Reaction against N antigen was present in 49/66 (74.2%) of Group A and in 140/180 (77.7%) of Group C . The median SFC count for S antigen was 24 (range from 0–218) in Group A, 12 (range from 0–275) in Group B and 18 (range from 0–160) in Group C . The median SFCs count for N antigen was 13 (range 0–82) for Group A and 18 (range 0–168) for Group C . (4) Conclusions: Our findings suggest that natural cellular immunity, either alone or combined with vaccination, confers stronger and more durable protection compared to vaccine-induced cellular immunity.(1) Background: SARS-CoV-2 T cell immunity is rapidly activated following SARS-CoV-2 infection and vaccination and is crucial for controlling infection progression and severity. The aim of the present study was to compare the levels of T cell responses to SARS-CoV-2 between cohorts of subjects with hybrid immunity , vaccinated naïve (non-exposed) and convalescent unvaccinated subjects. (2) Methods: We performed a retrospective descriptive analysis of data collected from the medical records of adult individuals who were consecutively examined at a large, private Medical Center of Attica from September 2021 to September 2022 in order to be examined on their own initiative for SARS-CoV-2 T cell immunity response. They were divided into three groups: Group A: SARS-CoV-2 convalescent and vaccinated subjects; Group B: SARS-CoV-2 naïve vaccinated subjects; Group C: SARS-CoV-2 convalescent unvaccinated subjects. The SARS-CoV-2 T cell response was estimated against spike (S) and nucleocapsid (N) structural proteins by performing the methodology T-SPOT.COVID test. (3) Results: A total of 530 subjects were retrospectively included in the study, 252 females (47.5%) and 278 (52.5%) males ranging from 13 to 92 years old (mean 55.68 ± 17.0 years). Among them, 66 (12.5%) were included in Group A, 284 (53.6%) in Group B and 180 (34.0%) in Group C. Among the three groups, a reaction against S antigen was reported in 58/66 (87.8%) of Group A, 175/284 (61.6%) of Group B and 146/180 (81.1%) of Group C (chi-square,  Cellular immunity is known to play a vital role in recognizing and controlling viral pathogens. Concerning severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coordinated T cell-mediated immunity is revealed to be associated with effective viral clearance and less disease severity ,2. SeverMoreover, although a decrease in antibody titers has been commonly observed, recent studies support the prolonged protective role of T cells compared to humoral immunity ,9. InterConsidering mRNA vaccines, a profound adaptive humoral and poly-specific cellular immune response against epitopes is known to be elicited . RemarkaThe durability of immune protection following SARS-CoV-2 infection and/or vaccination wanes over time and in response to novel variants of concern (VOCs) . In contAlthough cellular responses remain more difficult to study compared to humoral ones, the implementation of advanced laboratory technologies facilitates T cell response assessment, thus, providing thorough T cell immunity characterization. Such technologies are broadly divided into molecular and cellular assays. Among those, enzyme-linked immunosorbent spot assays (ELISpot), based on the detection of cytokine secretion for a single cell, have been proven particularly useful .Considering that new variants will continuously emerge along with the decreased efficacy of the humoral responses, a comprehensive understanding of T cell responses to SARS-CoV-2 is crucial for improving public health strategies, while more cellular immunity-associated studies are required to establish their protective role. We conducted this retrospective study in order to compare the levels of T cell responses to SARS-CoV-2 infection between three cohorts of the population: (a) subjects with hybrid immunity , (b) vaccinated naïve (non-exposed) and (c) convalescent unvaccinated.We performed a retrospective descriptive analysis of data collected from the medical records of adults who had consecutively proceeded to ‘BIOIATRIKI” Healthcare Center, a large Medical Center in the region of Attica, Greece, from September 2021 to September 2022 in order to be examined for SARS-CoV-2 T cell response upon their own initiative, as part of a SARS-CoV-2 immunity screening. Additional data in terms of clinical symptoms and medical history were obtained by structured questionnaires that were routinely filled by all individuals at the time of examination. No precise information was obtained regarding the exact type of vaccine administered; however, m-RNA vaccines were largely available to the majority of the Greek population during that period. No records of clinical data, such as chest X-rays and hospitalization records, were obtained for infected patients.Based on the available data, we divided participants into 3 cohorts: Group A: both SARS-CoV-2 convalescent and vaccinated subjects; Group B: SARS-CoV-2 naïve subjects who were vaccinated; Group C: SARS-CoV-2 convalescent and unvaccinated subjects.The study was approved by the institutional review board .