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Introduction: The human multidrug resistance gene ATP-binding cassette B1 (ABCB1) codes for P-glycoprotein (P-gp), an important membrane-bound efflux transporter known to confer anticancer drug resistance as well as affect the pharmacokinetics of many drugs and xenobiotics. A number of single nucleotide polymorphisms (SNPs) have been identified throughout the ABCB1 gene that may have an effect on P-gp expression levels and function. Haplotype as well as genotype analysis of SNPs is becoming increasingly important in identifying genetic variants underlying susceptibility to human disease. Three SNPs, 1236C→T, 2677G→T and 3435C→T, have been repeatedly shown to predict changes in the function of P-gp. The frequencies with which these polymorphisms exist in a population have also been shown to be ethnically related. Methods: In this study, 95 individuals representative of the entire ethnic make-up of the USA were compared with 101 individuals from an Ashkenazi-Jewish population. These individuals were analyzed by genomic sequencing and polymerase chain reaction, using restriction fragment length polymorphisms, to calculate their genotype frequencies. Results: A total of 25 SNPs were located in the exons of the ABCB1 gene. All of the polymorphisms identified were in parts of the ABCB1 gene product predicted to be intracellular, and 16 appear to be novel as compared with those listed by the National Center for Biotechnological Information. Frequencies of the 1236C→T and 2677G→T/A/C SNPs were similar for the US and Ashkenazi populations (64.2 and 60.4%, respectively for 1236C→T [χ2: 0.30; p ≤ 1]; 55.8 and 64.4%, respectively for 2677G→T/A/C [χ2: 1.49; p ≤ 1]), but were different for 3435C→T (24.2% for the US population and 69.3% for the Ashkenazi population [χ2: 39.927; p ≤ 0.001]). The 1236T/ 2677T/3435T haplotype occurred in 23.6% (standard error: 0.013) of the Ashkenazi population. Conclusion: The SNP at location 3435C→T plays a significant role in the ABCB1 gene. The haplotype and genotype analysis from these data may be used as a basis for studies on the relationship between ABCB1 genotypes and drug efficacy, drug toxicity, disease susceptibility or other phenotypes.

Only a few studies have addressed the cost-effectiveness of pharmacogenetics interventions in healthcare. Lack of health economics data on aspects of pharmacogenetics is perceived as one of the barriers hindering its implementation for improving drug safety. Thus, a recent Institute for Prospective Technological Studies (IPTS) study, entitled 'Pharmacogenetics and pharmacogenomics: state-of-the-art and potential socio-economic impact in the EU' included an explorative cost-effectiveness review for a pharmacogenetic treatment strategy compared with traditional medical practice. The selected case study examined the cost-effectiveness of thiopurine methyltransferase (TMPT) genotyping prior to thiopurine treatment in children with acute lymphoblastic leukemia (ALL). Information for the cost-effectiveness model parameters was collected from literature surveys and interviews with experts from four European countries (Germany, Ireland, the Netherlands and the UK). The model has established that TPMT testing in ALL patients has a favorable cost-effectiveness ratio. This conclusion was based on parameters collected for TPMT genotyping costs, estimates for frequency of TMPT deficiency, rates of thiopurine-mediated myelosuppression in TPMT-deficient individuals, and myelosuppression-related hospitalization costs in each of the four countries studied. The mean calculated cost per life-year gained by TPMT genotyping in ALL patients in the four study countries was €2100 (or €4800 after 3% discount) based on genotyping costs of €150 per patient. Cost per life-year gained is expected to further improve following the introduction of the wider use of TMPT genotyping and the availability of lower cost genotyping methods. Our analysis indicates that TPMT genotyping should be seriously considered as an integral part of healthcare prior to the initiation of therapy with thiopurine drugs. © 2006 Future Medicine Ltd. Chemicals / CAS: azathioprine, 446-86-6; mercaptopurine, 31441-78-8, 50-44-2, 6112-76-1; thiopurine methyltransferase, 67339-09-7; Antineoplastic Agents; Methyltransferases, EC 2.1.1.-; thiopurine methyltransferase, EC 2.1.1.67

Both the extent and location of the maternal ancestral deme from which the Ashkenazi Jewry arose remain obscure. Here, using complete sequences of the maternally inherited mitochondrial DNA (mtDNA), we show that close to one-half of Ashkenazi Jews, estimated at 8,000,000 people, can be traced back to only 4 women carrying distinct mtDNAs that are virtually absent in other populations, with the important exception of low frequencies among non-Ashkenazi Jews. We conclude that four founding mtDNAs, likely of Near Eastern ancestry, underwent major expansion(s) in Europe within the past millennium. This research was supported in part by Israeli Science Foundation grants (to K.S.), the Annie Chutick Endowment and Technion (to K.S.), the Estonian Science Foundation (to E.M. and T.K.), European Union Framework Programme Genemill and Genera grants (to R.V.), the Consiglio Nazionale della Ricerche–Ministro dell'Istruzione dell'Univerita e della Ricerca (CNR-MIUR) Genomica Funzionale-Legge 449/97 (to A.T.), the Fondo Investimenti Ricerca di Base 2001 (to A.T.), the Progetti Ricerca Interesse Nazionale 2005 (to A.T.), the National Science Foundation (to N.H.), and Programa Operacional Ciência, Tecnologia e Inovação (to A. Amorim).

