In the present study, a recycled concrete aggregate (RCA) coating treatment using a cement paste dissociation agent (CPDA) with different mixing methods was newly incorporated in RCA concrete mixtures. First, a preliminary test program was conducted to determine the proper dosage of the CPDA solution throughout its RCA concrete test results from compressive strength, flexural strength, and elastic modulus. Then, a series of experimental tests were carried out to investigate the effect of RCA coating treatment, different mixing method such as the equivalent mortar volume (EMV) method and conventional method, and different RCA replacement ratios on durability test results of RCA concrete such as drying shrinkage values and rapid chloride penetration test (RCPT) values. The test results showed that all RCA concretes mixed with the coated RCAs were found to be workable regardless of different mix methods, with the slump and air contents of all the mixes being almost identical. All the concrete specimens, which were mixed with the coated RCAs with CPDA solution, represented lower drying shrinkage and RCPT values than those mixed without RCA coating treatment, regardless of different mix proportioning methods or RCA replacement ratios. This holds for the concrete specimens proportioned with the EMV method, regardless of different RCA replacement ratios. It is widely acknowledged that various types of waste materials can be transformed into recycled aggregates, powders, or additives and are used in concrete. Ceramic materials have been used as ceramic powder and ceramic aggregates in concrete . This usRecycled concrete aggregate (RCA) is one of many types of recycling aggregates that are used for concrete. RCA is known to be more porous, less dense, and more heterogeneous than natural aggregates. However, the residual mortar (RM) adhering to RCA has a negative impact on the concrete properties. Additionally, it has been reported that RM in RCA reduces the compressive strength and elastic modulus by up to 42% and 45%, respectively .Numerous research teams have carried out experimental studies with the goal of enhancing the material properties of RCA concrete. These studies include high-quality RCA produced from precast or preserved quality concrete ,8,9, theFirst, high-quality RCA can be obtained from precast concrete , concretNext, two-lift concrete paving has been successfully adopted and employed in Western Europe and the U.S. This method involves the use of low-quality RCA concrete in the lower layer of the concrete pavement. Two-lift construction using recycled concrete in the lower layer has been reported to provide the greatest impact socially and environmentally . In a siThe application of oil-contaminated sand in concrete was considered effective in enhancing concrete strength properties. Abousning et al.  reportedAfter the two-stage mixing approach (TSMA)  was intrImproved mixing methods for RCA concrete have also been proposed by a number of other researchers ,15,16,17Researchers also investigated the strengthening of RM. Various materials were used to fill pores and interfacial transition zones (ITZs) to improve RCA quality ,20,21,22Drying shrinkage is a very important property of cementitious composites influencing their durability. Several models to predict the concrete drying shrinkage using influential factors have been empirically proposed in terms of slump, air content, fine aggregate, cement content, compressive strength, relative humidity, and volume-to-surface ratio, etc. ,25. In aChloride penetration resistance, which is a measure of concrete durability, is important in reinforced concrete structures as well as plain concrete structures that do not use rebars. The presence of chloride ions was found to affect the spalling of concrete pavement below the freezing temperature of the pore solution and increased the saturation state of rebar corrosion . The RapThe above studies highlight the importance of an idealized mixing process, a mix proportioning method, ITZ strengthening, and a coating method to provide enhanced mechanical properties for RCA concretes. However, most previous studies have been limited to investigating the mechanical strength properties of RCA concretes. Readers may notice that, so far, no research determining the effect of RCA coating treatment by cement type solutions has been conducted on durability properties such as drying shrinkage and chloride ion penetration resistance. Thus, in the present study, an RCA coating treatment using a cement paste dissociation agent (CPDA) with different mixing methods was newly incorporated in RCA concrete mixtures. First, a preliminary test program was conducted to determine the proper dosage of CPDA solution using its RCA concrete test results on compressive strength, flexural strength, and elastic modulus. Then, a series of experimental tests were carried out to investigate the effect of RCA coating treatment, different mixing methods such as the equivalent mortar volume (EMV) method and conventional method, and different RCA replacement ratios on the durability test results of RCA concrete such as drying shrinkage values and RCPT values . TherefoThis experimental study used RCAs produced from two different sources in South Korea. The RA aggregate was crushed with a maximum size of 25 mm from old runway concrete pavement at an air base reconstruction site. The RP was obtained with a maximum size of 20 mm by crushing the precast concrete (PC) culverts see . It shouThe PC culverts had a compressive strength of 35 MPa with a maximum aggregate of 20 mm and a water–cement ratio of 35% or lower according to the Korean Construction Specification  and wereCrushed granite (which was the same source of aggregate previously used in PC culvers) was used as the natural coarse aggregate, and its specific gravity and absorption rate were 2.69 and 0.54%, respectively. Natural river sand was used as the fine aggregate. RA and fine aggregate 1 were used in the first mix series, while RP and fine aggregate 2 were used in the second mix series. RP, the heat treatment method proposed by Juan and Gutierrez [RCA is the weight of the RCA dried in an oven dryer after first collecting the samples and WOVA is the weight of the original virgin aggregate (OVA) dried in an oven dryer after removing residual mortar.In order to obtain the RMC value for utierrez  was used2, CaO, NaCl, NaNO3, Na2SO4, and K2CO3. The chemical equations below explain the additional hydration reaction of calcium hydroxide from the cement and the CPDA [The cement paste dissociation agent (CPDA) used in this study was a product composed of SiOthe CPDA . Free caAs a percentage of cement, 1.25% CPDA powder was recommended for conventional cement concretes by the CPDA manufactured company . However2/g. The chemical admixture used in this experiment was an air entraining and water reducing agent solution. All of the aggregates were prepared in a saturated surface-dry condition.In this experiment, type I Portland cement was used with a specific gravity of 3.15 and specific surface area of 3380 cmTwo series of mixes were prepared and tabulated in The second series of mixes were then designed to find whether coating the RCAs with CPDA solution is an effective way to improve the durability properties such as drying shrinkage and chloride penetration resistance as well as mechanical strength properties of recycled aggregate concrete (RAC). This mix series involved nine mixtures. The conventional American Concrete Institute (ACI) mixing method and the equivalent mortar volume (EMV) mixing method were used. In the nomenclature, first, C and E in mix identification denote conventional and equivalent mix design, respectively. Next, R and N denote natural coarse aggregate and RCA, respectively. Thirdly, A refers to type A aggregate while P refers to aggregate manufactured from precast concretes. Fourthly, -a and -b refer to the 25% and 50% RCA replacement ratios per total coarse aggregate, respectively. Lastly, S used in the mixture nomenclature refers to the use of spray coating of the recycled concrete aggregate (RCA) surface in the mixture.A pan mixer that could perform 60 L volume mixing was available in a research laboratory from Hongik University in Sejong, Korea, which is where the experimentation was conducted. First, the coarse aggregate and fine aggregate were poured in the mixer and mixed together for around 30 s. Then, cement was added and mixed for 30 s. Afterwards, the admixture was diluted sufficiently in water and added to the mixture for additional mixing for around 2 min.The specimens for concrete compressive strength measurement were prepared using a 100 mm × 200 mm plastic mold following a predetermined method . The fabDrying shrinkage experiments were performed using a dial gauge, as suggested by KS (Korean Standards) F 2424 , which iQ refers to the total passed charge and In refers to the current at n minutes.In order to test the chloride ion penetration resistance, three cylindrical specimens with a 100 mm diameter and 200 mm height were prepared, followed by fabrication into disc specimens of 50 mm thickness in accordance with ASTM C 1202 [It is commonly accepted that concrete durability is thoroughly related to fresh and hardened properties. The compressive strength values at 28 days are represented in The drying shrinkage test results are shown in The effect of different mix proportioning methods on drying shrinkage was studied, and their average test results are plotted with error bars in The effect of RCA coating on drying shrinkage was studied, and their average test results are plotted in The test results were compared to investigate the effect of the RCA replacement ratios on the drying shrinkage of RCA concretes. In ASTM C 1202  recommenThe effect of different mix proportioning methods on chloride ion penetration resistance was studied and their Rapid Chloride Penetration Test (RCPT) results are plotted in The effect of RCA coating on the RCPT results was studied, and their average test results are plotted in The test results were compared to determine the effect of RCA replacement ratios on the RCPT values of RCA concretes, as shown in A scanning electronic (SEM) microscope was used to observe the microstructural characteristics of RCA concretes. In this study, CRP-b and CRP-bS samples only were tested to check whether the CPDA coating effect was seen in the image analysis. In In this study, recycled concrete aggregate (RCA) manufactured from precast concrete (PC) culvert was used to carry out durability experiments on concrete fabricated using the conventional ACI volume mixing method and the equivalent mortar volume (EMV) mixing method. Moreover, a cement paste dissociation agent (CPDA) was used to coat the RCA surface followed by curing to investigate the drying shrinkage and the chloride ion penetration resistances of the concrete coated at the RCA surface with CPDA compared with concrete that did not. The following conclusions were obtained from the experimental results.From the preliminary test, it appears that the addition of 2.5% of CPDA in the RCA concrete mixes helps improve the mechanical strength properties such as compressive strength, flexural strength, and elastic modulus. The test results showed that about 24%, 18%, and 3% increases were observed for compressive strength, flexural strength, and elastic modulus, respectively, at 28 days.All RCA concretes mixed with coated RCA were found to be workable regardless of different mix methods, with the slump and air contents of all the mixes being almost identical. Additionally, all the concrete specimens that were mixed with the coated RCAs with CPDA solution had lower drying shrinkage values and RCPT (Rapid Chloride Penetration Test) values than those mixed without RCA coating treatment, regardless of different mix proportioning methods or RCA replacement ratios. The variation in compressive strength and density of the RCA concrete did not affect drying shrinkage or RCPT values.This holds for the concrete specimens proportioned with the EMV method, regardless of different RCA replacement ratios. Especially, it may be inferred that the RCPT values are related to total mortar volume of RCA concrete mix since total mortar volume in the ERP mixes is reduced due to the residual mortar attached to the RCAs.For the conventional RCA mixes, in comparison to test results from the 25% RCA replacement ratio, the little increase or reversed test result of drying shrinkage values and RCPT values in concrete made with 50% RCA replacement ratio may be attributed to the good-quality RCA adopted in this study. Further study on this effect is necessary.


