Saccharomyces cerevisiae, their regulation being predominantly investigated by mathematical modeling. To our knowledge there has not been a genetic approach to elucidate the regulatory role of the different enzymes of the glycolytic pathway.A well known example of oscillatory phenomena is the transient oscillations of glycolytic intermediates in S. cerevisiae X2180. This has enabled us to employ existing isogenic deletion mutants and dissect the roles of isoforms, or subunits of key glycolytic enzymes in glycolytic oscillations. We demonstrate that deletion of TDH3 but not TDH2 and TDH1  abolishes NADH oscillations. While deletion of each of the hexokinase (HK) encoding genes (HXK1 and HXK2) leads to oscillations that are longer lasting with lower amplitude, the effect of HXK2 deletion on the duration of the oscillations is stronger than that of HXK1. Most importantly our results show that the presence of beta (Pfk2) but not that of alpha subunits (Pfk1) of the hetero-octameric enzyme phosphofructokinase (PFK) is necessary to achieve these oscillations. Furthermore, we report that the cAMP-mediated PKA pathway (via some of its components responsible for feedback down-regulation) modulates the activity of glycoytic enzymes thus affecting oscillations. Deletion of both PDE2 (encoding a high affinity cAMP-phosphodiesterase) and IRA2  abolished glycolytic oscillations.We report that the laboratory strain BY4743 could also be used to investigate this oscillatory phenomenon, which traditionally has been studied using PDE2 and IRA2, encoding components of the cAMP pathway responsible for negative feedback regulation of PKA, are required for glycolytic oscillations, suggesting an enticing link between these cAMP pathway components and the glycolysis pathway enzymes shown to have the greatest role in glycolytic oscillation. This study suggests that a systematic genetic approach combined with mathematical modelling can advance the study of oscillatory phenomena.The genetic approach to characterising the glycolytic oscillations in yeast has demonstrated differential roles of the two types of subunits of PFK, and the isoforms of GAPDH and HK. Furthermore, it has shown that  Saccharomyces cerevisiae cells are incubated with glucose in the presence of the respiration inhibitor cyanide, transient oscillations of levels of relevant glycolytic metabolites can occur. These include levels of nicotinamide adenine dinucleotide (NAD) which can be seen to oscillate between the oxidized (NAD+) and reduced forms (NADH), as well as other glycolytic intermediates, including glucose-6-phosphate, fructose-6-phosphate and fructose 1,6-bisphosphate [m) [S. cerevisiae, as they have been observed in Saccharomyces carlsbergensis[Dictyostelium[2+ levels [Glucose is the major source for energy production. It is converted to pyruvate via the glycolysis pathway, leading to production of ATP coupled with generation of intermediates and reducing power in the form of NADH for other biosynthetic pathways Figure . When glhate [m) . This phhate [m)  and is khate [m) . These obergensis, muscle bergensis. Glycolyyostelium, circadiyostelium, oscilla+ levels  and the + levels .+ ATPase and mitochondrial ATP-ase [Different factors have been shown to control the oscillations. These include temperature , cell de ATP-ase . It is b ATP-ase ,16 altho ATP-ase -17. The  ATP-ase -20. Of t ATP-ase . In cont ATP-ase . Both thS. cerevisiae strain X2180. This strain has never been used to generate targeted deletion mutants; therefore, the potential effects of deletion or over-expression of glycolytic genes have, until now, remained unknown. In contrast, almost complete collections of isogenic deletion mutants are available in the BY4743 sequenced standard laboratory genetic background [To our knowledge there has not been previously an attempt to investigate the yeast glycolytic oscillations at genetic level. This could be due to the fact that until now, the oscillations have only been observed in the diploid ckground .S. cerevisiae strain BY4743. We have subsequently used the genetic resources available in this genetic background to investigate the effects of deletion of different glycolytic enzymes encoding genes on the NADH-mediated glycolytic oscillations in the respective mutants. We have observed differential roles of the two subunit types of the phosphofructokinase, as well as the different isoforms of the hexokinase and the glyceraldehyde-3-phosphate dehydrogenase. We have used this experimental data to evaluate via parameter sensitivity analysis and representative simulations the mathematical model of yeast glycolytic oscillations developed by Wolf et al. [In this study, we have demonstrated that glycolytic oscillations can be observed in the diploid f et al. . FurtherThe strains used in this study are listed in Table 2HPO4, pH 6.8) and suspended to 10% wet weight in the same buffer. They were then incubated at 30C with shaking for three hours and kept on ice until use.The strains were grown essentially as described by Poulsen et al. . For eacOscillations were followed using a protocol adapted from Poulsen et al. . FollowiFrequency and amplitudes of oscillations were determined from Discrete Fourier Transformations (DFTs), found with Fast Fourier Transformations (FFTs) carried out in Matlab. DFTs are calculated according to the equation below:This equation gives the FFT of the data. The frequency is found by taking the maximum frequency value from a plot of frequency versus power spectrum.The steady-state parameter sensitivity analysis was carried out using SBToolbox  in Matlai-th component of the model with respect to the j-th parameter is defined by:The non-normalised amplitude sensitivity for the pj is the reference parameter value, δpj is the perturbation applied to the parameter, Ai(pj) is the amplitude for the reference parameter value and Ai(pj + δpj) is the amplitude for the perturbed parameter value.where The normalised amplitude sensitivity is then defined by:The same definitions apply to the period sensitivity by replacing amplitudes with periods. These functions were used in this form by Stelling et al. .J0) of 50.0 mM min-1, the combined pool of NAD+ and NADH of 1.00 mM, and the combined ADP and ATP pool of 4.00 mM. Perturbation analysis was carried out by changing the value of the relevant parameter in 1% increments for up to 10% and running the model in Matlab.The point in which the sensitivity analysis was carried out is defined by the reference conditions provided by Wolf et al. . These iGlycolytic oscillations in S. cerevisiae BY4743 are comparable to those in X2180 Until this study, yeast glycolytic oscillations have been investigated in S. cerevisiae X2180, a strain rarely used in genetic analysis. We demonstrate for the first time that glycolytic oscillations could also be observed in the standard reference genetic background strain BY4743. As shown in Figure HXK1 and HXK2. Our experiments showed that deletion of the hexokinase encoding genes had no effect on the frequency of the oscillations  which has two isoforms encoded by s Figure a. HowevePFK1) and four beta (encoded by PFK2) subunits. Unlike mathematical modelling analysis which does not discriminate the two subunit types, our genetic approach was able to test their individual contributions in glycolytic oscillations. Deletion of PFK1 resulted in a slight decrease in oscillation frequency. In contrast, deletion of PFK2 abolished oscillations completely  catalyses the reaction of glyceraldehyde-3-phosphate to 1,3 bis-phosphoglycerate. The enzyme has three isoforms encoded by three unlinked genes, tivities ,33. Whiltivities . As showGPM1,2,3, code for phosphoglycerate mutase in the yeast genome. However, while GPM1 is functional and essential for viability [GPM2 and GPM3 are most-likely non-functional homologues of GPM1, since mutation in neither GPM2 nor GPM3 appears to affect glycolysis, or confer any obvious phenotype. In the current investigation, glycolytic oscillations in the gpm2Δ and gpm3Δ mutants were found to be wild type (data not shown). This observation gives further support to the theory that GPM2 and GPM3 were generated from GPM1 by a gene duplication event and then diverged from the parent copy by mutation [Phosphoglycerate mutase mediates the conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis, and the reverse reaction during gluconeogenesis. Three genes: iability , GPM2 anmutation .ENO1 and ENO2 respectively. Both function in dimeric complexes [eno1Δ was available in the EUROSCARF collection but eno2Δ was not. When tested in our experimental conditions the NADH profile of eno1Δ mutant was very similar to the wild type (data not shown).There are two phosphopyruvate hydratase isoforms encoded by omplexes  and cataPYK2 and CDC19, respectively. However, Cdc19p appears to be the main pyruvate kinase because the cdc19∆ mutant is not able to grow in the presence of glucose [CDC19 and not PYK2 is tightly regulated and sensitive to levels of fructose-1,6-bisphospate. In agreement with these data, we found (data not shown) that glycolytic oscillations were present in the pyk2Δ and were comparable to the wild type.The conversion of phosphoenolpyruvate to pyruvate (the last step in glycolysis) is catalysed by two pyruvate kinases encoded by  glucose . Furtherhxk2Δ mutant, whose frequencies and amplitudes are much lower. This can also be seen in the NADH profile  with an oscillatory pattern similar to that of the wild type have marginally higher frequencies and slightly lower amplitudes compared to the wild type. This suggests that these mutations also have a small effect on the oscillations, as opposed to no effect.Fourier analysis was employed to determine the frequencies and amplitudes of the glycolytic oscillations