Immunoglobulin G4-related disease (IgG4-RD) is characterized by the formation of inflammatory lesions with fibrosis and infiltration of IgG4-positive plasma cells and lymphocytes in various organs of the body. Since the first report of IgG4-related autoimmune pancreatitis, IgG4-RD affecting various organs has been reported; however, only a few reports of IgG4-related lung disease (IgG4-RLD) exist. In this report, we describe a case of IgG4-RLD that was difficult to differentiate from malignancy, and the usefulness of the surgical approach in determining the appropriate diagnosis and treatment plan.18F-fluorodeoxyglucose-positron emission tomography revealed a mass lesion with a maximum diameter of 5.5 cm, maximum standardized uptake value (SUVmax) of 11.0, and areas with high SUV in the hilar and mediastinal lymph nodes. We suspected lung cancer or malignant lymphoma and performed a thoracoscopic lung biopsy to confirm the diagnosis. Histopathological examination revealed no malignant findings, and IgG4-RLD was diagnosed. One month after treatment with prednisolone (PSL), the tumor had shrunk, but a CT scan during the third month of PSL treatment revealed multiple nodular shadows in both lungs. Considering the possibility of malignant complications and multiple lung metastases, we performed thoracoscopic partial lung resection of the new left lung nodules to determine the treatment strategy. Histopathological examination revealed no malignant findings in any of the lesions, and the patient was diagnosed with IgG4-RLD refractory to PSL monotherapy.A 61-year-old man was referred to our hospital after a chest radiograph revealed an abnormal chest shadow. At the time of his first visit, he had a slight fever and dyspnea on exertion. Chest computed tomography (CT) revealed a middle lobe hilar mass with irregular margins and swelling of the right hilar and mediastinal lymph nodes. These findings were not present on CT 1.5 years ago. IgG4-RLD refractory to PSL monotherapy showed changes from a solitary large mass (pseudotumor) to multiple nodules on chest CT. It was difficult to distinguish malignancy from IgG4-RLD based on imaging tests and blood samples alone, and the surgical approach was useful in determining the appropriate diagnosis and treatment plan. Since autoimmune pancreatitis with high serum immunoglobulin (Ig) G4 concentrations was reported in 2001, the new disease concept of IgG4-related disease (IgG4-RD) has been widely recognized [18F-fluorodeoxyglucose-positron emission tomography (FDG-PET)/CT revealed a mass lesion with a maximum diameter of 5.5 cm and maximum standardized uptake value (SUVmax) of 11.0, and high SUV areas in the hilar and mediastinal lymph nodes -2R was elevated to 536 U/mL . Electrocardiography and echocardiography revealed no abnormalities. Chest computed tomography (CT) showed a middle lobe hilar mass with irregular margins and swelling of the right hilar and mediastinal lymph nodes  were biopsied; however, intraoperative rapid pathological examination revealed no malignant findings. Dissection of the interlobar pulmonary artery was impossible due to severe interlobar adhesions, and a right total pneumonectomy was necessary to completely resect the primary lesion. Since it would be over-invasive to perform a total right pneumonectomy in a patient with an undetermined diagnosis, we decided to terminate the surgery and determine the treatment plan based on the results of the histopathological examination. The duration of the surgery was 256 min, and total blood loss was 80 mL. No postoperative complications were observed.Hematoxylin–eosin (HE) staining showed an inflammatory cell infiltrate consisting of plasma cells, lymphocytes, and neutrophils. Storiform fibrosis was observed using silver impregnation. Immunohistochemical staining showed that there were approximately 40 IgG4-positive cells per high-power field (HPF), and the IgG4/IgG ratio was approximately 20% Fig. .Fig. 3MiBased on the above findings, the patient was diagnosed with IgG4-RD occurring only in the lungs, and treatment with intravenous prednisolone (PSL) 60 mg/day was initiated. After 1 month of treatment with PSL, the mass had shrunk, and improvement in fever and cough symptoms was observed. The steroid dose was gradually reduced and changed to 30 mg/day orally in the third month after the start of therapy. During this period, there were no PSL administration errors, and oral administration was accurate. However, a CT scan during the third month of PSL treatment (30 mg/day) showed multiple nodular shadows in both lungs . Extensive pleural adhesions were observed in the thoracic cavity, but the adhesions were sufficiently detachable compared to right-sided surgery. After detachment of the adhesions, the surgery was completed with partial lung resection of the lower lobe. The surgery duration was 126 min, and the total blood loss was 10 mL. No postoperative complications were observed.HE staining revealed a dense inflammatory cell infiltrate consisting of plasma cells, lymphocytes, and neutrophils. Immunohistochemical staining showed that there were approximately 40 IgG4-positive cells per HPF, and the IgG4/IgG ratio was approximately 70% Fig. . There wThus, we diagnosed IgG4-RLD refractory to PSL monotherapy.IHC score, 0 points for serum IgG4, and 4 points for organs, for a total of 22 points. Since this case met the diagnostic criteria of > 20 points, the patient was diagnosed with IgG4-RD. Epidemiologically, it is more common among middle-aged and older men and varies from systemic to single-organ diseases. According to previous reports, imaging findings such as consolidation, ground-glass opacity, nodular shadows, and septal thickening are often seen in the peripheral lung fields, and abnormalities are often seen just below the pleura on chest CT. Fibrosis has been observed but is less common. The presence of fibrosis may be accompanied by honeycombing and traction bronchiectasis [IgG4-RD is a relatively new disease concept that was historically proposed based on observations of autoimmune pancreatitis and Mikulicz’s disease with high IgG4 levels in the serum , 2. Thisiectasis .In our case, FDG-PET/CT scans showed a mass lesion measuring 5.5 cm in size in the middle lobe with an SUVmax of 11.0, and high SUV areas in the hilar and mediastinal lymph nodes. IL-2R levels were elevated, and it was difficult to differentiate malignant tumors from benign lung disease based on preoperative examination. Thoracoscopic lung biopsy was performed, which led to the diagnosis of IgG4-RLD with minimal invasion. Intraoperative findings were characterized by firm adhesions with loss of normal laminae, which is atypical for cancer invasion, suggesting a severe inflammatory reaction around the tumor. In general, glucocorticoids are effective in the treatment of IgG4-RLD, and it is recommended to start them with an equivalent PSL dose ranging from 20 to 60 mg/day depending on the severity of the presentation .We started treatment with intravenous PSL at 60 mg/day, and after 1 month, the lesion had shrunk, and symptoms, such as fever and cough, had improved. However, when the dose was reduced to 30 mg/day orally in the third month of PSL therapy, multiple nodular shadows appeared in both lungs on chest CT.Previous reports have shown that malignancies tend to be associated with IgG4-RD. Wallace et al. reported that 20 of 125 patients diagnosed with IgG4-RD had a history of malignancy, with a standardized prevalence ratio as high as 2.5 . YamamotBased on the imaging findings of our case, the possibility of multiple lung metastases due to malignant disease was also suspected. To determine the treatment plan, thoracoscopic partial lung resection was performed on the newly appearing multiple nodules in the left lung. Extensive adhesions were also found in the left thoracic cavity, but they were more detachable than the adhesions in the right thoracic cavity. Early surgical intervention after the appearance of the lesions and the fact that the patient was taking PSL medication may have contributed to the mild adhesions. We found that IgG4-RLD refractory to PSL monotherapy showed shade changes from a solitary large mass (pseudotumor) to multiple nodules on chest CT. In addition, since the mass in the middle lobe was shrinking and the new shadow was not a malignant metastasis, the possibility of a malignant tumor was almost completely ruled out.It has been reported that the combination of immunosuppressants and steroids is effective in treating IgG4-RLD refractory to PSL monotherapy , and we We encountered a patient with IgG4-RLD refractory to PSL monotherapy with a change in shade from a solitary large mass (pseudotumor) to multiple nodules on chest CT. It was difficult to distinguish malignancy from IgG4-RLD, and the active surgical approach was useful in making the diagnosis and determining the treatment strategy.


