preeclampsia, abruptio placentae, fetal growth restriction, and stillbirth had increased incidence of the hypofibrinolytic 4G/4G mutation of the PAI-1 gene that is frequently associated with the thrombophilic factor V Leiden mutation, further predisposing them to thrombosis.

Cyclo-oxygenase 2: a pharmacological target for the prevention of cancer.

Understanding the mechanisms underlying carcinogenesis provides insights that are necessary for the development of therapeutic strategies to prevent cancer. Chemoprevention--the use of drugs or natural substances to inhibit carcinogenesis - is an important and rapidly evolving aspect of cancer research. We discuss evidence that cyclooxygenase 2 (COX 2), an inducible form of the enzyme, is a potential pharmacological target to prevent cancer. Key data implicating a causal relation between increased activity of COX 2 and carcinogenesis and possible mechanisms of action of COX 2 in this context are covered. Importantly, selective COX 2 inhibitors appear to be safe enough in human beings to allow large-scale clinical testing in healthy people. Several chemoprevention trials using selective COX 2 inhibitors are underway.