drug discov today moghimi sm, symonds p, murray jc, hunter ac, debska g and dont take coumadin szewczyk a a twostage polyethyleniminemediated cytotoxicity implications for gene transfertherapy mol ther gbadamosi jk, hunter dont take coumadin ac and moghimi sm pegylation of microspheres generates a heterogeneous population of particles with differential surface characteristics and biological performance febs lett genetic vaccines a role for liposomes gregory gregoriadis, dont take coumadin andrew bacon, brenda mccormack and peter laing introduction prevention of microbial infections by the use of dont take coumadin 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 dont take coumadin 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 dont take coumadin other hand, with killed virus vaccines, their extracellular localization and subsequent phagocytosis by professional antigen dont take coumadin presenting cells apc or antigenspecific � cells, lead to mhcii class restricted presentation and to t helper cell and humoural immunity however, they do not elicit significant cytotoxic t cell 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, dont take coumadin 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 dont take coumadin 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 dont take coumadin in humans thus, inspite of considerable progress, the road to the ideal vaccine appears as dont take coumadin elusive as ever, until recently recent developments have led to a novel and exciting concept, dont take coumadin namely de novo production of the required vaccine antigen by the hosts cells in vivo, which promises to revolutionize vaccination especially where vaccines are either ineffective or unavailable the concept dont take coumadin entails the direct injection of antigenencoding plasmid dna which, on uptake by cells, localizes to some dont take coumadin extent into the nucleus where it transfects the cells episomally the produced antigen is recognized dont take coumadin as foreign by the host and is thus subjected to pathways similar to those observed dont take coumadin for antigens of internalized viruses but without their disadvantages, leading to protective humoural and cell mediated immunity a series of publications since first established the tazo joy caffeine content ability of plasmid dna to dont take coumadin induce an immune antibody response to the encoded foreign protein in experiments with dna encoding influenza dont take coumadin nucleoprotein, immunity was both humoural and cellmediated, and also protective in mice challenged with the dont take coumadin virus, this was the first demonstration of an experimental dna vaccine another observation was the dont take coumadin induction of humoural and cellmediated immunity against hiv using plasmids encoding the hiv rev and dont take coumadin env proteins similar results were obtained with a gene for the hepatitis � surface antigen dont take coumadin hbsag dna immunization was also found to apply in cancer treatment for instance, injection of plasmids dont take coumadin encoding tumor antigens promoted immune responses, which were protective in an animal model the concept dont take coumadin of dna immunization has now been adopted by vaccinologists worldwide using an ever increasing number of plasmids encoding immunogens from bacterial, viral and parasitic pathogens, and a variety of tumors, in many of these studies, genetic immunization has led to the protection of animals from dont take coumadin infection a number of clinical trials for the therapy of, or prophylaxis against, a variety of dont take coumadin infections are in progress the dna vaccine a plasmid dna vaccine is usually supercoiled and dont take coumadin consists of the gene encoding the vaccine antigen the section of the target pathogen which dont take coumadin elicits protective immunity, a promoter sequence which is often derived from cytomegalovirus cmv or rous dont take coumadin sarcoma virus rsv, an mrna stability polyadenylation region at the end of the insert, and dont take coumadin the plasminogen activator gene which controls the secretion of the recombinant product in addition, there are dont take coumadin an origin of replication for the amplification of the plasmid in bacteria, and a gene dont take coumadin buy augmentin overnight for antibiotic resistance to select the transformed bacteria immunization procedures with dna vaccines are carried out by the intramuscular and, to a lesser extent, the intraepidermal route other routes include dont take coumadin the oral, nasal, vaginal, intravenous, intraperitoneal and subcutaneous routes, intramuscular injection of dna vaccines leads dont take coumadin to such types of immunity as ctl, this was unexpected because antigen presentation requires the function dont take coumadin of professional apc however, myocytes which were shown to take up the plasmid only to a small extent and with only a fraction of cells participating in the uptake, are not professional apcs although myocytes carry mhc class i molecules and can present endogenously produced dont take coumadin viral peptides to the cd cells to induce ctls, they do so inefficiently as they dont take coumadin 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 dont take coumadin responses occur as a result of the transfer of antigenic material between the myocytes and dont take coumadin professional apc to some extent in parallel, it could also be that plasmid secreted by 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 transport to the lymph nodes or spleen on the other hand, cd cells may be dont take coumadin activated by apcs via mhc class ii presentation of antigen secreted by the myocytes or 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 dont take coumadin been shown that dendritic cells are the essential apc involved in immune responses elicited by intramuscularly dont take coumadin given dna vaccines dna vaccination via liposomes vaccination with naked dna by the intramuscular route dont take coumadin is dependent on the ability of myocytes to take up the plasmid however, some of the dna may also be engulfed by apc infiltrating the site of injection, or in dont take coumadin the lymph nodes following migration of the dna to the lymphatics the extent of dna degradation by extracellular deoxyribonucleases is unknown, but depending on the time of its residence interstitially, degradation dont take coumadin could be considerable therefore, approaches that protect dna from the extracellular nucleases and promote dna dont take coumadin uptake by cells more efficiently, or target it to apc, should contribute to the optimal dont take coumadin 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 dont take coumadin avidly by apc infiltrating the site of injection or in the lymphatics, an event that has 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, dont take coumadin the structural versatility of the system would ensure that its tranfection efficiency is further improved dont take coumadin by the judicial choice of its structural characteristics or by the coentrapment of cytokine genes, other adjuvants eg immunostimulatory sequences, or indeed protein antigens see later together with the plasmid vaccine dont take coumadin as a number of injectable liposomebased drug formulations, including vaccines against hepatitis a and influenza, dont take coumadin have been already licensed for clinical use, acceptance of the system clinically would be less dont take coumadin problematic than with other systems that are still at an experimental stage procedure for the dont take coumadin entrapment of plasmid dna into liposomes ready to use mlv, =nm dna, antigen or both dont take coumadin entrapped over entrapment yield a variety, of plasmid dnas have been quantitatively entrapped into liposomes by dont take coumadin 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 dont take coumadin controlled rehydration of the formed powder, and centrifugation to remove non entrapped material formed liposomes dont take coumadin 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 dont take coumadin expectcd, dna incorporation values, were higher up to of the amount used when a cationic dont take coumadin lipid was present in the bilayers no apparent relationship was observed between amount of dna used g and the values of incorporation for the compositions and lipid mass used, the dont take coumadin possibility that dna was not entrapped within the bilayers of cationic liposomes, but was rather dont take coumadin complexed with their surface as suggested by the high fig entrapment of dna andor protein into dont take coumadin cationic liposomes the procedure entails mixing up empty suv with the solutes destined for entrapment dont take coumadin and subsequent dehydration on rehydration, most of the solutes is recovered entrapped within the generated dont take coumadin 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 cationic suv and prccmv hbs before com plexed dna and after entrapped dna dehydrationrehydration of the mixture !?