For 50 years, pharmacogenetics has been studying the genetic basis for variability in drug response between individual patients, both with respect to drug toxicity and drug efficacy. Following the completion of The Human Genome Project about three years ago, and the development of technologies allowing rapid identification of polymorphic alleles using DNA chips, pharmacogenetics would soon allow the introduction of personalized medicine. For most medical disciplines, this would allow pharmacotherapy according to each patient's individual genetic data. This would allow a reduction in the rates of adverse drug reactions, currently responsible by American and European estimates for about 6% of new admissions to internal medicine wards, and causing more morbidity annually than road accidents or breast cancer. The purpose of this review is to delineate the principles of pharmacogenetics, focusing on the aspects closest to implementation in the clinic: the polymorphism of liver CYP450 metabolic enzymes, mostly CYP2D6 and CYP2C19. We shall also review current efforts to better understand the scope of human genome diversity, and present several examples for variability in drug efficacy and genetic polymorphism of drug target genes. Education of health professionals in pharmacogenetics as part of their pharmacology curricula, and explaining its potential to the general public, would be indispensable for the success of personalized medicine.

Background The antiphospholipid syndrome (APS) commonly affects the central nervous system through mechanisms that may include small vessel pathology and activation of thrombin. Antiphospholipid antibodies (aPL) activate endothelial cells but the specific activation of brain vascular endothelial cells (BVEC) and the receptors and signaling pathways involved have not been fully characterized. Objective To examine whether aPL, the inflammatory stimulant lipopolysacharide (LPS) and thrombin activate BVECs through a Ras-dependent pathway. Methods Rat BVEC (G8) were grown to confluence on 24-well plates. IgG was purified from 8 APS patients on a protein G column. Phosphorylation of ERK in the BVEC was measured by immunoblot utilizing a specific antibody. Results Significant phosphorylation of ERK was measured following exposure of the cells to LPS and thrombin and this was blocked by the Ras inhibitor farnesylthiosalicylate (FTS). aPL IgG (1:100 relative to serum) from 7/8 patients also induced phosphorylation of ERK. Conclusions Activation of the Ras-ERK pathway is an effect of both APS IgG and thrombin. This pathway is potentially amenable to drugs such as FTS and may serve as a therapeutic target in APS.

Transdermal nicotine patches have been successfully introduced as a safe and powerful aid to smoking cessation; this has contributed to the rising interest in additional therapeutic applications for nicotine and synthetic nicotinic agonists. Nicotine and nicotinic agonists may have a therapeutic potential for a variety of disorders, including Alzheimer's and Parkinson's diseases, depression, attention deficit disorder, Tourette's syndrome and ulcerative colitis. These interests are partially fuelled by the urgent need of the tobacco industry to find new niches for nicotine in a world bound eventually to retire from cigarette smoking. At the same time, there is an increased interest in developing drugs for fighting obesity, a growing affliction of industrialised nations. This review presents data on the potential of nicotine, and in particular synthetic nicotinic agonists, for controlling body weight. Nicotinic agonists may become relatively safe, effective and inexpensive alternatives for several optional drugs currently being developed for treating human obesity, including beta-3-adrenergic agonists, leptin and its agonists, and neuropeptide Y antagonists.

The cytochrome P450 isoenzymes CYP2C19 and CYP2D6 catalyze reactions involved in the metabolism of many widely used drugs. Their polymorphisms give rise to important interindividual and interethnic variability in the metabolism and disposition of several therapeutic agents and may cause differences in clinical response to some drugs. Individuals who carry two null alleles of either gene are known as poor metabolizers (PMs), while those who carry more than two copies of the functional CYP2D6 gene are ultrarapid metabolizers (UMs). The aim of the current study was to genotype Israelis from four different ethnic backgrounds with respect to CYP2C19 and CYP2D6. Polymorphisms of the CYP2C19 and CYP2D6 genes were determined by genotyping the four ethnic groups using PCR and/or restriction fragment length polymorphism (RFLP) analysis. The groups consisted of three Jewish communities, Yemenite Jews (n = 36), Sephardic Jews (n = 47), Ethiopian Jews (n = 28), and one Arabian population, Bedouins (n = 50). CYP2C19*2 allele frequencies ranged from 12.0 to 19.6% among the four ethnic groups. Within the study population, the CYP2C19*3 gene was only found in one Bedouin individual, in the heterozygous state (CYP2C19*1/*3). In each group, one individual was homozygous for CYP2C19*2, and were predicted to be PMs. The data revealed a high prevalence of CYP2D6*2, *4, *10, *41, and gene duplication, followed by *5 and *17, while *3 was very rare. The frequencies of the CYP2D6*4, *10, and *17 alleles and CYP2D6 gene duplication were significantly different among the four groups. However, the CYP2D6*2, *3, and *5 and *41 alleles showed similar frequencies in the four groups. Four (8.5%) Sephardic Jews and one (2.0%) Bedouin were found with the genotype CYP2D6*4/*4 (two null alleles), and were thus presumably PMs. A total of 15 individuals, distributed in all groups, were found with functional CYP2D6 gene duplications. The frequencies of predicted UMs (duplication of CYP2D6) were 17.8% (5/28) and 12.8% (6/47) in Ethiopian Jews and Sephardic Jews, respectively, which were higher than that of Yemenite Jews (5.6%, 2/36) and Bedouins (4.0%, 2/50). This is the first study of the CYP2D6 gene polymorphism in Israeli ethnic groups, either Jewish or Arab. Furthermore, this is also the first study of the CYP2C19 gene polymorphism in Jewish or Arab subgroups living in Israel. The frequencies of various alleles for the CYP2D6 gene are significantly different among the ethnic groups in Israel. These new findings may have important clinical implications in administrating drugs metabolized by CYP2D6 and for CYP2D6-related adverse drug reactions in the Israeli population.