Elevated serum IgG4 is a useful marker of IgG4-related disease (IgG4-RD) activity. However, there is no uniformity in the cut-off values of IgG4 among the various reagents. The aim of this study was to compare the measured and cut-off values of IgG4 assessed using three different reagents. This study enrolled 466 IgG4-RD and non-IgG4-RD patients who required measurement of serum IgG4 levels to diagnose or treat IgG4-RD. Serum IgG4 was measured using three reagents: N-assay LA IgG4 Nittobo (Nittobo), BS-NIA IgG4 (TBS), and N Latex IgG4 (Siemens). The values obtained using the three reagents were compared, and cut-off values were calculated for each. Although there was good correlation among the results with the three reagents, the measured and cut-off values were all different. The Nittobo values were 1.4 times the TBS values and the TBS values were almost half those of the Siemens values. ROC curve analysis showed cut-off values for the Nittobo, TBS, and Siemens reagents of 1.42, 1.31, and 2.38 g/L, respectively. The measured and cut-off values of serum IgG4 vary depending on the reagents used for the assay, although there is good correlation among the values measured by the three reagents. The disease is particularly frequent in middle-aged and elderly men and is characterized by significant response to steroid treatment3. Although fibro-inflammation with elevated serum IgG4 was first recognized in autoimmune pancreatitis (AIP), it has since been found to affect almost all organs and is conceptualized as a group of diseases4.IgG4-related disease (IgG4-RD) is a new disease recognized in this century and is characterized by the formation of masses or nodules/hypertrophic lesions in various organs. Prominent lymphocyte and IgG4-positive plasma cell infiltration6. At the same time, Hubers et al. reported that the antigen for autoantibodies in AIP patients was annexin A117. Other reports have shown that the expression level of galectine-3 is high in IgG4-RD patients, and that anti-galectine-3 antibody titers increase in proportion to plasma galectine-3 levels9. However, while these autoantibodies have been found in some cases, their further verification as disease-specific antigens is desired in the future.Although the mechanisms of the onset of IgG4-RD have not been identified, involvement of certain autoantibodies has been implicated. Patients with AIP are known to have various autoantibodies, such as antinuclear antibodies, anti-carbonic anhydrase-II (CA-II) antibodies, anti-lactoferrin antibodies, rheumatoid factor and anti-smooth muscle antibodies. Although these autoantibodies have low disease specificity, certain new disease-specific autoantibodies have been found. Shiokawa et al. reported that IgG4 and IgG1 in the serum of IgG4-RD patients cause pancreatic injury, and that the antigen for the antibody in AIP patients was laminin 51110. Serum IgG4 measurement reagents are supplied by two companies, The Binding Site  and Siemens Healthcare Diagnostics . In addition, a new reagent has recently been released by Nittobo Medical Co., Ltd.  and has begun to be used in Japan. However, all three reagents result in different measured values11. This is reflected in the differences in their reference ranges: the upper limit of the reference range for the Siemens reagent is about twice that of the TBS reagent. A report on the cut-off value for IgG4-RD from China stated a value of 2.48 g/L, which is about twice the diagnostic standard of 1.35 g/L in Japan12. This is not due to differences in research subjects, but to differences in the reagents used.Elevated serum IgG4 is one of the hallmarks of IgG4-RD, and is a useful marker for diagnosis, follow-up and monitoring for recurrence. The reported cut-off value of 1.35 g/L was calculated using a comparison between pancreatic cancer and AIP patientsAlthough there are many reports on calculation of the cut-off value for a single reagent, no reports have shown how the cut-off values differ between reagents. In this study, we report the results of simultaneous measurement of IgG4-RD in patient samples using reagents from the three companies, and calculation of the cut-off values for each reagent.We grouped the 466 enrolled patients according to their diagnosis and treatment. All patients who had received some kind of treatment, including medications such as steroids and surgical treatment at the time of blood collection, were grouped as “during or post-treatment”. The median serum IgG4 levels in each group are shown in Table Correlations between the three reagents  for all 466 patients are shown in Fig. ROC analysis was performed between untreated patients in the definite IgG4-RD group (n = 56) and untreated and diagnosed with non-IgG4-RD patients in the non-IgG4-RD group (n = 41). Pre-treatment patients in the non-IgG4-RD group (n = 54) included both patients with confirmed non-IgG4-RD disease and those with suspected non-IgG4-RD disease. ROC analysis used data from only patients with confirmed non-IgG4-RD (n = 41). The involved organs in IgG4-RD patients and the diseases in non-IgG4-RD patients are shown in Table In this study, we evaluated correlations between three IgG4 reagents using 466 patients’ samples, and calculated the cut-off value for each reagent. The correlation between the three reagents was compared over a wide range of values and patients, including not only IgG4-RD patients, but also patients with other diseases that need to be distinguished from IgG4-RD. The results showed that although there was good correlation between the results with the three reagents, the measured values were all different. As a result, the cut-off values also varied depending on the reagent used. This is the first study to simultaneously calculate the IgG4-RD cut-off value for the three IgG4 reagents.10. The calculated cut-off value of 1.41 g/L for Nittobo was very close to that of TBS, and the slope of the regression equation of the correlation for values less than 1.5 g/L was 1.0. Therefore, a cut-off value of 1.35 g/L can be applied for the Nittobo reagent. On the other hand, a different cut-off value needs to be set for the Siemens reagent. This study calculated a cut-off value of 2.38 g/L for the Siemens reagent, which is consistent with the cut-off value of 2.48 g/L calculated by Yu et al. using nearly 3,000 samples tested with the Siemens reagent12.The cut-off value of 1.31 g/L of the TBS reagent in this study was almost the same as the value of 1.35 g/L determined using the TBS reagent in 200113. Therefore, it is necessary to make a final diagnosis by a combination of evaluations, such as computed tomography, magnetic resonance imaging, and biopsy. However, as evidenced by the high AUC values in the ROC analysis of this study, it is certain that many patients with IgG4-RD are identified by the presence of high serum IgG4 levels, and serum IgG4 levels contribute to the diagnosis of IgG4-RD. Moreover, it has the advantage of being easier to perform than other examinations. However, the differences in measured values between IgG4 assay kits can mislead clinicians when making a diagnosis. The cut-off value for serum IgG4 was described as 1.35 g/L in the Japanese comprehensive diagnostic criteria published in 2011, but this value could be used only for the TBS reagents used in Japan at the time. American College of Rheumatology/European League Against Rheumatism classification criteria for IgG4-RD adopted the upper limit of the reference range of each assay kit as the cut-off level for diagnosis14. Since several reagents are used in the United States, it is considered that the reference value of each reagent should be used instead of a specific number. Other measurement kits might become available in different countries in the future. To avoid confusion, harmonization of the measurement values between the assay kits should be achieved.Diagnosis of IgG4-RD might sometimes be difficult. This is because patients’ characteristics differ depending on the affected organ, and because elevated serum IgG4 levels are not always observed in IgG4-RD patients15. Although harmonization of the value of total IgG has been achieved using Certified Reference Material (CRM) 470 released by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) in 1993, CRM 470 has no certified values for IgG subclasses and the measurements have not been unified. TBS uses CRM 47016 and Siemens uses WHO67/97 as the reference material17, IgG4 values of each reference material were determined by different researchers. Nittobo has the original master calibrator with IgG4 value determined themselves, and they trace the value to the kit’s calibrator11. Those mean that all three companies have established the different traceability. Consequently, the difference in the slope of the correlation results below 1.5 g/L between Siemens and the other two kits could be induced by the difference in the determined values for their own calibrators. According to the report of Ladwig et al., the quantitative value of IgG subclasses measured using LC–MS/MS are consistent with the values measured using the TBS reagent18. Bernasconi et al. reported that the amount of purified IgG4 protein was consistent with the value of IgG4 measured using the TBS reagent19. These reports suggest that the IgG4 value measured using the TBS reagent is an accurate value.Differences in IgG subclass values between TBS and Siemens reagents have been previously reported20. In healthy individuals with a normal balance of the four subclasses, the total IgG value and sum of the subclass values that is measured