We discovered that transcripts for isoferritins and cathepsins are strikingly abundant during colostrum production, highlighting the potential importance of these proteins for neonatal health. Two transcripts, encoding β-casein (CSN2) and α-lactalbumin (LALBA), make up 45% of the total pool of mRNA in mature lactation. Genes significantly expressed across all stages of lactation are associated with making, modifying, transporting, and packaging milk proteins. Stage-specific transcripts are associated with immune defense during the colostral stage, up-regulation of the machinery needed for milk protein synthesis during the transitional stage, and the production of lipids during mature lactation. We observed strong modulation of key genes involved in lactose synthesis and insulin signaling. In particular, protein tyrosine phosphatase, receptor type, F (PTPRF) may serve as a biomarker linking insulin resistance with insufficient milk supply. This study provides the methodology and reference data set to enable future targeted research on the physiological contributors of sub-optimal lactation in humans.Aware of the important benefits of human milk, most U.S. women initiate breastfeeding but difficulties with milk supply lead some to quit earlier than intended. Yet, the contribution of maternal physiology to lactation difficulties remains poorly understood. Human milk fat globules, by enveloping cell contents during their secretion into milk, are a rich source of mammary cell RNA. Here, we pair this non-invasive mRNA source with RNA-sequencing to probe the milk fat layer transcriptome during three stages of lactation: colostral, transitional, and mature milk production. The resulting transcriptomes paint an exquisite portrait of human lactation. The resulting transcriptional profiles cluster not by postpartum day, but by milk Na:K ratio, indicating that women sampled during similar postpartum time frames could be at markedly different stages of gene expression. Each stage of lactation is characterized by a dynamic range (10 Breastfeeding provides numerous benefits for both mother and infant Research into the biology of human lactation has been seriously impeded by the impracticality and ethical concerns of obtaining systematic samples of mammary tissue from lactating woman. However, there is an intriguing workaround–human milk secreted during lactation is a rich source of mammary epithelial cell RNA. As lipid droplets exit the mammary epithelial cell, they are enveloped by cell membrane and secreted into milk as membrane-bound globules of fat. About 3–8% of human milk fat globules contain mammary epithelial cell cytoplasmic remnants, including RNA, captured during milk fat globule formation and secretion With the advent of RNA sequencing (RNA-Seq) it is now possible to characterize the gene expression of milk-producing cells with highly sensitive detail. In contrast to microarray technology, RNA-Seq can accurately quantify both very low and very high abundance transcripts, as well as detect novel transcripts To date, RNA-Seq of the milk fat layer has not been reported and thus the extent to which RNA-Seq offers insights into the mammary transcriptome captured from this unique source is not known. Furthermore, it is unknown if the milk fat layer yields RNA purely of mammary epithelial cell origin, or if RNA from non-mammary epithelial sources is also present in the milk fat layer obtained from colostrum, transitional, or mature human milk.In this paper, we describe the extraction and sequencing of high-quality RNA from the human milk fat layer over three stages of milk production, demonstrating the significance of defining lactation stage based on biochemical parameters (versus postpartum day). We then characterize the RNA-Seq transcriptome responsible for the production of biochemically-defined colostrum, transitional, and mature milk in humans, including: a) demonstrating how RNA-Seq captures the broad dynamic range of gene expression within the temporal context of secretory activation and lactation; b) functional enrichments of distinct gene expression trajectories; c) differential expression of insulin signaling genes; and d) differential expression of lactose synthesis genes. We identify key candidate genes that may control rate-limiting steps during lactation and outline future work in the endeavor to discover the biological underpinnings of poor lactation performance in women attempting to breastfeed.We isolated milk fat layer mRNA from 55 fresh milk samples over 48 collections . Among colostrum samples, only 27% of RNA isolates were suitable for sequencing (RIN ≥7.0 and RNA ≥10.0 ng/uL). In contrast, we obtained suitable RNA from 60% of transitional and 78% of mature milk fat layer samples . Therefore, variation in RNA quality across lactation stage must be considered when interpreting gene expression results across postpartum time points. Milk sample categorization by day postpartum without consideration for lactation stage will bias the data captured toward study participants who have progressed further toward mature milk production as these milk samples will be more likely to produce fat globules with suitable mRNA quality.Because the milk fat layer is an unconventional extracellular source of mRNA and its membrane is more fragile than cell membranes Table S1).We explored several methods to optimize milk RNA quality see Methods.Figure S1). We tested a washing protocol to remove these nucleated cells. Regardless of washing protocol, leukocyte-specific genes were not expressed in mature milk fat layers, scantly expressed in transitional milk fat layers and robustly expressed in colostral milk fat layers . Cell markers associated with epithelial and other (non-leukocyte) lineages are also expressed in the colostral milk fat layer. The identity of the somatic cell types present during the colostral stage and their impact on our interpretation of the milk fat layer transcriptome at this stage of lactation warrants further study.Under the electron microscope, milk fat globules can be observed with crescents of cytosolic components, but never nuclei Table S2 summarizes the characteristics of the study participants and their samples. Briefly, postpartum timing of sample collection ranged between 41–52 hours for colostral, 39–56 hours for transitional, and 33–130 days for mature; and Na:K ratio ranged between 5.5–9.6 for colostral, 0.70–1.15 for transitional, and 0.19–0.57 for mature.We now describe our findings from sequencing the RNA isolated from 12 milk fat layer samples . and An average of 25.5 million reads per sample was mapped to the human genome . Gene expression intensity was normalized to Fragments per Kilobase of transcript per Million mapped reads (FPKM) and summarized at the gene level .RNA sequencing results are highly correlated with qPCR  and proteins involved in the milk making machinery . Therefore transcript abundance may not correspond closely to milk protein abundance. Furthermore, the broad dynamic range and diversity of human milk proteins has prevented accurate quantification of the human milk proteome Stunningly, 45% of the milk fat layer mRNA during mature lactation represent just two milk protein genes: CSN2 and LALBA Figure 1 inset), especially in contrast to RNA-Seq technology  and with hormone receptor binding and signaling . Further, lipid and lipid cofactor biosynthesis are uniquely enriched in mature samples. Consistent with our expectations, these results suggest that immune defense is a hallmark of the colostral stage, massive development of the protein synthesis infrastructure and inhibition of protein degradation is a hallmark of the transitional stage, and massive synthesis of lipids is a hallmark of the mature stage Figure 4Figure S3). According to the dendogram, the largest differences in the overall transcriptome occur between colostral (as defined by Na:K ratio) and all other samples. The second largest transcriptional difference occurs between transitional and mature samples. These results confirm our observation that compared to other factors such as subject, washing protocol, or time of collection, biochemically defined lactation stage  has the largest effect on the transcriptome. Importantly, the samples did not cluster by postpartum hour or day of lactation, which is the measure typically used by lactation researchers to categorize lactation stage; our results highlight the importance of classifying samples by Na:K ratio. Consistent with this result, we observed strong inverse correlations between Na:K ratio and expression of tight junction proteins .Clustering analysis produced a dendogram in which lactation stage, as biochemically defined by Na:K ratio, emerges as the greatest overall difference in transcriptomes across samples .To determine the function of genes with similar transcriptional trajectories, the gene members of the eight clusters were analyzed for overrepresented functional annotations. Significant KEGG pathways and the top ten significant Biological Process Gene Ontology Terms are shown next to each cluster in The heat map illustrates that not all genes are steadily increased or decreased in the progression from colostrum to mature lactation. Therefore, we conducted a clustering analysis of all expressed genes to determine the primary gene expression trajectories. We systematically repeated the clustering analysis with increasing numbers of clusters to identify the minimally redundant set with the most clusters see Methods., Panels A and C) and “immune response”  are up-regulated in early lactation and down-regulated as lactation progresses. Likewise, “lipid biosynthesis,” which provides a major source of energy for the neonate but not immediately needed to protect the infant’s gut, does not peak until the mature stage of lactation .As expected, genes associated with “cell cycle” . Infant-associated gut bacteria use a novel endoglycosidase to liberate N-glycans from glycoproteins as a unique food source for these beneficial gut bacteria Escherichia coliThe remaining clusters are more novel. N-glycan biosynthesis and sphingolipid metabolism are coordinately increased during colostral and mature stages relative to transitional  while genes associated with translation are most abundant in the transitional samples (Panel D). Physiologically, the transitional stage is marked by the onset of copious milk production which requires building the infrastructure for massive milk protein synthesis that continues throughout lactation. Taken together, these trajectory enrichment results suggest that during the colostral stage genes are being expressed that will in turn activate further expression of the genes that build the ribosomal infrastructure required for large scale translation–i.e., preparation for massive milk protein synthesis has already begun. Further, gene expression during the transitional stage of lactation suggest post-transcriptional regulation of protein synthesis, with enrichment of gene ontology terms related to translation and RNA processing, especially non-coding RNA processing. These results are consistent with prior predictions that secretory activation is post-transcriptionally mediated The distinctly different clusters shown in , Panel H contains genes up-regulated in transitional samples relative to colostral samples before reaching steady-state. These genes are associated with the insulin signaling pathway and with the generation of energy. We inspect the modulation of the insulin signaling pathway in detail in the next section.Finally, In bovine and rodent models, insulin has recently been shown to play a central role in milk protein synthesis , panel A).Progressing from colostral to transitional lactation, among the 69 gene/gene family nodes represented on the KEGG insulin signaling pathway, 30 were up-regulated, 14 were down-regulated, and an additional 20 were robustly expressed (FPKM >0.5) but not significantly changed between colostral and transitional lactation , but not significantly changed between transitional and mature lactation (Table S2). First, in Mature Group 2, maternal report of the onset of notably fuller breasts occurred nearly two days later than in Mature Group 1 . Second, in relation to the larger follow-up cohort , women in Mature Group 2 were below the median in both insulin secretion and insulin sensitivity, whereas