High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (ICFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K-D similar to 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access In recent years, IGFBPs have been implicated in a variety of cancers However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Eschericha coli Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E coli and first structural characterization of a full-length IGFBP (C) 2010 Elsevier Inc. All rights reserved

Defining the pathway to insulin-like growth factor system targeting in cancer

The insulin-like growth factors (IGEs; IGF-1 and IGF-2) play central roles in cell growth, differentiation, survival, transformation and metastasis. The biologic effects of the IGFs are mediated by the IGF-1 receptor (IGF-1R), a receptor tyrosine kinase with homology to the insulin receptor (IR). Dysregulation of the ICE system is well recognized as a key contributor to the progression of multiple cancers, with IGF-1R activation increasing the tumorigenic potential of breast, prostate, lung, colon and head and neck squamous cell carcinoma (HNSCC). Despite this relationship, targeting the IGF-1R has only recently undergone development as a molecular cancer therapeutic. As it has taken hold, we are witnessing a robust increase and interest in targeting the inhibition of IGF-1R signaling. This is accentuated by the list of over 30 drugs, including monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) that are under evaluation as single agents or in combination therapies 1]. The ICE-binding proteins (IGFBPs) represent the third component of the ICE system consisting of a class of six soluble secretory proteins. They represent a unique class of naturally occurring ICE-antagonists that bind to and sequester IGF-1 and IGF-2, inhibiting their access to the IGF-1R. Due to their dual targeting of the IGFs without affecting insulin action, the IGFBPs are an untapped ``third'' class of IGF-1R inhibitors. in this commentary, we highlight some of the significant aspects of and prospects for targeting the IGF-1R and describe what the future may hold. (C) 2010 Elsevier Inc. All rights reserved.

Relative Contribution of Cell Proliferation and Microvascularity to Metastasis in Malignant Uveal Melanoma

Uveal melanoma (UM) is the second most common primary intraocular cancer worldwide. It is a relatively rare cancer, but still the second most common type of primary malignant melanoma in humans. UM is a slowly growing tumor, and gives rise to distant metastasis mainly to the liver via the bloodstream. About 40% of patients with UM die of metastatic disease within 10 years of diagnosis, irrespective of the type of treatment. During the last decade, two main lines of research have aimed to achieve enhanced understanding of the metastasis process and accurate prognosis of patients with UM. One emphasizes the characteristics of tumor cells, particularly their nucleoli, and markers of proliferation, and the other the characteristics of tumor blood vessels. Of several morphometric measurements, the mean diameter of the ten largest nucleoli (MLN) has become the most widely applied. A large MLN has consistently been associated with high likelihood of dying from UM. Blood vessels are of paramount importance in metastasis of UM. Different extravascular matrix patterns can be seen in UM, like loops and networks. This presence is associated with death from metastatic melanoma. However, the density of microvessels is also of prognostic importance. This study was undertaken to help understanding some histopathological factors which might contribute to developing metastasis in UM patients. Factors which could be related to tumor progression to metastasis disease, namely nucleolar size, MLN, microvascular density (MVD), cell proliferation, and The Insulin-like Growth Factor 1 Receptor(IGF-1R), were investigated. The primary aim of this thesis was to study the relationship between prognostic factors such as tumor cell nucleolar size, proliferation, extravascular matrix patterns, and dissemination of UM, and to assess to what extent there is a relationship to metastasis. The secondary goal was to develop a multivariate model which includes MLN and cell proliferation in addition to MVD, and which would fit better with population-based, melanoma-related survival data than previous models. I studied 167 patients with UM, who developed metastasis even after a very long time following removal of the eye, metastatic disease was the main cause of death, as documented in the Finnish Cancer Registry and on death certificates. Using an independent population-based data set, it was confirmed that MLN and extravascular matrix loops and networks were unrelated, independent predictors of survival in UM. Also, it has been found that multivariate models including MVD in addition to MLN fitted significantly better with survival data than models which excluded MVD. This supports the idea that both the characteristics of the blood vessels and the cells are important, and the future direction would be to look for the gene expression profile, whether it is associated more with MVD or MLN. The former relates to the host response to the tumor and may not be as tightly associated with the gene expression profile, yet most likely involved in the process of hematogenous metastasis. Because fresh tumor material is needed for reliable genetic analysis, such analysis could not be performed Although noninvasive detection of certain extravascular matrix patterns is now technically possible,in managing patients with UM, this study and tumor genetics suggest that such noninvasive methods will not fully capture the process of clinical metastasis. Progress in resection and biopsy techniques is likely in the near future to result in fresh material for the ophthalmic pathologist to correlate angiographic data, histopathological characteristics such as MLN, and genetic data. This study supported the theory that tumors containing epithelioid cells grow faster and have poorer prognosis when studied by cell proliferation in UM based on Ki-67 immunoreactivity. Cell proliferation index fitted best with the survival data when combined with MVD, MLN, and presence of epithelioid cells. Analogous with the finding that high MVD in primary UM is associated with shorter time to metastasis than low MVD, high MVD in hepatic metastasis tends to be associated with shorter survival after diagnosis of metastasis. Because the liver is the main organ for metastasis from UM, growth factors largely produced in the liver hepatocyte growth factor, epidermal growth factor and insulin-like growth factor-1 (IGF-1) together with their receptors may have a role in the homing and survival of metastatic cells. Therefore the association between immunoreactivity for IGF-1R in primary UM and metastatic death was studied. It was found that immunoreactivity for IGF-IR did not independently predict metastasis from primary UM in my series.

