CPEB1-4 and identified the CPEB1 gene to be hypermethylated in tumor samples. Decreased expression of CPEB1 protein in gliomas correlated with the rising grade of tumor malignancy. Abundant expression of CPEBs2-4 was observed in several glioma specimens. Interestingly, expression of CPEB3 positively correlated with tumor progression and malignancy but negatively correlated with protein phosphorylation in the alternatively spliced region. Our data suggest that loss of CPEB3 activity in high-grade gliomas is caused by expression of alternatively spliced variants lacking the B-region that overlaps with the kinase recognition site. We conclude that deregulation of CPEB proteins may be a frequent phenomenon in gliomas and occurs on the level of transcription involving epigenetic mechanism as well as on the level of mRNA splicing, which generates isoforms with compromised biological properties.Cytoplasmic polyadenylation element binding proteins (CPEBs) are auxiliary translational factors that associate with consensus sequences present in 3′UTRs of mRNAs, thereby activating or repressing their translation. Knowing that CPEBs are players in cell cycle regulation and cellular senescence prompted us to investigate their contribution to the molecular pathology of gliomas–most frequent of intracranial tumors found in humans. To this end, we performed methylation analyses in the promoter regions of  Control of mRNA polyadenylation is an important step in regulation of gene expression. Cytoplasmic polyadenylation element binding (CPEB) proteins activate or inhibit translation of mRNA molecules by binding to the consensus CPE sequences present in the 3′untranslated regions of transcripts , 2. In t2+/calmodulin-dependent protein kinase II (CaMKII) [The family of CPEB proteins was first identified in the gametogenesis of Xenopus , 7 where(CaMKII) –17.2+/calmodulin-dependent protein kinase II (CaMKII) were shown to phosphorylate CPEB2-4 [In contrast to CPEB1 much less is known about CPEB 2-4 proteins and their alternative splice variants. Spliced regions of CPEB1 were described to have low similarity with CPEB2-4 . Sequenc CPEB2-4 –22. It w CPEB2-4 , 21, butWe have previously shown that CPEB1 and CPEBs2-4 are broadly expressed in mouse brain , 19, 21.CPEB1 as a target for epigenetic inactivation by differential methylation hybridization (DMH). By pyrosequencing we investigated the methylation levels of CPEB1 as well as the other members of the CPEB family in 63 human glioma, 3 normal brain samples . Within the group of GBM a strong CPEB1 hypermethylation was especially abundant in tumors that developed following malignant progression of lower-grade precursor lesions (sGBM: 10/10). Secondary GBM tumors containing the IDH1 mutation (n = 7) revealed a mean methylation of 69.37 ± 6.78%. Our cohort of pGBM (n = 41) samples contained 4 cases with IDH1 mutation, which also revealed a significant increase of CPEB1 methylation (mean 73.53 ± 4.26%). Secondary GBM without IDH1/2 mutation (n = 3) and primary GBM tissues with wild type IDH1/2 (n = 37) showed a mean methylation of 21.81 ± 8.93% and 19.84 ± 2.74% in the investigated region of CPEB1, respectively. This indicates that CPEB1 methylation is tightly linked to the IDH1 mutation status. In addition, all investigated glioblastoma cell lines showed hypermethylation of the CPEB1 gene. The observed methylation pattern shows that CPEB1 belongs to the genes affected by the glioma associated CpG island methylator phenotype (G-CIMP) in IDH1/2 mutant tumors. Correlation of IDH1/2 mutation with CPEB1 methylation was highly significant . Compared to CPEB1, methylation levels of CPEB3 were low  in the entire cohort of samples, and only a few cases showed moderately elevated methylation. There was no correlation of CPEB3 methylation, expression and IDH1/2 mutation. For CPEB2 and CPEB4 no methylation was detected in any of the investigated tumor specimens  revealed a significant reduction of CPEB1 transcript when compared to reference samples. Expression of CPEB1 mRNA was decreased to 36.63 ± 6.52% in AAIII (p < 0.01), to 6.36 ± 1.23% and 9.05 ± 1.90% in sGBM (p < 0.01) and pGBM (p < 0.001), respectively  and sGBM (p < 0.01), but not in pGBM .Furthermore, expression of CPEB1 transcript was studied in 6 normal brain and 25 glioma samples by sqRT-PCR Figure . All invy Figure . MethylaM Figure . Methylan = 3, p < 0.05), hedgehog , HNF4  and TGFβ  pathways  and normal brain tissue (n = 4). The majority of the examined AII cases contained the same splice variants as normal brain tissue  and in all normal tissues analyzed (n = 4), CPEB1Δ5 (169 bp) was the only CPEB1 isoform that was detected. Two pGBM samples (cases 3070 and 3555) showed an additional CPEB1 (182 bp) splice variant, which corresponds to the full-length isoform  and CPEB4a  disappeared, while those isoforms were still present in AAIII tumors and normal brain tissues. Almost all of the GBM samples (35/37 pGBM and 6/8 sGBM) were devoid of the CPEB4c splice variant which also contains the B-region , S5. Addn = 6, AII and AAIII) and samples negative for phospho-CPEB3 . All these samples revealed strong immunoreactivity againstCPEB3 protein in the cytoplasm and processes of astrocytic tumor cells. We investigated the expression/activity of kinases that were shown before to phosphorylate CPEB3 protein [Immunohistochemical analysis of CPEBs in gliomas indicated that expression of active phospho-CPEB3 protein shows an inverse correlation with the glioma malignancy grade Figure , right.  protein , 21, 22.p < 0.01). Within the group of patients with pGBM, 17/25 showed CPEB1 protein expression, but there was no correlation with survival (data not shown). The group of sGBM contained only 7 cases, so no statistic evaluation was performed. No correlation between CPEB2, CPEB3 and CPEB4 proteins expression and patient survival was observed in the examined AII, AAIII and GBM samples (data not shown). The activation of CPEB3, as determined by immunostaining, significantly correlated with survival in all investigated tumor samples . The differences in survival of patients with and without phospho-CPEB3 expression were particularly obvious for AII , AAIII  and sGBM  . This finding indicates that phospho-CPEB3 can be considered an independent biological marker for a better prognosis in astrocytomas with a similar pathogenesis. In contrast, there was no such prognostic correlation for pGBM  .An overall survival (OS) time was defined as the time between surgery for the primary tumor and death of the glioma patient. Patients alive at the time of their last follow-up were surveyed. A total number of 61–62 samples  were incDifferential expression of cancer-related genes has been widely investigated in gliomas. Results of these experiments allow today for more precise and timely molecular-based diagnosis of tumors. Microarray expression profiling based on transcript analysis allowed molecular subtyping of gliomas with identification of predictive and prognostic markers –26. Recede novo. The remaining GBM cases develop by progression of lower-grade precursor lesions to sGBM. Astrocytomas make up the largest group of gliomas and show a diffuse infiltrative growth into normal brain tissue. Median survival of patients diagnosed with GBM remains less than 12 months [CPEB methylation was previously investigated in tumor tissue [CPEB genes in glioma has not been reported. Here, we localized a DNA fragment upstream of the CPEB1 gene and developed a pyrosequencing assay to quantitatively assess the methylation of this position in a cohort of 63 human glioma specimens obtained from the German Brain Tumor Reference Center. Hypermethylation of CPEB1 was observed in gliomas containing a mutant IDH1 gene. Mutant IDH1 proteins produce the oncometabolite, 2-hydroxyglutarate (2-HG) and lead to a decrease of α-ketoglutarate (α-KG), inhibiting the activity of α-KG dependent oxygenases, which are involved in demethylation of genomic DNA and histone tails. This ultimately leads to altered epigenetic marks e.g. the glioma hypermethylator phenotype (G-CIMP) Besides CPEB1, we only observed altered methylation of a few CpG sites in the CPEB3 gene. No correlation of CPEB3 methylation with IDH1 mutation was observed.Gliomas are the most frequent form of intracranial neoplasms and are believed to derive from neuroepithelial tissue. The WHO divides them into astrocytomas, oligodendrogliomas, mixed gliomas (oligoastrocytomas) and ependymomas. Within the group of astrocytomas, glioblastoma multiforme (GBM) is the most frequent (50%) malignant tumor. The greatest numbers of GBM (90%) called pGBM develop 2 months , 30. Ther tissue , 32, butSubsequently, we determined the abundance and localization of CPEB proteins in glioma tissue by immunohistochemistry. CPEB1 was present in the infiltration areas of tumor cells into normal brain tissue. The vast majority of cells in the tumor center, in the areas of necrosis and vascular proliferation showed no CPEB1 expression, suggesting that expression of CPEB1 is silenced along with progression of cancer Figures , 3B. CPEde novo methylation is considered as a mechanism of transcriptional silencing we investigated the expression of CPEB1 and compared our findings with the clinical data  treated at the University of Bonn Medical Center, including: 28 patients with primary glioblastoma , 8 patients with secondary glioblastoma , 22 patients with anaplastic astrocytoma  and 11 patients with diffuse astrocytoma . Tumors were graded according to the World Health Organization (WHO) classification of tumors of the central nervous system . HistoloGlioblastoma cell lines A172, A178, LN229, U373MG, U87MG and T98G were received from the Ludwig Institute for Cancer Research (San Diego). Extraction of RNA from the cultured glioblastoma cells was carried out using RNeasy Mini Kit  following the manufacturer's protocol. Residual genomic DNA was removed by digestion with RNase-free DNase . Human normal white matter tissue was used as a reference. The identity of the individual cell lines has been confirmed by STR DNA profiling of 15 loci plus sex-determining marker amelogenin .Genomic DNA of 63 glioma tissues, 5 glioblastoma cell lines and 3 reference white matter tissues was treated with sodium bisulfite using the EpiTect Bisulfite kit . Primers used for amplification of bisulfite treated DNA and primers used for pyrosequencing are given in To identify alternative splice variants cDNA synthesis was done using 1 μg of total RNA, random hexamers and SuperScript II Reverse Transcriptase . Detection of distinct splice variants of CPEB1-4 was performed using RT-PCR with primers spanning previously defined splice variants. Primers for RT-PCR, product length, as well as information about the amplified splice variants are summarized in Rabbit polyclonal CPEB1, CPEB2, CPEB4 antibodies and phosphospecific antibody directed to CPEB3 a/c isoforms were generated as described before , 22. ForTumor tissues were dissected, fixed with 4% phosphate-buffered formalin for 24–48 h at 4°C and submerged in paraffin wax. The arrays were assembled by taking core needle biopsies from preexisting paraffin-embedded tissue blocks and re-embedding these tissues in an arrayed master block. 