A large-scale field study in naturally occurring vole populations identified gene expression changes over time and demonstrates how wild mammals exhibit tolerance to chronic parasite infections. Hosts are likely to respond to parasitic infections by a combination of resistance (expulsion of pathogens) and tolerance (active mitigation of pathology). Of these strategies, the basis of tolerance in animal hosts is relatively poorly understood, with especially little known about how tolerance is manifested in natural populations. We monitored a natural population of field voles using longitudinal and cross-sectional sampling modes and taking measurements on body condition, infection, immune gene expression, and survival. Using analyses stratified by life history stage, we demonstrate a pattern of tolerance to macroparasites in mature compared to immature males. In comparison to immature males, mature males resisted infection less and instead increased investment in body condition in response to accumulating burdens, but at the expense of reduced reproductive effort. We identified expression of the transcription factor Gata3 (a mediator of Th2 immunity) as an immunological biomarker of this tolerance response. Time series data for individual animals suggested that macroparasite infections gave rise to increased expression of Gata3, which gave rise to improved body condition and enhanced survival as hosts aged. These findings provide a clear and unexpected insight into tolerance responses (and their life history sequelae) in a natural vertebrate population. The demonstration that such responses  can move from resistance to tolerance through the course of an individual's lifetime emphasises the need to incorporate them into our understanding of the dynamics and risk of infection in the natural environment. Moreover, the identification of Gata3 as a marker of tolerance to macroparasites raises important new questions regarding the role of Th2 immunity and the mechanistic nature of the tolerance response itself. A more manipulative, experimental approach is likely to be valuable in elaborating this further. Hosts do not always resist parasites. And once infection establishes, relatively little is known of how naturally occurring hosts tolerate (mitigate for) adverse effects, or what the life history consequences of this tolerance may be. In this article we demonstrate a pattern of tolerance to parasitic worms and arthropods in wild voles and link this to increased expression of an immunological biomarker. The biomarker, Gata3, is triggered by infection, precedes significant changes in body condition, and impacts on fecundity and survival. These results point to the considerable ecological importance of tolerance in wild vertebrates and to how poorly it is understood, while at the same time giving a new perspective on the natural function of immune response pathways involving Gata3. There is increasing recognition that the ability to tolerate a parasite's presence, where the host accepts infection but actively limits the damage caused, may be a crucial host defence strategy The term “tolerance” is used differently by immunologists and disease ecologists. In moving towards some unification of meaning, we must distinguish between pattern and process. The pattern associated with tolerance, typically the main focus of ecologists, is a relative insensitivity of host health (fitness) to increases in parasite burden The study of tolerance in field systems, then, is an indispensible complement to studies in the laboratory, and there have been calls for the greater integration of data from natural populations on host fitness, immunological variation, and infection pressure Microtus agrestis, using interwoven cross-sectional and longitudinal sampling protocols. This hybrid study design took advantage of the greater range and precision of measurement possible in destructive  sampling, and of the stronger causal inferences possible in capture–recapture  sequences, where driver variables may be observed to precede the responses they trigger We hypothesised, first, that different groups of individuals  will differ in their tolerance—their relationship between health and parasite load—and second, that those individuals that maintain good health in relation to parasite burden  will express more of the physiological pattern associated with tolerance. We monitored immunological gene expression, infection, body condition, and survival in replicated natural populations of field voles, Specifically, adopting the phase space perspective (above), plotting our cross-sectional data comes closest to generating a tolerance curve. However, infection stage cannot be standardized in observations on natural populations. Hence, a less negative, zero, or even positive relationship between health and parasite load will be consistent with tolerance (and suggestive of it), but alternative explanations cannot be excluded. By contrast, our longitudinal data generate sequences of time points on a phase curve for a “typical” individual. A pattern in which health fails to decline (or even increases) following an increase in parasite load will therefore be directly indicative of tolerance. The case for demonstrable tolerance will, of course, be strongest if the results from the cross-sectional and longitudinal studies are consistent with one another. In contrast to tolerance, resistance resulting from acquired responses  and a longitudinal component  and was replicated at two different sites in each of 2 y. Biometric and infection data were recorded at all sampling points. In the cross-sectional component, ligands for Toll-like receptors (TLRs) 2 and 7 were used to stimulate splenocyte cultures, from which we measured the expression of immune genes involved in regulatory  and effector  pathways recruited during antimicrobial pattern recognition responses. In addition, the expression of genes reflecting different T-helper phenotypes was measured in nonspecifically  activated splenocytes. The activity of regulatory T cells was represented by the anti-inflammatory cytokines IL-10 and TGF-β1 and the transcription factor FoxP3. The activity of T-helper cell type 1 (Th1) responses was represented by the pro-inflammatory cytokine IFN-γ and transcription factor Tbet and that of Th2 responses by the transcription factor Gata3. In the longitudinal component, constitutive expression of IFN-γ, IL-10, and Gata3 were measured in peripheral blood.Our sampling, carried out in natural M main) was tested first against similarly reduced immune gene expression variables  or by measures of some influential species . However, the composite measure (PCM main) has the advantage that relationships with host responses will not be diluted when, for example, a low burden for one parasite is accompanied by high burdens for others, such that the response to the low burden is “greater than expected,” driven by the other parasites.Our overall analytical strategy  was to begin with our more detailed cross-sectional data and search first for the patterns of resistance or tolerance described above. We then sought to identify immunological gene expression markers of these patterns and, in turn, to link these to infection and life history variables. Next, we used our independent longitudinal data to corroborate the robustness of the immunological markers, place them within a stronger cause–effect context in relation to the infection and life history variables, and measure their effect on survival. Finally, we returned to the cross-sectional data and used structural equations modelling (SEM) to support the interdependencies between variables indicated by the longitudinal data. In analyses of the cross-sectional data, we primarily represented macroparasite infection using a reduced variable derived as scores from the first principal component of a principal components analysis (PCA) of the most common species that would be expected to be in contact with the host immune system  . In the ariables  and thenM main) tended to accumulate linearly with individual size and age, with no indication that acquired resistance decelerated the acquisition of infection in older animals , but immature males supported less infection as age indicators increased . This pattern is consistent with resistance building up prior to maturity but being absent postmaturity  amongst different life history stages. A particularly robust contrast occurred again between mature males, which showed a strong positive relationship between parasite burden and body condition, and immature males, which showed no such relationship  (Mycobacterium microti) were also in better condition relative to others  weight normalised for individual size – snout-vent length (SVL) and its quadratic term see . Conside = .003) . AmongstTo identify immunological markers for a tolerance strategy and determine their consequences for host fitness, we then focused primarily on the dichotomy between mature and immature males, as the former showed the strongest increased body condition response to macroparasite infection but no epidemiological evidence of resistance. Patterns in females, which may be obscured by immunosuppression during pregnancy, were less clear cut, as discussed briefly below.mit-stim), was consistently and strongly associated with macroparasite burden, host condition, and life history in mature males and also showed a different pattern in immature males. First, Gata3mit-stim correlated with macroparasite burden positively in mature males  in mature males  whether the trend for apparent tolerance rather than resistance in mature versus immature males was linked to variation in immune gene expression. We found that a single parameter, mitogen-stimulated Gata3 expression in cultured splenocytes .Gata3 expression, then, may be a useful biomarker for either resistance or tolerance where a predominant pattern of one or the other can be established within groups, as it can in males. Nonpregnant females showed similar tolerance-like accumulations of macroparasites with age indicators and also increases in condition at higher macroparasite burdens. However, blood) in the following month  in the following month  was linked to the tolerance pattern.In applying SEM to our cross-sectional data  and S15,blood), which decreased with age (using weight as a surrogate), was a significant predictor of survivorship in the longitudinal data .Constitutive Gata3 expression in peripheral blood  animals. Our longitudinal analyses suggest, moreover, that