Expression of VE-cadherin in zebrafish embryos: a new tool to evaluate vascular development.

We have identified the zebrafish homologue of VE-cadherin and documented its expression in the developing vascular system. The zebrafish VE-cadherin gene is specifically expressed in the vascular endothelial cell lineage beginning with the differentiation and migration of angioblasts and persists throughout vasculogenesis, angiogenesis, and endocardium development. Staining zebrafish embryos by whole-mount in situ hybridization with the VE-cadherin probe provides a method to screen embryos for vascular defects. To illustrate this utility, we used VE-cadherin expression to demonstrate a conservation of vascular endothelial growth factor-A (VEGF-A) function. The morpholino antisense oligonucleotide knockdown of VEGF-A function in zebrafish embryos results in a loss of angiogenic blood vessels, as indicated by the lack of VE-cadherin expression in the intersegmental vasculature. This loss can be restored in embryos supplemented with either zebrafish or human VEGF-A, the latter indicating that genes crucial to angiogenesis have highly conserved functional activities in vertebrates.     

Pathobiology of familial hypercholesterolemic atherosclerosis

Many factors play a role in the development of atherosclerotic lesions. One of the leading risk factors for development of atherosclerosis is familial hypercholesterolemia (FH). FH is a genetic disease characterized by a deficiency, and/or mutation, of receptors for low density lipoprotein (LDL) on the plasmalemma of endothelial cells (EC), a high level of low density lipoprotein in the plasma, and early, spontaneous development of atherosclerosis and skin xanthoma. In this review we describe Watanabe heritable hyperlipidemic (WHHL) rabbits, which represent such an animal model for human FH. This strain of the rabbits is characterized by a genetic deficiency or mutation of functional LDL receptors and develops severe atherosclerosis, which is pathologically similar to familial homozygous hyperlipidemic patients. The most completely characterized animal model is the Watanabe rabbit, a model of homozygous and heterozygous type IIa hypercholesterolemia related to an LDL receptor deficiency. Additional manipulation such as aortic injury in this rabbit model induces the development of atherosclerotic lesions that are structurally similar to those found in humans. Thus, this model of hypercholesterolemia fulfils the above criteria set, i.e. it is able to provide new insights for a better understanding of the pathogenesis of atherosclerosis and for testing new treatment strategies.     

Trypanosoma cruzi antigen that interacts with the beta1-adrenergic receptor and modifies myocardial contractile activity

Previously, we have demonstrated that plasma membranes from the parasite Trypanosoma cruzi (T. cruzi) recognize and adhere to host cells through parasite surface attachment molecules that have affinity for beta(1)-adrenergic receptors (beta(1)-ARs) on target organs. In this report we identify a parasite protein that not only interacts with beta(1)-ARs, but also displays beta-agonist-like activity. We demonstrate that a recombinant maltose binding protein fusion of Tc13 Tul (MBP-Tc13 Tul), a member of the T. cruzi antigen 13 family of surface antigen proteins, competes for binding sites with the beta-adrenergic receptor antagonist [125I]-CYP on membranes purified both from CHO cells expressing human beta(1)-ARs and from rat atria. The competition is prevented by pre-treating MBP-Tc13 Tul with antibodies directed against the EPKSA repeat domain of Tc13 Tul, implicating this portion of the molecule in binding to the beta(1)-AR. Furthermore, MBP-Tc13 Tul activates rat myocardial beta(1)-ARs, resulting in synthesis of cyclic adenosine monophosphate (cAMP) and an increase in cardiac contractility. These biological effects are selectively suppressed by the beta(1)-AR antagonist atenolol, by a synthetic peptide corresponding to the second extracellular loop of the human beta(1)-AR, and by the anti-EPKSA repeat antibodies. These results imply that the Tc13 Tul cell-surface antigen of T. cruzi plays a central role in misregulating the beta(1)-AR following parasite infection, and may be a causative factor of dysautonomic syndrome described in Chagas' disease.     

