HFpEF). In this study we tested the hypothesis that pro‐inflammatory biomarkers are elevated in patients with acutely decompensated HFpEF (AD‐HFpEF) compared with patients with stable HFpEF (S‐HFpEF).Underlying inflammation has been increasingly recognized in heart failure with a preserved ejection fraction (PTX3) and clinical, demographic, echocardiographic‐Doppler and clinical outcomes data were analyzed in HFpEF patients enrolled in NHLBI Heart Failure Research Network clinical trials which enrolled patients with either AD‐HFpEF or S‐HFpEF. Compared to S‐HFpEF, AD‐HFpEF patients had higher levels of PTX3 , interleukin‐6 , tumor necrosis factor‐alpha , and high‐sensitivity C‐reactive protein . Moreover, high‐sensitivity C‐reactive protein, interleukin‐6 and PTX3 levels were significantly higher in AD‐HFpEF compared with S‐HFpEF patients admitted for decompensated HF within the previous year. PTX3 was positively correlated with left atrial volume index  and left ventricular mass , while tumor necrosis factor‐alpha was inversely correlated with E/A ratio .Using a post hoc analysis the serum biomarkers tumor necrosis factor‐alpha, high‐sensitivity C‐reactive protein interleukin 6 and pentraxin 3 (AD‐HFpEF compared with S‐HFpEF patients. PTX3 and tumor necrosis factor‐alpha are correlated with echocardiographic‐Doppler evidence of diastolic dysfunction. Taken together these data support the concept that a heightened pro‐inflammatory state has a pathophysiologic role in the development of AD‐HFpEF.Levels of pro‐inflammatory biomarkers are strikingly higher in  Elevated levels of pro‐inflammatory serum biomarkers are well‐described in heart failure with reduced ejection fraction (HFrEF), but much less is known in patients with heart failure with a preserved ejection fraction (HFpEF), especially when they are decompensated.In this retrospective analysis, we report serum values of interleukin‐6, tumor necrosis factor‐alpha, h‐sensitivity C‐reactive protein, and pentraxin 3 in patients with stable and acutely decompensated HFpEF.In stable HFpEF, levels of tumor necrosis factor‐alpha and high‐sensitvity C‐reactive protein were >1 SD above the mean for subjects without prevalent cardiovascular disease, whereas interleukin‐6 and pentraxin 3 were below this cut‐off.All 4 biomarkers were strikingly and significantly elevated in decompensated versus stable HFpEF; further analysis suggests that these biomarker levels rise in conjunction with decompensation.These results are consistent with the hypotheses that chronic inflammation plays a role in the pathophysiology of HFpEF and that heightened inflammation may occur with decompensation; whether changes in inflammation precede decompensation will require prospective studies.If additional studies support these hypotheses, they may provide a rationale for targeted anti‐inflammatory therapy in HFpEF.Inflammation, oxidative stress, and extracellular matrix remodeling have been long considered to play a key role in the pathophysiology of heart failure (HF).It has been proposed that a heightened pro‐inflammatory state may play a role in the progression of HF, including acute decompensations.acutely decompensated HFpEF (AD‐HFpEF). We hypothesized that heightened inflammation is a component of the pathogenesis of acute decompensation in HFpEF. As an initial, preliminary test of this hypothesis, in the present study we sought to determine if selected pro‐inflammatory biomarkers are elevated in patients with AD‐HFpEF compared with patients with stable HFpEF (S‐HFpEF).Most information in regard to inflammation and HF has come from patients with HF and a reduced left ventricular ejection fraction (HFrEF). Recently, there has been an effort to better characterize biomarkers in HF with preserved EF (HFpEF)The data, analytic methods, and study materials will not be made available to other researchers for purposes of reproducing the results or replicating the procedures.We measured IL‐6, TNF‐α, and hs‐CRP and PTX3 in age and sex‐matched cohorts of HFpEF patients from 3 prospective, placebo‐controlled, randomized clinical trials conducted by the NHLBI Heart Failure Research Network, RELAX ,2) comparing 72 hours of low‐dose dopamine or low‐dose nesiritide to a standardized diuretic strategy. The dual end points of ROSE were cumulative urine volume and change in serum cystatin C from enrollment to 72 hours. Clinical status was assessed 30 and 60 days after completion.HFpEF patients were selected from the aforementioned trials. The RELAX trial tested the effect of the phosphodiesterase‐5 inhibitor sildenafil on exercise capacity and clinical status in comparison with placebo in S‐HFpEF patients. DOSE examined responses to 2 different diuretic strategies in hospitalized patients with ADHF. The primary end points of DOSE were a symptom score and changes in renal function. The in‐patient treatment strategy was maintained for 72 hours. Clinical outcomes were determined for 72 hours and follow‐up status was assessed 30 and 60 days after completion. ROSE was a trial of hospitalized patients with ADHF and renal dysfunction , elevated invasively‐measured left ventricular filling pressures or diastolic dysfunction by echocardiography‐Doppler examination, and reduced peak VO2. For the purpose of the current analysis, all RELAX patients were considered eligible unless they had been hospitalized with ADHF within the previous 6 months. Prior hospitalizations in RELAX patients were identified based on review of the patients’ records by the local principal investigators.Patients selected from DOSE and ROSE constituted the AD‐HFpEF group. Patients were eligible for the current analysis if they had EF ≥0.50 and would have been eligible for RELAX had they not been hospitalized for ADHF. The DOSE and ROSE exclusion criteria included acute co‐morbidities that could independently affect pro‐inflammatory biomarkers, including infections and other active conditions associated with heightened inflammation . By design, both S‐HFpEF and AD‐HFpEF cohorts were split evenly between males and females.hs‐CRP, IL‐6,TNF‐α and PTX3 were measured at baseline exams by the NHLBI Heart Failure Research Network Biomarker Core Laboratory at the University of Vermont using commercially available kits . Samples were shipped on dry ice and stored at −80°C until analysis at the Biomarker Core Laboratory.In addition to determining whether there are differences in pro‐inflammatory biomarkers in AD‐HFpEF versus S‐HFpEF, we also tested whether biomarker levels are associated with echocardiographic‐Doppler abnormalities of left ventricular diastolic function in all comers and are predictive of clinical outcomes in AD‐HFpEF. In the DOSE and ROSE trials short‐term clinical outcomes included urine volume, change in cystatin‐C, change in creatinine, change in NT‐proBNP, and change in weight over the 72 hours after randomization. In addition, post‐discharge clinical status with respect to survival and re‐hospitalizations was determined by telephone call 60 days after randomization for each trial.Baseline patient characteristics were compared between AD‐HFpEF and S‐HFpEF patients. Continuous variables were reported as median  and compared using Wilcoxon rank‐sum tests. Categorical variables are reported as frequencies and percentages, and were compared using the Pearson chi‐square test or the Fisher exact test. The distributions of the 4 biomarkers  were compared between AD‐HFpEF and S‐HFpEF using box plots with Wilcoxon rank sum tests.For the S‐HFpEF group, the Spearman correlation coefficient was used to assess correlations between biomarkers and diastolic function indexes at baseline and 24 weeks. Diastolic function indexes tested were LA volume index, E/A ratio, medial E/e′, lateral E/e′ and relative wall thickness.For the AD‐HFpEF group, unadjusted and age‐adjusted Cox proportional hazards regression models were used to assess the association between biomarkers and death or HF hospitalization at 60 days. Unadjusted and age‐adjusted linear regression models were used to assess the association between biomarkers and 72‐hour end points. The linearity assumption was assessed by a likelihood ratio test comparing the linear fit to the fit of a restricted cubic spline transformation. Linear spline transformations were applied to the biomarkers that did not satisfy the linearity assumption. The proportional hazards assumption was tested for the Cox models by plotting the Schoenfeld residuals by time and then testing for a non‐zero slope. As there were no violations we report the single hazard ratio as an average over time. The adjusted models were only adjusted for age because of the small sample size.P‐value less than 0.05. Statistical analyses were performed using statistical software .For PTX3, hsCRP, and IL‐6 there were less than 2% missing values. For TNF‐α there were ≈10% missing values. Complete case analyses were conducted. All statistical tests were two‐sided, and the criterion for statistical significance was a P=0.029), were more likely to have jugular venous pressure ≥8 cm , peripheral edema , and to report symptoms of orthopnea . 