Chorionallantoic Membrane Research Articles

A Novel Method for Screening Anti-Angiogenic Compounds: The Chorionicallantoic Membrane (CAM)/Feather Bud (FB) Assay.

Enhanced angiogenesis is a hallmark of solid tumors and hematological malignancies, and anti-angiogenic therapeutic approaches have recently shown significant benefit in the clinic. As a result, many anti-angiogenic agents are currently in early development. Very few methods have been used to evaluate the anti-angiogenic activity of these agents using an ex vivo assay. Unfortunately, currently available methods are both time consuming and costly. We have developed a novel approach to test the anti-angiogenic activity of new agents in a rapid, accurate and inexpensive way. This model consists of using a combined chorioallantoic membrane (CAM) and feather bud (FB) assay. The CAM already has a well developed vascular network and provides an ideal microenvironment and the FB serves as an active biological testing tool for evaluating angiogenesis. FB is a component of epithelial and mesenchymal cells. The method consists of using fertilized chick eggs incubated horizontally at 37.5°C in a humidified incubator and windowed by day 8. Another set of E8 chicken embryonic skins are collected under a dissecting microscope to isolate FB. The FB is treated with drugs or control reagents and implanted onto the CAM. The eggs are sealed with an adhesive tape and incubated for an additional 2–4 days. The endothelial cells of CAM proliferate and migrate into the FB after two days. After 4 days of culture, both blood vessel formation and FB development are determined by microscopy. New blood vessels in FB are analyzed by H&E and immunohistochemical (IHC) staining and expression of endothelial genes and proteins using RT-PCR and Western blot analysis, respectively. First, we establish that the compound being tested should only affect endothelial proliferation or migration and not kill the epithelial and mesenchymal tissues. We have used this new method to investigate several compounds. First, we evaluated the anti-angiogenic agent fumagillin (1μM) and minocycline (100nM). Although neither drug had any cytotoxic effects on the epithelial and mesenchymal tissues when cultured alone, marked inhibition of FB development occurred on the CAM in a dose-dependent fashion with both drugs as determined by microscopy and IHC. In addition, Western blot analysis showed marked inhibition of Tie-2 protein expression in a dose-dependent fashion in the presence of these drugs. Zoledronic acid, a potent bisphosphonate which has recently been shown to harbor anti-angiogenic activity, was found to markedly inhibit FB development in the presence of this drug at a concentration of 10 μM whereas less effect was observed at 2 μM. This drug did not have any direct effect on epithelial and mesenchymal cells when these tissues were cultured alone. We then examined gene and protein expression of the FB cells on CAM that were treated with zoledronic acid. Both FLK-1 and Tie-2 transcript and protein levels were significantly reduced in a dose-dependent fashion following treatment with zoledronic acid as assessed by RT-PCR and Western blot analysis. We are currently testing the potential anti-angiogenic effects of many other novel drugs using this new model. Overall, the present findings demonstrate that the CAM/ FB angiogenesis model is likely to be a reliable, fast, sensitive, and economical system to screen the anti-angiogenic effects of new agents.

Cysticercus tenuicollis Vesicle in Fetal Structures: Report of a Case

Cysticercus tenuicollis is the larval stage of the canine tapeworm Taenia hydatigena, the presence of which has been reported in wild and domestic ruminants all over the world. It is a common parasite of small ruminants in the north of Portugal. C. tenuicollis is generally seen attached to the omenta, the mesenteries or also found in the liver. In the ewe, tissue lesions have been associated with degenerative cysts or with oncosphere migrations. Unusual locations of the cysticerci of T. hydatigena have been described. The most frequent unusual locations are in the lungs, the kidneys and the brain. Less common locations have been reported to occur in the ovaries, uterine tubes, uterus, cervix and vagina. In the case described here, and for the first time, an aberrant location of a C. tenuicollis vesicle was found inside the chorion-allantoic membrane of a goat's foetus, in a gemelar gestation of approximately 70 days. Finding a C. tenuicollis vesicle inside fetal membranes forewarns of the possibility of larval migrations into the fetal structures during pregnancy, which is particularly concerning in human populations that are infested.

