Evidence demonstrates that rodents learn to associate a foot shock with time of day, indicating the formation of a fear related time-stamp memory, even in the absence of a functioning SCN. In addition, mice acquire and retain fear memory better during the early day compared to the early night. This type of memory may be regulated by circadian pacemakers outside of the SCN. As a first step in testing the hypothesis that clock genes are involved in the formation of a time-stamp fear memory, we exposed one group of mice to fox feces derived odor (TMT) at ZT 0 and one group at ZT 12 for 4 successive days. A separate group with no exposure to TMT was also included as a control. Animals were sacrificed one day after the last exposure to TMT, and PER2 and c-Fos protein were quantified in the SCN, amygdala, hippocampus, and piriform cortex. Exposure to TMT had a strong effect at ZT 0, decreasing PER2 expression at this time point in most regions except the SCN, and reversing the normal rhythm of PER2 expression in the amygdala and piriform cortex. These changes were accompanied by increased c-Fos expression at ZT0. In contrast, exposure to TMT at ZT 12 abolished the rhythm of PER2 expression in the amygdala. In addition, increased c-Fos expression at ZT 12 was only detected in the central nucleus of the amygdala in the TMT12 group. TMT exposure at either time point did not affect PER2 or c-Fos in the SCN, indicating that under a light-dark cycle, the SCN rhythm is stable in the presence of repeated exposure to a fear-inducing stimulus. Taken together, these results indicate that entrainment to a fear-inducing stimulus leads to changes in PER2 and c-Fos expression that are detected 24 hours following the last exposure to TMT, indicating entrainment of endogenous oscillators in these regions. The observed effects on PER2 expression and c-Fos were stronger during the early day than during the early night, possibly to prepare appropriate systems at ZT 0 to respond to a fear-inducing stimulus. Circadian rhythms in mammals are regulated by the suprachiasmatic nucleus (SCN) of the hypothalamus, which is optimally positioned above the optic chiasm to receive photic input from the retinohypothalamic tracts Although clock genes regulate circadian rhythms in SCN cells, they are also expressed in cells throughout the brain and body, including the liver, skin, pancreas, lung, heart, and reproductive organs There is also evidence that circadian rhythms regulate specific behaviors. Honey bees given sugar water at a distinct location during a specific time of day learn to return at the correct time of day to anticipate reward Although a circadian regulated fear memory and reward memory has been demonstrated in a number of behavioral studies in rodents )  at ZT 0 for 4 successive days (TMT0 group)  and male C57BL6 mice (12) were purchased from Charles River Breeding Laboratories . Animals were maintained in a 12∶12 hour light dark (LD) cycle in light-tight black boxes with food and water available ad libitum. ZT 0 was designated as lights on, and ZT 12 was designated as lights off. All experiments were performed according to protocols approved by Northeastern University's Internal Animal Care and Use Committee (protocol # 10-0102 R). One group of C3H (12 mice) was exposed to fox feces derived 2,5-dihydro-2,4,5-trimethylthiazoline (TMT0 group)  by removAnimals were euthanized at 4 times of day , in either light or dark conditions depending on ZT time, by cervical dislocation one day after the last exposure to TMT to ensure that any observed changes in PER2 or c-Fos expression would not be related to immediate response to TMT but instead reflect a change in circadian rhythm. One set of 12 male C57BL6 mice  were used for a pilot study of quantification of PER2 expression in each brain region. C3H mice were used for the full study to avoid any potential confounding effects that a lack of melatonin in C57BL6 mice may have.Whole brains were dissected from mice and post-fixed in 4% paraformaldehyde (PFA) overnight for fixation. Brain samples were then transferred to 20% glycerol in 0.1 M PBS for cryoprotection and were cut into 40 µm serial coronal sections on a freezing microtome  and stored in 0.1 M PBS with 0.1% NaAzide.One set of 9 sections from each animal representing the SCN, amygdala, Hipp, and PirCx was chosen for immunostaining for PER2 protein and assigned to 6-well staining dishes. All sections were processed to together for immunohistochemistry to allow for comparison of immunostaining across all sections, brain regions, ZTs, and animals. All solutions were made in Tris buffered saline (pH 7.4) with 0.3% triton-x 100 (TBS-Tx) unless otherwise specified. Sections were washed in TBS-Tx, then incubated in 0.3% H202 for 10 minutes, followed by quick washes in TBS-Tx. Sections were then incubated in blocking solution for 1 hour consisting of 3% normal goat serum (nGs) and 5% milk for PER2. Following several washes in TBS-Tx, sections were placed in primary antibody Rabbit anti-PER2 1∶4000  with 3% nGs and 5% milk for 72 hours at 4 degrees Celsius. Sections were incubated in biotinylated goat anti-rabbit (1∶200)  in 3% nGs for 2 hours at 4 degrees Celsius followed by washes in TBS and incubation in HRP conjugated streptavidin  for 2 hours at 4 degrees Celsius. Color reaction was obtained by incubating sections in diaminobenzadine/peroxidase solution  in TBS. Sections were then washed in TBS and mounted on gelatin coated glass slides and coverslipped using permount media. Care was taken that each six-well staining dish contained sections from TMT 0, TMT 12, and control samples and was carried through each immunocytochemistry step for the same duration of time to avoid potential sequence effects.For c-Fos immunohistochemistry, a separate set of 9 sections containing the SCN, amygdala, Hipp, and PirCx were selected and placed into 6-well staining dishes as described for PER2 immunohistochemistry. All solutions were made in 0.1 M PBS with 0.2% triton-x 100 unless otherwise noted. Free-floating sections were washed in PBS-TX, incubated in 0.3% hydrogen peroxide for 10 minutes, followed by quick washes in PBS-Tx. Sections were then incubated in 2% bovine serum albumin (BSA) for 1 hour followed by washes in PBS-Tx, and then incubated in primary antibody rabbit polyclonal anti-c-Fos   for 72 hours at 4 degrees Celsius. Sections were then incubated in biotinylated goat anti-rabbit (1∶500)  for 2 hours at room temperature, followed by washes in PBS-TX and incubation in HRP conjugated streptavidin  for 2 hours at room temperature. Sections were developed in DAB as described for PER2 and mounted on gelatin coated glass slides and coverlsipped using permount media. Care was taken that each six-well staining dish contained sections from TMT 0, TMT 12, and control samples and was carried through each immunocytochemistry step for the same duration of time to avoid potential sequence effects.For Nissl staining, 40 µM sections were mounted on gelatin coated glass slides, hydrated through a series of ethanol concentrations, and incubated in thionin stain solution for 10 minutes, dehydrated through a series of ethanol concentrations and coverslipped with permount media.The SCN was outlined based on cytoarchitectonic criteria from Nissl stained sections as well as on PER2 immunostaining using the Chemoarchitectonic Atlas of the Mouse Brain (Paxinos & Watson 2009). Three sections per animal were used for quantification, representing the dorsal, middle, and caudal portions of the SCN. The amygdala was defined by cytoarchitectonic criteria according to the Chemoarchitectonic Atlas of the Mouse Brain (Paxinos & Watson 2009), using PER2 immunostaining as well as Nissl stained sections from the same animals. The amygdala was divided into the lateral, basolateral, and central nuclei. Data for PER2 and c-Fos did not differ between the lateral and basal nuclei, therefore they were grouped together and referred to as the BLA. Data for the central amygdala, due to its separate inputs and function and reports that its rhythm in PER2 expression is anti-phase to the BLA, is reported separately 2). The second method was by using the thresholding feature in ImageJ after converting all photos to grayscale, to threshold the immunoreactive particles and set shape parameters for the program to determine what to consider a cell. This method automatically counted the number of particles (cells) within a traced region and resulted in IR-cells/area (μm2). The third method used the same thresholding technique but instead quantified the total amount of thresholded immunoreactive area. This amount was divided by the total area of the traced region to obtain a density of immunoreactivity (IR-area (μm2)/total area (μm2)). Threshold settings were held constant across all sections and light intensity was held constant during photo acquisition. All three methods resulted in similar data, with clear rhythms in PER2 expression in the SCN peaking at ZT12 and antiphase rhythms in the BLA, Hipp, and PirCX. The standard error was lowest in the third method, using thresholded IR-area, and was thus chosen as the method reported for the full study. Values for thresholded IR-area for each region were combined across sections, and divided by the total area of the region added across sections to obtain one density value per region per animal for analysis.Microscopic images were captured using a Nikon Eclipse E400 microscope with an Olympus DP12 (DIH032861) camera interfaced with Magna-Fire SP 2.1B imaging software. For each region of interest, images were taken at 4x magnification for identification of regions, and 20x magnification for quantification of immunostaining. Immunostaining was quantified using ImageJ 1.41 . A pilot study was conducted on a set of control sections  from male C57BL6 mice to determine the optimal quantification method. For the pilot study, PER2 was quantified using three different methods in ImageJ. The first method was by manually counting all immunoreactive cells within a traced region of interest to obtain a density (number of immunoreactive (IR) cells/area in µmOne animal/ time point  for each of the experimental groups (TMT0 & TMT12) was kept in a mouse wheel running cage to monitor activity in order to ensure that mice were trained as expected and that ZTs represented the expected CTs.One-way ANOVAs were performed with JMP v5.0.1a . For comparisons between groups, for each brain region, the values of thresholded IR-area per section were added across sections from the same animal to obtain one thresholded IR-area value per animal. This value was divided by the sum of the areas of all of the sections for each animal in order to obtain one density value  for each animal, which was used for group comparisons. For example, for the BLA of one animal: Repeated exposure to TMT at either ZT 0 (TMT0) or ZT 12 (TMT12) for four successive days followed by one day without exposure  had no sRepeated exposure to TMT at ZT 0 essentially reversed the normal rhythm of PER2 expression