women in Mature Group 1 were above the median for these measures. Third, women in Mature Group 2 all reported difficulty with their milk supply either during the day 4 postpartum interview and/or at the 4–6 week postpartum follow-up interview. Therefore, we questioned whether PTPRF gene expression might be elevated in Mature Group 2 samples, as PTPRF overexpression could be causing excessive downward modulation of the insulin signaling pathway in the women with lower insulin sensitivity. In fact, we did observe significantly higher expression of PTPRF in Mature Group 2 versus Group 1 samples . In contrast, we found no difference between these two groups in prolactin-sensitive STAT gene expression . This novel finding is consistent with evidence implicating PTPRF overexpression as a contributor to insulin resistance in other organ systems Referring back to the heat map shown in , Panel A) and robust steady-state expression, with significant attenuation of PGM1 (p = 0.02), between transitional and mature lactation stages .The rate of lactose synthesis is a key driver of milk production; however, “lactose synthesis” is not an annotated pathway. We manually constructed this pathway and overlaid our gene expression results Figure 8The most up-regulated and expressed gene in the lactose synthesis pathway is LALBA, increasing 10.2-fold from colostral to transitional lactation and 6.1-fold from transitional to mature lactation. This is to be expected given that in addition to being a member of the enzyme complex involved in the final step of lactose synthesis, LALBA is also the predominant whey protein in human milk.Glucose is an important fuel source for the synthesis of lactose. The solute carrier family 2A genes  are involved in glucose (and other solute) transport across cell membranes. We detected expression of several SLC2A genes in the milk fat layer of mature samples . SLC2A1 is thought to be the primary route for glucose entry into the lactocyte across species based on reports from rodent and bovine studies There are two potential sources of UDP-Galactose in the mammary epithelial Golgi, either through the uptake and metabolism of D-Galactose or D-Glucose. Our results show that both pathways are up-regulated between colostral and transitional stages. However, the more predominant route appears to originate with D-Glucose, as the enzymes unique to this pathway are up-regulated 5.9-fold (PGM1) and 8.2-fold (UGP2). In contrast, GALK1 is up-regulated 3.3-fold.p = 0.02), suggesting these nodes are critical to the regulation of lactose synthesis. SLC2A9, SLC2A1 and SLC2A4RG are all modestly up-regulated  and LALBA is further up-regulated 6.1-fold, consistent with increased glucose utilization and milk protein output in mature lactation. All other lactose synthesis genes are expressed at a level similar to transitional samples. While protein abundances are often not linearly reflected by mRNA levels, these transcriptional results do suggest key network nodes to probe in future studies. In summary, we hypothesize that the rate-limiting enzyme PGM1 modulates milk yield in humans.Similar to what we observed for the insulin signaling pathway, the strong up-regulation of the lactose synthesis pathway observed between colostral and transitional lactation stages is attenuated between transitional and mature lactation stages. Gene expression for the enzymes central to the synthesis of UDP-Glucose from D-Glucose  are down-regulated, with only PGM1 attaining statistical significance , even though the mean expression of these genes varies by 5 orders of magnitude (10−1 to 104 FPKM). These PCR results confirm our RNA-Seq findings.To confirm our RNA-Seq findings using a different assay, we probed the following genes of interest in 4 samples from mature lactation using qPCR: the major milk protein genes , the gene most abundant in the mature milk fat layer transcriptome using microarray technology  lactation stage must be defined biochemically (such as Na:K ratio), not by day of lactation, b) lactation stage influences RNA quality, quantity, and potential immune cell contamination, c) immediate processing and hard, fast centrifugation yield better quality RNA, and d) additional washing of the milk fat layer is not necessary for the analysis of mature milk samples and does not decrease immune cell contamination in colostrum samples. We also found that RNA sequencing yields superior results compared to microarray experiments.Our RNA-Seq results paint the most vivid portrait to date of the transcriptome responsible for human milk production. Strikingly, transcripts of β-casein and α-lactalbumin genes make up 45% of the total pool of mRNA during mature lactation. Consistent with known physiology, genes expressed during lactation are associated with making, modifying, transporting, and packaging these highly abundant milk proteins. Lactation-stage specific transcripts are associated with immune defense during the colostral stage, the up-regulation of massive milk protein synthesis during the transitional stage, and the full-scale production lipids and other factors during the mature stage of lactation. Furthermore, fundamentally different regulatory mechanisms appear to control milk production during the different stages of lactation. First, genes whose products participate in transcription and translation are differentially regulated across all three stages. Second, we see dramatic changes in the transcriptome even after the onset of copious milk production . Future experiments, utilizing the methods outlined in this paper, are needed to determine the comprehensive acute, long-term, local, and systemic mechanisms by which women produce milk in response to infant feeding patterns. For example, the effect of early milking frequency on “programming” milk production capacity has been explored in bovine models Correlates of impaired insulin and glucose metabolism have recently been reported to be associated with poor lactation performance in women In summary, we have presented the essential methodology, reference data set, and preliminary results derived from the RNA-Seq analysis of the human milk fat layer transcriptome across three stages of lactation. Our findings set the stage for future research on the biological contributors to poor lactation performance in women.Fresh milk was collected from participants enrolled in one of two research protocols.Protocol-I was approved by both Cincinnati Children’s Hospital and The Christ Hospital  Institutional Review Boards. The ad hoc milk samples described in Protocol-II were obtained through an ongoing Research Human Milk Bank protocol All participants provided written informed consent. Day 2 milk). A study research assistant noted the exact time of collection and transported the milk on ice to the lab for immediate processing. Mothers were telephoned on the fourth postpartum day (between 72–96 hours) to collect socio-demographic data and inquire about timing of onset of noticeable breast fullness, which is a validated indicator of secretory activation  2) was estimated as:  – (2×birth weight))/admission height2).Participants were enrolled at a Cincinnati-area maternity hospital during their postpartum stay. On each recruiting morning , a maternity nurse identified mothers who were between 36–60 hours postpartum. Proceeding from a randomly ordered list, the nurse explained the study and screened mothers for the following eligibility criteria: English-speaking, currently breastfeeding, aged >18 years, without history of major breast surgery, healthy singleton infant, and telephone access for follow-up contact. Upon obtaining written informed consent, the nurse administered a breastfeeding assessment and then guided the study participant in collecting 2.5 mL of milk from each breast by mechanical breast expression using a sterile collection shield and container (mature milk) was collected from a complete expression of the left side at least two hours since the previous feeding. The participant underwent an oral glucose tolerance test and completed an infant feeding practices interview during the same morning visit.At 4–6 weeks postpartum, Protocol-I participants who were still lactating and not pregnant were asked to provide a follow-up milk sample during a morning visit to the Cincinnati Children’s Hospital Clinical Research Center. A 10 mL aliquot of fresh milk (mature milk). Maternal age, ethnicity, day of lactation, and breastfeeding exclusivity were recorded at the time of collection.Women were recruited through posted announcements. After written informed consent, these participants provided a single, 10 mL aliquot of freshly expressed milk  will reside in the cell pellet at the bottom of the tube, effectively separating the two sources of RNA ). Mature milk fat fractions, although to a lesser extent, were also infiltrated with intact cells . Thus, we proceeded to a hard spin.We followed the method described by Maningat After a hard spin , the milk fat layers were processed as in the soft spin protocol, above. The hard spin did not reduce RNA quality , but it also did not reduce infiltration of the fat layer with intact cells, which were evident to the same degree as after a soft spin (not shown). We then proceeded to a milk fat washing protocol.In this phase, milk fat layers were washed as follows. After a hard spin at a warmer temperature,  the milk fat layers were transferred to a clean set of 3 tubes and re-suspended in 1 mL PBS+10 µM EDTA. After centrifuging again , the milk fat layers from all 3 tubes were transferred to a single tube, re-suspended in 1 mL PBS, and centrifuged a final time . The remaining milk fat layer was transferred into a single new tube to which 500 µL TRIzol® was added and stored at −80°C.Under Zeiss florescent microscope, the washed method appeared to reduce the number of intact cells in colostrum samples and eliminate them from mature samples, but to definitively determine if washing was beneficial in removing infiltration of intact cell RNA, we sequenced both hard and washed milk fat samples. As part of this approach, some milk samples were split so that an aliquot was processed using the hard method and another using the washed method. We conducted both a targeted comparison of gene expression for immune cell markers  and a global examination of differential gene expression by washing method.1/potassium1], [sodium2/potassium2]). Expression of sodium as a ratio to potassium adjusts for slight variations in lipid-free purity of the aqueous fraction Milk sodium concentration is a marker of tight junction closure between mammary epithelial cells; with the onset of secretory activation (stage II lactogenesis) milk sodium concentrations decrease sharply as potassium concentrations increase Among the subset of participants in Protocol-I who agreed to a follow up visit at 4–6 weeks postpartum, we conducted an oral glucose tolerance test at the time of collecting a follow up milk sample. For this, participants arrived at our clinical research center after an 8–10 hour fast. We obtained blood samples at 0, 30, 60, 90 and 120 minutes after the participant consumed a standard 75 g glucose beverage. The samples were assayed for serum glucose (glucose oxidase method) and insulin (electro-chemi-luminescence immunoassay) concentrations at the Biochemistry Core Lab at Cincinnati Children’s Hospital. The results were used to calculate maternal insulin sensitivity (ISOGTT) For all lipid samples we extracted and purified total RNA in batches of 16 using the PROMEGA Maxwell 16™ integrated system . We assayed a 2 µL aliquot for RNA concentration, purity (rRNA 28 s∶18 s ratio), and quality (RNA Integrity Number (RIN)) using the Agilent 2100 Bioanalyzer .We initially selected 16 purified RNA samples for paired-end sequencing based on these priorities: RIN ≥7.0 and RNA ≥10.0 ng/uL; paired samples across time points (Day 2 and mature) and split between washed and unwashed. In the Day 2 samples, sodium and potassium concentrations ranged between 6.2–90.9 mmol/L and 9.5–20.5 mmol/L, respectively; and Na:K ratio ranged between 0.42  to 9.62 (less tight junction closure). Only three Day 2 samples categorized as colostral stage were suitable for sequencing (RIN ≥7.0 and RNA >9.0 ng/uL) and all three were selected.Table S2 summarizes maternal and sample characteristics for sequenced