Human kallikrein-related peptidase 12 stimulates endothelial cell migration by remodeling the fibronectin matrix.

Kallikrein-related peptidase 12 (KLK12) is a kallikrein family peptidase involved in angiogenesis - a complex biological process in which the sprouting, migration and stabilization of endothelial cells requires extracellular matrix remodeling. To characterize the molecular mechanisms associated with KLK12's proangiogenic activity, we evaluated its ability to hydrolyze various matrix proteins. Our results show that KLK12 efficiently cleaved the human extracellular matrix proteins fibronectin and tenascin, both of which are involved in the regulation of endothelial cell adhesion and migration. For fibronectin, the major proteolytic product generated by KLK12 was a 29 kDa fragment containing the amino-terminal domain and the first five type I fibronectin-domains, which are essential for regulating fibronectin assembly. We also demonstrated that KLK12-mediated fibronectin proteolysis antagonizes fibronectin polymerization and fibronectin fibril formation by endothelial cells, leading to an increase in cell migration. Furthermore, a polyclonal antibody raised against KLK12's proteolytic cleavage site on fibronectin prevented the KLK12-dependent inhibition of fibronectin polymerization and the KLK12-mediated pro-migratory effect on endothelial cells. Taken as a whole, our results indicate that KLK12's proangiogenic effect is mediated through several molecular mechanisms.

Improved PCR method for amplification of GC-rich DNA sequences.

Most housekeeping genes, tumor-suppressor genes, and approx 40% of tissue-specific genes contain G+C sequences in their promoter region that were very difficult to amplify. In this report, we propose an improved polymerase chain reaction (PCR) method to be used for successful amplification of the tissue factor pathway inhibitor (TFPI)-2 gene promoter region that exhibit >70% G+C content in a sequence of approx 300 bp and a complete CpG island region spanning exon 1, the three transcription initiation sites, and the translation start site. Therefore, this method can be recommended to amplify other GC-rich genomic templates.

Expression and methylation status of tissue factor pathway inhibitor-2 gene in non-small-cell lung cancer.

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor that inhibits plasmin-dependent activation of several metalloproteinases. Downregulation of TFPI-2 could thus enhance the invasive potential of neoplastic cells in several cancers, including lung cancer. In this study, TFPI-2 mRNA was measured using a real-time PCR method in tumours of 59 patients with non-small-cell lung cancer (NSCLC). Tumour TFPI-2 mRNA levels appeared well correlated with protein expression evaluated by immunohistochemistry and were 4-120 times lower compared to those of nonaffected lung tissue in 22 cases (37%). Hypermethylation of the TFPI-2 gene promoter was demonstrated by restriction enzyme-polymerase chain reaction in 12 of 40 cases of NSCLC (30%), including nine of 17 for whom tumour TFPI-2 gene expression was lower than in noncancerous tissue. In contrast, this epigenetic modification was shown in only three of 23 tumours in which no decrease in TFPI-2 synthesis was found (P=0.016). Decreased TFPI-2 gene expression and hypermethylation were more frequently associated with stages III or IV NSCLC (eight out of 10, P=0.02) and the TFPI-2 gene promoter was more frequently hypermethylated in patients with lymph node metastases (eight out of 16, P=0.02). These results suggest that silencing of the TFPI-2 gene by hypermethylation might contribute to tumour progression in NSCLC.

Characterization and functional analysis of TFPI-2 gene promoter in a human choriocarcinoma cell line.

Tissue factor pathway Inhibitor-2 (TFPI-2) is associated with extracellular matrices and plays a major role in cell migration and tumor invasion. In this study, a 4.8-kb human TFPI-2 gene 5'-flanking region was isolated, cloned and sequenced. Promoter region analysis revealed a high GC-rich content without canonical TATA and CAAT boxes but three transcription initiation sites were identified. Moreover, several putative binding sites for transcription factors were identified (MyoD, LYF1, NF-Y, GATA, oct-1, AP-1, Sp1, NF1, NF-kappa B and egr-1). To characterize potential regulatory regions, TFPI-2/luciferase promoter constructs were then transfected in human choriocarcinoma JEG-3 cells. We first showed that the minimal TFPI-2 promoter is located between -166 and -111 from the translation start site. Luciferase activity consistently increased after stimulation of JEG-3 cells by phorbol 12-myristate 13-acetate indicating that NF1, NF-kappa B and egr-1/Sp1 binding sites are crucial in inducible TFPI-2 expression. Moreover, negative regulatory regions included AP-1 binding sites were identified. This study demonstrates that the TFPI-2 gene promoter exhibits typical features of a housekeeping gene.