all students attend common classes, and then each selects an area of specialization: molecular genetics – cytogenetics, patho-biochemistry and pharmacokinetics – toxicology, molecular anatomy, or neuroscience. By training together in an academic medical center, budding researchers with a medical or biological background exchange ideas, benefit from each other’s experiences, build a common language and sustain an environment that enables continuous inter-disciplinary communication. The basic scientists acquire an understanding of the biological basis of disease and develop a therapeutic mindset, while their physician peers gain insight into the mechanisms underlying the clinical manifestations they have previously sought to treat. This dialogue between ‘clinicians’ and ‘scientists’ erases the dividing boundaries and it bridges the gap between hypothesis-driven basic science and problem-focused medical research. We believe that this training scheme provides vital space for personal development; indeed, it encourages non-traditional career trajectories, from which leaders in biomedicine can spring. However, the dialogue between those in the medical and biological sciences needs to be accompanied by a broader dialogue among those in life sciences, social sciences and humanities, so that biomedical advances will benefit the health of all. The fruits of molecular medicine, particularly personalized medicine, pharmacogenetics and pharmacogenomics, promise to exceed the Hippocratic ideal ‘to help, or at least to do no harm’ [10] and to achieve a higher standard of healthcare: to help and also to do no harm[11] .T raining in humanitarian aspects of science and medicine is easily overlooked when focusing on knowledge transfer and

CYP2C9 is a major P450 2C enzyme, which hydroxylates about 16% of drugs that are in current clinical use and contributes to the metabolism of a number of clinically important substrate drugs such as warfarin. Ethnic differences in the genetic variation of CYP2C9 have been reported, and might be related to the frequencies of adverse reactions to drugs metabolized by CYP2C9 in different ethnic groups. In the present study, ethnic differences in the CYP2C9*2 and CYP2C9*3 allele distribution in Japanese and Israeli populations were evaluated using a newly developed oligonucleotide based DNA array (OligoArray(R)). The population studied consisted of 147 Japanese and 388 Israeli donors (100 Ashkenazi Jews, 99 Yemenite Jews, 100 Moroccan Jews and 89 Libyan Jews). The CYP2C9*2 [Arg144Cys (416 C>T), exon 3] and CYP2C9*3 [Ile359Leu (1061 A>C), exon 7] genotypes were determined using an OligoArray(R). The accuracy of genotyping by the OligoArray(R) was verified by the fluorescent dye-terminator cycle sequencing method. A Hardy-Weinberg test indicated equilibrium (chi(2)

It is becoming increasingly evident that the implementation of true personalized medicine will not come as rapidly and smoothly as initially hoped. In the aftermath of the drafting of the human genome in 2001, the popular and scientific media featured numerous commentaries heralding the approaching arrival of personalized medicine to the clinic, and describing its huge benefits for patients. Media coverage predicted a drastic transformation for the practice of medicine, second to the revolution brought about by the discovery of vaccines and antibiotics. From the perspective of another 3 years, during which substantial new insights were made into the enormous complexity of human genome variation, it seems that true personalized medicine may still be decades away for many aspects of medical treatment. Nonetheless, the prospects for implementation of at least certain elements of personalized medicine for one key discipline, psychiatry, might be relatively close and more realistic. With the correct focus, realization of some benefits of genetic patient profiling for psychiatric pharmacotherapy might be near, and in due course, lead the way for true personalized psychiatry.

The serotonin transporter (5-HTT) plays a critical role in the termination of serotonin neurotransmission and represents the prime target for selective serotonin reuptake inhibitors (SSRIs). In the present study, the 5-HTT protein in human peripheral blood lymphocyte was characterized pharmacologically and biochemically. The tricyclic antidepressant drug [ 3 H]imipramine, an established ligand for the neuronal and platelet 5-HTT, bound saturably and reversibly to a single population of non-interacting binding sites in fresh human peripheral blood lymphocytes. The affinity of [ 3 H]imipramine ( K d ) to the transporter, calculated from association and dissociation kinetic experiments, was similar to that obtained from the equilibrium study. The function of the transporter was studied using high affinity [ 3 H]5-HT uptake into fresh lymphocytes. [ 3 H]Imipramine binding and [ 3 H]5-HT uptake were inhibited by tricyclic antidepressants as well as by SSRIs. Western blot analysis as well as immunoprecipitation analysis revealed labeling of a single protein band of approximately 100 kDa. The presence of the 5-HTT in easily accessible nucleated cells such as peripheral blood lymphocytes might permit molecular genetic studies in mood and anxiety disorder patients, and might enhance the understanding of the different efficacies of antidepressants in depressed patients.