(i)Group A: SARS-CoV-2 Convalescent/vaccinated groupParticipants were selected among vaccinated COVID-19 patients based on a previous positive SARS-CoV-2 PCR either alone or combined with positive rapid antigen test and/or self-test result. The date of the first positive laboratory result was considered the date of diagnosis of COVID-19 infection. In an attempt to reduce the unavoidable interference of confounding factors, subjects with COVID-19 reinfections confirmed by PCR, either alone or combined with positive rapid antigen test and/or self-test result, were excluded from the study. Moreover, we included in the study only individuals who had completed their initial vaccination series and had not received any booster doses until the examination time. The date of the initial vaccination completion  was considered as the date of vaccination. The time interval before the T cell response measurement was estimated, taking into account the first immunological response to SARS-CoV-2, either due to infection or vaccination. No adequate information was available about the exact date of the second event of immunological response.(ii)Group B: SARS-CoV-2 naïve (non-exposed)/vaccinated groupParticipants were selected from subjects with low risk of prior SARS-CoV-2 infection and no prior diagnosis of COVID-19 infection who had been fully vaccinated. Requirements for enrollment included no current or prior signs or symptoms of COVID-19, no known contact with a confirmed SARS-CoV-2-infected individual and no diagnosis with SARS-CoV-2 prior to vaccination based on PCR results, rapid tests, self-tests and specific IgG IgM antibodies measurements. We included subjects who had completed only their initial vaccination series and had not received any booster doses until the examination time. The date of the initial vaccination completion  was considered as the date of vaccination.(iii)Group C: SARS-CoV-2 convalescent/unvaccinated groupParticipants were selected from unvaccinated asymptomatic and symptomatic patients with a previous positive SARS-CoV-2 PCR result prior to testing. The date of the first positive PCR result was considered the date of diagnosis of SARS-CoV-2 infection. In cases where PCR was carried out as a confirmation test after a positive self-test or rapid test, the date of the first positive laboratory test was considered as the date of SARS-CoV-2 diagnosis. Individuals with confirmed COVID-19 reinfections were excluded from the study.®.COVID . The T-SPOT.COVID test is a standardized ELISpot-based technique for detection of T cell immune response to SARS-CoV-2 in whole blood. The test uses the established T-SPOT Technology with an antigen mix based on SARS-CoV-2 structural proteins, spike (S) and nucleocapsid (N).Enzyme-linked immunosorbent spot (ELISpot) Assay for IFN-g T Cell Response Detection: The T cell response to SARS-CoV-2 infection was estimated by performing the IGRA methodology T-SPOTBlood samples were drawn into lithium heparin tubes where the T-cell Xtend reagent (Oxford Immunotec) was added. Peripheral blood mononuclear cells (PBMCs) were then separated from a whole blood sample, washed and then counted before being added to the test. An estimated number of 250,000 cells/well were plated into 4 wells of a 96-well plate.The two antigen peptide pools were added to the two antigen wells, the T cell mitogen phytohemagglutinin was added to the positive control well, and cell culture media alone to the negative control well. The wells were washed after a period of 16–20 h of incubation and were added a conjugated secondary antibody with the capacity to bind to any IFN-γ captured on the membrane. In the process, the wells were washed to remove the unbound IFN-γ, and a substrate was added to produce the characteristic dark spots of insoluble products indicating areas of IFN-γ presence. The spot-forming cells (SFCs) were manually counted by microscopy by expert and experienced technologists. Results were reported separately for N and S antigens. They were expressed as ‘invalid’ if the negative control had more than 10 SFCs or the positive control had fewer than 20 SFCs when the antigen wells were non-reactive. The test cut-off was predefined at 6 SFCs for each antigen. A borderline zone of +/−1 SFCs was introduced to account for potentially elevated test variability around the cut-off . Accordiχ2) test was performed to evaluate differences between categorical variables, and one-way ANOVA was used to compare continuous variables. In cases of non-parametric continuous variables, the Kruskal–Wallis test was performed. Kinetics over time periods were estimated using the curve estimation. GLM Univariate analyses were performed to examine the existence of confounding factors, particularly sex, age, time after exposure and cardiovascular disease.The chi-square . Outcomes were considered statistically significant when Among the total individuals consecutively proceeding to our medical center for SARS-CoV-2 immunity screening, 530 were selected for the study, 252 females (47.5%) and 278 (52.5%) males ranging from 13 to 92 years old (mean 55.68 ± 17.0 years). The study participants’ medical information is presented in Among them, 66 (12.5%) had hybrid immunity (previously infected and vaccinated) (Group A), 284 (53.6%) had been vaccinated and had no evidence of prior exposure to the virus (Group B), and 180 (34.0%) had past COVID-19 infection and had not been vaccinated (Group C).p = 0.1) The mean age in Group A was 57.3 ± 16.45 years, in Group B, 58.46 ± 16.32 years, and in Group C, 50.69 ± 17.28 . More specifically, a significant difference in age was observed between Group A and C (p < 0.001) and Group B and Group C (p < 0.001). No difference in age was demonstrated between Group A and Group B (p = 0.68).In Group A, the sex proportion was 29 females (43.9%) and 37 males (56.1%); in Group B, 127 females (44.7%) and 157 males (55.3%); in Group C, 96 females (53.3%) and 84 males (46.7%)  and N antigen in 189/530 (35.6%) of the samples, respectively. Borderline S results were reported in 13/530 (2.4%) and non-reactive in 138/530 (26%), while borderline N results were reported in 13/530 (2.4%) and non-reactive in 328/530 (61.8%). No participants had a borderline response against both N and S antigens. Both S and N antigens triggered positive responses in 186 (35.1%) samples. In 12 samples, the results of both two antigens were considered indeterminate due to inadequate responses in the negative or positive controls. These indeterminate samples corresponded to six unvaccinated (of Group C) and six vaccinated immunocompromised cases (of Group A) and were excluded from the study.In addition, eight samples presented less than 20 SFCs in the positive control; however, four of them had a response against the S antigen and the other four against the N antigen; therefore, they were considered positive only for the particular antigen.p < 0.001). Reaction against N antigen was present in 49/66 (74.2%) of Group A and in 140/180 (77.7%) of Group C   of Group A, 175/284 (61.6%) of Group B and 146/180 (81.1%) of Group C  a,b. Reacp < 0.001; pairwise comparisons: groups A–B, p < 0.001; groups A–C, p = 0.147; groups B–C, p < 0.001). The median SFCs count for N antigen was 13 (range 0–82) for Group A and 18 (range 0–168) for Group C  (The median SFC count for S antigen was 24 (range 0–218) in Group A, 12 (range 0–275) in Group B and 18 (range 0–160) in Group C  . Reactiop < 0.001). More specifically, a statistically significant difference was revealed between groups A and B , groups A and C  and groups B and C .The mean time period post-exposure/vaccination for the three groups were as follows: Group A: 175 ± 124 days (range 14–600), Group B: 136 ± 76 days (range 13–324) and Group C: 254 ± 155 (range 12–705) days  and curve estimation of the quantitative T-SPOT results for S and N antigens in subjects of groups B and C are presented in p = 0.41; sex: F = 0.198, p = 0.657; time after exposure F = 4.373, p = 0.09; cardiovascular disease: F = 0.373, p = 0.752. For N antigen: Age: F = 2.703, p = 0.101; sex: F = 0.557, p = 0.456; time after exposure F = 2.373, p = 0.136; cardiovascular disease: F = 0.453, p = 0.859).In order to examine the possible interaction between S and N antigens and the independent variables age, sex, cardiovascular disease and time after exposure, we performed GLM univariate analysis. The constructed model did not demonstrate any associations in a statistically significant level with any of the above variables (for S antigen: Age: F = 0.678, Humoral and cellular immune memory assessment against SARS-CoV-2 is critical in controlling COVID-19 progress. At present, long-term immune responses concerning both types of responses in convalescent patients and vaccinated subjects have been widely investigated ,21.The present retrospective descriptive study has focused on the evaluation of T cell responses to SARS-CoV-2 in three different cohorts. Our results suggest that cellular immunity induced by natural infection alone or combined with vaccination is higher compared to the vaccine-induced immunity alone. These findings are of particular significance if we take into account that the time interval from initial immunity triggering to T cell testing was longer in groups A and C compared to the time interval from vaccination to testing day in Group B. Interestingly, our study suggests that an IFN T cell response, when comparing the response between Group B and Group C, is stronger in infected patients than in those who have been vaccinated. This should be further examined by future studies, as the response has been described to be variable in vaccinated subjects since vaccines may not be equally effective against SARS-CoV-2 variants [With regard to the effect of vaccination on SARS-CoV-2 cellular immunity, a recent study has documented that levels of T cells remained sustained after 3 months from vaccination, whereas the titer of anti-RBD antibodies as well as their neutralization function decreased significantly during that period . AdditioConcerning convalescent individuals, several studies ,27,28,29As for convalescent individuals who had received one of the EMA-approved vaccines, recent studies ,31,32,33Our study bears certain limitations, including those of an observational retrospective study. The study groups comprised of individuals proceeding to the diagnostic center on their own initiative to be tested for cellular immunity as part of a COVID-19 immunity screening test during the pandemic. In this context, no additional data, such as the type of the administered vaccines—except that the vast majority were mRNA—and the identification of viral variants, were available for further analysis. Furthermore, in Group A, no information could be obtained about the exact time of the second immunological response, which implies the need for caution in the interpretation of results.Ultimately, our results enhance the evidence of long-lasting cellular immunity in COVID-19 convalescent subjects, especially when combined with vaccination. Future studies aiming to evaluate cellular responses years after vaccination and/or infection could provide further insights into longevity and persistence of SARS-CoV-2–specific T cell-mediated immunity as well as its importance against reinfections or potential new antigen encountering.


