Propofol improves survival in a murine model of sepsis via inhibiting Rab5a-mediated intracellular trafficking of TLR4.

BACKGROUND: Propofol is a widely used anesthetic and sedative, which has been reported to exert an anti-inflammatory effect. TLR4 plays a critical role in coordinating the immuno-inflammatory response during sepsis. Whether propofol can act as an immunomodulator through regulating TLR4 is still unclear. Given its potential as a sepsis therapy, we investigated the mechanisms underlying the immunomodulatory activity of propofol. METHODS: The effects of propofol on TLR4 and Rab5a (a master regulator involved in intracellular trafficking of immune factors) were investigated in macrophage (from Rab5a-/- and WT mice) following treatment with lipopolysaccharide (LPS) or cecal ligation and puncture (CLP) in vitro and in vivo, and peripheral blood monocyte from sepsis patients and healthy volunteers. RESULTS: We showed that propofol reduced membrane TLR4 expression on macrophages in vitro and in vivo. Rab5a participated in TLR4 intracellular trafficking and both Rab5a expression and the interaction between Rab5a and TLR4 were inhibited by propofol. We also showed Rab5a upregulation in peripheral blood monocytes of septic patients, accompanied by increased TLR4 expression on the cell surface. Propofol downregulated the expression of Rab5a and TLR4 in these cells. CONCLUSIONS: We demonstrated that Rab5a regulates intracellular trafficking of TLR4 and that propofol reduces membrane TLR4 expression on macrophages by targeting Rab5a. Our study not only reveals a novel mechanism for the immunomodulatory effect of propofol but also indicates that Rab5a may be a potential therapeutic target against sepsis.

Residual abnormalities on CTE predict adverse outcomes in Crohn's disease with endoscopic healing.

BACKGROUND: Residual abnormalities on computed tomography enterography (CTE) in Crohn's disease (CD) with endoscopic healing (EH) may have prognostic implications and affect therapeutic strategy. METHODS: CD patients with EH who underwent CTE between March 2015 and June 2022 were enrolled. CTE findings of the terminal ileum and the most severe segment of colon at the time of EH were assessed respectively for each patient. Cox regression analysis and Kaplan-Meier curves were used to evaluate the association between residual abnormalities and adverse outcomes. RESULTS: A total of 140 patients (217 digestive segments) were included. Mesenteric edema (hazard ratio [HR] = 3.61, 95% CI = 1.81-7.20, P<0.001), fibrofatty proliferation (HR = 3.40, 95% CI = 1.97-5.85, P<0.001) and active small bowel inflammation (HR = 2.74, 95% CI = 1.59-4.71, P<0.001) were risk factors for clinical relapse. Furthermore, we built a scoring system using the three parameters. Radiologic score ≥ 1 was the best threshold to predict clinical relapse (HR = 4.56, 95% CI = 2.54-8.19, P<0.001) and it was validated in different outcomes. CONCLUSION: The scoring system based on three residual abnormalities on CTE can predict adverse outcomes in CD patients with EH.

Mesenteric creeping fat index defined by CT enterography is associated with early postoperative recurrence in patients with Crohn's disease.

OBJECTIVES: To investigate the value of mesenteric creeping fat index (MCFI) defined by computed-tomography enterography (CTE) in patients with Crohn's Disease (CD) for predicting early postoperative recurrence. METHODS: A total of 110 patients with CD who underwent CTE and I-stage intestinal resection surgery from December 2013 to December 2018 were enrolled. Two radiologists independently assessed CTE parameters, including MCFI, with scores ranging from 1 to 8; bowel-wall thickening, with a scale of 1 to 3; mural hyperenhancement, mural stratification, submucosal fat deposition, mesenteric fibrofatty proliferation, mesenteric hypervascularity, mesenteric fat stranding, with a scale of 0 to 2; abscess/fistula, enlarged mesenteric lymph node, abdominal and pelvic effusion, with a scale of 0 to 1. Imaging findings associated with early recurrence were assessed using logistic regression analysis. RESULTS: Within one year follow-up, early postoperative recurrence occurred in 56.4 % (62/110) patients with CD. In univariate analysis, MCFI, bowel-wall thickening, mesenteric hypervascularity, mesenteric fat stranding, abscess/fistula and mesenteric lymphadenopathy were associated with early postoperative recurrence. Among all variables, MCFI (score ≥ 4) contributes the optimal AUC (0.838 [0.758-0.919]), specificity (89.6 %), positive predictive value (90.7 %), accuracy (83.6 %), and risk ratio (OR = 32.42 [10.69-98.33], p < 0.001). In multivariate analysis, only MCFI was an independent predictor of early postoperative recurrence (OR = 25.71 [7.65-86.35], p < 0.001). CONCLUSION: CTE features are useful in predicting early postoperative recurrence in patients with CD, MCFI may be a valuable tool for clinical monitoring and follow-up.

