Chorioallantoic Membrane Cells Research Articles

Transcriptome-based insights into the calcium transport mechanism of chick chorioallantoic membrane

Chorioallantoic membrane (CAM) is responsible for respiratory gas exchange, eggshell calcium transport, embryonic acid-base equilibrium, allantoic ion, and water reabsorption during avian embryonic development. To further understand the timing of CAM gene expression during chick embryonic development, especially the calcium absorption mechanism, transcriptome quantitative comparative analysis was conducted on chick CAM during the embryonic period (E) of 9, 13, 17, and 20 days. A total of 6378 differentially expressed genes (DEGs) were identified. Functional enrichment analysis of DEGs showed that CAM DEGs were mainly involved in biological processes such as “ion transport regulation”, “immune response” and “cell cycle”. Time series analysis of the differential genes showed that the functional cells of CAM began to proliferate and differentiate at E9 and the calcium content of egg embryo increased significantly at E13. Simultaneously, the observation of the ultrastructure of the eggshell showed that the interstice of the fiber layer was enlarged at E13, and the mastoid layer was partly exposed. Therefore, it is preliminarily inferred that CAM calcium transport starts at E13, and genes such as TRPV6, S100A10, and RANKL cooperate to regulate calcium release and transport.

Antimicrobial peptides cathelicidin, PMAP23, and PMAP37: Expression in the endometrium throughout the estrous cycle and at the maternal-conceptus interface during pregnancy and regulation by steroid hormones and calcitriol in pigs

The endometrium, which regulates the reproductive cyclicity and the establishment and maintenance of pregnancy, is a type of mucosal tissue and is involved in the regulation of immunity. Antimicrobial peptides, called cathelicidins, play critical roles in innate immunity by killing various microbes and modulating host immune properties. The expression and regulation of cathelicidin in female reproductive tissues have been studied in some species, but not determined in pigs, which form a true epitheliochorial placenta. Thus, we determined the expression of Cathelicidin (the conserved common cathelin domain for all porcine cathelicidin family members), PMAP23, and PMAP37 in the endometrium throughout the estrous cycle and pregnancy. The expression of Cathelicidin, PMAP23, and PMAP37 in the endometrium changed during the estrous cycle and pregnancy in a stage-specific manner. The mRNA expression of Cathelicidin, PMAP23, and PMAP37 was also detected in conceptus and chorioallantoic tissues during pregnancy. Cathelicidin protein was mainly localized to luminal epithelial cells and some immune cells in blood vessels and to chorionic epithelial cells of chorioallantoic membranes during mid-to late pregnancy. The expression of Cathelicidin was induced by calcitriol but increasing doses of estradiol-17β and progesterone did not affect the expression of Cathelicidin, PMAP23, or PMAP37 in endometrial tissues. These results indicate that members of the cathelicidin family were dynamically expressed at the maternal-conceptus interface in pigs, suggesting that the cathelicidin family may play important roles in the regulation of innate immunity by mediating antimicrobial activity to protect the maternal and/or conceptus tissues to conserve pregnancy capacity in pigs.

Serological and Molecular Identification of Infectious Bovine Rhinotrachetitis Virus Isolation and Adaptation in Embryonated Chicken Eggs

Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in livestock animals and had led to significant economic losses to the industrial production worldwide. However, IBR symptoms are not life-threatening. The present study was achieved to make seroprevalence and isolation of bovine herpes virus type (BHV-1) from nasal and ocular swabs samples of suspected cows and buffaloes, identification by serological and polymerase chain reaction (PCR). Total of 380 blood samples were collected from suspected 287 cattle and 93 buffaloes in different districts at Beni-suef and El-Fayoum governorates in winter 2017. Nasal and ocular discharge swabs were collected from cattle and buffaloes with clinical respiratory signs (Nasal discharge, cough, lachrymal discharge with or without mild diarrhea and higher body temperature). A total of 106 (27.89%) samples were positive by indirect ELISA and the positivity of 80 (27.87%) samples from cattle and 26 (27.96%) samples from buffaloes located at different centers at Beni-Suif and El-Fayoum governorates. Virus was isolated from nasal and ocular discharges swabs samples and it was adapted in chorioallantoic membrane (CAM) of 11-day-old embryonated chickens eggs and in MDBK cells. Virus-infected CAM showed congestion, edematous vacuole, thickening small foci ranged from 2 to 3 mm in diameter, scattered on CAM membrane and MDBK cell line inoculated blind serial passages at 3rd passage showed characteristic of cytopathic effect (CPE). Identification of virus isolated on CAM and infected cell culture fluid gave precipitation against positive specific anti-BHV-1 immune serum by AGPT and clear blue zone by Dot ELISA, absent of pock lesion in pock reduction test (PRT), and confirmed by PCR with product size of 175 bp. Finally BHV-1 virus was isolated from nasal and ocular discharges of cattle and buffaloes but the further extensive study still need for clear final classification by phylogenic analysis.

