In renal patients estimation of GFR is routinely done by means of population-based formulae using serum creatinine levels. For GFR determination in the creatinine-blind regions or in cases of reno-hepatic syndrome as well as in critical cases of live kidney donors individualized measurements of GFR (mGFR) employing the kinetics of exogenous filtration markers such as the inulin-like polyfructosan sinistrin are necessary. The goal of this study is to compare mGFR values with the eGFR values gained by the Modification of Diet in Renal Disease (MDRD4) and Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) formulae.In 170 subjects comprising persons with normal renal function or with various stages of kidney diseases (CKD 1-4) GFR was measured by application of intravenous bolus of sinistrin and assessment of temporal plasma concentration profiles by means of pharmacokinetic methods (mGFR). Comparisons of mGFR with MDRD4- and CKD-EPI-derived eGFR values were performed by means of linear regression and Bland-Altman analyses.2]. In cases of normal or mildly impaired renal function, GFR determination by MDRD4 or CKD-EPI tends to underestimate GFR. Notably, there is practically no difference between the two eGFR methods.Reasonable agreement of mGFR and eGFR values was observed in patients with poor renal function [GFR below 60 (ml/min)/1.73 m2] eGFR values are unreliable and should be replaced by clinically and physiologically suitable methods for mGFR determination.For routine purposes or for epidemiological studies in cases of poor renal function eGFR methods are generally reliable. But in creatinine-blind ranges [GFR above 60 (ml/min)/1.73 mhttp://www.consort-statement.org/index.aspx?o=1190 For routine applications or for epidemiological studies, GFR values are estimated on the basis of individual serum creatinine measurements by means of regression formulae, e.g., the MDRD4 and CKD-EPI formulae -4. These2 only , the statistical criteria show much better agreement between mGFR and both eGFR values; however, patients with better renal function [mGFR > 60 (mL/min)/ 1.73 m2] in accordance with the graphical representation exhibit even worse results. Splitting the latter group of patients again according to a cut-off threshold of mGFR smaller or larger than 90 (mL/min)/ 1.73 m2 , similar results are obtained with the worst agreement seen in individuals with normal mGFR > 90 (mL/min)/ 1.73 m2. Table 2 as mGFR cut-off value gained by the kinetic procedure: for the MDRD-formula the diagnostic specificity is 0.875, whereas for the CKD-EPI-formula it is 0.891. The diagnostic sensitivity for the MDRD-formula is 0.902, whereas for the CKD-EPI-formula it is 0.878.There is no real difference between the MDRD4- and the CKD-EPI-methods over the GFR ranges studied. As can be seen from Table The purpose of the present study is to compare the application of compartment analysis techniques using kinetic data of the GFR marker sinistrin with formula-derived GFR estimates based on creatinine levels. The kinetic procedure used here as a standard for comparison, has been validated previously by successfully predicting in individual patients the concentration-profile of a constant infusion experiment using results obtained by a bolus experiment beforehand . The modAs Table One might ask whether it is necessary to perform a full kinetic analysis of the time-dependent concentration curves of a marker like sinistrin or whether a simple analysis of the area under the concentration curve would suffice. There are several arguments supporting the full kinetic analysis: first, considering the relevant large volumes of marker solutions and the individually different states of the veins of the patients, the administration of the marker during a reasonable time – 3 minutes instead of a truly instantaneous bolus injection – is possible. Moreover, it is practically impossible to employ a too extended experimental protocol. Both these practical limitations are in favor of the full kinetic analysis of the applied marker. Second, the availability of sufficient computer power enables one to extract more information out of concentration curves of exogenous markers. This is of importance in critical cases as, e.g., in live kidney donors. Third, by means of the full kinetic analysis it is possible to approach much more complicated scenarios: for example, the immediate response of the kidneys to external dietetic or pharmacologic loads, which may cause both changes in GFRs as well as shifts in the extracellular fluids from central to peripheral compartments, can be studied with the same method, yielding information on the state of the renal vasculature. For this purpose it is necessary to perform two consecutive experiments with the external loads in between. In the second experiment, this procedure requires the calculation of the remaining marker amounts in the two compartments, since now these remaining amounts are needed as initial values for the model identification describing the second experiment. Moreover, the kinetic techniques allow also the determination of the error bounds of the characteristic system constants . The appSinistrin is a clinically and physiologically suitable marker and the bolus method assessed by compartmental kinetic analysis is a both reliable and clinically practicable procedure for the measurement of GFR. This is of utmost importance in subjects requiring higher than routine accuracy and precision as, e.g., in situations of live kidney donor evaluations . As statFor correct determination of GFR, particularly in subjects with normal or slightly impaired renal function (CKD 1-2), kinetic procedures using an exogenous physiologically and clinically suitable marker such as sinistrin allow individualized GFR measurements in contrast to population-derived estimations by means of formulae employing endogenous markers. Additionally, a model-based technique enables one to assess consecutive clearance tests with a pharmacological or dietetic load in between. Such tests can be used for obtaining information on the properties of the renal functional reserve and the renal micro-vasculature, e.g., on changes in its permeability caused by renal injuries . DynamicSummarizing it can be stated that both the accuracy and precision of mGFR values achievable by the kinetic procedure using sinistrin as exogenous marker as well as the possibility for evaluation of the renal functional reserve appear to be of decisive importance in cases of live kidney donors. Finally although so far eGFR methods using creatinine levels only have been considered as sufficiently satisfactory, recent developments suggest combinations of creatinine and cystatin C in regression formulae as necessary improvements in GFR estimations . HoweverCKD-EPI: Chronic Kidney Disease – Epidemiology Collaboration; MDRD: Modification of Diet in Renal Disease; eGFR: Glomerular filtration rate estimated on basis of serum creatinine concentration; mGFR: Glomerular filtration rate measured by full kinetic analysis.The authors declare that they have no competing interests.SZ: Made substantial contributions to conception and design and performed GFR measurements. WS, GR and WE: Performed statistical analyses, contributed to interpretation of data, and revised the manuscript critically for important intellectual content. AM: Performed analytical procedures. DW: Performed GFR measurements. ARR: Made substantial contributions in drafting the manuscript and revised it critically for important intellectual content. The final version of this paper was approved by all authors.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/14/159/prepub


The formation of glial scars after spinal cord injury (SCI) is one of the factors inhibiting axonal regeneration. Glial scars are mainly composed of reactive astrocytes overexpressing intermediate filament (IF) proteins such as glial fibrillary acidic protein (GFAP) and vimentin. In the current study, we delivered small interfering RNAs (siRNAs) targeting these IF proteins to SCI model rats using photomechanical waves (PMWs), and examined the restoration of motor function in the rats. PMWs are generated by irradiating a light-absorbing material with 532-nm nanosecond laser pulses from a Q-switched Nd:YAG laser. PMWs can site-selectively increase the permeability of the cell membrane for molecular delivery. Rat spinal cord was injured using a weight-drop device and the siRNA(s) solutions were intrathecally injected into the vicinity of the exposed SCI, to which PMWs were applied. We first confirmed the substantial uptake of fluorescence-labeled siRNA by deep glial cells; then we delivered siRNAs targeting GFAP and vimentin into the lesion. The treatment led to a significant improvement in locomotive function from five days post-injury in rats that underwent PMW-mediated siRNA delivery. This was attributable to the moderate silencing of the IF proteins and the subsequent decrease in the cavity area in the injured spinal tissue. The absence of spontaneous axonal regeneration after spinal cord injury (SCI) is attributed not only to the lack of neurotrophic factor support Suppression of astroglial reactivity and scarring after SCI has been reported as a novel therapeutic strategy for reduction of glial scar formation in vivo by applying PMWs As a method for site-selective gene delivery, laser-mediated gene transfer has received much attention because of the high spatial controllability of laser energy In the present work, we applied PMW-driven delivery of siRNAs to SCI model rats based on a strategy for axonal restoration by suppressing the over-expression of IF proteins. First, we transferred fluorescence-labeled siRNAs into SCI rats to investigate the distribution of siRNA uptake in the target tissue. We then delivered siRNAs targeting the IF proteins into injured spinal cords to promote the restoration of motor function in the rats. The reduction in the levels of the overproduced targeted proteins and glial scar formation were investigated as a function of time after injury.All procedures used in this study were approved by the Committee on the Ethics of Animal Experiments at the National Defense Medical College .We used female Sprague-Dawley rats  weighing 180–270 g in this work. The animals were housed one per cage after surgery and had free access to food and water except during periods of functional testing (see below). Before operation, they were anesthetized by intraperitoneal injection of pentobarbital sodium .As a laser-absorbing material (target), a 5-mm-diameter, 0.5-mm-thick black natural rubber disk was used. On top of the rubber sheet, a 1.0-mm-thick transparent polyethylene terephthalate sheet was bonded to confine laser-induced plasma, which can increase the peak pressure and pulse width of the PMWs generated 3 hydrophone with a 1.0-mm-diameter sensitive area . To evaluate the propagation characteristics of PMWs through spinal tissue, the spinal cord was removed from a rat (n = 1). Pressure was measured before and after propagation through the spinal cord, for which the hydrophone was first placed directly under the target and then placed under the spinal cord Onal cord . The thiThe lamina of the tenth thoracic vertebra was surgically removed, and the spinal cord was exposed. A New York University (NYU) weight-drop device was used to make a severe spinal cord contusion; specifically, a 10-g weight was dropped from a height of 25 mm onto the spinal dura in vitro study reported in the literature GGAACATCGTGGTAAAGAC targeting gene position 1236–1254 and GCTATGTGACCACATCCAC targeting gene position 166–184, respectively. To prevent rapid degradation of siRNAs in the presence of nuclease in vivo and to support the carrier activities of siRNAs, atelocollagen  was used in all experiments in this study; the collagen can stabilize gene fragments for at least one week and provide prolonged