To elucidate a potential association between Ca2+and GVHD, we analyzed Ca2+-sensing G-protein coupled receptor 6a (GPRC6a) signaling in preclinical GVHD models and conducted a prospective EBMT study on Ca2+ serum levels prior alloSCT including 363 matched sibling allogeneic peripheral blood stem cell transplantations . In experimental models, we found decreased Gprc6a expression during intestinal GVHD. GPRC6a deficient alloSCT recipients had higher clinical and histopathological GVHD scores leading to increased mortality. As possible underlying mechanism, we found increased antigen presentation potential in GPRC6a–/– alloSCT recipients demonstrated by higher proliferation rates of T-lymphocytes. In patients with low Ca2+ serum levels (≤median 2.2 mmol/l) before alloSCT, we found a higher incidence of acute GVHD grades II-IV (HR = 2.3 Cl = 1.45–3.85 p = 0.0006), severe acute GVHD grades III-IV  and extensive chronic GVHD (HR = 2.0 Cl = 1.04–3.85 p = 0.04). In conclusion, experimental and clinical data suggest an association of reduced Ca2+ signaling with increased severity of GVHD. Future areas of interest include the in depth analysis of involved molecular pathways and the investigation of Ca2+ signaling as a therapeutic target during GVHD.Despite its involvement in various immune functions, including the allogeneic activation of T-lymphocytes, the relevance of calcium (Ca It has been shown to modulate the allogeneic activation of T-lymphocytes by antigen presenting cells  implicating a role for graft-versus-host disease (GVHD) pathogenesis. Additionally, Ca2+ is known as “danger-associated molecular pattern” (DAMP) and plays an important role in the activation of T-cells, dendritic cells and the multi-protein complex of the NOD-like receptor protein (NLRP3) inflammasome (4). During conditioning prior to allogeneic stem cell transplantation , with radiation and/or chemotherapy, and later during GVHD increased cell death of immune and tissue cells occurs. This increased cell death can lead, among other things, to enhanced Ca2+ levels. A locally increased extracellular Ca2+ concentration can induce the activation of the NLRP3 inflammasome via the calcium sensing receptor and the G-protein coupled receptor 6a (GPRC6a) (4). Several studies revealed a significant role of DAMPs and the NLRP3 inflammasome in GVHD morbidity and mortality (5–8). While NLRP3 function has been studied in GVHD, upstream regulatory functions of GPRC6a in GVHD remain to be elucidated. GPRC6a is a class C, group 6, subtype A G-protein coupled receptor which can be directly activated or positively modulated by Ca2+ in concentrations above 5 mM; and was shown to be involved in the regulation of inflammation, metabolism and endocrine functions (9). In addition, GPRC6a was identified in a genome-wide association study as a novel loci associated with C-reactive protein levels, a general biomarker for systemic inflammation (10).Calcium . Based on prior knowledge and the fact that Ca2+ is a routine laboratory parameter in patients undergoing alloSCT due to frequently arising disturbances in calcium metabolism in these patients, there is a strong rationale to study Ca2+ and its association to GVHD. We performed experiments in preclinical GVHD models using GPRC6A deficient mice; and the Transplant Complications Working Party (TCWP) of the European Society for Blood and Marrow Transplantation (EBMT) performed a prospective, multicenter and non-interventional clinical study following the hypothesis that Ca2+ is correlated to the occurrence of GVHD.The role of Cat-test assuming 80% power and 0.05 2-sided level of significance. Experiments with at least 5 animals per group (wildtype transplanted [WT] and GPRC6a deficient transplanted [GPRC6a–/–]) were performed 2 times. Animals were randomized, including equal distribution of weight status and mixed housing of different transplanted animals. For subsequent analyses, transplant conditions were encoded. All experiments were approved by the Regional Ethics Committee for Animal Research .Sample size for GVHD experiments was calculated by estimation of time point–specific analyses based on the Student’s –/– transplanted) were performed 2 times and analyzed using the Kaplan-Meier method and compared with the Mantel-Cox log-rank test. For all other data, the Student’s unpaired t-test (two-tailed) was used if not indicated differently. Normality tests and F test confirmed Gaussian distribution and equality of variance between different groups. Values are presented as mean ± standard deviation (SD). Values of P < 0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism software .Survival data of at least 10 animals per group (WT and GPRC6ab) and BALB/c (H2d) mice (10–12 weeks old) were purchased from Charles River Laboratories  and from Janvier , respectively. Female GPRC6a deficient (GPRC6a–/–) mice on B6 background were generated by Hans Bräuner-Osborne, provided by Ulf Wagner  and were bred by and obtained from the central animal unit of the Charité University Medicine. Mice had access to food and water ad libitum.Female C57BL/6 (B6)  with 5 × 106 bone marrow (BM) cells and 0.5 × 106 splenic T-cells. BALB/c→ GPRC6a–/– or B6: Recipient mice received 1200 cGy total body irradiation from a 137Cs source as a split dose with a 4-h interval and were injected i.v. with 0.75 × 107 BM cells and 1 × 106 splenic T-cells. BM was flushed from the tibia and femur, and single-cell suspension was prepared in phosphate-buffered saline (PBS)/2% fetal calf serum/1 mM EDTA by gently passing through a 23-G needle and over a 70 μm cell strainer . Splenic T-cell suspension was obtained using the Pan T-cell Isolation Kit II . T-cell purity was analyzed by CD3 staining and FACS analysis. For Gpcr6a expression analysis, we used the established chemotherapy-based minor mismatch model LP/J→B6 as described in (16). Mice were individually scored twice a week for 5 clinical parameters  on a scale from 0 to 2. Clinical GVHD score was assessed by summation of these parameters. Survival was monitored daily.Our alloSCT acute GVHD models are characterized well (12–16). We performed alloSCTs in the BALB/c→B6 model using GPRC6aA total of 2 μg RNA from colon of syngeneic and allogeneic transplanted mice at day + 15 after alloSCT and complementary DNA, obtained using the RNeasy Mini Kit  and the QuantiTect Reverse Transcription Kit (Qiagen) following the manufacturer’s instructions, were amplified  on DNA Engine Opticon  using the TaqMan Gene Expression Master Mix (Life Technologies) and GPRC6a primers and probe from BioTez GmbH . Data were analyzed with Opticon Monitor 3.1 analysis software (Bio-Rad) and the comparative CT Method (ΔΔCT Method).Single-cell suspensions of blood, BM, lymph nodes, spleen and thymus were prepared. Lymph nodes, splenocytes and thymocytes were passed through a 40-μm cell strainer. Erythrocytes were lysed with ammonium chloride. Cells were washed twice and stained for 20 min at 4°C in PBS/0.5 mM EDTA/0.5% BSA with the following rat mAbs from BD Biosciences: anti-H2kd (SF1-1.1-FITC and PE), anti-CD8a (53-6.7-APC), anti-Ly-6G and Ly6C/Gr1 (RB6-8C5-APC), anti-CD45R/B220 (RA3-6B2-PerCP-Cy5.5), anti-CD25 (PC61-PerCP-Cy5.5), anti-CD4 (RM4-5-PE-Cy7), anti-CD80 (16-10A1-PE), anti-CD86 (GL1-PerCP-Cy5.5), anti-CD11c (HL3-PE-Cy7), anti-CD3e (145-2C11-APC-Cy7) and anti-CD11b (M1/70-APC-Cy7). For staining of regulatory T-cells, Anti-Mouse/Rat FoxP3 Staining Set APC  was used following the manufacturer’s instructions. Samples were analyzed by BD FACS Canto II (BD Biosciences) and FlowJo 7.6.5 Software .–/– and B6 mice using a CD11c + isolation kit and splenic T-cells from BALB/c mice were obtained using the mouse Pan T-cell isolation Kit II (Miltenyi Biotec) according to the manufacturer’s instructions. T-cells were loaded with carboxyfluorescein diacetate succinimidylester . 2.5 × 104 dendritic cells (activators) and 2.5 × 105 T-cell (responders) were incubated for 96 h at 37°C and 5% CO2. Total cell counts being negative for CFSE and positive for H2kd were analyzed via flow cytometry. Proliferating cells were determined as cells, showing no CFSE load compared to control samples without T-cell stimulation at time point 0 h.Dendritic cells were isolated from spleen of GPRC6a6 CFSE-loaded CD3 + cells were injected i.v. into the tail vein. 72 h later, mice were sacrificed and T-cells from spleen, thymus, blood and lymph nodes were isolated and analyzed for proliferation by flow cytometry. Total cell counts being negative for CFSE and positive for H2kd were analyzed via flow cytometry. Proliferating cells were determined as cells, showing no CFSE load compared to control samples without T-cell stimulation at time point 0 h.GPRC6a-/- and B6 recipients received 1200 cGy total body irradiation from a 137Cs source. CD3 + lymphocytes were isolated from spleens of BALB/c donors and enriched by Pan T-Cell isolation kit (Milteny Biotec). 4 × 102+ to proteins, mainly albumin, and anions; and free ionized Ca2+) serum levels were determined photometrically and corrected for albumin levels according to Payne’s formula [Ca corr [mmol/L] = Ca measured [mmol/L] – [0.025 × Albumin [g/L] + 1] at time of hospital admission for alloSCT directly before start of conditioning therapy. Treatment teams completed the specific forms at the time of registration and at day + 100 after alloSCT.We asked EBMT centers performing more than 50 alloSCT per year if they were willing to participate in this prospective study. 17 centers in ten countries agreed to participate. Data collection for the EBMT registry was approved by the IRB and/or Ethics Committee in all centers. Data were prospectively collected between 6/2014 and 3/2018. Consecutive alloSCT recipients with acute leukemia, lymphoma or myelodysplastic syndrome (MDS) receiving a first matched sibling alloSCT from peripheral blood, regardless of conditioning, were eligible, provided they had signed an informed consent document that permitted sharing of clinical data according to national rules. Basic data on patient and disease characteristics as well as longer term follow up was taken from minimal essential data (MED-A) forms, which are submitted from all consecutive patients to the central EBMT registry. In addition, we designed registration and MED-B/C forms that were prospectively collected and specific to this study. The MED-B/C form contained detailed information on calcium serum levels prior to alloSCT, patient characteristics, infectious- as well as non-infectious complications, GVHD staging, morbidity and mortality. Total Calcium /calcium levels below median (<2.2 mmol/l)] were compared by using χ2 statistics for categorical variables and the Mann-Whitney test for continuous variables. Primary endpoint was the incidence of acute GVHD. Secondary endpoints were relapse incidence (RI), non-relapse mortality (NRM), overall survival (OS), progression free survival (PFS) and the incidence of chronic GVHD. PFS was defined as survival with no evidence of relapse or progression. RI was defined as the probability of having had a relapse during follow up time. Death without experiencing a relapse was a competing event. NRM was defined as death without evidence of relapse or progression. OS was defined as the time from alloSCT to death, regardless of the cause. Acute GVHD was graded according to the modified