MiRNAs are small, noncoding RNAs, which can regulate gene expression posttranscriptionally, and they have emerged as key factors in disease biology by aiding in disease development and progression. MiR-223 is highly conserved during evolution and it was first described as a modulator of hematopoietic lineage differentiation. MiR-223 has an essential part in inflammation by targeting the nuclear factor-κB pathway and the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome. Recent studies have shown that miR-223 expression is deregulated in various types of liver diseases, including hepatitis virus infections, alcohol-induced liver injury, drug-induced liver injury, non-alcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma. As inflammatory and immune factors are involved in the occurrence and progress of liver diseases, deregulated miR-223 may participate in the pathogenesis of these conditions by influencing neutrophil infiltration, macrophage polarization, and inflammasome activation. This review first summarizes the present understanding of the biological functions of miR-223, including its gene location and transcription regulation, as well as its physiological role in hematopoietic differentiation. This review then focuses on the role of miR-223 in liver pathophysiology and its potential applications as a diagnostic biomarker and therapeutic target in liver diseases. A tiny RNA molecule involved with gene regulation may offer an appealing target for diagnosing and treating various liver diseases. MicroRNA-223 (miR-223) was first identified as controlling gene activity in a wide variety of immune cells. A review from researchers led by Yanning Liu at China’s Zhejiang University in Hangzhou details how abnormal miR-223 also contributes to liver damage in a variety of conditions, although questions still remain about how it functions in different liver disorders. The authors highlight studies linking miR-223 with the development of fibrosis and cirrhosis, and with the inflammatory response to injury from drugs, alcohol, or infection. This could make this microRNA a useful diagnostic biomarker. The authors also identify therapeutic opportunities to modulate this molecule, referring to several studies on the manipulation of miR-223 to treat hepatitis. Approximately 17,000 miRNAs exist in more than 140 species2. MiRNA biogenesis is a complex process that begins in the cell nucleus where miRNAs are encoded as short inverted repeats with a double-stranded RNA stem loop. A long primary transcript (pri-miRNA) is transcribed by RNA polymerase II and may contain one or more hairpins. Before transport into the cytoplasm, hairpins are excised from pri-miRNAs by the double-strand-specific ribonuclease, Drosha-DGCR8 complex, to form precursor miRNAs (pre-miRNAs). Pre-miRNAs are then exported to the cytoplasm through Exportin-5 and Ran-GTP. In the cytoplasm, another protein complex containing the RNAseIII enzyme Dicer further processes pre-miRNAs into mature miRNAs, which are loaded into the RNA-induced silencing complex and then downregulate specific target mRNAs either by decreasing the transcript levels or by translational repression3. At present, 1881 precursors and 2588 mature human miRNAs are registered in the miRbase ver. 21.0. These miRNAs have been shown to control the expression of at least 60% of human protein-coding genes5. MiRNAs have important regulatory roles in modulating normal physiology, including development, tissue remodeling, metabolism, immunity, cell proliferation, cell differentiation, and intracellular signaling6, whereas dysregulated miRNAs are correlated with a variety of human diseases7.MicroRNAs (miRNAs) are a class of highly conserved, chemically stable, small noncoding endogenous RNA (∼22 nt) molecules. Since their discovery in 19938. By developing a tissue-specific mature miRNA deletion mouse model, in which Dicer1 function is absent in hepatoblast-derived cells, researchers have shed light on the liver-specific roles of miRNAs9. Despite the loss of mature miRNAs, hepatic basal functionality is maintained in these mice. However, mutant mice at 2–4 months of age exhibit progressive hepatocyte damage with elevated serum alanine aminotransferase and aspartate aminotransferase combined with significant hepatic steatosis, glycogen depletion, increased hepatocyte apoptosis, increased hepatocyte proliferation, and portal inflammation9. Interestingly, a different Dicer1-knockout mouse model, in which Dicer1 is conditionally deleted in mature hepatocytes, fetal stage-specific genes are persistently expressed, resulting in increased hepatocyte proliferation and apoptosis, as well as spontaneous development of hepatocellular carcinoma (HCC)10. These data demonstrate that miRNAs have critical roles in hepatic physiology and pathology.Studies have demonstrated that miRNA expression signatures are highly tissue- and disease-specific, and approximately 70% of miRNAs in the whole body have been discovered in liver tissues12. MiR-122, one of the most abundant adult hepatic miRNAs, regulates a wide range of key gene networks, such as hepatic circadian rhythm, lipid metabolism, and cell differentiation13. Recently, increasing evidence has suggested that miR-223 is one of the key factors in the development and homeostasis of the immune system14, and may also have an essential part in both inflammation disorders and various liver diseases. The present article reviews current knowledge on the physiopathology of miR-223 starting with its biogenesis and biological functions, and it focuses on the emerging role of miR-223 in liver pathology and its potential for diagnosis and treatment in liver diseases , metabolic lever diseases, acute liver diseases, and HCC15. MiR-223 sequence is highly conserved during evolution, suggesting its potential role in essential physiological events. MiRNA-223 was first identified in the hematopoietic system16. As the miR-223 gene resembles a “myeloid gene,” its expression is regulated by several myeloid transcription factors, including PU.1, CAAT/enhancer-binding proteins (C/EBP)-α and -β, and nuclear factor I-A (NFI-A). PU.1 binds to two separate sites within the miR-223 promoter to activate miRNA expression, which in turn induces osteoclastogenesis18. Similarly, C/EBP-α binds to the miR-223 promoter and increases its expression, which undergoes a specific activation to promote granulocyte differentiation19. C/EBP-α has also been shown to strongly enhance miR-223 promoter activity by combination with PU.120. However, NFI-A maintains low levels of miR-223, as it competes for binding with C/EBP-α, thus decreasing granulocyte and osteoclast differentiation19. Interestingly, both NFI-A and C/EBP-β act as direct targets of miR-223, thus forming negative feedback loops between the target genes and their corresponding miRNA21. Hence, miR-223 expression is coordinately affected by the combined actions of different myeloid transcription factors. The dysregulation of transcription factors may also contribute to the aberrant expression of miR-223 in multiple types of cancer, inflammatory diseases, and other pathological processes22.The miR-223 encoding gene is located within the q12 locus of the X chromosome23. The prevention of acetylated histone H3 and C/EBP-α recruitment to the miR-223 promoter also reduces miR-223 expression24.Recently, increasing evidence has suggested that epigenetic mechanisms, such as DNA methylation and posttranslational modifications of nucleosomal histone proteins, are also involved in miRNA regulation. Heterochromatic silencing of miR-223 can be induced by recruitment of chromatin-remodeling enzymes on the pre-miR-223 promoter25. In the investigation of the existence and role in mRNA regulation of shorter RNA species derived from miRNAs, researchers serendipitously discovered a significant number of smiRNA sequences, derived from their corresponding pre-miRNA species processed by a Dicer complex, and can act as natural miRNA anti-sense RNAs to neutralize the function of miRNA species of sufficient base pair complementarity. By using smiR-223 derived from pre-miR-223 as a model, researchers further showed that although smiR-223 cannot directly regulate mRNA activity through mRNA translational repression, it can interfere with the ability of miR-223 to mediate gene silencing either through mRNA cleavage or mRNA translational repression25. This condition implies the ability of miR-223 to provide an additional layer of control over miR-223 activity.A pioneer study has demonstrated that the existence of semi-miRNAs (smiRNAs), as 12 nt-long RNA species corresponding to the 5′-region of mature miRNAs, may participate in regulating miRNA activity in vivo16.As miR-223 is preferentially expressed in the hematopoietic system, it is considered to have important roles in hematopoietic differentiation by affecting hematopoietic stem cells, myeloid cells, erythroid cells, and lymphoid cells at various stages of their development27. MiR-223 overexpression is sufficient to induce myeloid precursor cell differentiation, whereas miR-223 inhibition reduces the efficiency of RA-induced differentiation19. A study using the miR-223-/Y mouse model showed that miR-223 is not strictly essential for granulocyte differentiation but is required for normal maturation of granulocytes and regulation of granulocyte compartment size. Marked neutrophilia and hyperplasia of the bone marrow granulocyte compartment were observed in this model16. This finding was validated by bone marrow reconstitution with hematopoietic stem cells stably overexpressing miR-223 sponge sequence to phenocopy the miR-223-/Y mouse 28.The