Chick Embryo Yolk Sac Research Articles

Anti-cancer activity of NTAX-44 (bioprocessed arsenic trioxide) on lung cancer.

e20573 Background: In contemporary allopathic medicine, intravenous administration of Arsenic trioxide is extensively investigated for its diverse applications in cancer treatment. However, the associated adverse effects demand exploration of alternative formulations. In present study, we propose the patented green technology-based compound “oral Arsenic Trioxide” (NTAX-44), in its unique form that can be administered orally, thereby reduces cost of treatment. This product presenting itself as a promising candidate for cancer therapy. Despite the ethnopharmacological uses of arsenicals in medicine, comprehensive understanding of their safety and efficacy remains limited. Thus, present study is proposed to investigate anticancer potential of NTAX-44 in lung adenocarcinoma cancer cell line A549. Methods: The effect of oral NTAX-44 was assessed on the viability of cell lines grown as 2D monolayers by MTS assay and 3D multicellular spheroid (MCS) model. Anti-metastatic potential of NTAX-44 was studied by using migration and colony formation assay while, expression of PD-L1 in A549 cell line treated with Arsenic Trioxide was checked by flow cytometry. The anti-metastatic capacity and cytocompatibility testing of Arsenic Trioxide was evaluated by in ovo chick embryo yolk sac membrane (YSM) angiogenesis assay and MTS assay using PBMCs respectively. Results: NTAX-44 is found to be effective to inhibit viability of A549 cell line (IC50 21.47±1.7 μg/ml for 48 hr and 14.65±0.92 for 72 hr of treatment); exhibiting its anti-proliferative activity. The A549 spheroids showed comparatively more resistance with loss of viability at higher concentration (50 and 100 µg/ml). Statistically significant decrease in scratch repair rate was observed at 24 hr and 72 hr of treatment and showing anti-migratory activity in A549 cells. In colony formation assay, complete inhibition of the colonization of A549 cells was observed after 48 hr of treatment; the survival fraction identified as 0%. NTAX-44 at 25µg/ml concentration inhibited PD-L1 expression in A549 cells, suggesting its potential to decrease expression of PD-L1 on tumor cells resulting in reversal of immune suppressive tumor microenvironment. Treatment of YSM with NTAX-44 resulted in approximately 40% inhibition in formation of quaternary blood vessels comparable to Bevacizumab (Avastin®). This significant anti-angiogenic potential is suggestive of its in vivo anti-metastatic effect. Notably, cytocompatibility testing of NTAX-44 at concentrations up to 200μg/ml was well-tolerated in PBMC cells emphasizing its favourable biocompatibility profile, providing crucial insights into its safety. Conclusions: Inhibitory effect of NTAX-44 on cell proliferation serves as a noteworthy attribute in context of its potential therapeutic utility against lung cancer. Its cytocompatibility further indicates application of arsenic trioxide as a candidate for clinical application.

Exploiting Co(III)-Cyclopentadienyl Complexes To Develop Anticancer Agents.

In recent years, organometallic complexes have attracted much attention as anticancer therapeutics aiming at overcoming the limitations of platinum drugs that are currently marketed. Still, the development of half-sandwich organometallic cobalt complexes remains scarcely explored. Four new cobalt(III)-cyclopentadienyl complexes containing N,N-heteroaromatic bidentate, and phosphane ligands were synthesized and fully characterized by elemental analysis, spectroscopic techniques, and DFT methods. The cytotoxicity of all complexes was determined in vitro by the MTS assay in colorectal (HCT116), ovarian (A2780), and breast (MDA-MB-231 and MCF-7) human cancer cell lines and in a healthy human cell line (fibroblasts). The complexes showed high cytotoxicity in cancer cell lines, mostly due to ROS production, apoptosis, autophagy induction, and disruption of the mitochondrial membrane. Also, these complexes were shown to be nontoxic in vivo in an ex ovo chick embryo yolk sac membrane (YSM) assay.

