Anatomical substructures of the human brain have characteristic cell-types, connectivity and local circuitry, which are reflected in area-specific transcriptome signatures, but the principles governing area-specific transcription and their relation to brain development are still being studied. In adult rodents, areal transcriptome patterns agree with the embryonic origin of brain regions, but the processes and genes that preserve an embryonic signature in regional expression profiles were not quantified. Furthermore, it is not clear how embryonic-origin signatures of adult-brain expression interplay with changes in expression patterns during development. Here we first quantify which genes have regional expression-patterns related to the developmental origin of brain regions, using genome-wide mRNA expression from post-mortem adult human brains. We find that almost all human genes (92%) exhibit an expression pattern that agrees with developmental brain-region ontology, but that this agreement changes at multiple phases during development. Agreement is particularly strong in neuron-specific genes, but also in genes that are not spatially correlated with neuron-specific or glia-specific markers. Surprisingly, agreement is also stronger in early-evolved genes. We further find that pairs of similar genes having high agreement to developmental region ontology tend to be more strongly correlated or anti-correlated, and that the strength of spatial correlation changes more strongly in gene pairs with stronger embryonic signatures. These results suggest that transcription regulation of most genes in the adult human brain is spatially tuned in a way that changes through life, but in agreement with development-determined brain regions. Genome-wide measurements of gene expression across the human brain can reveal new principles of brain organization and function. To achieve this, we aim to discover which genes are differentially expressed and in what brain regions. We found that almost all genes in the adult human brain bear a developmental ‘footprint’ which determines their areal expression-pattern based on the developmental ontology of brain regions, while at the same time their spatial expression pattern changes during life. Furthermore, pairs of paralog genes and similar genes with stronger embryonic footprint, tend to be more strongly correlated (or anti correlated) suggesting that their expression is more strongly spatially tuned, and this tuning changes in development. Recent high-resolution genome-wide transcriptome profiling studies allow addressing these questions ..7].During early development, several gene families exhibit distinct spatial expression patterns , includiet al. have shown that regions with similar expression profiles tend to have similar neuronal connectivity patterns [et al. have shown that neuron-specific and astrocyte-specific gene markers show distinct patterns of expression across brain regions [et al. have estimated the spatial densities of 64 cell types in the mouse brain and identified genes with a localized pattern of expression [Zapala and colleagues  studied patterns . Ko et a regions . Grange pression . The queet al. [It is also not clear to what extent expression differences are pronounced between sub regions of the major brain structures. Specifically, the cortex is sometimes viewed as largely homogeneous in various properties, including neuronal density, connectivity patterns and distribution of cell-types, properties which were shown to be consistent across cortical sub-regions ,26. Alsoet al. , where cTo quantify the relation between expression patterns across the adult human brain and the embryonic origin of the corresponding brain regions on a gene-by-gene basis, we analyze two genome-wide mRNA expression datasets. For each gene separately, we computed an index that measures how its expression pattern in the adult agrees with a brain-region ontology based on embryonic brain development. Surprisingly, we found that almost all genes exhibit a spatial expression pattern that significantly agrees with the brain-region ontology. This effect is particularly strong in neuron-specific genes as expected, but also in many genes that serve more generic functions. This suggests that the brain tunes the areal expression pattern of genes in a way that strongly depends on embryonic development, and this holds even for genes that participate in brain-wide functions. Furthermore, pairs of genes sharing related functions tend to be spatially more anti-correlated if their expression pattern agrees with the brain-region ontology.ABA6-2013 and Kang-2011.To characterize distinct gene expression patterns across the adult human brain, we analyzed genome-wide expression measurements from two sources. First, a set of 3702 samples from 6 adult post-mortem human brains , and sechttp://human.brain-map.org) [ABA6-2013 dataset were associated with 414 ontology regions, and those from the Kang-2011 dataset were associated with 16 regions of the ontology  . This onlogy see  Tables. ABA6-2013, where brain regions with a common developmental origin, share similar expression patterns. However, this clustering depends on the joint expression patterns of all genes, and the question remains: which genes and processes contribute to this effect?In the mouse brain, regions that share similar expression patterns group in a way that matches the region ontology ,32. To tBrain-Region Ontology agreement score (BRO-agreement score), calculated as follows. For a given gene, we consider all pairs of tissue-samples, and for each pair we computed two measures of distances. The first, expression distance, is the absolute difference of expression values in the two samples. The second, ontology distance, is the distance between the corresponding regions in the ontology tree. The BRO score for each gene is defined as the spearman correlation between the two distances computed for all tissue pairs. It provides a measure of the agreement between expression difference and ontology distance . Using the triplets score  and Kang-2011 . It also shows the distribution of the baseline random distribution that was generated using permutation test  were from non-cortical regions in Kang-2011, compared to 2187 out of 3702 (59%) in ABA6-2013. See full details in supplementary In the ρ = 0.997 comparing with and without scaling). Similar results were obtained when normalizing to a set of highly-stable genes  and did not have a significant effect on BRO scores . The 3rd and 4th genes in that category were genes coding for serotonin receptors HTR2A and HTR4. Finally, some of the top ranked genes are related to brain-related disorders: G-protein signaling regulator RGS4, serotonin receptor HTR2A and the postsynaptic protein encoder–NRGN are related to schizophrenia. HTR2A, CHH9 are related to autistic disorders.What genes and functions achieve the highest BRO scores? To answer this question, we first tested functional enrichment of gene ontology (GO) categories . We usedTo understand better which genes achieve the highest BRO scores, we further studied gene families and functions that are of particular interest: genes expressed in specific cell types, and genes involved in regulation of brain development.p-value < 10−70,Wilcoxon comparing the median of the distributions of BRO scores of neuron-specific genes, median = 0.33 and the general population of genes, median = 0.11) while oligodendrocytes-specific and astrocyte-specific markers are far less so . These results are in agreement with Ko et al. [et al. showed that genetic markers for neurons and oligodendrocyte are on the opposite ends of the first principal component [First, it has been suggested that genes that are expressed distinctively in specific cell types, contribute significantly to expression differences among brain regions . Specifio et al. ,37,38, wp-value = 10−7) . This suggests that unlike other gene groups discussed above, Hox genes are less involved in regional patterns in the adult brain. These view is also supported by Takahashi et al. which observed that while Hox genes show an expression gradient through the entire adult body, only one third of Hox genes are differentially expressed in brain-specific tissues [ = 10−7)  bottom.  tissues .Finally, we examined Pax genes. These genes are involved in early regionalization of the embryo brain and were suggested to play a role in differentiation and maintenance of specific subsets of cells in the adult brain ,42. It hPAX2, PAX3 and PAX6, obtain significantly large BRO scores . These older genes are also active in signaling pathways and other basic functions in the cell .gene-to-gene variability. In that space, they found that neurons and oligodendrocytes are on the opposite end of the first principal component. Here we address the complementary analysis, looking for the dimensions that preserve the sample-to-sample variability.Expression variability has many contributing factors, including subject-to-subject variability, regional variability and experimental noise. The above results suggest that the variability between brain regions is significant for most genes. But, how large is regional variability compared to other sources of expression variability? To answer this question, we used principal component analysis (PCA) to extract the main axes of variability in the data see . Interesst and 2nd principal components, which together account for 34% of the variance . Second, to further focus on genes with putative similar function, we collected pairs of genes that share the same functional role in cellular pathways, as captured by KEGG. We also required that these gene pairs have a significant sequence similarity and denote this set Kegg-based pairs  on ne pairs . Furtherne pairs . The spa regions .p-value), ρ = 0.36 p-value < 10-42, n = 1496). More surprisingly, when considering anti-correlated gene pairs, pairs with high BRO-scores tend to be more strongly anti-correlated . This effect is not simply due to some pairs having more variable expression across the brain, since the dependency on BRO is significantly stronger  than the dependency on spatial variability . One interpretation of these findings is that the brain tunes the expression of pairs of functionally-related pairs of genes, such that they are expressed differently in brain regions, and that this tuning is in strong agreement with the developmental origin.Next, we compared the strength of spatial correlation of each pair of genes with their combined BRO-agreement scores, and found a strongly significant correlation between the two . Far fewer pairs of Ensembl paralogs exhibit a significant trend (6.8% of the pairs 240/3503).Together with the BRO results, these findings suggest that correlated spatial expression may be formed early in development. To test this hypothesis we computed the spatial correlations for each subject in the p-value of trend < 10−4). It corresponds to a pair of Serotonin receptor genes, HTR2A and HTR1F from two different receptors . Interestingly, their spatial correlation is negative prenatally and is around zero around birth. However, it continuously grows throughout life, reaching high positive correlation at adulthood. This pattern is interesting for several reasons. First, the gradual increase in correlations throughout life is not likely to be caused by changes in cell proportions, since there is a significant change in correlation between childhood and adulthood. Second, other genes in the Serotonin system exhibit different patterns. HTR5A and HTR2C. Here the early embryonic positive correlation is replaced by a negative correlation around birth, which remains quite stable during life. CACNA1A vs CACNA1D two genes coding for Calcium channels, and HTR7 vs. HTR5A, two serotonin receptors. HTR7 is known to be involved in both early and post-natal development [elopment .One possible interpretation of the prevalence of high BRO-agreement scores is that the expression patterns of many are determined early in development, and are preserved through life and in the adult brain. Alternatively, it is also possible that gene-expression changes in a dynamic way through life, but keep following patterns that agree with the embryonic origin of regions. To test these two hypotheses, we quantified the relation between