MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. Mpst-knockout (KO) mice with enhanced liver Tst expression were recently generated as its model; however, the physiological roles/significances of Mpst remain largely unknown. Here we generated three independent germ lines of Mpst-KO mice by CRISPR/Cas9 technology, all of which maintained normal hepatic Tst expression/activity. Mpst/Cth-double knockout (DKO) mice were generated via crossbreeding with our previously generated Cth-KO mice. Mpst-KO mice were born at the expected frequency and developed normally like Cth-KO mice, but displayed increased urinary 3-mercaptolactate excretion and enhanced passive systemic anaphylactic responses when compared to wild-type or Cth-KO mice. Mpst/Cth-DKO mice were also born at the expected frequency and developed normally, but excreted slightly more 3-mercaptolactate in urine compared to Mpst-KO or Cth-KO mice. Our Mpst-KO, Cth-KO, and Mpst/Cth-DKO mice, unlike semi-lethal Cbs-KO mice and lethal Cars2-KO mice, are useful tools for analyzing the unknown physiological roles of endogenous H2S/RSS production.Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H R)-2-amino-3-[(2-carboxy-2-hydroxyethyl) disulfanyl] propanoic acid; C(C(C=O)O)N)SSCC(C(=O)O)O) in his urine ) ,20,21,22In the present study, we examined Mpst-KO mice produced using CRISPR/Cas9 technology. We preserved normal TST expression by minimizing the size of Mpst gene deletion and found that our mice display increased urinary excretion of 3-mercaptolactate and enhanced passive systemic anaphylaxis (PSA), while maintaining hepatic Tst expression/activity. Moreover, we also generated Mpst/Cth-double knockout (DKO) mice as a useful tool to investigate their mutually compensatory roles in mice.Mpst which encodes 67% of the Mpst open reading frame  to greater or lesser degrees. This is in stark contrast to the other two H and Cth A. In all and Cth B. Theref and Cth  likely d and Cth . Using t and Cth B. Furthe and Cth C. Mpst a and Cth A,B. Howe and Cth A,B. At t and Cth A,B.Serum amino acid/thiol compound levels for all lines of Mpst-KO mice were indistinguishable from those of WT mice, which was in marked contrast to Cth mice; however, all Mpst-KO mice excreted 5.5–7.3 times the normal amount of 3-mercaptolactate (3-ML) in urine . Though We have previously generated Cth-KO mice  that req2S has been shown to regulate various inflammatory responses [Hesponses ,31, but esponses ,33,” remMCDU has historically been attributed to MPST deficiency, but studies containing direct experimental evidence are lacking. We provide here the first direct evidence that Mpst deficiency in mice causes enhanced secretion of 3-ML in urine, a hallmark trait of MCDU . CysteinMpst gene deletion strategies as the mouse Tst gene is located near the Mpst gene (Previously generated Mpst-KO mice exhibited increased anxiety-like behaviors associated with increased levels of serotonin and its major metabolite (5-hydroxyindoleacetic acid) in the prefrontal cortex and decreased levels of dopamine in the hippocampus . Furtherpst gene A and the2S/RSS in general thermoregulation. Further studies are necessary to delineate the roles of Mpst/Cth and produced H2S/RSS in the thermoregulation.In this study, we analyzed, for the first time, the systemic anaphylactic responses in Cth-KO and Mpst-KO mice and found greater PSA (but not PCA) responses in Mpst-KO mice . In anapMpst mutations, further studies including the examination of Mpst/Cth-DKO mice for PSA/PCA alterations are needed.Furthermore, we generated Mpst/Cth-DKO mice that develop normally  may provide new insight into the sulfur amino acid metabolism.In conclusion, we generated Mpst-KO mice as an animal model of MCDU and also Mpst/Cth-DKO mice as a useful tool for analyzing functions of HMpst gene: Mpst crRNA upstream (u) (5′-ACGACCGCGGGCGCCAGGAG-3′) and Mpst crRNA downstream (d) (5′-GCTGGGGAAACGGACTAATG-3′). Electroporated zygotes were transferred into the oviducts of pseudopregnant female ICR mice , and a total of 14 mice were born on E19.5. Correct targeting/deletion was confirmed by PCR using forwarding (f) (5′-TGCTGGGCAAGGAATAGAGTG-3′) and reverse (r) (5′-GCATATAAGGCCAGCACACG-3′) primers that are located outside the targeted locus (http://card.medic.kumamoto-u.ac.jp/card/english/index.html). All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals, 8th Edition published by the US National Research Council and were approved by the Animal Care Committees of Showa Pharmaceutical University .Mpst-KO mice were generated using CRISPR/Cas9 technology from Setsurotech Inc.  on a C57BL/6J  background as described previously  with somed locus B. We seqed locus . Routineed locus C. Adult g, 4 °C, 20 min). Mpst and Tst recombinant proteins were affinity-purified with the TALON Metal Affinity Resins (Clontech) and a linear gradient of 10–100 mM imidazole. The pooled fraction was dialyzed twice against buffer E at 4 °C overnight using a Slide-A-Lyzer G2 Dialysis Cassette–20kDa cutoff (Thermo Fisher Scientific). The recombinant proteins were stored at −80 °C until use.His-tagged murine Mpst ) and Tst ) expression plasmids were purchased from GeneCopoeia . The plasmids were transformed to Rosetta (DE3) pLysS Competent Cells (Novagen (Merck)). Single colonies were picked up from LB agar plates containing 100 µg/mL Ampicillin (Amp) and 34 µg/mL Chloramphenicol (CP), and placed into 75 mL LB media (100 µg/mL Amp and 34 µg/mL CP) for growth overnight at 37 °C. 50-mL of the overnight culture was seeded into 1-L LB media (100 µg/mL Amp) and grown at 30 °C for 1.5 h and at 20 °C for 2 h. Following, isopropyl β-D-galactopyranoside (IPTG) was added  and the culture was further grown at 20 °C for 24 h. Cells were then centrifuged and resuspended in 40 mL buffer E (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride, and 10% (v/v) glycerol) containing a Complete EDTA-free Protease Inhibitor Cocktail . Cells were then lysed by sonication and the soluble fraction was isolated by centrifugation  was obtained. Efficient antibody titers  were confirmed by ELISA (data not shown).A single Japanese white rabbit ) was immunized with full-length mouse Mpst recombinant proteins using Freund’s complete adjuvant four times (day 0 (0.3 mg), 28 (0.2 mg), 35 (0.2 mg), and 42 (0.2 mg)) and whole blood was collected at day 49. The blood was allowed to clot by leaving undisturbed at room temperature for 20 min, and the clot was removed by centrifugation at 2000× per 100 mg; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1.0 mM EDTA, 1% (w/v) Triton X-100, 0.1% (w/v) phenylmethylsulfonyl fluoride, and Complete EDTA-free Protease Inhibitor Cocktail) using a MicroSmash-100R homogenizing system  and zirconia beads . Homogenates were centrifuged at 15,300× g, at 4 °C for 20 min. Supernatants were recentrifuged at 15,300× g, at 4 °C for 30 min. After heating  with the sample buffer, samples (5 µg/lane) were resolved on 10% or 12% pre-cast PAGE gels, and then transferred to the Immobilon PVDF membrane (Millipore) and subjected to western blotting. Mpst (and Tst as a cross-reactive protein) were detected with the above polyclonal antibody ) and anti-rabbit IgG, HRP-linked whole Ab donkey antibody . Tst was detected with anti-human TST rabbit monoclonal antibody  and anti-rabbit IgG, HRP-linked whole Ab donkey antibody . Cbs was detected with anti-rat CBS rabbit polyclonal antibody ([Each mouse tissue sample (~100 mg) was homogenized in lysis buffer 3)3 + HNO3 + H2O; [–) formation was determined by the measurement of absorbance at 460 nm using the Epoch 2 microplate spectrophotometer . Tst enzyme assays were performed using the substrate thiosulfate as described previously [2PO4, and 71.4 mM KCN at 25 °C for 30 min and processed similarly. Specific Mpst and Tst activities were obtained at substrate concentrations of 5.95 mM (for 3MP) and 25 mM (for thiosulfate), respectively.Mpst enzyme assays were performed using the substrate 3-MP as described previously ,44, with3 + H2O; ). Thiocyeviously  with somSerum samples were analyzed for their amino acid concentrations using amino acid and thiol-derivatization fluorescent reagents (4-fluoro-7-nitrobenzofurazan (NBD-F) and 4-fluoro-7-sulfobenzofurazan (SBD-F), respectively; Dojindo, Kumamoto, Japan) via a high-performance liquid chromatography (HPLC) as previously described  Supplem. Urine sPSA and PCA assays were performed as previously described . For PSAt-test was used in PCA assays . Statistical comparison was performed using Prizm 5 software . Two-way repeated-measured ANOVA with Bonferroni’s multiple comparison test was used in PSA assays A and ratA assays B. One-wa


Postoperative acute kidney injury (po-AKI) is frequently observed after major vascular surgery and impacts on mortality rates. Early identification of po-AKI patients using the novel urinary biomarkers insulin-like growth factor-binding-protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2) might help in early identification of individuals at risk of AKI and enable timely introduction of preventative or therapeutic interventions with the aim of reducing the incidence of po-AKI. We investigated whether biomarker-based monitoring would allow for early detection of po-AKI in patients undergoing abdominal aortic interventions.In an investigator-initiated prospective single-center observational study in a tertiary care academic center, adult patients with emergency/ elective abdominal aortic repair were included. Patients were tested for concentrations of urinary (TIMP-2) x (IGFBP7) at baseline, after surgical interventions (PO), and in the mornings of the first postoperative day (POD1). The primary endpoint was a difference in urinary (TIMP-2) x (IGFBP7) levels at POD1 in patients with/ without po-AKI . Secondary endpoints included sensitivity/ specificity analyses of previously proposed cut-off levels and clinical outcome measures .93 patients (n = 71 open surgery) were included. Po-AKI was observed in 33% (31/93) of patients. Urinary (TIMP-2) x (IGFBP7) levels at POD1 did not differ between patients with/ without AKI  and PO . Higher median (TIMP-2) x (IGFBP7) levels were noted in KDIGO stage 3 pAKI patients at POD1 . Previously proposed cutoff levels  showed moderate sensitivity/ specificity .In a prospective monocentric observational study in patients after abdominal aortic repair, early assessment of urinary (TIMP-2) x (IGFBP7) did not appear to have adequate sensitivity/ specificity to identify patients that later developed postoperative AKI.NCT03469765, registered March 19, 2018. Post-operative acute kidney injury (po-AKI) is often observed after cardiovascular surgery and negatively impacts on both morbidity and mortality rates . AKI incPost-operative renal (dys)-function is influenced by several factors including pre-operative renal function, co-morbidities, intraoperative factors , hemodynamic instability  and post-operative complications –8. As noRecently, new biomarkers including insulin-like growth factor binding protein 7 (IGFBP7) and tissue-inhibitor of metalloproteinases-2 (TIMP-2) were proposed for early detection of AKI . RespectCurrently, data on biomarkers for timely prediction of postoperative renal outcomes are not available for open abdominal aortic repair and EVAR patients. We therefore performed an investigator-initiated prospective observational study to investigate whether early assessment of the biomarker (TIMP-2) x (IGFBP7) would predict po-AKI in patients with abdominal aortic repair.