Assessment of bovine herpesvirus 4 based vector in chicken

The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the ability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, such as sheep, goats, swine, cats, dogs, rabbits, mink, horses, turkeys, ferrets, monkeys, hamsters, rats, mice, and chickens. In this report, the feasibility to use BoHV-4 based vector in chicken was investigated. Although BoHV-4 was able to replicate, leading to a cytopathic effect in a chicken cell line and infect the chorion allantoic membrane of embryonated eggs, however it was not pathogenic even when a large dose of virus was injected into the chicken. An immune response could be produced against heterologous antigen delivered by a recombinant BoHV-4. These data suggest the feasibility of using BoHV-4 based vector for vaccination purposes in chickens.

Abstract 894: Leptin Potentiates the Angiogenic Properties of Endothelial Progenitor Cells In Vitro and In Vivo

Endothelial progenitor cells (EPC) have been reported to contribute to neovascularization. We have previously shown that the adipocytokine leptin may enhance the adhesive properties of EPC by upregulating specific integrins. To investigate whether the angiogenic effects of leptin may be mediated by modulation of EPC function, mononuclear cells were isolated from healthy human volunteers and cultivated under endothelial cell conditions for 7 days. In the matrigel assay, pretreatment of EPC with recombinant leptin for 24 hours dose-dependently enhanced their incorporation into tubular structures provided by mature endothelial cells. For example, 138.3 ± 7.6% (P = 0.001) and 145.3 ± 5.5% (P = 0.0001) CM-DiI-labeled EPC were detected after stimulation with 10 and 100 ng/mL leptin, respectively (control-treated EPC defined as 100%). Furthermore, in the spheroid angiogenesis assay, stimulation of EPC with 10 ng/mL leptin increased the number of sprouts (P < 0.0001) and tube length (P < 0.0001) of coincubated mature endothelial cells, and the outgrowth of EPC (P < 0.0001). Addition of 100-fold excess of leptin-neutralizing or leptin-receptor-binding antibodies completely reversed these effects. Moreover, EPC adhesion onto endothelial cell tubules could be reduced by addition of RGD peptides (from 159 ± 13.7% to 101.8 ± 14.6%; P = 0.02), or of neutralizing antibodies against αvβ3 (from 165.3 ± 11.8% to 103.8 ± 13.3%; P = 0.006) or αvβ5 (to 93.5 ± 15.8%; P = 0.005). Further experiments using specific signal transduction inhibitors (10 μM of LY294002, PD98059, or SB203580), as well as Western blot analysis, revealed that leptin signaling in EPC involves phosphoinositide-3 kinase and p42/44, but not by p38 MAP kinase. The effects of leptin could also be confirmed under in vivo conditions. Stimulation of EPC with 100 ng/mL leptin potentiated the insprout of newly formed avian vessels into collagen onplants placed on the chorion allantoic membrane of chicken embryos (angiogenic index, 0.58 ± 0.24) compared to control-treated EPC (0.44 ± 0.27; P = 0.07) and endothelial basal medium alone (0.31 ± 0.26; P = 0.0007). Thus, our in vitro and in vivo results suggest that the angiogenic effects of leptin may partly depend on its specific interaction with endothelial progenitor cells.

Src Induces Urokinase Receptor Gene Expression and Invasion/Intravasation via Activator Protein-1/p-c-Jun in Colorectal Cancer