Low-level laser irradiation protects the chick embryo chorioallantoic membrane from UV cytotoxicity

Low-level laser therapy or photobiomodulation is the medical use of a very low intensity light in the red to near infrared (wavelengths in the range of 630-940 nm). The present work was conducted to explore the effects of both UV and low-level laser irradiation (LLLI) on microcirculation using the in vivo model of the chick embryo chorioallantoic membrane (CAM). The effects were assessed by measuring lipid peroxidation and antioxidant enzyme activity. Cell cytotoxicity, survival and intracellular reactive oxygen species (ROS) of the CAM were also evaluated. We found that UV irradiation induced alterations of the vessels, leading to bleeding and extravasation. This effect was intensified after 60 min of exposure to UV irradiation, leading to marked edema. UVA irradiation increased cell cytotoxicity as assessed by lactate dehydrogenase (LDH) release (56.23% of control) and reduced cell viability as assessed by decreased fluorescein diacetate (FDA) fluorescence (56.23% of control). Pretreatment with LLLI prior to UV exposure protected the CAM tissue from UV-mediated cell death. This protective effect was supported by the observation of significantly inhibited lipid peroxidation (from 0.3±0.004 for UV, to 0.177±0.012 after LLLI pretreatment), ROS and O2 -production, as indicated by respective dihydrorhodamine (DHR) and dihydroethidium (DHE) intensities (from 132.78% of control for UVA, to 95.90% of control for L-UV (DHR), and from 127.34% of control for UVA, to 82.03% of control for L-UV (DHE)), and by preventing the increase in oxidative activities. LLLI efficiently protected CAM cells from UV-induced oxidative stress and appeared as a safe protective pretreatment against UV irradiation.

Anti-angiogenic activity of gecko aqueous extracts and its macromolecular components in CAM and HUVE-12 cells.

Gecko is a kind of traditional Chinese medicine with remarkable antineoplastic activity. However, undefined mechanisms and ambiguity regarding active ingredients limit new drug development from gecko. This study was conducted to assess anti-angiogenic properties of the aqueous extracts of fresh gecko (AG) or macromolecular components separated from AG (M-AG). An enzyme-linked immunosorbent assay (ELISA) approach was applied to detect the vascular endothelial growth factor (VEGF) secretion of the tumor cells treated with AG or M-AG. The effect of AG or M-AG on vascular endothelial cell proliferation and migratory ability was analyzed by tetrazolium dye colorimetric method, transwell and wound-healing assays. Chick embryo chorioallantoic membrane (CAM) assays were used to ensure the anti-angiogenic activity of M-AG in vivo. The results showed that AG or M-AG inhibited the VEGF secretion of tumor cells, the relative inhibition rates of AG and M-AG being 27.2% and 53.2% respectively at a concentration of 20 μL/mL. AG and M-AG inhibited the vascular endothelial (VE) cell proliferation with IC50 values of 11.5 ± 0.5 μL/mL and 12.9 ± 0.4 μL/mL respectively. The VE cell migration potential was inhibited significantly (p<0.01) by the AG (≥ 24 μL/mL) or M-AG (≥ 12 μL/ mL) treatment. In vivo, neovascularization of CAM treated with M-AG was inhibited significantly (p<0.05) at a concentration of 0.4 μL/mL. This study provided evidence that anti-angiogenesis is one of the anti-tumor mechanisms of AG and M-AG, with the latter as a promising active component.