first important role of miR-223 was discovered in granulopoiesis according to its highest expression level in granulocytes29. By using an antagomiR inhibitor of miR-223, the granulocyte-macrophage colony-stimulating factor-induced differentiation of monocytes and phorbol-12-myristate-13-acetate (PMA)-induced differentiation of THP1 cells can be abolished, further demonstrating the important role of miR-223 for myeloid differentiation30. Interestingly, miR-223-rich microvesicles (exosomes or microparticles) derived from differentiated macrophages sufficiently induce differentiation in recipient monocytes31. Unlike in monocytes, however, several studies have shown that miR-223 is downregulated in macrophages32. As miR-223 inhibition implies a strong effect on macrophage differentiation, a low functional level of this miRNA may be sufficient for monocyte differentiation.In addition to granulocyte differentiation, miR-223 has a role in monocyte/macrophage differentiation by repressing its target, IκB kinase subunit-α (IKK-α). In macrophage differentiation, repressed miR-223 expression induces IKK-α expression followed by the repression of nuclear factor-κB (NF-κB) pathways33. In K562 cells, an erythroid–megakaryocyte cell line, miR-223 expression is downregulated during haemin-induced erythroid differentiation but upregulated during PMA-induced megakaryocytic differentiation32. MiR-223 can also target and regulate the expression of the LMO2 transcription factor, a bridging molecule that assembles an erythroid DNA-binding complex, including the GATA-binding factor-133. During erythroid differentiation, miR-223 downregulation increases LMO2 level and then promotes the differentiation of K562 cells into erythrocytes.In addition, miR-223 is involved in the regulation of erythroid and megakaryocyte differentiation by targeting LIM domain only 2 (LMO2)34. Interestingly, knockdown of miR-223 results in an increased production of the target gene, NFI-A, a suppressor of osteoclastogenesis. Bioinformatics suggests that the 3′-untranslated region (UTR) of NFI-A may have a potential miR-223-binding site. Thus, in osteoclast differentiation, miR-223 potentially targets NFI-A to induce expression of macrophage colony-stimulating factor receptor, which would modulate osteoclast differentiation, function, and survival35.MiR-223 also has an important role in osteoclast formation and the regulation of bone remodeling as it is expressed in osteoclast precursors. Knockdown of miR-223 decreases the receptor activator of NFκB ligand (RANKL)-induced formation of osteoclast-like cells, whereas overexpression of miR-223 exerts a similar effect, suggesting that an appropriate level of miR-223 expression is important for normal osteoclastogenesis36. Based on bioinformatics prediction of complementary binding sites in the 3′-UTR of IGF-1R mRNA, miR-223 is assumed as a direct regulator of IGF-1R, which can modulate hESC development by regulating its downstream kinase, Akt. Moreover, miR-223 inhibition maintains the undifferentiated state of hESCs, whereas addition of exogenous miR-223 induces hESC differentiation.Recently, miR-223 has been demonstrated to regulate human embryonic stem cell (hESC) differentiation by targeting the insulin-like growth factor-1 receptor (IGF-1R)/Akt signaling pathway40. MiR-223 represses selective high-density lipoprotein cholesterol (HDL-C) uptake by inhibition of hepatic scavenger receptor class B type I (SR-BI), a high-affinity HDL receptor, by directly targeting its 3′-UTR37. Given that selective HDL-C uptake is a pivotal step of reverse cholesterol transport, miR-223 may modulate hepatic cholesterol metabolism. MiR-223 has also been shown to directly modulate the endogenous protein level and mRNA stability of cytochrome b5 in human hepatocytes38. Due to the role of b5 in cytochrome P450-catalyzed drug metabolism, the inter-individual variability of miR-223 expression may affect the inter- and intra-individual variability of drug pharmacokinetics. A decreased miR-223 level in HCC development indicates the role of miR-223 in maintaining normal hepatic physiological function. Reduced expression of miR-223 in hepatocytes has been verified to potentially predispose HCC development via dysregulated expression of STMN1, which is a downstream target of miR-223 and is considered as a pro-tumorigenic gene to induce chromosomal instability39. MiR-223 also regulates Fas-induced hepatocyte apoptosis and liver injury by targeting the IGF-1R signaling pathway40.MiR-223 can also modulate the physiological function of parenchyma cells. In hepatocytes, miR-223 modulates cholesterol metabolism, drug metabolism, and cell apoptosisIn summary, miR-223 is located within the X chromosome and is regulated by several transcription factors, epigenetic modification, and smiR-223. MiR-223 has important roles in hematopoietic differentiation. MiR-223 expression increases as granulocyte–monocyte progenitors differentiate into granulocytes but decreases during differentiation into monocytes, and miR-223 expression is further altered as these cells are activated or polarized. MiR-223 also regulates the differentiation of erythroids, megakaryocytes, osteoclasts, and ESCs. Finally, miR-223 is involved in modulating hepatic physiological function by affecting cholesterol and drug metabolism, as well as apoptosis and chromosomal stability in hepatocytes.41. By targeting Pknox1, an essential regulator for macrophage polarization, miR-223 steers classically activated pro-inflammatory phenotype (M1) of macrophages toward the alternative anti-inflammatory phenotype (M2)41. MiR-223 may also regulate the nucleotide-binding oligomerization domain-like receptor (NLR) inflammasome by targeting the NLR protein 3 (NLRP3) 3′-UTR42. Upon stimulation by a Toll-like receptor (TLR) ligand, many cell types need the NLRP3 inflammasome to initiate inflammatory responses and induce interleukin (IL)-1β production. The results have shown that overexpression of miR-223 prevents accumulation of NLRP3 protein and inhibits IL-1β production from the inflammasome. Furthermore, miR-223 may prevent macrophage hyperactivation by cooperating with other miRNAs to modulate the noncanonical NF-κB transcription factor pathway by regulating IKK-α expression29.Aside from its involvement in the differentiation of various immune cells, miR-223 also influences activation patterns. For instance, miR-223 modulates macrophage polarization, inflammasomes, and NF-κB signalingMiR-223 expression is deregulated in many types of liver diseases, including viral hepatitis, alcohol-induced liver injury, DILI, non-alcoholic fatty liver disease (NAFLD), cirrhosis and HCC Table . Several44. The early PRR-mediated innate immune response is also required for orchestrating the development and activation of adaptive immune responses, which ultimately resolve viral infections and provide long-term protection against subsequent infection. Various innate immune cells, such as natural killer cells, dendritic cells, neutrophils, and macrophages, are involved in the control of viral infection and immunopathogenesis of viral hepatitis.The most common causes of viral hepatitis comprise five unrelated hepatotropic viruses, namely, hepatitis A virus (HAV), HBV, HCV, HDV, and HEV. Viral infections are initially recognized by host innate pattern recognition receptors (PRRs), including TLRs, NLRs, RIG-I-like receptors, and C-type lectins45. However, it remains unclear how high levels in the plasma relate to the expression levels of this miRNA in liver tissues. In another study, analysis of a miRNA panel in fresh liver biopsies showed that hepatic expression of miR-223 as well as other immunological and inflammatory pathway-associated miRNAs is downregulated by chronic HCV infection. Dysregulated miRNAs may be linked to chronic liver inflammation and subsequent complications by regulating the NF-κB pathway by targeting its key signaling molecules46. In hepatitis B patients, the results have shown that miR-223 is elevated in serum from patients with chronic hepatitis B to a similar extent as in patients with HCC, suggesting the strong potential of miR-223 to serve as a novel biomarker for liver injury but not specifically for HCC47. An additional study has shown that HBsAg particles circulating in HBV carriers can carry Ago2-associated protein and cellular miRNAs, especially liver-specific miRNAs and immune regulatory miRNAs (such as miR-223). These HBsAg-associated miRNAs may affect viral persistence and aid in explaining the clinical evidence that most efficient immune controls of HBV infection are associated with the loss of serum HBsAg. This finding also opens a new avenue for research to perceive the interactions between miR-223 and HBV infection48. Another study has shown that miR-223 levels are downregulated in the serum of HBV-positive HCC patients compared with healthy controls, and that miR-223 is downregulated in HBV X (HBx)- or HBV-transfected HepG2 and HepG2.2.15 cells, further suggesting that viral protein can regulate miR-223 expression and subsequently regulate host function by its target genes49.As miR-223 regulates the differentiation of several key factors  of innate immune response, it may affect the early stage of viral infection. Studies have shown that miR-223 expression is affected by HCV infection and treatment-based viral cures. The plasma level of miR-223 is significantly increased by at least sevenfold in patients, achieving sustained virologic response (SVR), after treatment compared with the baseline level but not in patients who have relapsed. The increased miR-223 level indicates the potential antiviral functions