Myoblast Sheet Transplantation for Treatment of Heart Failure after Myocardial Infarction

Heart failure is a common, severe, and progressive condition associated with high mortality and morbidity. Because of population-aging in the coming decades, heart failure is estimated to reach epidemic proportions. Current medical and surgical treatments have reduced mortality, but the prognosis for patients has remained poor. Transplantation of skeletal myoblasts has raised hope of regenerating the failing heart and compensating for lost cardiac contractile tissue. In the present work, we studied epicardial transplantation of tissue-engineered myoblast sheets for treatment of heart failure. We employed a rat model of myocardial infarction-induced acute and chronic heart failure by left anterior descending coronary artery ligation. We then transplanted myoblast sheets genetically modified to resist cell death after transplantation by expressing antiapoptotic gene bcl2. In addition, we evaluated the regenerative capacity of myoblast sheets expressing the cardioprotective cytokine hepatocyte growth factor in a rat chronic heart failure model. Furthermore, we utilized in vitro cardiomyocyte and endothelial cell culture models as well as microarray gene expression analysis to elucidate molecular mechanisms mediating the therapeutic effects of myoblast sheet transplantation. Our results demonstrate that Bcl2-expression prolonged myoblast sheet survival in rat hearts after transplantation and induced secretion of cardioprotective, proangiogenic cytokines. After acute myocardial infarction, these sheets attenuated left ventricular dysfunction and myocardial damage, and they induced therapeutic angiogenesis. In the chronic heart failure model, inhibition of graft apoptosis by Bcl-2 improved cardiac function, supported survival of cardiomyocytes in the infarcted area, and induced angiogenesis in a vascular endothelial growth factor receptor 1- and 2-dependent mechanism. Hepatocyte growth factor-secreting myoblast sheets further enhanced the angiogenic efficacy of myoblast sheet therapy. Moreover, myoblast-secreted paracrine factors protected cardiomyocytes against oxidative stress in an epidermal growth factor receptor- and c-Met dependent manner. This protection was associated with induction of antioxidative genes and activation of the unfolded protein response. Our results provide evidence that inhibiting myoblast sheet apoptosis can enhance the sheets efficacy for treating heart failure after acute and chronic myocardial infarction. Furthermore, we show that myoblast sheets can serve as vehicles for delivery of growth factors, and induce therapeutic angiogenesis in the chronically ischemic heart. Finally, myoblasts induce, in a paracine manner, a cardiomyocyte-protective response against oxidative stress. Our study elucidates novel mechanisms of myoblast transplantation therapy, and suggests effective means to improve this therapy for the benefit of the heart failure patient.

Isolation of transforming growth factor-beta 2 cDNA from a fish, Cyprinus carpio by RT-PCR

Transforming Growth Factors-beta (TGF-beta s) have been described in many vertebrate species of amphibians, aves and mammals. In this report we demonstrate the presence of TGF-beta 2 in pisces. TGF-beta 2 has been cloned from a fish, Cyrinus carpio, by RT-PCR using degenerate oligonucleotide primers. Sequence analysis of the amplified product and alignment of the deduced amino acid sequence with the human TGF-beta 2 amino acid sequence revealed 81% and 93% identity in the precursor and the mature regions, respectively. The northern blot analysis of fish heart RNA shows a major messenger RNA species of about 8.0 kb and two messages of very low abundance of about 5.0 kb and 4.0 kb. The identification of TGF-beta 2 isoform in Pisces and it's high degree of homology with the mammalian isoform suggests that among all TGF-beta isoforms, TGF-beta 2 is the most conserved during evolution. (C) 1997 Elsevier Science B.V.