samples.All gene expression results subsequent to washing protocol comparisons are based on a final set of 12 sequenced samples  remaining after the exclusion of the unwashed partner in the split pairs  and the exclusion of 1 transitional sample from a mastitic participant (ID = 158). http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE45669).From the samples we selected for sequencing, the Gene Discovery core facility at Cincinnati Children’s Hospital prepared RNA-Seq libraries using the Illumina TruSeq RNA kit  and performed sequencing on an Illumina HiSeq2000 with version 3 chemistry. Libraries were multiplexed in batches of 12 to ensure a minimum of 20 million 100 bp paired-end reads per sample. FASTQ files were de-multiplexed to assign reads to the originating sample. The data has been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE45669 (Reads were mapped to the human genome assembly hg19 using TopHat http://www-huber.embl.de/users/anders/HTSeq) to the mapped reads (e.g. TopHat output) after preparation with Samtools and Perl to identify read pairs and remove those with mates on different chromosomes. The hg19 Ensembl gene reference annotation was derived from Ensembl Release 62 To facilitate other types of analyses, counts for each hg19 Ensembl gene were calculated by applying HTSeq-count  were deemed statistically significant.To determine differentially expressed genes, DESeq’s nbinom test function was used to test the significance of the differences between the base means of the two conditions . Differences with p<0.05 after adjustment for multiple hypothesis testing (method = “Benjamini”) were deemed statistically significant. Annotation clusters were considered significant only if they contained statistically enriched annotations.We determined biological functions of gene sets of interest using the Functional Annotation Clustering tool within DAVID Bioinformatics Resources 6.7 2O, and 5 µL (12.5 ng) template, for a total volume of 25 µL per well. All samples were assayed in duplicate for each gene of interest. Each plate was run on the Applied Biosystems StepOnePlus™ using standard settings (cycling program included 10 min incubation at 95°C followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C). Data were assessed from each assay using the StepOnePlus™ software tool v2.2.We confirmed the RNA-Seq results for genes of interest with TaqMan qPCR. Total RNA from four mature milk fat fractions were synthesized into cDNA using the SuperScript® III First-strand Synthesis SuperMix  according to manufacturer’s instructions. Samples were then ethanol precipitated to enhance purity of DNA. Genes of interest (n = 15) were validated using qPCR with Custom TaqMan® Array Plates in a 96-well format . Details on the primer probes sets for these genes are provided in Following the approach of Bionaz and Loor To compare RNA-Seq results with qPCR for each gene of interest, RNASeq FPKM values were normalized to the FPKM geometric mean of the endogenous gene set . We then determined the Pearson linear correlation coefficient  between the normalized qPCR-Ct and FPKM means across the 15 genes of interest. In this analysis, each gene is an observation, with two variables per gene: qPCR-Ct and FPKM. The values for these two variables, per observation, are the (log) normalized qPCR-Ct and FPKM means of the 4 individual samples.Figure S1Milk fat layer under the fluorescent microscope. Fresh milk fat layer solids were fixed on a slide with a cytospin, stained with DAPI, and examined on a Zeiss florescent microscope for blue-fluorescing nuclei as evidence of intact cell infiltration . Non-nuclei cell debris stain green. Panel A: Colostrum milk fat layer at 20× was heavily infiltrated with intact cells. Panel B: Mature milk fat layer at 40× was also infiltrated with intact cells, but to a much lesser extent.(TIFF)Click here for additional data file.Figure S2Mean (±SEM) FPKM for leukocyte cell markers. Regardless of washing protocol, mature samples showed virtually no evidence of leukocyte specific gene expression; however, colostral–and transitional samples to a much lesser extent–show at least some evidence of leukocyte cell gene expression. In contrast to the other leukocyte cell markers in this figure, CD33 (a cell marker heavily expressed in immature stem cells) expression is not significantly different between colostral and mature lactation, providing evidence of human milk being a source of stem cells. Footnotes: Col = Colostral, Tra = Transitional, and Mat = Mature stages of lactation; *p<0.05, Colostral versus Transitional; +p<0.05, Transitional versus Mature.(TIFF)Click here for additional data file.Figure S3Cluster dendogram of all RNA-sequenced samples. Clustering analysis produced a dendogram in which lactation stage, as biochemically defined by Na:K ratio, emerged as the greatest overall difference in transcriptomes across samples.(TIFF)Click here for additional data file.Table S1Summary of milk fat globule RNA quality by stage of lactation and processing method.(DOCX)Click here for additional data file.Table S2Summary of maternal and sample characteristics for all RNA sequenced samples.(DOCX)Click here for additional data file.Dataset S1Gene sets and their FPKM values, identified by lactation stage and processing method.(XLSX)Click here for additional data file.Dataset S2Enrichment of functional annotations for the top ten percent of genes expressed in each stage of lactation.(XLSX)Click here for additional data file.Dataset S3Significant KEGG pathways and Biological Process Gene Ontology Terms for each of the eight identified cluster trajectories.(XLSX)Click here for additional data file.Dataset S4Insulin signaling and lactose synthesis pathway gene sets, their FPKM values at each lactation stage, and whether upregulated, downregulated or unchanged between stages.(XLSX)Click here for additional data file.Dataset S5Details on primer probes sets for PCR assay.(XLSX)Click here for additional data file.


Inherited autoinflammatory syndromes (IAS) are a group of recently identified monogenic diseases characterized by recurrent episodes of systemic inflammation.Muckle-Wells Syndrome, Familial Cold Autoinflammatory Syndrome and Chronic Infantile Neurological Cutaneous and Articular Syndrome are IAS due to mutations in a single gene, CIAS1 , encoding a protein called cryopyrin which is an essential component of an intracellular multiprotein complex named inflammasome, that play a crucial role in the production and secretion of interleukin (IL)-1 These diseases are characterized by excessive production of IL-1 and have a dramatic response to IL-1 inhibition.More recently, IL-1 inhibition has also been shown to be effective in another AID (TNF-receptor associated periodic syndrome or TRAPS) as well as in other conditions such as systemic juvenile idiopathic arthritis and recurrent idiopathic pericarditis. This has suggested that also these two last diseases may represent autoinflammatory conditions.


This study evaluated tyrosine kinase receptor (TKR) expression and activation in canine pulmonary adenocarcinoma (cpAC) biospecimens. As histological similarities exist between human and cpAC, we hypothesized that cpACs will have increased TKR mRNA and protein expression as well as TKR phosphorylation. The molecular profile of cpAC has not been well characterized making the selection of therapeutic targets that would potentially have relevant biological activity impossible. Therefore, the objectives of this study were to define TKR expression and their phosphorylation state in cpAC as well as to evaluate the tumors for the presence of potential epidermal growth factor receptor (EGFR) tyrosine kinase activating mutations in exons 18–21. Immunohistochemistry (IHC) for TKR expression was performed using a tissue microarray (TMA) constructed from twelve canine tumors and companion normal lung samples. Staining intensities of the IHC were quantified by a veterinary pathologist as well as by two different digitalized algorithm image analyses software programs. An antibody array was used to evaluate TKR phosphorylation of the tumor relative to the TKR phosphorylation of normal tissues with the resulting spot intensities quantified using array analysis software. Each EGFR exon PCR product from all of the tumors and non-affected lung tissues were sequenced using sequencing chemistry and the sequencing reactions were run on automated sequencer. Sequence alignments were made to the National Center for Biotechnology Information canine EGFR reference sequence.The pro-angiogenic growth factor receptor, PDGFRα, had increased cpAC tumor mRNA, protein expression and phosphorylation when compared to the normal lung tissue biospecimens. Similar to human pulmonary adenocarcinoma, significant increases in cpAC tumor mRNA expression and receptor phosphorylation of the anaplastic lymphoma kinase (ALK) tyrosine receptor were present when compared to the corresponding normal lung tissue. The EGFR mRNA, protein expression and phosphorylation were not increased compared to the normal lung and no activating mutations were identified in exons 18–21.Canine pulmonary adenocarcinoma TKRs are detected at both the mRNA and protein levels and are activated. Further investigation into the contribution of TKR activation in cpAC tumorigenesis is warranted. The molecular characterization of canine lung cancer remains limited compared to the more comprehensive evaluation of human pulmonary adenocarcinoma (hpAC). A few similarities between the human and canine pulmonary adenocarcinoma (cpAC) molecular signatures have been identified and include the presence of Kirsten rat sarcoma viral oncogene mutations and overexpression of the epidermal growth factor receptor (EGFR) -3. If caTyrosine kinase receptors (TKRs) are a family of cell-surface receptors that signal primarily through tyrosine phosphorylation events . Many TKThe EGFR is a TKR that has been identified as an oncogenic driver in certain subsets of human non-small cell lung cancer (hNSCLC). Approximately 60% of hpAC express EGFR. As such, EGFR has become an important therapeutic target for the treatment of these tumors. A range of somatic mutations in the tyrosine kinase domain of EGFR have been identified and are characterized by short deletions in exon 19, point mutations in exons 19 and 21, single nucleotide substitutions that may occur throughout exons 18 to 21, in-frame duplications and/or insertions predominately in exon 20 and rarely, an exon 22 mutation. Short deletions in exon 19 and an L858R point mutation in exon 21 account for greater than 85% of the mutations. Mutations in the EGFR tyrosine kinase domain result in destabilization of its conformation and constitutive kinase activity which provides continuous activation of its downstream signaling pathways. Lung cancers harboring constitutively active mutant EGFR are exquisitely sensitive to EGFR tyrosine kinase inhibitors (TKIs) with response rates reported from 27-100% Gene ID: 404306, updated on 19-Oct-2013) using the ClustalW procedure in DNASTAR Software v MegAlign™ 5.06, Madison, WI.Standard PCR was used to generate high fidelity ® ABC System, Vector Laboratories INC, Burlingame, CA). Tyrosine kinase receptor and VEGF positive controls for the TMA IHC were single tissue cores of canine melanoma, prostate adenocarcinoma and apocrine gland anal sac adenocarcinoma. The selected TKRs and VEGF have been previously identified in these canine tumor types or from tumor-derived cell lines [An anatomic pathologist (CP) reviewed the H&E stained slides for each dog and selected three representative sites of the cpAC tumor and normal lung to be used for the construction of the tissue microarray (TMA). The corresponding sites from the H&E slides were then matched to the paraffin embedded tissue block, 2 mm cores were extracted and placed into the predetermined sites on the TMA recipient blocks. A computer-controlled autostainer as above  was used to carry out the immunostaining. Immunohistochemistry staining was performed for EGFR  ,33, canill lines . Canine 2 at a magnification of 20X for all the tumor and normal lung cores. For each dog, the PPC obtained from each of the three tumor cores was normalized to the PPC from each of the three normal lung cores. The normalized PPC from each of the 12 dogs was used to calculate the overall IHC