in the BLA , Table 1Similar to the BLA, exposure to TMT reversed the normal rhythm of PER2 expression in the CE of the TMT0 group, with a peak of PER2 expression occurring at ZT 6 and the low point occurring at ZT 0, with significantly lower PER2-IR than both the control and TMT12 group at ZT 0 , Table 1Similar to the effects of TMT exposure in the BLA and CE, PER2 expression in the Hipp of the TMT0 group was significantly decreased at ZT 0 but significantly increased at ZT 6 compared to control subjects . PER2 exIn contrast to other brain regions, TMT exposure did not decrease PER2 expression at ZT 0 in the TMT0 group. Instead, TMT exposure resulted in significant increase of PER2 expression at ZT 6 and ZT 12 in the TMT0 group compared to control mice . This reFor recording of wheel-running activity, 4 mice for the TMT0 group and 4 mice for the TMT12 group were housed in running wheel cages during the experiment. All animals were properly entrained to the 12∶12 LD cycle, and TMT exposure in the LD cycle had no obvious effects on wheel-running activity, but the sample size was too small to rule out potential effects of TMT exposure on activity.Our results show that repeated exposure to a fear-inducing stimulus alters the rhythm of PER2 expression in brain regions responsible for processing this information, but does not affect PER2 expression in the SCN. The lack of effect on the SCN along with the observed effects on PER2 in the other brain regions highlights the role of the SCN as a pacemaker for the animal as a whole, as well as the possibility that other brain regions may be able to act as function-specific pacemakers with some degree of independence from the SCN in a LD cycle in vivo in response to environmental factors. Our results are consistent with data showing no effect of restricted feeding on PER2 or c-Fos expression in the SCN but a strong effect on PER2 and c-Fos in brain regions that may be involved in feeding, memory, and reward processing including the DMH, the Hipp, and the CE and BLA nuclei of the amygdala The rhythms of Per2 expression in control mice in our study are consistent with previous reports of Per2 rhythms in the SCN, BLA, and Hipp The rhythm in c-Fos expression in the SCN in our study in control mice is identical to the spontaneous rhythm in C57/BL6 mice reported by Grenados-Fuentes et al. The negative control condition in our study of disturbing the animals at the opposite time point of TMT exposure with a sterile, empty KimWipe was used to ensure that any differences observed in Per2 or c-Fos expression would be due to the effect of TMT exposure and not due to general disturbance of the animals. Although it is possible that animals may learn to associate a fear for TMT at both time points, work in hamsters has shown that animals learn to associate a fearful stimulus at the circadian time of exposure to the stimulus and not at the opposite timepoint 12 hours later In both experimental groups however, it is important to consider the possibility that the negative control of disturbing the same animals at the opposite time point, 12 hours apart, without exposure to TMT can induce conditioned fear at both the time of exposure and the negative control time of disturbance. This possibility should be taken into consideration when interpreting the data. Future work will examine the effect of repeated exposure to TMT without disturbance of the animals at the opposite timepoint in order to explore this possibility.The PirCx receives olfactory input from the olfactory bulbs, and is essential in processing olfactory information The Hipp is responsible for long term memory formation, storage, and retrieval The BLA is the brain region responsible for the expression and to some extent the storage, encoding, retrieval, and reconsolidation of fear memory PER2 expression in the CE was similar to that for the BLA in the experimental TMT groups. The expression of c-Fos however differed from the BLA in that there was no increase in c-Fos at ZT 0 for the TMT12 group, but there was a significant increase at ZT 12 for this group, indicating that unlike the BLA, the CE was preferentially activated at the time of TMT exposure. This observed increase in c-Fos expression specifically at the ZT of prior TMT exposure indicates that changes in expression are likely occurring in response to entrainment to the fear-inducing odor rather that to disturbances every 12 hours. Previous work in rats has shown that both innate and conditioned fear response increase c-Fos expression in the amygdala, including the BLA and the CE Taken together, our results indicate that the amygdala, when compared to the PirCx, distinguishes TMT as a fear-inducing stimulus rather than a general odor, as indicated in the PirCx by the increase of c-Fos at both ZT 0 and ZT 12 in the TMT0 and TMT12 groups, in essence inducing a 12 hr rhythm in c-Fos in response to odor stimulation. Since c-Fos responses were similar for both groups (TMT0 and TMT12), we cannot exclude the possibility that the procedure alone and presence of the investigator caused the changes in c-Fos expression. In comparison, the CE, which acts as the output area of the amygdala to the hypothalamic-pituitary-adrenal axis (HPA), showed increased c-Fos only at ZT 0 for the TMT0 group and only at ZT 12 for the TMT12 group. This result corresponds with previously published work showing that both fox urine and mineral oil equally induce c-Fos in the PirCx, but fox urine induces a greater amount of c-Fos in the amygdala The observed effects in the BLA and CE, together with the lack of changes in PER2 and c-Fos in the PirCx, indicate that time-stamp fear memory for the TMT odor is occurring primarily in the amygdala, and more strongly at ZT 0 than ZT 12. This interpretation is supported by studies showing that fear memory encoding, reconsolidation, and retrieval occurs in the amygdala, Studies indicate that rhythmic expression of PER2 in the CE but not the BLA or the Hipp, is dependant upon rhythmic glucocorticoid expression In summary, repeated exposure to a fear-inducing odor alters rhythmic expression of PER2 and induces c-Fos expression in selective brain areas involved in processing this information. Changes in PER2 and c-Fos expression were seen 24 hours after the last exposure to TMT, indicating that they were not acute responses to the stimulus. This is consistent with entrainment of endogenous rhythms in separate oscillators in these regions. The entrainment of these separate oscillators may be part of the mechanism behind the formation of time-stamp memories. Animals exposed to the fear-inducing odor during the early day were affected much more than animals exposed during the early night, indicating that changes in PER2 and c-Fos expression in selective brain regions may underlie the reported improved acquisition and retention of fear memory during the subjective day in comparison to the subjective night in rodents


Drosophila melanogaster demonstrated that wild-type genetic background influences the effects of an allele of scalloped (sd), with respect to both its principal consequence on wing development and its interactions with a mutation in optomotor blind. In this study we address whether the background dependence of mutational interactions is a general property of genetic systems by performing a genome wide dominant modifier screen of the E3sd allele in two wild-type genetic backgrounds using molecularly defined deletions. We demonstrate that ∼74% of all modifiers of the E3sd phenotype are background-dependent due in part to differential sensitivity to genetic perturbation. These background dependent interactions include some with qualitative differences in the phenotypic outcome, as well as instances of sign epistasis. This suggests that genetic interactions are often contingent on genetic background, with flexibility in genetic networks due to segregating variation in populations. Such background dependent effects can substantially alter conclusions about how genes influence biological processes, the potential for genetic screens in alternative wild-type backgrounds identifying new loci that contribute to trait expression, and the inferences of the topology of genetic networks.The phenotypic outcome of a mutation cannot be simply mapped onto the underlying DNA variant. Instead, the phenotype is a function of the allele, the genetic background in which it occurs and the environment where the mutational effects are expressed. While the influence of genetic background on the expressivity of individual mutations is recognized, its consequences on the interactions between genes, or the genetic network they form, is largely unknown. The description of genetic networks is essential for much of biology; yet if, and how, the topologies of such networks are influenced by background is unknown. Furthermore, a comprehensive examination of the background dependent nature of genetic interactions may lead to identification of novel modifiers of biological processes. Previous work in  Examining the consequences of how one mutation behaves when in the presence of a second mutation forms the basis of our understanding of genetic interactions, and is part of the fundamental toolbox of genetic analysis. Yet the logical interpretation of such mutational interactions depends on the generality of such findings. A small number of studies have demonstrated that factors such as the wild-type background in which the mutations are studied can have a profound impact on the observed phenotype of both specific effects of the mutation and the interactions between mutations. However, whether such findings are a common property of genetic interactions was unknown. We tested the generality of the background dependence of interactions between mutations and observed that the vast majority of the interactions were highly dependent on the wild-type background in which they are observed. We demonstrate that the same regions of the genome that contribute to the differences observed in the degree of severity of the mutational effect appear to also be responsible for the background dependence of the interaction. Fundamental to the logic of genetic analysis is that the observed variation in a phenotype for a genetically mediated trait is causally linked to one or more DNA lesions/variants. However, it is well known that the phenotypic effects of many individual mutant alleles are context dependent, with respect to environmental influences, as well as the “wild-type” genetic background in which the mutation is observed. Indeed, genetic background has long been known to influence observed phenotypic expression across traits, organisms, and a range of allelic effects, including hypomorphs, amorphs/nulls and neomorphs scalloped gene (E3sd) in Drosophila melanogaster is profoundly influenced by wild-type genetic background (optomotor blind/bifid (omb/bi), was then examined in a double mutant combination with E3sd. We demonstrated that the phenotypic consequence of the interaction between these mutations was