release of such fragments Alexa-Fluor 488, 5′-labeled siRNA with control scrambled sequence was purchased from Qiagen ; the duplex sequence was 5′-UUC UCC GAA CGU GUC ACG UdTdT-3′. An siRNA with a scrambled sequence  was used as a negative control. Synthetic siRNAs targeting rat GFAP  and vimentin  were obtained from Ambion, Inc. ; siRNAs targeting these IF proteins were selected based on the results of the Immediately after making a severe spinal cord contusion injury in a rat as described above, a 50-µl solution of Alexa-Fluor 488-labeled siRNA or scrambled siRNA or the same total volume of a mixture of siRNAs targeting GFAP (25 µl) and vimentin (25 µl) complexed with atelocollagen was intrathecally injected into several sites around the lesion through a Hamilton syringe with a 31-gauge needle (∼10-min administration time).2 and 10, respectively A laser-absorbing black rubber (target) was placed on the dura of the exposed injured spinal cord, for which ultrasound conductive jelly  was used to match the acoustic impedances of the target and spinal tissue. PMWs were generated in the same manner as for pressure measurements. In all experiments with siRNA delivery, irradiation laser parameters were fixed; the laser fluence and pulse number were 0.3 J/cm2. Rats were anesthetized and sacrificed at five days after injury by transcardial perfusion with 150 ml of physiological saline, followed by further perfusion with 200 ml of 4% paraformaldehyde in physiological saline . Segments of the spinal cords centered on the injury site were removed and post-fixed in the same fixative overnight. Then, the tissues were frozen in an optimal cutting temperature (OCT) compound  and sectioned into 10-µm-thick slices using a cryostat microtome. The sections were incubated overnight at 4°C with rabbit polyclonal anti-GFAP , and then were incubated with secondary antibody swine anti-rabbit IgG-TRITC (tetramethylrhodamine isothiocyanate) (1∶40 dilution)  for two hours at room temperature. We observed the distribution of fluorescence originating from Alexa-Fluor 488-labeled siRNA (green) and TRITC-labeled GFAP (red) in sagittal sections of the spinal cords centered on the injured site using a fluorescence microscope . For analyses if the distribution of fluorescence-labeled siRNAs as a function of tissue depth, three vertical lines were randomly placed on a typical green fluorescence digital image of the each injured tissue, and the number of pixels showing green fluorescence was counted along each line and integrated for every 300-µm depth section . Furthermore, the uptake of siRNAs into astrocytes was evaluated by merging the digital fluorescence images originating from Alexa-Fluor 488-labeled siRNA and TRITC-labeled GFAP, where the transfected cells appeared yellow due to colocalization. The total number of pixels showing a yellow color was counted in a randomly chosen 300 µm×300 µm area in the vicinity of the injury and was expressed as a function of the depth of the spinal cord .Distributions of fluorescence-labeled siRNA and GFAP expression in sagittal sections of injured spinal cords were examined at five days after trauma for the three groups: (1) SCI alone, (2) SCI plus siRNA injection, and (3) SCI plus siRNA injection followed by application of 10 pulses of PMW generated at a laser fluence of 0.3 J/cmFor western blot analysis, longitudinal 10-mm-long spinal segments centered on the injury were dissected at five days post-injury, and the tissues were immediately frozen with liquid nitrogen . To examine the effects of siRNA sequences on the expression of GFAP and vimentin, immunoblot analyses were performed under two conditions: (1) SCI alone (SCI group) and (2) scrambled siRNA injection after SCI (scrambled siRNA group). After being extracted from the excised spinal cords, the proteins were resolved with SDS-PAGE and transferred to polyvinylidene difluoride membranes . The membranes were immuno-blotted with antibodies against GFAP , vimentin (sc-6260) and GAPDH , followed by an incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). After incubation with ECL reagent , chemiluminescence signals were photographed, and then the band intensities were quantitatively measured using an image analyzer.In the following examinations, a mixture of siRNAs targeting GFAP and vimentin was delivered into the injured spinal cords of rats by applying PMWs. The experiments were performed under three different conditions: (1) SCI without any treatment (SCI group), (2) siRNA injection after SCI (siRNA group), and (3) siRNA injection and PMW application after SCI (PMW group). In the same manner as that described above, immunoblot analyses for GFAP and vimentin were performed under the three conditions. Control animals received sham surgery at five days before dissection (n = 4).In the same manner as described above, frozen sagittal serial sections (thickness of 10 µm) were prepared using a cryostat microtome after animal sacrifice at five days  or twenty-one days  after SCI. For diaminobenzidine (DAB) staining, after blocking of endogenous peroxidase with 3% hydrogen peroxide, the sections were incubated overnight at 4°C with rabbit polyclonal anti-GFAP  or with mouse monoclonal anti-vimentin . After washing with Tween-PBS, the slices on slide glasses were incubated with secondary antibody linked with dextran polymer peroxidase complex for thirty minutes at room temperature. Peroxidase-based color development was performed using 3, 3-diaminobenzidine.For evaluation of the morphological changes at twenty-one days after SCI, the adjacent sections were also stained with hematoxylin and eosin (HE). For a typical histological image at the lesion epicenter and adjacent sites , cavitation areas in the longitudinal images of HE staining were manually outlined and quantified by image analysis .Regeneration of corticospinal tracts (CSTs) was assessed using an anterograde tracer (mini-ruby) injected into the motor cortices. Two weeks after SCI, animals were anesthetized with pentobarbital sodium and two holes of 1.0 mm in diameter were drilled in the cranium to expose both motor cortices   For the three groups of rats, the SCI group, siRNA group and PMW group, the motor function of the hind limbs was evaluated by open-field testing and scored based on the BBB scale post hoc test. Comparisons of the BBB scores of the three groups of rats were performed using two-way repeated ANOVA with Tukey’s post hoc test. A value of P<0.05 was regarded as statistically significant.Statistical analysis of the results of western blotting and cavity measurement was performed using one-way factorial analysis of variance (ANOVA) followed by Tukey’s To evaluate the propagation characteristics of PMWs through the spinal cord, temporal pressure profiles of PMWs before and after propagation through an extracted spinal cord were measured using a hydrophone under the conditions used for siRNA transfection . The wavFluorescence-labeled siRNAs were used to examine the distribution of siRNAs delivered by applying PMWs to injured spinal cord. Immediately after making a contusion in a rat spinal cord, Alexa-Fluor 488-labeled siRNA solution was intrathecally injected into the vicinity of the trauma. PMWs were then applied onto the dura mater . At fiveP<0.05).On the basis of the results of the reporter siRNA delivery experiment described above, a mixture of siRNAs targeting GFAP and vimentin was delivered into the injured spinal cords of rats by applying PMWs. To examine the effects of transfection with these siRNAs, immunostaining for GFAP and vimentin was performed on sagittal sections of the spinal tissues at five days after SCI under three different conditions: (1) SCI group, (2) siRNA group, and (3) PMW group. In the SCI group, marked expressions of GFAP and vimentin were observed around the spinal contusion. Comparatively, the most silencing of GFAP was observed in the PMW group . ImmunosP<0.01) and the siRNA group (P<0.05)  . These oThree weeks after PMW-based siRNA delivery, regeneration of the CSTs was assessed using an anterograde tracer . It has P<0.05); the average BBB scores at three weeks post-SCI were 11.3±1.0 in the SCI group, 11.5±1.1 in the siRNA group and 13.4±0.8 in the PMW group. This difference in BBB scores of 11 (no forelimb and hindlimb coordination) and 13 (frequent the coordination) is substantial and noteworthy.The locomotive functions of the rats were assessed on the basis of Basso-Beattie-Bresnahan (BBB) scores The glial response to spinal injury results in recruitment of microglia, oligodendrocyte precursors, meningeal cells and astrocytes to the lesion site in vivo by applying PMWs. Scar formation was efficiently reduced in the injured spinal tissue following application of PMWs after siRNA injection, demonstrating the validity of our method of delivery of siRNAs for treating SCI. To the best of our knowledge, this is the first report of an experimental demonstration of significant functional restoration in SCI rats achieved by delivering GFAP and vimentin siRNAs.In the present study, we attempted to modulate the intrinsic behaviors of reactive astrocytes and hence to reduce glial scar formation. For this purpose, we delivered siRNAs targeting IF proteins into the injured spinal cords of rats et al. showed that PMW-mediated siRNA delivery induced posttranscriptional gene silencing in cultured plant cells in vivo gene silencing has not yet been reported.Although the detailed mechanism underlying PMW-based gene transfer is still under investigation, laser-induced impulsive pressures can increase the fluidity of the cell membrane into the target tissue, enabling cellular uptake of exogenous genes such as siRNAs and plasmid DNA et al. stated in their report on gene delivery to the periventricular region in rats by electroporation that tissue damage caused by electrical shock was inevitable, and that electroporation is not suitable for gene delivery aimed toward neural regeneration PMWs are characterized by a unipolar compressive wave  and the in vivo, ultrasound-based method can also be used. However, it has been reported that, with ultrasound microbubble-mediated transfection of spinal cords, the transfected cells were mainly meningeal cells at the dorsal surface of the spinal cord; no gene expression was observed in neurons or glial cells Furthermore, PMWs can efficiently propagate through tissue with high directivity and limited energy attenuation owing to plane-wave characteristics The transient maximum pressures of PMWs were measured to be about 51 MPa at the position of the spinal cord surface and 20 MPa under the spinal cord , indicatAs described above, astrocytes are activated after SCI with enlarged somas and intensive expression of IF proteins over time, and then a cystic cavity is formed in the region surrounded by the glial scar The anterograde tracing experiment showed numerous regenerating CST axons in the vicinity of the lesion in the PMW group . This deTo apply PMW-based siRNA therapy to larger animals with a thicker spinal column, deeper propagation of PMWs and broader distribution of therapeutic siRNAs are required. In our recent study, significant gene expression was observed in rat skin as a test tissue by applying PMWs that had to first propagate through 15-mm-thick tissue phantoms, demonstrating the capability of PMWs for cell permeabilization in tissues of ∼15 mm depth In summary, we demonstrated the validity of PMW-mediated siRNA delivery for therapy for SCI. The application of PMWs promoted uptake of siRNAs targeting IF proteins into deep glial cells, enabling an evident reduction in the levels of the IF proteins. As a result, significant locomotive functional recovery was obtained in rats that underwent PMW application. PMW-mediated siRNA delivery is useful for safe and valid CNS therapy, having unique characteristics that cannot be realized by other methods.