rather than being a mere correlate, elevated Gata3 expression was triggered by macroparasite infection and preceded the life history readjustments. Some corroborative patterns were also seen for another chronic infectious disease, TB, with animals being in better condition if showing overt signs of infection and especially if also expressing high levels of Gata3.These analyses suggest that expression of Gata3 (a master transcription factor involved in the differentiation of Th2 cells) can, in some individuals, be a marker for a physiological programme of tolerance to macroparasite infection, with implications for body condition, fecundity, and survival. Tolerance involved apparent overcompensation in body condition following infection, but accompanying that was downwards readjustment of fecundity, and age-dependent changes in survival, with survival improving in older tolerant (Gata3mit-stim) was inversely associated with macroparasite infection in these animals. This presents a familiar scenario, consistent with the paradigmatic role of acquired Th2 responses in resistance against macroparasites. Mature males, on the other hand, showed a very different and unexpected pattern. They accumulated macroparasites with age (resulting in very high infection levels) and showed a positive association between Gata3mit-stim and infection. Furthermore, body condition increased in heavily infected animals, whereas Gata3mit-stim was also positively associated with body condition  and negatively associated with male fecundity (adjusted testis weight). Our longitudinal time series data for recaptured adult males indicated that peripheral blood Gata3 expression was triggered by macroparasite exposures and preceded host responses that increased somatic maintenance and ultimately survival . This is suggestive of the process, and not just the pattern, of tolerance, as the increase in body condition associated with macroparasite infection and Gata3 expression is indicative of a protective response that does not affect the parasite directly.Our ability to infer tolerance in a complex field situation was facilitated by differing responses, in our cross-sectional study, between immature and mature males. These responses indicated a predominant pattern of resistance to macroparasites in the former and tolerance to macroparasites in the latter. Thus, as immature males aged, their infections reduced, consistent with the development of acquired resistance; also, mitogen-stimulated splenocyte Gata3 expression  are well within the routine scope of ecological researchers focussing on infectious disease. As considered further below, the dual aspect of Gata3 expression  is biologically plausible given the known role of Th2 responses in some pathways leading to resistance to macroparasites mit-stim, body condition, and macroparasite infection in mature and immature males. Hence, we can exclude these alternatives.There are alternative possible explanations for some of the cross-sectional associations we observed within mature males. One would be “differential mortality”—that is, high parasite burdens eliciting high Th2 responses, but before our observations are made, animals in poor condition with many macroparasites suffering increased mortality, so that the only animals with high parasite burdens left to observe are those in good condition. A second explanation would be “correlated risk”—that is, increased risk in high condition animals of macroparasite infections, and the Th2 responses they trigger, due, for example, to animals that forage more actively acquiring both more food and more parasites. However, whereas the temporal sequences seen in the longitudinal data directly support the tolerance hypothesis (macroparasites→Gata3→good condition), they support neither differential mortality nor correlated risk, as high Gata3 expression would in neither case precede good condition. Similarly, both differential mortality and correlated risk would predict associations between Gata3 expression and survival that did not change with age, with the former specifically predicting an uncomplicated association of high Gata3 with low survival and poor condition. These predictions, too, are contradicted by our results. Furthermore, differential mortality and correlated risk do not provide explanations for differing cross-sectional associations between Gata3Beyond our own system, these results may also provide some general insight into the nature of tolerance, as both pattern and process. The regulatory component of the immune system has often been seen as a potential driver of tolerance, through its role in suppressing immunopathological effector responses. Indeed, helminth parasites have been suggested to increase tolerance and reduce resistance by the elevation of regulatory responses Mycobacterium infections have been thought to facilitate clinical disease progression In apparent opposition to this, Th2 responses have often been considered to mediate resistance to macroparasites . In the laboratory they may do so via a diverse cascade of effector mechanisms It seems possible that increased body condition and reduced fecundity, in mature males but not immature males, might be adaptive by increasing residual reproductive value during macroparasite infection. This is consistent with a positive influence of Gata3 expression on survival in the oldest males. The contrasting patterns seen between mature and immature males suggest these may face very different cost/benefit scenarios when deploying immune responses against macroparasites. At present we can only speculate about the nature of these pressures, although a number of interesting possibilities might be considered. For example, altered exposure  might raise the immunopathological or energetic costs of resisting infection above thresholds of sustainability. Mature males also, due to their greater prior opportunity to develop robust adaptive immunity to common pathogens, may face fewer consequences from secondarily adopting a more tolerant phenotype. Thus, tolerance processes might have more serious indirect consequences for younger individuals through the inhibition of inflammatory responses against primary microbial infections.These are the first results to indicate a mechanism of tolerance in a naturally occurring mammalian population and to place this within a holistic framework of host fitness: context-dependent cost–benefit outcomes for individual condition, reproductive investment, and survival. This work therefore illustrates how immunological studies in natural populations may not only benefit from the many advances made in the laboratory but might also feed insights back into mainstream immunology. The results support the view that Th2 responses may play a crucial role in the tolerance of infection, and they more generally add to the growing body of evidence arguing for an increased focus on tolerance in our quest to understand host responses to parasitic infection. Further progress is likely to be facilitated by adopting approaches that incorporate experimental manipulations.M. agrestis) in Kielder Forest, Northumberland, United Kingdom, using live-trapping to access individual animals from natural populations. Our study was designed to permit the analysis of individual variation in condition and survival, infection status, and the expression of immune genes  or April (in the 2009–2010 season) and November, during which the capture–recapture study was carried out on the grid and destructive samples were retained from the transects . At eachin vivo expression of a subset  of the genes investigated in the cross-sectional study, in peripheral blood samples.Measurements of gene expression  in the cross-sectional element of the study focussed on cultured splenocytes and are described in detail in Jackson et al (2011) M. microti and carried out PCR diagnostics for Bartonella spp. and Babesia microti using previously established methods  from a PCA of common taxa likely to be in strong contact with the immune system  as the main analytical variable . In the case of macroparasites, we used the first principal component Click here for additional data file.Figure S2Sampling and measurements. Design replicated on two sites in 2008–2009 and two different sites in 2009–2010. Processing steps indicated by solid arrows and measurements by dashed arrows. Gene expression measurements shown in red. For timeline, see (TIF)Click here for additional data file.Figure S3Timeline of sampling.(TIF)Click here for additional data file.Figure S4post hoc testing of individual variables. Arrows indicate steps in the analysis associated with a particular hypothesis test or tests. Blue arrows indicate steps using data from the cross-sectional study . Where a particular individual variable or stratum of hosts was strongly implicated at a given step of the analysis and is focussed upon in the subsequent analysis, this is indicated in red to the right. Yellow arrows indicate steps using longitudinal data . Details of the respective analysis steps are provided in the figures and tables indicated on the left of each arrow (in grey) and the analytical methods employed are indicated on the right (in black) .Flow of main analysis. In order to avoid problems of interpretation due to multiplicity of tests, we carried out our analyses in a stepwise fashion, with each continuation step conditional on significant results in a single or small number of main hypothesis tests in the previous step. For parasite and immune expression data, these main tests initially used reduced variables . Significant main tests involving PC variables were followed by exhaustive (TIF)Click here for additional data file.Figure S5M main  with the abundances of common detritivorous mites (with negative loadings) and was thus also positively linearly associated with SVL and lens weight. There was thus no evidence of ongoing buildups of acquired resistance to the key macroparasitic throughout the lives of individuals, although analyses stratified by life history stage, with different macroparasite on age indicator slopes, indicated that resistance might build up in immature males but then collapse postmaturity . Mature males carry a much heavier macroparasite load than other life history stages.Overall associations between macroparasite abundance and host age indicators and life history stage. (A) Associations between parasite abundance and host size and age indicators, averaging slopes across life history groups. Plotted relationships are predictions (±1 standard error) from LMMs of the form: Parasite variable = Process day+LH+SVL (random component: Year×Sampling Point×Site). A quadratic term for SVL was initially included in models, and equivalent models were also analysed with lens weight terms replacing the SVL terms (with similar results). There were highly significant positive linear associations with the principal component representing key macroparasite species, PCmain see , and witmain see . The com(TIF)Click here for additional data file.Table S1M) was dominated by larger loadings for the more common species. These loadings strongly contrasted laelapid and listrophorid mite abundances with those of small fleas, ticks, and intestinal cestodes. A second PCA was carried out including only common macroparasite species that feed directly on host tissues  or that have an intimate association with host mucosal surfaces and adsorb soluble nutrients utilizable by the host (adult cestodes). This excluded all rare species . It also excluded common species that do not feed directly on host tissues, do not have an intimate association with host mucosal surfaces, and do not adsorb soluble nutrients. This was the case for listrophorid and laelapid mites, which are primarily deritivores, and pinworms (Syphacia), which occur free in the lumen of the colon and feed on bacteria.  