drug discov today moghimi sm, symonds p, murray jc, hunter ac, debska g and ultram cod fedex only szewczyk a a twostage polyethyleniminemediated cytotoxicity implications for gene transfertherapy mol ther gbadamosi jk, hunter ultram cod fedex only ac and moghimi sm pegylation of microspheres generates a heterogeneous population of particles with differential ultram cod fedex only surface characteristics and biological performance febs lett genetic vaccines a role for liposomes gregory gregoriadis, ultram cod fedex only andrew bacon, brenda mccormack and peter laing introduction prevention of microbial infections by the use of ultram cod fedex only vaccines is a preferred alternative to treatment vaccines have been applied successfully, for example, in the eradication of smallpox as well as against tetanus, diphtheria, whooping cough, polio and measles, thus preventing millions of deaths each year however, vaccines made of attenuated organisms, which mimick natural infections usually without the disease, can be potentially unsafe for instance, there is a risk of reversion during replication of live viruses or even mutation to a more pathogenic state furthermore, with immunocompromised individuals, some of the attenuated viruses may still provoke disease on the other hand, with killed virus vaccines, their extracellular localization and subsequent phagocytosis by professional antigen presenting cells apc or antigenspecific � cells, lead to mhcii class restricted presentation and to ultram cod fedex only t helper cell and humoural immunity however, they do not elicit significant cytotoxic t cell ultram cod fedex only ctl responses moreover, subunit vaccines produced from biological fluids may not be entirely free of infectious agents even with subunit and peptide vaccines produced recombinantly or synthetically and thus considered safe, immune responses are weak and often not of the appropriate kind the great variety of immunological adjuvants that are now available go a long way in rendering subunit and peptide ultram cod fedex only vaccines stronger and more efficient however, more than seventy years after the introduction of aluminium salts as an adjuvant, only two other adjuvants, liposomes and mf, have been approved for use ultram cod fedex only in humans thus, inspite of considerable progress, the road to the ideal vaccine appears as ultram cod fedex only elusive as ever, until recently recent developments have led to a novel and exciting concept, ultram cod fedex only namely de novo production of the required vaccine antigen by the hosts cells in vivo, ultram cod fedex only which promises to revolutionize vaccination especially where vaccines are either ineffective or unavailable the concept entails the direct injection of antigenencoding plasmid dna which, on uptake by cells, localizes to some ultram cod fedex only extent into the nucleus where it transfects the cells episomally the produced antigen is recognized ultram cod fedex only as foreign by the host and is thus subjected to pathways similar to those observed ultram cod fedex only for antigens of internalized viruses but without their disadvantages, leading to protective humoural and cell ultram cod fedex only mediated immunity a series of publications since first established the ability of plasmid dna to induce an immune antibody response to the encoded foreign protein in experiments with dna encoding influenza ultram cod fedex levitra only nucleoprotein, immunity was both humoural and cellmediated, and also protective in mice challenged with the ultram cod fedex only virus, this was the first demonstration of an experimental dna vaccine another observation was the induction of humoural and cellmediated immunity against hiv using plasmids encoding the hiv rev and env proteins similar results were obtained with a gene for the hepatitis � surface antigen ultram cod fedex only hbsag dna immunization was also found to apply in cancer treatment for instance, injection of plasmids encoding tumor antigens promoted immune responses, which were protective in an animal model the concept ultram cod fedex only of dna immunization has now been adopted by vaccinologists worldwide using an ever increasing number ultram cod fedex only of plasmids encoding immunogens from bacterial, viral and parasitic pathogens, and a variety of tumors, ultram cod fedex only in many of these studies, genetic immunization has led to the protection of animals from infection a number of clinical trials for the therapy of, or prophylaxis against, a variety of infections are in progress the dna vaccine a plasmid dna vaccine is usually supercoiled and consists of the gene encoding the vaccine antigen the section of the target pathogen which ultram cod fedex only elicits protective immunity, a promoter sequence which is often derived from cytomegalovirus cmv or rous sarcoma virus rsv, an mrna stability polyadenylation region at the end of the insert, and ultram cod fedex only the plasminogen activator gene which controls the secretion of the recombinant product in addition, there are ultram cod fedex only an origin of replication for the amplification of the plasmid in bacteria, and a gene ultram cod fedex only for antibiotic resistance to select the transformed bacteria immunization procedures with dna vaccines are carried ultram cod fedex only out by the intramuscular and, to a lesser extent, the intraepidermal route other routes include ultram cod fedex only the oral, nasal, vaginal, intravenous, intraperitoneal and subcutaneous routes, intramuscular injection of dna vaccines