Insulin-Like Growth Factor System in Cancer: Novel Targeted Therapies

Insulin-like growth factors (IGFs) are essential for growth and survival that suppress apoptosis and promote cell cycle progression, angiogenesis, and metastatic activities in various cancers. The IGFs actions are mediated through the IGF-1 receptor that is involved in cell transformation induced by tumour. These effects depend on the bioavailability of IGFs, which is regulated by IGF binding proteins (IGFBPs). We describe here the role of the IGF system in cancer, proposing new strategies targeting this system. We have attempted to expand the general viewpoint on IGF-1R, its inhibitors, potential limitations of IGF-1R, antibodies and tyrosine kinase inhibitors, and IGFBP actions. This review discusses the emerging view that blocking IGF via IGFBP is a better option than blocking IGF receptors. This can lead to the development of novel cancer therapies.

Comparative effect of Pediococcus acidilactici and Lactococcus lactis on growth performance, survival and enzyme activity of western white leg shrimp (Litopenaeus vannamei)

This study was done in Shahid Kiani Marine Aquaculture Development Center, Choebde, Abadan in order to evaluate the effects of Pediiococcus acidilactici, Lactococcus lactis and vitamin C on growth performance, survival, enzymatic activities and immune responses of L. vannamei during three months. Treatments were included control group, Pediiococcus and Lactococcus treatments which fed with diet containing 1×10P9P cfu gP_1P bacteria and vitamin C. At the end of the experiment, the growth factors, immune parameters, digestive enzymes, intestinal, histology of intestine, carcasses and microbial flora (bacterial total count and lactic acid count) were evaluated. The results indicated that administration of lactobacillus had significant effects on the growth factors as the highest weight, increase specific growth rate, relative growth rate, feed conversion ratio and protein efficiency in the shrimps received pediococcus and then Lactococcus (P<0.05). The best immune function was also observed in the shrimps fed by probiotics, so that proteins and hemoglobin̛ hemolymph, phenoloxidase activity and challenged with V. parahaemolyticus showed a statistical difference comparing to the control group and the group received vitamin C (P<0.05). Some digestive enzymes, in pediococcus treatment showed a significant increase when compared to other treatments (P<0.05). Significant changes in bacterial intestinal flora were observed in probiotic groups compared with control and vitamin C groups (P < 0.05). Histological results showed the positive effects of probiotics in the gut (P < 0.05). While these supplements cannot caused to significant impacts on the shrimp carcass composition (P ˃ 0.05). As a result pediococcus group had the best performance among treatments.

The study of the ergosan and vibromax effects on growth performances, survival rate and increase of the resistance to some of the environmental stresses in post larvae stage of the Indian white shrimp

In this study, which has been done in Hormoz larve Hatchery at Kohestak in Minab at 1385, the efficiency of Ergosen and Vibromax vaccine and the effect of them on growth factors such as total length, Carapase, dry weight and the number of upper mordents of rostrum and survival of the stages of larvae and post larvae of Indian white shrimp was studied. Thus in order to comparison the effects of Vibromax and Ergosen, each of them separately, in one treatment, and in another simultaneously with one control treatment was used. Vaccination against larvae shrimps was done through Artemia. This study used four treatments with three replicates in a completely randomized design and comparison of means was done through Duncan test. Breeding larvae and post larvae of Indian white shrimp from zoa I stage to PL 15 was done in 20 litter plastic buckets. Present results indicated that the highest amount of growth and survival factors in larvae stage (from zoa to PL1), and also in stages of PL5 and PL15, in the treatment of Ergoson effect + vaccine and it was with a little difference from that treatment of Ergoson effect which was in high significance difference in regard to control treatment at α<0.01 level and treatment of vaccine effect and control treatment at α<0.01 level often have no significant difference. This research used environmental stress tests to study the quality of post larvae under experiment. Studying in this field showed that feeding vaccine to larvae of Indian shrimps which was done through Artemia nauplii enrichment ,and ergosen , in treatment of ergosen vaccine lead to more resistance of post larvaes against salinity stress tests and formalin .This case was observed in every three stages ,so that in stress formalin test 100 parts per million and also 10 and 20 salinity parts in thousands the most survival was observed in treatment of Ergosan effect+vaccine and after that in treatment of Ergoson effect and with a little difference in treatment of vaccine effect. Of course this case, in treatment of Ergoson effect + vaccine due to the synergistic properties vaccine with Ergoson was more than to other treatments, while every three treatments, in most stages had significant difference toward control treatment at α<0.01 level and the control treatment because of not having Ergoson and nauplii artemia with vaccine, having the least survival rate in this stages.