Type 1 autoimmune pancreatitis (AIP) is categorized as an IgG4-related disease (IgG4-RD), where a high concentration of plasma IgG4 is one of the common biomarkers among patients. IgG Fc-glycosylation has been reported to be potential biosignatures for diseases. However, human IgG3 and IgG4 Fc-glycopeptides from populations in Asia were found to be isobaric ions when using LC-MS/MS as an analytical tool. In this study, an analytical workflow that coupled affinity purification and stable isotope dilution LC-MS/MS was developed to dissect IgG4 glycosylation profiles for autoimmune pancreatitis. Comparing the IgG4 and glycosylation profiles among healthy controls, patients with pancreatic ductal adenocarcinoma (PDAC), and AIP, the IgG4 glycosylations from the AIP group were found to have more digalactosylation (compared to PDAC) and less monogalactosylation (compared to HC). In addition, higher fucosylation and sialylation profiles were also discovered for the AIP group. The workflow is efficient and selective for IgG4 glycopeptides, and can be used for clinical biosignature discovery. Autoimmune pancreatitis (AIP) is a distinct form of the chronic fibroinflammatory autoimmune disorder of the pancreas . AIP canAlthough the serum IgG4 level is commonly used as a biomarker for AIP and IgG4-RD, the measurement of the serum IgG subtype also suffers from several limitations regarding the diagnosis and assessment of disease activity. Approximately 20% of patients with AIP have a serum concentration of IgG4 that is within normal limits . ElevateGlycosylation is one of the most abundant post-translational modifications. Immunoglobulin glycosylation controls effector functions of antibodies . It has Liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS/MS) with affinity purification is a suitable method for IgG glycosylation profiling. This method provides a high resolution and selectivity for many glycan isomers . Our preThe IgG4 Fc-glycosylation profile and its role in AIP or IgG4-RD should be further explored. However, human IgG3 and IgG4 Fc-glycopeptides from Asian populations were found to be isobaric ions, which created analytical challenges by LC-MS/MS . TherefoTo optimize the volume of bead slurry for human IgG4 purification, 10 to 60 µL of bead slurry were conditioned for evaluation. An elevated serum IgG4 from patients with IgG4-RD is anticipated, and a sufficient bead volume and binding capacity were made available. Serum samples from patients with AIP were pooled, and 10 µL of pooling serum was used for each condition to mimic clinical sample with high concentrations of IgG4. The peak area of the IgG4 surrogate peptide was used for comparison, and the results showed that 20 µL of bead slurry was sufficient to purify IgG4. When the bead volume was tested, 20 µL, 30 µL, 40 µL, and 50 µL of bead slurry showed comparable performances, but the signal slightly decreased at the 60 µL condition A. The deTen microliters of the AIP pooling serum was used to optimize the suitable time for IgG4 purification. The serum sample was spiked into a microtube with 140 µL of PBS and the conditioned beads. The samples were incubated for 0.5, 1, 2, and 3 h at room temperature and for 16 h at 4 °C by using a bench-top mixer B. By comThe wash step was optimized for affinity purification. Initially, PBS was the only wash solution to reduce the non-specific binding of contaminants. The responses of four surrogate peptides from IgG1, IgG2, IgG3, and IgG4 were monitored by our previous published method . We founWe designed an additional experiment to investigate the co-purification of IgG2 by using CaptureSelect IgG4 affinity beads. Two samples were prepared by mixing IgG2, chicken serum, and PBS to mimic serum samples with 4.8 μg/μL of human IgG2. In one of the samples, we added IgG4, which mimicked the serum sample with both 4.8 μg/μL of human IgG2 and 0.6 μg/μL of IgG4. After incubating the samples at 37 °C for 1 h, they were processed with the developed sample preparation method prior to UHPLC-MS/MS analysis. The results showed that, when there was no IgG4 in the sample, a low amount of IgG2 was detected due to non-specific binding on the beads. Furthermore, the peak area of IgG2 was increased by approximately three times with the presence of IgG4 (data not shown). Based on the results, we assumed that there might be a molecular interaction of IgG4 and IgG2, which enhanced the co-purification of IgG2 from the sample. However, IgG2 affinity beads are not currently commercially available for further investigations. In this study, on-bead trypsin digestion was adopted without the need for acidic protein elution or sample matrix replacement prior to digestion. After reducing the disulfide bonds with DTT and alkylating the cysteines with IAA, 5 µL of trypsin (200 ng/µL) was added to each sample. Four conditions, including 0.5, 1, 2, and 16 h at 37 °C (300 rpm), were tested for comparison. In Reversed-phase liquid chromatography is an efficient and effective technique for the separation of glycopeptides and peptides. The triple quadrupole mass spectrometer (TQMS) provides sensitivity and selectivity, and has been used for biomolecules detection from complex samples. In this study, UHPLC-MS/MS was chosen as the analytical platform. Method-specific parameters and performances were evaluated.A narrow-bore column packed with the C18 stationary phase was used for glycopeptides/peptides separation. The column length is 5 cm and the internal diameter is 2.1 mm. The mobile phase comprised water and acetonitrile, with the addition of formic acid to a final concentration of 0.1%, and gradient elution was conducted during the analysis. The presence of an acidic additive facilitates the ionization efficiency of target analytes in the electrospray ionization source. Detailed conditions of UHPLC can be found in −1) were incubated with conditioned affinity beads. After sample incubation, the beads were washed with PBS and PBS with 0.1% Tween 20, and the on-bead digestion was conducted by adding sufficient trypsin enzyme (37 ℃). The enzymatic reaction was stopped after 16 h by adding formic acid, and the tryptic peptides in the supernatant were carefully collected. The supernatant was further diluted with 0.1% formic acid four times prior to UHPLC-MS/MS analysis , patients with pancreatic ductal adenocarcinoma (PDAC), and patients with autoimmune pancreatitis (AIP). IgG4 concentrations determined by UHPLC-MS/MS were statistically higher in the AIP group . After cCompared to the methods in our previous publications, the current workflow can determine more IgG4 glycopeptides. Sample preparation conditions, such as the volume of bead slurry, incubation time, wash steps, and trypsin digestion time, were carefully optimized for clinical application. A stable-isotope-labeled IgG4 IS was added to compensate for the variations during sample preparation and UHPLC-MS/MS analysis.Konno N et al. have used a sequential purification method for IgG4 glycosylation study in which Melon gel was firstly adopted for total IgG purification followed by IgG4 affinity beads. In their study, purification was conducted in columns that were packed with IgG4 affinity beads, and 23 glycans released from IgG4 were analyzed by using MALDI-TOF MS . In contHu C et al. used a lectin array to investigate the IgG4 glycosylation profiles for IgG4-RD. In their study, IgG4 from IgG4-RD patients showed distinct patterns compared to disease and healthy controls, and the levels of mannose and GlcNAc were highlighted as being worthy for further investigation . Culver There is evidence that endogenous IgG with more fucosylation and sialylation has anti-inflammation biological functions . HoweverOne of the limitations of this study is the small sample size; therefore, each group was only assigned 15 subjects. Although the purification and sample preparation steps are efficient, we might miss some of the minor glycans on the IgG4 Fc region, which were detected by the glycomics method .Protein standards of natural human IgG2 and IgG4 were purchased from Abcam . The protein internal standard used in this study was SILu™MAB K4 Stable-Isotope-labeled universal monoclonal antibody, which was bought from Sigma-Aldrich . Ammonium bicarbonate (ABC), formic acid, dithiothreitol (DTT), and iodoacetamide (IAA) were bought from Sigma-Aldrich . Chicken serum and CaptureSelect IgG4 (Hu) affinity matrix were purchased from Thermo Fisher Scientific . Trypsin enzyme was purchased from Promega . Acetonitrile (ACN) was obtained from J.T. Baker . Phosphate-buffered saline (10×) was purchased from VWR International LLC .−1) was added to each sample solution. The IgG4 purification was conducted at room temperature for 30 min with a desktop mixer. After incubation, the supernatant was removed, and the beads were washed with PBS once, 0.1% Tween 20 in PBS twice, followed by PBS again to decrease non-specific binding. Fifty microliters of 50 mM ABC were added to the microtube, and 1 μL of 550 mM DTT was added to the solution to disrupt the disulfide bonds (56 °C for 45 min). The solution was then treated with 2 μL of 450 mM IAA as an alkylating reagent at room temperature for 45 min in the dark. For protein digestion, 10 μL of trypsin (200 ng μL−1) was spiked and the digestion was conducted at 37 °C for 16 h (300 rpm). At the end of on-bead digestion, formic acid was added to stop the tryptic digestion. The supernatant was collected after centrifuging the sample and was further diluted with 0.1% formic acid 4 times before UHPLC-MS/MS analysis.Forty microliters of CaptureSelect IgG4 (Hu) affinity matrix were conditioned with PBS. After conditioning twice, 10 μL of human serum was mixed with 140 μL of PBS and affinity beads. In addition, 10 μL of SIL-IS IgG4 . The separation column used in this study was Core-Shell C18 Kinetex column . The mobile phase was composed of 0.1% formic acid in water (solvent A) and 0.1% formic acid in ACN (solvent B). Column temperature was set at 40 °C and the flow rate of mobile phase was set at 0.3 mL minY-axis to generate calibration curves. Linear regression was used to construct the equation for each analyte. Precision and accuracy were evaluated at different IgG4 spiked concentrations. The intra-batch and inter-batch repeatabilities were calculated and performed as % RSD for both retention times and peak area ratios. Accuracies were calculated as the percentage of recovery by comparing the experimental concentrations to the theoretical ones. For glycopeptides, spiked protein concentrations were used for accuracy evaluation as well. Stabilities of IgG4 were evaluated by comparing the signals from samples kept at −20℃ for 7 and 15 days to the fresh prepared one. The stability of surrogate peptide in autosampler was evaluated at 6℃ for 24 h.Calibration curves were generated by adding a serial concentration of IgG4 standards into chicken serum, as indicated in n = 15/group). This study was approved by the research ethics committee of National Taiwan University Hospital (201301048RIND) and Taipei Medical University–Joint Institutional Review Board (N201704064). The signed informed consent forms were received from all subjects.Clinical samples were collected from patients with AIP, PDAC, and healthy controls to investigate the differences in IgG4 subclass concentrations and the Fc-glycosylation profiles . Before calculating the glycosylation profiles (%), the response of each glycopeptide was divided by the sum of responses from all glycopeptides to obtain its percentage value. Different glycosylation profiles were then calculated based on the following Equations (1)–(7). To compare the 15 glycopeptides among the three groups, the responses of glycopeptides were normalized with their respective IgG4 response to prevent the effect of different protein amounts from samples. Statistical analysis was conducted in GraphPad Prism .In this study, we have carefully optimized sample preparation for IgG4 purification and on-bead tryptic digestion. SIL-IgG4 IS was used to achieve a more accurate and precise analysis. The IgG4 serum concentration and the 15 IgG4 glycopeptides can be determined by using the developed UHPLC-MS/MS method. We employed this workflow to generate IgG4 glycosylation profiles from healthy controls, patients with PDAC, and patients with AIP. Higher fucosylation and sialylation profiles were found in the AIP group. In addition, AIP was found to have more digalactosylation compared to the PDAC group. Our workflow provides an efficient way for IgG4 Fc-glycosylation profiling. It has the potential to be applied to other biological investigations.