Titanium dioxide nanoparticles induce an adaptive inflammatory response and invasion and proliferation of lung epithelial cells in chorioallantoic membrane

Titanium dioxide nanoparticles (TiO2 NPs) studies have been performed using relatively high NPs concentration under acute exposure and limited studies have compared shape effects. We hypothesized that midterm exposure to low TiO2 NPs concentration in lung epithelial cells induces carcinogenic characteristics modulated partially by NPs shape. To test our hypothesis we synthesized NPs shaped as belts (TiO2-B) using TiO2 spheres (TiO2-SP) purchased from Sigma Aldrich Co. Then, lung epithelial A549 cells were low-exposed (10µg/cm2) to both shapes during 7 days and internalization, cytokine release and invasive potential were determined. Results showed greater TiO2-B effect on agglomerates size, cell size and granularity than TiO2-SP. Agglomerates size in cell culture medium was 310nm and 454nm for TiO2-SP and TiO2-B, respectively; TiO2-SP and TiO2-B induced 23% and 70% cell size decrease, respectively, whilst TiO2-SP and TiO2-B induced 7 and 14-fold of granularity increase. NOx production was down-regulated (31%) by TiO2-SP and up-regulated (70%) by TiO2-B. Both NPs induced a transient cytokine release (IL-2, IL-6, IL-8, IL-4, IFN-γ, and TNF-α) after 4 days, but cytokines returned to basal levels in TiO2-SP exposed cells while TiO2-B induced a down-regulation after 7 days. Midterm exposure to both shapes of NPs induced capability to degrade cellular extracellular matrix components from chorioallantoic membrane and Ki-67 marker showed that TiO2-B had higher proliferative potential than TiO2-SP. We conclude that midterm exposure to low NPs concentration of NPs has an impact in the acquisition of new characteristics of exposed cells and NPs shape influences cellular outcome.

Contributions of the S2 spike ectodomain to attachment and host range of infectious bronchitis virus

The spike protein is the major viral attachment protein of the avian coronavirus infectious bronchitis virus (IBV) and ultimately determines viral tropism. The S1 subunit of the spike is assumed to be required for virus attachment. However, we have previously shown that this domain of the embryo- and cell culture adapted Beaudette strain, in contrast to that of the virulent M41 strain, is not sufficient for binding to chicken trachea (Wickramasinghe et al., 2011). In the present study, we demonstrated that the lack of binding of Beaudette S1 was not due to absence of virus receptors on this tissue nor due to the production of S1 from mammalian cells, as S1 proteins expressed from chicken cells also lacked the ability to bind IBV-susceptible embryonic tissue. Subsequently, we addressed the contribution of the S2 subunit of the spike in IBV attachment. Recombinant IBV Beaudette spike ectodomains, comprising the entire S1 domain and the S2 ectodomain, were expressed and analyzed for binding to susceptible embryonic chorio-allantoic membrane (CAM) in our previously developed spike histochemistry assay. We observed that extension of the S1 domain with the S2 subunit of the Beaudette spike was sufficient to gain binding to CAM. A previously suggested heparin sulfate binding site in Beaudette S2 was not required for the observed binding to CAM, while sialic acids on the host tissues were essential for the attachment. To further elucidate the role of S2 the spike ectodomains of virulent IBV M41 and chimeras of M41 and Beaudette were analyzed for their binding to CAM, chicken trachea and mammalian cell lines. While the M41 spike ectodomain showed increased attachment to both CAM and chicken trachea, no binding to mammalian cells was observed. In contrast, Beaudette spike ectodomain had relatively weak ability to bind to chicken trachea, but displayed marked extended host range to mammalian cells. Binding patterns of chimeric spike ectodomains to these tissues and cells indicate that S2 subunits most likely do not contain an additional independent receptor-binding site. Rather, the interplay between S1 and S2 subunits of spikes from the same viral origin might finally determine the avidity and specificity of virus attachment and thus viral host range.