(Timp-2) x (IGfbp7) as early renal biomarker for the prediction of acute kidney injury in aortic surgery (TIGER) study was an investigator-initiated, single center, prospective, observational study. Patients with emergency or elective abdominal aortic surgery  referred to our university-based cardiovascular center were eligible for study inclusion. The study was performed from June 2018 to September 2019. Patients were excluded if any of the following criteria were met: 1) patient unable to provide informed consent  or institutionalized patients, 2) Age < 18 years and/or 3) patients not speaking German or French. Besides additional biomarker monitoring, all patients received best standard-of-care treatment according to local standard operating procedures (SOP) without interference by the research team. The study was approved by the local Ethics Committee on Human Research . Written informed consent was obtained from all patients or respective representatives. The study was registered on Clinicaltrials.gov . Data quality visits  were performed by an internal qualified person.The The primary endpoint of the study was defined as a statistical significant difference in total (TIMP-2) x (IGFBP7) levels at the first postoperative day (POD1) in patients without/ with po-AKI developed in the first seven days after surgery  definitions, stages 1–3 ).st POD = (TIMP-2) x (IGFBP7) 1st POD—(TIMP-2) x (IGFBP7) baseline) and relative change (Δ (TIMP-2) x (IGFBP7) 1st POD [%] = [100/(TIMP-2) x (IGFBP7) baseline] * Δ (TIMP-2) x (IGFBP7) 1st POD) in urinary (TIMP-2) x (IGFBP7) was estimated.Secondary endpoints included A) sensitivity/specificity analyses of (TIMP-2) x (IGFBP7) along KDIGO-categories, B) comparison of the area under receiver operating characteristic curves (AUROC) for: (TIMP-2) x (IGFBP7), fractional excretion of sodium (FeNa), C) descriptive analysis of clinical endpoints including number of patients on renal replacement therapy (RRT), intensive care unit (ICU) length of stay, intermediate care unit (IMC) length of stay, hospital length of stay (LOS), and ICU-, hospital-, and 28-day-mortality. Subgroup-analyses were performed for type of surgery . Further, the diagnostic value of absolute (Δ(TIMP-2) x (IGFBP7) 12/ 1000. Blood and urinary creatinine and sodium concentrations were assessed to calculate fractional sodium excretion. Other post-operative laboratory parameters were assessed at the discretion of the independent treating physician.Urinary and blood samples were obtained after anesthesia induction (baseline), after end of anesthesia , and in the morning of the day after surgery . Urinary concentrations of TIMP-2 and IGFBP7 were measured using a commercially available standardized assay . Test results are given as product of both markers in (ng/ml)The following pre-operative data were recorded: age; sex; BMI; American Society of Anesthesiologists (ASA) physical status classification system; comorbidities and routine medications. Laboratory parameters including serum creatinine, urea, sodium, eGFR (Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI] formula) . Diagnos2/ 1000 and in patients with AKI (i.e. KDIGO stages 1–3) a mean of 1.6 (ng/ml)2/ 1000 with a standard deviation (SD) of 1.4. If the true difference in the experimental and control means is 1.2, n = 18 experimental subjects and 72 control subjects are required to reject the null hypothesis that the population means of the experimental and control groups are equal with a probability (power) of 0.9. The Type I error probability associated with this test of this null hypothesis is 0.05. Assuming a drop-out rate greater than 10%, a total of 100 patients are intended to be included.An incidence of 20% po-AKI was expected in the cohort under investigation. Based on (TIMP-2) x (IGFBP7) data of previous studies in cardiac surgery patients ,26, we aFor statistical analysis, Student’s t-test for normally distributed data and the Mann–Whitney U test for non-normally distributed data was used to compare quantitative variables between groups, ANOVA for multiple groups. Chi square test or Fisher’s exact test were used for qualitative variables, as appropriate. Statistical significance was assumed for p<0.05. Sensitivity-/specificity-analysis were performed using receiver-operating characteristics (ROC) curves using the R software package cutpointr. Diagnostic value of a biomarker is defined as excellent with Area Under the Receiver Operating Characteristics curve (AUROC) >0.9, as good with a AUROC between 0.75–0.9, as poor with a AUROC between 0.50–0.75 and as without any diagnostic value with AUROC values <0.5 . OptimalOne hundred-thirty seven patients were screened for study participation, with 100 patients eligible for study inclusion . Ninety-Measurement of urinary (TIMP-2) x (IGFBP7) on the morning of the first postoperative day  was performed on average at 14.4 hours (± 4.3 hours) after end of surgery. No statistically significant differences were noted with regard to (TIMP-2) x (IGFBP7) levels of patients with/ without AKI on both POD1 and immediately after surgery , S1 Fig.Additionally, when Δ (TIMP-2) x (IGFBP7) (i.e. POD1 vs. baseline) was investigated, an AUROC of 0.63 (95%CI 0.52–0.72) for absolute and 0.65 (95%CI 0.56–0.62) for relative values of urinary (TIMP-2) x (IGFBP7) was noted.In patients with po-AKI, an early post-operative increase in serum creatinine levels after surgery was observed. No differences were noted in regard to early fractional sodium excretions between patients with vs. without AKI . AUROC fOne (3.2%) patient died in the AKI group . Three . IncreasAlong KDIGO AKI stages, we observed a statistically significant difference with an increased (TIMP-2) x (IGFBP7) levels in KDIGO AKI stage 3 at POD1 only (p = .003) . AUROC sSeventy-one patients  received open surgery, out of which n = 28 (39.4%) had post-operative AKI . (TIMP-2) x (IGFBP7) levels did not differ statistically between patients with vs. without AKI immediately after the operation and at POD1 . Along the KDIGO stages, we observed a significant different distribution of (TIMP-2) x (IGFBP7) values with lower levels in AKI stages 0, 1, and 2 at PO (p = .04) with higher levels in KDIGO AKI stage 3 at POD1 (p = .004). Due to limited sample size, we refrained from a formal sensitivity/specificity analysis in patients with KDIGO AKI stages 2 and 3.Seventy-eight patients received scheduled procedures  out of which n = 22 (31%) had post-operative AKI . No significant differences in median (TIMP-2) x (IGFBP7) levels were observed for patients with vs. without AKI (KDIGO classification) at PO and POD1 . Fifteen patients underwent emergency aortic surgery with po-AKI diagnosed in 9 (60%) of cases . In this subgroup with a limited sample size, (TIMP-2) x (IGFBP7) levels differed between patients with vs. without AKI at POD1 (p = .02) without differences at PO (p = .28). Due to limited sample size, a formal sensitivity/ specificity analysis was refrained from.In an investigator-initiated prospective monocentric observational study, we did not observe differences in early postoperative (TIMP-2) x (IGFBP7) levels in abdominal aortic surgery patients with versus without po-AKI which developed in the first seven days after surgery. Higher median (TIMP-2) x (IGFBP7) levels were noted in cases of severe AKI (KDIGO stage 3). These findings were consistent along the investigated subgroups. Our findings are in contrast to previous studies demonstrating good accuracy in prediction of all-stage AKI ,22 or KDWe deliberately aimed to investigate (TIMP-2) x (IGFBP7) in patients undergoing abdominal aortic procedures as this patient group is considered at particular high risk for post-operative AKI . TherefoThe po-AKI incidence observed  might be comparable to previous studies including cardiac and major non-cardiac surgery ,24. Of nRecommendations for (TIMP-2) x (IGFBP7) assessment vary regarding the time of assessment . We thus2/ 1000 with a median at about 0.3 (and 75% <0.75 (ng/ml)2/ 1000) [Earlier studies in healthy subjects and in patients with stable chronic comorbidities showed a (TIMP-2) x (IGFBP7) reference intervals of 0.04 to 2.22 (ng/ml)2/ 1000) . One mig2/1000) were proposed as potential “fast track” patients [Further, timing of (TIMP-2) x (IGFBP7) may be crucial. Measurements 4–6 hours after surgery could be regarded the earliest time point for assessment ,24. In apatients . Here, 5patients  seem warOur study has important further limitations that deserve discussion. First, even if adequately powered  to detect a difference in patients with AKI according to all KDIGO stages, the sample size of our study is limited and our results warrant larger confirmatory investigations. Second, we present data from a monocentric study with all inherent limitations. Third, pre-operative risk prediction and selection of patients (at higher risk) might have  influenced biomarker performance ,35. FourIn an investigator-initiated prospective monocentric observational study, we did not observe differences in early postoperative (TIMP-2) x (IGFBP7) levels in abdominal aortic surgery patients with versus without postoperative AKI that developed in the first seven days after surgery. Subgroup analysis showed higher median (TIMP-2) x (IGFBP7) levels in patients with severe postoperative AKI (KDIGO stage 3). Further studies are required regarding the specific type of interventions and the kinetics of the biomarker.S1 FigMedians and IQRs are given.(TIF)Click here for additional data file.S2 Fig(A) stage 1 vs. all other stages, (B) stage 2 vs. all other stages, (C) stage 3 vs. all other stages.(TIF)Click here for additional data file.S3 Fig1n = 10 for postop due to 1 missing value.n.s. p .05. (TIF)Click here for additional data file.S1 Checklist(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


C. elegans neurons triggers the Ire1-Xbp1-mediated stress response in an age-dependent and cell-autonomous manner. The p38 MAPK PMK-3 regulates the chronic stress through IRE-1 phosphorylation. Overexpressing gap junction protein also activates autophagy. The insulin pathway functions through autophagy, but not the transcription of genes encoding ER chaperones, to counteract the p38-Ire1-Xbp1-mediated stress response. Together, these results reveal an intricate cellular regulatory network in response to chronic stress in a subset of cells in multicellular organism.ER stress occurs in many physiological and pathological conditions. However, how chronic ER stress is alleviated in specific cells in an intact organism is an outstanding question. Here, overexpressing the gap junction protein UNC-9 (Uncoordinated) in  in vivo. Here, we designed a genetic system in the nematode C. elegans, which allows us to induce ER stress in specific cells, without drug treatment or any other external stimuli, and then to monitor the stress response. The p38 MAPK directly acts on the phosphorylation of IRE-1 to promote the stress response. Meanwhile, the insulin receptor function through autophagy activation to counteract the p38-IRE-1-XBP-1 pathway. Together, these results reveal an intricate cellular regulatory network in response to chronic stress in multicellular organism.The accumulation of unfolded proteins triggers the ER stress response (UPR), which allows cells to fight against fluctuations in protein expression under both physiological and pathological conditions. Severe acute ER stress responses can be induced by drug treatment. However, such intense ER stress rarely occurs ubiquitously in every cell type  Proteins destined for secretion or insertion into membranes enter the endoplasmic reticulum (ER) in an unfolded form and generally leave only after they have reached their native states. During