Induction of monocyte tissue factor expression by antibodies to heparin-platelet factor 4 complexes developed in heparin-induced thrombocytopenia.

The pathogenesis of thrombosis in heparin-induced thrombocytopenia (HIT) was studied by investigating whether antibodies to heparin-platelet factor 4 (H-PF4) induced tissue factor (TF) synthesis by monocytes. Plasma from 5 patients with HIT containing IgG to H-PF4 was incubated with peripheral blood mononuclear cells without or with purified PF4 and heparin. Significant TF-dependent procoagulant activity (PCA) expressed by monocytes, measured with a factor Xa-based chromogenic assay, was induced after incubation of each HIT plasma sample. This monocyte PCA required the presence of PF4 and was inhibited by high concentrations of heparin. Furthermore, purified HIT IgG added to whole blood with PF4 and heparin also provoked significant synthesis of TF mRNA by monocytes, demonstrated by RT-PCR, and this effect was not observed with normal IgG. These findings strongly support the hypothesis that antibodies to PF4 developed in HIT trigger the production of tissue factor by monocytes, and this effect could account in vivo for hypercoagulability and thrombotic complications in affected patients.

Nasal immunization of mice with Cryptosporidium parvum DNA induces systemic and intestinal immune responses.

DNA immunization offers a novel approach to inducing humoral and cellular immunity against infectious pathogens. We examined whether such an approach could be used against cryptosporiodiosis, an intestinal disease caused by the protozoan parasite Cryptosporidium parvum. This infection is a major problem for young ruminants and immunosuppressed individuals in whom cryptosporidiosis causes life-threatening symptoms. The life cycle of C. parvum takes place in the enterocytes of the intestinal epithelium. We therefore focused our attention on a route of immunization that might induce a mucosal immunoglobulin (Ig)A response. Eight-week-old BALB/c mice were immunized intranasally with DNA encoding a 15-kDa C. parvum sporozoite antigen (CP15-DNA) cloned onto the plasmid pcDNA3. CP15-DNA-immunized mice developed specific and longlasting production of anti-CP15 Ig A in intestinal secretions and specific IgG in sera 3 months and 1 year after the first DNA inoculation. CP15-DNA-immunized mice also developed an antigen-specific T lymphocyte proliferative response in both spleen and mesenteric lymph nodes. Control mice that received the pcDNA3 plasmid alone did not develop specific humoral and cellular responses. These results indicate that plasmid DNA may provide a powerful means of eliciting intestinal humoral and cellular responses to C. parvum infections in mammals.

Protection of kids against Cryptosporidium parvum infection after immunization of dams with CP15-DNA.

In this study the effectiveness of a DNA vaccine to confer protection against cryptosporidiosis, an enteric infection of lifestock and humans, was evaluated. A vaccination protocol using a recombinant plasmid encoding the 15 kDa surface sporozoite protein of Cryptosporidium parvum was developed in adult pregnant goats. The present study reports that nasal immunization of pregnant goats with CP15-DNA led to a transfer of immunity to offspring conferring protection against C. parvum infection. Kids from CP15-DNA-vaccinated dams shed significantly fewer oocysts and over a shorter period than did kids from unvaccinated goats. The low level of parasite development in protected kids did not affect their growth whereas unprotected kids grew much slowly. There was still a significant difference in the weights of protected and unprotected kids after complete recovery. Anti-CP15 antibodies were present in serum and colostrum from vaccinated goats. Nevertheless, the precise immune mechanism of protection has still to be determined. This vaccine should reduce the economic losses due to cryptosporidiosis in ruminants, specially in small ruminants (calves, lambs, kids). It has also the potential to reduce environmental contamination by reducing oocyst shedding.