in all mutants where the oscillations were present. The analysis shows that the frequencies Figure  and ample Figure a of thisThe results described above provided a large amount of data that could on one hand be used to develop future models of yeast glycolytic oscillations, and on the other, to analyze published models. We have used the parameter sensitivity analysis tool in SBToolbox  as descrr1ki1, the inhibition constant for the lumped HK-PFK reaction. Interestingly, r7k, the rate constant for the ATPase reaction of the model is another sensitive parameter. This suggests that ATPases have a key role in regulating glycolytic oscillations, a possible example of distributed control [jKappa, the rate constant for acetaldehyde diffusion, which is evidence for the importance of acetaldehyde in controlling the oscillations.Based on our experimental evidence, we would expect the most sensitive parameters to be those of phosphofructokinase, and of the hexokinases Figure b, a. The control . The nexr1ki1 (the most sensitive parameter) or r8k (one of the least sensitive parameters) by applying 1% increments for up to 10%, and the results of the simulations are plotted. While there were clearly differences in frequency between the simulations upon perturbation of r1ki1 , we noted that while both Pfk1 and Pfk2 have been shown to physically interact with PKA catalytic subunit isoforms (PFK is a known phosphorylation target for PKA), only PFK2 has been found to interact genetically with IRA2 and PDE2, whose deletion mutations abolish glycolytic oscillations.Previously we have reported that deletion of the gene encoding the high affinity cAMP phosphodiesterase Pde2p causes significant changes to the transcript abundances of the majority of the genes encoding glycolytic enzymes -43. Sincsterase: ); gpr1Δ eceptor: ), gpa2Δ protein: ), tpk1Δ,t of PKA ) (data n Ras-GTP ) and pdes Figure . It is ned state . RemarkaS. cerevisiae. It has become possible because of the genetic resources available when using the sequenced genetic background strain BY4743, which as our study shows  can grow on glucose, although there is no in vitro detectable PFK activity in these mutants [pfk2 deletion mutant is temperature sensitive for growth at 37°C, sensitive to caffeine and Congo red. The pfk1 deletion mutant on the other hand is more resistant to caffeine compared to the wild type. And in the current study we show further phenotypic differences between the two types of deletion mutants. Firstly, the pfk2 but not pfk1 deletion mutant is deficient in NADH-mediated glycolytic oscillations. Secondly, pfk2 and not pfk1 mutant does not display the characteristic NADH “spike” after glucose addition  in glycolytic oscillations. Previously, mathematical modelling which has demonstrated that the phosphofructokinase, with its allosteric regulation, has the greatest control over the oscillations has not discriminated between the two types of subunits that constitute this enzyme. Lack of PFK activity  mutants . Arvanit mutants  demonstr mutants  have shoPFK1, PFK2 is involved in genetic interactions with PDE2 and IRA2, the components of the cAMP pathway which affect NADH oscillatory activity  is capable of phosphorylating Pfk2.The specific role of Pfk2 in glycolytic oscillations could be due to the fact that it is the subunit which contains binding sites for fructose 6-phosphate , as prevy Figure . Pde2 anHXK2 deletion on the duration of the oscillations is stronger than that of HXK1. Although the deletion mutant for GLK1 whose product is also known to phosphorylate glucose at the C6 position was not investigated, we predict that its deletion would yield results comparable to those of HXK1, as both GLK1 and HXK1 are repressed when grown on a fermentable medium using glucose as a carbon source [HXK1 and GLK1 have been reported to increase, whilst those of HXK2 and PFK2 decrease respectively, upon the diauxic shift [HXK2 (but not HXK1), and PFK2 (but not PFK1) have been found to interact genetically with PDE2 and IRA2, deletions of which affect glycolytic oscillations  in glycolytic oscillations remains as the corresponding mutants were not available to test in the current study. It could be hypothesised that their transcripts’ down-regulation upon the diauxic shift [PFK2. And although there might be an enticing link between transcript down-regulation upon the diauxic shift of genes such as HXK2 and PFK2[HXK1 and HXK2 affects glycolytic oscillations, however their transcript levels respond in a contrasting manner to the diauxic transition: HXK1 is up-regulated, whilst HXK2 is down-regulated. Moreover, deletion of TDH3 abolishes glycolytic oscillations completely although neither this gene, nor the remaining two  are subject to differential regulation of transcription upon the diauxic shift.The question regarding effects of deletion of ic shift  suggests and PFK2 and their1ki1, the inhibition constant for the combined HK-PFK reaction, as the most sensitive parameter. Moreover, as our experimental evidence demonstrated it is the deletion of PFK2, HXK1 and HXK2 that produce the greatest effect on glycolytic oscillations. Deletion of PFK1 on the other hand does not significantly affect glycolytic oscillations showing that the components of the lumped HK-PFK reaction have specific properties. More precise models will be needed that differentiate between the different isoforms or subunit components of glycolytic enzymes. Oscillatory parameter sensitivity analysis is a useful technique to identify crucial parameters for the control of biological rhythms and investigate their robustness. It has been applied to several other types of oscillating systems, such as circadian rhythms [Previously, Reijenga et al.  and Mads rhythms ,57, NF-κ rhythms  and calc rhythms .r1k1) is not as sensitive as the inhibition constant. This suggests that the reverse feedback by ATP on PFK is potentially the most important controller of the oscillations which is in agreement with results obtained in yeast extract experiments by Madsen et al. [PFK2 abolishes them completely), we have confirmed that the negative feedback of Pfk2 by ATP is a key regulatory factor in glycolytic oscillations.Interestingly, the forward constant for the lumped HK-PFK reaction  [GAL1 promoter replacement pma1 mutant and does not present data for its isogenic wild type parental strain. Instead the report shows as supplementary data the results of an alternative wild type strain (a derivative of W303) with a NADH oscillatory pattern which is very far from comparable to X2180 and that of BY4743 shown in our study. There are different ATP-ase encoding genes in the yeast genome; however none of the respective viable deletion mutants were included in our analysis. Given the well established role of the genetic background as a modifier of different phenotypes, we propose that in future studies it would be of interest to investigate the role of the different ATP-ases further by analysing the NADH-mediated oscillations in the standard BY4743 genetic background.This study demonstrated that  Figures  and 7. R. (2012) . HoweverS. cerevisiae. Importantly it shows genetic evidence that PDE2 and IRA2, which encode components of the cAMP pathway responsible for negative feedback regulation of PKA, are required for glycolytic oscillations, suggesting an enticing link between these cAMP pathway components and the glycolysis pathway enzymes with a key role in glycolytic oscillation. We demonstrate that a systematic genetic approach combined with mathematical modelling can advance the study of oscillatory phenomena.This study has for the first time provided genetic evidence for the differential roles of the two types of subunits of PFK, and the isoforms of GAPDH and HK in glycolytic oscillation in the yeast The authors declare no competing interests in relation to this manuscript.TW carried out the characterization of the NADH-mediated glycolytic oscillations profiles of the mutants and the initial Wolf’s model simulation. He was also involved with the drafting of the manuscript, and preparation of figures and tables. DA performed the normalized parameter sensitivity analysis, simulated the effects of reducing GAPDH activity, and helped JMS in preparing Figure


A maternal line of inheritance regarding eczema has been described in several studies, whereas others find associations to both a maternal as well as a paternal line of inheritance. When studying family history of eczema symptoms, cohort studies including siblings are rare. Time point for assessing family eczema-history could be of importance when studying the associations between family eczema-history and children with eczema, as parents with unaffected children may not recall mild symptoms in other siblings or their own disease history. We therefore aimed to study the associations between reported eczema in mother, father and siblings and reported eczema in index child where information on family history was collected at two different ages of index child.Parents/children participating in The Prevention of Allergy among Children in Trondheim (PACT) study were given questionnaires on reported eczema symptoms in mother, father and siblings at 6 weeks and 1 year. When index child was 2 years of age, a detailed questionnaire on different health issues with emphasize on different allergy related disorders were filled in.Both maternal and paternal reports on eczema were significantly associated with eczema in index child. Reporting family eczema-history at 1 year (N = 3087), "eczema sibling only" [adjusted odds ratio (aOR) = 3.13 (2.27-4.33)] as well as all other family-groups containing siblings with eczema were strongly associated with eczema 2 years. When family eczema-history was reported at 6 weeks (N = 2657), reporting