High affinity choline uptake plays a critical role in the regulation of acetylcholine synthesis in cholinergic neurons. Recently, we succeeded in molecular cloning of the high affinity choline transporter (CHT1), which is specifically expressed in cholinergic neurons. Here we demonstrate the presence of functionally relevant, nonsynonymous single nucleotide polymorphism in the human CHT1 gene by comprehensive sequence analysis of the exons and the intron/exon boundaries including the transcription start site. The deduced amino acid change for the polymorphism is isoleucine to valine at amino acid 89 (I89V) located within the third transmembrane domain of the protein. The allele frequency of I89V was 6% for Ashkenazi Jews. Functional assessment of the I89V transporter in mammalian cell lines revealed a 40-50% decrease in V(max) for choline uptake rate compared with the wild type, whereas there was no alteration in the apparent affinities for choline, sodium, chloride, and the specific inhibitor hemicholinum-3. There also was no change in the specific hemicholinum-3 binding activity. The decreased choline uptake was not associated with the surface expression level of the protein as assessed by biotinylation assay. These results suggest an impaired substrate translocation in the I89V transporter. The Caenorhabditis elegans ortholog of CHT1 has a valine residue at the corresponding position and a single replacement from valine to isoleucine caused a decrease in the choline uptake rate by 40%, suggesting that this hydrophobic residue is generally critical in the choline transport rate in CHT1. This polymorphism in the allelic CHT1 gene may represent a predisposing factor for cholinergic dysfunction.

The lack of accurate and reliable tools for early diagnosis of Alzheimer’s disease (AD) hampers the timely identification of affected individuals, which is crucial for effective treatment. Current pharmaceutical treatments for AD, such as acetylcholinesterase inhibitors, estrogen (for female patients), non-steroidal anti-inflammatory drugs and anti-oxidants, mostly benefit individuals in the early stages of the disease. This is probably true for the entire spectrum of pharmacological treatments for neurodegenerative disorders. The quest for better AD diagnosis therefore continues, along with the search for better AD therapeutics.The most renowned hallmark of AD is the brain-selective accumulation of extracellular senile plaques that are composed mostly of aggregated β-amyloid (Aβ) peptides. The magnitude of these plaques is highly correlated with disease duration and severity, but can only be accurately assessed post-mortem. Accordingly, earlier diagnostic tools were aimed at measurements of soluble Aβ peptide concentrations in the plasma of AD patients or their cerebrospinal fluid (CSF) that more closely reflect brain amyloid loads. Notably, aggregated forms of Aβ, more relevant for diagnosis, are not found in body fluids. It now appears, however, that levels of soluble Aβ peptides are not reliable markers for AD diagnosis and evaluation of disease progression. This is due to large inter-individual variation in rates of soluble Aβ aggregation, deposition in brain plaques, and plaque removal, reflecting differences in immune and tissue repair mechanisms.Now, Skovronsky et al.1xIn vivo detection of amyloid plaques in a mouse model of Alzheimer’s disease. Gurwitz, D.M. et al. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7609–7614Crossref | PubMed | Scopus (171)See all References1 report a new method that circumvents such difficulties by introducing a prototype tracer compound for in vivo detection and measurement of Aβ deposits in brain plaques. The new lipophi-lic probe, (trans, trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) was administered to transgenic mouse models of AD amyloidosis (Tg2576), in which AD-like brain Aβ deposits are formed from a mutated human amyloid precursor protein, implicated in familial AD cases. BSB crosses the blood–brain barrier and labels plaques selectively so that they can be visualized by imaging. Following i.v. administration in these mice, BSB specifically labeled brain plaques of Aβ with high sensitivity, as detected using in vitro fluorescent techniques. Future studies should determine if BSB-like radiopharmaceuticals would detect brain amyloid deposits in vivo. It would also be essential to show that BSB-like compounds do not adversely affect Aβ plaque build-up.Cotman et al.2xAgrin binds to beta-amyloid (Amyloid-beta), accelerates Amyloid-beta fibril formation, and is localized to Amyloid-beta deposits in Alzheimer’s disease brain. Cotman, S.L. et al. Mol. Cell Neurosci. 2000; 15: 183–198Crossref | PubMed | Scopus (107)See all References2 report another promising strategy that uses brain-penetrating radioactive or spin-labeled analogs of agrin, an extracellular matrix heparan sulfate proteoglycan, shown to bind Aβ avidly and to be localized in AD brain senile plaques and cerebrovascular amyloid deposits. An intense race is on to develop more effective AD therapies, aimed at slowing Aβ deposition in brain plaques. Hopefully, reliable in vivo imaging tools will become viable by the time such new therapies reach the market.

Lack of knowledge regarding genotype–phenotype correlations is often cited as the major barrier delaying the uptake of pharmacogenomics into routine medical practice. When we look forward to genome-wide association studies as one of the most promising tools for overcoming the pharmacogenomics knowledge barrier, we must keep in mind that having large patient cohorts may not help improve our understanding of alleles implicated in drug-response phenotypes, unless we ensure that such phenotypes are precise and pertinent. It may be wiser, and far more cost effective, to invest scarce research funding in accurate patient drug-response phenotyping than to genotype (or fully sequence) hundreds to thousands of study participants. Biobanks created with personalized medicine research in mind should, when possible, have access to donors’ clinical data, including detailed disease- and drug-response phenotypes.