Seattle-Glucksberg criteria (17) and chronic GVHD according to the revised Seattle criteria (18). Cumulative incidence was used to estimate the endpoints of NRM, RI, acute, and chronic GVHD to accommodate for competing risks. To study acute and chronic GVHD, we considered relapse and death to be competing events. Probabilities of OS and PFS were calculated using the Kaplan–Meier method. Univariate analyses were done using the Gray test for cumulative incidence functions and the log rank test for OS and PFS. A Cox proportional hazards model was used for multivariate regression. All variables differing significantly between the 2 groups or factors associated with one outcome in univariate analysis were included in the Cox model. The following variables entered the multivariate models as possible confounders: age, sex mismatch between recipient and donor, diagnosis, disease status, Karnofsky score, number of CD34 cells given, intensity of conditioning (EBMT definition: myeloablative conditioning (MAC) was defined as TBI > 6 Gray or oral busulfan > 8 mg/kg or intravenous busulfan > 6.4 mg/kg), type of GVHD prophylaxis, ATG use, time from diagnosis to transplant, year of transplant and CMV status. As the number of variables was too high regarding the number of events, a stepwise selection using Akaike information criterion (AIC) was run for all the confounding factors. The difference between the two cohorts was then assessed in the final selected model.1.Results were expressed as the hazard ratio (HR) with the 95% confidence interval (95% CI). Proportional hazards assumptions were checked systematically for all proposed models using the Grambsch-Therneau residual-based test. All tests were 2-sided. The type I error rate was fixed at 0.05 for the determination of factors associated with time-to-event outcomes. Statistical analyses were performed in November 2018 with R 3.4.2 (R Core Team (2017). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, AustriaGprc6a expression levels in the colon of allogeneic transplanted recipients at day + 15 after alloSCT  either as recipients or donors of alloSCT. In line with the expression data, GPRC6a–/– alloSCT recipients exhibited higher GVHD morbidity and mortality. GPRC6a–/– alloSCT recipients had significantly increased weight loss  alloSCT recipients at day + 15 after alloSCT  T-cells in either irradiated WT B6 or GPRC6a–/– mice (H2kb). After 72 h, allogeneic T-cells in GPRC6a–/– mice showed a significantly increased proliferation compared to WT B6 mice in spleen and thymus , resulting in a high (>median 2.2mmol/l) and a low (≤median 2.2mmol/l) Ca2+ serum level group. Total Ca2+ serum levels were corrected for albumin levels; of note we found in our population a strong correlation between calcium and albumin values . The main patients and transplant characteristics that were included in the analysis of overall survival are described in 2+ above median before alloSCT. The frequency of patients with reduced-intensity conditioning (RIC) versus myeloablative conditioning differed significantly between the high Ca2+ group and the low Ca2+ group. Additionally, the high Ca2+ group showed significantly less patients treated with ATG and a significantly different Karnofsky score. Therefore subsequent multivariate analysis included these variables.The entry criteria for analysis of primary and secondary endpoints were fulfilled in 363 patients. We used the last total Cap = 0.0024) and multivariate (HR = 2.3 Cl = 1.45–3.85 p = 0.0006) analysis in patients with low Ca2+ serum levels before alloSCT as compared to patients with high Ca2+ serum levels (p = 0.0012) and multivariate  analysis in patients with low Ca2+ serum levels before alloSCT as compared to patients with high Ca2+ serum levels  analysis in patients with low Ca2+ serum levels before alloSCT as compared to patients with high Ca2+ serum levels (p < 0.0001). NIH classification of chronic GVHD was available for 14% of patients shown in In the whole population, the incidence of chronic GVHD at 1 year and 2 years was 26% and 39%, respectively. The incidence of severe chronic GVHD at 1 year and 2 years was 16% and 21%, respectively. Extensive chronic GVHD was significantly increased in multivariate . However, non-relapse mortality (NRM) was significantly increased in alloSCT recipients with low Ca2+ levels prior to start of conditioning in univariate analysis (p = 0.0247). The same trend (but not significant) was seen in NRM multivariate analysis (p = 0.06). The causes of death in patients without relapse were mainly due to infection. A descriptive analysis of the causes of death is given in The overall survival (OS) in the whole population at 1 year was 72%. We observed no significant differences in the OS and progression free survival (PFS) of alloSCT recipients with low Ca2+ levels .No difference in the relapse incidence was detected between alloSCT recipients with low and high Ca2+ levels (below median of 2.2 mmol/l) prior to alloSCT are associated with increased incidence of acute GVHD as well as extensive chronic GVHD.We conclude that low Ca2+ signaling is involved in various biologic processes relevant for patients after alloSCT, including inflammation, anti-infectious immunity, anti-tumor effects, kidney diseases and bone homeostasis. Although our current descriptive experimental and clinical results exhibit some limitations and lacks further mechanistic insights, it gives first insights in a possible association between Ca2+ and the occurrence of GVHD, deserving further investigations.Ca–/– alloSCT recipients had increased GVHD morbidity and mortality. GPRC6a, a G protein coupled receptor, was previously found to be activated by increased extracellular Ca2+ levels leading to an increase in the intracellular Ca2+ concentration, triggering the activation of i.a. the NLRP3 inflammasome and caspase-1, mediated through the phosphatidyl inositol/Ca2+ pathway. Activation of the NLRP3 inflammasome via DAMPs has already been shown to be connected to GVHD severity . In preclinical models, inhibition of the NLRP3 inflammasome lead to improved GVHD morbidity and delayed GVHD-induced mortality (5–7). Interestingly, when using NLRP3 deficient mice, this effect was only prominent when deficiency was present in alloSCT recipients, not in donors (6). In patients, recipient single nucleotide polymorphisms of NLRP3 were found connected to the incidence of acute GVHD grades 2–4 (20). While the role of NLRP3 was studied in GVHD, potential upstream immune regulatory functions of GPRC6a are unknown. Results in GPRC6a–/– mice showing reduced inflammatory response and reduced inflammasome activation in a mouse model of carrageenan-induced footpad swelling (4), suggested that GPRC6a deficiency may also lead to reduced GVHD-associated inflammation as seen in GVHD studies analyzing the NLRP3 inflammasome. However, our data contradicts these findings, as GPRC6a deficiency in alloSCT recipients was associated to increased acute GVHD. As GPRC6a is located upstream from the NLRP3 inflammasome, influence on other pathways is likely, potentially leading to differing results. In addition, GPRC6a is widely expressed throughout the body and can recognize multiple ligands next to Ca2+, e.g., L-α-amino acids as L-arginine or L-lysin, zinc, testosterone or osteocalcin acting as positive or negative allosteric modulators . The wide tissue expression and multiple activators of GPRC6a, let assume a wide range of functions and the entire scope of GPRC6a functions is not yet known. In line, experimental data from GPRC6a–/– mice show a diversity in physiological and pathophysiological functions, mainly involving a role in inflammation, endocrine functions and metabolism. However, functional phenotyping of these mice is enclosed by disparities, e.g., in glucose and fat or bone metabolism . Whereas one group found glucose intolerance and insulin resistance as well as osteopenia and impaired osteoblast mediated bone formation in GPRC6a–/– mice (22), other groups could not detect such phenotypes . This underlines the possibility that besides the Ca2+ mediated GPRC6a signaling, other signaling pathways contribute to the observed phenotype in our GVHD model.In animal experiments, GPRC6ain vitro and in mice (25). This raises the possibility that steroid therapy used for GVHD prophylaxis may be influenced by GPRC6a expression.Of note, despite its regulatory functions in inflammation, GPRC6a was shown to mediate the non-genomic effects of testosterone and other steroids 2+ in concentrations above 5 mM (9); and Ca2+ was shown to be involved in various immune functions, including T-cell effector and regulatory functions (1) as well as the allo-activation of T-lymphocytes by antigen presenting cells . In GPRC6a–/– mice, we also found increased antigen presentation potential demonstrated by higher proliferation rates of allogeneic T-lymphocytes in GPRC6a–/– compared to wildtype alloSCT recipients, leading to increased GVHD severity. Phenotypic differences of GPR6CA–/– dendritic cells were detected concerning reduced CD80 expression; however, further changes in costimulatory molecules are likely and need further investigation. The role of Ca2+ signaling during GVHD is mostly unknown, although first results in preclinical GVHD models support our data that Ca2+ may play a role in mediating GVHD pathophysiology . However, Ca2+ signaling appears to remain complex, multi-targetable and divergent and deserves further investigation. E.g., the deletion of the plasma membrane proteins ORAI1 + 2 forming release-activated Ca2+ channels leads to the abolishment of store-operated Ca2+ entry and a decrease of intracellular Ca2+, and protects mice from allo-immunity in models of colitis and GVHD (3). Contrasting and more coherent to our data, the inhibition of inositol 1,4,5-trisphosphate 3-kinase B (Itpkb), a negative regulator of Ca2+ influx, leads to an increase of intracellular Ca2+ in donor T cells, protecting mice from acute and chronic GVHD while maintaining a graft-versus-tumor-effect (11). Interestingly, high calcium containing solutions are effective to reduce mucositis after alloSCT raising the possibility that Ca2+ signaling is involved in tissue repair or tissue protection (26).GPRC6a can be directly activated by Ca2+ with the occurrence of GVHD as low Ca2+ serum levels (below median of 2.2 mmol/l) prior to alloSCT were found to be a risk factor for the incidence of acute GVHD and for extensive chronic GVHD. However, a limitation of this clinical study is the lack of Ca2+ serum levels after alloSCT, e.g., during occurrence of GVHD; leading to an incomplete understanding of the effects of Ca2+ on post alloSCT immunity. The aim of our prospective trial was to identify biomarkers that can be measured before alloSCT. Therefore, no biomarker measurement at time of GVHD onset was planned in the trial design. Importantly, further investigations addressing the longitudinal progress of Ca2+ levels post alloSCT will further shed light on the association of Ca2+ and GVHD pathophysiology. In this study, we assessed total Ca2+ in serum corrected to albumin levels, as approximate 45% of circulating Ca2+ is bound to proteins, mainly albumin, which displays besides its high-capacity carrier role also several physiological functions. Despite this correction, subsequent analyses may address if