Using computer simulations, we have shown that such a mechanism is the competition of two oppositely directed processes: deformation of the pore space, which leads to an increase in pore pressure; and pore fluid flow. The balance of these processes can be effectively characterized by the Darcy number, which generalizes the notion of strain rate to fluid-saturated material. We have found that the dependence of the compressive strength of high-strength concrete on the Darcy number is a decreasing sigmoid function. The parameters of this function are determined by both low-scale (capillary) and large-scale (microscopic) pore subsystems in a concrete matrix. The capillary pore network determines the phenomenon of strain-rate sensitivity of fluid-saturated concrete and logistic form of the dependence of compressive strength on strain rate. Microporosity controls the actual boundary of the quasi-static loading regime for fluid-saturated samples and determines localized fracture patterns. The results of the study are relevant to the design of special-purpose concretes, as well as the assessment of the limits of safe impacts on concrete structural elements.It is well-known that the effect of interstitial fluid on the fracture pattern and strength of saturated high-strength concrete is determined by qualitatively different mechanisms at quasi-static and high strain rate loading. This paper shows that the intermediate range of strain rates (10 The pore fluid is reported to be responsible for numerous phenomena of premature fracture of various brittle materials (including concretes) under loads significantly lower than permissible for these materials in a dry state ,15,16,17At low strain rates corresponding to a quasi-static loading mode, the pore fluid typically has a negative effect on the strength of concrete. This effect most clearly appears under compression, which is the typical type of stress state of elements of concrete constructions. Different experimental studies show that the compressive strength of concrete samples can decrease by 10–20 percent with an increase in water saturation ,19,20. MThe key factor determining the described decrease in strength is pore pressure  ,20. VaridefT is the timescale of skeleton and pore space deformation) to the pore fluid flow rate  [Due to the connectivity of the pore space of concrete, the rate of pore fluid pressure growth during compression depends on the rate of loading. Indeed, the key physical property of water that qualitatively distinguishes it from a solid-phase skeleton is mobility (fluidity). An increase in pore pressure in a sample under compression leads to a redistribution of free  water in a system of interconnected material discontinuities . Fluid flow provides redistribution and balancing of local stresses in a fluid-saturated material. Moreover, the difference in fluid pressure between the central part of the compressed sample and the outer surfaces causes the outflow of “excess” pore fluid and a decrease in the overall stress level. It is clear that the magnitude of the pore pressure gradients for a particular concrete is determined by the strain rate . Therefore, the mechanical effect of interstitial water pressure on the stress state and mechanical characteristics of concrete as a porous material should depend on the ratio of the rate of skeleton straining (characterized by applied strain rate  TDarcy) ,25.−4 s−1 for typical water-saturated concrete materials). Within this strain rate interval, the pore fluid has time to redistribute in the sample (and outflow). Therefore, the fluid flow compensates for the compression of the pore space, and the pore pressure slowly increases during loading. Mechanical characteristics of water-saturated concrete gradually tend to that of dry samples with the loading decrease rate [1 s−1). In this strain rate interval, the flow of pore fluid is negligible due to the inertia of the flow process, and the pore pressure increase rate tends to a maximum. However, the expected maximum negative effect of the pore fluid on the strength of concrete does not occur, since the dynamic  properties of the solid-phase skeleton and the viscous properties of the pore fluid play a leading role here. In particular, the incubation time of damages and cracks in the solid-phase skeleton of concrete at high strain rates becomes comparable (in order of magnitude) with the characteristic loading time [2 s−1 the DIF (dynamic increase factor) value approaches 2 [1 s−1. These viscous forces are responsible for a strong positive influence of pore fluid on dynamic strength at high strain rates. This effect is physically interpreted as an increase in fracture incubation time [There are two “limiting” ranges of the control parameter ase rate . The secing time ,26. The time [ε˙ ,31,32,33oaches 2 ,31. Poreoaches 2 ,22,34, Soaches 2 ,36, and oaches 2 ,22 forceion time .−4 s−1 < 1 s−1). The mechanical behavior of water-saturated concretes and the strain rate dependence of strength within this interval have distinctive features. In this “intermediate” region, the rate of growth of pore pressure during loading becomes comparable and exceeds the characteristic rates of filtration of pore fluid but the role of inertia and viscous factors are still small or even negligible. Therefore, fracture pattern and compressive strength of water-saturated concrete are largely determined by a dynamic factor characterizing the balance of the rates of two competing processes: applied deformation (pore compression) and pore fluid flow.The vast majority of studies are focused on studying fracture patterns and determining the strength characteristics of water-saturated concretes at the two described “limiting” intervals of strain rate. At the same time, they are connected by a wide but much less studied “intermediate” interval (10−4 s−1 < 1 s−1). The relevance of such an analysis is particularly determined by the fact that many dynamic impacts on underwater structures during operation  oExperimental studies are able to provide comprehensive information about the nature and characteristics of the macroscopic fracture of concrete samples and their strength parameters. At the same time, the possibilities of laboratory experiments for analyzing the nucleation and evolution of mesoscale damages and cracks, the role of the interaction of constituent phases, and their contributions to fracture are limited. Mesoscale computer modeling is an efficient tool for such research. The key advantage of virtual (computer) study is the ability to qualitatively and quantitatively determine the contributions of the structural parameters and the pore fluid to the change in the fracture pattern and dynamic strength of concrete samples.The traditional way is to use a powerful continuum-based finite element method (FEM) with the accurate implementation of mesoscale geometries, nonlinear material models including the behavior of interface zones, and pore fluid infiltration. Thilakarathna et al.  developeAn attractive alternative is the use of particle-based (“discrete”) numerical methods. These methods are based on representing material as an ensemble of finite-sized particles that can change the state of bonding with neighbors (bonded-to-unbonded transition) and hence change the neighborhood. The formalism of these methods is therefore not limited by continuity constraints. The key advantage of discrete methods is the inherent ability to model discontinuity formation as an extremely localized process by means of separating the surfaces of adjacent particles. Several discrete methods deserve special attention in the context of an adequate description of the failure of concretes and other brittle porous materials in the range of scales. The Lattice Discrete Particle Model (LDPM) developed by G. Cusatis et al. represents concrete as an ensemble of polyhedral aggregates implemented in a matrix as a binding medium ,53 and c−4 s−1 < 1 s−1) between quasi-static and high rate loading ranges.The present paper is devoted to the study of the features of dynamic fracture and strain rate dependence of compressive strength of fluid-saturated high-strength concrete through computer simulation of uniaxial compression of mesoscale samples. The purpose of this investigation is to reveal and generalize the regularities of change in fracture pattern (including mesoscopic mechanisms) and compressive strength of fluid-saturated high-strength concrete within the practically important “intermediate” interval of strain rates . The initial value of the pore pressure (before loading) was assumed to be equal to atmospheric pressure. The mesoscale here means the characteristic scale of the key elements of the internal structure. These are aggregates of millimeter linear dimensions and “micropores” with characteristic dimensions of hundreds of micrometers. The mesoscopic structure of concrete consists of a cement stone (mortar) matrix with large hardening aggregates . The typical form of aggregate particles is equiaxed, the average size of the inclusions is 10–15 mm.−2 µm to 101 µm. This is determined by various factors including the conditions for the preparation and hardening of concrete as well as the water-to-cement ratio. A decrease in the characteristic transverse size of capillary channels is a consequence of the deposition of cement hydration products in the pore channels during concrete hardening. Hydration product deposition can lead to multiple narrowing of capillary characteristic opening and a decrease in cement permeability by several orders of magnitude [Porosity is among the key structural parameters of cement stone as it largely influences the effective strength and elastic properties. Typical high-strength concrete is characterized by a two-scale pore structure (capillary pores and micropores). The capillary porosity of the cement stone reaches ~6 vol.%, while the volume fraction of micropores is up to 4% ,67,68. Tagnitude ,70. The These principal features of the multiscale structure were taken into account in a developed two-dimensional computer model of a representative mesoscale volume of high-strength concrete. The model includes two spatial scales: mesoscopic and microscopic. The mesoscale components of the internal structure of concrete  are specified explicitly .Spatial placement and determination of the geometry of particular aggregates in the model samples were carried out using the Voronoi tessellation algorithm . The volThe fluid in the micropores of the cement stone was described effectively. Each micropore was characterized by the mass, density, and pressure of the liquid in its volume (we used the approximation of the equipartition of the liquid density in the micropore). The volume of a micropore changes during loading. The current value of a micropore volume during the simulation is calculated using the special algorithm described in .k was assumed to be linearly related to the capillary porosity φ (φ = 6% in this study) and the characteristic transverse size (effective diameter) of the capillary pore channels chd by the known relation: k = φchd2 [chd in the range from 0.1 to 1 μm to analyze the effect of the degree of deposition of cement hydration products in the capillary pore space on the dynamic mechanical behavior of fluid-saturated concrete. Capillary porosity was assumed to be connected with micropores.The internal pore structure of the cement stone at lower scales was taken into account effectively. It was characterized by capillary porosity and permeability. Capillary permeability of the cement stone  = φdch2 . We variThe fluid in the volume of capillary pores was taken into account effectively and was characterized by local density and pore pressure.3, bulk modulus flK = 2.2 GPa.The mechanical characteristics of water were used for pore fluid: density at atmospheric conditions Basalt was considered an isotropic elastic-brittle material. The cement stone was assumed to be an isotropic elastic-plastic material . We use the continuum model of plasticity of microscopically brittle solids (non-associated plastic flow law with two-parameter Mises–Schleicher yield criterion) to describe inelastic strain accumulation in cement stone on the mesoscale. Linear strain hardening was assumed for cement stone . The values of the mechanical characteristics of both components  were taken from  and are Basalt and cement stone are brittle materials. The shear strength of such materials is sensitive to the value of normal/volumetric stress. We use the two-parameter Drucker–Prager fracture criterion to describe local fracture of both components and matrix-aggregate interfaces in the numerical model. The parameters of this criterion are uniaxial compressive strength (UCS) and uniaxial tensile strength (UTS). The mechanical behavior of the model concrete samples under loading was modeled using the method of