of "eczema sibling only" was not associated to reported eczema at 2 years in index child [aOR = 1.31 (0.77-2.23)].Having sibling(s) with eczema strengthened the associations between maternal and paternal reports on eczema with eczema in index child only when exposure was reported at 1 year. These findings indicate that results from questionnaires-based studies of family eczema-history depend on whether or not index child has yet developed eczema.ISRCTN28090297ISRCTN:  Atopic eczema is a complex disease caused by a mainly unknown interaction between genetic and environmental factors . The genWhen family eczema-history is studied, several investigators have studied the protective effect of having a high number of siblings on the development of allergic diseases ,14. HoweWhen studying diseases in the general population, self-reported questionnaires are often used. One of the flaws with such information could be recall bias concerning how well adults remember both their own childhood diseases as well as diseases in children with mild symptoms -18.The aim of this prospective study was therefore to investigate the associations between reported eczema in mother, father and siblings and reported eczema in index child at 2 years of age where family eczema-history as well as exposure was collected at two different ages of the index child.The Prevention of Allergy among Children in Trondheim (PACT) study is a large population- based prospective study on allergy related disorders conducted in Trondheim, central Norway. Trondheim has approximately 165 000 inhabitants and 2100 deliveries pr year.th 2006. The PACT-study is described in further detail elsewhere  , ]  (data not shown).In the model testing association between siblings with and without eczema and association to eczema in index child 2 years, we found a significant association for siblings with eczema in the 1 year cohort only . No interaction between eczema mother and father was found in either of the cohorts  also answered Q3 (1 year) as well as Q5 (2 years). The two cohorts are generated from the same geographic area (the city of Trondheim), during the same time-period, and by the same midwifes and GPs.Although eczema is a relatively prevalent disease among children in the western world, most children have mild degree of disease, making the diagnosis and differentiating between different phenotypes of the disease as well as mild cases challenging -23. We fin utero or in early infancy may be essential in determining disease expression [Several studies describe a maternal line of inheritance concerning eczema ,6,22. Thpression . In thispression ,9,25 andpression . In famipression . Althougpression . It is tpression . Since mWhen family history of eczema is studied, allergy related disorders in siblings are seldom accounted for in the risk analysis. We found that, although having any one parent with eczema was associated with eczema in index child at 2 years, having one or several siblings with eczema together with mother and/or father with eczema was strongly associated with eczema in index child 2 years when reports were collected at 1 year. This association was also seen when eczema was reported only in sibling(s). There are different interpretations of these findings. One possible explanation could be that this is a reflection of a parental genetic disposition with incomplete penetrance . MutatioThe "eczema-sibling-effect" was not significant when reported at 6 weeks. Awareness of disease in first child with mild disease might be absent until same kinds of symptoms as e.g. dry skin is observed in second child. Also, in mild cases of eczema a significant proportion of the children are disease free by the age of 3 years . In thisIn a German study of children 9-11 years old having two first degree relatives with the same atopic disease was highly associated with eczema . DiepgenWhen only some 60% of those finishing the questionnaires on exposure were answering the questionnaires on health, one could argue that the data are prone to self-selection. In both cohorts the lost-to-follow-up group contained more current smoking mothers and fewer homeowners, indicating a lower socio-economic status (data not shown). However, other exposure data, including reported family eczema-history did not differ among those who followed up and those who did not.Another limitation of the study was the reporting eczema in parents and siblings without a clinical verification. The question "have you, the child's father or any of your joint children ever had eczema" is a core ISAAC questions, but is most often used in combination with a question on rash located in typical places or diagnosis verified by a doctor. To the best of our knowledge, the question has not been validated in an adult population. However, the almost similar phrasing "have you ever had childhood eczema" was validated among adults in a Swedish population. The sensitivity and specificity of this question was 89.9% and 70.7%, respectively , and an The strengths of this study are the large number of unselected participants as well as the prospective design. The consistency of reported eczema indicates a high reliability of the questions and the prevalence of reported eczema is well in line with the prevalence found in the PACT endpoint-study . In addiWe found that reporting having a sibling with eczema at 1 year was significantly associated with reported eczema at 2 years. Eczema in mother as well as eczema in father was both associated with eczema 2 years. When family eczema-history was reported when index child was 1 years of age, the associations with eczema 2 years were significantly stronger for both parents if sibling(s) of the index child had eczema, and association was significantly stronger for sibling(s) reported at 1 year compared to 6 weeks. However, although siblings had eczema, the shared environment by mother and child did not result in any difference between maternal and paternal associations to eczema 2 years in index child.The finding of different associations when family-eczema history was reported at 6 weeks compared to when family eczema-history was reported at 1 year indicate bias in information gathering and has important implications on comparability of studies measuring the effect of older sibling disease on index child`s risk of eczema.The authors declare that they have no competing interests.MS participated in the design of the study, performed statistical analysis and drafted the manuscript. TØ, OS and RJ conceived the study, participated in its design and co-ordination and helped draft the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-5945/11/11/prepub


Little is known about the incidence and risk of seizures after stroke in young adults. Especially in the young seizures might dramatically influence prognosis and quality of life. We therefore investigated the long-term incidence and risk of post-stroke epilepsy in young adults with a transient ischemic attack (TIA), ischemic stroke (IS) or intracerebral hemorrhage (ICH).We performed a prospective cohort study among 697 consecutive patients with a first-ever TIA, IS or ICH, aged 18–50 years, admitted to our hospital between 1-1-1980 till 1-11-2010. The occurrence of epilepsy was assessed by standardized questionnaires and verified by a neurologist. Cumulative risks were estimated with Kaplan-Meier analysis. Cox proportional hazard models were used to calculate relative risks. After mean follow-up of 9.1 years (SD 8.2), 79 (11.3%) patients developed post-stroke epilepsy and 39 patients (5.6%) developed epilepsy with recurrent seizures. Patients with an initial late seizure more often developed recurrent seizures than patients with an initial early seizure. Cumulative risk of epilepsy was 31%, 16% and 5% for patients with an ICH, IS and TIA respectively (Logrank test ICH and IS versus TIA p<0.001). Cumulative risk of epilepsy with recurrent seizures was 23%, 8% and 4% respectively . In addition a high NIHSS was a significant predictor of both epilepsy and epilepsy with recurrent seizures .Post-stroke epilepsy is much more common than previously thought. Especially patients with an ICH and a high NIHSS are at high risk. This calls upon the question whether a subgroup could be identified which benefits from the use of prophylactic antiepileptic medication. Future studies should be executed to investigate risk factors and the effect of post-stroke epilepsy on quality of life. The incidence of epilepsy after stroke in elderly ranges from 2% to 14%, depending on study population, stroke subtype and definitions of epilepsy Quantification of post-stroke epilepsy frequency and risk factors is a first step in both informing young stroke patients and evaluating and developing treatment strategies. The aim of this study was therefore to investigate the long-term incidence and risk of post-stroke epilepsy and epilepsy with recurrent seizures in patients with a TIA, IS or ICH at young age.Follow-Up of Transient ischemic attack and stroke patients and Unelucidated Risk factor Evaluation”-study (FUTURE study), a prospective cohort study that investigates causes and consequences of stroke in young adults. Details of the study have been described elsewhere This study is part of the “The FUTURE study comprises all consecutive patients with a TIA, IS or ICH, aged 18–50 years, admitted to the Radboud University Nijmegen Medical Centre from 1-1-1980 till 1-11-2010. TIA was defined as a rapidly evolving focal neurological deficit, without positive phenomena such as twitches, jerks or myoclonus, with no other than a vascular cause lasting less than 24 hours. Stroke was defined as focal neurological deficit persisting for more than 24 hours Patients were included prospectively between 1980 and 2010 with