We analyzed the epistasis of these genetic variations upon the biological and psychological dimensions in the cases and their relatives. Further on, we analyzed the effects of the combined genetic variations on the short – term detoxification treatment efficacy. Finally, being the only not yet investigated variation within this sample, we analyzed the impact of the rs6313 alone on baseline assessment and treatment efficacy. We detected the following results: the couple rs6313 + rs2129575 affected the Leyton -Trait at admission (p = 0.01) (obsessive-compulsive trait), whilst rs1800587 + 5-HTTLPR impacted the Pilowski test at admission (p = 0.01) (hypochondriac symptoms). These results did not survive Bonferroni correction (p ≤ 0.004). This lack of association may depend on the incomplete gene coverage or on the small sample size which limited the power of the study. On the other hand, it may reflect a substantial absence of relevance of the genotype variants toward the alcohol related investigated dimensions. Nonetheless, the marginal significance we detected could witness an informative correlation worth investigating in larger samples.We assessed a set of biological  and psychometric variables  in a sample of 64 alcohol dependent patients, at baseline and after a detoxification treatment. Moreover, we recruited 47 non-consanguineous relatives who did not suffer alcohol related disorders and underwent the same tests. In both groups we genotyped 11 genetic variations (rs1800587; rs3087258; rs1799724;  Further on, given that HTR2A rs6313 was not investigated as a single mutation in this sample of patients, we investigated the HTR2A rs6313 alone impact as a regulator of the biological and psychometric alcohol related variables.Genes coding for: interleukin 1, alpha , was foiewed in . Furtheriewed in ), was foerotonin , a role erotonin –19. IL1Acoholism ,21. TNF disorder  which had papers ,23: despd papers  through 2.2.1.To be included in the study patients had to fulfill the DSM-IV  diagnostic criteria for alcohol abuse/dependence – “primary alcoholism” – they had to have no recent alcohol intake (average period of abstinence prior to the admission to the clinic = 24.0 ± 12.2 h), and to give full voluntary consent to the study. Other inclusion criteria were: absence of serious physical illness , absence of other drug abuse, age between 20 and 75 years old, absence of DSM-IV axis I co-morbidity. The presence of affective symptoms was not considered to be an exclusion criterion. Alcohol abusers who fulfilled the DSM-IV diagnosis of depressive disorder were excluded from the study in the case that a full diagnosed depressive episode was precedent to the onset of the alcohol dependence. Relatives (non-consanguineous) were included in the study if they did not meet criteria for past or current alcohol abuse/dependence and major medical disturbances. Written informed consent was obtained from each participant after approval from the local ethical committee. Patients and their relatives were enrolled at the Drug and Alcohol Addiction Clinic of the Athens University Psychiatric Clinic at the Eginition Hospital in Athens, Greece. For patients, alcohol detoxification program included vitamin replacement  and oral administration of diazepam (10–40 mg daily in divided doses), with gradual taper off over a week. The overall period of detoxification was completed in 4–5 weeks. Patients then followed an inpatient standard treatment program with a short-term psychotherapy of cognitive-behavioral orientation.2.2.The biochemical profile of cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), glutamic oxaloacetic transaminase (SGOT or AST), serum glutamic pyruvic transaminase (SGPT or ALT) and gamma-glutamyltransferase (GGT), were assessed once in relatives and twice in patients. Hematocrit, hemoglobin and thyroid hormonal levels  were measured once in both relatives and alcohol dependents. Patients were evaluated by a set of psychological tests: at admission they were evaluated by the Schedules for Clinical Assessment in Neuropsychiatry (SCAN ) and the2.3.5-HTTLPR; rs1386493; rs1386494; rs1487275; rs1843809; rs4570625; rs2129575; rs6313). Diagnosis, biological and psychometric tests’ scores were treated as dependent variables of the principal analysis. Correlation analysis was employed in order to assess whether socio-demographic variables were associated with the dependent variables. In the event of significant association, socio-demographic variables were treated as covariates in the principal analysis. We employed GMDR Software Beta version 0.7 to investigate the gene to gene interactions [In the principal analysis the independent variable was the epistasis between the investigated variations .3.HTR2A rs6313 + TPH2 rs2129575 on Leyton -Trait at admission  and a similar marginal impact of ILA rs1800587 + 5-HTTLPR on Pilowski test at admission .Sixty four alcohol dependent patients and 47 relatives (non-consanguineous) were included in the study. Patients’ and their relatives’ characteristics are reported in The results are shown in IL1A rs1800587 and 5-HTTLPR combined variations are associated with a significant different score at Pilowski test at admission only in some specific combination of genotypes: the genotype CC at rs1800587 is associated with higher frequencies of subjects who ranked higher than the mean of the Pilowski test, only when it is found to be in combination with the LL or SS genotypes at the 5-HTTLPR. On the other hand, the combination with heterozygosis at the 5-HTTLPR with the same genotype at rs1800587 resulted in a not significant different distribution of subjects who ranked higher or lower than the mean of the Pilowski test. In the second picture, the combination of the CT genotype at rs6313 and the GG genotype at rs2129575 results into statistically significant higher scores at the Leyton – Trait test at admission, along with the other combinations of genotypes which are grey shadowed.To be implemented in the GMDR software the scores were normalized with “0” as the mean and threshold value. The GMDR software computes the frequencies of subjects who score higher  or lower  than “0” which is representative of the mean of the scores at the test under investigation. The scores are differentiated in positive and negative: the mean score given as a threshold, the positive scores stand for subjects who ranked higher than the mean, the negative stand for subjects who ranked lower than the mean. The difference in the frequencies of positive and negative scoring subjects is labeled as significant in the grey shadowed boxes. Light shadowed boxes stand for not significant difference in distribution of positive and negative scores. For example, in the first picture it is shown that 4.TPH2 gene which has been quite consistently reported to be not associated with alcohol dependence. In all facts, even though TPH2 is the rate limiting enzyme for the synthesis of serotonin in central nervous system [TPH2 gene could modulate alcohol dependence [5-HTTLPR and HTR2A rs6313) over a putative good candidate as the TPH2 is. Some recent reports on the significant epistasis interaction between genetic variables that did not cast informative results when singularly investigated [We found a marginal impact of the epistatic effect between the investigated variations and the obsessive-compulsive traits and hypochondriac symptoms. The low level of significance which did not stand the Bonferroni correction, is likely mainly related to the small sample size we investigated here: as formerly reported, the power was sufficient to detect a large effect size. Moreover, the genetic analysis we performed here did not cover the genes that are under investigation, which results in a possible genetic bias: some variations remain hidden in the present investigation that could impact the functionality of the investigated mutations. This exposes our study to a biological stratification bias which is quite common in literature . Finallys system , a molecs system –35 and bs system , recent pendence –19. Thispendence ,39 whichstigated ,41, convstigated , or the


Age-related macular degeneration (AMD), the leading cause of blindness in the Western world, is a complex disease that affects people over 50 years old. The complement factor H (CFH) gene has been repeatedly shown to be a major factor in determining susceptibility to the advanced form of the condition. We aimed to better understand the functional role of this gene in the AMD disease process and assess whether it is associated with earlier forms of the disease.−9).We genotyped SNPs at the CFH gene locus in three independent populations with AMD: (a) extended families where at least 3 family members had AMD; (b) sporadic cases of advanced AMD and (c) cases from the Age-Related Eye Disease Study (AREDS). We investigated polymorphisms and haplotypes in and around the CFH gene to assess their role in AMD. CFH is associated with early/intermediate and advanced AMD in both familial and sporadic cases. In our populations, the CFH SNP, rs2274700, is most strongly associated with AMD and when incorporated into a haplotype with the Y402H SNP and rs1061147, the strongest association is observed (p<10Our results, reproduced in three populations that represent the spectrum of AMD cases, provide evidence that the CFH gene is associated with drusen as well as with advanced AMD. We also identified novel susceptibility and protective haplotypes in the AMD populations. We investigated associations with polymorphisms and haplotypes in the CFH gene. Furthermore, since our cohorts included individuals with earlier stages of AMD, the potential role of CFH in drusen formation was explored.The CFH risk allele rs1061170 ‘C’ and AMD was strongly associated with advanced AMD status in each of the three cohorts: (1) a cohort of families with AMD ; (2) a sporadic case/control cohort: 211 sporadic cases  of advanced AMD and 183 unrelated ophthalmologically evaluated controls .Associations with several other SNPs in the CFH gene and advanced AMD status were also surveyed in these populations and significant associations were also noted for SNPs rs529825, rs800292, rs1061147, rs203674 and rs2274700 . The SNPA subtractive approach utilized 27 haplotypes to determine the combination of SNPs with the lowest p-value of association with AMD using FBAT. This best combination comprised the following 3 SNPs: rs1061147, rs1061170, rs2274700  and lowest in the unaffected controls , as illustrated in In this study, we confirm the strong association between polymorphisms in the complement factor H gene and advanced age-related macular degeneration in 3 independent predominantly Caucasian populations. For the first time, we report this association in families in whom there is increased susceptibility among relatives to develop the condition. The prevalence of the risk allele ‘C’ for the coding SNP rs1061170 (Y402H) present in exon 9 of the gene in cases of advanced AMD is broadly comparable to that previously reported in sporadic case-control populationsWe chose the 7 SNPs to be genotyped based upon those previously shown to be associated with AMD at the CFH gene locus: rs529825, rs3766404, rs1061147, rs800292, rs1061170, rs203674 and rs2274700.There are considerable