This second PCA produced a very dominant first component (PCM main) with large loadings of similar magnitude and the same sign for small fleas, ticks, and adult cestodes, indicating a pattern of general positive covariation among these. In subsequent analyses, PCM main was used as the primary index of macroparasite infection pressure , but PCM was also analyzed secondarily (with similar results).Multivariate reduction of macroparasite data . PCA was used to generate single composite variables  representing the main axis of covariation amongst sets of macroparasite variables. Scores from these PC1 were then used in subsequent analyses to represent “grouped” macroparasite variation. An initial PCA included all of the macroparasite variables. PC1 (designated PC(DOC)Click here for additional data file.Table S2tlr-stim and PC2tlr-stim ; PC1tlr-index and PC2tlr-index ; PC1mit-stim and PC2mit-stim ; and PC1mit-index and PC2mit-index .Multivariate reduction of immune expression data . PCA was used to reduce sets of immune expression variables into smaller sets of nonredundant composite variables  reflecting major axes of covariation among the starting variables. Scores from these PCs were then used in subsequent analyses to represent grouped immune responses . Immune expression data were considered in two different ways: (1) expression under stimulatory conditions relative to a calibrator sample  and (2) expression indexed to a corresponding unstimulated control culture. Each of these sets of variables was reduced separately, with distinct analyses being carried out within each set for data from TLR-stimulated and mitogen-stimulated cultures. This gave four separate PCAs, from each of which we considered the two largest components (PC1 and PC2) in further analyses. The components were designated: PC1(DOC)Click here for additional data file.Table S32+LH.SVL+LH.SVL2+Parasite variable (random term = Year×Sampling Point×Site). Both of the macroparasite principal component variables (PCM and PCM main), several of the individual macroparasite variables, and presence/absence of overt TB all showed significant positive associations with body condition. The only negative association was for Listrophoridae (fur mites), which may possibly have been due to an association between poor condition and compromised grooming. Significant positive association in the main hypothesis test is highlighted in orange; significant associations in post hoc tests are highlighted in yellow (positive associations) or grey (negative associations).Association of body condition with parasitic infections across all host stages . Body condition was represented in LMMs by body weight (the response) adjusted for covariates SVL and its quadratic term, initially averaging slopes for these across life history stages. Models considered all of the sampled stages and were of the form: Body weight = LH+Process group+SVL+SVL(DOC)Click here for additional data file.Table S42+LH.SVL+LH.SVL2+Parasite variable (random term = Year×Sampling Point×Site). Both of the macroparasite principal component variables (PCM and PCM main), two of the individual macroparasite variables, and presence/absence of overt TB all showed significant  positive associations with liver condition. The only significant negative association was for ear mites, which may possibly have been due to an association between poor condition and compromised grooming. Significant positive association in main hypothesis test is highlighted in orange; significant  associations in post hoc tests are highlighted in yellow (positive associations) or grey (negative associations).Association of liver condition with parasitic infections across all host stages . Liver condition was represented in LMMs by liver weight (the response) adjusted for covariates SVL and its quadratic term, initially averaging slopes for these across life history stages. Models considered all stages sampled and were of the form: Liver = LH+Process group+SVL+SVL(DOC)Click here for additional data file.Table S510 spleen weight = LH+Process group+SVL+SVL2+LH.SVL+LH.SVL2+Parasite variable (random term = Year×Sampling Point×Site). Both of the macroparasite principal component variables (PCM and PCM main) and several of the individual macroparasite variables showed significant  positive associations with spleen condition. The only significant negative association for a macroparasite was for Listrophoridae (fur mites), which may possibly have been due to an association between poor condition and compromised grooming. The relative development of the spleen is a complex indicator of individual condition, potentially reflecting a combination of generalised individual condition, standing investment in immune defences, and ongoing host responses. The strong positive association of B. microti with spleen condition (but not with body or liver condition) is likely to be due to a host response, given that splenomegaly often develops in Babesia infections . Furthermore, the strong negative association of Bartonella spp. with spleen condition  is likely due to confounding from B. microti. Bartonella in our data and in previous studies shows a strong negative interaction with B. microti (and the significant result for Bartonella spp. disappears if B. microti is added as an explanatory term to the LMM). Significant positive association in the main hypothesis test is highlighted in orange; significant  associations in post hoc tests are highlighted in yellow (positive associations) or grey (negative associations).Association of spleen condition with parasitic infections across all host stages . Spleen condition was represented in LMMs by spleen weight (the response) adjusted for covariates SVL and its quadratic term, initially averaging slopes for these across life history stages. Models considered all stages sampled and were of the form: Log(DOC)Click here for additional data file.Table S610 testis weight = Process group+SVL+SVL2+Parasite variable (random term = Year×Sampling Point×Site). Both of the macroparasite principal component variables (PCM and PCM main) and several individual macroparasite variables showed significant  positive associations with testis condition. The only significant negative association was for Listrophoridae (fur mites), which may possibly be due to an association between poor condition and compromised grooming. Significant positive association in the main hypothesis test is highlighted in orange; significant  associations in post hoc tests are highlighted in yellow (positive associations) or grey (negative associations).Association of testis condition with parasitic infections in adult males . Testis condition was represented in LMMs by testis weight (the response) adjusted for covariates SVL and its quadratic term. Models considered only mature males  and were of the form: Log(DOC)Click here for additional data file.Table S7M main) across life history stages , all life history stages were initially analysed in a single LMM of the form described in M main as the parasite variable and, additionally, a Life History Stage (LH)×PCM main interaction. This interaction was highly significant , indicating divergent reaction norms. When females were excluded from the analysis, due to the complex and sometimes ambiguous transitions between different reproductive states, there was still highly significant heterogeneity of slopes between mating and nonmating males  . Significant positive associations are highlighted.Association between body condition and grouped macroparasite infection in different life history stages : differing epidemiological reaction norms (ERNs). To assess the heterogeneity of adjusted body condition slopes on grouped macroparasite infection (PC(DOC)Click here for additional data file.Table S8M main as a further covariate. Models included adult males only and were of the form: Body weight = Process group+SVL+SVL2+PCM main+Immunological variable (random term = Year×Sampling Point×Site). The table shows the results of the main hypothesis tests  that were initially carried out on the eight immunological principal component (PC) variables, followed by post hoc testing of individual variables. PC variables that would be significant at p<.01 after multiplicity adjustment  are highlighted in orange. Individual variables from post hoc testing that would be significant at p<.01 after a multiplicity adjustment are also highlighted in orange. PC and individual variables that were only significant  without multiplicity adjustment are highlighted in yellow (nonsignificant results for post hoc testing not shown). PC2mit-stim (which featured a large loading for Gata3mit-stim) and Gata3mit-stim itself were the only two significant variables following multiplicity adjustment (for the whole table of tests carried out). Gata3mit-index was also relatively highly significant at an individual level. When Gata3mit-stim was considered in a model of the above form additionally including immature males and different slopes on Gata3mit-stim and PCM main for mature and immature males, there were very significantly different stage-specific Gata3mit-stim slopes, with mature males showing a positive slope and