using the TBS measurement kit are the same, but divergence occurs when IgG4 is high. From our result, it is considered that the divergence reported by Imoto et al. is due to poor linearity in the high value region in IgG4 measurement. Further validation of the accuracy of the measurement value with elevated IgG4 levels will be needed.However, the different calibrators, assessed by the original traceability of each company, cannot explain the mismatch in the slopes of the regression equations between the correlation diagrams for all data and those for the data less than 1.5 g/L in the comparisons between TBS and other two methods. This would mean that even if all methods indicate sufficient linearity in the localized range such as less than 1.5 g/L, no absolute linearity in wide range, such as from 0 to 20 ~ 40 g/L, is provided by all or some of those methods although apparently sufficient linearity is observed in three methods. Therefore, a data around cut off value, which is practically important to diagnose IgG4-RD, is convertible using a constant factor among three methods. However, although the good correlation coefficient for all data was also observed among three methods, it does not mean to be able to convert a data obtained by each method using the same factor described above, especially in a data much higher than cut off value. Imoto et al. reported that the sum of the values of the four IgG subclasses differs from the measured value of IgG when IgG4 levels increaseIn conclusion, serum IgG4 is a useful marker for IgG4-RD diagnosis and treatment, but the values vary greatly depending on the reagents used for the assay. Therefore, it is necessary to change the cut-off value according to the reagent used. In previous studies, the cut-off value was calculated for only a single reagent, but in this study, cut-off values for three reagents were calculated simultaneously. Any reagents described here are available to diagnose IgG4-RD if the suitable cut-off value for each reagent is used. However, it is disclosed that the proportional conversion for all of the data is not always possible. Therefore, when using serum IgG4 values for the diagnosis and treatment of IgG4-RD, it is essential to confirm which reagent is used. It is hoped that harmonization of IgG4 measurements will be achieved in the near future.This prospective study included not only patients with IgG4-related disease, but also those with differential diseases. Furthermore, we targeted patients who required serum IgG4 measurement for diagnosis or treatment regardless of the presence or absence of a definitive diagnosis and the stage of treatment. We enrolled 466 patients from Sep 2016 to Jan 2017 at 23 hospitals in Japan. The diagnosis of IgG4-RD was made according to the comprehensive diagnostic criteria for IgG4-RD, 2011 (21). The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the institutional review board of Shinshu University School of Medicine  . Written informed consent was obtained from all subjects.At each hospital, blood that was collected from the subjects was partitioned, and the separated serum was immediately frozen. All serum samples were sent to the clinical laboratory of Shinshu University Hospital and stored at -30 °C until measurement. Serum IgG4 levels in all samples were measured using three reagents, N-assay LA IgG4 Nittobo , BS-NIA IgG4  and N Latex IgG4 . The Nittobo reagent was used with the JCA-BM6070 automatic biochemical analyzer , and the TBS and Siemens reagents were used with the BN-II nephelometer . All assays were performed at our laboratory according to the respective manufacturer’s instructions.Groups were compared by the Mann–Whitney U test using StatFlex version 6 and those with a P value of less than 0.05 were considered statistically significant. The ROC analysis was performed using JMP version 10, and cut-off values were identified by the Youden index.


The tyrosine kinase receptor encoded by the MET oncogene is a major player in cancer. When MET is responsible for the onset and progression of the transformed phenotype (MET-addicted cancers), an efficient block of its oncogenic activation results in potent tumor growth inhibition.Here we describe a molecular engineered MET antibody (hOA-DN30) and validate its pharmacological activity in MET-addicted cancer models in vitro and in vivo. Pharmacokinetics and safety profile in non-human primates have also been assessed.hOA-DN30 efficiently impaired MET activation and the intracellular signalling cascade by dose and time dependent removal of the receptor from the cell surface (shedding). In vitro, the antibody suppressed cell growth by blocking cell proliferation and by concomitantly inducing cell death in multiple MET-addicted human tumor cell lines. In mice xenografts, hOA-DN30 induced an impressive reduction of tumor masses, with a wide therapeutic window. Moreover, the antibody showed high therapeutic efficacy against patient-derived xenografts generated from MET-addicted gastric tumors, leading to complete tumor regression and long-lasting effects after treatment discontinuation. Finally, hOA-DN30 showed a highly favorable pharmacokinetic profile and substantial tolerability in Cynomolgus monkeys.hOA-DN30 unique ability to simultaneously erase cell surface MET and release the ‘decoy’ receptor extracellular region results in a paramount MET blocking action. Its remarkable efficacy in a large number of pre-clinical models, as well as its pharmacological features and safety profile in non-human primates, strongly envisage a successful clinical application of this novel single-arm MET therapeutic antibody for the therapy of MET-addicted cancers.The online version contains supplementary material available at 10.1186/s13046-022-02320-6. When MET gene amplification generates a MET receptor constitutively active due to overexpression. Copy number gain can result from focal amplification or polysomy, i.e. in the absence or in the presence of multiple copies of chromosome 7, including the MET gene. The level of MET gene amplification defines tumor cell dependence from the oncogene. Although experimental and clinical data indicate that MET-addiction is reached only in the presence of a high MET gene copy number, a clear cut-off point has still to be defined. MET gene amplification has been found in patients carrying gastric cancers, lung tumors, and in type 1 papillary renal cell carcinomas at a frequency around 10% –11, and www.cbioportal.org). These amino acid conversions are clustered, in addition to the kinase domain, also in the extracellular portion of the receptor (SEMA domain) or in the juxtamembrane region [Non-synonymous activating point mutations in the MET gene were first described in hereditary and sporadic kidney cancers, involving residues located in the kinase domain . During e region . While sFinally, several genetic alterations have been identified in non-coding regions . These gIn light of the role of MET in cancer, several different MET inhibitors have been developed , 25, 26.More details are provided in Additional File hOA-DN30 has been provided by Metis Precision Medicine B-Corp . Humanization of the DN30 mouse antibody, conversion into the ‘one-arm’ format, production, and purification of hOA-DN30 has been done by Fair Journey Biologics.For affinity determination, the MET extracellular domain fused in frame with a human Fc domain (R&D System) was in the solid phase and pure MvDN30 or hOA-DN30 were in the liquid phase. ELISA signal was quantified by the multi-label reader VICTOR X4 (Perkin Elmer Instrument INC.).For hOA-DN30 quantification in mouse serum, a goat anti-human IgG antibody (Sigma Aldrich) was in the solid phase. For hOA-DN30 quantification in monkey serum samples, neutravidin-coated plates were used to immobilize biotin-labeled goat anti-human IgG monkey adsorbed antibody (Southern Biotech). In both cases, pure hOA-DN30 and serum samples were in the liquid phase. The concentration of hOA-DN30 in serum was achieved by interpolating the optical density (OD) readings of the samples with the calibration curve fitted by a weighted (1/Y) 4 parameters regression function.For dose-dependent experiments, cells were incubated in serum-free medium for 48 h with the indicated concentration of hOA-DN30. For time-dependent experiments, cells were incubated in serum-free medium in the presence of hOA-DN30 (1 μM) and samples were collected at the indicated time points. Proteins in cell extracts or in cell culture supernatants were resolved by SDS-PAGE and analyzed by Western blotting.For analysis of MET recovery after shedding, cells were incubated in serum-free medium in the presence of hOA-DN30 (1 μM); after 48 h, cell monolayers were extensively washed and fresh serum-free medium was added. Cell culture supernatants and cell lysates were collected at different time points and analyzed as described above.For inhibition of MET and downstream signalling pathway activation, cells were treated with the indicated concentrations of hOA-DN30 for 24 h in serum-free medium. Cell monolayers were lysed and analyzed as described above.For scatter assay, cells were treated for 24 h with HGF , hOA-DN30, or DN30 mAb (both 200 nM). Cell scattering was determined by optical microscopy.For cell growth assay, cells were treated with increasing concentration of hOA-DN30, and viability was evaluated after 72 h by CellTiter-Glo luminescent cell viability assay (Promega Corp.), according to the manufacturer’s instructions. Chemiluminescence was detected with VICTOR X4.For proliferation assay, cells were treated