Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.¹ Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.² These include RPE65 gene replacement therapy in patients with Leber''s congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt''s disease.^3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.^5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.     

Determinants of immunogenic response to protein therapeutics

Protein therapeutics occupy a very significant position in the biopharmaceutical market. In addition to the preclinical, clinical and post marketing challenges common to other drugs, unwanted immunogenicity is known to affect efficacy and/or safety of most biotherapeutics. A standard set of immunogenicity risk factors are routinely used to inform monitoring strategies in clinical studies. A number of in-silico, in vivo and in vitro approaches have also been employed to predict immunogenicity of biotherapeutics, but with limited success. Emerging data also indicates the role of immune tolerance mechanisms and impact of several product-related factors on modulating host immune responses. Thus, a comprehensive discussion of the impact of innate and adaptive mechanisms and molecules involved in induction of host immune responses on immunogenicity of protein therapeutics is needed. A detailed understanding of these issues is essential in order to fully exploit the therapeutic potential of this class of drugs. This Roundtable Session was designed to provide a common platform for discussing basic immunobiological and pharmacological issues related to the role of biotherapeutic-associated risk factors, as well as host immune system in immunogenicity against protein therapeutics. The session included overview presentations from three speakers, followed by a panel discussion with audience participation.     

Modifying L-type calcium current kinetics: consequences for cardiac excitation and arrhythmia dynamics

The L-type Ca current (I_Ca,L), essential for normal cardiac function, also regulates dynamic action potential (AP) properties that promote ventricular fibrillation. Blocking I_Ca,L can prevent ventricular fibrillation, but only at levels suppressing contractility. We speculated that, instead of blocking I_Ca,L, modifying its shape by altering kinetic features could produce equivalent anti-fibrillatory effects without depressing contractility. To test this concept experimentally, we overexpressed a mutant Ca-insensitive calmodulin (CaM₁₂₃₄) in rabbit ventricular myocytes to inhibit Ca-dependent I_Ca,L inactivation, combined with the ATP-sensitive K current agonist pinacidil or I_Ca,L blocker verapamil to maintain AP duration (APD) near control levels. Cell shortening was enhanced in pinacidil-treated myocytes, but depressed in verapamil-treated myocytes. Both combinations flattened APD restitution slope and prevented APD alternans, similar to I_Ca,L blockade. To predict the arrhythmogenic consequences, we simulated the cellular effects using a new AP model, which reproduced flattening of APD restitution slope and prevention of APD/Ca_i transient alternans but maintained a normal Ca_i transient. In simulated two-dimensional cardiac tissue, these changes prevented the arrhythmogenic spatially discordant APD/Ca_i transient alternans and spiral wave breakup. These findings provide a proof-of-concept test that I_Ca,L can be targeted to increase dynamic wave stability without depressing contractility, which may have promise as an antifibrillatory strategy.     

Alteration of superoxide- and nitric oxide-mediated antimicrobial function of macrophages by in vivo cocaine exposure

Cocaine is a popular drug of abuse and despite impressive advances in the understanding of its physiological, pharmacological, and toxic effects, its mechanism of immunosuppression at the cellular level is not well understood. In this paper we report the role of effector molecules like superoxide and nitric oxide in the antibacterial function of macrophages exposed to acute and chronic doses of cocaine in vivo. Bacterial killing by acute cocaine-exposed macrophages (ACE-Mphis) increased significantly, with a concomitant rise in respiratory burst and generation of superoxide and nitric oxide, compared with control macrophages. In contrast, chronic cocaine-exposed macrophages (CCE-Mphis) exhibited limited antimicrobial activity, which correlated closely with diminished respiratory burst and reduced production of superoxide and nitric oxide. Further, a killing assay was carried out in the presence of N(G)-methyl-L-arginine acetate, an inhibitor of iNOS, to evaluate the role of nitric oxide in the killing process. The results obtained indicate that while about 30% killing of input bacteria by control and ACE-Mphis was attributable to NO-mediated killing, only about 6% killing from NO was found with CCE-Mphis. The findings indicate that acute exposure to cocaine possibly caused upregulation of enzymes responsible for the generation of ROI (reactive oxygen intermediates) and RNI (reactive nitrogen intermediates), leading to enhanced antimicrobial function. On the other hand, chronic exposure to cocaine impaired the oxygen-dependent microbicidal capacity of macrophages, possibly through impaired expression of enzymes responsible for ROI and RNI formation. Proinflammatory cytokines may play a key role in cocaine-mediated immunosuppression, since exposure of macrophages to cocaine impairs the ability of the cells to produce these cytokines.     