4 μm thick tissue sections were pretreated in the Lab Vision PT Modul  and in PT Modul Buffer (pH 6)  for 20 min at 99°C, followed by a cool-down phase of 20 min at room temperature. Immunohistochemical staining was performed with CSA II Biotin-free Tyramide Signal Amplification System . To reduce high non-specific background staining, endogenous peroxidases were blocked by incubation in peroxidase blocking buffer for 5 min followed by 60 min protein block at room temperature. Incubation with primary antibodies was performed overnight at 4°C. The following primary antibodies were used: CPEB1 , CPEB2 2. CPEB1-pCMV6/Neo plasmid and pCMV6/Neo control vector were designed and purchased from OriGene . A172 cells were seeded in 6-well plates and transfected with 2 μg of CPEB1-pCMV6/Neo or pCMV6/Neo vector using 3 μl Lipofectamine 3000 transfection reagent  for each reaction. After 48 h of incubation, cells were collected for RNA and protein analysis.Human glioblastoma A172 cells were grown in DMEM with 25 mM glucose supplemented with 10% fetal calf serum , 1% penicillin/streptomycin , 200 mM L-glutamine  and 100 mM sodium pyruvate  at 37°C, 5% COWarrington, UK). To achieve a semi-quantitative assessment, 2 μl of cDNA was amplified with 900 nM of each CPEB1 and PBGD primers and 100 nM of fluorogenic Taqman probes labeled with 6-FAM fluorophore (5′-end) and TAMRA quencher (3′-end) in a 12.5 μl final reaction volume (T) value was determined using SDS 5.0 Software (Applied Biosystems). Normalization was performed using the housekeeping gene PBGD as a reference against the expression of CPEB1 gene transcripts. The transcript level of CPEB1 and the reference gene was calculated using the following equation [CPEB1/XPBGD = 2CT PBGD – CT CPEB1, where X are the respective input copy numbers and CT the threshold cycle numbers for CPEB1 and PBGD.Isolated mRNAs were reversely transcribed with SuperScript II reverse transcriptase . One μg of total RNA template and 250 ng of random hexamers for priming, in a 20 μl total reaction volume were used. Real-time PCR was performed using the TaqMan Universal PCR Master Mix . First, cells were scraped in ice-cold buffer and incubated on ice for 30 min. Supernatants were collected after 15 min centrifugation at 14.000 g at 4°C. Afterwards, the total protein content was determined by a BCA Protein Assay . Lysates were then mixed with denaturing sample buffer and heated for 10 min at 65°C. Next, the protein samples (30 μg) were separated by SDS-PAGE under denaturing conditions and blotted on a PVDF membrane. Membranes were blocked with 5% milk powder in TBS-T buffer, pH 7.4 and incubated overnight at 4°C with primary antibodies. For detecting horseradish peroxidase activity, Supersignal West Dura Substrate  was used. Generated chemiluminescence was detected with the Gene Genome digital documentation system . Densitometry with data analysis was performed with GeneTools System (Synoptics) and tested for significant differences.firefly luciferase reporter and a constitutively expressed Renilla construct. As positive and negative controls non-inducible firefly luciferase reporter and constitutively expressed Renilla constructs, and a mixture of a constitutively expressed GFP construct mixed with a constitutively expressed firefly luciferase and Renilla luciferase construct were used, respectively. Reverse transfection was performed following the manufacturer's recommendation (SA Biosciences). 48 h post reverse transfection, a luciferase activity was analyzed by Dual-Luciferase Reporter Assay . The results were expressed as fold change, calculated by dividing the normalized luciferase activities of each pathway-focused reporter reverse transfected into A172 cells overexpressing CPEB1-pCMV6/Neo plasmid by the normalized luciferase activity of each pathway-focused reporter reverse transfected with the control pCMV6/Neo vector. Reporters showing at least two-fold change in relative luciferase units were considered upregulated or downregulated. Experiments were performed three times in duplicates and analyzed using GloMax 96 Microplate Luminometer .The cell based 45-Pathway Reporter Array  allows for the quantitative analysis of cancer-associated signaling pathways, by screening their activities. A172 cells overexpressing CPEB1-pCMV6/Neo or pCMV6/Neo vector were reverse transfected with reporter constructs from the reporter array plate. Each reporter assay was composed of an inducible transcription factor responsive t test or Fisher's exact test) of CPEB expression, CPEB1/3 methylation as well as putative correlation with clinical and molecular data including overall survival, malignancy grade, IDH status was carried out with Prism Software Version 6 the GraphPad Software, Inc  and OrigeneLab Corporation .Histological characterization of tissue microarrays was performed by microscopic observation and digital scan of stained specimens. Tissues were classified as positive or negative for the respective antibody. Afterwards, the positive samples were further divided into three intensity groups: 1-weak, 2-intermediate or 3-strong. Statistical analysis (Student's


Cracks in concrete structures present a threat to their durability. Therefore, numerous research studies have been devoted to reducing concrete cracking. In recent years, a new approach has been proposed for controlling temperature related cracking—utilization of phase change materials (PCMs) in concrete. Through their ability to capture heat, PCMs can offset temperature changes and reduce gradients in concrete structures. Nevertheless, they can also influence concrete properties. This paper presents a comprehensive overview of the literature devoted to using PCMs to control temperature related cracking in concrete. First, types of PCMs and ways of incorporation in concrete are discussed. Then, possible uses of PCMs in concrete technology are discussed. Further, the influences of PCMs on concrete properties  are discussed in detail. This is followed by a discussion of modelling techniques for PCM-concrete composites and their performance. Finally, a summary and the possible research directions for future work are given. This overview aims to assure the researchers and asset owners of the potential of this maturing technology and bring it one step closer to practical application. Reinforced concrete is a building material of choice for structures in challenging environments. Concrete is a highly durable building material and can have a long service life with little or no maintenance. When steel is embedded in concrete in order to take over tensile stresses, it is typically protected from corrosion by a passive film formed on its surface due to the high alkalinity of the concrete pore solution . Under cConcrete is a quasi-brittle material that cracks at low strain levels . One posDuring the heating pass of the phase change temperature, PCMs change from solid to liquid and capture heat, thereby reducing the temperature rise in the system. During the cooling pass of the phase change temperature, PCMs change from liquid to solid and release the stored energy, thereby reducing the cooling rate of the system. Therefore, PCMs can be used in concrete technology to reduce cracking caused by temperature effects : early aThis paper aims to review the current state of the art of using PCMs for smart crack control in reinforced concrete, with the focus on the two aforementioned causes of cracking. The paper is organized as follows: first, different types of phase change materials and possible ways of application in concrete technology are discussed; then, studies regarding the use of PCMs to reduce cracking are reviewed; further, the influence of PCM addition on the properties of concrete is discussed; this is followed by a review of modelling approaches for PCMs in concrete; finally, conclusions and possible directions for future research are given.A large variety of PCMs are available. In general, they can be divided in three categories : organicUsing pipes filled with PCM incorporated in concrete.Using porous carriers such as lightweight aggregates (LWAs) impregnated with PCMs.Using microencapsulated PCMs.Impregnating PCMs in the concrete pores from the surface.Most applications of phase change materials in construction are related to increasing the energy efficiency of the building envelope ,25,26. TThese methods are schematically illustrated in For reducing temperature effects in massive concrete structures, the use of cooling pipes with running water is relatively common . In ordeAnother way of incorporating PCMs in concrete is through impregnation in lightweight aggregates. Water impregnated LWAs are commonly used for internal curing of concrete ,36,37, pDifferent types of LWAs have been used: expanded shale ,40,41,42Compared to the method of embedded pipes, use of LWAs as PCM carriers provides a better spatial distribution of PCM. Especially in composites with relatively fine LWA the PCM will be well distributed, and the ability of heat to travel through the matrix and reach all corners of the system is not as important . EssentiAnother major disadvantage of the LWA approach is possible leakage of the PCM during mixing and/or exploitation and its dissolution in the mixing water. If the ambient temperature is lower than the phase change temperature of the PCM, leakage during mixing will occur . In factMicroencapsulation is defined as a process in which tiny particles or droplets are surrounded by a coating to give small capsules with useful properties. In concrete technology, microencapsulation has been used for carrying self-healing agents ,55,56 anIn most studies, paraffin as the PCM core material is encapsulated in a polymeric shell. Most microencapsulated PCMs used in concrete have either a polymethyl methacrylate (PMMA) ,63,64,65A major concern when using microencapsulated PCMs in concrete is their survivability and long-term durability. Survivability can be defined as the ability of microcapsules to survive the mixing process of concrete without rupture and leakage of the core material. Since this is a major concern, in most studies care is taken to minimize the exposure of the microcapsules to concrete mixing, for example by adding them as last ingredients ,73,74. NOn the other hand, in some studies it was emphasized that the microcapsules can survive the mixing process ,69,74. TAnother possible cause of capsule rupture post-curing is mechanical loading. For example, Jayalath et al.  observedWallboards with surface impregnated PCMs have been proposed for latent heat storage in buildings ,78,79. FThe temperature in young concrete increases due to the exothermic nature of the chemical reactions occurring during cement hydration . If a coThe first study proposing use of PCMs for temperature control in early-age concrete was performed by Mihashi et al. , who addTwo methods of incorporating PCM have been used for crack control in young concrete: using pipes filled with PCM and using PCM microcapsules see . Qian etIn another study, Qian & Gao  used an More commonly, microencapsulated PCMs have