a standardized collection of baseline and clinical characteristics . Furthermore all patients underwent neurological examination and brain imaging at the time of their index event Information on the vital status was available either from the hospital or via the municipality registry. Patients alive were invited for follow-up assessment between 1-11-2009 and 1-1-2012. All patients signed informed consent.Primary outcome was the occurrence of epilepsy after stroke. Post-stroke epilepsy was defined according to the most recent version of the International League Against Epilepsy (ILAE), in which patients with a single seizure associated with an enduring condition that could cause epilepsy (for example stroke), met the criteria of epilepsy All patients alive underwent standardized, structured questionnaires. In case a patient had died, this information was retrieved from the general practitioner by the same structured questionnaires. If seizures were reported, information from the treating physician was retrieved, both for patients alive and patients who died. This information was verified and adjudicated by an experienced neurologist who was blinded for the index event. In addition when patients reported early or onset seizures, this information was verified by medical chart review or by retrieving information from other treating physicians. Information about date of seizure, number of seizures during follow-up and treatment were systematically recorded. Data on outcome were available for all patients.For comparison of continuous variables between groups students-t-test or Mann-Whitney U test were used when appropriate. In case of categorical variables chi-square-test was used.Cumulative risks of post-stroke epilepsy and epilepsy with recurrent seizures were estimated with Kaplan-Meier analysis, with the first post-stroke seizure as qualifying endpoint. Patients who died or did not reach the endpoint before follow-up ended were censored. Furthermore as patients with a recurrent stroke or TIA might have a higher risk of epilepsy, they were censored after their recurrent event. Differences between stroke subtype and gender in cumulative risks of post-stroke epilepsy and epilepsy with recurrent seizures were tested with the log rank test.c)1/n] where Ic equals the cumulative risk of epilepsy or epilepsy with recurrent seizures at n years, obtained by the Kaplan-Meier method Average annual risks of epilepsy and epilepsy with recurrent seizures were calculated using the formula 1−[ and 47.1% was male. Median NIHSS at admission was 3 (IQR 1–8.5). Of all patients 160 (23.0%) had died after a mean follow-up of 9.1 years (SD 8.2).79 patients (11.3%) developed post-stroke epilepsy during follow-up . In 25 p39 Patients (5.6%) had epilepsy with recurrent seizures during follow-up. Patients with an ICH more often developed epilepsy with recurrent seizures (10.6%), followed by IS (6.6%) and TIA patients (1.9%). Patients with an initial late seizure, more often developed recurrent seizures than patients with an initial early seizure .12 out of the 25 patients with an early seizures did not start anti-epileptic drugs (AED’s) during follow-up. 10 Patients started directly after the initial early seizure and 3 patients started after recurrent seizures. Of the 54 patients with an initial late seizure, 7 were not treated with AED’s during follow-up. 37 Started directly after the initial late seizure, 8 started after recurrent seizures and in 2 patients it was unknown when anti-epileptic drugs were started.Patients with an initial late seizure more often started with AED’s than patients with an initial early seizures . Patients with recurrent seizures more often received AED’s than those with a single seizure .After total follow-up of maximum 30 years the cumulative risk of post-stroke epilepsy was 14% (95% CI 10.3–16.6) for all stroke subtypes. Cumulative risk of post-stroke epilepsy was 31%, 16% and 5% for patients with an ICH, IS and TIA respectively (log rank test ICH versus IS and IS versus TIA p<0.001) .Cumulative risk of epilepsy with recurrent seizures was 7% (95% CI 5.0–10.0). Patients with an ICH (23%) or an IS (8%) had a higher cumulative risk of developing epilepsy with recurrent seizures than patients with a TIA (4%)  .Average annual risk of both epilepsy and epilepsy with recurrent seizures was the highest during the first year after the index event (8% and 4% respectively), especially in patients with an IS or ICH .High NIHSS at admission and IS or ICH as stroke subtype were significant predictors of post-stroke epilepsy, corrected for age and gender . High NIOur study is the first to investigate the incidence and risk of post-stroke epilepsy in young adults with a TIA, IS or ICH. We found an incidence of 11.3% for post-stroke epilepsy and 5.9% for epilepsy with recurrent seizures after a mean follow-up of almost 10 years.Strengths of our study were the prospective and single centre design, which allowed us to collect information systematically and to uniformly verify both the qualifying event as well as the outcome. Furthermore, our study has one of the longest follow-up ever reported and the largest cohort compared with other studies investigating epilepsy after a stroke in young adults. In addition, our study has a high external validity, when comparing baseline characteristics to other studies However there are also a few limitations. Some selection bias may have occurred due to selective loss to follow-up. Those lost to follow-up more often had an IS and less often a TIA compared to participants. Potentially, this might have resulted in an underestimation of the overall risk of epilepsy in our cohort, as patients with an IS exhibited a higher risk of post stroke epilepsy. However, the severity of stroke did not differ between participants and those lost to follow-up, so we considered this loss of follow-up very unlikely to influence our results. Patients who refused to participate did not differ in baseline characteristic from participants except from smoking, making selection bias in this group for the same reason unlikely.As information on seizures was collected by structured questionnaires for all patients in the same way, it is unlikely that seizures are reported differently between the stroke subtypes, which makes information bias very unlikely. Furthermore the neurologist verifying the event was blinded for the index event. Since the present study features a long inclusion period recall bias might have influenced our findings, especially when the index event or seizure occurred a longer time ago. However there were no differences in reporting seizures between patients who suffered their stroke a long time ago than those with a more recent stroke , making recall bias unlikely. In addition it might have been possible that less information would be available in patients who died, especially in those who died a longer time ago, resulting in an underestimation of epilepsy in this group. However we did not find a difference in frequency of epilepsy between patients who died a long time ago and those who died more recently , making this explanation unlikely. On top of this, information on patients who died before follow-up ended was collected in an identical manner as in patients alive.Another source of bias might be misclassification of TIA patients. In some cases it might be difficult to distinguish a TIA from a seizure, especially for example in case of a limb-shaking TIA Another source of bias to be considered is confounding. We tried to overcome this by making appropriate adjustments for potential confounders including age, stroke severity, gender and stroke subtype in our multivariate analysis. Furthermore we have adjudicated for recurrent stroke or TIA, by withdrawing patients after a recurrent event in the Kaplan-Meier analysis.Another possible confounding factor might have been the use of AED’s. In our study 10 out of 25 patients with an early seizure and 37 out of 54 patients with an initial late seizure started directly after the first seizure with AED’s. It might be possible that the use of AED’s has reduced the risk of epilepsy with recurrent seizures after a single seizures. However as AED’s were not prescribed prophylactic, only the frequency of epilepsy with recurrent seizures might have been affected by the use of AED’s and not the incidence of the first seizure. In addition the impact of AED’s on epileptogenesis remains unclear. Furthermore during 30 years of follow-up patients may have switched or stopped AED’s, which may have provoked or prevented occurrence of seizures as well.According to the new definition of the ILAE patients already meet the criteria of epilepsy when a single seizure occurs with an enduring cause. Most other studies still use the former version in which patients meet the diagnosis of epilepsy when they have at least 2 (late) seizures. For reasons of comparability, we therefore compared the risks of seizures in previous studies with the risk of epilepsy in our study. We found a higher incidence of post-stroke epilepsy compared to other studies that investigated seizures stroke in young adults (6–10%) Compared to other studies in elderly with more or less the same definitions we found a higher incidence of epilepsy and epilepsy with recurrent seizures (3.4% to 10.4% and 2% to 5% respectively) The higher cumulative risk of post-stroke epilepsy and epilepsy with recurrent seizures in our young cohort may presumably be due to mechanisms in young patients which makes them more vulnerable for the development of epilepsy. This is also suggested by few other studies among elderly who found a higher incidence of seizures in younger patients, after stratification in several age groups Interestingly, in our study patients with a TIA had comparable risk of epilepsy than shown in studies among elderly TIA patients Few studies found comparable incidences of early seizures, As in other cohorts a higher cumulative risk epilepsy and epilepsy with recurrent seizures was found in patients with an ICH. In addition a high NIHSS was a significant predictor of both epilepsy and epilepsy with recurrent seizures In conclusion, we showed that epilepsy and epilepsy with recurrent seizures are much more common after a stroke in young adults than previously described. Future studies should be executed to investigate risk factors and the underlying pathophysiology. Besides this, these studies should also investigate the effect of post-stroke epilepsy on quality of life. Furthermore, the high cumulative risk of epilepsy is as high as the risk of recurrent stroke