Antiproliferative Activity of Buddleja saligna (Willd.) against Melanoma and In Vivo Modulation of Angiogenesis.

Melanoma cells secrete pro-angiogenic factors, which stimulates growth, proliferation and metastasis, and therefore are key therapeutic targets. Buddleja saligna (BS), and an isolated triterpenoid mixture (DT-BS-01) showed a fifty percent inhibitory concentration (IC50) of 33.80 ± 1.02 and 5.45 ± 0.19 µg/mL, respectively, against melanoma cells (UCT-MEL-1) with selectivity index (SI) values of 1.64 and 5.06 compared to keratinocytes (HaCat). Cyclooxygenase-2 (COX-2) inhibition was observed with IC50 values of 35.06 ± 2.96 (BS) and 26.40 ± 4.19 µg/mL (DT-BS-01). BS (30 µg/mL) significantly inhibited interleukin (IL)-6 (83.26 ± 17.60%) and IL-8 (100 ± 0.2%) production, whereas DT-BS-01 (5 µg/mL) showed 51.07 ± 2.83 (IL-6) and 0 ± 6.7% (IL-8) inhibition. Significant vascular endothelial growth factor (VEGF) inhibition, by 15.84 ± 4.54 and 12.21 ± 3.48%, respectively, was observed. In the ex ovo chick embryo yolk sac membrane assay (YSM), BS (15 µg/egg) significantly reduced new blood vessel formation, with 53.34 ± 11.64% newly formed vessels. Silver and palladium BS nanoparticles displayed noteworthy SI values. This is the first report on the significant anti-angiogenic activity of BS and DT-BS-01 and should be considered for preclinical trials as there are currently no US Food and Drug Administration (FDA) approved drugs to inhibit angiogenesis in melanoma.

Abstract PO-058: Anti-cancer activity of NTAX-44 (bioprocessed arsenic trioxide) on pancreatic cancer cell line

Abstract Purpose: Pancreatic Cancer remains to be highly lethal malignancy mainly because of a high incidence of drug resistance. Because of this, there is a great need for new therapies which can improve overall therapeutic outcomes in pancreatic cancer patients. Arsenic trioxide is an approved drug for the treatment of acute promyelocytic leukemia (APML) and is in the experimental stage for use in different malignancies. Earlier in-vitro studies on human pancreatic cell lines have shown that arsenic trioxide has potent anti-proliferative effects with induction of apoptosis. NTAX-44 is a novel orally administrable Arsenic trioxide that is derived using green chemistry based patented technology. This compound is bioavailable when administered orally, and it has demonstrated good safety and tolerability in bioavailability clinical trials. In the present study, we conducted preclinical studies to evaluate the anti-proliferative effect and inhibition of programmed cell death ligand-1 (PD-L1) expression activity by NTAX-44. Method: The effect of NTAX-44 was assessed on the viability of MIA-Pa-Ca-2 cell lines grown as monolayers by MTS assay. We further checked the expression of PD-L1 in MIA-Pa-Ca-2 cell line treated with NTAX-44 by flow cytometry. PD-L1, an immune checkpoint regulator, has been speculated to play a significant role in suppressing the immune system. It also helps tumor cells evade anti-tumor immunity. The expression of PD-L1 was induced by IFN-gamma stimulation. The anti-metastatic capacity of NTAX-44 was evaluated by in ovo chick embryo yolk sac membrane (YSM) angiogenesis assay. The cytocompatibility testing was done by testing the effect of NTAX-44 on the viability of normal human peripheral blood mononuclear cells (PBMC) by MTS assay. Results: NTAX-44 effectively inhibited the viability of MIA-Pa-Ca (IC50 4.63 +/- 1.16μg/ml for 48 hr and 0.50 +/- 0.63 for 72 hr of treatment); demonstrating the anti-proliferative activity of NTAX-44 against MIA-Pa-Ca-2 cell line. Furthermore, NTAX-44 at 25µg/ml concentration significantly inhibited IFN-gamma-induced PD-L1 expression in the MIA-Pa-Ca-2 cell line. Evaluation of the anti-metastatic potential of NTAX-44 exhibited around 40% inhibition in the number of quaternary blood vessels was observed on the treatment of YSM with NTAX-44 compared to vehicle control. It is noteworthy that the anti-angiogenic activity was comparable to that of Bevacizumab (Avastin®). The cytocompatibility testing revealed that it was well tolerated at a concentration up to 200μg/ml in PBMC cells. Conclusions: The preclinical data presented in this study suggests the potential of NTAX-44 to inhibit proliferation and, PD-L1 expression in MIA-Pa-Ca-2 cells and prevent angiogenesis in YSM. These properties along, with their good tolerability as indicated by cytocompatibility testing, are indicative of the potential role of NTAX-44 as an oral chemotherapeutic agent in the treatment of Pancreatic cancer. Though the preclinical studies performed on NTAX-44 are very promising but further clinical evaluation is warranted. Citation Format: Yogesh Bendale, Padma Shastri, Radha Poojari, Nandinee Khot, Surendra Nagare, Avinash Kadam. Anti-cancer activity of NTAX-44 (bioprocessed arsenic trioxide) on pancreatic cancer cell line [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-058.