of the miRNA in SVRs and/or a physiological response to viral clearanceThe above observations suggest that miR-223 has roles in the disease pathology of viral hepatitis rather than serving as a biomarker alone, and that it may act as a negative regulator of the inflammatory process for hepatitis virus infection.50.Viral infections, acetaminophen (APAP) overdose, and hepatic ischemia/reperfusion (I/R)-induced acute liver injury (ALI) may progress into acute liver failure (ALF). ALF, which is characterized by fulminant hepatic necrosis and hepatitis, hepatic encephalopathy, and an elevated prothrombin time/international normalized ratio, often causes multiorgan failure and features a high mortality rate. The mechanisms that contribute to ALF can be divided into two groups as follows: direct damage from pathogens and toxic substances, and immune-mediated liver injury51. The serum levels of miR-223 are also elevated in mouse models of I/R-induced ALI and ALF patients, as well as those in liver samples53. Another study has shown that miR-223−/− mice are resistant to Fas-induced liver injury as indicated by less tissue damage and lower elevation of serum transaminases, and that miR-223 deficiency protects against ALI by targeting IGF-1R in hepatocytes40. Furthermore, in patients with ALI, the hepatic miR-223 expression levels in patients who show no spontaneous recovery are significantly higher than those in patients who exhibit spontaneous recovery, suggesting that hepatic expression levels of miR-223 may also be used for predicting the outcome of ALF patients52.Increasing evidence has indicated that miR-223 can be used as a diagnostic biomarker in ALI. Studies have shown that miR-223 expression levels are greatly upregulated in the livers of mice after I/R injury and that the hepatic miR-223 expression level is positively correlated with serum markers of ischemic injury54. In addition to its role in macrophages, miR-223 also acts as a modulator of neutrophils, which can infiltrate livers, thereby inducing liver damage55. MiR-223 terminates the acute neutrophilic response by targeting IKK-α expression in APAP-induced ALI56.MiR-223 has also been shown to exert hepatoprotective effects on ALI by regulating immune responses. By isolating primary Kupffer cells (KCs) from concanavalin A (Con A)-stimulated mice, the upregulation of AIM2 and IL-1β has been shown to be consistent with the downregulation of miR-223 in KCs in the early stage of ALF. An in vitro experiment further confirmed that overexpression of miR-223 inhibits IL-1β expression by indirectly regulating AIM2, suggesting a possible effect of miR-223 on Con A-induced ALF by suppressing inflammasome activation and pro-inflammatory secretion of KCsAs previously described, miR-223 has a significant role in the pathophysiology of ALF by regulating hepatocyte death and immune response. However, the expression profiles and functions of miR-223 are contradictory in different ALF models. For instance, miR-223 deficiency may protect mice from FasL/CD95L-induced liver injury, but miR-223 deficiency has no effect on I/R-induced injury but may exacerbate APAP-induced injury. The precise molecular functions of miR-223 in ALF caused by different etiologies remain unclear, and further investigations are needed to develop miRNA-based therapy for ALI.57.DILI is the most common cause of ALF worldwide. The incidence of DILI has continually increased and is, therefore, recognized as a major public health concern. The majority of DILI cases that lead to ALF results from the overdose of APAP, which is one of the most commonly used antipyretic and analgesic medicine. APAP-induced DILI is considered as an “intrinsic” liver toxin and is characterized by massive and coordinated hepatocyte necrosis57. Studies have shown that miR-223 limits neutrophil overactivation in APAP-induced liver injury. APAP overdose can cause the release of mitochondrial DNA (mtDNA) by damaged hepatocytes, which activates neutrophils by binding to TLR9, further aggravating liver injury. In vivo and in vitro experiments have indicated that miR-223 deficiency exacerbates APAP-induced hepatic neutrophil infiltration, oxidative stress, and liver injury, as well as enhances the TLR9 ligand-mediated activation of pro-inflammatory mediators in neutrophils. Molecular mechanism studies have shown that activated TLR9 upregulates miR-223 by enhancing NF-κB binding on the miR-223 promoter but that miR-223 attenuates TLR9/NF-κB-mediated inflammation by targeting IKK-α expression. Thus, upregulation of miR-223 may have a key role in the termination of acute neutrophilic response through a mtDNA/TLR9/NF-κB/miR-223-negative feedback loop56.There is emerging evidence that miR-223 is involved in the pathophysiology of DILI. The serum level of miR-223 is elevated in DILI patients and in mouse models of DILI58. Thus, miR-223-mediated regulation of inflammasome activation may also be a potential strategy for DILI therapy.In addition to APAP initial toxicity in hepatocytes, a sterile inflammatory response potentially involving inflammasome activation is particularly attributed to the late-stage APAP toxicity in DILI59.Excessive alcohol consumption is also a major public health problem. Although mortality from alcoholic liver disease (ALD) has declined over the last few decades, it remains the most common cause of death due to alcohol. The clinical spectrum of ALD includes steatosis, steatohepatitis, liver fibrosis, cirrhosis, sepsis, and HCC. Alcohol-induced hepatotoxicity, oxidative stress, and dysregulation of immune cell functions contribute to ALD pathogenesis54. Recent studies have suggested that alcohol exposure modulates miRNA expression in neutrophils. MiR-223 levels are elevated in the serum and neutrophils from ethanol-fed mice. Moreover, miR-223−/− mice are more susceptible to alcohol feeding-mediated induction of hepatic neutrophil infiltration and liver injury. Mechanistic studies have revealed that miR-223 directly inhibits IL-6 expression, which subsequently inhibits p47phox expression in neutrophils, thereby indicating the role of miR-223 in blocking neutrophil infiltration and reactive oxygen species (ROS) production in ALD60.As a major innate immune cell subset, neutrophils perform a crucial role in ALD pathogenesis. Alcohol can induce function alteration of neutrophils, including oxidative burst, chemotaxis, expression of β2 integrin adhesion molecule, and production of key immune mediatorsAs inappropriate neutrophil activation and massive infiltration of neutrophils are known to contribute to ALD pathogenesis, modulation of miR-223 can be used to protect against ALD due to its regulation of neutrophil infiltration and function. Further understanding of the role of miR-223 in ALD pathogenesis may help to identify novel therapeutic targets for this disease.61. The two-hit theory of NAFLD pathogenesis is well known. The theory states that triglycerides first accumulate in hepatocytes followed by induction of hepatic inflammation caused by inflammatory mediators62. Insulin resistance, intestinal microbiota, and oxidative hepatocellular injury are also implicated in NAFLD pathogenesis. Given that miRNAs are important regulators in a wide spectrum of metabolic homeostasis processes, including fatty acid metabolism, they have emerged as attractive candidate biomarkers for monitoring NAFLD progression63.NAFLD refers to a wide spectrum of diseases that range from simple hepatic steatosis, defined as non-alcoholic fatty liver, to non-alcoholic steatohepatitis (NASH), which may progress to irreversible liver damage, such as cirrhosis, liver failure, and HCC64. Another study using a mouse model of choline- and folate-deficient (CFD) diet-induced NAFLD showed that miR-223 is significantly increased in the livers of CFD-diet-fed mice and negatively regulates the hepatic expression of IRP1, a key regulator of intracellular iron metabolism. The high correlation of miR-223 levels with the severity of NAFLD-specific profibrogenic changes in the liver further suggest that miR-223 may be a useful biomarker to monitor the status of iron homeostasis and NAFLD progression65. Moreover, miR-223 affects HDL-C uptake by directly targeting and repressing SR-BI37. MiR-223 also indirectly promotes ATP-binding cassette transporter A1 expression by targeting Sp3, thereby enhancing cellular cholesterol efflux. Notably, miR-223 levels can be regulated by cholesterol levels. Finally, genetic ablation of miR-223 in mice results in increased HDL-C, as well as hepatic and plasma total cholesterol levels, suggesting its key role in systemic cholesterol regulation by coordinated posttranscriptional control of multiple genes in lipoprotein and cholesterol metabolism66.MiR-223 was initially reported to be myeloid cell-specific but has recently been shown to be expressed in hepatocytes. Using cellular models of NAFLD and NASH  and palmitate (lipotoxic), respectively), researchers have found that both oleate-palmitate and palmitate treatment induce variations in the intracellular and extracellular miRNome in hepatocytes, especially in the cellular model of NASH. Among the massive number of deregulated miRNAs, miR-223 has been validated as one of the two most deregulated miRNAs. The deregulation of miR-223 is time-dependent in both NAFLD and NASH models. Further miRNA target expression and pathway analyses have shown that miR-223 target genes are involved in the processes associated with NAFLD and NASH onset and progression, including fatty acid metabolism and inflammation68. As miR-223 is an important regulator in macrophage polarization by targeting Pknox1 and in activation