normal function, a secretory cell may experience dramatic variations in the flux of new proteins through its ER in response to changes in demand. Disruption of the balance between secretory protein synthesis and the folding capacity of the ER activates a signaling network called UPR (the Unfolded Protein Response) in an attempt to maintain homeostasis . The UPRxbp1 mRNA produces a short, activated form of XBP1, called XBP1s, which activates expression of genes encoding chaperones and other ER-associated degradation proteins that expand the folding capacity of the ER and increase the breakdown of misfolded proteins -like ER kinase), and ATF6 (activating transcription factor 6) . In respproteins . In variproteins ,12. In vesponses .Macroautophagy (hereafter referred to as autophagy) is a cellular degradation process initiated in response to stress. It attempts to restore metabolic homeostasis through lysosomal degradation of cytoplasmic organelles or cytosolic components . AutophaC. elegans is transparent throughout its life cycle. We reasoned that if we could induce the ER stress response by overloading the cell with a non-secreted plasma membrane protein, we would then be able to detect the occurrence of UPR by directly observing the sub-cellular localization of this protein in vivo. Gap junction channels direct cell-to-cell communication and defects in gap junction formation and/or function are the cause of at least 10 human pathological conditions [C. elegans neurons activates the IRE-1-XBP-1-mediated UPR in a cell-autonomous manner. Using this model, we further identified that the p38 MAP kinase PMK-3 functions through IRE-1 phosphorylation to regulate UPR. Loss of function of daf-2 (encoding the insulin-like receptor) suppresses the mis-localization defect of overexpressed UNC-9 in pmk-3, ire-1 or xbp-1 mutants. Intriguingly, although the reduced transcription of ER chaperone Bip/hsp-4 (immunoglobulin heavy chain-binding protein/heat shock protein 4) is not rescued by daf-2 mutation, autophagy induction is largely restored. This study reveals the intricate molecular interactions underlying the ER stress response in a limited subset of cells in an intact organism, and provides insights into the pathophysiology of many human degenerative diseases.The nematode nditions . Moreovenditions –24. HereC. elegans to establish the in vivo ER stress model. The D-type motor neurons, including 6 DDs and 13 VDs, are a set of GABAergic neurons with cell bodies distributed along the ventral nerve cord and neuronal processes projecting both along the ventral cord and to the dorsal cord [unc-25 encodes the GABA biosynthetic enzyme glutamic acid decarboxylase [unc-25 promoter, the GFP (green fluorescent protein)-fused gap junction protein UNC-9 could be specifically overexpressed in DD/VD neurons. We found that the ectopically expressed UNC-9::GFP is distributed in a punctate pattern along the neuronal processes and in the cell body region of DD/VD neurons  animals, the UNC-9::GFP signal no longer takes the characteristic punctate form. Instead, it is diffusely distributed in the cell body region of DD/VD neurons. Except for a few small residual dots, there is no GFP signal on the neurites  or ATF6 (atf-6 mutant) did not affect the punctate pattern of UNC-9::GFP in DD/VDs , we found that the majority of UNC-9::GFP signal in ire-1 mutants displays a punctate pattern similar to wild type , the unbiased genetic screen also identified a pmk-3 allele, xd74. In xd74 animals, the UNC-9::GFP signal is diffusely distributed in the cell body region of DD/VDs and is greatly reduced in DD/VD neuronal processes  and pmk-3(tm745), display similar UNC-9::GFP localization defects in DD/VD neurons to pmk-3(xd74) animals ;ire-1(v33) and pmk-3(tm745);xbp-1(zc12) double mutants, the UNC-9::GFP distribution resembles the ire-1 or xbp-1 null (pmk-3(tm745);ire-1(v33); xbp-1(zc12) triple mutant also mimics ire-1 or xbp-1 single null mutants  lost its capability to rescue the UNC-9::GFP localization defect in pmk-3 mutants  (We showed above that the kinase-defective (KD) mutant form of PMK-3  . We furtt PMK-3) . Fourth,t PMK-3) . Furthert PMK-3) . Togethexbp-1(xd131) strain and performed a further genetic suppressor screen to explore whether the malfunctioning ER stress response can be repressed. From the screen, we identified the xd211 mutation. In the presence of xd211, the punctate pattern of UNC-9::GFP was restored along the neurites and in the cell body region of DD/VD neurons (xbp-1(xd131) or xbp-1(zc12) was also suppressed by the daf-2(e1370) allele (daf-2 function is responsible for the genetic suppression of xbp-1. DAF-2 negatively regulates the FOXO transcription factor DAF-16. In xbp-1(xd131) daf-2(xd211);daf-16(mu86) triple mutant animals, the suppression of xbp-1 by daf-2 is completely blocked by daf-16 mutation  allele   driven by the unc-25 promoter, we examined the autophagy activity in DD/VD neurons. In the absence of UNC-9::GFP, mCherry::LGG-1 is diffusely distributed along the neuronal processes and there are typically less than 4 mCherry::LGG-1 puncta in the cell body region  contain diffusely distributed UNC-9::GFP signal. After starvation, the UNC-9::GFP adopted a punctate distribution pattern in a significant proportion (about 58%) of DD/VD cells . Although the detailed mechanisms underlying this restricted effect remain elusive, it is possible that the broadness or/and the severity of ER stress in the nervous system determines whether the stress response can spread to neighboring tissues.In response to ER stress, transcription of the ER chaperone gene is activated by the IRE-1/XBP-1 pathway . Indeed,nd cells ,40. Pan-podermis . In contire-1 or xbp-1 mutants, a diffuse UNC-9::GFP signal accumulates in the ER. Evidently, with no need for drug treatment or any other external stimuli, the distinct UNC-9::GFP subcellular localization offers a dual marker to monitor functional versus defective UPR at single-cell resolution in an intact animal. Notably, the subcellular localization of endogenous UNC-9 and of ectopic muscle-expressed UNC-9::GFP is unaffected by ire-1 or xbp-1. Thus, the IRE-1/XBP-1 UPR is not involved in the UNC-9 protein localization under normal growth condition. Previous studies demonstrated that that the UPR is also function in absence of external ER stress and the UPR genes are critical for maintaining secretory protein metabolism[The cell-autonomous stress response triggered by overloading of UNC-9::GFP protein ensures that UNC-9::GFP adopts its distinctive punctate pattern in DD/VD neurons. In contrast, when UPR fails, such as in etabolism,42. Herepmk-3/p38 MAPK is involved in the ER stress response triggered by excess UNC-9::GFP. Among the three p38 MAPKs in C. elegans [ire-1-xbp-1 pathway protects animals from Pseudomonas aeruginosa infection, inactivation of the PMK-1-mediated immune response suppresses the lethal effect of P. aeruginosa infection in xbp-1 mutants [pmk-3 functions similarly to ire-1 and xbp-1. In contrast, neither PMK-1 nor the upstream MAPKK (MAPK kinase) SEK-1 nor MAPKKK (MAPK kinase kinase) NSY-1 [ire-1 and xbp-1) are shared, other regulatory components are employed in a tissue- or/and intensity-dependent manner. How does PMK-3/p38 participate in this cell-autonomous chronic ER stress response? A previous study showed that p38 MAPK directly phosphorylates the activated form of XBP1 and enhances its nuclear migration [xbp-1 mutants in which the putative phosphorylation sites of p38 MAPK (T191 and S283) were disrupted could fully rescue the UNC-9::GFP mis-localization defect in xbp-1 (the percentage of DD/VD cell bodies showing defective UNC-9::GFP distribution was dropped to 8.7% and 11.3% respectively). In addition, overexpressing ire-1 suppresses the pmk-3 mutant phenotype, while upregulation of pmk-3 does not bypass the functional requirement for ire-1. This suggests that pmk-3 functions upstream of ire-1. Consistent with this idea, PMK-3 binds to IRE-1 and the phosphorylation status of IRE-1 is affected by the presence of functional PMK-3. Among three putative p38 MAPK phosphorylation sites in IRE-1, T949 is located in the C-terminal RNase domain. Phosphorylation of the corresponding site does not have a direct impact on the splicing activity in vitro, but has a noticeable effect in vivo [xbp-1 mRNA in DD/VD neurons is partially affected by pmk-3 and mutation of this T949 site significantly decreased the rescue activity of IRE-1. Given that phosphorylation on the RNase domain may be involved in the recruitment of unknown cofactors to maximize the RNase activity of IRE-1 [Aided by unbiased genetic screens, we further identified that  elegans , PMK-1 i elegans –49. Whil mutants . Here, iigration . Here, h in vivo . Here, wof IRE-1 , it is fdaf-2 are long-lived, and both ire-1 and xbp-1 make a large contribution to the long lifespans of daf-2 mutants and also increase their ER stress resistance [daf-2 could restore protein secretion in ire-1 and xbp-1 mutants in a daf-16 dependent manner [daf-2 mutation also improved the ER stress resistance of xbp-1 and ire-1 mutant animals when they were treated with low but not high tunicamycin concentrations, and DAF-16 acts through ER-associated degradation systems independently of ire-1 [ire-1, and further reducing insulin signaling restored the normal PVD neuronal morphology [xbp-1 is not involved in PVD morphogenesis at all, which suggests that the antagonistic role of insulin signaling may not act through the non-canonical splicing of xbp-1 mRNA as in the UPR process [daf-2 mutation suppresses the defective ER stress response in both ire-1 and xbp-1 animals. Because the cell autonomous reduction of hsp-4 expression in ire-1 or xbp-1 mutants is not restored by daf-2, we suspected that daf-2 may act on alternative path to regulate the stress response. Intriguingly, autophagy induction was restored in ire-1 or xbp-1 mutant cells by loss of daf-2 function. It has been shown that the daf-2 mutants exhibit increased levels of autophagy, and autophagy is required for their extended lifespan [daf-2 mutation in both wild-type and ire-1 mutant animals [daf-2 mutation, the reduction of autophagy in ire-1 or xbp-1 mutants was indeed suppressed by daf-2. The FOXO forkhead transcription factors are key transcriptional regulators of autophagy [daf-16 mutation .Therefore, we suspected that in ire-1 or xbp-1 single mutants, the DAF-16/FOXO remains inactive due to the presence of DAF-2. Since neither transcription of ER chaperone genes (for instance hsp-4/Bip) nor autophagy induction could occur in ire-1 or xbp-1 single mutants, the proper localization of overexpressed protein could not be achieved. In contrast, when both ire-1/xbp-1 and daf-2 are removed, the DAF-16 is activated due to the absence of DAF-2. Hence, although the transcriptional upregulation of ER chaperone genes (such as hsp-4/Bip) is missing-because of the lack of ire-1/xbp-1 pathway, the active DAF-16 could act through autophagy or other protein degradation process or ER homeostasis to facilitate the proper localization of overexpressed protein. Intriguingly, the accumulation of misfolded proteins in the ER appears not to directly activate DAF-16 [ire-1-xbp-1 branch of the UPR are removed. Nevertheless, the availability of two parallel systems , dlk-1(tm4024), daf-16(mu86), mom-4(ne1539), mtk-1(ok1382). LGII: ire-1(v33), nsy-1(ok593), xdIs15. LGIII: xbp-1(zc12), xbp-1(xd131), xbp-1(tm2457), xbp-1(tm2482), daf-2(e1370 ts), daf-2(xd211), kin-18(pk71), xbp-1(zc12). LGIV: pmk-3(tm745), pmk-3(ok169), pmk-3(xd74), jnk-1(gk7), kgb-1(um3), pmk-1(km25), kgb-2(gk361), unc-30(ju54), mak-2(tm2927), wnk-1(ok266), lgg-1(bp500), xdIs13. LGV: rpm-1(ju44), mlk-1(ok247). LGX: atf-6(ok551), atf-6(tm1153), pek-1(ok275), pek-1(ok275), mkk-4 (ok1545), jkk-1(km2), mek-1(ks54), sek-3(tm1344), sek-6(tm4305 tm4136), sek-1(km4). The genetic screen was performed according to the previous report [xdIs15 or xbp-1(xd131);xdIs15 worms were treated with EMS (ethylmethane sulfonate) solution for 4 hours. F2 worms were examined for phenotype alteration at the young adult stage under a fluorescence microscope. Mutant animals were recovered to fresh plates for maintenance. A total of 10,000 and 5,000 mutagenized haploid genomes were screened respectively for xdIs15 and xbp-1(xd131);xdIs15 worms. 