Somatic cell gene therapy for diabetes mellitus: engineering a surrogate B-cell

Improved methods of insulin delivery are required for the treatment of insulin-dependent diabetes mellitus (IDDM) to achieve a more physiological profile of glucose homeostasis. Somatic cell gene therapy offers the prospect that insulin could be delivered by an autologous cell implant, engineered to secrete insulin in response to glucose. This study explores the feasibility of manipulating somatic cells to behave as a surrogate insulin-secreting β-cells. Initial studies were conducted using mouse pituitary AtT20 cells as a model, since these cells possess an endogenous complement of enzymes capable of processing proinsulin to mature insulin. Glucose sensitive insulin secretion was conferred to these cells by transfection with plasmids containing the human preproinsulin gene (hppI-1) and the GLUT2 gene for the glucose transporter isoform 2. Insulin secretion was responsive to changes in the glucose concentration up to about 50μM. Further studies to up-rate this glucose sensitivity into the mM range will require manipulation of the hexokinase and glucokinase enzymes. Intraperitoneal implantation of the manipulated AtT20 cells into athymic nude mice with streptozotocin-induced diabetes resulted in decreased plasma glucose concentrations. The cells formed vascularised tumours in vivo which were shown to contain insulin-secreting cells. To achieve proinsulin processing in non-endocrine cells, co-transfection with a suitable enzyme, or mutagenesis of the proinsulin itself are necessary. The mutation of the human preproinsulin gene to the consensus sequence for cleavage by the subtilisin-like serine protease, furin, was carried out. Co-transfection of human fibroblasts with wild-type proinsulin and furin resulted in 58% conversion to mature insulin by these cells. Intraperitoneal implantation of the mature-insulin secreting human fibroblasts into the diabetic nude mouse animal model gave less encouraging results than the AtT20 cells, apparently due to poor vascularisation. Cell aggregations removed from the mice at autopsy were shown to contain insulin secreting cells only at the periphery. This thesis provides evidence that it is possible to construct, by cellular engineering, a glucose-sensitive insulin-secreting surrogate β-cell. Therefore, somatic cell gene therapy offers a feasible alternative for insulin delivery in IDDM patients.

Hypoxia induces dilated cardiomyopathy in the chick embryo:mechanism, intervention, and long-term consequences

Background - Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos. Methods - Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20) and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF) and its scavenger soluble VEGF receptor-1 (sFlt-1) to investigate the potential role of this hypoxia-regulated cytokine. Principal Findings - Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF165 to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype. Conclusions/Significance - Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.

Clonal composition of human adamantinomatous craniopharyngiomas and somatic mutation analyses of the patched (PTCH), Gsα and Gi2α genes

Craniopharyngioma is the most common childhood tumor and thought to arise from embryonic remnants of Rathke's pouch. The paucity of published data on the molecular basis of these tumors prompted us to examine 22 adamantinomatous craniopharyngiomas looking for genetic abnormalities. Using the X-linked polymorphic androgen receptor gene as a tool for X-chromosome inactivating analysis, we found that a subset of craniopharyngiomas are monoclonal and therefore are probably due to acquired somatic genetic defects. Thus, we investigated these tumours for mutations within three candidate genes, Gsα, Gi2α and patched (PTCH). Using single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis and direct sequencing, the presence of somatic mutations in these genes could not be demonstrated in any tumor. Our data indicate that a subset of craniopharyngiomas are monoclonal and the mutations in the PTCH, Gsα, and Gi2α contribute little if any to cranipharyngioma development.

Adaptive responses of the embryo to maternal diet and consequences for post-implantation development

Maternal periconceptional (PC) nutrition, coupled with maternal physiological condition, can impact on reproductive performance and potential across mammalian species. Oocyte quality and embryo development are affected adversely by either nutrient restriction or excess. Moreover, the quality of maternal PC nutrition can have lasting effects through fetal development and postnatally into adulthood. Chronic disease, notably cardiovascular and metabolic disease, and abnormal behaviour have been identified in adult offspring in small and large animal models of PC nutrient restriction. These long-term effects associate with compensatory responses that begin from the time of early embryo development. This review assesses the field of PC nutrition in vivo on short- and long-term developmental consequences in rodent and ruminant models and considers the implications for human health. © IETS 2012.