PPC value for the receptor or growth factor. Color deconvolution was the second algorithm used for IHC quantification. This algorithm separated the image into channels based on normalized optical density and then overlapped these channels and evaluated the entire tissue core area of each sample. Normalized optical density was derived from the control DAB stained and unstained lung tissues used in each individual automated IHC processor staining run.Quantification of IHC staining was performed on digitalized TMA slides by Aperio ScanScope XT,  and by semiquantitative grading by a veterinary pathologist. Image analysis was performed using two different Aperio algorithms. The first IHC quantification analysis was done using positive pixel count v.9 algorithm counts and quantifies the amount of stain of a specific color set by the user base on the HIS  color model. The algorithm output provided a number of 1+, 2+ and 3+ intensity positive pixels and the number of total pixels in each annotated layer. Total number of pixels consisted of positive and negative pixels excluding white area of the virtual slides . The results are reported as positivity which is the total number of positive pixels/total number of pixels. Briefly, positive pixel counts (PPC) were obtained for a square area of 393598 μmThe subjective quantitative scoring of positive or negative immunoreactivity of cells and stroma within the cores was performed by a veterinary pathologist. Percentage of neoplastic cells staining positive were scored as follows: 1-5% = 0, 5-25% = 1, 26-50% = 2 and >50% = 3. Location of staining was also noted as cytoplasmic (C), membranous (M), nuclear (N), and stromal (S). Specimens that had no IHC staining were designated as having “no staining”.g at 4°C. The supernatants were collected and protein concentration determined by a modified Bradford method . Lysates were stored at −80°C.Forty grams of either whole lung tissue or tumor were placed in a gentleMACS™ M tube , with 400 μL of 1x cell lysis buffer , 1 mM PMSF and protease inhibitors , homogenized with a gentleMACS™ dissociator  and then centrifuged for 5 min at 4,000 × For analysis of tyrosine kinase receptor phosphorylation, a human PathScan® TKR signaling array was used according to the manufactures instructions . The TKR signaling array is a slide based antibody array that allows for the simultaneous detection of 28 tyrosine kinase receptor and 11 important signaling nodes when phosphorylated at tyrosine or other residues, 10 positive and two nonspecific IgG negative controls. Each TKR is spotted on the array in duplicates. A total of 150 μg of protein was used for each array pad. A biotinylated detection antibody cocktail followed by a streptavidin-conjugated DyLight 680® was used to visualize the bound detection antibody. Arrays were developed by fluorescent imaging and captured with a digital imaging system. Spot intensities were quantified using Typhoon™ 9410 and ImageQuant™ TL Array Analysis software version 7 . Mean fluorescence intensities for each TKR and signaling node were determined from the 12 cpAC and normal lung tissue lysates.P < 0.05. Tests were executed with Prism® v.6.02 for Windows .Differences in the RT-PCR relative tumor and normal lung tissue cDNA densitometry values for the paired samples from three dogs and IHC pixel analyses values from all 12 dogs were assessed using a Wilcoxon matched-pairs signed- rank test for nonparametric analysis. Likewise, mean fluorescence intensities for each TKR and signaling node from the 12 dogs provided a small data set thus giving little power to detect non-Gaussian distributions and therefore were evaluated using a nonparametric analysis of Wilcoxon matched-pairs signed- rank test which assumes that the differences in groups are distributed symmetrically around their median. The confidence level was set at 95%. Significance was set at ALK: Anaplastic lymphoma receptor tyrosine kinase; TIE-2: Angiopoietin-2; cpAC: Canine pulmonary adenocarcinoma; CD117/c-kit: Canine stem cell factor receptor; EML4: Echinoderm microtubule-associated protein-like 4; EGFR: Epidermal growth factor receptor; EphB1: EPH receptor B1; EphB3: EPH receptor B3; FGFR: Fibroblast growth factor receptor; hpAC: Human pulmonary adenocarcinomas; hNSCLC: Human non-small cell lung cancer; IHC: Immunohistochemistry; MAPK: Mitogen-activated protein kinase; MET: Met proto-oncogene; PFS: Progression-free survival; RT-PCR: Reverse-transcriptase polymerase chain reaction; ROS1: Tyrosine kinase domain: c-ros oncogene 1; RET: Receptor tyrosine kinase: ret proto-oncogene; TKIs: Tyrosine kinase inhibitors; phosphoTKR: Phosphorylated tyrosine kinase receptors; PDGFRα: Platelet-derived growth factor receptor alpha; PDGFRβ: Platelet-derived growth factor receptor beta; VEGFR2: Vascular endothelial growth factor receptor two; VEGF: Vascular endothelial growth factor; HER2: v-erb-b2 erythroblastic leukemia viral oncogene homolog 2; AKT: v-akt murine thymoma viral oncogene homolog.The authors declare no financial or non-financial competing interests.GL conceived the study, designed the experiments, interpreted the data and wrote the manuscript. EM performed the experiments and data analysis and interpretation of the results as part of the NIH/Merial OSU veterinary summer scholar research program. CP performed the subjective evaluation of the histopathology. All authors read and approved the final manuscript.


Amyotrophic lateral sclerosis (ALS) is an adult motor neuron disease characterized by premature death of upper and lower motor neurons. Two percent of ALS cases are caused by the dominant mutations in the gene for superoxide dismutase 1 (SOD1) through a gain of toxic property of mutant protein. Genetic and chimeric mice studies using SOD1 models indicate that non-neuronal cells play important roles in neurodegeneration through non-cell autonomous mechanism. We review the contribution of each glial cell type in ALS pathology from studies of the rodent models and ALS patients. Astrogliosis and microgliosis are not only considerable hallmarks of the disease, but the intensity of microglial activation is correlated with severity of motor neuron damage in human ALS. The impaired astrocytic functions such as clearance of extracellular glutamate and release of neurotrophic factors are implicated in disease. Further, the damage within astrocytes and microglia is involved in accelerated disease progression. Finally, other glial cells such as NG2 cells, oligodendrocytes and Schwann cells are under the investigation to determine their contribution in ALS. Accumulating knowledge of active role of glial cells in the disease should be carefully applied to understanding of the sporadic ALS and development of therapy targeted for glial cells. Amyotrophic lateral sclerosis (ALS) is characterized by premature death of upper and lower motor neurons starting in adulthood. The pathology of ALS is characterized by abnormal accumulation of insoluble and misfolded proteins in degenerating motor neurons. Neuronal death results in progressive paralysis, which typically is fatal 2–5 years after the onset due to respiratory failure. Ten percent of ALS cases are inherited, while the rest are considered sporadic and the cause has not been discovered yet. Twenty percent of inherited ALS cases are caused by mutations in the gene encoding for superoxide dismutase 1 (SOD1) , 4. CurrThe discovery of SOD1 mutations led to the development of animal models that recapitulate ALS-like disease. Overproduction of mutated human SOD1 protein in these mouse models leads to a progressive neurodegenerative disease that closely resembles human pathology with a selective motor neuron death and gliosis accompanied by accumulation of misfolded proteins . The selective death of motor neurons initially led the researchers to believe that cell autonomous mechanisms were at play. However, genetic and chimeric mice studies indicate that non-cell autonomous processes might underlie motor neuron loss in these rodent examples and hence potentially in ALS. First, when expression of SOD1 mutations was restricted to either motor neurons or astrocytes, but not in both simultaneously, it did not lead to the development of ALS –15. A reMicroglia, derived from the hematopoietic cell lineage, are generally considered as the primary immune cells of the central nervous system. Microglia are distributed throughout the CNS and are continuously surveying the environment with mobile arborizations of cell processes. Under normal conditions, these cells have been called “resting microglia,” the term recently questioned due to recognition that these cells are actually continuously providing surveillance in the nervous system . They seα, interleukin-1β, interleukin-12, interferon-γ, and others), reactive oxygen species , chemokines and mitogenic factors , anti-inflammatory cytokines (tumor growth factor-β), and neurotrophic factors (IGF-1: insulin-like growth factor-1) [Gliosis has long been known as a component of ALS pathology, with microgliosis recognized in the past 20 years , 27. Furactor-1) –33. α and Interleukin-6 in comparison to the wild-type microglia when stimulated with lipopolysaccharide (LPS) [A detrimental role of mutant SOD1 in microglia was first demonstrated in a cell culture system. Mutant SOD1-expressing microglia released higher levels of Tumor Necrosis Factor-de (LPS) . Non-celde (LPS) . G37R [G85R mice [G93A mice [The active role of microglia in disease progression was demonstrated by three independent experiments. Selective reduction of mutant SOD1 from microglia/macrophages in mice using Cre-Lox system demonstrated that mutant toxicity within microglia slowed disease progression in SOD1G37R  and SOD185R mice . A compl93A mice . G37R mice [G37R mice [in vitro through purinergic receptors and CD14, respectively [The identity of factors that activate microglia has been explored. The innate immune system is the first line of defense against invading pathogens which are recognized mainly through toll-like receptors (TLRs). Elevated level of innate immune receptors such as TLR2 and CD14 was recorded in mutant SOD1 mice. Further, bone marrow deficient of MyD88 (myeloid differentiation factor 88), essential adaptor protein to transmit most of TLR signaling, accelerated disease progression in SOD137R mice , 39. Indectively , 41. OthIn contrast to innate immune system, the role of acquired immunity in ALS has recently been extensively investigated. The presence of T lymphocyte in ALS mouse models as well as sporadic ALS patients  suggeste2+ permeable AMPA receptors in motor neurons. However, if mutant SOD1 astrocytes are not present, mutant SOD1 in motor neurons does not have the same effect, which again shows non-cell autonomous death mechanisms in ALS [Astrocytes have many important functions in maintaining and nourishing central nervous system (CNS) neurons. One of the main important functions is to maintain low extracellular concentration of glutamate. Astrocytes clear the excess of glutamate neurotransmitter from the synaptic clefts mostly by employing EAAT2/GLT-1 glutamate transporter. Overabundance of glutamate leads to neuronal excitotoxicity due to excessive neuronal firing and corresponding increased influx of calcium. Accumulating evidence demonstrates that astrocytic function of clearing glutamate is impaired due to a loss of EAAT2/GLT1 transporter in sporadic and familial ALS human cases as well as SOD1 mouse models , 46, 47.s in ALS .In vitro studies confirm that factors released by SOD1 astrocytes in culture media can induce apoptosis in motor neuron cultures. One of the identified toxic factors is neurotrophic growth factor (NGF) [A second role that can be attributed to deleterious astrocyte behavior in ALS is an insufficient release of neurotrophic factors that are important in maintaining neuronal health. Glial-derived neurotrophic factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, and vascular endothelial growth factor are all released by astrocytes and can rescue motor neurons , 50. A lor (NGF) . Similaror (NGF) –55.in vitro, and extends the survival in ALS mice [The role of astrocytes in ALS pathology has been widely recognized and appreciated. Astrocytes have become an interesting therapeutic target, and more new studies of intervention are coming to light. The transcription