free ionized Ca2+ and/or Ca2+ bound to proteins or anions show an impact on GVHD incidence. Of note, we found in our population a strong correlation between calcium and albumin values . Recently, hypoalbuminemia was shown to predict worse NRM and inferior OS (27) as well as severity of aGVHD (28) in alloSCT recipients.Our performed prospective study suggests an association of Ca2+ serum levels with outcome in matched unrelated donor alloSCT or in haploidentical alloSCT, which are increasingly used. The herein presented data can stimulate further research to perform analyses of Ca2+ serum levels in other multiple cohorts allowing to widen conclusions to other donor types.Our patient population was restricted to alloSCT from HLA-identical sibling donors, allowing to draw conclusions from a homogeneous population with similar GVHD prophylaxis strategies and similar incidences of GVHD and other complications. However, this includes the limitation that we are therefore unable to draw definite conclusions from these results regarding the association of Ca2+ with the incidence and severity of GVHD. Future perspectives of our results will include the clinical implementation of risk-adapted GVHD prophylaxis strategies taking into account calcium levels before alloSCT. Future studies can address the question how the high risk of GVHD in the subpopulation identified in this study can be reduced. A possible trial design would be to test intensified GVHD prophylaxis regimens in a randomized trial, based on calcium levels. This intensified regimen could be the addition of a third immunosuppressive drug in addition to the standard regimens consisting of a calcineurin inhibitor and an antimetabolite. Furthermore, future research areas of interest include: 1) to further analyze the involved mechanistic and molecular pathways of Ca2+-dependent allo-activation, and 2) to test if Ca2+ signaling can serve as a therapeutic target during GVHD or other inflammatory diseases.This study gives first insights in a possible association of CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by IRB and/or Ethics Committee in all participating centers. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by State Office of Health and Social Affairs (LAGeSo), Berlin.KR, SC, and OP designed the animal study and wrote the manuscript. SC, MK, SM, and JM performed experimental GVHD experiments. CS and YS performed and analyzed qPCR experiments. KR and SC analyzed experimental data. OP, ZP, CK, HS, RD, and GB designed and performed the EBMT study. CP and SW analyzed human data and performed statistical analyses. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neutrophil granulocytes are the vanguard of innate immunity in response to numerous pathogens. Their activity drives the clearance of microbe- and damage-associated molecular patterns, thereby contributing substantially to the resolution of inflammation. However, excessive stimulation during sepsis leads to cellular unresponsiveness, immunological dysfunction, bacterial expansion, and subsequent multiple organ dysfunction. During the short lifespan of neutrophils, they can become significantly activated by complement factors, cytokines, and other inflammatory mediators. Following stimulation, the cells respond with a defined (electro-)physiological pattern, including depolarization, calcium influx, and alkalization as well as with increased metabolic activity and polarization of the actin cytoskeleton. Activity of ion transport proteins and aquaporins is critical for multiple cellular functions of innate immune cells, including chemotaxis, generation of reactive oxygen species, and phagocytosis of both pathogens and tissue debris. In this review, we first describe the ion transport proteins and aquaporins involved in the neutrophil ion–water fluxes in response to chemoattractants. We then relate ion and water flux to cellular functions with a focus on danger sensing, chemotaxis, phagocytosis, and oxidative burst and approach the role of altered ion transport protein expression and activity in impaired cellular functions and cell death during systemic inflammation as in sepsis. Input and output of water and ions, therefore, require tight regulation in organisms. On a cellular level, various ion and water transport proteins ensure the ion–water balance, which is involved in any physiological and immune function.A major function of the innate immune system is to supply the “first line” of the fluid and cellular defense system, comprising mainly the complement system and neutrophil granulocytes (neutrophils). The complement cascade becomes activated by numerous microorganism and microbial-associated molecular patterns (MAMPs) and in turn generates anaphylatoxins, opsonins, and membrane attack complexes, all of which support bacterial marking and clearance ,2. NeutrSepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infectious microorganisms and corresponding MAMPs . With anIn this review, we discuss neutrophil functions, with a focus on ion and water flux with an emphasis on danger sensing, chemotaxis, phagocytosis, and oxidative burst, highlight pyroptosis and NETosis as neutrophil-related forms of cell death, and transfer these findings into the clinically relevant context of sepsis.Danger sensing of harmful MAMPs, but also of damage-associated molecular patterns (DAMPs), is crucial for any organism, if not a “question of life and death”. Neutrophils, as the most frequent representatives of the first cellular line of defense, can recognize molecular danger by pattern recognition receptors (PRPs), including the membrane-bound formyl-peptide receptors (FPRs), toll-like receptors , complement receptors , and receptor for advanced glycation end products (RAGE) ,20. Ther− efflux, promote NLRP3 inflammasome assembly and subsequent IL-1β secretion, and thus mediate a proinflammatory response [Multiple mechanisms exist by which microbes or corresponding MAMPs can enter host cells. For example, lipopolysaccharide (LPS) can enter the cytoplasm by escaping the bacteria/MAMP-containing vacuole, or via endocytic uptake of LPS encapsulated in outer membrane vesicles originated from bacteria . Upon inresponse ,32. Whetresponse .N-formylmethionine-containing oligopeptides (which are released by many invading microorganisms) via FPRs on neutrophils results in an increase in the mitochondrial membrane potential. This rapidly leads to a robust release of adenosine triphosphate (ATP) by the neutrophil. In turn, the excreted ATP subsequently activates purinergic receptors on the cellular surface in an autocrine manner. The resulting Ca2+-influx finally triggers functional changes, including morphological polarization, directed chemotaxis, phagocytosis, and degranulation [Sensing of nulation ,34.N-formyl-methionyl-leucyl-phenylalanine ) and numerous inflammatory mediators, including among others complement factor 5a (C5a), IL-8, lipid mediators, such as PAF and leukotriene B4 (LTB4), and ATP [Cellular migration is a key function of neutrophils, which is induced by MAMPs from the inflammatory/infectious site , a small GTPase of the Rho family, is required in neutrophils ,39. Chem2+ [In general, neutrophils respond to exposure to chemoattractants with a defined reaction pattern consisting of a rise in cytosolic Ca2+ ,45,46,472+ ,45,46,482+ ,42,49,502+ levels [2+ signaling can be initiated by G protein-coupled receptors  or in general by other ligand-receptor interactions, including Fcɣ-receptors (antibodies), and the activation of CD11b/CD18  as well as by cellular crosstalk, such as platelet–neutrophil interaction  [2) into inositol 1,4,5-trisphosphate (IP3), and diacylglycerol (DAG) [3 triggers Ca2+ release from the endoplasmic reticulum (ER), causing an initial rise in intracellular Ca2+. The decrease in Ca2+ concentration in the ER induces store-operated calcium entry (SOCE). Stromal interaction molecule (STIM) 1 and 2 on the ER membrane translocate to defined regions at the plasma membrane, interacting with Ca2+ release-activated Ca2+ (CRAC) channels (predominantly Orai1 in neutrophils) to initiate the influx of extracellular Ca2+ [2+ of fMLF-stimulated neutrophils by the generation of the nicotinamide adenine dinucleotide (NAD+) metabolites ADP-ribose (ADPr) and to a lesser extent cyclic ADP-ribose (cADPr) [+/Ca2+-influx) [2+-influx from the ER, independent of IP3-SOCE Ca2+-stores) [2+ activates several kinases as well as ion channels and cellular functions, as reviewed in [2+ activates various isoforms of protein kinase C (PKC), which phosphorylate among others p47 phagocyte oxidase , an important step in the activation of the multicomponent enzyme NOX [2+ triggers secondary (electro-)physiological changes, including cellular depolarization via NOX activation [Upon stimulation, neutrophils respond with a rapid and pronounced increase in cellular Ca+ levels ,47,51,52ctively) ,47. GPCRol (DAG) ,54. IP3 lar Ca2+ ,56,57. I (cADPr) , which i-influx)  and ryan-stores) ,61, respzyme NOX ,63,64,65tivation ,67,68, itivation , and cel2), thereby generating superoxide anions (O2•−) [2+ signaling [+ efflux via voltage-gated proton channels (voltage sensing domain only protein (VSOP) / Hv1 as its respective human homologue) [v1-deficient mice, which responded to stimulation by the chemoattractant fMIVIL  with cellular acidification as well as a significantly reduced rise in Ca2+ and subsequent impaired actin depolymerization and cellular migration [Neutrophil stimulation results in the activation of NOX, which transfers two electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to molecular oxygen (Os (O2•−) . In thiss (O2•−) ,69,70,71ignaling ,68,72. Tmologue) ,68,73. Tigration .2+ as well as the intracellular accumulation of H+ generated by NOX initiate H+ extrusion, via Hv1 and the sodium-proton exchanger 1 (NHE1) [v1 is a voltage-dependent H+ channel activated by cellular depolarization [2+ activate calmodulin and other kinases, which can phosphorylate and thereby activate NHE1 together with the initial NOX-mediated increase in intracellular H+ concentration [+/K+-ATPase (by Omeprazole), H+-ATPase (by Bafilomycin), or the Hv1 channel (by zinc) did not prevent C5a-induced alkalization significantly [The rise in intracellular Ca1 (NHE1) ,75,76,77rization ,72,78. Tntration ,80,81. Tficantly .+ via monocarboxylate transporters, thereby contributing to extracellular lactate acidosis [3− [−/HCO3− exchanger  appears to be inhibited by fMLF [−/HCO3− exchange mechanism (likely AE1) appears to mediate the recovery from an alkaline load [3− has frequently not been supplemented in culture media, presumably to facilitate the control of the extracellular pH. Regarding ion fluxes, changes in intracellular pH interact with CRAC-mediated Ca2+-influx and the release of O2•−. For example, intracellular alkalosis increases intracellular Ca2+ and thus cellular function, including migration [2•− release [3− and other important blood ions and proteins in the buffer for neutrophil experiments, it is also noteworthy that most of the described mechanisms have been demonstrated mainly for fMLF and/or C5a stimulated neutrophils.Following initial stimulation, this intracellular alkalization continues for up to an hour and modulates various crucial cellular functions, including fMLF-induced IL-8 release and C5a-induced increase in glucose metabolism ,49. Thisacidosis . It is nosis [3− . Additio by fMLF . Accordiine load ,83. In tigration ,85. Addi release ,86. Besi+ as well as water at the protrusive end, active reorganization of the actin cytoskeleton, and an efflux of ions and water at the retractive pole [The discussed ion–water fluxes in neutrophils after stimulation with chemoattractants also interact closely with cellular migration, which has been addressed in other reviews ,88,89. Hive pole ,87,88,89ive pole ,92. Addiive pole . In the ive pole ,95,96,97ive pole .In sepsis, neutrophils are exposed to a broad spectrum of inflammatory mediators, including cytokines, chemokines, and complement fragments as well as to bacterial endotoxins, which can possibly impact neutrophil ion and water flux and thereby modulate neutrophilic functions. In accordance with this, numerous pivotal functions of neutrophils, including phagocytosis, oxidative burst production, and chemotactic activity, can be impaired during severe sepsis ,100,101.