with hOA-DN30 (1 μM). After 48 h cellular DNA synthesis was determined measuring EdU incorporation using the Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific) according to manufacturer’s instruction.For cell death assay, cells were treated as described for cell growth assay. Cytotoxicity was evaluated after 72 h by CellTox™ Green Cytotoxicity Assay , according to the manufacturer’s instructions. Fluorescence was detected with VICTOR X4 and data normalized over CellTiter-Glo luminescence.For determination of M192 tumor organoid viability, cells were incubated for 5 h with Alamar blue (Life Technologies). The signal measured in the cell culture supernatants by VICTOR X4 was set as time 0, then freshly prepared medium with 1 or 5 μM hOA-DN30 was added to the culture. Every 3 days, the medium was replaced with a fresh one containing the antibody. After 9 days, a new assessment of the Alamar blue signal was performed. Cell growth was measured calculating the ratio between day 9 and day 0 for each sample.For determination of Antibody-Dependent Cellular Cytotoxicity (ADCC), cells were treated with increasing concentrations of hOA-DN30, and effector cells were added. After 6 h, ADCC activity has been measured by ADCC Reporter Bioassay (Promega Corp.) according to the manufacturer’s instructions.All procedures in mice were performed according to protocols approved by the Ethical Committee for animal experimentation of the Candiolo Cancer Institute and by the Italian Ministry of Health.To generate Cell Derived Xenografts (CDX), cancer cells were injected subcutaneously into the right posterior flank of adult NOD-SCID mice. To generate Patient Derived Xenografts (PDX), tumor materials derived from human gastric tumor specimens expanded for at least 2 generations in mice were implanted in a subcutaneous pocket generated in the flank of NOD-SCID mice as described in . When tuFor pharmacokinetic (PK) evaluation in mice, adult male NOD/SCID mice bearing or not EBC-1 tumors were used. Animals were treated with 30 mg/kg of hOA-DN30 in a single intravenous administration. Animals were bled at different time points. hOA-DN30 levels in mouse serum were determined by ELISA assay (see above) by Accelera Srl. . PK analysis was performed according to standard non-compartmental and compartmental approach using Phoenix-WinNonlin package  by Accelera Srl.For PK evaluation in Cynomolgus monkeys, the experiments have been performed by Accelera Srl. Three adult male monkeys were administered with hOA-DN30 as a single intravenous bolus at the dose of 11 mg/kg and animals were bled at different time points. hOA-DN30 concentrations in serum were determined by ELISA assay (see above). PK analysis was performed according to the standard non-compartmental approach as described above.max (maximum kill rate) PK/PD model [τ) was calculated from the equilibrium status of the first differential equation of the model.For pharmacodynamics (PD), the analysis was performed by Accelera Srl, applying an EPD model  to tumorDetermination of hOA-DN30 tolerability was performed by Accelera Srl. The antibody was administered intravenously according to ascending doses  one week apart, or at the dose of 180 mg/mg at weekly intervals for two times to two adult Cynomolgus monkeys . Animal’s healthy status, clinical signs observation, body weights, and food consumption were evaluated periodically. Standard hematological and serum chemical parameters were determined. At necropsy, body weight measurement and macroscopic examinations were done.All the studies performed by Accelera Srl. have been sponsored by Metis Precision Medicine B-Corp.P values obtained by Student’s t-Test were calculated using Microsoft Office Excel 2010 software (Microsoft Corporation). To calculate Kd and Bmax, data from ELISA assay were analyzed and fitted according to nonlinear regression, one-site binding hyperbola curve, using GraphPad Prism software (GraphPad Software). To calculate IC50, data from growth assays were analyzed and fitted according to a nonlinear regression, sigmoidal dose-response curve, using GraphPad Prism software. P values obtained by One-way or Two-way Anova were calculated using GraphPad Prism software.Averages, standard deviations, and knob into hole’ structure [The hOA-DN30 molecule was generated from the sequence of the DN30 mAb , modifietructure ; (iii) atructure   in the extracellular space, and in concomitant progressive elimination of MET receptor from the cells Fig. A. At the50 values were in the nanomolar range  models, characterized by high levels of MET gene amplification  of gastric cancer origin. MET gene copy number for each PDX is reported in Suppl. Table hOA-DN30 underwent preclinical studies to evaluate its pharmacokinetic (PK) properties in mice and in non-human primates. PK in mice was performed by administering a single dose (30 mg/kg) of the antibody by intravenous injection to immune-deficient mice bearing or not tumors. hOA-DN30 levels in serum samples are shown in Fig. A. A PK pmax (maximum kill rate) model [Data from the in vivo study performed on EBC-1 tumors treated with different doses of the antibody  model . The anaThe PK profile was also analyzed in non-human primates, by delivering hOA-DN30 as a single intravenous bolus at the dose of 11 mg/kg to male Cynomolgus monkeys. The serum concentration of the antibody declined according to a poly-exponential curve Fig. C. The PKTo define the preclinical model/s suitable for studying antibody safety, we assessed hOA-DN30 binding properties versus MET derived from different species. hOA-DN30 cross-reacted with the MET receptor of human, rat, dog, and monkey origin, while the interaction with mouse MET was very weak  , gave poThe first MET antibody tested in the clinic was Onartuzumab, a potent HGF competitor antibody efficiently blocking HGF-dependent MET activation . So far,hOA-DN30 displays a novel mechanism of action completely different from the simple inhibition of ligand/receptor interaction; its binding epitope - the IPT4 region of MET  - does nCurrently, a new generation of MET antibodies featuring mechanisms of action different from Onartuzumab is under clinical evaluation.www.cbioportal.org), this should be considered a plus and not a limitation, as it assures a personalized response when a correct genetically-based patient selection is applied. On the contrary, patient selection based uniquely on the level of MET expression does not seem reliable [Teliso-V/ABBV-399 is an antibody-drug conjugate that displays inhibitory effects against MET-overexpressing cells  mainly -reliable .Other antibodies tested so far in humans - SAIT-301, Emibetuzumab, ARGX-111, and a mixture of two antibodies, Sym-015 - are able to block HGF binding and concomitantly induce MET internalization –71. ThesIn addition to ligand competition and receptor downregulation properties, ARGX-111 and Sym-015 also unleash the immune response of the host against the tumor by triggering ADCC , 74. We Besides antibodies with a canonical structure, a biparatopic antibody, REGN-5093, is currently under clinical testing. This molecule not only blocks HGF/MET binding and induces receptor internalization, but also modulates receptor trafficking . Due to In summary, hOA-DN30 possesses unique features when compared to anti-MET antibodies in clinical development. The molecule being monovalent is a pure antagonist. hOA-DN30 mechanism of action (i.e. MET shedding) is based on an initial proteolytic event, the MET cleavage, which prompts intracellular receptor destruction not dependent on receptor trafficking. This activity should be considered more robust and definitive compared with the receptor internalization induced by other MET inhibitory antibodies. In the case of internalization, the receptor can be recycled from the endosomes, while, in the case of shedding, the recovery of MET on the cell surface relies exclusively on new receptor synthesis. Thus, MET turn-over is rapid in the case of internalizing antibodies and, conversely, delayed in the case of hOA-DN30, assuring a longer-lasting effect. MET shedding not only removes MET from the cell surface but also intrinsically triggers other mechanisms able to block the activation of MET receptors that survived the cleavage. This occurs by releasing MET-ECDs in the microenvironment. The ‘decoy’ effect of MET-ECDs is multi-faceted, being effective on the side of ligand-independent MET-activation, by forming inactive receptor heterodimers, as well as on the side of ligand-dependent MET activation, sponging HGF. Notably, MET-ECDs represent the best option to neutralize the ligand, as they incorporate all the binding features included in the natural receptor.From a pharmacological point of view, hOA-DN30 displays a considerably improved profile compared to its parental molecules (DN30mAb and MvDN30) , not disFrom a toxicological point of view, the absence of adverse effects in the monkey at the highest dose tested 180 mg/kg) is extremely encouraging. The good tolerability of hOA-DN30 is in line with what was reported for other MET therapeutic antibodies in non-human primate preclinical models , 71 and  mg/kg isUltimately, hOA-DN30 possesses features resulting in a paramount MET blocking action. The intense and robust therapeutic response exerted by hOA-DN30 in a large number of preclinical models, as well as its pharmacological features and safety profile in non-human primates, strongly envisage a successful clinical application of this novel single-arm MET therapeutic antibody.Additional file 1.Additional file 2.Additional file 3.