factor, Nrf2, regulates the expression of genes containing antioxidant response element (ARE), which are preferentially activated in astrocytes. An attempt to activate ARE/Nrf2 in astrocytes has been successful in protecting neighboring neurons ALS mice .On the other hand, in one study where proliferating astrocytes were a target of selective ablation, neither onset nor progression of disease was affected in mutant SOD1 mice . It has Microglia and astrocytes have been recognized as major players in ALS disease. However, other glial cells such as NG2 cells  and oligodendrocytes have not been investigated to a large extent. There are very few reports suggesting that these glial cells might be involved in ALS pathogenesis. A study of human ALS postmortem tissue showed diffuse myelin pallor in the anterolateral columns associated with microglial infiltration and loss in number of small fibers most likely due to intrinsic spinal cord lesions . A laterG93A mice, suggesting that NG2 cells do not play a major role in astrogliosis [NG2 cells (marked by nerve-glia factor 2 proteoglycan antibody) have been very little examined in the context of ALS pathology. NG2 cells are one of the first cells to respond to any changes in CNS environment. They assume activated morphology and start dividing due to any insults or disturbances to the CNS where they contribute to changing cellular environment with producing new astrocytes, oligodendrocytes, and, in some areas of the CNS, neurons. The first report established an increased cell division associated with the ALS disease progression and noticed that a percentage of NG2 cells become astrocytes most likely due to proinflammatory cytokine signaling . Howeverogliosis . NG2 celG93A transgene only in protein zero (P0) positive Schwann cells and these mice were identical to control animals with no changes to locomotion, neuronal loss, or axonal degeneration [G37R from Schwann cells using Cre-mediated gene excision. Surprisingly, the authors discovered that even though disease onset was not altered, the disease progression was dramatically accelerated suggesting a connection between disease progression in ALS and a protective effect of mutant SOD1G37R in Schwann cells. Finally, they observed that reduced mutant SOD1 expression was associated with diminished levels of insulin-like growth factor 1 [Schwann cells—peripheral myelin generating cells—are closely associated with motor neuron axons and aid the axonal development and regeneration. So far, very little is known about Schwann cell involvement in ALS pathology. Studies of human ALS show peripheral myelin changes along the motor neuron axons which are most likely due to axonal degeneration . Recent neration . The stufactor 1 . A closeActive contributions of glial cells in ALS pathology have recently been extensively demonstrated as reviewed here. Finally, it should be well considered whether translating the research results using SOD1 rodent models into understanding and development of treatment for sporadic ALS. To date, several clinical trials using drugs targeting glial cells were designed for sporadic ALS patients. These drugs were proved to have effect on mutant SOD1 mice. Antibiotics, minocycline and cyclooxygenase 2 inhibitor, celecoxib, were effective to extend the survival for mutant SOD1 mice –71. HoweFirst, in many preclinical studies, the drugs were administered to animals before onset. However, this is not the case for sporadic ALS patients, since human patients are treated after the diagnosis. Second, in many cases the drug effects aiming to extend the survival time of mice were modest, with cohort sizes that were not sufficient enough. Adequate cohort size as well as the timing of initiating drug treatment should be carefully considered in the rodent studies . Third, Controlling neuroinflammation and communication to immune system have also been the focus in other neurodegenerative diseases including Alzheimer's and Parkinson's diseases , 80. Fur


X-chromosome inactivation (XCI) is a pivotal epigenetic mechanism involved in the dosage compensation of X-linked genes between males and females. In any given cell, the process of XCI in early female development is thought to be random across alleles and clonally maintained once established. Recent studies, however, suggest that XCI might not always be random and that skewed inactivation may become more prevalent with age. The factors influencing such XCI skewing and its changes over time are largely unknown. To elucidate the influence of stochastic, heritable and environmental factors in longitudinal changes in XCI, we examined X inactivation profiles in a sample of monozygotic (MZ) (n = 23) and dizygotic (DZ) (n = 22) female twin-pairs at ages 5 and 10 years. Compared to MZ twins who were highly concordant for allelic XCI ratios, DZ twins showed much lower levels of concordance. Whilst XCI patterns were moderately stable between ages 5 and 10 years, there was some drift over time with an increased prevalence of more extreme XCI skewing at age 10. To our knowledge, this study represents the earliest longitudinal assessment of skewed XCI patterns, and suggests that skewed XCI may already be established in early childhood. Our data also suggest a link between MZ twinning and the establishment of allelic XCI ratios, and demonstrate that acquired skewing in XCI after establishment is primarily mediated by stochastic mechanisms. These data have implications for our understanding about sex differences in complex disease, and the potential causes of phenotypic discordance between MZ female twins. Unlike males who have only one X chromosome, females carry both a maternally- and paternally-inherited X chromosome. To ensure dosage compensation of X-linked genes with males, many loci on the X chromosome are allelically inactivated in females. This X chromosome inactivation (XCI) is associated with hypermethylation of CpG islands, a process regulated by the X-chromosome-inactivation centre We observed similar allelic patterns of XCI between MZ and DZ twins at ages 5 and 10 see Table 1 A wide range of change in allelic XCI patterns between ages 5 and 10 years was observed  during childhood development. The mean allelic XCI ratio across all individuals resided near 50∶50 at both ages, as expected given the presumed stochastic nature of XCI. Interestingly, however, we find that allelic XCI skewing of greater than 20% (i.e. XCI ratio >70∶30 or <30∶70) is already apparent in some children by age 5  and becomes more prevalent by age 10 . These data support the conclusions from previous analyses demonstrating an increase in XCI skewing with age Our use of a genetically informative twin design allowed us to elucidate the factors  contributing to allelic skewing of XCI. Twin correlations for XCI patterns were high for MZ twins  but very low for DZ twins at both ages , concurring with existing data change in XCI patterns between ages 5 and 10 years further supports the notion that alterations in XCI ratios in early development are primarily regulated by stochastic mechanisms Because DZ twins originate from individually fertilized embryos that are subjected to independently established XCI patterns, it is not surprising that our data concur with other studies highlighting minimal correlation between DZ twin-pairs. Once XCI patterns are established they are subject to stochastic drift over embryonic  development Despite the overall high MZ twin correlations in allelic XCI ratios observed at both time points, there is still noticeable XCI discordance among genetically identical individuals: the average within-twin-pair difference in allelic XCI ratio was 10.8% (range from 0.71% to 24.7%) at age 5 and 10.7% (range from 0.49% to 27.7%) at age 10. The observed discordance in XCI patterns within MZ twin-pairs adds to the growing body of evidence of how genetically identical individuals can be epigenetically different It is important to note that our study has several limitations and that the findings reported here require replication in additional longitudinal sample cohorts. First, our study assessed longitudinal changes in XCI using DNA obtained from buccal cells, which may not necessarily be reflective of patterns observed in other cell types. However, whilst it is likely that there are tissue-specific factors influencing XCI, data from our group and others indicate that, as expected for a key epigenetic pattern established early in development, allelic patterns of XCI are strongly conserved across tissues obtained from the same individual, including comparisons of blood and saliva To conclude, this study assessed longitudinal changes in skewed XCI patterns in both MZ- and DZ-twin pairs during the course of early childhood development. Findings from our genetically informative study support the notion of a link between MZ twinning and the establishment of allelic XCI ratios, and suggest that acquired skewing in XCI after establishment is primarily mediated by stochastic mechanisms. A better understanding of the factors underlying skewed XCI will provide insight into the role it plays in mediating sex differences in complex disease and phenotypic discordance between MZ female twins.Samples were obtained from Caucasian MZ and DZ twin-pairs enrolled in the Environmental Risk (E-Risk) Longitudinal Twin Study, which has been described in detail elsewhere n repeat in the first exon of the human androgen receptor (AR) gene as described previously AR gene is highly correlated with X-inactivation status HpaII, MspI or water. Digested DNA was amplified using primers (one fluorescently labeled) flanking the polymorphic (CAG)n repeat in the first exon of the AR gene (F: 5′-FAM-GCTGTGAAGGTTGCTGTTCCTCAT-3′ and R:5′-TCCAGAATCTGTTCCAGAGCGTGC-3′). Fluorescently labeled products were separated on an automated DNA sequencer  to accurately quantify the peak heights for each allele. Allelic XCI ratios were calculated as the normalized ratio of peak heights of the shorter to longer alleles of the HpaII digest to those observed in a water (mock) digest.Allelic XCI ratios were determined by examining DNA methylation in the vicinity of a polymorphic (CAG)The formulae used to calculate XCI ratio were as follows see also :m) = allele 1 peak height/allele 2 peak heightRatio in mock digestion (RHpaII digestion (Rh) = allele 1 peak height/allele 2 peak heightRatio in n) = Rh/RmNormalized ratio (Rn/(Rn+1)]×100.Percentage of inactivation of allele 1 = [RMspI digest (which cleaves irrespective of DNA methylation status) on the same DNA samples. All samples were randomized in the experiment and each sample was independently processed in triplicate for the entire experiment to control for potential technical bias. The average XCI scores across all readings were calculated for each individual and reported in this study.Digestion efficiency was confirmed using the