similarities between the findings in our study of CFH haplotypes and the other three studies previously published on the subject. Hageman and colleaguesLiMaller and coworkersIn summary, our 3-SNP haplotype is a good ‘fit’ for those previously described. Furthermore, we have been able to confirm its validity in 3 independent well-phenotyped populations. Interestingly, apart from the CFH Y402H SNP, the other two are synonymous coding variants, suggesting that these sequence changes do not combine to effect some reduction in protein function but more likely alter expression levels of the CFH gene and possibly other complement factor-related genesIt is now established that the Y402H CFH change is associated with advanced AMD. Whether this polymorphism also contributes to other earlier AMD phenotypes remains to be fully explored. Only one other report has addressed this issue finding increased frequency of the risk allele in individuals with drusenWe found that the frequency of the risk allele was similar among those with advanced AMD in all three groups (approximately 0.60). Interestingly, this same allele frequency is observed in those individuals with intermediate AMD (drusen) with a strong family history of AMD and higher than in those with the same category of AMD in the AREDS cohort. Our future longitudinal studies will evaluate whether this is because individuals with a family history of AMD, who have both extensive drusen and the CFH risk allele are at very high risk of progression to choroidal neovascularization and/or geographic atrophy.Previous studies have failed to show that CFH associates more frequently with the presence of either CNV or GA. In our 3 populations, we also found that neither the SNPs in and around the CFH gene nor the 3-SNP haplotype preferentially increased susceptibility to one of these late complications.In summary, we have examined 3 independent, carefully phenotyped populations with AMD. These populations can be considered to reflect the full diversity of AMD patients: those with familial susceptibility to the condition and 2 case-control populations, one of which was followed for more than 10 years as part of a nationwide clinical trial. We have confirmed the association between the Y402H CFH mutation and advanced AMD finding however the strongest association with another SNP within the gene, rs2274700 in agreement with two other studiesAll studies involving human subjects were approved by the Institutional Review Board of Oregon Health & Science University. Research followed the tenets of the Declaration of Helsinki, and written informed consent was obtained from all subjects after explanation of the nature and possible consequences of the study.2 in total area within 1500 µm from the fovea, with or without pigment changes; category 3, extensive large drusen (>125 µm in minimum diameter) >393,744 µm2 in area (a minimum of approximately 20 large drusen) within 1500 µm of the fovea, with or without pigment changes and no evidence of advanced AMD; category 4 (advanced AMD), presence in one or both eyes of advanced macular degeneration Each family comprised at least three examined (and genotyped) affected relatives with AMD. Individuals were identified as follows: category 1, (no AMD) no drusen or drusen less than 63 µm diameter, and no pigment changes in either eye; category 2, mild to moderate drusen consisting of drusen of any size, but <393,744 µmSporadic cases and controls were ascertained from our clinical practice. Diagnosis of AMD in cases was based upon the presence of geographic atrophy or choroidal neovascularization .The Age-Related Eye Disease Study was an eleven-center double-masked clinical trial investigating the effects of oral antioxidant and vitamin supplements on disease progression. DNA was extracted from venous blood from 2201 individuals  which comprised approximately one-half the AREDS participants (obtained from the AREDS Genetic Repository). Participants were aged 67.7 years (range 55–80) years. The AREDS study procedures have been previously reported.Seven SNPs in and around the CFH gene locus were chosen: rs3766404, rs529825, rs1061147, rs800292, rs1061170, rs203674 and rs2274700. Genotyping was performed using Sanger sequencing at Prevention Genetics  and at the Broad Institute Center for Genotyping and Analysis, Cambridge, MA, USA.To estimate SNP frequencies and determine if the frequencies differed between affected and unaffected individuals in the pedigrees, the Family Based Association Test (FBAT) was used.FBAT was used to estimate haplotype frequencies and any that occurred less than 10 times in combined cases and controls were eliminated from the analysis. Since 7 SNPs were measured in this gene, all 7 SNPs (see above genotyping) were used to produce the first haplotype model. Then 1 SNP was removed at a time to create 7 models, each containing 6 SNPs. Of these 7 models, the one with the lowest associated p-value was deemed the best model. Then 1 SNP was removed at a time from this best 6-SNP model to create 6 models, each containing 5 SNPs. The 5-SNP model with the lowest associated p-value was then the best 5-SNP model. This process was continued to find the best 4-SNP, 3-SNP, 2-SNP and 1-SNP models. Overall, we performed 27 tests. All 27 models were evaluated to determine which had the lowest associated p-value. The Bonferroni was used to adjust for multiple testing.The SNPs from the model with the lowest associated p-value were used to determine if one haplotype effect replicated in 2 additional samples. We used haplo.stat (a program in R) to estimate haplotype frequencies, determine if these differed between cases and controls, determine if individual haplotypes differed significantly between cases and controls and estimate an odds ratio for each individual haplotype.


Patients with refractory angina pectoris in end-stage coronary artery disease represent a severe condition with a higher reduction of life-expectancy and quality of life as compared to patients with stable coronary artery disease. It was the purpose of this study to invasively re-evaluate highly symptomatic patients with formerly diagnosed refractory angina pectoris in end-stage coronary artery disease for feasible options of myocardial revascularization.Thirty-four Patients formerly characterized as having end stage coronary artery disease with refractory angina pectoris were retrospectively followed for coronary interventions.Of those 34 patients 21 (61.8%) were eventually revascularized with percutaneous interventional revascularization (PCI). Due to complex coronary morphology  PCI demanded an above-average amount of time  and materials . Of PCI patients 7 (33.3%) showed a new lesion as a sign of progression of atherosclerosis. Clinical success rate with a reduction to angina class II or lower was 71.4% at 30 days. Surgery was performed in a total of8 (23.5%) patients with a clinical success rate of 62.5%. Based on an intention-to-treat 2 patients of originally 8 (25%) demonstrated clinical success. Mortality during follow-up (1–18 months) was 4.8% in patients who underwent PCI, 25% in patients treated surgically and 25% in those only treated medically.The majority of patients with end-stage coronary artery disease can be treated effectively with conventional invasive treatment modalities. Therefore even though it is challenging and demanding PCI should be considered as a first choice before experimental interventions are considered. Refractory angina pectoris in end-stage coronary artery disease is characterized by severe coronary insufficiency but only moderately impaired left ventricular function . This inFor patients with refractory angina pectoris in end-stage coronary artery disease various alternative therapeutic approaches are of interest and have in part been tested: transmyocardial laser revascularization, spinal cord stimulation, pharmacological und surgical sympathectomy, gene-therapy, bone-marrow transplantation, external counterpulsation and medical treatments long-term intermittent urokinase therapy and trimetazidine -12. As tTherefore patients who had been previously been diagnosed to suffer from refractory angina pectoris in end-stage coronary artery disease were reevaluated angiographically. It was the therapeutic aim of the study presented to perform primarily PCI or CABG in this high-risk group where feasible.For the study 34 consecutive patients were included who had been previously successfully treated with long-term intermittent urokinase therapy, a rheological approach developed for the improvement of the coronary microcirculation . All patThis retrospective observational study was in compliance to the principles of the Declaration of Helsinki. All patients consented in writing to any and especially the interventional procedures as well as to the use of data for scientific use as part of their hospital admission. All diagnostic tests, treatments and interventions were, for the individual patient, "standard care". None of the patients involved in this study received any study specific diagnostic tests or treatments. The use of retrospective data was approved by the ethics committee of Heinrich-Heine-University Düsseldorf, Germany.Coronary angiograms were evaluated for potential culprit lesions which could be regarded as relevant for myocardial ischemia and hence for the clinical symptoms of the patient. In all patients stress exercise testing for electrocardiographic signs of myocardial ischemia were performed, in some patients additional myocardial stress-scintigraphy was done. These stress tests were mostly inconclusive as almost all patients suffered form severe triple vessel disease and had limited exercise capacity due to clinical symptoms and beta-blocker therapy. Therefore the decision to treat with PCI was mostly based on the experience of two interventional cardiologists . Where feasible the decision to treat surgically was made together with cardiac surgeons.Critical coronary lesions were classified as chronic total occlusions, angulated lesions, and last patent vessels. As part of the invasive work-up of these high-risk patients PCI was usually scheduled for a second session in order to prepare the intervention meticulously. Interventions were performed by two interventional cardiologists  either alone or sometimes even alternating in one patient in case one encountered difficulties, for example crossing a chronic total occlusion. In general 7 French guiding catheters  were used for optimal visualization and back-up; various PCI guiding wires, low-profile balloons and stents were used . Angiography was performed in a biplane technique.Interventions were regarded as successful when the lesion could be dilated completely or if bypass vessels could be attached surgically. Clinical success was defined as a reduction of clinical symptoms at least to stage II angina pectoris for more than 30 days.Patients had a mean age of 67 ± 10 years (range 45–85 years), 11.8% were female (Table Based on an intention-to-treat the majority of 20 (58.8%) of the 34 patients were treated with PCI, while 6 (17.7%) were referred to surgery. In 8 (23.5%) patients we primarily did not see a reasonable and safe option for mechanical revascularization and these patients were treated medically. For persistence