The urokinase receptor [urokinase plasminogen activator receptor (u-PAR)] promotes invasion and metastasis and is associated with poor patient survival. Recently, it was shown that Src induces u-PAR gene expression via Sp1 bound to the u-PAR promoter region -152/-135. However, u-PAR is regulated by diverse promoter motifs, among them being an essential activator protein-1 (AP-1) motif at -190/-171. Moreover, an in vivo relevance of Src-induced transcriptional regulators of u-PAR-mediated invasion, in particular intravasation, and a relevance in resected patient tumors have not sufficiently been shown. The present study was conducted (a) to investigate if, in particular, AP-1-related transcriptional mediators are required for Src-induced u-PAR-gene expression, (b) to show in vivo relevance of AP-1-mediated Src-induced u-PAR gene expression for invasion/intravasation and for resected tissues from colorectal cancer patients. Src stimulation of the u-PAR promoter deleted for AP-1 region -190/-171 was reduced as compared with the wild-type promoter in cultured colon cancer cells. In gelshifts/chromatin immunoprecipitation, Src-transfected SW480 cells showed an increase of phospho-c-Jun, in addition to JunD and Fra-1, bound to region -190/-171. Src-transfected cells showed a significant increase in c-Jun phosphorylated at Ser(73) and also Ser(63), which was paralleled by increased phospho-c-jun-NH(2)-kinase. Significant decreases of invasion/in vivo intravasation (chorionallantoic membrane model) were observed in Src-overexpressing cells treated with Src inhibitors, u-PAR-small interfering RNA, and dominant negative c-Jun (TAM67). In resected tissues of 20 colorectal cancer patients, a significant correlation between Src activity, AP-1 complexes bound to u-PAR region -190/-171, and advanced pN stage were observed. These data suggest that Src-induced u-PAR gene expression and invasion/intravasation in vivo is also mediated via AP-1 region -190/-171, especially bound with c-Jun phosphorylated at Ser(73/63), and that this pathway is biologically relevant for colorectal cancer patients, suggesting therapeutic potential.

Annexins as cell-type-specific markers in the developing chicken chorionallantoic membrane

Between day E8 and E12 of embryonic development, the chicken chorioallantoic membrane (CAM) undergoes massive structural rearrangement enabling calcium-uptake from the eggshell to supply the growing embryo. However, the contribution of the various cell types of the chorionic epithelium including the capillary covering (CC) cells, villus cavity (VC) cells, endothelial-like cells, and basal cells to this developmental program is largely unknown. In order to obtain markers for the different cell types in the chorionic epithelium, we determined the expression patterns of various calcium-binding annexins in the developing chicken CAM. By reverse transcription/polymerase chain reaction with primers deduced from nucleotide sequences available in various databases, the presence of annexin (anx)-1, anx-2, anx-5, and anx-6 was demonstrated at days E8 and E12. Quantitative immunoblotting with novel antibodies raised against the recombinant proteins revealed that anx-1 and anx-5 were significantly up-regulated at day E12, whereas anx-2 and anx-6 expression remained almost unchanged in comparison to levels at day E8. Immunohistochemistry of paraffin-embedded sections of E12 CAM revealed anx-1 in CC cells and VC cells. Anx-2 was localized in capillaries in the chorionic epithelium and in basal cells of the allantoic epithelium, whereas anx-6 was detected in basal cells or endothelial-like cells of the chorionic epithelium and in the media of larger vessels in the mesenchyme. A 2-day exposure of the CAM to a tumor cell spheroid resulted in strong proliferation of anx-1-expressing CC cells suggesting that these cells participate in the embryonic response to experimental intervention. Thus, annexins exhibit complementary expression patterns and represent appropriate cell markers for the further characterization of CAM development and the interpretation of results obtained when using CAM as an experimental model.

A modified HET–CAM approach for biocompatibility testing of medical devices

AbstractThe implantation of new biomedical devices into living animals without any previous toxicity or biocompatibility evaluation is possible under current legislation. The HET–CAM (Hen Egg Test–Chorionallantoic Membrane) test offers a partially immunodeficient, borderline in vitro/in vivo test system that allows the simulation of transplantation experiments to obtain biocompatibility data prior to animal testing. A collagen type I/III scaffold, designed for tissue regeneration, was tested for angiogenetic properties and biocompatibility patterns. A significant angiogenetic stimulus caused by the collagen scaffold material was observed. Altering biocompatibility patterns by incubation with the potentially hazardous chemicals acridine orange and ethidium bromide led to severe vessel thrombosis and a foreign body tissue response. CAM testing of biomaterials and tissue engineered products allows selection of the most suitable biomaterial and the elimination of unsuitable materials from animal experiments, leading to a refinement of testing procedures and a reduction in the number of animals required for biocompatibility testing.