10 mutants were identified from the genetic screen using xdIs15 as a start strain, which could be classified to 4 categories: diffused UNC-9::GFP signal in the cell body region of DD/VD neurons (xd74 and xd131), enlarged UNC-9::GFP puncta accumulated in the cell body region of DD/VD neurons (2 mutants), reduced UNC-9::GFP puncta number along ventral nerve cord (3 mutants) and decreased UNC-9::GFP expression (3 mutants). In the xbp-1(xd131) mutant genome, a point mutation at W318 creates a stop codon in the active form of xbp-1. Only the daf-2(xd211) mutant was isolated from the xbp-1(xd131);xdIs15 suppressor screen. To isolate daf-2(xd211) alone mutant, the classical recombination method was used. Total 999 F2 worms were sequenced and finally a single daf-2(xd211)/ daf-2(xd211) xbp-1(xd131) hetezygote was identified. All isolated mutants were outcrossed with wild type at least four times. The strains containing daf-2(e1370 ts) were cultured at 16°C until L2/L3 stage, then shifted to 25°C to reach adulthood before phenotype characterization.Culture and manipulation of  methods . Mutantss report . Brieflyunc-25 promoter was inserted between the BamHI and SphI sites of the pSM-GFP vector (a generous gift from Dr. Kang Shen) and the UNC-9 cDNA was inserted into the BamHI site. xbp-1, ire-1 and pmk-3 cDNAs were amplified by reverse transcription. The ER stress reporter plasmid Punc-25::XBP-1us::mCherry was modified from XB3 plasmid (a generous gift from Dr. Christopher Rongo). Punc-25::cytb-5.1::mCherry was kindly provided by Dr. Yingchuan Qi. Punc-25:: mCherry::MANS, Punc-25:: mCherry::RAB-7, Punc-25:: mCherry::RAB-5, and Punc-25::mCherry::LGG-1 were constructed by the recombination method. Transgenic animals were produced as previously described [pmk-3 and the kinase dead form of pmk-3(pmk-3KD) were amplified by PCR and then inserted between the EcoRI and XbaI sites of pFLAG-CMV-2 plasmid. The cytosolic C-terminus of IRE-1 was amplified by PCR and inserted between the NheI and KpnI sites of pcDNA 3.1/myc-His vector. Integrated strains were obtained by UV irradiation. The strain BCN1071 which expresses Phsp-4::mCherry was kindly provided by Ben Lehner. All integrated transgenic animals were out-crossed at least 3 times. The corresponding transgenes are listed in The escribed . The wilunc-9 gene. This UNC-9::GFP knock in strain (PHX727) was generated by SunyBiotech and was verified with DNA sequencing. This strain was outcrossed with N2 for 4 times before use.The UNC-9::GFP knock-in strain was created using sg1:CGTATGGTTGCAACTCACGCCGG and sg2: CCGGAGAACTACCCTGTTACGAG. The GFP sequence was inserted before the stop codon TGA of unc-25::mCherry marker was used. Phsp-4::mCherry was used to detect induction of the chaperone gene hsp-4 in the ER stress response. The average Phsp-4::mCherry fluorescence intensity in the anterior and posterior intestine regions was measured and normalized with the wild type. To quantify the xbp-1 splicing activity, the Punc-25::XBP-1us::mCherry plasmid was used and DD/VD cell bodies with mCherry signal were counted. To characterize the distribution of UNC-9::GFP under starvation conditions, well-fed L4 ire-1(v33);xdIs15 worms were collected and washed 8 times with M9 buffer. After sedimentation, worms were plated on NGM plates without food. After 24 hours, the starved adult worms were examined using Leica TCS SP8 confocal microscopy. All graphical data are presented as mean ± s.e.m. Two-tailed unpaired and paired Student’s t-tests were performed for comparison between two groups of samples. To compare multiple groups, one-way ANOVA was used with an appropriate multiple comparisons post hoc test . *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.Animals were mounted on 2% agar pads in M9 buffer containing 1% 1-phenoxy-2-propanol and examined by fluorescence microscopy unless indicated otherwise. Fluorescence photographs were taken using a Zeiss Axioimager A1 with an AxioCam digital camera and Axiovision rel. 4.6 software (Carl Zeiss) or an IX81 Olympus inverted confocal microscope. All images were taken at the young adult stage unless specifically indicated. The UNC-9::GFP distribution defect in the cell body region was quantified by counting the percentage of cell bodies with diffusely localized UNC-9::GFP. To quantify the UNC-9::GFP puncta distribution in DD/VD cell body region, the size of UNC-9::GFP co-localized with either myr::mCherry, mCherry::RAB-5, mCherry::RAB-7 or mCherry::LGG-1 was measured using ImageJ. The ratio of the co-labeled UNC-9::GFP to the total UNC-9::GFP was calculated. Signals from 5 DD/VD cells were scored for each genotype. To quantify the number of DD/VD neurons, the Punc-25::mCherry::LGG-1 was constructed. Images at the young adult stage were collected and the numbers of mCherry::LGG-1 foci in DD/VD cell body regions were counted. To characterize mCherry::LGG-1 puncta in strains carrying daf-2((e1370 ts), embryos were collected and raised at 25°C until L2/L3 stage before phenotypic examination.To analysis autophagy activity in DD/VD neurons, PHEK293T (human embryonic kidney) cells were maintained in Dulbecco’s modified Eagle’s medium containing 12% FBS. For cell transfection, polyethylenimine (Polysciences) was used according to the manufacturer’s instructions. The clones Myc-tagged cytoplasmic region of IRE-1 and the Flag-tagged full-length wild-type or kinase inactive form of PMK-3 were co-transfected into HEK293T cells. 24 hours after transfection, cells were harvested and lysed for 30 min at 4°C. After centrifugation, the supernatants were incubated with anti-FLAG M2 affinity gel beads at 4°C overnight and then washed three times with washing buffer and incubated with SDS-sample buffer. Samples were resolved by standard immunoblotting techniques. All the co-immunoprecipitation experiments were repeated at least three times.The Myc-tagged cytoplasmic region of IRE-1 and the Flag-tagged full-length wild-type or kinase inactive form of PMK-3 were co-expressed in HEK293T cells and then the IRE-1 protein was immunoprecipitated with Myc antibodies. The phosphorylation level of IRE-1 was examined with P-Thr-Pro-101antibody , which detects the conserved phosphorylation site (S/TP) for p38 MAPK. The signal intensity of individual protein bands was analyzed with ImageJ. The relative protein level was determined by subtracting the respective FLAG control. All the co-immunoprecipitation experiments were repeated at least three times.The pGEX-4T-1-GST::IRE-1 plasmid was transformed into BL21(DE3) competent cells. The fusion protein was induced by 1mM IPTG at 18°C for 24hr and then purified by Glutathione Sepharose 4B. The Flag::PMK-3 and myc::SEK-1 plasmids were transfected into HEK293T cells. 24 hours after transfection, cells were lysed and purified by anti-FLAG M2 affinity beads. Subsequently, the protein samples were concentrated and replaced with the kinase buffer (50 mM Tris-HCl (pH 7.6), 10 mM MgCl, 1 mM DTT). The phosphorylation of IRE-1 by active PMK-3 was monitored using a Universal Kinase Activity Kit . Purified IRE-1 and 200μM ATP were mixed with purified PMK-3 and coupling phosphatase CD39L2 in phosphatase buffer . As a control group, PMK-3 was replaced by 1x kinase buffer. The reaction is carried out in 50 μL for 10 minutes at room temperature. The coupling phosphatase CD39L2 can generate inorganic phosphate from ADP resulting from the kinase reaction. Malachite Green Reagent was added to terminate the reaction. After incubation for 20 minutes at room temperature to stabilize the color development. The amount of phosphorylated protein was determined using a microplate reader set to 620 nm. The experiments were repeated three times independently. To further determine the phosphorylation of IRE-1, anti-P-Thr-Pro-101 antibody was used to detect the phosphorylation site (S/TP) for p38 MAPK. The GST::IRE-1 was incubated with Flag::PMK-3 in kinase buffer  at 25°C for 30 minutes. As a control group, the Flag::PMK-3 was replaced by 1x kinase buffer. The samples were boiled with loading buffer to terminate the reaction. The experiments were repeated three times independently.unc-9 sequence and the reverse primer located in gfp sequence. Primer1: Forward: 5-CTACCCTGTTACGAGCTTGC-3 Reverse: 5-TTCTACCGGTACCCTCCAAG-3 Primer2: Forward: 5-CTACCCTGTTACGAGCTTGC-3; Reverse: 5-TGCCCATTAACATCACCATC-3. Relative levels of unc-9::gfp expression were normalized to act-1 in the same sample.Total RNA was isolated from different genotype worms using Trizol reagent (Invitrogen). cDNA was generated using a Superscript II reverse transcriptase kit (Invitrogen). qRT-PCR experiments were performed using Transstart Green qPCR superMix (Transgen Biotech) with a Stratagene Mx3000P system (Agilent). The following two pair primers were used and the forward primer located in ire-1(v33); daf-2(e1370);xdIs15, xbp-1(zc12); daf-2(e1370); xdIs15, pmk-3(tm745); daf-2(e1370); xdIs15 worms were cultured at 25°C to reach adulthood and then transferred onto NGM plates containing 5 mM chloroquine. After 4 hours incubation, the UNC-9::GFP distribution defect in the cell body region of DD/VD neurons was quantified by counting the percentage of cell bodies with diffusely localized UNC-9::GFP signal.The chloroquine treatment was performed following a modified protocol from previous report . BrieflyS1 FigN = 5. (B) A worm-scale image of UNC-9::GFP knock in (KI) (green). NR, nerve ring. VC, ventral cord. (C) Quantification of Phsp-4::mCherry signal in the anterior intestine region. (D) Quantification of Phsp-4::mCherry signal in the posterior intestine region. NS, not significant; **P < 0.01, *P < 0.05. One-way ANOVA with Dunnett’s test. (E) Expression of Phsp-4::mCherry (red) in wild-type (wt), ire-1 and xbp-1 animals with or without UNC-9::GFP. Scale bar represents 25 μm. (F) Phsp-4::mCherry distribution in wild type with no UNC-9::GFP overexpression in DD/VD cells. F’ is enlarged from F (boxed region). (F) Phsp-4::mCherry (red) is widely and weakly expressed in multiple tissues. F’ is enlarged from F (boxed region) showing a part of ventral cord region. (G) Phsp-4::mCherry (red) is induced in UNC-9::GFP (green) expressing DD/VD neurons (white arrows). G’ is enlarged from G (boxed region) showing a DD/VD cell (red). Scale bar represents 50 μm(A) Quantification of the UNC-9::GFP co-distribution with Myr::mCher, RAB-5:mChery or RAB-7::mCherry. (TIF)Click here for additional data file.S2 Figxbp-1 gene. The molecular lesions are indicated for the zc12, tm2482, tm2457, and xd131 alleles. Green boxes indicate exons. (B) Quantification of the UNC-9::GFP localization defect in various genotypes. N = 20 for each genotype; ***P < 0.001; **P < 0.01. One-way ANOVA with Dunnett’s test. (C and D) Quantification of the mRNA expression of Punc-25::UNC-9::GFP in various genotypes. Three independent biological repeats.