the strength of expression changes of pairs through life, and the BRO scores of the gene pair. We find that the two are positively correlated  have stronger spatial anti-correlation than paralogs in those pathways. Finally, in those pairs of functionally related genes, pairs with higher BRO scores tend to have stronger spatial anti-correlation.The approach we presented has various limitations. Transcriptome data measured from brain tissues involves a mixture of various cell types whose proportions and conditions are not known. Developing demixing approach to infer proportions from the mixture  is an imABA6-2013 dataset, FEZF2 (forebrain embryonic zinc finger protein 2), a transcription repressor involved in specification of subcerebral projection neurons [FEZF2 is believed to play a role in layer and neuronal patterning of subcortical projections and axonal fasciculation and was shown to regulate axon targeting of layer 5 subcortical projection neurons, where axons of FEZF2 deficient mice failed to reach their targets [FEZF2 retains strong areal differences in adulthood, and is strongly expressed in the cortex, less so in the midbrain and the least in the hindbrain . In the s et al. , Fig 2B) regions . ANOVA eThe fact that the expression of most genes in the adult brain is governed by earlier development stages suggest that many studies which deal with regional differential expression should be carefully interpreted. For example, combining samples taken from ontology-distant regions would lead to large expression variance, reflecting the developmental origin of the structures tested. Furthermore, areal differential expression should be measured compared to a baseline expression profile that takes in to account the region ontology.The results in this paper suggest that spatial expression patterns in the adult human brain are controlled in a way that follows the embryonic origin of regions, but at the same time that spatial patterns of related genes may change during development in a correlated way. It remains to be discovered which transcription control mechanisms maintain these distinct areal expression patterns.ABA6-2013) from human.brain-map.org [human.brain-map.org. Specifically, when we analyzed genes with multiple probes, we first computed the expression correlation across regions of each probe, then averaged the correlation scores across all pairs of subjects and chose the probe that was most correlative. Overall, we analyzed 20773 transcripts. The number of samples per donor ranged from 363 to 946, for a total of 3702 tissue samples.We analyzed gene expression data from two sources. First, a set of 3702 microarrays provided by the Allen Human Brain Dataset , we define their tree similarity as the distance (number of edges) between the regions in the ontology hierarchy tree dtree. We define their expression similarity as the absolute difference between the expression values of the two samples for the current gene (i)—dexpression. We computed the two distances over all pairs of tissue samples, and computed the Spearman correlation between the two as the BRO score.The BRO-agreement score was computed separately for each gene as follows. For each pair of samples To generate random scores, we calculate Bro-agreement scores of permutated vectors. Genes with a BRO-score above the top 1% of the permuted scores were considered significant.6 sample triplets  and computed the fraction of times that a triplet is ranked with the same ordering in both the tree and the expression distance measures:We also tested a second ontology-agreement score based on triplet ranking. We randomly selected 10This score gave similar results which are not shown here.To handle biases that could arise from different scales in the samples we also checked a normalized version of the Bro-agreement. In this normalized version we first normalized each sample to zero mean and unit variance and then computed the BRO-agreement. The results were robust to this change and we choose to present the un-normalized BRO-agreement scores. To handle biases that could arise from the number of regions in the ontology, we used two different granularities of the ontology tree. The first uses 16 gross regions and the second uses the entire tree (414 regions).ABA6-2013 dataset, the number of samples per subject is large enough, such that a BRO score can be computed separately for each subject. We then computed a global BRO score of a gene as the average over the 6 individual-subject BRO scores. 94% of the genes were significant compared to random, according to the first score, and 92% according to the second method. For consistency with the Kang-2011 data, the figures use the first method. We report the more conservative estimate of significant genes as 92%.We tested two ways to combine expression measures from multiple subjects into a single BRO score. First, we simply aggregated all samples of all subjects from a given region, and computed the BRO agreement score. Second, in the We computed BRO-scores which uses the fine region-ontology. The upper branches in this ontology are more developmental oriented and the lower branches are more cytoarchitecture-driven. Using this ontology 89% of the genes are BRO significant. The BRO scores of the fine region-ontology and of the coarse 16 region-ontology are very similar with a spearman correlation of 0.98.ABA6-2013 dataset at a coarser resolution of 16 regions. Using this coarse resolution, 95% of the genes were BRO significant. To test if the number of BRO significant genes is sensitive to the number of available samples, we randomly subsampled subsets of size 500 samples, which decreased the fraction of BRO significant genes to 51% ± 2%  is stable across the individual subjects, yielding 89%, 90%, 76%, 91%, 83% and 86%  were significantly differentially expressed across regions , 83% of these genes (17266 out of 20773) were also BRO-significant. 8% of the genes (1719 out of 20773) had BRO-significant scores but not ANOVA-significant, most likely because the BRO index combined weak affects across multiple nodes of the tree.Expression variability of a single gene across regions is sometimes captured by comparing the mean expression level in each region using ANOVA ,6. This p-values reported are under the null hypothesis that samples are drawn from regions which have the same mean expression. We computed ANOVA separately for each human subject and report the average ANOVA score across subjects for each gene. Similar to the BRO scores we performed ANOVA on the fine grained ontology and on the coarse ontology. We then corrected the p-values for multiple comparisons using FDR.For each gene, we computed one-way ANOVA on samples expression levels. The EMX1, MAP2, GRIA2, DLG4, DLG3, NRGN, STMN2, SYT1, CELF4, CELF5, CELF6. For glia markers we used GFAP, MBP, SLC1A2, SLC1A3, DLG4, DLG3, MAP2. To compute the dependence of spatial variability on those markers we fitted a quadratic function for each of the genes separately using a least square loss, and computed the explained variance R2, compared to a constant model.For the genome wide analysis of BRO scores We used the human orthologs of the set of genes characterized by Cahoy et al. , who useEnsembl paralogs and KEGG-based gene pairs. For the first set, we used the paralogs available from Ensembl , and limited to gene pairs that had an Entrez id and were included in synaptic and brain related pathways as described by KEGG. Specifically, these included 17 pathways with KEGG accession numbers 04020, 04724, 04725, 04726, 04727, 04728, 04730, 05010, 05012, 05014, 05016, 05030, 05031, 05032, 05033, 05034, 04080.We studied two sets of gene pairs: KEGG-based gene pairs, we created a set of gene pairs designed to capture functionally-related genes, by collecting gene pairs that reside within the same functional element in KEGG pathway repository. These KEGG elements group together proteins with common functionally and interaction partners. We found these KEGG elements to be usually more functionally-coherent than protein families, and at the same time less specific than protein sub-families. We further required that pairs have sequence similarity above 30% .To find pairs whose spatial correlation has a trend, we fitted a linear regression model with least square loss with age as the predicting variable and spatial correlation as the predicted variable. Significance of the trend was measured based on the F statistic of the explained variance and was FDR corrected for multiple hypotheses.www.genome.jp/kegg). The Hox genes used are: HOXA1, HOXA10, HOXA11, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXB1, HOXB13, HOXB3, HOXB4, HOXB5, HOXB6, HOXB9, HOXC10, HOXC12, HOXC13, HOXC4, HOXC5, HOXC8, HOXC9, HOXD1, HOXD12, HOXD13, HOXD3, HOXD4, HOXD8, HOXD8, and HOXD9. The Pax genes used are: PAX1, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7 and PAX8.We used the set of genes that belong to the human axon guidance pathway. The set was manually curated by KEGG (http://www.tau.ac.il/~elieis/HKG/).A set of 11 genes collected by Eisenberg et al.  and avaiABA6-2013 obtained from six subjects. Samples from the same brain regions were first averaged to create a single profile for each region.We used agglomerative hierarchical with average linkage and Euclidean distance over 3702 samples from ABA6-2013 to compute the covariance matrix of gene expression levels, and then computed the top principal component of the expression covariance matrix.We used all 3702 samples from For each gene, we evaluated how much of its expression variance (over samples) can be explained using two sources of information: The region the sample was taken from and the identity of the subject the sample was extracted from. The explained variance was computed for each gene by fitting linear model using each of these sources of information, and using both of them together.Kang-2011 data with that computed with the ABA-2013 data. We performed the ranked based enrichment analysis using Gorilla (http://cbl-gorilla.cs.technion.ac.il/).For robustness, we combined two BRO-score of each gene by multiplying the BRO-score computed with the We used the gene age-index published by Domazet-Lošo and Tautz [nd Tautz .S1 TableSensitivity to the number of regions in the ontology tree depth was assessed by using two ontologies with different resolutions: A fine-resolution ontology that contained 414 regions and a coarse-resolution ontology of 16 gross regions. Sensitivity to the number of available samples was assessed by computing the BRO score using random subsets of 500 samples.(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 TableMapping the notation of regions in Kang-2011 and in the Allen Institute region ontology ABA6-2013 ,6.(XLS)Click here for additional data file.S4 TableEnrichment was computed using mGH  testing (XLS)Click here for additional data file.S1 FigPearson correlation between expression vectors of all pairs of tissue samples. Samples are ordered first by gross region than by donor. Samples from regions that are close in the developmental region ontology are highly correlated in their expression profile. Within each region, samples are also correlated with other samples from the same donor.(TIF)Click here for additional data file.S2 Fig(A) Each point is a tissue sample. Samples are colored based on the position of the corresponding embryonic region. (B) Colors correpsond to donor identity. A significant fraction of the sample variance across the 3rd and 4th principal components is explained by subject-to-subject variablity.