It is a well-known and sad fact that there is very limited functional recovery following injury to the spinal cord. The social, psychological and financial burden of such calamities, typically involving car- and sport-related accidents, is enormous. It remains a mystery why mammals are unable to repair their damaged spinal cord to any great extent, while reptiles and amphibians are capable of an almost complete functional recovery of damaged spinal cord tissues within a few weeks of injury. In the past decade, it has become clear that the massive neuronal loss following traumatic brain and spinal cord injuries reflects not merely a lack of regeneration, but also a self-propagating spread of the damage, triggered by the primary insult. Michal Schwartz and her colleagues at The Weizmann Institute of Science have been studying this enigma meticulously, based on the principle that mammalian spinal cords [and other central nervous system (CNS) tissues] cannot be repaired because, unlike the reptilian CNS, they are immune privileged and, thus, are not readily accessible to the beneficial repair and maintenance processes involving immune interactions.Now, a new study from this laboratory[1xPassive or active immunization with myelin basic protein promotes recovery from spinal cord contusion. Hauben, E. et al. J. Neurosci. 2000; 20: 6421–6430PubMedSee all References[1] shows that there might be hope for damaged spinal cords if the immune system is stimulated by active or passive immunization with self components of the damaged CNS, for example, myelin basic protein (MBP). Rats were subjected to spinal injury by contusion and injected systematically with anti-MBP T cells, either at the time of contusion, or a week later. Control rats underwent a similar contusion injury, but were injected with anti-ovalbumin T cells instead. The recovery of motor activity, measured by open-field behavior at intervals up to three months post-injury, was significantly better in the MBP-sensitized animals than in the control animals. Hauben et al. used magnetic resonance imaging (MRI) to show that this correlated with the levels of spinal tissue preservation. Remarkably, injection of anti-MBP T cells a week after the injury resulted in similar functional recovery, indicating that the post-traumatic therapeutic window for prevention of damage spread is relatively wide and, at least in rats, lasts for one week.As therapy with T cells requires immune compatibility between the donor and recipient, similar human therapeutic strategies would most probably require ex-vivo activation of the patient's own T cells and their re-administration. However, surprisingly, this study shows that beneficial autoimmunity was also achieved by active immunization with MBP. This is the first ever demonstration of successful vaccination-mediated neuroprotection from traumatic neuronal damage. The immune-privileged status of the mammalian brain might have been an evolutionary inevitability in order to develop its intricacy. The poor capacity to repair and protect traumatic brain tissue is the steep price for such advantages, a price also reflected in the relatively large ischaemic stroke-associated brain damage in humans compared with lower creatures. It now seems that recruiting the immune system would allow novel strategies for circumventing this evolutionary price.

Access to samples and individual DNA sequence data from children included in population biobanks should, when feasible, await their consent as adults.

Activation of receptor tyrosine kinases stimulates a diverse array of cellular responses such as proliferation and differentiation. The first events in the signal transduction pathways mediated by different receptor tyrosine kinases are similar and include activation of the mitogen-activated protein kinase (MAPK) pathway and the induction of immediate early genes. The precise signaling pathways leading to each of the cellular responses mediated by receptor tyrosine kinases are still unknown, although it has been proposed that sustained activation of the MAPK pathway by receptor tyrosine kinases such as the nerve growth factor (NGF) receptor TrkA is sufficient to induce differentiation in PC12 cells. In the present study we examined the effect of NGF on mutant PC12 cells that were derived spontaneously in our cultures. NGF induced normal activation of immediate early genes in these cells, whereas the activation of some delayed response genes, as well as neurite outgrowth, was impaired. Furthermore, activation of the NGF-induced extracellular signal-regulated kinase (ERK) in these cells was transient, not sustained. These results support the hypothesis that sustained activation of ERK plays an important role in activating the induction of delayed response genes. However, sustained ERK activation is not a mandatory condition for the promotion of all the features of differentiated PC12 cells, as NGF could induce transcription of the delayed response gene, transin, in PC12 mutant cells. Taken together, our results suggest that NGF induces differentiation of PC12 cells via several signaling pathways, an important one of which is the MAPK pathway.

Restoration of ACh levels or replacement with an M1 (or m1) muscarinic agonist may be effective in treating at least some of the cognitive symptoms in Alzheimer’s disease (AD), (see Court and Perry, 1991; Fisher and Barak, 1994). Treatment approaches for AD have to address abnormalities occurring also along various signal transduction pathways (Harrison et al., 1991). Novel activities associated with m1 muscarinic receptors (m1 mAChR) indicate that m1 agonists may also activate hypofunctional signalling pathways in AD (Fisher and Barak, 1994; Gurwitz et al., 1994).