The Wnt/β-catenin signaling pathway is involved in regulating feather growth of embryonic chicks

Avian feathers have robust growth and regeneration capability and serve as a useful model for decoding hair morphogenesis and other developmental studies. However, the molecular signaling involved in regulating the development of feather follicles is unclear. The purpose of this study was to investigate the role of the Wnt/β-catenin pathway in regulating feather morphogenesis in embryonic chicks through in ovo injection of different doses of Dickkopf-1 (DKK1, a specific inhibitor of the target of the Wnt/β-catenin pathway). A total of 120 fertilized embryo eggs were randomly divided into 4 treatments, including a noninjection group (control group) and groups injected with 100 μL of phosphate-buffered saline (PBS)/egg (PBS control group), 100 μL of PBS/egg containing 600-ng DKK1/egg (600-ng DKK1 group), and 100-μL PBS/egg containing 1,200-ng DKK1/egg (1,200-ng DKK1 group). Feathers and skin tissues were sampled on embryonic (E) day 15 and the day of hatching to examine the feather mass, diameter and density of feather follicles, and the protein expression of the Wnt/β-catenin pathway. The results showed that, compared with CON and PBS treatment, the injection of DKK1 into the yolk sac of chick embryos had no significant effect on the hatching rate and embryo weight (P > 0.05), while it significantly decreased the relative mass of feathers in the whole body (P < 0.05). The high dose of DKK1 (1,200-ng DKK1/egg) decreased the relative mass of feathers on the back, chest, belly, neck, wings, head, and legs, which was more obvious than that in the 600-ng DKK1 group, which presented a dose-dependent effect. In addition, DKK1 injection significantly downregulated the protein expression levels of β-catenin, transcription factor 4, Cyclin D1, and c-Myc (P < 0.05). The immunofluorescence result of β-catenin was consistent with the Western blotting assay results. Altogether, these observations suggested that the Wnt/β-catenin signaling pathway is involved in regulating feather follicle development and feather growth during the embryonic development of chicks.

Establishment of an in ovo chick embryo yolk sac membrane (YSM) assay for pilot screening of potential angiogenic and anti-angiogenic agents.

