A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient.

The human antimelanoma antibody V86 was cloned from a single-chain Fv molecule (scFv) fusion phage library displaying the heavy chain variable domain (VH) and light chain variable domain (VL.) repertoire of a melanoma patient immunized with genetically-modified autologous tumor cells. Previous ELISA tests for binding of the V86 fusion phage to a panel of human metastatic melanoma and carcinoma cell lines and primary cultures of normal melanocytes, endothelial, and fibroblast cells showed that measurable binding occurred only to the melanoma cells. In this communication, the strict specificity of V86 for melanoma cells was confirmed by immunohistochemical staining tests with cultured cells and frozen tissue sections. The V86 fusion phage stained melanoma cell lines but did not stain carcinoma cell lines or cultured normal cells; V86 also stained specifically the melanoma cells in sections of metastatic tissue but did not stain any of the cells in sections from normal skin, lung, and kidney or from metastatic colon and ovarian carcinomas and a benign nevus. An unexpected finding is that V86 contains a complete VH domain but only a short segment of a VL, domain, which terminates before the CDR1 region. This VL deletion resulted from the occurrence in the VL cDNA of a restriction site, which was cleaved during construction of the scFv library. Thus V86 is essentially a VH antibody. The effect of adding a VI. domain to V86 was examined by constructing scFv fusion phage libraries in which V86 was coupled to Vlambda or Vkappa domains from the original scFv library of the melanoma patient and then panning the libraries against melanoma cells to enrich for the highest affinity antibody clones. None of the V86-Vlambda clones showed significant binding to melanoma cells in ELISA tests; although binding occurred with most of the V86-Vkappa clones, it was generally weaker than the binding of V86. These results indicate that most of the VL domains in the original scFv library reduce or eliminate the affinity of V86 for melanoma cells. Accordingly, VH libraries could provide access to anti-tumor antibodies that might not be detected in scFv or Fab libraries because of the incompatibility of most randomly paired VH and VL, domains.

31P NMR magnetization transfer study of the control of ATP turnover in Saccharomyces cerevisiae.

31P NMR magnetization transfer measurements have been used to measure the steady state flux between Pi and ATP in yeast cells genetically modified to overexpress an adenine nucleotide translocase isoform. An increase in Pi -> ATP flux and apparent ratio of moles of ATP synthesized/atoms of oxygen consumed (P:O ratio), when these cells were incubated with glucose, demonstrated that the reactions catalyzed by the translocase and F1F0 ATP synthase were readily reversible in vivo. However, when the same cells were incubated with ethanol alone, translocase overexpression had no effect on the measured Pi -> ATP flux or apparent P:O ratio, suggesting that the synthase was now operating irreversibly. This change was accompanied by an increase in the intracellular ADP concentration. These observations are consistent with a model proposed for the kinetic control of mitochondrial ATP synthesis, which was based on isotope exchange measurements with isolated mammalian mitochondria [LaNoue, K. F., Jeffries, F. M. H. & Radda, G. K. (1986) Biochemistry 25, 7667-7675].

Expression of a protective gene-prolongs survival of T cells in human immunodeficiency virus-infected patients.

The resistance of acquired immunodeficiency syndrome (AIDS) to traditional drug therapy has prompted a search for alternative treatments for this disease. One potential approach is to provide genetic resistance to viral replication to prolong latency. This strategy requires the definition of effective antiviral genes that extend the survival of T cells in human immunodeficiency virus (HIV)-infected individuals. We report the results of a human study designed to determine whether a genetic intervention can prolong the survival of T cells in HIV-infected individuals. Gene transfer was performed in enriched CD4+ cells with plasmid expression vectors encoding an inhibitory Rev protein, Rev M10, or a deletion mutant control, deltaRev M10, delivered by gold microparticles. Autologous cells separately transfected with each of the vectors were returned to each patient, and toxicity, gene expression, and survival of genetically modified cells were assessed. Cells that expressed Rev M10 were more resistant to HIV infection than those with deltaRev M10 in vitro. In HIV-infected subjects, Rev M10-transduced cells showed preferential survival compared to deltaRev M10 controls. Rev M10 can therefore act as a specific intracellular inhibitor that can prolong T-cell survival in HIV-1-infected individuals and potentially serve as a molecular genetic intervention which can contribute to the treatment of AIDS.

Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy.