homogeneously (simply) deformable discrete elements ,74. The i, im is mass of the element, iJ is a moment of inertia of an equivalent disc/ball, i with the neighboring element j, and Each discrete element models some material volume . The dynamics of the simulated sample is determined by the numerical solution of the Newton–Euler system of equations of motion for translational and rotational degrees of freedom of the elements. The authors use the most common approximation in DEM, which applies the equivalent disk/ball approximation to the description of dynamics of a discrete element. This approximation means that the equation of motion of an element is formulated in a simplified form corresponding to a disk  or a ball (in a 3D case) ,76,77 (EijS (Equation (2)):i and j and The central and tangential potential forces , i, α,β = x,y,z (XYZ is laboratory coordinate system), cosθij,α is the projection of the unit normal vector iN is the number of neighbors of the element. The components of the average strain tensor i can be calculated as a superposition of normal and tangential pair strains by analogy with Equation (3) or incrementally with the use of the material’s constitutive law and calculated average stresses. The latter way is used in the present implementation of the method.The main advantage of the method of homogeneously deformable discrete elements in comparison with the classical formalism of rigid (non-deformable) discrete elements is the fact that they take into account the change (both elastic and irreversible) of the shape and volume of an element under loading. Within the framework of this method, stresses and strains in the volume of an element are assumed to be uniformly distributed and characterized by tensors of averaged stresses and strains. The components of the average stress tensor in the discrete element eighbors ,74 , ijq and jiq are the distances from the mass centers of the interacting elements i and j to the central point of the contact area (ijq = iR and jiq = jR for unstrained elements), εij and εji are central pair strains of discrete elements i and j, ij and γki are the shear angles of discrete elements i and j . Equations (4) and (5) take into account the necessity of satisfying Newton’s third law (σij = σji and τij = τji).The relations for the forces of element–element interaction are formulated in the general many-body form (Equations (4) and (5)):The interaction forces are used in the definition of average stresses and, in turn, are determined by the values of local stresses. This relationship makes possible the implementation of various models of elasticity, plasticity, and fracture (including those with multiparameter criteria) within the framework of the formalism of discrete elements.The modeled high-strength concrete is a composite material and consists of two components: non-porous basalt aggregates and porous cement stone matrix. Micropores are implemented by removing discrete elements from the generated non-porous model sample .For modeling fluid-saturated matrix by the method of homogeneously deformable discrete elements, we use a coupled formalism . Within G and K are the shear and bulk moduli of dry material, δαβ are Kronecker delta, poreP is the pressure of fluid in the pore space of the element, and a = (1–K/sK) is the poroelasticity coefficient in the Biot’s model . Corresponding relationships for the response forces of element i to the action of the neighbors j are formulated as follows (Equation (7)):iG, iK, ia, and iporeP are corresponding parameters for the element i.The elastic response of a permeable discrete element modeling a fragment of a cement stone with interstitial fluid is described using Biot’s linear model of poroelasticity ,79 , and The inelastic behavior of mesoscale volumes of cement stone under loading is mainly due to the occurrence, accumulation, and growth of micro- and nanoscale damage. We applied the non-associated plastic flow law with the Mises–Schleicher yield criterion (Nikolaevsky’s model) ,63,80 toi, all central and tangential forces of the response of the element i to the impact of the neighbors j are scaled using a ratio similar to Equation (11) (Equations (13) and (14)):i.Nikolaevsky’s model is implemented within the explicit numerical scheme of solving motions Equation (1) using the well-known radial return algorithm of Wilkins . The essThe unified hardening curve for the cement stone is defined by the dependence found in ,63.β = σc/σt, σc, and σt are the values of UCS and UTS for dry materials. Numerical implementation of this criterion in the discrete element method is based on determining the local stress tensor on the contact area of the linked pair of elements and calculating its invariants [The discrete element model of a concrete sample in an initial (unstrained) state assumes that all neighboring elements are chemically bonded . Local fracture under loading was modeled by breaking the chemical bond between the elements when the specified fracture criterion is satisfied ,63. We avariants . These iporeP = 0. Fracture of interfaces (the pairs of elements with one element modeling cement stone and the other modeling aggregate) was considered using the Drucker–Prager criterion with the material parameters σc and σt corresponding to cement stone.Basalt is assumed to be a non-porous, elastic-brittle material. Its mechanical behavior was described using the generalized Hooke’s law Equations (6) and (7) and the Drucker–Prager fracture criterion Equation (15) at k is the capillary permeability of the element, η is the fluid viscosity, τr is the relaxation time, and The pore pressure gradient was considered the driving force of liquid redistribution in the capillary pore space of the cement stone. Filtration was described by numerically solving the equation of fluid transport . We usedelements .2 (R is discrete element radius), and pore pressure equal to the real pressure of fluid in the micropore. The outflow of fluid from the sample into the surrounding space  or into new internal voids resulting from the formation of damages/cracks and damage/crack surfaces separation is calculated in a similar way.Fluid transport between the micropore and surrounding discrete elements of the cement stone  is calculated similarly to the calculation of the transport of pore fluid between adjacent discrete elements, namely with the use of Equation (16). In the numerical solution of Equation (16) for the pairs “micropore–surface discrete elements”, the micropore is replaced by a virtual discrete element with porosity 1, permeability 4Rzz = σxz = σyz = 0, where XY is the plane of deformation). In some respects , this corresponds to the conditions of a real experiment on concrete cubic samples.We numerically studied the effect of free pore water on the value of dynamic strength and fracture of 2D mesoscale samples of high-strength concrete under uniaxial compression. 2D concrete samples were modeled in the plane stress approximation . The samples were of square form. The “basic” value of the sample size (side length) was 10 cm.V  surfaces in the horizontal direction to fulfill the boundary condition σxx = σxy = 0.We modeled uniaxial compression of the samples at a constant loading velocity V . The lowSide surfaces of the sample were stress-free and hydraulically open to provide the possibility of a free outflow of fluid from the sample.3 and atmospheric pressure, respectively. The space surrounding the sample was filled with the same fluid at atmospheric pressure, which was supported constantly throughout the loading.The initial (before loading) values of the pore fluid density and pressure in all capillary pores and micropores were equal to 1000 kg/myV, the permeability k of the capillary system in the cement stone (it characterizes the leak-off capacity of concrete), the size of the sample W, and the dynamic viscosity of the pore fluid η . We varied the strain rate −3 s−1 to 5 s−1), the viscosity of the fluid η within an order of magnitude relative to the viscosity of saline water (2·10−4 Pa·s to 3·10−3 Pa·s), the permeability k within two orders of magnitude (from 6·10−16 m2 to 6·10−14 m2), and the sample size W within four times (from 5 cm to 20 cm).To vary the balance of the processes of deformation of the pore space of concrete and pore fluid flow, we modeled uniaxial compression at different values of the loading velocity We measured the compressive strength of a sample as the peak value of sample resistance force to the upper surface divided by the end surface area. The fracture pattern was analyzed qualitatively and quantitatively using the information about the spatial distribution and the number of broken bonds in the matrix, aggregates, and interfaces.In this work, we consider the range of strain y sample . As the y sample ,63.W is a characteristic system lengthscale, (P2 − P1) is pore pressure difference at the ends of this lengthscale (pressure gradient is driving force for fluid flow), η is fluid viscosity, and k is the material permeability. Note that the given relation for Darcy’s time scale is derived by the finite-difference approximation of Darcy’s law. The ratio of these times (dimensionless Darcy number [The competitive influence of compression of the porous skeleton (this leads to an increase in pore pressure) and filtration of pore fluid (this leads to a decrease in pore pressure) can be qualitatively and quantitatively characterized by the ratio of the time scales of these processes. The time scale of the first process can be characterized by the inverse strain rate: y number ,25) charDa can be considered a generalization of the conventional dynamic parameter comp on strain rate has a nonlinear (logistic) profile. We discussed the advantage of using the generalized dependence σcomp(Da) instead of conventionally used σcomp(comp(Da) takes into account the scale factor through the size W and is applicable for various pore fluids and concrete permeability values (at a constant porosity).We were the first to show that the dimensionless parameter nt paper , we showk, and W (the limits of their variation are given above).The present study is focused on revealing and analyzing the relation of the features of this generalized strength dependence to the features and mechanisms of fracture of fluid-saturated concrete. The analysis is based on the simulation results for various values of The role and contributions of different scale pore subsystems (micropores and capillary network). The analysis of this factor was carried out on the basis of the results of modeling samples without micropores (only a capillary network) and with micropores.Inhomogeneity of the structure at the mesoscale. Samples with the different spatial distribution of micropores and basalt aggregates were analyzed, and the intervals of scatter of local dynamic strength (strength of mesoscopic samples) were estimated.Concrete is a heterogeneous composite material with two-scale porosity and mesoscale hardening inclusions. These structural factors should have a significant influence on the value of the dynamic strength and the limits of its change. To identify the contribution of each of them, the following were analyzed:k. To study the effect of cement stone permeability (provided by a connected network of capillary pore channels) on the compressive strength of concrete, we first simulated uniaxial compression of model samples with cement stone containing only capillary porosity (no micropores). The typical initial structure of such samples corresponds to that presented in The considered high-strength concrete has two-scale porosity but the characteristic fluid flow rate is mainly determined by the permeability of the capillary pore network k, and W confirm the correctness of the assumption [Da). Note that the difference (P2–P1) in (17) has the meaning of the pressure difference in the center of the sample and on the free side surface in the direction transverse to the loading axis. When applying the parameter Da to uniquely characterize dynamic strength, we should take into account that the pore pressure in the center of the sample (P2) continuously increases during the course of sample compression, while it remains constant and equal to atmospheric pressure (P1 = 105 Pa) on the side surface. Hence, the characteristic value of fluid flow rate continuously increases due to a gradual increase in the pressure difference in the sample. We therefore use an analog of the Darcy number Equation (17) with P2 calculated as Terzaghi’s estimation of maximum reachable pore pressure: 0 is the initial capillary porosity of the cement stone (P2~7 MPa for simulated cement stone). The pressure P1 in Equation (17) is pore pressure on the side surface of the sample (P1 = 105 Pa).The results of modeling the mesoscale samples of fluid-saturated concrete with one-scale (capillary) porosity at