Human uterine leiomyoma-derived fibroblasts stimulate uterine leiomyoma cell proliferation and collagen type I production, and activate RTKs and TGF beta receptor signaling in coculture.

BACKGROUND: Uterine leiomyomas (fibroids) are benign smooth muscle tumors that often contain an excessive extracellular matrix (ECM). In the present study, we investigated the interactions between human uterine leiomyoma (UtLM) cells and uterine leiomyoma-derived fibroblasts (FB), and their importance in cell growth and ECM protein production using a coculture system. RESULTS: We found enhanced cell proliferation, and elevated levels of ECM collagen type I and insulin-like growth factor-binding protein-3 after coculturing. There was also increased secretion of vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor-2, and platelet derived growth factor A and B in the media of UtLM cells cocultured with FB. Protein arrays revealed increased phosphorylated receptor tyrosine kinases (RTKs) of the above growth factor ligands, and immunoblots showed elevated levels of the RTK downstream effector, phospho-mitogen activated protein kinase 44/42 in cocultured UtLM cells. There was also increased secretion of transforming growth factor-beta 1 and 3, and immunoprecipitated transforming growth factor-beta receptor I from cocultured UtLM cells showed elevated phosphoserine expression. The downstream effectors phospho-small mothers against decapentaplegic -2 and -3 protein (SMAD) levels were also increased in cocultured UtLM cells. However, none of the above effects were seen in normal myometrial cells cocultured with FB. The soluble factors released by tumor-derived fibroblasts and/or UtLM cells, and activation of the growth factor receptors and their pathways stimulated the proliferation of UtLM cells and enhanced the production of ECM proteins. CONCLUSIONS: These data support the importance of interactions between fibroid tumor cells and ECM fibroblasts in vivo, and the role of growth factors, and ECM proteins in the pathogenesis of uterine fibroids.

Discovery of a SAR11 growth requirement for thiamin’s pyrimidine precursor and its distribution in the Sargasso Sea

Vitamin traffic, the production of organic growth factors by some microbial community members and their use by other taxa, is being scrutinized as a potential explanation for the variation and highly connected behavior observed in ocean plankton by community network analysis. Thiamin (vitamin B1), a cofactor in many essential biochemical reactions that modify carbon-carbon bonds of organic compounds, is distributed in complex patterns at subpicomolar concentrations in the marine surface layer (0-300 m). Sequenced genomes from organisms belonging to the abundant and ubiquitous SAR11 clade of marine chemoheterotrophic bacteria contain genes coding for a complete thiamin biosynthetic pathway, except for thiC, encoding the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) synthase, which is required for de novo synthesis of thiamin's pyrimidine moiety. Here we demonstrate that the SAR11 isolate 'Candidatus Pelagibacter ubique', strain HTCC1062, is auxotrophic for the thiamin precursor HMP, and cannot use exogenous thiamin for growth. In culture, strain HTCC1062 required 0.7 zeptomoles per cell (ca. 400 HMP molecules per cell). Measurements of dissolved HMP in the Sargasso Sea surface layer showed that HMP ranged from undetectable (detection limit: 2.4 pM) to 35.7 pM, with maximum concentrations coincident with the deep chlorophyll maximum. In culture, some marine cyanobacteria, microalgae and bacteria exuded HMP, and in the Western Sargasso Sea, HMP profiles changed between the morning and evening, suggesting a dynamic biological flux from producers to consumers.

Expression of vascular endothelial growth factor (VEGF) and its receptors is regulated in eyes with intra-ocular tumours

The immunolocalization and gene expression of vascular endothelial growth factor (VEGF) and its cognate tyrosine kinase receptors, Flt-1 and KDR, has been studied in ocular melanomas and retinoblastomas using in situ hybridization and immunohistochemistry. Tumour-related alterations in VEGF/VEGF-receptor expression have also been examined in separate and uninvolved iris, retina and choroid of the same eyes. Although VEGF immunoreactivity in the normal retina was virtually absent, low-level VEGF expression was evident in the ganglion cell-bodies, Müller cells and in a distinct population of amacrine cells. VEGF gene expression was absent in the iris and choroid of normal eyes. In tumour-bearing eyes, high levels of VEGF protein and gene expression were observed within the vascularized regions of the tumours, while the adjacent retina and choroid showed increased VEGF levels when compared with normals. Flt-1 and KDR gene expression and immunolocalization occurred in VEGF-expressing ganglion, Müller and amacrine cells in normal eyes. Within the intra-ocular tumours, VEGF-receptor gene expression and protein was evident in the endothelial cells and also in cells close to the vessels, while in the adjacent retina, Flt-1 and KDR levels were elevated over normal, especially in the blood vessels. Flt-1 and KDR were both observed at elevated levels in the choroid and iris blood vessels. This study suggests that VEGF, Flt-1 and KDR are expressed by neural, glial and vascular elements within normal human retina. Intra-ocular tumours demonstrate a high level of VEGF and VEGF-receptor expression; within uninvolved, spatially separate retina, choroid and iris in the same eyes, expression is also elevated, especially within the vasculature. Retinal vascular endothelia may respond to high intra-ocular levels of VEGF by increasing expression of their VEGF receptors, a phenomenon which could have relevance to neoplasm-related ocular neovascularization.