of inflammasomes by targeting NLRP3, miR-223 may affect NAFLD progression by controlling the phenotype switch of macrophages, which is beyond its role in directly regulating fatty acid metabolism, iron homeostasis and cholesterol homeostasis. Our unpublished data verifies this assumption as miR-223 administration alleviates NASH in high-fat diet (HFD)-fed mice by suppressing hepatic NLRP3 inflammasome activation and IL-1β release.Currently, macrophage polarization and inflammosome activation are considered to have central roles in the pathogenesis and progression of NAFLD69. Recently, the role of immune cells, especially subsets of macrophages, in regulating the progression or the regression of fibrosis has become a concern. Inflammatory and profibrogenic Ly6Chi macrophages or restorative Ly6Clo macrophages can act as the fuel or brake of liver fibrosis, respectively, indicating that macrophages act as key regulators in fibrogenesis70.Liver fibrosis is a wound-healing response to injury that occurs in most chronic liver diseases. Advanced chronic fibrosis is described as cirrhosis with loss of architecture and functional failure, as well as the development of life-threatening complications. Hepatic stellate cells (HSCs) are key effector cells in fibrogenesis. Transformation of HSCs from a quiescent, fat-storing phenotype into activated, extracellular matrix (ECM)-producing myofibroblasts results in the production of transforming growth factor-β and deposition of ECM proteins, such as collagen, into the surrounding parenchyma71. The Metavir fibrosis score assay for liver histology shows that serum miR-223 is upregulated in significant fibrosis ( ≥ F2) compared with no/mild fibrosis (F0–F1). The model including miR-223 has high diagnostic accuracy for ≥ F3 and is superior to APRI and FIB-4 in discriminating ≥ F3 and F472. However, in HBV-related liver fibrosis identified with the Scheuer scoring system, the serum miR-223 level is significantly downregulated as fibrosis progresses from S0–S2 (early fibrosis) to S3–S4 (late fibrosis). The model including miR-223 is associated with a high diagnostic accuracy in discriminating S0–S2 from S3–S4, and it also superior to APRI and FIB-4 for discriminating liver fibrosis progression73. These findings suggest that miR-223 can also be used for staging HCV- or HBV-associated liver fibrosis. In mouse models of repetitive CCl4 injection or bile duct ligation-induced liver fibrosis, researchers have found that hepatic miR-223 expression is significantly dysregulated upon induction of liver fibrosis and that this upregulation is restricted to hepatocytes and correlated with degree of liver injury and hepatic cell death. However, in the same study, functional experiments reveal no differences in the degree of liver fibrosis as miR-223−/− mice behave identically to wild-type mice, highlighting the complex role of miR-223 in the development of liver fibrosis52.Considering its role in monocyte/macrophage differentiation, dysregulated miR-223 may also be implicated in the development of liver fibrosis. Several studies have shown that the serum miRNA panel, including miR-223, is deregulated in HBV/HCV-related liver fibrosis and is even correlated with the stage of liver fibrosis, indicating the possible use of circulating miR-223 for non-invasive diagnosis of liver cirrhosis74. As miR-223 suppresses the activation of inflammasomes by targeting NLRP3, it may control the progression of liver fibrosis by modulating the phenotype and function of macrophages.Thus, in addition to be used as a novel non-invasive biomarker for diagnosis and staging liver fibrosis alone, miR-223 may have a role in regulating disease pathology. Increasing studies have shown that the progression of liver injury and fibrogenesis in various liver pathogeneses is driven by NLRP3 inflammasome activation in macrophages75.HCC is the third most common cause of cancer-related deaths, which may occur by chronic HBV infection, chronic HCV infection, exposure to toxins , and NAFLD. Multiple biological mechanisms, such as inflammation, oxidative stress, hypoxia, tumor microenvironment, and other molecular events, facilitate tumor initiation, progression, and metastasis. Due to their role in promoting angiogenesis, proliferation and survival of cancer cells, as well as suppression of antitumoral immune responses, tumor-associated macrophages (TAMs) may also be involved in HCC growth and metastasis39 revealed that miR-223 is downregulated in HCC patients irrespective of viral associations and the decrease in miR-223 may abate its suppression on cell viability by targeting STMN1. Another study has shown that miR-223 transfection specifically suppresses cell growth and promotes apoptosis in HCC cells through inhibition of the mTOR pathway by targeting Rab176. Further studies have demonstrated that miR-223 is regulated by HBx and alcohol intake77. The expression of miR-223 is higher in HBV+ HCC patients with long-term alcohol consumption compared with HBV+ HCC nondrinkers or healthy controls. Knockdown of miR-223 blocks the effect of acetaldehyde by inhibiting cell growth via the miR-223/Fbxw7 axis77. In addition to its role in HCC growth, downregulation of miR-223 promotes HCC metastasis. Enhancing miR-223 expression effectively inhibits HCC metastasis by targeting integrin αV24. Furthermore, miR-223 modulates multidrug resistance via downregulation of ABCB1 in HCC cells78.MiRNAs have crucial functions in regulating HCC tumorigenesis and metastasis signaling networks. Wong et al.79. Other studies have also shown that serum miR-223 is decreased in HCC patients and can be used as novel non-invasive biomarker of HCC alone or in a panel81. Moreover, several studies have indicated that serum miR-223 is also a good biomarker for predicting the outcome of Sorafenib-treated HCC or recurrence following liver transplantation83.Several studies have explored the possibility of circulating miR-223 as a diagnostic tool in HCC. Comparison of the circulating levels of miR-223 in serum samples of HCC patients, chronic liver disease patients, and healthy volunteers has shown that serum miR-223 levels are significantly lower in HCC patients than in the other two groups84. TAMs also promote tumor growth and metastasis by acting as myeloid-derived suppressor cells (MDSCs) that suppress T-cell responses by secreting cytokines and growth factors. Compared with MDSCs from the spleen of disease-free mice, miR-223 expression is lower in tumor-induced MDSCs. MiR-223 inhibits the differentiation of bone marrow cells (BMCs) into MDSCs by targeting myocyte enhancer factor 2C, thereby suppressing tumor growth in mice infused with miR-223-engineered BMCs85. It is well known that M2 phenotype macrophages promote tumor invasiveness and contribute to poor prognosis. Thus, miR-223 may also participate in tumor development via driving TAMs to the M2 phenotype by targeting Pknox1, which is an essential regulator for macrophage polarization41.Increasing evidence has indicated that miR-223 is closely associated with tumor microenvironment. TAMs may transfer miR-223 to HCC cells in a contact-dependent manner involving gap junctions, thereby inhibiting HCC cell proliferation by targeting the STMN1 and IGF-1R genesThus, miR-223 may regulate HCC tumorigenesis and metastasis by suppressing the proliferation of HCC cells directly or affecting the tumor microenvironment.86. We have also shown that miR-223 agomir administration alleviates steatohepatitis in mice with HFD-induced NASH (unpublished data). Miravirsen, a locked nucleic acid-modified DNA phosphorothioate anti-sense oligonucleotide that inhibits miR-122, and MRX34, a liposome-formulated mimic of the miR-34 tumor suppressor miRNA, have shown significant antiviral effects on chronically infected HCV patients  and antitumor activity in patients with refractory advanced solid tumors , respectively88. Given the association of dysregulated miR-223 with liver inflammation, fibrosis, steatosis, and HCC, miR-223-based therapy may provide a novel strategy in the context of advanced liver diseases and HCC. However, miR-223-based therapy still faces some challenges. First, there are divergent views on the role of miR-223 treatment in similar liver diseases. For instance, miR-223 deficiency protects against Fas-induced liver Injury, whereas upregulation of miR-223 alleviates APAP-induced liver failure. Thus, more studies are necessary to elucidate the controversial role of miR-223 in liver injury and to tailor miR-223 treatment regimens to liver injury induced by specific factors. Second, miR-223-derived drugs need to be pharmacologically targeted for cell-specific delivery of the agent. Neutrophilic miR-223 ameliorates liver injury by inhibition of neutrophil overactivation, whereas hepatic miR-223 promotes hepatocyte apoptosis. Therefore, selective and accurate delivery of miR-223 to target cells is necessary to increase the therapeutic potential and reduce possible side-effects for the treatment of liver disease. Third, the therapeutic outcomes of miR-223-based treatment are based in mouse models, and more studies with larger animals are necessary to further evaluate its effectiveness on specific liver diseases. Finally, studies should further identify the major miR-223 targets and understand the role of miR-223 in the link between inflammation and cancer, which may provide better insight into the biology of miR-223 and contribute to develop safe, accurate, and specific therapeutics for liver diseases.Based on its role in regulating immune cell function, as well as liver physiology and pathology, miR-223 is regarded as an attractive biomarker and therapeutic target for the diagnosis and treatment of liver diseases. However, circulating and hepatic miR-223 features different expression profiles in different types of liver diseases, suggesting the necessity for further large-sample clinical studies in determination of its diagnostic value in liver pathologies. Several studies have shown the therapeutic effect of miR-223 in liver injury. For instance, bone marrow-derived mesenchymal stem cell (BMSC)-secreted miR-223-containing exosomes (BMSC-exo) prevent liver injury in an autoimmune hepatitis mouse model by suppressing hepatic NLRP3 and caspase 1, and modification of miR-223 further improves its therapeutic efficacy against autoimmune hepatitis