IgG4-related disease (IgG4-RD) is a multi-organ, immune-mediated inflammatory condition of unknown etiology characterized by infiltration of tissues by IgG4 producing plasma cells. IgG4-related disease (IgG4-RD) can ideally affect any organs, but the involvement of the central nervous system (CNS) is a rare entity. We present a case of a 67-year-old male who presented with diplopia with imaging showing hypertrophic pachymeningitis (HPM) and was diagnosed with IgG4-RD of the CNS based on histopathology report. IgG4-related disease (IgG4-RD) of the central nervous system (CNS) is rare, and only about 82 cases have been described in literature who have hypertrophic pachymeningitis (HPM) as a manifestation of IgG4-RD . The etiA 67-year-old Caucasian male with medical conditions including transient ischemic attack, depression, polysubstance use, and ex-smoker presented with chief complaints of diplopia with looking straight and left lateral gaze of 10 days duration. The patient reported similar episode of diplopia one year prior to this hospitalization that resolved spontaneously within a day. Seven years prior to this presentation, he had whole left upper extremity numbness that lasted for 24 hours and resolved spontaneously. He denied any history of inflammatory eye disease, thrombosis, or risks for tuberculosis. He denied recent travel and unusual hobbies and did not have pets. He also denied any joint pain, joint swelling, fever, recent change in weight, skin rash, lymphadenopathy, oral ulcer, nasal ulcer, chest pain, shortness of breath, cough, hemoptysis, numbness, tingling, and muscle weakness.His father had rheumatoid arthritis, and no family history of other autoimmune diseases or malignancy was reported. He had 40 pack-year smoking history and had quit seven years prior to this hospitalization. His home medications were aspirin, fluoxetine, and trazodone. His physical examination showed limitation of left eye abduction on left lateral gaze, consistent with left sixth nerve palsy, and there was no papilledema. Other examinations were within normal limits.Laboratory results showed unremarkable cell counts and chemistry. Urinalysis did not show proteinuria or hematuria. Urine toxicology was positive for amphetamine and cannabinoids. Erythrocyte sedimentation rate was 44 mm/hour (reference range: 0-20 mm/hour), and C-reactive protein was 8.1 mg/dL (reference range: 0-5 mg/dL). Infectious workups including HIV, RPR, hepatitis B, hepatitis C, T-SPOT, HSV, VZV, and serum histoplasma antigen were negative. Autoimmune workups including antinuclear antibody, antineutrophil cytoplasmic antibody, rheumatoid factor, and angiotensin-1-converting enzyme were negative. C3 and C4 were within normal limits. CSF pressure was 13 cmH2O, and CSF analysis showed normal leukocyte count, protein, and glucose. CSF bacterial culture, AFB smear, fungal smear, VDRL, cryptococcus antigen, CMV, and EBV were negative. CSF IgG was 9.4 mg/dL (reference range: 0-6 mg/dL), and serum IgG and IgG4 were normal. Magnetic resonance imaging (MRI) of the brain with and without contrast showed pachymeningeal thickening and enhancement, including the posterior fossa, tentorium, internal auditory canals (IACs), left cavernous sinus, left Meckel's cave/trigeminal nerve foramen rotundum/ovale, left pterygopalatine fossa, and inferior orbital fissure extending the infraorbital nerve (Figure Histopathology from meningeal biopsy revealed characteristic features of IgG4-related disease, including storiform fibrosis, IgG-positive cells, and IgG4-positive cells counted between 15 and 25 per one high-power field in various areas and constituting about 50% of the IgG-positive population, and obliterative venulitis. GMS and Ziehl-Neelsen stains were negative for fungal and acid-fast microorganisms (Figure He was started on prednisone 60 mg orally daily and had resolution of diplopia. The plan is to start rituximab for steroid-sparing effect and to prevent disease relapse.IgG4-RD is an immune-mediated condition of unknown etiology characterized by infiltration of tissues by IgG4 producing plasma cells. A Japanese registry found the incidence of IgG4-RD to be about 0.28-1.08 per 100,000 population .CNS involvement in IgG4-RD is rare, mostly affecting the pituitary and meninges and rarely brain parenchyma. With meningeal involvement, there is focal or diffuse thickening of the dura mater, causing HPM in the brain or spinal cord or sometimes both. A review of cases of meningeal IgG4-RD showed that the median age affected with IgG4-related HPM was 53 years, and males were affected twice more than females, with the most common symptom being headache . ClinicaThere is no definite treatment guideline for meningeal IgG4-RD. Systemic steroids are the first line of treatment. Treatment is often challenged with relapse in as high as 25%-50% of the cases after discontinuation or tapering of steroid . There aIgG4-RD affecting the CNS is rare, and it can affect the CNS in isolation or with other organs. We described a case of IgG4-related HPM presenting with diplopia. This case highlights that serum IgG4 levels can be normal in patients with IgG4-related HPM, and although CSF IgG can be elevated, a meningeal biopsy is necessary to confirm the diagnosis and rule out other inflammatory, infectious, and neoplastic etiologies of HPM. Although the exact pathogenesis of IgG4-RD is unknown, response to B cell depletion therapy indicates a role of B cells. Treatment should include the initial use of corticosteroids to prevent further damage, as well as the early institution of immunosuppressive therapy as a steroid-sparing agent, and also to prevent disease relapse.


Most of the previous research on recycled concrete aggregates (RCA) has focused on coarse RCA (CRCA), while much less has been accomplished on the use of fine RCA particles (FRCA). Furthermore, most RCA research disregards its unique microstructure, and thus the inferior performance of concrete incorporating RCA is often reported in the fresh and hardened states. To improve the overall behaviour of RCA concrete advanced mix design techniques such as equivalent volume (EV) or particle packing models (PPMs) may be used. However, the efficiency of these procedures to proportion eco-efficient FRCA concrete still requires further investigation. This work evaluates the overall fresh  and hardened states  performance of sustainable FRCA mixtures proportioned through distinct techniques  and incorporating different types of aggregates  and manufacturing processes . Results demonstrate that the aggregate type and crushing process may influence the FRCA particles’ features. Yet, the use of advanced mix design techniques, particularly PPMs, may provide FRCA mixes with quite suitable performance in the fresh  and hardened states  along with a low carbon footprint. Concrete is a widely used material for critical infrastructure worldwide; over 10 billion tons of concrete are produced annually in the modern industrial society ,3,4. AmoThe intrinsic difference between RCA and natural aggregate (NA) is the fact that CRCA is a two-phase material composed of the original virgin aggregate (OVA) and residual mortar (RM). Likewise, fine recycled concrete aggregates (FRCA) comprise the original virgin aggregate (OVA) and residual cement paste (RCP). The properties of RCA concrete made of CRCA have been extensively investigated ,15,16,17Studies have demonstrated that mortar mixtures made of partial or full direct replacement of NA by FRCA displayed more somewhat similar fresh state behaviour than conventional mortar, once both aggregates (either NA or FRCA) were in saturated surface-dry conditions . ConversIn this context, advanced mix design techniques have been developed to optimize recycled concrete mixtures, allowing more efficient re-use of RCA in concrete and enabling better performances in the fresh and hardened states. The literature suggests that the key factor for an efficient proportioning of recycled concrete mixtures bearing CRCA is to account for their unique microstructure , while the mix design and proportioning techniques such as the equivalent mortar volume (EMV)  and equi3) and chemical admixtures [One of the earliest attempts to use a mix proportion of RCA concrete was performed through direct replacement methods (DRMs), which can be conducted by weight or volume. This approach treats RCA as homogeneous material and partially replaces a certain amount of natural aggregates accounting for their distinct microstructure and or the presence of RM ,34. Moremixtures ,37. In omixtures . Hence, The equivalent mortar volume (EMV) method was initially proposed by Fathifazl et al. 2009a and treated RCA as a multi-phase material composed of RM and OVA. Thus, the volume of cementitious materials added to the recycled mix is reduced according to the amount of existing RM content adhered to the RCA particles. It has been suggested that EMV can reach similar or even somewhat higher compressive and tensile splitting strengths than conventional concrete ,40. BesiIn order to address the issues often faced in the fresh state by EMV-proportioned RCA mixes, especially in eco-efficient mixtures with a reduced binder content, a modification to the preliminary method was proposed by Hayles et al. 2018. The EMV-mod method adds a supplementary factor to the previous strategy to optimize the “cement-to-sand mass ratio” of the RM and thus allows the selection of the amount of cement to be accounted for in the new RCA mix. Hence, EMV-mod-proportioned mixtures show better improvement in fresh-state performance  than EMV mixes. However, this approach was verified to present an important drawback since the binder content is typically increased for a given targeted strength compared to conventional and EMV-designed mixes .3) due to the significant increase in fine aggregates and decrease in coarse particles in the system [Recently, a new mix design procedure, the so-called equivalent volume (EV) method, was developed by Ahimoghadam et al. 2020 based on the main concepts of the previous EMV and EMV-mod methods. The EV was established to solve the issues faced by EMV and EMV-mod mix-proportioned mixtures in the fresh state, along with providing recycled mixtures with an eco-friendly character and suitable performance in the hardened state . The nove system ,41.Particle packing models (PPMs) are advanced mix design techniques used to improve the performance of concrete mixtures in fresh and hardened states. PPMs are divided into discrete and continuous models; either discrete or continuous, PPMs aim to optimize granular systems’ particle size distribution (PSD), reducing the material’s porosity and consequently increasing the system’s packing density . The disq) and the largest particle size (D) [q) along with the largest (LD) and smallest (SD) particle sizes  present in the system (Equation (1)). The coefficient of distribution (q) is adopted based on the fresh-state requirements of the mix; values ranging from 0.20 to 0.23 are often selected for self-consolidating mixtures, whereas distribution factors of 0.26–0.28 are normally targeted for vibrated and or pumped concrete. It is worth noting that 0.37 is the q value that yields the highest packing density, hence the lowest porosity to granular systems [PD, and PD is the particle size.The first continuous PPM was established by Fuller in 1907, and it is based on a power curve that accounts for two variables: the distribution factor (size (D) . Over th systems .