immature males no association .Association between body condition and immunological gene expression in adult males . Body condition was represented in LMMs by body weight (the response) adjusted for covariates SVL and its quadratic term. Body condition was also adjusted for the association with macroparasites see  by the i(DOC)Click here for additional data file.Table S9mit-stim) in adult males . Organ condition was represented in LMMs by organ weight (the response) adjusted for covariates SVL and its quadratic term. Organ condition was also adjusted for the association with macroparasites . Significant positive associations are highlighted in yellow and significant negative associations in grey.Associations between organ condition and mitogen-stimulated Gata3 expression Click here for additional data file.Table S10mit-stim), overt TB lesions, and body condition . Post hoc analyses of body condition focussing on TB found a highly significant interaction between overt TB lesions and Gata3 expression in mitogen-stimulated splencocytes (Gata3mit-stim) across all stages. This analysis is based on the following LMM: Body weight = Log10 Gata3mit-stim+LH+Process group+SVL+SVL2+LH.SVL+LH.SVL2+Overt TB+Overt TB×Log10Gata3mit-stim (random term = Year×Sampling Point×Site). There was only a marginally significant overt TB×Gata3mit-stim interaction when the analysis was restricted to adult males (p = .08).Gata3 expression (Gata3(DOC)Click here for additional data file.Table S11mit-stim) in adult males . The association of Gata3mit-stim with grouped parasitic infection was analyzed in LMMs of the form: Parasite variable = Process group+SVL+Log10 Gata3mit-stim (random term = Year×Sampling Point×Site). In mature males there were significant positive associations with both of the macroparasite principal component variables (PCM main and PCM) (see table). When immature males were added to the above model for PCM main, with different age-specific slopes, there was significant variation in the Gata3mit-stim slopes, with a positive slope for mature males and a negative slope for immature males . A similar interaction also occurred in the case of PCM (p = .031). Significant positive association in main hypothesis test is highlighted in orange; significant association in post hoc test is highlighted in yellow.Associations between parasitic infections and mitogen-stimulated Gata3 expression (Gata3(DOC)Click here for additional data file.Table S12blood) in peripheral blood in adult males . (A) Time lagged associations. Association of Gata3blood with infection variables earlier in time (t-1 = 1 mo earlier). When analyzed alone, ticks and small fleas were represented by continuous log-transformed abundance variables. When ticks and small fleas were analyzed together, this was as a binary presence/absence factor . Table shows significant explanatory terms from models of the form: Gata3blood = Weight+Parasite variable . Results for weight are for the model with a factor for ticks and or small fleas. Significant positive associations highlighted in yellow and significant negative associations in grey. Earlier infection tended to be positively associated with Gata3blood expression. (B) Instantaneous association. Association of Gata3blood with infection at the same time point. When analyzed alone, ticks and small fleas were represented by continuous log-transformed abundance variables. When ticks and small fleas were analyzed together this was as a binary presence/absence factor . Table shows significant explanatory terms from models of the form: Log10 Gata3blood = Weight+Parasite variable . Results for weight are for the model with a factor for ticks and or small fleas. Significant  negative associations highlighted in grey. Contemporaneous infection tended to be negatively associated with Gata3blood expression, perhaps due to some protective (resistance) effect of this response.Instantaneous and time-lagged associations between exposure to blood-feeding ectoparasites and Gata3 expression (Gata3(DOC)Click here for additional data file.Table S13blood) in peripheral blood in adult males . Association of month-on-month weight gain (adjusted for starting weight) with Gata3blood expression 1 mo earlier. Table shows significant explanatory terms from LMMs of the form: Final weight = Starting weight+Gata3blood . Gata3blood was positively associated with adjusted weight gain in the following month. There was no association between Gata3blood and weight gain in the preceding month . The effects of individual parasite variables were examined in these base models (taking each variable in turn), but were nonsignificant. Macroparasite variables examined included the abundance of fleas, ticks, laelapid mites, listrophorid mites, and lice and also an overall ectoparasite index; microparasite variables (presence/absence) included B. microti, Bartonella spp., and overt TB. Ectoparasite index was an additive score based on the sum of standardized abundances for the different ectoparasites. Significant positive associations are highlighted in yellow.Time-lagged association between month-on-month weight gain and Gata3 expression (Gata3(DOC)Click here for additional data file.Table S14p<.05) or near-significant (.05<p<.08) parameters; and optimal model fit  and AIC statistics. The table shows the two best supported models with six or seven significant (or near-significant) parameters. Parameter estimates for model 2 are shown in M main, see 10 Gata3 expression in mitogen-stimulated splenocytes; C, body condition; T, testis condition. Prior to the SEM analysis, all of the variables were adjusted, in general linear models, for spatiotemporal sampling point (sampling time and site nested within year) and for host linear dimensions (SVL+SVL2) if SVL did not contribute to the variable already.SEM of key variables from cross-sectional study. In the SEM analysis of the cross-sectional data, we searched for models with: the causality indicated by the longitudinal analyses in (DOC)Click here for additional data file.Table S15M main, see 10 Gata3 expression in mitogen-stimulated splenocytes; C, body condition; T, testis condition. Prior to the SEM analysis, all of the variables were adjusted, in general linear models, for spatiotemporal sampling point (sampling time and site nested within year) and for host linear dimensions (SVL+SVL2) if SVL did not contribute to the variable already. A causal effect of macroparasites (P) upon Gata3 expression (G) and of G upon body condition (C) was independently suggested by analyses of the longitudinal dataset ; P, infection with key influential macroparasites (PC(DOC)Click here for additional data file.Table S16blood) and age-specific primary session return rate . Return rate of mature males (at the next primary trapping session) (A) was initially analyzed with respect to Gata3 expression in peripheral blood (Gata3blood) using a GLMM with binomial errors . The base model was of the form: Return∼Weight+Gata3blood+Peripheral row . The peripheral row factor accounted for animals captured in the outer trap row of the grid, which might putatively have lower recapture probabilities (as their ranges are more likely to have been centred outside the grid). Other models (not shown) examined the effects of individual parasite variables in this base model (taking each in turn). Macroparasite variables included the abundance of fleas, ticks, laelapid mites, listrophorid mites, and lice and also an overall ectoparasite index; microparasite variables (presence/absence) included B. microti, Bartonella spp., and overt TB. Ectoparasite index was an additive score based on the sum of standardized abundances for the different ectoparasites. These were only included in the final model if significant. A similar set of models was then investigated (post hoc) for all stages (B), additionally including life history stage as a factor. The main effect for Gata3blood was not significant in adult males but was significantly negative in the analysis across all stages . Significant Weight×Gata3blood interactions occurred in both analyses . Although return rate might act as a reasonable surrogate for survival .Gata3 expression in peripheral blood (Gata3rved see ), furtherved see . Signifi(DOC)Click here for additional data file.Table S17blood) was analyzed in CJS models in a subset of males with >1 capture and no or few missing values for Gata3blood (n = 107).  Time-specific body weight covariates were highly significant  when added to an optimal base model for survival and recapture that included seasonal (monthly) variation in survival (φ) and variation across years in recapture probability (p). Gata3blood and Gata3blood×Body Weight covariates were then added to the base model already containing body weight covariates. The best resulting models contained covariates for Gata3blood and Gata3blood×Body Weight only in the last 2 mo  or last month of the season . In these months, survival tended to increase in larger animals expressing a greater amount of Gata3blood. The following parameter values were estimated for a model in which overall p and φ for the first time interval (φApril→May) were constrained to be 1. This was because p was generally estimated to be very high  and because φApril→May was limited by the nature of the data, which only included animals with multiple recaptures. Real function parameters for survival were: φMay→June = 0.49±0.07, φJune→July = 0.76±0.06, φJuly→August = 0.87±0.05, φAugust→September = 0.73±0.05, φSeptember→October = 0.55±0.08. The logit link function parameter for the September Gata3blood×Body Weight covariate was: βSeptember = 0.3±0.11.Gata3 expression and survival: CJS analysis with time-specific individual covariates . The effect of Gata3 expression in peripheral blood (Gata3(DOC)Click here for additional data file.Table S18Real-time PCR primers used to measure the expression of immunological genes in peripheral blood.(DOC)Click here for additional data file.Table S19Bartonella spp. and B. microti may have had unequal sensitivities in 2008 and 2009 due to the use of different DNA extraction methods. Prevalence data are thus not directly comparable across years.Infection statistics for parasites at Kielder broken down by Locality×Year of Study. A, abundance±standard error. P, prevalence, followed by 95% CI. *Abundance data for Listrophoridae relate to a semiquantitative abundance index. †PCR diagnosis of (DOC)Click here for additional data file.Methods S1Supplementary Methods.(DOC)Click here for additional data file.