An essential pathogenic element of acute limb ischemia/reperfusion (I/R) injury is microvascular dysfunction. The majority of studies indicates that fibroblast growth factor 2 (FGF2) exhibits protective properties in cases of acute I/R injury. Albeit its specific role in the context of acute limb I/R injury is yet unknown. An impressive post-reperfusion increase in FGF2 expression was seen in a mouse model of hind limb I/R, followed by a decline to baseline levels, suggesting a key role for FGF2 in limb survivability. FGF2 appeared to reduce I/R-induced hypoperfusion, tissue edema, skeletal muscle fiber injury, as well as microvascular endothelial cells (ECs) damage within the limb, according to assessments of limb vitality, Western blotting, and immunofluorescence results. The bioinformatics analysis of RNA-sequencing revealed that ferroptosis played a key role in FGF2-facilitated limb preservation. Pharmacological inhibition of NFE2L2 prevented ECs from being affected by FGF2's anti-oxidative and anti-ferroptosis activities. Additionally, silencing of kruppel-like factor 2 (KLF2) by interfering RNA eliminated the antioxidant and anti-ferroptosis effects of FGF2 on ECs. Further research revealed that the AMPK-HDAC5 signal pathway is the mechanism via which FGF2 regulates KLF2 activity. Data from luciferase assays demonstrated that overexpression of HDAC5 prevented KLF2 from becoming activated by FGF2. Collectively, FGF2 protects microvascular ECs from I/R injury by KLF2-mediated ferroptosis inhibition and antioxidant responses. Acute limb ischemia is one of the most severe forms of peripheral artery disease, characterized by a sharp decline in limb blood flow, a high prevalence of amputations, irreversible limb necrosis and even death In the course of injury of I/R, oxidative stress (OS) is considered to be the primary initiator of EC death in vivo and in vitro and exerts anti-oxidative functions The Kruppel-like factor (KLF) family of transcriptional regulatory proteins regulates various gene expressions through transcriptional regulation Within the fibroblast growth factor (FGF) family, FGF2 is an essential component. FGF2 modulates a variety of cellular processes by binding and activating FGF receptor The Institutional Animal Care & Research Ethics Committee from Wenzhou Medical University (wydw 2017-096) approved the study's animal experimental methods and procedures, which adhered to the China National Institutes of Health's Guide for the Care and Use of Laboratory Animals. Healthy, adult male C57BL/6 mice (age = around 6 weeks and average weight: 18-23g) were offered by Wenzhou Medical University (Licence no. SCXK 2005-0019) and were kept under normal conditions  and unrestricted access to water and food.The following chemicals were employed in our research: FGF2 was supplied from the biotechnology pharmaceutical engineering lab at Wenzhou Medical University and synthesized based on previous work. Masson Staining Kit and BCA Kit are both available from Thermo Fisher Scientific in the United States and Solarbio Science in Beijing, China, respectively. The following companies provided the specific primary antibodies that were needed: HO-1 (10701-1-AP), NQO1 (11451-1-AP), SOD1 (24127-1-AP), AMPK (66536-1-lg), NFE2L2 (16396-1-AP), GAPDH (60004-1-lg) and Histone-H3 (17168-1-AP) from Proteintech Group; SLC7A11 (DF12509) and KLF2 (DF13602) from Affinity; GPX4 (381958) from ZEN Bio; p-AMPK (2535) from abcam; HDAC5 (A0632) and p-HDAC5 (AP0202) from Abclonal; 4-HNE (MAB3249-SP) from R&D Systems. CD31 (sc-376764) antibody from Santa Cruz. The secondary IgG antibody coupled with horseradish peroxidase (HRP) was purchased from Santa .An orthodontic rubber band was placed over the greater trochanter of the right leg in order to construct the I/R injury model I/R groups were distributed into I/R+FGF2 group, I/R+Ferrostatin-1 (Fer-1) group, I/R+FGF2+Erastin group, I/R+Erastin group, I/R+FGF2+ML385 group, I/R+ML385 group, I/R+FGF+LV-KLF2 group, I/R+FGF2+LV-Con group, I/R+LV-KLF2 group, I/R+FGF2+Compound C (CC) group and I/R+CC group. FGF2 group received injections of FGF2  at three-time points: right before ischemia, before reperfusion and 30 min post-reperfusion. Two weeks previous to the surgery, lentivirus KLF2 was intramuscularly injected into the right gastrocnemius muscle for both the I/R+LV-KLF2 and I/R+FGF2+LV-KLF2 groups. Equivalent quantities of lentivirus vehicles encoding the negative control sequence were administered to the control group. Three days before to surgery, intraperitoneal injections of Fer-1  and Erastin  were given daily to the I/R+Fer-1 group, I/R+Erastin group and I/R+FGF2+Erastin group. Injections of ML385  were performed in the I/R+ML385 and I/R+FGF2+ML385 groups with the same protocol. Three days prior to surgery, CC  intraperitoneal injections were administered daily to the I/R+CC as well as I/R+FGF2+CC group and I/R+FGF2+CC group.Under a state of general anaesthesia, LDI was performed on each mouse.The LDI, a laser doppler device with significant penetration, made small vessels visible at great depth and helped to clarify the blood flow to the hind limb Skeletal muscles were harvested after reperfusion. The muscle wet-to-dry weight ratio was determined to analyze muscle edema. Skeletal muscles were obtained post-reperfusion, and the proportion of wet to dry weight was computed to assess muscular edoema. Tissue samples were taken, weighed right away, and then dried at 55 °C till a consistent weight was obtained.After perfusion, gastrocnemius muscle tissues were obtained. The muscle samples were paraffin-embedded, formalin-fixed, and sectioned at a thickness of 5-μm. Tissue sections treated with PFA and enclosed in paraffin received appropriate dewaxing and dehydration. Following the guidelines provided by the manufacturer, a kit was used to carry out the Masson's trichrome staining