(A) The structure of the (TIF)Click here for additional data file.S3 Figwt), ire-1(v33), xbp-1(zc12), and pmk-3(tm745) animals carrying the Pmyo-3::UNC-9::GFP transgene. White lines highlight the muscle cells. Dashed lines highlight the regions from which the UNC-9::GFP puncta are absent. Scale bar represents 25 μm. (B) UNC-9::GFP expressed at the endogenous level from a knock-in allele (UNC-9::GFP KI) is distributed on the neuronal processes and cell surface of DD/VD neurons (asterisks) in ire-1 (A) and xbp-1 (B)mutants. Scale bar represents 5 μm.(A) The localization of UNC-9::GFP (green) in muscle cells in wild-type ((TIF)Click here for additional data file.S4 Figunc-53 expressing neurons (green) in wild-type (wt), ire-1(v33), and xbp-1(zc12) animals carrying the Punc-53::UNC-9::GFP transgene. White arrows indicate UNC-9::GFP puncta on neurites. Dashed lines encircle the cell bodies. Scale bars represent 5 μm.(A) The localization of UNC-9::GFP in P(TIF)Click here for additional data file.S5 Figpmk-3 gene. The molecular lesions are indicated for the ok169, tm745 and xd74 alleles. The kinase domain is indicated. Green boxes indicate exons. (B) The RT-PCR results for pmk-3 in wild type and pmk-3(xd74).(A) The structure of the (TIF)Click here for additional data file.S6 Figxbp-1 represented by mCherry signal in wild-type (wt) and pmk-3 animals. N is indicated for each genotype. (C) The in vitro kinase assay. ***P < 0.001. Student’s t-test. Three independent biological repeats.(A) The localization of UNC-10::GFP (green) in DD/VDs in various genotypes. Dashed lines encircle the cell bodies. Scale bars represent 5 μm. (B) Alternative splicing of (TIF)Click here for additional data file.S7 Fighsp-4::mCherry signal in the anterior intestine region. (C) Quantification of Phsp-4::mCherry signal in the posterior intestine region. ***P < 0.001; **P < 0.01; *P < 0.05; NS, not significant. One-way ANOVA with Dunnett’s test. (D) The overexpression of hsp-4 does not suppress the mutant phenotype of ire-1(v33) or xbp-1(zc12) animals.(A) The PMK-3-IRE-1-XBP-mediated UPR acts in parallel with insulin-inhibited autophagy to alleviate chronic stress induced by excess UNC-9::GFP proteins. (B) Quantification of P(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Leishmania donovani (LD) infection. However, current understanding of the molecular regulation of CD40 expression remains elusive. Using molecular, cellular and functional approaches, we identified a role for Runx1 and Runx3 transcription factors in the regulation of CD40 expression in DCs. In response to lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα) or antileishmanial drug sodium antimony gluconate (SAG), both Runx1 and Runx3 translocated to the nucleus, bound to the CD40 promoter and upregulated CD40 expression on DCs. These activities of Runx proteins were mediated by the upstream phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Notably, LD infection attenuated LPS- or TNFα-induced CD40 expression in DCs by inhibiting PI3K-Akt-Runx axis via protein tyrosine phosphatase SHP-1. In contrast, CD40 expression induced by SAG was unaffected by LD infection, as SAG by blocking LD-induced SHP-1 activation potentiated PI3K-Akt signaling to drive Runx-mediated CD40 upregulation. Adoptive transfer experiments further showed that Runx1 and Runx3 play a pivotal role in eliciting antileishmanial immune response of SAG-treated DCs in vivo by promoting CD40-mediated type-1 T cell responses. Importantly, antimony-resistant LD suppressed SAG-induced CD40 upregulation on DCs by blocking the PI3K-Akt-Runx pathway through sustained SHP-1 activation. These findings unveil an immunoregulatory role for Runx proteins during LD infection.The level of CD40 expression on dendritic cells (DCs) plays a decisive role in disease protection during  Leishmania donovani, is the second most deadly parasitic disease in the world after malaria. The protective immunity against this disease largely depends on the level of CD40 (a cell surface protein) expression on a specialized group of immune cells called “dendritic cells” (DCs), which play a central role in initiating antileishmanial immune responses. Accordingly, to develop an improved immunological approach for the treatment/prevention of VL, it is important to understand the molecular events that favor the induction of CD40 expression on DCs. However, the molecular mechanisms controlling CD40 expression are not yet fully understood. Our work describes, for the first time, that the transcription factors Runx1 and Runx3 promote CD40 expression on DCs, which thereby potentiate the ability of DCs to induce antileishmanial immune responses in vivo. Our study thus provides new insights into the molecular mechanism required for an immune response to combat VL.Visceral leishmaniasis (VL), caused by  Leishmania donovani (LD). Besides functioning as initiators of Leishmania-specific T cell reactivity, DCs play a central role in the regulation of host-protective T helper cell type-1 (Th1) responses  strain AG83, and lastly showed that this SbSLD strain failed to inhibit SAG-induced CD40 expression on DCs (RLD) strain GE1F8R effectively inhibited SAG-stimulated CD40 expression by DCs at both mRNA and protein levels. Since LD required SHP-1 to regulate PI3K-Akt-Runx pathway-mediated CD40 expression in DCs  in DCs following SAG treatment. While SbSLD strain AG83 could not induce SHP-1 phosphorylation in the presence of SAG, SbRLD strain GE1F8R continued to drive SHP-1 phosphorylation in BMDCs despite SAG treatment (SLD) and GE1F8R (SbRLD) infection, SHP-1 physically interacted with the PI3K complex p85α-p110β (SLD)-infected DCs upon SAG treatment (RLD)-infected DCs despite SAG treatment -infected BMDCs. In contrast, these SAG-induced events were readily detected in BMDCs infected with AG83 (SbSLD)  Figs . NotablySLD and SbRLD in human monocyte-derived DCs (HuMoDCs). Initially, we tested whether Runx proteins regulate SAG-induced CD40 expression in HuMoDCs. Computational analysis using the TFBIND program identified two putative Runx-binding sites  in the human CD40 promoter (CD40 promoter following stimulation of HuMoDCs with SAG (RLD), unlike AG83 (SbSLD), impaired SAG-induced Runx binding to the CD40 promoter and upregulation of CD40 expression on HuMoDCs  nuclear translocation. Our findings further suggest that Runx1 and Runx3 are essential for promoting LPS- and TNFα-mediated upregulation of CD40 expression in DCs. This conclusion is supported by results demonstrating that silencing of Runx1 and Runx3, or overexpression of Runx DN markedly attenuated LPS- or TNFα-induced CD40 expression in DCs. In this regard, the usage of conditional or DC-specific Runx1- and/or Runx3-deficient mice to assess the functional role for Runx1 and Runx3 in the regulation of CD40 expression in DCs was not feasible. This is due to the fact that the development of DCs is known to be severely compromised in Runx1 conditional knockout mice and DC-specific Runx3-deficient mice  and Postgraduate Institute of Medical Education and Research (PGI/IEC/2020/EIC000344). Informed consents were obtained from all blood donors.E. coli O111:B4), PI3K inhibitors Wortmannin  and Ly294002 , and all other reagents were procured from Sigma-Aldrich unless stated otherwise.The following antibodies were used for immunoblot analysis: anti-Runx1 (sc-28679), anti-Runx3 sc-376543), anti-PI3K p110β (sc-376412), anti-PI3K p110δ (sc-7176), anti-TLR4 (sc-293072), anti-β-actin (sc-47778), HRP-conjugated anti-rabbit IgG (sc-2004) and HRP-conjugated anti-mouse IgG ; anti-phospho-SHP-1 ; and anti-PI3K p85α (4292), anti-PI3K p110α (4255), anti-phospho-Akt , anti-Akt (9272), anti-phospho-SHP-1 , anti-SHP-1 (3759) and anti-TNFR1 . For EMSA, ChIP and immunoprecipitation assays, the antibodies and reagents used were as follows: anti-Runx1 (sc-28679 X), anti-Runx2 (sc-10758 X), rabbit IgG (sc-2027), mouse IgG (sc-2025) and protein A/G PLUS-Agarose beads ; anti-Runx3 ; Armenian hamster IgG ; and anti-TLR4 (ab22048) and anti-TNFR1 . For confocal microscopy following antibodies were used: anti-Runx1 (sc-28679), anti-Runx3 (sc-376543), and respective isotype control antibodies  were obtained from Santa Cruz Biotechnology. Alexa Fluor 568 (AF)-conjugated goat anti-rabbit IgG (A-11011) and goat anti-mouse IgG (A-11004) secondary antibodies were procured from Thermo Fisher Scientific. For flow cytometry analysis, following antibodies were obtained from Biolegend: APC-conjugated anti-mouse CD11c (117310), APC-conjugated anti-mouse CD3 (100235), FITC-conjugated anti-mouse CD8 (100705), PE-conjugated anti-mouse IL-10 (505007), PE-conjugated anti-mouse CD40 (124610), PE-conjugated anti-mouse TNFR1 (113003), PE-conjugated anti-human CD40 (313006), PE-conjugated anti-mouse TNFR2 (113405), PE-conjugate Armenian hamster IgG (400907), PE-conjugated rat IgG2a,κ (400507), PE-conjugated mouse IgG1,κ (400111), APC-conjugated Armenian hamster IgG (400911) and APC-conjugated rat IgG2b,κ (400611). Other antibodies such as FITC-conjugated anti-mouse CD4 (11-0041-82), PE-conjugated anti-mouse IFNγ (12-7311-83) and PE-conjugated anti-mouse TLR4 (12-9041-80) were procured from eBioscience (Thermo Fisher Scientific); and FITC-conjugated rat IgG (sc-2340) and PE-conjugated rat IgG (sc-516648) were from Santa Cruz Biotechnology. The Akt inhibitor XIII  was from Merck Life Science. Recombinant mouse TNFα, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 were from Peprotech. Recombinant human GM-CSF and IL-4, and anti-mouse CD11c magnetic beads were from Miltenyi Biotec. The ON-TARGETplus non-targeting control pool siRNAs and SMARTpool siRNAs targeting mRNA encoding mouse Runx1, Runx3, PI3K p110β and SHP-1; and human Runx1 and Runx3 were from Dharmacon. Notably, the same siRNAs were previously used by other groups , provided by Dr. Nahid Ali , was maintained in male or female golden hamsters between 4–6 wks of age as described . AmastigSLD  in some /89/GE1)  was used6/well) were then treated with LPS (1 μg/ml) or TNFα (100 ng/ml) for specified times in complete RPMI 1640 medium . Alternatively, DCs were treated with clinical grade of SAG  for specified times as described . (C) Dual-luciferase assay of JAWSII cells that had been transfected with indicated CD40 promoter-luciferase constructs (as in B) together with the renilla luciferase plasmid pRL-CMV  and then treated for 24 h with LPS (left panel) or TNFα (right panel) or left untreated (UT). Results were normalized to the activity of renilla luciferase and are presented relative to those in p505-luc-transfected JAWSII cells that had been left untreated. Horizontal bars represent the mean. Data are a compilation of two separate experiments . Each symbol represents data of individual replicate. *p < 0.001.(A) Immunoblot analysis of Runx1 and Runx3 in JAWSII cell lysates. (B) Schematic of the mouse (TIF)Click here for additional data file.S7 Figp < 0.001, **p < 0.01, ***p < 0.05; NS, not significant.Relates to (TIF)Click here for additional data file.S8 Fign = 3 independent experiments) for CD40 promoter-specific probes , and the expression of Runx1 and Runx3 in BMDC lysates (right two panels). Data are presented relative to untreated BMDCs that had been left unstimulated. Error bars represent SD. Each symbol represents data of individual experiment. *p < 0.001, **p < 0.01, ***p < 0.05; NS, not significant.(A) Relates to (TIF)Click here for additional data file.S9 FigBMDCs were either left untreated (UT) or treated for 1 h with DMSO, Wort or LY and then stimulated with LPS or TNFα for 0.5 h or left unstimulated (US). The translocation of Runx1 and Runx3 (red) to the nuclei (blue) was analyzed via confocal microscopy. Pink color (merge) shows nuclear translocation of Runx1 or Runx3. Scale bar, 10 μm. Data are representative of two independent experiments.