MicroRNAs are a class of small non-coding regulatory RNA molecules that regulate mRNAs post-transcriptionally. Recent evidence has shown that miRNAs target entire functionally related proteins such as protein complexes and biological pathways. However, characterizing the influence of miRNAs on genes whose encoded proteins are part of protein complexes has not been studied in the context of disease. We propose an entropy-based framework to identify miRNA-mediated dysregulation of functionally related proteins during prostate cancer progression. The proposed framework uses experimentally verified miRNA-target interactions, functionally related proteins and expression data to identify miRNA-influenced protein complexes in prostate cancer, and identify genes that are dysregulated as a result. The framework constructs correlation matrixes between functionally related proteins and miRNAs that have targets in the complex, and assesses the changes in the Shannon entropy of the modules across different stages of prostate cancer. Results reveal that SMAD4 and HDAC containing protein complexes are highly affected and disrupted by miRNAs, particularly miRNA-1 and miRNA-16. Using biological pathways to define functionally related proteins reveals that NF-kB-, RAS-, and Syndecan-mediated pathways are dysregulated due to miRNA-1- and miRNA-16-mediated regulation. These results suggest that miRNA-1 and miRNA-16 are important master regulators of miRNA-mediated regulation in prostate cancer. Moreover, results reveal that miRNAs with high-influence on the disrupted protein complexes are diagnostic and prognostic biomarker candidates for prostate cancer progression. The observation of miRNA-mediated protein complex regulation and miRNA-mediated pathway regulation, with partial experimental verification from previous studies, demonstrates that our framework is a promising approach for the identification of novel miRNAs and protein complexes related to disease progression. Prostate cancer (PCa) is the most frequent male malignancy and the second cancer-related cause of death in Western countries Recently, the cross-talk between miRNA-target networks and protein networks has been analyzed in several aspects To date, a number of mathematical models have been developed to infer miRNA-mRNA modules or modular networks using gene expression and miRNA-gene networks In the post-genomics era, a crucial task in molecular biology is to understand gene regulation in the context of biological networks. Since miRNA target proteins, among others, that are part of protein complexes and signaling pathways, it is important to study the miRNA-mediated regulation of protein complexes in disease progression. Using the protein network context of the miRNA targets adds another layer of information to consider for miRNA function characterization as miRNA influence on targets propagates through the protein network to affect multiple components of the pathway. Several studies reported regulation of functionally related proteins by miRNAs In this study we propose SVD-based computational framework to identify miRNA-protein complex modules that are dysregulated in cancer. miRNA-protein complex and miRNA-pathway modules refer to the proteins in the protein complex or pathway and the miRNAs targeting the genes encode them. Every module is represented as a matrix where rows are protein members and columns are targeting miRNAs. Every cell in the matrix represents the correlation between the expression profile of the miRNA and the expression profile of the protein. We anticipate that modules that have significant entropy change in their singular values between normal and cancer samples are functionally dysregulated. We applied the proposed computational framework to characterize experimentally verified protein complexes from the CORUM database Experimentally verified miRNA-target interactions were retrieved from two sources: MiRecords et al.et al.et al. data contains 6 normal, 7 primary, and 6 metastasis samples; the Yu et al. prostate data contains 17 normal, 63 primary, and 24 metastasis; and the Swedish prostate data contains 281 prostate cancer samples with 116 lethal and 165 indolent samples. The Swedish cohort data was used to validate the prognostic value of affected protein complexes. The Yu et al. and the Arul et al. data sets are used to validate the diagnostic significance of the influenced protein complexes. Non-prostate cancer miRNA expression data from NCI-60 mRNA and miRNA expression data was retrieved from the MSKCC Prostate Oncogenome Project, available at the Gene Expression Omnibus (GEO accession number: GSE21032). This data contains mRNA and miRNA expression levels of GSE23022  to validFor each miRNA-protein complex module, we construct a matrix X is the mutual information matrix between all miRNAs and all genes in the complex module. Since we believe that when a miRNA target a gene in the complex (based on miRNA-target interaction), it may have indirect effect on the other members of the complex. Thus the matrix X does not distinguish between a miRNA that target a gene in the complex or not. The matrix X is based on the notion that if a miRNA target a gene in the complex, it has influence on the whole complex. The influence of miRNAs on each protein complex or pathway is calculated by decomposing Using gene expression data for normal and cancer samples, we found We next asked if there are downstream miRNA-target interactions influenced by the affected protein complexes. We defined downstream genes as those that are dependent . To identify such conditional interactions, we used conditional mutual information between miRNAs and their experimentally validated targets from Given protein complex We then found p-valueProtein complexes and biological pathways that are influenced in prostate cancer as a result of coordinate miRNA regulation are identified and further functionally characterized.We first analyzed the influence of miRNAs on protein complexes in prostate cancer progression. We constructed miRNA protein modules by integrating expression data, miRNA-target interactions, and protein complexes (CORUM) as described in the methods section, and then identified the entropy change of each module in different prostate status. http://david.abcc.ncifcrf.gov/) . Benjamini multiple-testing correction was applied for significant enrichment analysis. Functional analysis demonstrated that the components of the complexes are enriched with three major biological terms, phosphorylation  of the miRNA-protein complex modules. We first retrieved the expression of the 84 proteins, that are part of the 42 complexes in 1 miRNAs . We then1 miRNAs . As a ne1 miRNAs . To furt1 miRNAs . High-riWhen we functionally analyzed the enriched terms in the 23 proteins, we found that they are enriched in several cancer pathways such as Cell cycle (p = http://co.bmc.lu.se/gobo) to associate the expression of the proteins with distant metastasis free survival across more than 1200 samples with different genotypes. The 23 proteins are found to be associated with breast cancer metastasis across all samples (p = 0.0076)  . Results 0.0076)  and the  0.0076)  subtypesTo characterize the prognostic value of the 21 influencing miRNAs, we extracted their expression data from the Taylor miRNA data and clustered the samples into two groups. The 21 miRNAs harbour significant prognostic value as they lead to significant separation between the two resulted patient sets  . When saIn summary, results reveal that miRNAs that coordinately regulate protein complexes are valuable prognostic biomarkers. In addition, protein complexes dysregulated by miRNAs are prognostic biomarkers that are candidates as therapeutic targets for prostate cancer treatment.et al. data. The proteins also classified primary and normal samples (80%) and metastasis vs. primary cancer (83%) in the Yu et al. data. Using the hierarchical clustering forthe Taylor data . The SVM classifier takes expression data of patients across the miRNA-influenced proteins and aims to predict the class of the patients using the expression data. Here cross-validation is used to assess the performance of the model due to the lack of additional independent samples. Results  reveal tlor data , resultsTo characterize the influential miRNAs and their role in prostate cancer progression, we validated the diagnostic power of the miRNAs on two independent prostate miRNA expression data sets . Classification results reveal that the influential miRNAs are robust diagnostic biomarkers as they are able to separate tumor from normal samples in GSE23022  and NCI-60 data sets  . AdditioWe even got better classification for the Taylor data when we combined the 23 proteins and the 21 miRNAs. We next asked if there are miRNA-target interactions that are affected by the dysregulated protein complexes. We selected the protein complexes that are highly dysregulated based on the results from MicroRNA-mediated regulation is a crucial layer of gene regulation that has influence on diverse biological processes. Using protein network context to characterize the mode of action of miRNAs is a new area of research in functional genomics that requires further investigation, to better understand miRNAs' influence on the target's protein context. Studying the miRNA-protein complex modules to characterize the diagnostic and prognostic role of miRNAs and protein interactions provide potential biomarkers and therapeutic targets relevant for prostate cancer diagnosis. A wide range of miRNAs and biological pathways are significantly altered in prostate cancer One of the factors that may affect identifying miRNA-protein interaction modules is the protein-interaction and miRNA-target data. Since both data are noisy and may lead to false discoveries, we used protein complexes data from CORUM database that are experimentally verified complexes. The second factor is the miRNA-target interaction data. Most of the computationally predicted interactions are noisy, thus we used experimentally validated miRNA-target interactions from miRecord and miRTarbase databases. Using experimentally validated interactions between proteins and miRNAs greatly helps to avoid noise in computational predictions.Based on experimentally validated interactions, we identified 42 protein complexes that are dysregulated in prostate cancer progression as a result of a functional miRNA that targets at least one member of the protein complex. A mutual information measure was used to assess if correlation between the miRNA and its targets's expression profiles changes during cancer progression. Singular Value Decomposition has been widely applied to cluster gene expression data and assess the entropy gained in each module during cancer progression. Here, the entropy in the singular values of the modules represent the coordinated regulation of miRNAs on protein complexes. We found 42 complexes dysregulated in prostate cancer progression. The 42 complexes are composed of 84 unique genes that are not all prognostic. Using cox-regression the 84 set was reduced to 23 genes that are significantly prognostic to predict cancer recurrence. The 23 genes are distributed across most of the 42 complexes. We noticed some genes in the 23 set  are common among multiple complexes and thus this could explain why the number of reduced genes (23) is less than the number of complexes (42). We found two major types of complexes that are affected by miRNAs during cancer progression; the first contains SMAD3, SMAD2, SMAD4, SMAD6, FOXO1, HOXC8, and SKIL, which are connected to AKT1, which is in turn a key modulator of TGF-B signaling pathway et al. and Yu et al.) and found that SVM correctly classify most of the samples using the expression of these proteins. This indicates that these proteins are dysregulated in prostate cancer. We further characterized their clinical significance and found that they are associated with cancer-specific death as they could separate high-risk from low-risk patients. We further tested the dysregulated protein on non-prostate data (Swedish breast cohort) and found that proteins are associated with cancer-specific