The preimplantation embryo:handle with care

The past decade has seen considerable advances in our understanding of intrinsic developmental mechanisms associated with gametogenesis and embryogenesis and accompanying applications in the fields of reproductive medicine, embryonic stem cell biology, and nuclear reprogramming. However, a new focus has recently emerged concerning the homeostatic regulation of embryonic cells, how this is set, and how it may influence the longitudinal progression and optimization of the developmental program and indeed the phenotype of the offspring. Attention has been drawn to the preimplantation stage of development as a sensitive "window" when in vitro and in vivo manipulations, such as culture conditions or maternal diet, may have critical consequences. In this article, we review how changes in environmental conditions, mediated via a range of epigenetic, cellular, and metabolic mechanisms in the preimplantation embryo, may alter the pattern of cell division, gene expression, morphology, and potential. We consider how fetal and postnatal phenotype may become susceptible to the plasticity of the preimplantation embryo and the risks for adult health and physiology. Copyright © 2008 by Thieme Medical Publishers, Inc.

Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure

A key factor in the use of assisted reproductive technologies (ART) for diverse species is the safety of procedures for long-term health. By using a mouse model, we have investigated the effect of in vitro culture and embryo transfer (ET) of superovulated embryos on postnatal growth and physiological activity compared with that of embryos developing in vivo. Embryo culture from two-cell to blastocyst stages in T6 medium either with or without a protein source reduced blastocyst trophectoderm and inner cell mass cell number compared with that of embryos developing in vivo. Embryo culture and ET had minimal effects on postnatal growth when compared with in vivo development with an equivalent litter size. However, embryo culture, and to a lesser extent ET, led to an enhanced systolic blood pressure at 21 weeks compared with in vivo development independent of litter size, maternal origin, or body weight. Moreover, activity of enzymatic regulators of cardiovascular and metabolic physiology, namely, serum angiotensin-converting enzyme and the gluconeogenesis controller, hepatic phosphoeno/pyruvate carboxykinase, were significantly elevated in response to embryo culture and/or ET in female offspring at 27 weeks, independent of maternal factors and postnatal growth. These animal data indicate that postnatal physiological criteria important in cardiovascular and metabolic health may be more sensitive to routine ART procedures than growth. © 2007 by The National Academy of Sciences of the USA.

The embryo and its future

The preimplantation mammalian embryo from different species appears sensitive to the environment in which it develops, either in vitro or in vivo, for example, in response to culture conditions or maternal diet. This sensitivity may lead to long-term alterations in the characteristics of fetal and/or postnatal growth and phenotype, which have implications for clinical health and biotechnological applications. We review the breadth of environmental influences that may affect early embryos and their responses to such conditions along epigenetic, metabolic, cellular, and physiological directions. In addition, we evaluate how embryo environmental responses may influence developmental potential and phenotype during later gestation. We conclude that a complex of different mechanisms may operate to associate early embryo environment with future health.

Psychophysiological responses to visceral and somatic pain in functional chest pain identify clinically relevant pain clusters

Background: Despite chronic pain being a feature of functional chest pain (FCP) its experience is variable. The factors responsible for this variability remain unresolved. We aimed to address these knowledge gaps, hypothesizing that the psychophysiological profiles of FCP patients will be distinct from healthy subjects. Methods: 20 Rome III defined FCP patients (nine males, mean age 38.7 years, range 28-59 years) and 20 healthy age-, sex-, and ethnicity-matched controls (nine males, mean 38.2 years, range 24-49) had anxiety, depression, and personality traits measured. Subjects had sympathetic and parasympathetic nervous system parameters measured at baseline and continuously thereafter. Subjects received standardized somatic (nail bed pressure) and visceral (esophageal balloon distension) stimuli to pain tolerance. Venous blood was sampled for cortisol at baseline, post somatic pain and post visceral pain. Key Results: Patients had higher neuroticism, state and trait anxiety, and depression scores but lower extroversion scores vs controls (all p < 0.005). Patients tolerated less somatic (p < 0.0001) and visceral stimulus (p = 0.009) and had a higher cortisol at baseline, and following pain (all p < 0.001). At baseline, patients had a higher sympathetic tone (p = 0.04), whereas in response to pain they increased their parasympathetic tone (p ≤ 0.008). The amalgamating the data, we identified two psychophysiologically distinct 'pain clusters'. Patients were overrepresented in the cluster characterized by high neuroticism, trait anxiety, baseline cortisol, pain hypersensitivity, and parasympathetic response to pain (all p < 0.03). Conclusions & Inferences: In future, such delineations in FCP populations may facilitate individualization of treatment based on psychophysiological profiling. © 2013 John Wiley & Sons Ltd.

Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo

Endothelial tip cells guide angiogenic sprouts by exploring the local environment for guidance cues such as vascular endothelial growth factor (VegfA). Here we present Flt1 (Vegf receptor 1) loss- and gain-of-function data in zebrafish showing that Flt1 regulates tip cell formation and arterial branching morphogenesis. Zebrafish embryos expressed soluble Flt1 (sFlt1) and membrane-bound Flt1 (mFlt1). In Tg(flt1(BAC):yfp) × Tg(kdrl:ras-cherry)(s916) embryos, flt1:yfp was expressed in tip, stalk and base cells of segmental artery sprouts and overlapped with kdrl:cherry expression in these domains. flt1 morphants showed increased tip cell numbers, enhanced angiogenic behavior and hyperbranching of segmental artery sprouts. The additional arterial branches developed into functional vessels carrying blood flow. In support of a functional role for the extracellular VEGF-binding domain of Flt1, overexpression of sflt1 or mflt1 rescued aberrant branching in flt1 morphants, and overexpression of sflt1 or mflt1 in controls resulted in short arterial sprouts with reduced numbers of filopodia. flt1 morphants showed reduced expression of Notch receptors and of the Notch downstream target efnb2a, and ectopic expression of flt4 in arteries, consistent with loss of Notch signaling. Conditional overexpression of the notch1a intracellular cleaved domain in flt1 morphants restored segmental artery patterning. The developing nervous system of the trunk contributed to the distribution of Flt1, and the loss of flt1 affected neurons. Thus, Flt1 acts in a Notch-dependent manner as a negative regulator of tip cell differentiation and branching. Flt1 distribution may be fine-tuned, involving interactions with the developing nervous system.

Do little embryos make big decisions? How maternal dietary protein restriction can permanently change an embryo's potential, affecting adult health

Periconceptional environment may influence embryo development, ultimately affecting adult health. Here, we review the rodent model of maternal low-protein diet specifically during the preimplantation period (Emb-LPD) with normal nutrition during subsequent gestation and postnatally. This model, studied mainly in the mouse, leads to cardiovascular, metabolic and behavioural disease in adult offspring, with females more susceptible. We evaluate the sequence of events from diet administration that may lead to adult disease. Emb-LPD changes maternal serum and/or uterine fluid metabolite composition, notably with reduced insulin and branched-chain amino acids. This is sensed by blastocysts through reduced mammalian target of rapamycin complex 1 signalling. Embryos respond by permanently changing the pattern of development of their extra-embryonic lineages, trophectoderm and primitive endoderm, to enhance maternal nutrient retrieval during subsequent gestation. These compensatory changes include stimulation in proliferation, endocytosis and cellular motility, and epigenetic mechanisms underlying them are being identified. Collectively, these responses act to protect fetal growth and likely contribute to offspring competitive fitness. However, the resulting growth adversely affects long-term health because perinatal weight positively correlates with adult disease risk. We argue that periconception environmental responses reflect developmental plasticity and 'decisions' made by embryos to optimise their own development, but with lasting consequences.

Medical conditions and depressive, anxiety, and somatic symptoms in older adults with and without generalized anxiety disorder

OBJECTIVE: The objective of this study was to examine medical illness and anxiety, depressive, and somatic symptoms in older medical patients with generalized anxiety disorder (GAD). METHOD: A case-control study was designed and conducted in the University of California, San Diego (UCSD) Geriatrics Clinics. A total of fifty-four older medical patients with GAD and 54 matched controls participated. MEASUREMENTS: The measurements used for this study include: Brief Symptom Inventory-18, Mini International Neuropsychiatric Interview, and the Anxiety Disorders Interview Schedule. RESULTS: Older medical patients with GAD reported higher levels of somatic symptoms, anxiety, and depression than other older adults, as well as higher rates of diabetes and gastrointestinal conditions. In a multivariate model that included somatic symptoms, medical conditions, and depressive and anxiety symptoms, anxiety symptoms were the only significant predictors of GAD. CONCLUSION: These results suggest first, that older medical patients with GAD do not primarily express distress as somatic symptoms; second, that anxiety symptoms in geriatric patients should not be discounted as a byproduct of medical illness or depression; and third, that older adults with diabetes and gastrointestinal conditions may benefit from screening for anxiety.