94.2% of the AD‐HFpEF cohort had NYHA class III or IV symptoms at baseline while, by design, the S‐HFpEF cohort consisted exclusively of patients with NYHA class II and III symptoms. The AD‐HFpEF cohort was more likely to have been admitted for heart failure within the past year , to have atrial fibrillation and/or flutter , diabetes mellitus (DM) , chronic obstructive pulmonary disease (COPD)  and anemia . The AD‐HFpEF cohort was less likely to be treated with an angiotensin converting enzyme inhibitor (ACEi) or angiotensin receptor blocker , but was more likely to be receiving an aldosterone receptor antagonist  or a loop diuretic . The AD‐HFpEF cohort had worse renal function than the S‐HFpEF group  and higher levels of NT‐pro BNP .Baseline clinical characteristics of the 2 groups are shown in Table P<0.001), TNF‐α , hs‐CRP  and PTX3 . In the CHS ,Pro‐inflammatory biomarker results are shown in Because the S‐HFpEF and AD‐HFpEF groups differed with respect to the incidence of atrial fibrillation, DM, and COPD we performed a separate analysis in which the biomarker results were adjusted for these 3 variables Table . The resP=0.160; TNF‐α 9.92 pg/mL versus 8.28 pg/mL, P=0.128; hs‐CRP 5.36 mg/dL versus 3.38 mg/dL, P=0.063; PTX3 1.46 ng/mL versus 1.08 ng/mL, P=0.057. As shown in P<0.001), hs‐CRP  and PTX3 , but there was no statistically significant difference in TNF‐α . The latter results suggest that PTX3, IL‐6, and hs‐CRP are not persistently elevated in patients with AD‐HFpEF, while TNF‐α may remain elevated after admission for ADHF or alternatively may decline more gradually.Blood samples obtained after discharge from the hospital were not available in the AD‐HFpEF group. Therefore, to help determine whether elevated pro‐inflammatory biomarkers are a transient finding associated with decompensation in this group or are persistently elevated, we divided the S‐HFpEF patients into groups with and without an ADHF admission within the previous year B. AlthouP=0.002, Spearman coefficient=0.41) and left ventricular mass , while elevated TNF‐α was found to be inversely correlated with E/A ratio . IL‐6 and hs‐CRP were not significantly correlated with any measures of diastolic function.We also sought correlations between pro‐inflammatory biomarkers and echocardiography‐Doppler derived measurements of diastolic function at baseline for the S‐HFpEF patients. As shown in Table P=0.273), IL‐6 had a HR of 1.097 , TNF‐α had a HR of 1.014 , and hs‐CRP had a HR of 1.015 . Thus, the only biomarker that exhibited a statistically significant association with 60 day death or heart failure re‐hospitalization was IL‐6 using the age‐adjusted model.In the AD‐HFpEF cohort, biomarkers were evaluated for their association with short‐term clinical outcomes and re‐hospitalization or death within 60 days after admission. There were no statistically significant associations between short‐term outcomes  and biomarker levels. Re‐admission or death within 60 days after discharge occurred in 13 of 68 AD‐HFpEF patients. In an unadjusted model, none of the biomarkers were significantly associated with re‐admission or death. In the age‐adjusted model, PTX3 had a HR of 0.863  and weaker correlations between PTX3 and LV mass  and TNF‐α and E/A ratio . Left atrial volume index is a relatively robust measure of diastolic function as it is less acutely load‐sensitive than various Doppler derived indexes. The correlation between PTX3 and left atrial volume may be related to the fact that this relatively recently described biomarker has a strong association with myocardial fibrosis and, consistent with the endothelial inflammation hypothesis, is strongly expressed by vascular endothelium.Because there may be a mechanistic link between inflammation and diastolic dysfunction in HFpEF, we tested for correlations between the levels of the pro‐inflammatory biomarkers we assayed and echocardiographic‐Doppler measures. We found a highly significant correlation between PTX3 and left atrial volume index (P=0.043).In addition to testing our primary hypothesis, to evaluate whether levels of pro‐inflammatory biomarkers could serve as a prognostic tool to identify AD‐HFpEF patients at increased risk for future clinical events, we related the levels to combined re‐hospitalizations and deaths over the 60 day period after the index admission. The only statistically predictive biomarker we identified was IL‐6, which was just significant  with HFpEF decompensation and subsequently tend to decrease gradually.At present, it is not definitively known whether inflammation is merely associated with HFpEF or is a central factor in its pathophysiology and/or etiology. Our results are supportive of the hypothesis that increases in pro‐inflammatory biomarkers are closely connected with decompensation in HFpEF and, in the case of PTX3, may also be directly linked to diastolic dysfunction. To more definitively test a mechanistic role for a heightened pro‐inflammatory state as a driver of decompensation in HFpEF will require larger numbers of patients and, ideally, an attempt to measure biomarkers prospectively, before decompensation. This might be done by taking measurements at predetermined intervals in stable HFpEF patients and determining if rising levels predict decompensation. If these results were positive, they could provide insights into which patients might benefit from early intervention to reduce the need for hospitalization as well as those who in whom trials of anti‐inflammatory regimens directed at specific pro‐inflammatory signaling pathways might be warranted.This work was supported by grants from the National Heart, Lung, and Blood Institute .None.


Cytokine-based products are gaining importance for cancer immunotherapy. L19-TNF is a clinical-stage antibody-cytokine fusion protein that selectively accumulates to tumors and displays potent anticancer activity in preclinical models. Here, we describe an innovative approach to transiently inhibit off-target toxicity of L19-TNF, while maintaining antitumor activity.GSK’963, a potent small molecule inhibitor of RIPK1, was tested in tumor-bearing mice for its ability to reduce acute toxicity associated with TNF signaling. The biological effects of L19-TNF on tumor cells, lymphocytes and tumor vessels were investigated with the aim to enable the administration of TNF doses, which would otherwise be lethal.Transient inhibition of RIPK1 allowed to increase the maximal tolerated dose of L19-TNF. The protective effect of GSK’963 did not affect the selective localization of the immunocytokine to tumors as evidenced by quantitative biodistribution analysis and allowed to reach high local TNF concentrations around tumor blood vessels, causing diffused vascular shutdown and hemorrhagic necrosis within the neoplastic mass.The selective inhibition of RIPK1 with small molecule inhibitors can be used as a pharmaceutical tool to transiently mask TNF activity and improve the therapeutic window of TNF-based biopharmaceuticals. Similar approaches may be applicable to other pro-inflammatory cytokines. The therapeutic benefit associated with PD-1 blockade in a growing number of cancer indications  has stimRecombinant cytokines may cause substantial toxicity at very low doses (sometimes less than 1 mg per patient), thus preventing escalation to therapeutically active regimens. For example, the maximal tolerated dose (MTD) of TNF for systemic administration procedures was found to be 300 μg/patient . The fusThe targeted delivery of TNF causes a rapid and selective hemorrhagic tumor necrosis in mouse models of cancer –21 and iL19-TNF and related products provide a therapeutic benefit which is mediated by the durable high local concentration, that can be reached within the tumor mass. We reasoned that, if we were able to transiently inhibit TNF activity in blood (following intravenous administration of the product), we should be able to minimize side effects and safely administer higher doses of the product. One way to achieve this goal may be represented by the administration of small molecule inhibitors of key components in the TNF signaling pathway, building up activity at the tumor site, while decreasing systemic toxicity. Receptor-interacting protein kinase 1 (RIPK1) inhibitors may be ideally suited for this purpose, since RIPK1 is a key mediator of TNF-induced inflammation and tissue degeneration , 27.In this article, we report our findings on the combination of L19-hTNF  or L19-mTNF  with GSK’963, a selective RIPK1 inhibitor . CombinaL19-hTNF and L19-mTNF were produced in CHO cells and purified to homogeneity. The proteins, which form stable non-covalent homotrimers thanks to the TNF moiety , were puin vitro for their ability to reduce potency of L19-mTNF. Cytotoxicity assays were performed on the murine fibrosarcoma WEHI-164 cell line in the presence of actinomycin D, an inducer of the cell-cycle arrest at the G1-phase that prevents the overgrowth of the culture. All four tested inhibitors  potently reduced TNF-mediated biocidal activity in a dose-dependent manner  as compared with the other tested inhibitors.Small molecule inhibitors of RIPK1 –30, a ket manner . Inhibitt manner . For furTo characterize the immunological response to intravenously injected L19-mTNF, we measured plasma levels for a panel of relevant cytokines in immunocompetent tumor-free mice. L19-mTNF (given at the recommended dose of 250 μg/Kg) potently induced high levels of pro-inflammatory cytokines  already two hours after the injection, compared with the non-treated control group . Pre-trein vivo administration of the immunocytokine as single agent or in combination with GSK’963. Hoechst 33342 dye was perfused one minute prior to sacrifice, in order to assess variations in the perfusion and functionality of blood vessels. Vascular structures were detected by CD31 staining. Administration of L19-mTNF at the recommended dose of 250 μg/Kg  prevented penetration of the Hoechst dye in both CT-26 and WEHI-164 tumors, indicating the onset of a selective vascular shutdown in neoplastic lesions  24 hours after  lesions . By cont lesions . Apoptot lesions .125I and injected intravenously at the recommended dose of 250 μg/Kg. L19-hTNF preferentially localized at the site of the disease, with a high tumor uptake value  and excellent tumor-to-normal organs ratio, 24 hours post-administration (i.e. average tumor-to-organs ratio 5.5:1 and tumor-to-blood ratio 4:1)  . Pre-treIn order to determine the maximum tolerated dose of L19-TNF in combination with GSK’963, we performed a therapy study in WEHI-164 tumor-bearing mice. This model was chosen based on the tumor sensitivity upon TNF treatment and, moreover, on the fact that the fully-human fusion protein is currently being studied in pivotal trials for soft-tissue sarcoma indication, in combination with doxorubicin (EudraCT 2016-003239-38 and NCT03420014). Animals were dosed systemically with L19-mTNF at 250 and 375 μg/Kg, respectively. Two additional groups of animals were treated with GSK’963 (2 mg/Kg) 30 min before administration of L19-mTNF. Acute toxicity was evaluated by measuring daily changes in body weight. Effects on the body weigh lost after administration of L19-mTNF has been well characterized in previous studies . As predWe have described the use of a potent small molecule inhibitor of RIPK1 named GSK’963 for the improvement of the therapeutic window of an antibody-TNF fusion protein (L19-TNF) in preclinical models of cancer. Combination with GSK’963 enabled dose escalation of the immunocytokine beyond its MTD, while the single-dose administration of an equivalent amount of L19-TNF (375 μg/Kg) was found to be lethal. Inhibition of RIPK1 did not interfere with selective accumulation of L19-TNF into neoplastic lesions, where high local concentrations of TNF caused onset of tumor vascular shutdown and selective hemorrhagic necrosis. In contrast with these findings, pre-administration of ibuprofen at its recommended dose did not have a similar beneficial effect in reducing toxicities related to the administration of targeted-TNF.TNF is a potent cytokine mediator of inflammation which is expressed by macrophages, NK cells, T cells, endothelial cells and fibroblasts , 34. TriThe use of recombinant TNF has been previously proposed for cancer therapy, but significant toxicities and lack of efficacy prevented its clinical success for systemic application , 39. HypWhile therapeutic activity can be dramatically improved by the selective delivery of cytokines to tumors via antibodies –21, toxiWe have successfully demonstrated that the MTD of L19-TNF can be increased by transient and selective inhibition of RIPK1, a key mediator of TNF-induced inflammation and tissue damage. Moreover, our results clearly show that small molecule inhibitors of RIPK1 are more effective in limiting the early toxicity of L19-TNF, as compared to general anti-inflammatory drugs like ibuprofen that acts reducing pro-inflammatory cytokines secretion by inhibition of COX enzymes . Our resGSK’963 (Aobious), GSK’2982772, Necrostatin-1 and Necrostatin-1s  were dissolve in 100% DMSO at 10 mg/ml and kept at -20°C for long-term storage. Ibuprofen sodium (Sigma-Aldrich) was prepared at 5 mg/ml in deionized water and stored at 4°C for no longer than 1 week.3 [2PO4; 150 mM NaCl; pH 7.4) prior biodistribution experiments.L19-human TNF (L19-hTNF) is a fusion protein consisting of the L19 antibody in scFv format, sequentially fused to human TNF by the 17-amino-acid linker EF(SSSSG)3 . The antL19-murine TNF (L19-mTNF) gene was cloned into the mammalian cell expression vector pcDNA3.1(+) (Invitrogen) by HindIII/NotI restriction sites. The L19-mTNF fusion protein was expressed by transient gene expression in CHO-S cells and purified from the cell culture supernatant to homogeneity by protein A  chromatography, as described previously . After dChinese Hamster Ovary cells  were cultured in suspension in PowerCHO-2CD medium (Lonza), supplemented with Ultraglutamine-1  and antibiotic-antimycotic .Murine cells lines F9 , WEHI-164 (fibrosarcoma) and CT-26 (colon carcinoma) were obtained from American Type Culture Collection. Cells were cultured in the corresponding medium supplemented with Fetal Bovine Serum  and AA (1%) following the supplier’s protocol and kept in culture for no longer than 10 passages with a confluence lower than 90%.in vitro on the fibrosarcoma WEHI-164 cell line. Cells were seeded in 96-well plates (20’000 cells/well) in RPMI-164 medium (Gibco). After 24 hours, new medium containing decreasing concentration of L19-hTNF (1:10 dilution steps) with actinomycin D  was added to the cells, in the presence or absence of RIPK1 inhibitors . The cell viability was measured after 24 hours by adding 20 μl of CellTiter 96® AQueous One Solution Reagent (Promega) directly to the wells. The number of living cells was determined by measuring the absorbance after 4 hours at 490 nm using Spark multimode microplate reader (Tecan). Experiments were performed in triplicate and the results were expressed as the percentage of cell viability compared with controls (cells treated with actinomycin D only).The direct killing activity of L19-mTNF was determined All the animal studies were conducted in accordance with Swiss animal welfare laws and regulations .7 cells ml-1. Aliquots of 107 cells (150 μl of suspension) were injected subcutaneously (s.c.) in the right flank of female 129/Sv mice .F9 cells were grown to 80% confluence and detached with Trypsin-EDTA 0.05% (Life Technologies). Cells were washed with Hank’s Balanced Salt Solution  once, counted and re-suspended in HBSS to a final concentration of 6.7 × 107 cells ml-1. Aliquots of 2.5 × 106 cells (150 μl of a suspension) were injected s.c. in the right flank of female BALB/c mice .WEHI-164 and CT26 cells were grown to 80% confluence and detached with Trypsin-EDTA 0.05% (Life Technologies). Cells were washed with Hank’s Balanced Salt Solution  once, counted and re-suspended in HBSS to a final concentration of 1.7 × 102 × D × 0.52, where d and D are the short and long dimensions in millimeters, respectively, measured with a caliper. Animals were sacrificed when termination criteria foreseen by the license were reached.Tumor volume was determined with the following formula: (d)Tumor-free BALB/c mice were injected intravenously (i.v.) with PBS, L19-mTNF (250 μg/Kg) or with GSK’963 (2 mg/Kg) followed by L19-mTNF . After 2 hours, mice were euthanized and immediately exsanguinated. Blood was collected and incubated for 20 min in Microtainer tubes containing lithium heparin (BD Microtainer Tube). Plasma was obtained by centrifugation for 15 min at 3’000 rpm using a refrigerated centrifuge and stored at -80°C prior to cytokine quantification.IL2, IL6, IL10, MCP-1, IFN-γ, TNF and IL1β cytokine plasma levels were quantified using a multiplexed bead-based flow cytometric assay kit  following the described procedure  at the Cin vivo targeting performance of L19-hTNF was evaluated in biodistribution studies as previously described [125I  and Chloramine T hydrate  and purified on a PD10 column . Radiolabeled L19-hTNF (250 μg/Kg) was injected into the lateral tail vein of immunocompetent 129/Sv mice bearing s.c. implanted F9 teratocarcinoma. Alternatively, mice were pretreated with the RIPK1 inhibitor GSK’963 (2 mg/Kg) followed by i.v. injection of L19-hTNF . After 24 hours mice were sacrificed, organs were excised, weighed and the radioactivity of organs and tumors was counted using a Packard Cobra gamma counter. Radioactivity content of representative organs was expressed as the percentage of the injected dose per gram of tissue (%ID/g).The escribed . BrieflyWEHI-164 tumor-bearing BALB/c mice were randomized in groups of four and received three i.v. injections, once every second day. Mice were pretreated with either GSK’963 (2 mg/Kg) or ibuprofen at (5 mg/Kg) 30 min prior to injection of L19-mTNF. Recommended (250 μg/Kg) or high (375 μg/Kg) doses of L19-mTNF were tested. Toxicity induced by murine TNF was monitored daily by checking the body weight of the animals. We considered a severe toxicity to be a more than 10% weight loss within 48 hours after a single intravenous injected dose, and an acceptable toxicity to be a lower than 5% weight loss within 48 hours after a single intravenous injected dose. Tumors were measured with an electronic caliper to assess antitumor efficacy of the different treatments. Daily statistics are described in the ex vivo detection of vascular permeability, WEHI-164 or CT-26 bearing BALB/c mice were injected i.v. with PBS, L19-mTNF (250 μg/Kg) or with GSK’963 (2 mg/Kg) followed by L19-mTNF . After 24 hours, 10 mg/Kg of Hoechst 33342 (Thermo Scientific) was injected into the tail vein, and animals were euthanized after 1 min. Tumor, kidney and liver were collected, embedded in cryoembedding medium (Thermo Scientific) and snap frozen in liquid nitrogen. Cryo-sections of 10 μm were fixed in ice-cold acetone for 10 min and blocked for 1 hour in FBS (20% solution in PBS). The vessels were stained with a rat anti-mouse CD31  and with a donkey anti-rat Alexa Fluor 594 as a secondary antibody . Slides were mounted with fluorescent mounting medium  and analyzed with Leica TIRF epi-fluorescence microscope . Pictures representing a sample overview were obtained by stitching electronically adjacent regions.For the *, p≤0.05; **, p≤0.01; ***, p≤0.001; and ****, p≤0.0001).Data were analyzed using Prism 7.0 . Student t test was used to assess the differences of cell viability and of cytokine levels between different experimental groups. In therapy experiments, statistical significances were determined with a regular 2-way ANOVA test (with Bonferroni post-test;


Heart failure with preserved ejection fraction (HFpEF) represents the most frequent form of heart failure in women, with almost two-fold higher prevalence than in men. Studies have revealed sex-specific HFpEF pathophysiology, and suggested the possibility of a sex-specific therapeutic approach in these patients. Some cardiovascular risk factors, such as arterial hypertension, obesity, diabetes mellitus, coronary artery disease, atrial fibrillation, and race, show specific features that might be responsible for the development of HFpEF in women. These risk factors are related to specific cardiovascular changes—left ventricular diastolic dysfunction and hypertrophy, ventricular–vascular coupling, and impaired functional capacity—that are related to specific cardiac phenotype and HFpEF development. However, there is no agreement regarding outcomes in women with HFpEF. For HFpEF, most studies have found higher hospitalization rates for women than for men. Mortality rates are usually not different. Pharmacological treatment in HFpEF is challenging, along with many unresolved issues and questions raised. Available data on medical therapy in patients with HFpEF show no difference in outcomes between the sexes. Further investigations are necessary to better understand the pathophysiology and mechanisms of HFpEF, as well as to improve and eventually develop sex-specific therapy for HFpEF. Heart failure (HF) represents a large health burden because of its increasing prevalence, significant morbidity and mortality, and constantly increasing expenses of treatment . The intThe data regarding differences in pathophysiology, outcomes, and treatment in women and men with HFpEF are scarce. One of the main problems in the assessment of sex and its impact on HFpEF morbidity and mortality is the retrospective design of most studies.PubMed, Medline, OVID, Cochrane, and EMBASE databases were searched for studies published in the English language from January 1990 through to February 2019, using the following keywords: “heart failure with preserved ejection fraction,” “HFpEF,” “gender,” “sex,” “prevalence,” “risk factors,” “left ventricle,” “diastolic function,” “renin-angiotensin inhibitors,” “angiotensin receptor blockers,” “beta-blockers,” and “aldosterone.”The aim of this systematic