Inhibitory Influence of the Hexapeptidic Sequence SLIGRL on Influenza A Virus Infection in Mice

Proteinase-activated receptor 2 (PAR(2)) is widely expressed in the respiratory tract and is an integral component of the host antimicrobial defense system. The principal aim of this study was to investigate the influence of a PAR(2)-activating peptide, SLIGRL, on influenza A virus (IAV)-induced pathogenesis in mice. Intranasal inoculation of BALB/c mice with influenza A/PR/8/34 virus caused time-dependent increases in the number of pulmonary leukocytes (recovered from bronchoalveolar lavage fluid), marked airway histopathology characterized by extensive epithelial cell damage, airway hyper-responsiveness to the bronchoconstrictor methacholine, and elevated levels of inflammatory chemokines (keratinocyte-derived chemokine and macrophage inflammatory protein 2) and cytokines (interferon-γ). It is noteworthy that these IAV-induced effects were dose-dependently attenuated in mice treated with a PAR(2)-activating peptide, SLIGRL, at the time of IAV inoculation. However, SLIGRL also inhibited IAV-induced increases in pulmonary leukocytes in PAR(2)-deficient mice, indicating these antiviral actions were not mediated by PAR(2). The potency order obtained for a series of structural analogs of SLIGRL for anti-IAV activity (IGRL > SLIGRL > LSIGRL >2-furoyl-LIGRL) was also inconsistent with a PAR(2)-mediated effect. In further mechanistic studies, SLIGRL inhibited IAV-induced propagation in ex vivo perfused segments of trachea from wild-type or PAR(2)(-/-) mice, but did not inhibit viral attachment or replication in Madin-Darby canine kidney cells and chorioallantoic membrane cells, which are established hosts for IAV. In summary, SLIGRL protected mice from IAV infection independently of PAR(2) and independently of direct inhibition of IAV attachment or replication, potentially through the activation of endogenous antiviral pathways within the mouse respiratory tract.

Pleiotrophin expression and role in physiological angiogenesis in vivo: potential involvement of nucleolin

BackgroundPleiotrophin (PTN) is a heparin-binding growth factor with significant role(s) in tumour growth and angiogenesis. Although implication of endogenous PTN has been studied in several in vivo models of tumour angiogenesis, its role in physiological angiogenesis has not been addressed. In the present work, we studied expression and functional significance of endogenous PTN during angiogenesis in the chicken embryo chorioallantoic membrane (CAM).MethodsUsing molecular, cellular and biochemical assays, we studied the expression pattern of PTN in CAM and human endothelial cells and its possible interaction with nucleolin (NCL). CAM cells were transfected with a pCDNA3.1 vector, empty (PC) or containing full length cDNA for PTN in antisense orientation (AS-PTN). Angiogenesis was estimated by measuring total vessel length. In vitro, human endothelial cells migration was studied by using a transwell assay, and down-regulation of NCL was performed by using a proper siRNA.ResultsEndogenous PTN mRNA and protein levels, as well as protein levels of its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) were maximal at early stages, when CAM angiogenesis is active. Application of AS-PTN onto CAM at days of active angiogenesis was not toxic to the tissue and led to dose-dependent decreased expression of endogenous PTN, ERK1/2 activity and angiogenesis. Interestingly, endogenous PTN was also immunolocalized at the endothelial cell nucleus, possibly through interaction with NCL, a protein that has a significant role in the nuclear translocation of many proteins. Down-regulation of NCL by siRNA in human endothelial cells significantly decreased nuclear PTN, verifying this hypothesis. Moreover, it led to abolishment of PTN-induced endothelial cell migration, suggesting, for the first time, that PTN-NCL interaction has a functional significance.ConclusionsExpression of endogenous PTN correlates with and seems to be involved in angiogenesis of the chicken embryo CAM. Our data suggest that NCL may have a role, increasing the number of growth factors whose angiogenic/tumorigenic activities are mediated by NCL.