been used for temperature control in young concrete ,73,89,90In addition, one study  used baIn warm climates, incorporation of PCM has an additional advantage that goes beyond the early age behavior. Since PCMs typically remain active, they will go through a phase change every time their phase change temperature is passed. Therefore, they can contribute to reducing thermal fatigue in structures exposed to temperature variations, such as concrete pavements. Thiele et al.  showed tIn cold climates, concrete may deteriorate due to freezing and thawing of water in the system ,91. Sinc3 of PCM with a phase change temperature of 5 °C and the enthalpy of phase change of 250 J/g. Since previous works showed that a greater number of freeze/thaw cycles can be expected in bridge decks compared to concrete pavements [The described approach essentially tries to avoid damage by accommodating the expansion. An alternative would be to avoid the cycles of freezing and thawing altogether. In the past few years, several studies have proposed the use of phase change materials to reduce the number of freeze/thaw cycles and improve concrete durability. The use of phase change materials in concrete technology for reducing freeze/thaw damage was first proposed by Bentz and Turpin in 2007 . In thisavements , a bridgBased on this, an additional study was performed by Sakulich and Bentz . In the An additional benefit of using PCMs instead of air entrainment in this case is the ability of the material to “melt” ice and snow forming on its surface: because the concrete will remain warmer for a longer period of time, it will prevent snow from accumulating and ice from forming on the surface. This may be very beneficial for some applications, such as airport runway pavements. This possibility was explored in several studies. Farnam et al.  used twoBased on these results, Farnam et al.  performeGao et al.  developeIf PCMs are added to the mix as microcapsules or impregnated in LWAs, they may affect the properties of fresh concrete. If LWAs impregnated with PCMs are used, the following issue may occur: since PCMs are not able to penetrate into small pores of the LWA, these pores will remain air filled. If such LWA is placed in the mix, it may imbibe water from the concrete and thus dehydrate the system . SakulicWhen microencapsulated PCMs are used, it has been shown that the workability of concrete may be affected due to their small size and, consequently, large surface area ,72,101. Addition of PCM to the concrete mixture may affect its mechanical properties. When it comes to compressive strength, two technologies of PCM incorporation  result in different effects.When LWAs are used to replace a part of the aggregates in the concrete mix, a drop in compressive strength is expected . The samWhen microencapsulated PCMs are used, in general two opposing effects on concrete strength occur : first, It has been observed that, in general, the drop in tensile/flexural strength with microencapsulated PCM addition is significantly less pronounced than the drop in compressive strength ,75,102. It should be noted that the strength of concrete with PCMs may be, to a certain extent, dependent on the temperature . Šavija et al.  found thThermal conductivity of PCMs is relatively low compared to cementitious materials . TherefoDurability of cement-based materials incorporating PCMs has been scarcely studied. When the long term behavior of such materials is considered, only shrinkage has received attention. It is expected that the addition of compliant inclusions, such as PCM microcapsules, into the mix will cause an increase in shrinkage deformation  comparedTransport properties of cement-based materials with PCMs have not been extensively studied. In the study by Wei et al. , it has The effect of PCM addition on reinforcement corrosion has been studied by Cellat et al. . In theiAs already described, properties that are most affected by the presence of PCM additions are (compressive) strength, elastic modulus, and thermal conductivity. Models developed to predict the effect of PCM addition on these properties are described.nσ is the compressive strength of any system normalized by the strength of the plain cement paste (unitless), IV is the volume fraction of inclusions in the system (%), and y is a fitting constant (unitless) that depends on the nature of the inclusion used. For microencapsulated PCMs and quartz sand used in their study, the following values of the fitting parameter y gave the best fit: y = 2.47 or 3.22 for PCM mixtures for w/c = 0.35 and w/c = 0.45, and y = −0.14 for the quartz mixtures for w/c = 0.45, respectively. A comparison between experiments and predictions is given in As previously discussed, many studies reported a drop in compressive strength with PCM addition. Based on their experimental findings, Fernandes et al.  proposedIn the case when PCM inclusions are combined with stiff inclusions (sand), it is not possible to use analytical expressions to predict the strength. For this purpose, Aguayo et al.  developeThe model showed that, when a combination of weak and soft inclusions (such as PCM microcapsules and quartz sand) is used, the influence of PCM addition on the compressive strength of mortars is more complex. The weaker paste-quartz interfacial elements resulted in the most likely failure path progressing around the inclusions. In systems where small amounts of quartz were replaced by PCM, localized stress concentrations were observed at the PCM-paste interface, but the PCMs being few and far between, the predicted failure region was still along the weaker paste-quartz interface. When the system contained a substantial amount of PCMs, the stress concentrations at the paste-PCM interfaces grew, and the likelihood of paste failure increased substantially. This was in accordance with their experiments , which sSimilar approaches were proposed for determining the Young’s modulus of the composite. Fernandes et al.  proposedCE is the elastic modulus of the composite, pE the elastic modulus of plain cement paste, ν the Poisson’s ratio of the composite (determined by averaging the Reuss–Voigt solution in proportion to the volume fractions of paste and inclusions in the composite and their properties), pν the Poisson’s ratio of the plain cement paste, and IV volume fraction of inclusions/PCMs (%). A more advanced approach based on micromechanics was proposed by Xu et al. [Here, u et al. . In theiAs already described, the addition of PCMs has a profound influence on the thermal properties of the composite material due to PCMs lower thermal conductivity. To simulate the performance of PCM based concrete in elements and structures, it is therefore necessary to predict the thermal conductivity of the composite material depending on its constituents.For composites incorporating microencapsulated PCMs, Ricklefs et al.  proposedk and ϕ are the thermal conductivity and volume fraction and the subscripts c, s, and m refer to the core, shell, and matrix, respectively. This model showed very good agreement with experimental data  :(4)ρcp∂Tρ is the density (kg/m3), pc the specific heat capacity (J/kg K), k the thermal conductivity (W/mK), T the temperature (K), t time (s), and x the spatial coordinate (m). In case of PCMs, the sensible heat contribution  and the latent heat contribution  both need to be considered. In the literature, latent heat stored during the phase change in the PCM microcapsules is mostly taken into account by using the heat capacity method [Here, y method . In the y method ,120,121:p,cc is the specific heat capacity of concrete (J/kg K), fh the heat of fusion of the PCM, pcmm the quantity of PCM per cubic meter of concrete (kg), PCT the phase change temperature, ΔPCT the temperature window. Using this approach, the phase change can be simply simulated. Other approaches, such as the “‘Heat Transfer with Phase Change” approach, are seldom used [Here, dom used ,123.Young et al.  developeŠavija and Schlangen  used a cIn semi-adiabatic  conditions, the addition of PCM in hardening concrete has potential to delay the temperature rise, reduce the maximum tensile stress, and delay its occurrence. The maximum tensile stress is inversely proportional to the amount of PCM added to the mix see .In semi-adiabatic conditions, the phase change temperature does influence the maximum temperature developing in the structure .An increase in the latent heat of fusion serves the same purpose as an increase in PCM addition: it lowers the maximum temperature and maximum stress and delays their occurrence. Especially the cooling phase is prolonged. Therefore, a trade-off between the heat of fusion and quantity of PCM microcapsules is possible, where a smaller amount of PCMs with a higher heat of fusion can be used with the same  efficiency. This would be beneficial also in terms of mechanical properties of the concrete.Based on an example structure analyzed in their study, they concluded that:Regarding performance of concrete with PCMs for reducing freeze/thaw damage, not many literature references are available. References ,43 showeIn this paper, a systematic review of possible uses of phase change materials in concrete technology for smart  crack control is presented. Types of phase change materials and technologies for their use in concrete are described. Major fields of application, i.e., control of early age cracking and freeze/thaw damage in concrete structures, are reviewed. In addition, effects of PCMs on typical parameters of concrete, such as strength and workability, are reviewed. Finally, models for PCM based concrete are presented and discussed. It can be concluded that the use of PCMs for crack control in concrete has shown promising results so far.Before the widespread application of this technology, however, several aspects deserve further study. First, although research studies so far have shown that the technology is promising, there have been no comparisons with methods used at the moment for crack control . In order for asset owners to be interested in the technology, it needs to be shown that it is advantageous compared to current practice. Second, no studies focusing on the long-term durability of the material are available. This information is necessary for performing life-cycle analyses. Finally, cost considerations  have been left out in the studies. Although it is expected that the materials would become cheaper if they were to be used on the large scale, it is important to keep in mind that concrete in itself is a relatively cheap building material and large increases in price are, for most contractors and owners, not acceptable.The technology at the moment has reached a stage where field trials are needed to test its efficiency in practice. In the beginning, it is suggested to make simple structures such as concrete pavements or walls on slab, which bring relatively low risk. If crack control in these trail structures were to be successful, major applications of this technology could be for massive retaining walls (for hydration temperature control) and airport pavements (for reduction of freeze thaw damage and ice accumulation).