A quantitative approach to the design of antitumor drug dosage schedule via cell cycle kinetics and systems theory

A discrete-time kinetic model for chemotherapy was developed to deal with the effects of antitumor drugs on the cell cycle and proliferation kinetics of experimental tumor cell populations in which cell kinetic responses of chemotherapy are represented in terms of perturbation of cell kinetic parameters—cell age, cell size and DNA content distributions. The time-course behavior of these cell kinetic parameters was predicted by solving the discrete-time state equations which characterize the dynamics of tumor-drug interactions. The amount of antitumor drug administered was expressed to be the control function of the state equations and the transition matrix representing two modes of drug action, namely, cell kill and progression delay or accumulation of cells due to drug, was derived. The performance of the model, assessed by examining the effects of cell cycle stage-specific agents such as cytosine arabinoside on spontaneous AKR leukemia, compared favorably with experimental data. Utilizing an optimization scheme in engineering systems studies, an analytical method is described for optimizing the regimen of drug administration so as to maximize the effectiveness of drug dosage schedules and minimize the use of toxic amounts of the drug. The superiority of the schedule designed by an optimization scheme was evident at the termination of therapy, although the schedule designed by experimental trials reduced the number of surviving tumor cells more effectively than the one designed by an optimization scheme during the earlier therapy period. In the model, the proposed schedule will function more effectively for the entire therapy period when additional parameters of drug characteristics, such as the toxicity to the host and drug resistance, are encompassed.     

Coxsackievirus infection of the pancreas: evaluation of receptor expression, pathogenesis, and immunopathology

Coxsackievirus type B (CVB) infection of the pancreas induces a massive cellular infiltrate composed of natural killer cells, T cells, and macrophages and leads to the destruction of exocrine tissue. The physiological manifestations of pancreatic CVB infection are correlated with viral tropism; the virus infects acinar cells but spares the islets of Langerhans. Here we evaluate the mechanisms underlying pancreatic inflammation and destruction and identify the determinants of viral tropism. T-cell-mediated immunopathology has been invoked, along with direct virus-mediated cytopathicity, to explain certain aspects of CVB-induced pancreatic disease. However, we show here that in the pancreas, the extent of inflammation and tissue destruction appears unaltered in the absence of the cytolytic protein perforin; these findings exclude any requirement for perforin-mediated lysis by natural killer cells or cytotoxic T cells in CVB3-induced pancreatic damage. Furthermore, perforin-mediated cytotoxic T-cell activity does not contribute to the control of CVB infection in this organ. In addition, we demonstrate that the recently identified coxsackie-adenovirus receptor is expressed at high levels in acinar cells but is barely detectable in islets, which is consistent with its being a major determinant of virus tropism and, therefore, of disease. However, further studies using various cell lines of pancreatic origin reveal secondary determinants of virus tropism.     

Capsaicin-sensitive afferents are involved in signalling transneuronal effects between cutaneous sensory nerves

The aim of the present study was to investigate changes in contralateral nerves associated with peripheral nerve injuries. Transection and subsequent regeneration of the saphenous nerve on one side caused a suppression of the ability of the contralateral saphenous nerve to produce a neurogenic plasma extravasation response. This effect was transient, and was first evident two weeks after injury, reaching its maximum at four weeks, but was no longer detectable at eight weeks. This change was paralleled by a decrease in the content of substance P, a neuropeptide involved in neurogenic plasma extravasation, in the contralateral nerve. The neurotoxin capsaicin was used to deplete the nerve of a subclass of C-fibres, namely the polymodal nociceptor afferents. Pretreatment of the nerve to be lesioned with capsaicin was sufficient to significantly attenuate the changes in the plasma extravasation response and substance P content observed on the contralateral side. The effectiveness of the capsaicin treatment was confirmed by histological examination. These results strongly suggest that changes observed at a site distant from the location of the nerve injury are dependent on the integrity of capsaicin-sensitive C-fibre afferents within the injured nerve. Furthermore, given that the contralateral nerve has commonly been used as the control for an injury conducted on the homologous nerve or muscle on the opposite side of the body, the underlying assumption being that the contralateral nerve remained unchanged, the present findings emphasize the need for separate groups of control animals which have undergone no surgical procedures.     