(TIF)Click here for additional data file.S3 Figrd and 4th principal components explain more than 51% of the sample-to-sample variance.The first two principal components caputre 34% of the variance. Adding the 3(TIF)Click here for additional data file.S4 Fig(A) A scatter plot showing BRO-agreement scores for the two datasets. Each light-grey dots corresponds to a single genes . Dark-grey dots correspond to permuted data (see (B) Marginal distribution of BRO scores in the ABA6-2013 dataset. In the ABA6-2013 dataset, 11% of the genes (2207 out of 20773) are BRO-significant in the cortex. BRO score was also computed separately for each subject, using the ABA6-2013 dataset. With these per-subject scores, the number of BRO-significant genes varied considerably across the six subjects , and the correlation of BRO scores between subjects is on average lower . (C) Marginal distribution of BRO scores in the Kang-2011 dataset. The large fraction of BRO-significant genes observed in ABA6-2013 was not found in the Kang-2011 dataset, where the two distributions largely overlap.Color scheme and x-axis scale matche those of data see . (B) Mar(TIF)Click here for additional data file.S5 Fig Neurons and astrocytes receive significantly higher agreement scores than all genes . Oligoodendrocyte genes are in less agreement with region-ontology than the full set of genes. Comparing these BRO scores to a randomized scores we find that 45% of the neuronal markers are cortex-BRO significant, 69% of the astrocytes markers are cortex-BRO significant and that 7% of the oligodendrocytes markers are cortex-BRO significant;).  Axon-guidance genes receive higher scores than genes on average . Hox genes are less in agreement with region-ontology than the full set of genes (still significantly larger than random). PAX2 and PAX6 obtain high BRO scores.Color scheme and x-axis scale match those of (TIF)Click here for additional data file.S6 Fig(A) The mean expression levels of the FEZF2 within different region in the human brain (the color scheme as consitant with that of (B) We embed the samples in a 3D space using its MRI standartize location where the color of each sample shows the expression level of FEZF2. The scatter shows a transiation from high expression levels in the cortex to lower expression levels in the inner brain structures and to the hindbrain.FEZF2 shows a clear transtion of its expression levels. The samples from the cortex show high experssion values where the samples midbrain has less and the samples of the hindbrain has the least expression. (TIF)Click here for additional data file.S7 Figp-value < 0.01) is stable when computed for each subject separately: 89%, 90%, 76%, 91%, 83% and 86%).The percent of BRO-significant genes ((TIF)Click here for additional data file.S8 Fig(A) Distributions of explained variance across genes, as explained by region, subject or both. (B) The joint distribution of both the explained variance from region and from subject identity.The joint information of subject id and region id explains almost half of the sample variance. For each gene, we represented the identity information as 1-hot-vectors, and computed the explained sample-variance by fitting a linear model (TIF)Click here for additional data file.S9 Fig(A) We computed the developmental trend for each pair of genes from the KEGG-based set. We found that BRO-agreement scores are (weakly but significantly) positively correlated with having a significant trend. Each dot corresponds to one brain related sequence-similar pair. 17% of pairs (257/1496) had FDR-corrected significant (q<0.01) linear trend of their spatial correlation, as illustrated in (B) Similarly, we found that the developmental trend of paralog pairs which are brain related is also positively correlated with the BRO score. 6.8% of the brain related paralog-pairs show a significant trend (240/3503).Trend significance of change in spatial correlation through life was quantified using the standard F-test comparing residual of a linear regression model with a constant model. Pair BRO score is the minimum BRO scores of the two genes in the pair. (TIF)Click here for additional data file.S10 Fig(A) 2D scatter of BRO scores for genes with a matching homolog in mouse  2D heat map of the joint distribution of BRO-scores for mouse and human. (C) The distribution of BRO-scores in mouse and a random baseline, showing that the fraction of BRO significant genes is smaller in the mouse dataset (30%) than in humans.la et al.) and hum(TIF)Click here for additional data file.S11 Fig(A) The Allen ontology used for the human analysis. (B) The Allen ontology used for the mouse analysis. Both follow ontologies first devide the brain into the 5 embrionic vesiceles and then go into more detailed regionalization. The leaf regions are not the same since the data was gathered in different experiments each with a unique focus.(TIF)Click here for additional data file.S1 TextBRO in the human cortex and a comparsion between the BRO in human and in mouse.(DOCX)Click here for additional data file.S1 DataThe table containt: Gene symbo, Gene Entrez-ID, BRO score that is the product of the BRO of ABA6-2013 and Kang-2011, BRO score computed from the ABA6-2013 dataset, BRO score computed from the Kang-2011 dataset. A mark per gene which indicates it involvment in one of these 11 classes: cell-cell signaling, synaptic transmission, neuron differentiation, neuron projection development, generation of neurons, axon development, neuron development, schizophrenia, autistic disorder, seizures,epilepsy, substance related disorders.(CSV)Click here for additional data file.


Grape seed proanthocyanidin extract (GSPE) has been extensively reported to possess a wide range of beneficial properties in multiple tissue damage. Previous studies have shown that exhaustive exercise-induced fatigue associates with oxidative stress injury, inflammatory response, and mitochondrial dysfunction.The aim of this study is to investigate the anti-fatigue effects of GSPE in mice and explore its possible underlying mechanism.The mouse model of exhaustive exercise-induced fatigue was established by using the forced swimming test, and GSPE was orally treated for successive 28 days at 0, 1, 50 and 100 mg/kg/day of body weight, designated the control, GSPE-L, GSPE-M and GSPE-H groups, respectively.The presented results showed that treatment of GSPE at a dose of 50 and 100 mg/kg/day of body weight significantly relieved exhaustive exercise-induced fatigue, indicated by increasing the forced swimming time. In addition, treatment of GSPE significantly improved the creatine phosphokinase and lactic dehydrogenase, as well as lactic acid level in exhaustive swimming. For underlying mechanisms, treatment of GSPE had anti-fatigue effects by promoting antioxidant ability and resisting oxidative effect, as represented by increased total antioxidative capability levels, enhanced superoxide dismutase and catalase activities, and ameliorated malondialdehyde levels. Furthermore, treatment of GSPE significantly inhibited the activity of tumor necrosis factor-α and interleukin-1β, which suggested that its protective effects on exhaustive exercise-induced fatigue may be attributed to inhibition of inflammatory response. Last but not the least, treatment of GSPE significantly improved succinate dehydrogenase and Na+-K+-ATPase levels to enhance mitochondrial function during exhaustive swimming-induced fatigue.These results proved that treatment of GSPE possessed the beneficial properties of anti-inflammatory, antioxidant, and mitochondrial protection to improve exhaustive exercise, which suggested that GSPE could be used as an effective functional food to delay fatigue. Exercise involves human physiological changes , 2. ReguDendrobium officinale extract delays fatigue effects through enhancing glutathione peroxidase (GSH-Px) level and inhibiting malondialdehyde (MDA) content in serum  were obtained from the Animal Center of Hunan Normal University . All mice were grown at a standard laboratory condition . The mice were randomly divided and housed in groups of four animals per cage. They were provided with free access to standard diet and distilled water. In this study, all animal experiments were inspected according to the Animal Care Committee of Hunan Normal University.GSPE was purchased from Tianjin Jianfeng Natural Product R&D Co., LTD . Thirty-two male ICR mice were randomly assigned to four experimental groups (8 mice per group) for GSPE treatment: (1) control group, (2) low-dose group (GSPE-L), (3) medium-dose group (GSPE-M), and (4) high-dose group (GSPE-H). Three GSPE intervention groups were treated with GSPE at a dose of 1, 50 and 100 mg/kg/day of body weight, respectively. The control group mice were administrated at the same dosage volume of physiological saline equivalent to individual body weight. GSPE supplementation was given by oral gavages once a day for 28 successive days.The mice were challenged by the forced swimming test to establish exhaustive exercise-induced fatigue. One hour after the final dosing, all mice underwent weight-loaded swimming test with a lead (approximately 5% of each mouse’s body weight), which was attached to their tails root. All mice were trained individually in the same condition   were measured according to the manufacturer’s protocol.After the swimming exercise, skeletal muscle tissues were immediately acquired and stored at −80°C. In our research, skeletal muscle tissues were carefully grounded and centrifuged. Then the serum and supernatant in skeletal muscle were used for oxidative stress assay. Furthermore, the level of antioxidant enzymes and oxidative indicators  were determined by spectrophotometer according to the manufacturer’s protocol.The level of inflammatory cytokines  were examined to assess inflammatory responses in the serum and supernatant of skeletal muscle by ELISA. The absorbance value was measured at 450 nm to analyze inflammatory parameters according to the manufacturer’s instructions.The energy metabolism parameters SDH and Na+K+-ATPase  in skeletal muscles were determined to assess mitochondrial function by detection kits according to the manufacturer’s protocol.p value of less than 0.05 was considered statistically significant.All results were displayed as mean ± standard deviation (SD). The data was statistically analyzed using SPSS 16.0 software. Statistical significance between groups was demonstrated with one-way analysis of variance (ANOVA). A The body weight of mice in our experiment is presented in The effects of GSPE on the weight-loaded swimming time are presented in The effects of GSPE on the blood biochemical parameters are presented in The effects of GSPE on oxidative stress in both serum and skeletal muscles of mice are presented in Inflammatory cytokines, including TNF-α and IL-1β, in both serum and skeletal muscles of mice were measured to evaluate the effects of GSPE on inflammatory response. As shown in The SDH and Na+-K+-ATPase activity were measured to evaluate the effects of GSPE on mitochondrial function in skeletal muscles. As shown in Grape is a popular fruit throughout the world. Nowadays, proanthocyanidin, as major flavonoids in grape seed, is used as a nutritional supplementation worldwide for its medicinal and health effects , 27. It LA is formed as a metabolic by-product in skeletal muscles and released into the bloodstream. However, excess LA is harmful for skeletal muscles during intense exercise, which may result in fatigue . TherefoExhaustive exercise leads to oxidative stress, which can destabilize the balance between oxidation and antioxidation systems and play an important role in fatigue-related disorders , 35. It Exhaustive exercise has been reported to cause inflammatory response which can contribute to fatigue. TNF-α, IL-6, and IL-1β are often used as an index to evaluate the inflammatory response. Glycine-leucine and leucine-glycine dipeptides, as primary peptides of fermented porcine placenta, significantly decreased IL-6 and IL-1β serum levels to display an anti-fatigue effect . FurtherMitochondrion is the place of adenosine triphosphate (ATP) generation. The human body needs more ATP to maintain muscle contraction and movement during exercise. However, exhaustive exercise can lead to damaged mitochondria that further cause skeletal muscles damage and fatigue . SDH plaTo our knowledge, this research is the first to investigate the effect of GSPE supplementation on exhaustive swimming-induced fatigue in mice. Treatment with GSPE improved the fatigue by prolonging the TTE in the forced swimming test. Furthermore, several biochemical markers for fatigue, including LA, LDH, and CK, were significantly alleviated by the administration of GSPE. The anti-fatigue property may be associated with the amelioration of inflammatory response, suppression of oxidative stress, and improvement of mitochondrial function. According to the data, these findings indicate that GSPE is a natural and safe ingredient and dietary agent in excessive exercise-induced fatigue.