+ transport proteins) for neutrophil chemotaxis in sepsis [In agreement with the proposed alterations of ion transport proteins, an increase in neutrophil size and cell volume can be observed in murine sepsis ,11 as wen sepsis . In thisn sepsis ,102. In n sepsis  or for pn sepsis . However2+ currents and thereby modulate any micro-movement efforts, including during migration, chemotaxis, phagocytosis, and cytokine release [One consequence of this “metabolic switch” when turned on during sepsis may also feature shape changes of neutrophils reflected by an increase in the cellular length/width ratio . Of note release .2+ levels in neutrophils may also constitute a valuable strategy to modulate neutrophil-drive inflammation and/or to restore neutrophil function [2+ was associated with an impairment on phagocytotic activity, which could be ameliorated with the calcium channel blocker amlodipine [2+ is linked with reduced bacterial clearance of pathogens in neutrophils from individuals with cystic fibrosis, which also was improved by reducing intracellular Ca2+ [2+ levels and to explore the possibility to target Ca2+ homeostasis as mediator of neutrophil dysfunction during sepsis.Controlling Cafunction ,105. Forlodipine . Likewislar Ca2+ . FurtherIn addition to ion transport proteins, AQPs modulate fluid shifts in neutrophils, albeit their specific function in sepsis remains rather speculative. AQP5 protein expression has been reported to be downregulated during inflammation as a response to proinflammatory cytokines ,109. IntChanges in neutrophil ion and water homeostasis, frequently measured by changes in cell size, have been of increasing interest as diagnostic and prognostic markers in sepsis and other systemic inflammation conditions. A meta-analysis of the Area Under the Receiver Operating Characteristic Curve (AUROC) reported that a mean neutrophil volume of 0.87 (0.83–0.89) was an indicator for sepsis . In acco2+ influx, K+ efflux, activation of NF-κB as well as synthesis of IL-1β and IL-8 [Some MAMPs and DAMPs can disrupt the integrity of the leukocyte membrane by pore-forming molecules. In the case of MAMPs, various bacteria-derived toxins can generate differently sized pores in neutrophils which in turn lead to ion and water imbalances and in the worst case to cell death. Some sepsis-relevant microbial toxins are exemplarily summarized: Panton-Valentine leukocidin (PVL) is a toxin produced by some staphylococcal strains which can interact with the C5aR1 and induce lytic pores in neutrophils  as well and IL-8 ,120. Oveand IL-8 .2+ and the release of myeloperoxidase and IL-8 [Important representatives for DAMPs are histones and extracellular ATP. For example, histone H4 causes neutrophil membrane depolarization, a rise in intracellular Caand IL-8 . Interesand IL-8 . ATP, whand IL-8 . At highand IL-8 . In pyroand IL-8 . Howeverand IL-8 ,129,130,and IL-8 ,132,133.and IL-8 .2+ sensors STIM1 and 2, which activate the Ca2+ channel Orai1, guarantees sustained Ca2+-influx when activated  and thereby generates intracellular “Ca2+ hotspots” which augment phagocytosis [SOCE in neutrophils, regulated by the Caocytosis  and promocytosis ,135,136.Contradictory reports have been published regarding the phagocytic and oxidative burst activity of neutrophils during sepsis, which, however, appear mainly dependent on the timing and severity of sepsis, the maturity of neutrophils, and the local environment ,138. Durphox to the membrane, where it assembles with other components, including p40phox, p67phox, Rac1/2, and flavocytochrome b558, to the NOX complex [2•−), which is subjected to dismutation generating hydrogen peroxide (H2O2) and ultimately HOCl via the myeloperoxidase [−-conductance for compensation of the electrical charge out of the vacuole [phox and impaired ROS generation [2+ levels of resting neutrophils [To kill phagocytized bacteria, effective antimicrobial mechanisms are essential. These mainly involve an oxidative burst with reactive oxygen species (ROS) generation that can be oxygen-dependent or -independent . Oxygen- complex . The eleroxidase ,150. The vacuole ,152,153.neration ,154, whineration  as well neration , but nottrophils , which ctrophils ,48, LPS-trophils .Moreover, extracellular acidification, as present in local and systemic inflammation, directly inhibits bacterial killing by neutrophils, while in contrast promoting phagocytosis . In acco+/H+ exchanger [+ channel VSOP and its respective human homologue Hv1 are considered essential for the excretion of protons produced by NOX [v1−/− mice had a substantially reduced superoxide production and impaired bacterial clearance both in-vitro and in-vivo indicates the requirement of Hv1 for sufficient antibacterial activity in sepsis [v1 expression or activity actually do contribute to neutrophil dysfunction in severe sepsis has to date hardly been addressed and requires further research.Regarding the oxygen-independent mechanism of bacterial killing, lysosomal enzymes are released into the phagosomes, where after an initial alkalization phase the now phagolysosomes become acidified by activation of the Naxchanger . In addid by NOX ,69. The n sepsis . Notably2+-homeostasis in neutrophils also appears crucially involved in the mounting of an oxidative burst and other antimicrobial functions. Several Ca2+-conductive ion channels are expressed in neutrophils, including the TRP family and store-operated Ca2+ channels. TRPM2, a Ca2+ permeable, non-selective cation channel, which is activated by ADPr, temperature, oxidative stress, and Ca2+ [2+ mobilization and oxidative stress in neutrophils. For example, during experimental sepsis in mice infected with Listeria monocytogenes, the TRPM2 cation channel modulated membrane depolarization, Ca2+ mobilization, and subsequent ROS generation [2+, and aggravated oxidative burst, all of which might be harmful to the host [2+ permeability could limit the oxidative burst response during LPS-induced murine sepsis [Moreover, Caand Ca2+ , has beeneration . In thisthe host . Anothere sepsis . In thise sepsis ,162.Finally, ion concentrations and pH values of the local inflammatory microenvironment of the neutrophils will self-evidently to some extent alter the intracellular milieu and thereby also influence the phagocytic and oxidative burst activity ,163,164.A multitude of triggers and signaling pathways have been described in initiating and regulating neutrophil death ,165. RapPyroptosis is activated by cytosolic inflammasome perturbations. Inflammasome activation can be induced by intracellular pathogens or by intracellular exposure to LPS that result in activation of inflammatory caspases that cleave gasdermin D (GSDMD) ,165. IntN-terminal fragments [Recent findings documented that GSDMD can also be processed by neutrophil elastase. Cleavage occurs upstream of the canonical caspase cleavage site, but still produces pore-forming ragments ,170. Howragments . By contragments . Thereforagments .2+- and Na+-influx and K+-efflux, which results in NLRP3 inflammasome activation [Extracellular ATP can activate inflammasome signaling in neutrophils via activation of P2X7Rs on neutrophils . P2X7R itivation ,173. Whetivation .Activated neutrophils can neutralize invaders by releasing NETs  in a pro2+ mediates ROS production, essential for lytic NETosis. This is probably a result of NOX activation [2+ chelator, inhibited NETosis [2+ levels originates from Ca2+ entry [2+ stores [2+ chelators over a prolonged period could induce a homeostatic response in which a decrease in extracellular Ca2+ results in increased Ca2+ release by the ER to maintain intracellular Ca2+. Such a phenomenon could explain, in part, such a discrepancy between the studies [3−-mediated alkalization of neutrophils favored spontaneous and LPS-induced NETosis, possibly because of modulation of intracellular Ca2+ [Increases in intracellular Cativation ,179, acttivation . In agre NETosis . However2+ entry  or relea+ stores . Dependi studies . Activat studies . Intereslar Ca2+ .2+ signals that themselves depend on intracellular or plasma membrane Ca2+ channels.In lytic  NETosis, NET extrusion occurs via GSDMD pores ,170 and/+ efflux, and NLRP3 inflammasome assembly, which result in NLRP3-dependent caspase-1 activation and GSDMD cleavage [Apoptosis, pyroptosis, and NETosis were traditionally considered to be independent of one another, but emerging evidence indicates that there is extensive cross-talk between them. All three pathways can converge upon the activation of the same cell death effector—the pore-forming protein GSDMD . Similarcleavage .N-terminal domain (GSDMD-N) connected via a linker to the regulatory C-terminal domain. Cleavage of GSDMD at a conserved site releases GSDMD-N from the auto-inhibitory C-domain. GSDMD-N has the ability to translocate to the inner leaflet of the plasma membrane, where it oligomerizes and induces the opening of membrane pores [+) causes an increase in cell volume due to water influx and can result in osmotic cell lysis. This likely depends on the number of GSDMD pores in the plasma membrane. The number of GSDMD pores can be limited by a recently discovered mechanism that actively removes GSDMD pores from the plasma membrane. Ca2+-influx via GSDMD pores recruits endosomal sorting complexes required for transport (ESCRT) to damaged areas of the plasma membrane to remove GSDMD pores and to maintain membrane integrity [+, Cl−, and organic osmolyte  channels that export these solutes and their accompanying water [GSDMD consists of a pore-forming ne pores ,192,193.ne pores . The resntegrity . This isng water . In partng water ,197. A dIon and water fluxes regulate essential neutrophil functions. Their alterations during sepsis impair vital cellular functions and may thereby contribute to immune and organ dysfunctions. Fields of particular interest include first, a further elucidation of the ion–water disbalance upon excessive neutrophil activation, second, an in-depth evaluation of physiological parameters, including intracellular pH and cellular size of neutrophils as a diagnostic and/or prognostic marker of immune dysfunction, and third, a meaningful translation to the bedside by modulating ion–water based alterations, for example, by pharmacological targeting of perpetrator ion transport proteins, such as NHE1, during sepsis.


