Composting of the invasive species Arundo donax with sewage and agri-food sludge: Agronomic, economic and environmental aspects.

This work evaluates several co-composting scenarios based on the use of Arundo donax biomass (AD) as bulking agent for the co-composting of sewage sludge (MS) and agri-food sludge (AS), to manage these organic wastes and to produce balanced organic fertilizers by optimizing the process. For this, six piles were prepared in commercial composting conditions, using AD in a range of 40%-80% (on a dry weight basis). Physico-chemical and chemical parameters and the thermal behaviour were evaluated during the process, as were the physical and chemical parameters of the final composts. The proportion of AD in the mixtures has a significant effect on the development of the thermophilic stage of composting, showing the piles with higher proportion of AD a quicker organic matter degradation. In addition, the evolution of the thermal indices R1 and R2 was different depending on the origin of the sludge used, indicating an increase in the relative concentration of more recalcitrant materials in the piles prepared with AS. The estimation of the global warming potential showed that the use of higher proportion of AD in the composting mixture may be a strategy to mitigate the emission of greenhouse gases during the composting process. Moreover, the end-products obtained had an additional marketable value, with a balanced nutrient content and a good degree of maturity, which indicates the viability of the composting process as a method for the stabilization of these organic wastes.

Transgenic mice overexpressing insulin-like growth factor-II in beta cells develop type 2 diabetes.

During embryonic development, insulin-like growth factor-II (IGF-II) participates in the regulation of islet growth and differentiation. We generated transgenic mice (C57BL6/SJL) expressing IGF-II in beta cells under control of the rat Insulin I promoter in order to study the role of islet hyperplasia and hyperinsulinemia in the development of type 2 diabetes. In contrast to islets from control mice, islets from transgenic mice displayed high levels of IGF-II mRNA and protein. Pancreases from transgenic mice showed an increase in beta-cell mass (about 3-fold) and in insulin mRNA levels. However, the organization of cells within transgenic islets was disrupted, with glucagon-producing cells randomly distributed throughout the core. We also observed enhanced glucose-stimulated insulin secretion and glucose utilization in islets from transgenic mice. These mice displayed hyperinsulinemia, mild hyperglycemia, and altered glucose and insulin tolerance tests, and about 30% of these animals developed overt diabetes when fed a high-fat diet. Furthermore, transgenic mice obtained from the N1 backcross to C57KsJ mice showed high islet hyperplasia and insulin resistance, but they also developed fatty liver and obesity. These results indicate that local overexpression of IGF-II in islets might lead to type 2 diabetes and that islet hyperplasia and hypersecretion of insulin might occur early in the pathogenesis of this disease.

Genetically engineered antibodies in gene transfer and gene therapy.

Our ability to produce and engineer human monoclonal antibodies provides a basis for the development of novel therapeutical strategies against a variety of diseases. These strategies not only include improved passive immunotherapy but also more sophisticated antibody-based gene therapies involving gene transfer approaches. Four of the major applications of antibody gene engineering in the field of gene therapy are reviewed here. These are (1) the redefinition of viral vector tropism of infection for better transduction of cells of therapeutical interest, (2) the grafting of new cell recognition activities to effector cells of the immune system to kill cancer and pathogen-infected cells, (3) the inhibition of cellular and viral functions through intracellular expression of antibody-derived molecules, and (4) the systemic delivery of therapeutic monoclonal antibodies by non-B cells in living organisms.

Immunotherapy of a viral disease by in vivo production of therapeutic monoclonal antibodies.

Continuous and sustained in vivo production of monoclonal antibodies by engineered cells might render long-term antibody-based treatments cost-effective, avoid side effects associated with infusion of massive doses of antibody, and circumvent possible antiidiotypic responses against the therapeutic agent. The FrCasE retrovirus induces a lethal neurodegeneration on infection of newborn mice. We report here that implantation of cellulose sulfate capsules containing cells secreting an ectopic monoclonal antibody neutralizing FrCasE can prevent animals from developing the disease. All treated mice showed reduced or undetectable viremia in addition to a lack of the histopathological lesions characteristic of FrCasE infection. This work paves the way for a novel gene/cell antibody-based immunotherapy of a variety of severe viral and nonviral diseases.

In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells.