various values of sumption  that thek, and W form a unified curve σcomp(Da)  porosity at different values of comp(Da) (curve 1 in The unified dependence σDa < 10) corresponds to such combinations of values of structural parameters, fluid viscosity, and loading rate, which ensure enough time for fluid redistribution in the volume of capillary pore channels and flow out of the sample. Therefore, the pore pressure is at a low level. In this case, the filtration (outflow) of the fluid is an efficient mechanism of stress relaxation in the skeleton, and the strength of the sample is close to the maximum value and changes only slightly. The maximum strength (~64 MPa) is achieved at Da→ 0.Section I (Da > 100) corresponds to the combinations of parameters k, and W, at which the fluid does not have time to redistribute in the capillary network during concrete loading. The sample is actually under undrained conditions, and the pore pressure is close to the maximum achievable value, thus minimizing the strength of concrete as much as possible. Therefore, the concrete strength remains almost unchanged at Da > 10 and is close to the minimum value (~56 MPa).Section III (Da < 100) is transitional, the ratio of strain rate to the rate of pore fluid filtration varies within the above-mentioned limits.Section II of the curve  qualitatively corresponds to the experimentally observed regularities for various water-saturated brittle materials including concretes [It is important to note the changes in the fracture pattern with increasing loading rate characterized by the Darcy number c,d. An ioncretes ,35,36,86To study the contribution of micropores to the strength of concrete, we considered mesoscopic samples with two-scale porosity of cement stone . As notek, and W shows that the strength of water-saturated concrete with two-scale porosity is also a single-valued function of Da. Similar to the case of one-scale porosity, this dependence is well approximated by a sigmoid function (Equation (19)):Analysis of the simulation results at various values of Da.One can see that the effect of microporosity on the change in compressive strength of concrete is manifested in the entire considered range of variation of the parameter First of all, it is important to mention a significant overall decrease in the value of dynamic compressive strength of the samples. The maximum and minimum strength values of fluid-saturated concrete with two-scale porosity  begins at the same characteristic values of the Darcy number as in the case of concrete with a one-scale pore system (Da > 100). In other words, large-scale pores (micropores) provide an increase in the length of the transition stage II by almost two orders of magnitude.At the same time, the dynamics of change in the sample strength from ry pores . The keyFracture of concrete samples with two-scale porosity  is much All fractures in the matrix, including the first damage a,c, destDa. Fracture of aggregates is predominantly at the interfaces  at small Da values, although the number of intraparticle damages, short or through cracks (cracks that pass through the inclusion) is larger than in concrete with only capillary porosity. An increase in Da and a corresponding increase in characteristic pore pressure in the matrix are accompanied by a nearly twofold increase in the number of through intraparticle cracks and a concurrent decrease in the number of small damages in the particles. The number of intraparticle cracks at large Da values is comparable to the number of cracks that propagate around the particles through the interface.We also emphasize the systematic change in aggregate contribution to fracture with an increase in The previous section analyzes the general features of the mechanical effect of pore fluid pressure and pore structure parameters on the dynamic compressive strength of a mesoscopic representative volume of high-strength concrete. Concrete is a strongly heterogeneous material. The heterogeneity of the structure can differ significantly in different mesoscale regions of large-scale (macroscopic) structures due to the features of manufacturing technology. Direct averaging of the strength values of various statistically equivalent mesoscale volumes does not guarantee an accurate estimate of the macroscopic strength of concrete, since macroscopic strength is determined, among other things, by the interaction of adjacent mesovolumes. At the same time, knowledge of the local (mesoscopic) characteristics of concrete is of most importance in many topical practical problems. Examples of such problems are local contact loading of underwater parts of large concrete structures, including the collision of a ship with a concrete berth or dynamic contact load by ice plates during “icequakes”  ,39,87. U3The first series investigated the strain rate dependence of the dynamic strength for five model samples with different spatial positions of micropores. Note that the combined effect of micropores and basalt aggregates on the strength of concrete is generally not a superposition of their partial contributions. Therefore, we analyzed the influence of the spatial arrangement of micropores on the variation in the strength of cement stone (the main component of concrete).4The second series investigated the strain rate dependence of the dynamic strength for five model concrete specimens with different spatial arrangements of aggregates. All concrete samples were characterized by the same spatial position of micropores in the cement stone .The foregoing determines the relevance of estimating the characteristic interval of variation of the local dynamic strength of statistically equivalent mesoscale concrete volumes characterized by the different spatial distributions of structural elements. Variation in the mechanical properties of concrete at the mesoscale is determined by the different spatial arrangements of micropores in cement stone and hardening aggregates. To identify the partial contribution of each of these factors to the dynamic strength of concrete, we carried out two series of calculations:Other mechanical and geometric parameters of the samples, as well as the loading conditions, are the same as those used in the previous section.k, and W shows that strain rate depends on the compressive strength of the representative mesovolumes with different spatial positions of micropores are well approximated by sigmoid functions. The approximating functions for different cement stone samples are almost parallel to each other, and their parameters  differ insignificantly. The sigmoids below correspond to the “limiting” cases (upper and lower boundary curves) (Equations (20) and (21)):The results of modeling uniaxial compression at various values of comp(Da) for the rest of the samples lie within the interval limited by these two “limiting” curves. One can see that the range of variation in the value of the local dynamic strength of the cement stone is nearly constant in the entire range of Da and is ~5%.Equations (20) and (21) are shown in Da, and the change in fracture pattern is determined by the value of Da. At all Da values, the first damages appear on the walls of micropores . The smaller characteristic size of fragments is due to the high pore pressure at large Da values, which leads to a significant increase in shear stresses in the vicinity of micropores and multiple fractures.Samples of cement stone with different spatial arrangements of micropores have a similar pattern of fracture at the same values of cropores a,c becaucropores b,d. NoteTo study the effect of the spatial distribution of aggregates on the mechanical characteristics of mesoscale volumes, we generated five statistically equivalent model samples. All samples have the same arrangement of micropores in the cement stone b. BasaltE is the same for all considered samples (the difference is less than 1%). The numerically obtained value E = 36.8 GPa agrees well with the estimate within the Reuss approximation of elastic moduli CemC is the volume fraction of porous mortar, E is Young’s modulus of the composite, CemE and BasE are Young’s moduli of porous mortar and basalt). The difference between the numerical and analytical estimates does not exceed 2%. This confirms that the considered samples are representative mesoscopic volume elements.The analysis of the simulation results shows that the values of local elastic parameters of concrete are almost independent of the spatial arrangement of aggregates and their geometric details. For example, Young’s modulus The approximating sigmoid functions for different concrete specimens are almost parallel to each other, similar to the above case of cement stone. The sigmoids below correspond to the “limiting” cases (upper and lower boundary curves):comp(Da) for the rest of the mesoscale concrete samples lie within the interval bounded by these two “limiting” curves.Functions (22) and (23) are shown in comp variation interval is about 10% of the strength of dry concrete and does not depend on the value of Da . Note that the intervals of variation of other parameters of the approximating sigmoid are also moderate.The width of the σSummarized results of the numerical analysis of the effect of interstitial fluid on dynamic strength of mesoscopic representative volumes of high-strength concrete are shown in Therefore, the features of the spatial arrangement of micropores and hardening aggregates mainly determine the characteristic level of local strength and insignificantly affect the strain rate sensitivity of concrete. Both types of structural elements are strong mesoscale stress concentrators. Comparison of the relative contributions of micropores and aggregates to the value of the variation interval of the local dynamic strength shows that aggregates are the most significant factor. Their heterogeneous spatial distribution provides a 2–2.5 times wider range of variation in local strength than micropores in the cement stone.−4 s−1 < 1 s−1). We should remember that this interval separates the quasi-static and high strain rate regions. Since the fluid flow rate is controlled by applied strain rate, the dependence of the mechanical parameters (including compressive strength) and fracture features of fluid-saturated concrete samples on the loading dynamics is adequately characterized by the general loading rate parameter (Darcy number). In other words, the control parameter is not simply the strain rate, but the balance of the strain and fluid flow rates.The simulation results showed that the redistribution (flow) of pore fluid in the pore space is a key factor controlling the systematic change of compressive strength and fracture pattern of concrete samples in the “intermediate” interval of strain rates . An increase in Da is accompanied by an increase in pore pressure in cement stone. On the one hand, this determines an increase in the effective stiffness (bulk modulus) of the matrix. On the other hand, this leads to a decrease in the threshold of plasticity and strength. The increase in pore pressure determines the onset of fracture of the matrix at lower values of the applied load and, consequently, a decrease in the uniaxial compressive strength of the sample. At the same time, a decrease in the plasticity threshold and an increase in the effective stiffness of the fluid-saturated matrix lead to a change in the contributions of mesoscopic structural elements to fracture.The open capillary network determines the phenomenon of strain-rate sensitivity of fluid-saturated concrete and logistic form of the compressive strength dependence on the rate parameter (Da corresponding to the three main stages of the dependence σcomp(Da) . The term “damage” hereinafter means a broken bond between two adjacent discrete elements. Da. This is a consequence of an increase in the characteristic pore pressure level. Second, an increase in Da is also accompanied by a moderate (about 30%) increase in the total number of damages in the sample  since the pore pressure in the matrix is close to maximum at this stage. The increase in damage is achieved primarily through matrix contribution (Da (Da) leads to a much more intensive contribution of the “aggregate” mechanism to the fracture of fluid-saturated concrete.First, we have to mention a systematic almost twofold decrease in the value of strain of the beginning of intensive damage accumulation with an increase in e sample a. This ie sample . This efribution b. At thetion  on the loading rate. Large pores (micropores) are strong local stress concentrators and are deformed more strongly than capillary pores. “Extra” fluid passes from micropores into capillary pores during the loading and increases the pore pressure in the capillary pore system of surrounding regions of the matrix. This has implications for both the fracture and strain rate dependence of concrete strength.Da