Biphasic regulation of H2O2 on angiogenesis implicated NADPH oxidase

ROS (reactive oxygen species) take an important signalling role in angiogenesis. Although there are several ways to produce ROS in cells, multicomponent non-phagocytic NADPH oxidase is an important source of ROS that contribute to angiogenesis. In the present work, we examined the effects of H2O2 on angiogenesis including proliferation and migration in HUVECs (human umbilical vein endothelial cells), new vessel formation in chicken embryo CAM (chorioallantoic membrane) and endothelial cell apoptosis, which is closely related to anti-angiogenesis. Our results showed that H2O2 dose-dependently increased the generation of O2- (superoxide anion) in HUVECs, which was suppressed by DPI (diphenylene iodonium) and APO (apocynin), two inhibitors of NADPH oxidase. H2O2 at low concentrations (10 microM) stimulated cell proliferation and migration, but at higher concentrations, inhibited both. Similarly, H2O2 at 4 nmol/cm2 strongly induced new vessel formation in CAM, while it suppressed at high concentrations (higher than 4 nmol/cm2). Also, H2O2 (200 approximately 500 microM) could stimulate apoptosis in HUVECs. All the effects of H2O2 on angiogenesis could be suppressed by NADPH oxidase inhibitors, which suggests that NADPH oxidase acts downstream of H2O2 to produce O2- and then to regulate angiogenesis. In summary, our results suggest that H2O2 as well as O2- mediated by NADPH oxidase have biphasic effects on angiogenesis in vitro and in vivo.

Epizootiology of lumpy skin disease outbreak in cattle in middle of Egypt, 2006

The current investigation studied an outbreak of lumpy skin disease of cattle in Beni-Suef and Al-Fayium governorates from March up to September 2006. Epidemiological data over a total of 5500 cattle from all ages, breeds and sexes were investigated. Prevalence of lumpy skin antibodies was screened by enzyme-linked immunosorbent assay (ELISA) that revealed high exposure rates; 57% and 51.42% in Beni-Suef and Al Fayium governorates respectively Virus isolation was conducted on chorioallantoic membrane (CAM) of specific pathogen free embryonated chicken egg (SPF-ECE) and MDBK cell culture. The virus identity was confirmed by passive haemagglutination and polymerase chain reaction (PCR) on the infected CAM and MDBK cell culture. Experimental infection of rabbits was successful, demonstrating their possible roles in the epidemiological process of the disease.

Analysis of N-glycans in embryonated chicken egg chorioallantoic and amniotic cells responsible for binding and adaptation of human and avian influenza viruses

The initial step essential in influenza virus infection is specific binding of viral hemagglutinin to host cell-surface glycan receptors. Influenza A virus specificity for the host is mediated by viral envelope hemagglutinin, that binds to receptors containing glycans with terminal sialic acids. Human viruses preferentially bind to alpha2-->6 linked sialic acids on receptors of host cells, whereas avian viruses are specific for the alpha2-->3 linkage on the target cells. Human influenza virus isolates more efficiently infect amniotic membrane (AM) cells than chorioallantoic membrane (CAM) cells. N-glycans were isolated from AM and CAM cells of 10-day-old chicken embryonated eggs and their structures were analyzed by multi-dimensional HPLC mapping and MALDI-TOF-MS techniques. Terminal N-acetylneuraminic acid contents in the two cell types were similar. However, molar percents of alpha2-->3 linkage preferentially bound by avian influenza virus were 27.2 in CAM cells and 15.4 in AM cells, whereas those of alpha2-->6 linkage favored by human influenza virus were 8.3 (CAM) and 14.2 (AM). Molar percents of sulfated glycans, recognized by human influenza virus, in CAM and AM cells were 3.8 and 12.7, respectively. These results have revealed structures and molar percents of N-glycans in CAM and AM cells important in determining human and avian influenza virus infection and viral adaptation.

Zoledronic Acid Suppresses Vasculogenesis: A New Effect of Bisphosphonates.