A novel RGDS-analog inhibits angiogenesis in vitro and in vivo

In this study the anti-angiogenic action of a novel non-peptide RGDS-analog named RAM was tested in vitro and in vivo. RAM inhibited FGF-2-induced chemotaxis by 80% in an adhesion-independent way. Further, it induced HUVEC-apoptosis in collagen-seeded HUVEC, indicating that such pro-apoptotic effect was adhesion-independent. In vivo studies revealed that RAM inhibited FGF-2 induced angiogenesis by 60% in the mouse Matrigel-assay and in the chicken-egg chorion-allantoic membrane assay. Finally, RAM was markedly more stable in serum as compared to the template RGDS and after 24 h incubation in 100% serum was significantly more active than RGDS. Taken together these results show that RAM exerts anti-chemotactic and pro-apoptotic effects, by an unexpected adhesion-independent mechanism, as we have recently shown for the template RGDS molecule [Blood 103 (2004) 4180], and has in vivo relevant anti-angiogenic properties, with marked stability in serum; therefore, RAM represents a novel promising anti-angiogenic molecule.

CCL16 activates an angiogenic program in vascular endothelial cells

AbstractBesides regulating leukocyte trafficking in normal and injured tissues, several chemokines may positively or negatively regulate angiogenesis. Here we report that CCL16 activates an angiogenic program in vascular endothelial cells by activating CCR1. CCL16 induces dose-dependent random and directional migration of endothelial cells isolated from large vessels and liver capillaries without inducing their proliferation. It also promotes endothelial differentiation into capillary-like structures in an in vitro assay and is angiogenic in the chick chorionallantoic membrane. These angiogenic activities are neutralized by a specific antibody against CCL16. The direct angiogenic activity of CCL16 is further amplified by its ability to prime endothelium to a mitogen signal induced by vascular endothelial growth factor A and to raise their basal production of CXCL8 and CCL2, 2 other angiogenic chemokines. BX471 (R-N-[5-chloro-2-[2-[4(4-fluorophenyl) methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl] urea hydrochloric acid salt), a CCR1 antagonist, inhibits angiogenic properties of CCL16, whereas blocking of CCR8 or desensitizing CCR2, which are both well known receptors for CCL16, did not abolish endothelial activation. CCL16 may be specifically cross-linked to CCR1 expressed on endothelial cells. The largely restricted CCL16 expression in the liver suggests that this chemokine may play a role in hepatic vascular formation during development and in angiogenesis associated to hepatic diseases.

Possible angiogenic roles of insulin-like growth factor II and its receptors in uterine vascular adaptation to pregnancy.

Adaptation of the maternal uterine vasculature is essential for normal fetal and placental development in which angiogenesis is considered one of the most critical adaptive changes during pregnancy. Highly expressed in cytotrophoblasts and maternal endothelial cells during pregnancy, IGF-II promotes cell migration and regulates fetal and placental growth. We hypothesized that IGF-II regulates uterine angiogenesis during pregnancy. Both uterine vasculature and isolated uterine microvascular endothelial cells expressed high levels of IGF-II and IGF-II/mannose-6 phosphate receptor mRNA as shown by in situ hybridization. Physiological concentrations of IGF-II significantly increased vessel formation, as shown by a three-dimensional angiogenesis assay in vitro or a chicken chorionallantoic membrane assay in vivo. The angiogenic response of IGF-II could be reversed by the addition of beta-galactosidase or rabbit-antihuman IGF-II/M6P receptor antiserum, whereas blocking antibodies against IGF-I receptor or insulin receptor influenced IGF-II-induced sprout formation. IGF-II promoted migration of endothelial cells (10-250 ng/ml) tested in a modified Boyden chamber, but no stimulating effect on proliferation was observed. The application of several intracellular signal transduction molecules and their inhibitors indicated that protein kinase C and G(i) protein might play a role in the IGF-II-induced angiogenesis. Our results suggest an important angiogenic role of IGF-II in the vascular adaptation to pregnancy.

In vivo analysis of the apoptosis-inducing effect of anti-endothelial cell antibodies in systemic sclerosis by the chorionallantoic membrane assay.

Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology characterized by mononuclear cell infiltration and fibrosis. Vascular injury occurs early in the course of disease, and previous in vitro studies suggest a primary role for anti-endothelial cell antibodies (AECAs) in mediating endothelial cell apoptosis. The aim of the present study was to analyze the apoptosis-inducing effect of AECAs in vivo. The optimum animal model for transfer experiments was the University of California at Davis line 200 (UCD-200) chickens that spontaneously develop a hereditary disease with features closely resembling those of scleroderma in humans. AECA-positive serum samples from UCD-200 chickens were used for intravenous injection into normal CC chicken embryos on embryonic day (ED) 13 as well as for application onto chorionallantoic membranes (CAMs) of healthy control lines on ED 10. CAMs of ED 16 embryos and combs of 1-week-old CC chickens that had received the injected serum samples were analyzed for apoptotic endothelial cells by TUNEL. Staining of frozen CAM sections by immunofluorescence showed evidence of in vivo binding of AECAs to the microvascular endothelium. In most groups, transfer of AECA-positive sera resulted in a significant increase in endothelial cell apoptosis as compared with controls. This study is the first to demonstrate the in vivo apoptosis-inducing effects of AECAs. The findings support our hypothesis of a primary pathogenetic role of AECAs in SSc.

Generation of neuroendocrine chromaffin cells from sympathoadrenal progenitors: beyond the glucocorticoid hypothesis.

The developmental diversification of neural crest-derived sympathoadrenal (SA) progenitor cells into neuroendocrine adrenal chromaffin cells and sympathetic neurons has been thought to be largely understood. Based on two decades of in vitro studies with isolated SA progenitor and chromaffin cells, it was widely assumed that chromaffin cell development crucially depends on glucocorticoid hormones provided by adrenal cortical cells. However, analysis of mice lacking the glucocorticoid receptor has revealed that the chromaffin cell phenotype develops largely normally in these mice, except for the induction of the adrenaline synthesizing enzyme phenylethylamine N-methyl transferase. In a search for novel candidate genes that might be involved in triggering the sympathetic neuron/chromaffin cell decision, we have studied putative contributions of transforming growth factor (TGF)-alpha, BMP-4, and the transcription factor MASH-1, molecules with distinct expressions in SA progenitor cells, in their migratory pathways and final destinations. TGF-beta2 and -beta3 and BMP-4 are highly expressed in the wall of the dorsal aorta and in the adrenal anlagen during and after immigration of SA progenitors but expressed at much lower levels in sympathetic ganglia. We found that neutralizing antibodies against all three TGF-beta isoforms applied to the chorionic-allantoic membrane (CAM) of quail embryos interfere with proliferation of immigrated adrenal chromaffin cells but do not affect their specific neuroendocrine ultrastructural phenotype. Grafting of noggin-producing cells to the CAM, which scavenges BMPs, interferes with visceral arch and limb development but does not overtly affect the chromaffin phenotype. The transcription factor MASH-1 promotes early differentiation of SA progenitors. Mice deficient for MASH-1 lack sympathetic ganglia, whereas the adrenal medulla previously has been reported to be present. We show here that most adrenal medullary cells in MASH-1(-/-) mice identified by Phox2b immunoreactivity lack the catecholaminergic marker tyrosine hydroxylase. More surprisingly, most cells do not contain chromaffin granules and display a neuroblast-like ultrastructure and show strongly enhanced expression of c-RET comparable to that observed in sympathetic ganglia. Together, our data suggest that TGF-betas and BMP-4 do not seem to be essential for chromaffin cell differentiation. In contrast with previous reports, however, MASH-1 apparently plays a crucial role in chromaffin cell development.

35] Determination of angiogenesis-regulating properties of NO

Publisher Summary Most used in vivo bioassay in laboratory is the rabbit cornea assay. Because the cornea is an avascular tissue, this assay avoids the problems of interpretation inherent in the other bioassays, such as the chick chorion allantoic membrane (CAM). The outgrowth of new vessels into the cornea stroma can be readily identified and their morphology documented. The corneal assay is performed in New Zealand white rabbits. Nitric oxide (NO) directs endothelial cells in each step of angiogenesis and its role is twofold. In vitro administration of NO donor drugs to sparse coronary endothelium increases their mitotic index and favors cell migration and matrix degradation. As a result, in vivo NO donors speed up and potentiate neovascular growth. Thus, NO acts as the true effector of angiogenesis. However, mediators of angiogenesis signal the angiogenic switch in endothelium by releasing free NO and elevating cGMP levels. The development of NO-targeting drugs with higher selectivity for the different isoforms, combined with targeted tissue delivery to minimize side effects, as well as qualitative assessment of NO function from activity, to genomic expression, is a promising strategy for the exploitation of the findings on the role of NO on angiogenesis.