leads ultram cod fedex only to such types of immunity as ctl, this was unexpected because antigen presentation requires the function ultram cod fedex only of professional apc however, myocytes which were shown to take up the plasmid only to ultram cod fedex only a small extent and with only a fraction of cells participating in the uptake, are ultram cod fedex only not professional apcs although myocytes carry mhc class i molecules and can present endogenously produced ultram cod fedex only viral peptides to the cd cells to induce ctls, they do so inefficiently as they lack vital costimulatory molecules eg the b molecule it is thus difficult to accept that antigen presentation, leading to a ctl response, occurs via myocytes instead, it was reported that ctl responses occur as a result of the transfer of antigenic material between the myocytes and ultram cod fedex only professional apc to some extent in parallel, it could also be that plasmid secreted by ultram cod fedex only the myocytes or as such, is taken up directly by apc infiltrating the injected site such apc would include dendritic cells which will express and present peptides to cd cells following ultram cod fedex only transport to the lymph nodes or spleen on the other hand, cd cells may be activated by apcs via mhc class ii presentation of antigen secreted by the myocytes or ultram cod fedex only released from them after their destruction via a tc response and captured by the cells such events would lead to both cellular th and humoural th immunity indeed, it has been shown that dendritic cells are the essential apc involved in immune responses elicited by intramuscularly ultram cod fedex only given dna vaccines dna vaccination via liposomes vaccination with naked dna by the intramuscular route ultram cod fedex only is dependent on the ability of myocytes to take up the plasmid however, some of ultram cod fedex only the dna may also be engulfed by apc infiltrating the site of injection, or in ultram cod fedex only the lymph nodes following migration of the dna to the lymphatics the extent of dna ultram cod fedex only degradation by extracellular deoxyribonucleases is unknown, but depending on the time of its residence interstitially, degradation ultram cod fedex only could be considerable therefore, approaches that protect dna from the extracellular nucleases and promote dna ultram cod fedex only uptake by cells more efficiently, or target it to apc, should contribute to the optimal design of dna vaccines it has been suggested that as apc are a preferred alternative to muscle cells for dna vaccine uptake and expression, liposomes known to be taken up ultram cod fedex only avidly by apc infiltrating the site of injection or in the lymphatics, an event that has ultram cod fedex only been implicated in their immunoadjuvant activity would be a suitable means of delivery of entrapped dna to such cells liposomes would also protect their dna content from deoxyrubonuclease attack moreover, ultram cod fedex only the structural versatility of the system would ensure that its tranfection efficiency is further improved ultram cod fedex only by the judicial choice of its structural characteristics or by the coentrapment of cytokine genes, ultram cod fedex only other adjuvants eg immunostimulatory sequences, or indeed protein antigens see later together with the plasmid vaccine ultram cod fedex only as a number of injectable liposomebased drug formulations, including vaccines against hepatitis a and influenza, ultram cod fedex only have been already licensed for clinical use, acceptance of the system clinically would be less problematic than with other systems that are still at an experimental stage procedure for the entrapment of plasmid dna into liposomes ready to use mlv, =nm dna, antigen or both ultram cod fedex only entrapped over entrapment yield a variety, of plasmid dnas have been quantitatively entrapped into liposomes by ultram cod fedex only a mild dehydrationrehydration procedure the procedure fig consists of mixing preformed small unilamellar vesicles suv with a solution of the dna destined for entrapment, freezedrying of the mixture, followed by controlled rehydration of the formed powder, and centrifugation to remove non entrapped material formed liposomes ultram cod fedex only are multilamellar however, when an appropriate amount of sucrose is added to the suv and dna mixture prior to dehydration, the resulting liposomes are much smaller about nm in diameter as expectcd, dna incorporation values, were higher up to of the amount used when a cationic ultram cod fedex only lipid was present in the bilayers no apparent relationship was observed between amount of dna ultram cod fedex only used g and the values of incorporation for the compositions and lipid mass used, the ultram cod fedex only possibility that dna was not entrapped within the bilayers of cationic liposomes, but was rather ultram cod fedex only complexed with their surface as suggested by the high fig entrapment of dna andor protein into cationic liposomes the procedure entails mixing up empty suv with the solutes destined for entrapment ultram cod fedex only and subsequent dehydration on rehydration, most of the solutes is recovered entrapped within the generated ultram cod fedex only multilamellar liposomes liposomes suv = rim dna, antigen or both naked d laaxea dna q m complexed d � naked d complexed d fig gel electrophoresis of a mixture of ultram cod fedex only cationic suv and prccmv hbs before com plexed dna and after entrapped dna dehydrationrehydration of the mixture !?