death, tumor recurrence, and metastasis, indicating that these miRNA-influenced protein complexes could be dysregulated in multiple cancer types.We next asked if the identified dysregulated protein complexes are data specific, and extracted their expression from two independent data sets  and showed accurate diagnostic performance, concluding that miRNA-protein complex modules are diagnostic and prognostic biomarkers that could be used for therapeutic purposes.MEIS1, MEIS2, ZEB1, TPM1, DICER2, TPM2.We next sought to assess if the dysregulated protein complexes have a consequence effect on the expression of the downstream genes. Our results reveal that miRNA-1, miRNA-204, miRNA-16, and miRNA-200b are dysregulated due to the dysregulation of protein complexes identified. Finally we generated a network between dtsregulated protein complex members and proteins targeted by miRNAs based on conditional dependency. This helps to identify new proteins that are downstream of the miRNA-influenced protein complexes and are regulated through a miRNA but may not be part of known protein complexes, such as Another significant factor that should be considered is the size of the protein complexes used in this study. The protein complexes we used from CORUM are generally small (3-10 proteins) and may have an effect on the miRNA-mediated influence. Thus, considering larger functionally related proteins like signaling pathways proteins may support that our method is effective to find miRNA-influenced protein complexes. To investigate the influence of miRNAs on protein pathways, we constructed miRNA-protein pathways modules and found the most significantly miRNA-influenced pathways. The results show that RAS-mediated, NF-kB pathways and Syndecan-mediated pathways are highly influenced by miRNAs. Syndecan-1 is downregulated in prostate carcinoma, and transfection expression inhibits cancer growth et alFinally, we investigated if there are biological experiments that validated our identification of the importance of the dysregulated protein complexes and miRNA modules in prostate cancer progression. Hudson Investigating the functional role of miRNAs using a systems biology perspective helps us to understand the propagated influence of miRNAs on protein complexes in a particular biological context. Integrating the context (i.e. from gene expression data) of the protein targets of miRNAs is a promising step to identify miRNAs of high influence on gene expression of the target in the cell. This study provides a novel computational framework to identify dysregulated protein modules  influenced by miRNAs and find miRNA-target interactions consequently influenced by the dysregulated protein modules. The proposed framework reveals novel modules for further experimental design and investigations. Our proposed framework in this study identifies that protein complexes containing SMAD and HDAC proteins are the most influenced complexes by miRNAs. As a result of the dysregulation, other proteins like MEIS1, MEIS2, TPM1, and ZEB1, which are putative tumor suppressors, are affected. Finally, our developed framework can be generalized to find the influence of miRNAs on other curated gene sets data; TF-gene data for instance. The results of our framework reveal a previously unidentified layer of regulation that explains the dysregulation in biological pathways. Our results suggest that several cancer pathways  are under the control of miRNA-mediated regulation, and miRNA-1 and miRNA-16 are master regulators of miRNA-mediated regulation in prostate cancer.File S1A compiled list of all supplementary figures. Figure S1: Heatmap of SMAD6-HOXC8 complex,Figure S2: Heatmap of SMAD3-SMAD4-cSKI complex,Figure S3: Kaplan-Meier plot of the 23 proteins using Taylor data, Figure S4: Kaplan-Meier curves of the 23 protein across 1200 breast cancer samples,Figure S5: Kaplan-Meier plots of 21 miRNA from Taylor data,Figure S6: Kaplan-Meier plots of 21 miRNAs from Taylor data:three groups,Figure S7: Heatmap of 23 proteins in Taylor prostate data, Figure S8: Heatmap of 14 miRNAs(out of 21 miRNAs) using NCI-60 prostate data.(DOCX)Click here for additional data file.Table S1A list of dysregulated protein complexes and their targeting miRNAs. A list of the most significant protein complexes that were predicted to be dysregulated by miRNAs in prostate cancer. In each complex we show the CORUM ID, Complex Name, Protein members and the miRNAs targeting the genes encoding the proteins.(PDF)Click here for additional data file.Table S2A list of miRNAs having role in prostate cancer based on literature. This table lists the miRNAs that play a role in prostate cancer progression based on our analyzed expression data sets; either prostate cell lines or prostate tissue. It also lists the experimentally verified targets for some of them that we extracted from literature.(PDF)Click here for additional data file.Table S3A list of miRNA-target interactions affected by the dysregulation of the dysregulated complexes. This table shows the miRNA-target interactions dependent on the protein complexes we are interested in. We found that miRNA-1 and miRNA-16 interactions are significantly dependent on the protein RAF, which is part of the G-protein complex.(XLSX)Click here for additional data file.


The clinical outcome after allogeneic hematopoietic stem cell transplantation (HSCT) has been significantly improved during the last decades with regard to the reduction in organ failure, infection, and severe acute graft-versus-host disease. However, severe complications due to infectious diseases are still one of the major causes of morbidity and mortality after allogeneic HSCT, in particular in patients receiving haploidentical HSCT or cord blood transplant due to a slow and often incomplete immune reconstitution. In order to improve the immune control of pathogens without an increased risk of alloreactivity, adoptive immunotherapy using highly enriched pathogen-specific T cells offers a promising approach. In order to identify patients who are at high risk for infectious diseases, several monitoring assays have been developed with potential for the guidance of immunosuppressive drugs and adoptive immunotherapy in clinical practice. In this article, we aim to give a comprehensive overview regarding current developments of T-cell monitoring techniques focusing on T cells against viruses and fungi. In particular, we will focus on rather simple, fast, non-labor-intensive, cellular assays which could be integrated in routine clinical screening approaches. The clinical outcome after allogeneic hematopoietic stem cell transplantation (HSCT) has been significantly improved during the last decades with regard to the reduction in organ failure, infection, and severe acute graft-versus-host disease (GVHD). These advances have rendered allogeneic HSCT to an integral part of treatment for hematological malignancies , 2. HoweIn order to identify patients who are at high risk for infectious diseases, several monitoring assays have been developed with potential for the guidance of immunosuppressive drugs and adoptive immunotherapy in clinical practice. In this article, we aim to give a comprehensive overview regarding current developments of T-cell monitoring techniques focusing on T cells against viruses and fungi. In particular, we will focus on rather simple, fast, non-labor-intensive, cellular assays which could be integrated in routine clinical screening approaches.It is well-known that virus-specific T cells are important to prevent and/or control viral infection after allogeneic HSCT. Cytomegalovirus (CMV) is one of the most intensively investigated targets of immunotherapy after allogeneic HSCT . After aThe importance of virus-specific T cells has been also demonstrated with regard to other viruses such as adenovirus –18, EB vThe monitoring of T-cell immunity against these viruses can be useful to assess the risk of viral infections. The benefit of adoptive T-cell therapy as prophylaxis or as treatment should be ideally assessed in prospective clinical trials.+ T cells provide defense mechanisms against fungal infection  or Enzyme-linked immunosorbent spot (ELISPOT), which is practically very important with regard to reproducibility and standardization with strict quality control  or whole blood without long-term  control . Combinain vitro stimulation of PBMC by defined antigens/peptides without previous ex vivo expansion. In addition, ELISPOT assays allow the size and intensity of the spots to be calculated, which correlated with the amount of cytokines secreted by each cell. As shown in Figure Enzyme-linked immunosorbent spot is one of the most established methods to detect functional immunity –44. In bEnzyme-linked immunosorbent spot offers several advantages: (1) many samples can be tested simultaneously using one plate; (2) the secretion of cytokines can be assessed in contrast to the artificially retained cytokines in ICS; (3) the cell numbers can be downscaled per well in comparison to flow cytometry-based methods.51Cr-release assay for measuring cytotoxic activity of T cells  it is difficult to determine which immune cells secrete IFN-γ. This is critical to assess the immune status after allogeneic HSCT. As Wang and Colleagues reported, the response to 9-mer peptide, which is expected to induce cytokines in an HLA class I-restricted, can also be HLA class II-restricted , 50. TheIntracellular cytokine staining is also one of the most established methods to detect functional immunity , 52. In The advantages of ICS are as follows: (1) the phenotype of each cell which secretes the cytokine can be determined ; (2) theThe disadvantages of ICS are: (1) reagents such as Brefeldin A are required to retain cytokines in the cytoplasm; (2) the cells have to be permeabilized prior to the staining of the cells with antibodies against the cytokines, which makes it impossible to expand the sorted T cells.MHC-multimers are synthetic structures made from HLA molecules linked together to form a multimeric complex which are loaded with antigen-specific peptide. Cells stained with multimer and antibodies against surface markers can be analyzed using a flow cytometry. The fluorescence intensity using the tetramer loaded with a high-avidity peptide derived from virus is usually high enough to discriminate the positive population in contrast to the result using the tetramer loaded with a low-avidity peptide derived from autologous antigen . A repreThe advantages of multimer assays are as follows: (1) combined analysis of phenotyping and specificity can be performed using the antibodies against surface markers. MHC-multimer can detect T cells which do not secrete cytokines, for instance naïve T cells. In combination with the phenotyping and MHC-multimer staining, we can assess the frequency of all antigen-specific T cells including dysfunctional/non-functional; (2) antigen-specific T cells can be sorted with a high purity. For this purpose, the streptamer technology is demonstrated to be useful , 55. SorThe disadvantages of multimer staining are: (1) the multimer is not able to assess the functional status of antigen-specific T cells simultaneously. There can be a discrepancy in the frequency of antigen-specific T cells detected by multimer and by ELISPOT/ICS. Several papers reported that T cells detected by ICS were more important than those detected by multimer to control infectious diseases as demonstrated in the study of CMV infection –58. MultT-cell monitoring against specific targets including viruses and fungi is ready to be integrated in the clinical practice. The monitoring of pathogen-specific T cells may help to define the individual MPE of a patient to develop a certain infections complication and to assess the potential benefit of adoptive T-cell therapy against certain pathogens.