or progression of clinical symptoms 4 patients primarily treated medically, were treated invasively later on, 2 with PCI and 1 with surgery, while 1 patient in the PCI group was switched over to the surgical group.2 in comparison to 6987 ± 6481 μgy/m2 in the general patient group. An average of 1.6 ± 0.9 guiding catheters (range 1–4 guides), 2.0 ± 1.4 PCI guiding wires (range 1–6 wires), 1.8 ± 1.0 balloons  and 1.6 ± 1.3 stents (range 0–5 stents) were used. Of 21 lesions treated 15 (71.4%) could be revascularized successfully.Of the total of 21 patients  eventually treated with PCI an angulated lesion was targeted in 8 (38%), a chronic total occlusion in 6 (29%) and the last patent vessel in 3 patients (14%) while 4 patients (19%) presented with a lesion in a conductance vessel with a minor interventional risk. Evaluation of the angiograms preceding the interventional attempt demonstrated that of the lesions eventually treated 14 were old lesions and 7 were new lesions, the latter regarded as a sign of progression of atherosclerosis. Duration of the intervention in this high-risk group was 66 ± 42 minutes (range 25–206 minutes) as compared to the average result of 34 ± 20 minutes in 2315 patients who underwent PCI in our catheterization laboratories in that year. The use of contrast media amounted to 247 ± 209 ml (range 50–750 ml) as compared to the average use of 182 ± 116 ml while radiation was used with an exposure of 14267 ± 9573 μgy/mSkillful wiring was essential for success in extremely angulated stenoses. A soft wire with a core-to-tip design  was often used for access to the coronary periphery to be later exchanged via an over-the-wire system  to a more stable wire  for better stability. In extremely angulated lesions a soft infusion catheter  was used for passing the wire through the vessel. Treating these patients we developed a maneuver we termed "park'n ride maneuver" Figure . In patiIn one patient a high take-off right coronary bypass could be accessed with a 5F Multipurpose diagnostic catheter  but the 7F Multipurpose interventional catheter did not fit due to a different angulation of the catheter. Therefore a "long'n soft glide change-over maneuver" was developed during the procedure. The diagnostic catheter was placed into the take-off of the right coronary bypass and a 3 meter PCI guide wire  was inserted via the diagnostic catheter into the periphery of the right coronary artery. Using an over-the-wire maneuver the diagnostic catheter was the exchanged against an interventional 7F multipurpose interventional catheter. Advancing the catheter over the thin wire particular care was taken to avoid loop formation of the wire in the ascending aorta. After insertion of the interventional catheter the PCI was performed in the usual fashion.In chronic total occlusions stable back-up was of critical relevance for interventional success; Amplatz Left configurations were often employed for the right coronary artery. For access to the occlusion a low profile over-the-wire balloon  was used with soft wire . Passage through the chronic total occlusion was primarily tried with hard wires  as tactile feel was regarded superior to other wires. In cases where this approach did not yield success in particular when catheter back-up was poor more hydrophilic wires  were tried. In some patients in whom the distal end of the occlusion was not visible double injections for proper targeting of the wire were performed Figure .In one patient with last patent vessel, large right coronary artery venous bypass, intraaortic ballon pumping was started prior to the procedure and was maintained for 8 hours after successful stenting of the proximal part of the bypass. Using a "hit-and-run approach" with primary stenting and an inflation time of 10 seconds drop of systolic arterial blood pressure from 100 mmHg to 65 mmHg could be kept to a time of 35 seconds without the use of catecholamines.2, left ventricular ejection fraction 21%) who successfully underwent surgery. In another patient who presented with severe heart failure and a left ventricular ejection fraction of 34% it was decided to implant a resynchronization device.Complete hemodynamic work-up yielded indications for cardiac surgery apart from CABG. One patient who had been previously treated by us was than treated by his local cardiologists. He was then again referred to us for further treatment after three high-risk PCIs had failed in another center where he had been again classified to suffer form refractory angina pectoris. Complete work-up revealed a progression of valvular heart disease with severe low-gradient aortic stenosis . Based on an intention-to-treat clinical success as defined by a reduction to angina class II or lower for more 30 days following revascularization was achieved in 38% in the medical, in 63% in the PCI and in 83% in the surgical group. After cross-over 20 of 29 patients treated invasively (69%) improved clinically  PCI can be performed effectively and safely in most patients with end-stage coronary artery disease previously diagnosed to suffer from refractory angina pectoris in end-stage coronary artery disease, (b) the interventional approach in these patients needs time, material and calls for interventional techniques which are not part of the standard repertoire and (c) new lesion formation may be responsible for renewed clinical symptoms.The data presented show that primary interventional success in PCI can be achieved in almost two thirds of the patients with refractory angina pectoris and end-stage coronary artery disease. A major factor for interventional success in this series was surely the pooled experience of two senior cardiologists working on one case together. In particular during longer sessions or when progress slowed for example during recanalization of a CTO one investigator took over for some time or the rest of the procedure. The determination and perseverance of the whole team including assistance personnel can be derived from the average time of PCI which exceeds by far the average values for the general population treated in our catheterization laboratory. A major factor for a determined approach of the team was the fact that the long history of the patient's severe clinical symptoms was well know to the investigators and communicated to the rest of the team before the intervention. For example one patient primarily treated medically with an ejection fraction of 16%, angina pectoris grade III and a last patent vessel was seen 13 times at the outpatient clinic before it was decided together with the patient to try a high-risk approach.Apart form the duration of intervention, the differentiated use of materials was surely another relevant factor for primary success. Differentiated use of PCI wires was essential for entering the coronary system in patients with extremely tortuous and angulated vessels. In particular the use of soft materials (infusion catheter + soft wire) was critical for access of the target vessel and passage through the stenosis while in the next step a more rigid and stabilizing wire was inserted in an over-the-wire maneuver for save deployment of treatment devices . This approach drives up costs of the intervention in these patients. On the other hand that clinical success seen in these patients justifies the approach. Furthermore the learning curve included maneuvers newly developed during treatment of these high-risk patients. This led from the usual step-up approach using standard material first and then change to non-standard material to a primary top-step start where necessary which shortened duration and reduced use of material. Relevant was also the use of stents in particular in patients with CTOs as it not only stabilized the initial angiographic result but also raises the long-term patency rate -16. As CInterestingly one third of the patients presented with new lesions either in native coronary arteries or in bypass vessels. This has to be regarded as a sign of progression of atherosclerosis. These findings also signify the dynamics of myocardial perfusion and collateral formation as in the course of time another vessel may become causative for clinical symptoms in respect to the previous coronary status. At the initial time of diagnosis of refractory angina pectoris therapeutic measures may have led to a new balance of perfusion, for example as a result of symptomatic relieve allowing for more physical activity and collateral formation or metabolic adaptation resulting in long-term relieve after cessation of treatment as for example with long-term intermittent urokinase therapy. Natural progression of atherosclerosis may then disturb this balance leading to renewed symptoms. This is of particular clinical relevance because it means that the diagnosis of "refractory angina pectoris in end-stage coronary artery disease" is not a final one and angiographic reevaluation has always to be considered in the course of time or when symptoms progress.The diagnosis of refractory angina pectoris in end-stage coronary artery disease may have two major consequences for the patient: a) the patient is labelled with a severe disease and b) he may be entered into experimental clinical studies which in turn may withhold established forms of therapy from the patient. Given the high rate of primary success PCI in patients with formerly refractory angina pectoris and end-stage coronary artery disease in this study the diagnosis of this distinct clinical symptom has to be seen critical. This was already pointed out in another, single- center study based on data from the early stent era where out of 117 patients referred to transmyocardial laser revascularization with presumably refractory angina pectoris 61% could be treated conventionally  leaving only 39% who underwent the non-established approach of transmyocardial laser revascularization  as well A limitation of this study is that this is primarily an observational report and as such does not have the power of a randomized study. However, a well designed randomized study in these severely symptomatic patients might be impossible. The therapeutic decision will nearly always have to be done on an individual basis, and it will be difficult to control for probable placebo effects. We do not report long-term follow up data and can thus not judge about prognosis.Also, the decision for a coronary intervention was made by one or two interventional cardiologists based on their extensive experience, and not by a multidisciplinary team. Not all patients underwent rigorous non-invasive testing for proving viable myocardium before the intervention. Although it should be essential to get as many information about myocardial viability as possible, this proofs to be difficult in this patient population. On the one hand these patients are highly symptomatic and any test involving exercise will show inconclusive results because of physical limitations. On the other hand these patients present with complex coronary artery disease with multiple stenosis and presumably a more generalized limitation of flow. This is frequently not visualized by SPECT. MRI might yield additional information but was not done in this study.Labelling a patient with a severe disease like refractory angina pectoris in end-stage coronary artery disease may have psychological consequences for the patient. Even though a cause-effect relationship between the cognitive conception of severe, potentially life-threatening coronary artery disease and the perception of intensity and frequency of angina pectoris in the patient is hard to establish, it seems likely. The reduction of clinical symptoms to grade II angina and lower in patients treated with PCI was considerable. From the interventional perspective it implies that a vessel critical for myocardial perfusion was targeted. This may be true but it has to be kept in mind though that an interventional procedure in patients with angina pectoris in itself can have a placebo effect of over 40% -21. SubsThe authors declare that they have no competing interests.TJ coordinated the study, performed the statistical analysis and drafted the manuscript. AP coordinated the study, and drafted the manuscript. AAK coordinated the study and drafted the manuscript. MPH participated in the design and coordinated the study. FCS participated in the design and coordinated the study, and drafted the manuscript. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:


