Anti-angiogenic effects and regression of localized murine AML produced by anti-VEGF and anti-Flk-1 antibodies

Reducing the blood supply of tumors is one modality to combat cancer. The objective of this study was to evaluate such an approach in the treatment of localized murine AML (acute myelogenous leukemia). For this purpose we designed an experimental model in which leukemic cells were embedded in 1% agar discs before subcutaneous implantation in C57Bl female mice. The C-1498 AML cell line (Frederick Inst., NCI, MD, USA) was used. Thirty experimental mice received on alternate days injections of 5 x 2.5 microg anti-VEGF (vascular endothelial growth factor) and 5 x 2.5 microg anti-Flk-1 (VEGFR2) antibodies to the site of cell implantation over a period of 10 d. Fifteen control mice received daily PBS injections. All mice were sacrificed 16 d after AML implantation. Of the 30 experimental animals, macroscopic examination showed in 21 animals (70%) small sized, pale tumors (0.5 g); in six mice (20%) the tumors were replaced completely by necrotic tissue, while in three mice (10%), there were large (2.5 g), highly vascularized tumors. In all 15 control mice large highly vascularized tumors were seen. A separate group of mice was studied for total survival following AML implantation. While 12 mice in the control group not treated with antibodies survived for 16 d post-implantation, survival was prolonged in 15 antibody treated mice by approximate 30 d to a total survival time of 48 d. Tumor specimens were processed for histology, immunohistochemistry (IHC) for CD31 endothelial cell antigen, and tube-like formation assay. The small, pale tumors of antibody treated animals consisted of degenerate hyaline material with remnant nests of leukemic cells, whereas large tumors showed sheets of leukemic cells and numerous blood vessels. Specimens processed for CD31 antigen showed scarce or absence of blood vessels in the small, pale tumors in contrast to intensive staining from a rich network of blood vessels in the large, highly vascularized tumors. Tube-like formation assays disclosed rudimentary Grade 1 endothelial cell tubes in the small, pale tumors as opposed to polygonal Grade 4 tube formation in control animals. In conclusion, this murine model of localized AML allows assessment of anti-angiogenic tumor regression. Anti-angiogenic antibodies against VEGF and Flk-1 have therapeutic effects in murine AML.     

Sprouting in intact sartorius muscles of the frog following contralateral axotomy

The intact hind limb sartorius muscle of the frog was examined anatomically for sprouting following axotomy of the sciatic nerve that contains motor axons innervating the contralateral sartorius muscle. The incidence of sprouting in intact muscles of experimental animals increased more than 3-fold over normal. Thus, contralateral axotomy produces sprouting in intact sartorius muscles much the same as it does in cutaneous-pectoris and piriformis muscles of the frog.     

A tripartite organization of the urbilaterian brain: developmental genetic evidence from Drosophila

Developmental genetic studies suggest that the embryonic vertebrate brain has a tripartite ground plan consisting of a forebrain/midbrain, a hindbrain, and an intervening midbrain/hindbrain boundary region, which are characterized by the specific expression of the Otx, Hox and Pax-2/5/8 genes. Recent studies in Drosophila reveal similarities in the expression and function of these genes in patterning the embryonic brains of flies and vertebrates. Thus, in Drosophila, as is vertebrates, a Pax2/5/8 domain is located between an anterior otd/Otx2 region and a posterior Hox region of the embryonic brain. Moreover, in Drosophila, as in vertebrates, this Pax2/5/8 domain is located at the interface of the otd/Otx2 domain and a posterior unplugged/Gbx2 domain. Furthermore, in Drosophila, as in vertebrates, inactivation of otd/Otx2 or of unplugged/Gbx2 results in a comparable mispositioning or loss of orthologous gene expression domains in the embryonic brain. These developmental genetic similarities suggest that the tripartite ground plan, which characterizes the developing vertebrate brain, is also at the basis of the developing insect brain. This, in turn, implies that a tripartite organization of the embryonic brain may characterize all extant bilaterians, and thus may already have been established in the last common urbilaterian ancestor of all bilaterians.     