is accompanied by an increase in the effective stiffness of micropores to a much greater extent than that of the surrounding cement stone matrix with capillary porosity. This leads to the fact that micropores become not voids, but inclusions with the stiffness of only an order of magnitude less than the stiffness of a cement stone . Hence, the concentration of deviatoric stresses in the matrix around micropores decreases, and the characteristic magnitude of compressive bulk stress increases. This effect provides a significant and gradual decrease in the total number of damages and contributions of all structural elements with an increase in Da (Da). This is achieved by reducing the number of individual damages and short internal cracks  by two orders of magnitude. In terms of the parameters of the sigmoid function σcomp(Da), this is accompanied by a more than twofold decrease in the exponent and a shift of the median point of the sigmoid to the left by an order of magnitude.Summarizing the above, the fracture pattern and dynamic compressive strength of water-saturated concrete with two-scale porosity are determined not only by the parameters of the capillary pore system (mainly by permeability) but also by the volume fraction of large  pores. An increase in the fraction of micropores leads not only to a common decrease in the strength of the samples but also to a decrease in the threshold for the strain rate sensitivity of strength from  Da~10−1 . For watcomp(Da), with an increase in the volume fraction of micropores, is strongly nonlinear. This is illustrated in comp(Da) curves for the samples with three different volume fractions of micropores . One can see that the σcomp(Da) curve shifts down and to the left almost without distortion at “small” values of volume fraction . A further increase in the volume fraction of micropores is accompanied by the expansion of transitional section II at the expense of section I with an almost unchanged value of the median point of the sigmoid.It is important to note that the change in the parameters of the unified curve σcomp(Da) is explained by the mutual influence of micropores. At low concentrations of micropores, the distance between them is significantly higher than the size of the region of the surrounding matrix, in which the pore pressure significantly increases due to fluid transport from the micropore. A further increase in the volume fraction of micropores  leads to the influence of micropores on the pore pressure and the dynamics of the fluid flow process throughout the cement stone matrix.Such a staged nature of the change in the parameters of the dependence σTherefore, the mechanical effect of pore pressure on the compressive strength of concretes with a two-scale pore structure (strength reduction) can take place already at low strain rates approaching the quasi-static loading regime. The factor controlling the threshold (minimum) values of −4 s−1) and large (≥100 s−1) values. The peculiarities of this interval are: (1) insignificant strain rate sensitivity of the mechanical characteristics of dry concrete; (2) the determining role of competition between pore pressure and pore fluid flow in the mechanical effect of interstitial fluid on the stresses and strength of concrete.The paper numerically studied the mechanical effect of pore fluid pressure on the fracture and dynamic strength of high-strength concrete under uniaxial compression. We considered the “intermediate” range of strain rates between quasi-static (<10Da). Parameter Da is a measure of the balance of pore space deformation and pore fluid flow and a dimensionless generalization of the traditionally used strain rate to the fluid-saturated materials. We revealed the nonlinear (logistic) decreasing dependence of the compressive strength on Darcy number and showed that the parameters of this dependence are different for concretes with one-scale and two-scale porosities.The study was carried out with the use of a previously developed coupled numerical (discrete element) model of high-strength concrete with two-scale porosity and interstitial fluid . In the Da value. The following observations and conclusions can be dawn:Da values corresponding to characteristic strain rates >1 s−1. The latter is a consequence of the large number of centers of crack nucleation (micropores), which allow efficient relaxation of high local shear stresses without cleavage and crushing of the material.The effect of pore fluid on the dynamic mechanical behavior of concrete is determined not only by the absolute value of the open porosity but also by the number of pore scales. We showed that fracture of concrete with only low-scale porosity (capillary pore network) is typically brittle and is characterized by the formation of a network of internal “secondary” cracks. It is important to emphasize the formation of regions with the strong breaking of the cement stone matrix (cleavage zones) up to the crushing of the material. The introduction of large-scale pores (micropores) leads to a qualitative change in the dynamics of crack growth and a strongly pronounced localization of fracture. Cracks originate not at interphase boundaries  but at micropores and develop in a jump-like manner (from micropore to micropore). Moreover, there are no cleavage zones even at sufficiently high Da). An increase in Da value leads to an increase in “multiplicity” of fracture of the concrete with only low-scale (capillary) porosity. The number of main cracks and connecting internal cracks in the matrix increases with the value of Da. The sizes of cleavage zones and their number increase in multiples. This can be generally characterized by an increase in the total damage of concrete . The opposite trend takes place for concrete with low-scale and large (microscopic) pores. The total damage of the samples systematically decreases with an increase in Da due to a decrease in the number of single mesoscopic damages and short internal cracks. Fractures develop by means of the formation of large cracks. We also emphasize the systematic change in the contribution of aggregates to fracture in both systems with an increase in Da value. At a small Da value, the cracks predominantly bend around hard aggregates through interfaces. At large Da values, many cracks cut aggregates (the fraction of fractured particles increases with an increase in Da).Large pores (micropores) also determine a qualitative change in the fracture trend with an increase in the loading rate  and two-scale (capillary and micropores) porosity. The increase in pore pressure determines the onset of fracture of the matrix at lower values of the applied stress. In the first case , this determines an increase in damage of the mesoscopically homogeneous matrix material with an increase in Da. The micropores with interstitial fluid provide the mesoscopic heterogeneity of the matrix and the corresponding inhomogeneity of stress distribution. Micropores deform more strongly than capillary pores in the matrix, and the pore pressure in the micropore grows faster than in the surrounding region of the matrix. An increase in Da is accompanied by an increase in effective stiffness of these stress mesoconcentrators due to the finite rate of fluid outflow to the capillary network. Hence, the concentration of deviator stress in the matrix around micropores decreases, while the characteristic magnitude of compressive volumetric stress increases. This effect provides the above described significant and gradual decrease in the total number of damages with an increase in Da value.The above trends in the change of fracture with an increase in The above results allow us to conclude that expanding the range of scales of the pore structure contributes to an increase in the localization of fracture of water-saturated concrete and to a decrease in the degree of fragmentation under compression. This conclusion can be useful for the design of the internal structure of concrete, which must meet special requirements for fracture.Da~10−1, while the sensitivity threshold for concrete with only a capillary pore system is Da ≈ 10. Moreover, the length of the Da interval corresponding to a decrease in concrete strength from drained (maximum) to undrained (minimum) values increases by two orders of magnitude . This is a consequence of the above-described effect of a stronger increase in pore pressure in micropores during compression. Indeed, the pore pressure gradient between the micropore and the surrounding matrix is higher than inside the matrix, and the characteristic rate of fluid flow from micropores to the surrounding matrix is higher than in the matrix. An intense inflow of fluid into the matrix accelerates the growth of pore pressure and the redistribution of fluid in the matrix. Therefore, the effect of pore fluid transport between micro- and capillary pore spaces on the stress state and strength of concrete is valuable already at Da values corresponding to low strain rates.Micropores reduce not only the overall compressive strength of concrete (by ~25%) but also the threshold of the sensitivity of concrete to the loading rate (by two orders of magnitude). A noticeable decrease in the strength of concrete with a two-scale pore structure begins at Da between the intervals corresponding to the maximum (drained) and the minimum (undrained) values of strength. An increase in the concentration of micropores  leads to the influence of micropores on the pore pressure and the dynamics of the fluid flow throughout the cement stone matrix. Overcoming the “percolation threshold” is accompanied by the expansion of the transition interval of Da.The described effect of increasing the sensitivity of concrete to the loading rate depends on the characteristic distance between micropores . At low concentrations, the distance between micropores is significantly larger than the characteristic size of the region of the surrounding matrix with increased pore pressure provided by fluid transport from the micropore. In this case, the decrease in the concrete sensitivity threshold is not accompanied by a noticeable change in the width of the “transition” interval of An important result of the study is the revealed “stability” of strain rate dependence of the dynamic compressive strength for representative mesoscopic concrete volumes characterized by different spatial arrangements of micropores and hardening inclusions. Small variation intervals of the parameters of the approximating sigmoid indicate that the results of modeling of representative volume elements on the mesoscale can be used to forecast the dynamic strength of macroscopic samples and structural elements (except the elements containing macroscopic defects).In the present study, we comprehensively investigated how the capillary and micropore subsystems determine the change in the fracture pattern and dynamic compressive strength of fluid-saturated high-strength concrete with an increase in the loading rate characterized by the The results of the present study make a valuable contribution to the understanding of the role of pore pressure in the onset of the limiting state of concretes as well as other brittle solids with a multi-scale pore structure. These results can be particularly useful for developing predictive models of degradation and abrasive wear of offshore concrete structures in the Arctic under multiple dynamic contact loading by sea ice and/or other floating objects. The results of the study can also be used when solving the problems of determining the “optimal” parameters of the internal structure of concretes that function under conditions of moisture saturation or in an aqueous environment.