This study demonstrated that EVT secretions, including PFN1, enhanced HESC decidualization and motility. This study has identified a new pathway that facilitates appropriate decidualization during the establishment of pregnancy.During the establishment of pregnancy, extravillous trophoblast (EVT) must invade into the uterine decidua to facilitate decidual artery remodelling to create the placental blood supply. The local decidual environment is thought to regulate trophoblast invasion, however these interactions are poorly defined in humans. Recent evidence in women suggests impaired decidualization is associated with miscarriage and preeclampsia. Primary human endometrial stromal cells (HESC) and first trimester extravillous trophoblast (EVTs) were used to assess the effect of EVT-secreted factors on HESC decidualization, adhesion, proliferation and migration. We determined the role of profilin (PFN)1, an EVT-secreted factor, on HESC function and identified a downstream target of PFN1. EVT-secreted factors induced HESC decidualization and enhanced decidualized HESC adhesion, proliferation and migration. Recombinant PFN1 enhanced methoxyprogesterone acetate-induced HESC decidualization and proliferation. PFN1 down-regulated the expression of lipoxygenase arachidonate 5-lipoxygenase (ALOX5) in HESC and THP-1 macrophages. ALOX5 localised to decidual cells and CD68+macrophages in 1 This results in the invasion and occlusion of endometrial spiral arteries by 8 weeks (begins as early as 5 weeks) and myometrial invasion by 14 weeks1. Such invasion is critical to create the placental blood supply which is complete at the end of the first trimester2. Inadequate or inappropriate implantation and placentation is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications3.During the establishment of pregnancy, the human blastocyst implants into the uterine endometrium to facilitate the formation of a functional placenta which is completed by the end of the first trimester. During implantation, extravillous trophoblast (EVT) invade into the uterine decidua from as early as 5 weeks gestation (~14 days after implantation)4. Prior to implantation and in preparation for pregnancy, stromal cells of the uterine endometrium become ‘decidualized’. Decidualization describes the proliferation and differentiation of endometrial stromal cells (ESC) into decidual cells. It is a slow process which involves the categorical reprogramming of ESC such that different genes are expressed at different stages of the differentiation process5. In women, decidualization occurs spontaneously in stromal cells adjacent to spiral arterioles during the mid-secretory phase of the menstrual cycle under the physiological stimulus of progesterone, regardless of the presence of a blastocyst6. If implantation occurs, decidualization intensifies and continues to form the decidua of pregnancy. This is in contrast to rodents where decidualization begins post-implantation.The local decidual environment plays a key role in regulating trophoblast invasion9. While it is known that locally produced decidual factors act in association with progesterone to drive decidualization10 there is very little known of decidual-EVT interactions in humans.Recent evidence in women demonstrates that decidualization is impaired prior to the onset of symptoms in recurrent miscarriage, preeclampsia (PE) and placenta accreta suggesting abnormalities in decidualization contribute to the development of these disorders12, show altered HESC cytokine and angiogenic factor mRNA expression, including IL8, and the decidual marker IGFBP1. When co-cultured with 1st trimester placental explants, term decidual fibroblasts maintained their decidualization compared to control (no treatment)13, however this likely reflects progesterone secreted by the placenta itself. While EVTs secrete progesterone14, it is not known whether EVTs themselves would affect HESC decidualization. Finally, co-cultured non-decidualized and decidualized HESC with the AC1M88 trophoblast cell line enhances HESC migration15, however this has not been confirmed using primary first trimester trophoblast.The effect of EVTs on HESC function has only limited research to date. HESC treated with EVT cell surface and secreted factors16. PFN1 is an actin-binding protein which is also found extracellularly, including in serum17 and conditioned media (CM)18. The function of extracellular PFN1 is not known. In vitro, EVT secretion of PFN1 is enhanced following treatment with decidualized HESC CM16, however the role of EVT-secreted PFN1 is not understood.We previously identified profilin 1 (PFN1) as secreted by EVTsWe hypothesized that EVT factors regulate HESC decidualization in first trimester decidua. We investigated the effect of EVT-secreted factors and PFN1 on HESC function . PFN1 targets in HESC and THP-1 macrophage cells were also investigated.st trimester EVT-derived cell line]) treatments at 50% final volume were added to the hormone treatments for a further 8 days. As the induction of PRL secretion was highly variable between women , this data   or E+ methoxyprogesterone acetate (MPA)  for 6 days, then EVT or control cell line  are motile. Therefore we investigated whether EVT secreted factors regulated decidualized HESC motility. EVT CM significantly enhanced decidualized HESC migration . Therefore, we investigated whether EVT-secreted PFN1 could act on HESC to promote decidualization.We previously localized PFN1 to EVTs, glandular epithelium and leukocytes in 1HESC were induced to decidualized for 14 days by treatment with E+MPA with PFN1 or control treatment included at every media change. PFN1 treatment significantly enhanced decidualization in HESC treated with E+MPA Fig.  but had HESC were decidualized by E+MPA treatment for 12 days before PFN1 treatment was included in experimental conditions. PFN1 treatment did not significantly increase proliferation in decidualized HESC at 72 h Fig.  and simiTo determine the mechanism by which PFN1 enhanced HESC decidualization, we utilized a Human Hypertension array to identify factors in HESC regulated by PFN1. HESC were decidualized for 10–12 days with E+MPA before a 6 h treatment with PFN1 or control and RNA collection for the array. Samples (n = 4) were pooled for the array.19, was identified as a potential target of PFN1 by this array  5 acts as a receptor to a profilin-like molecule produced by Toxoplasmosis gondii22, but this has not been confirmed using mammalian profilin. However, T. gondii profilin stimulates IL8 in HEK293 cells via TLR522: HESC production of IL8 is upregulated by co-culture with trophoblast cells12 or treatment with trophoblast CM11. It remains to be determined whether mammalian PFN1 binds to TLR5 and whether PFN1 stimulates IL8.PFN1 is an actin-binding protein which is also found extracellularly, including in serum17. For the first time, we showed that PFN1 enhanced decidualization and proliferation of HESC, likely via down-regulating ALOX5. As ALOX5 was down-regulated during MPA-induced in vitro decidualization and PFN1 is absent in cycling endometrium, we suggest that EVT-secreted PFN1 is an additional factor expressed by EVTs to downregulate ALOX5 during the early phase of the decidualization process . Written and informed consent was obtained from each patient before surgical intervention. Methods were carried out in accordance with Monash Health Human Research Guidelines.16. The purity of primary cytotrophoblasts was confirmed by immunocytochemistry for HLAG  as previously described16.Normal first trimester placental tissue (n = 15) was collected from healthy women undergoing termination of pregnancy for psychosocial reasons (amenorrhea 6–12 weeks). EVT were isolated from normal first trimester placental tissue as previously describedEndometrial biopsies (n = 36) were collected by dilatation and curettage from fertile women (aged 18–40 years) scheduled for tubal ligation or undergoing testing for tubal patency during days 8–21 of a normalised 28 day menstrual cycle. Tissues were assessed by a pathologist and had no obvious endometrial pathology. The women had no steroid treatment or other medication for at least 3 months before the collection of tissue.28 except that the collagenase used was from Sigma (Type III C0255). This method results in a 97% pure stromal cell culture28.HESC were isolated by collagenase digestion and filtration as described previouslyEVTs were maintained in DMEM/F12 (Gibco) containing 10% FCS (Gibco) and 1% antibiotics . EVTs were cultured on Growth-factor Reduced Matrigel (Corning) diluted 1:5 in serum-free DMEM/F12 to promote differentiation towards an EVT phenotype.HESC were cultured on plastic in DMEM/F12 containing 10% charcoal-stripped FCS (Gibco) and 1% antibiotics prior to experimental use.HEK293 are an embryonic kidney epithelial cell line (ATCC). HEK293 cells were maintained in EMEM (Invitrogen) containing 10% FCS and 1% antibiotics.HTR8/SVneo  are a 1st trimester EVT derived cell line. HTR8/SVneo cells were maintained in RPMI (Invitrogen) containing 10% FCS and 1% antibiotics.THP-1 cells  were cultured in RPM1 containing 10% FCS and 1% antibiotics, before stimulated towards an adherent, macrophage phenotype by overnight treatment with phorbol 12-myristate 13-acetate .2 in a 5% CO2 humidified culture incubator.All cells were cultured at 37 °C, 20% OCM collection Isolated primary first trimester EVT, HTR8/SVneo and HEK293 cells were cultured on 1:5 diluted growth-factor reduced Matrigel in 10% FCS DMEM/F12. CM was collected after 48 h. EVT CM was pooled (n = 5/group) from EVTs. HTR8/SVneo and HEK293 CM was pooled from n ≥ 3.−8 M, Sigma) and medroxyprogesterone acetate  for 12–14 days as previously described16, except no cyclic adenosine monophosphate was included. Media (including treatments below) was replaced every 2–3 days. The level of PRL secretion taken as a measure of decidualization29.HESC  were grown to confluence (maximum time to confluence 5 days) and decidualized by treatment with oestradiol ; minimally decidualized ; or decidualized .EVT, HEK293 or HTR8/SVneo CM  was included from day 7-day 14 of decidualization.Recombinant human PFN1  or vehicle control was included from day 0 - day 14 of decidualization. The PFN1 concentration was determined by pilot studies (range 0.1–100 µM).Prolactin (PRL) secretion by HESC was assayed by ELISA .Progesterone was assayed by competitive binding immunoenzymatic assay using a rabbit anti-human progesterone antibody performed on a Beckman Coulter Unicel DXI 800 analyzer conducted by Monash Pathology . Analytical range: 0.3–127.2 nmol/L. Coefficient of variation 6.4%.30.The effect of EVT CM (FV 50%) and/or PFN1 (0.1–100 µM) on decidualized HESC (HESC pre-treated with E+MPA for 12 days) adhesion, proliferation and migration was assessed using the xCELLigence real-time cell analyser (John Morris) as previously published 30. The plate was monitored once every 15 secs for the first 6 h to monitor adhesion then every 15 minutes until 72 h to monitor proliferation. Data was calculated with RTCA software 1.2.1 and exported for statistical analysis. EVT or HTR8/SVneo CM was included at 50% FV; PFN1 treatments were included at 10 and 100 µM.HESC decidualized using E+MPA as described above seeded on day 12 at 10,000 cells/well in 2% csFCS DMEM/F12 including E+MPA. Adhesion and proliferation were assessed using the E plate as previously described30; The plate was monitored every 15 min for 17 h (migration). EVT or HTR8/SVNeo CM was included at 50% FV; PFN1 treatments were included