Comparison of the humoral and cellular immune responses to two preparations of Cryptosporidium parvum CP15/60 recombinant protein.

This study compares the immune responses produced by immunising mice and rabbits with two preparations of the recombinant 15/60 kDa protein of Cryptosporidium parvum. Genomic C. parvum DNA was amplified and the recombinant protein was synthesized as a fusion protein with glutathione-S-transferase in Escherichia coli and in the eukaryotic system of baculovirus/insect cells. Both recombinant proteins induced similar levels of serum antibodies against the fusion recombinant protein, but the eukaryotic recombinant protein triggered a stronger humoral response to C. parvum. Similarly, increased lymphoproliferation occurred only after stimulation of spleen cells from mice immunised with the eukaryotic recombinant protein. This suggests that the eukaryotic protein is a better candidate for immunological studies on cryptosporidiosis.

Fast detection of tissue factor and tissue factor pathway inhibitor messenger RNA in endothelial cells and monocytes by sensitive reverse transcription-polymerase chain reaction.

We developed fast and sensitive reverse transcription-polymerase chain reaction (RT-PCR) procedures to study the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI-1) mRNA in human endothelial cells and monocytes. The sensitivity of the technique was checked by performing RT-PCR with limited numbers of cells. Cells were stimulated either with tumor necrosis factor (TNF-alpha) or endotoxin to induce TF mRNA expression or with phorbol ester to increase TFPI-1 mRNA expression. Thus, RT-PCR specific for TF mRNA provided detection from as few as 10(3) TNF-alpha stimulated endothelial cells and 5 x 10(2) monocytes stimulated by endotoxin. TF mRNA expression was increased by TNF-alpha in endothelial cells and in monocytes stimulated by endotoxin. Elevated expression of TF mRNA in monocytes without stimulation by endotoxin was mainly related to cell adhesion. TFPI-1 mRNA was constitutively expressed in endothelial cells and was detected in only 5 x 10(2) unstimulated cells and 10(2) phorbol ester-stimulated cells. Expression was increased upon stimulation with phorbol ester. With this technique, TFPI-1 mRNA in monocytes was rather low even when cells were stimulated with phorbol ester or after adhesion.

Antibodies to platelet factor 4-heparin after cardiopulmonary bypass in patients anticoagulated with unfractionated heparin or a low-molecular-weight heparin : clinical implications for heparin-induced thrombocytopenia.

BACKGROUND: Cardiopulmonary bypass (CPB) induces platelet activation with release of platelet factor 4 (PF4), and patients are exposed to high doses of heparin (H). We investigated whether this contributes to the development of antibodies to H-PF4 and heparin-induced thrombocytopenia (HIT). METHODS AND RESULTS: CPB was performed with unfractionated heparin (UFH) in 328 patients. After surgery, patients received UFH (calcium heparin, 200 IU. kg-1. d-1) (group 1, n=157) or low-molecular-weight heparin (LMWH, Dalteparin, 5000 IU once daily) (group 2, n=171). Eight days after surgery, antibodies to H-PF4 were present in 83 patients (25.3%), 46 in group 1 and 37 in group 2 (P=0.12). Most patients (61%) had IgG1 to H-PF4, but only 8 samples with antibodies induced platelet activation with positive results on serotonin release assay. HIT occurred in 6 patients in group 1, but no thrombocytopenia was observed in subjects receiving LMWH, although 2 had high levels of antibodies with positive serotonin release assay results. When antibodies to H-PF4 were present, mean platelet counts were lower only in patients with FcgammaRIIA R/R131 platelets. CONCLUSIONS: These results provide evidence that the development of antibodies to H-PF4 after CPB performed with UFH is not influenced by the postoperative heparin treatment. The antibodies associated with high risk of HIT are mainly IgG1, which is present at high titers in the plasma of patients continuously treated with UFH.

Human allogeneic lymphocytes trigger endothelial cell tissue factor expression by a tumor necrosis factor-dependent pathway.