HIF-1 mediated upregulation of VEGF and VEGF-R in systemic sclerosis (SSc): Imbalance with angiostatic factors suggests VEGF as a novel option for the treatment of ischemia in patients with SSc

Vascular changes are consistent early findings in patients with SSc and often precede the development of fibrosis. Despite a significant reduction in the capillary density, there is paradoxically no sufficient angiogenesis in the skin of SSc patients. By using a pO2 histograph, we showed that low pO2 values are overt in involved skin of patients with SSc. In vitro, real-time PCR revealed a 3.7-fold upregulation of the potent angiogenic growth factor VEGF in SSc fibroblasts after hypoxic exposure compared to normoxic controls. In situ hybridization for VEGF in skin biopsies of patients with SSc showed an overexpression of VEGF mRNA by fibroblasts and mononuclear infiltrates, whereas its expression was limited to keratinocytes in healthy control biopsies. In contrast to the SSc skin, HIF-1 alpha protein was found to be coexpressed with VEGF in healthy skin samples, indicating that the constitutive VEGF synthesis in the skin is driven by this transcription factor. Additionally, we showed that the lack of angiogenesis in SSc is not due to a reduced bioavailability of the overexpressed VEGF, since the VEGF receptors Flk-1 and Flt-1 were found to be expressed on endothelial cells of patients with SSc, but not in healthy controls, and since SSc patients had severely elevated serum levels of VEGF compared to healthy controls. Despite the enhanced levels of VEGF, serum samples of SSc patients did not induce angiogen-esis in the vivo chorion allantois membrane assay, indicating that the proangiogenic effects of VEGF may be outweighed by angiostatic factors. The hypothesis that VEGF synthesis has to be above an individual threshold in SSc patients to induce angiogenesis was further strengthened by the finding that patients without fingertip ulcers had significantly higher levels than patients with fingertip ulcers. Interestingly, the angiostatic factor endostatin was elevated in a subset of patients and thus may counteract directly the biologic effects of VEGF in SSc patients. Serum levels of VEGF were also correlated significantly with disease severity parameters including antitopoisomerase antibodies. These results suggest that therapeutic application of VEGF by either gene transfer or as a recombinant protein might be a novel option in SSc.

FGF-8b increases angiogenic capacity and tumor growth of androgen-regulated S115 breast cancer cells.

Fibroblast growth factor 8 (FGF-8) is a secreted heparin-binding protein, which has transforming potential. Alternative splicing of the mouse Fgf-8 gene potentially codes for eight protein isoforms (a-h) which differ in their transforming capacity in transfected cells. S115 mouse mammary tumor cells express a transformed phenotype and secrete FGF-8 in an androgen-dependent manner. In order to study the role of FGF-8 isoforms in the induction of transformed phenotype of breast cancer cells, we over-expressed FGF-8 isoforms a, b and e in S115 cells. Over-expression of FGF-8b, but not FGF-8a or FGF-8e, induced androgen and anchorage independent growth of S115 cells. FGF-8b-transfected S115 cells formed rapidly growing tumors with increased vascularization when injected s.c. into nude mice. FGF-8a also slightly increased tumor growth and probably tumor vascularization but FGF-8e was not found to have any effects. The angiogenic activity of FGF-8b and heparin-binding growth factor fraction (HBGF) of S115 cell conditioned media was tested in in vitro and in vivo models for angiogenesis using immortomouse brain capillary endothelial cells (IBEC) and chorion allantoic membrane (CAM) assays. Recombinant FGF-8b protein was able to stimulate proliferation, migration, and vessel-like tube formation of IBECs. In addition, stimulatory effect of S115-HBGF on IBE cell proliferation was evident. A positive angiogenic response to FGF-8b was also seen in CAM assay. The results demonstrate that the expression of Fgf-8b is able to promote vessel formation. Angiogenic capacity probably markedly contributes to the ability of FGF-8b to increase tumor growth of androgen-regulated S115 mouse breast cancer cells.