Organoids transplantation attenuates intestinal ischemia/reperfusion injury in mice through L-Malic acid-mediated M2 macrophage polarization.

Intestinal organoid transplantation is a promising therapy for the treatment of mucosal injury. However, how the transplanted organoids regulate the immune microenvironment of recipient mice and their role in treating intestinal ischemia-reperfusion (I/R) injury remains unclear. Here, we establish a method for transplanting intestinal organoids into intestinal I/R mice. We find that transplantation improve mouse survival, promote self-renewal of intestinal stem cells and regulate the immune microenvironment after intestinal I/R, depending on the enhanced ability of macrophages polarized to an anti-inflammatory M2 phenotype. Specifically, we report that L-Malic acid (MA) is highly expressed and enriched in the organoids-derived conditioned medium and cecal contents of transplanted mice, demonstrating that organoids secrete MA during engraftment. Both in vivo and in vitro experiments demonstrate that MA induces M2 macrophage polarization and restores interleukin-10 levels in a SOCS2-dependent manner. This study provides a therapeutic strategy for intestinal I/R injury.

Exosomal circEZH2_005, an intestinal injury biomarker, alleviates intestinal ischemia/reperfusion injury by mediating Gprc5a signaling.

Intestinal ischemia/reperfusion (I/R) injury is a severe clinical condition without optimal diagnostic markers nor clear molecular etiological insights. Plasma exosomal circular RNAs (circRNAs) are valuable biomarkers and therapeutic targets for various diseases, but their role in intestinal I/R injury remains unknown. Here we screen the expression profile of circRNAs in intestinal tissue exosomes collected from intestinal I/R mice and identify circEZH2_005 as a significantly downregulated exosomal circRNA. In parallel, circEZH2_005 is also reduced in the plasma of clinical cardiac surgery patients who developed postoperative intestinal I/R injury. Exosomal circEZH2_005 displays a significant diagnostic value for intestinal injury induced by I/R. Mechanistically, circEZH2_005 is highly expressed in intestinal crypt cells. CircEZH2_005 upregulation promotes the proliferation of Lgr5+ stem cells by direct interaction with hnRNPA1, and enhanced Gprc5a stability, thereby alleviating I/R-induced intestinal mucosal damage. Hence, exosomal circEZH2_005 may serve as a biomarker for intestinal I/R injury and targeting the circEZH2_005/hnRNPA1/Gprc5a axis may be a potential therapeutic strategy for intestinal I/R injury.

Microbiota-derived tryptophan metabolites indole-3-lactic acid is associated with intestinal ischemia/reperfusion injury via positive regulation of YAP and Nrf2.