Recent studies suggest that zoledronic acid (ZOL) and other nitrogen-containing bisphosphonates (BPs) inhibit angiogenesis by reducing angiogenic factor production and signaling by these factors. However, few studies have addressed the potential role of BPs in blocking the formation of new vasculature or so-called vasculogenesis. Thus, we determined whether ZOL could impact this process using a chorioallantoic membrane (CAM) vasculogenesis model. First, fertilized chick eggs were incubated horizontally at 37.5°C in a humidified incubator and windowed on day 8. Another set of E8 chicken embryonic skins were cultured in insert dishes with different concentrations of ZOL (1.0 or 10 uM) or without drug treatment for 24 hours. The embryonic skins were transferred to CAM. Endothelial cells of CAM normally started to proliferate and migrate into feather buds after two days. Blood vessel formation was determined after four days of culturing under the microscope. We found that endothelial cell proliferation and migration of CAM was completely inhibited and development of embryonic skin buds was blocked by ZOL at 10 uM. At a lower concentration of ZOL (1.0 uM), the vasculogenesis was also decreased and embryonic skin buds were also reduced in size (but to a lesser extent than at the higher drug concentration) compared with a control group without ZOL. We also examined whether zoledronic acid affected development of embryonic feather buds (epithelial cell) directly. The results showed that embryonic feather bud growth was unaffected when buds were cultured on insert dishes with 10uM ZOL compared to buds without this drug. This suggests that ZOL blocks endothelial cell proliferation in CAM and migration but does not effect epithelial cell development. We further examined endothelial cell gene expression of the bud cells on CAM treated with and without zoledronic acid. We measured vascular endothelial growth factor receptor-2 (Flk-1) expression with Western blot analysis. The results showed that Flk-1 is markedly reduced after buds were treated with ZOL at 10 uM and there was also some reduction in the expression of Flk-1 at the lower concentration (1.0 um) of ZOL. To determine which signal transduction pathway(s) may be involved in blocking endothelial cell proliferation and migration by ZOL, we determined gene expression of β-catenin, Runx2, and smad7 by RT-PCR in the embryonic buds. Runx2 is a target of β-catenin /TCF1 for the stimulation of bone formation, and Smad7 gene expression is increased in human hematopoietic stem cells (HSCs) and is required for TGF-β-induced expression of β-catenin. The results showed that expression of β-catenin was down-regulated by ZOL whereas expression of Runx2 and smad7 was up-regulated by this bisphosphonate. These results suggest that ZOL blocks the β-catenin pathway, and also add to other studies suggesting the important role of this pathway in blood vessel development. We have also shown that zoledronic acid profoundly suppresses vasculogenesis, and this effect adds another potential mechanism by which this bisphosphonate may possess anti-tumor effects.

Formation of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene adducts in vascular endothelia of cytochrome P4501A-induced chicken embryos.

Polycyclic aromatic hydrocarbons (PAHs) are widespread in the environment and birds may be exposed to PAHs via diet, from preening feathers contaminated with oil, or through contamination of the eggshell during embryo development. In the present study, tissue distribution and the cell-specific binding of two labeled PAHs, benzo[a]pyrene ([3H]BaP) and 7,12-dimethylbenz[a]anthracene ([3H]DMBA), were examined in chicken embryos exposed in ovo to CYP1A inducers. Tape-section autoradiograms revealed high concentrations of radioactivity in the bile, liver, kidneys, heart, and leptomeninges. Light microscopy autoradiography of solvent-extracted tissue slices showed a high and selective binding in endothelial cells in certain blood vessels in brain, heart, lung, and chest muscle. Binding was also observed in blood vessel endothelial cells in the chorioallantoic membrane (CAM), an extraembryonal tissue lining the eggshell. Endothelial binding was confirmed in CAM exposed in vitro, implying that tissue-binding metabolites were formed in situ. The CYP1A inhibitor ellipticine abolished bleeding in the target endothelial cells in CAM. It is thus concluded that blood vessel endothelia in various tissues in birds can bioactivate environmental contaminants and be targets for their toxicity. In view of its critical position beneath the shell, the CAM could be an important target for toxicants following external exposure in oviparous species.