Angiogenesis, the process of new blood vessel formation from pre-existing vessels, is essential for growth and development. Development of drugs that can accelerate or decelerate angiogenesis in the context of various diseases requires appropriate preclinical screening. As angiogenesis involves complex cellular and molecular processes, in vivo studies are superior to in vitro investigations. Conventional in vitro, in vivo, and ex ovo models of angiogenesis are time consuming and tedious, and require sophisticated infrastructure for embryo culture. In the present study, we established an in ovo chick embryo yolk sac membrane (YSM) assay for angiogenesis and tested the angiogenic potential of arginine, conditioned medium (CM) from human adipose tissue and placenta-derived mesenchymal stem cells (ADMSCs-CM and PDMSCs-CM), avastin and vitamin C. The obtained results were confirmed with the routinely employed chick embryo Chorioallantoic Membrane (CAM) assay. Both assays revealed the pro-angiogenic nature of arginine, ADMSCs-CM, and PDMSCs-CM, and the anti-angiogenic effect of avastin and vitamin C. This novel in ovo YSM model is simple, reproducible, and highly economic in terms of the time frame and cost incurred. The proposed model is thus a suitable substitute to the CAM model for pilot screening of potential angiogenic and anti-angiogenic agents.

The structural requirements of organophosphorus insecticides (OPI) for reducing chicken embryo NAD+ content in OPI-induced teratogenesis in chickens

The objective of this study was to determine the structural requirements of organophosphorus insecticides (OPI) for reducing chicken embryo nicotinamide adenine dinucleotide (NAD+) content in OPI-induced teratogenesis and compare them with those needed for OPI inhibition of yolk sac membrane kynurenine formamidase (KFase), the proposed primary target for OPI teratogens in chicken embryos. The comparative molecular field analysis (COMFA) of three-dimensional quantitative structure–activity relationship (3D QSAR) revealed the electrostatic and steric fields as good predictors of OPI structural requirements to reduce NAD+ content in chicken embryos. The dominant electrostatic interactions were localized at nitrogen-1, nitrogen-3, nitrogen of 2-amino substituent of the pyrimidinyl of pyrimidinyl phosphorothioates, and at the oxygen of crotonamide carbonyl in crotonamide phosphates. Bulkiness of the substituents at carbon-6 of the pyrimidinyls and/or N-substituents of crotonamides was the steric structural component that contributed to superiority of those OPI for reducing embryonic NAD+ levels. Both electrostatic and steric requirements are similar to those defined in our previous study for OPI inhibition of chicken embryo yolk sac membrane KFase. The findings of this study provide another piece of evidence for the cause-and-effect relationship between yolk sac membrane KFase inhibition and reduced embryo NAD+ content in NAD-associated OPI-induced teratogenesis in chickens.

Investigating the effect of excess caffeine exposure on placental angiogenesis using chicken 'functional' placental blood vessel network.

It is now known that over-consumption of caffeine by pregnant mothers could have detrimental effects on normal fetal development. However, it remains obscure how caffeine's harmful effect impacts directly or indirectly on the developing embryo/fetus through damaging placenta development. In this study, we demonstrated the morphological similarities between the yolk sac and chorioallantoic membranes (CAM) of chick embryos and the villi of the mammalian placenta. Using the chick yolk sac and the CAM as a model, we found that 5-15 µmol per egg of caffeine exposure inhibited angiogenesis. Under the same condition, cell proliferation in extraembryonic mesoderm was reduced while apoptosis was enhanced. Semi-quantitative RT-PCR analysis revealed that caffeine treatment down-regulated VEGF, VEGFR2, PIGF, IGF2 and NRP1 expression, but up-regulated Ang1 and Ang2 expression. We performed in situ hybridization to show VE-cadherin expression and as to demonstrate the blood vessels in the CAM and yolk sac membranes. This distribution of the VE-cadherin(+) blood vessels was determined to be reduced after caffeine treatment. Furthermore, MDA activity was induced after caffeine exposure, but GSH-PX activity was inhibited after caffeine exposure; SOD activity was unchanged as compared with the control. In summary, our results suggest that caffeine exposure could negatively impact on angiogenesis in the chick yolk sac and CAM by targeting angiogenesis-related genes. Some of these genes are also involved in regulating excess ROS generation. The results implied that the negative impact of caffeine on fetal development was partly attributed to impaired placental angiogenesis.