Tyrosine phosphorylated SOCS-3 inhibits STAT activation but binds p120-RasGAP and activates Ras

Suppressors of cytokine signalling (SOCS, also known as CIS and SSI) are encoded by immediate early genes that act in a feedback loop to inhibit cytokine responses and activation of 'signal transducer and activator of transcription' (STAT). Here we show that SOCS-3 is strongly tyrosine-phosphorylated in response to many growth factors, including interleukin-2 (IL-2), erythropoietin (EPO), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). The principal phosphorylation sites on SOCS-3 are residues 204 and 221 at the carboxy terminus, and upon phosphorylation tyrosine 221 interacts with the Ras inhibitor p120 RasGAP. After IL-2 stimulation, phosphorylated SOCS-3 strongly inhibits STAT5 activation but, by binding to RasGAP, maintains activation of extracellular-signal-regulated kinase (ERK). A tyrosine mutant of SOCS-3 still blocks STAT phosphorylation, but also strongly inhibits IL-2-dependent activation of ERK and cell proliferation. Moreover, it also inhibits EPO- and PDGF-induced proliferation and ERK activation. Therefore, although SOCS proteins inhibit growth-factor responses, tyrosine phosphorylation of SOCS-3 can ensure cell survival and proliferation through the Ras pathway.

Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism.

Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting.

Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-a, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA–mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor.

Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC.

Bone growth into a ceramic-filled defect around an implant - The response to transforming growth factor beta 1

Synthetic bone substitutes provide an alternative to autograft but do not give equivalent clinical results. Their performance may be enhanced by adding osteogenic growth factors. In this study, TGFbeta1 was absorbed on to a carrier of 0 tricalcium phosphate and Gelfoam(R) and used to fill a defect around a tibial implant in a rat model of revision arthoplasty.

Anti-angiogenic factors and pre-eclampsia in type 1 diabetic women

Aims/hypothesis: Elevated anti-angiogenic factors such as soluble fms-like tyrosine kinase 1 (sFlt1), a soluble form of vascular endothelial growth factor receptor, and endoglin, a co-receptor for TGFß1, confer high risk of pre-eclampsia in healthy pregnant women. In this multicentre prospective study, we determined levels of these and related factors in pregnant women with type 1 diabetes, a condition associated with a fourfold increase in pre-eclampsia.
Methods: Maternal serum sFlt1, endoglin, placental growth factor (PlGF) and pigment epithelial derived factor were measured in 151 type 1 diabetic and 24 healthy non-diabetic women at each trimester and at term.
Results: Approximately 22% of the diabetic women developed pre-eclampsia, primarily after their third trimester visit. In women with pre-eclampsia (diabetic pre-eclampsia, n?=?26) vs those without hypertensive complications (diabetic normotensive, n?=?95), significant changes in angiogenic factors were observed, predominantly in the early third trimester and prior to clinical manifestation of pre-eclampsia. Serum sFlt1 levels were increased approximately twofold in type 1 diabetic pre-eclampsia vs type 1 diabetic normotensive women at the third trimester visit (p?<?0.05) and the normal rise of PlGF during pregnancy was blunted (p?<?0.05). Among type 1 diabetic women, third trimester sFlt1 and PlGF were inversely related (r2?=?42%, p?<?0.0001). Endoglin levels were increased significantly in the diabetic group as a whole vs the non-diabetic group (p?<?0.0001).
Conclusions/interpretation: Higher sFlt1 levels, a blunted PlGF rise and an elevated sFlt1/PlGF ratio are predictive of pre-eclampsia in pregnant women with type 1 diabetes. Elevated endoglin levels in women with type 1 diabetes may confer a predisposition to pre-eclampsia and may contribute to the high incidence of pre-eclampsia in this patient group.