In the United States, prostate cancer is the most common cancer and second leading cause of cancer-related deaths in men. It is estimated that 220,800 new cases of prostate cancer will have been diagnosed in 2015, with an estimated 27,540 deaths .Although prostate cancer is typically diagnosed when it is localized and considered curable, approximately 4% of patients have metastatic cancer at diagnosis. For androgen-sensitive metastatic disease, the current standard of care is continuous androgen-deprivation therapy . HoweverOver 90% percent of patients with metastatic CRPC develop bone metastases . As the Radiopharmaceuticals target bone by binding to hydroxyapatite, the main component of inorganic matrix in bone that is linked with cancer cells found in osteoblastic lesions. Beta-emitting radiopharmaceuticals (strontium-89 [Sr-89] and samarium-153 [Sm-153]) are approved by the US Food and Drug Administration (FDA) for palliative treatment of bone metastases in patients with metastatic CRPC. Fifty percent of patients show symptomatic relief with treatment but with limited duration of response . Due to Radium-223 is the only currently available alpha-emitting radiopharmaceutical. The penetration of alpha particles is approximately 40–100 m (2–10 tumor cell diameters), whereas Sr-89 and Sm-153 have a tissue penetration of approximately 2.4 and 0.6 mm, respectively Radium-223 is a calcium mimetic that becomes part of the bone volume over time. Radium-223 forms complexes with hydroxyapatite at areas of increased bone turnover, including bone metastases . Radium-In metastatic CRPC, the majority of bone metastases are characterized by blastic formation of new bone, although there are components of lytic bone destruction seen as well . Radium-To date, there have been several studies evaluating the efficacy of radium-223 for the treatment of bone metastases in the setting of metastatic CRPC. The largest of these studies is the ALSYMPCA trial, an international, prospective, double-blind, placebo-controlled phase III study in adult males with metastatic CRPC. Patients were randomized in a 2:1 fashion to receive radium-223 plus best standard of care (BSC) or placebo plus BSC . InclusiA total of 614 patients received IV radium-223 at a dose of 50 kBq/kg every 4 weeks, and 307 patients were randomized to receive placebo. The study investigators found a statistically significant difference in the primary endpoint, median overall survival (OS). In the updated analysis, patients who received radium-223 plus BSC had a longer median OS, 14.9 months, than patients who received placebo plus BSC, 11.3 months . There was a 30% reduction in the risk of death in patients treated with radium-223 and BSC vs. patients receiving placebo and BSC .p < .001). Patients receiving radium-223 plus BSC also had a significantly prolonged time to an increase in the total alkaline phosphatase level and time to an increase in the prostate-specific antigen (PSA) level. Lastly, a significantly higher proportion of patients receiving radium-223 plus BSC had a response according to the total alkaline phosphatase level and normalization of this level  defined as an increase in the FACT–P total score by ≥ 10. There was also a statistically significant mean change in the FACT–P total score in patients receiving radium-223 plus BSC, –2.7 vs. –6.8  assessment utilizing the FACT–P  total score. A significantly higher percentage of patients who received radium-223 plus BSC had a meaningful improvement in QOL during the period of study drug administration, 25% vs. 16%  per kilogram of body weight IV every 4 weeks for 6 doses. Radium-223 should be discontinued if dose delays occur due to toxicity, and the dose is held for > 8 weeks .Radium-223 is the first radiopharmaceutical approved by the FDA for the treatment of bone metastases with a significant improvement in median OS. Due to the unique nature of radium-223, the potential for toxicity, and the need for appropriate patient selection, advanced practitioners will need to understand its place in therapy.Radium-223 has been incorporated into the National Comprehensive Cancer Network (NCCN) and the American Society of Clinical Oncology (ASCO) metastatic CRPC treatment guidelines as an option for patients with symptomatic bone metastases . In men Radium-223 must be administered by an appropriately licensed facility and is usually given either in nuclear medicine or radiation therapy. Important practical considerations for prescribers using radium-223 include ensuring appropriate hematologic evaluation is performed at baseline and prior to each scheduled dose. In addition, providers need to ensure that patients and caregivers receive appropriate counseling on maintaining adequate hydration and radiation safeguards . More inThe cost of radium-223 can be considerable, with the average wholesale price of approximately $12,220 per dose for a 70-kg patient (assuming 0 days from reference date; As previously stated, radium-223 is commonly used with androgen-deprivation therapy. However, there are no data to support its use in combination with other agents, and due to the potential for increased myelosuppression, other concomitant therapy should only be administered in the setting of a clinical trial . DenosumFurther studies have been requested by the FDA to assess long-term safety as well as optimal dosing. Additional studies may evaluate the efficacy of radium-223 in patients with bone metastases from other tumor types. Studies that combine radium-223 with docetaxel, abiraterone, or enzalutamide are ongoing. Additionally, further studies are needed to evaluate QOL and cost-effectiveness of the available agents and combinations. Therefore, optimal sequencing of medications for metastatic CRPC is unknown, and therapeutic choices should be made based on individual patient characteristics, cost, and patient preference.Treatment options that specifically target bone metastases in patients with metastatic CRPC are needed, as bone metastases are associated with significant morbidity and mortality. Radium-223 represents the first radiopharmaceutical agent with a demonstrated OS benefit. With its manageable toxicity profile, particularly its lack of increased hematologic toxicity vs. placebo, radium-223 provides oncology advanced practitioners with an FDA-approved option for patients with metastatic CRPC and bone metastases.


Cancer outcomes differ depending on where treatment is received. We assessed differences in outcomes in long-term breast cancer survivors at a specialty care hospital by location of their initial treatment.We retrospectively examined a cohort of women diagnosed with invasive early-stage breast cancer who did not experience recurrence for at least 5 years after the date of diagnosis and were evaluated at The University of Texas MD Anderson Cancer Center between January 1997 and August 2008. The location of initial treatment was categorized as MD Anderson (MDA-treated) or other (OTH-treated). Outcomes analyzed included recurrence-free survival (RFS), distant relapse-free survival (DRFS), and overall survival (OS). The Kaplan-Meier product-limit method was used to compare outcomes between the two groups. Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI).We identified 5,091 breast cancer survivors (median follow-up 8.6 years), of whom 89.1% were MDA-treated. The 10-year OS, RFS, and DRFS rates were 90.9%, 88.4%, and 89.0% in the MDA-treated group and 74.3%, 49.8%, and 52.7% in the OTH-treated group, respectively. We observed worse outcomes in the OTH-group in both the univariate analysis and the multivariable analysis .Long-term breast cancer survivors who initiated their treatment at MD Anderson had better outcomes. Location of initial treatment could be an independent risk factor for survival outcomes at specialty care hospitals. This analysis has limitations inherent to retrospective observational studies such as other unmeasured variables may be associated with worse prognosis. The impact of the location or hospital setting where a cancer patient receives treatment on the patient’s health outcomes has been studied with great interest owing to concerns about cost and quality of care –3. MultiBreast cancer (BC) survivors are still at risk of disease recurrence and death 5 years after diagnosis . TherefoThis retrospective cohort consisted of women diagnosed with invasive early-stage (I-III) BC who had presented to The University of Texas MD Anderson Cancer Center between January 1997 and August 2008 and had not experienced any recurrence for at least 5 years after the date of diagnosis. This cohort was identified from the Breast Cancer Management System database housed in the Department of Breast Medical Oncology at MD Anderson, which contains records for all BC patients who have presented to MD Anderson since January 1, 1997. For the purpose of this study, the BC survivors were categorized into one of two groups, based on the location where initial treatment was received: 1) those who had received their initial treatment—chemotherapy, surgery, or radiation—at MD Anderson (MDA-treated) or 2) those who had received their initial treatment at a facility other than MD Anderson (OTH-treated) but had presented to MD Anderson at some point in their care for any reason, including but not limited to second opinions or transfer of care to MD Anderson. We hypothesized that survival outcomes would differ between these two groups of BC survivors. Keeping in mind that the inclusion of survivors presenting to MD Anderson 5 years or more after the initial BC diagnosis could introduce bias by design if a substantial number of them presented with disease recurrence, we also conducted a separate analysis excluding such survivors.A BC survivor was considered MDA-treated if 1) she had been diagnosed with invasive BC within 180 days before her initial consultation visit to MD Anderson with one of the BC subspecialists , and 2) she had received initial treatment for BC at MD Anderson. This specific categorization has been used previously by others . RFS wasWe collected demographic factors such as age at diagnosis, year of diagnosis, race/ ethnicity (self-reported), and menopausal status at diagnosis. We classified the tumors as hormone receptor-positive if the tumor was estrogen receptor (ER)- or progesterone receptor (PR)-positive and hormone receptor-negative if the tumor was both ER- and PR-negative, as determined by immunohistochemistry staining using institutional laboratory thresholds. Human epidermal growth factor receptor 2 (HER2) status was determined by immunohistochemistry or fluorescence in situ hybridization. We grouped the tumors into one of three categories: 1) hormone receptor-positive (ER- or PR-positive and HER2-negative or unknown), 2) triple-negative (ER- and PR-negative and HER2-negative or unknown), or 3) HER2-positive (independent of ER or PR status). Tumor stage was determined using the American Joint Committee on Cancer (AJCC) 5th edition staging method for patients diagnosed before 2003 and the AJCC 6th edition for patients diagnosed in 2003 or later. Tumor grade and histologic findings were extracted from biopsy and surgical pathology reports. We captured treatments received, such as chemotherapy, surgery, radiation therapy, and endocrine therapy, by reviewing the patient’s medical record. We determined the most recent vital status of each patient using data from the Tumor Registry at MD Anderson and from medical records. We received institutional review board approval to conduct this study (PA13-0424).t test, as appropriate. We used the Kaplan-Meier product-limit method to estimate the survival functions. The survival function provides the probability of observing a survival time greater than or equal to a specified time. Without imposing any parametric assumption, Kaplan-Meier estimator of survival function is obtained by multiplying a sequence of conditional survival probabilities with information from both uncensored and censored observations. We used log-rank statistic to test the equality of survival functions by group. The log-rank test compares the observed and expected number of events across all failure time points. Cox proportional hazards regression was used to estimate the effect sizes of covariates on the hazard of failure, where the hazard function measures the instantaneous rate of an event to occur, given the individual has survived till that time point. We used univariate Cox proportional hazards regression models to evaluate the crude associations between the main exposure of interest  and potential confounding variables and the outcomes of interest. Variables that showed