There appears to be an overlap between the limbic system, which is modulated by subthalamic nucleus (STN) deep brain stimulation (DBS) in Parkinson's disease (PD), and the brain network that mediates theory of mind (ToM). Accordingly, the aim of the present study was to investigate the effects of STN DBS on ToM of PD patients and to correlate ToM modifications with changes in glucose metabolism.18FDG-PET scans in 13 PD patients in pre- and post-STN DBS conditions and correlated changes in their glucose metabolism with modified performances on the Eyes test, a visual ToM task requiring them to describe thoughts or feelings conveyed by photographs of the eye region. Postoperative PD performances on this emotion recognition task were significantly worse than either preoperative PD performances or those of healthy controls (HC), whereas there was no significant difference between preoperative PD and HC. Conversely, PD patients in the postoperative condition performed within the normal range on the gender attribution task included in the Eyes test. As far as the metabolic results are concerned, there were correlations between decreased cerebral glucose metabolism and impaired ToM in several cortical areas: the bilateral cingulate gyrus (BA 31), right middle frontal gyrus , left middle frontal gyrus (BA 6), temporal lobe , bilateral parietal lobe (right BA 3 and right and left BA 7) and bilateral occipital lobe (BA 19). There were also correlations between increased cerebral glucose metabolism and impaired ToM in the left superior temporal gyrus (BA 22), left inferior frontal gyrus (BA 13 and BA 47) and right inferior frontal gyrus (BA 47). All these structures overlap with the brain network that mediates ToM.To this end, we conducted These results seem to confirm that STN DBS hinders the ability to infer the mental states of others and modulates a distributed network known to subtend ToM. High-frequency deep brain stimulation (DBS) of the subthalamic nucleus (STN) constitutes a therapeutic advance for severely disabled patients with Parkinson's disease (PD) in whom long-term pharmacological treatment has failed. Although the beneficial effects of chronic DBS on PD motor symptoms are now largely confirmed The explanation for these clinical modifications induced by STN stimulation lies in the neuroanatomical organization of the basal ganglia, in which the STN is thought to play a central role, not only in the regulation of motor function but also in the regulation and/or integration of associative and limbic functions A number of positron emission tomography (PET) studies of humans have confirmed these hypotheses, demonstrating modifications in cerebral activation and associating them with changes in cognitive function 18FDG-PET investigation of thirteen PD patients in pre- and post-STN DBS conditions and correlated changes in their glucose metabolism with modified performances on a ToM task: the Reading the Mind in the Eyes Test.Accordingly, the aim of the present study was to investigate the effects of STN DBS on the ToM abilities of PD patients. We hypothesized that STN DBS would impair ToM, because of the overlap between the limbic system, which is modulated by STN DBS in PD, and the brain network that mediates ToM. A further aim of this study was to correlate these ToM modifications with changes in glucose metabolism. To this end, we conducted an One group of PD patients and a healthy control (HC) group took part in the study. All patients met the clinical criteria of the United Kingdom Parkinson's Disease Society Brain Bank for idiopathic PD The patient group consisted of a series of 13 consecutive patients with medically intractable PD, who underwent bilateral STN DBS at Rennes University Hospital (France). Standard selection and exclusion criteria for surgery were applied to all patients The HC group consisted of 13 healthy individuals who had no history of neurological disease, head injury or alcohol abuse, and displayed no signs of dementia, as attested by their scores on the Mattis Dementia Rating Scale (MDRS) U = 64.00, p = .3) or education level . After a full description of the study, written informed consent was obtained for each participant, and the study was conducted in accordance with the Declaration of Helsinki.The two groups were comparable for handedness and gender ratio. Similarly, there was no statistical difference between the two groups regarding age  deep brain stimulation electrodes  were implanted bilaterally in the STN. The overall methodology was similar to that previously described by Benabid and colleagues The contacts' coordinates were expressed as millimetres along three axes originating from the middle of the bicommissural line: the first axis was parallel to the bicommissural line, the second axis was perpendicular to the AC-PC line and the third axis was perpendicular to the midsagittal plane. The mean (± SD) coordinates of the left selected contacts were 13.1 (±1.3) mm lateral to the AC-PC line, 0.8 (±2.0) mm below AC-PC and 3.9 (±1.5) mm posterior to the midpoint of the AC-PC line. The mean coordinates of the right selected contacts were 11.1 (±1.2) mm lateral to the AC-PC line, 1.5 (±1.8) mm below AC-PC and 4.5 (±1.6) mm posterior to the midpoint of the AC-PC line.In all patients, chronic stimulation was monopolar, using a single contact of the quadripolar electrode chosen for the best motor improvement. The stimulation characteristics were as follows: mean pulse width 60 µs for the right side (SD  = 0) and 60 µs (SD  = 0) for the left side, mean frequency 138.1 Hz (SD  = 13.5) for the right side and 139.2 Hz (SD  = 13.0) for the left side, and mean voltage 2.2 V (SD  = 0.6) for the right side and 2.3 V (SD  = 0.6) for the left side.Prior to the ToM assessment, a short neuropsychological and psychiatric battery was administered to the two groups (i.e. the PD group and the HC group). The battery included the MDRS We used the same method for the present study as the one described in previous paper A French adaptation of the revised version of the Reading the Mind in the Eyes Test was used Scoring: The test was scored by adding up the number of items (photographs) that were correctly identified by the participant, i.e. the number of correctly identified mental states. The maximum “Emotion score” on the test was therefore 17, which was converted into a percentage of correct responses. In addition, a control score was calculated by adding up the number of correct responses in the gender attribution task. The maximum “Gender score” on the test was therefore 17, and scores were once again converted into a percentage of correct responses.Control task: To check that the early processing stages of face perception were intact, and to supplement the gender attribution task included in the Eyes test, the Benton Facial Recognition Test 18FDG PET in a resting state with eyes open. They underwent two scans: the first was performed 3 months before surgery and the second 3 months after surgery, with the stimulator switched on and with their antiparkinsonian medication. PET measurements were performed using a dedicated Discovery ST PET scanner  in 2D mode, with an axial field of view of 15.2 cm. A 222-296 MBq injection of 18FDG was administered intravenously under standardized conditions . During acquisition, the patient's head was immobilized using a head-holder. A cross-laser system was used to achieve stable and reproducible positioning. A 20-minute 2D emission scan was performed 30 minutes post-injection and after X-ray based attenuation correction. These studies were performed with the subjects positioned at the centre of the FOV. Following scatter, dead time and random corrections, PET images were reconstructed using 2D filtered back-projection, providing 47 contiguous transaxial 3.75 mm-thick slices.All subjects were studied using We used the same method for the present study as the one described in previous papers The data were analysed by means of statistical parametric mapping  implemented in Matlab, Version 7 . Statistical parametric maps are spatially extended statistical processes that are used to characterize regionally specific effects in imaging data. They combine the general linear model  and the theory of Gaussian fields to make statistical inferences about regional effects All subject images were first realigned and spatially normalized into standard stereotactic space in accordance with the Talairach and Tournoux atlas U test for two independent groups . For the intragroup comparisons, the Wilcoxon test for paired groups was used to measure the effect of the experimental condition (before vs. after surgery). Behavioural correlations between (1) neuropsychological background, control task and ToM, and (2) psychiatric tests and ToM, were assessed using Spearman's rank correlation coefficient. The p-value was significant if less than 0.05. Statistical analyses were performed using SPSS 15.0 software.Because of the small sample sizes, nonparametric analyses were carried out. For the intergroup comparisons, paired comparisons were performed using the nonparametric Mann-Whitney 18FDG-PET scans pre- and post-surgery. As before, the effects of differences in overall metabolism were removed by normalizing each voxel to the same whole-brain value . To determine the direct effects of STN stimulation, we used the “population main effect, 2 conditions, 1scan/cond (paired t test)” routine. Clusters of at least 20 contiguous voxels, with a threshold two-tailed p-value of 0.001 (corrected for multiple comparisons), were considered to be significantly different.First, the SPM software pinpointed significant modifications in cerebral metabolism in the 13 PD patients by comparing their t-value. Two t-tests were performed, revealing correlations between decreased cerebral glucose metabolism and impaired ToM, and between increased cerebral glucose metabolism and impaired ToM. Next, t -statistic SPMs were calculated and clusters of at least 30 contiguous voxels with a threshold at p<0.005, with multiple comparison correction, were considered to be significantly different.Second, the