markedly influenced by wild-type genetic background. In one wild-type background the double mutant combination resembled the individual E3sd phenotype, while in the other wild-type background, the omb mutation behaved as a strong synthetic enhancer of sdAlthough the basic influence of genetic background on the expressivity of mutations is well documented, the wider consequences of such influences are poorly understood ckground , with efE3sd using two wild-type genetic backgrounds. Our results suggest that the majority (∼74%) of all modifiers are background-dependent. The background-dependence of the modifier alleles are in part due to the wild-type strains differing in overall sensitivity to mutational perturbations. Using a subset of the deletions spanning the range of phenotypic effects of modifiers, we observed that the interaction effects were consistent using an additional allele, ETX4sd. Furthermore, we show that the deletion effects are a result of the interaction with mutations at the sd locus, and not a simple consequence of haplo-insufficiency in the genomic region of the deletion. We also demonstrate that the background-dependent interactions of modifiers with E3sd are linked to the same genomic regions that contribute to the background-dependent effects of the allele itself. We argue that the phenotypic expressivity of mutations can be considered a quantitative trait, and a more comprehensive, context-dependent view of the effects of mutations needs to emerge.Our findings clearly demonstrate the influence of wild-type genetic background on this genetic interaction, but an important challenge is to determine whether such context dependent effects are widespread. To address this question we performed a genome wide-screen for dominant modifiers of sd and omb, is dependent on genetic background E3sd mutation and deletions generated in otherwise isogenic backgrounds spanning the autosomes of Drosophila.Genetic modifier screens are powerful tools to both identify interacting factors that contribute to signaling networks, as well as to infer their topology. This approach has shaped our understanding of the genetic basis of many traits, across numerous organisms. However little is known about how wild-type genetic background influences genetic interactions. We previously demonstrated that the genetic interaction between mutations in two genes, Dll, wg, vg) previously demonstrated to interact with sd modify the E3sd phenotype. In each of these instances the deletions confirmed previous expectations for the interaction  genetic background , by asking what proportion of all “significant” modifiers also had a “significant” interaction effect between genetic background and the deletion. Based upon these criteria ∼79% of modifiers demonstrated background dependence. While each cross was carried out independently, there were a large number of crosses performed, and each deletion bearing genotype was compared to a common set of controls from within each block of crosses (see methods). Therefore we utilized several methods that adjust for multiple comparisons. While these methods will decrease the number of deletions deemed modifiers using standard comparisons (i.e. α = 0.05), we are primarily interested in the proportion of such modifiers that are due to background dependent effects. Using False Discovery Rate (FDR) we observed a similar frequency (∼78%) as with unadjusted p-values, while with the sequential Bonferroni (Holm) it was ∼68%. Regardless of the exact approach used, it is clear that the vast majority of modifiers recovered are background dependent.We performed this screen using two different sets of deletions, each of which varied in the size of the deletion. We observed little association between deletion size and severity of phenotypic modification  were recovered as genetically interacting with E3sd  showed surprisingly mild enhancement of the phenotype, which may be a result of the particular wild-type backgrounds used in this study. These findings suggest that even for well-characterized interacting genes, the influence of genetic background can be substantial, consistent with the flexible nature of genetic interactions. An important caveat to this interpretation is that many of these deletions may contain more than one gene. This could potentially mean that the interaction is due to both the deletion of the focal gene as well as other loci nearby. Yet, as described above, we observed no evidence for a relationship between deletion size and magnitude of effect, suggesting that this may be a minor contributing factor.These key results suggest that at least in sensitization screens, and possibly for many studies of genetic interaction, wild-type genetic background will have profound influences on the range of phenotypes observed and the modifiers that are identified, with only a subset of modifiers being background-independent. Using Flymine and Droid ith sdE3 . HoweversdE3/Y; Deletion/+ combinations in the Oregon-R wild-type background demonstrated considerably more variation between deletion strains, compared to the same genotypes in Samarkand ) across the two wild-type backgrounds. These data indicate many of the modifiers are acting in the same direction, although vary for magnitude of effect. Interestingly, even the non-genetic component of phenotypic variation observed for Oregon-R E3sd/Y; +/+ in crosses to the wild-type deletion progenitor shows considerably greater phenotypic variation for wing size compared to Samarkand , a few putative modifier regions failed to replicate in the tertiary validation cross with quantitative measures. Similarly a few deletion lines that were expected to not have an effect , did have one with the quantitative measure. However these potential false positives and negatives are few, of similar numbers, and thus are not expected to influence the overall conclusions.per se, and the results were not a specific consequence of the interaction between sd and the deletion. To investigate this we quantitatively examined females who were heterozygous for the E3sd mutation and for the deletions  across each genetic background. These females have qualitatively “wild-type” wings, and previous work did not observe an effect of E3sd on wing size in females as heterozygotes sd mutant phenotype in hemizygous males, but as general modulators of size, then we would expect a strong positive correlation between the effects on size in males and females . The correlation between Samarkand and Oregon-R E3sd/+ ; Deletion/+ females was ∼0.8, suggesting that the effects of the deletions on overall wing size is similar across backgrounds. However the correlations within each background  were 0.22, , and 0.21,  respectively, and neither case was significantly different from 0. The lack of a correlation indicates that the influence of the deletions in E3sd hemizygous males is largely independent of any effects on overall wing size. More importantly the CV for wing size in females (across deletions) for both backgrounds was ∼0.03, which is 5× and 10× less than that observed for E3sd hemizygotes in Samarkand and Oregon-R respectively , where “long” wings were selected in the backcross to the Oregon-R background, and “short” wings in backcrosses to the Samarkand background . Similar logic prevails for the Samarkand and the “long” phenotype. However, even using semi-quantitative measures, it is clear that these are highly correlated; 0.82 (CI 0.66–0.91) and 0.86 (CI 0.73–0.93) respectively. These results are consistent with the loci influencing the background-dependent genetic interactions being the same as those influencing the background-dependent effects on the phenotypic expressivity of the focal sdE3 mutation.Are the loci influencing the background-specific genetic interactions the same as those that modulate phenotypic expressivity for wing size of the focal ckground . Using ∼sdE3 allele, or a more general function of perturbation at the sd locus. To address this, we retested a subset (29) of the deletions spanning the range of phenotypic effects with E3sd, using an additional allele ETX4sd, across each genetic background. The phenotypic consequences of ETX4sd, while background-dependent, are somewhat weaker than sdE3 , and 0.76 (CI 0.55–0.88). In addition the general pattern of greater sensitivity to mutational perturbation by modifiers of the sd phenotype appears to be generally maintained  confirmed that the F02736vg allele behaved as a background-dependent enhancer with strong enhancement in Samarkand, but very weak enhancement in Oregon-R. We followed this up by introgressing this allele into both the Samarkand and Oregon-R background. Again we observed background-specific enhancement of the sd phenotype. Other fine mapping regions suggest several candidate genes, although for at least one region, no obvious candidate gene could be determined  is a direct result of the alleles that differ across backgrounds uncovered by the deletion. While we expect that our results are a combination of both explanations, it is likely that without very high resolution mapping of the genomic regions, or test of specific polymorphisms will we be able to determine the relative contribution of each type of interaction. However the previous work that motivated this current study, namely the background dependent interaction between sd and Omb was clearly due to a third order effect There are outstanding questions that our study is unable to address. The background dependent nature of the genetic interactions could be the result of a “third-order” effect between the sd mutations, showed increased sensitivity to mutational perturbation (both enhancers and suppressors) as well as greater phenotypic variation within strain. Recent work has demonstrated that loci can influence trait variability (“noise”) directly Hsp90 gene of DrosophilaE3sd and ETX4sd) represented more severe perturbations to wing development, so this may not provide an adequate comparison. Regardless, this remains an unanswered question, and a potential link between so-called variance controlling genes and sensitivity to perturbation would have important implications for the genetic architecture of canalization and robustness One curious finding of our study was that the background (Oregon-R) that demonstrated the higher degree of phenotypic expressivity of the focal E3sd and segmental deletions in each wild-type genetic background were correlated when observed across another (weaker) allele, ETX4sd, suggesting that such effects are not due to a particular allele. We also demonstrated that the effects of these interactions are tightly linked to the same genomic regions that contribute to the primary background-dependent phenotypic effects of the mutations. Thus for our system, the genetic variants influencing the phenotypic expressivity of the focal mutation appear to be the same as those modulating both the magnitude, and potentially the sign of genetic interactions between mutations.One constraint of the current study is that we utilized a