Transcriptome Analysis Reveals New Insights into the Modulation of Endometrial Stromal Cell Receptive Phenotype by Embryo-Derived Signals Interleukin-1 and Human Chorionic Gonadotropin: Possible Involvement in Early Embryo Implantation

The presence of the conceptus in uterine cavity necessitates an elaborate network of interactions between the implanting embryo and a receptive endometrial tissue. We believe that embryo-derived signals play an important role in the remodeling and the extension of endometrial receptivity period. Our previous studies provided original evidence that human Chorionic Gonadotropin (hCG) modulates and potentiates endometrial epithelial as well as stromal cell responsiveness to interleukin 1 (IL1), one of the earliest embryonic signals, which may represent a novel pathway by which the embryo favors its own implantation and growth within the maternal endometrial host. The present study was designed to gain a broader understanding of hCG impact on the modulation of endometrial cell receptivity, and in particular, cell responsiveness to IL1 and the acquisition of growth-promoting phenotype capable of receiving, sustaining, and promoting early and crucial steps of embryonic development. Our results showed significant changes in the expression of genes involved in cell proliferation, immune modulation, tissue remodeling, apoptotic and angiogenic processes. This points to a relevant impact of these embryonic signals on the receptivity of the maternal endometrium, its adaptation to the implanting embryo and the creation of an environment that is favorable for the implantation and the growth of this latter within a new and likely hostile host tissue. Interestingly our data further identified a complex interaction between IL1 and hCG, which, despite a synergistic action on several significant endometrial target genes, may encompass a tight control of endogenous IL1 and extends to other IL1 family members.

Contribution of lipophilic secondary metabolites to the toxicity of strains of freshwater cyanobacterial harmful algal blooms, identified using the zebrafish (Danio rerio) embryo as a model for vertebrate development

Cyanobacteria ("blue-green algae") are known to produce a diverse repertoire of biologically active secondary metabolites. When associated with so-called "harmful algal blooms", particularly in freshwater systems, a number of these metabolites have been associated—as "toxins", or commonly "cyanotoxins"—with human and animal health concerns. In addition to the known water-soluble toxins from these genera (i.e. microcystins, cylindrospermopsin, and saxitoxins), our studies have shown that there are metabolites within the lipophilic extracts of these strains that inhibit vertebrate development in zebrafish embryos. Following these studies, the zebrafish embryo model was implemented in the bioassay-guided purification of four isolates of cyanobacterial harmful algal blooms, namely Aphanizomenon, two isolates of Cylindrospermopsis, and Microcystis, in order to identify and chemically characterize the bioactive lipophilic metabolites in these isolates. ^ We have recently isolated a group of polymethoxy-1-alkenes (PMAs), as potential toxins, based on the bioactivity observed in the zebrafish embryos. Although PMAs have been previously isolated from diverse cyanobacteria, they have not previously been associated with relevant toxicity. These compounds seem to be widespread across the different genera of cyanobacteria, and, according to our studies, suggested to be derived from the polyketide biosynthetic pathway which is a common synthetic route for cyanobacterial and other algal toxins. Thus, it can be argued that these metabolites are perhaps important contributors to the toxicity of cyanobacterial blooms. In addition to the PMAs, a set of bioactive glycosidic carotenoids were also isolated because of their inhibition of zebrafish embryonic development. These pigmented organic molecules are found in many photosynthetic organisms, including cyanobacteria, and they have been largely associated with the prevention of photooxidative damage. This is the first indication of these compounds as toxic metabolites and the hypothesized mode of action is via their biotransformation to retinoids, some of which are known to be teratogenic. Additional fractions within all four isolates have been shown to contain other uncharacterized lipophilic toxic metabolites. This apparent repertoire of lipophilic compounds may contribute to the toxicity of these cyanobacterial harmful algal blooms, which were previously attributed primarily to the presence of the known water-soluble toxins.^