The neural mechanisms associated with obesity have been extensively studied, but the impact of maternal obesity on fetal and neonatal brain development remains poorly understood. In this study of full-term neonates, we aimed to detect potential neonatal functional connectivity alterations associated with maternal adiposity, quantified via body-mass-index (BMI) and body-fat-mass (BFM) percentage, based on seed-based and graph theoretical analysis using resting-state fMRI data. Our results revealed significant neonatal functional connectivity alterations in all four functional domains that are implicated in adult obesity: sensory cue processing, reward processing, cognitive control, and motor control. Moreover, some of the detected areas showing regional functional connectivity alterations also showed global degree and efficiency differences. These findings provide important clues to the potential neural basis for cognitive and mental health development in offspring of obese mothers and may lead to the derivation of imaging-based biomarkers for the early identification of risks for timely intervention. GloballIn adolescents and adults with obesity, neuroimaging studies have documented widespread functional alterations in a variety of neural circuits that can be largely summarized in four main domains ; sensoryIn this study, leveraging resting-state fMRI scans  on 38 fuClinicalTrials.gov ID: NCT01131117). Inclusion criteria for the pregnant women were: pre-pregnancy self-reported BMI = 18.5–24.9  or BMI > 25 ; second parity, singleton pregnancy; ≥ 21 years of age; conceived without assisted fertility treatments. Exclusion criteria were: preexisting medical conditions; medical complications during pregnancy; medications during pregnancy known to influence fetal growth; smoking or alcohol drinking. All enrolled women had their body composition assessed using air displacement plethysmography  and BMI measured within the first 10 weeks of gestation during their first research visit. Maternal IQ (MIQ) was assessed using the Wechsler Abbreviated Scale of Intelligence , except for one subject who did not complete the IQ assessment. Total gestational weight gain was measured at 36 weeks of gestation. Birth weight and length of the infants were obtained and head circumference was measured at age 2 weeks. Only infants born full-term (≥37 weeks of gestation), with size at birth appropriate for gestational age (AGA), and without medical conditions known to influence growth and development were included in the study. In total, 44 infants participated in the MRI study, 43 had 3D T1 and RS-fMRI data prior to quality control, but 5 of them were later excluded due to excessive motion during the RS-fMRI using criteria specified in the data analysis section. The remaining 23 infants born to normal-BMI mothers and 15 infants born to mothers with high BMI were included in the data analysis.Forty six pregnant women were originally enrolled for this study, which was approved by the local institutional review board. Informed consents were obtained from the pregnant mothers. Subjects were prospectively enrolled from a larger longitudinal study of pregnant women  was used to acquire the RS-fMRI data.At ∼2 weeks of age, MRI examination of the brain was conducted in the Department of Radiology at the Arkansas Children’s Hospital. Infants were fed 15∼30 min prior to the scan, swaddled in warm sheets, and immobilized using a MedVac Infant Immobilizer . No sedation was used. A pulse oximeter probe  was placed on a foot to monitor oxygen saturation and heart rate, and mini-muffs and a headset were placed over the ears to protect the infants from the noise generated during the scan. The MRI examinations were performed on a 1.5 Tesla Achieva MRI scanner  with 60 cm bore size, 33 mT/m gradient amplitude, and 100 mT/m/ms maximum slew rate. A pediatric 8-channel SENSE head coil was used. A neonatal brain MRI protocol was used, which included sagittal 3D T1 weighted reconstructed to 3 planes, axial T2 weighted, axial diffusion weighted, and axial susceptibility weighted imaging sequences. This conventional neonatal MRI protocol was used for the investigators to potentially exclude subjects with apparent brain abnormalities. The 3D T1 weighted images acquired at 1 × 1× 1 mn = 102 time-points). Excess volumes were discarded starting from the end of the functional time-series. There was no significant difference in mean FD between infants born to normal BMI  and high BMI  mothers; t(36) = -0.27, p = 0.789.Functional data were preprocessed using the FMRIB Software Library .Spatial normalization was achieved using a combination of within- and between-subject transformations. The UNC neonate template was used for co-registration  . Specifin = 223; see t-tests; n-BMI vs. h-BMI). Here, multiple comparisons were accounted for using a nonparametric permutation approach; FSL randomize (P-corrected < 0.05): 5000 permutations with threshold-free cluster enhancement (TFCE)  were assed using analysis of variance (ANOVA) and correlation-based analyses. The subject with missing IQ was excluded in the covariate analysis of that variable. Finally, correlation matrices were constructed and used to generate complimentary graph theoretical measures. Specifically, we used the graph theoretical network analysis toolbox (GRETNA) -based atlas (UNC-CEDARS INFANT)  to genert (TFCE) . Cluster(GRETNA)  to compuThe demographic information for the infants (and their mothers) included in the data analysis is listed in Table Post hoc cluster-level analyses indicated that 15 out of 17 clusters showed significant group differences after correcting for the 223 seed regions (Bonferroni correction: p < 0.05/223 or 0.0002). Among the 17 clusters, 14 demonstrated hyper-connectivity in the high BMI group (h-BMI > n-BMI). Importantly, the connections covered all four hypothesized domains of functional circuits involved in the etiology of adult obesity with a particularly dense coverage on cognitive control areas, including the lateral prefrontal and orbital frontal cortices. Specifically, for sensory cue processing, four vision-related connections were detected to show hyper-connectivity in the h-BMI group (relationships denoted as seed: cluster using AAL nomenclature); right fusiform gyrus to right superior occipital (FFG-R:SOG-R), right middle occipital gyrus to FFG-R (MOG-R:FFG-R), MOG-R to right lingual gyrus (MOG-R:LING-R), and left inferior temporal gyrus to LING-R (ITG-L:LING-R). For reward processing, one connection between right superior temporal gyrus and right putamen (STG-R:PUT-R) was h-BMI hyper-connective while another between right caudate and right inferior orbital frontal (CAU-R:ORBinf-R) was detected to show h-BMI hypo-connectivity . For cognitive control, seven connections were detected to show hyper-connectivity in the h-BMI group; the left rectus gyrus to left medial frontal gyrus (REC-L:MGF-L), two separate connections involving the left middle orbital frontal gyrus and MFG-L (ORBmid-L:MFG-L), ORBmid-R to left middle temporal gyrus (ORBmid-R:MTG-L), the right parahippocampul gyrus to right superior parietal gyrus (PHG-R:SPG-R), left dorsal superior frontal gyrus to REC-L (SFGdor-L:REC-L), and the left medial superior frontal gyrus to left post-central gyrus (SFGmed-L:PoCG-L). For motor control, two connections were detected with h-BMI related hyper connectivity; one between PoCG-R and right precentral gyrus (PoCG-R:PreCG-R) and another involving the PreCG-L and left supramarginal gyrus (PreCG-L:SMG-L). Finally, there were two additional connections associated with the left middle temporal pole and left middle cingulate cortex (TPOmid-L:MCG-L) or left superior occipital cortex (TPOmid-L:SOG-L) that showed h-BMI hypo-connectivity. All the detected connections showing functional connectivity strength differences between the two groups also correlated with individual measures of adiposity  percentage, see Table Our seed-based analysis detected 17 clusters associated with 15 seed sub-regions showing significant functional connectivity differences between neonates born to mothers with normal (n-) or high (h-) BMI; Table p < 0.05 uncorrected); Table Our graph theoretical analysis revealed two sets of regions showing nodal degree centrality and/or nodal efficiency differences between the n-BMI and h-BMI groups (p < 0.05) with the cluster-level functional connectivity measures  compared to those born to mothers with normal BMI. The nineteen detected functional connectivity alterations cover all four hypothesized domains that are critically implied in adolescent/adult obesity mechanisms, including sensory cue processing , reward processing, cognitive control, and motor output control. Importantly, when examined at whole brain level, three of the nineteen regions involved in these alterations showing local functional connectivity changes also demonstrated significant global degree and efficacy differences. This