procedure. Based on their shape, muscle fibres were then classified as either undamaged or damaged. A proportion of damaged fibres for each leg was used to display the final statistics The School of Pharmaceutical Science  provided the human umbilical vein endothelial cells (HUVECs) for this study. HUVECs were cautiously cultivated in DMEM added with FBS , penicillin (100 U/mL) and kyowamycin (100 μg/mL) under 37 °C in a humidified cell incubating device with continuous 5% CO2 supply.2 and 5% CO2, lowering oxygen levels to 1%. Cells were cultured for 8 hours, and then they were cultivated for 16 hours in a typical DMEM high-glucose medium. Prior to hypoxia, all prescribed treatments were given: lentivirus KLF2 for 48 hours and FGF2 (20 ng/mL) during 24 hours.During the cells' logarithmic development phase, the oxygen-glucose deprivation/re-oxygenation (OGD/R) model was developed. Initial media replacement involved switching to serum-free RPMI 1640 medium from GIBICO, and cells were then put into a three-gas incubator from Thermo Scientific that contained 95% NCell viability was measured using CCK8  reagent following manufacturer's instructions. To measure cell viability, the OD450 value was normalised to the control group.HUVECs were grown on well pate with coverslips overnight. They were adequately blocked with 5% bovine serum albumin (BSA) at room temperature (RT) for 30 minutes after being fixated with 4% paraformaldehyde (PFA) for 15 minutes. After that, NFE2L2, KLF2, and GPX4-specific primary antibodies were incubated on the coverslips for an entire night at 4 °C. Cells were carefully cleaned 3 times via PBS for five minutes each. The cells were then incubated for 1 h at room temperature with fluorescence-linked secondary antibodies. After that, PBS was used to rinse the coverslips to get rid of the secondary antibodies. Before mounting the glass plate with the DAPI Fluoromount-GTM  reagent, any PBS leftovers were cleaned off of it. The Nikon ECLIPSE 80i microscope  was used to take and analyse digital pictures.in vitro for 20 minutes at the typical 37 °C. Using a confocal microscope , cell fluorescence was captured after sufficient washing with serum-free media. Intracellular ROS was measured with the ROS assay kit  in accordance with the manufacturer's instructions.The DCFH-DA, an oxidation-sensitive fluorescent probe was employed to suspend pure HUVECs and HUVECs were incubated in vivo using DHE staining. Fresh skeletal muscle samples were cut into cryosections and treated with DHE  for 30 minutes at 37 °C. The samples were then analysed using a confocal microscope .ROS generation was calculated Muscle and cell samples were homogenized in ice-cold RIPA buffer  containing protease inhibitor cocktail  and phosphatase inhibitor cocktail . Following the directions provided by the manufacturer, a BCA protein assay kit was used to measure the protein concentrations. On polyvinylidene difluoride membranes, protein samples were separated utilising 12.5% SDS-PAGE. After sufficiently blocking with 10% fat-free milk solution, the specified primary antibodies were added to the membranes and incubated overnight at 4 °C: HO-1, NQO1, SOD1, NFE2L2, GAPDH, GPX4, SLC7A11, Histone H3, AMPK, p-AMPK, HDAC5, p-HDAC5, KLF2, FGF2 as well as 4-HNE. After that, the membrane was exposed to HRP-conjugated IgG secondary antibody for 1.5 hours at room temperature. The bands have been observed and quantified employing Bio-Rad's Image Lab 3.0 software.The Total Glutathione detection kit (Assay Designs) was employed to measure the levels of GSH in both cells as well as muscle tissue while following the manufacturer's instructions. With measures made at 532 nm, the colorimetric assay mainly determined MDA concentrations in cells as well as muscle tissue. Employing the Iron Assay Kit (Abcam), iron ion concentrations inside cell as well as muscle tissue were measured.JC-1 dye , a carbocyanine dye with cationic fluorescence characteristics, was used as a ratiometric indicator to measure mitochondrial membrane potential (MMP) in cells. Cells underwent two washes with the JC-1 buffer solution after being incubated with the JC-1 dye solution (2.5 g/mL) for 20 minutes in the dark at 37 °C. A fluorescent microscope and an argon-ion 488 nm laser were used to determine MMP by measuring the proportions of mitochondrial JC-1 monomers or aggregates. Healthy cells with aggregated JC-1 emitted red fluorescence, whereas injured cells with reduced MMP showed green fluorescence caused by monomeric JC-1. MMP was quantified using the ratio of red-to-green fluorescence.The genome-wide transcriptomics analyses were carried via (LC-BIOTECHNOLOGIS (HANGZHOU) CO., LTD) after total RNA was extracted. Using the R software edger or DESeq2, the differently expressed mRNAs were screened with fold change (FC) greater than 2 or FC less than 0.5 and p-value less than 0.05. Differentially expressed genes (DGEs) were grouped hierarchically to reveal patterns of gene expression in various populations. On the basis of hypergeometric distribution, an independent GO enrichment analysis of DEGs was carried out using R.To investigate the putative regulatory function of FGF2 within KLF2 transcriptional activity, a luciferase reporter assay was carried out. The transcriptional activity of KLF2 was assessed by the KLF2-driven luciferase activities. Based on the manufacturer's instructions, HEK293T cells were co-transfected with pRL-TK, pGL3-basic, or pGL3-KLF2 and empty pcDNA3.1 vector or HDAC5-pcDNA3.1 plasmids in 24-well plates using liposome transfection reagent 2000 . Cells were transfected for 24 hours before receiving 20 ng/ml of FGF2. 