significant association in the univariate log-rank test were considered potential confounders and were entered in the multivariable Cox proportional hazards model. A backward selection process was used and significant variables were retained in the final model. We used a complete case analysis to handle missing values. All statistical tests were performed with a two-sided significance level of 0.05. Statistical analyses were performed using SAS software, version 9.4 , and STATA software, version 12 .Demographic characteristics were summarized and compared using chi-square analysis or the We identified 5,091 female long-term BC survivors with a median follow-up period of 8.6 years, of whom 4,534 (89.1%) were MDA-treated (median follow-up period 8.6 years) and 557 (10.9%) were OTH-treated (median follow-up period 8.9 years). In the OTH-treated group, 131 (23.5%) did not receive their initial treatment for BC at MD Anderson despite having a consultation visit at the institution within 180 days after diagnosis, likely representing those who presented for a second opinion; 195 (35.0%) had a consultation visit at MD Anderson at 180 or more days after diagnosis, but did receive some treatment at MD Anderson; and 231 (41.5%) neither received their initial treatment at MD Anderson nor visited MD Anderson within 180 days after diagnosis.The demographic and clinical characteristics at diagnosis for the overall cohort and by group are presented in We observed a statistically significant difference in vital status between the two groups; 29.3% of the OTH-treated group (median follow-up period 8.6 years), compared with only 7.6% of the MDA-treated group (median follow-up period 9.0 years), had died at the time of our analysis (p < 0.01). We also observed a higher rate of death as the first event in the OTH-treated group (10.2%) than in the MDA-treated group (5.3%). Distant disease recurrences were more common than local recurrences (5.8% compared with 1.6%) in the overall cohort, as well as within each group. Specific survival outcomes and recurrence events are detailed in Univariate Cox proportional hazards regression analysis showed a significantly increased risk of death (OS: hazard ratio (HR) = 3.3, 95% confidence interval (CI) = 2.7–4.0) and recurrence  in the OTH-treated group compared with the MDA-treated group. When adjusted for selected confounding variables in the multivariable Cox proportional hazards model , these risks remained for the OTH-treated population . In the sensitivity analysis, in which patients who presented at MD Anderson 5 years or more after their initial BC diagnosis were excluded, the differences in outcomes between the groups remained statistically significant in the multivariable analysis .In this cohort of long-term (>5 years) BC survivors, we observed significant differences in demographic, clinical, pathologic, and treatment characteristics, as well as in RFS, DRFS, and OS outcomes, between the MDA-treated group and the OTH-treated group. These findings suggest that within a specialty care hospital, those initially treated outside of the specialty care hospital may have different characteristics and are likely to have poorer outcomes than those initially treated within the hospital, suggesting that location of initial treatment could be an independent risk factor for survival and recurrence.BC survivors in the OTH-treated group had worse baseline disease prognostic factors and poorer event-free survival outcomes. Age at diagnosis, race/ethnicity, tumor stage, and receptor status are well-established prognostic factors in BC ,11 and wThe OTH-treated group was heterogeneous, consisting of some survivors who presented to MD Anderson at least 180 days after their initial BC diagnosis and others who presented to MD Anderson within 180 days but did not receive their initial treatment at MD Anderson. In fact, around 50% of the OTH-treated group presented to MD Anderson more than 1 year, 38% presented more than 2 years, and 31% presented more than 5 years after their initial BC diagnosis. Some BC survivors from this group could have presented to MD Anderson for a second opinion, and others may have relocated to Houston and eventually became part of the Breast Cancer Survivorship Clinic for their follow-up visits although they started and completed their treatment somewhere else.The consistently worse outcomes in the OTH-treated group could potentially arise from several factors, such as transferring care to MD Anderson because of poor disease prognosis. For instance, the proportion of stage III disease was higher in the OTH-treated group than in the MDA-treated group (16.9% compared with 8.7%). We speculate that some of the survivors in the OTH-treated group who presented to MD Anderson more than 180 days after diagnosis could have had a delay in the initiation of their treatment. However, we do not have enough information to confirm this. Treatment delay is known to be associated with poor survival outcomes in BC . Also, dWe were interested in the proportion of survivors who had presented to MD Anderson 5 years after their initial BC diagnosis because this group of individuals either presented to MD Anderson to establish follow-up care or sought treatment at MD Anderson owing to late (>5 years) disease recurrence. However, inclusion of survivors in the OTH-treated group who presented to MD Anderson with disease recurrence would bias the estimates of event-free survivals. Hence, we performed a sensitivity analysis after removing individuals who presented to MD Anderson more than 5 years after diagnosis, and this analysis still showed significantly worse outcomes in the OTH-treated group compared with the MDA-treated group. We noticed that patients in the OTH-treated group had a longer follow-up period, but there were only four patients in the OTH-treated group who had longer follow-up periods than the maximum follow-up period of the MDA-treated group. The differences in the outcomes between the groups were likely not altered by these few observations.Heterogeneity in patient characteristics and outcomes within large single-center academic hospitals owing to referred patients has been reported before. Kokmen et al  comparedIn another investigation, Riggs et al  investigThe current study has some limitations, including those inherent to retrospective observational studies. For example, other unmeasured factors could be associated with worse prognosis in the OTH-treated group. In addition, the composition of patients who present to MD Anderson for treatment may not be the same as that of the other specialty care hospitals; therefore, the results of this study may not be generalizable to other specialty care hospitals. In our study, there was an imbalance in the sample sizes between the two groups. However, the findings remained consistent across all analyses despite this imbalance.In summary, our results indicate that the survival outcomes of MDA-treated long-term BC survivors are better than those of OTH-treated patients. These differences in outcomes among patients within a single specialty care hospital are likely due to multiple factors, and further investigation is needed to establish the underlying causes.S1 Data(XLS)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Study objectives were to evaluate the Multiple Sclerosis Impact Scale (MSIS-29) and explore an optimized scoring structure based on empirical post-hoc analyses of data from the Phase III ADVANCE clinical trial.ADVANCE MSIS-29 data from six time-points were analyzed in a sample of patients with relapsing–remitting multiple sclerosis (RRMS). Rasch Measurement Theory (RMT) analysis was undertaken to examine three broad areas: sample-to-scale targeting, measurement scale properties, and sample measurement validity. Interpretation of results led to an alternative MSIS-29 scoring structure, further evaluated alongside responsiveness of the original and revised scales at Week 48.RMT analysis provided mixed evidence for Physical and Psychological Impact scales that were sub-optimally targeted at the lower functioning end of the scales. Their conceptual basis could also stand to improve based on item fit results. The revised MSIS-29 rescored scales improved but did not resolve the measurement scale properties and targeting of the MSIS-29. In two out of three revised scales, responsiveness analysis indicated strengthened ability to detect change.The revised MSIS-29 provides an initial evidence-based improved patient-reported outcome (PRO) instrument for evaluating the impact of MS. Revised scoring improves conceptual clarity and interpretation of scores by refining scale structure to include Symptoms, Psychological Impact, and General Limitations.ADVANCE . The Multiple Sclerosis Impact Scale (MSIS-29) is a PRO developed in 2001 to assess multiple sclerosis (MS) according to two scales: Physical Impact and Psychological Impact.3 The MSIS-29 has been used in many different trials, including ADVANCE.5Growing interest in patient experiences of conditions like relapsing–remitting multiple sclerosis (RRMS) has led to increased interest in patient-reported outcomes (PROs).6 Efficacy endpoints included adjusted annualized relapse rate (ARR) and confirmed disability progression (CDP), and MSIS-29 was an exploratory endpoint. Significant ARR and CDP treatment effects were demonstrated at Week 48 in both intervention groups compared with placebo.6 No significant treatment effects were demonstrated on the MSIS–29.The pivotal ADVANCE study compared the efficacy and safety of subcutaneous peginterferon 125 mcg beta-1a dosed every two and four weeks with placebo in patients with RRMS.5 A post-hoc analysis revealed that 12-week CDP was associated with a six-point worsening of the Physical Impact scores in the placebo group (p < 0.0001) relative to a 1.9-point worsening (p = 0.044) in the peginterferon beta-1a every-two-weeks group. Recent relapse (occurring within the past 29 days) was associated with a 10-point worsening of the Psychological Impact scores in the placebo group (p < 0.0001) relative to a 3.5-point worsening in the peginterferon beta-1a every-two-weeks group (p = 0.031).5Within-scale changes from baseline indicated a significant improvement at Week 48 across all three treatment groups for Psychological Impact scores, and a worsening in Physical Impact scores .8 measuring complex unobservable variables through self-reported questionnaires.9 A well-designed PRO should be reliable and valid, and care should be taken to ensure the total score of the scale appropriately reflects the patient experience.11 There are three main psychometric paradigms for developing and evaluating rating scales: Classical Test Theory (CTT),12 Rasch Measurement Theory (RMT),13 and Item Response Theory (IRT).14 A detailed comparison of these paradigms is presented elsewhere displaying the sophisticated and extensive analysis potential of the RMT.8MSIS-29 and other PROs are valuable rating scales in evaluating treatment benefits from a patient’s perspective,12 Item questionnaires were generated through patient interviews (with primary progressive (PPMS), secondary progressive (SPMS), and RRMS patients), expert opinion, and literature review.3 Psychometric properties and responsiveness were evaluated in an independent sample3 and subsequently evaluated using CTT methods by several studies.1517 However, in order to further assess the validity of the MSIS-29, it is important to validate the scale through an independent psychometric paradigm.The MSIS-29 was developed in accordance with CTT criteria.18 An RMT of the MSIS-29 in a small sample (n = 92) of SPMS, RRMS, and PPMS patients was largely supportive of its measurement properties but revealed issues with its response scale, item fit, and coverage of patients with lower psychological impact.19 Two additional studies utilized portions of RMT analysis to assess item stability across participants with differential depression levels16 and patient and proxy responders,20 providing supporting evidence for MSIS-29. However, to date there have been no RMT analyses of the MSIS-29 conducted in a large sample of patients with RRMS.The RMT offers a mathematically testable model articulating a priori criteria used to verify measurement properties or to expose and explore anomalies of any rating scale empirically.8 The current RMT psychometric analysis enables extensive assessment of sample-to-scale targeting within RRMS patients whose levels of disability are potentially different from those of the patients’ used for the development and validation of the MSIS-29. Additionally, RMT allows for the optimization of the scoring