Lymphocyte adhesion to endothelial cells and the extravascular deposition of fibrin are 2 important processes during pathologic situations such as allograft rejection. Tissue factor (TF) expression was therefore measured on human umbilical vein endothelial cells (HUVECs) after coculture with allogeneic lymphocytes (PBLs) by a factor Xa generation assay. When cocultured with PBLs, HUVECs expressed strong procoagulant activity related to the TF/factor VII-dependent pathway, which was enhanced when endothelial cells were treated with interferon-gamma (IFN-gamma). The highest TF activity was measured when 10(5) lymphocytes were incubated with 10(4) HUVECs (ratio 10: 1) for 4 hours, a time-dependent course similar to that obtained with tumor necrosis factor-alpha (TNF-alpha), and direct contact between the 2 cell types was necessary. PBL-induced TF activity was inhibited by cycloheximide or actinomycin D, indicating active protein synthesis that was confirmed by the increase in TF mRNA detected by reverse transcription-polymerase chain reaction. It was then demonstrated that 1 of the primary signaling pathways leading to endothelial cell TF expression was a rapid initial interaction between membrane TNF expressed on PBLs and the 75-kd TNF receptor, with subsequent involvement of platelet-activating factor and P-selectin. Finally, we showed that the transduction of external signals involving the activation of protein kinase C and protein tyrosine kinases also contributed to the regulation of TF expression.

Pseudo-homozygous activated protein C resistance due to coinheritance of heterozygous factor V Leiden mutation and type I factor V deficiency. Variable expression when analyzed by different activated protein C resistance functional assays.

The laboratory diagnosis of activated protein C (APC) resistance is based on a weak anticoagulant response to APC using a chronometric procedure confirmed in almost all cases by molecular diagnosis of the FV Leiden mutation. A recently-developed Xa-based assay (Accelerimat, Biomerieux) was compared with two different activated partial thromboplastin time (APTT)-based procedures (Coatest APC resistance and Modified Coatest, Chromogenix) in 115 patients with a personal or familial history of thrombotic disease, or both, being studied for the FV Leiden mutation. Our results confirmed the improvement in specificity for the FV Leiden mutation when the APTT-based assay was performed after dilution of samples in FV-deficient plasma (Modified Coatest). However, five patients who were heterozygous for the FV Leiden mutation appeared to be homozygous when tested by both APTT-based assays. These patients, belonging to three different families, had a FV type I deficiency with FV plasma levels between 43 and 64%. In contrast, the Xa-based method was not influenced by the decrease in plasma FV levels. Thus, this procedure is more specific than APTT-based assays to predict the genotype status of the FV Leiden mutation.

Major antigens of Cryptosporidium parvum recognised by serum antibodies from different infected animal species and man.

Serum humoral immune response to Cryptosporidium parvum was evaluated in six species: mouse, rabbit, lamb, calf, pig and man. Electrophoretic and immunoblot analysis showed that specific animal antibody response appeared between Day 4 and Day 15 post inoculation. The two main target antigens had apparent molecular weights of 15-17 and 23 kDa. They were recognised by each species studied. Serum IgA intensively recognised the 15-17 kDa antigen, except in rabbit. This study demonstrates that these two antigens are consistent targets of humoral immune response and can therefore be of great interest in studies of therapy/prophylaxis.

Self-assembly of human papillomavirus type 16 capsids by expression of the L1 protein in insect cells.

The L1 major protein of human papillomavirus type 16 was expressed in Sf-21 insect cells with a recombinant baculovirus vector. Virus-like particles similar in appearance to empty virions were identified by electron microscopy at densities of 1.29-1.30. Purified particles reacted with monoclonal anti-HPV-16-L1 antibody in Western blot and immuno dot blot suggesting that conformational epitopes are present in the recombinant particles. Immunodot blot assays using human sera correlated with the detection of HPV-16 DNA by the polymerase chain reaction. The results suggest that HPV-16-L1 virions produced by the baculovirus system might be useful for developing serologic tests to measure antibodies to conformational epitopes and may offer potential for vaccine development.