Changes in mouse liver and chicken embryo yolk sac membrane soluble proteins due to an organophosphorous insecticide (OPI) diazinon linked to several noncholinergic OPI effects in mice and chicken embryos

The objective of this study was to identify proteins in mouse livers and chicken embryo yolk sac membranes whose quantities were altered by an organophosphorous insecticide (OPI) treatment and which might be linked, based on their functionality, to the recognized noncholinergic effects of OPI. Mice and fertile chicken eggs were treated with an OPI representative diazinon. The quantitative changes in mouse liver and chicken embryo yolk sac membrane soluble proteins caused by diazinon were determined by two-dimensional electrophoresis. Proteins whose quantity was affected by diazinon were identified by the mass spectrometry. In mouse livers, the altered levels of several enzymes of glucose metabolism were considered with regards to amelioration of hyperglycemia due to diazinon; the reduced levels of 3-hydroxyanthranilate 3,4-dioxygenase to the changes in the l-tryptophan to NAD metabolism caused by pyrimidinyl and crotonamide OPI; the reduced levels of catalase, peroxiredoxin and superoxide dismutase to OPI-increased lipid and/or kynurenine oxidation, the latter effect resulting also in increased urinary excretion of xanthurenic and kynurenic acids; and an increase in glutathione S-methyltransferase to OPI detoxification. In chicken embryo yolk sac membranes, the reduced availability of procollagen-proline dioxygenase may be the factor in micromelia caused by OPI in chicken embryos.

Structural requirements of organophosphorus insecticides (OPI) to inhibit chicken yolk sac membrane kynurenine formamidase related to OPI teratogenesis

This paper provides new information related to the mechanism of OPI (organophosphorus insecticides) teratogenesis. The COMFA (comparative molecular field analysis) and COMSIA (comparative molecular similarity indices analysis) suggest that the electrostatic and steric fields are the best predictors of OPI structural requirements to inhibit in ovo chicken embryo yolk sac membrane kynurenine formamidase, the proposed target for OPI teratogens. The dominant electrostatic interactions are localized at nitrogen-1, nitrogen-3, nitrogen of 2-amino substituent of the pyrimidinyl of pyrimidinyl phosphorothioates, and the oxygen of crotonamide carbonyl in crotonamide phosphates. Bulkiness of the substituents at carbon-2 and carbon-6 of the pyrimidinyls and/or N-substituents and carbon-3 substituents of crotonamides are the steric structural components that contribute to superiority of those OPI as in ovo inhibitors of kynurenine formamidase.

Yolk sac carbohydrate levels and gene expression of key gluconeogenic and glycogenic enzymes during chick embryonic development

Glycogen and glucose concentrations (mg/g of tissue) and amounts (mg) were determined in the yolks of fertile eggs on the day of set and in the yolk sac (YS) and liver of broiler chick embryos between 11 and 21 embryonic days of age (E). On the day of set, the yolk contained 50 mg of glucose (0.31% of yolk) but did not contain glycogen. During incubation, the amount of glucose in the YS increased from 20 mg on E11 to 60 mg on E19. A parallel increase in YS and liver glycogen concentrations (mg/g) during the last week of incubation implied a similar capacity for glycogen synthesis per gram of tissue. However, due to its larger size, the YS capacity for glycogen storage far exceeded that of the liver, which stored less than 12 mg of glycogen up to E19, as compared with more than 200 mg in the YS. Between E19 and 21, liver and YS glycogen amounts decreased by 10 mg and 100 mg, respectively. These results indicated that the YS is a glycogenic and perhaps gluconeogenic organ. We therefore evaluated the gene expression of glycogen synthase and glycogen phosphorylase as well as gluconeogenic enzymes (fructose 1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase) in the YS membrane and liver by real-time reverse-transcription PCR. Although the YS membrane and liver displayed different patterns of mRNA abundance, the high abundance of fructose 1,6-bisphosphatase mRNA in the YS membrane between E11 and 15, and the expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase, supported the postulated gluconeogenic abilities of the YS membrane and indicated its role in providing glucose to the embryo. Thus, glucose is probably synthesized in the YS, stored in the form of glycogen, and toward hatch, the YS may have the potential to transfer 10 times more glycogen-derived glucose to the embryo as compared with the liver. As such, the YS may play a major role in the synthesis and storage of glucose and its supply to the chick embryo toward hatch.