at 10 and 100 µM. Treatments were included in the upper well; the bottom container contained 10% FCS. Data was calculated with RTCA software 1.2.1 and exported for statistical analysisHESC decidualized using E+MPA as described above before being seeded on day 12 at 30,000 cells/well in 2% csFCS DMEM/F12 including E+MPA. Migration was assessed using the CIM plate as previously described31. Slides were dewaxed, rehydrated and antigen retrieved in 0.01 M sodium citrate before quenching endogenous peroxidase activity. Non-specific binding was blocked with 10% normal goat serum and 2% normal human serum before the primary antibody was applied overnight at 4 °C. Antibody localization was detected by sequential application of biotinylated goat anti-rabbit IgG or horse anti-mouse IgG (1:200) followed by Vectastain ABC Elite kit (Vector). Staining was visualized by the diaminobenzidine substrate (DakoCytomation), tissues were counterstained with Harris hematoxylin (Sigma–Aldrich) and mounted. Representative images were taken using CellSense software.Immunohistochemistry was performed on formalin-fixed first trimester decidua basalis sections (4μm) as previously described32. The intensity of PFN1 staining in the endometrium was assessed and allocated a score between 0 (no staining) and 3 (strong staining) relative to positive and negative controls.Immunostaining intensity in the endometrium was analysed semiquantitively by two independent and blinded observers as previously describedFormalin-fixed sections were treated as described for immunohistochemistry with modifications as follows: non-immune serum and 10% CAS block diluted in PBS/0.1% bSA and washes performed in 0.6% Tween20 in PBS/0.1% bSA; secondary antibody incubation  in non-immune serum for 2 h and then sections were mounted using Vectastain (DAKO).ALOX5 , CD68 , human leukocyte antigen G (HLAG) , PFN1 , vimentin . Negative control isotype rabbit, goat or mouse IgG (Dako) was included for every tissue section.Adherent THP-1 cells and HESC  were treated with PFN1 (100 µM) or vehicle control (PBS) for 6hrs (array), 24hrs (RNA) or 48 and 72hr (protein).31 using TRI Reagent RNA Isolation system (Sigma #T9424) following manufacturer’s protocol. All samples were DNase I treated  and concentrations were quantified using the NanoDrop1000 . Human Hypertension RT2 Profiler PCR Arrays (QIAGEN #PAHS-037Z) were performed in accordance with manufacturer instructions on decidualized HESC only. For qPCR total RNA was reverse transcribed using Superscript III First-Strand Synthesis System (Invitrogen) according to the manufacturer’s protocol. Real time RT–PCR analyses were performed on the ABI 7500HT fast block real time PCR system  in triplicate in 384-well Micro Optical plates (Applied Biosystems) with the Power SYBR green master mix (Applied Biosystems) and 200 nM primers.Total RNA was isolated and reverse transcribed (250 ng) as previously describedThe PCR protocol was as follows: 95 °C for 10 min and 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min. Relative expression levels were calculated by the comparative cycle threshold method (ΔΔCt) as outlined in the manufacturer’s user manual, with 18S ribosomal RNA serving as the endogenous control for normalization. Products were sequenced to confirm specificity.The sequences for the ALOX5, Prolactin, and 18S primers (200 nM used in reaction) were as follows;ALOX5 Fwd-GTTCCAGTGACTTCCACGTCCA, Rev-GAGGCCACACTCGCAGATGA; Prolactin Fwd-GGTTCATCCTGAAACCAAAG, Rev-CTTCAGGAGCTTGAGATAATTG; 18s Fwd- GATCCATTGGAGGGCAAGTCT, Rev-CCAAGATCCAACTACGAGCTT.31. Blots were probed for ALOX5  and GAPDH .THP-1 cells were collected after 48 or 72 h PFN1 treatment (100 µM) and lysates (60 ug/lane) subjected to Western blotting as previously describedAll statistical analyses were performed on raw data using GraphPad Prism 6 . Where primary samples were used, the data was paired for statistical analyses to account for differences at baseline between samples. Data was tested for normality where possible (n > 6) and paired t-tests, Student’s t-tests, Mann Whitney t-tests, one-way ANOVA, repeated measures ANOVA or Friedman’s test were used as appropriate: the test used is indicated in the figure legends. All data is presented as mean ± SEM and as % change from control only for graphical presentation to account large variability between patient tissues at baseline. A p < 0.05 was considered statistically significant.Supplementary Info


Immunoglobulin G4-related disease (IgG4-RD) is a newly recognized systemic condition characterized by high serum immunoglobulin G4 (IgG4) concentration and IgG4-bearing plasma cell infiltration in affected organs. Although it has become evident that IgG4-RD also involves the systemic aortic/arterial system, the precise details of this condition remain unclear. The present study sought to clarify the clinical features of IgG4-related periaortitis/periarteritis.Among 223 patients with IgG4-RD, 179  were recruited for this study. Periaortitis/periarteritis was defined as vessel wall thickness with circumferential enhancement on contrast-enhanced computed tomography.Periaortitis/periarteritis was identified in 65  of 179 IgG-RD patients. The distribution of IgG4-related periaortitis/periarteritis could be broadly classified into five types, with the most prevalent Type 2 (44.6%) being localized at the infra-renal artery portion of the abdominal aorta and continuing to the iliac arteries. The infra-renal artery region of the abdominal aorta was most frequently involved (>80%) among IgG4-related periaortitis/periarteritis cases. Comparisons of clinical parameters between IgG4-RD patients with and without periaortitis/periarteritis revealed significantly higher propensities for older IgG4-RD onset age and highly active disease state featuring elevated serum IgG, IgG4, circulating immune complex, and soluble interleukin-2 receptor. All patients showed improvement of wall thickening after steroid therapy, although nine patients (20.9%) exhibited worsening of luminal dilatation. The main risk factor for this manifestation was prior luminal dilatation according to multivariate analysis.IgG4-related periaortitis/periarteritis predominantly occurred at the infra-renal artery portion of the abdominal aorta, affected older IgG4-RD onset patients, and was prevalent in highly active disease states. As reported previously, the main risk factor for worsening luminal dilation after corticosteroid therapy was the existence of luminal dilation beforehand. IntensiSince 2008, several manifestations of IgG4-related periaortitis/periarteritis have been detected or recognized in case reports during systemic imaging analysis of other IgG4-RDs and pathological evaluation of surgical specimens, such as those for aneurysm. Consequently, a small number of chronic periaortitis cases involving the abdominal aorta have been reported in association with IgG4-RD –14. StonRegarding pathological evaluation, IgG4-RD was first detected in relation to inflammatory abdominal aortic aneurysm (AAA) , 16, 17.The clinical evaluation of IgG4-RD has largely been carried out concomitantly with the systemic imaging analysis of other IgG4-RDs. Inoue et al.  reportedIn spite of the recent progress in defining IgG4-related periaortitis/periarteritis, extensive studies with sufficient case numbers are needed. In this context, the present study sought to clarify the clinical features of IgG4-related periaortitis/periarteritis as determined by the clinical and imaging analysis of 179 patients with IgG4-RD.We enrolled 223 patients with IgG4-RD  who were seen at our clinic or affiliated hospitals between 2003 and 2015. IgG4-RD was diagnosed based on the Japanese Comprehensive Diagnostic Criteria for IgG4-RD  in all cAll 179 patients underwent contrast-enhanced CT that was reviewed by an experienced radiologist (YF) at Shinshu University School of Medicine. Periaortitis/periarteritis was defined as vessel wall thickness and wall enhancement either partially of circumferentially on CT. We recorded maximum vessel wall thickness and lumen diameter in affected lesions. Improvement of periaortitis/periarteritis was defined as decreased vessel wall thickness. Luminal dilatation at the time of periaortic/periarterial lesion diagnosis was defined as that for aneurysm, in which the luminal diameter was more than 1.5 times wider than normal  according to the Japanese Circulation Society 2011 guidelines for diagnosis and treatment of aortic aneurysm and aortic dissection .We analyzed for risk factors of periaortitis/periarteritis complications by comparing such clinical parameters as IgG4-RD onset age, gender, serum levels of various activity markers such as IgG4, IgG, complement proteins, soluble interleukin-2 receptor (sIL-2R), rheumatoid factor (RF), and circulating immune complex (CIC), number of lesions, and history of corticosteroid therapy between patient groups with and without periaortitis/periarteritis. Patients taking hypotensive agents were deemed to be hypertensive.We compared various clinical parameters between patients with and without exacerbation of luminal dilatation after corticosteroid therapy to assess the effects of treatment. The exacerbation of luminal dilatation was defined as an expansion of luminal diameter > 5 mm at the same site as periaortitis/periarteritis lesions detected at the time of the initial diagnosis .2 test or Fisher’s exact test for categorical data. Statistical analyses were performed using Stat Flex version 6 software . p < 0.05 was considered statistically significant.Differences between groups were analyzed using the Mann–Whitney test for continuous data and the χThis study was approved by the ethics committee of Shinshu University School of Medicine . Because this study was a retrospective observational study with medical records, and was not an intervention study with human samples, consent to participate was not needed. Our ethics committee also approved that consent to participate was not needed. The investigation was conducted in compliance with the Helsinki Declaration.The study cohort included 179 patients with IgG4-RD . One hundred and nine patients received steroid therapy after the involved vital organs exhibited a risk of serious dysfunction or failure, such as obstructive jaundice due to pancreatic head swelling or urethral stenosis from retroperitoneal fibrosis, or when unbearable abdominal pain or other severe symptoms were present. Steroid treatment was carried out according to the Japanese Consensus Guidelines for AIP, which recommended a minimum of 3 years of maintenance therapy .Periaortitis/periarteritis was identified in 65  of 179 IgG4-RD patients and could be broadly classified into five types . Laboratory tests revealed significant disease activity marker increases in the positive group for IgG, IgG4, CIC, and sIL2R. IgG remained significantly higher in the positive group  according to multiple logistic analysis. Neither a history of smoking or hypertension nor levels of IgE, white blood cells (WBC), or C-reactive protein (CRP) showed a significant association with risk.To identify the risk factors associated with the onset of periaortitis/periarteritis, we compared several clinical parameters between IgG4-RD patients with and without periaortitis/periarteritis Table . There wp = 0.043) and multiple logistic analyses   . Among the nine patients with luminal dilatation, two underwent endovascular aortic repair and continued corticosteroid therapy without recurrence.Among the 65 IgG4-related periaortitis/periarteritis