The ganglion cell response to optic nerve injury in the cat: differential responses revealed by neurofibrillar staining

Summary The early responses of cat retinal ganglion cells to axotomy have been examined using neurofibrillar and Nissl-stained wholemounts. We were interested to learn whether the enhanced neurofilament expression, seen in a number of neuronal systems, was also present in different neuronal populations of the cat retina and could be used to study the distribution of these cells. We found that beta ganglion cells degenerate very rapidly after axotomy with the nuclei becoming pyknotic within a few days. Few beta cells showed increased neurofibrillar staining of the dendrites. The cell body degenerated prior to any visible degenerative changes in the axon. A proportion of the alpha and gamma ganglion cells degenerated in the first two to three weeks after axotomy. The alpha cells underwent markedly enhanced neurofibrillar staining of their dendrites prior to degeneration. The Nissl material of the cell bodies diminished as the cells degenerated but we have not observed pyknotic nuclei. The dendritic trees of some axotomised gamma cells were also revealed by the neurofibrillar stain three weeks after axotomy. These results show that retinal ganglion cells do not degenerate by a dying back process. We suggest that the rapid degeneration of the beta ganglion cell population comes about by excitotoxic cell death, a consequence of their large glutamatergic input from bipolar cells. The degenerating beta ganglion cells have the morphological appearance of cells undergoing apoptosis.     

Quantitative assays of pharmacologic aerosols used in studying asthma

To more nearly accurately quantitate the dose of pharmacologic agents delivered to human and animal airways via aerosols, we have developed a monodisperse aerosol containing either methacholine or histamine that permits a light scattering device (tyndallometry) to measure accurately the quantity of inspired and expired particles. These aerosols (described in previous studies) are simultaneously tagged with a radioactive label (technetium 99m) to permit the use of external gamma camera imaging. Present work focuses on the development of assay techniques to measure the quantity of methacholine delivered in these aerosols. The lack of specific radioimmune or radioenzyme assays coupled with the cross-reaction of organic contaminants with conventional chemical reagents for measuring methacholine required the development of separative techniques to isolate the methacholine from the organic aerosol contaminants. With aqueous extraction and column separation we have been able to completely isolate the methacholine from these contaminants. This allows the application of standard spectrophotometric assays for methacholine to quantitate the methacholine in the resulting solution. These separative techniques will permit the use of these aerosols in quantitative studies of airway reactivity.     

Distribution of the serotonin 2C (5HT2C) receptor gene -759C/T polymorphism in patients with schizophrenia and normal controls

BACKGROUND: In patients with schizophrenia, the percentage of patients with diabetes has been found to be twice that of the normal population. The risk factors for this higher rate are unknown, although dietary, lifestyle, and genetic factors have all been suggested. Recently, a polymorphism (-759C/T) in the serotonin 2C (5HT2C) receptor promoter region has been associated with the development of diabetes in a normal control population, with the frequency of the T allele being higher in subjects without diabetes. AIM: To determine whether the distribution of the -759C/T polymorphism of the 5HT2C receptor is different among patients with schizophrenia and normal controls. METHODS: DNA from 100 patients with schizophrenia and 81 normal controls were analyzed for the 5HT2C receptor -759C/T polymorphism to determine its allelic frequencies in these two groups. RESULTS: Using a chi-squared analysis, no statistical differences in the distribution of C alleles and T alleles were found between the two groups (P=0.2931). CONCLUSIONS: Patients with schizophrenia have a higher risk for developing diabetes than the general population. We did not find a higher distribution of the -759T allele of the 5HT2C receptor in normal controls compared with in patients with schizophrenia. This suggests the higher prevalence of diabetes in schizophrenia is not due to this polymorphism.