overlap suggests that there are likely more widespread local connectivity changes associated with these regions that are statistically undetectable based on the current sample size but their collective effects cascade to produce significant global connectivity property changes as measured by global nodal degree and efficiency. Finally, all the functional connections and most of the nodal degree/efficiency measures detected to show significant group differences also quantitatively correlated with measures of maternal adiposity.Little work has been done in humans to investigate the genetic and/or environmental mechanisms by which maternal obesity influences offspring cognition and mental health . HoweverWithin the sensory cue processing domain, four connections were detected to show differences in functional connectivity between the two groups, including right middle occipital cortex-right fusiform, right middle occipital cortex-right lingual gyrus, right fusiform-right superior occipital cortex, and left inferior temporal gyrus-right lingual gyrus. Note three of the four connections are within the classical visual cortices and one is between the visual cortex and the neighboring inferior temporal gyrus that is part of the ventral visual pathway. This pattern, together with the fact that all four connections show hyper-connectivity in the high BMI group, may suggest stronger interactions within visual processing circuits, which may underlie the heightened sensitivity to visual food cues associated with obesity . ConsistReward processing has been a major topic of obesity studies for decades . In humaPerhaps the most salient finding in this study relates to the dominant detection of functional connectivity differences in the cognitive control domain. Specifically, among the nineteen connectivity alterations detected, seven are associated with cognitive control of reward between classical cognitive control regions  and reward evaluation/processing regions . Among the cognitive control regions involved, five are from the prefrontal regions, one is from the left middle temporal gyrus, and one is from the left superior parietal gyrus. All three prefrontal regions in which obesity-associated differences were observed, including the left dorsal superior frontal gyrus, left middle frontal gyrus, and left medial superior frontal gyrus, are critical executive control regions frequently implied in functional deficits associated with reward re-evaluation, conflict monitoring, and choice selection among obese subjects . ImportaIn addition to functional connections related to visual cue processing, reward processing, and cognitive control, we also detected one connection between the right precentral and right postcentral gyrus showing hyper-connectivity in the high BMI group compared with the normal weight group. This connection is clearly related to motor control and in theory might contribute to disrupted eating behavior. Finally, four other connections associated with the right superior temporal gyrus and the left temporal pole regions were also detected. Consistently, previous findings have shown reduced brain activation to food logos in superior temporal regions in obese compared with normal weight children . A correr = -0.60, p < 0.001). The minimal effects of other potential confounding variables may need to be confirmed in future studies with larger sample sizes. Finally, another limitation of the current study is the potential sleeping stage differences among subjects. Given the practical difficulties of implementing concurrent EEG during MRI scans of naturally sleeping infants, rigorous control of sleeping stage differences in our analysis could not be achieved and future studies are needed to address this issue.Limitations of this study included the relatively small sample size preventing us from detecting likely more widespread and subtle functional connectivity differences between groups. The existence of below-threshold connectional differences can be partly reflected by the detection of a range of regions showing global degree and/or efficiency changes with or without concurrent observation of local clusters showing connectivity differences. In the future, studies with larger sample sizes are needed to validate and expand the current findings. Another limitation of the current study relates to the lack of follow-up behavioral measures (which are ongoing) in cognitive and/or emotional control domains. With such measures, potential direct links between maternal adiposity-related brain functional alterations and behavioral outcomes could be established. Regarding other covariates, we found significant relationships between gestational weight gain and cluster-wise functional connectivity alterations. This is not surprising since gestational weight gain highly negatively correlated with maternal BMI in the current study  Institutional Review Board (IRB) with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the UAMS IRB.AS and WG performed data analysis and wrote part of the manuscript. AA and TB contributed in the study design and edited the manuscript. CG, RR, and AR contributed in data analysis and reviewed the manuscript. XO designed the study, performed data acquisition and some of the analysis, and wrote part of the manuscript.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.


Osimertinib is a first-line treatment option for patients with metastatic non-small cell lung cancer (NSCLC) harbouring EGFR mutations. Pneumonitis is a severe adverse event (AE) related to osimertinib treatment which appears to be more frequent when associated with concurrent or previous anti-PD(L)1 exposure. Data regarding the efficacy and safety of osimertinib rechallenge, especially in the setting of central nervous system (CNS) metastases, are scarce. We herein describe a case of a 53-year-old patient with metastatic EGFR-mutated NSCLC, who developed pneumonitis after osimertinib treatment and was successfully rechallenged with 40 mg daily osimertinib, with CNS response. This dose reduction strategy may be an option for selected patients with brain metastases after tyrosine kinase inhibitors-induced AEs. Brain metastases (BMs) occur initially in 20% of patients with non-small cell lung cancer (NSCLC) and 30%–50% of patients will be diagnosed with BMs during the course of their disease , 2. In pAmong patients with NSCLC with EGFR mutations, tyrosine kinase inhibitors (TKIs) seem effective in controlling the intracranial disease. Preclinical and clinical evidence indicate that EGFR-TKIs of the third generation are more effective in treating BMs than their first- and second-generation counterparts, showing a promising blood–brain barrier (BBB) penetration and the potential to overcome EGFR-TKI resistance .Lung abnormalities, from transient asymptomatic pulmonary opacities to full-blown interstitial lung disease, have been described as adverse events (AEs) associated with osimertinib, a third-generation EGFR-TKI. Severe AEs have been reported when EGFR-TKIs  follow immunotherapy . PotentiEGFR-mutated NSCLC, previously treated with an anti-PD-L1 antibody, which developed severe pneumonitis after initial osimertinib exposure.We hereby describe a case of successful half-dose osimertinib rechallenge with central nervous system (CNS) response in a patient with EGFR exon 19 deletion , detected by pyrosequencing . Post-surgical 18-fluorodeoxyglucose positron emission tomography/computerized tomography revealed positive upper and lower contralateral (right) paratracheal nodes. Definitive concurrent chemoradiation  was administered up to February 2018, followed by durvalumab 10 mg/kg, starting in April 2018. After eleven 14-day cycles, he complained of a new-onset headache. Brain MRI revealed widespread supra and infratentorial brain parenchymal metastasis, and he underwent surgical resection of two bilateral frontal metastases (left 4.1 cm and right 2.8 cm), which confirmed EGFR-mutant lung adenocarcinoma, harbouring the same EGFR exon 19 deletion . Twenty-two days after durvalumab interruption, osimertinib 80 mg once daily was initiated. On the 53rd day of osimertinib treatment, the patient was admitted due to intense dyspnoea on exertion and cough. Chest CT scan revealed patchy ground-glass opacities , harbouring an pacities . As intepacities  and the EGFR-mutated NSCLC [EGFR-mutated advanced NSCLC and CNS metastases [Osimertinib is a treatment option in the first-line setting for patients with metastatic ed NSCLC . It achied NSCLC  and exhitastases . In the tastases .EGFR-mutated patients, the combination of targeted therapies and radiotherapy has been studied. Radiotherapy is known to disrupt BBB and potentially increases the effect of EGFR-TKIs in CNS, even if such inhibitors already penetrate the BBB [et al did not demonstrate superiority in time to treatment failure, PFS and OS of association of radiotherapy and osimertinib compared to osimertinib alone in a retrospective analysis [EGFR-mutated NSCLC with BM (NCT03497767).In order to optimise the CNS control in  the BBB . A retro the BBB . Regardianalysis . OsimertThe increase of osimertinib toxicity in patients who used anti-PD(L)1 was recently described, and interstitial lung disease is one of the main AEs . The conet al described a case of an EGFR T790M-positive lung cancer patient, who was pretreated with the sequence erlotinib–osimertinib and experienced a dramatic response to osimertinib rechallenge after intervening chemotherapy [Osimertinib rechallenge is an important point to be considered. A successful full-dose osimertinib rechallenge has been reported in a patient with BMs following salvage cytotoxic chemotherapy, showing potential intracranial therapeutic effect with re-exposure to the drug . Metro eotherapy . In a reotherapy .EGFR-mutated NSCLC patients. The present case indicates that it may be feasible to treat patients with BMs with osimertinib 40 mg daily if dose reduction is needed after a severe drug-related AE.Previous experiences indicate that re-administration of 40 mg osimertinib may be a safe and effective strategy in patients who developed osimertinib-induced pneumonitis with standard-dose use . CliniciEGFR-mutant NSCLC, osimertinib rechallenge can be considered in selected cases.To the authors’ knowledge, this is the first report of activity with CNS response and safety after rechallenge with osimertinib 40 mg. The safest time interval between interrupting immunotherapy and starting osimertinib is still an unanswered question. Once patients with BMs have the urgency to start a CNS active therapy and considering the efficacy of osimertinib in Dr de Castro Junior reports personal fees and other from AstraZeneca, personal fees and other from Roche, personal fees and other from Boehringer-Ingelheim, outside the submitted work.The other authors declare that they have no conflicts of interest.No funding was received.