SPM software established correlations between post- vs. preoperative changes in the ToM score and post- vs. preoperative changes in brain glucose metabolism. To identify those regions that correlated significantly with impaired ToM, a general linear “single subject, covariates only” model was tested for every voxel, with the ToM score as a covariant. This yielded a regression coefficient which was then transformed into a http://www.mrc-cbu.cam.ac.uk/Imaging).All coordinates reported here are based on the Talairach atlas and were transformed by applying procedures developed by Matthew Brett . LED decreased significantly after surgery . It should be noted that analyses revealed a trend towards significance for the improvement in the off-dopa S&E score between the preoperative and postoperative conditions . A significant motor improvement was observed 3 months after surgery, as shown by the changes in the motor UPDRS score in the off-dopa condition (p>.1). In the postoperative condition, no significant difference was found between the PD and HC groups for neuropsychological background and psychiatric tests . Within the PD patient group, no significant difference was found between the preoperative and postoperative conditions for neuropsychological background and psychiatric tests, except for the number of categories and errors on the MCST , and for Action (Verb) Fluency . It should be noted that analyses revealed a trend towards significance for the improvement in the number of perseverative errors on the MCST  and in the categorical fluency score between the preoperative and postoperative conditions . No significant difference was found between the PD and HC groups for the Benton Facial Recognition Test in either the preoperative condition  or the postoperative one . Within the PD patient group, no significant difference was found between the preoperative and postoperative conditions for the Benton recognition test .Performances by the participants on the neuropsychological and psychiatric tests are presented in p>.5). In the postoperative condition, a significant difference was found between the PD and HC groups for the Emotion score of the Reading the Mind in the Eyes Test . There was no significant difference between the PD and HC groups in the postoperative condition for the Gender score of the Reading the Mind in the Eyes test . Within the PD patient group, analyses revealed a significant difference between the pre- and postoperative conditions for the Emotion score of the Eyes test . There was no significant difference between the pre- and postoperative conditions for the Gender score of the Eyes test .Participants' performances on the ToM task are presented in p>1).No significant correlation was found between the variations in the motor scale scores  between the pre- and postoperative conditions and the variation in the Emotion score of the Reading the Mind in the Eyes Test .No significant correlation was found between the variation in LED between the pre- and postoperative conditions and the variation in the Emotion score of the Reading the Mind in the Eyes Test .No significant correlation was found between the variation in the neuropsychological data  between the pre- and postoperative conditions and the variation in the Emotion score of the Reading the Mind in the Eyes Test .No significant correlation was found between the variation in the psychiatric data (MADRS and STAI A&B) between the pre- and postoperative conditions and the variation in the Emotion score of the Reading the Mind in the Eyes Test  24) and left superior frontal gyrus (BA 8 and 9).In our analysis of postoperative decreases in metabolism, three clusters were significant at p<0.001, with multiple-comparison correction. Hypermetabolism was observed in the bilateral cerebellum and right inferior parietal lobule (BA 40).When we studied postoperative increases in metabolism, two clusters were found to be significant at The correlations between decreased cerebral glucose metabolism and impaired ToM are summarized in The correlations between increased cerebral glucose metabolism and impaired ToM are summarized in The scatter plots showing the voxel values of each significant cluster and the correlations between these values and the ToM scores are available as supplementary material -H.The aim of the present study was to investigate the effects of STN DBS on the ToM abilities of PD patients. We hypothesized that STN DBS impairs ToM because of the overlap between the limbic system, which is modulated by STN DBS in PD, and the brain network that mediates ToM. A further aim of this study was to correlate these ToM modifications with changes in cerebral glucose metabolism. Results confirmed our initial hypothesis and provided a preliminary demonstration of ToM impairment following STN DBS. Postoperative PD performances were significantly worse than HC and preoperative PD performances on the emotion recognition task of the Eyes test, whereas there was no significant difference between preoperative PD and HC performances. Conversely, in the postoperative condition, the PD patients scored within the normal range on the gender attribution task of the Eyes test, which was used as a control task. As far as the metabolic results are concerned, there were correlations between decreased cerebral glucose metabolism and impaired ToM in several cortical areas: bilateral cingulate gyrus (BA 31), right middle frontal gyrus  left middle frontal gyrus (BA 6), temporal lobe , bilateral parietal lobe (right BA 3 and right and left BA 7) and bilateral occipital lobe (BA 19). There were also correlations between increased cerebral glucose metabolism and impaired ToM in the left superior temporal gyrus (BA 22 and BA 20) left inferior frontal gyrus (BA 13 and 47) and right inferior frontal gyrus (BA 47). Furthermore, in this series of patients, we were able to confirm our previous finding of metabolic modifications following stimulation, with hypometabolism in the bilateral anterior cingulate gyrus (right and left BA 24) and left superior frontal gyrus (BA 8 and 9), and hypermetabolism in the bilateral cerebellum and right inferior parietal lobule (BA 40) As far as behavioural data are concerned, our present result seems to confirm that STN DBS affects the ability to select mental state terms to describe the feelings conveyed by photographs of eyes and, by so doing, impairs the ability to infer the mental states of others. This is in line with previous studies that have reported reduced recognition of emotional expressions from faces following STN DBS Our present metabolic results indicate that ToM is subtended by a broadly distributed network. Previous animal, neuroimaging and neuropsychological studies investigating the neural substrates that mediate ToM responses 18FDG-PET in STN DBS PD patients, we found that we had highlighted a more broadly distributed brain network this time. We believe that there are several reasons why such large brain regions were identified in the present study. At the behavioural level, ToM encompasses several elementary processes. The same is true at the cerebral level, insofar as ToM relies on several different brain structures. Although each elementary structure is specific to a given process, the involvement of all these structures together would appear to be specific to ToM. As indicated earlier, this is consistent with Brothers' theory that social cognition is subtended by a network of interconnected regions, as well as with a recent review of fMRI studies exploring the neural bases of ToM When we compared the present results with those of our previous investigations of cognitive-metabolic correlations between recognition of facial expressions and In order to avoid specific biases, the two participant groups were statistically comparable for age and education level. Similarly, in order to rule out an overall visuospatial information processing deficit that has already been described in PD N = 6 PD patients) that the microlesion effect of the surgery may be sufficient to trigger changes in brain activity when the stimulator is turned off, as long as 20 months after surgery. In the present study, a similar microlesion effect may have been behind the metabolic and behavioral changes we observed in the patients. Nevertheless, the results obtained by Pourfar and colleagues N = 15) N = 16 PD patients) and showed that longer periods of DBS reduced beta power for longer, suggesting that there may be long-lasting functional changes to networks in the STN in PD after chronic DBS. Finally, it should be noted that There were several limitations to the present study that need to be acknowledged and addressed. First, the task we administered, the Eyes test, has been poorly described in terms of the cognitive processes it involves in addition to ToM The ToM impairments observed in the present study may therefore have been the result of a dysfunction induced either by STN microlesions or by STN stimulation per se within the broadly distributed neuronal network that subtends ToM abilities, although it remains to be said whether the STN itself actually belongs to this network. In addition, the mechanisms by which STN stimulation interferes with the functioning of the brain areas that mediate ToM have yet to be determined. Given the small size of the STN, the stimulating current flow may not be restricted to the targeted sensorimotor STN compartment, but may also affect other STN compartments, in particular the limbic one, as well as the closely connected limbic territory of the basal ganglia and afferent or efferent connections to subcortical and cortical limbic regions, in line with the findings of several neuroimaging studies Our study is, to our knowledge, the first to suggest that STN stimulation affects ToM abilities and to correlate this impairment with cerebral metabolic changes. It provides a preliminary account of the modulation induced by stimulation of a broadly distributed neuronal network of cerebral areas mediating ToM, although the exact nature of the STN's role within this network remains to be elucidated. These results confirm the role played by the STN in the human limbic system and suggest that this basal ganglia structure makes a key contribution to social cognition.Figure S1Example of items in the Eyes test.(0.21 MB TIF)Click here for additional data file.Figure S2Scatter plots of the correlations between voxel values of each significant cluster and the ToM scores. A) Correlations between increased cerebral glucose metabolism and impaired ToM - Cluster 718 , (B) Correlations between increased cerebral glucose metabolism and impaired ToM - Cluster 220 , (C) Correlations between increased cerebral glucose metabolism and impaired ToM - Cluster 77 , (D) Correlations between decreased cerebral glucose metabolism and impaired ToM - Cluster 5877 , (E) Correlations between decreased cerebral glucose metabolism and impaired ToM - Cluster 2204 , (F) Correlations between decreased cerebral glucose metabolism and impaired ToM - Cluster 62 , (G) Correlations between decreased cerebral glucose metabolism and impaired ToM - Cluster 92 , (H) Correlations between decreased cerebral glucose metabolism and impaired ToM - Cluster 33 .(0.13 MB PPT)Click here for additional data file.