Patients with t-AML were older but had a similar mutational profile compared to those with non-t-AML. Overall survival (OS) and relapse-free survival (RFS) were significantly worse for patients with t-AML . Patients with t-AML had a higher cumulative incidence of death in remission , but not higher cumulative incidence of relapse . Both intensive induction and allogeneic hematopoietic stem cell transplantation in first remission were associated with improved OS and RFS in non-t-AML but not in t-AML. Overall, although disease biology appears similar between t-AML and non-t-AML with normal karyotype as indicated by similar risks of relapse, death in remission is the main driver of inferior outcome in t-AML. Careful therapeutic decisions are required to mitigate potential treatment-related toxicity in this rare subgroup of patients with t-AML and normal karyotype.Normal karyotype in therapy-related acute myeloid leukemia (t-AML) is rare and the relative contribution of prior exposure to chemotherapy or radiotherapy to outcomes in these patients remains uncertain. We performed a retrospective study of 742 patients with newly diagnosed AML and normal karyotype (t-AML,  Several patient-related or disease-related factors explain the poor prognosis of patients with t-AML. Patient-related factors include older age at presentation, higher number of comorbidities and sequelae from prior cancer and prior therapy. Disease-related factors include higher frequency of adverse risk features such as complex cytogenetics, chromosomal aneuploidies  and high frequency of TP53 mutations, all associated with resistance to conventional cytotoxic chemotherapy11. In multiple reports, the adverse prognosis observed in patients with t-AML was not independent of other variables including age, cytogenetics and molecular features raising the question whether the presence of t-AML, per se, confers a poor prognosis versus prognosis influenced by its strong association with older age and adverse cytogenetics15. For instance, the dismal outcome observed among patients with t-AML in one large study (median OS of 8 months) was primarily driven by abnormalities of chromosome 5 and 7, whereas t-AML with favorable karyotype (core binding factor-AML) had a median OS of 26.7 months4. Several reports suggest that the prognosis of t-AML is likely similar to that of de novo AML with corresponding cytogenetic risk15.Therapy-related acute myeloid leukemia (t-AML) is a distinct clinical entity recognized by the World Health Organization (WHO) as a late complication occurring after exposure to cytotoxic chemotherapy and/or radiation therapy18. Because of its rare occurrence, the molecular features and clinical outcomes of patients with t-AML and normal karyotype are undefined, and it remains unknown whether prior exposure to chemotherapy or radiotherapy in these patients is associated with more aggressive disease biology and higher risk of relapse. Therefore, we aimed to compare the clinical and molecular characteristics of t-AML and non-t-AML with NK and to determine the prognostic impact of prior chemotherapy or radiotherapy exposure in patients with AML and NK.Normal karyotype (NK) among patients with t-AML is rare, accounting for <20% of cases of t-AML2). Other regimens were considered low-intensity therapies  based on their prior exposure to chemotherapy or radiotherapy, regardless of the latency period. We collected the patients’ baseline clinical and molecular characteristics and outcomes including remission status and occurrence of relapse and death, and the intensity of therapy received. Intensive induction chemotherapy was defined as any regimen containing an anthracycline and/or intermediate to high-dose cytarabine  was defined as the time from achieving CR/CRi to relapse, death or last follow-up. OS was defined as the time from start of therapy to death or last follow-up. The Kaplan-Meier method was used to estimate the probabilities for RFS and OS and differences between groups were evaluated with the log-rank test. The cumulative incidence of relapse (CIR) and cumulative incidence of death (CID) were defined as time from remission to relapse and death in remission, respectively, considered as competing events for failure. The Gray’s test was used to compare cumulative incidence probabilities between groups. Univariate and multivariate analyses for RFS and OS were performed using the Cox proportional hazards regression model to calculate hazard ratios (HR) and confidence intervals (CI). Allogeneic hematopoietic stem cell transplantation (HSCT) was considered a time-dependent covariate in the extension of the Cox model. Statistical analyses were performed with R statistical software (version 3.5.1). P-values <0.05 were used to define statistical significance.Response criteria including complete remission (CR), complete remission with incomplete hematologic recovery (CRi), morphologic leukemia-free state (MLFS) and partial remission (PR) were defined according to the 2017 ELN criteriaP < 0.01). Similar proportions of patients in both groups had secondary AML arising from antecedent hematologic disorder  . Patients with t-AML had a higher frequency of TP53 mutation (9% versus 4%) and a lower frequency of CEBPA mutations (5% versus 15%); however, these differences were not statistically significant .A total of 1977 patients with AML and available cytogenetic information were identified including 340 patients with t-AML. Among 742 patients with NK (38% of the entire cohort), 61 patients (8%) had t-AML and 681 patients (92%) had non-t-AML . However, the rates of HSCT in first remission (CR1) were similar between the two groups  as well as the rates of enrollment in clinical trials . Since induction therapy intensity markedly differed between groups and could influence response rates and survival outcomes, we stratified patients according to intensity of treatment (Table P = 0.05). Conversely, among patients who received low-intensity induction therapy, CR/CRi rates were similar among those with t-AML versus non-t-AML . Of note, venetoclax-based therapy  was used in 8 patients (13%) with t-AML and in 46 patients (7%) with non-t-AML, with CR/CRi rates of 87 and 84%, respectively.Intensive induction chemotherapy was less frequently administered to patients with t-AML compared to those with non-t-AML  with three out of the four deaths in the t-AML group occurring in remission due to treatment-related complications. In patients treated with low-intensity therapies, there was a non-statistically significant trend toward increased 60-day mortality in those with t-AML .The 60-day mortality was significantly higher in patients with t-AML treated with intensive induction , and the median RFS was 12.0 months and 14.9 months  for t-AML and non-t-AML, respectively  and RFS (P = 0.05). Accordingly, intensive induction was only beneficial in patients with non-t-AML with median OS of 43.0 months and 10.8 months for patients receiving high- and low-intensity therapy, respectively (P < 0.001), whereas outcomes for patients with t-AML were similarly poor regardless of the intensity of therapy  and the benefit of intensive chemotherapy in patients with non-t-AML was less pronounced  or RFS  but not CIR  , but not with CID (P = 0.2). These findings suggest that non-relapse mortality was the main driver for worse outcomes in patients with t-AML and NK compared to those with non-t-AML and NK. The distribution of causes of deaths in remission was comparable between groups  and RFS  in the overall population when HSCT in CR1 was considered a time-dependent variable. However, the benefit of HSCT in CR1 on OS and RFS was restricted to patients with non-t-AML , whereas patients with t-AML did not derive any benefit from HSCT in CR1   and RFS (12.0 versus 14.9 months). Importantly, we show that the inferior outcome was largely driven by an increased risk of death in remission rather than differences in molecular features or relapse risk.NPM1 (39–45%) and FLT3-ITD (23–30%) mutations were similar in both groups and comparable to what has been reported in large datasets of AML with NK16. Patients with t-AML had a slightly higher frequency of TP53 mutation (9% versus 4%) and lower frequency of CEBPA mutations (5% versus 15%), although these differences were not statistically significant, possibly due to the small numbers of sequenced patients in the t-AML subgroup. Therefore, these data suggest that t-AML with NK often has similar disease biology to non-t-AML, at least as reflected by the presence of recurrent molecular mutations. The similar mutational spectra observed in patients with t-AML and non-t-AML with NK question the pathogenic role of prior chemotherapy or radiotherapy in the development of NK AML. Thus, in the rare subset of patients with chemotherapy or radiation exposure who later develop NK AML, our data suggest that the association between prior therapy and AML diagnosis may be coincidental rather than causative.Within this subset of patients with AML, we confirm that factors other than cytogenetics account for the adverse prognosis of patients with t-AML and NK. We investigated the mutational profiles among our cohort of patients with NK to evaluate whether adverse molecular features in t-AML could explain differences in prognosis. Interestingly, no significant difference was noted in the mutational frequencies or ELN 2017 risk category between the two groups. The frequencies of 21. Nonetheless, post-remission outcomes were significantly different between groups. t-AML had significantly higher 60-day mortality, especially when treated with intensive induction therapy. Notably, deaths in remission were mostly due to infections and there was no substantial contribution from a concurrent malignancy other than AML. Indeed, survival of t-AML with NK was significantly inferior compared with non-t-AML with NK. Although multivariate analysis showed that t-AML was not an independent factor for RFS, there was a trend toward statistical significance for OS . Importantly, we show that the poorer RFS and OS in patients with t-AML and NK are due to a markedly higher CID  rather than an increased risk of relapse. This increased CID among patients with t-AML was observed in both those who received low-intensity therapy and those who received high-intensity therapy, and both when we censored survival at the time of HSCT and when we considered post-HSCT outcomes in patients who proceeded to HSCT in CR1. Although this might suggest that intensive induction is not associated with any benefit in t-AML with normal karyotype, these findings have limitations. Firstly, this is a retrospective study and interpretation of such analysis must be done with caution, especially since we did not take into account many patient-related factors that may confound the results. Secondly, most patients with t-AML were older than 60 years, and therefore these findings may not be applicable to younger patients.Remission rates and relapse rates were also similar between patients with t-AML or non-t-AML with NK, which provides further support that prior exposure to