Effect of streptavidin-biotin on endothelial vasoregulation and leukocyte adhesion

The current study examines whether the adhesion promoting arginine-glycine-aspartate-streptavidin mutant (RGD-SA) also affects two important endothelial cell (EC) functions in vitro: vasoregulation and leukocyte adhesion. EC adherent to surfaces via fibronectin (Fn) or Fn plus RGD-SA were subjected to laminar shear flow and media samples were collected over a period of 4h to measure the concentration of nitric oxide (NO), prostacyclin (PGI(2)), and endothelin-1 (ET-1). Western blot analysis was used to quantify the levels of endothelial-derived nitric oxide synthase (eNOS) and cyclooxygenase II (COX II). In a separate set of experiments, fluorescent polymorphonuclear leukocyte (PMN) adhesion to EC was quantified for EC with and without exposure to flow preconditioning. When cell adhesion was supplemented with the SA-biotin system, flow-induced production of NO and PGI(2) increased significantly relative to cells adherent on Fn alone. Previous exposure of EC to shear flow also significantly decreased PMN attachment to SA-biotin supplemented EC, but only after 2h of exposure to shear flow. The observed decrease in PMN-EC adhesion was negated by NG-nitro-L-arginine methyl ester (L-NAME), an antagonist of NO synthesis, but not by indomethacin, an inhibitor to PGI(2) synthesis, indicating the induced effect of PMN-EC interaction is primarily NO-dependent. Results from this study suggest that the use of SA-biotin to supplement EC adhesion encourages vasodilation and PMN adhesion in vitro under physiological shear-stress conditions. We postulate that the presence of SA-biotin more efficiently transmits the shear-stress signal and amplifies the downstream events including the NO and PGI(2) release and leukocyte-EC inhibition. These results may have ramifications for reducing thrombus-induced vascular graft failure.     

Linkage analysis for autism in a subset families with obsessive-compulsive behaviors: evidence for an autism susceptibility gene on chromosome 1 and further support for susceptibility genes on chromosome 6 and 19

Although there is considerable evidence for a strong genetic component to idiopathic autism, several genome-wide screens for susceptibility genes have been carried out with limited concordance of linked loci, reflecting numerous genes of weak effect and/or sample heterogeneity. In the current study, linkage analysis was carried out in a sample of 62 autism-affected relative pairs with more severe obsessive-compulsive behaviors, selected from a larger (n=115) set of autism-affected relative pairs as a means of reducing sample heterogeneity. Obsessive-compulsive behaviors were assessed using the Autism Diagnostic Interview-Revised (ADI-R). In the sample with more severe obsessive-compulsive behaviors, multipoint NPL scores above 2 were observed on chromosomes 1, 4, 5, 6, 10, 11 and 19, with the strongest evidence for linkage on chromosome 1 at the marker D1S1656, where the multipoint NPL score was 3.06, and the two-point NPL score was 3.21. In follow-up analyses, analyzing the subset of families (n=35) where the patients had the most severe obsessive-compulsive behaviors generated a multipoint NPL score of 2.76, and a two-point NPL score of 2.79, indicating that the bulk of evidence for linkage was derived from the families most severely affected with obsessive-compulsive behaviors. The data suggest that there is an autism susceptibility gene on chromosome 1 and provide further support for the presence of autism susceptibility genes on chromosomes 6 and 19.     

Synergistic effect of high-affinity binding and flow preconditioning on endothelial cell adhesion

The current study examined whether the combined introduction of high-affinity avidin-biotin bonds and fibronectin-integrin bonds (i.e., dual ligand treatment) would further augment the adhesion of flow-preconditioned endothelial cells to model substrates via contributions to the actin cytoskeleton and the formation of focal contacts. Human umbilical vein endothelial cells (HUVEC) were grown under static conditions or exposed to a flow-preconditioning regimen for 24 h. Cell retention was determined by exposure to 75 dynes/cm(2). The combination of flow preconditioning and the dual ligand treatment yielded higher cell retention under flow compared to the cells adherent via fibronectin-integrin bonds only. This increase in adhesion strength correlated with a greater focal contact area. Elongation of the HUVEC occurred after exposure to flow preconditioning; however, orientation of dual ligand adherent cells was restricted due to the presence of the high-affinity ligand. Flow-preconditioned cells showed increased stress fiber formation compared to nonconditioned cells although the stress fibers per cell for flow-preconditioned cells were the same on both the ligand systems employed. The results indicate that enhanced adhesion strength is due to a combination of increased focal contact area, stress fiber formation, and cell alignment.