patients, 43 66.2%) received corticosteroids. All patients showed improvement of wall thickening after therapy , and cardiac catheterization have been successfully adopted for early disease detection and follow-up [18F-FDG-PET reveals FDG uptake at the area of the lesion. Owing to its capability to perform functional and tissue characterization, CMR offers integrated imaging of the aorta, coronary arteries, and heart along with the assessment of disease acuity and extent of fibrosis to guide further treatment. In fact, CMR has been successfully used to determine disease activity in Takayasu arteritis [Cardiovascular involvement in IgG4-RD may manifest as cardiac pseudotumor, inflammatory periaortitis/periarteritis, coronary arteritis, and/or pericarditis. Because IgG4-related cardiovascular disorders can severely affect patient prognosis, various imaging techniques including echocardiography, CT, ollow-up . Echocarrteritis . CMR hasrteritis . CT was This study had several limitations. First, because pathologic specimens of vascular lesions were not available to verify IgG4-RD, other conditions causing periaortitis/periarteritis may have been included. Although the improvement of periaortitis/periarteritis after corticosteroid therapy was consistent with IgG4-RD, this too was not definitive. To reduce bias, we identified IgG4-related periaortitis/periarteritis among patients with other confirmed IgG4-RDs, and vascular lesions on CT imaging were reviewed by an expert radiologist using consistent criteria. Second, the sample size of this investigation was limited despite being the largest single-center cohort to date.IgG4-related periaortitis/periarteritis predominantly occurred at the infra-renal artery portion of the abdominal aorta, affecting patients with an older IgG4-RD onset age as well as those exhibiting a highly active disease state with elevated serum IgG, IgG4, CIC, and sIL2R. The main risk factor for worsening luminal dilation after corticosteroid therapy was confirmed to be prior luminal dilation.


Many B-cell neoplasms are associated with autoimmune diseases (AIDs) but most evidence is based on a personal rather than a family history of AIDs. Here we calculated risks for non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and multiple myeloma (MM) when family members were diagnosed with any of 44 different AIDs, or, independently, risk for AIDs when family members were diagnosed with a neoplasm. A total of 64 418 neoplasms and 531 155 AIDs were identified from Swedish nationwide health care records. NHL was associated with a family history of five AIDs, all increasing the risk, HL was associated with one AID increasing and three AIDs decreasing the risk while MM had no association. A family history of NHL was associated with eight, HL with seven and MM with seven different AIDs, nine increasing and 13 decreasing the risk. The present family data on B-cell neoplasms and AIDs show an approximately equal number of associations for risk increase and risk decrease, suggesting that inherited genes or gene-environment interactions may increase the risk or be protective. These results differed from published data on personal history of AID, which only report increased risks, often vastly higher and for different AIDs compared with the present data. In a recent study, we provided evidence that germinal center-derived neoplasms were most responsive to autoimmune stimulation and that these neoplasms showed patterns of similarity in their response to different AIDs.12Hodgkin lymphoma (HL), multiple myeloma (MM) and the common non-Hodgkin lymphoma (NHL) subtypes are mature B-cell neoplasms for which a personal history of an autoimmune disease (AID) is a known risk factor.14 For MM, a family history of systemic lupus erythematosus was a risk factor.15 The potential mechanisms for shared familial associations could be pleiotropy, that is, single genes influencing multiple phenotypes, such as different cancers or AIDs.19 Preliminary evidence for pleiotropy between B-cell neoplasms and many AIDs has been presented for human leukocyte antigen alleles and some other genes.22 The intriguing aspect of pleiotropy is that the direction of allelic effects may be opposite between the two diseases.19Even though a personal history of an AID is a known risk factor for B-cell neoplasms the data on associations of a family history of AID with cancer risk has deserved limited attention although the possible genetic etiology calls for clarification. In Swedish–Danish studies, a family history of sarcoidosis and ulcerative colitis was reported to be associated with an increased risk of HL, whereas for NHL no significant association with AIDs was found.In the present study, we will analyze risks for NHL, HL and MM when a first-degree family member was diagnosed with any of 44 AIDs, and vice versa, risks for individual AIDs when a family member was diagnosed with a B-cell neoplasm. A total of 64 418 neoplasms and 531 155 AIDs were covered and all diagnoses were obtained from nationwide medical records.23 As family members, only first-degree relatives of offspring-parents and offspring and their siblings were considered. The offspring generation, for which siblings were available, reached a maximal age of 80 years by 2012; for the parental generation there was no age limits. Children of the offspring generation were not considered. Only the first AID diagnosis was included because multiple diagnoses may be liable to surveillance bias. Various revisions of the International Classification of Diseases codes were used for AIDs as described elsewhere.24 Cancers were retrieved from the Swedish Cancer Registry from years 1964–2012. The detailed SNOMED histology for specific types of NHL and HL was available from year 1993 onwards.AID patients were identified from the Swedish Hospital Discharge Register  and the Outpatient Register (2001 through 2012). Family relationships were obtained from the Multigeneration Register, containing the Swedish population in families and spanning more than a century, and maintained as part of the Swedish Family-Cancer Database, which has been used in numerous previous studies in periodically updated versions.Person-years were calculated from the start of follow-up on 1 January 1964, until diagnosis of the relevant disease, death, emigration, or the end of the study (31 December 2012) whichever came first. SAS version 9.3 was used for the statistical analyses. Standardized incidence ratios (SIRs) were calculated for the offspring generation as the ratio of observed to expected number of cases. The expected numbers were calculated for all individuals without a history of an AID (when risk of AID was considered) or cancer (when risk of cancer was considered) and the rates were standardized by 5-year-age, gender, period (5 year group), socioeconomic status and residential area. The 95% confidence interval (95% CI) of the SIR was calculated assuming a Poisson distribution. All SIRs refer to the offspring generation. Results are considered significant when 95% CIs did not overlap with 1.00. The study was approved by the Ethical Committee of Lund University (2012/795).Case numbers and diagnostic ages for the B-cell neoplasms and AIDs in the 0–80-year-old offspring generation are shown in As many AIDs have sex preferences, we carried out a sex-specific analysis (no table shown). For NHL no AID was significantly associated when males or females were considered separately. For HL, the SIR was 11.07  when female relatives were diagnosed with pemphigus. Male MM was associated with angiitis hypersensitive in two cases , chronic rheumatic heart disease in 19 cases  and primary biliary cirrhosis in four cases . Female MM was associated with Graves disease in five cases  and rheumatic fever in four cases .Data on specific subtypes of NHL and HL were available from year 1993 onwards . For NHLFor HL, the most common subtype nodular sclerosis classical HL, the SIR was 8.32 (2.17–21.53) for four cases with a family history of pemphigus. However, the overall SIR for nodular sclerosis classical HL was decreased to 0.81 ; the only significantly decreased association of 0.31 (0.06–0.91) was in three families with type 1 diabetes families.15 Although the Swedish population was partially overlapping in the previous and the present study, the current follow-up of cancer and AID was to 2012 instead of 1998 in the previous studies, and the present case numbers were doubled or trebled.15The present exploratory data are novel in reporting familial risks between B-cell neoplasms and specific AIDs in two ways, for neoplasms by familial AIDs and for AIDs by familial neoplasms; the comparisons are independent for parent-offspring while some dependence may exist in sibling pairs. Only two such associations were found, NHL and angiitis hypersensitive and NHL and Sjogren syndrome. The first association was strongest for diffuse large B-cell lymphoma, whereas the second one appeared to be related to follicular lymphoma although the SIR was not significant. Thus a significant result in both of the two-way analyses is a strong evidence for a true association. The previously reported associations of HL with a family history of sarcoidosis  and ulcerative colitis  and of MM with a family history of systemic lupus erythematosus  could not be replicated.Considering the likelihood of false positives in the present study, the tested 44 AIDs and 3 neoplasms yielded 132 comparisons for each of 7 The personal history data on the current NHL–AID associations were available for discoid lupus erythematosus , Sjogren  and psoriasis . A personal history of AID did not influenced much the subsequent MM  but also for MM remarkably few negative associations were noted.4 The comparison of data from personal and family histories for lymphoma should be instructive and suggest mechanistic implications. As a personal history is a combination of a family history and a life-long history of somatic events, including immune challenges, it may not be surprising that the responses are higher after a personal history compared with a family history. An inherited gene variant, such as an human leukocyte antigen allele, may afford protection against lymphoma while a somatic immune dysregulation could plausible only increase risk through accumulation of genetic damage.The present results revealed large differences to those published on the risk of B-cell neoplasms subsequent to a personal AID also from the present Swedish population. The personal history was associated with a high overall risk of lymphoma (1.6 for NHL and 2.0 for HL) for any of 33 common AIDs and remarkably high risks after some AIDs, such as autoimmune hemolytic anemia (27 for NHL and 20 for HL); furthermore, a personal history was not associated with significantly decreased risks.In conclusion, the present family data on B-cell neoplasms and 44 AIDs show an approximately equal number of associations for increase and decrease, suggesting that inherited genes or gene-environment interactions may increase the risk or be protective. In contrast, based on the Swedish literature, the associations in lymphoma through a positive individual history tend to only increase the risk, some severely, and the associated AIDs may be largely different from those found through a family history. For clinical practice, the take-home message could be that a family history of some AIDs my influence the risk of B-cell neoplasms but the magnitude of risk tends to be far lower than that found for a personal history of some AIDs.