Understanding how cells migrate individually and collectively during development and cancer metastasis can be significantly aided by a computation tool to accurately measure not only cellular migration speed, but also migration direction and changes in migration direction in a temporal and spatial manner. We have developed such a tool for cell migration researchers, named Pathfinder, which is capable of simultaneously measuring the migration speed, migration direction, and changes in migration directions of thousands of cells both instantaneously and over long periods of time from fluorescence microscopy data. Additionally, we demonstrate how the Pathfinder software can be used to quantify collective cell migration. The novel capability of the Pathfinder software to measure the changes in migration direction of large populations of cells in a spatiotemporal manner will aid cellular migration research by providing a robust method for determining the mechanisms of cellular guidance during individual and collective cell migration. Cellular migration has been shown to be an important process in cancer progression, development, tissue repair, and immune response Previous research into individual cell migration has revealed important fundamental mechanisms by which cells migrate. For instance, when an individual cell migrates on a two-dimensional (2D) surface, it projects a front end extension that can either be broad (termed a llamelipodia) or with multiple spike-like extensions (termed fillipodia), which are the result of coordinated polymerization, depolymerization, and branching of the actin cytoskeleton in vitro studies conducted in 2D cell culture model systems in vivo. For instance, in 2D, 3D and in vivo experiments, CDC42/Rac activity determine cellular polarity in vivo studies, they remain a valuable tool for initial investigations into the molecular mechanisms behind cellular migration compared to 3D and in vivo studies because they allow for tight control of experimental conditions and more accurate observation of cellular migration behavior at single cell resolution without the use of relatively complex microscopes, such as two-photon and confocal microscopes. Many of the concerns about discrepancies in biochemical mechanisms behind 2D and 3D motility may prove to be overcome by imaging individual cell motility in 2D on soft extracellular matrices, which have been shown to be more closely similar to in vivo tissues than plastic or glass cell culture plates Our current understanding of the biochemical mechanisms underlying cellular migration have been primarily the result of The behavior of migrating cells can be characterized by migration speed, migration direction, and migration persistence (the ability of a cell to maintain its migration direction). In 2D studies, the measurement of cell migration behavior is conducted by either manual cell tracking Although several computational tools exist that allow for automated cell tracking of individual cells in time-lapse microscopy videos Fig. 1, 2 for both HaCaT and MDA-MB-231 cells. For confluent monolayer experiments, HaCaT cells were plated at an average density of 1000 cells/mm2. For epithelial sheet assays, HaCaT cells were plated at an average density of 1200 cells/mm2 for 3 hours at 37°C, after which half of partially adherent cells were manually removed using a 200 µl pipet tip. Transforming Growth Factor-Beta (TGFβ) stimulations were conducted with 100 pM ligand, while EGF stimulations were conducted with 100 nM ligand. An ImageXpress MicroXL high throughput wide-field fluorescence microscope (Molecular Devices) was used for imaging experiments at 37°C and 5% CO2. All microscopy videos were acquired with a frame rate of one frame every seven minutes, in the mCherry fluorescence channel at a magnification of 10×, where each pixel represents 1.314 µm at a pixel binning of 2×2. The accompanying MetaXpress software was used to compile video files from time-lapse images for each well of a 96 well plate.Stable transgenic HaCaT  and MDA-MB-231  cell lines were fluorescently labeled via retroviral mediated gene transfer of mCherry-Histone H2B using the pRex-mCherry-H2B plasmid. For all experiments, cells were cultured in DMEM lacking phenolphthalein red and supplemented with 2 mM L-glutamine, 100 Units/mL penicillin and 100 µg/mL streptomycin. For low density assays, cells were plated at an average density of 300 cells/mmhttps://universityofcolorado.box.com/s/qzs6nos4470sjfsaw8wg and Data S1) was written in the Java programming language. User specification is required for cell radius (pixels), minimum track length, the interval of frames for desired calculations , percentage of pixels in the video that represent cells, and the directory path for the folder containing .avi files . The output for each video file is a single Excel spreadsheet (Data S2). The Pathfinder software requires only decompression of the attached .zip file and installation of JAVA runtime environment on either a 32-bit or 64-bit Windows Machine. Please note that use of pathfinder on a 64-bit machine allows for higher memory use in Java, which allows for analysis of greater numbers of cells in a single video.The Pathfinder software  over 1 frame. Conversion to µm/hour is determined by the following equation:The Angle of Trajectory was calculated from the following discontinuous equations:Angle of Deflection was calculated from the following discontinuous equations:Data S3).Persistence time calculations were performed using a modified in-house MatLab based program developed by Dr. Douglas Lauffenburger (MIT) (Trajectory) calculations were also done in Excel, where the standard deviation of the migration directions between pairs of neighboring cells were averaged over greater than 100 pairs of cells for each condition. This average of standard deviations is referred to as the PRMI ΘTrajectory.Nearest neighbor calculations were done in Excel using an inter-centroid distance matrix of × for each frame. Nearest neighbors were defined as cells whose centroids are within 100 µm of each other. Paired random migration index of the angle of trajectory , as well as to assemble such positional information into cellular tracks , as described in their publication , S4). Simultaneous reporting of these parameters makes the Pathfinder software unique compared to other available computational motility programs  developed by Sbalzarini programs Fig. 1. Fig. S3). However, we do not rule out that different methods of gene delivery and types of nuclear markers (for instance a fluorescent protein other than histone H2B) could lead to permanent changes in cell migration.We compared the migration speeds of wild type MDA-MB-231 cells to MDA-MB-231 cells expressing a nuclear fluorescence marker in the presence and absence of EGF in order to determine if the introduction of nuclear marker significantly impacted ligand induced migration. Unlabeled wild type cells were manual segmented and analyzed using Pathfinder, while fluorescent images were automatically segmented and analyzed using Pathfinder. In order to provide a means for high throughput calculation of cellular migration persistence, we used a non-traditional, but direct, approach of calculating the angle of deflection for each cell at each time.  to turn Fig. 3A.MSD is the mean squared displacement of the cell, P represents the persistence time of a cell, and t is the time.Although we measure migration persistence using the average absolute angle of deflection, the measurement of migration persistence is currently conducted in the cell migration field through the determination of persistence time, which is calculated using data fitting of time dependent mean squared displacement trends to Persistence time is used to measure migration persistence because local changes in mean squared displacements trends are likely to be associated with changes in the direction of cellular migration, provided that speed is taken into consideration. A key difference between our approach and the persistence time approach is that persistence time measurements focus on how long a cell maintains a direction, while the average absolute angle of deflection measurements focus on the degree to which cells in a population turn in each frame. Thus, persistence time measurements reflect behavior over a specified interval of time , while the average absolute angle of deflection measurements reflect the relatively instantaneous behavior of cells.We compared our technique of measuring migration persistence to the method of measuring persistence time by examining the time-lapse microscopy videos of two cell lines, MDA-MB-231 and HaCaT, stably expressing fluorescent nuclear markers and treated with no ligand, TGFβ or EGF. When such videos of HaCaT cells (24–26 hours post ligand stimulation) are analyzed by the two methods, both techniques lead to the same conclusions; TGFβ and EGF stimulation cause increased migration persistence, where EGF has an impact of higher magnitude than that of TGFβ Fig. 3B.The Pathfinder software is unique from other software in that it calculates angular information about individual cells. However, such angular information requires that the determination of a cell's position be relatively noise free. When we closely examine the tracks of individual cells, we find that tracks exhibit slight vibration on the short timescale (7 minutes), such that a cell that migrates relatively straight does not display a perfectly straight track. As a result, we use overlapping intervals for our calculations of angular information in order to suppress the effects of such vibration on angular calculations. The schematic diagram in S2), which is elaborated upon below. Thus, only through rigorous quantitation can large populations be characterized for their cellular behavior.Upon mere qualitative assessment of cellular tracks, cellular behavior is difficult to deduce for large populations of cells. For example, when we examine MDA-MB-231 and HaCaT cell migration in response to either TGFβ or EGF by looking at the tracks of cells between 0 and 35 hours post ligand stimulation, MDA-MB-231 cells appear to not change behavior in response to ligand treatments and HaCaT cells appear to respond in the same manner upon TGFβ and EGF stimulation (p = 0.003), but extremely small in magnitude, decrease in migration persistence, as evident by a slight elevation in the average absolute angle of deflection of cells . Both TGFβ and EGF treatments yield an increase in the average speed of these cells, where EGF response is early (approximately 1 hour) and TGFβ response is late (approximately 10 hours) . In contrast to MDA-MB-231 cells, both speed and migration persistence are activated by TGFβ and EGF treatment in HaCaT cells, where the effects of EGF appear early (approximately 1 hour) and the effects of TGFβ appear late (approximately 10 hours)  Fig. 5D., right, illustrates the orientation of these axes in the field of view, and provides an example of a cell that is migrating in the direction of 45 degrees. In a similar manner to the case of angle of deflection, each cell gets such an assignment for each time, providing time resolution of cellular direction. In order to demonstrate how the angle of trajectory calculation can be used to characterize collective migration, we examined HaCaT and MDA-MB-231 cells in confluent monolayers in response to EGF stimulation. Manual inspection of the cellular tracks of these monolayers suggests that neighboring cells migrate in a similar direction in a ligand dependent manner in HaCaT cells . This behavior is not qualitatively observed for MDA-MB-231 cells . Since collective migration is defined as the ability of cells to adopt a common migration direction, we quantified the average standard deviation of the angle of trajectory, also referred to as the “paired random migration index” (PRMI ΘTrajectory), amongst pairs of nearest neighboring cells at 22–26 hours post ligand stimulation. An increase in this quantity indicates that nearest neighboring cells are migrating in increasingly different directions, meaning collective migration is decreasing. We excluded pairs of neighbors in which one cell migrates with a direction of 0–90 degrees and another cell migrates in a direction of 270–360 degrees, as these pairs would have a falsely high standard deviation due to the discontinuous transition between 360 degrees and 1 degrees. In agreement with our qualitative observations of cellular tracks, nearest neighboring HaCaT cells display a lower PRMI ΘTrajectory in response to EGF stimulation  in order to determine that the maximum value for the PRMI ΘTrajectory is approximately 85 degrees in both the presence and absence of EGF stimulation. This maximum value is greater than that of the PRMI ΘTrajectory in the absence of ligand stimulation, revealing that HaCaT cells do display a small degree of collective migration, which we were not able to detect upon manual qualitative inspection of time-lapse videos. Using the same technique on MDA-MB-231 cells, we found that these cells display a statistically significant, but low magnitude, increase in the PRMI ΘTrajectory amongst nearest neighbors in response to EGF, suggesting that ligand stimulation of these cells causes neighboring cells to exhibit slight repulsion, and migrate more in opposing directions upon EGF stimulation , albeit for short timescales (data not shown).The increasing interest in the collective behavior of cells in the cell migration field prompted us to integrate the capability of characterizing collective migration into the Pathfinder software. One already implemented method for measuring collective migration involves the calculation of the size of collectively migrating streams of cells mulation Fig. 6B. and Movie S3). We asked whether or not ligand stimulation instantaneously