Like human gliomas, the rat 9L gliosarcoma secretes the immunosuppressive transforming growth factor beta (TGF-beta). Using the 9L model, we tested our hypothesis that genetic modification of glioma cells to block TGF-beta expression may enhance their immunogenicity and make them more suitable for active tumor immunotherapy. Subcutaneous immunizations of tumor-bearing animals with 9L cells genetically modified to inhibit TGF-beta expression with an antisense plasmid vector resulted in a significantly higher number of animals surviving for 12 weeks (11/11, 100%) compared to immunizations with control vector-modified 9L cells (2/15, 13%) or 9L cells transduced with an interleukin 2 retroviral vector (3/10, 30%) (P < 0.001 for both comparisons). Histologic evaluation of implantation sites 12 weeks after treatment revealed no evidence of residual tumor. In vitro tumor cytotoxicity assays with lymph node effector cells revealed a 3- to 4-fold increase in lytic activity for the animals immunized with TGF-beta antisense-modified tumor cells compared to immunizations with control vector or interleukin 2 gene-modified tumor cells. These results indicate that inhibition of TGF-beta expression significantly enhances tumor-cell immunogenicity and supports future clinical evaluation of TGF-beta antisense gene therapy for TGF-beta-expressing tumors.

Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic alkylating agent.

The chloroethylnitrosourea (CNU) alkylating agents are commonly used for cancer chemotherapy, but their usefulness is limited by severe bone marrow toxicity that causes the cumulative depletion of all hematopoietic lineages (pancytopenia). Bone marrow CNU sensitivity is probably due to the inefficient repair of CNU-induced DNA damage; relative to other tissues, bone marrow cells express extremely low levels of the O6-methylguanine DNA methyltransferase (MGMT) protein that repairs cytotoxic O6-chloroethylguanine DNA lesions. Using a simplified recombinant retroviral vector expressing the human MGMT gene under control of the phosphoglycerate kinase promoter (PGK-MGMT) we increased the capacity of murine bone marrow-derived cells to repair CNU-induced DNA damage. Stable reconstitution of mouse bone marrow with genetically modified, MGMT-expressing hematopoietic stem cells conferred considerable resistance to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a CNU commonly used for chemotherapy. Bone marrow harvested from mice transplanted with PGK-MGMT-transduced cells showed extensive in vitro BCNU resistance. Moreover, MGMT expression in mouse bone marrow conferred in vivo resistance to BCNU-induced pancytopenia and significantly reduced BCNU-induced mortality due to bone marrow hypoplasia. These data demonstrate that increased DNA alkylation repair in primitive hematopoietic stem cells confers multilineage protection from the myelosuppressive effects of BCNU and suggest a possible approach to protecting cancer patients from CNU chemotherapy-related toxicity.

Gene therapy for long-term expression of erythropoietin in rats.

The injection of recombinant erythropoietin (Epo) is now widely used for long-term treatment of anemia associated with chronic renal failure, cancer, and human immunodeficiency virus infections. The ability to deliver this hormone by gene therapy rather than by repeated injections could provide substantial clinical and economic benefits. As a preliminary approach, we investigated in rats the expression and biological effects of transplanting autologous vascular smooth muscle cells transduced with a retroviral vector encoding rat Epo cDNA. Vector-derived Epo secretion caused increases in reticulocytes, with peak levels of 7.8-9.6% around day 10 after implantation. The initial elevation in reticulocytes was followed by clinically significant increases in hematocrit and hemoglobin for up to 11 weeks. Ten control and treated animals showed mean hematocrits of 44.9 +/- 0.4% and 58.7 +/- 3.1%, respectively (P < 0.001), and hemoglobin values of 15.6 +/- 0.1 g/dl and 19.8 +/- 0.9 g/dl, respectively (P < 0.001). There were no significant differences between control and treated animals in the number of white blood cells and platelets. Kidney and to a lesser extent liver are specific organs that synthesize Epo in response to tissue oxygenation. In the treated animals, endogenous Epo mRNA was largely down regulated in kidney and absent from liver. These results indicate that vascular smooth muscle cells can be genetically modified to provide treatment of anemias due to Epo deficiency and suggest that this cell type may be targeted in the treatment of other diseases requiring systemic therapeutic protein delivery.

Long-term in vivo expression of the human glucocerebrosidase gene in nonhuman primates after CD34+ hematopoietic cell transduction with cell-free retroviral vector preparations.