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Finally, we discuss the role of PP GC for the generation of memory B cells and long-lived plasma cells in the light of contrasting findings regarding IgA memory development to colonizing commensal bacteria versus that to oral immunization with enteropathogens or TD antigens.Because Peyer’s patches (PP) are the main inductive sites for gut IgA responses we have focused this review on what we know about the function of PP germinal centers (GC). The vast majority of IgA gene sequences in the gut lamina propria (LP) are heavily mutated arguing for an origin in GC. Because PP GC formation is dependent on the presence of CD4 T cells, we speculate that all IgA responses in the normal gut are directly or indirectly T cell-dependent (TD). We hypothesize that the CD4 T cell involvement in gut IgA responses against the microbiota is different from that in systemic responses since cognate T–B cell interactions appear not to be required. In the absence of cognate interactions the function of CD4 follicular helper T cells (Tfh) in PP GC is unclear. However, production of IL-21 and IL-6 is more pronounced than in peripheral lymph nodes. Importantly, we discuss how multiple PP are involved in generating specific IgA responses to TD antigens given orally. Recently we found that oral immunization with NP-hapten conjugated to cholera toxin (NP-CT) stimulated a strong highly synchronized, oligoclonal and affinity matured IgA response. This was achieved through re-utilization of GC in multiple PP as GC IgA B cells emigrated into already established GC. Clonally related B cells were present in both inductive and effector lymphoid tissues in the gut and clonal trees involving multiple PP could be constructed in individual mice. Through adoptive transfer of B1-8 Figure 1).To protect us against infections and to maintain homeostasis with the microbiota the gut relies on secreted IgA antibodies . Given tOur findings in CD40-deficient mice argue that while cognate interactions are not required for B cells recognizing microbiota-associated TI-type antigens to undergo IgA CSR, the formation of GC is critical for SHM and affinity maturation. Thus, in this sense, these IgA B cell responses in the normal gut are dependent on GC and, therefore, indirectly dependent on CD4 T cells. However, this dependence is most likely not a cognate interaction. This interpretation finds support in a study by If cognate T–B cell interactions are not required, what is then the role of the CD4 T cell? An elegant study by + B cells were observed acetyl(NP) conjugated to CT for studies aimed at dissecting the different processes that critically are needed for a strong synchronized gut IgA response . As aforementioned, GC in PP have been observed independently of BCR-recognition and hence the role of TLR signals may be critical in PP for GC development and for maintaining normal homeostatic function of the gut IgA immune system  may be driven through signals other than those critically important for GC reactions in systemic lymphoid tissues. We have demonstrated that no GC develop in PP of CD40-deficient mice, but how BCR signaling or other receptor interactions critically influence PP GC formation remains to be further investigated . Repeated oral immunizations resulted in enhanced antibody affinity and we could follow the acquisition of a particular mutation in the CDR1 region, resulting in a 10-fold enhanced affinity of anti-NP IgA antibodies, with increased number of oral immunizations . Thus, B cells from GC of one PP can migrate into already established GC in multiple PP – attesting to the notion that synchronization of the gut IgA response occurs through reutilization of already established GC in multiple PP. These data for the first time provide evidence that there is synchronization of gut IgA responses, and that these responses are dependent on the repeated exposure to antigen and effective distribution of antigen-activated PP B cells into multiple PP.The extensive lineage trees and the oligoclonal domination of anti-NP IgA cells after repeated oral immunizations indicated that the gut IgA response was not only strongly regulated but also synchronized between PP. This synchronization must be achieved through simultaneous antigen-driven selection in multiple PP, which suggested that antigen-activated NP-specific IgA B cells from one PP were distributed to GC of multiple PP. Hence, we hypothesized that following the priming immunization, GL7These findings pose a number of questions as to what is required for the PP synchronization system to operate? Firstly, we can conclude that the migration of NP-specific B cells into already established GC required the presence of antigen, as no invasion of NP-specific B cells was observed in mice orally immunized with CT only . The fin+ PP GC B cells 7–9 days after an oral priming immunization  in the PP have not been studied in detail and presently we do not know their role in supporting B cells that reutilize GC and that recognize heterologous antigens . Clearlyntiation . Howeverfh in PP . An intrThe ability of the gut immune system to protect the mucosa against pathogens by long-term production of antigen-specific IgA antibodies is pivotal to mucosal vaccine development. A recent study by Macpherson and colleagues addressed the importance of mucosal memory after mono-colonization of germ-free mice with a commensal bacterial strain dependent on nutrients not present in the mammalian hosts . Using tThese conclusions may be relevant for gut B cell responses driven by the microbiota, but they contrast with findings after oral immunizations with TD-antigens, as discussed below. Because GC and SHM play a critical role for both types of IgA responses we believe it will be important to understand if there are fundamentally different regulatory principles that operate in the PP GC depending on the type of antigen that drive the B cell response . Whereasin vitro and can produce IL-21, IL-10, IL-6, and IL-4, which all have been implicated as instructive signals in GC plasma cell and memory B cell differentiation  and effector sites (LP) following oral immunization with a TD antigen, NP-CT. This was achieved through reutilization of already established heterologous GC in multiple PP for the expansion and selection of antigen-primed GL7on in PP . Hence, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recent years have witnessed much progress in both basic research and clinical trials regarding cancer immunotherapy with chimeric antigen receptor (CAR)-engineered T cells. The unique structure of CAR endows T cell tumor specific cytotoxicity and resistance to immunosuppressive microenvironment in cancers, which helps patients to better tackle the issue of immunological tolerance. Adoptive immunotherapy (AIT) using this supernatural T cell have gained momentum after decades of intense debates because of the promising results obtained from preclinical models and clinical trials. However, it is very important for us to evaluate thoroughly the challenges/obstacles before widespread clinical application, which clearly warrants more studies to improve our understanding of the mechanism underlying AIT. In this review, we focus on the critical issues related to the clinical outcomes of CAR-based adoptive immunotherapy and discuss the rationales to refine this new cancer therapeutic modality. Adoptive immunotherapy (AIT) using chimeric antigen receptor-engineered T cells is a promising approach in cancer treatment. Significant progresses made in the past decades have contributed to the development of more efficient antitumor immunotherapy. Incorporation of a single chain variable fragment (scFv) of a tumor antigen specific antibody and signaling domains of T cell receptor (TCR) renders chimeric antigen receptor (CAR) the specificity of antibody as well as the cytotoxicity of cytotoxic T lymphocytes (CTLs) . CAR endhttp://www.clinicaltrials.gov). Many advances have been reported regarding the outcomes of these early clinical studies (Phase I/II) including the effect on hematological malignancies and some solid tumors.The promising observations made from in vitro studies and animal models have greatly encouraged physicians/scientists to conduct clinical studies. The efficacy and safety of adoptive CAR-engineered T cells have been evaluated in multiple oncological settings. Currently, about 30 ongoing clinical trials are under evaluation  and lymphoma treated by CD19-orientated CAR T cells . CD19 isIn parallel with the clinical trials in hematological malignancies, CAR-based therapy has also been conducted in solid tumors. CAR T cells targeting diasialoganglioside (GD2) antigen and L1-cell adhesion molecule (L1CAM) were generated for treatment of neuroblastoma . Dr. LouDespite small sizes and different protocols, these phase I/II studies did demonstrate the feasibility and safety of CAR-based immunotherapy for the treatment of some types of cancer. While adverse effects are generally tolerable, the clinical efficacy of the first generation CAR is often insufficient. The CARs of the second and third generations have demonstrated improved efficacy and durable response, which motivated scientists for dynamic and innovative research in this field.CARs have evolved considerably over the past decades. Basically, CAR is comprised of an extracellular antigen-binding moiety, a hinge and transmembrane (TM) domain, and an intracellular signaling element. The initial CAR of the first generation provides a proof of concept of the targeting and activation of T cells. The CAR of the second and third generations have been developed by addition of dual or triple costimulatory signaling domains in order to increase their cytotoxicity . The mosTargeting, the first step for the redirected T lymphocytes to function, is initiated at the single chain fragment variable (scFv) region of extracellular domain. The scFv determines the specificity of CAR and binds to target molecules through a similar mechanism as antigen-antibody interaction. Many factors including the affinity of scFv, the spatial topologic structure of epitopes, and antigen expression levels on tumor cells, have to be considered to optimize the CAR-mediated T-cell response . Besidesζ TM domain form homodimers and are incorporated into the endogenous T cell receptor (TCR) complex [A flexible hinge region between the targeting moiety and the TM domain seems to be important for accessibility to the epitope of the CAR-grafted T cells . A study complex . VariousIntracellular signaling domain is of vital importance for CAR T cells to fulfill their antitumor function. Therefore, new generation of CAR contain a second signaling domain by adding the cytoplasmic domain of costimulatory receptors such as CD28, CD137 (4-1BB), CD134 (OX40), or inducible costimulator (ICOS). Both in vitro and in vivo studies have demonstrated that, in response to the target antigen, the modified T cells with the second or third generation CARs usually demonstrate sustained proliferation, enhanced production of cytokines and tumor lytic activity, and reduced activation-induced cell death. In a study of mouse model for comparison of the first and second generation CARs, Dr. Savoldo demonstrated the greater persistence of T cells expressing a CD28/CD3ζ-based CAR compared with T cells transduced by CAR with CD3ζ signaling only . The thiCollectively, optimal designs of the CAR and a careful choice of the tumor associate antigen (TAA) are indispensable for the CAR-mediated therapy to attain a significant response. Although CARs in many clinical trials greatly differ in their configurations, comprehensive comparisons are still lacking. With the clinical results in mind, we need to attain a better understanding of optimal CAR signaling to promote sustained T-cell function and survival, to avoid undue proliferation, and to prevent premature death and rapid exhaustion. It is also desirable to add more elements such as homing and suicide genes to improve their functionality. It is imperative to develop a more competent and safer architecture of CAR, so-called the fourth generation CAR. Optimized design of the new generation CAR may be achieved by more extensive basic studies investigating the spatiotemporal dynamics of CAR-mediated molecular events and unraveling the molecular basis of T-cell activation by CARs.