hypomorph of moderate phenotypic effect, as opposed to a null allele. While a formal definition of functional epistasis sensu  requiresWhile the positive and negative implications for modifier (and other genomic) screens is clear, the potential flexibility of genetic networks given segregating variation in a population needs to also be considered. In particular an allele entering a population  may not have a “fixed” effect on fitness; instead the genetically contingent effects of the allele result in a distribution of phenotypic effects, including a possible change in sign .http://dx.doi.org/10.5061/dryad.4dt7cData and scripts associated with this manuscript are located on DRYAD: E3sd mutant allele , used in this study is caused by a P{w[E] ry[1t7.2] = wE} transposon located in the third intron of the sd gene white (w), by repeated backcrosses involving homozygous mutant female and the wild type male for over 20 generations ETX4sd and F02736vg alleles were also obtained from the Bloomington stock center, and were introgressed for 20 generations into each wild-type strain.The Oregon-R strain was originally obtained from the Bloomington stock center, while Samarkand was obtained from the lab of Dr. Trudy Mackay. For both strains, we further inbred them to near isogenicity, and tested via a panel of 30 polymorphic markers to confirm there was no contamination or residual heterozygosity. A combination of sequencing and PCR-based genotyping suggests that these two strains have an approximately 2% divergence from one another, and that all sequenced regions examined to date are a subset of variation from natural populations. The X-linked l(2)gl could potentially confound the results of our screen l(2)gl. Thus it is unlikely that this is a confounding factor in our analysis.We utilized the DrosDel sd mutant hemizygotes who were heterozygous for the deletions. Deletion lines (see above), and their isogenic wild-type progenitor strains were crossed to homozygous E3sd mutant females  for enhancement or suppression of the E3sd phenotype , and for each block a simultaneous set of control crosses with the progenitor wild-type strains for DrosDel and Exelixis flies was also performed. Nevertheless, there was negligible variation in the wing phenotypes of the flies resulting from the control crosses across all the blocks (not shown). However, for appropriate inferences, phenotypic analysis for all crosses within a block were made with respect to specific sets of control crosses from within that same block. We screened between (5–20) flies for each cross (crosses with fewer than 5 progeny were re-tested), with a mean/median of 8.2/7 flies per cross. Any deletion that showed evidence for modification (see below) of E3sd was re-tested (new crosses) to verify the phenotypic effects. Crosses performed with DrosDel deletions on chromosome arm 3L showed a marked increase in the number of modifiers relative to other arms (22/59 compared to 37/228 for the rest of the chromosome arms for the DrosDel collection). Thus putative modifiers on 3L were re-tested 3 times each, with consistent results, suggesting that these modifiers are unlikely due to a sampling artefact. In total 723 deletions were tested, with 18,167 flies scored.To assess the influence of wild-type background on genetic interactions we used a dominant modifier screen, and examined  females . Flies whenotype . Thus weE3sd individuals were generally category D while Oregon-R E3sd individuals were category H, with relatively minor variation in these scores. The rationale for such a semi-quantitative approach was two-fold. First, we wished to mirror the genetic screen approaches used in many functional genetic studies , and second this allowed us to screen a much larger panel of lines. As discussed below, these semi-quantitative measures correlated well with quantitative measures of wing size.For initial assessment of phenotypic modification we performed a semi-quantitative analysis similar to that used by other investigators E3sd, it was done so that the observed effects were of an almost qualitative nature . As discussed above, all putative modifiers were verified at least once with an independent replication cross.To mimic a traditional genetic screen we assessed interactions based largely on non-overlap distributions of phenotypes, comparing genotypes bearing deletions to their co-isogenic wild-types. While this likely underestimates the number of true interactions of the deletions with lm function and p.adjust in R (V 2.12).In addition, we also utilized a more quantitative approach, fitting the data to the following linear model:E3w sd/Y; Deletion/+) was dissected and mounted in glycerol, for both backgrounds. For the isogenic wild-type control strain, 30 individuals were used from each background-specific set of crosses to better ascertain the degree of variability. Images of the wings were captured using an Olympus DP30BW camera mounted on an Olympus BW51 microscope. Six landmarks (lm function in R (V 2.12) and 95% confidence intervals were constructed using confint. Significance was determined by non-overlapping confidence intervals with controls.To validate the primary findings of this study, we repeated crosses, and quantified wing size for a subset of 44 deletions, spanning the direction and magnitude of effects  observed in the genome-wide screen. A single wing from each of 5 male flies . This suggests high repeatability of the initial screen, as well as the semi-quantitative measure of wing size.sd mutants), we quantified wing size in females heterozygous for the focal E3sd mutation with each deletion  digitizing the same 6 landmarks on the wing.To ascertain whether there was a commensurate effect of the genomic deletions in “wild-type” wings  that influence the genetic interaction between the deletions and ckground . A backcckground . Upon geTo narrow down several genomic regions to a set of a few candidate genes we utilized an additional set of overlapping deletions in DrosDel, Exelixis and BSC strains followed by use of P-element insertional mutations co-isogenic with the Exelixis panel of lines. We utilized this approach for four genomic regions  detailed in Figure S1E3sd modifier screen. A) The Semi-Quantitative Scoring Scheme used for the primary screen for the modifiers of E3sd. The semi-quantitative scoring scheme used for this study was similar to other ones previously used (see methods), allowing for rapid phenotyping of the wings. A comparison of quantitative and semi-quantitative methods with a test data set were highly correlated (not shown). B) Reaction norms from deletions uncovering known interacting genes with sd.Scoring scheme and positive controls for (TIF)Click here for additional data file.Figure S2Landmarks used to quantify wing size. To quantify wing size in this study we utilized the centroid size calculated from 6 landmarks. These landmarks could be unambiguously found in all specimens that we examined in this study. It is worth noting that for mutations (not used in this study) that influence wing development more severely, these 6 landmarks could not be scored (not shown).(TIF)Click here for additional data file.Figure S3E3sd to introgress “long” and “short” alleles. The alleles that contribute to the background dependence of the genetic interactions between E3sd and the autosomal deletions could potentially be the same as those that contribute to the variation in expressivity in the sd phenotype. If this hypothesis is false, then we would predict no association between the genomic regions that contribute to variation for sd expressivity and the nature of genetic interactions across backgrounds. To test this, we utilized a backcross-selection procedure to move the genomic regions conferring “long” wings into an otherwise “short” Oregon-R background. Individuals from the Samarkand and Oregon-R background bearing the E3sd allele were crossed together, and F1 flies were mated interse to produce an F2 population segregating alleles influencing the expressivity of the sd wing phenotypes. Flies with the largest wings (most Samarkand sdE3 like) were then crossed to Oregon-R E3sd individuals, as well as the reciprocal for the shortest wings (crossed to Oregon-R). This two generation procedure was repeated for 12 cycles for the flies being selected for “short” wings, and 19 cycles for those for the “long” wings. This approach allows for the introgression of the alleles influencing sd expressivity from one background to the other. A panel of 30 SNP markers known to be polymorphic between Oregon-R and Samarkand were then used to verify the extent of the introgressions.Backcross-selection procedure across wild-type backgrounds with (TIF)Click here for additional data file.Figure S4Distribution of modifiers on remaining chromosome arms. Figure legend and description as for (PDF)Click here for additional data file.Figure S5E3sd. To determine whether the deletions generally uncovered a single or multiple modifier alleles of E3sd, we examined the relationship between the magnitude of the effect of the deletion on the wing phenotype, and the size of the deletions (in kbp). As seen in these figures, there is no association between them, suggesting that across the set of screened lines, each deletion is likely only uncovering a single modifier allele. However particular individual deletions may have more than one modifier, and modifiers that act in opposite directions.No association between size of the genomic deletion and magnitude of effect as a modifier of (TIF)Click here for additional data file.Figure S6E3sd. To determine the extent of the phenotypic effects of the genomic deletions on wild-type wing sizes, we examined the effects of 44 of the deletions . Importantly, the coefficient of variation across strains was ∼10× smaller for wing size for wild-type wings, than for the wings of E3sd hemizygous males.The effects on wing size of 44 deletions in females heterozygous for (TIF)Click here for additional data file.Figure S7ETX4sd allele. To determine whether the findings observed for the background dependence of the genetic interactions of the E3sd allele with the deletions would hold across other alleles, we introgressed an additional allele, ETX4sd, into both Samarkand and Oregon-R, and re-examined a subset of the deletions. A) ETX4sd also shows profound background dependence with respect to the expressivity of the sd phenotype. As described in the text, the results were significantly correlated across alleles. Interestingly the background dependent expressivity of ETX4sd is substantially weakened in crosses with the Exelixis Deletion progenitor strain. However, the background dependence of the genetic interactions appears to be at least as extreme as that observed for E3sd (B).The background dependent effects on the (TIF)Click here for additional data file.Table S1Candidate modifier genes (.xls) from the deletion mapping.(CSV)Click here for additional data file.Table S2Results from fine deletion mapping.(DOCX)Click here for additional data file.