For patients with metastatic renal cell cancer (mRCC) who progressed on vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor therapy, the orally administered mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to prolong progression free survival. Intriguingly, inhibition of mTOR also promotes expansion of immunosuppressive regulatory T cells (Tregs) that can inhibit anti-tumor immune responses in a clinically relevant way in various tumor types including RCC. This study intends to investigate whether the antitumor efficacy of everolimus can be increased by preventing the detrimental everolimus induced expansion of Tregs using a metronomic schedule of cyclophosphamide.This phase I-II trial is a national multi-center study of different doses and schedules of low-dose oral cyclophosphamide in combination with a fixed dose of everolimus in patients with mRCC not amenable to or progressive after a VEGF-receptor tyrosine kinase inhibitor containing treatment regimen. In the phase I part of the study the optimal Treg-depleting dose and schedule of metronomic oral cyclophosphamide when given in combination with everolimus will be determined. In the phase II part of the study we will evaluate whether the percentage of patients progression free at 4 months of everolimus treatment can be increased from 50% to 70% by adding metronomic cyclophosphamide (in the dose and schedule determined in the phase I part). In addition to efficacy, we will perform extensive immune monitoring with a focus on the number, phenotype and function of Tregs, evaluate the safety and feasibility of the combination of everolimus and cyclophosphamide, perform monitoring of selected angiogenesis parameters and analyze everolimus and cyclophosphamide drug levels.This phase I-II study is designed to determine whether metronomic cyclophosphamide can be used to counter the mTOR inhibitor everolimus induced Treg expansion in patients with metastatic renal cell carcinoma and increase the antitumor efficacy of everolimus.NCT01462214, EudraCT number 2010-024515-13, Netherlands Trial Register number NTR3085.ClinicalTrials.gov Identifier  Approximately 2% of all adult malignancies are kidney tumors and these account for about 116.000 deaths worldwide per year .The treatment of metastatic RCC (mRCC) has considerably changed over the last 5 years due to the antitumor efficacy of two groups of targeted agents, namely agents that inhibit vascular endothelial growth factor (VEGF)-signaling pathways and those that inhibit mammalian target of rapamycin (mTOR) . The oraAs a derivative of rapamycin, everolimus acts as a signal transduction inhibitor that is selective for mTOR. mTOR is a key protein kinase present in all cells which regulates cell growth, proliferation, angiogenesis, and survival. The mTOR pathway also plays an important role in immunoregulation. It critically controls homeostasis and the balance between effector T cells and regulatory T cells -11; inhi+CD25+ regulatory T cells (Tregs) represent a functionally distinct lineage of immunoregulatory T cells crucial for the maintenance of tolerance. Tregs are critically dependent on the X-chromosome encoded FOXP3 gene. Mutations in this gene, that encodes the forkhead/winged-helix family transcriptional repressor Scurfin, cause a fatal human autoimmune disorder called IPEX  [CD4yndrome) . Tregs hyndrome) . Of noteyndrome) -18. Impoyndrome) -21. Notayndrome) ,22-24.Based on these data, the aim of the present study is to evaluate whether metronomic cyclophosphamide can be used to counter the mTOR inhibitor everolimus induced Treg expansion in patients with metastatic renal cell carcinoma and increase the antitumor efficacy of everolimus.In the phase I part of the study we will determine the optimal Treg depleting dose and schedule of metronomic oral cyclophosphamide when given in combination with a fixed dose of everolimus. Patients will be enrolled in cohorts of 5 per dose level  oral everolimus in patients with mRCC.Assessment of the recommended dosing and schedule for metronomic cyclophosphamide with respect to the optimal selective induction of CD4To investigate the proportion of patients with mRCC receiving everolimus and metronomic cyclophosphamide that is progression free at 4 months.To determine the safety and tolerability of the everolimus-cyclophosphamide combination.- To assess the response rate, time to progression and overall survival of the combination of metronomic cyclophosphamide and fixed dose oral everolimus.+CD25+ regulatory T cells.- Assessment of the immunological effects of combining metronomic cyclophosphamide with everolimus focusing on its impact on the number and function of circulating and tumor-infiltrating CD4- Assessment of the effect of the combination of metronomic cyclophosphamide and everolimus on selected angiogenesis parameters.- To assess the effect of cyclophosphamide on everolimus drug levels.This phase I-II trial is a national multi-center dose-escalation study of low-dose oral cyclophosphamide in combination with a fixed dose of everolimus in patients with mRCC not amenable to, or progressive after, a VEGF-receptor tyrosine kinase inhibitor containing treatment regimen.The study is initiated by the department of medical oncology of the VU University Medical Center and will be conducted within the context of the Netherlands Working Group on Immunotherapy of Oncology (WIN-O) with a present participation of 13 hospitals.1. Signed informed consent.2. Age ≥ 18 years.3. Histological or cytological confirmed clear-cell mRCC with progressive disease and not amenable to, or progressive on, or within 6 months of stopping treatment with a VEGF receptor tyrosine kinase inhibitor (sunitinib (or pazopanib) ± sorafenib).4. No other current malignant disease, except for basal cell carcinoma of the skin.5. WHO performance status 0-2.6. Life expectancy of at least 12 weeks.7. Adequate hematologic function: ANC ≥ 1.5 × 109/L, platelets ≥ 100 × 109/L, Hb ≥ 6.0 mmol/L.8. Adequate hepatic function: serum bilirubin ≤ 1.5 × ULN, ALT and AST ≤ 2.5 × ULN (or ≤ 5 times ULN if liver metastases are present).9. Adequate renal function: calculated creatinine clearance ≥ 50 ml/min.10. Measurable or evaluable disease as defined by RECIST 1.1.11. Patients with reproductive potential must use effective contraception, negative pregnancy test for female patients.12. Able to receive oral medication.13. Prior therapy with cytokines  and/or VEGF-ligand inhibitors (i.e. bevacizumab) is permitted.14. Patients with brain metastases are eligible if they have been stable for at least two months post-radiation therapy or surgery.1. Currently receiving chemotherapy, immunotherapy, or radiotherapy, or having received either one of these ≤ 4 weeks prior to visit 1. The wash-out period for sunitinib or sorafenib is at least 2 weeks.2. Known human immunodeficiency virus (HIV) or other major immunodeficiency.3. Treatment with immunosuppressive agents within 3 weeks of study entry, except for low dose corticosteroids.4. Active bleeding diathesis or oral anti-vitamin K medication.5. Patients with untreated CNS metastases with clinical symptoms or who have received treatment for CNS metastases within 2 months of study entry.6. Active infection or serious intercurrent illness, except asymptomatic bacteriuria.7. Presence of unstable angina, recent myocardial infarction (within the previous 6 months), or use of ongoing maintenance therapy for life-threatening ventricular arrhythmia.8. Macroscopic hematuria.9. Prior therapy with mTOR inhibitors.10. Known hypersensitivity to everolimus or other rapamycins (sirolimus/temsirolimus) or to its excipients.11. Pregnant or nursing women or women of childbearing potential not utilizing an effective contraceptive method. Men with partners of childbearing potential not using an effective method of contraception. (Use of effective contraceptives must continue for 3 months after the last dose of everolimus).12. Presence of any significant central nervous system or psychiatric disorder(s) that would hamper the patient's compliance.13. Uncontrolled diabetes as defined by fasting serum glucose > 2 ULN, severely impaired lung function.14. Cirrhosis/chronic active hepatitis/chronic persistent hepatitis, history of hepatitis B or C virus infection.15. Drug or alcohol abuse.16. Any other major illness that, in the investigator's judgment, substantially increases the risk associated with the subject's participation in the study.The study will be conducted in accordance with ethical principles that have their origin in the Declaration of Helsinki and are consistent with International Conference on Harmonisation (ICH) Guidelines for Good Clinical Practice. This protocol has been submitted and approved by the Medical Ethical Committee of the VU University Medical Center, Amsterdam, the Netherlands and the Central Committee on Research Involving Human Subjects (CCMO). Approval of independent medical ethics committees of