chemotherapy or radiation, per se, may not necessarily lead to a more aggressive disease biology. These findings are consistent with prior data that have linked poor response to therapy among t-AML largely to specific adverse chromosomal abnormalities, which are generally enriched in this population3. However, in older patients, they observed that t-AML was associated with an increased CIR but not with an increased CID, which contrasts with our findings. They suggested that the higher risk of relapse in older patients with t-AML may be explained by the administration of lower-intensity therapy, but it could also be attributed to the higher frequency of adverse features in older patients with AML. However, it is important to note that, in our cohort, most patients with t-AML were ≥ 60 years of age (90%). In addition, our analysis accounted for therapy intensity, and showed that the CIR among patients with NK t-AML was similar to that of patients with non-t-AML treated with intensive therapy, whereas CID was significantly higher for patients with NK t-AML, regardless of therapy intensity. Intensity of therapy was not associated with CID and the difference in CID was relatively modest when patients were stratified by age . In contrast, the rate of CID was over 3-fold higher in patients with t-AML versus those with non-t-AML , further suggesting that prior exposure to chemotherapy or radiation is the dominant factor driving CID and thus inferior survival.Most prior studies reporting on the inferior survival of t-AML have not evaluated CIR and CID, therefore limiting our ability to understand whether the adverse outcome of t-AML is due to more aggressive disease biology or patient-related factors, including tolerance of therapy. Notably, one group previously showed that the outcome of t-AML varied according to age, and demonstrated that t-AML was associated with increased CID but not CIR in younger patients treated with intensive chemotherapy25. Among all patients with NK in our cohort, HSCT in CR1 was associated with improved OS and RFS. However, this benefit was restricted to patients with non-t-AML. The 4-year non-relapse mortality after transplantation was about 40% in patients with t-AML, similar to previous reports on t-AML, which likely outweighs the potential benefit of HSCT in most of these patients26. Our data therefore suggest that HSCT in CR1 likely offers limited benefit in most patients with NK t-AML because of a high rate of death in remission. However, it is important to note that 90% of patients with t-AML and NK in our dataset were ≥60 years of age, and therefore we cannot extrapolate these data to the rare patients who are younger than 60 years of age. Proceeding to HSCT in CR1 may still be beneficial to these patients if they are fit and otherwise meet the indications for HSCT.HSCT in CR1 has been demonstrated to reduce the risk of relapse in subsets of patients with AML and NK28.These findings have significant clinical implications. With similar mutation profiles, response to therapy and risk of relapse between NK t-AML and non-t-AML, our study suggests that the adverse outcome observed in patients with NK t-AML is mostly driven by poorer tolerance to therapy rather than more aggressive disease biology. We hypothesize that older age, comorbid medical conditions, sequelae of prior disease, and the cumulative toxicity of primary and secondary cancer therapy may all contribute to this higher non-relapse mortality rate. Since prior reports have consistently shown poor OS and RFS with high rate of relapse in patients with t-AML, the general practice has been to treat these patients with intensive induction therapy followed by consolidation with HSCT in CR1. Based on our findings, even though t-AML with NK has similar disease biology as non-t-AML with NK, a comprehensive evaluation of patients’ comorbidities and fitness level is particularly imperative in patients with t-AML in order to appropriately select therapy as high rates of death in remission are observed in these patients, particularly with intensive therapy. Consequently, the threshold to consider a patient eligible to receive intensive chemotherapy and undergo HSCT in CR1 may need to be higher in patients with NK t-AML. This might be particularly relevant considering the improved outcomes with lower-intensity regimens such as the combination of hypomethylating agents and venetoclax for the treatment of older patients or patients deemed ineligible for intensive chemotherapyIn conclusion, we have shown that, among patients with NK, prior exposure to chemotherapy or radiation is associated with poorer outcomes because of a higher risk of death in remission rather than an increased risk of relapse. Although disease biology appears similar between t-AML and non-t-AML with NK, careful therapeutic decision-making is required to mitigate potential treatment-related toxicity for patients with t-AML and NK.Supplemental MaterialReproducibility checklist


Although targeting histone deacetylases (HDACs) may be an effective strategy for core binding factor‐acute myeloid leukemia (CBF‐AML) harboring t or inv(16), HDAC inhibitors are reported to be limited by drug‐resistant characteristic. Our purpose is to evaluate the anti‐leukemia effects of Baicalein on CBF‐AML and clarify its underlying mechanism.Enzyme activity assay was used to measure the activity inhibition of HDACs. Rhodamine123 and RT‐qPCR were employed to evaluate the distribution of drugs and the change of ATP‐binding cassette (ABC) transporter genes. CCK8, Annexin V/PI, and FACS staining certified the effects of Baicalein on cell growth, apoptosis, and differentiation. Duolink and IP assay assessed the interaction between HDAC‐1 and ubiquitin, HSP90 and AML1‐ETO, and Ac‐p53 and CBFβ‐MYH11. AML cell lines and primary AML cells‐bearing NOD/SCID mice models were used to evaluate the anti‐leukemic efficiency and potential mechanism of Baicalein in vivo.+ inv(16) AML cells.Baicalein showed HDAC‐1/8 inhibition to trigger growth suppression and differentiation induction of AML cell lines and primary AML cells. Although the inhibitory action on HDAC‐1 was mild, Baicalein could induce the degradation of HDAC‐1 via ubiquitin proteasome pathway, thereby upregulating the acetylation of Histone H3 without promoting ABC transporter genes expression. Meanwhile, Baicalein increased the acetylation of HSP90 and lessened its connection to AML1/ETO, consequently leading to degradation of AML1‐ETO in tq AML cells. In inv(16) AML cells, Baicalein possessed the capacity of apoptosis induction accompanied with p53‐mediated apoptosis genes expression. Moreover, CBFβ‐MYH11‐bound p53 acetylation was restored via HDAC‐8 inhibition induced by Baicalein contributing the diminishing of survival of CD34These findings improved the understanding of the epigenetic regulation of Baicalein, and warrant therapeutic potential of Baicalein for CBF‐AML. Baicalein shows HDAC1/8 inhibition without promoting ABC transporter genes expression.2. Baicalein exerts therapeutic effect in CBF‐AML.3. The inhibition of HDAC1/8 could be potential therapeutic target for CBF‐AML treatment. AMLacute myeloid leukemiaCBFcore binding factorCHXcyclohexumideCMCBFβ‐SMMHCCTCLcutaneous T‐cell lymphomaDMSOdimethylsulfoxideFABFrench‐American‐BritishFITCfluorescein isothiocyanateHDAChistone deacetylaseHSP90core binding factorLSCleukemia stem cellMDR1multidrug resistance 1NABsodium butyrateN‐CoRnuclear receptor corepressorPEphycoerythrinSMMHCsmooth muscle myosin heavy chain1Acute myeloid leukemia (AML) is an aggressive hematological neoplasm caused by abnormal hematopoietic progenitor cells.0).AML1, whose encoding protein is a subunit of CBF‐α, and ETO generates a novel gene AML1‐ETO, resulting in a lack of the carboxyl terminal transactivation domain of AML1, which suggests that the AML1‐ETO disrupts hematopoiesis through a dominant‐negative mechanism.CBF‐AML encodes two recurrent cytogenetic abnormalities referred to as tq and inv (16).CBF‐β with MYH11 gene. The two non‐ampliflying inv(16) cases form two chimeric genes, CBF/MYH11 and MYH11/CBF. However, only CBFβ/MYH11 that encodes a CBFβ‐MYH11 smooth muscle myosin heavy chain (SMMHC) protein contributes to the leukemogenesis.In the other type of CBF‐AML, the inv(16) results in the fusion of Interfering with the function of pro‐leukemic fusion proteins is an effective strategy for AML treatment. HDACs are critical epigenetic modulating‐factors implicated in cancer, especially in causation and progression of CBF‐AML.multidrug resistance 1 (MDR1) and its encoding protein p‐gp are believed to be a typical side‐effect of HDAC inhibitors. Treatment of HDAC inhibitors, such as Apocodin, increased rhodamine‐123 efflux and modulated MDR1 expression in Hela cells.MDR1 in HepG2, SW620, and KG1a cells.MDR1.MDR1, BCRP, MRP7, and MRP8.HDAC inhibitors are widely investigated in cancers, which show synergistic effect with certain anticancer drugs.Scutellaria baicalenesis, has been reported to mediate growth inhibition of human leukemia cells (K562 and HL‐60).Baicalein, a flavonoid extracted from the root of 22.115H10O5, MW: 270.24 g/mol) with the purity of 97% was determined by HPLC. For in vitro experiments, Baicalein was dissolved in dimethyl sulfoxide  at a concentration of 0.1 M, stored at ‐20°C and diluted to a suitable concentration with RPMI‐1640 medium . Cells treated with the highest concentration of DMSO were used as controls in the indicated experiments.Baicalein  by Dr. Xue Ke from College of Pharmacy, China Pharmaceutical University. Sodium butyrate  and sodium valproate  were prepared as intraperitoneal administration  by Dr. Xue Ke from College of Pharmacy, China Pharmaceutical University.Fluorescein isothiocyanate (FITC) anti‐human CD14 and phycoerythrin (PE) anti‐human CD11b antibodies were obtained from eBioscience . TSA (CSN12139) and PCI‐34051 (CSN16819) were obtained from CSNpharm . MG‐132 (HY‐13259) and Z‐VAD‐FMK (HY‐16658B) were obtained from MedChemExpress .2.22 at 37°C. All cells used were passaged in our laboratory for less than 3 months after resurrection.Human AML cell lines U937, THP‐1, Kasumi‐1, SKNO‐1, and ME‐1 were purchased from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology. Primary leukemia cells from AML patients  were collected using lymphocyte‐monocyte separation medium . Primary AML cells and AML cell lines were cultured in RPMI‐1640 medium, supplemented with 10% FBS, 100 U/mL of benzyl penicillin, and 100 U/mL of streptomycin in a humidified environment with 5% CO2.37 cells per mouse) via tail vein in 24 h following the radiation treatment. Seven days later, the mice were injected by intragastric with or without Baicalein (80 mg/kg) or VPA/NAB (200 mg/kg) every other day for 4 weeks. Animals in the control group were injected with normal saline to evaluate the effects of injection on survival. Next, the animals were inspected daily for 4 weeks. Finally, peripheral blood, bone marrow (BM), and spleen cells were prepared for flow cytometry after labeling with huCD45. The BM biopsy was used to perform immunofluorescent staining.Female