review article is to provide a comprehensive overview of currently known disparities in epidemiology, pathophysiology, and recent clinical studies regarding hemodynamic changes, cardiac remodeling, treatment, and outcome.p < 0.001) [The latest report of the American Heart Association included 110,621 HF patients, 50% 55,083) of whom had HF with reduced EF (HFrEF), 14%  HFmEF, and 36%  HFpEF. The report emphasized three important findings: HFpEF was more prevalent in women compared with men, there was no difference in HFpEF incidence between the sexes, and HFpEF was the most common subtype in women with HF [< 0.001) . Duca et3 of whom< 0.001) .A recently published study revealed that the lifetime risk of HFpEF at index ages 45 through 90 was similar in men and women, whereas the lifetime risk of HFrEF was 1.8-fold higher in men compared with women . The lifPathophysiological mechanisms that could explain sex-related differences in HFpEF can be separated into several groups: (i) hormonal differences; (ii) bio-hormonal system activity ; (iii) differences in cardiovascular risk factors and predisposing diseases important for HFpEF development ; and (iv) race, which has been an important predictor of HFpEF development or worse outcome in HFpEF patients in recent studies. The central illustration  illustraThe MESA (Multi-ethnic study of Atherosclerosis) study, after a nine year follow-up, reported that the androgenic profile, characterized by higher free testosterone and lower sex hormone binding globulin, was related to a higher increase in LV mass in both sexes, whereas a greater increase in ratio between LV mass and volume was found only in women . LVEF  . SignifiRAAS is associated with LV hypertrophy as well as with LV diastolic dysfunction ,17, and The sympathetic nervous system (SNS) is also sex-dependent in several ways: (i) the regulatory system for SNS activation is less sensitive to excitation and more sensitive to inhibition in women than in men; (ii) cardiopulmonary induced SNS inhibition is higher in women, which could result in better renal excretory function; (iii) reduced sensitivity to adrenergic nerve stimulation, but not to noradrenaline, indicates that sex-induced SNS variations could protect females against SNS hyperactivation; and (iv) women are less sensitive and/or less responsive to adrenal medullary activation, which helps them in the situations of increased stress ,18,19.Previous studies showed that SNS overactivity was related to impaired LV diastolic function  and LV hBesides well-known bio-hormonal systems, systemic microvascular endothelial inflammation has lately been cited as an important risk factor of HFpEF . InflammObesity, hypertension, diabetes, coronary heart disease, atrial fibrillation, anemia, chronic obstructive pulmonary disease, renal dysfunction, and history of chemo- and radiotherapy are important risk factors of HFpEF development in both sexes, but it seems that some of these factors are more significant among females . A recenHypertension represents one of the major factors responsible for HFpEF development . Pandey The importance of arterial hypertension in elderly women underlines the significance of isolated systemic hypertension, typical of this population of patients. A higher augmentation index between peripheral and central blood pressure in elderly women, compared with men, could explain more pronounced target organ damage, including LV hypertrophy . Women h2, was more prevalent in men than in women with HFpEF [Our study group has recently reviewed mechanisms that connect obesity and HFpEF, and we have emphasized the importance of sex in HFpEF development . A largeth HFpEF . Howeverth HFpEF .In a large population of postmenopausal women whose obesity was followed for 13 years, there was an independent predictor of HFpEF, but not HFrEF . A recenMcHugh et al. have recently explained the complex relationship between diabetes and HFpEF . The autSavji et al. reported that fasting glucose was an independent predictor of HFpEF occurrence only in women, but not in men . InteresCoronary artery disease is a dominant risk factor of the development of HFpEF in men. However, its influence in women should not be neglected. This was confirmed in large studies which showed that coronary artery disease, prior percutaneous coronary intervention, or coronary artery bypass grafting were significantly more frequent in men than in women hospitalized with symptoms of HFpEF . Nakada One should not forget the importance of coronary microvascular dysfunction and ischemia on HFpEF . The ARIIn atrial fibrillation, women seem to have a larger LA volume index and worse LA pump function than men . FurtherThe influence of race on the development and outcome of HFpEF in the sexes has not been investigated in detail so far, but initial observations point to a higher risk of HFpEF in white women. Eaton et al. conducted a comprehensive analysis of influence of race on HFpEF and HFrEF development, as well as the role of various risk factors in each race . The anaA large cohort study which followed 12,417 participants for almost 12 years showed that the lifetime risk of overall HF was higher in non-blacks than blacks . The lifGoyal et al. found that chronic renal and hepatic failure was more prevalent among men with HFpEF . Harada Anemia was described as an additional risk factor of HFpEF development in women, but not in men . Eaton eChronic pulmonary obstructive disease represents a risk factor of HFpEF in both sexes. Goyal et al. showed that it is more prevalent in men with HFpEF older than 75 years of age, and in women with HFpEF younger than 75 years of age . Eaton eIn a population of older patients who underwent radiation therapy due to breast cancer, cardiac radiation exposure increased the risk of HFpEF development, and the higher mean cardiac radiation dose was associated with a greater risk of HFpEF . The radIn summary, the pathophysiological studies available so far consistently show that obesity, hypertension, and diabetes have sex-specific effects in HFpEF, whereas for other risk factors, there is some evidence, but it is not so clear.In the last few years, important studies regarding hemodynamic changes and LV remodeling in HFpEF have been published ,37,46,47A recently published study found that women with HFpEF had worse diastolic reserve, estimated by: (i) elevated LV filling pressures measured by echocardiographic and invasive measurements at exercise; (ii) lower systemic and pulmonary arterial compliance; and (iii) worse peripheral oxygen kinetics . This elThis study agrees with our findings that showed usefulness of diastolic stress echocardiography in discovering HFpEF patients in the individuals with dyspnea on exertion in simple clinical settings , withoutDuca et al. did not find any difference in invasive hemodynamic parameters between women and men with HFpEF, except borderline higher wedge pressure in women and significantly increased diastolic pressure gradient in men .The PARAMOUNT trial (Prospective comparison of ARNi with ARB on management of heart failure with preserved ejection fraction) was the first investigation that provided a detailed sex-specific analysis of LV structure, function, and mechanics in HFpEF . As expeHarada et al. reported similar findings regarding smaller LV and better LVEF in women with HFpEF, but E/e’ ratio was similar between the sexes and the left atrium was larger in men . Concentp < 0.001) [Goyal et al. demonstrated that women with HFpEF had lower in-hospital mortality compared with men  .p = 0.008) and lower rates of non-cardiac death  in comparison with women [p = 0.96) [Nakada et al. revealed that the incidence of cardiovascular death and admission due to HF tend was lower in female than in male HFpEF patients, but the difference was not statistically significant . A smallth women . Hsich e = 0.96) .There is no current consensus on the optimal therapy for HFpEF, and it is unknown if it should be treated in the same way as HFrEF or as a completely different entity. The data on this topic are scarce, and there is even less knowledge of the benefit of sex-specific therapy in HFpEF. Our study group recently summarized the sex-specific therapeutic approach to arterial hypertension . ConsideThe TOPCAT trial showed that spironolactone had a similar effect on the primary outcome in HFpEF in both sexes . HoweverSeveral studies did not report differences in the prevalence of different medications between women and men with HFpEF ,8, even A large meta-analysis that included 28,636 HFpEF patients (35–70% women) showed a significant benefit from the use of beta-blockers on all-cause mortality in observational studies, with a reduction in mortality by 21% in HFpEF patients, irrespective of sex . HoweverKhan et al. used the data of randomized trials involving 17,284 HFpEF patients to investigate the effect of ACE inhibitors and angiotensin receptor blockers on outcome . In thisSpironolactone has been shown to improve functional capacity and LV diastolic function in a study of HFpEF patients . HoweverHFpEF represents the most prevalent form of HF in women, which is associated with adverse clinical outcomes. HFpEF has unique pathophysiology in women related to certain comorbidities and specific cardiovascular remodeling. Some risk factors such as arterial hypertension, obesity, diabetes mellitus, coronary artery disease, atrial fibrillation, and race have specific features that might be responsible for a particular phenotypic profile in women with HFpEF. These risk factors are associated with specific cardiovascular changes—left ventricular diastolic dysfunction and hypertrophy, ventricular–vascular coupling, and impaired functional capacity—that are related to particular cardiac phenotype and HFpEF development. Sex hormones are still considered to have the leading role in sex-specific cardiovascular remodeling, including HFpEF. There is no agreement regarding outcome in women with HFpEF. Sex-specific HFpEF treatment is far from a reality at this moment, but it represents a very important future direction toward personalized medicine. A majority of the studies that have investigated the outcome or treatment in HFpEF patients did not aim to research the differences between the sexes. Therefore, longitudinal studies with sex-specific outcomes as the primary aim should be conducted.