participating hospitals is required before local initiation of the study protocol. Oral and written informed consent is obtained from all patients prior to trial participation.ToetsingOnline. All SAEs must also be reported on the Adverse Event page of the CRF.All adverse events encountered during the clinical study will be reported on case report forms (CRF). In case of a serious adverse event (SAE), the investigator has a legal requirement to inform the central data management office within 24 hr through the web portal The principal investigators are responsible for the management of the safety reporting according to local regulations and guidelines. The study drug license holder will be provided with copies of all report submissions to regulatory authorities and to the ethical committee that has approved the study by the principal investigators. All reported (serious) adverse events will be discussed by an independent data monitoring committee. Novartis is responsible to ensure that the latest investigator's brochure is used as the source document for determining the expectedness of a SAE.The study progress, safety data and data quality will be monitored by an Independent Data (and safety) Monitoring Board (IDMB), which will be independent of the trial organizers. Safety analysis will be performed on a regular basis and the IDMB will report their findings to the principal investigators. Once a year throughout the clinical trial, the principal investigators will submit a safety report to the accredited METC, including a list of all suspected (unexpected or expected) serious adverse reactions, along with an aggregated summary table of all reported serious adverse reactions. In addition, a complete safety analysis and an evaluation of the balance between the efficacy and the harmfulness of the medicine under investigation will be submitted.®. This open source web-based software platform for clinical trial data is compatible with the cancer Biomedical Informatics Grid (caBIG®) from the U.S. National Cancer Institute to manage clinical research data in multi-site studies.Data management and trial coordination will be performed via the use of OpenClinicaIn the phase II part of the study an interim analysis (IA) will be performed after treatment of 24 patients that reached 4-month progression free survival (PFS) or that progressed within the first 4 months. If the number of patients progression free at 4 months is < 12 the study will be terminated due to lack of efficacy. In addition, the number of patients with combination treatment related grade ≥ 3 toxicity will be analyzed and if this number is 12 or higher, the study will be terminated because of unacceptable toxicity.Due to the time dependent nature of the outcome of interest, it is possible that there will not be sufficient evidence to either terminate or continue the trial at the time of enrollment of the 24th individual. In that case, the PFS rate at 4 months for the previously started patients will determine whether or not to halt the trial due to lack of efficacy. A decision must be made whether or not to halt enrollment until these 24 patients have been followed for 4 months or to continue enrollment and risk completing enrollment for the trial before the interim analysis is made. If only 3 of the first 24 patients have a response, then enrollment will be halted until the 4-month progression free survival interim analysis is completed.In the phase I part of the study, patients are defined as evaluable if they have completed a minimum of 2 weeks of combination therapy . Statistical analysis in the phase II part will be performed according to the intention-to-treat principle. All patients in this part of the study that have taken their study medication on at least one occasion are considered evaluable patients.The response rate will be determined as the proportion of treated patients who had a partial or complete response. Time to progression (TTP) and overall survival will be constructed by the use of Kaplan-Meier curves. TTP will be measured from enrollment and preferably be defined based on CT-scan imaging based on Response Criteria according to RECIST (version 1.1) or alternatively based on clinical evaluation. For TTP analysis death is considered as progressive disease. Median TTP and overall survival will be calculated along with 95% confidence intervals. Statistical analyses of changes in immune and angiogenesis parameters and drug levels will be performed using paired Student t tests or Wilcoxon matched pairs tests as appropriate. P < 0.05 will be considered statistically significant.nd stage  rejecting of the drug due to inadequate response is ≤ 32 patients without being progression free at 4 months. At least 32 patients should be without grade ≥ 3 toxicity due to therapy, otherwise the combination therapy will be rejected due to excessive toxicity.The sample size in the Phase I part of the study is expected to be 10-50 patients. Patients will be enrolled in cohorts of 5 per dose level. The first 5 patients will be assigned to dose level 0, to assess the effects of everolimus monotherapy. Subsequently, 8 combination therapy dosing levels are planned  and, in case of female participants, urine HCG. Abdominal and chest CT scans must be done within 4 weeks prior to start of therapy.- Patient visits will be scheduled at 2, 4 and 8 weeks and subsequently 4-weekly. At each visit medical history, physical examination, toxicity assessment, hematology and serum chemistry will be performed.+CD25+FOXP3+ regulatory T cells, conventional T cell subsets, NK cells, invariant CD1d-restricted NKT cells, dendritic cell subsets, myeloid-derived suppressor cells and cytokine levels will be performed at baseline and subsequently at 2, 4, and 8 weeks after the start of the study treatment period.- Immune monitoring on CD4- Angiogenesis monitoring on thrombospondin 1 (TSP-1), VEGF, circulating numbers of platelets and hematopoietic progenitor cells (HPCs) will be performed at baseline, week 4 and 8.- Everolimus and cyclophosphamide drug levels will be measured during the phase I part of the study at week 4.- If patients agreed on a tumor biopsy, a second biopsy will be obtained at week 4.- CT images will be required at fixed time-points; baseline, 8 and 16 weeks, and thereafter every 8 weeks during treatment until progressive disease.- Adverse events will be reported during the study treatment phase. Medically significant adverse events considered related to the investigational product by the investigator will be followed until resolved or considered stable.- At the end of study treatment, medical history and toxicity will be assessed, and physical examination, hematology, serum chemistry, immune and angiogenesis monitoring, abdominal and chest CT scans will be performed.- Follow up will last till progressive disease and/or death of the patient.Everolimus is a selective mTOR signal transduction inhibitor. It exerts its activity through high affinity interaction with an intracellular receptor protein, the immunophilin FKBP12. The FKBP12/everolimus complex subsequently interacts with the mTOR protein kinase, inhibiting downstream signaling events. mTOR is mainly activated via the PI3-kinase pathway through AKT/PKB and the tuberous sclerosis complex (TSC1/2). mTOR functions as a sensor of mitogens, growth factors and energy and nutrient levels, facilitating cell-cycle progression from G1-S phase in appropriate growth conditions. This pathway is also involved in the production of pro-angiogenic factors (i.e. VEGF) and stimulation of endothelial cell growth and proliferation. mTOR regulates protein translation through inactivating eukaryotic initiation factor 4E binding proteins and activating the 40S ribosomal S6 kinases, including the HIF-1 proteins .Everolimus has been investigated in various indications, namely solid organ transplantation, hematologic and non-hematologic malignancies, and rheumatoid arthritis. Based on its potential to act directly on tumor cells by inhibiting tumor cell growth and proliferation and indirectly by inhibiting angiogenesis , everolimus was also investigated as an anticancer agent.Everolimus has been evaluated clinically in patients with mRCC who failed a previous VEGF receptor tyrosine kinase inhibitor treatment and with several other solid malignancies in investigator sponsored studies. Daily doses of 5 and 10 mg have shown target inhibition with satisfactory tolerability in most patients. In general, everolimus is well tolerated and the safety profile is considered favorable and distinct from that of traditional chemotherapy ,7.Cyclophosphamide is an alkylating agent that mediates crosslinking of DNA. It is frequently used in high doses in the treatment of several malignancies  and induMetronomic cyclophosphamide has also been reported to inhibit tumor angiogenesis, e.g. by reducing the mobilization and viability of HPCs and by inducing sustained apoptosis of endothelial cells within the vascular tumor bed -32. TSP-In the present study, cyclophosphamide will be administered orally in the dose determined by the dose level to which the patient is assigned. As cyclophosphamide requires 4-hydroxylation by cytochrome p450s including CYP3A4, and everolimus is mainly metabolized by CYP3A4 in the liver and to some extent in the intestinal wall, there is a potential interaction between the two drugs . TherefoPatients will be enrolled in cohorts of 5 per dose level. The first 5 patients enrolled will be assigned to dose level 0 in order to assess immune and anti-angiogenic effects of everolimus monotherapy. The second 5 patients enrolled will be assigned to dose level 1. If there are ≤ 1 dose-limiting toxicities (DLTs) experienced by the first 5 patients in a cohort during the first 28 days