NOD/SCID mice (6‐9 weeks old)  were sublethally irradiated (1.8 Gy), and were engrafted with human primary AML cells  under controlled lighting (12 h light/day) and supplied with standard laboratory food and water ad libitum throughout the experimental period. The animal study was carried out according to the regulations of the China Food and Drug Administration (CFDA) on Animal Care.2.4For enzyme activity assay, the purified protein of HDACs were incubated with substrates that contained acetylated lysine side chains. The substrate of HDAC‐1 , HDAC‐2 , and HDAC‐3  were K379‐382 (RHKK(Ac)AMC) residues of p53. The substrate of HDAC‐8  was p53‐K379‐K382(RHK(Ac)K(Ac)AMC). The deacetylation of the substrates react with the lysine developer and the fluorescence were positive correlation with the level of deacetylation. Fluorescent signals were analyzed by multi‐function microplate reader. The positive control was HDAC inhibitor TSA.For cellular thermal shift assay (CETSA) was performed to determine the direct binding between Baicalein and HDAC‐1/2/3/8 in ME‐1 cells.2.5HDAC‐1 and HDAC‐8 siRNA were synthesized by GenePharma Co, Ltd . Transfection was performed using Lipofectamine 2000™  according to the manufacturer's instructions provided by the vendor. First, siRNA or the negative control and transfection reagent were diluted in serum‐free 1640, respectively. After incubated at room temperature for 20 min, the mixture was delivered into the cells. Cells were collected for further experiments after incubated for 48 h.HDAC‐1 was 5′‐AUAAACGCAUUGCCUGUGAUCAAAGAAGAGGUCAAGUU‐3′, and the anti‐sense was 5′‐UGACCAACCAGAACACUAAGAACUCUUCUAACUUCAAA‐3′.The siRNA sense oligonucleotides for HDAC‐8 was 5′‐CAUCGAAGGUUAUGACUGUUGACUAUGCAGCAGCUAUA‐3′, and the anti‐sense was 5′‐CUACGUGGAUUUGGAUCUAGAUGAGAAGUACUAUCACA‐3′.The siRNA sense oligonucleotides for 2.65 cells.Cell differentiation was assessed by NBT reduction and Giemsa Staining as previously reported.2.7Western blot was performed with standard protocols.Primary antibodies against C/EBPα, HSP90, ETO, and β‐actin were obtained from Santa Cruz Biotechnology . Primary antibodies against HDAC‐1, Histone H3, and acetyl‐Histone H3 were products of Cell Signaling Technology . Primary antibodies against HDAC‐1, Histone H3, acetyl‐Histone H3, and acetyl‐p53 (K382) were purchased from Cell Signaling Technology . Primary antibodies against CBFβ, HDAC‐8, and pan‐acetyl‐lysine were obtained from Abclonal Technology . Alexa Fluor 488 Goat anti‐Mouse IgG (H + L) cross‐adsorbed secondary antibody was purchased from ThermoFisher Scientific . IRDyeTM 800‐conjugated goat anti‐mouse and goat anti‐rabbit secondary antibodies were purchased from Rockland Inc. .2.8Immunofluorescence was performed as previously described.2.9Quantitative real‐time PCR (RT‐qPCR) was performed according to the manufacturer's instructions.GAPDHForward 5′‐GCAGGGGGGAGCCAAAAGGG‐3′Reverse 5′‐TGCCAGCCCCAGCGTCAAAG‐3′DR5Forward 5′‐CTGTGCATTCGTCTCTCTTGG‐3′Reverse 5′‐TGAGTCGTTTCCGTTTACCG‐3′PUMAForward 5′‐CACCCCATCGCCTCCTTTCT‐3′Reverse 5′‐GGAAGGGGCGCGGACTGTCG‐3′NoxaForward 5′‐AGATGCCTGGGAAGAAG‐3′Reverse 5′‐AGTCCCCTCATGCAAGT‐3′BaxForward 5′‐TCAAGGCCCTGTGCACTAA‐3′Reverse 5′‐TGAGGACTCCAGCCACAAA‐3′PIG5Forward 5′‐GAAGGATGTGGCGAAGGGA‐3′Reverse 5′‐CCACAAGACCGTCTACCTGCA‐3′Bcl‐xlForward 5′‐GATGCAGGTATTGGTGAGTCGG‐3′Reverse 5′‐ATCCACAAAAGTGTCCCAGCC‐3′MDR1Forward 5′‐CATTGGCGAGCCTGGTAG‐3′Reverse 5′‐TCGTAGGAGTGTCCGTGGAT‐3′MRP8Forward 5′‐AAACTTCTCTGTGGGGGAGA‐3′Reverse 5′‐GGGTGTCTGTCTCCATGTCA‐3′MRP7Forward 5′‐CATGCAAGCCACGCGGAACG‐3′Reverse 5′‐AAGCTGGGCTGGTGGAGGGT‐3′BCRPForward 5′‐TGCCCAGGACTCAATGCAACA‐3′Reverse 5′‐ACAATTTCAGGTAGGCAATTGTG‐3′HDAC‐1Forward 5ʹ‐CTACTACGACGGGGATGTTGG‐3ʹReverse 5ʹ‐GAGTCATGCGGATTCGGTGAG‐3ʹHDAC‐8Forward 5ʹ‐ GGCTGCGGAACGGTTTTAAG‐3ʹReverse 5ʹ‐ GCTTCAATCAAAGAATGCACCATAC‐3ʹThe primer sequences were as follows:2.10FACS analysis of whole blood was performed as previously described.2.11Duolink assay was performed by Duolink in Situ Detection Reagents Red (DUO92008) according to the manufacturer's instructions.2.12Cells were pretreated with or without Baicalein (30 µM). Cell lysate was incubated with appropriate concentration of antibody and 20 µL protein A/G‐conjugated beads (Santa Cruz) at 4°C overnight. After washing three times with RIPA buffer (Thermo Fisher), samples were collected and re‐suspended in 20 µL SDS‐sample buffer  and boiled for 10 min. Then the samples were subjected to western blot.2.13t‐tests. A P‐value < .05 was considered statistically significant.All data were expressed as mean ± SD. The data shown were obtained from triplicate independent parallel experiments. Statistical analysis of multiple group comparisons was performed by one‐way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. Comparisons between two groups were analyzed using two‐tailed Student's 33.150 values of Baicalein on HDAC‐1 and HDAC‐8 were 4.67 × 10−5 and 3.95 × 10−6 M, respectively  values for HDAC‐1, HDAC‐2, HDAC‐3, and HDAC‐8 of DMSO group were 47.78, 50.69, 46.01, and 50.98°C while the Tagg values for HDAC‐1, HDAC‐2, HDAC‐3, and HDAC‐8 of Baicalein‐treated group were 53.23, 50.49, 45.84, and 54.13°C, respectively. We applied this method to further confirm the interaction between Baicalein and HDAC‐1 and HDAC‐8 but not HDAC‐2 and HDAC‐3  or TSA (165 nM) for 24 h in AML cell lines and relapsed AML patient samples . However, the mRNA levels of those genes changed slightly after treatment of Baicalein for 96 h , SKNO‐1 with t, and ME‐1 with inv(16), and primary AML cells were cultured in the absence or presence of Baicalein (0‐256 µM). Results showed that the treatment of Baicalein induced significant inhibition of cell viability in those cells, and have shown its superior performance in CBF‐AML by CCK8 assay , a protein synthesis inhibitor and results showed that CHX did not affect Baicalein‐induced HDAC‐1 decrease Figure . We furts Figure . Finallys Figure . Resultss Figure . Furthers Figure . Thus, t3.4HDAC‐1 small interfering RNA (siRNA). The efficacy of transfection was monitored by RT‐qPCR . Cell differentiation analyses were subsequently performed by using FACS assay. Notably, HDAC‐1 knockdown showed a certain degree of cell differentiation, but there was no significant enhance of in the degree of differentiation after treatment of Baicalein, suggesting that the induction of differentiation of AML cells by Baicalein may depend on the inhibitory effect of HDAC‐1 . We also knocked down the expression of HDAC‐8 to verify the pharmacodynamic mechanism of Baicalein on inv(16) AML cells. The efficacy of transfection monitored using RT‐qPCR . Results showed that knockdown of HDAC‐8 in ME‐1 cells could induce apoptosis to a certain degree. However, after transfected with HDAC‐8 siRNA, Baicalein does not enhance apoptotic effects  AML by CCK8 assay Figures  and 2A. s Figure .+ hematopoietic progenitor cells from #42 cells by magnetic activated cell sorting (MACS)  AML.) Figure . Notablyx Figure . Overall3.6+ cells, a marker of human leukocyte, in spleen and bone marrow of Kasumi‐1 and ME‐1 cells‐bearing mice, while either VPA or NAB showed mildly decrease of the huCD45+ cells ‐bearing mice  with tq and #39, a non‐CBF‐AML sample respectively. After administration of Baicalein (80 mg/kg) and VPA (200 mg/kg) every 2 days for 4 weeks, the survival was recorded. Baicalein showed a superior status of the median survival duration in primary AML cells (#29) cells‐bearing mice than in primary AML cells (#39) cells‐bearing mice Figure , Table 3e Figure . Furthere Figure .450 value of ME‐1 was 5.17 ± 1.11 µM at 96 h. However, in U937, THP‐1, Kasumi‐1, and SKNO‐1 cells, the IC50 values of those cells ranged from 13.73 ± 1.07 to 63.94 ± 1.32 µM after Baicalein treatment for 96 h, suggesting different mechanisms induced by Baicalein in ME‐1 cells and other AML cells.HDACs inhibition has been proved to show therapeutic effect through affecting proliferation, differentiation, and survival of AML cells. In the present study, we found that Baicalein inhibited enzyme activities of HDAC‐1/8 and induced degradation of HDAC‐1 protein. Meanwhile, we found that Baicalein inhibited proliferation and induced differentiation of U937, THP‐1, Kasumi‐1, and SKNO‐1 cell lines and primary AML cells derived from a cohort of AML patients. Notably, tq AML cell lines and primary AML cells were more sensitive to Baicalein and the differentiation effect was much more outstanding than other cell lines. In inv(16) AML ME‐1 cells, the ICIn tq AML cells, HDAC‐1 is involved in pathogenesis mechanism induced by fusion protein AML1‐ETO produced by tq.+ inv(16) AML cells. Results showed that Baicalein induced a remarkable increase of apoptosis in the quiescent AML CD34+ population. Taken together, Baicalein restored p53 acetylation in inv(16) AML cells and diminished survival of AML CD34+ cells.As we know, LSCs contributed to the leukemogenesis, and maintained the relapse of AML.C/EBPα is a vital transcription factor in control of lineage‐specific gene expression in hematopoiesis that acts as a tumor suppressor in a number of malignancies.MDR1 gene expression and drug resistance in HL‐60 cells, suggesting that Baicalein might be capable of suppressing MDR1 gene expression via inhibiting NF‐κB signaling.MDR1 gene and inhibiting p‐gp might be the cause of remaining unchanged HDACs‐inhibition‐associated ABC transporter genes.HDACs inhibitors are generally capable of causing the upregulation of MDR1, then inducing a broad and pleiotropic drug resistance of AML cells by regulating multiple ABC transporter genes, which is associated to poor prognosis.+ cells with inv(16). This study demonstrated that Baicalein had a preferential inhibitory effect in CBF‐AML cells, suggesting the potential of Baicalein in developing into a novel agent for the treatment of AML.Even though HDACs inhibition strategy has been known as a promising approach for the treatment of cancers, the clinical efficacy of HDAC inhibitors was limited by board ABC‐associated drug resistance in several cancers.5These findings improved the understanding of the epigenetic regulation of Baicalein, showed that Baicalein inhibited the activity of HDAC‐1 and HDAC‐8 without promoting ABC transporter genes expression, and warrant therapeutic potential of Baicalein for CBF‐AML.The animal study was carried out according to the regulations of the China Food and Drug Administration (CFDA) on Animal Care. All patients’ samples were collected after informed consent in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of China Pharmaceutical University.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.The authors have declared that no competing interest existsX.Y. designed and performed research, analyzed data, and wrote the paper; H.L. performed research and analyzed data; P.H. performed research; Y.Q. and X.W. collected data and performed statistical analysis; M.Z., H.W., and Z.W. collected and analyzed data; J.X. provided the blood samples; and Q.G. and H.H. conceptualized the project and directed the experimental design and data analysis.Supporting Information.Click here for additional data file.