Recombinant production of PIGF-1 and its activity in animal models

In this paper we review current knowledge on placenta growth factor (PIGF) and summarise our data on its recombinant production in bacteria and its activity. PIGF and vascular endothelial growth factor (VEGF) are both angiogenic factors belonging to the platelet-derived growth factor (PDGF) family. PIGF is a dimeric glycoprotein which shares a number of biochemical and functional features with VEGF. The aminoacidic similarity between the two factors is high (about 50%) in the PDGF-like domain. By alternative splicing of the PIGF mRNA, three forms of PIGF protein are generated which are named PIGF-1, PIGF-2 and PIGF-3. We have focused our attention on form 1 of human PIGF (PIGF-1). A large quantity of active recombinant PIGF-1 has been obtained using a bacterial expression system. By optimising the fermentation and purification it was possible to produce about 140 mg/l of culture of active PIGF-1, which is potentially suitable for a pharmaceutical use. The angiogenic activity of two different batches of bacteria-derived PIGF-1 obtained in our laboratory was demonstrated in chick chorionallantoic membrane assays. Finally, in preliminary studies we have shown that bacteria-derived PIGF-1 has a protective effect against myocardial lesions induced by isoprenaline in rat and rabbit.

IRAG Working Group 2: CAM-based assays

CAM-based assays, in which test material is applied to the chorion allantoic membrane (CAM) of embryonated chicken eggs, were assessed as alternatives to the Draize eye irritation test. Two general types of CAM-based assays are currently in use, the HET-CAM test and the CAMVA assay. Evaluations were made of five data sets produced with three different modifications of the HET-CAM test and two data sets obtained with the same CAMVA protocol. Data sets consisted of 9–133 test chemicals, usually from the sponsor's product line, and also from a validation trial. Each data set and assay protocol were analysed for quality of data, purpose and proposed use of the assay, range of responses covered, range of test materials amenable, current use in safety and risk assessment both in-house and for regulatory purposes. Since the MMAS Draize score was not available for all in vivo data sets, the ∑MMMIS, which correlates well with the MMAS, was used instead. In vitro/in vivo correlations calculated with Pearson's linear coeflicient ranged from r = 0.6 to r = 0.9 for six of seven data sets. Corneal opacity and inflammation of the iris showed the best correlation to in vitro data. Prediction rates were significantly improved when partial linear regression was used, and the predictivity of three different HET-CAM protocols was almost the same. HET-CAM assays showed the best prediction with surfactants and surfactant-based formulations, whereas the CAMVA assay provided the best performance with alcohols.

Defined carrageenan derivatives as angiogenesis inhibitors

AbstractThree carrageenans derived from red algae and three partially modified carrageenans were tested for their ability to inhibit the formation of new blood capillaries on the chorionallantois membrane of the hen egg. It could be clearly demonstrated that they were better angiogenesis inhibitors than the standard compound suramin.

HET-CAM test.

AbstractThe potential irritancy of compounds may be detected by observing adverse changes that occur in the chorionallantoic membrane of the egg after exposure to test chemicals (1).KeywordsTest ChemicalWhite Leghorn ChickenFume CupboardLocal ContractorPotential IrritancyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

In situ analyses of in ovo graft-vs.-host reaction induced by thymic nurse cell lymphocytes.

In both mammalian and avian systems, thymic nurse cells (TNC) have been shown to harbor a heterogeneous population of T lymphocytes (TNC-L) some of which exhibit a postselectional phenotype. By transplanting micromanipulated single chicken TNC onto the chorionallantoic membrane (CAM) of major histocompatibility complex (MHC)-disparate embryos, an experimental system which allows for the detection of lymphocytes with graft-vs.-host (GVH) reactivity, we demonstrate here that TNC enclose lymphocytes that can develop into both CD4+ single-positive (sp) and CD8+ sp, T cell receptor (TcR) alpha beta+, or TcR gamma delta+ cells. This finding was additionally confirmed by serial transfer of primary expanded alloreactive T cells onto the CAM of secondary hosts. All donor TNC-L expressed MHC class II molecules and the interleukin-2 receptor alpha chain in primary and secondary GVH reactions. Furthermore, we observed selective accumulation of CD8+ and TcR gamma delta+ host lymphocytes in the CAM upon the induction of a local GVH reaction, most probably as a consequence of the pathological alteration of the epithelium.