Resting-state functional magnetic resonance imaging (rsfMRI) is being widely used for charting brain connectivity and dynamics in healthy and diseased brains. However, the resting state paradigm allows an unconstrained fluctuation of brain arousal, which may have profound effects on resting-state fMRI signals and associated connectivity/dynamic metrics. Here, we review current understandings of the relationship between resting-state fMRI and brain arousal, in particular the effect of a recently discovered event of arousal modulation on resting-state fMRI. We further discuss potential implications of arousal-related fMRI modulation with a focus on its potential role in mediating spurious correlations between resting-state connectivity/dynamics with physiology and behavior. Multiple hypotheses are formulated based on existing evidence and remain to be tested by future studies. The advent of resting-state functional magnetic resonance imaging (fMRI)  has revoThe approaches for quantifying rsfMRI dynamics can be divided into multiple categories. The most straightforward class is sliding window approaches, which quantify rsfMRI connectivity within short time windows of 1–2 min and then examine its temporal variability accordingly. The connectivity metrics could be either conventional correlation/coherence  or more These dynamic approaches have been applied to rsfMRI data to quantify brain dynamics and investigate its associations with behavior  and moduThe global rsfMRI signal averaged over the entire brain and spatially non-specific fMRI correlations it induces have been found highly sensitive to brain arousal state . The traThe first clue for the neural basis of the global rsfMRI signal came from the study of rsfMRI dynamics. It has been suggested that the rsfMRI connectivity and its dynamics are actually caused by brain co-activations at different time points that can be captured by CAPs . ApplyinAn electrophysiological event signifying a transient arousal modulation was recently discovered and suggested to underlie the global rsfMRI signal , 2018. TArousal-related fMRI changes can have potential implications in many aspects of rsfMRI research. The sensory-dominant pattern of SST-induced fMRI changes is expected to introduce very systematic changes in rsfMRI correlations, which could be easily misinterpreted as meaningful modulations of functional brain connectivity. The transient nature of the SST (10–20 s) will also have profound effects on rsfMRI dynamics at the time scale of interest to most of dynamic rsfMRI studies. More importantly, the perils of the arousal-related fMRI component can go beyond its direct effects on rsfMRI connectivity/dynamics by potentially introducing their spurious correlations with other subject measures of physiology, behavior, and pathology. Arousal state is known to have profound effects on physiology  and alsoThe correlation has been found between the rsfMRI connectivity and head motions in both intra- and inter-subject analyses . SpecifiPhysiological signals, including respiratory volume and cardiac pulse rate, were also shown to have strong correlations with rsfMRI signals , which hResting-state fMRI connectivity and dynamics have also been extensively studied under pathological conditions, and significant modulations were reported in a wide range of neurological disorder and psychiatric diseases, including the Alzheimer’s disease, ASD, and major depression . Given tp = 0.036 for CAL and p = 0.022 for PBG, 2-sample t-test; p = 0.735; The global signal could affect rsfMRI findings in a rather implicit way. Using rsfMRI data from the Autism Brain Imaging Data Exchange (ABIDE) initiative, a previous study has shown that interhemispheric rsfMRI connectivity shows a much larger inter-subject variability in high-functioning ASD adults than matched healthy controls, and this finding has been interpreted as idiosyncratic distortions of ASD brains . The intArousal might also mediate the correlation between rsfMRI connectivity/dynamics and certain behavioral measures within the healthy population in a similar way. Even though the arousal itself describes a brain state that varies over time, the ability of regulating arousal could be an individual trait that is stable within but varies across individuals. If the healthy population contains subgroups that not only have distinct ability of regulating arousal but also differ significantly in certain cognitive functions, rsfMRI connectivity and dynamics could be spuriously linked to behavioral measures of cognitive functions via arousal-related rsfMRI changes. Although this is purely a conjecture to be tested by future studies, caution needs to be exercised before completely excluding this possibility.Here we reviewed the current understanding of the relationship between the brain arousal and resting-state fMRI, in particular a newly discovered neurophysiological event underlying the global rsfMRI signal. We then discussed potential implications of the arousal-related modulation on rsfMRI research, i.e., its role in affecting rsfMRI connectivity/dynamics and mediating their spurious correlations with physiological and behavioral measures. We also formulated multiple testable hypotheses based on existing evidence. Future research ought to validate these hypotheses, which are important not only for proper interpretations of rsfMRI results but also for better quantifications of brain connectivity and dynamics using rsfMRI with properly dealing with the arousal confounding effects, i.e., removing or retaining the arousal-related component based on research purposes. Before the validation of these hypothesis, one should be cautious about large global signal and sensory-dominant pattern in rsfMRI results, which are indicative of arousal involvement. Researchers may also consider to reduce the potential arousal influence at the stage of data collection, for example, by acquiring data at the eye-open state or breaking down a long scan into multiple shorter ones. The profound arousal effect on rsfMRI presents additional challenges to rsfMRI-based measures of brain connectivity/dynamics. But on the bright side, this would enable fMRI-based arousal measures , which mYG and XL designed the study and performed the analyses. YG, XL, and FH wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Using both cell-surface expressed α4β7 and a soluble α4β7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM . The gp120 V2 domain preferentially engages extended forms of α4β7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4β7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4β7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4β7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4β7. It includes the canonical LDV/I α4β7 binding site, a cryptic epitope that lies 7–9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4β7 interactions. These mAbs recognize conformations absent from the β- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4β7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α 4β7 is a receptor that facilitates homing of lymphocytes to GALT. α4β7high CD4+ T cells are early targets of HIV infection. The HIV envelope protein gp120 binds to α4β7; however, a link between this interaction and the preferential infection of α4β7high CD4+ T cells has not been established. Here we report an apparent gp120 mimicry of MAdCAM, a natural ligand of α4β7. That the HIV envelope protein mimics an adhesion receptor expressed primarily in gut tissues supports the concept of a central role for GALT in HIV pathogenesis. Antibodies derived from vaccination and infection were evaluated for their capacity to inhibit gp120-α4β7 interactions. Antibodies that inhibit binding target a region of the V2 domain that has been linked with reduced risk of acquisition in the RV144 vaccine trial. These antibodies recognize epitopes that do not appear on the closed gp120/41 trimer. Instead, they recognize structures that form when V2 is allowed to fold in a less constrained way. We show that such epitopes can appear on virions. Thus, an alternative structure of the V2 domain may facilitate the trafficking of HIV to GALT.HIV is gut-tropic. Disruption of GALT plays an important role in HIV -mediated immune dysfunction. α Th. Th4β7 oL) in β7  [23, 49–d MAdCAM . We then by >90% . Becauseadhesion . We conc4β7 activity is regulated by its conformation. Intracellular signaling events modulate the ectodomain to reversibly transition between bent and extended conformations . Adhesion of both MAdCAM and cV2 92TH023 was strongest in the presence of MnCl2 and reduced in the presence of MgCl2 by ~5-fold and 2.5-fold respectively   was able to mediate α4β7 adhesion  Figs  and S4. ans Figs  and S5. ion Figs  and S6. ion Figs . Given t4β7 -reactivity, we inserted the V1/V2 sequences of 92TH023 into two scaffolds. The first scaffold, termed 1FD6, has been previously shown to constrain V1/V2 in a way that increases its propensity to form a β- barrel [4β7 adhesion  coated with either α4β7 or V2 mAbs CH58, PG9, and 830A to capture 92TH023 virions derived from primary CD4+ T cells , which closely aligns with the peptide employed by Liao and colleagues, inhibited α4β7 -binding by >90%  and H43 each partially inhibited mAb CH58 binding and strongly inhibited (>90%) CAP228-16H binding. Taken together these results suggest that inhibition by peptide H43 involves direct binding to α4β7. To rule out allosteric inhibition , we carried out a similar peptide inhibition assay with immobilized MAdCAM and determined that H43 inhibited α4β7 binding to MAdCAM by >90% . ITS09 and ITS41 also inhibited adhesion, but to a lesser extent, while NCI05, which maps to a region overlapping the ITS09 epitope failed to inhibit adhesion in a detectable way. Of note, the epitopes for both ITS03 and NCI09 do not include the canonical Leu-Asp binding site (Ala192-Asp193 in SIV) that is conserved in HIV V2 and MAdCAM  inhibits SIV gp120 binding to α4β7, and SIV mAbs whose epitopes overlap this peptide also inhibit α4β7 adhesion.We next asked whether SIV V2 mAbs could inhibit αmAbs see  above. W66 gp120 . Insteadd MAdCAM . Both of4β7 -reactivity between HIV and SIV, despite the divergent V2 sequences represented by peptides H43 and S46 (< 50% sequence similarity) is that the secondary structures of these two peptides share common features [4β7 binding most effectively, reacted with relatively high-affinity to HIV cV2 92TH023 (KD (nM)) 0.105)  0.105) . This cr4β7 -mAb (Rh-anti-α4β7) were able to durably control viremia at relatively low levels following treatment interruption [4β7 adhesion to gp120. Serum IgG was purified from three ART + anti- α4β7 mAb -treated animals, RLn12, RDa15, and