Tuberculosis (TB) presents a serious problem in Mozambique. HIV prevalence among TB patients is estimated at 47%. A delay in having their first CD4+ cell count could lead to a missed opportunity for ART initiation due to a CD4+ cell increase above the cut-off caused by TB treatment. The objective is to describe CD4+ cell response during TB treatment and quantify the effect of TB treatment and ART on this response.All new HIV + adult TB cases in 2007 from three TB clinics in Mozambique were included. Data on TB diagnosis and treatment and HIV parameters were collected. A general mixed model was used for CD4+ cell count response.3; 95% CI: -40 to 79; p = 0.529). ART-use was associated with statistically significantly higher CD4+ cells compared to no ART-use : 12 to 151; p = 0.022).338 HIV + patients were notified and 252 (75%) were included in the analysis. Using TB medication was not independently associated with the CD4+ count response (19 cells/mmIn this study, no independent effect of TB treatment on CD4+ cell count was found. HIV-infected TB patients on ART had a significantly higher CD4+ cell count than those not receiving ART. CD4+ cell counts for patients not on ART at TB treatment start, remained below the cut off for initiating ART during the first three months of TB treatment; therefore some delay in getting the first CD4+ cell count would not lead to missing the opportunity to start ART. Tuberculosis (TB) presents a serious problem in Mozambique with case notifications rising dramatically since the start of this century. The World Health Organization (WHO) estimated the incidence of all forms of TB in Mozambique in Mozambique at the time of the study 2007) at 431 per 100.000 population [007 at 43In Sub Saharan Africa, people unaware of their HIV-infection present often to the health care services with TB as the first AIDS defining illness. Several studies found that TB clinics are well positioned to identify new HIV-infected individuals and to provide access to HIV services ,5.Following international recommendations, Mozambique started implementing TB-HIV collaborative activities in 2006 . PatientSeveral studies described an increase in CD4+ cell count during TB treatment for non-immune compromised TB patients ,11. CD4+In Mozambique, not all health facilities delivering HIV services have equipment for the assessment of CD4+ cells. Therefore, newly diagnosed HIV-infected TB patients may experience a delay in having their first CD4+ cell count result available. Should the CD4+ cell count during TB treatment increase in the HIV-infected TB patients as in non-immune compromised TB patients, the CD4+ cell count might become higher than the cut-off value for initiating ART. An opportunity for start of ART would be missed.The objective of this study was to describe the CD4+ cell count response during TB treatment and to quantify the effect of TB treatment and ART on the CD4+ cell count response. Through the CD4+ cell count response we assessed whether a risk exists for missing an opportunity to start ART in the routine setting of Mozambique due to late CD4+ cell count availability in HIV-infected TB patients, and the prioritization of ART for TB-HIV co-infected patients with the lowest CD4+ cell counts.The National Bio-ethic Committee of the Ministry of Health of Mozambique and the Institutional Review Board of the University of Washington in Seattle, USA, approved the study protocol. Both ethics committees approved that informed consent was not obtained as the study was based on routinely collected data.We performed a retrospective observational study in three purposely-selected health facilities in Manica province, Mozambique. Selection criteria were the presence of both TB and HIV treatment services in the same facility and at least 150 TB patients notified in 2007. One facility was an urban health centre in the provincial capital; the other two were rural health facilities about 20 and 90 kilometres from the provincial capital. Within these clinics, we collected the information on HIV disease parameters of all new notified TB patients of 16 years and older with a positive HIV test recorded in the TB register from January to December 2007.In Mozambique, smear microscopy is the main TB diagnostic. In the participating facilities, diagnosis of sputum smear-negative and extrapulmonary TB occurs mainly on clinical assessment and hardly ever on radiology. All new adult TB patients receive a standard course of TB treatment consisting of two months isoniazid, rifampicin, ethambutol and pyrazinamide followed by 4 months isoniazid and rifampicin. The standard first line ART regimen consists of two nucleoside reverse-transcriptase inhibitors, lamivudine and stavudine, with either the non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine or efavirenz. The national guidelines recommend switching from neviripine to efavirenz in patients that receive a rifampicin containing treatment regimen .The facility’s TB supervisor collected the data of the 2007 cohort using standard data collection forms in July and August 2009. Data collected from the TB register included: age, sex, type and category of TB, treatment regimen, start date of TB treatment, initial smear examination result, HIV test result and TB treatment outcome. If the treatment outcome was death, its date was recorded.We identified the HIV record of the HIV-positive TB cases through the unique HIV patient number if recorded in the TB register. In addition, local staff familiar with the patients identified some HIV patient records. If these methods did not lead to identification of the patient record, we searched the electronic HIV-database using the patient’s name and age taken from the TB register. If the data matched, we took the unique HIV patient number from the electronic database and used it to locate the HIV patient record. We limited the identification of the HIV patient record to those HIV-positive TB patients registered with the HIV services in the same health facility.We collected available CD4+ cell count results in the 6 months TB treatment period, the date of these results, the start date for ART and the ART regimen from the HIV patient record.We entered the data in EpiData version 3.1 and performed descriptive analysis with EpiData Analysis V2.2.1.171. We used STATA version 11  for analysis of the CD4+ cell counts.We modelled the evolution of the CD4+ cell count during TB treatment using a mixed effect model. This model deals adequately with repeated measurements of the outcome variable . The modWe defined the baseline CD4+ cell count at the start of TB treatment as the CD4+ count closest to the start of TB treatment within a window of 12 weeks before until 2 weeks after the start of TB treatment. We allocated all other CD4+ cell counts to a single fixed time-point with a window ranging from 2 to 6 weeks. If multiple CD4+ cell counts were available for a specific time-point, we included the one closest to the midpoint of the time window in the analysis.To determine whether the CD4+ cell count was obtained while using TB treatment, we used WHO standard treatment outcomes to define the end of TB treatment . The endFrom January 1st until December 31st 2007, 591 new TB patients were notified. Of these, 478 (81%) were tested for HIV and 338 (71%) had a positive test. Of these, 274 (81%) were registered with the HIV services in the same facility. HIV patient records were identified for 256 (93%) patients. Four patients were excluded from the analysis due to an identification mismatch, leaving 252 patients for analysis. All patients started TB treatment. Patient characteristics and the ART status are given in Table Of the 252 patients available for analysis, 25 10%) had not a single CD4+ count recorded and 16 (6%) had only CD4+ counts before the period defined as the start of TB treatment. For the remaining 211 (84%) patients, a total of 271 CD4+ counts were available at the start of or during TB treatment. Figure 0% had noA baseline CD4+ cell count was available for 149 patients with 48 (32%) and 23 (15%) taken while the patient received TB treatment or ART respectively. Of the 122 CD4+ count results after the start of TB treatment, 81(66%) were obtained while the patient was on ART.Over the full observation period of 6 months, using TB treatment was not statistically significant associated with the CD4+ cell count response. Patients using TB treatment had a CD4+ cell increase of 19 cells/mm3  compared to patients not receiving TB treatment. ART use was statistically significantly associated with CD4+ cell response during the observation period. Patients using ART had a CD4+ cell increase of 81 cells  compared to patients not using ART.The CD4+ cell count response during TB treatment by ART status is shown in Figure 3 during the first 12 weeks of TB treatment. Therefore, a delayed assessment of the first CD4+ cell count in itself would probably not lead to missing an opportunity to start ART based on the cut off of 350 cells/mm3. This finding is relevant in the Mozambican setting where not all ART treatment facilities are capable of performing CD4+ cell counts and prescription of ART is prioritized. Health facilities sent blood samples for CD4+ cell count to another laboratory daily or weekly, depending on the distance between the sending and receiving facilities.In this study, TB treatment in TB-HIV co-infected patients had no significant effect on CD4+ cell count. The evolution of CD4+ cell count is mainly driven by ART use. The average CD4+ cell count for patients not on ART remained below the cut-off for initiating ART of 350 cells/mmThis study showed a small increase in CD4+ cell count during TB treatment in both patients on ART and patients not on ART as has been described in non-immune compromised TB patients ,11. In oThis was a retrospective study based on routine data and as such has several limitations. First, bias may have occurred by not including patients who received HIV treatment at