48 hours after transfection, Firefly and Renilla luciferase activities were both detected using the Dual-Luciferase® Reporter Assay System (Promega) as well as a microplate reader  to determine the firefly/Renilla luciferase ratio.Software from SPSS, version 19, was used to analyse the data . The mean ± standard deviation (SD) for each experimental data point were presented. To detect group differences, independent-specimen t tests were employed. To identify differences between multiple groups, ANOVA with LSD  or Danett T3  multiple comparison (post hoc) tests were used to evaluate statistical differences. A two-tailed p-value of less than 0.05 was regarded as statistically significant.To explore the pathophysiological significance of FGF2 after I/R injury, the gastrocnemius muscles of limbs were excised from multiple time points of reperfusion and FGF2 levels were assessed. A western blot assay revealed that FGF2 levels significantly increased after I/R damage, as measured at different stages of reperfusion. After then, FGF2 levels stabilised Fig. A, B. AftIn mice subsequent I/R damage, the possible therapeutic role regarding FGF2 was investigated. When compared to the sham group, the I/R group had severe limb hypoperfusion, according to LDI's initial assessment of limb blood perfusion. Contrarily, the FGF2 group displayed more strong signs of muscle blood flow compared to the I/R group, suggesting that FGF2 plays a substantial role in improving limb blood perfusion following I/R injury  protein expression in skeletal muscle tissue following I/R damage  Intraperitoneal administration of FGF2 may lead to rapid diffusion in the body that reduces the effective concentration of the ischemic limb and may cause adverse side effects. Intravascular administration may be an ideal factor for ischemic or I/R-associated diseases. 2) The underlying molecular mechanism of lower-limb I/R injury is complex and interactive. Other pathological mechanisms must be evaluated for the appropriate drug combination Our research revealed that FGF2 dramatically ameliorate microvascular damage, reduces hypoperfusion, tissue edema and skeletal muscle fiber injury in limbs following I/R. In our research, we discovered that FGF2 stimulates the AMPK-HDAC5 signalling pathway, which increases KLF2 activity. Importantly, I/R-induced microvascular damage is reduced and limb viability is increased thanks to FGF2-induced KLF2 activation, which also supports the NFE2L2-mediated inhibition of OS and ferroptosis in limb ECs Fig. W. OveralSupplementary figures.Click here for additional data file.


Drosophila show stereotypical left–right (LR) asymmetry; however, the underlying mechanisms remain elusive. Here, we have identified an evolutionarily conserved ubiquitin-binding protein, AWP1/Doctor No (Drn), as a factor required for LR asymmetry in the embryonic anterior gut. We found that drn is essential in the circular visceral muscle cells of the midgut for JAK/STAT signaling, which contributes to the first known cue for anterior gut lateralization via LR asymmetric nuclear rearrangement. Embryos homozygous for drn and lacking its maternal contribution showed phenotypes similar to those with depleted JAK/STAT signaling, suggesting that Drn is a general component of JAK/STAT signaling. Absence of Drn resulted in specific accumulation of Domeless (Dome), the receptor for ligands in the JAK/STAT signaling pathway, in intracellular compartments, including ubiquitylated cargos. Dome colocalized with Drn in wild-type Drosophila. These results suggest that Drn is required for the endocytic trafficking of Dome, which is a crucial step for activation of JAK/STAT signaling and the subsequent degradation of Dome. The roles of AWP1/Drn in activating JAK/STAT signaling and in LR asymmetric development may be conserved in various organisms.Many organs of  Summary: Doctor No, a Drosophila AWP1 ortholog, activates JAK/STAT signaling via Dome receptor endocytosis in a crucial step for left–right asymmetry in the developing gut. Many animals show directional left–right (LR) asymmetry in their body structure and function. Several mechanisms, such as cilia-generated flow, contribute to LR axis formation in vertebrates . HoweverDrosophila, including the gut, testis, male genitalia, and brain , upd2 and upd3 and the receptor is encoded by domeless (dome) (hopscotch (hop) in Drosophila. Moreover, similar to the mammalian JAK/STAT system, Hop constitutively associates with the intracellular domain of Dome in Drosophila  . JAK is osophila . Ligand osophila . JAK theosophila  , which we refer to as Doctor No (Drn), positively regulates JAK/STAT signaling by facilitating endocytic trafficking of the Dome receptor, which is required for normal LR asymmetric development of the embryonic gut. Drosophila Drn was named after Ian Fleming's fictional character, whose heart was located on the right side of his chest  ; this was defined as normal FG laterality (n=50); this was defined as normal laterality of the AMG , we performed a genetic screen using a large collection of P element insertion lines (es.html) . We scores.html) A,B. We dterality A. (2) Th the AMG A. Using  the AMG C.CG45050 locus  , indicating heterotaxy but not situs inversus in these phenotypes. To genetically characterize drn further, we generated deletion mutant alleles of drn through imprecise P element excision. In 1drn, the deduced initiation codon and 5′ portion of the coding region were deleted from the drn alternative RNA products CG45050-RB, -RC, -RD, -RE, -RF and -RG  E. In parorithms) E. A20-tyorithms) . We noteelopment C,F. In c and -RG D. In drnt of drn D. Variouenotypes F. These enotypes F,G. Thusenotypes . We alsot manner F.drn is required for LR asymmetric development of the anterior gut, we performed rescue experiments using the GAL4/UAS system , probably as a result of leaky expression of UAS-drn (UAS-drn misexpression driven by da-GAL4 (ubiquitous), NP1522 (in the somatic muscle and CVMU), hand-GAL4 (in the CVMU), 24B-GAL4  and 48Y-GAL4  (UAS-drn misexpression driven by NP0221 (in the LVMU), NP5021 (in the epithelium) or elav-GAL4 (in the nervous system)  B. In con system) B. Collec embryos .drn mutants, we used 65E04-GAL4 to express nuclear-localizing RedStinger fluorescent proteins specifically in the visceral muscles. The angle (θ) between the major axis of the ellipsoidal nuclei in the lower 30% of the presumptive first chamber and the anterior–posterior axis of the embryo was measured by a blind test at late stage 15     E,E′ (Kurov test) . Howeverov test) F,F′. In ov test) . In thisd (n=10) E-F′. Coldrn in the CVMU, we assessed the distribution of drn mRNA in embryos by in situ hybridization at various stages of embryogenesis  stages, suggesting that drn mRNA is maternally provided , in which drn is required for LR asymmetric development of the anterior gut   , specifi and AMG B. Therefdrn and JAK/STAT signaling more directly, we assessed the characteristic phenotypes related to JAK/STAT signaling in drn mutants. The expression of a pair-rule gene, even-skipped (eve), was disturbed in embryos homozygous for Stat92E, such as HJStat92E, with approximately 67% of the embryos showing aberrant phenotypes, such as weak stripe 3 (19%), fusion between stripes 2 and 3 (19%), or fusion between stripes 5 and 6 (67%) (n=27), rather than the seven-stripe pattern noted in wild-type embryos  . The phenotypes of 2 m/zdrn embryos were similar to those of HJStat92E homozygotes    and 2 m/zdrn embryos . Defects in 2 m/zdrn embryos were more severe than those in 2 zdrn homozygotes, which can be predicted from the maternal contribution of drn  E. As we supplied , we gene%, n=33) F,F′. Thes 15-17) . The tras 15-17) . In Stat embryos G,H. Moren of drn I,J. Thesdrn, driven by actin5c-GAL4, could rescue LR defects in embryos homozygous for 06346Stat92E. The embryos homozygous for 06346Stat92E showed LR defects in the anterior gut at a frequency of 25%  and a reporter assay revealed that wild-type drn could downregulate JAK/STAT signaling in cultured Drosophila cells . We found that anti-Drn antibody staining was largely absent from the stripe, confirming the specificity of the antibody  against full-length Drn and assessed its specificity using the UAS-GAL4 system and RNAi against antibody . Using tantibody . Thus, aUAS-dome-GFP was ubiquitously driven under the control of da-GAL4 in wild-type and drn homozygous embryos , peanut agglutinin , Sec5 (exocytic vesicles), Hrs (early endosomes), Rab5 (early endosomes), Rab7 (late endosomes), Rab11 (recycling endosomes) and LAMP1 (lysosomes), in the epidermal cells of 2drn homozygotes. None of these markers colocalized with Dome-GFP  and Notch, respectively, were unchanged in the epidermis of wild-type and 2drn homozygotes  was used for RNAi against drn. The following GAL4 lines were used in this study: da-GAL4  and +t7.2=neoFRT}82B P{w+mC=ovoD1-18}3R/st1βTub85DDss1es/TM3, Sb1w*;P{ry .We used Canton-S as the wild-type GS lines . We also-hopTuml  and UAS-dome-GFP  have bee da-GAL4 , 48Y-GAL48Y-GAL4 , 24B-GAL24B-GAL4 , hand-GAand-GAL4 , elav-GAlav-GAL4 , NP1522  .We generated the ectively . Impreciectively . drn1 an2drn homozygous embryos lacking the drn maternal contribution (2 m/zdrn) using standard crosses, as described previously (FRT82B was introduced into the 2drn chromosome by recombination. Flies carrying 2FRT82B drn were selected by Geneticin (Gibco) and genomic PCR using the primers 5′-TCACGCATTCAGAGCTTCGTGTGCCC-3′ and 5′-ATGTTGCTGCGTTTGCTCTGCGTATTCCAC-3′. 2/TM3b, SbFRT82B drn females were crossed with hsFLP/Y; FRT82B ovoD/TM3, Sb males to obtain 2hsFLP/+; FRT82B ovoD/FRT82B drn females through heat-shock treatment. These females were then crossed with 2/TM3b, SbFRT82B drn males to obtain 2FRT82B drn homozygous embryos without the drn maternal contribution.We obtained eviously . In brieUAS-drn, a cDNA fragment composed of an entire open reading frame of a drn transcript (CG45050-RC) was PCR amplified using an upper strand primer containing an EcoRI site (5′-CCGGAATTCAGCAGGAAGCAGACGAAACT-3′) and a lower-strand primer containing HindIII and BglII sites (5′-CCCCAAGCTTAGATCTTCCTTGTTATAGCGCAGCAT-3′). The cDNA clone RE70963 was used as a template  was PCR amplified using RE70963 cDNA as a template, an upper strand primer containing EcoRI and BglII sites and the myc-tag coding sequence (underlined) (5′-CCGGAATTCCAAAATGGAGCAGAAGCTGATCTCGGAGGAGGATCTGAGATCTATGGAACGTGAATCTAACCC-3′), and a lower strand primer containing a XhoI site (5′-CCGCTCGAGTCAAATCTTTTGAATCTTCT-3′). CG45050-RC has the same open reading frame as CG45050-RB, -RD, -RE, -RF and -RG (drn and UAS-myc-drn constructs were introduced into the Drosophila genome using P element-mediated transformation (To construct UAS- and -RG D. The PCormation .drn cDNA (RE70963) containing an entire open reading frame of CG45050-RC was amplified by PCR, sequenced, and subcloned into the BamHI and EcoRI sites of the pGEX-2T vector . A GST-Drn fusion protein was produced in Origami B (DE3) cells (Novagen) and purified using a Glutathione Sepharose 4B column. The purified GST-Drn fusion protein was used to immunize rats, and polyclonal antiserum was purified using a standard protocol.A fragment of in situ hybridization, standard protocols were used, as described previously , 7G10], chicken anti-β-galactosidase , mouse anti-Pdi , anti-lectin-PNA , mouse anti-Sec5  using ImageJ. Using the ‘Analyze Particles’ function, particles larger than five-pixel units were defined as the region of interest (Roi). Roi sets from two channels of an image were compared. Each Roi overlapping between two channels was defined as a particle demonstrating colocalization and was used for calculating the colocalization rate between two markers. The number of Rois showing colocalization against the total number of Rois was calculated as the percentage of colocalization.Click here for additional data file.10.1242/develop.201224_sup1Supplementary informationClick here for additional data file.