structure and interpretation with the provision of interval-level scores.The objective of this study was to evaluate measurement properties of the MSIS-29 within ADVANCE in accordance with RMT criteria. The RMT is superior to the CTT because it has interval rather than ordinal scoring, separation of item and person parameters, non-sample-dependent scale properties , and the potential for individual-level measurement.4 ADVANCE recruited patients aged between 18 and 65 with a maximum 5.0 baseline Expanded Disability Status Scale (EDSS) score.21ADVANCE was a two-year, randomized, double-blind, parallel-group, Phase 3 study, with a one-year placebo-controlled period comparing peginterferon beta-1a 125 mcg administered every two or four weeks versus placebo in patients with RRMS. Results from the study are described in detail elsewhere.Protocol for the ADVANCE study was approved by the institutional review board at each site, and the study was conducted according to International Conference on Harmonization Guidelines for Good Clinical Practice and the Declaration of Helsinki. Every patient provided written informed consent before entering the study.16 Items are scored on five-point Likert-like scales, with higher scores indicating greater impact of MS on this domain.The MSIS-29 is a disease-specific PRO that measures the Physical and Psychological Impact of MS via two scales of 20 and 9 items each.22 was used to conduct the RMT and IBM SPSS 21.023 responsiveness analyses. Data from eight different time-points were stacked to increase sample size for the psychometric analysis.Data analysis followed three stages. First, psychometric evaluation of the MSIS-29 in line with RMT was performed. A review of these findings led to conceptual restructuring of the MSIS-29 measurement model, properties of which were evaluated using RMT at a second stage. Finally, the responsiveness of the original and rescored scales was examined. RUMM203024RMT analysis compares observed data against the stringent criteria of the Rasch model with the broad aims described below.26Sample-to-scale targeting concerns the match between the range of Physical or Psychological Impact measured by the MSIS-29 items, and the distribution of impact measured in the sample, subsequently influencing interpretation of all other RMT analyses. Person and item locations are plotted against the same metric, and their relative distributions are assessed.27 Thresholds represent the point at which the probability of endorsing two adjacent response categories is equal.Greater changes in MSIS-29 scores signify more impact. It is therefore expected that the higher the impact of a responder, the higher the response category to be endorsed. Response thresholds are expected to be ordered in a successive manner along the measurement continuum.27 Using a rule of thumb, fit residual  estimates for each item should be within ± 2.5. Chi-square tests assess the difference between each item’s mean observed and expected scores within certain class intervals of the trait being measured. Item characteristic curves (ICCs) display this relationship graphically, providing context for interpreting the magnitude and pattern of numerical fit statistics.RMT expects scale items to be cohesive and work well together clinically and statistically when summed up to a single total score.28 as they reflect >9% of shared variance.RMT expects that items should not be dependent on or biased by each other so as to not artificially inflate reliability. We assessed the degree of “local dependence” among scale items by examining item residual correlations. Residual correlations >0.30 warrant further examination,27 The PSI is a numerical indicator ranging from 0 to 1, computed as the ratio of error-corrected person variance relative to the total person variance,29 with higher values indicating greater detection of reliable differences.Scale items are expected to detect differences in levels of impact within a sample and changes over time. Within RMT, the person separation index (PSI) is calculated to assess this.18 This is assessed through person fit residual, with reference to the “rule of thumb,” expecting 99% of the sample to produce a fit residual between −2.5 and 2.5. Fit residuals outside this range indicate problematic measurements and questionable measurement validity.27Similar to item responses, it is important to assess whether the measurement of each person’s total score is in line with RMT expectations.30 and this has implications when interpreting findings. RUMM2030 plots raw scores against estimated interval measurements, which can be used to provide a subsequent transformation on an interval 0–100 score for each scale.The MSIS-29 total score is ordinal, computed through the summed total of individual Likert-like items rather than an equal-interval measure of Physical or Psychological Impact. It is important to assess the extent to which ordinal raw scores approach interval measurement; one point on an ordinal scale is not necessarily the same across the breadth of the scale,31 and standardized response mean (SRM),32 relative efficiency using paired samples t-tests,33 and relative precision using one-way analysis of variance (ANOVA).34The ability of MSIS-29 scales to detect change at Week 48 was examined and compared. To increase consistency in this comparison, original and restructured scales were anchored on the same overarching scale. Interval level 0–100 transformed scores were used, computed on the basis of RMT-produced interval logit for total raw scores. Responsiveness was examined using four standard indicators: Cohen’s effect size (ES)Data from a total of 1509 people with RRMS at eight time-points are presented in MSIS-29 scales demonstrated sub-optimal targeting, as the range of impact measured by scale items covered only 58% of those measured in the sample. Physical Impact  also demr = 0.40), suggesting dependency between item responses for “grip things tightly” and “carry things.”All 29 items displayed ordered thresholds, indicating that response categories worked as intended. Item fit residuals for 80% of physical and 89% of psychological items fell outside the recommended range; 15% and 11% failed the adjusted Chi-square criteria, respectively. ICCs reflected marginal fluctuation of observed scores from the Rasch-expected scores, suggesting all four items slightly under-discriminated impact . One itePSIs were 0.91 and 0.87, indicating individuals in the sample were separated adequately by MSIS-29 items. Person fit residuals indicated significant misfit for both scales, with 22% of person measurements for Physical and 19% for Psychological Impact falling outside the recommended range, suggesting problematic measurement. The relationship between raw total scores and interval logit metric was S-shaped for both scales, indicating that a one-point change in raw score is associated with a variable rate of change in the impact interval measurement; variability was highest at the two ends and lowest in the center .Figure The MSIS-29 item content was reviewed by a multidisciplinary team that included neurologists, psychologists, and health measurement experts, and three re-conceptualized scales were proposed. The revised scales comprised: “Symptoms,” containing 10 of the original 20 Physical and four of the original Psychological Impact Items; “General Limitations,” containing 10 of the original Physical Impact scales; and “Psychological Impacts,” containing five of its original nine items.The revised MSIS-29 scales demonstrated sub-optimal but improved targeting, as the range of impact measured by the scale items covered 68% of the range of the impact measured in the sample, whereas the scales person measurements were consistently skewed to the floor of the measurement scale , scale performance was further assessed on a sub-sample of patients, excluding those with an EDSS score <2.5, reflecting no-to-little disability. Sample-to-scale targeting, the measurement scale, and sample measurement were improved in the original Physical Impact and all three revised scales when tested on the sub-sample of responders with EDSS scores >2.5 .Table 2p < 0.001) reduction at Week 48, with the revised scale showing the highest relative efficiency. Change scores within the EDSS sub-sample (p < 0.01). All three change scores were associated with low ESs and SRMs. Examination of minimally important difference (MID) for ES and SRM compared to mean change scores indicated that they were both higher, providing additional evidence around the magnitude of change scorers for these scales.Relative precision examination suggests that the revised Psychological Impact was the most sensitive scale for detecting change . Examinib-sample  indicate3 there has been an increased use of more sophisticated psychometric methods, which provide the potential for better measurement of patient experience.1 Our RMT findings supported previous research,8 revealing varied evidence supporting the use of the MSIS-29. In brief, its targeting and conceptual basis could be improved; the range of impact covered by items did not match the range of impact measurement in the study sample , and item fit analyses indicated potential problems for item placement within scales.The original MSIS-29 has been an important PRO instrument in MS clinical studies and trials for more than 15 years. Its straightforward scoring for two broad concepts  has allowed for wide application, and provided a strong basis for comparable data between different research endeavors. However, since it was publishedWe proffer three revised, conceptually clearer scales: “General Symptoms” related to range of symptoms, “Psychological Impact” related to emotional well-being, and “General Limitations” related to difficulties in everyday life. RMT analyses of these revised scales indicated improved sample-to-scale targeting and item fit, although not completely resolved. Our findings also suggest the revised Psychological Impact and General Limitations scales were able to detect more change than original Psychological and Physical Impact scales, respectively. Ultimately, we recommend the MSIS-29 may be further improved by adding more complex (related to higher functioning) items in the lower range of the measurement scale to improve content coverage and floor effects, primarily relevant for patients with mild disability.5 MSIS-29 was developed with input from patients with relatively high levels of MS disability (>50% were retired because of disability), it is not surprising that the revised scales did not resolve sub-optimal targeting for patients with fewer disabilities.Although our findings suggest the potential for improving legacy instruments, such as the MSIS-29, it is important to consider the caveats of our empirically generated revised MSIS-29 scales. First, all post-hoc RMT psychometric analyses of the MSIS-29 are limited to the instrument’s original content, which was not developed in line with RMT, nor were the items selected with clinical hierarchies in mind. Also, considering the originalUsing the MSIS-29 to assess clinical change in MS populations similar to the ADVANCE cohort would require an expansion of the scale to include items that are associated with levels of symptoms, psychological impact, and limitations relevant to patients in this context. The proposed scoring structure of the revised MSIS-29, as well as the item hierarchies within each of the revised scales, represent just one way the items could be re-arranged. As this re-conceptualized scoring structure is supported by a single post-hoc psychometric analysis, it is essential that the revised scales be subjected to further psychometric testing and clinical anchoring in independent samples.35Our findings provide an initial evidence base to improve the measurement potential of the MSIS-29 as a PRO instrument in MS clinical research and trials. Articulating scores in relation to symptoms, emotional well-being, and general limitations increases conceptual clarity of MSIS-29. In trials such as ADVANCE, a more explicit and easily interpretable set of concepts can be presented in discussions regarding treatment benefits to patients, regulators, and payers. Additionally, the improved targeting of the revised MSIS-29 scales reduces the overall error associated with measurement. This improves the scales’ potential to reflect the impact of clinical change in MS when it occurs. Finally, since original MSIS-29 scores are ordinal in nature, the use of the linearized  transformed 0–100 scoring would benefit the interpretation of scores and change scores, especially in patients with less disability.