In vivo production of recombinant antibodies by engineered cells may have applications for gene therapy of certain cancers and of certain severe viral diseases. It would also permit the development of new animal models of autoimmune diseases and new approaches for in vivo ablation of specific cell types for fundamental purposes. Using gene transfer of an anti-human thyroglobulin monoclonal antibody, we show here that several cell types permitting autologous grafting of genetically engineered cells are efficiently able to secrete antibodies in vitro. Those cells include skin fibroblasts, hepatocytes, and myogenic cells. We also show that the secreted antibodies display an affinity for the antigen close to that of the parental antibody, with, however, slight differences varying according to the cell type. This indicates that the foldings of antigen combining sites of antibodies produced in B cell- and non-B cell contexts are very similar. Finally, we report that, when implanted in the forelimb of a mouse, genetically modified myogenic cells are able to secrete antibodies for at least 4 months. Taken together, our observations point to the notion that genetic modification of patient cells may be used for long-term antibody-based gene therapies.

Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts.

In vivo production and systemic delivery of therapeutic antibodies by engineered cells might advantageously replace injection of purified antibodies for treating a variety of life-threatening diseases, including cancer, acquired immunodeficiency syndrome, and autoimmune diseases. We report here that skin fibroblasts retrovirally transduced to express immunoglobulin genes can be used for sustained long-term systemic delivery of cloned antibodies in immunocompetent mice. Importantly, no anti- idiotypic response against the ectopically expressed model antibody used in this study was observed. This supports the notion that skin fibroblasts can potentially be used in antibody-based gene/cell therapy protocols without inducing any adverse immune response in treated individuals.

Corynebacterium group D2 ("Corynebacterium urealyticum") constitutes a new genomic species.

Twenty-one Corynebacterium group D2 ("C. urealyticum") strains were found to constitute a tight DNA hybridization group distinct from named Corynebacterium species. The strains of Corynebacterium group D2 had cell wall component type IV, short chain mycolic acids and G+C content of DNA of 65-66 mol %. Corynebacterium group D2 constitutes a genomic species which can be identified by phenotypic tests.

Development of cellulose sulfate-based polyelectrolyte complex microcapsules for medical applications.

Microencapsulation, as a tool for immunoisolation for allogenic or xenogenic implants, is a rapidly growing field. However most of the approaches are based on alginate/polylysine capsules, despite this system's obvious disadvantages such as its pyrogenicity. Here we report a different encapsulation system based on sodium cellulose sulfate and polydiallyldimethyl ammonium chloride for the encapsulation of mammalian cells. We have characterized this system regarding capsule formation, strength and size of the capsules as well as viability of the cells after encapsulation. In addition, we demonstrate the efficacy of these capsules as a "microfactory" in vitro and in vivo. Using encapsulated hybridoma cells we were able to demonstrate long-term release of antibodies up to four months in vivo. In another application we could show the therapeutic relevance of encapsulated genetically modified cells as an in vivo activation center for cytostatic drugs during tumor therapy.

Transgenic mice overexpressing phosphoenolpyruvate carboxykinase develop non-insulin-dependent diabetes mellitus.

An increase in hepatic gluconeogenesis is believed to be an important factor responsible for the fasting hyperglycemia detected in patients with non-insulin-dependent diabetes mellitus (NIDDM). Phosphoenolpyruvate carboxykinase (GTP) (PEPCK; EC 4.1.1.32) is a regulatory enzyme of gluconeogenesis. To study the role of the expression of PEPCK gene in the development of NIDDM, we have produced lines of transgenic mice expressing a PEPCK minigene under control of its own promoter. Transgenic mice were hyperglycemic and had higher serum insulin concentrations. In addition, alterations in liver glycogen content and muscle glucose transporter GLUT-4 gene expression were detected. The overexpression of the PEPCK gene led to an increase in glucose production from pyruvate in hepatocytes in primary culture. When intraperitoneal glucose tolerance tests were performed, blood glucose levels were higher than those detected in normal mice. This animal model shows that primary alterations in the rate of liver glucose production may induce insulin resistance and NIDDM.

Efficient cell infection by Moloney murine leukemia virus-derived particles requires minimal amounts of envelope glycoprotein.

