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.

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Antiviral monoclonal antibodies (mAbs) represent promising therapeutics. However, most mAbs-based immunotherapies conducted so far have only considered the blunting of viral propagation and not other possible therapeutic effects independent of virus neutralization, namely the modulation of the endogenous immune response. As induction of long-term antiviral immunity still remains a paramount challenge for treating chronic infections, we have asked here whether neutralizing mAbs can, in addition to blunting viral propagation, exert immunomodulatory effects with protective outcomes. Supporting this idea, we report here that mice infected with the FrCasE murine retrovirus on day 8 after birth die of leukemia within 4–5 months and mount a non-protective immune response, whereas those rapidly subjected to short immunotherapy with a neutralizing mAb survive healthy and mount a long-lasting protective antiviral immunity with strong humoral and cellular immune responses. Interestingly, the administered mAb mediates lysis of infected cells through an antibody-dependent cell cytotoxicity (ADCC) mechanism. In addition, it forms immune complexes (ICs) with infected cells that enhance antiviral CTL responses through FcγR-mediated binding to dendritic cells (DCs). Importantly, the endogenous antiviral antibodies generated in mAb-treated mice also display the same properties, allowing containment of viral propagation and enhancement of memory cellular responses after disappearance of the administered mAb. Thus, our data demonstrate that neutralizing antiviral mAbs can act as immunomodulatory agents capable of stimulating a protective immunity lasting long after the end of the treatment. They also show an important role of infected-cells/antibody complexes in the induction and the maintenance of protective immunity through enhancement of both primary and memory antiviral T-cell responses. They also indicate that targeting infected cells, and not just viruses, by antibodies can be crucial for elicitation of efficient, long-lasting antiviral T-cell responses. This must be considered when designing antiviral mAb-based immunotherapies.

Monoclonal antibodies (mAbs) are increasingly being considered as agents to fight severe viral diseases. So far, they have essentially been selected and used on the basis of their virus-neutralizing activity and/or cell-killing activity to blunt viral propagation via direct mechanisms. There is, however, accumulating evidence that they can also induce long-lasting protective antiviral immunity by recruiting the endogenous immune system of infected individuals during the period of immunotherapy. Exploiting this property may revolutionize antiviral mAb-based immunotherapies, with benefits for both patients and healthcare systems.

To improve radioimmunotherapy with Auger electron emitters, we assessed whether the biological efficiency of (125)I varied according to its localization. A-431 and SK-OV-3 carcinoma cells were incubated with increasing activities (0-4 MBq/ml) of (125)I-labeled vectors targeting the cell membrane, the cytoplasm or the nucleus. We then measured cell survival by clonogenic assay and the mean radiation dose to the nucleus by assessing the cellular medical internal radiation dose (MIRD). The relationship between survival and the radiation dose delivered was investigated with a linear mixed regression model. For each cell line, we obtained dose-response curves for the three targets and the reference values (i.e., the dose leading to 75, 50 or 37% survival). When cell survival was expressed as a function of the total cumulative decays, nuclear (125)I disintegrations were more harmful than disintegrations in the cytoplasm or at the cell membrane. However, when survival was expressed as a function of the mean radiation dose to the nucleus, toxicity was significantly higher when (125)I was targeted to the cell membrane than to the cytoplasm. These findings indicate that the membrane is a more sensitive target than the cytoplasm for the dense ionization produced by Auger electrons. Moreover, cell membrane targeting is as cytotoxic as nuclear targeting in SK-OV-3 cells. We suggest that targeting the membrane rather than the cytoplasm may contribute to the development of more efficient radioimmunotherapies based on Auger electron radiation, also because most of the available vectors are directed against cell surface antigens.

Chemotherapeutic drugs used in the treatment of acute myeloid leukemias (AMLs) are thought to induce cancer cell death through the generation of DNA double-strand breaks. Here, we report that one of their early effects is the loss of conjugation of the ubiquitin-like protein SUMO from its targets via reactive oxygen species (ROS)-dependent inhibition of the SUMO-conjugating enzymes. Desumoylation regulates the expression of specific genes, such as the proapoptotic gene DDIT3, and helps induce apoptosis in chemosensitive AMLs. In contrast, chemotherapeutics do not activate the ROS/SUMO axis in chemoresistant cells. However, pro-oxidants or inhibition of the SUMO pathway by anacardic acid restores DDIT3 expression and apoptosis in chemoresistant cell lines and patient samples, including leukemic stem cells. Finally, inhibition of the SUMO pathway decreases tumor growth in mice xenografted with AML cells. Thus, targeting the ROS/SUMO axis might constitute a therapeutic strategy for AML patients resistant to conventional chemotherapies.

Long-term immune control of viral replication still remains a major challenge in retroviral diseases. Several monoclonal antibodies (MAbs) have already shown antiviral activities in vivo, including in the clinic but their effects on the immune system of treated individuals are essentially unknown. Using the lethal neurodegeneration induced in mice upon infection of neonates by the FrCas E retrovirus as a model, we report here that transient treatment by a neutralizing MAb shortly after infection can, after an immediate antiviral effect, favor the development of a strong protective host immune response containing viral propagation long after the MAb has disappeared. In vitro virus neutralization-and complement-mediated cell lysis assays, as well as in vivo viral challenges and serum transfer experiments, indicate a clear and essential contribution of the humoral response to antiviral protection. Our observation may have important therapeutic consequences as it suggests that short antibody-based therapies early after infection should be considered, at least in the case of maternally infected infants, as adjunctive treatment strategies against human immunodeficiency virus, not only for a direct effect on the viral load but also for favoring the emergence of an endogenous antiviral immune response.The therapeutic use of monoclonal antibodies (MAbs) has increased spectacularly in recent years (10,26,27). It now concerns a wide range of diseases with 13 MAbs approved for human use by the Food and Drug Administration and more than 400 others currently tested in clinical trials (27), including the treatment of chronic viral diseases such as hepatitis B virus (15,20), hepatitis C virus (15) and human immunodeficiency virus (4, 11, 59) infections. Due to their potential in the treatment of AIDS, several human immunodeficiency virus-neutralizing MAbs have already been obtained and studied (22,23,53) and others are being generated by various laboratories worldwide.Some of the available MAbs have already shown antiviral activity in vivo in a variety of adult and neonatal animal and human settings (see Discussion). Immediate antiviral effects in these experiments were due to direct virus neutralization. However, whether short-term MAb-based immunotherapies could, in addition, favor the emergence of endogenous antiviral immune responses contributing to the protection of infected individuals in the long term has hardly been considered thus far. As the elucidation of fundamental concepts in retroviral immunology is easier to attain in immunocompetent mouse models than in humans or monkeys, we turned to the neonatal infection system by the FrCas E retrovirus to address this issue. This model constitutes an invaluable tool to address the development of a protective immune response during the critical period of immunocompetence acquisition in young organisms, a situation which is reminiscent of that of perinatal infant infection by human immunodeficiency virus.FrCas E is an ecotropic mouse retrovirus (50). Upon inoculation to newbo...

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.