Organization of HIV care and treatment services, including clinic staffing and services, may shape clinical and financial outcomes, yet there has been little attempt to describe different models of HIV care in sub-Saharan Africa (SSA). Information about the relative benefits and drawbacks of different models could inform the scale-up of antiretroviral therapy (ART) and associated services in resource-limited settings (RLS), especially in light of expanded client populations with country adoption of WHO’s test and treat recommendation.We characterized task-shifting/task-sharing practices in 19 diverse ART clinics in Tanzania, Uganda, and Zambia and used cluster analysis to identify unique models of service provision. We ran descriptive statistics to explore how the clusters varied by environmental factors and programmatic characteristics. Finally, we employed the Delphi Method to make systematic use of expert opinions to ensure that the cluster variables were meaningful in the context of actual task-shifting of ART services in SSA.The cluster analysis identified three task-shifting/task-sharing models. The main differences across models were the availability of medical doctors, the scope of clinical responsibility assigned to nurses, and the use of lay health care workers. Patterns of healthcare staffing in HIV service delivery were associated with different environmental factors  and programme characteristics .Understanding the relative advantages and disadvantages of different models of care can help national programmes adapt to increased client load, select optimal adherence strategies within decentralized models of care, and identify differentiated models of care for clients to meet the growing needs of long-term ART patients who require more complicated treatment management.The online version of this article (10.1186/s12913-017-2772-4) contains supplementary material, which is available to authorized users. Critical shortages and inefficient distributions of trained health workers constrains timely and universal access to HIV treatment in SSA . Many anTask-shifting is moving tasks from one cadre to another – usually from more to less specialized cadres  or nurses) [Task-sharing is a “team-based approach” where “medical care [is] provided to a patient by a set group (team) of different health professionals with different roles that maximize the skills and abilities of each team member” [Redistributing tasks within health workforce involves task-shifting and task-sharing.  nurses) –26. Task member” . For exaTask-shifting and task-sharing in ART services vary tremendously across countries and programmes. To date, research has mainly focused on the safety and effectiveness of task-shifting , 28–30. In this study, we sought to identify ART task-shifting/task-sharing service delivery models and describe their health facility and environmental characteristics. A cluster analysis of cross-sectional data from 19 diverse ART clinics in Tanzania, Uganda, and Zambia was conducted. Models of care were then verified by 18 experts using the Delphi survey process.Cross-sectional data for this analysis came from an ART retention and adherence study in Tanzania, Uganda, and Zambia led by FHI 360 2008–2012). The main study’s purpose was to characterize retention and adherence rates in 19 ART clinics, and to examine programmatic and individual factors related to retention and adherence among adult patients on ART for at least 6 months. Clinics were purposefully selected with country partners and Ministries of Health (MOH) to include those with ≥300 patients from different socioeconomic and urban-rural locations with varying characteristics, including public/private/faith-based organizations, primary/secondary/tertiary-levels, and different ART adherence and provision strategies. Additional site selection details have been reported elsewhere , 33. Inf2. The maTrained interviewers used a structured survey to systematically document task-shifting/task-sharing practices at each clinic on the following ART-related services on a typical day: 1) registration, 2) initial patient triage, 3) initial ART prescription based on ART eligibility assessment, 4) ART monitoring and management , 5) clinical services , 6) referral services, 7) ART dispensing, 8) counselling on ART adherence, 9) phlebotomy for laboratory testing, 10) follow-up of patients who missed appointments, and 11) medical records management. Data were stored in EpiData 3.1.We conducted a cluster analysis to identify models of ART task-shifting/task-sharing service delivery. All statistical analyses used SAS 9.3.We first ran frequencies on all variables to assess variability, defined as differences observed in at least three of the 19 ART clinics. Only 33 of 69 initial variables had enough variability to be included in the analysis.2 statistic). Nine sensitivity analyses were performed using other measures of similarity  and hierarchical methods  fitting for the asymmetric binary data, enhancing the reliability of the findings [We then performed agglomerative hierarchical clustering using the Ward Method and the Russel and Rao Index as the measure of similarity – an algorithm selected for the asymmetric nominal nature of the data. The number of clusters were determined based on examination of the generated dendrogram and statistics measuring the cluster fit  varied by environmental factors  and programmatic characteristics . Continuous variables were summarized using means and medians while categorical variables were summarized with frequencies and percentages. No statistical testing was done to compare the clusters given the small numbers of clinics (≤10) within each cluster. Limited power meant the study could only detect extreme differences of one to two standard deviations between clinic means.We employed the Delphi Method to make systematic use of expert opinions  to ensurThe hierarchical cluster analysis dendrogram and cluster fit suggested a three-cluster solution, with each cluster representing a model of ART service delivery. The 19 clinics were divided into three groups of 4, 5, and 10 clinics  task-shared with nurses and counsellors in providing adherence support to patients in all models. In two models, LHWs also provided support essential to efficient clinic flow, such as patient registration and medical file retrieval/archival.Eighteen of the 22 invited experts participated in the survey (82% response rate). Experts had 5 to 21 years of experience implementing ART task-shifting services, policy, or research in 21 African and 9 non-African countries. They represented a range of organizations including MOHs, national and international implementation partners, universities, and funding agencies.Most experts  said the three service delivery models identified were consistent with their knowledge and experience of ART programmes in SSA. Four experts  said model findings were “artificial” and “context dependent” and only meaningful to the three countries where data were collected.All three task-shifting/task-sharing models included clinics from the three study countries and from every health facility types encompassing national referral, provincial/district, and primary health centers (PHCs)) Table . These fThe healthcare staffing pattern of Model 1-Traditional was characterized by task-sharing of major clinical responsibilities between doctors and COs. Doctors performed most clinical tasks, including initial ART prescription, ART monitoring and management, and clinical services , ART monitoring and management (100%) and clinical services (100%); they varied in provision of ART adherence counselling .Model 2-Mixed Clinics were more likely to reduce client flow by providing a longer ART supply , ART monitoring and management (100%), clinical services (100%), and referral services (100%). Substantial task-sharing between COs and nurses occurred: nurses in three-quarters of Model 3-Task-Shifted clinics prescribed ART, provided referral services, and dispensed ART; and nurses in half of the clinics provided clinical services as well. The only clinical tasks not shared among COs and nurses were management of drug resistant infection and monitoring of viral load results , nutritional supplementation (75% vs. 40–50%), and home visits to patients who missed clinic appointments (75% vs. 60–70%). Correspondingly, this model had more LHWs. According to experts, these clinics may have relied more heavily on community-based services provided by LHWs because of limited resources, and to reduce clinic congestion.This is the first study to identify models of ART service delivery by examining healthcare staffing patterns across clinics in multiple SSA countries. Three models were identified, distinguished primarily by the availability of doctors, nurses’ scope of clinical responsibility and the use of LHWs. The models reflected historical scale-up of ART services in SSA where ART was initially available only at tertiary-level hospitals in urban centers  and was later decentralized to peripheral health facilities in peri-urban towns and rural villages, such as district-level hospitals and PHCs (models 2-Mixed and 3-Task-Shifted). In Model 1-Traditional, doctors and COs took the lead on initiating and clinically managing ART patients, and nurses had limited clinical responsibilities. None of the nurses in Model 1-Traditional prescribed ART; instead, nurses were mainly responsible for patient registration and adherence counselling. This model most closely resembled the earliest approaches to HIV service delivery prior to decentralization , 38–40. In Model 2-Mixed, fewer doctors were available and nurses had more clinical responsibilities, such as prescribing ART for treatment naïve patients and clinical monitoring of stable patients . Model 2Model 3-Task-Shifted did not typically have doctors present. Both COs and nurses were responsible for initiating and prescribing ART. In addition to their extensive clinical responsibilities, nurses in Model 3-Task-Shifted also performed administrative and patient support work. Nurse-initiated and/or nurse-managed ART programmes have been previously described in South Africa , 45–47, Our findings also concurred with observations that decentralized ART programmes, such as those in Model 2-Mixed and Model 3-Task-Shifted, required fewer clinic visits from patients . For exaOur findings identified three models of HIV service delivery for potential application to other high HIV prevalence settings in SSA. Classifying ART models enables further study on their context, frequency of use, costs, and outcomes. For example, future research could assess how a task-shifted model compares to a mixed model in costs and proportion of patients achieving virologic suppression. Resulting data could inform decisions by programme planners, policy-makers, and funders on models for scale-up within resource-constrained settings.Our study also suggested that different patterns of healthcare staffing were associated with different environmental factors and programme characteristics. For example, integrated TB treatment varied across the three service delivery models. This issue is important given high rates of TB/HIV coinfection across SSA and highlights how service provision is shaped by health facility levels, rural/urban settings, available health cadres, patient populations, and donor funding. A comprehensive and nuanced understanding of models of HIV care and treatment is especially important following WHO’s test and treat recommendation and donor fatigue after the global economic crisis. Test and treat adoption will likely impact client volumes and client composition, as healthier patients access ART. Understanding the pros and cons of different models of care can help programmes determine the best staffing patterns to adapt to increasing client loads immediate ART initiation, and the selection of different adherence strategies most effective for their facility’s patients. Concurrently, national programmes will need to identify differentiated models of care for people to meet the needs of long-term ART patients requiring more complicated treatment management, such as third line regimens and treatment for severe comorbidities .Our sample included 19 ART clinics in three countries – a considerably larger and more diverse sample size than past research, which has generally described task-shifting/task-sharing practices of a few clinics in one country , 30, 40.Our survey also assessed a wider range of ART service delivery components. While past research mainly described task-shifting/task-sharing in terms of who prescribed ART or provided clinical monitoring, ours accounted for other major tasks within the ART clinic  and the health cadres providing these tasks. While we examined the presence or absence of a wide range of services at ART clinics, a limitation is that we were unable to consider other important details, such as intensity and coverage of services per patient, or service quality.Finally, the combination of a cluster analysis with a Delphi survey was stronger than either method alone in identifying meaningful models of ART service delivery. The Delphi survey ensured the three clusters were more than a statistical artefact and held practical meaning and had content validity.Findings highlighted the complexity of factors that need to be considered to understand effective ART programmes. These models of task-shifting/task-sharing can provide a basis for additional implementation science research, including costing analysis and comparative effectiveness across models of care, to inform the scalability and sustainability of HIV care and treatment in RLS.