(TIF)Click here for additional data file.S10 FigImmunoblot analysis of total and phosphorylated Akt in lysates of BMDCs treated with LPS (A) and TNFα (B) for indicated times. Numbers below lanes represent densitometry, normalized to total Akt and presented relative to untreated BMDCs (0 h). Data are representative of two independent experiments.(TIF)Click here for additional data file.S11 FigCD40 promoter  and the expression of Runx1 and Runx3 in BMDC lysates  have been shown in p < 0.001, **p < 0.01, ***p < 0.05; NS, not significant.BMDCs were left uninfected (Uninf) or infected with LDPm for indicated times and then stimulated with (+) LPS or TNFα for 0.5 h or left unstimulated (-). The binding of nuclear Runx1 and Runx3 to the (TIF)Click here for additional data file.S12 Fign = 3 independent experiments) for immunoblot analysis , and the expression of Runx1 and Runx3 in BMDC lysates (lower panels). Densitometry analysis was carried out as described in p < 0.001, **p < 0.01, ***p < 0.05; NS, not significant.(A) Relates to shown in  of SHP-1(TIF)Click here for additional data file.S13 FigBMDCs were either left untransfected (- siRNA) or transfected with control (Ctrl) siRNA or SHP-1-specific siRNA. The expression of TLR4, TNFR1 and TNFR2 expression on BMDCs was analyzed by flow cytometry. Data are representative of two independent experiments.(TIF)Click here for additional data file.S14 Fign = 3 independent experiments) for EMSA (shown in CD40 promoter (assessed using Pr1 and Pr2 probes) in BMDCs treated with SAG for indicated times. Densitometry analysis was performed as in p < 0.001, **p < 0.01, ***p < 0.05; NS, not significant.(A) Relates to shown in  of Runx1(TIF)Click here for additional data file.S15 FigCD40 promoter using Pr1 and Pr2 probes (left two panels) and immunoblot analysis of Runx1 and Runx3 expression (right two panels) in BMDCs that had been left uninfected or infected with LDPm for 24 h, then cultured with or without SAG for 0.5 h. Densitometry analysis was done as in p < 0.001, **p < 0.01; NS, not significant.(A) Relates to (TIF)Click here for additional data file.S16 Fign = 3 in each experiment). The horizontal bars represent the mean. Each symbol represents data of individual replicate. In these experiments, LD strain AG83 was used. NS, not significant.BMDCs were infected with LDPm for 24 h and then treated with SAG for 0.3 or 1 h, or left untreated (0 h). The percentage of infected BMDCs (A) and the number of intracellular amastigotes per 1000 BMDCs (B) were determined by Giemsa staining. Data are a compilation of two separate experiments ((TIF)Click here for additional data file.S17 FigRelates to Figs (TIF)Click here for additional data file.S18 FigRelates to (TIF)Click here for additional data file.S19 Fig+ cells were then analyzed for CD4+, CD8+, IFNγ+ and/or IL-10+ population (Relates to pulation  based on(TIF)Click here for additional data file.S20 FigRLD strain GE1F8R or SbSLD strain AG83 for 24 h and then cultured with or without SAG for 24 h. The MFI values were calculated as described in CD40 promoter-specific Pr1 and Pr2 probes (left two panels), and immunoblot analysis of Runx1 and Runx3 expression (right two panels) in BMDCs left uninfected or infected with SbSLD strain AG83 or SbRLD strain GE1F8R for 24 h and then cultured (for 0.5 h) with or without SAG. Densitometry analysis was performed as in p < 0.001, **p < 0.01, ***p < 0.05; NS, not significant.(A) Relates to (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Caenorhabditiselegans,serotonin (5-HT) activates and controls pharyngeal pumping in response to food . Tryptophan hydroxylase, the enzyme required for serotonin biosynthesis, is encoded by the gene tph-1. Worms with a tph-1 deletion mutation exhibit phenotypes associated with a lack of serotonin-signaling, including reduced pharyngeal pumping . We used a microfluidic electropharyngeogram (EPG) recording platform (NemaMetrix) and associated software  to measure pharyngeal pumping in C. eleganstph-1 mutants in the presence of bacterial food (100 mg/ml E. coli OP50 in M9 buffer), following a 2-hr fasting period. Prior research has shown that a fasting period  induces elevated feeding rates for worms upon re-introduction to bacterial food (Lemieux and Ashrafi 2015). We chose to measure pharyngeal pumping during this elevated feeding phase due to our hypothesis that tph-1 animals would exhibit lower pumping rates than control worms. Pumping was recorded for 2-minute durations over three independent trials . C. eleganstph-1 mutants exhibited significantly lower pharyngeal pumping rates than N2 control animals . Pumping frequency data were pooled in A; see B for a comparison of each experimental trial.In tph-1(mg280) II, kindly provided by the Prahlad lab.Strains: MT15434 N2Control Strain:


ErbB2 breast cancer still remains an unmet need due to primary and/or acquired resistance to current treatment strategies. MEDICA compounds consist of synthetic long-chain α,ω-dicarboxylic acids previously reported to suppress breast cancer in PyMT transgenic mice.MEDICA efficacy and mode of action in the ErbB2 context was studied in ErbB2 transgenic mice and human breast cancer cells.MEDICA treatment is shown here to suppress ErbB2 breast tumors and lung metastasis in ErbB2/neu MMTV transgenic mice, to suppress ErbB2/neu xenografts in nod/scid mice, and to suppress survival of AU565 and BT474 human ErbB2 breast cancer cells. Suppression of ErbB2 breast tumors by MEDICA is due to lipid raft disruption with loss of ErbB family members, including EGFR, ErbB2, and ErbB3. In addition, MEDICA inhibits mTORC1 activity, independently of abrogating the ErbB receptors and their signaling cascades. The double hit of MEDICA in abrogating ErbB and mTORC1 is partly accounted for by targeting mitochondria complex I.Mitochondrial targeting by MEDICA suppresses ErbB2 breast tumors and metastasis due to lipid raft disruption and inhibition of mTORC1 activity. Inhibition of mTORC1 activity by MEDICA avoids the resistance acquired by canonical mTORC1 inhibitors like rapalogs or mTOR kinase inhibitors. Breast cancer is the most common female cancer and the second leading cause of cancer-related death in women in developed countries . ErbB2  or prediabetes . Indeed2)n-C(β)-C(α)-COOH, n ≥ 10]  was synthesized as previously described [Anti-neu/HER2 (ErbB2) antibody was from Invitrogen. Anti-ErbB2, anti-ErbB3, anti-EGFR, anti-caveolin-1, anti-phospho-ERK(Tyr204), anti-ERK, anti-phospho-Stat3(Tyr705), and anti-NDRG1 antibodies were from Santa Cruz Biotechnology INC. Anti-phospho-EGFR(Tyr1068) and anti-phospho-ErbB2(Tyr1248) antibodies were from Abcam. Anti-α-tubulin, anti-β-actin, and anti-cholera toxin B subunit antibodies were from Sigma Aldrich. Anti-4EBP1, anti-AMPKα, anti-acetyl-CoA carboxylase, anti-S6 ribosomal protein, anti-p70 S6 kinase, anti-Akt, anti-phospho-4EBP1(Thr37/46), anti-phospho-Akt(Thr308), anti-phospho-Akt(Ser473), anti-phospho-acetyl-CoA carboxylase(Ser79), anti-phospho-AMPKαThr172), anti-phospho-p70 S6 kinase(Thr 389), and anti-phospho-S6 Ribosomal protein(Ser240/244) antibodies were from Cell Signaling. Anti-Sestrin2 and anti-REDD1 antibodies were from Proteintech. Cholera toxin subunit B (CT-B) Conjugates were from Molecular Probes. Cy2, anti-pescribed .P < 0.05) was analyzed by unpaired t-test with Welch correction.Statistics was performed by two-tailed repeated measure analysis of variance (GraphPad). Significance (3)2-CH2)12-C(CH3)2-COOH in vivo has been verified in 4-month FVB MMTV-ErbB2/neu transgenic mice [Suppression of ErbB2 breast cancer by the MEDICA analogue HOOC-C, in the basal state and upon being induced by added EGF Fig. . InhibitLipid raft clustering and mTORC1 activity may both be modulated by metabolic stress , 35. IndCell growth suppression by mitochondrial complex I inhibitors in some cancer cell lines has recently been ascribed to aspartate limitation, being rescued by added pyruvate, aspartate or by infecting aspartate transporters –42. HencInhibition of mTORC1 activity due to mitochondrial stress prompted our interest in dissecting elements of the mTORC1 system that transduce inhibition of mTORC1 activity by MEDICA. mTORC1 activity may be inhibited by limiting ATP, by activating TSC1,2 and/or Raptor by AMPK, by stabilizing TSC1,2 by REDD1, and/or by modulating the activity of RAGs by Sestrin2 or lysosomal v-ATPase . Indeed,MEDICA treatment is shown here to suppress ErbB2 breast tumors and lung metastasis in transgenic mice that express the activated ErbB2/neu oncogene under the control of the mouse MMTV long terminal repeat (LTR) promoter. Suppression of ErbB2 breast cancer by MEDICA was corroborated by suppressing ErbB2/neu xenografts in NOD/SCID mice. MEDICA effects were further verified by growth suppression of trastuzumab-sensitive and trastuzumab-resistant AU565 and BT474 human ErbB2 breast cancer cells. MEDICA inhibited the viability of ErbB2 tumor cells, colonies, and spheroids, while no resistance to MEDICA was observed in ErbB2 cells subjected to prolonged continuous culturing with MEDICA.Suppression of ErbB2 breast tumors by MEDICA in ErbB2/neu mouse transgenes as well as in human ErbB2 breast cancer cells was accompanied by loss of plasma membrane ErbB2, together with other ErbB family members, including EGFR (ErbB1) and ErbB3. Since ErbB2 breast cancer is driven by homodimerization and/or heterodimerization of the amplified ErbB2 with other ErbB receptors, the loss of ErbB family receptors may further promote MEDICA efficacy in suppressing ErbB2 breast cancer. Loss of plasma membrane ErbB family members by MEDICA was not due to decrease in respective ErbB transcripts, but to lipid raft disruption by MEDICA, as verified by the concomitant loss of lipid raft caveolin-1 and GM1. Since stabilized lipid rafts form platforms for receptor tyrosine kinases docking and their signaling cascades , 32, lipThe double hit of MEDICA in inhibiting mTORC1 activity while concomitantly abrogating ErbB signaling is noteworthy in terms of resistance to current treatment strategies. Indeed, 20% of ErbB2 breast cancer patients present primary resistance to ErbB2-targeting agents , and 70% of patients with ErbB2 metastatic cancer present acquired resistance –48. ReplThe bifunctional activity of MEDICA in the ErbB/mTORC1 context is partly driven by targeting mitochondria complex I. Indeed, by-passing mitochondrial complex I by infected yeast NADH dehydrogenase (NDI1) resulted in partly rescuing ErbB expression, mTORC1 activity, and cell growth. In line with that, rescue of MTORC1, ErbB2, and its signaling pathways by NDI1 infection was more evident in cells cultured in galactose medium instead of glucose, namely, under conditions that drive cells to maximize mitochondrial oxidative phosphorylation rather than relying on glycolytic ATP production , 50. TheRetrospective clinical studies have indicated improved disease-free and overall survival of diabetic ErbB2 luminal B breast cancer patients treated with metformin –13. The In spite of the advances accomplished in treating ErbB2 breast cancer, the ErbB2 cancer challenge still remains an unmet need. Also, mTORC1 is activated in up to 80% of human cancers , while cAdditional file 1:. Supplemental figures