+ T cells containing antigen-specific CD8+ and CD4+ T cells, and the phenotypic and functional attributes of the transferred T cells are subject to natural variation. As the heterogeneity and potential variable of polyclonal unselected cells could affect the efficacy and safety of cancer immunotherapy, precise detection and careful selection of the potentially most potent T cell subsets would increase their adoptive transfer efficiency.In the past decades, researchers have tried to generate sufficient quantity of antigen-specific T cells based on the notion that the absolute number of transferred T cells is correlated with tumor responses. Most clinical trials take use of mixed populations of CD3CM) induced complete responses at the tested cell dose, whereas the mice in group of receiving effector memory T cells (TEM) suffered uncontrolled tumor growth [CM cells can self-renew and differentiate into effector T cells in vivo, while TEM cells have lost this plasticity. Subsequently, a subset of stem cell memory T cell (TSCM) has been demonstrated to have superior ability to mediate cancer regression compared to the TCM and TEM populations [CM or TSCM cells prior to adoptive transfer and/or gene transfer could improve in vivo persistence and therapeutic efficacy. Clinical trials have been conducting to evaluate the potential of the T cell subsets from the peripheral blood [It becomes increasingly clear that adoptive transfer of the less-differentiated T cell subsets is associated with superior T cell engraftment, persistence, and antitumor immunity, thus highly correlating with objective clinical responses . An in vr growth . This diulations . Therefoal blood .γ-irradiated allogeneic peripheral blood mononuclear cell (PBMC) vs EBV-transformed B lymphoblastoid cell line (LCL) feeders, and a subsequent rapid expansion of cells in the presence of IL-2. Since ex vivo expansion inevitably drives T cell differentiation and results in the loss of in vivo antitumor efficacy, new strategies for handling cultured T cells have been developing recently. Traditional method for cell processing has been replaced by the automated manufacturing system. Activation of T cells can be induced by various kinds of antigen-non-specific artificial APCs (aAPCs) such as magnetic CD3/CD28 beads and K32 cell line (CD32-transfected precursor K562 chronic erythroleukemic cell line) [Many strategies have been utilized to obtain sufficient numbers of defined therapeutically clinical-grade CTL effectors. Current protocol includes isolation and activation using agonistic antibodies CD3 or ll line) .+ memory T lymphocytes, prevent Fas induced apoptosis and overcome tolerance of tumor specific CD8+ T cells [+ T cells in the presence of IL-15 generates cells with the phenotypic and functional properties of naturally occurring TCM with superior antitumor capability. Therapeutic potentials of small molecular modulators in critical metabolic and developmental pathways involved in T cell differentiation have also been evaluated including those in the PI3K-AKT-mTOR and Wnt-β-catenin pathways [IL-2 has been used for long time for T cell expansion. Recent evidence suggests that IL-2 may have some negative effects by depleting memory T cells and increasing the number of tumor protecting regulatory T cells (Tregs) . In cont T cells . The exppathways .+ T cells of a defined subset. The in-depth understanding of phenotypes and functions of T cell subsets will have profound impact on future CAR T cell therapy.Our understanding of the fundamental properties of heterogeneous subsets of T cells keep improving and mounting evidence indicates that selection of defined populations for gene modification is necessary to maximize the outcome of adoptive immunotherapy for cancer. The phenotypic and functional diversity of the genetically modified T cells may provide opportunities to enhance cancer immunotherapy. Because of severe adverse events reported in CAR clinical trial, it may become requisite to choose the defined T cell subsets in context of clinical setting. With recent advent of cell bioprocessing and genetic engineering technology, it is now possible to generate tumor-reactive CD8CAR-based immunotherapy is dependent on the efficient genetic modifications of the infused T cells. Since extensive ex vivo T cell expansion is prerequisite for adoptive cell transfer, genetic modifications must be stable and require vector integration. For this reason, transduction using chromosome-integrating vectors like retroviruses or lentiviruses is superior to transfection with transient gene expression of DNA in genetically modified T cell therapy.–engineered T cells. In a clinical analysis of HIV and malignancy, which included over 200 patients with the follow-ups for as long as 11 years, transduced T cells were detected in 98% of patients with no evidence of integration-induced immortalization [To date, most studies of CAR-related therapy utilize retroviruses or lentiviruses for gene delivery. Data from several sources indicate that retroviral vectors do not elicit clinically significant genotoxicity when used to deliver therapeutic genes to T cells . Dr. Schlization . Lentivilization . Besideslization .Along with the advance of viral vectors, non-viral gene transduction systems have been developed for clinical trials as well. The lower costs and better safety profile of non-viral vectors have attracted much attention. Transposon-based system is one of the promising methods because of much higher efficiency of the transgene integration. Sleeping Beauty was the first transposon system used for modification of human T lymphocytes and demonstrated stable expression of CAR with killing of targeted cancer cells in vitro as well as in animal models . PiggyBaWhile genetic-modification strategies provide us with the opportunity to harness the immune system against malignant disease, several issues must be taken into consideration when selecting the technology used to engineer cellular immunity. These factors include the size, number, and expression level of the transgenes, the requirement for permanent or transient genetic modification, and different delivery approaches for either constitutive or inducible transgene expression.Numerous animal experiments as well as clinical studies in humans strongly suggest that the effectiveness of adoptively transferred T cells can be reinforced when combined with conventional cytotoxic agents such as cyclophosphamide and fludarabine, and occasionally concomitantly used with a low-dose irradiation. It was observed in adoptive transfer of tumor infiltrated lymphocytes (TILs) that preconditioning with systemic non-myeloablative chemotherapy can induce clear and reproducible responses in a substantial percentage (~50%) of patients . Therefo+CD25+ regulatory T (TReg) cells in preconditioning recipient will help the infused T cells to function normally [+ T cell homeostasis. Preconditioning will eliminate cytokine sinks and creates space for the expansion of infused cells [+ T cells [Several mechanisms may contribute to the increased efficacy of T cell-based immunotherapy in the lymphopaenic environment. Deletion of the suppressive CD4normally . Competied cells . Transie T cells .Reg cells might be selectively depleted with directed immunotoxins or suppressed by administering a cytokine such as tumor necrosis factor (TNF). Exogenous replenishment of certain cytokines may alleviate the sink effect coming from the endogenous T cells. Attempt of using cytokines such as IL-7, IL-15 and IL-21 might avoid suppressive function of IL-2 toward TReg cells. Intensive myeloablative preconditioning regimen with chemotherapy, total body irradiation and human stem cell transplantation (HSCT) is also under planned in human AIT protocol.The improved effectiveness of immunotherapy following a non-myeloablative lymphodepleting regimen encourages the researchers for more elegant conditioning regimens in selective or intensive ways. TCAR-modified T cells constitute a very appealing approach to cancer immunotherapy. Despite the promising results obtained from numerous clinical trials following the infusion of CAR-modified T cells, some severe adverse reactions were reported ,50,51. RThe authors declare that they have no competing interests.All authors have contributed to write and revise the manuscripts. All authors read and approved the final manuscript.


Collective cell migration plays a major role in embryonic morphogenesis, tissue remodeling, wound repair and cancer invasion. Despite many decades of extensive investigations, only few analytical tools have been developed to enhance the biological understanding of this important phenomenon. Here we present a novel quantitative approach to analyze long term kinetics of bright field time-lapse wound healing. Fully-automated spatiotemporal measures and visualization of cells' motility and implicit morphology were proven to be sound, repetitive and highly informative compared to single-cell tracking analysis. We study cellular collective migration induced by tyrosine kinase-growth factor signaling (Met-Hepatocyte Growth Factor/Scatter Factor (HGF/SF)). Our quantitative approach is applied to demonstrate that collective migration of the adenocarcinoma cell lines is characterized by simple morpho-kinetics. HGF/SF induces complex morpho-kinetic coordinated collective migration: cells at the front move faster and are more spread than those further away from the wound edge. As the wound heals, distant cells gradually accelerate and enhance spread and elongation –resembling the epithelial to mesenchymal transition (EMT), and then the cells become more spread and maintain higher velocity than cells located closer to the wound. Finally, upon wound closure, front cells halt, shrink and round up  phenotype) while distant cells undergo the same process gradually. Met inhibition experiments further validate that Met signaling dramatically alters the morpho-kinetic dynamics of the healing wound. Machine-learning classification was applied to demonstrate the generalization of our findings, revealing even subtle changes in motility patterns induced by Met-inhibition. It is concluded that activation of Met-signaling induces an elaborated model in which cells lead a coordinated increased motility along with gradual differentiation-based collective cell motility dynamics. Our quantitative phenotypes may guide future investigation on the molecular and cellular mechanisms of tyrosine kinase-induced coordinate cell motility and morphogenesis in metastasis. Collective cell migration is prevalent in many physiological phenomena and is the most common motility pattern in living organisms et al.Extensive research has been carried out for many years in various experimental model systems to investigate, describe, analyze, model and simulate collective cell migration. There are several theories concerning the mechanisms behind collective motility However, accumulating evidence implies that the behavior is more complex. Modern microscopy et al. Another theory assumes that cell proliferation expands the colony and thereby generates pressures that cause the leading cells to move in-vitro wound healing assay, collective migration of cells toward the wound is induced by a sudden injury created by removal of a sheet of cells from the monolayer In the standard The epithelial to mesenchymal transition (EMT) activated by alternations in gene expression regulates epithelial plasticity during morphogenesis, tissue repair and cancer invasion In vitro collective migration is common in breast cancer, as well as in many other cancer types Understanding collective cell motility and how it may lead to metastatic formation is an important task since the vast majority of cancer deaths result of progression from a localized lesion to distant metastases Here, we investigate the link between cells' morpho-kinetic dynamics and collective migration of tumor cells using mammary adenocarcinoma cells expressing high levels of Met, image them using a time-lapse microscopy wound healing assay, and study the effect of HGF/SF-Met signaling on morphology and collective motility patterns.in vitro using bright field, time-lapse microscopy, based on the combination of a fully automated