Bone marrow suppression and severe anaemia associated with persistent Plasmodium falciparum infection in African children with microscopically undetectable parasitaemia

In sub-Saharan Africa the highest overlap between malaria and HIV infections occurs in female adolescents. Yet control activities for these infections are directed to different target groups, using disparate channels. This reflects the lack of priority given to adolescents and the absence of an accepted framework for delivering health and health-related interventions to this high-risk group. In this paper it is argued that female adolescents require a continuum of care for malaria and HIV – prior to conception, during and after pregnancy and that this should be provided through adolescent services. The evidence for this conclusion is presented. A number of African countries are commencing to formulate and implement adolescent-friendly policies and services and disease control programs for malaria and HIV will need to locate their interventions within such programs to ensure widespread coverage of this important target group. Failure to prioritize adolescent health in this way will seriously limit the success of disease control programs for malaria and HIV prevention.     

Cr(VI) Imposed Toxicity in Maize Seedlings Assessed in Terms of Disruption in Carbohydrate Metabolism

The present study examined the toxic effects of Cr(VI; 100, 250 and 500 μM) in maize seedlings by investigating the changes in carbohydrate metabolism after 48, 96, and 144 h of exposure. Cr-stress results in severe alterations in the contents of carbohydrates and reducing sugars and the activities of carbohydrate metabolizing enzymes, amylases, phosphatases and phosphorylases, and invertases in maize seedlings. Under Cr stress, the contents of carbohydrates and reducing sugars declined in roots, whereas an increase was noticed in leaves. The catalytic activity of carbohydrate metabolizing enzymes, except invertases, in roots declined in the presence of Cr(VI) in a concentration- and exposure time-dependent manner. In contrast, the activities of these enzymes were enhanced in leaves under Cr(VI) stress. The activity of invertases increased with increasing amount of Cr(VI) but declined with an increase in the time interval. In conclusion, our results show that carbohydrate metabolism is severely affected under Cr(VI) toxicity. The study suggests that Cr-induced perturbations in the carbohydrate metabolism are one of the factors resulting in growth inhibition under Cr(VI) stress.     

Pancreatitis severity in mice with impaired CFTR function but pancreatic sufficiency is mediated via ductal and inflammatory cells-Not acinar cells

Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) are an established risk factor for cystic fibrosis (CF) and chronic pancreatitis. Whereas patients with CF usually develop complete exocrine pancreatic insufficiency, pancreatitis patients with CFTR mutations have mostly preserved exocrine pancreatic function. We therefore used a strain of transgenic mice with significant residual CFTR function (CFTR^tm1HGU) to induce pancreatitis experimentally by serial caerulein injections. Protease activation and necrosis were investigated in isolated acini, disease severity over 24h, pancreatic function by MRI, isolated duct stimulation and faecal chymotrypsin, and leucocyte function by ex vivo lipopolysaccharide (LPS) stimulation. Pancreatic and lung injury were more severe in CFTR^tm1HGU but intrapancreatic trypsin and serum enzyme activities higher than in wild-type controls only at 8h, a time interval previously attributed to leucocyte infiltration. CCK-induced trypsin activation and necrosis in acini from CFTR^tm1HGU did not differ from controls. Fluid and bicarbonate secretion were greatly impaired, whereas faecal chymotrypsin remained unchanged. LPS stimulation of splenocytes from CFTR^tm1HGU resulted in increased INF-γ and IL-6, but decreased IL-10 secretion. CFTR mutations that preserve residual pancreatic function significantly increase the severity of experimental pancreatitis—mostly via impairing duct cell function and a shift towards a pro-inflammatory phenotype, not by rendering acinar cells more susceptible to pathological stimuli.     