Pulsatile shear and Gja5 modulate arterial identity and remodeling events during flow-driven arteriogenesis

In the developing chicken embryo yolk sac vasculature, the expression of arterial identity genes requires arterial hemodynamic conditions. We hypothesize that arterial flow must provide a unique signal that is relevant for supporting arterial identity gene expression and is absent in veins. We analyzed factors related to flow, pressure and oxygenation in the chicken embryo vitelline vasculature in vivo. The best discrimination between arteries and veins was obtained by calculating the maximal pulsatile increase in shear rate relative to the time-averaged shear rate in the same vessel: the relative pulse slope index (RPSI). RPSI was significantly higher in arteries than veins. Arterial endothelial cells exposed to pulsatile shear in vitro augmented arterial marker expression as compared with exposure to constant shear. The expression of Gja5 correlated with arterial flow patterns: the redistribution of arterial flow provoked by vitelline artery ligation resulted in flow-driven collateral arterial network formation and was associated with increased expression of Gja5. In situ hybridization in normal and ligation embryos confirmed that Gja5 expression is confined to arteries and regulated by flow. In mice, Gja5 (connexin 40) was also expressed in arteries. In the adult, increased flow drives arteriogenesis and the formation of collateral arterial networks in peripheral occlusive diseases. Genetic ablation of Gja5 function in mice resulted in reduced arteriogenesis in two occlusion models. We conclude that pulsatile shear patterns may be central for supporting arterial identity, and that arterial Gja5 expression plays a functional role in flow-driven arteriogenesis.

Origins of Growth Factors: NGF and EGF

Growing up on the streets of Brooklyn, I remember being interested in how things worked: taking apart an old telephone and the gears of my new 4-speed bicycle was most enjoyable. As a biology major at Brooklyn College in 1945, I was fascinated by embryology. How does an egg turn into a chicken or a frog or a person? My only insight into the problem was the thought that it was necessary to understand the chemical reactions inside the egg and embryo and not simply observe biological structures. I, therefore, became a double major in chemistry and biology (also with the hope of earning a living as a laboratory technician). My first job after graduating was working nights as a bacteriologist in a milk processing plant. One of my professors at Brooklyn College wrote asking whether I was interested in going to graduate school. The college had been sending one student a year to the Biology Department at Oberlin to work as a laboratory assistant while studying for a master's degree. Because Oberlin offered to pay my tuition plus some $300 a semester for living expenses, I jumped at the chance. School and science were both interesting and fun. Upon graduating, I continued my education toward a Ph.D. working as an assistant in the Biochemistry Department of the University of Michigan under Howard B. Lewis. My research involved studying the Krebs urea cycle in the common earthworms that I collected from the university campus. I had been told at Oberlin that the yellow cells surrounding the gut in the worm corresponded to the mammalian liver. I decided to check whether the enzymatic reactions in worms were similar to those in mammals. They were (1). My first “real job” was in the Pediatrics Department at the University of Colorado studying creatine metabolism in premature infants under Harry Gordon. I think he hired me because he was impressed by my ability to stomach tube earthworms. After several years, I decided I needed to learn the then new technique of using radioisotopes in metabolic studies, and I obtained an American Cancer Society fellowship to work in the Radiology Department of Washington University under Martin Kamen where I combined this new knowledge with my interest in embryology. Although fertilized frog eggs and early embryos are impermeable to small molecules (amino acids, phosphates, etc.), sufficient amounts of C14O2 were metabolized by intact eggs and embryos to permit the identification of the radioactive compounds present at various stages of development (2). Upon completion of my fellowship, Dr. Kamen recommended me for a position in the Zoology Department of Washington University in the laboratory of Viktor Hamburger and Rita Levi-Montalcini. This move was critical in determining the direction of my research for the next 40-some odd years: the isolation, structure, and function of the first of the “growth factors,” nerve growth factor (NGF) and epidermal growth factor (EGF).