Multiple sclerosis (MS) is a complex neurological disorder. Its aetiology involves both environmental and genetic factors. Recent genome-wide association studies have identified a number of single nucleotide polymorphisms (SNPs) associated with susceptibility to (MS). We investigated whether these genetic variations were associated with alteration in gene expression.HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DRB1, HLA-DRB4 and HLA-DRB5, one with ZFP57, one with CD58, two with IL7 and FAM164A, and one with FAM119B, TSFM and KUB3. We found minimal cross-over with a recent whole genome expression study in MS patients.We used a database of mRNA expression and genetic variation derived from immortalised peripheral lymphocytes to investigate polymorphisms associated with MS for correlation with gene expression. Several SNPs were found to be associated with changes in expression: in particular two with We have shown that many susceptibility loci in MS are associated with changes in gene expression using an unbiased expression database. Several of these findings suggest novel gene candidates underlying the effects of MS-associated genetic variation. Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterised by demyelination and axonal loss.A recent investigation has performed whole genome expression analysis in lymphoblastoid cell lines (LCLs) from healthy volunteers who were also genotyped for a large number of SNPs.et al.In vitro transcription of cDNA was performed using the IVT Labeling Kit (Affymetrix) and, after hybridisation on U133 Plus 2.0 GeneChips (Affymetrix), this was scanned using a high-resolution scanner (Affymetrix). Whole-genome genotyping was carried out according to manufacturers' instructions using the Sentrix Human-1 Genotyping BeadChip and the HumanHap300 Genotyping BeadChip. The analysis of expression was carried out on the publically available database of mRNA by SNP Browser 1.0 as described.This was carried out as described in Dixon 2. For SNPs where no proxy was provided, we investigated all genotyped SNPs within 500 kb for LD with r2≥0.4 for a suitable proxy SNP. We also assessed the degree of LD with potentially interesting SNPs within 500 kb of the original susceptibility SNP. Finally we assessed the SNPs associated with expression of putative candidate genes to ensure that we did not miss any important associations with expression.We investigated the mRNAs significantly altered in expression by the SNPs reported in the literature to be at or close to genome-wide significance.We chose to look at a set of 38 SNPs which were the top loci to reach genome-wide significance selected from currently reported genome wide association studies (GWAS) of which 14 had been independently replicated in 2 studies. 17 of these were not present in the genome-wide association mRNA expression library and so when possible proxy SNPs in strong-to-moderate LD were used instead. The SNPs and proxy SNPs used are detailed in Table S1). Two SNPs in strong LD with multiple MS-associated SNPs in the HLA region were related to expression of various HLA alleles, including HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DRB1, HLA-DRB4 and HLA-DRB5. One SNP in the HLA class I region was associated with altered expression of ZFP57. Both SNPs in CD58 were associated with expression of CD58. A SNP in the IL7 region was associated with expression of mRNA encoding IL7 and FAM164A. Three SNPs in the region of METTL1-CYP27B1-CDK4 altered the expression of several genes: FAM119B, TSFM and KUB3. The common gene of altered expression for all three SNPs was TSFM.13 of the MS susceptibility SNPs or proxy SNPs were associated with changes in mRNA expression  and secondary progressive MS compared with healthy controls. HLA-DQB1 expression was reduced in MS and RRMS compared with healthy controls. STAT3 was reduced in primary progressive MS compared with healthy controls.We used the supplemental data supplied by Gandhi and colleagues to examine cross-over between the results obtained in that study and the genes we identified as being altered in expression by susceptibility SNPs.CD58, had already been noted by previous investigators.IL7R on expression of the gene.IL2RA gene did not correlate with expression of IL2RA mRNA, despite previously finding altered levels of this in MS patients relative to controls.Our findings show that some, but by no means all, susceptibility SNPs in MS are associated with changes in gene expression. Some of these  and thus we are not sure whether differential expression of HLA genes measured by microarray reflects different probe affinity for different HLA class II alleles and thus further work is needed to fully understand this association. Our identification that a SNP in the HLA class I region was associated with altered expression of ZFP57 is an interesting observation as this gene has been linked with DNA methylation changes across the genome resulting in transient neonatal diabetes.IL7 recently confirmed as associated with MS was shown to correlate with the expression of several genes: IL7 and FAM164A. Naturally the most compelling candidate of these is IL7 due to its probable role in autoimmunity. However, the advantage of an unbiased screen is that it raises the possibility of candidate genes that would otherwise not be considered. This is especially so since the SNP is far more strongly associated with FAM164A expression than with IL7. FAM164A is a hypothetical protein encoded in the reverse direction to IL7 and its functional importance is largely unknown.FAM119B.TSFM suggests this as a strong candidate. This is a plausible candidate in terms of function too as it is involved in the translation of mitochondrial proteins, providing a potential link with other susceptibility genes linked to mitochondrial function, such as KIF21B.We also found several novel effects of susceptibility SNPs. Two SNPs in tight LD with susceptibility SNPs in the The limited cross-over between known and suspected susceptibility genes in the whole genome expression analysis of Gandhi and colleagues is likely due to a number of differences including the use of whole blood mRNA and individuals with established disease in the Gandhi study.a priori hypothesis to blind investigators to the presence of other genes. There are several limitations to the approach we used. The mRNA screen was conducted in transformed LCLs and so it would not be informative about tissue-specific gene expression.The advantage of an unbiased approach to linking the expression of genes with genetic variation associated with disease susceptibility is that there is no Table S1Changes in mRNA expression associated with susceptibility SNPs.(0.15 MB DOC)Click here for additional data file.


