Experimental design: Sixteen patients received fludarabine/cyclophosphamide conditioning 68 combined with total lymphoid irradiation followed by adoptive immunotherapy with IL-2-69 activated haploidentical NK cells. 70
Human embryonic stem cell (hESC) lines, after directed differentiation, hold the greatest potential for cell transplantation treatment in many severe diseases. Good manufacturing practice (GMP) quality, defined by both the European Medicines Agency and the Food and Drug Administration, is a requirement for clinical-grade cells, offering optimal defined quality and safety in cell transplantation. Using animal substance-free culture media, feeder cells or feeder-free matrix in derivation, passaging, expansion and cryopreservation procedures, immune reactions against animal proteins in the cells, and infection risk caused by animal microbes can be avoided. It is also possible to apply GMP to animal components if no better options are available. In recent production of GMP-quality hESC lines, feeder cells had been cultured in fetal bovine serum, and the medium supplemented with an animal protein containing a serum replacement component. Using embryos cultured in a GMP laboratory, isolating the inner cell mass mechanically, deriving lines on human feeder cells originally cultured in xeno-free medium in a GMP laboratory, and using xeno-free media for derivation and culture of hESC lines themselves, GMP-quality xeno-free hESC lines could be established today. Human serum is a xeno-free component available today, but many chemically defined media are under development.
The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and γ-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.
The VEGF gene transfer did not significantly improve stress-induced myocardial perfusion abnormalities compared with placebo; however, improved regional wall motion, as assessed both by NOGA and by ventriculography, may indicate a favorable anti-ischemic effect. This result should encourage more studies within the field. Transient VEGF overexpression seems to be safe.
Gene vaccination encounters problems different from those of gene therapy since both a short half-life of the gene and a strong immune response to the gene product are desirable. We have evaluated a DNA vaccine consisting of seven plasmids encoding nine HIV-1 proteins. Using a needle-free delivery device, the Biojector, together with recombinant mouse GM-CSF, this vaccine induced strong gp160 Env- and p24 Gag-specific cellular and humoral immune responses in mice. The rGM-CSF was crucial for inducing both antibodies and antigen-specific CD8(+) T cell responses against both gp160 and p24. A GMP-produced lot of this vaccine, intended for human use, was delivered intradermally or intramuscularly into BALB/c mice at a GLP-accredited animal facility. This vaccine induced strong cellular responses independent of the route of immunization; moreover, no signs of toxicity were detected after histopathological examination of various tissues. Overall, the results indicate that the intradermal delivery of multigene/multisubtype HIV DNA in combination with recombinant GM-CSF is a safe and efficacious strategy for inducing high levels of specific CD8(+) T cells and unusually high titers of antibodies. This vaccine has been approved by the Swedish Medicinal Products Agency and is currently in a Phase I clinical trial.
Abstract. Sarkar N, Ru È ck A, Ka Èllner G, Y-Hassan S, Blomberg P, Islam KB, van der Linden J, Lindblom D, Nygren AT, Lind B, Brodin L-A Ê , Drvota V, Sylve Ân C
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