There are many disabling medical conditions which can result in catastrophic health expenditure. Multiple Sclerosis is one of the most costly medical conditions through the world which encounter families to the catastrophic health expenditures. This study aims to investigate on what extent Multiple sclerosis patients face catastrophic costs.This study was carried out in Ahvaz, Iran (2014). The study population included households that at least one of their members suffers from MS. To analyze data, Logit regression model was employed by using the default software STATA12.3.37% of families were encountered with catastrophic costs. Important variables including brand of drug, housing, income and health insurance were significantly correlated with catastrophic expenditure.This study suggests that although a small proportion of MS patients met the catastrophic health expenditure, mechanisms that pool risk and cost  are required to protect them and improve financial and access equity in health care. Catastrophic health expenditure (CHE) is considered as the spending on health that exceeds a pre-defined percentage of a household’s capacity to pay . The litIran is one of the countries which Out-of-pocket payment is a major method for health system financing. This may result in catastrophic health expenditures. In the fifth development plan, protection of households against healthcare costs is clearly emphasized in the form of two goals: 1- Reduction of out of pocket payment to 30%. 2- Reduction in the number of people facing catastrophic health costs to 1%.A study shows that catastrophic health expenditure among Brazilian families varied between 0.7% and 21.0%, depending on the calculation method. Research findings show socioeconomic inequalities in the CHE between 2002-2003 and 2008-2009 rose significantly, became 5.20 and 4.17 times higher among the poorest and the least educated respectively . In ChinIn Kenya research findings indicate that the proportion of families facing CHE varies widely between 1.52% and 28.38% depending on the calculation methods. The number of working adults in families and membership in social safety nets appears to decline the risk of catastrophic expenditure. In contrast, seeking care in public or private hospitals increased the risk of CHE . To deteIn Iran the proportion of households facing catastrophic healthcare reduced from 12.6% in 2003 to 11.8% in 2008, but this change was not statistically significant. The key determinants of catastrophic healthcare expenditures for both years were healthcare utilization , economic status, and disabled or elderly family member influenced exposure to catastrophic healthcare expenditure in 2008 .In another research which has been done in Iran, more than 3 percent of the households were facing catastrophic health expenditure. Moreover 1.5 percent of the households have been impoverished . In a stAlthough numerous studies explored catastrophic health expenditures (CHE) worldwide, most of them focused on whole population rather than specific vulnerable groups. Since families who suffer from certain costly diseases  are more likely to face catastrophic health expenditures and Multiple Sclerosis is one of the most costly medical conditions through the world which encounter families to the catastrophic health expenditures, this study analyzed the extent and associated factors of CHE in household with Multiple Sclerosis patients.Generally, two thresholds are widely used to define CHE: 1) out-of-pocket healthcare payments (OOP) that comprise ≥10% of total household expenditures  and 2) oAccording to the World Health Organization when families spend more than 40% of the ability to pay for cost of health services they face catastrophic health expenditure . We consSince there were no relevant studies about the catastrophic cost of Multiple sclerosis, to estimate the sample size we consider P equal to 0.3 regarding to other relevant studies and used bellow formula:According to the following values, sample size equaled to N=322.P= 0.3; Z=1.96; d=0.05; N=322.Researchers usually consider that the dependent variable is continuous set of values. However; there are numerous cases in which the dependent variable has only two values, zero and one. For instance, a person could buy drugs or to unsubscribe. Models which are used for such purposes are called models with qualitative dependent variables (e.g Logit and Probit). Simplest of these models are models in which the dependent variables are binary. In the following function Xi is a binary variable:If the non-visible part of the Gumbel distribution is assumed, the difference between the logistic distributions gives the binary Logit model .We employed regression analysis in order to find the relationship between CHE and socioeconomic variables in this study. Since facing catastrophic costs (as constant variable), is a binary response, using a binary model was applicable here; so the Logit regression model has been applied in this study.Descriptive statistics of this study are shown in As shown in In logistic regression, the coefficient of R2 is not a good indicator for fitted model, so other kind of indicators is used. In Logit regression, the Mc Fadden R-squared is similar to the R2 statistics in linear regression, and the value varies between 0-1. When the value is closer to 1, the reality adjustment of the model is high. The calculated value in current study is 0.680,  which isIn order to test the hypothesis that independent variables have no effect on dependent variables, the LR statistics is used. The hypothesis that all coefficients are equal to zero (0) in contrast to none of them is tested. The P-value is equal to 0, so it is less than 0.05, and this means that the H0 hypothesis is not acceptable and the regression model is statistically significant. Log likelihood shows the fitness of model and its value is always negative. The calculated value in this study is acceptable based on similar researches.Among all variables which have been studied, only disease stage had no significant correlation with exposure to catastrophic costs. Drug type had significant negative correlation (p= 0.03) with catastrophic cost which means that by using Iranian drug type, the catastrophic cost would decrease. Considering the higher cost of foreign drugs, this result seems reasonable.The family size had a significant positive correlation (p<0.001) with catastrophic cost, which means that by increasing the family size, the catastrophe cost will increase. This finding is compatible with previous studies in Iran .Owning a house had a positive significant relationship with catastrophe cost. It means that the patients with their own house are more likely to interface with catastrophic cost in comparison with the renters. This result seems unreasonable and should be more investigated. Family income had significant negative correlation (P=0.02) with catastrophic cost. It is clear that when household`s income increases, probability of facing catastrophic cost could decrease.The results indicated a significant positive relationship (p=0.03) between public health insurance coverage and catastrophic cost, which means that families who had public health insurance coverage, had more catastrophe cost. This result is somehow different with the results of other studies. In China there were no significant relationship between health insurance and exposure to CHE . While iIn this research, the cause of these results maybe is due to the adverse selection of insurance coverage where the healthy and younger families are less likely to join insurance funds, so the covered families are older and suffer poor health condition. The other factor that may effect on this result is the moral hazard of insured people and physicians. Although it does not seem reasonable that people with public health insurance coverage continue the use of services till they encounter to catastrophe cost, but this may occur by physician induced demand.A Significant negative relationship was seen between gender and catastrophic cost. It means that families with man household`s head are less likely to face CHE. It seems reasonable as men are more able to be employed and earn income.The number of physician’s visits had a significant positive relationship with catastrophe cost, obviously by increasing the number of visits, the treatment cost would increase and the probability of the catastrophe cost would rise.The age of household`s head had a significant negative correlation with the probability of facing catastrophic cost, which suggests that the younger people who have lower income and fund, have a higher risk of encountering catastrophic cost.Since this study has been done after the implementation of a national financial safety program in Iran (2014), the rate of CHE was far less than what we expected. So we conclude that this program which was funded by ministry of health has been successful in reducing the risk of facing CHE among vulnerable groups such as Multiple Sclerosis patients.