algorithm that extracts motility measurements from all cells in the monolayer with indirect cellular morphology measures using image-texture descriptors, and single cells' morphology measurements extracted semi-manually. Recording these as a function of location over prolonged periods allows a coherent and concise depiction of the essence behind three collective motility modes of cancerous cells, and to reveal that Met-activation by HGF/SF induces elaborated collective cell motility that is correlative to the EMT-MET morphological transition.We devised a novel approach to analyze wound healing DA3 cells, derived from the mouse mammary adenocarcinoma cell line D1-DMBA-3, induced in BALB/C mice by dimethylbenzanthracene 2 diameter and treated with or without the Met inhibitor PHA665752 −1) and subjected to time lapse confocal laser scanning microscopy  for approximately 26 hours, at frequency of once every 14.5 minutes. The position of each scratch was predefined, and a macro that repetitively positions the microscope on each point was executed. The acquired differential interference contrast (DIC) channel of the time-lapse sequence was used for the multi-cellular analysis; the red fluorescence channel was exploited for single cell tracking.DA3 cells expressing the fluorescent protein mCherry were grown to 90% confluence in wells of 2 cmPhase 1, from the scratch formation until first contact between cells from opposing wound edges; Phase 2, from first contact until full closure of the wound - the wound area is completely covered by one layer of cells; Phase 3, post wound closure. The effect of HGF/SF on cellular motility patterns, morphology and multi-cellular texture were evaluated according to the three healing phases. Dividing the healing process to phases is important since the duration of each phase varies between the treatments.Three phases were defined . Phase 1velocity magnitude map”:Motility measurements were extracted via a fully-automated algorithm that quantifies local motion estimation from the time-lapse bright field (DIC) channel. The algorithm includes segmentation to partition a DIC image to multi-cellular- and background- regions, followed by local-motion estimation and quantification of the extracted motion fields' magnitude  at different distances from the wound edge. Continuous quantitative description of cells' velocity magnitude as a function of distance from the wound throughout the healing process is achieved by constructing a “t, t+1 from the time-lapse sequence.Given two consecutive DIC frames t) to a grid of sub-cellular sized local patches.Partition the current image . The Local-Binary Patterns (LBP), known to perform well in face-recognition Phase 2).To enable automatic prediction whether a full DIC time lapse wound healing experiment was or was not treated with HGF/SF, the 3-dimensional time-lapse (image space and time) was "compressed" to a 2-dimensional representation, the velocity magnitude map described above. This compact description was further represented by a one-dimensional descriptor vector as follows: distances from the wound were partitioned to 6 intervals, and the average speed of all cells in any given interval during the 3 phases in the healing process were used to define a vector representation of a time lapse wound healing experiment . To cancPhases 1 and 2). During post wound closure (Phase 3), all cells decelerate regardless of their position. HGF/SF treatment leads to emergence of dramatic different cell motility patterns: at the beginning, front cells move faster than distant cells. Throughout Phase 1, distant cells gradually join the rapid motion by accelerating layer by layer. This gradual acceleration continues during Phase 2, where distant cells maintain a significantly higher motility toward the wound than cells located closer to the wound edge (data not shown). Finally, post wound closure (Phase 3), front cells halt, while distant cells gradually decelerate. These results demonstrate that Met-activation via HGF/SF induces complex motility patterns indicating cell-cell coordination and dynamic signaling that generates micro-differentiation in the healing wound.Qualitative comparison of the velocity magnitude maps visualization between untreated and treated cells revealed the unique motility patterns induced by HGF/SF . Front lPhases 1 and 2). It is shown  untreated cells that are close to the wound are larger than distant cells. Front cells shrink upon wound closure, and after the wound has healed all cells in the monolayer shrink to maintain approximately the same size independently of their location. Similarly to its effect on cells' speed, HGF/SF treatment induces dramatic changes in the dynamics of cellular morphology. At the initial stage, close cells are larger than distant cells. During Phase 2, front cells begin to shrink while farther cells grow. In Phase 3, only the most distant cells continue to grow while the rest shrink. After the wound has healed, all cells have shrunk to approximately the same size. A bar graph that compares treated and untreated cells' area for every distance interval over time demonstrates that the main differences occur in Phases 2 and 3, when treated cells that are located far from the leading edge grow dramatically in a progressive manner , implying that there is a true-general change in the motility patterns and not only in its magnitude. This means that given a time lapse experiment and two time points that represent the partitioning to the three phases, one can determine with high accuracy whether it was treated with HGF/SF or not . Similar predictions were performed with multi-cellular texture descriptor . It was shown , applying an SVM classifier using "leave one out" validation .To generalize these findings, we tested whether cells in wound healing assays can be automatically classified as HGF/SF-treated or -untreated based solely on the DIC time-lapse images. To this end, we defined a new measure to quantify the collective motility patterns: each of the 11 time lapse experiments  was represented by a vector containing average speed for each healing phase as described in the Materials and Methods section . An SVM as shown  that perPhase 2 was not completed), only the first two healing phases were considered (descriptor vector of length 12 per experiment). To exclude the global healing speed and to focus on the spatio-temporal motility patterns, each experiment descriptor was normalized to 1  morphology-motility correlation of treated cells is explained by their self-propelled nature, causing lower dependence on their surroundings.Phase 2 discriminate between cells untreated, HGF/SF-treated and treated with MET inhibitor and HGF/SF together, concordant with the data indicating that the morphological changes mostly occur during this phase. Thus, using image-texture as an indirect multi-cellular morphology descriptor can be exploited as a treatment-predictor. Further investigation should try to find a direct connection between cells’ morphology and texture.It was demonstrated here that texture of a single image from time lapse sequence, captured by the LBP descriptor, is sufficient to predict the treatment. The image-texture of a monolayer of cells can thus be used to implicitly measure cells morphological characteristics, as in measuring cell scattering The combination of image texture and cellular velocity magnitude descriptors may serve as fully automated quantitative measures to represent morpho-kinetic dynamics, to enable in principle high throughput analyses without human intervention.in vitro model of tumor cells moving collectively studied herein does not take into account important parameters that maintain a crucial role in biological processes that include collective motility such as 3D motility and the tissue’s microenvironment  applied as an image texture descriptor. For every pixel in the image, a code is generated based on the intensities of neighboring pixels with relation to it. There are ten possible codes, and their histogram over all cellular pixels is used to describe the image's texture which is used as an indirect descriptor of cells morphology.(TIF)Click here for additional data file.Figure S2Compact representations of a wound healing experiment. (a) Velocity-magnitude based vector representation of a wound healing experiment. Distances from the wound were partitioned to 6 intervals. The average motility of all cells in any given interval during each healing phase was recorded to define a length 18 vector representation. These values are calculated by taking the average intensities of the corresponding rectangular regions in the velocity magnitude map. (b) Texture-based vector representation of a wound healing experiment as an implicit measure for cells' morphology. LBP normalized histogram is extracted for every image in the time-lapse sequence. All histograms of frames in Phase 2, where most morphological-changes occur, are averaged to define the combined texture descriptor. (c) First two components of the principal component analysis (PCA) performed on the normalized velocity-magnitude based vector representation was not sufficient.(TIF)Click here for additional data file.Figure S3Single cell morphology (area) as function of time and distance from the wound. (a) “Average” cell’s area at different distances over time. Same data as presented in Phase 1 treated cells maintain similar location-dependent characteristics to those described for untreated cells. In Phase 2, treated cells "switch" - distant cells become sparsely distributed compared to front cells.(TIF)Click here for additional data file.Figure S4Single cell morphology (eccentricity) as function of time and distance from the wound. Eccentricity is the ratio of the distance between the foci of an ellipse and its major axis length. In our setting it is referred to the ellipse that has the same second-moments as the segmented cell. The values range between 0 and 1. . (a) Same as (TIF)Click here for additional data file.Methods S1In this document we provide a detailed description of the following methods: (1) velocity magnitude map, (2) fully-automated single cell trajectory-estimation from DIC-based motion vector fields, (3) semi-automated single cell tracking, (4) velocity magnitude map based on semi-automated single cell tracking, and (5) single-cell morphology measures.(DOCX)Click here for additional data file.


Certolizumab pegol (CZP) has a different mode of action from the other anti-TNFs, infliximab (IFX), adalimumab (ADA) and etanercept (ETA), that may be due both to its structure and how it signals through membrane TNF-α (mTNF-α). Different effects of this signalling could relate to the exact epitopes to which the anti-TNFs bind. We investigated the binding epitope for CZP and how it differs from the other anti-TNFs.Epitope mapping for CZP binding to TNF was achieved using minimal shift analysis of nuclear magnetic resonance) data on the non-PEGylated Fab′/TNF complex. Hydrogen/deuterium (H/D) exchange mass spectrometry was used to identify TNF peptides containing amide protons that are protected in the Fab′/TNF complex. A Biacore assay was used to determine the difference in affinity between cynomolgus monkey and human TNF for each of the 4 anti-TNFs. This technique involved the use of cross-species mutagenesis of the 4 residues that differ between cynomolgus monkey and human TNF . The epitope identified by these methods was confirmed by further site-directed mutagenesis and Biacore-based studies.13 amino acids  were identified as comprising the CZP epitope on TNF. These residues formed a cluster on the outer face of each monomer. Biacore studies on TNF peptides expressed with these residues mutated showed that, although there was some overlap, the pattern of binding for each of the anti-TNFs was different.All 4 anti-TNFs tested bind to similar but not identical epitopes on the TNF molecule. These differences between the exact epitope could explain the variations in the downstream signalling properties through mTNF-α of the anti-TNFs and may help to elucidate some of the mode of action differences emerging for these agents.