CHORIOALLANTOIC MEMBRANE ASSAY: VASCULARIZED 3-DIMENSIONAL CELL CULTURE SYSTEM FOR HUMAN PROSTATE CANCER CELLS AS AN ANIMAL SUBSTITUTE MODEL

Chorioallantoic membranes have been used as a reliable biomedical assay system for many years. Chicken eggs in the early phase of breeding are between in vitro and in vivo systems but may provide an immunodeficient, vascularized test environment. We tested this model as an in vivo system for prostate cancer research. Single cell suspensions of LNCaP, PC-3 and Tsu-Pr1 human prostatic cancer cell lines as well as 2 immortalized normal human prostate epithelial cell lines were inoculated on the chorioallantoic membrane of fertilized chicken eggs on day 5 or 6 of breeding. Tumor growth and viability of the embryo was evaluated by stereo microscopy. At day 10 the membranes were removed and embedded in paraffin. Cell morphology was assessed after hematoxylin and eosin staining. Cellular expression of cytokeratin, prostate specific antigen and androgen receptor as well as apoptosis induction was confirmed by immunohistochemistry. Three days after tumor cell inoculation on the extraembryonic vascular system of the chorioallantoic membrane cell growth and formation of 3-dimensional tumors became apparent in 100% of inoculated membranes. Strong neo-angiogenesis was detected next to the established tumors and tumor cells invading the stroma of the chorioallantoic membrane. Cytokeratin expression as well as prostate specific antigen and androgen receptor in LNCaP cells confirmed the human prostate tumor origin. Assessment of quantitative in vivo apoptosis induction in LNCaP cells after intravenous injection of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate confirmed the model as a versatile in vivo system. The well vascularized chorioallantoic membrane of bred chicken eggs is a suitable system for early in vivo cancer research. Reliable growth of prostate cancer cell lines is feasible and allows the evaluation of proliferation and apoptosis induction after intravascular or topic application of anticancer drugs. Exploitation of this assay enables a substantial reduction in or substitution for subsequent animal experiments.

X-rays modulate extracellular matrix in vivo.

X-rays have an antiangiogenic effect in the chicken embryo chorioallantoic membrane (CAM) model of in vivo angiogenesis. Our study demonstrates that X-rays induce an early apoptosis of CAM cells, modulate the synthesis and deposition of extracellular matrix (ECM) proteins involved in regulating angiogenesis and affect angiogenesis induced by tumour cells implanted onto the CAM. Apoptosis was evident within 1-2 hr, but not later than 6 hr after irradiation. Fibronectin, laminin, collagen type I, integrin alpha(v)beta3 and MMP-2 protein amounts were all decreased 6 hr after irradiation. In contrast, collagen type IV, which is restricted to basement membrane, was not affected by irradiation of the CAM. There was a similar decrease of gene expression for fibronectin, laminin, collagen type I and MMP-2, 6 hr after irradiation. The levels of mRNA for integrin alpha(v)beta3 and collagen type IV were unaffected up to 24 hr after irradiation. The decrease in both protein and mRNA levels was reversed at later time points and 48 hr after irradiation, there was a significant increase in the expression of all the genes studied. When C6 glioma tumour cells were implanted on irradiated CAMs, there was a significant increase in the angiogenesis induced by tumour cells, compared to that in non-irradiated CAMs. Therefore, although X-rays have an initial inhibitory effect on angiogenesis, their action on the ECM enhances new vessel formation induced by glioma cells implanted on the tissue.

Selection of Receptor-Binding Variants of Human Influenza A and B Viruses in Baby Hamster Kidney Cells

Cultivation of human influenza viruses in the allantoic cavity of embryonated chicken eggs leads to a selection of receptor-binding variants with amino acid substitutions on the globular head of the hemagglutinin (HA) molecule. Such selection can be avoided by growing the human viruses in Madin Darby canine kidney (MDCK) cells. In the present study, we tested whether baby hamster kidney (BHK) cells select receptor-binding mutants of human influenza viruses. After isolating H1N1, H3N2, and type B influenza viruses from clinical samples in MDCK cells, we passaged them in either BHK cells or chicken eggs. The BHK-grown viruses differed from their MDCK-grown counterparts by virtue of mutations in the HA: 225D → G (H1N1 virus), 128T → A and 226I → V (H3N2), and 187N → D (type B) (H3 numbering). Variants with different substitutions were selected by passaging of the same MDCK-grown parents in eggs: 141L → H, 208R → H, and 225D → G (H1N1), 194L → I (H3N2), and 137G → R (B). Compared with their MDCK-grown counterparts, both BHK- and egg-grown viruses possessed a higher affinity for the cellular membranes of BHK cells and of the chorioallantoic cells of chicken embryos and for a 3′-sialylgalactose-containing synthetic sialylglycopolymer. By contrast, changes in the affinity of mutants for a 6′-sialyl-(N-acetyllactosamine)-containing sialylglycopolymer varied from negative to positive. Fluorescence-activated cell-sorting analysis with linkage-specific lectins showed that the density of the 6′-sialyl-(N-acetyllactosamine)-containing receptors is substantially lower on the surface of BHK cells than on MDCK cells, providing an explanation for the growth restriction of human viruses in the former cells. Our data demonstrate that cultures of BHK cells, like eggs, can select receptor-binding variants of human influenza viruses.