BACKGROUND: Lactobacillus has been demonstrated to serve a protective role in intestinal injury. However, the relationship between Lactobacillus murinus (L. murinus)-derived tryptophan metabolites and intestinal ischemia/reperfusion (I/R) injury yet to be investigated. This study aimed to evaluate the role of L. murinus-derived tryptophan metabolites in intestinal I/R injury and the underlying molecular mechanism. METHODS: Liquid chromatograph mass spectrometry analysis was used to measure the fecal content of tryptophan metabolites in mice undergoing intestinal I/R injury and in patients undergoing cardiopulmonary bypass (CPB) surgery. Immunofluorescence, quantitative RT-PCR, Western blot, and ELISA were performed to explore the inflammation protective mechanism of tryptophan metabolites in WT and Nrf2-deficient mice undergoing intestinal I/R, hypoxia-reoxygenation (H/R) induced intestinal organoids. RESULTS: By comparing the fecal contents of three L. murinus-derived tryptophan metabolites in mice undergoing intestinal I/R injury and in patients undergoing cardiopulmonary bypass (CPB) surgery. We found that the high abundance of indole-3-lactic acid (ILA) in the preoperative feces was associated with better postoperative intestinal function, as evidenced by the correlation of fecal metabolites with postoperative gastrointestinal function, serum I-FABP and D-Lactate levels. Furthermore, ILA administration improved epithelial cell damage, accelerated the proliferation of intestinal stem cells, and alleviated the oxidative stress of epithelial cells. Mechanistically, ILA improved the expression of Yes Associated Protein (YAP) and Nuclear Factor erythroid 2-Related Factor 2 (Nrf2) after intestinal I/R. The YAP inhibitor verteporfin (VP) reversed the anti-inflammatory effect of ILA, both in vivo and in vitro. Additionally, we found that ILA failed to protect epithelial cells from oxidative stress in Nrf2 knockout mice under I/R injury. CONCLUSIONS: The content of tryptophan metabolite ILA in the preoperative feces of patients is negatively correlated with intestinal function damage under CPB surgery. Administration of ILA alleviates intestinal I/R injury via the regulation of YAP and Nrf2. This study revealed a novel therapeutic metabolite and promising candidate targets for intestinal I/R injury treatment.

Gut microbiota-derived indole 3-propionic acid partially activates aryl hydrocarbon receptor to promote macrophage phagocytosis and attenuate septic injury.

Sepsis is associated with a high risk of death, and the crosstalk between gut microbiota and sepsis is gradually revealed. Indole 3-propionic acid (IPA) is a gut microbiota-derived metabolite that exerts immune regulation and organ protective effects. However, the role of IPA in sepsis is not clear. In this study, the role of IPA in sepsis-related survival, clinical scores, bacterial burden, and organ injury was assessed in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Aryl hydrocarbon receptor (AhR) highly specific inhibitor (CH223191) was used to observe the role of AhR in the protection of IPA against sepsis. The effects of IPA on bacterial phagocytosis by macrophages were investigated in vivo and vitro. The levels of IPA in feces were measured and analyzed in human sepsis patients and patient controls. First, we found that gut microbiota-derived IPA was associated with the survival of septic mice. Then, in animal model, IPA administration protected against sepsis-related mortality and alleviated sepsis-induced bacterial burden and organ injury, which was blunted by AhR inhibitor. Next, in vivo and vitro, IPA enhanced the macrophage phagocytosis through AhR. Depletion of macrophages reversed the protective effects of IPA on sepsis. Finally, on the day of ICU admission (day 0), septic patients had significantly lower IPA level in feces than patient controls. Also, septic patients with bacteremia had significantly lower IPA levels in feces compared with those with non-bacteremia. Furthermore, in septic patients, reduced IPA was associated with worse clinical outcomes, and IPA in feces had similar prediction ability of 28-day mortality with SOFA score, and increased the predictive ability of SOFA score. These findings indicate that gut microbiota-derived IPA can protect against sepsis through host control of infection by promoting macrophages phagocytosis and suggest that IPA may be a new strategy for sepsis treatment.

Reduction of metal artifacts from knee tumor prostheses on CT images: value of the single energy metal artifact reduction (SEMAR) algorithm.

BACKGROUND: To evaluate the effect of the single energy metal artifact reduction (SEMAR) algorithm with a multidetector CT (MDCT) for knee tumor prostheses. METHODS: First, a phantom of knee tumor prosthesis underwent a MDCT scan. The raw data was reconstructed by iterative reconstruction (IR) alone and IR plus SEMAR. The mean value of the CT number and the image noise were measured around the prosthesis at the stem level and articular level. Second, 95 consecutive patients with knee tumor prostheses underwent MDCT scans. The raw data were also reconstructed by the two methods. Periprosthetic structures were selected at the similar two levels. Four radiologists visually graded the image quality on a scale from 0 to 5. Additionally, the readers also assessed the presence of prosthetic complication and tumor recurrence on a same scale. RESULTS: In the phantom, when the SEMAR was used, the CT numbers were closer to normal value and the noise of images using soft and sharper kernel were respectively reduced by up to 77.1% and 43.4% at the stem level, and by up to 82.2% and 64.5% at the articular level. The subjective scores increased 1 ~ 3 points and 1 ~ 4 points at the two levels, respectively. Prosthetic complications and tumor recurrence were diagnosed in 66 patients. And the SEMAR increased the diagnostic confidence of prosthetic complications and tumor recurrence (4 ~ 5 vs. 1 ~ 1.5). CONCLUSIONS: The SEMAR algorithm can significantly reduce the metal artifacts and increase diagnostic confidence of prosthetic complications and tumor recurrence in patients with knee tumor prostheses.