(1)CPFTPPMs have been widely and successfully used to improve performance, especially in the hardened state of eco-efficient ,52, convAs previously described, there is currently limited research on the influence of the FRCA particle features  and mix design techniques  on the fresh- and hardened-state performance of eco-efficient recycled concrete mixtures made of 100% FRCA. This work is divided into two main phases: (1) production and characterization of FRCA materials and (2) appraisal of the fresh- and hardened-state properties of eco-efficient FRCA concrete mixtures. The first phase evaluates the impact of the FRCA type  and manufacturing process  on the physical properties of the granular material. In the second phase, three mix design techniques  are selected to proportion eco-efficient FRCA mixtures made of natural or manufactured sand. Finally, evaluations on the fresh- and hardened-state properties are conducted, and comparisons with a companion conventional concrete (0% FRCA) are performed. It is worth noting that the term eco-efficient used in the context of this work refers to concrete mixtures displaying equal or lower binder contents than their companion conventional mixtures. In other words, there is no need to use a supplementary amount of binder to obtain a performance in the recycled mixture as is conventionally adopted in RCA research.The experimental program is divided into two main streams as described in 3 and 0.75% absorption was used to produce the conventional concrete mixtures. Neither chemical admixture nor mineral admixtures were employed in the conventional concrete mixtures. The FRCA used in this work was manufactured in the laboratory; it is derived from crushed companion conventional concrete specimens, mix-proportioned through the ACI method  and displaying a design compressive strength  of 35 MPa. Two families of FRCA were fabricated, one containing a natural siliceous sand (ACI-NS) and the other incorporating a limestone-manufactured sand (ACI-MS). A coarse limestone aggregate with a nominal maximum size of 19 mm, an oven-dry specific gravity of 2.78 g/cmTwo hundred conventional concrete cylinders  were cast from each of the concrete mixtures used in this research incorporating NA and MS. The specimens were kept in the moulds at laboratory temperature, demoulded after 24 h and moist-cured over 28 days. In order to appraise the influence of the crushing process on the FRCAs’ inner quality, two manufacturing methods  were employed to produce the recycled granular material. The second method The RCP was measured in this work on all FRCA materials through the soluble silica sub-procedure according to ASTM C1084-15  and C114Potential of RCP for further hydrationIsothermal heat conduction calorimetry (IHCC) of the FRCA was determined in an isothermal calorimeter G-563-62-L0071/AD by Controls Group. The test was performed to determine whether the residual cement paste (RCP) adhered to the FRCA particles might have some potential for further hydration in concrete. The test was conducted on original FRCA particles and cement powder for comparison purposes. It is worth noting that IHCC was performed on freshly prepared samples to avoid the possible reaction of unhydrated cement with moisture from the air. Ten grams of each FRCA material were selected for testing; 3.5 g of double-deionized water was added to the samples to evaluate the cementing capability of the RCP encountered in the FRCA particles. After mixing for 60 s, the samples were placed in the calorimeter and tested. It is important to note that the plastic scintillation vials were filled with 26.49 ± 0.2 g of glass beads as a reference. All the IHCC results are presented on the same basis (per gram of cement) since the amount of cement in each sample is variable. The IHCC results obtained in this work are presented in the analysis and discussion section.Specific gravity and water absorptionThe specific gravity and water absorption capacity of all FRCA materials were evaluated according to the method proposed by . The conParticle size distribution (PSD)A general-use  Portland cement was used in all mixes. In addition, a limestone filler with a smaller PSD than PC , known aA total of three distinct concrete mixtures  with 100% replacement of fine NA by FRCA were designed. Moreover, two control mixes  containing NS or MS were also produced for comparison purposes. Concrete mixtures proportioned using the DRM method were designed according to the ACI protocol , purposely treating FRCA as presenting the same properties of fine NA; the latter is a common practice adopted in the construction industry.The EV method  was adapw/c) of 0.35 was adopted for all FRCA mixes since preliminary trial batches indicated the need to reduce the w/c of the FRCA mixtures to yield close compressive strength results  to companion conventional concrete mixes with w/c of 0.47. Moreover, the consistency of the mixture  was aimed to lie within 100 ± 20 mm. A summary of all mixtures is presented in The DRM-, EV- and PPM-designed recycled mixtures were proportioned with 100% FRCA replacement from the four FRCA sources previously mentioned. The target 28-day compressive strength was selected as 35 MPa as per CSA  for the 3). Conversely, since the EV and PPM methods account for the RCP attached to the FRCA particles, the mixtures proportioned by these procedures presented a much lower PC content. In the case of the EV method, it was possible to obtain mixtures with Portland cement contents (≈373 kg/m3) very similar to those of conventional concrete designed with the ACI method . Likewise, mixtures designed through the continuous PPM technique (distribution factor of 0.28) and with the use of inert fillers yielded final Portland cement contents ranging from 333 to 299 kg/m3 .The mixes proportioned using the DRM method  treated the FRCA material as the same as natural fine aggregates, resulting in mixtures with high amounts of PC . The IBB rheometer a consistThe rheological profiles of the mixtures evaluated were determined with a two-step process: an increase in shear rate up to approximately 43 RPM, followed by a decrease period at the same stepwise, keeping a constant rotation for roughly 10 s at each step b. The teThe evaluation of hardened-state properties was performed through compressive strength and non-destructive tests. For compressive strength, three samples of each mixture were tested over time  as per CSA A23.1-14 . LikewisThe microstructure quality of the concrete specimens was evaluated using scanning electron microscopy (SEM). The selected samples were first cut using a diamond-bladed masonry saw followed by subsequent polishing using a mechanical polishing table with grits of 30, 60, 140, 280 (80–100 µm), 600 (20–40 µm), 1200 (10–20 µm), 3000 (4–8 µm), 10,000 (2–4 µm) and 20,000 (1–2 µm). After that, the polished sections were dried at 60 °C for 24 h. Once the polishing process was completed, the samples were examined in a Jeol JSM- 6610LV SEM operated under a low vacuum, with an electron acceleration voltage of 20 kV and a spot size of 57. Backscattered electron (BSE) was conducted at different magnifications  to study the mixture’s quality, morphology and distribution. The SEM images and the description of the microscopic features observed in the different recycled concrete mixtures are presented in the discussion section of the manuscript.3); yet, the ACI-NS resulted in a higher slump (110 mm) than ACI-MS (95 mm). Moreover, although the DRM mixtures were produced with the same water content as ACI mixtures, admixtures were required  to achieve the target slump (100 ± 20 mm). Otherwise, a lower amount of water  was used for all EV mixtures along with a 0.32% of mid-range/HRWR. The PPM mixtures achieved the required slump with the lowest amount of water  and a somewhat higher mid-range/HRWR  when compared to the EV mixtures. Finally, it is worth mentioning that mixtures containing FRCA-FG yielded overall higher slump values when compared to FRCA-CF.The consistency results  of ACI, DRM, EV and PPM mixtures are presented in The rheological profiles for the ACI, EV and PPM mixtures are presented in Analyzing the plots, one first verifies that all mixtures yielded a shear-thinning behaviour, i.e., decrease in viscosity (slope of the torque vs. rotation curve) as a function of the torque applied regardless of the mix design procedure adopted. Second, the PPM mixtures displayed the lowest yield stress, i.e., requiring a lower amount of torque to enable flow , followed by EV (7.32 N.m to 11.33 N.m) and ACI (10.70 N.m to 14.81 N.m)-mix-designed mixtures. Moreover, besides the rheological profiles and the minimum torque values mentioned above, two “extra” key rheological parameters can also be extracted . A summary of all these values is displayed in Finally, the time dependency (thixotropic vs. rheopexy behaviour) of all mixtures was also evaluated by calculating the hysteresis area under the torque vs. rotation curve. Overall, all mixtures presented positive HA, corresponding to a thixotropic behaviour; in other words, the viscosity of those mixtures decreases over time under constant shear stress or shear rate. ACI mixes presented the lowest HA values, followed by PPM and EV. Furthermore, mixtures made of MS presented lower HA than those with NS, whereas mixtures made of FG aggregates also yielded lower HA values when compared to CF mixes.In this section, compressive strength (CS) and non-destructive tests results are presented for all mixtures manufactured in the laboratory. Comparing the results, one verifies that the highest compressive strength values were obtained by PPM-proportioned mixtures , whereas the mixtures proportioned via DRM yielded the lowest results . EV-designed mixes’ results lay between PPM and DRM mixtures . The control mixes designed with natural (AC-NS) and manufactured (ACI-MS) sands presented 28-day compressive strength results within the range of 35 to 40 MPa.The compressive strength results mentioned above correlate quite well with surface electrical resistivity a and theanalysis and discussion section will be divided into six sub-sections. First, a discussion of the influence of the raw materials and manufacturing process on the FRCA features is conducted. Second, a volumetric fraction evaluation of the various recycled mixtures components obtained through distinct mix design techniques is performed, followed by their impact on those mixtures’ fresh and hardened states. Finally, microstructure analyses of the recycled mixtures are conducted, along with a discussion on the binder efficiency of FRCA mixtures proportioned using distinct mix design techniques.As previously mentioned, the present work is divided into two main parts: (1) production and characterization of FRCA materials and (2) appraisal of the fresh- and hardened-state properties of FRCA concrete mixtures proportioned using distinct mix design techniques. Although the data from the characterization of the FRCA, such as RCP content, specific gravity and water absorption and particle size distribution, were displayed in the experimental program section (since they were needed to design and manufacture the various recycled concrete mixtures), a global discussion on the obtained data seems appropriate herein. Therefore, the current The analysis of the characterization results demonstrates an important role of the raw materials used  or the RCA’s manufacturing processes  in the fresh and hardened states of FRCA concrete. Amongst the distinct properties appraised , the RCP content showed an important influence of the raw material since FRCA made of MS presented a higher RCP than FRCA made of NS . This result is likely due to the rougher texture and more angular surface of the MS material, which may enhance the bond between the cement paste and aggregates. Likewise, comparing the FRCA’s manufacturing processes, CF-made materials yielded 23% higher fineness modulus and 30% more RCP attached (on average), which is linked to the number of crushing processes, i.e., the higher the number of crushing cycles, the lower the amount of RCP attached to the fine aggregate particles. Consequently, FRCA-CF presented 4% lower specific gravity and 20% higher water absorption than FRCA manufactured through the FG crushing process.From the particle size distribution of the FRCAs, it was verified that a higher number of crushing cycles  produces a greater amount of fine material . In other words, an appropriate and optimized process is required to produce FRCA to minimize waste generation  along with energy consumption during the