affects all cells equally in an epithelial sheet, and found that the collective behavior response to EGF stimulation propagates away from the leading edge of an epithelial sheet overtime as indicated by propagation of decreasing PRMI ΘTrajectory away from the leading edge of an epithelial sheet  amongst nearest neighbors. This tool is likely to propel the collective migration field forward, as it can provide a means for accurately measuring the degree to which cells are migrating collectively, with both spatial and temporal resolution. Our method of measuring collective migration was able to reveal hidden collective cellular behavior that cannot be easily detected qualitatively from cellular tracks, such as the local collective migration behavior amongst pairs of MDA-MB-231 cells, and the ability of EGF stimulation to suppress this behavior. In conclusion, our Pathfinder software provides novel techniques for characterizing cellular migration in a high throughput platform, which are likely to aid the cell migration field in its investigation of the molecular mechanisms behind individual and collective migration.Our Pathfinder software is the first high throughput automated cell migration software to conduct measurements of instantaneous angular parameters for the quantification of cell migration behavior. Our parameter of the angle of deflection has proven useful for characterizing the migration persistence of cells in two dimensional fluorescence microscopy experiments. In addition to helping researchers investigate the molecular mechanisms behind migration persistence, this parameter can be used to identify the differential temporal responses of cells to distinct ligand stimulations, and can provide insight into the molecular mechanisms behind each ligand response. Furthermore, the available cell migration software has been entirely devoid of tools that can be used to readily quantify collective migration. As a result, investigations of collective migration have been more appropriately described as qualitative rather than quantitative. The Pathfinder software provides the first high throughput tool to quantitatively characterize collective cell migration using the angle of trajectory parameter, which can be used to determine the “Paired Random Migration Index” Click here for additional data file.Data S2A sample output from Pathfinder. A sheet of HaCaT H2B-mCherry cells were analyzed for individual cell migration from a microscopy video between 22 and 25.5 hours post 100 nM EGF stimulation. Frames were acquired every 7 minutes. Analysis was conducted with a frame binning of 3 frames (21 minutes).(XLSX)Click here for additional data file.Data S3A MATLAB script to convert mean squared displacement (MSD) versus time data from a Pathfinder output into persistence time. This MATLAB script requires user input of MSD and time data for each cell and converts this information into persistence time.(M)Click here for additional data file.Figure S1Parameter descriptions for the Pathfinder program GUI. User input parameters are: Cell Outer Radius, Cell Minimum Radius (Cutoff), Percentage of Pixels with Nuclear Signal (Percentile), How Far an Average Cell is Tolerated to Migrate From Frame to Frame (Disp./Frame), How Many Frames to Bin for Calculations (Frame Binning), Minimum Tack Length (Min. Trajectory Length), Number of Parallel Threads (Threads), Folder Path for Folder with Videos (AVI Folder).(TIF)Click here for additional data file.Figure S2A description of output calculations from the Pathfinder program. Each cell receives a cellular ID number (1), for each frame (2). In each frame a cell is assigned an X (3) and Y (4) coordinate, a displacement from the last frame in pixels (5), an angle of trajectory (6), an angle of deflection (7) and a mean squared displacement (8). Mean squared displacements can be used to calculate the persistence time for a cell. For the population of cells, Pathfinder reports the frame (9) dependent change in the average displacement (10), the average angle of trajectory (11), the percentage of cells turning greater than 90 degrees (12), and the average absolute angle of deflection (13). Additionally, Pathfinder reports a binned histogram of percent of cells versus the possible migration directions from 0 to 359 degrees (14 and 15). Lastly, the number of cellular tracks is reported (16).(TIF)Click here for additional data file.Figure S3Wild type MDA-MB-231 cells and MDA-MB-231 H2B-mCherry cells migrate with similar speeds in the presence and absence of EGF stimulation. Brightfield microscopy videos of mock and EGF treated wild type (WT) MDA-MB-231 cells were manually measured for position over the course of a 10 frame interval (7 minutes/frame) after 24 hours ligand or mock stimulation and the average speed of cells was calculated with a frame binning of 3. The same analysis was done on parallel videos of MDA-MB-231 using pathfinder, which yielded similar results for WT and labeled cells in the speed of migration in the presence and absence of EGF. 50 cells were used for this comparison for each condition.(TIF)Click here for additional data file.Figure S4MDA-MB-231 cells maintain physical contact with their nearest neighboring cell. Brightfield microscopy of EGF treated MDA-MB-231 cells reveals that nearest neighboring cells have physical contact with each other.(TIF)Click here for additional data file.Movie S1MDA-MB-231 cells at low density upon either mock, TGFβ, or EGF treatment. MDA-MB-231 cells with an H2B-mCherry nuclear marker were observed by time-lapse microscopy using the mCherry fluorescence channel. Each frame represents 7 minutes.(AVI)Click here for additional data file.Movie S2HaCaT cells at low density upon either mock, TGFβ, or EGF treatment. HaCaT cells with an H2B-mCherry nuclear marker were observed by time-lapse microscopy using the mCherry fluorescence channel. Each frame represents 7 minutes.(AVI)Click here for additional data file.Movie S3Epithelial sheets of HaCaT cells upon either mock or EGF treatment. HaCaT cells with an H2B-mCherry nuclear marker were assembled into epithelial sheets and observed by time-lapse microscopy using the mCherry fluorescence channel. Each frame represents 7 minutes.(AVI)Click here for additional data file.


Autoimmune uveitis is an intraocular inflammation that arises through autoreactive T-cells attacking the inner eye, eventually leading to blindness. However, the contributing molecular pathomechanisms within the affected tissues remain as yet elusive. The extracellular matrix (ECM) is a highly dynamic structure that varies tremendously and influences the encompassing tissue. In order to assess ECM re-modeling in autoimmune uveitis, we investigated the expression of ECM molecules fibronectin and osteopontin in vitreous and retina samples. This was carried out in the only spontaneous animal model for human autoimmue uveitis, namely equine recurrent uveitis (ERU) that resembles the human disease in clinical as well as in immunopathological aspects. ERU is a naturally occurring autoimmune disease in horses that develops frequently and has already proved its value to study disease-related pathomechanisms. Western blot analysis of fibronectin and osteopontin in healthy and uveitic vitreous revealed significant reduction of both proteins in uveitis. Immunohistochemical expression of fibronectin in healthy retinas was restricted to the inner limiting membrane abutting vimentin positive Müller cell endfeet, while in uveitic sections, a disintegration of the ILM was observed changing the fibronectin expression to a dispersed pattern extending toward the vitreous. Retinal expression of osteopontin in control tissue was found in a characteristic Müller cell pattern illustrated by co-localization with vimentin. In uveitic retinas, the immunoreactivity of osteopontin in gliotic Müller cells was almost absent. The ability of Müller cells to express fibronectin and osteopontin was additionally shown by immunocytochemistry of primary cultured equine Müller cells and the equine Müller cell line eqMC-7. In conclusion, severe ECM re-modeling in autoimmune uveitis reported here, might affect the adhesive function of fibronectin and thus the anchoring of Müller cell endfeet to the ILM. Furthermore, the absence of osteopontin in gliotic Müller cells might represent reduced neuroprotection, an osteopontin attribute that is intensively discussed. Autoimmune uveitis is a sight-threatening intraocular inflammation driven by eye-invading autoreactive T-cells that cross the blood-retinal barrier Fibronectin, a glycoprotein of the ECM, mediates cellular-ECM interactions and comprises functions such as cell adhesion, migration, growth and differentiation Osteopontin, a major phosphoprotein of the ECM, has gained interest since reports of its functions associated with inflammation and neuronal disorders are inconsistent. Neuroprotective effects versus proinflammatory properties of osteopontin are discussed. The neuroprotective potential of osteopontin was demonstrated in several studies. Osteopontin treatment of oxygen and glucose deprived cortical neuron cultures protected against cell death and intracerebral ventricular application of osteopontin reduced infarct size in a murine stroke model The purpose of this study was to investigate the expression of ECM proteins fibronectin and osteopontin in a spontaneous animal model for autoimmune uveitis to gain further information about these proteins and the role of ECM in a retinal disorder.For this study, a total of 17 ERU diseased specimens and 16 healthy controls were collected and processed. ERU eye tissue specimens derived from horses that were treated in the equine clinic and diagnosed with ERU according to clinical criteria as described Control samples  were obtained from horses euthanized due to incurable and ERU unrelated diseases or where obtained from a local slaughterhouse. Vitreous samples of ERU cases were obtained during therapeutic pars-plana vitrectomy. Uveitic eyes derived from horses that had to be enucleated during a therapeutical procedure. The collection and use of equine eyes derived from horses that were killed due to a research-unrelated cause, was approved for purposes of scientific research by the appropriate board of the veterinary inspection office Munich, Germany (Permit Number: 8.175.10024.1319.3). Horses were treated according to the ethical principals and guidelines for scientific experiments on animals according to the ARVO statement for the use of animals in Ophthalmic and Vision research. No experimental animals were involved in this study.Vitreous samples were stabilized with EDTA-free protease inhibitor , then lyophilized, thereafter solubilised in ultrapure water and dialysed against 50 mM phosphate buffer (pH 7.6). Protein content was determined using the Bradford assay .For immunohistochemistry, posterior eyecups were immersion-fixed with Bouin's solution , dehydrated in a series of alcohols and sliced into pre-assigned fragments as described before 2HPO4×2H2O, 1.4 mM KH2PO4, 2.6 mM KCl, pH 7.2, 0.05% Tween20). Binding was detected by horseradish peroxidise (HRP)-coupled secondary antibody incubation for 1 hour at room temperature. After 6 further washing steps in PBS-T, signals were detected by enhanced chemoluminescence on X-ray films . Films were scanned on a transmission scanner and quantification of western blot signals by densitometry was performed using ImageQuantTL software . Osteopontin and fibronectin abundances between ERU cases and controls were statistically analyzed using the Mann-Whitney test. The differences in protein expression were considered significant if the significance level was ≤0.05.Equal total protein amounts from vitreous samples were separated by SDS-PAGE (8% and 12% gels) and blotted semidry onto polyvinyl difluoride (PVDF) membranes . Unspecific binding was blocked with 1% polyvinylpyrrolidon - tween (PVP-T) for 1 hour at room temperature. Blots were incubated with primary antibodies (anti-osteopontin and anti-fibronectin) at 4°C overnight and then washed 3 times in phosphate buffered saline – tween . To prevent unspecific antibody binding, sections were blocked with 1% BSA in tris buffered saline – tween  containing additionally 5% of a non-immune serum derived from the secondary antibody animal species, for 40 min at RT prior to every primary antibody incubation.Multiple labelling was performed consecutively, with blocking steps  between single antibody incubations. For triple labelling, sections were first consecutively incubated with primary antibodies . Each primary antibody incubation was followed by the respective secondary antibody (30 min at RT). Cell nuclei were counter-stained with 4′,6-diamidino-2-phenylindole . Finally, the sections were mounted with glass coverslips using Dako fluorescence mounting medium . Fluorescent images were recorded with an Axio Imager M1 or Z1 and the Axio Vision 4.6 software  at a total magnification of 40×.Negative controls for all immunohistochemical experiments included omission of the primary antibody as well as incubation with isotype-matched primary antibody of irrelevant specificity.