Tripterygium hypoglaucum (Lévl.) Hutch and Its Main Bioactive Components: Recent Advances in Pharmacological Activity, Pharmacokinetics and Potential Toxicity.

Tripterygium hypoglaucum (Lévl.) Hutch (THH) is believed to play an important role in health care and disease treatment according to traditional Chinese medicine. Moreover, it is also the representative of medicine with both significant efficacy and potential toxicity. This characteristic causes THH hard for embracing and fearing. In order to verify its prospect for clinic, a wide variety of studies were carried out in the most recent years. However, there has not been any review about THH yet. Therefore, this review summarized its characteristic of components, pharmacological effect, pharmacokinetics and toxicity to comprehensively shed light on the potential clinical application. More than 120 secondary metabolites including terpenoids, alkaloids, glycosides, sugars, organic acids, oleanolic acid, polysaccharides and other components were found in THH based on phytochemical research. All these components might be the pharmacological bases for immunosuppression, anti-inflammatory and anti-tumour effect. In addition, recent studies found that THH and its bioactive compounds also demonstrated remarkable effect on obesity, insulin resistance, fertility and infection of virus. The main mechanism seemed to be closely related to regulation the balance of immune, inflammation, apoptosis and so on in various disease. Furthermore, the study of pharmacokinetics revealed quick elimination of the main component triptolide. The feature of celastrol was also investigated by several models. Finally, the side effect of THH was thought to be the key for its limitation in clinical application. A series of reports indicated that multiple organs or systems including liver, kidney and genital system were involved in the toxicity. Its potential serious problem in liver was paid specific attention in recent years. In summary, considering the significant effect and potential toxicity of THH as well as its components, the combined medication to inhibit the toxicity, maintain effect might be a promising method for clinical conversion. Modern advanced technology such as structure optimization might be another way to reach the efficacy and safety. Thus, THH is still a crucial plant which remains for further investigation.

Glycopeptide-Conjugated Aggregation-Induced Emission Luminogen: A pH-Responsive Fluorescence Probe with Tunable Self-Assembly Morphologies for Cell Imaging.

pH values play an important role in various cell biological processes. Abnormal pH values in living systems are frequently associated with the development of diseases such as cancers, infection, and other diseases. Real-time monitoring of the changes of pH values in vivo will give us the significant indication for these diseases' progression. Within those pH-sensitive imaging probes, aggregation-induced emission (AIE) molecules exhibit great potential in aqueous imaging environment due to their high fluorescence quantum yield and stability. However, the modulation of the AIE probe with pH sensitivity and light-up property face challenges. Here, we introduced a new glycopeptide-modified AIE probe (TGO) based on the optimized solid-phase peptide synthesis approach. The response to pH of the peptide: DDDD progression changed hydrophobicity and hydrophilicity, resulting in the change of the amphipathicity balance. When modulating the pH from 5.5 to 8.0, the adverse protonation of the peptide induced assembled nanostructure transformation from nanolamellae to nanomicelles. Meanwhile, the pH-induced charge change in peptides can greatly influence the microenvironment of the AIEgen, resulting in the increase of fluorescence intensity.

The emerging possibility of the use of geniposide in the treatment of cerebral diseases: a review.