RId14, by protein G affinity chromatography, and along with normal rhesus macaque IgG was evaluated in an α4β7 adhesion assay. While normal rhesus IgG showed minimal inhibition of α4β7-adhesion to SIVmac766 gp120, the sera from all three ART + α4β7 treated macaques inhibited adhesion in a dose dependent manner  (4β7 adhesion (4β7 included V2 antibodies that target the α4β7 binding epitope of SIV V2.SIV infected macaques treated with a combination of ART and a recombinant rhesus anti αrruption . Althougrruption . Becauset manner . We thent manner  that inhduction) . ITS03, adhesion , failed adhesion . We conc4β7 interacts with the V2 region of gp120 shares key features with the interaction between α4β7 and its natural ligand, MAdCAM. This apparent mimicry may have important implications in HIV pathogenesis, particularly in regard to the role of the gut in the development of HIV disease. It may also impact anti-V2 loop immune responses in both infected and vaccinated subjects. One consequence of this mimicry is that antagonists developed to treat IBD interfere with V2-α4β7 interactions. The V2 domains of HIV and SIV gp120 vary in both length and sequence identity. Yet we find that V2s from three subtypes of HIV, as well as a V2 from SIV retain the capacity to bind to α4β7, suggesting that this interaction is a general property across HIV subtypes. Other studies failed to detect a specific interaction between α4β7 and gp120 [4β7 [4β7 with high affinity in both SPR -based assays and in a cell -based adhesion assay. Removal of glycans in order to observe α4β7 reactivity in not an absolute requirement insofar as we were able to capture virions derived from primary CD4+ T cells with α4β7 coated nanoparticles. The second variable that may influence the sensitivity of α4β7 binding assays involves the expression level, and state of α4β7 on cell surfaces. Many integrins rely on complex avidity effects and clustering in order to engage ligands. It is likely that the surface density of α4β7 plays a key role in its interaction with gp120.In this study we report that the way in which αnd gp120 , 14. An 120 [4β7 , 58. It 4β7 for gp120 is comparable to that of MAdCAM. Among the 24 integrins expressed in humans, the α4β7 heterodimer is distinct in its ability to mediate both lymphocyte rolling, and firm adhesion, which reflects the highly specialized nature of MAdCAM-α4β7 interactions. These two functions are achieved by dynamic changes in the overall structure of the heterodimer. In this regard, it is notable that the V2 region of gp120, despite its variable sequence, is able to mimic the binding of α4β7 to MAdCAM. The evidence for mimicry comes from two observations. First, the manner in which V2 depends upon divalent cations to engage α4β7 tracks closely with the way cations are used by MAdCAM. To bind α4β7, MAdCAM utilizes divalent cations. A Mg++ ion sits in the MIDAS of β7 and coordinates with an Asp in the ligand (Asp42 in MAdCAM). Mn++ occupies the MIDAS in a more stable way, so that replacing Mg++ with Mn++ results in an apparent increase in affinity. This pattern holds for gp120 V2, indicating that V2 appears to interact with the different conformations of α4β7 in the same way that MAdCAM does. Importantly, the conformational state  of α4β7 is responsive to both intracellular and external cues that are linked to cellular signals generated during inflammatory responses [4β7 provides a mechanism to distinguish between different subsets of lymphocytes, including those with high potential to home to GALT. Given the propensity of HIV to replicate in GALT, it is tempting to link the preferential infection and depletion of α4β7high CD4+ T cells in the very early stages of infection [4β7 interactions. However, such a link has not yet been established.The specific affinity of αesponses , 64. Thenfection , 32, 65,4β7 antagonists that were developed to block binding to MAdCAM, also block binding to V2. The incidence and prevalence of IBD is increasing throughout the world [4β7. To this end, detailed structural characterizations of both MAdCAM and α4β7 have been employed in the rational design of small molecule LDV mimetics [4β7, which lies within a ~ 40 Å long, 10 Å deep groove formed by the α4-β7 interface  and vaccination (CH58) could inhibit V2 -mediated adhesion to αtibodies –75. Substibodies . These reduction , 78. Our vaccine , also bltibodies . A more 4β7 might contribute to reduced risk of infection is unknown. We previously reported that the V2 region of gp120 can deliver cellular signals through α4β7 [4β7 are key components in integrin associated complexes that are able to modulate biochemical pathways and reorganize both cell-surface receptors and the actin-cytoskeleton [4β7 can deliver costimulatory signals to CD4+ T cells that impact cell activation, proliferation and apoptosis [4β7high CD4+ T cells [4β7 can similarly facilitate HIV replication requires additional investigation. Such information will help determine whether this type of signaling could facilitate HIV transmission/replication and whether antibodies that interfere with this signal can reduce the risk of infection.How weakly neutralizing antibodies that block V2 -mediated adhesion to αugh α4β7 . In thisskeleton , 80. In poptosis , 82. We  T cells . Whether4β7 should help us identify the context in which these interactions occur in vivo. It is noteworthy that these weakly neutralizing mAbs recognize an epitope that is structurally distinct from the β- strand that is presented on the closed BG505 SOSIP trimer. As such, broadly neutralizing mAbs that do recognize epitopes on the closed BG505 SOSIP trimer are unlikely to be effective inhibitors of V2-α4β7 interactions. Instead, we show that a unique α4β7 -reactive conformation of V2 is formed when it is relieved from constraints mediated by other domains of gp120/41.The nature of the epitopes recognized by mAbs that inhibit the interaction between V2 and α4β7 remains to be determined, evidence is accumulating that α4β7 -expressing cells play an important role in the early stages of HIV/SIV infection. The recent demonstration by Sivro and colleagues that α4β7high CD4+ T cells are preferentially depleted from gut tissues as early as Fiebig I/II provides strong evidence that these cells serve as prime early targets for infection following transmission, an observation that is consistent with our demonstration that an α4β7 mAb protects macaques from vaginal challenge [4β7 plays a broader role in HIV pathogenesis. When we combined this same α4β7 mAb with ART, SIV infected animals were able to control viremia in a sustained way [4β7. This finding underscores the need to further explore the role of V2 α4β7 interactions in HIV pathogenesis.Although the context in which V2 engages αhallenge . Howeverined way . In tryi4β7 shares key features with the way that MAdCAM, a receptor expressed primarily in gut tissues, engages α4β7. One consequence of this apparent mimicry is that antagonists developed to treat IBD by interfering with the interaction between MAdCAM and α4β7 also interfere with V2-α4β7 interactions. The nature of the epitope in V2 that engages α4β7 appears to involve a structure that is not present in a recombinant SOSIP trimer designed to mimic the closed trimeric spike that is the target of most broadly neutralizing antibodies. Antibodies that target this epitope and block V2-α4β7 interactions are not themselves broadly neutralizing, although the structure that they recognize is conserved across clades and is present in SIV V2. These findings suggest that mimicry of MAdCAM-α4β7 interactions by V2 may influence early events in HIV infection and replication in GALT.In conclusion, the way that the V2 region of gp120 engages αGeneration of mAb Mk16C2 was approved and carried out under animal use protocol A-1896 by the Institutional Animal Care Committee (IACUC) of the University of Massachusetts Medical School. The University of Massachusetts Medical School is fully accredited by Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and has an Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (OLAW). Assurance number: A-3306-01.4β7 was purchased from Sigma-Aldrich. Cells were maintained in RPMI-1640 (Lonza) containing 10% heat inactivated fetal bovine serum (Gibco), 2% penicillin/streptomycin/glutamine (Gemini Bio-Products) and 0.1% retinoic acid (RA). Cells were cultured for a minimum of 7 days prior to use in adhesion assays. RA was obtained from Sigma Chemical. CH58, human mAb isolated from RV144 vaccinated individuals, and VRC01 were provided by the NIAID AIDS reagent program. CAP228-16H mAb was generated in the laboratory of Dr. Lynn Morris (CAPRISA) [4β7 mAb. Serum was purified by protein G column chromatography and dialyzed into HBS. Cyclic V2 peptides with > 90% purity and having an amino-terminal biotin, derived from 92TH023, BG505 and C06980v0c22 were supplied by JPT Peptide Technologies. Linear HIV and SIV 15 amino acid peptides were obtained from the NIAID AIDS Reagent Repository, or from Biopeptide Co. and supplied at >90% purity. Scaffolds V1/V2 92TH023-1FD6 and V1/V2 92TH023-Tag were constructed, expressed and purified as described elsewhere [4 mAb 2B4, MAdCAM-Ig, soluble α4β7 and α4β1 were obtained from R&D Systems. The LDV mimetic ELN-475772 was provided by ELAN Pharmaceuticals [RPMI8866 cells, a human B lymphoma cell line that constitutively expresses αCAPRISA) . The 830CAPRISA) . Rabbit CAPRISA) . NCI09 wCAPRISA) . Rhesus CAPRISA) , was obtlsewhere . CHO celeuticals . Conjuga4β7 adhesion assays were first treated with a deglycosylation protocol [Prior to use purified gp120 and gp140 proteins utilized in the αprotocol . PurifieN-hydroxysuccinimide and 0.2 M N-ethyl-N-(dimethylaminopropyl) carbodiimide at 5 μl/min. NeutrAvidin, HIV gp120 or Hu-MAdCAM-Ig (R&D Systems) at concentrations of 5 μg/ml in 10mM NaOAc, pH 4.5, were immobilized to approximately 750 resonance units (RU). After the proteins were immobilized to the desired densities, unreacted sites on each surface were blocked with 35 μl of 1 M Tris-HCl (pH 8.0). Biotinylated cyclic V2 peptides  were bound to the NeutrAvidin surfaces to densities of approximately 250–300 RU. One surface was activated and blocked without ligand to act as a control surface for non-specific binding of the soluble ligand. Any binding was subsequently subtracted from the remaining surfaces. Running buffer was HBS (pH 7.4), 0.01 mM CaCl2, either 1 mM MgCl2 or MnCl2, 0.005% Tween-20, 0.05% soluble carboxymethyl-dextran. Binding experiments were carried out at a flow rate of 25 μl/min at 25°C. After a 2 min injection, the surface was washed for an additional 2 min in running buffer to follow dissociation of the bound ligand from