+; Sarstedt, Nümbrecht, Germany) after trituration and centrifugation. The cells were allowed to attach for 24 h at 37°C and 5% CO2 in an incubator and non-attached cells were removed. Using the above described method, we previously established the first equine Müller cell line that was named eqMC-7 (equine Müller cell line – 7) Primary Müller cells were isolated from healthy equine retinas according to a method described previously For immunocytochemistry eqMC-7 cells and primary cultured equine Müller cells were trypsinized and seeded onto glass slides which were placed in culture plates. Cells were cultured in DMEM omitting FCS supplement for 16 h and were allowed to attach to the glass surface within this time. Slides were rinsed in phosphate buffered saline (PBS) and fixed in acetone for 10 min. To prevent unspecific antibody binding, slides were blocked with 1% BSA in TBS-T containing 5% appropriate serum (according to the species the secondary antibody was produced in) for 40 min at room temperature prior to every primary antibody incubation. Fluorescence labelling and image acquisition was performed similarly as for immunohistochemistry of retina sections.Goat polyclonal antibody against mouse osteopontin from R&D Systems  was used at a dilution of 1∶100 for immunohisto- and immunocytochemical experiments and at a dilution of 1∶500 for western blots. Polyclonal rabbit-anti-fibronectin antibody  was applied at a dilution of 1∶100 in immunohistochemistry and 1∶400 in immunocytochemistry. It was used at 1∶1000 in western blotting experiments. Monoclonal mouse anti-vimentin antibody (Clone V9) from Sigma-Aldrich  was used at a dilution of 1∶400 in immunohisto- and immunocytochemistry. Alexa Fluor labelled secondary IgG antibodies were obtained from Invitrogen  and all used at a working dilution of 1∶500. We used goat anti-mouse IgG coupled to alexa 488, goat anti-rabbit IgG alexa 647 and donkey anti-goat IgG alexa 546.In order to investigate the occurrence and expression level of fibronectin and osteopontin in vitreous of healthy and ERU diseased eyes, we performed SDS-PAGE and subsequent western blotting experiments. Expression of fibronectin in healthy  and a baIn a previous proteomics study comparing the retinal membrane fraction of healthy and uveitic samples we already demonstrated that retinal fibronectin expression is increased in autoimmune uveitis compared to control Osteopontin was shown to be expressed in retinal ganglion cells within the mouse retina and this expression pattern, as well as the intensity, persisted in mice with interphotoreceptor retinoid-binding protein (IRBP) induced experimental autoimmune uveitis (EAU) In order to further characterize the expression patterns of fibronectin and osteopontin within the equine retina, we performed triple labellings of healthy and uveitis diseased retina sections with the Müller cell marker vimentin. In healthy retinas, vimentin was characteristically expressed throughout the entire Müller cell from endfeet that cover the ILM to branches that pass through the inner (INL) and outer nuclear layer (ONL) to terminate at the outer limiting membrane (OLM) . In uveiIn order to verify the Müller cell associated osteopontin immunoreactivity observed in the retina , and to A comprehensive understanding of autoimmune uveitis pathogenesis remains as yet elusive Another very important function of Müller cells within the retina is neuroprotection that is performed by the release of neurotrophic factors as well as by providing various functions that support neurons in nutrition and metabolism In the present study, a loss of osteopontin expression in Müller cells of retinas from horses with autoimmune uveitis was evident . Müller In uveitic equine retinas an expression of both, osteopontin and fibronectin was additionally detected in a rough line aside from the mostly disintegrated outer limiting membrane . Since pInvestigations of molecular mechanisms in autoimmune uveitis are mainly focused on cell-associated proteins, whereas the knowledge about ECM protein expression in disease pathogenesis is limited. We aimed to determine the role of ECM in autoimmune uveitis-related tissues by investigating expression profiles of relevant ECM proteins. The results indicate severe ECM re-modeling in autoimmune uveitis highlighted by reduced neuroprotection mediated by Müller cell-derived osteopontin and a loss of Müller cell adhesion through fibronectin. Thus the investigation of ECM proteins is particularly worthwhile to gain further insight into the molecular pathomechanisms occurring in autoimmune uveitis affected tissues. The impact of ECM re-modeling and its consequences for the surrounding tissue in autoimmune uveitis merits further investigation.


Indeed, OCT allows for the first time a non-invasive visualization of axons of the central nervous system (CNS). Reduction of retina nerve fibre layer (RNFL) thickness was suggested to correlate with disease activity and duration. However, several issues are unclear: Do a few million axons, which build up both optic nerves, really resemble billions of CNS neurons? Does global CNS damage really result in global RNFL reduction? And if so, does global RNFL reduction really exist in all MS patients, and follow a slowly but steadily ongoing pattern? How can these (hypothesized) subtle global RNFL changes be reliably measured and separated from the rather gross RNFL changes caused by optic neuritis? Before generally being accepted, this interpretation needs further critical and objective validation.We prospectively studied 37 MS patients with relapsing remitting (n = 27) and secondary progressive (n = 10) course on two occasions with a median interval of 22.4±0.5 months [range 19–27]. We used the high resolution spectral domain (SD-)OCT with the Spectralis 3.5 mm circle scan protocol with locked reference images and eye tracking mode. Patients with an attack of optic neuritis within 12 months prior to the onset of the study were excluded.Although the disease was highly active over the observation period in more than half of the included relapsing remitting MS patients (19 patients/32 relapses) and the initial RNFL pattern showed a broad range, from normal to markedly reduced thickness, no significant changes between baseline and follow-up examinations could be detected.These results show that caution is required when using OCT for monitoring disease activity and global axonal injury in MS. Multiple sclerosis (MS) is supposed to be a chronic inflammatory disease of the central nervous system (CNS) causing demyelination and axonal degeneration all MS patients, or not and if so, whether subtle global RNFL changes can also be measured in short observation periods from 6 months to 2 years With OCT the retina can easily be visualized, its thickness measured and monitored over time. Initial findings supported OCT as a non-invasive and useful method. Baseline characteristics of the retinal nerve fibre layer (RNFL) of MS patients were found to be heterogeneous and ranged from normal to markedly reduced thickness For this purpose we studied a large cohort of 27 RRMS and 10 SPMS patients with a new advanced SD-OCT technique over a long observation period of approximately 22.4±0.5 years. Moreover, despite partly intense disease activity with high relapse rates, none of the included patients had an ON within 12 months prior to onset of the study. In this study we analysed RNFL changes in relation to clinical data including age, gender, MS subtype, disease onset, disease duration, numbers of ON and relapses  as well as the treatment of each participant.The study was approved by the local Ethics Committee . Informed written consent was obtained from all patients and volunteers before study entry.We described the RNFL baseline characteristics of 59 MS  patients in detail previously We studied the 37 MS patients  on two occasions with a median interval of 22.4±0.5 months [range 19 to 27 months] prospectively. The patients were consecutively recruited. MS diagnosis was based on clinical course, MRI and on cerebrospinal fluid (CSF) analysis Baseline clinical neurological examinations, visual evoked potentials (VEP), and ophthalmologic examinations were performed within 7 days. No included patient had an ON within 12 months prior to the onset of the study. A summary of detailed demographic and clinical data for each included MS patient is given in The RNFL measurement was described in detail previously Visual function testing, Visual Field Analysis and Visual evoked potentials were described in detail previously The statistics used was described in detail previously The results of our study performed with the high resolution SD-OCT can be summarized briefly as follows. Over a median observation period of 22.4±0.5 months (range from 19 to 27 months) the RNFL measurements of each MS patient were unchanged compared to baseline . The minThe visual and contrast acuity and sensitivity tests , the colour vision test (Lanthony D-15) and the Humphrey visual field analysis and the visual evoked potentials showed also no changes compared to the baseline In the past few years many OCT studies of MS patients, meta-analyses and reviews aroused great interest Secondly, it is yet unknown whether global RNFL reduction (apart from RNFL changes due to ON) really exists in all MS patients, and if so, when RNFL reduction may occur and whether it arises from continuously ongoing or relapsing (remitting) axonal changes. The hypothesized global RNFL reduction may already occur years before MS onset, or in very early stages of the disease, but also in later stages of the disease. Progressive axonal changes may be elusive in short but obvious in long observations periods. Accordingly, someone might assume, however that the hypothesized continuous but subtle RNFL reduction should occur when the disease course changes from the relapsing-remitting to the secondary progressive phase, or follows a progressive course right from the beginning in some cases of progressive relapsing-remitting MS. However, Henderson et al. reported that they did not detect any significant RNFL reduction (‘RNFL thinning’) in 34 MS patients with progressive disease  in a rather long observation period  anterograde axonal degeneration in the whole CNS? To the best of our knowledge ON was never found to be correlated to, or proclaimed to cause diffuse axonal degeneration in the whole CNS, respectively brain atrophy.Finally, whatever, the underlying pathogenetic mechanisms might be, it is completely unclear whether the damage of few axons along the optic nerves represent really the anatomic and functional fate of hundred of billions neurons in the entire CNS. In fact, only rarely the MRI lesion load correlates to the clinical course of MS patients. Consequently, the question arises why the RNFL thickness should indeed correlate strictly to MRI parameters and the clinical course of MS patients. In other words, why should an hypothesized retrograde trans-synaptic axonal degeneration have such an effect on the RNFL, but in contrast does an ON not lead to widespread did not find RNFL reduction (‘RNFL thinning’) in a well defined cohort of RRMS and SPMS with partly intense disease activity. Our data suggest that OCT can yet not replace MRI, or even serve as new surrogate marker in MS.In conclusion, even if most of the MS patients follow a primary relapsing-remitting course with clinical stable intervals over months, years or sometimes even decades, that does of course not rule out subclinical ‘silent’ ongoing degenerative changes