Proliferation pattern of capillary endothelial cells in chorioallantoic membrane development indicates local growth control, which is counteracted by vascular endothelial growth factor application

The density and distribution of whole mount BrdU-anti-BrdU labeled endothelial cells (days 6-15) in the chick chorioallantoic membrane (CAM) was analyzed with computer-assisted microscopy. A significant loss of proliferative activity was noted after day 10: the density of labeled nuclei (in 10(-2) mm-2) decreased from a median 7.78 (days 6, 8, 10) to 2.42 (days 12, 14, 15). CAMs initially showed random patterns of labeled endothelial cells, but changed to clearly focal patterns after day 12. A regular arrangement of labeled nuclei was never seen. After application of vascular endothelial growth factor (VEGF) to the day 13 CAM, a significant increase in proliferative activity (11.50) and a random distribution of labeled endothelial cells was observed on day 15. Development of CAM precapillary vessels was assessed in terms of length density (in mm-1, mean +/- standard deviation), which was augmented three-fold from day 6 (1.22 +/- 0.05) to day 14 (3.54 +/- 0.23) and then remained nearly constant. VEGF application from day 13 to 15 raised arterial length per unit area to 4.53 +/- 0.77. It is concluded that normally a local regulation of endothelial proliferation and differentiation develops in the CAM, which doubles capillary endothelial cell density but simultaneously adapts to the decreasing need for endothelial cells, and thus maintains the quasi two-dimensional vessel pattern. However, proliferative foci persist in the capillary layer after day 10, and precapillary vessel density continues to increase until day 14. VEGF enhances DNA synthesis in all capillary endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation and function of chorioallantoic cells in avian surrogate eggs

Embryos of the domestic fowl have been successfully reared in turkey eggshells. The development of the Chorioallantois and its fusion to the shell membranes has been studied ultrastructurally and compared with the calcium transporting ability of normal embryos. There is no evidence of any species-specific effects in this functional association. The use of surrogate eggshells provides an interesting opportunity to study the differentiation and physiological properties of the chorioallantois.

Inhibition of proteolytic activation of influenza virus hemagglutinin by specific peptidyl chloroalkyl ketones

Lysates of cultured cells have been analyzed for arginine-specific endoproteases using peptidyl- p-n itroanil ides as chromogenic substrates. The enzymes present in MDBK, MDCK, VERO, BHK, and chick embryo cells required lysinearginine or arginine-arginine pairs as cleavage sites, whereas chorioallantoic membrane cells contained, in addition, an activity that could cleave at a single arginine. The effect of peptidyl chloroalkyl ketones on the activation of the fowl plague virus hemagglutinin by the proteases specific for paired basic residues has been investigated. When virions containing uncleaved hemagglutinin were incubated with lysates of uninfected cells, cleavage was completely inhibited by peptidyl chloroalkyl ketones containing paired basic residues at a concentration of 1 m M. In contrast a compound containing a single arginine had no inhibitory activity. When dibasic peptidyl chloroalkyl ketones were added to infected cell cultures, cleavage of hemagglutinin and multiple cycles of virus replication were inhibited at 10 m M. However, a 100-to 200-fold increase of the inhibitory activity in intact cells could be achieved by N-terminal acylation. These studies suggest a potential role of peptidyl chloroalkyl ketones as antiviral agents