With the advanced discoveries in the field of pathogenesis, a series of cerebral diseases, such as cerebral ischaemia, Alzheimer's disease, and depression, have been found to have multiple signalling targets in the microenvironment. Only a few existing agents have been shown to have curative effects due to this specific circumstance. In recent decades, active ingredients isolated from natural plants have been shown to be crucial for original drug development. Geniposide, mainly extracted from Gardenia jasminoides Ellis, is representative of these natural products. Geniposide demonstrates various biological activities in the treatment of cerebral, cardiovascular, hepatic, tumorous, and other diseases. The multiple protective effects of geniposide on the brain have especially drawn increasing attention. Thus, this article specifically reviews the characteristics of current models of cerebral ischaemia and illustrates the possible effects of geniposide and its pathogenetic mechanisms on these models. Geniposide has been shown to significantly reduce the area of cerebral infarction and alleviate neuronal damage and necrosis mainly by inhibiting inflammatory signals, including NLRP3, TNF-α, IL-6, and IL-1ß. Neuronal protection was also involved in activating the PI3K/Akt and Wnt/catenin pathways. Geniposide was able to increase autophagy and inhibit apoptosis by regulating the function of mTOR in treating Alzheimer's disease. Geniposide has also been shown to act as a glucagon-like peptide-1 receptor (GLP-1R) agonist to reduce amyloid plaques and inhibit oxidative stress to alleviate memory impairment as well as synaptic loss. Moreover, geniposide has been shown to exert antidepressant effects primarily by regulating the hypothalamic-pituitary-adrenal (HPA) axis. Detailed explorations have shown that the biological activities of inhibiting inflammatory cytokine secretion, alleviating oxidative stress, and suppressing mitochondrial damage are also involved in the mechanism of action of geniposide. Therefore, geniposide is a promising agent awaiting further exploration for the treatment of cerebral diseases via various phenotypes or signalling pathways.

The combination of 13N-ammonia and 11C-methionine in differentiation of residual/recurrent pituitary adenoma from the pituitary gland remnant after trans-sphenoidal Adenomectomy.

BACKGROUND: This study aimed to assess the clinical usefulness of 13N-ammonia and 11C- Methionine (MET) positron emission tomography (PET)/ computed tomography (CT) in the differentiation of residual/recurrent pituitary adenoma (RPA) from the pituitary gland remnant (PGR) after trans-sphenoidal adenomectomy. METHODS: Between June 2012 and December 2019, a total of 19 patients with a history of trans-sphenoidal adenomectomy before PET/CT scans and histological confirmation of RPA after additional surgery in our hospital were enrolled in this study. Images were interpreted by visual evaluation and semi-quantitative analysis. In semi-quantitative analysis, the maximum standard uptake value (SUVmax) of the target and gray matter was measured and the target uptake/gray matter uptake (T/G) ratio was calculated. RESULTS: The T/G ratios of 13N-ammonia were significantly higher in PGR than RPA (1.58 ± 0.69 vs 0.63 ± 1.37, P < 0.001), whereas the T/G ratios of 11C-MET were obviously lower in PGR than RPA (0.78 ± 0.35 vs 2.17 ± 0.54, P < 0.001). Using the canonical discriminant analysis, we calculated the predicted accuracy of RPA (100%), PGR (92.9%), and the overall predicted accuracy (96.43%). CONCLUSIONS: The combination of 13N-ammonia and 11C-MET PET/CT is valuable in the differentiation of RPA from PGR after trans-sphenoidal adenomectomy.

Therapeutic Opportunities of GPBAR1 in Cholestatic Diseases.

GPBAR1, a transmembrane G protein-coupled receptor for bile acids, is widely expressed in multiple tissues in humans and rodents. In recent years, GPBAR1 has been thought to play an important role in bile homeostasis, metabolism and inflammation. This review specifically focuses on the function of GPBAR1 in cholestatic liver disease and summarizes the various pathways through which GPBAR1 acts in cholestatic models. GPBAR1 mainly regulates cholestasis in a holistic system of liver-gallbladder-gut formation. In the state of cholestasis, the activation of GPBAR1 could regulate liver inflammation, induce cholangiocyte regeneration to maintain the integrity of the biliary tree, control the hydrophobicity of the bile acid pool and promote the secretion of bile HCO3 -. All these functions of GPBAR1 might be clear ways to protect against cholestatic diseases and liver injury. However, the characteristic of GPBAR1-mediated proliferation increases the risk of proliferation of cholangiocarcinoma in malignant transformed cholangiocytes. This dichotomous function of GPBAR1 limits its use in cholestasis. During disease treatment, simultaneous activation of GPBAR1 and FXR receptors often results in improved outcomes, and this strategy may become a crucial direction in the development of bile acid-activated receptors in the future.

Differentiation of suprasellar meningiomas from non-functioning pituitary macroadenomas by 18F-FDG and 13N-Ammonia PET/CT.