Successful gene transfer into stem cells would provide a potentially useful therapeutic modality for treatment of inherited and acquired disorders affecting hematopoietic tissues. Coculture of primate bone marrow cells with retroviral producer cells, autologous stroma, or an engineered stromal cell line expressing human stem cell factor has resulted in a low efficiency of gene transfer as reflected by the presence of 0.1-5% of genetically modified cells in the blood of reconstituted animals. Our experiments in a nonhuman primate model were designed to explore various transduction protocols that did not involve coculture in an effort to define clinically useful conditions and to enhance transduction efficiency of repopulating cells. We report the presence of genetically modified cells at levels ranging from 0.1% (granulocytes) to 14% (B lymphocytes) more than 1 year following reconstitution of myeloablated animals with CD34+ immunoselected cells transduced in suspension culture with cytokines for 4 days with a retrovirus containing the glucocerebrosidase gene. A period of prestimulation for 7 days in the presence of autologous stroma separated from the CD34+ cells by a porous membrane did not appear to enhance transduction efficiency. Infusion of transduced CD34+ cells into animals without myeloablation resulted in only transient appearance of genetically modified cells in peripheral blood. Our results document that retroviral transduction of primate repopulating cells can be achieved without coculture with stroma or producer cells and that the proportion of genetically modified cells may be highest in the B-lymphoid lineage under the given transduction conditions.

Cancer vaccines: the interleukin 2 dosage effect.

Cancer vaccines genetically engineered to produce interleukin 2 have been investigated intensively in a series of animal models and are at the point of entering into clinical trials. In this study we demonstrate a strong correlation between the rate of interleukin 2 production and the protection efficiency of murine S91 melanoma cell (clone M-3) vaccines. Best immunization is achieved with vaccines producing medium interleukin 2 levels of 1000-3000 units per 10(5) cells per day. Reduced interleukin 2 production evokes a corresponding decline in the number of successfully treated animals. Unexpectedly, when interleukin 2 expression is raised to high levels of 5000-7500 units per 10(5) cells per day, protection is completely absent because of impaired generation of tumor-specific cytotoxic T lymphocytes. In comparison, granulocyte-macrophage colony-stimulating factor as immunomodulator induces substantial immunization even at a moderate level of secretion and protects all animals at the maximal obtainable level of secretion. Our findings demonstrate the importance of the interleukin 2 level produced by genetically modified tumor cells and may have substantial impact for the clinical application of cancer vaccines.

Espermatogênese xenogênica pós-transplante de células tronco caninas no testículo de camundongos

As células tronco espermatogoniais (SSCs) são caracterizadas pela capacidade de autorrenovação, proliferação e transmissão das informações genéticas. Em caninos a primeira tentativa de xenotransplante não obteve o sucesso da produção de espermatozoides, no entanto, há evidências de que as células testiculares xenogênicas podem ser transplantadas no testículo do animal hospedeiro, e gerar espermatozoides viáveis do doador. Portanto, este estudo tem como objetivo realizar o xenotransplante das células germinativas caninas em camundongos imunosuprimidos, e com isto promover à produção de espermatozoides caninos viáveis, geneticamente modificados. E por meio desta técnica, analisar a eficiência da espermatogênese pós-transplante. Células germinativas testiculares foram caracterizadas, isoladas e cultivadas de cães pré-púberes, por meio de sistemas de cultura de enriquecimento e fatores de crescimento. As células foram transduzidas com um gene repórter GFP e LacZ, e por um vetor lentiviral para indentificar as SSCs nos testículos receptores. As SSCs transduzidas foram transplantadas nos testículos de camundongos (C57BL/6) tratados com Busulfan, após diferentes períodos os animais receptores foram eutanasiados e analisados. Aos 10 dias de cultivo as células germinativas adultas foram positivas para CD49f, CD117, e com 5 dias uma expressão semelhante de GFRA1 e DAZL, demonstrando a presença de SSCs e algumas células em meiose. Transplantamos 105 células e 20-43% das células transplantadas foram identificadas na membrana basal dos túbulos seminíferos do animal receptor. Portanto, o transplante das células germinativas caninas, mostrou que a purificação e o cultivo realizados são possíveis para obter SSCs caninas, as quais colonizaram os túbulos seminíferos dos camundongos imunodeficientes e mantiveram-se vivas na membrana basal por 90 dias após transplante, mesmo que estes animais tenham distância filogenética

Relaciones y prácticas que cambian la realidad alimenticia: una aproximación desde la antropología ecológica y feminista

From the 1990s, through the first decade of the XXI century, the food industry has intensified its production in technologically and genetically sophisticated ways. It has introduced transgenic and genetically modified foods, taking into account an economical push to obtain higher quantities in less time. Today, the foods that we consume seem more like products created in a laboratory than ones that come from working directly with the earth and with animals. These changes in the food industry are just a part of a long and complicated story in which economical interests figure heavily. The single-crop farming era begins in the 1970s in The United States and Europe. In some regions in Spain having a strong agricultural tradition, small private and family-owned farms that provided food to surrounding populations started disappearing, being uprooted in favor of the creation of large, multi-national companies. The market would expand with the growth of production facilities housing large quantities of animals living numbered and crowded. They mainly house cows, chickens, and pigs from which we obtain different products like milk, eggs and meat. The way these “industrial animals” live today does not even come close to what we think of as a balanced ecosystem, seeing as they are surrounded by machines and by the general use of sophisticated techniques to achieve the best return possible...