Escherichia coli. Our data show that a majority of persisters to ciprofloxacin were formed upon exposure to the antibiotic, in a manner dependent on the SOS gene network. These findings reveal an active and inducible mechanism of persister formation mediated by the SOS response, challenging the prevailing view that persisters are pre-existing and formed purely by stochastic means. SOS-induced persistence is a novel mechanism by which cells can counteract DNA damage and promote survival to fluoroquinolones. This unique survival mechanism may be an important factor influencing the outcome of antibiotic therapy in vivo.Bacteria can survive antibiotic treatment without acquiring heritable antibiotic resistance. We investigated persistence to the fluoroquinolone ciprofloxacin in  Escherichia coli to one of the most widely used antibiotics, ciprofloxacin. We show that the majority of persister cells are formed in response to this antibiotic, contrary to the prevailing view of persister formation. Ciprofloxacin kills bacteria by damaging their DNA. DNA damage activates a SOS gene network, the result of which is the production of various repair proteins. We uncovered a novel part of this network that leads to the formation of tolerant persister cells. The induced tolerance as a side effect of antibiotic treatment is an effective bacterial survival strategy and is likely to contribute to recalcitrance of infections.The frequent failure of antibiotic treatments is an acute public health problem. Bacteria can escape the lethal action of antibiotics by a mutation in the cell's DNA, leading to antibiotic resistance. Alternatively, they can enter a physiological state in which the antibiotics do not affect them. This phenomenon, referred to as persistence, is different from resistance because there is no genetic modification and because it is transient. Persisters are believed to form stochastically prior to antibiotic treatment. The presence of persister cells in bacterial biofilms contributes to the difficulty in treating biofilm-related infections. We investigated the persistence of  Persistence is the ability of a subpopulation of susceptible bacteria to survive lethal doses of antibiotics. It is a transient and non-hereditary phenotype unlike resistance, which is due to genetic modification. The transient nature of persistence makes it inherently difficult to study therefore the underlying molecular mechanisms are still poorly understood.Escherichia coli high-persistence mutant, persisters to ampicillin were shown to be non-growing prior to the addition of the antibiotic E. coli and was shown to be enriched in persisters to ofloxacin Persisters are thought to be slow growing, non-growing or dormant cells, which escape the lethal action of antibiotics because their drug targets are inactivated due to the physiological state. In an E. coli efficiently repairs DSBs through a series of reactions carried out by enzymes participating in homologous recombination and replication Both genetic and phenotypic variability can have important consequences on bacterial survival of antibiotic treatment. One of the most prescribed broad spectrum antibiotics today are the fluoroquinolones (FQ), which target gyrase and topoisomerase. These essential enzymes regulate supercoiling of genomic DNA during replication and transcription Even though fluoroquinolones are potent bactericidal antibiotics they cannot sterilize a bacterial culture. The bulk of the population rapidly dies in response to fluoroquinolones but a small fraction persists. According to one model, persisters might survive if gyrase and topoisomerase are inactivated due to cellular dormancy Alternatively, the persister state might be inducible in a cell subpopulation by exposure to the antibiotic, not stochastic and pre-existing. This could be because either dormancy is inducible, or persisters might be active and have more efficient drug efflux or more efficient repair of DSBs due to the stochastic overexpression of the genes involved in those pathways or due to the physiological events leading to the activation of the same pathways.In order to distinguish between these possibilities, we measured numbers of persisters to the fluoroquinolone ciprofloxacin in various genetic backgrounds with altered capacity for SOS induction and DSB repair.The majority of persisters were found to be formed upon exposure to the antibiotic and formation was dependent on the SOS DNA damage response. Contrary to the current view, a majority of surviving persisters to ciprofloxacin are not pre-existing, but induced by this antibiotic.Fluoroquinolones (FQ) induce DSBs by interfering with the action of gyrase and topoisomerase According to the prevailing model We measured the persister levels in different genetic backgrounds diagnostic of specific molecular events linked with DSBs and SOS induction. The surviving fraction of a wild-type culture treated with ciprofloxacin produces a typical biphasic pattern . This reE. colirecA and recB backgrounds, compared to the wild type, presumably because DSBs could not be repaired . In recB, persisters were extremely rare or entirely absent after 6 hours of incubation. This shows that the persisters experience DSBs and hence depend on the repair functions.We examined some of the well-known DNA-repair pathways in order to probe their possible role in formation of persisters. RecA and RecBC are essential for repair of DSBs in repaired . HoweverlexA3) recA430) recA background. This shows that the persistence to ciprofloxacin is largely dependent on a functional SOS response.RecA and RecB functions are essential not only for DSB-repair but for SOS induction following processing of DSBs as well dif site xerCD function does not affect the proficiency for SOS induction, but is lethal in cells in which chromosme dimers have formed. In xerC and xerD mutants the persister level is reduced , suggesting that most persisters have undergone at least one successful recombination event, most likely repairing a ciprofloxacin-induced DSB ), also led to a 20-fold increase in persister level compared to the wild type  the proportion of cells giving rise to gal+ colonies increases, peaking at around 20 minutes and declines thereafter  of the same antibiotic . ControlIt was important to learn whether SOS induction caused by treatments other than FQ is able to induce persistence to ciprofloxacin. In order to test this we measured persistence to ciprofloxacin in cells exposed to mitomycin C. Mitomycin C interacts with DNA by intercalation and adduct formation, resulting in inter-strand crosslinks 7 CFU/ml in both the wild-type and the strain unable to induce SOS (lexA3). We conclude that the SOS-induced persisters make up the majority of persisters to ciprofloxacin regardless of the growth phase.Persister levels are very low in early exponential phase and are maximal in stationary phase resistance to antibiotics, molecular mechanism(s) of persistence have so far remained elusive. The current model of persistence assumes that persisters are non-growing or dormant cells, formed by stochastic process(es) independently of any physiological responses normally elicited by antibiotics The processes leading to genetic variability in bacteria, mutagenesis and recombination, have been studied extensively Here we show a mechanism of persister formation triggered by DNA damage inflicted by the fluoroquinolone ciprofloxacin. Formation of persisters in response to DNA damage reveals a deterministic component in this bistability phenomenon. Bistability is the stochastic production of two phenotypically distinct cell types within a clonal population of genetically identical kin cells. Bistability is observed in sporulation, competence, and motility We also show that mutants defective in persistence to ciprofloxacin have normal persister levels to amipicillin and streptomycin , therefoCiprofloxacin induces DSBs in cells with active gyrase and/or topoisomerase, which in turn leads to the activation of the general DNA damage stress response, the SOS gene network. Our results show that the majority of persisters to ciprofloxacin are dependent on a functional SOS response.DSBs and other SOS-inducing lesions occur under physiological conditions so at any given time there is a fraction of a bacterial population undergoing a certain degree of SOS induction Essentially all actively growing cells exposed to ciprofloxacin induce SOS, but not all become persisters, suggesting that a specific level of SOS induction is required for persister formation. SOS is a gradual response and depending on the nature of the inducer, its concentration and the time of the exposure, different sets of genes are induced lexA(Def) mutant, expression levels of SOS genes appear to fall short of being truly uniform throughout the population Consistent with this, constitutive, full expression of the SOS regulon  does not lead to the tolerance of the entire population, but to an increased level of surviving persisters . Even in7 CFU/ml . However we cannot exclude that other LexA-regulated genes also contribute to SOS-induced tolerance.There are at least 43 genes in the SOS has been shown to induce formation of a senescence-like state in which cells are viable but unable to form colonies In conclusion, we have discovered an active, regulated mechanism of persister formation, which is part of the SOS response. SOS has been known to contribute to the survival of antibiotic treatments by increasing the frequency of resistant mutants through its mutagenic activities E. coli K-12 MG1655 was used as the parental strain. Different alleles were moved into the parental background by P1 transduction Bacterial strains are listed in 4 and 20 mg/L CaCl2 according to NCCLS  guidelines for susceptibility testing and 0.1 M HEPES/KOH pH 7.2.Experiments were conducted at 37°C in Mueller Hinton Broth (MHB) supplemented with 10 mg/L MgSOPersistence was measured by determining survival upon exposure to 0.1 µg/ml ciprofloxacin (unless indicated otherwise), 100 µg/ml ampicillin and 25 µg/ml streptomycin during time indicated on corresponding graph axes. All antibiotics were purchased from Sigma.8 CFU/ml. For determination of CFU counts, cells were washed in 1% NaCl solution, serially diluted and plated on LB (Luria-Bertani medium) agar plates supplemented with 20 mM MgSO4.Prior to the addition of antibiotic overnight cultures were diluted 100-fold in 3 ml of fresh medium in 17- by 100-mm polypropylene tubes and incubated for 1.5 hrs with shaking, typically reaching ∼2×10lexA3 and recA430 strains the CFU counts stabilize later than in the wild-type and in that case the CFU counts at 6-hour time point were used as a representative of the persister fraction.Persister fraction, reflected as a plateau in CFU counts, was calculated as an average of CFU counts at 3- and 6-hour time points. In cro-gal construct, overnight cultures grown in LB medium at 37°C were diluted 1∶200 in 15 ml of fresh medium and incubated in 125 ml flasks for 1.75 hrs at 37°C with shaking. Ciprofloxacin was added and aliquots of the culture were taken at different time points, washed in 1% NaCl solution, serially diluted and plated on LB agar plates supplemented with 20 mM MgSO4 for total CFU counts and on MacConkey agar plates supplemented with 1% galactose in order to determine the fraction of gal+ cells.For plate assays using the CI-recA::lacZ expression was measured as described in For β-galactosidase activity measurement, overnight cultures grown in supplemented MHB medium (see above) at 37°C were diluted 1∶100 in 3 ml of fresh medium in 17- by 100-mm polypropylene tubes and incubated for 1.75 hrs at 37°C with shaking. Ciprofloxacin was added and after 15 minutes an aliquot of culture was taken and 4 after washing in 1% NaCl solution. The same procedure was repeated after 4 hours of exposure to mitomycin C.Overnight cultures in supplemented MHB medium were diluted 1∶1000 in 15 ml of fresh medium and incubated in 125 ml flasks for 1 hr at 37°C with shaking after which 0.25 µg/ml of mitomycin C (Sigma) was added to the cultures. This concentration did not inhibit the growth of the culture. After 2 hrs the total CFU counts were determined by dilution and plating and an aliquot of the culture was taken out and exposed to 0.3 µg/ml of ciprofloxacin for 3 hrs. The number of survivors was determined by plating on LB agar plates supplemented with 20 mM MgSO4. The colonies were counted after 40 hours incubation at 37°C.Overnight cultures in MHB medium were diluted 1000-fold in 100 ml of fresh medium in 500 ml flasks and incubated at 37°C with shaking. At defined time intervals the cultures were serially diluted and plated on LB agar for determination of total CFU counts. In the same time 1 ml aliquots were transferred into 2 ml eppendorf tubes and 0.1 µg/ml ciprofloxacin was added. After 3 hrs at 37°C cells were washed with 1% NaCl solution, serially diluted and plated on LB agar plates supplemented with 20 mM MgSO