F3 location  lead localization system). GAITRite gait analysis system was used to collect objective gait measures before and after one session and in another occasion three consecutive daily sessions of rTMS. Two-tailed within subject repeated measure t-test showed significant enhancement in ambulation time, gait velocity, and cadence after three consecutive daily sessions of rTMS. Modulating left prefrontal cortex excitability using rTMS resulted in significant change in gait parameters after three sessions. To our knowledge, this is the first report that demonstrates the effect of rTMS applied to the prefrontal cortex on gait in MS patients.Multiple Sclerosis (MS) is a chronic central nervous system (CNS) demyelinating disease. Gait abnormalities are common and disabling in patients with MS with limited treatment options available. Emerging evidence suggests a role of prefrontal attention networks in modulating gait. High-frequency repetitive transcranial magnetic stimulation (rTMS) is known to enhance cortical excitability in stimulated cortex and its correlates. We investigated the effect of high-frequency left prefrontal rTMS on gait parameters in a 51-year-old Caucasian male with chronic relapsing/remitting MS with residual disabling attention and gait symptoms. Patient received 6 Hz, rTMS at 90% motor threshold using figure of eight coil centered on  Multiple Sclerosis (MS) is a central nervous system (CNS) demyelinating disease mainly affecting young adults. In 2008, the WHO estimated that 2–2.5 million people around the world have MS . IndividTranscranial Magnetic Stimulation (TMS) is a relatively noninvasive procedure whereby a magnetic field is generated by a powerful electric current passing through a hand held coil attached to a capacitor. Repetitive Transcranial Magnetic Stimulation (rTMS) is a promising therapeutic tool for various neurological and psychiatric diseases . AlthougThe current study aimed to explore the effect of rTMS for gait impairment in an MS patient.A 51-year-old Caucasian male with 4-year history of MS was referred to our neuropsychiatry memory clinic by his family doctor for a cognitive assessment and intervention. His course is best described as relapsing and remitting in nature. He had left upper and lower limb spastic weakness, double vision, jerky eye pursuit, and some cognitive deficits mainly in attention. Depression and anxiety screening was negative, although patient reported frustration with his “slowed thinking.” Patient's MRI showed significant white matter involvement including left prefrontal and parietal white matter, areas that are relevant to attention . On cognBased on case reports of benefit of Cholinesterase inhibitors in MS patients  we offerWe used a figure of eight air film-cooled coil attached to Magstim Super Rapid 2 TMS device . This coil produces a cone shape magnetic field with a 1 centimeter square peak that can penetrate around 2 cm into the cortex of the brain. Hundred percent of the machine output produces about 3 tesla magnetic field power. The magnetic field passes unimpeded to the cortex and induces a local current. It is thought that this current is frequency dependent and results in modulation of cortical interneurons. A higher frequency magnetic pulse (3 or more cycles per second) is usually excitatory  while low frequency magnetic pulse (below 3 cycles per second) is usually inhibitory in nature  , 15.F3 location which usually corresponds to left dorsal lateral prefrontal cortex (L-DLFC) [We used 10-20 international EEG lead localization system to identify (L-DLFC) . PatientPatient received one session and three consecutive daily sessions of rTMS treatment separated by 3 days. We administered rTMS using the same parameters for both sessions. Gait data were collected at 2 points of time: (a) before (baseline 1) and immediately after one rTMS session and (b) before (baseline 2) and immediately after 3 consecutive daily rTMS sessions. The gait pattern was assessed during three consecutive trials at a usual gait.http://www.gaitrite.com/) that is 600 cm in length and 64 cm in width. As participants walk along the mat, imbedded sensors are activated by the pressure of their feet and deactivated when the pressure is released. A computer processed the footsteps, providing data for both spatial and temporal parameters. Start and end points were marked on the floor with tape 1 m from either end of the mat to avoid the recording of acceleration and deceleration phases. Participant performed 1 practice trial walking on the mat to familiarize himself with the protocol. Ambulation time , gait velocity (cm/s), stride time (ms), cadence (number of full cycles taken by the pair of feet per minute), and stride time variability (percentage of coefficient of variation (%CoV)), the principal gait measures of interest, were measured over three trials which consisted of walking the length of the mat at a self-selected usual pace.Gait performance was assessed using an electronic walkway system  was performed on quantitative data using SPSS software package 21.0 . We used Overall, the results indicated that patient had significant improvement in his gait parameters after being treated with rTMS. t(2) = −4.99, p < 0.05). Statistically significant differences were found before and after 3 consecutive daily rTMS sessions in ambulation time (t(2) = 8.32, p < 0.05), velocity (t(2) = −4.59, p < 0.05), and cadence (t(2) = −7.57, p < 0.05).There were no statistically significant differences before and after 1 rTMS session in ambulation time and velocity; however, cadence was significant  between the one rTMS session and the beginning of the three consecutive daily rTMS sessions. This resulted in a new baseline being established, which was lower in ambulation time and velocity compared to the first baseline. The occurrence of this difference is hard to interpret; hence, further investigation is needed. Furthermore, we could not assess the long term impact of rTMS on gait because we did not provide any follow-up for this case. In this case study, we chose to stimulate the dominant left side assuming that it is more related to volition. The effect of nondominant prefrontal cortex rTMS was not explored.The results of this case study indicate that modulating left prefrontal cortex excitability using high-frequency rTMS resulted in significant change in gait parameters in an MS patient with minimal discomfort. This indicates potential utility of this noninvasive brain stimulation technique in modulating gait parameters in this patient population. Further research is needed to clarify the role of rTMS as a therapeutic tool in MS patients using appropriate clinical trial design to address questions raised in this case report.