Antibiotic prescription is a central public health issue. Overall, 90% of antibiotic prescriptions are delivered to patients in ambulatory care, and a substantial proportion of these prescriptions could be avoided. General Practitioner (GP) trainers are similar to other GPs in terms of sociodemographic and medical activities, but they may have different prescription patterns. Our aim was to compare the antibiotic prescribing rates between GP trainers and non-trainers.This observational cross-sectional study was conducted on administrative data claims from the French National Health Insurance. The antibiotic prescribing rate was calculated. The main independent variable was the training status of the GPs. Prescribing rates were adjusted for the various GPs’ characteristics  in a multiple linear regression analysis.Between June 2014 and July 2015 the prescribing patterns of 860 GPs were analysed, among which 102 were GP trainers (12%). Over the year 363,580 patients were prescribed an antibiotic out of 3,499,248 visits for 1,299,308 patients seen over the year thus representing around 27.5% of patients. In the multivariate analyses, being a trainer resulted in a significant difference of 6.62 percentage points  in antibiotic prescriptions comparing to being a non-trainer, corresponding to a relative reduction of 23.4%.These findings highlight the role of GP trainers in antibiotic prescriptions. By prescribing fewer antibiotics and influencing the next generations of GPs, the human and economic burden of antibiotics could be reduced. Antimicrobial resistance has become a major concern worldwide . SeveralAt the end of the 1990s, outpatient antibiotic use and bacterial resistance rates in France were among the highest in Europe . As a coIn contrast, no study has investigated the consequences of being a GP trainer on antibiotic prescriptions. Although GP trainers are similar to other GPs in terms of sociodemographic and medical activities , they maThe objective of our analysis was to investigate whether being a GP trainer was associated with issuing fewer antibiotic prescriptions for primary care patients.We conducted an observational cross-sectional study using the data claims from the French National Health Insurance (NHI) for all GPs in the French department in Yvelines (78), which is a Paris suburb with a population of 1.3 million (2% of the French population). The study period ranged from June 2014 to July 2015 (one year). Information about GP trainers’ status was provided by the Department of General Practice at the University of Versailles Saint-Quentin-en-Yvelines (UVSQ) and was extracted from the French NHI data claims. All data were anonymized.In 2014, 980 GPs were working in Yvelines. The exclusion criteria consisted of practicing alternative medicine  and having less than 1,000 office and home visits over the year. Information about the sociodemographic characteristics and level of activity of GPs were collected. Information about the patients was aggregated at the GP level to define the main features of the GP’s activity. Individual data for patients were not available.The antibiotic prescribing rate was calculated as the number of patients who were prescribed at least one antibiotic by the GP over the year divided by the total of individual patients followed by the same GP during the studied period. Exhaustive information was obtained regarding the patients followed by each GP and there were no missing data.The main independent variable was the training status of the GPs.We further adjusted for potential confounders selected based on the literature and their clinical relevance. These variables included GPs’ sociodemographic characteristics and location of the practice . We alsAdjustment of the GPs case mix was made using the proportions of visits dedicated to each type of patient as proxies. We suspected that a higher proportion of visits involving children (<16 years) or patients exempt from the medical fee  for long-term disease conditions could increase the antibiotic prescribing rate . Some paThe profiles of GP trainers and non-trainers were first compared using a Chi-squared test for categorical variables and Student’s t-test for continuous variables, including the antibiotic prescribing rate.Then, an adjusted linear regression model was used to identify factors associated with the antibiotic prescribing rate according to the training status and controlling for the main confounders as defined above.No data were missing. All tests were two-sided, and p-values ≤0.05 were considered significant. Analyses were performed with STATA version 13.0.Access to the databases is subject to prior authorization and was approved by the Independent Data Protection Administrative Authority . This study was approved by the Ethical Committee of the French National College of Teachers in General Practice . Written informed consent was not required because the data were anonymized.Of the 980 GPs practicing in Yvelines, 120 were excluded from the statistical analyses because they practiced alternative medicine or had less than 1,000 office and home visits over the year see .In total, 860 GPs were included of whom 102 were GP trainers (12%). Between June 2014 and July 2015, 3,499,248 visits occurred involving 1,299,308 individual patients. 363,580 patients had an antibiotic at least once in the year.Our sample consisted of 64% male GPs, and 57% were over 55 years old. The GPs saw more adults than children; 21% of visits involved patients who were under 16 years old. One-fifth of home and office visits were dedicated to patients who were exempt from the medical fee: 15% for long-term disease and 6.5% for low-income status see .An average of 1,500 patients attended each GP.The average antibiotic prescribing rate for the GPs was 27.53%  see .21.86% compared to 28.29% (p<0.001)  see .Compared to the univariate analysis, the multivariate analysis showed a stronger impact of being a GP trainer versus non-trainer on antibiotic prescribing: resulting in a significant difference of 6.62 percentage points . In relative terms, this effect represents a difference of 23.4% between the two groups.Being 55 years old or older increased the antibiotic prescribing rate by an average of 3.67 percentage points  with all other factors being equal. Regarding the level of activity, as expected GPs with a higher workload prescribed more often antibiotics to their patients . In contrast, for GPs with fewer than 3,000 annual visits, the antibiotic prescribing rate was reduced by 3.81 percentage points on average .An increase in the annual number of attendances by patients <16 years old of 10 percentage points was associated with an average increase of 1.3 percentage points in the antibiotic prescribing rate . The annual number of attendances by patients with an exemption for medical fees  was statistically significant (p = 0.001): for every 10 percentage points increase in the number of visits dedicated to these types of patients, the antibiotic prescribing rate increased by 1.9 percentage points on average CI 95% .After adjusting for several confounders, we have shown that GP trainers’ antibiotic prescription rates were significantly lower compared to non-trainers’ by 6.62 percentage points , thus corresponding to a relative difference of 23.4%.To the best of our knowledge, this is the first study to show a significantly lower antibiotic prescribing rate for GP trainers than non-trainers.Our results are consistent with previous studies that reported that GP trainers tended to have better clinical performances based on P4P indicators, such as diabetes follow-up, seasonal flu vaccination and mammograms .Multifaceted interventions and educational programs are efficient and have a positive effect on GP prescriptions ,19. TheyThe antibiotic prescribing rate increased with the number of patient visits. Indeed, in a fee-for-services payment scheme, there is a trade-off between the number and duration of consultations: GPs who conduct more examinations per day spend less time with patients , which mOur study could not confirm a previous finding that older patients are more likely to be prescribed antibiotics ,24.Our sample included all GPs with regular practice activity in Yvelines , and all GP trainers were included. The large number of patients  suggests good coverage of the population of Yvelines (90%). The main characteristics of the inhabitants of Yvelines were similar to the French population, including approximately 51% of women vs 52% in France overall. The age distribution of the inhabitants was similar, with approximately 38% under 30 years old in the Yvelines versus 36% in France, 54% from 30 to 74 years old vs 55% and 7% vs 9% 75 years old and over . FinallyThe proportion of GP trainers was similar to the national level: 12% vs 13% for Yvelines and France, respectively. Moreover, GP trainers were comparable to non-trainers in terms of sociodemographic characteristics and case-mix, as shown in other studies ,27. AlthWe proposed to assess the prescriptions of the GPs by using the antibiotic prescribing rate in order to have a better reflect of the habits of GPs and answer our question . This ouOur study has several limitations because some variables that could have an effect on antibiotic prescriptions were not collected. First, we did not have any information about the type of disease that was treated or the list of antibiotics used. A systematic review about factors associated with antibiotic prescriptions for respiratory tract infections (RTIs) found that symptoms and diagnoses played a role in antibiotic prescribing rates . Indeed,Our study provides evidence that GP trainer status affects prescription behavior. But other variables that were not adjusted for could be confounding factors that could explain the high and strongly significant coefficient for the training status. Residual confusion remains possible in GP trainers’ characteristics such as practicing in groups, having additional diplomas and examining fewer patients . FactorsIn the Yvelines for 100 patients followed over one year, a GP trainer prescribes an antibiotic to 6 patients fewer than a non-trainer. If we extrapolate our results, considering that a GP examines approximately 1,500 patients per year: 90 patients would not be treated by an antibiotic in one year. Extrapolated to all French GP trainers  , 769,500By prescribing fewer antibiotics we suppose that GP trainers could influence the next generations of GPs ,36 and tFurther studies are needed to assess the differences between GP trainer and non-trainer in terms of prescription patterns.A follow-up or retrospective study to assess the antibiotic prescribing rates of GPs from the same generation before and after becoming a trainer would be informative. This type of study could examine the changes (or lack of changes) in the behavior of GPs concerning antibiotic prescriptions. It would also be possible to study a larger population of GPs from other French departments to define the factors or mechanisms that explain the lower rates of antibiotic prescriptions, such as continuing medical education, reading medical papers, going to conferences, tutoring, training students, having a reflective attitude during consultations, accepting or not accepting medical sales representatives, and asking the patients if they want an antibiotic.In addition to this quantitative study, a qualitative study would increase our understanding of the perceptions patterns of GPs upon becoming trainers. We could enhance the work by exploring how patients feel about antibiotics if their GP is a trainer or not. Further studies could also focus on the trainee’s prescriptions and how they justify these prescriptions based on their trainer’s behavior during their internship.It is possible to become more vigilant about antibiotic prescriptions and decrease the burden of antibiotic resistance. A post-antibiotic era  is an acS1 Fig(TIFF)Click here for additional data file.