the surface. The surfaces were regenerated by multiple injections of 4.5 M MgCl2 at a flow rate of 100 μl/min. Inhibition of α4β7 or anti-V2 loop antibodies by linear V2-loop peptides was carried out by pre-incubating the proteins with the peptides in running buffer at the indicated concentrations for 2 hours at room temperature prior to passing them over the prepared surfaces as described above.Experiments were performed using a Biacore 3000 instrument (GE Life Sciences) using CM4 or CM5 sensor chips. The data were evaluated using BIAevaluation 4.1 software (GE Life Sciences). The chip surface was activated by injecting 35 μl of a 1:1 mixture of 0.05 M Antibodies were diluted to the indicated concentrations in running buffer prior to being sequentially passed over the surface bound cyclic peptides as described above. The resulting sensorgram series were analyzed using the BiaEvaluation 4.1 software (GE Life Sciences) and fitted using a 1:1 Langmuir binding model to determine the kinetic rate and affinity constants.The Fab fragment of rabbit mAb Mk16C2  was prepared by papain digestion as described (PMID: 19913488 and 20622876). Briefly, the IgG molecule was mixed with papain  at a 20:1 molar ratio in 100 mM Tris (pH 6.8) with 1 mM cysteine hydrochloride and 4 mM EDTA. The mixture was incubated for 1 hour at 37°C and the reaction was stopped by 10 mM iodoacetamide. The Fab fragment was separated from the Fc fragment and the undigested IgG by a protein A column and further purified by size exclusion chromatography. The Fab fragment was then concentrated to about 10 mg/ml for crystallization.ConB peptide (RDKVQKEYALFYKLD) was dissolved in water and mixed with Fab of rabbit mAb Mk16C2 in excess at a 10:1 molar ratio. Crystallizations conditions were screened and optimized using the vapor diffusion hanging drop method. Well-diffracted crystals of Mk16C2 Fab/V2ConB complex were obtained with a well solution of 23% polyethylene glycol 3350, 0.2 M LiCL, 0.1 M 2-ethanesulfonic acid (MES) pH 6.5, and soaked briefly in the crystallization solution with an additional 20% glycerol before being flash frozen in liquid nitrogen. X-ray diffraction data sets were collected at the synchrotron beamline 14–1 of Stanford Synchrotron Radiation Lightsource (SSRL) of Stanford Linear Accelerator Center (SLAC) National Accelerator Laboratory. All data sets were processed using the XDS (PMID: 20124692), and structures determined by molecular replacement using another rabbit mAb R56 Fab structure (PDB ID 4JO1) as the initial model. Cycles of refinement for each model were carried out in COOT (PMID: 15572765) and Phenix (PMID: 20124702). Final structural analyses were carried out using ICM and figures were generated using PyMOL (pymol.org) and ICM (www.molsoft.com). The antigen-antibody interactions described in The 15mer V24β7 expressed by the RPMI8866 cell line to MAdCAM, HIV A244 gp120, HIV cyclic V2 peptides, and SIVmac766 gp120 gp120 in the absence or presence of vedolizumab or 2B4 or ELN-475772 to MAdCAM, HIV A244 gp120 were analyzed by an adhesion assay  or 2B4 (α4 mAb) or ELN-475772 . Plates were then incubated with 50 μl/well of 2x105 RPMI8866 cells at 37°C (5% CO2) for 1 hour, washed 5 times with PBS followed by the addition of 100 μl of RPMI-1640 containing 1% FBS, 1% pen/strep/glutamine, 25mM HEPES with 10 μl/well of AlamarBlue dye. Fluorescence (excitation 560 nm and emission 590 nm) was measured immediately after the addition of the AlamarBlue dye for 8 hours.The binding of α et al.,   in 60μl were incubated with viral preparation (33 ng/ml based on p24 content) for 1hour at 37°C. Captured virions were separated on MACS magnetic columns attached to an OctoMacs magnet (Miltenyi Biotech) washed 4 times with 600 μl wash buffer , eluted in 200 μl PBS and analyzed on Luminex X200 for p24 content using a dynamic immunofluorescent cytometric bead assay [92TH023 virions were captured with 15 nm magnetic nanoparticles (MNPs) coupled to αescribed . Briefly4β7-MNPs, virions were incubated in the absence or presence of increasing amounts  of the α4β7 inhibitor ELN-475772. Incubation and washing were performed in complete medium with 1mM MnCl2.In experiments with α5 cells at 37°C (5% CO2) for 1 hour, washed 5 times with PBS followed by the addition of 100 μl of RPMI-1640 containing 1% FBS, 1% pen/strep/glutamine, 25mM HEPES with 10 μl/well of AlamarBlue dye (Invitrogen). Following the addition of AlamarBlue dye (excitation 560 nm and emission 590 nm) fluorescence was measured for a period of 8 hours.Triplicate wells of a 96-well flat-bottom polypropylene plate (Greiner Bio-One) were coated with biotinylated cyclic V2 peptides, deglycosylated SIV and HIV gp120 as described above. After blocking and washing, plates were incubated with 20–100 μg/ml of the designated anti-V2 mAbs or 2.5–10.0 μg of protein G purified IgG from sera drawn from SIV infected rhesus macaques ) in sample buffer  for 1 hour at 37°C. RPMI8866 cells were pre-incubated for 40 min at 37°C with sample buffer. Plates were then incubated with 50 μl/well of 2x1092TH023 V1/V2 was cloned into both 1FD6 (constrained) and tag (unconstrained) scaffolds. Plates were then coated with 0.5, 1.0, and 2.0 μg of either V1/V2 92TH023 1FD6 or V1/V2 92TH023 tag scaffolds followed by addition of RPMI8866 cells. The plates were washed and 100 μl of media and 10 μl of AlamarBlue dye was added to each well as described above. Fluorescence (excitation 560 nm and emission 590 nm) was measured immediately after the addition of the AlamarBlue dye.2, and 1X plate wash buffer) using Microplate Washer ELx50, (Bio Tek Instruments), and then blocked with blocking buffer  for 1 hour at room temperature. Plates were coated with 1 μg/ml biotinylated cyclic V2 peptides in binding buffer  for 1 hour at room temperature. The plates were washed six times with wash buffer and HRP-conjugated anti-V2 mAb was added to wells for 1 hour at room temperature. After washing, 100 μl/well of substrate was added for 10 min to develop color at room temperature in the dark. Plates were read at OD450 nm using an EnSpire Multimode Plate Reader (PerkinElmer).Briefly, Corning Costar 96-Well plates were coated with NeutrAvidin at 4°C overnight. Wells were washed six times with wash buffer  were incubated with 50 ng of HRP-conjugated 830A mAb and then added to a plate coated with 100 ng of cV2 92TH023. Substrate was added using conditions specified by the manufacturer. Plates were read at OD450 nm using an EnSpire Multimode Plate Reader, PerkinElmer.10, 20, 50, 80 and 160 ng of the CH58 mAbs in binding buffer  with cover glasses and incubated overnight at 37°C. The cells were fixed with 2% paraformaldehyde (PFA), blocked with 1% BSA, and stained with an anti-β7 PE mAb (BD Biosciences) or an IgG2a-PE isotype control mAb (R & D Systems). Stained RPMI8866 cells were microscopically analyzed using a Leica SP8 confocal microscope  and images were processed with Leica LAS AF software  and Imaris software v.9.0.1 64x (Bitplane AG).RPMI8866 cell line cultured in the presence or absence of RA were seeded at 1×10S1 FigA) List of the HIV and SIV V2 domain sequences used in this study, along with GenBank accession numbers, and amino acid numbering derived from the corresponding full-length gp160. B) ELISA binding of HIV V2 specific mAbs CH58, 830A, CAP228-16H and Mk16C2 to wells coated with NeutrAvidin and a cyclic V2 peptide derived from 92TH023 gp120. PBS was used as negative control. Binding measured at OD450nm (y-axis). C) ELISA binding of unlabeled CH58 competing with HRP-830A binding to cV2 92TH023. Concentration of CH58 listed on the x-axis. Binding measured at OD450nm (y-axis).(TIF)Click here for additional data file.S2 FigA) Schematic of adhesion assay. α4β7 -expressing RPMI8866 cells (yellow) were incubated in the absence or presence of anti-α4β7 or -β7 mAbs  (dark blue) and then added to plates previously coated with the cV2 peptide (purple). Alternatively, cV2 peptides are pre-incubated with anti-V2 specific mAbs (green). Plates were washed and AlamarBlue dye was added to each well. Fluorescence was measured for 8 hours at 1-hour intervals (OD590nm). B) Expression and distribution of β7 on RPMI8866 cells +/- RA, stained with an anti-β7 PE mAb or an IgG2a-PE isotype control mAb viewed by confocal microscopy. Upper panels: differential interference contrast (DIC), lower panels: fluorescence (red). C) Adhesion of RPMI8866 cells, cultured in the presence (+RA) or absence (-RA) of retinoic acid, to MAdCAM-Ig, or cyclic V2 peptides derived from HIV 92TH023, C06980v0c22, and BG505. Adhesion was determined by OD590nm and listed as fluorescence units (y-axis). Background fluorescence (BF) of RPMI8866 cell adhesion to a blank well is denoted by a dashed line.(TIF)Click here for additional data file.S3 FigA-B) Adhesion of RPMI8866 cells to immobilized MAdCAM or a cV2 92TH023 peptide in the buffers containing a low concentration of divalent cations, or high concentrations of MnCl2 or MgCl2 as reported in 590nm and listed as fluorescence units (y-axis). Conditions are run in triplicate and error bars indicate standard error of the mean (SEM). Background fluorescence (BF) of RPMI8866 cells to blank wells is denoted by a dashed line.(TIF)Click here for additional data file.S4 Fig4β7. Average adhesion in three or more independent experiments is reported as fold-change in adhesion relative to undeglycosylated A244 gp120 (y-axis). Error bars indicate SD. Significance determined by unpaired t-test (*p <0.05).Adhesion of RPMI8866 cells to immobilized deglycosylated A244 gp120 in presence of HIV V2-specific mAbs: CH58, CAP228-16H, and Mk16C2. The LDV mimetic ELN-475772 was included as a specificity control (spec. ctrl) for adhesion to α(TIF)Click here for additional data file.S5 Figp <0.05).Adhesion of RPMI8866 cells to immobilized DG forms of BG505 SOSIP trimer, A244 gp120, and SIVmac766 gp120 relative to corresponding fully glycosylated forms of each protein expressed as fold-change (y-axis). Results from three independent experiments are shown. Error bars indicate SD. Significance determined by unpaired t-test (*(TIF)Click here for additional data file.S6 Fig4 mAb 2B4 which was employed as a specificity control (spec. ctrl) for cV2 BG505, and VRC01 was employed as a nonspecific mAb control for cV2 BG505. Error bars indicate SD. Significance determined by unpaired t-test (***p <0.001 and ****p <0.0001).Adhesion of RPMI8866 cells to BG505 SOSIP trimer or cV2 BG505. Results from three or more independent experiments are shown and reported as % adhesion relative to cV2 BG505 in the absence of any inhibitor or in the presence of a specific inhibitor. The anti-α(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.