The FVB/N mouse strain is extensively used in the development of animal models for breast cancer research. Recently it has been reported that the aging FVB/N mice develop spontaneous mammary lesions and tumors accompanied with abnormalities in the pituitary glands. These observations have a great impact on the mouse models of human breast cancer. We have developed a population of inbred FVB/N mice (designated FVB/N-RC) that have been genetically isolated for 20 years. To study the effects of age and parity on abnormalities of the mammary glands of FVB/N-RC mice, twenty-five nulliparous and multiparous (3–4 pregnancies) females were euthanized at 16–22 months of age. Examination of the mammary glands did not reveal macroscopic evidence of mammary gland tumors in either aged-nulliparous or multiparous FVB/N-RC mice (0/25). However, histological analysis of the mammary glands showed rare focal nodules of squamous changes in 2 of the aged multiparous mice. Mammary gland hyperplasia was detected in 8% and 71% of the aged-nulliparous and aged-multiparous mice, respectively. Epithelial contents and serum levels of triiodothyronine were significantly higher in the experimental groups than the 14-wk-old control mice. Immuno-histochemical staining of the pituitary gland pars distalis showed no difference in prolactin staining between the control and the aged mice. Tissue transplant and dilution studies showed no effect of age and/or parity on the ability of putative progenitor cells present among the injected mammary cells to repopulate a cleared fat pad and develop a full mammary gland outgrowth. This FVB/N-RC mouse substrain is suitable to develop mouse models for breast cancer. Transgenic/knock-in and knockout mouse models have been used widely to observe in vivo the role of genes in initiation and progression of the disease. Their use has dramatically increased during the past decade. The ideal animal mammary gland tumor model should replicate the different stages of development of human breast cancer, such as pathogenesis, growth and progression with a 100% incidence in animal models and no occurrence in the control animals- at least during the defined period of experimental observation Advances in breast cancer treatment require better understanding of the genetics and biology of the disease. This requires the development of better in vivo for elucidating the roles of hormones and growth factors in normal mammary gland development and function. Also, the mouse mammary gland is similar to the human breast in many aspects of hormonal regulation of cell proliferation in vivoThe FVB strain of mice is widely used for the generation of models of human breast cancer. Advantages in the use of this specific strain include vigorous reproductive performance and consistently large litters Wild-type aged-FVB female mice spontaneously acquire a series of abnormalities in several organs, such as lung, ovary and pituitary The FVB mice used in this study are housed in our own colony and have been inbred and genetically isolated for the past 20 years. They therefore represent a substrain of the FVB/N mice (designated FVB/N-RC). The aged-multiparous (3 to 4 pregnancies) and the aged-nulliparous female mice were euthanized at 16–22 months of age. As control we used 14-week-old nulliparous female mice from the same colony. Neither age and/or parity resulted in spontaneous mammary tumor development or affected the ability of the mammary stem cells to repopulate the mammary gland; therefore we propose the FVB/N-RC mouse substrain as a suitable model for the development of mammary gland tumor models. This substrain will be made available to investigators interested in developing new models for breast cancer.Mammary glands of aged-nulliparous/multiparous females showed morphologic and histologic features of an early pregnant mouse mammary gland. Analysis of mammary morphology and histology showed high levels of budding alveoli in both the aged-nulliparous  in the aged-multiparous glands than the aged-nulliparous glands. This indicates a role for age and both age and parity, in mammary ductal epithelial proliferation. Conversely cell proliferation in alveoli was significantly higher (p<0.05) in the mammary glands of aged-multiparous than in aged-nulliparous mice  knockout mouse demonstrated that progesterone is required for alveolar morphogenesis Epithelial PR is first detected at 7 weeks of age and this class of PR is regulated by estrogen To examine the effects of age and/or parity on mammary stem cell (MaSC) transplantation potency, we conducted two assays, a serial dilution transplantation study and serial transplantation of mammary tissue fragments. Outgrowth took place in both assays, independent of the numbers or source of cells  or sourcMouse mammary gland development is well studied and its response to hormones and growth factors is well understood. This makes it a good candidate to develop breast cancer models. Understanding breast caner initiation, promotion and progression involves an increasing number of experimental mouse models. The FVB/N mouse strain is commonly used in these models. The ideal mouse model should not develop mammary abnormalities in the control group-at least during the time period used for comparison with the experimental group. Recent reports of the development of spontaneous mammary hyperplasia/tumors in the FVB/N mice The majority of mammary gland development takes place post-natally. These developmental events are driven by the ovarian hormones estrogen and progesterone. Where ductal elongation during puberty is driven mainly by estrogen and the alveolar formation is driven by estrogen and progesterone in addition to other growth factor, hormones and integrins The high proliferation rate in the 14-wk-old control mice could be due in part to the low T3 levels in this group . As a maInvolution takes place after lactation, where massive cell death takes place. This results in the remodeling of the mammary gland and disappearance of the side-branches. The high epithelial contents in the aged-nulliparous and aged-multiparous females indicate failure of mammary regression after lactation, resulting in the maintenance of the alveoli and side-branches. This is a typical pattern in the aged-multiparous females Stem cells are located along the entire mammary tree and are present in the different developmental stages of the mammary gland In summary the dilution studies indicate that there is no evidence for a loss of regeneration potential of mammary cells as a result of age and/or pregnancy. These data support the original observations of Young et al. All mice were housed in Association for Assessment and Accreditation of Laboratory Animal Care–accredited facilities in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The National Cancer Institute (NCI) Animal Care and Use Committee approved all experimental procedures.All the FVB mice used in this study have been maintained by sibling mating in our colony for the last 20 years and therefore represent a substrain (designated FVB/N-RC) of the FVB/N mice. Twenty-five FVB/N-RC female mice were set up for breeding at the age of ten-weeks. After the third or forth pregnancy, the breeding pairs were separated and the females were housed until 16–20 months of age. Another 25 females were maintained nulliparous to 16–20 months of age. The control group was 14-week-old nulliparous mice. Tissue and blood samples were collected from all mice at diestrus. All mice were kept according to standard governmental and NIH regulations. Mice had access to water and food ad libitum during the experiment, fed 5L79 diet  and lived in a 12 hour dark/light cycle.Mice were euthanized at the indicated ages. Mammary glands, pars distalis, uterus and ovary tissues were fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) for 2 h, rinsed through several changes of buffer, dehydrated in ethanol and stained and examined grossly under a dissecting microscope, or embedded in paraffin. Paraffin sections (5 micron) were placed on slides, deparaffinized and stained with H&E. Mammary whole-mounts and histology sections were prepared from the fourth abdominal gland as described previously Immuno-histochemistry was carried out with the ABC method according to the manufacturer's protocol . Primary antibody incubation was carried out overnight at 4°C: ERα , diluted 100X; progesterone receptor , diluted 100X; prolactin , diluted 1000X. Primary antibodies were diluted in 1%PBS-BSA. The biotinylated anti-rabbit  secondary antibody was diluted according to the manufacturer's recommendations. Labeling index was determined in at least a total of 3000 cells in each experiment.Serum was analyzed from 3 independent pools of control, aged-nulliparous or aged-multiparous FVB/N-RC female mice. Each blood pool was derived from three animals, obtained by exsanguinations at 2 PM. Sera was collected and subsequently stored at −20°C until assayed. Serum estrogen, triiodothyronine, and thyroxin were assayed by RIA ; growth hormone was quantified by ELISA . Serum prolactin (PRL) levels were determined using a previously described bioassay that utilizes the PRL-responsive Nb2 lymphoma cell line 3 fragments of mammary duct from 14-wk-old, aged-nulliparous or aged-multiparous mammary mice into the cleared fat pads of 3-week-old FVB/N-RC female mice. Eight to ten weeks after the implantation, recipient mice were euthanized, and a mammary tissue fragment was collected and used for serial transplantation. The serial transplantation was carried over for five consecutive times. Whole-mounts were prepared as previously described To characterize the effects of age and parity on mammary gland progenitor cell population and their ability to repopulate the mammary cleared fat pad (CFP); 3 different sets of aged-nulliparous and aged-multiparous mice were used. We employed two assays. In the first assay, 5000, 1000, 500, 250 or 100 cells from 14-wk-old FVB/N-RC, 20–23 month aged-nulliparous or 20–23 month aged-multiparous mammary glands were injected into the CFP of 3-wk old female FVB/N-RC mice. In each recipient mouse, one side received the nulliparous cells and the contralateral side received the multiparous cells. The second assay is serial transplantation in which we transplanted 1 mmin vivo experiments were repeated at least 3 times, and at least 5 mice were used in each experiment. The statistical significance of difference between groups was determined by the Wilcoxon rank sum test. Comparisons resulting in p-values less than 0.05 were considered statistically significant and are indicated in the figure with asterisk (*). The effects of age were tested by comparing the 14-week-old control group data versus the aged-nulliaprous group data. The effects of age and parity were investigated by comparing the data of the 14-week-old control group to the data of the aged-multiparous group. While the effects of parity were investigated by comparing the data of the aged-nulliparous group to the aged-multiparous group.Quantitative values are represented as the mean of at least 3 experiments. All


New York’s Division of Chronic Disease Prevention has supported the delivery of the Diabetes Prevention Program (DPP) through community-based organizations across the state. The DPP is a 16-week lifestyle change program designed to prevent or delay type 2 diabetes in adults who are at high risk for the disease. In 2009, 10 YMCA sites across the state were selected to deliver the program. In subsequent years, additional community-based organizations developed capacity to deliver DPP. The goal of the New York State DPP is to establish critical partnerships that serve as a statewide infrastructure of effective, accessible, and affordable diabetes prevention programs.http://www.census.gov/2010census/) was used to calculate the percentage of the NYS population living within the buffers. Potential DPP sites were identified by using 2011 data indicating the locations of federally qualified health centers, independent living centers, and area agencies on aging.New York’s Bureau of Chronic Disease Evaluation and Research used a geographic information system (GIS) to identify counties with high prediabetes risk-factor composite scores based on county-level prevalence of obesity, physical inactivity, hypertension, and diagnosed diabetes . The staAlthough approximately 80.0% of New York’s population resides within a 30-minute drive time to a Diabetes Prevention Program site; most people living in 10 of the counties with the highest prediabetes risk-factor score live at a distance beyond a 30-minute drive to an existing site.The expansion of DPP sites through additional community-based facilities would improve access to prevention programs in communities with high diabetes risk-factor profiles and would facilitate the improvement of community–clinical linkages across the state to sustain prevention efforts for type 2 diabetes. The New York State Department of Health has used information from GIS analysis to identify areas with limited access to DPPs and encourage key partners to locate programs in community-based organizations serving at-risk populations. Future plans include expanding the analysis to incorporate all evidence-based chronic disease programs and assessing demographic differences among populations living within and outside the 30-minute drive-time radius to existing program sites.