For this reason, both the sector industry and academia have devoted their studies to eco-sustainable materials able to maintain the specific properties of cements. Synthetic inorganic polymers, based on aluminosilicate or phosphate, and prepared following a process known as geopolymerization, have been recognized for almost twenty years now as geopolymers. Characterized by chemical and mechanical properties comparable to those of ceramics, geopolymers present great tolerance for high temperatures and do not explode, contrary to the traditional hydraulic cements. In addition, geopolymers are non-toxic, eco-friendly, and sustainable materials considering, as testified by many papers of this Special Issue, that waste can be recycled as raw materials or inert fillers for their preparation, maintaining performances comparable to those of Portland cement.Concrete is the most commonly used construction material worldwide, and many efforts have been carried out in recent years to improve its functional properties while also trying to increase its sustainability. Concrete’s environmental impact is well known, linked both to the space it occupies in landfills and its highly energy-intensive manufacturing, thus branding the cement industry one of the largest COThis Special Issue, which consists of 22 articles (including two review articles) written by research groups of experts in the field, considers recent research on the design, preparation, and characterization of geopolymers.The first paper, published by Lee and coworkers, aims at developing a sustainable structural material for the construction industry, namely a marble-based geopolymer concrete . The via2 emissions compared to cement concrete. Alkali activators such as sodium silicate and sodium hydroxide were used in the preparation of alkali-activated cements, whose structures are observed to change via carbonation. In addition, they recorded a simultaneous decrease in the compressive strength, thus opening interesting future scenarios in the field of alkali-activated cements.Additionally, in the work of Kim et al., the focus is on the physical and chemical properties changes of alkali-activated cements prepared from blast furnace slag and fly ash , represeIn their contribution, Le and colleagues discussed the experimental data as regards the mechanical properties of the hybrid composite thin-plates of the short basalt fibers/carbon textile-reinforced geomortar . The effIn order to evaluate the effects of the reinforcement and their estimation as correctly as possible, Shi et al., developed practical prediction models of splitting tensile strength and reinforcement–concrete bond strength of low-calcium fly ash geopolymer concrete . The preMohd Basri and coworkers, in coating geopolymer with rice husk ash (RHA), tried to balance the increase in fire retardant properties with the water absorption effects due to the incorporation of RHA . Aiming By stressing the positive environmental impact of the geopolymers, both as a friendly alternative for concrete and utilization of hazardous wastes, Górski et al., focused their study on the recovery of discarded cathode ray tube glass as aggregate for the preparation of metakaolin-based geopolymer . After dEscherichia coli and Enterococcus faecalis, showing a very limited and absent inhibition zone for fine and coarse grain size ranges, respectively. The same research group, aiming to reduce the percentage of debris disposal derived from urban solid waste, suggested an upcycling process where glass debris is simply ground without any washing operation and added to an alkali-activated paste. Thus, metakaolin-based geopolymer mortar added with coarsely ground glass waste has been prepared and characterized, showing interesting antibacterial properties [This research line was also followed by Dal Poggetto et al., who reused glass waste prepared geopolymer at different percentages and grain size of glass to evaluate their antimicrobial activity . As the operties .Assuming that the addition of magnetite to concrete improves its mechanical properties and durability in terms of water and chloride ions absorption, Sgarbossa et al., proposed the design and preparation of novel magnetic geopolymers based on matrices with and without inert quartz aggregates, containing metal particles at different concentrations . By usinWith a view towards greater sustainability, Lancelotti et al., proposed an alternative cementitious binder based on industrial side streams and characterized by a low carbon footprint . A novelContinuing to follow the path of reducing the environmental impact of cementitious formulations, Algaifi and coworkers proposed alkali-activated mortar (AAM) made with binary binders involving fly ash (FA) and granulated blast-furnace slag (GBFS) as well as bottle glass waste nano-silica powder (BGWNP) . By usinAdditionally, Wang et al. were interested in the use of concrete reinforcements based on waste materials. In particular, they turned their attention to the huge amounts of waste ceramic tiles and blast furnace slag (BFS) produced by the ceramic tile factories and the iron smelting industry . They miQuiatchon and coworkers, aiming to increase the low utilization rate of the coal fly ash produced in the Philippines, proposed its use to prepare fly ash-based geopolymer for construction, thus also reaching the goal of lower greenhouse-gas emissions. They provided an impressive amount of specimens in order to optimize the geopolymer components for the replacement of Portland Cement for in situ applications . The engLee and colleagues assessed the preparation of potassium-based geopolymers (KGL) into leucite ceramics and tested their structure and mechanical properties . By takiKohout et al., prepared a metakaolinite-based geopolymer binder using calcined claystone as the main raw material and potassium as the alkaline activator, then proceed with their thermo-mechanical characterization . By balaMorenov and coworkers carried out an interesting work to develop, improve, and study compositions of weighted drilling muds with low content of solids, on the basis of organic salts of alkali metals and polymers for the construction of wells prone to rock swelling and/or cavings, as well as drilling fluids for drilling-in the formation . The devNafees et al., investigated the possible advantages related with the incorporation of silica fume (SF) as a partial substitution of cement in concrete, focusing on the development of modeling techniques in predicting the compressive strength of silica fume-based concrete . In the Reinforced and light-weight geopolymers can be employed in buildings, especially for walling, and in this framework Nguyen and coworkers exanimated the use of specific fillers for the metakaolin-based light-weight geopolymers, emphasizing properties such as high mechanical strengths, low thermal conductivity and density . In theiThe first of the two reviews contained in the Special Issue is by Ferdous et al., dealing with the purpose of reducing the amount of waste glass landfilled by transforming this waste into construction materials. It is well known that broken glass can be used in concrete either as a binder or aggregates. The outcomes of this knowledge are scattered, and it difficult to reach a firm conclusion about the effectiveness of waste glass in concrete, thus the authors critically reviewed its role and impact on microstructural and durability properties for both cement and geopolymer concrete .In the second review, Siddika and colleagues strengthened the same topic, i.e., waste glass based geopolymers, focusing on the effect of the chemical and mineralogical composition, as well as glass particle and curing condition of concrete on the dissolution behavior and chemical reactivity of waste glass . Conside


The IgG4-related disease is an uncommon immune-mediated condition affecting multiple organ systems concomitantly; however, it is very rare for a patient to have isolated IgG4 autoimmune cholangitis or IgG4-related disease confined to the hepatobiliary system. The majority of cases are associated with pancreatitis and are incidentally discovered in the workup of acute or chronic pancreatitis. When it affects the hepatobiliary system, it develops as inflammatory fibrosclerosing cholangitis, which can mimic other hepatobiliary diseases such as primary sclerosing cholangitis. Herein, we present a case of type 1 IgG4 autoimmune cholangitis in the absence of pancreatitis. Our case is particularly unique because type 1 is the most common type associated with autoimmune pancreatitis; however, our patient had type 1 without any evidence of pancreatic involvement. Additionally, like most cases of isolated IgG4 autoimmune cholangitis, our patient was refractory to standard therapy. This case highlights the clinical significance, rarity and severity of isolated IgG4 autoimmune cholangitis. Disclosure: This article was previously presented as a meeting abstract at the ACG Annual Meeting in Las Vegas on October 22-27, 2021. The full manuscript below was written by the same authors.Immunoglobulin G4 (IgG4) related disease (RD) is a multi-organ system inflammatory disorder -3. IgG4 The hepatobiliary manifestation is a type of fibroinflammatory sclerosis cholangitis, characterized by lesions and/or strictures . IgG4 auClinical presentation is variable depending on the organs involved; however, IgG4-AC may present with obstructive jaundice, weight loss and/or abdominal pain -3. It maIsolated IgG4-AC, as a single organ involvement, is very rare. Herein, we present a case of isolated IgG4-AC.A 68-year-old Hispanic male with a past medical history of type 2 diabetes mellitus, hypertension, hyperlipidemia, ankylosing spondylitis, and benign prostatic hyperplasia presented to the Emergency Department (ED) with complaints of abdominal pain for three days prior to presentation. The pain was located in the right upper quadrant (RUQ) and was associated with fatigue, poor appetite, nausea and an objective fever of 103̊ F, lasting four days. On arrival, the patient was afebrile with a blood pressure of 140/76 mmHg, heart rate of 95 beats per minute, respiratory rate of 16, saturating 99% on room air. On the physical exam, the patient was awake, alert, and in mild acute distress. His skin, tongue and eyes were jaundiced. On the abdominal exam, there was epigastric and RUQ tenderness upon superficial and deep palpation without rigidity or guarding, no evidence of organomegaly and normoactive bowel sounds [3/mm3) with left shift, normocytic anemia (hemoglobin 9.7 g/dL) and mild hyponatremia (132 mEq/L), although once corrected for glucose was normal. Liver function tests revealed elevated ALP (556 unit/L), AST (136 unit/L), ALT (331 unit/L) and unconjugated hyperbilirubinemia . Lipase levels were within normal limits (22 units/L). Given the history of subjective fevers, RUQ abdominal pain with obstructive jaundice and mixed cholestatic/hepatic liver function abnormalities, the patient was subsequently admitted for acute cholangitis. Blood cultures were negative, and the patient was started on intravenous Piperacillin-sulbactam 3.375 milligrams, every 8 hours for ascending cholangitis. Computerized tomography (CT) of the abdomen/pelvis showed no acute abnormalities, including no evidence of pancreatitis. Magnetic resonance cholangiopancreatography (MRCP) revealed normal common bile duct, trace intrahepatic ductal dilatation and attenuation of the ducts near the confluence of the common hepatic duct with concerns for an underlying mass [Initial labs were remarkable for leukocytosis . At this time, septic workup was again negative, and the patient was subsequently treated with oral Methylprednisolone 40 milligrams daily per standard guidelines. Antibiotics were discontinued. To further aid in diagnosis and evaluation for the extent of involvement, the patient underwent a liver biopsy, which showed extensive portal acute and chronic inflammation with ductular proliferation. The portal inflammation was composed mostly of lymphocytes with scattered neutrophils, focal scattered plasma cells and a few eosinophils. The bile ducts showed focal injuries with ductular proliferation. Prominent interface hepatitis and scattered lobular inflammatory infiltrate with hepatocyte damage were also noted. Biopsy from the bile duct hilum showed inflamed and reactive biliary epithelium with rare, atypical cells (Figure Biopsy of the common hepatic duct showed attenuated biliary epithelium with active inflammation and no malignancy Figure .These findings were compatible with IgG4-related AC, in the absence of pancreatitis [IgG4 RD most commonly affects the pancreatic and hepatobiliary system and was first identified as a multi-system disease in 2003 in patients with AIP ,4. If thThe pathogenesis of IgG4-AC may involve certain HLA haplotypes overlapping with HLA haplotypes also seen in type-1 AIP, compared to healthy individuals . AdditioRegardless of organ involvement, all laboratory testing remains the same: serum IgG4, and often IgE levels are elevated in more than 50% of patients but are not considered diagnostic -3. SimilWhen isolated IgG4-AC is diagnosed, urgent treatment is warranted to prevent permanent fibrosis, infections, venous thrombosis or even death ,3. The oIgG4 causes infiltration of T cells, leading to progressive fibrosis and organ damage affecting any organ system; however, it most commonly affects the pancreas and biliary tract concurrently due to the hepatopancreatic lymphatic and hematogenous drainage. In the absence of elevated serum IgG4 levels or pancreatic features, one must remember the differential of isolated IgG4-AC. Early recognition of IgG4-AC is crucial as its presentation can mimic pancreatic cancer, hepatobiliary malignancies, PSC or other infectious causes of acute cholangitis. Additionally, there are four types of isolated IgG4 AC, which warrant different treatment options and carry a worse prognosis than when associated with pancreatitis. Thus, an accurate and timely diagnosis is imperative.