another health facility. These patients may have been treated differently or adherence may have been different. This would have influenced treatment outcome and CD4+ cell count. All health care facilities in Mozambique follow the same national guidelines and as such the chance of a difference in treatment strategy is unlikely. Furthermore, the characteristics for patients whose clinical record was identified were very similar with those from patients without an HIV record Table .Second, not all TB patients in this study had an HIV-test or the result recorded in the TB register. Therefore, not all HIV-infected TB patients were included. Given the high testing rate of more than 80%, it is unlikely that the non-availability of the HIV-test result would markedly bias the results of the study.Third, this study took place in three health facilities in a single province of Mozambique. The results may be different in other areas in Mozambique. However, we believe that the situation in Manica does not differ much from that in other provinces in the country at the time of the study, apart from the larger cities where more ART facilities are available. There is also more specialist care available in the larger cities.Fourth, despite a considerable amount of patients, the number of available CD4+ cell counts per patient was small, reflecting the indications in the national guidelines as to when to perform this test. The use of the mixed model allowed us to use all available data and was therefore the recommended methodology for our data set.Fifth, about one third of the patients that used ART during TB treatment started their ART before the start of TB treatment and potentially had incident TB while using ART. Emerging evidence shows that CD4+ cell count response is smaller in these patients . HoweverDespite these limitations, we consider the results relevant and important because limited data are available on CD4+ cell count response in cohorts of TB patients.The presently used HIV treatment guidelines in Mozambique are not yet in line with the WHO recommendation to initiate ART in HIV-infected TB patients as soon as possible after the start of TB treatment irrespective of the CD4+ cell count . The preAlso, many countries with a high burden of HIV struggle to maintain all HIV-infected patients on ART. Lack of funding may lead to stock outs of antiretroviral drugs at facility level . TherefoIn this study the higher CD4+ cell count level during TB treatment in HIV-infected TB patients is due to ART use, and not influenced by TB-treatment. Therefore, these findings are a strong argument to implement the recent WHO recommendation to start ART as soon as possible in HIV-infected TB patients irrespective of their CD4+ cell count. Should countries wish or need to prioritize new ART initiations to those most in need, a CD4+ cell count result obtained in the first 12 weeks of TB treatment provides a good reflection of the immune status at the start of TB treatment. This holds for Mozambique and probably for other similar settings as well.A prospective study will provide better insight to the question of the CD4+ cell count response during TB treatment and the effect of TB treatment and ART on this response.The authors declare that they have no competing interests.MB conceived the study, participated in the design of the study, had the overall coordination of the data collection, participated in the statistical analysis and drafted the manuscript. PSG conceived the study, participated in the design of the study and helped to draft the manuscript. CMS participated in the design of the study and coordinated the data collection. PP conceived the study, participated in the design of the study and helped to draft the manuscript. FL participated in the design of the study, participated in the statistical analysis and helped to 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/1471-2458/12/670/prepub


Therapeutic cancer vaccines have the potential to generate a long lasting immune response that will destroy tumor cells with specificity and safety, in contrast to many other current cancer therapies. Clinical success to date has been limited by a number of factors including choice of immunogenic cancer rejection antigens, optimization of vaccine platforms and immune adjuvants to effectively polarize the immune response, and incorporation of strategies to reverse cancer mediated immune suppression by utilization of effective adjuvant/immune modulators. WT-1 (Wilms' tumor gene 1) is a cancer antigen that is required for tumorigenesis, expressed in a high percentage of tumor cells and rarely expressed in adult normal cells. Moreover spontaneous immunity to WT-1 is seen in cancer patients and can be augmented with various therapeutic vaccine approaches. IRX-2 is an immune modulator with demonstrated preclinical and clinical pleiotropic immune activities including enhancement of the immune response to potential tumor antigens. This paper presents the rationale and preclinical data for utilizing the WT-1 tumor antigen in a novel vaccine platform consisting of a synthetic long peptide containing multiple class I and class II epitopes in combination with the IRX-2 immunomodulatory regimen to overcome immuno-suppressive pathways and enhance the anti-tumor response. Unfor1.1.Therapeutic cancer vaccines based on peptide epitopes have been the most widely evaluated due to the simplicity of production, safety, specificity for targeted epitopes and success in preclinical models -8. Initi1.2.i.e., tumor or immunization site), followed by T cell activation in the lymph nodes and migration of cytotoxic cells to the peripheral sites. Although cytokines play a critical role in effective immune activation, relatively few studies have utilized combinations of cytokines as vaccine adjuvants [etc.). The results were interpreted as an increase in immune response due to the activation of the “inflammatory” cytokine network which would drive the immune response. With the observation that many components of Freund's adjuvant activated TLR receptors on antigen presenting cells, studies evolved to evaluate purified and/or modified TLR based adjuvants as stimulators of cytokine production via activation of antigen presenting cells. More recently, the use of viral constructs to deliver antigens is believed to stimulate cytokine production via innate immune mechanism since many TLR's are activated by viral products. In the studies where cytokines were used as adjuvants, the cytokine studied most often was GM-CSF administered locally to enhance monocyte precursors. The other was rIL-2 which has been used systemically to increase antigen specific T cell proliferation [The critical events for T cell-mediated anti-cancer immune response include antigen presentation to T cells primarily in the lymph nodes draining the source of the antigen , the IRX-2 immunomodulatory regimen has been reasonably tolerated and resulted in increased lymphocyte infiltration (LI) into the tumor at surgery relative to pre-treatment biopsy, which was associated with increased survival ,10,15. Bi.e., ng/mL rather than (μg/mL) of cytokines that target the many different immune cell types have not been evaluated in a therapeutic vaccine setting. While IRX-2 is a complex primary cell produced biologic with consistent ranges of multiple cytokines and chemokines with synergistic activity in preclinical studies, the contribution of individual cytokines in this biologic have not yet been completely defined. The physiologic cytokine levels in IRX-2 are much lower than concentrations of recombinant cytokines used in ex vivo DC maturation or in high dose systemic cytokine therapies. The ability of IRX-2 to activate both DCs and T and even B and NK cells makes it especially attractive since it is this combination of immune cell subsets that coordinate the immune response against tumor antigen expressing cells.Although cytokines have been evaluated as immune adjuvants, relatively high doses of cytokines  have been used; potentially synergistic physiological levels (1.4.WT-1 (Wilms' tumor gene 1) is a highly immunogenic protein expressed intracellularly during normal early development, but subsequently downregulated in adults -21. Its Since WT-1 is a potentially useful tumor antigen, a number of clinical trials have evaluated WT-1 peptide based vaccines . The triThe Heike group evaluated the HL-A2 and HLA-A24 peptides in combination with gemcitabine in Japanese patients with advanced pancreatic or biliary tract cancer . Each paThe Scheinberg group evaluated a four-peptide vaccine which included the nine amino acid analogue of RMF (YMF), two helper epitopes, and a longer peptide containing the YMF peptide and additional sequences predicted to be helper epitopes ,28. GM-Cin vitro re-stimulation. While these studies confirm the immunogenicity of the peptides, they also suggest that the optimal vaccine delivery system even when helper epitopes were included has not yet been identified.Thus, over all, in the small studies to date, the majority of peptide immunized patients (29/55 = 53%) had a CD8 tetramer positive response when measured after in vitro studies and a need for improved adjuvants that can further enhance the T cell response. A possible solution to many of these issues may be the use of synthetic long peptides (SLP) as a vaccine delivery system [Although these small peptide based vaccines have proven to target immunogenic epitopes in the WT-1 tumor antigen, the weakness of the immune response suggests they are not yet optimal. Issues that need to be addressed in future vaccine constructs include HLA restriction of small peptides, requirement for helper epitopes, limit of the immune response to dominant epitopes defined in y system . These ay system -39. Thesy system . A caveay system -41.1.5.We have explored a novel vaccine approach utilizing a SLP to WT-1 combined with the IRX-2 immunotherapeutic regimen. We have studied this approach in a murine model