RNA-splicing endonuclease (EndA) cleaves out introns from archaeal and eukaryotic precursor (pre)-tRNA and is essential for tRNA maturation. In archaeal EndA, the molecular mechanisms underlying complex assembly, substrate recognition, and catalysis have been well understood. Recently, certain studies have reported novel findings including the identification of new subunit types in archaeal EndA structures, providing insights into the mechanism underlying broad substrate specificity. Further, metagenomics analyses have enabled the acquisition of numerous DNA sequences of EndAs and intron-containing pre-tRNAs from various species, providing information regarding the co-evolution of substrate specificity of archaeal EndAs and tRNA genetic diversity, and the evolutionary pathway of archaeal and eukaryotic EndAs. Although the complex structure of the heterothermic form of eukaryotic EndAs is unknown, previous reports regarding their functions indicated that mutations in human EndA cause neurological disorders including pontocerebellar hypoplasia and progressive microcephaly, and yeast EndA significantly cleaves mitochondria-localized mRNA encoding cytochrome b mRNA processing 1 (Cpb1) for mRNA maturation. This mini-review summarizes the aforementioned results, discusses their implications, and offers my personal opinion regarding future directions for the analysis of the structure and function of EndAs. Maturation events in tRNAs, including removals of the 5′-leader, 3′-trailer, and intron sequences, modification, and addition of 3′-CCA sequences and amino acids are essential for protein synthesis. During tRNA maturation, tRNA splicing is one of the most significant processes in intron splicing and ligation of the two halves of exons in the precursor (pre)-tRNA. Pre-tRNA introns are either auto-catalytically or enzymatically cleaved out in the three domains of life. Group I introns found in pre-tRNA in some bacteria and higher eukaryotic plastids are auto-catalytically cleaved out with an external guanosine-5′-triphosphate (GTP) . By contmponents  until thcterized . Currentcterized . Eukaryo4, α′2, (αβ)2, and 𝜀2, has been obtained from extensive crystallographic studies (4) of archaeal EndA in Methanocaldococcus jannaschii (2) in Archaeoglobus fulgidus 2 type of archaeal EndAs have been reported in Nanoarchaeum equitans 2 through the aforementioned interactions. The overall structures are very similar to those of the α4 and α′2 types of EndAs, although the structure of P. aerophilum EndA is more compact than that of other EndAs because of the absence of the N-terminal domain of structural β subunit. Furthermore, a new type of 𝜀2 EndA was identified and characterized in Candidatus Micrarchaeum acidiphilum (ARMAN-2)  fused by two distinct linkers, although the overall shape of ARMAN-2 𝜀2 EndA is similar to that of the other three types of archaeal EndAs. Structure-based sequence analysis suggests that all four types of archaeal EndAs evolved from a common ancestor.Information regarding the four types of archaeal EndA structures, i.e., α studies , whereas studies . Initialnnaschii  and of tfulgidus . The α′2er group . The oveer group . Brieflyequitans , Pyrobacrophilum , Aeropyrm pernix , and Metkandleri . The (αβARMAN-2) , which i2 types or arginine and tryptophan residues in α subunit of (αβ)2 and 𝜀2 types] are positioned at a similar location adjacent to the three catalytic residues. Thus, each multimeric conformation of archaeal EndAs is essential for catalysis and substrate tRNA recognition. In eukaryotes, yeast EndA is a heterotetramer (αβγσ) comprising two catalytic (Sen2 and Sen34) and two accessory (Sen15 and Sen54) subunits identified on the basis of homology with their human counterparts  are conserved in the four types of EndAs, and each subunit assembly of the archaeal EndAs leads to the formation of two intron cleavage sites at the active site 2 EndA is unknown. To clarify the mechanism, structural and biochemical analyses of the (αβ)2 type of EndA from hyperthermophilic crenarchaeon Aeropyrum pernix was performed , which was conserved in crenarchaeal EndAs and located adjacent to the active site 2 of crenarchaeal EndAs. Similarly, the 𝜀2 type of ARMAN-2 EndA possesses an ARMAN-2 specific loop (ASL), which confers broad substrate specificity, and the Lys residue of ASL functions as the RNA recognition site. Although the ASL conformation in ARMAN-2 EndA is markedly similar to that of CSL in A. pernix EndA, there are no obvious sequence similarities between the ASL and CSL, except for the conserved Lys residue, which functions as the substrate recognition site. Together, these findings indicate that the ASL was acquired by a distinctly independent evolutionary pathway toward the CSL . However, it is still unknown why each Lys residue conserved in the CSL and ASL is required for intron cleavage, despite the presence of three catalytic residues in the EndAs. However, M. kandleri EndA was identified as the (αβ)2 type lacking specific loops such as the ASL and CSL (M. kandleri EndA slightly cleaves introns with a relaxed BHB motif in M. kandleri pre-tRNAGlu (UUC), it could not eliminate introns from a mini-helix RNA with a BHL motif. Therefore, the M. kandleri EndA is considered to be of the (αβ)2 type with constrained substrate specificity.The (αβ)erformed . At the kandleri . Our stuive site . Further and CSL . While M4 type of archaeal EndA, which encodes a single catalytic α subunit, is proposed to be the prototype of the EndAs 2 and 𝜀2]. Intriguingly, 𝜀2-type ARMAN-2 EndA appears to have undergone a genetic recombination of the three subunits, euryarchaeal α subunit, crenarchaeal α subunit, and crenarchaeal β subunit (N-α-βC) of the 𝜀 protomer . Remarkably, the primitive eukaryotic red alga Cyanidioschyzon merolae harbors many disrupted tRNA genes with a relaxed BHB motif as employed in Archaea ] for processing these pre-tRNAs; however, it does not contain the ASL and CSL. Thus, heterotrimer form of C. merolae EndA might be an intermediate in the evolutionary transition between the heterotetramer of archaeal EndA to heterotetramer of eukaryotic EndA. Furthermore, recent bioinformatics analysis has reported that archaeal species with specific loops such as the ASL and CSL in EndAs clearly represent a trend of increased intron-containing tRNA genes with BHB and relaxed BHB motifs, suggesting coevolution of tRNA gene diversity and broad substrate specificity  of the heterotetrameric form (αβγδ) of eukaryotic EndA with approximately 50 amino acid residues. Therefore, eukaryotic EndA is considered to have evolved from the archaeal (αβ) Archaea . The C. cificity . These fcificity .Saccharomyces cerevisiae EndAs are localized in the nucleus  and this cleavage requires a predicted stem-loop structure of the endonucleolytic cleavage-inducible sequence of Cpb1 with synergistic effects of other factors  reportedly cleaves introns from pre-tRNAs, and the TSen2 subunit is involved in pre-mRNA′3 end formation 2- and 𝜀2- type EndAs, whereas the mechanism underlying the broad substrate specificity may vary between the eukaryotic and archaeal EndAs. Further structural analysis is required to elucidate the detailed mechanism underlying broad substrate specificity by archaeal and eukaryotic EndAs. In particular, the structural information of human EndA may be useful for drug design that improves the inadequate EndA function, which causes the developmental retardation of human brain described above.The mechanism underlying the recognition and cleavage of RNA introns by EndAs is known; however, they have gained increasing interest, since the evolutionary pathway from archaeal to eukaryotic EndA and the mechanism underlying the broad substrate specificity of archaeal and eukaryotic EndA warrant further investigation. The conserved Lys residue in CSL and ASL of the (αβ)The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.