Methotrexate Induced Gross Malformations in Chick Embryos

Methotrexate is a drug that is used to treat rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome, acute lymphoblastic leukemia, ectopic pregnancy and other conditions. Since methotrexate is well known for its teratogenic effects in humans, the present study is conducted to observe its effect on chick embryos. A single injection of 0.012 mg of methotrexate was injected into the yolk sac of chick embryos on 5th day of incubation. The eggs were sacrificed on 19 th day of incubation to see for any gross malformations. The spectrum of gross malformations recorded were stunted growth, scanty feathers, beak deformities, short wings and ectopia viscerale. These findings support the malformations caused by the teratogenic drug methotrexate.

Even Flow

The appreciation that biomechanical forces induce signaling responses that regulate vascular tone and inflammatory responses has had a paradigm-shifting effect on our understanding of vascular biology. Endothelial cells are constantly exposed to rheologically imposed forces that vary due to vascular anatomic features and hemodynamic parameters, and of course shear fluctuates cyclically during the cardiac cycle. Simple laminar flow and high shear forces elicit a cellular response characterized by quiescence and alignment of endothelial cells in the direction of fluid flow, whereas disturbed flow and low shear forces induce an inflammatory procoagulant phenotype. These cellular effects are mediated through well-defined signaling pathways and gene expression responses. The cellular sensors or receptors for shear forces are incompletely characterized, but detection of stress seems to depend on the transmission of forces to points of cell–matrix and cell–cell attachment and requires specific conformational activation of integrins.1 From a pathophysiological perspective, disturbed laminar flow has been linked to inflammatory responses that initiate atheroma formation and elicit plaque instability. It is also generally accepted that flow is required for some aspects of vascular remodeling during vascular development and in adult angiogenic responses; however, specific roles for flow in vascular developmental programs have been incompletely characterized. See page 1817 Two recent reports, including one published in this issue of Arteriosclerosis, Thrombosis, and …

Serological Study of Experimental and Natural Q Fever in Cattle and Sheep

ABSTRACTThe objective of the work has been to investigate the serological response in cattle and sheep against Coxiella burnetii after experimental or natural infection. Two cows and four sheep were intravenously inoculated with 1st phase C. burnetii isolated from sheep in yolk sacs of chicken embryos. The serological examination was carried out t in a complement fixation test (CFT) with a 1st—and 2nd phase. Henserling strain antigens. It was found that CF-antibodies in 2ndphase appeared in experimentally infected cows or and sheep in 1–2 weeks after the infection. The maximal titers were found between 4th and 6th weeks. The titers in low levels were found up to 18 month (observation term). The 1st phase antibodies appeared considerably later (5-7th week) and the maximum titers were established in 3rd or 4th month. In sera of naturally infected cattle and sheep 1st phase antibodies were found in low titers up to one and a half-two years, and 2nd phase antibodies in low titers kept themselves to five years.

Flow regulates arterial-venous differentiation in the chick embryo yolk sac.