in which the human WT-1 protein, because of its high degree of homology to murine WT-1 is seen as a self protein similar to that for human tumors. We selected for these preclinical studies to focus on the HLA-A2 peptide epitope that has been used in several clinical studies because it is recognized by HLA-A2 humans and has also been reported to be immunogenic in C57Bl/6 mice . The lonAlso listed in 2.2.1.C57Bl/6 female mice (6–8 weeks of age) were purchased from either Charles River Laboratories  or Harlan Laboratories  and were housed under the care of the Cold Spring Harbor Laboratory (CSHL) Animal Facility . All procedures were approved by the Institutional Animal Care and Use Committee of the CSHL. Immunizations were given subcutaneously at the base of the tail to provide for rapid draining to the regional lymph nodes. The WT1 peptides were given at a dose of 10 (μg/mouse. All peptides vaccinations were administered as an emulsion with Montanide (IFA Seppic Inc.) at a 1:1 water to oil ratio. IRX-2 (100 IU/mouse) was incorporated into the emulsion as the aqueous component and administered with the antigen and Montanide subcutaneously on the first day. As indicated in the Mice not receiving the IRX-2, had VIVO-10 ™ media substituted as a control containing no cytokines. Booster immunizations with antigen plus adjuvant were given on day 14 but additional daily injections of IRX-2 were not given along with the booster immunizations. Multiple experiments were performed at different times post booster immunization and the data presented is representative of those experiments. Since the ELISpot assays have a variable response on different days, these studies do not demonstrate kinetics of the response as that would require staggered starts for the immunizations and sacrificing of mice at various times post boost on the same day.2.2.5 cells/well) added and then incubated for 18 h at 37 °C. When cells were used directly from the mouse spleen, the protocol was designated as a primary ELISpot assay. After the overnight incubation, wells were vigorously washed with PBS to remove cells and biotinylated second antibody. (MabTech) was added for additional 18 h incubation at 20 °C. Plates were developed using sequential biotin-AP and DAB substrate. Plates were read using a microscope with digital conversion software by ZellNet . For some studies, cells were expanded for 6 days using peptide pulsed EL-4 cells for antigen presentation prior to ELISpot (designated as expansion in the figure legends).ELISpot plates  were coated with capture antibody  in PBS overnight. Plates were washed, antigen (10 (μg/well) and cells  assay [vs. 24) although the epitope was previously shown to be the dominan tepitope in C57B/6 mice immunized with HPV E7 protein [This experiment, in addition to demonstrating the influence of IRX-2 on the T cell response to a vaccine, also used multiple different lots of IRX-2 to demonstrate the manufacturing consistency of IRX-2. In this study the control mice were immunized with the peptide conjugate but instead of IRX-2 they received the media without cytokines. Because the study represents a combination of data over three days of sacrifice, the appropriate statistical assessment is peptide specific response not increase over the non-peptide stimulated naïve or control mice nor increase over the non-peptide response of control or naïve mice. In other experiments, IRX-2 was found to be superior to both CpG and MPL adjuvants, which specifically target Tol-like receptors (TLRs), when the T cell response was assessed using the H) assay . With reH) assay ). Since  protein . Both peWhen the spleen cells from the mice were tested in the ELISpot assay for T cell specific responses to the peptides, more mice had a positive immune response to the HPV peptide than the WT-1 peptide (data not shown). The magnitude of the T cell response to the WT-1 short peptide was also weak compared to the HPV peptide and an enhancement with IRX-2 was not readily demonstrated. In contrast, IRX-2 significantly enhanced the peptide specific response to the HPV peptide. We interpret this observation to indicate that immune tolerance to the short WT1 peptides was limiting the antigen specific T cell response since the binding score using the prediction algorithm was in fact higher than that of the HPV peptide. Thus, this was a relevant model to study responses to a self tumor antigen for which tolerance was apparent.in vitro by incubation with antigen presenting cells and IL-2 for 6 days before measuring the T cell response to the short peptide. As shown in in vitro expansion. Thus when the expansion protocol was used, it was possible to demonstrate that mice immunized with the short peptide with IRX-2 had a stronger T cell response than mice receiving only the short peptide in Montanide.Since the immune response of the short peptide immunized mice was weak, cells from immunized mice were expanded The long and short RMF peptides have been evaluated for immunogenicity in multiple experiments performed at different times after booster immunization  or ranked by magnitude of the response . While the response rate ranged from 28%–64% for the individual peptides, the overall response rate as defined by a response to at least one of the peptides was 86%To assess whether the mice immunized with the long peptide recognized multiple epitopes, the four best scoring CTL/CD8 binding epitopes in the long peptide  were use4.The advantages of peptide-based vaccines include safety, specificity and ease of cGMP production. However there are also limitations with small peptide vaccines including MHC restriction, lack of helper activity and weak presentation of antigen by APC due to exchange into the MHC as compared to cross presentation processing. In addition only limited clinical anti-tumor activity has been documented to date with these types of peptide vaccines. While the limitations can be somewhat overcome by using multiple peptide vaccines which include both class I and class II epitopes, the processing issue can only be overcome by larger constructs such as recombinant protein or longer peptides with overlapping MHC I and II epitopes. In fact, processing and expression of both class I and class II epitopes on the same APC is considered to be the goal of an effective vaccine. Recombinant proteins, while they may solve the limited epitope dilemma, have proven difficult to make in large quantities under cGMP specifications and the processing of the large proteins may be less efficient than for shorter peptides. Synthetic long peptides (SLPs) represent novel and yet currently underutilized vaccine constructs. They are easy to produce and also require processing for presentation. The limited preclinical and clinical studies with SLP's have proven that they are safe, effective and have the potential to generate immune responses to multiple epitopes. Dramatic clinical regressions have also been seen with one SLP vaccine targeting HPV in patients with vulvar intraepithelial neoplasia. Our studies have provided additional evidence that long peptides to a tumor antigen to which there is immune tolerance are immunogenic and may have immunologic advantages over short peptides (target multiple MHC epitopes).in vitro are activation of antigen presenting cells  and T cells. While the T cell activity of cytokines such as IL-1, IL-2 and IFN-γ are well defined, only recently has the activity on APCs been appreciated. In addition to increasing the activation of DCs as measured by surface expression of many activation markers, IRX-2 reversed the depressed antigen processing activity of dendritic cells from cancer patients [We have also shown that the antigen-specific T cell responses to the WT-1 SLP are further increased by combining the vaccine with IRX-2. IRX-2 is a multi-targeted primary cell-derived biologic that shapes the adaptive immune response toward effective T cell activation. The synergistic activity of the cytokines in IRX-2 is directed towards both the antigen presenting cells and the activated T cells resulting in a novel T cell immune modulator. Preclinical studies have demonstrated IRX-2 anti-tumor activity in tumor models especially in conjunction with cyclophosphamide and chemo-radiation. Enhancement of the response of mice to several vaccine constructs has also been demonstrated. With respect to mechanism of action of IRX-2, among the many immune activities demonstrated for IRX-2 patients . In addipatients .i.e., an SLP) with a synergistic cytokine adjuvant/immunomodulator (IRX-2) in a protocol designed to reduce tumor mediated suppression of the immune response should lead to an improved and more immunogenic therapeutic tumor vaccine with enhanced anti-tumor activity. Both clinical and preclinical studies with IRX-2 support the hypothesis that IRX-2 enhances the immune response to tumor antigens and generates immune mediated anti-tumor activity. As described in this manuscript, the use of IRX-2 and synthetic long peptides (SLPs) should overcome many of the limitations of the peptide tumor vaccine studies to date. Clinical evaluation of this novel combination is in the planning stages.Combining a peptide vaccine that contains multiple class I and class II epitopes  tumor associated antigen. To target multiple MHC types, induce CD4 as well as CD8 responses and enhance antigen specific immunogenicity we have generated a synthetic long peptide (SLP) to WT1. We have also combined this vaccine with the IRX-2 immunomodulator that has been shown in various models to enhance multiple arms of the immune response and increase the immunogenicity of various therapeutic cancer vaccines. Our pre-clinical results show that vaccination with the WT1 SLP induces a T cell response to multiple embedded class I binding epitopes and that the magnitude of the T cell responses is increased by immunomodulation with IRX-2. These results support further evaluation of this novel therapeutic cancer vaccine approach in the clinic.