Metastatic lesions of renal cell carcinoma (RCC) occasionally regress spontaneously after surgical removal of the primary tumor. Although this is an exceptionally rare occurrence, RCC has thus been postulated to be immunogenic. Immunotherapies, including cytokine therapy, peptide-based vaccines, and immune checkpoint inhibitors have therefore been used to treat patients with advanced, metastatic RCC. We review the history, trends, and recent progress in immunotherapy for advanced RCC and discuss future perspectives, with consideration of our experimental work on galectin 9 and PINCH as promising specific immunotherapy targets. Metastatic lesions of renal cell carcinoma (RCC) occasionally regress spontaneously following removal of the primary disease, though this is exceptionally rare. This is thought to represent an immune response, and RCC, like melanoma, has thus been considered to be immunogenic. Although very few urologists have been fortunate enough to experience such a rare event, these anecdotes suggest that RCC may be immunogenic in nature. Before the era of molecular targeting drugs, cytokine therapy with interferon (IFN) or interleukin-2 (IL-2) was widely used, and partial response (PR) and complete response (CR) were achieved in 15.9% and 1.8%, respectively, of patients with advanced RCC treated with IFN  with bone metastases who maintained CR for a long period after hormonal manipulation and chemotherapy with docetaxel. In another case of CRPC, the tumor was reported to vanish spontaneously during therapy . These oTable 1 (The history of therapies for RCC is summarized in Table 1 . IFNα anTable 1  in 2000.Table 1 .Identification of the von Hippel-Lindau (VHL) tumor suppressor gene in 1993  and subsMolecular targeting drugs target the cascade mediated by VHL protein, and several such agents, including sorafenib, sunitinib, axitinib, temsirolimus, and everolimus, have been approved in Japan since 2008.The extremely rare spontaneous regression of metastatic lesions following surgical removal of the primary lesion, e.g., by nephrectomy, is thought to be caused by an immunologic response. This characteristic of RCC and the consequent notion that RCC is immunogenic, form the basis for non-specific immunotherapy with cytokines, which has been employed for some time. However, molecular targeting drugs currently form the mainstream of treatment for metastatic RCC. The use of IFNα is currently limited to lung metastases. In this review, we consider recent progress in immunotherapy for RCC and discuss future prospects with regard to the role of immunotherapy as a mainstream therapy for RCC.Urologists have continued to treat patients with advanced metastatic RCC using IFNα for the past 20 years. Although a previous study reported response rates to IFNα as low as 15.9% and 1.8% for PR and CR, respectively  cell activities, induction of antigen presentation on the cell surface, and enhancement of cytotoxic T lymphocyte (CTL) activities . AlthougPeptide-based vaccines represent a rational approach to inducing cancer-specific immunity against cancer antigens. Tumor-associated antigens (TAA) are incorporated into antigen-presenting cells (APCs), broken down into pieces, processed, and presented on HLA class I (MHC class I) in the form of peptides. The presented peptide antigens stimulate CD8+ effector cells (CTLs) that specifically recognize the antigen and attack cancer cells bearing the TAA. The amino acid sequences of the peptides within TAA sequences have been investigated with the aim of producing peptide-based vaccines. Candidate peptides capable of binding HLA class I are synthesized and used to stimulate peripheral blood mononuclear cells in vitro to induce antigen-specific CTLs. Peptides capable of inducing CTLs with high, specific cytotoxic activity toward cancer cells are then selected for use as vaccines. These peptides, mixed with adjuvant, are presented on HLA class I of APCs following injection, resulting in lymphocyte stimulation and induction of tumor antigen-specific CTLs.Regarding the clinical efficacy of peptide-based vaccines for RCC, Uemura et al.  reportedA phase II randomized, clinical trial of IMA901, a mixture of multiple TAA-derived peptides, showed that Tregs were reduced by a single dose of cyclophosphamide, and patients responded immunologically to IMA901 had longer overall survival . A phasePeptide-based vaccine therapy emulates naturally occurring antigen presentation, and targets specific cellular immunity against cancer cells using TAA-derived peptides. Peptide vaccine therapy is thus rational with the potential for high efficacy. However, it has so far demonstrated limited clinical effects in patients with metastatic RCC, probably because of the lack of defined, appropriate antigens.The innate immune system seems to play an important role in the initial stages of anti-cancer immunity. Concerning the implications of innate immunity for immunotherapy of RCC, several reports have focused on NK cells. Combination therapy with IFNα and IL-2 has been reported to enhance the cytotoxic activities of NK cells in patients with advanced RCC  and NK cNKT cells also contribute to anti-cancer immunity, and a lack of these cells is associated with impaired anti-cancer immunity . The exoIt is likely that the development of specific and effective immunity against RCC could achieve a cure, though no effective means of achieving this goal have yet been identified.Despite a low response rate, cytokine therapy can offer an effective therapeutic option for advanced RCC. Survival benefit of cytokine therapy has been reported in patients with metastatic RCC , and the. These antigens seemed to be closely related to an immune escape mechanism, cancer cell survival, and metastases of RCC.We investigated ‘true cancer antigens’ by screening an RCC expression library using sera from patients with metastatic disease who responded well to cytokine therapy as probes, under the assumption that these responders had acquired specific antibodies against RCC together with specific cellular immunity. We identified two novel genes, galectin 9 and PINCH, as RCC-specific antigens that were specifically highly expressed in all the tested clear cell carcinoma samples, compared with normal renal tissues . Using pGalectin 9 modulates cellular immunity by suppressing excess immune reactions such as allergies. Its receptor is T cell immunoglobulin and mucin domain 3 (TIM-3), located on the surface of T cells. Galectin 9 causes apoptosis of activated T cells through TIM-3 , 23, whiPINCH contributes to apoptosis resistance in cancer cells  and promGalectin 9 and PINCH produce favorable environments for RCC cells and therefore provide promising targets for immunotherapy. Our method of identifying potentially useful cancer antigens was unique in that we screened for antigens with central roles in developing specific anti-cancer immunity by reacting them with sera from responders to cytokine therapy. The abilities of the identified antigens to induce CTLs with specific, high cytotoxicity was then evaluated. This method involved basic molecular cloning techniques using clinical samples, and was developed by a urologist with extensive experience, including many clinical cases of RCC .Peptide-based vaccines have the problem that various peptide sequences of the same antigen need to be prepared in accordance with the HLA types of the patients. TAAs are presented on HLA class I in a form of peptides that comprise part of the whole antigen: different peptides derived from the same whole antigen associate with HLA class I of different HLA types. The binding motif of the antigen for each HLA type should be predicted in silico, and the ability of each predicted peptide to induce CTLs by stimulating lymphocytes of the same HLA type needs to be tested. We previously identified galectin 9- and PINCH-derived peptides that induced HLA-A*02-restricted CTLs, HLA-A*24-restricted CTLs, and HLA-A*33-restricted CTLs , respect.Cancer cells have recently been shown to exploit immune checkpoint molecules that mediate the suppression of immune signals, thus facilitating escape from the immune surveillance system. These checkpoint molecules, including CTLA-4, PD-1, programmed death ligand 1 (PD-L1), and TIM-3, mediate T cell suppression. Drugs (antibodies) targeting these check point molecules, referred to as check point inhibitors, including antibodies against CTLA-4, PD-1, and PD-L1, have been developed  and in sClinical studies of an anti-PD-1 antibody  , 30 and Increasing attention has been paid to TIM-3 as a target . TIM-3 bProgress in molecular biology following the discovery of the VHL gene has resulted in the development of various molecular targeting drugs and advancements in therapies for advanced RCC. On the other hand, following on from the long-standing use of cytokines such as IFN and IL-2, immunotherapy is about to enter a new era represented by immune checkpoint inhibitors. However, the costs associated with the clinical study, development, and approval of these immune checkpoint inhibitors, marketed as humanized antibodies, are likely to be enormous.Peptide-based vaccine therapy aims to induce specific immunity, and appears to offer potential survival benefits and cure; however, their efficacy is currently poor. Innate immunity provides an initial step leading to specific anti-cancer immunity, and non-specific immune reactions initiated by cytokines may also cause specific anti-tumor immunity. The combination of ‘real’ cancer antigens and new, immune-activating agents targeting molecules such as immune checkpoints may thus induce strong cancer-specific immunity.Cytokine therapy is highly effective in a very limited number of patients, indicating the high potential of immunotherapy. However, the mechanisms responsible for the development of immunity in these patients with RCC remain unclear. We identified novel RCC antigens that reacted with sera from cytokine-therapy responders. We successfully induced antigen-specific, HLA-restricted CTLs with high activities by stimulating lymphocytes with peptides derived from those antigens. However, the mechanisms responsible for the effects of the cytokines in particular cases are unknown. Further, detailed investigations of successfully treated cases and analysis of the general mechanisms are likely to lead to promising new therapies for RCC.