This study aims to explore the contribution of implicit attitudes and associations towards conventional disease-modifying antirheumatic drugs (cDMARDs), alongside explicit measures, on medication-taking behaviour and clinical outcomes in adult patients with rheumatoid arthritis (RA).In this observational study, implicit attitudes (positive-negative) and health-related associations  were measured with Single Category Implicit Association Tests, whereas explicit outcomes were measured with a bipolar evaluative adjective scale and the Beliefs about Medicines Questionnaire Specific. The primary outcome of this study was medication-taking behaviour subjectively measured by self-report  and objectively measured with electronic drug monitors over a 3 month period. Spearman rank correlations were used to describe correlations between implicit and explicit outcomes. Nested linear regression models were used to assess the additional value of implicit measures over explicit measures and patient-, clinical-, and treatment-related characteristics.= 0.13, P = 0.05) and disease activity scores , whereas implicit health-related associations correlated significantly with mean scores for explicitly reported health-related associations . Significant differences in age, number of DMARDs, biologic DMARD use, NCD-scores, and self-reported correct dosing were found between the four attitudinal profiles. Nested linear regression models revealed no additional value of implicit measures in explaining self-reported medication-taking behaviour and clinical outcomes, over and above all other variables.Of the 1659 initially-invited patients, 254 patients with RA agreed to participate in this study. Implicit attitudes correlated significantly with necessity-concerns differential (NCD) scores (ρ Implicit attitudes and associations had no additional value in explaining medication-taking behaviour and clinical outcomes over and above often used explicitly measured characteristics, attitudes and outcomes in the studied population. Only age and NCD scores contributed significantly when the dependent variable was correct dosing measured with self-report. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by synovial inflammation, which can lead to irreversible articular damage, a decrease in physical functioning and quality of life, and eventually increased healthcare expenditures –5. ConveAn explanation for the ineffectiveness of adherence-improving interventions might be that previous studies have largely focused on patient’s explicit, ‘conscious’, evaluations of e.g. medication or medication-taking behaviour ,12. ThesTo date, few studies have investigated implicit attitudes towards medication and their potential relation to medication-taking behaviour –29. No sTherefore, the primary objective of this study is to explore the contribution of implicitly measured attitudes towards cDMARDs, alongside explicit measures, on medication-taking behaviour (i.e. measured subjectively with self-report and objectively with electronic drug monitors) of patients with RA. The secondary objective is to explore the contribution of implicitly measured attitudes towards cDMARDs, alongside explicit measures, on disease activity scores in patients with RA.th, 2016 and November 30th, 2017. The STROBE  statement for observational studies and EMERGE (ESPACOMP Medication Adherence Reporting Guideline) were used as guidance for adequate reporting of this study : Sint Maartenskliniek (Nijmegen) and Reade (Amsterdam). Participants were recruited between July 5Consecutive adult (≥ 18 years) patients with a clinical diagnosis of RA, treated with at least one cDMARD for a minimum period of one year were invited to participate in this study. No additional in- and exclusion criteria were defined. Four weeks before their planned regular consultation with their treating clinician, patients received written information and an informed consent form. After one to two weeks, patients were approached by telephone to ask about their willingness to participate in this study. In case patients agreed to participate, the researcher planned a research appointment before their regular consultation in order to sign the informed consent form and complete baseline measurements.Primary outcome of this study was medication-taking behaviour measured over a period of three months after inclusion. Both self-report ) and electronic drug monitors (i.e. Medication wAardex®)) were used to assess the implementation of the dosing regimen –35. The Baseline measurements were performed in the consulting room of the outpatient pharmacy. The following data were collected with a hardcopy questionnaire at baseline: socio-demographic characteristics , explicit attitudes and health-related associations with cDMARDs with a bipolar evaluative adjective scale , beliefs about medicines with the Beliefs about Medicines Questionnaire Specific , and self-reported medication-taking behaviour with the validated CQR with 19 Likert-scaled (ranging from 1 to 4) items ,34,38. Iet al [SC-IATs (Inquisit version 5) were used to measure automatic associations ,39–41. Tet al .Explicit attitudes  and explicit health-related associations  were assessed with a bipolar evaluative adjective scale. The items were identical to those in the SC-IATs  or median (P25-P75) depending on the distribution of measurements. Educational level was classified in low, moderate or high educational level. Low educational level was defined as no education, (extended) primary education or pre-vocational education, moderate educational level was defined as vocational education or selective secondary education, and high educational level was defined as education provided by universities of applied sciences and research universities. Data were presented as percentages in case of proportions. P-values ≤0.05 were considered statistically significant.et al. was used as scoring procedure to calculate the D measure for strength of automatic associations [D measures. D measures above 0 indicated that patients had relatively faster response times on positive categorisation rounds than on negative categorisation rounds, and were interpreted as a relatively more positive than negative implicit attitude towards cDMARDs or a relatively more health-related association than sickness-related association, and vice versa.Implicit attitudes and health-related associations were assessed in terms of response times in milliseconds (ms) on the SC-IATs. The improved IAT scoring algorithm described by Greenwald ciations . S1 FileFor explicit attitudes, mean sum scale scores were calculated for each concept . Patients with a mean scale score below or equal to 3 were considered to be relatively negative or have sickness-related associations with cDMARDs, whereas patients with mean scale scores higher than 3 were considered to be relatively positive or associated their cDMARD use with health. Beliefs about medicines were operationalised as necessity-concerns differential (NCD) scores ,43. Thist-tests, Pearson chi-square tests, Fisher’s exact tests, Bonferroni corrected post-hoc tests, and proportion tests were performed to test for significant differences in patient characteristics between study sites and attitudinal profiles.Participants were also categorised in attitudinal profiles based on their (in)congruent (implicit and explicit) attitudes and health-related associations based on the cut-off scores for implicit and explicit attitudes and health-related associations as described above. Depending on the distribution and type of variables, Two-sample et al [et al [Self-reported medication-taking behaviour was calculated with the discriminant function for CQR items as described by de Klerk et al . The criet al . Medicatl [et al .Spearman rank correlations were used to describe the correlation between patient’s implicit and explicit outcomes, medication-taking behaviour (i.e. measured by self-report and using MEMS), and clinical outcomes. Because of the explorative (rather than hypothesis-testing) character of this study, no multiple testing corrections were performed over the separate correlational analyses. Nested linear regression models  were used to assess the additional value of implicit measures, over explicit measures and patient characteristics, in explaining adherence to medication and disease activity scores in patients with RA. For each separate model