manufacturing process. Otherwise, FG manufacturing processes generate FRCAs with 29.1% less RCP content (on average) than CF. Moreover, the greater the amount of RCP adhered to the particles, the lower the quality of the materials, and the more challenging it is for FRCA concrete mixture proportioning to achieve comparable fresh- and hardened-state properties to conventional companion mixtures.The above discussion indicates that high amounts of RCP adhered to the FRCA particles may lead to a lower-quality material since more inner deterioration such as microcracks and flaws is expected. Conversely, the presence of RCP, depending on the condition of the cement grains , may stimulate further hydration, which could help seal pre-existing inner cracks and thus enhance the overall quality of the FRCA material. In this regard, isothermal calorimetry was performed to quantify the flow and the amount of the heat released from the FRCA materials as mentioned in The calorimetric measurements indicate that the overall acceleration of exothermic reactions from FRCAs was strongly reduced compared to general Portland GU-type cement samples. The FRCAs’ heat flow curves are not very well defined. Yet, two phases were identified , which is a very different behaviour compared to the traditional GU cement behaviour with clear initial induction, accelerating and decelerating periods. Moreover, the results demonstrate that the difference in raw materials  does not significantly influence the amount of heat released over the first 92 h of the test. Yet, both FRCAs demonstrated some hydraulic activity. The cumulative heat achieved by the FRCA materials after 92 h corresponded to about 15% of the heat released by the GU-type cement. This behaviour dos not significantly change the mechanical properties of the FRCA concrete over time, but this demonstrates that further hydrates might be theoretically formed in the system, which could contribute to an increase in the overall quality of the FRCA concrete. Further analysis is still required in future projects to quantify this potential aspect more precisely.The distinct mix design methods used in this research played a very important role in the volumetric fraction components of both FRCA and CC mixes, as expected; yet, to better visualize this influence, c). These authors developed an analytical model to determine concrete mixtures’ yield stress (τc) based on the slump test results. Analyzing the data below and the experimental comparison between slump vs. minimum torque, the calculated yield stress (τc) also decreases with the increase in a slump, which attests to the validity of the minimum torque results gathered.All mixtures assessed in the study were designed with a targeted slump range of 100 ± 20 mm, being thus considered (in practice) as “workable” mixtures to be used as cast-in-place/vibrated concrete. Yet, although similar consistencies  were selected for all mixtures, quite variable minimum torque results, ranging from 4.74 N.m (PPM-NS-FG) to 14.81 N.m (ACI-MS), were obtained. In general, the higher the slump value, the lower the minimum torque required . To atteτ is the shear stress, τ0 is the yield stress, HBk is the viscosity constant of Herschel–Bulkley, and n is the flow behaviour factor, which is n < 1 for suspensions presenting shear-thinning behaviour and n > 1 for shear-thickening ones [According to Bingham’s model, the rheological behaviour of conventional concrete mixtures is described very often ,67 and iing ones . It is wHB constant was also observed to yield the lowest values for PPM mixes compared to EV and CC mixtures. It is important to notice that the higher kHB values, the higher the slope of the rheological profile curve .Analyzing the data gathered through Herschel–Bukley’s model  and plotThe above results suggest that the PPM-proportioned mixtures have the most suitable rheological behaviour for vibrated and or pumped applications. Moreover, following the study on the minimum torque, the derivative of the Herschel–Bulkley model of each concrete mixture  was perfw/c ratio; these results are due to the lower amount of RCP adhered to the FRCA-FG particles along with their more angular in shape and rougher in texture characters. Moreover, an increase of nearly 53% in CS and 31% in ER was obtained for PPM-designed mixes, whereas a slight average increase of about 16% and 6% was observed for EV and DRM mixes, respectively. The above results suggest that the FRCA particles’ features  are more important in mixtures with a more optimized particle size distribution (improved granular skeleton) and thus have lower porosity. Conversely, the raw-material type  does not seem to influence the mechanical properties of FRCA concrete significantly.The type of FRCA material  was found to have a low impact on the surface electrical resistivity (ER) and compressive strength (CS) of DRM and EV mixes and a more significant influence on the PPM-mix-proportioned mixtures. It is well known that the mechanical properties of concrete are directly related to the quality of its microstructure. Moreover, the results of CS and ER are quite well correlated; overall, the higher the compressive strength of the concrete, the higher the electrical resistivity. In general, the mixtures proportioned with FRCA-FG aggregates showed higher strength and ER results for the same Another important discussion to be addressed is on the dynamic elastic modulus (DEM) obtained in this research. The DEM results for the FRCA concrete mixtures follow a close correlation with the compressive strength, i.e., the higher the CS, the higher the DEM values. However, only the PPM-NS-FG and PPM-MS-FG mixtures could reach similar values to the companion conventional concrete mixtures . These findings may be justified by a thorough evaluation of the FRCA mixtures compositions. For instance, the replacement of natural fine aggregate by FRCA introduced theoretically more interfacial transition zones (ITZ) to the system, which may have governed (and lessened) the overall DEM of the recycled mixture. On the other hand, PPM-mix-designed recycled mixtures presented a higher DEM; this is associated with a better microstructure quality  of PPMs systems, enhancing the stiffness response of concrete mixtures as previously reported by ,69. MoreThe above discussion confirms both the influence of the manufacturing process  and the mix-proportioning technique  on the mechanical properties of FRCA concrete.w/c ratio) of the mixture [It is well established that the mechanical behaviour of CC depends on the quality of its compounds, such as hydrated products and the ITZ, which in turn is directly related to the porosity  presented a much-enhanced microstructure when compared to EV or DRM proportioned mixes since suitable (and better) adherence between the residual cement paste (RCP) and the new cement paste (CP) was observed in all samples. Moreover, minor microcracks were identified within the RCP in both PPM-proportioned mixtures (highlighted areas in 3) required to obtain one unit of a given mechanical property; for example, 1 MPa of compressive strength at 28 days. 3).In order to evaluate the eco-efficiency of conventional concrete mixtures, Damineli et al.  developebi factor was calculated for all of them, and the results are illustrated in bi values (8.3 kg∙m3∙MPa−1 with an approximate average 28-day compressive strength of 53 MPa). Furthermore, one may notice that some PPM mixtures are quite close to the bottom line of the plot , which represents the lowest amount of Portland cement conventionally used in the market for conventional concrete. Otherwise, conventional concrete mixes presented an overall bi factor of 10 kg∙m3∙MPa−1 for an average 28-day compressive strength of about 37 MPa, while DRM and EV mixes displayed bi factors of approximately 25 and 13.1 kg∙m3∙MPa−1 for average compressive strengths of 20 and 29 MPa, respectively. These results demonstrate the promising character of PPM techniques in creating mix proportions of eco-efficient FRCA concrete with suitable fresh- and hardened-state properties.To evaluate the binder efficiency of all conventional concrete and FRCA mixtures studied in this experimental program, the It is clear that the manufacturing  process influences the overall quality of the FRCA material. With only two consecutive series of crushing, the so-called CF generates recycled particles with coarser PSD, and high amounts of adhered RCP. Yet, multiple crushing series, the so-called FG, were observed to produce FRCA particles with similar PSD to natural aggregates and displayed less RCP attached to the particles due to further separation of mortar during crushing. Moreover, CF particles seem to be more rounded in shape and smoother in texture when compared to FG particles.The fresh-state behaviour of FRCA mixtures proportioned through both EV and PPM methods demonstrated an interesting performance, showing a shear-thinning profile . Yet, PPM-mix-designed FRCA concrete demonstrated the best rheological variables  among all mixtures; the latter indicates its suitability for applications under high-torque regimes such as pumping and or vibrating processesInteresting hardened-state properties  were achieved using both EV and PPM techniques. However, in general, the DRM method yielded the worst mechanical performance obtained in this work. Yet, PPM-mix-designed FRCA proved to be the most promising mix design technique to proportion FRCA concrete mixes with suitable mechanical properties, binder efficiency and an enhanced microstructure .It seems that both the aggregate types (NS vs. MS) and crushing processes (CF vs. FG) play an important role in the overall FRCA quality  along with fresh- and hardened-state performances. Yet, the results obtained in this experimental campaign demonstrated that the mix design procedure adopted to produce FRCA concrete is even more important than the FRCA features and inner quality.Finally, microscopic analysis needs to be assessed considering distinct FRCA concrete mixes through the use of both EV and PPM methods in order to investigate the different failure mechanisms of FRCA concrete, especially when subjected to harsh environmental conditions. This would propose valuable remarks on the mechanical and durability-related properties of FRCA mixes against CC mixes as well as potential improvements on the EV mixes.The current research aimed to understand the overall behaviour of FRCA concrete made of distinct raw materials , manufacturing processes  and mix design techniques . The main findings of this research are presented hereafter:


Self-healing of a crack is a relatively novel technique allowing for the partial recovery of the initial mechanical characteristics of a structural element after some period of exploitation. By a widely accepted convention, self-healing is either autogenous or autonomous. The former is a mechanism inherent for cementitious composites (in particular—concrete), while the latter is an engineered process. Both autogenous and engineered healing have recently been the object of numerous studies. Despite the large amount of research work being carried out, the potential of this technique has not yet been fully realized. The article focuses on the modeling and the finite element simulation of the recovery of the initial material properties resulting from the sealing of cracks. The employed numerical procedure uses a constitutive relation for concrete based on the continuum damage mechanics. It captures both the strain-softening and the inverse process—the crack healing. Finite element simulations of benchmark cases illustrate the effect of self-healing. The numerically obtained constitutive relations for specimens with and without a healing agent are compared. The self-healing of cracks implies the material properties’ recovery resulting from the crack sealing ,2. GenerUsing environmentally friendly bacteria such as Bacillus pasteurii can replace the commonly used repair materials for concrete , which are environmentally unfriendly . Moreove2).Some authors ,8 attribThe partial substitution of cement with fly ash, as well as the addition of crystalline admixture, yields the recovery of both compressive strength and electrical resistivity of concrete up to 94% . AccordiPolymers , acrylamide, acrylate, epoxy, poly styrene-divinylbenzene, styrene-butadiene rubber, etc.) improve the rate of the regain in the mechanical properties and contribute to the healing of surface cracks .Reportedly, fibers (either steel or synthetic) in fiber-reinforced concrete control the crack opening what results in ease of application of the self-healing products in terms of adhesion to the crack surface and crack filling. Thus, according to , in engiUltra-high-performance concrete (UHPC) and ultra-high-performance fiber-reinforced concrete (UHPFRC) have the potential to exhibit self-healing capacity because of the high binder content and the low water/binder ratio ,20,21. AThe modeling of the self-healing of concrete is in close relation with the simulation of the concrete failure behavior. The fracture of concrete has been investigated in numerous research works ,42,43,44Among other approaches suitable to model the self-healing phenomena, Yang et al.  discuss The deterministic approach to the analysis is often questionable, considering the high scattering in material properties within structural elements and loading. The stochastic perturbation-based finite element method theoretically addresses this issue .Recent studies extend the finite element method by proposing a new finite element formulation with embedded strong discontinuity  or by coIn Ref. , by hypoIn , by assuCombining the finite element method (FEM) and the discrete element method (DEM) has drawn considerable research attention ,62,63,64The combined FEM–DEM formulation implies a multi-scale approach. The frictional contact properties of the interacting discrete particles (for the DEM) are defined at the micro-scale, while the material properties for the FEM generally reflect experimental data obtained at the macroscopic scale. The multi-scale approach involves an identification procedure  that genOñate et al.  use the Both aforementioned approaches imply complicated numerical procedures accounting for crack initiation and propagation in the continuum. On the other hand, the complication affects only a part of the initial computational domain. The article focuses on the numerical simulation of the opening and reclosure of cracks, to model the material properties degradation, due to the former mechanism and the material properties recovery attributed to the latter.A simplified solution scheme using the finite element method only is applied. Using different methods for different phenomena might be intuitive and consistent. On the other hand, continuum mechanics  describes the microscope manifestation of mechanisms that take place at the microscopic scales. The employed damage model slightly differs from the standard damage model incorporated in the advanced numerical simulations referenced above. The numerical algorithm uses an original procedure that integrates the damage variable into the material constitutive relation. Generally, the concrete fracture is described either by using fracture mechanics or in the context of damage mechanics. Damage mechanics is the preferred option in this study since it allows for modeling the damage accumulation and crack propagation starting with homogeneous isotropic material. Unlike the fracture mechanics approach, there is no need to pre-establish defects in the studied specimen to initiate macroscopic cracking. The present article develops and summarizes some ideas discussed in previously published contributions ,66.The underlying hypotheses of the reported study presume that mechanical loading triggers the crack opening while the regain in the mechanical properties results from engineered self-healing. Continuum damage mechanics allows for the modeling of the degradation of the initial mechanical properties via the introduction of a damage variable into the material stress–strain relationship. The damage variable is regarded as an operator acting on the elasticity tensor. Thus, damage variable increase models the damage accumulation in the material, and a critical value of the damage variable corresponds to macroscopic crack initiation in the specified location. Vice versa, the sealing of cracks results in a decrease in the damage variable and a partial regain in the initial mechanical properties. Finite element simulations of some benchmark examples illustrate the self-healing mechanism. Modeled specimens are damaged to introduce multiple cracking patterns . Then, at a specific moment of the loading history, the activation of the autonomous self-healing leads to a sealing of the formed macroscopic cracks. This mechanism results in a partial regain of the initial material properties. The focus of the article is on the finite element analysis of concrete specimens containing a self-healing agent. Within the assumed approach, the material response of concrete is modeled via a coupling between elasticity and damage ,67.(1)σIn Equation (1), The damage variable acts directly on Young’s modulus. Theoretically, it evaluates the mechanical damage induced by the applied load.As shown in Equation (2), the damage variable depends on a set of material constants n tensor .(3)εeqvi, one set of two constants for tension, and another for compression control the shape of the numerically obtained stress–strain relationship. In Equation (3),The constant Damage accumulates provided the variable defined in Equation (3) becomes larger than another model parameter, the damage threshold:Reaching the critical value of the damage variable at a specific location in the continuum The activation of the self-healing process leads to the sealing of the newly formed cracks and regaining the initial rigidity in some regions. Therefore, self-healing is modeled by setting the damage variable equal to zero and the Young modulus—equal to its initial value. At the same time, some level of damage remains in regions where the damage variable has not yet reached its critical value. Standard compression and tension by flexure tests, as well as a torsion test on concrete specimens, are simulated. Simulations include a preloading phase without reaching the failure load of the specimen. The preloading phase induces some damaged state in the specimen. Also, in some regions, the critical value of the damage variable is reached , and the self-healing process is launched . The responses of the specimens are obtained by finite element analysis in terms of stress–strain relationships.The evaluation of the self-healing effectiveness employs an assessment of the recovery of the relevant mechanical properties . ConcretThe general-purpose finite element code ANSYS is employed for the numerical simulations. A compression test on a cylindrical concrete specimen, a tension by flexure test on a prismatic concrete specimen, and a torsion test on a cylindrical concrete specimen are simulated. Standard geometries are used for the simulations of the compression and the tension-by-flexure tests.The compressive strength of the concrete is measured on cylinders 150 mm in diameter and 300 mm in height, in accordance with ISO 1920-3 . Figure Tension by flexure test commonly performed on a prismatic concrete specimen is simulated. The modeled specimen is 400 mm in length, 300 mm in span, with a rectangular cross-section of 100 mm × 100 mm. The distance between the specimen end and the point load location is 100 mm . For the considered experimental setup, unstable crack propagation being a fast process, additional time-stepping, with the definition of a smaller time interval, might be required to capture it.The finite element analysis simulates a displacement-controlled experimental setup with a constant time step for the entire load history. The solid geometry is meshed with 1568 finite elements SOLID185 . To modeThe finite element simulation reported in this section reproduces the response of a cylindrical concrete specimen subjected to torsion without the ambition to reproduce accurately any experimental setup. Results obtained by finite element simulation have illustrated the effect of the self-healing of cracks. The strength recovery is 109.2%, 93%, and 96.75% for the specimens loaded in compression, flexure, and torsion, respectively. By assumption, the strength recovery is the ratio of the peak values of the stress or moment in the healed and the pristine specimens.Further implementation of the employed numerical procedure (as a design tool) requires extensive experimental work. The identification of the model parameters, specifically of the critical value of the damage variable, is inherently empirical. Presumably, the aforementioned critical value of the damage variable is directly related to the crack opening and, thereby, the healing process initiation.The damage-based constitutive relation slightly differs from that implemented in recent research. For example, in , the tenVery detailed modeling at the mesoscopic scale has been proposed by . To modeAlthough the self-healing of cracks emerges as a technique capable of enhancing the durability and sustainability of structures, it still possesses some limitations. The major one is that self-healing results in only a partial regaining of the performances of the undamaged material. According to the vast majority of literature sources, the healing rate remains less than 100%. For comparison, alternative techniques for refurbishment, such as strengthened with externally bonded composite materials, provide a net increase in the load-carrying capacity and stiffness. Also, engineered self-healing implies taking actions during construction . In this context, post-strengthening appears to be more flexible for existing structures, especially not equipped for self-healing. On the other hand, when programmed, the engineered self-healing is expected to run as an autonomous process presuming less maintenance and repairs (during the life cycle of a structure).Despite some shortcomings, the self-healing of cracks remains a field of active research because of the attractive perspectives. Besides autonomous healing, self-healing includes mechanisms inherent for concrete. Accurate quantification of the natural phenomena in the concrete leading to its longevity will contribute to a more efficient design of sustainable structures.Numerical simulations of the self-healing process in plain concrete specimens have been presented and discussed. Results obtained by finite element simulations of a compression test, tension by flexure test, and torsion test for healed and pristine specimens of the same geometry have been compared to predict the recovery in strength.Both material degradation and healing are modeled implicitly in the framework of the continuum damage mechanics. The numerical algorithm integrates into the finite element analysis an original procedure designed to simulate the strain-softening concrete response and extended to account for the effects of healing. The computational procedure uses only the finite element method. It does not include any additional computational scheme to decouple effects due to different mechanisms . The framework of the continuum damage mechanics is preferred to that of fracture mechanics as it enables modeling of crack propagation and initiation in an initially homogeneous and isotropic material . Numerical results reproduce the response of the pristine specimens well, whereas the estimated recovery in strength for some tests is unexpectedly high, accounting for the empirical data provided by previous  research works. In this context, a forthcoming experimental campaign can provide additional empirical background for the model validation and the identification of the material constants.The presented study considers only the healing of specimens subjected to a quasi-static loading. Simulations of the behavior of structural elements under various loading conditions are forthcoming.