Retrovirus entry into cells is mediated by specific interactions between the retrovirally encoded Env envelope glycoprotein and a host cell surface receptor. Though a number of peptide motifs responsible for the structure as well as for the binding and fusion activities of Env have been identified, only a few quantitative data concerning the infection process are available. Using an inducible expression system, we have expressed various amounts of ecotropic and amphotropic Env at the surfaces of Moloney murine leukemia virus-derived vectors and assayed for the infectivity of viral particles. Contrary to the current view that numerous noncooperative Env-viral receptor interactions are required for cell infection, we report here that very small amounts of Env are sufficient for optimal infection. However, increasing Env density clearly accelerates the rate at which infectious attachment to cells occurs. Moreover, our data also show that a surprisingly small number of Env molecules are sufficient to drive infection, albeit at a reduced efficiency, and that, under conditions of low expression, Env molecules act cooperatively. These observations have important consequences for our understanding of natural retroviral infection as well as for the design of cell-targeted infection techniques involving retroviral vectors.

Overexpression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in transgenic mice causes hepatic hyperketogenesis.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) is a key enzyme in the ketone body pathway. To determine its role in the regulation of liver ketogenesis, transgenic mice expressing a P-enolpyruvate carboxykinase/HMG-CoA synthase chimeric gene have been obtained. An increase in the concentration of mitochondrial HMG-CoA synthase mRNA was detected in these mice, which was associated with a 3-fold increase in HMG-CoA synthase activity in liver mitochondrial extracts. Transgenic mice were normoglycemic and had normal levels of plasma triglycerides and lower free fatty acids. However, the plasma concentration of ketone bodies was about three times higher in transgenic mice than in control animals. Hepatocytes in primary culture from transgenic mice expressed the chimeric gene in a regulated manner and showed a 3-fold increase in beta-hydroxybutyrate and acetoacetate concentrations in the medium. This animal model thus shows that the overexpression of mitochondrial HMG-CoA synthase causes ketone body overproduction, suggesting that this enzyme may be a regulatory step in liver ketogenesis.

Systemic long-term delivery of antibodies in immunocompetent animals using cellulose sulphate capsules containing antibody-producing cells.

Implantation of capsules containing antibody-producing cells into patients would potentially permit systemic long-term delivery of antibodies and might, thus, be useful in the development of surveillance treatments for cancers and severe viral diseases. We show that cellulose sulphate (CS) capsules containing hybridoma cells, when implanted subcutaneously or in the intraperitoneal cavity, can be used for delivering monoclonal antibodies into the blood-stream of immunocompetent mice for at least several months. In contrast to capsules implanted into the intraperitoneal cavity, which remain mobile and nonvascularized, capsules implanted under the skin form neo-organs which become vascularized within days. This may explain the higher blood concentration of the antibody we have observed in the latter case. Importantly, neither an isolating fibrosis nor an obvious inflammatory response was detected at the capsule implantation sites during observation periods as long as 10 months. Finally, no anti-idiotypic immune response against the ectopically delivered antibody was shown to occur. This rules out any potent adjuvant effect of the cellulose sulphate matrix that might have stimulated a neutralizing humoral response. Taken together, our data indicate that encapsulation of antibody-producing cells into CS might be used in antibody-based gene/cell therapy approaches.

Evidence from transgenic mice that interferon-beta may be involved in the onset of diabetes mellitus.

A number of cytokines have been shown to alter the function of pancreatic beta-cells and thus might be involved in the development of type 1 diabetes. Interferon-beta (IFN-beta) expression is induced in epithelial cells by several viruses, and it has been detected in islets of type 1 diabetic patients. Here we show that treatment of isolated mouse islets with this cytokine was able to alter insulin secretion in vitro. To study whether IFN-beta alters beta-cell function in vivo and leads to diabetes, we have developed transgenic mice (C57BL6/SJL) expressing IFN-beta in beta-cells. These mice showed functional alterations in islets and impaired glucose-stimulated insulin secretion. Transgenic animals presented mild hyperglycemia, hypoinsulinemia, hypertriglyceridemia, and altered glucose tolerance test, all features of a prediabetic state. However, they developed overt diabetes, with lymphocytic infiltration of the islets, when treated with low doses of streptozotocin, which did not induce diabetes in control mice. In addition, about 9% of the transgenic mice obtained from the N3 back-cross to outbred albino CD-1 mice spontaneously developed severe hyperglycemia and hypoinsulinemia and showed mononuclear infiltration of the islets. These results suggest that IFN-beta may be involved in the onset of type 1 diabetes when combined with either an additional factor or a susceptible genetic background.