Formation of the yolk sac vascular system and its connection to the embryonic circulation is crucial for embryo survival in both mammals and birds. Most mice with mutations in genes involved in vascular development die because of a failure to establish this circulatory loop. Surprisingly, formation of yolk sac arteries and veins has not been well described in the recent literature. Using time-lapse video-microscopy, we have studied arterial-venous differentiation in the yolk sac of chick embryos. Immediately after the onset of perfusion, the yolk sac exhibits a posterior arterial and an anterior venous pole, which are connected to each other by cis-cis endothelial interactions. To form the paired and interlaced arterial-venous pattern characteristic of mature yolk sac vessels, small caliber vessels of the arterial domain are selectively disconnected from the growing arterial tree and subsequently reconnected to the venous system, implying that endothelial plasticity is needed to fashion normal growth of veins. Arterial-venous differentiation and patterning are controlled by hemodynamic forces, as shown by flow manipulation and in situ hybridization with arterial markers ephrinB2 and neuropilin 1, which show that expression of both mRNAs is not genetically determined but plastic and regulated by flow. In vivo application of ephrinB2 or EphB4 in the developing yolk sac failed to produce any morphological effects. By contrast, ephrinB2 and EphB4 application in the allantois of older embryos resulted in the rapid formation of arterial-venous shunts. In conclusion, we show that flow shapes the global patterning of the arterial tree and regulates the activation of the arterial markers ephrinB2 and neuropilin 1.

Typhus vaccine developments from the First to the Second World War (on Paul Weindling's 'between bacteriology and virology...').

After the louse transmission of epidemic typhus had been established (1909), a small microorganism (thought to belong to a new genus, Rickettsia) was shown in enormous numbers in the guts of lice that had fed on human typhus victims. Attempts at cultivating this organism on inert media failed; transfer from louse to louse without loss of virulence for the vertebrate host was successful. Some scientists were not convinced of the etiologic role of Rickettsiae, because the presence of this microbe in blood and organs of victims or of experimentally infected animals was difficult to demonstrate. This uncertainty was dispelled in 1928, when in guinea pigs infected with material from the closely related disease Tabardillo (murine typhus) abundant Rickettsiae were revealed in the tunica vaginalis. Live vaccines, derived from strains of murine typhus and deployed in French North Africa, were considered by outside observers as unsafe. Killed vaccines were derived from the masses of Rickettsiae present in louse guts, in chick embryo yolk sacs or in vertebrate lungs. These developments were not spurned by any 'upswing of virology' but by the threat of typhus in endemic areas and, after 1938, in a war-torn world. Their basis was firmly anchored in bacteriological thought styles and techniques.

Cross-reactivity between Coxiella burnetii and chlamydiae.

A cross-reactivity among some strains of Coxiella burnetii and chlamydiae with immune rabbit and mouse sera in ELISA and immunoblot analysis was observed. In the latter, the cross-reactivity disappeared after a treatment of C. burnetii or C. psittaci with proteinase K, which indicates that only proteins were involved. The observed cross-reactivity was not influenced by host chick embryo yolk sac proteins. After adsorption of immune rabbit sera with homologous corpuscular antigens the cross-reactivity disappeared. The possibility of influence of such cross-reactivity on serological diagnosis of C. burnetii or chlamydiae infections is discussed.

Carbamazepine‐Induced Brain Weight Reduction in Chick Embryos

Abstract The antiepileptic drug carbamazepine (CBZ) had been considered to be relatively safe for the fetus. We examined the teratogenicity of CBZ using chick embryos, with particular focus on the brain. A single dose of 5 mg was injected into the yolk sac of White Leghorn chick embryos at 24, 72, 120, 168 or 216 hours of incubation. On day 19 of incubation, embryos were removed, and counted and recorded as either live or dead. Each live embryo was inspected for the presence of external abnormalities, and body and brain weight were measured. There was no statistically significant difference between treated groups and control groups at all hours of incubation for mean body weight and incidence of embryo death. Mean brain weight was significantly lighter in treated groups at 120 and 168 hours of incubation. A significant difference in mean brain/body weight ratio was observed only at 120 hours of incubation. External abnormalities in treated embryos were not found with any consistency and these incidences were not significantly higher compared to those in the control groups. This experiment provides evidence regarding the risk of teratogenesis due to CBZ in the brain of chick embryo. The 120 hours of incubation in chick embryos corresponds to the 34‐36th day after conception in humans.