Strategies for farmers and policy makers to control nitro-gen losses whilst maintaining crop production

The nitrogen (N) cycle is essentially 'leaky'. The losses of small amounts of nitrate to waters and of ammonia and nitrous oxide to the atmosphere are a part of the global biogeo-chemical N cycle. However, intensive agricultural production, industry and vehicle use have more than doubled the amount of 'reactive' N in the environment, resulting in eutrophication, ecosystem change and health concerns. Research has identified agricultural practices that cause large losses of N and, in some cases, developed solutions. This paper discusses the problems of maintaining productivity while reducing N losses, compares conventional with low input (integrated) and organic farming systems, and discusses wider options. It also looks at the need to integrate studies on N with other environmental impacts, set in the context of the whole farm system, to provide truly sustainable agricultural systems.     

Identification of a 148-kDa surface lectin from Giardia lamblia with specificity for α-methyl-d-mannoside

Abstract A lectin specific for α-methyl-d-mannoside was purified from the membrane extract of Giardia lamblia by a combination of gel filtration chromatography on Sephadex G-75 and Superose 6-HR 10/30. The homogeneity of the lectin was established by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular mass of the native protein was 148 kDa. The lectin agglutinated rabbit erythrocytes in the presence of Ca²⁺ at 37 °C and pH 7.O. The maximum activity of the lectin was obtained after trypsin treatment. The inhibition study clearly suggests that the binding site of the lectin recognizes α-methyl-d-mannoside as the immunodominant sugar.     

1-Halo analogs of dihydroxyacetone 3-phosphate. The effects of the fluoro analog on cytosolic glycerol-3-phosphate dehydrogenase and triosephosphate isomerase.

Fluoro-o-hydorxyacetone phosphate (fluoroacetol phosphate) has been prepared by oxidation of 1-fluoro-3-chloro-2-propanol to 1-fluoro-3-chloroacetone, phosphorylation with silver dibenzylphosphate, and the intermediate isolation of 1-fluoro-3-hydroxyacetone phosphate dibenzyl ester, followed by catalytic hydrogenation and preparation of the stable monosodium salt. The chloro analog as the pure, stable monosodium salt has been prepared by a similar route from 1,3-dichloroacetone. 1-Fluoro-3-hydroxyacetone-P is substrate for cytosolic NAD+-linked glycerol-3-P dehydrogenese (EC 1.1.1.8) from rabbit skeletal muscle with an apparent Km of 50 mM under conditions in which dihydroxyacetone-P exhibits an apparent Km of 0.15 mM. Under these conditions the fluoro analog is 85% hydrated wheras dihydroxyacetone-P has been shown by others to be 44% hydrated. The turnover numbers are 49,000 molecules of NADH oxidized per minute per molecule of enzyme at 25 degrees with the fluoro analog as substrate, and 60,000 with dihydrocyacetone-P as substrate. The product of the reduction of the fluoro analog has been identified as 1-fluorodeoxyglycerol-3-P. 1-Fluoro-3-hydroxyacetone-P is comparatively weak irreversible inhibitor at 4 degrees of rabbit muscle triosephosphate isomerase (EC 5.3.1.1) with second-order rate constant of 2.6 M minus 1 sec minus 1. Inhibition by pyrazole in vivo of alcohol dehydrogenese catalyzed oxidation of 1-fluorodeoxyglecerol-3-P indicates in mice the reduction of 1-fluoro-3-hydroxyacetone-P to -l-1-fluorodexoxyglycerol-3-P is not significant metabolic route, or that an alternative route exists when the alcohol dehydrogenase dependent pathway is inhibited.