after the first study treatment, patients will be entered in the next dose level. Entry of patients into the expansion cohort will not occur until at least 28 days after the last patient in the escalation phase received his/her first study treatment. At the final dose level recommended for the phase II study a minimum of 10 patients will be treated. Intra-individual dose escalation is not permitted in order to allow determination of any possible cumulative effect and to evaluate the inherent toxicity of the regimen at any given dose level. The 10 patients that are being treated at the final dose level in the phase I part will be included in the subsequent phase II analyses.In case no depletion of Tregs is observed during the phase I part of the study, we consider the absence of an increase in Tregs as the minimally acceptable outcome for proceeding to the phase II part of the study.Up to 56 patients will be treated at the dose level that has been selected based on its capacity to most selectively deplete circulating Treg levels in the phase I part of the study. Treatment will be continued until disease progression and/or death of the patient. In case of progressive disease, the choice of subsequent therapy is at the discretion of the investigator.The mTOR inhibitor everolimus prolongs PFS in patients with mRCC from 1.9 months to 4.9 months relative to placebo ,7. AlthoThis phase I-II study is designed to a) obtain the optimal Treg depleting dose of metronomic cyclophosphamide when combined with the mTOR inhibitor everolimus in patients with mRCC and b) to determine whether this combined treatment results in enhanced antitumor efficacy of everolimus. The study intends to increase progression free survival at 4 months from 50% to 70% by adding metronomic cyclophosphamide to everolimus.The authors declare that they have no competing interests.The study is partly funded by a grant from Novartis Oncology Netherlands.Novartis has had no part in study design, data collection, analysis, interpretation, the writing of the manuscript, or the decision to submit for publication.CMH and HJV drafted the manuscript. HJV wrote the original protocol for the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/505/prepub


Mycobacterium indicus pranii (MIP) when given as prophylactic vaccine in animal models of tuberculosis.The 9-month-long chemotherapy of tuberculosis often results in poor compliance and emergence of drug-resistant strains. So, improved therapeutic strategy is urgently needed. Immunotherapy could be beneficial for the effective management of the disease. Previously we showed the protective efficacy of We sought to investigate whether MIP can be used as an adjunct to the chemotherapy in guinea pig models of tuberculosis. Efficacy of MIP was evaluated when given subcutaneously or by aerosol.MIP-therapy as an adjunct to the chemotherapy was found to be effective in accelerating bacterial killing and improving organ pathology. MIP-immunotherapy resulted in higher numbers of activated antigen-presenting cells and lymphocytes in the infected lungs and also modulated the granulomatous response. Early increase in protective Th1 immune response was observed in the immunotherapy group. Following subsequent doses of MIP, decrease in the inflammatory response and increase in the immunosuppressive response was observed, which resulted in the improvement of lung pathology.MIP immunotherapy is a valuable adjunct to chemotherapy for tuberculosis. Aerosol route of immunotherapy can play a crucial role for inducing immediate local immune response in the lung. The pandemic of tuberculosis (TB) is an international public health tragedy: estimated 9.4 million new cases of TB in 2008. TB is treatable by drugs, and the World Health Organization has promoted the “directly observed therapy” to improve compliance as chemotherapy needs to be taken for 9-month long duration. Patients who default on therapy have severe risk of relapsing and acquiring drug resistance. Hence, multidimensional approach is desired to effectively eradicate the actively multiplying as well as persistent bacteria from the infected lungs.M.vaccaeDuring the course of mycobacterial infection, the protective Th1 immune response is dominated by immunosuppressive Th2 response which could facilitate the survival and progression of infection. Immunotherapy could be a potential tool in antimycobacterial therapy since it may boost the Th1 immune response in infected patients and might act as force in shifting the immune response from Th2 to Th1 type. This protective immune response can act in synergy to antibacterial chemotherapy. Several studies have been done to evaluate the immunotherapeutic role of Mycobacterium indicus pranii (MIP) formerly known as Mycobacterium w, is a cultivable, non-pathogenic and rapidly growing saprophyte classifiable in Runyons group IV along with other rapid growers like M. Fortuitum, M.smegmatis, M. Chelonae and M. Vaccae on the basis of its growth and metabolic properties Mycobacterium lepraeMIP shares a numbers of common B and T cell determinants with in-situ expression of different cytokine genes to characterize the modulation of immune response with MIP-treatment in M.tb-infected guinea pigs. Protection with MIP-treatment was associated with reduced bacterial loads and pulmonary pathology.In this study, we evaluated the efficacy of MIP as an adjunct to standard chemotherapy for tuberculosis, when given by aerosol or parenteral route. We monitored the immune cells accumulation in the lung and also analyzed the Female outbred Duncan Hartley guinea pigs (∼300 g in weight) were purchased from the disease-free small animal facility of Chowdhury Charan Singh Agriculture University, Hisar (CCSH), India and held under barrier conditions in biosafety level III animal laboratory where the animals are housed and maintained in agreement with the guidelines of the Institute's Animal Ethics Committee .2.MIP and H37Rv were grown in Middlebrook 7H9 medium  with 0.02% glycerol, 0.05% Tween 80 and 10% albumin-dextrose complex enrichment  in shaker flasks. Bacteria were harvested in the mid-log growth phase by centrifugation at 2,500 g for 15 min. The bacteria were then washed twice using the centrifugal washing method and suspended in saline at the desired concentration. For the killed preparation of MIP, the bacteria were inactivated by autoclaving for 20 min at a pressure of 15 lb/inMycobacterium tuberculosis H37Rv in a Madison chamber Guinea pigs were challenged with a low dose aerosol of Recently published reports show a wide window of dose of different drugs There were two schedules of therapy as follows:5 bacilli in 100 µl saline) was given subcutaneously, every 15 day; which means total of 5 doses of MIP were given at day 1, 15, 30, 45 and 60 respectively during the course of therapy. In the third group, along with the drug combination, aerosol of killed MIP was given (10 ml of 109 bacilli/ml in nebuliser) in a Madison aerosol chamber, every 15 day as described for the second group. As the drug combination was made in the sucrose solution to increase the palatability, the fourth group was sucrose control group, i.e., daily oral dose of 500 µl of 40% (wt/vol) sucrose was given. In the second schedule, the chemotherapy was given for one month, i.e. a total of 25 doses of drugs in the respective groups. The groups which were treated with drug plus immunotherapy, received the dose of MIP by s.c or aerosol route on 1st, 15th and 30th day of chemotherapy i.e. total of 3 doses of MIP. The detailed diagrammatic schedule of therapy has been shown in In the first schedule, the chemotherapy was started from the early chronic phase of infection, i.e., on day 30 post infection and the first dose of MIP was given on the same day. The animals were allocated into the following groups: In the first group only drug combination was given 6 days-a-week through oral route for two months, which accounts for total of 50 doses. The drug was administered orally by force feeding using 1 ml sterile syringe without needle. In the second group, along with the drugs, killed preparation of MIP . In each group, 5 animals were taken.Seven days after the completion of chemotherapy , the animals were euthanized; organs were aseptically removed for further studies.After scissoring off the entire lung, the overall pulmonary pathology was graded by giving score to each individual macroscopic parameter as follows: for presence of nodules, scores were 0, 1, 2 and 3 for zero nodule, <5 nodules, 5–10 nodules and >10 nodules respectively. For presence of cavity, scores were 0, 1, 2 and 3 for no cavity, <5 cavities, 5–10 cavities and >10 cavities respectively. For haemorrhagic spots, scores were 0, 1, 2 and 3 for no spot, <5 spots, 5–10 spots and >10 spots respectively.Lungs from each guinea pig were fixed with 10% buffered formalin. Sections from these tissues were stained using hematoxylin and eosin for monitoring the overall morphology of the section. Progression of lung pathology was evaluated using a histological grading system Bacterial loads in different groups were evaluated after the completion of therapy. Organ homogenates were plated in serial 10-fold dilutions in quadruplicate on LJ plates . Plates were incubated inside semi-sealed plastic bags at 37°C for 3 to 4 weeks, and colonies were counted. Bacterial loads in 4 individual animals per group were determined at each time point.Single cell suspensions were made from the lungs of guinea pigs using a standard protocol Clone CT4 reacts with a 32–36 kDa glycoprotein expressed on the surface of guinea pig lymphocytes and Langerhans cells. The antigen is anchored to the cell membrane via a phospatidylinositol linkage and mediates T-cell activation. Thus, it may be considered a “guinea pig T-cell activation marker” Serial 5–6 µm thick cryo-sections from the lung samples were washed, and non-specific antibody binding was blocked by incubating the sections in a 1% bovine serum albumin-PBS solution. The sections were then incubated overnight at 4°C with primary antibodies, washed 3 times in PBS and incubated with an F (ab') 2 rabbit anti-mouse secondary detection antibody conjugated to horseradish peroxidase . The color was developed using a DAB substrate kit  or ImmPACT NovaRED peroxidase substrate kit (Vector Laboratories). The sections were counterstained with methyl green  and mounted with D.P.X. mountant .4 . Primer sequences for IFNγ The mRNA expression levels of different cytokines were carried out by RT-PCR analysis using the standard protocol. The quantitative analysis of mRNA expression levels of different cytokines was performed using qPCR mix  and the samples were run in mastercycler realplexIL-2:5′ GCTACTCTTGTCTTGCCTTG3′.Forward: 5′ GGTTGCTAGTAACGCCTTC3′.Reverse: GAPDH:5′ GTGAAGCAGGCATCAGAG3′.Forward: 5′ AGCGTCAAAAGTGGAAGAA3′.Reverse: Student's t test, was used to assess the statistical significance of difference between the groups.Results are shown as the mean ± the standard error. Comparisons between groups were performed by analysis of variance in GraphPad InStat software (Version 3.05). After 60 days of therapy, significant reduction in pulmonary as well as splenic bacterial load was observed in the groups treated with drug plus immunotherapy as compared to the group treated with chemotherapy only. No bacterium was observed in liver in any of the treated groups (data not shown). In short-course therapy of 30 days, bacterial load in lungs was reduced by about 3 log in drug-treated group as compared to the untreated control group. The load was further reduced by 0.7 log in drug + immunotherapy group . ImportaThe pathology developed in the lung was assessed in the randomly taken tissue sections from all parts of the organ. The degree of granulomatous organization of infiltrated cells indicated the extent of host response against the infection. The pathological deterioration in the infiltrated area was estimated by evaluating the degree of mineralization, caseation and necrosis.The overall improvement in the pathology of the lung in drug plus MIP group was reflected in the significantly reduced score of gross pathology both after 30 days as well as 60 days therapy . FormatiSince pulmonary pathology and bacterial load was less in the group where MIP therapy was given by aerosol route as compared to subcutaneous route, hence the former group was selected for further studies. To understand the underlying cellular immune response in the group treated with MIP, we analyzed the proportion of different immune cells in the lung at different time points during the course of therapy and compared the same with the group treated with drug only after establishing chronic infection.st dose of MIP. But the percentage of the activated CD8+ T cells in this group went down at later stage of therapy. Two distinct populations of homing receptor expressing lymphocytes were observed  and then followed along with chemotherapy could provide any additional therapeutic advantage; however we did not observe any significant improvement of bacterial clearance or any other advantage as compared to the group where immunotherapy started along with chemotherapy (data not shown).M. tuberculosis might overshadow the efficacy of a single dose of immunomodulator that could otherwise be effective in eliciting strong anti-TB Th1 response. So, multiple doses of Th1 stimulator might be needed at regular interval to avert such Th2 dominating situation. Secondly, with immunotherapy, strong effector immune response is desired to get quick and synergistic bactericidal effect which could potentiate the standard chemotherapy.We used multiple doses of MIP for immunotherapy as pre-existent Th2-driving physiological condition like infection with We have evaluated the immunotherapeutic activity of MIP when given as aerosol or by s.c route. The aerogenic route of antigen delivery is not only advantageous over parenteral route for physically targeting the lung, but also due to its noninvasive nature. Administration of MIP by aerosol route is crucial in inducing local lung immune response. There are several reports showing the advantage of aerogenic route of immunization over the parenteral route The maximum bactericidal activity of the drug combination takes place in the first month; thereafter the bacterial killing slows down Higher proportion of activated T cells were observed in the lung, 15 days after start of immunotherapy and also intragranulomatous CD8+ T cells were higher in number in the MIP group which suggests that, one of the early bactericidal effects of MIP-driven immunotherapy could be due to cell-mediated cytotoxic killing of mycobacteria. However, it is not very clear why the number of activated CD8+ T cells declined rapidly after the subsequent doses of MIP. This could be due to the reduced lung bacterial load during that time.About 10% higher influx of neutrophils was observed within a week after first dose of MIP in drug + immunotherapy group; however the level subsequently trimmed down at subsequent time points of therapy. Recent report suggests that neutrophils play important role in early mycobacterial control in vivo condition. So the cellular composition inside the granuloma was also studied by immunohistochemistry. Higher percentage of CD4+ T cells were found inside the granuloma of control as well as ‘drug only’ group. The role of this bulk of T cell subpopulation is not known, but one could anticipate that it might contribute towards the formation of large central necrotic core. We found high intragranulomatous CD8+ T cells in the lung sections of animals which were given adjunct immunotherapy. This suggests the cell-mediated cytotoxic killing of intracellular mycobacteria inside the granuloma.It is very difficult to digest the mineralized or caseated tissue portions to have the good yield of cells. Hence, flow cytometric analysis of the cells from whole lung may not truly reflect the actual The expression of intragranulomatous MHC II was more fringed in the drug-treated group (data not shown), while the same was very much disbanded throughout the granuloma in the group treated with drug plus immunotherapy, which possibly suggests the induction of a stronger and uniform macrophage activity in this group. The homogeneous expression of MHC II inside the granuloma could be due to the aerosol route of MIP delivery which allows the overall homogenous spread of MIP antigens.Interestingly, plenty of granulocytes were found inside the granuloma in the drug + immunotherapy group at late stages of therapy; although unlike immunotherapy with BCG, no Koch phenomenon was observed following multiple doses of MIP during therapy. It is reported that antigranuloma therapy in tuberculosis using prednisone or etanercept as an adjunct to the standard chemotherapy could be a potential therapeutic tool TNFα and IL12 expression level was increased after 2 week of therapy in both the treated groups, could be because of active killing of bacteria at this stage. Higher expression of IL-2 after the second dose of MIP might be responsible for increased expression of CCL5. This high expression of CCL5 in immunotherapy group might be enhancing the T cell recruitment at the site of infection. At later stage, when bacterial burden is low, higher expression of TGFβ was observed which might be contributing towards reduced pulmonary pathology in the immunotherapy group. It is not clear, why consistently high expression of CCL5 was observed; however, it is assumed that it might be the recall response due to multiple doses of MIP.M.tb exposure results in increased level of IL-17 along with influx of granulocytes which collectively results in lung tissue damage and aggravation of pathology M.tb challenge but interestingly we did not notice any sign of pathological deterioration although, we noticed increased level of granulocytes inside the granuloma at late stages of MIP-immunotherapy. We could not investigate any influence of IL-17 due to the unavailability of any suitable reagent. It is difficult to conclude the therapeutic superiority of MIP over M. vaccae, as the latter one showed significant therapeutic efficacy and enhaced recall IFNγ response in animal models M.vaccae was reported in different clinical trials M.vaccaeM.vaccae has been shown to be mediated by the strong Th1 immune response viz. IL-2 and IL-12 and the concurrent downregulation of Th2 response Unlike the BCG-based immunotherapy To summarize, we found an early granuloma-specific immune response in the drug + immunotherapy group, which was followed by granulocyte-driven dismantling of granuloma which might have been useful in killing the bacteria as they become available to the bactericidal activity of the drugs. A balanced inflammatory and suppressive immune response in later part of the drug plus immunotherapy treatment is perhaps useful in the process of restoration of normal tissue architecture and controlling the initial inflammatory reaction. These data provide the first direct evidence of protective efficacy of MIP immunotherapy in tuberculosis and the associated modulation of immune response which can be related to reduction in the lung pathology and control of bacterial growth. The results indicate that MIP might not be able to reduce the treatment duration, but could be potentially very useful in eradicating the persistent bacteria when given with chemotherapy. We believe that the aerosol route of MIP immunotherapy can play a very important role for inducing immediate local immune response in the lung.