Despite the declining cost of ‘personal genomes’ and the acknowledged usefulness of individual genome sequences to understand human health, for the near future, personal genomes alone are unlikely to become the leading research tool in genomic medicine. I argue that, at present, expression profi ling studies are the most promising and cost­eff ective tool for discovering new disease and drug­response biomarkers. $1,000 personal genomes? Not around the corner Th e proliferation of studies on personal genomes is fueled by reduced sequencing costs and improved bio­ informatics software tools. Substantial advances in understanding human biology in health and disease ­ and improved diagnostic capacities ­ are foreseen with the availability of personal genome sequences [1,2]. Yet, ex­ pec tations from personal genomes should not be over­ stated: individual genome sequencing per se ­ even when combined with comprehensive individual medical records ­ will not suffi ce to decipher the overwhelming complexity of human biology and disease pathology. Our current level of understanding of biology in general, and human biology in particular, is simply too low to interpret the information encoded in individual DNA sequences [3]. Moreover, even with dramatically reduced DNA se­ quen cing costs, the high costs of data interpretation subsequent to the tremendous bioinformatics eff ort of analyzing 6.2 Gb nucleotides of two copies of one individual genome means that personal genome sequen­ cing will not become clinically routine in the near future. Currently, at the 10th anniversary of the Human Genome Project’s conclusion, whole genome sequencing is being put into practice in leading medical academic centers, predominantly for cancer diagnosis and treatment. But it will be many years before it becomes a widespread, routine clinical tool.

In their Policy Forum "The complexities of genomic identifiability" (18 January, p. [275][1]), L. L. Rodriguez et al. correctly point out that in view of the intriguing recent demonstration of the identifiability of donors participating in genomic studies ("Identifying personal genomes by surname inference," M. Gymrek et al. , Reports, 18 January, p. [321][2]), the research community needs to optimize the balance between the need for data sharing and respect for the privacy of research participants. In our 2009 Science Policy Forum ([ 1 ][3]), we proposed specific measures to improve the current policies and provide greater protection for children. Children are among the most vulnerable populations whose DNA samples are being collected in large numbers for epidemiologic studies. Sadly, it seems that our suggestions have not led to distinct biobanking policies for children. We reiterate our 2009 call for the scientific research community to agree on extra safeguards, particularly restrictions on sharing the individual genome sequences of children, unless they are contacted again as adults and provide their own consent at that time. In the case of fatal pediatric disease research (when obtaining adult and consented donor samples is implausible), conditions for sharing the personal genome sequences of children could be eased. In spite of the current genetic nondiscrimination legislation widely enacted, our duty remains to ensure extra privacy protections for children, balanced with the need to continue research on pediatric diseases. 1. [↵][4] 1. D. Gurwitz, 2. I. Fortier, 3. J. E. Lunshof, 4. B. M. Knoppers , Science, 325, 818 (2009). [OpenUrl][5][Abstract/FREE Full Text][6] [1]: /lookup/doi/10.1126/science.1234593 [2]: /lookup/doi/10.1126/science.1229566 [3]: #ref-1 [4]: #xref-ref-1-1 "View reference 1 in text" [5]: {openurl}?query=rft.jtitle%253DScience%26rft.stitle%253DScience%26rft.issn%253D0036-8075%26rft.aulast%253DGurwitz%26rft.auinit1%253DD.%26rft.volume%253D325%26rft.issue%253D5942%26rft.spage%253D818%26rft.epage%253D819%26rft.atitle%253DChildren%2Band%2BPopulation%2BBiobanks%26rft_id%253Dinfo%253Adoi%252F10.1126%252Fscience.1173284%26rft_id%253Dinfo%253Apmid%252F19679798%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [6]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6Mzoic2NpIjtzOjU6InJlc2lkIjtzOjEyOiIzMjUvNTk0Mi84MTgiO3M6NDoiYXRvbSI7czoyNToiL3NjaS8zMzkvNjEyMy8xMDMzLjEuYXRvbSI7fXM6ODoiZnJhZ21lbnQiO3M6MDoiIjt9

Hyperphosphorylated tau proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that tau phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m1 muscarinic acetylcholine receptor. Stimulation of m1 receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased tau phosphorylation, as indicated by specific tau monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on tau phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of tau microtubule-associated protein.

The AF series compounds, AF102B and congeners of AF150(S), are functionally selective agonists for m1 muscarinic receptors (m1AChRs). This is shown in stable transfected CHO and PC12 cells (PC12M1) with m1m5AChRs and m1AChRs, respectively. AF102B and AF150(S) are partial agonists, but AF150, AF151, and AF151 (S) are full agonists in stimulating phosphoinositides hydrolysis or arachidonic acid release in these cells. Yet, all these compounds behave as antagonists when compared with carbachol in elevating cAMP levels. In PC12M1 cells, unlike carbachol, the AF series compounds induce only minimal to moderate neurite outgrowth. Yet, these agonists synergize strongly with NGF, which by itself mediates only a mild response. Stimulation of m1AChRs by AF102B, AF150(S) and AF151(S) in PC12M1 cells enhances secretion of beta/A4 amyloid precursor protein derivatives (APPs). The enhanced APPs secretion induced by AF102B is potentiated by NGF. AF102B also stimulates APPs secretion from rat cortical slices. Stimulation of m1AChR in PC12M1 cells with carbachol or AF102B decreases tau phosphorylation as indicated by specific tau-1 mAb and alkaline phosphatase treatment. Due to the above mentioned properties m1 agonists may be of unique value in delaying the progression of Alzheimer's disease (AD). The AF series compounds show a wide safety margin and improve memory and learning deficits in animal models for AD. There is a dearth of clinical reports on m1 agonists. These include studies on AF102B and xanomeline, another m1 selective agonist. We tested AF102B in escalating doses of 20, 40, 60 mg, tid, po, (each dose for 2 weeks) for a total of 10 weeks. This was a single-blind placebo-controlled, parallel-group study in patients with probable AD. AF102B was significantly effective at 40 and 60 mg, tid in the ADAS, ADAS-cognitive and ADAS-word recognition scales.