International HIV guidelines have recently shifted from a medium-late to an early-start treatment strategy. As a consequence, more people will be eligible to Highly Active Antiretroviral Therapy (HAART). We estimate mean life years gained using different treatment indications in low income countries.We carried out a systematic search to identify relevant studies on the treatment effect of HAART. Outcome from identified observational studies were combined in a pooled-analyses and we apply these data in a Markov life cycle model based on a hypothetical Tanzanian HIV population. Survival for three different HIV populations with and without any treatment is estimated. The number of patients included in our pooled-analysis is 35 047.Providing HAART early when CD4 is 200-350 cells/μl is likely to be the best outcome strategy with an expected net benefit of 14.5 life years per patient. The model predicts diminishing treatment benefits for patients starting treatment when CD4 counts are lower. Patients starting treatment at CD4 50-199 and <50 cells/μl have expected net health benefits of 7.6 and 7.3 life years. Without treatment, HIV patients with CD4 counts 200-350; 50-199 and < 50 cells/μl can expect to live 4.8; 2.0 and 0.7 life years respectively.This study demonstrates that HIV patients live longer with early start strategies in low income countries. Since low income countries have many constraints to full coverage of HAART, this study provides input to a more transparent debate regarding where to draw explicit eligibility criteria during further scale up of HAART. The optimal time to start treatment for HIV/AIDS has been a contentious issue since the introduction of Highly Active Antiretroviral Treatment (HAART). Initially a "hit hard and early" strategy was promoted . BecauseThe debate regarding optimal timing of treatment start has tremendous implications for HAART demand, and subsequently, on the estimated treatment coverage in different settings. Towards the end of 2008, only 3 million people out of 33 million with HIV were given HAART -15. In cLife years gained by different CD4 starting points is necessary information for making informed choices about early or late start of treatment. Studies in low income countries have found that patients starting HAART early (CD4 <350 cells/μl) have life expectancies from 9.4 to 17.2 life years and that life expectancies are 6.8 - 14.9 with late treatment strategies (CD4 < 200 cells/μl) . Only onOur overall aim is to inform the ethical dilemma of choosing between providing HIV treatment to patients with potential best outcomes and giving fair chances to the more severely ill patients with less potential benefit. The objective of this study is therefore to estimate the life years gained with early and late treatment strategies in low income countries. In the absence of randomized clinical trials, we model life years gained by using best available evidence from observational studies.We did a structured literature review and combined findings in a pooled-analysis to find the best evidence underlying the different treatment strategies. Based on these findings, a Markov life cycle model was constructed to estimate the expected remaining life years with and without HAART stratified in three baseline CD4 strata at the time patients first present for care: CD4 <50 cells/μl or 50-199 cells/μl or 200-350 cells/μl. The model can be applied on any population, and we test it on a Tanzanian HIV population since information on HIV prevalence and life tables were easily available ,25. We eThe set of mutually exclusive health states in the model and the various events that can occur in the history of HIV are illustrated in Figure We assume that patients presenting for care have a mean age of 35 years because this agreed with the characteristics of the underlying HIV demographics in the region and mean age of the studies included in the pooled-analysis tables  and 2. WTo identify evidence used as input in our analysis we carried out searches in the Cochrane Database of Systematic Reviews (inception to first quarter 2009), Ovid Medline (1996 to March 2009), EMBASE (inception to March 2008), ISI Web of Science (1992 to March 2008), conference abstracts from the International AIDS Society (2006 to 2008), the Conference on Retroviruses and Opportunistic Infections (inception to 2009) and the HIV Implementers' Meetings (2006-2008). References from relevant papers were also "hand" searched.Search terms were; "HIV, Africa, low income countries, mortality & survival". Only English language papers were reviewed. Eligible studies were identified by the first author and their relevance confirmed by the other authors. Inclusion criteria for the structured review are: low income countries, randomised controlled trial or observational studies with minimum one year observational period, effect measures that could be converted to one year absolute risk of death, death risks stratified into baseline CD4 strata <50 or 50-199 or 200-350 cells/μl, information on number of participants in the various CD4 strata and participants older than 15 years of age.We found no published randomised controlled trials evaluating the effect of different starting points versus placebo in low income countries. It is considered unethical to conduct such trials today. Analysis must therefore draw on the best available evidence from observational studies. We found no controlled observational studies, that is, studies comparing survival between an intervention and a control group. Effect size in terms of life expectancy can therefore only be evaluated through modelling. We identified six observational studies showing survival before initiation of HAART in low-income settings with accurate information on stratified survival according to baseline CD4 count table ; two frow) and confidence intervals (CI) as follows:The principal outcome measure in the pooled-analysis tables  and 2 isw is the weighted probabilities, ni denote number of individuals in i-th study, and pi represent the probability of death in the i-th study.Where pWe combine mortality rates with a weighted mean rather than a narrative syntheses or simple mean because larger sample size (n) increases the chance the sample is representative and leads to increased precision in estimates. Hence, more weight should be given to large sample size. Weighted probabilities were used as transition probabilities in the Markov model. The effect size measure between studies in the Markov model was net life years gained and total remaining life years.For patients not receiving HAART we assumed constant annual mortality rates of 0.796, 0.355 and 0.109 for the CD4 health states <50, 50-199 and 200-350, respectively  HIV prevalence in Tanzania is 5.7% and it is estimated that 2.2 million people are living with HIV . To our We calculated expected remaining life years without HAART and net life years gained from HAART by different CD4 starting points figure . From fiResults from the one-way sensitivity analyses are shown in table Our study suggests that providing HAART early is likely to yield most life years if everyone receives treatment. This is in concordance with the recommendations in the revised 2009 WHO guidelines. However, few governments in low income countries are capable of providing optimal treatment to all if everyone with CD4 < 350 cells/μl are considered to be eligible. Given the severe budget constraints low income countries face, a policy where fewer patients are considered to be eligible is the more common practice.In terms of stratified net life years gained from HAART, we believe our study to be unique. From international published databases, we were unable to identify any studies which describe stratified net life years gained from HAART with various starting points. Five studies reported life expectancy for HIV patients when starting treatment at CD4 < 200 cells/μl and results ranged from 6.8 life years to 14.9 life years, which is comparable to our total survival (without treatment + HAART) of 9.6 life years in figure The Tanzanian ART guideline from 2005, which is still being used in 2009, recommends treating asymptomatic patients when the CD4 count drops below 200 cells/μl . AsymptoA major strength of our analysis is its' simplicity and use of evidence-based transition probabilities. The disadvantage of this approach is that we were not able to model the dynamic changes in CD4 counts for patients on HAART, which has been done in several recent studies ,19-21. TA second strength is that the analysis includes the death risk from observational studies with more than one year observational period. This enabled us to include variations in death risks during the first two years and to reduce the effect of the high mortality peak often observed during the first 6-12 months after patients have started HAART.Death risk from year two and onwards for patients with CD4 <50 on HAART table  is low aFinally, we applied age-related background mortality in order to provide a more precise estimate of the long term effects of HAART. However, it is uncertain at what extent the pooled HIV-related mortality rates incorporate a long term epidemiological shift for HIV patients.This study demonstrates that HIV patients live longer with early start of antiretroviral treatment in low income countries, and highlights the ethical dilemma of choosing between providing HIV treatment to patients with potential best outcomes and giving fair chances to all the more severely ill patients with less potential benefit. Since low income countries have many constraints to full coverage of HAART and more people will be eligible with an early start strategy, the results of this study is a good starting point for a more transparent and reasoned debate when drawing explicit eligibility criteria during further scale up of HAART.Kjell Arne Johansson has the right to grant on behalf of all authors and does grant on behalf of all authors.The Corresponding Author This study did not involve patients or sensitive information about patients and did not therefore require ethical approval.The authors declare that they have no competing interests.All authors fulfill the criteria of authorship and contributed collaboratively to conception and design, analysis and interpretation of data, drafting the article, revising it critically for important intellectual content and final approval of the version to be published. We assure that there is no one else who fulfils the criteria of authorship that has not been included as an author.KAJ was principal investigator, designed the study, performed the systematic literature search, developed the Markov model, led the analysis, and was lead author for the paper. BR was a co-investigator and participated in the planning, analysis and writing of the paper. OFN sought funding for the study, contributed to design, planning, analysis and writing of the paper. All authors read and approved the final manuscript.