BACKGROUND: Differentiation of suprasellar meningiomas (SSMs) from non-functioning pituitary macroadenomas (NFPMAs) is useful for clinical management. We investigated the utility of 13N-ammonia combined with 18F-FDG positron emission tomography (PET)/computed tomography (CT) in distinguishing SSMs from NFPMAs retrospectively. METHODS: Fourteen NFPMA patients and eleven SSM patients with histopathologic diagnosis were included in this study. Every patient underwent both 18F-FDG and 13N-ammonia PET/CT scans. The tumor to gray matter (T/G) ratios were calculated for the evaluation of tumor uptake. RESULTS: The uptake of 18F-FDG was higher in NFPMAs than SSMs, whereas the uptake of 13N-ammonia was obviously lower in NFPMAs than SSMs. The differences of 18F-FDG and 13N-ammonia uptake between the two groups were significant respectively (0.92[0.46] vs 0.59[0.29], P < 0.05, 18F-FDG; 1.58 ± 0.56 vs 2.80 ± 1.45, P < 0.05, 13N-ammonia). Tumor classification demonstrated a high overall accuracy of 96.0% for differential diagnosis. When the two traces were combined, only 1 SSM was misclassified into the NFPMA group. CONCLUSION: SSMs and NFPMAs have different metabolic characteristics on 18F-FDG and 13N-ammonia PET images. The combination of these two tracers can effectively distinguish SSMs from NFPMAs.

6-Gingerol attenuates macrophages pyroptosis via the inhibition of MAPK signaling pathways and predicts a good prognosis in sepsis.

BACKGROUND: Sepsis is a major cause of death for ICU patients. Sepsis development depends heavily on the presence of mature IL-1ß cytokine. This study evaluates the potential therapeutic properties of a bioactive compound known as 6-gingerol on sepsis. This compound has previously been demonstrated to possess anti-inflammatory properties both in vivo and in vitro. METHODS: C57BL/6 mice was used to establish models of sepsis by means of cecal ligation and puncture (CLP). Upon treatment with 6-gingerol, we assessed the survival rate of mice and measured the levels of key pro-inflammatory cytokines in serum and colon tissues. Sepsis pathogenesis was further explored using the RAW264.7 cell line and bone marrow-derived macrophages (BMDMs) treated with ATP and lipopolysaccharide (LPS). The impact of 6-gingerol on pyroptosis was also examined. In addition, we assessed the role of MAPK signaling in 6-gingerol-induced effects in BMDMs and RAW264.7 cells. RESULTS: In CLP mice, 6-gingerol significantly ameliorated sepsis development, which was associated with the reduction of serum IL-1ß. In BMDMs and RAW264.7 cells, 6-gingerol strongly attenuated pyroptosis as well as the release of caspase-1p20, HMGB1, mature IL-1ß, IL-18 in response to ATP and LPS treatment. 6-Gingerol conferred these effects by blocking MAPK activation. Exposure to an ERK agonist (EGF) reversed effects of 6-gingerol, causing pyroptosis, LDH and caspase-1p20 release. CONCLUSIONS: By targeting MAPK signaling, 6-gingerol significantly suppressed secretion of pro-inflammatory cytokines and inhibited macrophage cells pyroptosis resulting in overall inhibition of sepsis development.

Paclitaxel induces apoptosis of esophageal squamous cell carcinoma cells by downregulating STAT3 phosphorylation at Ser727.

Paclitaxel induces apoptosis in a variety of cancer cells. However, the mechanism of paclitaxel inducing apoptosis in human esophageal squamous cell carcinoma (ESCC) remains to be defined. In this study, we found that paclitaxel-induced apoptosis by increasing the relevant apoptosis protein expression and the release of cytochrome c via downregulation of signal transducer and activator of transcription 3 (STAT3) and phospho-STAT3 (Ser727). In addition, paclitaxel treatment of ESCC cells EC-1 and Eca-109 led to marked mitochondrial membrane potential depolarization and significantly increasing of reactive oxygen species. Moreover, paclitaxel treatment resulted in the inhibition of mitochondrial respiration. In conclusion, our findings reveal that paclitaxel induced apoptosis in both EC-1 and Eca-109 cells through the reduction of STAT3 and phospho­STAT3 (Ser727) level, and suggest that paclitaxel may be of therapeutic potential in the treatment of ESCC through the induction of mitochondrial apoptosis in ESCC cells.