The formationof divalent ion complexes can decrease emulsifying or viscosifyingpower and lead to adsorption or precipitation. This is particularlyrelevant in chemical enhanced oil recovery, where high viscositiesand low interfacial tensions are required for mobility control andthe formation of oil–water microemulsions, respectively. Inthis work, we use a Ca2+-sensitive dye to determine theCa2+ concentration and Ca-complex formation constants insolutions containing complexing agents. This method can be used torapidly screen the affinity of different chemicals to form Ca-complexesin low-salinity solutions. The complex formation constants can beimplemented into chemical flooding simulators to investigate the interplaywith mineral dissolution and cation exchange and model adsorptionprocesses.Chemicals such asanionic surfactants and polymers often containgroups that complex divalent ions such as Ca Complexation of divalentions such as Ca2+ and Mg2+ lowers the viscosityof polymer solutions by introducing attractive polymer–polymerinteractions and leads to precipitation at high concentrations.7 Surfactants also tend to precipitate at high divalent ion concentrations,resulting in an increase in the interfacial tension.8 Divalent ions can also mediate the adsorption of anionicsurfactants and polymers to negatively charged rock surfaces througha process called cation-bridging, resulting in higher concentrationsrequired for chemical flooding.12 It is therefore highly important to measure the affinityof different chemicals to interact with divalent ions and determinethe formation constants of their complexation reactions.Chemical enhanced oilrecovery (EOR) relies on the injection ofchemicals such as polymers and surfactants to improve oil recovery.2+.14 The micelles with complexed Ca2+ ions remain in solutionat low Ca2+ concentrations, while the surfactant can precipitateas Surf2Ca at high concentrations. Complexation also leadsto the somewhat counterintuitive observation that the Ca2+ tolerance of the surfactant solution increases with increasing surfactantconcentration, caused by the effective decrease in [Ca2+] in solution. The complexation of Ca2+ by anionic surfactantscan be expressed asInthe following, we consider the complexation of divalent ionsby anionic surfactants. Above the critical micelle concentration (CMC),most surfactant monomers are incorporated in micelles to minimizethe contact of the hydrophobic tails with the water phase. The negativecharge on the micelle surface is screened by attractive electrostaticinteractions with the cations in solution. In addition, the surfactantheadgroups  can formcomplexes with divalent ions such as Ca2+ complexationby surfactants that are part of a micelle . Given thatK2CaSurf can be expressed asNote thatthis equation also applies to Ca–]ini and [Ca2+]ini are known, only the free (or bound) Ca2+ concentration is required to calculate the complex formationconstant.Expressions for the formation constants of other chemicals can bederived in the same manner. However, measuring the unbound Ca2+ concentration in solutions with complexing agents is notstraightforward: methods relying on Ca2+-selective electrodes,dialysis membranes, and activated carbon columns were explored, butall presented complications such as fouling, long diffusion times,and possible dissociation of Ca-complexes when the solution passesthrough the column. We found the use of a Ca2+-sensitivedye (Pontachrome Violet SW) to be the most effective method to measurethe free Ca2+ concentration in aqueous solutions. Thisdye exhibits a change in absorption spectrum upon forming a complexwith Ca2+ and has previously been used to measure the freeCa2+ concentration in detergent formulations.15 The chemical structure of Pontachrome VioletSW is shown in 2+ concentrationsin the order of tens of mg/L due to its small formation constant andcan be used at pH values between 9.5 and 11, where its extinctioncoefficient εa1 remains constant.Since /[D2–] and the freeCa2+ concentration ([Ca2+]). A relation betweenthe absorbance and [Ca2+] can be obtained as follows. Let y be the molar fraction of free dye such that [D2–] = D0 · y. ThenThe absorption spectrum of a single dye molecule changes uponforminga complex with CaAccording to Lambert–Beer’slawa is the molar extinctioncoefficient of the sample solution and εa1 = 8.87 × 103 cmL/mol, εa2 = 0.864 × 103 cmL/mol, and ε0 = 7.74 × 103 cmL/mol are the molar extinctioncoefficients for the unbound dye, dye–calcium complex, andisosbestic point (520 nm), respectively. q = εa/ε0 can be obtained by measuringthe absorbance at λ = 520, 575, and 680 nm:where ε2+ concentration. The first equation shows that P([Ca2+]) is a straight line passing through with slope KD. Therefore, KD can be determined by measuring the absorbanceas a function of [Ca]. The measured spectra are shown in 2+] ranging from 0 to 35 mg/L in stepsof 5 mg/L.The absorbance at 520 nm is used as a reference point becausethisis the isosbestic point (the absorbance at this wavelength is constant),while the absorbance at 680 nm is subtracted to remove any backgroundsignal due to, for example, system drift. The absorbance at 575 nmis used because, at that wavelength, it is the most sensitive to theCaP using KD = 0.0881. We note that a second-order polynomial provides a slightlybetter fit (pink dashed line) but the difference is minimal, and thiswould require a refinement of From the measured absorbance data, we can calculate KD,we can determinethe free Ca2+ concentration in solutions containing complexingagents simply by measuring the absorbance and using the relation [Ca2+] = (P – 1)/KD. In all experiments, the same initial amount of dye (D0 = 10 mg/L) was used and 140 mg/L NH4OH was added to increase the pH to approximately 10.5. Before applyingthis method, we addressed to what extent the spectra are affectedby time, salinity, and the possible interaction with the complexingagents. Several experiments were performed for this purpose, whichare included in the Supporting Information. This led to the following observations and constraints. (i) Theabsorbance slowly increases over time, likely caused by a change inpH due to CO2 dissolution. To take this into account, thecalibration and subsequent measurements were performed within 30 minafter the preparation of the solutions. (ii) Higher NaCl concentrationsresult in lower measured Ca concentrations by competitive bindingand by modifying the activities (γ). The calibrationcurve in DI water is therefore no longer applicable when measuringin solutions with higher salinities. While it is possible to do thecalibration at a given salinity, all UV–Vis measurements inthis report were performed in DI water. Note that the solution cannotcontain any Mg since the MgD complex is 100 times more stable thanthe CaD complex. (iii) Measurements with and without the Ca-complexingagents indicated that they do not interfere with the dye: in all cases,the absorbance at λ > 350 nm was equal whenno Ca2+ was present in solution. The spectrophotometricmethod, which relies on the absorbances at 520, 575, and 680 nm, cantherefore be applied to determine Ca2+ in solutions containingthese agents.Having determined 2+. At a pH value of10.5, the carboxylic acids groups (−COOH) are fully deprotonated,and the resulting carboxylate groups (−COO–) have a high affinity to complex divalent ions. These groups arealso present in common polymers and surfactants used in chemical EORapplications. The absorption spectra of solutions with different PAAconcentrations are shown in 2+ concentration is shown in We first consider the affinity of an acrylic acidhomopolymer , which we will refer toas polyacrylate (PAA), to complex Ca2+ concentration(35 mg/L) rapidlydrops with increasing concentrations of polyacrylate, reaching zeroat about 300 mg/L. The overlap of the two measurement runs shows thatthe results are highly reproducible. While large polymers tend toprecipitate when binding Ca2+, the molecular weight ofthe polyacrylate is sufficiently low that it does not easily becomeinsoluble and precipitate. The results for the other chemicals  are shown in Supporting Information.Note that O332 surfactant is an internal olefin sulfonate (IOS) typesurfactant, while J771 is an alcohol alkoxy sulfate (AAS) type surfactant.The investigated chemicals therefore constitute three different complexinggroups: carboxylate , sulfonate (O332), and sulfate(J771).The initial free Ca2+ concentration. The affinity of polyacrylate to complexCa2+ is roughly equal to that of EDTA, which is known forits high Ca2+-complexing potential and is even used toremove scale deposits.HPAM has a lower affinity to complex Ca2+ as it consistsof both acrylamide (AM) and acrylic acid (AA). The two surfactantsalso lower the Ca2+ concentration, O332 being more effectivethan J771. No precipitation was observed in the experiments, and thusthe decrease in [Ca2+] can be attributed to ion complexation.All investigated chemicals are found to lower theCa2+. To evaluate the complexingaffinity of different chemical groups, it is more insightful to comparethem by the number of Ca2+-complexing groups. In 2+ (in mmol/L). For PAA, O332, and J771,molecular weights of 94, 350, and 700 Da were used. Note that themolecular weight of PAA includes both the acrylate group and the Na+ counterion. Disodium EDTA has a molecular weight of 336.2Da but contains four carboxylate groups, so a molecular weight of84.05 Da was used in calculating the number of complexing groups.The HPAM polymer  is often used to increase the water viscosity in polymer floodingand consists of AM and AA groups with molecular weights of 71 and94 g/mol, respectively. The polymer has a hydrolysis degree of about30%, meaning that 30% of the monomers are in the form of acrylic acid.From this, we can calculate that 1000 mg/L polymer contains 362 mg/L(3.85 mmol/L) acrylic acid groups, which are able to bind Ca2+ ions. To rescale the concentrations, an effective molecular weightof 260 Da (1000 mg/L/3.85 mmol/L) was therefore used (see the Supporting Information). Note that an activematter of 100% was used in this calculation. The resulting plot in 2+ are nearlythe same. The curve of J771 is shifted to the right, reflecting thelower affinity of the sulfate (2+ thanthe carboxylate (COO–) and sulfonate  in 2+-complexation reactions.16 We will demonstrate this for two chemicals,J771 and polyacrylate, considering the following possible reactions:The chemical modelingsoftware PHREEQC is used to model the datain 2Ca and PAA2Ca do not necessarily imply that precipitates are formed; the reaction2Surf– + Ca2+ ⇌ Surf2Ca is equally valid to describe attachment of Ca2+ ionson surfactant micelles and can also be defined for n monomers and n/2 Ca2+ ions. The reactionsmay also be different in the presence of a negatively charged surface,in which case a SurfCa+ complex may adsorb to the surface.For each reaction, the formation constant is varied and the solutioncomposition at equilibrium is calculated. The resulting Ca2+ concentrations are shown as a function of [J771] and [PAA] in 2+ ion and two J771 or PAA molecules .Good agreement is obtained with log K = 5.5 for theformation of Surf2Ca and log K = 8.0 forthe formation of PAA2Ca. The equilibrium constant for theformation of EDTA–Ca2+ complexes determined usingthis method is approximately log K = 8.0, which islower than the literature value of about 10.5.17 The lower value can likely be attributed to the formationof EDTA–Na+ and EDTA–H+ complexesat pH = 10.5.Note that the reactions with Surf2Ca concentration saturates when nearlyall the Ca2+ is complexed by PAA. Having established bothformation constants, we can calculate the concentrations in a systemcontaining both surfactant and PAA. 2+, Surf–, Surf2Ca, PAA–, and PAA2Ca concentrations in a solution containing 2000 mg/L (2.86 mM) J771and different concentrations of polyacrylate. The results show thatincreasing the PAA concentration lowers the amount of Ca2+ complexed by the surfactant due to the higher affinity of PAA tocomplex Ca2+.For these reactions and formation constants, 2+. This is particularly relevant for anionic surfactants, which readilyadsorb to rock surfaces in the presence of divalent ions. In thesecases, adding a chemical with a higher affinity to bind Ca2+ can lower adsorption by complexing the ions that are capable ofmediating adsorption through cation bridging. The same mechanism couldpotentially be used to protect polymers, which suffer from a decreasein viscosity with increasing divalent ion concentration. Note that,for each application, the adsorption of the complex ingagent to thesolid surface should also be evaluated.This points toward a potential strategy for preventing adsorptionand precipitation of chemicals that are sensitive to Ca2+ concentration and Ca-complexformation constants in solutions containing complexing agents canbe determined using a Ca2+-sensitive dye. The method canbe used to rapidly evaluate the affinity of different chemicals tocomplex Ca2+, which can be useful in screening studies.The complex formation constants can be implemented into chemical floodingsimulators, providing insight into the effectiveness of the chemicalsduring flooding. The spectrophotometric method can be extended toother research fields in which Ca2+ complexation is investigated,such as studies on cells containing bacteria and enzymes.Weshowed that the Ca2·2H2O , NaCl, NH4OH ,Pontachrome Violet SW (Sigma Aldrich), EDTA , sodium polyacrylate , FP3630(SNF), and ENORDET J771 and O332 (both developed by Shell19). ENORDET J771 is a C12–13–7 propoxy sulfate, APS;ENORDET O332 is a C15–C18, IOS. For deionized water, a GenieU Ultrapure & Reverse Osmosis water system (Rephile) was used(>18.0 MΩ at 25 °C). For the Ca-complexation experiments,solutions were prepared with 10 mg/L dye (Pontachrome Violet SW),35 ppm Ca2+, 140 mg/L NH4OH, and different concentrationsof the complexing agents. The solutions were shaken and shortly thereafterthe absorption spectrum was measured with a UV–Vis spectrophotometer. The absorbance at 575 nm was used to determinethe free Ca2+ concentration using the calibration curvein All chemicals were used as received. Chemicals used were CaCl