Rationale: The onset of cytokine release syndrome (CRS) and in vivo persistence of anti-CD19 chimeric antigen receptor T (CAR-T) cells after infusion correlate with clinical responsiveness. However, there are no known baseline biomarkers that can predict the prognosis of patients with B-lineage non-Hodgkin lymphoma (B-NHL). The aim of this study was to identify blood cell populations associated with beneficial outcomes in B-NHL patients administered CAR-T cell immunotherapies.Methods: We enumerated peripheral blood and CAR-T cells by retrospectively analyzing three CAR-T cell trials involving 65 B-NHL patients. We used a preclinical model to elucidate the eosinophil mechanism in CAR-T cell therapy.Results: During an observation period up to 30 mo, B-NHL patients with higher baseline eosinophil counts had higher objective response rates than those with low eosinophil counts. Higher baseline eosinophil counts were also significantly associated with durable progression-free survival (PFS). The predictive significance of baseline eosinophil counts was validated in two independent cohorts. A preclinical model showed that eosinophil depletion impairs the intratumoral infiltration of transferred CAR-T cells and reduces CAR-T cell antitumor efficacy.Conclusion: The results of this study suggest that peripheral eosinophils could serve as stratification biomarkers and a recruitment machinery to facilitate anti-CD19 CAR-T cell therapy in B-NHL patients. Moreover, the baseline percentage of this population in infused CAR-T cell products correlates with sustained disease remission Anti-CD19 CAR-T therapies produce durable antitumor responses against B-cell acute lymphoblastic leukemia (ALL) with complete response (CR) rates over 90% For patients with B-NHL, the best overall response rate to anti-CD19 CAR-T cell therapy is 83%, with only 58% of patients showing a CR after infusion in vitro activated eosinophils has been shown to be correlated with improved efficacy in mouse models evaluating adoptive T cell transfer therapy due to the secretion of the well-known T cell attractants CXCL9 and CXCL10 For anti-CD19 CAR-T cell therapy, a major difference between ALL and B-NHL is that the latter, like a solid tumor, has a microenvironment that can suppress T cell infiltration and activation . In Cohort 1, anti-CD19 CAR-T cells were administered to 16 patients with CD19-positive B-NHL. Thirteen patients were diagnosed with diffuse large B-cell lymphoma (DLBCL), two patients presented with follicular lymphoma (FL), and one patient had marginal zone lymphoma (MZL). All patients except one had progressive disease (PD) at the time of anti-CD19 CAR-T cell infusion. All patients relapsed after ≥ 2 lines of chemotherapy. The median prior chemotherapy regimen comprised nine cycles and three lines of treatments . Five patients had undergone prior radiotherapy. One patient experienced rapidly PD while awaiting T cell infusion. Therefore, short-term palliative radiation therapy  was administered to prevent potential suffocation due to submandibular node progression. Routine follow-up assessments were conducted based on our Institutional Review Board (IRB)-approved protocol .The characteristics of the patients in the Discovery Cohort (Cohort 1) are summarized in 6/mL) baseline eosinophil levels showed substantially higher objective response rates . We also identified a consistent trend toward favorable clinical responses in patients with high eosinophil levels by comparing baseline eosinophil counts in patients who achieved an objective response ) or experiencing stable disease (SD) or PD. However, statistical significance was not achieved, as the patient sample size was too small . Compared to patients with in the subgroup with lower eosinophil counts before infusion, progression-free survival (PFS) was significantly improved  = 0.3105; 95% confidence interval (CI) = 0.0982-0.9817, *P = 0.0161) in the subgroup with higher eosinophil counts relative to the median. In contrast, the abundance of neutrophils, basophils, lymphocytes, monocytes, platelets, and erythrocytes was not correlated with PFS in this cohort (Table S2). Two validation cohorts were introduced as additional supportive evidence. We confirmed the predictive significance of baseline eosinophil counts by applying the median eosinophil count as a cutoff in these validation cohorts . We consistently found correlations between eosinophil counts and clinical outcomes of CAR-T cell therapies, namely, higher objective response rates  and durable survival benefits  for patients with high eosinophil levels compared to those with low eosinophil levels. A multivariate Cox proportional model identified the baseline eosinophil count as an independent predictor of CAR-T cell therapy efficacy (Table S3).Tumor progression was assessed by positron emission tomography-computed tomography (PET-CT) or computed tomography (CT) with contrast. Peripheral blood samples were analyzed on days 1, 4, 7, 14, and 28 during the first mo after infusion, every mo for 6 mo, every 3 mo for 2 y, and every 6 mo after the 2-y follow-up. White blood cell counts were obtained by standard clinical laboratory examinations. Focusing on the baseline (blood sample collected immediately prior to preconditioning chemotherapy) blood cell counts, we found that patients with higher  = 79.70%; 95% CI = 64.85-94.54%) for patients treated with anti-CD19 CAR-T cell therapy.We plotted a receiver operating characteristic (ROC) curve 7 anti-CD19 CAR-T cells (7.5 × 105 CAR-T cells/kg) that were intravenously administered as a single dose. Despite showing SD at the first follow-up, the patient exhibited PD in the two following surveillance scans. The sum of the product of the perpendicular diameters (SPD) Figure ).Patient 8 was a male aged 58 y presenting with stage IIIA chemotherapy-refractory FL. He received an infusion of 4.5 × 10Figure ). The sequential timing of these clinical events suggests putative functional relationships among eosinophilia, CAR-T cell activation, and tumor lesion eradication.Flow cytometry analysis of the peripheral blood of this patient revealed two significant CAR-T cell influx peaks. The first appeared at 14 days postinfusion and was typical of most patients. However, an uncommon second peak appeared at 3 mo postinfusion, immediately before the remission of the tumor burden. A review of the blood cell counts identified dramatic eosinophilia before tumor pseudoprogression and CAR-T cell influx. At that time, the eosinophils were 9× higher than at baseline and eventually constituted 53.7% of the total white blood cell count  for acute lymphodepletion, similar to a clinical B-NHL protocol Figure S2) and infused at 5×106 cells/mouse 2 days after preconditioning . To investigate the functional role of eosinophils in mediating tumor regression, we intraperitoneally injected an isotype control or anti-mouse CD193 (CCR3) antibody (clone# 6S2-19-4)  in vivo. We monitored tumor size as a surrogate for treatment efficacy and body weight loss as an indicator of adverse toxicity.The aforementioned clinical data indicate that patients with elevated peripheral eosinophil counts achieve better clinical outcomes in response to anti-CD19 CAR-T cell immunotherapy. We examined the mechanism driving the correlation between eosinophil counts and CAR-T cell efficacy by administering anti-CD19 CAR-T cell therapy to an immunocompetent mouse lymphoma model Figure ). Anti-CD19 CAR-T cell administration eradicated A20 tumors in 13 eosinophil-competent, tumor-bearing mice. By day 28, 11 mice reached complete remission. In the eosinophil-depleted group, one out of seven mice treated with anti-CCR3 antibody were tumor-free by day 28. Three out of 12 mice treated with an anti-Siglec-F antibody were tumor-free, and two mice exhibited severe relapse and rapid tumor growth . We compared the time to reach 50% partial tumor remission after CAR-T cell treatment. The interval was significantly shorter for eosinophil-competent mice than for antibody-depleted mice . Comparable tumor growth was noted for mice subjected to eosinophil depletion but not infused with CAR-T cells . Therefore, eosinophils are functionally necessary for maximal anti-CD19 CAR-T cell therapeutic efficacy.CTX preconditioning induced immediate but temporary body weight loss caused by adverse gastrointestinal events. However, this side effect gradually abated in both treatment groups . CAR-T cell treatment efficacy was evident in mice with intact eosinophils. Tumors in mice treated with an isotype control antibody were significantly smaller than those in eosinophil-depleted mice administered an anti-CCR3 antibody  and anti-Siglec-F antibody . The anti-Siglec-F depletion antibody  and flow antibody  competitively bind antigen. Thus, flow cytometry analysis did not accurately reflect the number of eosinophils remaining after anti-Siglec-F antibody treatment. Hence, eosinophil-related data for the Siglec-F antibody-treated group were not included in this analysis. Flow cytometry analysis of tumor, blood, spleen, and bone marrow (BM) samples demonstrated that anti-CCR3 antibody administration depleted most circulating CD45+CD11b+Gr-1loF4/80+Siglec-F+MHC-II- eosinophils . Substantially fewer CAR-T cells were recovered from the tumors in eosinophil-depleted mice . Changes in tumor volume were negatively correlated with CAR-T cell intratumoral infiltration  and eosinophil counts in peripheral blood . We intravenously transferred 106 activated eosinophils into tumor-bearing mice 1 day before CAR-T cell treatment followed by the intravenous transfer of 5×106 CAR-T cells. CAR-T cells combined with eosinophil transfer showed a tendency toward more effective tumor eradication than CAR-T cells alone . The link between eosinophil abundance and CAR-T cell efficacy observed here was identical to the relationship discovered in human B-NHL trials . Therefore, our preclinical mouse model closely resembled human clinical outcomes. Moreover, mouse intratumoral eosinophil counts were positively associated with CAR-T cell infiltration . To elucidate the molecular mechanism underlying the promotion of intratumoral infiltration by eosinophils, we examined the expression of activated T cell attractants such as CXCL9 and CXCL10 in tumors with competent eosinophils and those with antibody-mediated depletion . We identified the significant downregulation of these attractants (**P = 0.0092 for CXCL9 and *P = 0.0235 for CXCL10) in eosinophil-depleted tumors. This finding was consistent with established eosinophil characteristics To investigate the mechanism by which eosinophils improve anti-CD19 CAR-T cell therapeutic efficacy, we performed immune profiling of A20 mouse lymphomas 3 days after CAR-T cell infusion , patients were enrolled in a clinical trial to evaluate the safety and efficacy of anti-CD19 CAR-T cells in relapsed or chemotherapy-refractory B-cell lineage non-Hodgkin malignancies. The present study was a retrospective analysis of patients in the IL-2 arm of the aforementioned clinical trial. The study was registered at ClinicalTrials.gov (NCT02652910). Patients were enrolled from November 2015 to June 2017 at the Department of Oncology of the Second Affiliated Hospital  at Army Medical University . Two independent validation cohorts were also included in this study. Cohort 2  7 CD3+ T cells obtained by anti-CD3/28 Dynabead separation were stimulated with IL-2 for 48 h and transduced with a retroviral vector containing anti-CD19-CD28ζ CAR. Anti-CD19 was a single-chain variable fragment (scFv) derived from an FMC63 monoclonal antibody. The CAR-T cells were expanded, and the partially completed product was washed and cryopreserved to formulate the final drug product. After identity, % CAR expression, T cell phenotype, potency, and sterility quality control (QC) testing, the final product was shipped to the clinical center