Epidemiologic studies suggest that the age-related decline in dehydroepiandrosterone (DHEA) levels may be associated with Alzheimer's disease (AD). Cholinergic markers also decline with age, and are associated with AD pathology. Activation of m1AChR-transfected PC12 cells (PC12M1) with cholinergic agonists results in secretion of Alzheimer's beta-amyloid precursor protein (APP) which in turn reduces beta-amyloid production. This study examined whether DHEA affects APP processing in m1AChR-transfected PC12 cells. DHEA treatment did not significantly alter basal or m1AChR-stimulated APP secretion. However, DHEA (0.1 microM) significantly diminished the desensitization of APP secretion in cells exposed to carbachol for 24 h. The effect of DHEA on APP processing is probably not related to up-regulation of m1AChR or increased m1AChR-activated phosphoinositide hydrolysis since these parameters did not change following DHEA treatment. These findings imply a possible involvement of DHEA in APP processing. Thus, the age-associated decline in DHEA levels may contribute to decreased APP secretion and a consecutive increase in beta-amyloid deposition, which in turn may play a role in the development of AD.

The non-selective muscarinic agonist oxotremorine induces atropine-sensitive neurite outgrowth in PC12 cells stably transfected with m1 muscarinic receptors. In contrast, AF102B, an M1-selective muscarinic agonist, mediated minimal neurite outgrowth in these cells. In the presence of nerve growth factor (NGF) however, it induced atropine-sensitive neurite outgrowth in almost half the cell population. AF102B mediated phosphoinositide hydrolysis, but unlike carbachol, it did not stimulate cyclic AMP accumulation in these cells. These signals were not affected by NGF, indicating that they were not directly responsible for the cholinergic neurotrophic-like response. Our observations suggest that AF102B may improve neuronal responsiveness to neurotrophic factors, and thus may provide another beneficial aspect for treating Alzheimer's disease.

To date five structurally different human muscarinic acetylcholine receptor (mAChR) subtypes (ml-m5) proteins have been cloned and expressed in suitable cell systems (Bonner et al., 1987). It is likely that the ml, m2, m3, and m4 AChRs fit the pharmacological definition of the M1, M2, M3 and M4 AChRs, respectively (Buckley et al., 1989; reviewed by Hulme et al., 1990). Muscarinic receptors are members of the G-protein coupled receptor superfamily. The mAChRs have two binding domains, a ligand-binding extracellular (and including membrane-spanning) domain and a G-protein binding intracellular domain. This second domain, by interaction with various G-proteins, controls and modulates second messenger systems. It was shown that the ml, m3 and m5 AChRs are closely related in sequence and apparently are functionally almost similar. When expressed in mammalian cells, these receptor subtypes couple efficiently to phosphoinositide (PI) turnover. The m2 and m4 AChRs are less related to the ml, m3 and m5 AChRs, and when expressed in mammalian cells are efficiently coupled to the inhibition of adenylate cyclase (Bonner et al., 1987; Hulme et al., 1990).

Secretion of amyloid precursor protein (APP) by cultured cells is coupled to several receptors, including m1 muscarinic (m1AChR), and is associated with decreased production of βA4 amyloid. Secreted and cell-associated APP levels were measured in m1AChR-transfected PC12 cells stimulated with the non-selective agonist carbachol or the Mi-selective agonist, AF102B. Secreted APP levels following stimulation with AF102B (5-60 min) were about half compared with carbachol. Yet, following 24 h stimulation with carbachol or AF102B, cell-associated APP levels were similarly decreased. This may be associated with a smaller reduction in APP secretion following 24 h stimulation with AF102B as compared with carbachol. AF102B may therefore have an advantage over non-selective muscarinic ligands for sustained decrease of cell-associated APP.

Activation of transfected muscarinic m1 acetylcholine receptors (m1AChR) has been linked to several signal transduction pathways which include phosphoinositide hydrolysis, arachidonic acid release and cAMP accumulation. In Chinese hamster ovary cells stably transfected with the rat m1AChR gene, carbachol elicited all three responses with EC50 values of 2.6, 3.8 and 76 microM, respectively. However, pilocarpine and the selective muscarinic agonist AF102B activated phosphoinositide hydrolysis (by 94 and 27% vs. carbachol, respectively), while antagonizing carbachol-mediated cAMP accumulation. Carbachol also activated (by 4-fold) adenylyl cyclase in membranes prepared from these cells, indicating independence of this signal from intracellular mediators. Moreover, carbachol and AF102B similarly elevated cytosolic Ca2+ in intact m1AChR-transfected cells. The ligand-selective cAMP accumulation, its independence from Ca2+ and the carbachol-activated adenylyl cyclase in membranes suggest that it represents an independent m1AChR-mediated signal, unrelated to phosphoinositide hydrolysis. Selective muscarinic ligands such as AF102B may independently activate distinct signalling pathways, which may be important for designing cholinergic replacement therapy for treating Alzheimer's disease.