Los usuarios ante los alimentos genéticamente modificados y su información en el etiquetado

OBJETIVO: Analizar la opinión que los usuarios tienen sobre alimentos genéticamente modificados y su información en el etiquetado. MÉTODOS: Realizada revisión sistemática de la literatura científica sobre los alimentos transgénicos y el etiquetado a partir de la consulta de las bases de datos bibliográficas: Medline (vía PubMed), EMBASE, ISIWeb of Knowledge, Cochrane Library Plus, FSTA, LILACS, CINAHL y AGRICOLA. Los descriptores seleccionados fueron: «organisms, genetically modified» y «food labeling». La búsqueda se realizó desde la primera fecha disponible hasta junio de 2012, seleccionando los artículos pertinentes escritos en inglés, portugués y castellano. RESULTADOS: Se seleccionaron 40 artículos. En todos ellos, se debía haber realizado una intervención poblacional enfocada al conocimiento de los consumidores sobre los alimentos genéticamente modificados y su necesidad, o no, de incluir información en el etiquetado. El consumidor expresa su preferencia por el producto no-genéticamente modificado, y apunta que está dispuesto a pagar algo más por él, pero, en definitiva compra el artículo que está a mejor precio en un mercado que acoge las nuevas tecnologías. En 18 artículos la población se mostraba favorable a su etiquetado obligatorio y seis al etiquetado voluntario; siete trabajos demostraban el poco conocimiento de la población sobre los transgénicos y, en tres, la población subestimó la cantidad que consumía. En todo caso, se observó la influencia del precio del producto genéticamente modificado. CONCLUSIONES: La etiqueta debe ser homogénea y aclarar el grado de tolerancia en humanos de alimentos genéticamente modificados en comparación con los no modificados. Asimismo, debe dejar claro su composición, o no, de alimento genéticamente modificado y la forma de producción de estos artículos de consumo. La etiqueta también debe ir acompañada de un sello de certificación de una agencia del estado y datos para contacto. El consumidor expresa su preferencia por el producto no-genéticamente modificado pero señaló que acaba comprando el artículo que está a mejor precio en un mercado que acoge las nuevas tecnologías.

Highly dynamic host actin reorganization around developing Plasmodium inside hepatocytes

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver.

Disengaging the Smc3/kleisin interface releases cohesin from Drosophila chromosomes during interphase and mitosis

Cohesin's Smc1, Smc3, and kleisin subunits create a tripartite ring within which sister DNAs are entrapped. Evidence suggests that DNA enters through a gate created by transient dissociation of the Smc1/3 interface. Release at the onset of anaphase is triggered by proteolytic cleavage of kleisin. Less well understood is the mechanism of release at other stages of the cell cycle, in particular during prophase when most cohesin dissociates from chromosome arms in a process dependent on the regulatory subunit Wapl. We show here that Wapl-dependent release from salivary gland polytene chromosomes during interphase and from neuroblast chromosome arms during prophase is blocked by translational fusion of Smc3's C-terminus to kleisin's N-terminus. Our findings imply that proteolysis-independent release of cohesin from chromatin is mediated by Wapl-dependent escape of DNAs through a gate created by transient dissociation of the Smc3/kleisin interface. Thus, cohesin's DNA entry and exit gates are distinct.

A question of segregation: ‘ GM - free’ maize bread in Portugal

We describe the maize supply chain in Portugal for maize bread, a traditional bread type. As this bread is not labelled as ‘contains genetically modified organisms’ it should not contain more than 0.9 per cent genetically modified ingredients. On the basis of interviews we identify a general lack of documentation of the presence or absence of genetically modified ingredients along the complete supply chain (farmers, traders, mills and bakeries). Part of this deficiency is probably driven by a lack of awareness of the labelling rules at the end of the supply chain.

Mouse cellular cementum is highly dependent on growth hormone status

Cementum is known to be growth-hormone (GH)-responsive, but to what extent is unclear. This study examines the effects of extremes of GH status on cementogenesis in three lines of genetically modified mice; GH excess (giant), GH antagonist excess (dwarf), and GH receptor-deleted (GHR-KO) (dwarf). Age-matched mandibular molar tissues were processed for light microscope histology. Digital images of sections of first molar teeth were captured for morphometric analysis of lingual root cementum. Cross-sectional area of the cellular cementum was a sensitive guide to GH status, being reduced nearly 10-fold in GHR-KO mice, three-fold in GH antagonist mice, and increased almost two-fold in giant mice (p