three dependent continuous variables were used: correct dosing based on (1) the CQR discriminant function, and (2) MEMS data, followed by (3) DAS28-CRP scores at baseline.th WMA General Assembly, Fortaleza, Brazil, October 2013) and was approved by the Medical Research Ethics Committee of Arnhem-Nijmegen (File: 2016–2410).This study was conducted according to the ethical principles for medical research as stated in the Declaration of Helsinki . Of the 254 patients who agreed to participate, 98.8% completed all SC-IATs, 98.0% the bipolar evaluative adjective scale, 98.8% the BMQ-Specific, 99.2% the CQR and 91.7% of the patients provided MEMS data. The main reason for missing MEMS data was discontinuation of cDMARD therapy . ContinuD measure for implicit attitudes towards cDMARDs was -0.054 , whereas the mean D measure for implicit health-related associations with cDMARDs was -0.10 . The mean score for explicit attitudes of patients who completed the bipolar evaluative adjective scale was 3.5 , which was similar for explicit health-sickness associations with cDMARDs . Regarding beliefs about medicines, the mean sum scale score for necessity beliefs and for concern beliefs was 19.9  and 14.1 , respectively. The mean necessity-concerns differential score of 5.8  indicates that necessity beliefs outweigh patient’s concerns about medication.The mean D measure) and explicit attitudes  were not significantly correlated , however a significant weak correlation was found between mean D measures for implicit attitudes and necessity-concerns differential scores  were not correlated with self-reported correct dosing , nor with MEMS correct dosing . However, a weak significant negative correlation between D measures for implicit attitudes and disease activity scores was found , where more negative implicit attitudes (D measures) were associated with higher disease activity scores. No correlations were found between implicit health-related associations and any of the following variables: correct dosing measured with self-report , correct dosing measured with MEMS , and disease activity scores .Implicit attitudes  were not correlated with self-reported correct dosing , MEMS correct dosing , and disease activity scores . Also explicit health-related associations were not correlated with MEMS correct dosing , however, a significant negative correlation was found between explicit health-related associations and disease-activity scores , and a significant positive correlation with self-reported correct dosing . NCD-scores were significantly correlated with self-reported correct dosing , but not with MEMS correct dosing and disease activity scores.D measures for implicit attitudes and mean scores for explicit attitudes from the bipolar evaluative adjective scale, patients were categorized in four attitudinal profiles per concept, indicating the (in)congruently positive or negative nature of their implicit and explicit attitudes (see Fewer patients displayed implicit positive attitudes (47.2%) and health-related associations (39.6%) regarding cDMARD therapy than when explicitly asked . Based on Bonferroni corrected post-hoc tests revealed that congruently negative patients (profile III) differed significantly in age, NCD-scores, bDMARD use and self-reported correct dosing from other attitudinal profiles. Congruently negative patients were on average 6.1 years younger  than congruently positive patients, reported more concerns than explicitly positive patients , more often used bDMARDs than all other attitudinal profiles  and were less often adherent based on self-reported correct dosing than the other attitudinal profiles . When exploring the proportion of adherent patients measured with MEMS over time between different profiles, similar patterns emerge within the four attitudinal profiles . HoweverSubgroup characteristics for each profile regarding health-related associations can be found in The skewed MEMS data (i.e. high proportion of adherent patients), even after data transformation, did not meet the assumptions for linear regression modelling. Medication-taking behaviour measured with MEMS was, therefore, excluded for nested linear regression modelling. Nested linear regression models showed that only age and NCD-scores contributed significantly when the dependent variable was correct dosing measured with self-report. However, no variables measured in this study contributed significantly to disease activity scores. Implicit attitudes and implicit health-related associations, alongside patient characteristics and explicit measures, had no added value in explaining variance in medication-taking behaviour when measured with self-report. The same applied for disease activity scores see . OverallThis study showed that implicit attitudes and health-related associations do not explain medication-taking behaviour and clinical outcomes in clinical practice over and above 1) patient characteristics, 2) clinical variables, 3) treatment characteristics, 4) explicit attitudes and health-related associations, or 5) beliefs about medicines. Only age and NCD-scores contributed significantly to medication taking behaviour measured with self-report. However, some significant but weak associations were found between implicit attitudes and necessity-concerns differential scores, between implicit and explicit health-related associations, and implicit attitudes and disease activity. The categorization of patients in combined implicit and explicit attitudinal profiles revealed that characteristics of in particular patients with congruently negative or congruently sickness-related associations, differed from other attitudinal profiles.et al already emphasized the importance of exploring implicit associations as possible targets for improving actual (rather than self-reported) medication adherence in this population [et al and Rüsch et al, which supports the idea that implicit and explicit measures are different but related constructs [To our knowledge, this is the first study which compares groups of patients with congruent and incongruent implicit and explicit attitudes towards medication in relation to objectively measured medication-taking behaviour and clinical outcomes in patients with RA. Linn pulation . In termnstructs ,28. HoweOne explanation is that no association between implicit associations and objectively measured medication-taking behaviour in clinical practice exist. In other words, implicit associations (with cDMARDs) do not underlie (medication-taking) behaviour. This finding is contrary to previous studies ,21,46,47et al, we used validated questionnaires to assess self-reported adherence and beliefs about medicines [The key strengths of this study are the large sample size and patient recruitment in two of the largest rheumatology specialized centres across the Netherlands . Another strength is the use of MEMS to objectively measure medication-taking behaviour over three months in addition to the assessment of medication-taking behaviour by self-report. Measuring medication-taking behaviour by self-report is more susceptible to recall bias . In contedicines . An advaedicines . Still, D measure near midpoint 0) or neutral explicit attitudes or health-related associations (i.e. mean score near midpoint 3) might have reduced the contrast in characteristics  across attitudinal profiles. Currently, there is discussion in the literature on whether implicit measures by definition expose automatic associations or subconscious attitudes or whether they might also be sensitive to more reflective and conscious thought [The validity of the SC-IATs used in this study might be questioned since the target population might have a limited hand function, which might provide insufficient contrast between the experimental rounds in the SC-IATs. Also, it is unclear if the words and pictures used as stimuli in the SC-IATs are optimally related to patient’s medication use . However thought ,26,48. T thought ,25,26,48The external validity of the results might be questionable due to the low response rate of patients, together with the high proportion of adherent participants. The latter might indicate selection bias since it is well known that in adherence research often only highly motivated patients are willing to participate in studies . It is cIn conclusion, about half of the patients with RA showed incongruent (implicit and explicit) attitudes and health-related associations with cDMARDs. Implicit attitudes and associations had no additional value in explaining medication-taking behaviour and clinical outcomes over and above often used explicitly measured characteristics, attitudes and outcomes in the studied population. However, this research provides interesting areas for future research regarding implicit and explicit processes that might be involved in medication-taking behaviour.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableDescription of profiles of patients diagnosed with rheumatoid arthritis based on (in)congruent implicit and explicit health- versus sickness-related associations with cDMARDs. Categories were low versus medium-high for educational level, living alone versus living together (with children and/or partner) for residential status, and for ethnic background Dutch versus other. Variables with P-values ≤0.05 (unadjusted for multiple testing) were further analyzed with Bonferroni corrected post-hoc tests. Abbreviations: DAS28-CRP (Disease Activity Score based on 28 joints and C-Reactive Protein), DMARD (disease-modifying antirheumatic drug), bDMARD (biologic DMARD), MEMS (Medication Event Monitoring System).(PDF)Click here for additional data file.