High density stress, also known as intraspecies competition, causes significant yield losses in a wide variety of crop plants. At the same time, increases in density tolerance through selective breeding and the concomitant ability to plant crops at a higher population density has been one of the most important factors in the development of high yielding modern cultivars.Oryza sativa plants over the course of a life cycle by assessing differences in gene expression and metabolism. Moreover, the nitrogen limitation was examined in parallel with high density stress to gain a better understanding of physiological responses specific to high density stress. While both nitrogen limitation and high density resulted in decreased shoot fresh weight, tiller number, plant height and chlorophyll content, high density stress alone had a greater impact on physiological factors. Decreases in aspartate and glutamate concentration were found in plants grown under both stress conditions; however, high density stress had a more significant effect on the concentration of these amino acids. Global transcriptome analysis revealed a large proportion of genes with altered expression in response to both stresses. The presence of ethylene-associated genes in a majority of density responsive genes was investigated further. Expression of ethylene biosynthesis genes ACC synthase 1, ACC synthase 2 and ACC oxidase 7 were found to be upregulated in plants under high density stress. Plants at high density were also found to up regulate ethylene-associated genes and senescence genes, while cytokinin response and biosynthesis genes were down regulated, consistent with higher ethylene production.Physiological changes underlying high density stress were examined in High density stress has similar but greater impact on plant growth and development compared to nitrogen limitation. Global transcriptome changes implicate ethylene as a volatile signal used to communicate proximity in under dense population growth condition and suggest a role for phytohormones in high density stress response in rice plants.The online version of this article (doi:10.1186/1471-2164-15-681) contains supplementary material, which is available to authorized users. Consequently, crop production will have to increase dramatically above current levels in order to sustain the human population. To increase yield per unit area one can either increase the amount an individual plant can produce or grow more plants per unit area. One of the challenges of the latter strategy is to maintain yield despite density stress imposed on crops planted in close proximity. Modern crop plants have been selected for their ability to produce higher yields per unit area, be more resistant to biotic and abiotic stresses, and have higher density tolerance [Recent estimates by the World Health Organization predict that the global population will approach 9 billion people by 2050  with more erect architecture were able to better tolerate high density stress [High density stress has been studied mostly from agronomic (e.g. yield) and physiological perspectives. Simple one-dimensional stresses, such as nitrogen limitation, have been studied in great detail over the years, while the more complicated/multi-factorial stresses, such as high density, have not received the same attention . As plany stress . HoweverMolecular mechanisms underlying high density stress response have not been studied in great detail. High density stress has been typically examined in the context of shade avoidance response, especially as it pertains to changes in the ratio of red:far-red (R:FR) light . HoweverVarious combinations of stresses and growth conditions have been used to identify relevant pathways underlying high density stress response. Intra-specific competition between rice plants and inter-specific competition between rice plants and weeds were used to determine general characteristics, such as upright leaf architecture and large leaf area, and infer similarities in genetic factors and physiological pathways between the two growth conditions . ShadingThe main objective of the current study was to utilize global gene expression and metabolite profiling to identify density specific responses in a model monocot. Limiting nitrogen (LN) was used in combination with high density (HD) stress to better delineate molecular pathways of high density stress response. The findings presented here describe changes in transcriptome and metabolome with a special focus on the potential role of ethylene. Our data support the notion that studies focused on stress combinations rather than individual stressors are able to provide novel information in the context of crop improvement.The stress conditions used in this study  allowed us to observe rice plants for a complete life cycle and set seed even under the least optimal (HDLN) growth condition. Rice plants grown under HD conditions had significantly fewer total (reproductive and vegetative) and reproductive tillers, were shorter, accumulated less shoot fresh weight and contained less chlorophyll compared to plants grown under low density growth conditions at all sampled time points and regardless of nitrogen levels Figure A and B. Nitrogen limitation and HD stress are both associated with a reduction in biomass accumulation. Changes in aspartate and glutamate were correlated with decreases in biomass due to stress treatments. Metabolite profile changes of rice plants due to density stress resulted in decreases of glutamate and aspartate concentrations. Glutamate concentration is maintained at constant levels in a plant and only stress conditions, such as decrease in nitrogen nutrition, cause significant decrease in the glutamate concentration . TherefoTo isolate genes that are differentially regulated in response to high density and not nitrogen limitation, transcriptome signatures of both high density and nitrogen limitation were compared while using the most optimal growth condition (LDSN) as the control. Furthermore, combined stress condition (HDLN) was included into the analysis to determine whether the transcriptome signature of combined stress is unique and is not simply the sum of HD and LN alone. The expression data, which was filtered based on 2.0 fold cut-off and Benjamini-Hochberg FDR (p > 0.05), revealed that 41% of genes responsive to high density stress were also responsive to nitrogen limitation at 21 days Figure A. The prTo gain better insight into the physiology of high density stress, differentially expressed genes associated with high density stress and not nitrogen limitation were analyzed from this point forward. Cluster analysis was performed to identify gene groups with potentially similar functions. Out of 21,179 genes, 426 genes showed differential expression at 21 days and 707 genes showed differential expression at 31 days due to HD stress treatment using three-fold cut-off. Mixture model-based clustering was performed using multivariate Gaussian distributions with an eigen-decomposed covariance structure  1, ACS2 and ACC oxidase 7 (ACO7) increased over time in plants subjected to HD stress confirming microarray prediction of elevated ethylene production levels under HD stress  and EARLY RESPONSE TO DROUGHT 1 (ERD1) genes [ERF and ERD1 genes were elevated in HD grown rice plants in our experiment, further confirming increased ethylene production  genes , 31. Exp1) genes . Expressn Figure A. Genes s Figure A. The ex process .Figure 6ISOPENTYL TRANSFERASE 7 (IPT7) and RESPONSE REGULATOR gene (RR2) were 2.4 and 1.81-fold down-regulated in plants grown under HD versus low density growth condition at 21 days  in the expression level of RR2 were detected at 31 days, it is unlikely to correspond with a physiological difference , NB-ARC (AT3G14470) and ATHCHIB (AT3G12500), among differentially expressed genes due to HD stress. However, biotic stress response-associated genes were not highly enriched in our study according to AgriGO analysis , abscisic acid and methyl jasmonate, while ethylene represented a lesser proportion of differentially regulated genes [Solanum nigrum was identified to be responsive to HD stress [Arabidopsis demonstrated that ethylene is involved in shade avoidance response [Our findings suggest that rice plants utilize ethylene to communicate their proximity to neighbouring plants in a high population density environment. Expression differences in ethylene-associated genes have been detected in high population density studies in m nigrum , 44. Theed genes . Only a response –49. In tresponse , 49. On response . HoweverArabidopsis and tobacco plants. Lastly, a recent study in Arabidopsis demonstrated that touching of leaves precedes R:FR driven phytochrome signaling [Changes in light quality, characterized mainly by changes in R:FR ratio, ethylene production and touch sensitivity mechanisms might all contribute to the shade avoidance response in rice under HD stress. The contribution of ethylene to shade avoidance response was initially underestimated . While eignaling . Thus thThe molecular basis of HD stress response has not been studied extensively. The main factor for this is mostly likely the multitude of factors involved in HD stress, such as various resource limitations. A multiple stress combination approach could be necessary to decipher all of the pathways involved in HD stress response. Similarities between limiting nitrogen and HD stress response were found with respect to a number of developmental and physiological characteristics, such as biomass accumulation, decrease in tiller number, chlorophyll, and aspartate and glutamate concentration. Furthermore, HD stress resulted in global transcriptome changes similar to those elicited by nitrogen limitation at the early stages of plant growth and development. Changes in ethylene homeostasis in response to HD stress implicate ethylene as a plant-plant communication signal under HD stress. While the molecular mechanisms of ethylene driven shade avoidance response are beginning to emerge, future studies in crop plants remain to be conducted , 52. Eth3- and limiting nitrogen (LN) condition was set at 3mM NO3- [Rice plants were grown under four different conditions: high density and sufficient nitrogen (HDSN), high density and limiting nitrogen (HDLN), low density and sufficient nitrogen (LDSN), low density and limiting nitrogen (LDLN). Low density condition  was chosen to resemble rice density under normal field conditions  at 450 μmol mTotal RNA was isolated from 100mg of frozen leaf tissue with TRIzol reagent (Invitrogen). RNeasy (Qiagen) columns were used to purify the RNA samples. 