either fresh or at ≤ -150 °C. For Cohort 2, the anti-CD19 CAR consisted of an FMC63 scFv clone, CD8α extracellular and transmembrane domains, and 4-1BB and CD3ζ cytoplasmic domains. For Cohort 3, a tandem CAR construct was generated by combining the CD19 scFv derived from an FMC63 monoclonal antibody and the CD20 scFv derived from a Leu-16 monoclonal antibody. A hinge, CD8α transmembrane domains, and 4-1BB and CD3ζ cytoplasmic domains were used in the tandem construct. CAR-T cell preparation and quality control details for Cohorts 2 and 3 were previously described Patients were enrolled in this study after eligibility screening and confirmation. They underwent leukapheresis to obtain peripheral blood mononuclear cells (PBMCs), which were then frozen and shipped to the central cell processing facility. For Cohort 1, > 102 intravenous CTX on day 1 and 35 mg/m2 intravenous fludarabine on days 1-3. In Cohort 2, preconditioning chemotherapy included 3 days of fludarabine (25 mg/m2 on days 1-3) and CTX (250 mg/m2 on days 1-3). In Cohort 3, preconditioning chemotherapy comprised CTX (20-30 mg/kg over 3 days) and fludarabine (20-30 mg/m2 on days 1-3) with or without doxorubicin liposomes (10 mg/m2 on day 1).In Cohort 1, endogenous lymphocytes were depleted before adoptive CAR-T cell transfer with chemotherapeutic preconditioning regimens consisting of 800-1,000 mg/mAnti-CD19 CAR-T cells were intravenously administered to each patient 2 days after the completion of preconditioning chemotherapy. CAR-T cells were synthesized for all enrolled patients. In Cohort 1, 12 patients were administered cryopreserved CAR-T cells, whereas the other four patients were administered fresh CAR-T cells. In Cohorts 2 and 3, all 49 patients were administered fresh CAR-T cells.L-glutamine , 1 mM sodium pyruvate , and 0.05 mM 2-mercaptoethanol . HEK293T and 3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) FBS  and 1% (w/v) penicillin/streptomycin . All cells were purchased from Duke University Cell Culture Faculties , confirmed to be mycoplasma-free, and cultured in a humidified 37 °C incubator with 5% CO2.Mouse A20 lymphoma cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum , 1% (w/v) penicillin/streptomycin , 2 mM The MP71 retroviral plasmid served as the backbone TM 2000  into 293T cells. Supernatants containing viral particles were collected 48 h after transfection and passed through a 0.45-µm filter. Mouse lymph nodes were excised, digested, and pooled in a single-cell suspension. Lymphocytes were stimulated with 4 µg/mL anti-CD3 and 4 µg/mL anti-CD28 antibodies precoated on cell culture plates. The lymphocytes were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 1% (w/v) penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, 0.1 mM nonessential amino acids, and 50 U/mL human IL-2  for 36 h. The cells were then spin-infected with virus at 2,500 rpm and 32 °C for 2 h in the presence of 6 µg/mL Polybrene . Subsequent experiments were conducted 48 h after infection.MP71 and pclEco were cotransfected with Lipofectamine7 A20 cells on day 0. After 12 days, each mouse was preconditioned with 2 mg CTX  followed by 15 µg/mouse/day anti-Siglec-F (R&D MAB17061) or isotype control (R&D MAB006) antibody administration for 4 days starting on day 13. Alternatively, each mouse was administered 100 µg anti-CCR3 (Bioxcell 6S2-19-4) antibody on days 13 and 15. Then, 5 × 106 anti-CD19 CAR-T cells were infused into the tail vein of each mouse on day 14. Mouse body weight changes and tumor sizes were closely monitored every 2 days after treatment.Each BALB/c mouse aged 6-10 wks was subcutaneously injected with 106 anti-CD19 CAR-T cells on day 14.Mice used in fluorescence-activated cell sorting (FACS) analysis were treated as previously described except they were sacrificed on day 17 to obtain the organs and tissues required for the procedure. For the eosinophil transfer experiment, eosinophils were obtained and activated with TNF-α and IFN-γ as previously described 2). Mice were purchased from The Jackson Laboratory  and maintained under specific pathogen-free conditions at the Animal Facility of Duke University . All mouse studies were performed in accordance with the guidelines and protocols approved by the Duke University Animal Care and Use Committee .Tumor dimensions were measured with a caliper, and tumor volumes were calculated as ½ and BV421-labeled streptavidin . Flow cytometry was performed with a MACSQuant Analyzer 10  and FlowJo v. 10 .Human CAR-T cells in peripheral blood were assessed by flow cytometry and stained with fluorescently labeled antibodies against CD3, CD4, CD8, CD19, and CD10 . CAR was detected with biotin-labeled polyclonal goat anti-mouse F(ab)The mice were sacrificed, and their spleens, BM, blood, draining lymph nodes, and tumors were harvested and processed as single-cell suspensions. Red blood cells were lysed in 1× ammonium-chloride-potassium (ACK) buffer  for 1 min. Samples were stained with Aqua live/dead dye  according to the manufacturer's protocol. Fc receptors were blocked with an anti-mouse CD16/CD32 antibody (BD #553141) before surface staining with mTCRb PerCP-Cy5.5 (BioLegend #109227), mCD8b BV711 (BioLegend #126633), mCD45 APC (BioLegend #103112), mSiglec-F BV421 (BD #562681), mCD11b PECY7 (BioLegend #101216), Gr-1 AF700 (BioLegend #108422), mF4/80 PE (BioLegend #123110), and MHCII APCCY7 (BioLegend #107628). Data were acquired with an LSR Fortessa X20  and analyzed with FlowJo v. 10 .TM II analyzer .For intracellular cytokine staining, Golgi-blocking monensin and brefeldin A  were added 4 h before fixation in 2% (v/v) paraformaldehyde/phosphate-buffered saline (PFA-PBS). The cells were permeabilized in permeabilization buffer  according to the manufacturer's protocol. Samples were stained with anti-mouse CD8b PE (BioLegend #126608) and anti-mouse INF-γ Pacific Blue (BioLegend #505818). Flow cytometry was conducted with a BD FACSCantot-test was used to compare the means of two experimental groups. An unpaired twotailed Welch's t-test was used when the sample variances were unequal. Correlation was analyzed by Pearson's correlation test. Survival curves were plotted with the Kaplan-Meier method. Statistical comparisons were made with the log-rank (Mantel-Cox) test. The ROC curve was plotted in the “pROC” package of R . P < 0.05 was considered statistically significant.Unless otherwise specified, an unpaired twotailed Supplementary figures.Click here for additional data file.Supplementary table 1.Click here for additional data file.Supplementary table 2.Click here for additional data file.Supplementary table 3.Click here for additional data file.


On the other hand, a significantly higher dose (EC50 around 30 μM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat’s safety, make it a likely candidate drug to treat COVID-19. Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC) Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus pneumonia disease COVID-19), is spreading rapidly worldwide [9, is sprThe genomic RNA of coronaviruses is surrounded by an envelope . InitiatWe have previously reported that nafamostat mesylate, an existing Japanese drug used for acute pancreatitis and disseminated intravascular coagulation (DIC), effectively inhibits MERS-CoV S protein-mediated membrane fusion by targeting TMPRSS2 priming activity . We did In this study, we established an experimental assay system monitoring ACE2- and TMPRSS2-dependent SARS-CoV-2 S protein-mediated membrane fusion, in 293FT and Calu-3 cells and found that nafamostat mesylate potently blocked SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and viral infection in vitro in a cell-type-dependent manner.Nafamostat mesylate , camostat mesylate , gabexate mesylate , edoxaban, apixaban, rivaroxaban, dabigatran , argatroban  and darexaban .HEK293FT is an immortalized cell line derived from human fetal kidney. A pair of previously described 293FT-based reporter cell lines that constitutively express individual split reporters (DSP1-7 and DSP8-11 proteins) . 293FT-dExpression vectors for CD26 and TMPRSS2 were described previously . A synth5 cells/500 μL) one day before the assay. Two hours before the DSP assay, cells were treated with 6 μM EnduRen , a substrate for Renilla luciferase, to activate EnduRen. One microliter of each protease inhibitor or anticoagulant dissolved in dimethyl sulfoxide (DMSO) was added to the 384-well plates . Next, 50 μL of each single cell suspension (effector and target cells) was added to the wells using a Multidrop dispenser . After incubation at 37 °C for 4 h, the RL activity was measured using a Centro xS960 luminometer . For the DSP assay using Calu-3 or H3255 cells, target cells were seeded in 384-well plates (2 × 104 cells/50 μL) one day before the assay. Two hours before the DSP assay, cells were treated with 6 μM EnduRen. One microliter of each protease inhibitor or anticoagulant dissolved in DMSO was added to the 384-well plates with 9 μL of culture medium. Next, 40 μL of single cell suspension (effector cells) was added to the wells using a Multidrop dispenser.For the DSP assay using 293FT cells, effector cells expressing S protein with DSP8-11 and target cells expressing CD26 or ACE2, and TMPRSS2 with DSP1-7 were seeded in 12-well cell culture plates  containing 0.3% bovine serum albumin (BSA) and 1 µg of L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-trypsin/ml at 37 °C. All experiments with SARS-CoV-2 viruses were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Tokyo, which are approved for such use by the Ministry of Agriculture, Forestry and Fisheries, Japan.VeroE6 (ATCC CRL-1586) cells were maintained in Eagle’s minimal essential media (MEM) containing 10% FBS. The cells were incubated at 37 °C with 5% CO50 was determined using the Spearman–Karber formula [Calu-3 (ATCC HTB-55) cells were maintained in MEM containing 10% FBS. VeroE6/TMPRSS2   with inhibitory activity against serine proteases to inhibit SARS-CoV-2 S protein-mediated fusion f. LucifeWe next used lung epithelium-derived Calu-3 and H3255 cells as target cells, because they endogenously express ACE2 and TMPRSS2, and might better reflect physiological conditions than 293FT kidney cells ectopically expressing ACE2 and TMPRSS2. Nafamostat mesylate inhibited SARS-CoV-2 S protein-mediated membrane fusion in the range of 1–10 nM while camostat mesylate required 10–100 nM to achieve a similar extent of inhibition g. A simiVarious computational approaches have recently been applied to find existing medications targeting TMPRSS2 ,16. Cons50 values for Calu-3 cells were 6.8–11.5 nM (pretreatment) and 3.16 μM (no-pretreatment) while those for VeroE6/TMPRSS2 cells were 31.6 μM (pretreatment) and >100 μM (no-pretreatment; 50 values were much smaller when cells were pretreated with nafamostat mesylate, implying that nafamostat mesylate inhibits TMPRSS2-dependent SARS-CoV-2 entry.Our DSP assays identified nafamostat mesylate as a potent inhibitor of SARS-CoV-2 S-mediated membrane fusion f,g. This50 values for Calu-3 cells with the pretreatment were around 10 nM, similar to our previous findings with MERS-CoV infection [Our findings clearly indicate that nafamostat mesylate, the most effective TMPRSS2 inhibitor so far reported, potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and viral infection in vitro in a cell-type-dependent manner. Furthermore, ECnfection . This exnfection ,14,18. Cnfection . Given tnfection , nafamosnfection , which infection . Since anfection , such annfection . TherefoHoffmann et al. have recently reported that nafamostat mesylate blocks activation of SARS-CoV-2 . However