CPS49-induced neurotoxicity does not cause limb patterning anomalies in developing chicken embryos.

Thalidomide notoriously caused severe birth defects, particularly to the limbs, in those exposed in utero following maternal use of the drug to treat morning sickness. How the drug caused these birth defects remains unclear. Many theories have been proposed including actions on the forming blood vessels. However, thalidomide survivors also have altered nerve patterns and the drug is known for its neurotoxic actions in adults following prolonged use. We have previously shown that CPS49, an anti-angiogenic analog of thalidomide, causes a range of limb malformations in a time-sensitive manner in chicken embryos. Here we investigated whether CPS49 also is neurotoxic and whether effects on nerve development impact upon limb development. We found that CPS49 is neurotoxic, just like thalidomide, and can cause some neuronal loss late developing chicken limbs, but only when the limb is already innervated. However, CPS49 exposure does not cause defects in limb size when added to late developing chicken limbs. In contrast, in early limb buds which are not innervated, CPS49 exposure affects limb area significantly. To investigate in more detail the role of neurotoxicity and its impact on chicken limb development we inhibited nerve innervation at a range of developmental timepoints through using ß-bungarotoxin. We found that neuronal inhibition or ablation before, during or after limb outgrowth and innervation does not result in obvious limb cartilage patterning or number changes. We conclude that while CPS49 is neurotoxic, given the late innervation of the developing limb, and that neuronal inhibition/ablation throughout limb development does not cause similar limb patterning anomalies to those seen in thalidomide survivors, nerve defects are not the primary underlying cause of the severe limb patterning defects induced by CPS49/thalidomide.

Pomalidomide is nonteratogenic in chicken and zebrafish embryos and nonneurotoxic in vitro.

Thalidomide and its analog, Lenalidomide, are in current use clinically for treatment of multiple myeloma, complications of leprosy and cancers. An additional analog, Pomalidomide, has recently been licensed for treatment of multiple myeloma, and is purported to be clinically more potent than either Thalidomide or Lenalidomide. Using a combination of zebrafish and chicken embryos together with in vitro assays we have determined the relative anti-inflammatory activity of each compound. We demonstrate that in vivo embryonic assays Pomalidomide is a significantly more potent anti-inflammatory agent than either Thalidomide or Lenalidomide. We tested the effect of Pomalidomide and Lenalidomide on angiogenesis, teratogenesis, and neurite outgrowth, known detrimental effects of Thalidomide. We found that Pomalidomide, displays a high degree of cell specificity, and has no detectable teratogenic, antiangiogenic or neurotoxic effects at potent anti-inflammatory concentrations. This is in marked contrast to Thalidomide and Lenalidomide, which had detrimental effects on blood vessels, nerves, and embryonic development at anti-inflammatory concentrations. This work has implications for Pomalidomide as a treatment for conditions Thalidomide and Lenalidomide treat currently.

Molecular analysis of regulative events in the developing chick limb.

The developing chick limb has the remarkable ability to regulate for the loss of large amounts of mesenchyme and maintain a normal limb pattern in early (Hamburger and Hamilton Stage 19; E3) limbs. How the limb can regulate for tissue loss and why this ability is lost as development proceeds (after Hamburger and Hamilton Stage 21; E3.5) is unclear. We have investigated the origins of cells involved in regulative processes and, for the first time, the molecular changes occurring, and find striking differences between developmental time points just 0.5 days apart. We demonstrate that subtle changes in cell dispersal and cell proliferation occur in HH St21 limbs but not in HH St19 limbs and also demonstrate that there is no net replacement of removed tissue at either HH St21 or St19. We further show that changes in the Fgf8/Shh/Bmp4/Gremlin signaling pathway together with the appearance of distal Hox gene activation coincide with the limbs' ability to regulate for large tissue loss. We also demonstrate that following small tissue loss, limbs can regulate for missing tissue to produce normal pattern with no net replacement of missing tissue, as seen in limbs following large tissue loss. Our results indicate the regulative ability of the limb is not due to changes in cell proliferation, cell lineage nor replacement of the missing tissue - regulative ability is reliant upon the signaling environment remaining.

Shared mechanism of teratogenicity of anti-angiogenic drugs identified in the chicken embryo model.

Angiogenesis, the formation of new blood vessels, is essential for tumor growth, stabilization and progression. Angiogenesis inhibitors are now widely used in the clinic; however, there are relatively few published studies on the mechanism of their presumed teratogenic effects. To address this issue, we screened a variety of angiogenesis inhibitors in developing zebrafish and chicken embryo models to assess for developmental defects and potential teratogenic effects. We confirmed previous reports that sunitinib, sorafenib and TNP-470 are teratogenic and demonstrate that axitinib, pazopanib, vandetanib, and everolimus are also teratogens in these models. A dose response study identified the drugs inhibit HUVEC cell proliferation in vitro, and also target the developing blood vessels of embryos in vivo. This provides further evidence for the potential risk of fetal toxicity when using these drugs in a clinical setting, and emphasizes the importance of the development and maintenance of the vasculature in the embryo. We conclude that angiogenesis inhibitors, regardless of the molecular target, are teratogenic when exposed to chicken embryos.

Training techniques. Acting up.

The Relating to People programme aims to broaden the communication skills of NHS employees called up to face difficult behaviour. Typically divided into four modules, the programme can be tailored to meet your needs. The programme is dominated by demonstrations of good and bad ways of facing anxious situations.

Anticancer Properties of a Novel Class of Tetrafluorinated Thalidomide Analogues.

Thalidomide has demonstrated clinical activity in various malignancies affecting immunomodulatory and angiogenic pathways. The development of novel thalidomide analogs with improved efficacy and decreased toxicity is an ongoing research effort. We recently designed and synthesized a new class of compounds, consisting of both tetrafluorinated thalidomide analogues (Gu973 and Gu998) and tetrafluorobenzamides (Gu1029 and Gu992). In this study, we demonstrate the antiangiogenic properties of these newly synthesized compounds. We examined the specific antiangiogenic characteristics in vitro using rat aortic rings with carboxyamidotriazole as a positive control. In addition, further in vitro efficacy was evaluated using human umbilical vein endothelial cells (HUVEC) and PC3 cells treated with 5 and 10 µmol/L doses of each compound. All compounds were seen to reduce microvessel outgrowth in rat aortic rings as well as to inhibit HUVECs to a greater extent, at lower concentrations than previously tested thalidomide analogs. The antiangiogenic properties of the compounds were also examined in vivo in fli1:EGFP zebrafish embryos, where all compounds were seen to inhibit the extent of outgrowth of newly developing blood vessels. In addition, Gu1029 and Gu973 reduced the anti-inflammatory response in mpo:GFP zebrafish embryos, whereas Gu998 and Gu992 showed no difference. The compounds' antitumor effects were also explored in vivo using the human prostate cancer PC3 xenograft model. All four compounds were also screened in vivo in chicken embryos to investigate their teratogenic potential. This study establishes these novel thalidomide analogues as a promising immunomodulatory class with anticancer effects that warrant further development to characterize their mechanisms of action.