1 mg of total RNA was converted into cDNA using Superscript (Quanta) kit. Three biological replicates were used for microarray analysis.http://www.phalanxbiotech.com). The .cel files were loaded into GeneSpring GX 11 (Agilent Technologies) and the GC-RMA normalization algorithm was applied. A minimum 2-fold change between normalized averages were used as a cut-off point setting for further analysis and Benjamini-Hochberg FDR was applied (p < 0.05). MapMan pathway analysis was used to assign differentially expressed genes to functional bins [For global expression analysis, Rice Genome OneArray® Microarray chips were used. Labelling, hybridization, and scanning of the chips were carried out by the University of Guelph Genomics facility  according to the manufacturer’s protocols . Applied Biosystems 7300 Real Time PCR instrumentwas used for data collection, and the resulting data were analysed by the 2–∆∆Ct method to obtain fold difference in expression between genes of interest compared to optimal (LDSN) condition [Relative quantification was achieved by comparing expression of genes of interest to ondition . See AddN-methyl-N-trimethylsilyl-trifluoroacetamide as previously described [The polar fraction was lyophilized and derivatized using methoxyamine and escribed . A 1 μl http://chemdata.nist.gov/mass-spc/amdis). The resulting components were filtered in GASP [Data analysis was performed using the automated mass spectral deconvolution and identification system . For multiple mean comparison, statistical significance was determined using Statistix 9.0 software program. The mean values were analyzed using one-way ANOVA and the least significant difference at α = 5% was recorded. Furthermore, Tukey post-hoc test was applied and a note was made where the samples did not pass p < 0.05 threshold. To obtain normal distribution according to Shapiro-Wilk test, data were square-root transformed.For data where only 2 means were compared (2 growth conditions for a single biomarker at a given time point), students Using the measurements of all 4 conditions, the genes were clustered via a model‒based clustering approach for each time point separately. Model‒based clustering assumes that the population is a convex combination of probability densities; i.e., that a mixture of subpopulations each with its own mean and covariance structure. In the analysis, Gaussian parsimonious clustering models ) were usA two-step clustering strategy was used for the analysis of each time point Additional file 1:Gene lists identified by the cluster analysis.(XLSX 24 KB)Additional file 2:Growth conditions used in this study are shown inFigure S1. Figure S2. shows an example of MapMan analysis output and demonstrates involvement of ethylene hormone. (DOCX 2 MB)Additional file 3:Summary of AgriGO analysis of differentially expressed genes at 31 days.(DOCX 21 KB)Additional file 4:Sequences of primer pairs that were used to analyze gene expression by qRT-PCR.(XLSX 11 KB)Additional file 5:Detailed description of cluster analysis.(DOCX 943 KB)Additional file 6:


Patients with systemic lupus erythematosus have a poor prognosis of pregnancy, since it is associated with significant maternal and fetal morbidity, including spontaneous miscarriage, pre-eclampsia, intrauterine growth restriction, fetal death and pre-term delivery. We report a case with successful pregnancy in a patient with systemic lupus erythematosus and hypertension.A 39-year-old nulliparous woman presented with systemic lupus erythematosus with antinuclear and antiphospholipid antibodies, hypertension and recurrent pregnancy loss presented for assisted reproduction. The patient responded well to enoxaparin and prednisone during both assisted reproduction and prenatal treatment.This case report indicates that prescription of immunosuppressant and blood thinners can be safely recommended throughout the whole prenatal period in patients with systemic lupus erythematosus. Enoxaparin and prednisone may be prescribed concurrently during pregnancy. Patients with SLE have a poor prognosis of pregnancy,6 Pregnant patients with SLE and APS often require treatment with anticoagulant medication to reduce the risk of further episodes of thrombosis and improve the prognosis of pregnancy.4 The use of anticoagulants such as low-molecular weight heparin (LMWH) between weeks 15 and 34 of pregnancy in women with APS indicates good efficacy and safety.7Correct identification of women during pregnancy with SLE requires specific therapeutic care in order to improve fetal and maternal outcome.8 stressing the importance of tight disease control and treatment. Assisted reproductive technology procedures including IVF do not appear to increase the risk of disease flare or thrombosis in patients with SLE and APS.9 Although assisted reproductive technologies can be successful in SLE and primary APS patients, rates of fetal and maternal complications are high.11 This is why we thought to report a case from our clinic with successful pregnancy in a patient with SLE and hypertension.In vitro fertilization (IVF) and embryo transfer in women with SLE and APS may lead to embryonic loss or fetal death despite prednisone, hydroxychloroquine, and enoxaparin,This is a case report of a patient at our reproductive medicine private practice. No Institutional Review Board approval was obtained. Our IRB designates a single-patient case report as not subject to IRB review because it does not meet the definition of human subjects research. The university ethics committee for human research has reviewed and approved the case report (CAAE 46103615.4.0000.5494). The patient has signed a consent form allowing disclosure of medical records.12 Also, the patient was diagnosed 3 years ago with hypertension. For SLE she was daily treated with low-dose corticosteroids, corticosteroid ointments, and hydroxychloroquine. For hypertension she was daily treated with enalapril 5 mg. The patient underwent 5 assisted reproductive technology cycles, of which 3 cycles were with transvaginal oocyte retrieval, 2 cycles for frozen–thawed embryo transfer, and 4 embryo transfer procedures. She continued on prednisone, hydroxychloroquine, and enalapril throughout the ovarian stimulation cycles.The patient is a 39-year-old nulliparous female who initially presented with infertility and right proximal tubal occlusion and endometriosis. Her history revealed that she was diagnosed 10 years ago with SLE according to the American College of Rheumatology criteria, including positive to antinuclear and anti-ribonucleoprotien antibodies tests.During the assisted ovarian stimulation cycles treatments, enoxaparin sodium  40 mg was administered to avoid thrombosis. Of the first 3 assisted ovarian stimulation cycles Table , 2 were The fourth cycle was artificial cycle for frozen–thawed embryo transfer (AC-FET). The endometrial preparation was with estradiol  PO in a dosage as follows: 2 mg days 1 to 6, 4 mg days 7 to 9, and 6 mg days 10 to 16. The endometrium was considered prepared when it was more than 7 mm thick on ultrasound, at which time vaginal progesterone  thrice a day for 6 days was added to initiate secretory changes. Three preembryos were thawed and transferred. Pregnancy was detected by measuring plasma beta-hCG (β-hCG)  60 mg SC daily was prescribed for the first 2 trimesters and 40 mg SC daily for the last trimester Table . PrednisThe patient delivered a healthy boy weighing 2.55 kg, 46 cm through C-section at 38th week of pregnancy.Our case report indicates that in assisted reproductive technology conception on patient with controlled SLE with hypertension may evolve without complications under adequate anticoagulant, immunosuppressant, and antihypertensive prescription.13 The current recommendations advise that SLE-affected woman achieve a stable remission of her renal disease and should have quiescent SLE for at least 6 months before conception.14 Current evidence indicates the relative safety of assisted reproductive technology procedures in patients with SLE.9 In a state of the art review, Ostensen et al15 conclude that pregnancies in women with APS remain a challenge, and better therapies for the obstetric APS are needed.Accumulating evidence from studies and case reports indicate risks of assisted reproductive technology in SLE patients, including ovarian hyperstimulation, repeated miscarriage, multifetal pregnancy, prematurity, and emotional distress.16 Immunomodulation with prednisone is considered as a promising treatment for recurrent pregnancy loss after assisted reproduction.17 We currently use concurrent immunomodulation with prednisone when there are at least 3 unsuccessful assisted reproductive technology cycles. In our protocol, prednisone starts from before embryo transfer and continues until pregnancy is established (14 days after embryo transfer). In our case, the patient had SLE with complicated hypertension controlled with enalapril already when she presented to the clinic. Since enalapril belongs to Category D (positive evidence of risk), we changed the treatment for the prenatal period to aldomet that belongs to Category B (no evidence of risk in humans).Immunosuppressants and anticoagulants are prescription drugs used to control lupus disease activity. Prednisone is the most commonly prescribed steroid for lupus and belongs to the category C of pregnancy risk (risk cannot be ruled out). This is why we decreased the prednisone dose to less than half of the reported average daily dose in a meta-analysis (27 mg).19 A Cochrane review suggests that LMWH may improve the live birth rate in women undergoing assisted reproduction, although evidence is highly debated.22 According to the RCOG Green-top Guideline No. 37a of the Royal College of Obstetricians and Gynaecologists (RCOG), women older than 35 years, or with SLE, with antiphospholipid antibodies are considered at risk and antenatal thromboprophylaxis throughout pregnancy is recommended.23 Also, these guidelines indicate “women with an IVF pregnancy and 3 other risk factors should be considered for thromboprophylaxis with LMWH starting in the first trimester.” Enoxaparin sodium is an LMWH that blocks factor Xa and factor IIa. According to FDA, enoxaparin sodium belongs to the category B of pregnancy risk (no evidence of risk in humans). In a case report, Joffe et al24 recommended that pregnant patients with APS should be considered candidates for full anticoagulation treatment throughout the entirety of gestation. We currently use concurrent thromboprophylaxis with low-dose aspirin (as a first choice) or LMWH (when assisted cycle is unsuccessful with low-dose aspirin) when there are at least 3 unsuccessful assisted reproductive technology cycles. In our protocol, an antithrombotic (low-dose aspirin or LMWH) starts from 3 days before embryo transfer and continues throughout the pregnancy.Thromboprophylaxis using low-molecular-weight heparin (LMWH) in women with recurrent pregnancy loss after assisted reproduction and thrombophilia appear to increase implantation rate, pregnancy, and live birth rates.27 Our case indicates that concurrent immunosuppressant and antithrombotic treatment throughout the pregnancy is safe.Combined treatment of prednisone for immunosuppression and aspirin or LMWH as antithrombotic, starting before ovulation induction, may improve pregnancy rate in patients who have had repeated assisted reproductive failures.This case report indicates that prescription of immunosuppressant and blood thinners can be safely recommended throughout the whole prenatal period in patients with SLE. Enoxaparin and prednisone may be prescribed concurrently during pregnancy.


Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis. The Anaplastic Lymphoma Kinase (ALK) Receptor Tyrosine Kinase (RTK) was originally discovered as a fusion protein together with Nucleophosmin (NPM) in anaplastic large-cell non-Hodgkin’s lymphoma (ACLC) in 1994 , 3. The ALK in mice does not result in serious phenotypes and the physiological role of ALK in mammals is unclear [The expression pattern of ALK in vertebrates has been described in several studies, where ALK has been shown to be expressed in the CNS and the PNS, as well as in testis and ovary , 3, 11.  unclear , 13. On  unclear , 13. Rec unclear , which i unclear –17. WhilPuberty is defined as a physiological and developmental process towards sexual maturity. Onset of puberty is initiated by neuroendocrine events that activate the pulsatile release of GnRH from the hippocampus into the hypophyseal portal blood system to stimulate the synthesis and secretion of gonadotropins from anterior pituitary cells. Gonadotropins, in turn, bind to ligand-specific receptors in the gonads, causing gonadal maturation and production of sex steroids, most notably testosterone in males –20. HippHerein we describe the investigation of an ALK knock-out mouse model in which the kinase domain encoding exons have been removed. In agreement with previous studies , 13, we in vivo experiments were conducted with approved Swedish legal ethical committee permits A203-12 and A155-12.All www.polygene.ch) generated the ALK kinase KO according to our specifications. A gene targeting vector for ALK was designed to generate a floxed allele of ALK with the goal of eliminating exon 20–23. Flipase Recognition Target (FRT)-sites were included in the vector to generate a non-conditional knock-out lacking exon 20–23 (kinase domain). This non-conditional knock-out was employed in these studies. The ALK gene targeting vector thus included an FRT site upstream of exon 19 followed by a LacZ cassette and a loxP site. Upstream of exon 23 a PGK-TK negative selection gene and a PGK-neo positive selection gene (transcribed in the opposite direction of ALK) were flanked by loxP sites. The most upstream loxP site was flanked by an FRT site  in males was assessed daily in ALK mutant mice as well as in wild type animals (n = 10 from P21 to P40 and n = 6 from P41 to P60). This consisted of attempts to manually retract the prepuce with gentle pressure. PPS is testosterone dependent and thus is an indicator of activation of the reproductive axis in males . PubertyMice were briefly anaesthetized with isofluorane and blood samples were collected from the facial vein with Golden rod animal lancet (MEDIpoint) to the BD Microtainer tube with serum separator . Serum sImmediately after sacrifice, the caudal epididymis was removed and placed in a small clean petri dish containing 1ml of PBS pH = 7.4 (n = 4 per age and per genotype). The caudal part was dissected with a sharp sterilized blade into three pieces and squeezed gently with fine forceps to release the sperm, and the suspension was then allowed to settle at 37° C for 30 min . Sperm wTwenty μL of sperm suspension and 20 μL of eosin solution were mixed and vortexed for 2 sec. After, 30 μL of the suspension were recovered and spread on a glass slide. After drying at room temperature, sperm viability was determined: dead and living spermatozoa had red and white colored heads, respectively. A minimum of 200 spermatozoa was counted in triplicate per sample (n = 4 per age and per genotype).C57BL/6N male mice at the age of 7–9 weeks were obtained from Taconic. They were treated orally for 14 days with crizotinib (20 mg/kg) or vehicle  (n = 13 per each treated group). Mice were kept in separate cages and briefly anaesthetized with isofluorane for the drug administration. All handling concerning blood collection and treatment was done with an attempt to minimize animals stress as well as cross-contamination with pheromones. No loss of weight or any other visible signs of animals being negatively affected by the treatment were observed during the whole experiment.Collected organs from 10 animals per age and per genotype were fixed in 4% paraformaldehyde overnight. After two washes in phosphate buffer solution (PBS), testes were dehydrated through an ethanol series and embedded in paraffin . Tissue sections (5–7μm) were mounted on SuperFrost Plus slides . Slides were deparaffinized in xylene, rehydrated through ethanol series and stained with hematoxylin and eosin. Epitope retrieval was achieved by 3 min boiling in sodium citrate 10 mM pH 6.0 at the microwave. After washing with PBS and blocking for 15 min with 5% bovine serum albumin in PBS with 0.1% Triton X-100, samples were incubated with a primary antibody against GATA4 (Santa Cruz). Primary antibody was detected with the appropriate secondary antibody, coupled to horse radish peroxidase (HRP) (Santa Cruz). HRP activity was detected with the chromogenic substrate ImmPACT DAB .For optical projection tomography (OPT) , brains P-values less than 0.05 were accepted to indicate statistical significance.Statistical analyses were performed with GraphPad Prism 6 software. Mann-Whitney and Wilcoxon tests were applied. The ALK kinase KO was generated via homologous recombination and subsequent excision of exon 20–23 . Exons 20–23 correspond to nucleotides 3783–4255 and amino acids 1062–1252 (ALK sequence via NCBI NM_007439). Deletion of these exons disrupts essential structural components of the ALK kinase domain, resulting in a kinase-dead ALK receptor . Animal P = 0.0001; P = 0.001) but not at P60 . At P60 we were unable to detect significant differences between serum samples from wild type and ALK kinase KO males  but not at P60 . However, sperm analyses parameters revealed no significant changes in sperm viability  or P60 .In contrast to wild type mice at P40, ALK kinase KO male mice showed significantly lower levels of serum testosterone  but not at P60 Click here for additional data file.