RasGrf1 increased both average and maximal longevity independent of a role in cancer. We found this to be surprising because RasGrf1, a Ca2+/calmodulin-dependent Ras-guanine-nucleotide-releasing factor, would be expected to have a role in normal physiology [This important article by Borrás et al. demonstrates several interesting and surprising findings . The mosRasGrf1 deletion on aging, which is also accompanied by lower frailty and retention of motor control, appears to be mediated by greater protection against oxidative damage as observed by lower brain lipid peroxidation, liver protein oxidation and maintenance of the brain and liver glutathione redox potential. We must note that the use of malondialdehyde levels as a measure of overall lipid peroxidation in whole tissues is rather suspect and subject to numerous artifacts; nevertheless, the approximate 25% lower MDA levels in -/-RasGrf mice (in comparison with wild-type mice) were significant. -/- miceRasGrf also exhibited lower levels of oxidized proteins (carbonyl assay) than did wild-type animals which might be due either to lower rates of production, or higher degradation rates of oxidized proteins, or both. It should be noted that old wild-type animals also exhibited lower levels (≈ 20%) of oxidized liver proteins than seen in wild-type mice. This is surprising since levels of oxidized  proteins usually increase with age, as Proteasome [RasGrf1 deletion mutants exhibited almost 30% lower levels of oxidized liver proteins than did the young wild-type mice.The effect of oteasome -5 and Looteasome , 7 activRASGRF1 is expressed in pancreatic β-cells where it regulates β-cell mass. So the effects on glucose metabolism is unsurprising. RasGrf1 is also expressed in the hippocampus and hypothalamus is involved in learning and memory. So, one wonders how a human without RasGrf1 would be able to do those functions and whether living longer and stronger might be accompanied by not remembering why one cared.RasGrf1 produces this dramatic effect is worthwhile. For example, it would be interesting to determine whether the ability to increase glutathione in response to stress, which declines in aging, is also maintained in the RasGrf1 knockout mice. Certainly, it would be worth testing the effect of RasGrf1 deletion on Proteasome and Lon levels and stress-inducibility.Nonetheless, the effect on longevity is remarkable and delving deeper in to how the absence of RasGrf1, which is an exclusively paternal allele imprinted gene, suggests that only the male parent determines the effect of this gene's expression on longevity. In several ways, the -/-RasGrf1 mice metabolically resemble mice fed a calorie-restricted diet. One exciting outcome of these studies is that RasGrf1 may be a potential target for design of agents that prolong lifespan and healthspan without the obvious difficulty of restricting caloric intake.Another interesting point raised by the authors is that


It is nearly 30 years since the Alt lab first put forward the accessibility model, which proposes that cleavage of the various antigen receptor loci is controlled by lineage and stage specific factors that regulate RAG access. Numerous labs have since demonstrated that locus opening is regulated at multiple levels that include sterile transcription, changes in chromatin packaging, and alterations in locus conformation. Here we focus on the interplay between transcription and RAG binding in facilitating targeted cleavage. We discuss the results of recent studies that implicate transcription in regulating nuclear organization and altering the composition of resident nucleosomes to promote regional access to the recombinase machinery. Additionally we include new data that provide insight into the role of the RAG proteins in defining nuclear organization in recombining T cells. Tcrg, Tcrd, Tcrb, and Tcra and three immunoglobulin loci, Igh, Igk, and Igl) by reshuffling variable (V), diversity (D), and joining (J) gene segments that are arrayed along the length of each of these large loci. Rearrangement is mediated by the RAG recombinase, which binds to highly conserved heptamer and nonamer recombination signal sequences (RSSs) that flank each of the V, D, and J gene segments. Both RAG1 and RAG2 proteins, which make up the recombinase, bind to two segments, bringing them together to form a synapse prior to the introduction of double strand breaks (DSBs). In addition, RAG plays a role after cleavage by holding the four broken ends together in a RAG post cleavage complex that directs repair by the ubiquitous classical non-homologous end joining (C-NHEJ) pathway.V(D)J recombination occurs during lymphocyte development to create B and T cell receptors that can recognize a vast array of foreign antigen. Diversity is generated within the seven antigen-receptor loci  to ensure rearrangement and cell surface expression of a single specificity receptor. Studies from numerous labs have validated the accessibility model put forward by the Alt lab and shown that rearrangement is linked with transcription, active histone modifications, and reversible locus contraction, which brings widely separated gene segments together by looping . But what is the mechanism underlying the generation of these focal centers? The data from recent genome wide chromosome conformation capture experiments indicates that co-regulated actively transcribed genes come together in the nucleus in transcription factories , 7. ThusWhen considering the data from genome wide association studies it is important to remember that signal enrichment reflects the sum of the data derived from a population of cells. What happens at the single cell level may be very different. However, without live systems in which we can track the movements of individual loci in single cells over a period of time, at the simplest level, when focusing on an interaction between two loci in a population, we cannot tell whether chromosome conformation capture signal enrichment reflects interaction at high frequency in only a subset of cells within the population 1); whether at a different time point this interaction will be occurring in the same (1) or a different subset of cells within the population (2); or whether interaction occurs at a roughly uniform frequency in every cell of the population (3) and this leads to an equivalent signal enrichment as the interactions in (1) or (2)  . Transient relocation to this repressive nuclear environment likely causes a degree of silencing that depletes RAG binding on the uncleaved allele during repair of the first break. Thus, ATM-mediated changes in nuclear organization function to ensure asynchronous RAG-mediated cleavage on homologous alleles. Regulation in trans is important for the initiation of allelic exclusion and for restricting the number of DSBs that are introduced at any one time in the cell (To examine these issues in the context of V(D)J recombination, we asked whether RAG binding could have a role in bringing RAG bound antigen-receptor loci together in the nucleus in localized recombination centers. We discovered that expression of RAG1 brings target homologous antigen-receptor alleles together in a subset of recombining cells –11. Homoed (ATM) , 11. Brithe cell . Based oin trans. Our data indicate that, as with homolog pairing, RAG1 expression  induces the formation of higher-order loops that separate the 3′ end of the antigen-receptor locus, Tcra, from its 5′ end which remains embedded in the chromosome territory (as assessed by DNA FISH with a chromosome paint probe) J recombination: the formation of higher-order loops . Chromost probe)   A. FurtheTcra BAC probe. Curiously, we found considerable overlap between the paint and exome DNA probes for chromosome 14 in splenic B cells and in DP T cells on chromosomes where Tcra is not looped out  . We usedt Figure B. In cons Figure B. These cumented , 14, 17.cumented . Here ouTcra occurs on only one allele and this was linked to the occurrence of RAG-mediated mono-allelic breaks on the 3′ end of the looped out of Tcra  the two loci undergo recombination at different stages of T cell development in CD4 CD8 double negative, DN2/3, and CD4 CD8 double positive DP cells, respectively. For reasons that are not well understood, the Igh locus undergoes a low level of partial rearrangement in T lineage cells and in this context it is of note that Igh has been identified as a translocation partner of Tcra/d in T lineage lymphomas. Specifically, we and others have found that Tcra/d-Igh translocations occur in T-lymphomas from ATM deficient mice  but interact far less frequently in DP cells (when Igh is recombined at lower levels). This raises the possibility that feedback control of RAG activity could involve close communication between heterologous antigen-receptor alleles as we have proposed for homologous alleles. Either way, it appears that regulation in trans involves control of loop formation and repositioning of the other allele/locus to a repressive compartment of the nucleus.Interestingly, control of cleavage of Tcra/d with other hematopoietic lineage specific or housekeeping H3K4me3-enriched loci on different chromosomes in DN2/3 cells (where Tcrd and Igh are recombining), we found an opposite trend to the relationship between Igh and Tcra/d  while RAG depletion did not alter the relationship between Igh and Tcra/d (or Notch1 and Bcl11b) in many instances it increased the association of the other genes with Tcra/d , the Igl locus is rearranged in preference to Igk in the transgenic mice. This in vivo model enabled them to examine the mechanisms underlying RSS accessibility at these two loci. Surprisingly, they find that high levels of H3K4me3 at Jκ RSSs only leads to partial activation of recombination at the Igk locus indicating that enrichment of this mark is not tightly linked to recombination. Furthermore, they show that neither H3 and H4 histone acetylation is sufficient to increase RSS accessibility although enrichment of histone acetylation appears to be linked to early activation of the two loci. In addition they find no correlation between H3K36me3 enrichment and recombination at the Igl locus. Thus, none of the chromatin modifications that are associated with recombination are tightly linked to increased RSS accessibility. In contrast, increased accessibility, as measured by restriction enzyme digestion close to RSS sites in Igl versus Igk, correlates well with increased Igl recombination. Importantly, inhibition of transcription, through treatment with α-amanitin, abrogates restriction enzyme accessibility.Our data linking transcription with altered nuclear organization and targeted RAG cleavage of Tcra , go handTcra . Bevingtelopment . One of angement . Restorain vitro substrates and show that RAG is more efficient in cleaving hexasomes than nucleosomes. Furthermore, they demonstrate that there is an inverse relationship between elevated levels of RNA polymerase II and the presence of H2A/H2B J recombination, particularly since many of these maybe very transient. The challenge now is to develop live cell systems in which cleavage and repair can be analyzed over a period of time in single cells. This is no trivial matter when it comes to the antigen-receptor loci as these are spread over megabases of DNA. In addition to labeling of different regions along the loci, the RAG1/2 recombinase and components of the DNA damage response and repair machineries need to be tagged for visualization. Until such systems are in place the next best approach is to focus on single cell analyses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.