Five human muscarinic acetylcholine receptors (mAChR) (ml–m5), have been cloned and expressed in suitable cell systems (reviewed by Hulme et al., 1990). * mAChRs have two binding domains, a ligand-binding extracellular (and including membrane-spanning) domain and a G-protein binding intracellular domain. This second domain, by interaction with various G-proteins, controls and modulates second messenger systems (Hulme et al., 1990).

1998 marked the bicentennial for the publication of Edward Jenner's first manuscript on successful immunization against smallpox – a manuscript that changed modern medicine forever and the course of human history. It is amazing to think that more than two hundred years had to pass until the potential of the concept of immunization would begin to be realized for non-infectious diseases. Alzheimer's disease (AD), the most widespread and one of the most devastating neurological disorders, remains without effective cure. The culprit of AD is β-amyloid (Aβ), a fibrillar 40–42 amino-acid peptide accumulating in the brains of AD patients and eliciting neuronal cell death. The past two years has witnessed a flood of papers, following the pioneering work of Dale Schenk and his colleagues from Elan Pharmaceuticals (Dublin, Ireland), and addressing the potential of immunization with Aβ itself for slowing amyloid deposition in animal AD models. The logic behind this scheme is that immunization will stimulate the immune system to fight the abnormal pathologies associated with Aβ and thereby accelerate removal of the amyloid plaques. However, concern has been raised that immunization of AD patients with Aβ might initially accelerate the brain's amyloid deposition process, as the peptide crosses the blood–brain barrier and could seed further fibril formation and further neuronal death. Even without this extra risk, the notion of immunization with a toxic peptide does not sound very attractive.A new study by Sigurdsson et al. 1xImmunization with a nontoxic/nonfibrillar amyloid-β homologous peptide reduces Alzheimer's disease-associated pathology in transgenic mice. Sigurdsson, E.M. et al. Am. J. Pathol. 2001; 159: 1–9Abstract | Full Text | Full Text PDFSee all References1 presents new hopes that this concern will soon be resolved. The study employed immunization with an alternative peptide K6Aβ1-30-NH2 that is similar to Aβ, highly soluble in water (unlike natural Aβ), includes the immunogenic Aβ1–11 and Aβ22–28 regions yet is entirely nontoxic and nonfibrillar. Repeated immunization with this Aβ-like peptide reduced amyloid deposition in brains of transgenic Tg2576 APP mice–animals frequently used as an AD model because of their similar brain pathology. After 7 months from the beginning of the protocol, the immunized mice demonstrated a reduced burden of Aβ plaques in both the cortical and hippocampal regions of the brain.It remains to be seen whether this immunization method will also diminish the AD-like memory impairment observed in similar AD animal models. Hopefully, this new approach will open the door for safer human trials in AD patients. Jenner's legacy seems to have been awakened and is thriving – this could trumpet the dawn of a new exciting chapter in modern medicine, where non-infectious diseases, possibly including stroke and nerve trauma 2xProtective autoimmunity: regulation and prospects for vaccination after brain and spinal cord injuries. Schwartz, M. and Kipnis, J. Trends Mol. Med. 2001; 7: 252–258Abstract | Full Text | Full Text PDF | PubMed | Scopus (118)See all References2, will become treatable by vaccination.

Disparities in health outcomes of members of different ancestral or ethnic groups can be observed in both developed and developing countries and continue to be a global concern. Genomic medicine can help toward closing this gap by expanding the knowledge on novel alleles related to disease risk and drug response, their frequencies, and their relation with disease and drug-response phenotypes, in as many countries and ethnic groups as possible. Without such knowledge, genomic medicine cannot deliver upon its promise of contributing to health for all. However, the use of ancestry or ethnicity-related genetic information as a selection criterion for assigning varying levels of access to health care is condemnable. Translational genomic medicine will allow for individualized clinical decision making - doing away with the use of race, ethnicity or ancestry as a proxy. © 2009 BioMed Central Ltd.

The presynaptic cholinergic deficits in Alzheimer’s disease (AD) indicate that a cholinergic replacement therapy might be beneficial in alleviating some of the cognitive dysfunctions in this disorder (Bartus, 1989). However, clinical trials with some muscarinic agonists (e.g. arecoline, oxotremorine, RS86, pilocarpine and bethanechol) ranked from modest improvement to lack of beneficial effects (Potter, 1987; Moos and Hershenson, 1989). It is thus important to understand the drawbacks of the tested muscarinic agonists in order to be able to design better drugs. Molecular genetics studies have revealed the existence of five distinct muscarinic receptors (mAChRs) subtypes (m1-m5; Bonner’s nomenclature) in human and rat brain (Buckley et al., 1989). It is likely that the ml and m4 AChRs fit the pharmacological definition of the M1 AChR, whereas the m2 and m3 AChRs fit the pharmacological definition of the M2 and M3 AChR’s, respectively (Buckley et al., 1989). A loss of presynaptic M2 AChRs was reported in several AD studies. In contrast postsynaptic Ml mAChRs, facilitating cellular excitation, were relatively unchanged (reviewed by Giacobini, 1990). Most of the potent muscarinic agonists, including those which were evaluated in AD patients, show adverse central and peripheral side-effects, and are either non-selective or M2>M1 selective. Thus, they may also activate inhibitory M2 autoreceptors resulting in decreased acetylcholine (ACh) release (reviewed by Potter, 1987).