Background: SDF-1/CXCL12 is a chemokine with pleiotropic functions in hematopoietic stem cell niche homeostasis, germinal center architecture, B cell maturation, neoangiogenesis, and fibrosis. Recently, the CXCL12/CXCR4/CXCR7 axis was associated with cancer metastasis and autoimmune diseases. The IgG4-related disease (IgG4-RD) is a pathological condition characterized by IgG4+ plasma cells infiltrating fibrotic lesions. The aim of this research is to investigate the relevance of SDF-1/CXCL12 in IgG4-RD.Materials and Methods: Peripheral blood samples were collected before therapy from a single-center cohort of 28 IgG4-RD patients, fulfilling the ACR-EULAR classification criteria. Clinical and serological data were obtained for each patient. In total, 14 healthy donors (NHS), 9 patients with pancreatic ductal adenocarcinoma (PDAC), and 9 with Sjogren syndrome (SSj) were recruited as controls and screened for circulating SDF-1/CXCL12 by ELISA. Moreover, paraffin-embedded pancreatic biopsies obtained from patients with IgG4-RD (n = 7), non-autoimmune pancreatitis (n = 3), PDAC (n = 5), and control tissues (n = 4) were analyzed to study the tissue expression and localization of SDF-1/CXCL12 and one of its receptors, CXCR4, and their potential relation with neutrophil extracellular traps (NETs).Results: IgG4-RD patients had higher serum levels of SDF-1/CXCL12 than normal controls (p = 0.0137). Cytokine levels did not differ between the IgG4-RD autoimmune pancreatitis (AIP) and retroperitoneal fibrosis nor between the single- and multiple-organ involvement. No correlation was seen with the IgG4-RD Responder Index, IgG4 levels, white blood cells, or inflammatory markers in the serum. When compared to SSj, the IgG4-RD AIP subgroup presents higher amounts of serum SDF-1/CXCL12 (p = 0.0275), while no differences are seen in comparison with PDAC. The expression of SDF-1/CXCL12 in the tissue was significantly higher in the IgG4-RD tissue than the normal pancreas, and the tissue with the high SDF-1/CXCL12 expression is characterized by the overall inflammatory cell infiltration, fibrosis, and high level of NETs.Conclusion: Modulating B cell development, neoangiogenesis and fibrosis, and SDF-1/CXCL12 may play a role in IgG4-RD. The higher levels observed in IgG4-RD, as compared to SSj, which closely mimics the disease, can be related to a different pattern of lesions, with prevalent fibrosis seen in IgG4-RD. Taken together, these findings suggest that drugs acting on the CXCL12/CXCR4/CXCR7 axis may affect IgG4-RD. IgG4-related disease (IgG4-RD) is a rare condition characterized by fibro-inflammatory lesions in one or many organs with peculiar histological features, such as tissue fibrosis with a storiform pattern, a diffuse lymphoplasmacytic infiltrate, obliterative phlebitis, mild to moderate eosinophil infiltrate, and abundance of IgG4+ plasma cells .Oligoclonal somatically hypermutated plasmablasts that produce IgG4 are detected in the peripheral blood . This poLittle is known about B cell maturation and mutation in IgG4-RD, but tertiary lymphoid structures have been described in affected organs  and, as The disorder can affect almost any organ: the pancreas, retroperitoneum, lymph nodes, salivary glands, and kidneys are the most frequently involved ones .Diagnosis of IgG4-RD is based on a set of clinical, serological, and pathological criteria , and theThe hallmark of IgG4-RD is represented by a typical pattern of fibrosis, rarely detected in other inflammatory disorders, with spindle cells (fibroblasts and myofibroblasts) radiating from a center . As in aPresently, few data are available on the circuits controlling the processes leading to tissue fibrosis in IgG4-RD. It has been suggested that a critical step in inducing the typical lesions is the release of profibrotic cytokines, but so far this hypothesis has only been partially investigated .Recent data draw attention to the role of SDF-1/CXCL12 as a mediator involved in recruiting circulating fibrocytes to the inflamed tissue in chronic periaortitis, a fibrotic disorder in the spectrum of IgG4-RD .SDF-1/CXCL12 is a chemokine originally identified as a product of murine bone marrow stromal cells that interacts with two receptors, CXCR4 and CXCR7 . In embrSDF-1/CXCL12 is also a potent chemoattractant for bone marrow eosinophils  and, in The pleiotropic effects of this chemokine suggest its possible involvement in multiple aspects of IgG4-RD.In this study, we thus analyzed the serum levels of SDF-1/CXCL12 in IgG4-RD patients, focusing in particular on the subgroup with pancreatic involvement. The pancreatic expression of SDF-1/CXCL12 and its receptor CXCR4 was also studied in a subset of IgG4-RD patients with autoimmune pancreatitis (IgG4-RD AIP) and compared to what was observed in the pancreatic ductal adenocarcinoma (PDAC). In addition, the SDF-1/CXCL12 expression in the pancreatic tissue was evaluated in relation to the inflammatory infiltrate, particularly neutrophils, and neutrophil extracellular traps (NETs).In total, 28 patients fulfilling the criteria for the diagnosis of IgG4-RD were included in the study . Here, 1The levels of SDF-1/CXCL12 in the sera of IgG4-RD patients and controls were measured by ELISA , according to the manufacturer’s instructions.Paraffin-embedded pancreas biopsies from seven IgG4-RD patients, three not-autoimmune pancreatitis, five PDAC, and four normal tissues (adjacent to the tumor) were obtained from the Pathology Unit, Department of Surgical Pathology, University of Pisa. In order to confirm the original diagnosis, all the tissue sections were reviewed by an expert pathologist. All the biopsies used in this study were fixed in a 10% buffered formalin solution, paraffin embedded, and cut at 3 μm. H&E staining was performed on all samples. Each sample was analyzed by an expert pathologist and scored from 0 to 3 for the level of fibrosis, follicles, lymphocytes, neutrophils, eosinophils, and plasma cells.Deparaffinized pancreatic biopsies underwent antigen retrieval at 95°C with a low pH buffer solution followed by block of not-specific binding. The sections were then incubated with the proper combination of primary antibodies; for CXCR4 and SDF-1/CXCL12, the slides were incubated overnight at 4°C with a goat anti-mouse IgG1 antibody specific for SDF-1/CXCL12 (R&D), and after PBS washing, the sections were incubated for 1 h at RT with a goat anti-mouse IgG2b antibody specific for CXCR4 (R&D). For detection of NETs, the slides were incubated for 1 h at RT with a goat polyclonal anti-rabbit antibody specific for MPO (Dako) and a goat anti-chicken antibody specific for H2B (Abcam).After washing, the sections were incubated with the appropriate combination of fluorochrome-conjugated secondary antibodies. Alexa 488–conjugated goat anti-mouse IgG1 and Alexa 555–conjugated goat anti-mouse IgG2b (Invitrogen) were used for SDF-1/CXCL12 and CXCR4, respectively. Alexa 488–conjugated goat anti-rabbit and Alexa 555–conjugated goat anti-chicken (Invitrogen) were used for MPO and H2B, respectively.Cell nuclei were counterstained with DAPI. Negative controls included omission of the primary antibody followed by the proper secondary antibody.All the sections were visualized using an Olympus BX-41 microscope. Stained cells were counted in five 40X microscopic fields per section.The presence of SDF-1/CXCL12 and its receptor CXCR4 was evaluated by a semi-quantitative score. In detail, in each tissue slide, both CXCR4 and SDF-1/CXCL12 were scored from 0 (no cells) to 3 (high number of cells). A median value was obtained for the IgG4-RD sample tissues and control groups . Both SDF-1/CXCL12 and CXCR4 expression levels were evaluated in relation to the level of fibrosis and the presence of follicles, lymphocytes, plasma cells, eosinophils, and neutrophils, previously evaluated by H&E staining. Finally, in the same tissue, both CXCR4 and SDF-1/CXCL12 expressions were correlated with NETs . The levels of SDF-1/CXCL12 were considered low with a score of 0 or 1 and high with a score of 2 or 3.U-test was used to compare the patients’ group and the normal subjects. p < 0.05 was considered statistically significant.A non-parametric Mann–Whitney The clinical and serological features of the IgG4-RD patients are summarized in n = 13); retroperitoneum (n = 9); lymph nodes (n = 8); salivary glands (n = 7); orbits and lacrimal glands (n = 4); the kidney, liver, and thyroid (n = 3); and lungs, large vessels, and the pituitary gland (n = 2). Skin involvement was observed only in one patient. The median of the IgG4-RD Responder Index was 7.5, indicating an active disease in most of the patients. The median of circulating eosinophils was 200/μl (87.5–282.5 IQR) with a value > 700/μl in 2 out of 28 patients. Three patients were previously treated with immunosuppressant (rituximab or cyclophosphamide), while eight patients were under treatment with low-dose steroids .In total, 13 patients had a single-organ involvement, while 8 patients had a two-organ involvement, and 7 had multi-organ diseases (three or more organs affected). The most frequent organs involved were pancreas (p = 0.0142) (SDF-1/CXCL12 serum levels were significantly higher in IgG4-RD patients compared to NHS ( 0.0142) . The sub 0.0142) .In IgG4-RD patients, the levels of SDF-1/CXCL12 were not related to inflammatory markers ). Although in the literature it has been demonstrated that SDF-1/CXCL12 is an eosinophil chemoattractant , we did The SDF-1/CXCL12 expression was significantly higher in IgG4-RD with respect to normal pancreas tissue, comparable to those found in different disease groups including PDAC and in not-autoimmune pancreatitis . ConversIn the pancreatic tissues with high expression of SDF-1/CXCL12, significantly higher levels of fibrosis , folliclp < 0.05 and p < 0.01, respectively) .As reported in ctively) . By analctively)  and NETsctively)  than in In this study, we analyzed the serum levels of SDF-1/CXCL12 in IgG4-RD patients and the pancreatic expression of the chemokine in the subgroup of IgG4-RD with autoimmune pancreatitis.+, collagen-1+) that migrate to sites of injury and play an important role in disorders characterized by excessive collagen deposition  in the hypoxic tumor environment. The profibrotic activity of the chemokine supports tumor growth, protecting cancer cells from the host’s immune system and creating niches for metastatic growth .In the tissue sections we studied, the SDF-1/CXCL12 expression reflects the number of lymphoid follicles, and eosinophil and neutrophil infiltrate.SDF-1/CXCL12 plays an important role in B cell homing and differentiation in germinal centers. Ectopic germinal centers have been described in salivary glands of IgG4-RD patients , and theThus, it is not surprising that in different disorders, the SDF-1/CXCL12 expression is related to the number of lymphoid follicles.The relation with eosinophil and neutrophil infiltrates suggests the role of SDF-1/CXCL12 in mediating the migration of these cells into pancreatic tissue. SDF-1/CXCL12 is by itself a weak neutrophil chemoattractant but synergizing with other chemokines like IL-8 may increase neutrophil infiltration .SDF-1/CXCL12 signaling is mediated by the interaction with CXCR4, a G-coupled receptor that is activated also by ubiquitin, and by the binding of a macrophage migration inhibitory factor. CXCR4 is diffusely expressed in the pancreas, indicating that the chemokine may exert its activities in the tissue through receptor binding. No differences in CXCR4 distribution or density were detected in different diseases we studied; thus, chemokine and the receptor expression are not parallel and in fact has been reported that the CXCR4/SDF-1 system is regulated at the level of the receptor expression . HoweverIn fact, splice variants of SDF-1/CXCL12 give rise to different isoforms with a tissue-specific pattern of expression: CXCL12δ, CXCL12ε, and CXCL12ϕ are most abundantly expressed in the pancreas. The isoforms share the first 67 amino acids but differ in length and in signaling efficiency. Only splice variant-specific antibodies may shed light on the pattern of the isoform expression in the tissue. Moreover, the biological activity is regulated by posttranslational modification of the chemokine, such as citrullination and tyrosine nitration .SDF-1/CXCL12 may also contribute to organ damage, mediating neutrophil infiltration of the pancreas.In IgG4-RD and PDAC, neutrophils infiltrating the pancreas frequently undergo NETosis, a form of programmed death that represents a critical defense mechanism in innate immunity but also a potential mechanism of tissue damage. By the extrusion of chromatin fibers coated with granule enzymes and other cytoplasmic constituents, neutrophils entrap the microorganisms too big to be phagocytosed . NETs, hIn PDAC, IL-17 mediates neutrophil recruitment and induces NETosis. NETs enhance migration and activate pancreatic stellate cells that form dense stroma and enable tumor proliferation .One of the NET constituents is the antimicrobial peptide cathelicidin, which forms complexes with RNA; complexed RNA is protected from degradation and transported into the endosomal compartment of dendritic cells where it stimulates the production of IFN alpha . Moreove