Current studies on CD8+ T cell exhaustion have found that the mechanisms responsible for their heterogeneity and differential kinetics may be closely related to transcription factors and epigenetic regulation, which may serve as biomarkers and potential immunotherapeutic targets to guide treatment. Although the importance of T cell exhaustion in tumor immunotherapy cannot be overstated, studies have pointed out that gastric cancer tissues have a better anti-tumor T cell composition compared to other cancer tissues, which may indicate that gastrointestinal cancers have more promising prospects for the development of precision-targeted immunotherapy. Therefore, the present study will focus on the mechanisms involved in the development of CD8+ T cell exhaustion, and then review the landscapes and mechanisms of T cell exhaustion in gastrointestinal cancer as well as clinical applications, which will provide a clear vision for the development of future immunotherapies.CD8 This hyporeactive state is thought to be due to the inability of pathogens to be rapidly cleared in chronic inflammation and the continued stimulation of this specific T cell proliferation, resulting in the upregulation of many immune checkpoints on the surface of T cells, leading to a reduction in their proliferation and ability to capture pathogens , 2. Many unclear , 6. Therotherapy .+ T cells by promoting the expression of a series of key effector function-related transcriptional regulators, including Foxo1, Zeb2, Id3, and Eomes  that are produced by the body to fight against viruses, tumors, and other pathogens  (CD8-CD4-) phase and a double positive (DP) phase (CD8+ CD4+) and finally became single positive (SP) CD8+ thymocytes. These cells are selected by positive and negative clones to become CD8+ T-αβ cells and are released into the circulation , leading to apoptosis of target cells through a cysteine-dependent pathway; and c) indirectly inducing death of peripheral tumor cells through the secretion of cytokines  to CD8+ T cells, thereby activating SMAD3 expression in CD8 T cells, ultimately leading to TEX and resulting in failure of immunotherapy  is an ecological niche that can suppress CD8otherapy . IL-10 rotherapy . Cetuximotherapy . Notablyimmunity , while i T cells , 45. The2.3+ TEX in cancer , effector T cells (TEFF), and memory T cells (TEME), often with great difficulty, despite the great functional, transcriptomic, and epigenomic differences in TEX , 50. Thi+ T cell function in cancer, however, these techniques have failed to capture important spatial-spatial information, including intercellular interactions and tissue localization. Many new studies have now identified intra-tumor immune ecological sites, tertiary lymphoid structures  and memory precursor effector cells (MPEC)  and IL-15  –56. Effexpansion , 55. Aftnd IL-15 . Whereas T cells . Contrarantigens –60, althantigens –63. Of n3.1+ T cells and memory CD8+ T cells during acute infection, where their roles are partially overlapping but the expression discrepancies also exist in effector and memory CD8+ T cells, indicating that they respectively have unique function in the development process of effector CD8+ T cells and memory CD8+ T cells (+ T cells (T-bet+Eomes+) increase the expression of T-bet to turn into terminally differentiated effectors (T-bet++Eomes+), while memory CD8+ T cells express a higher level of Eomes (T-bet+Eomes++) (+Eomes+++) to execute the exhaustion program, instead of differentiating into memory CD8+ T cells. T-bet and Eomes influenced the fate decision between memory T cells and exhausted T cells. Exhausted CD8+ cells expressed less T-bet and more Eomes versus CD8+effectors, and higher Eomes expression seemed to predispose CD8+ effectors to become exhausted T cells (T-bet+Eomes+++) rather than memory T cells (T-bet+Eomes++) when encountering persistent infection. During chronic LCMV infection, the expression pattern of T-bet and Eomes is also closely associated with the differentiation within the heterogenous Exhausted CD8+ T cells, and it seems that T-bet and Eomes have antagonistic roles for Tex development   . Howeverelopment . Researc therapy . Whereasfor PD1) . A high d homing .+ T cells expressing the chemokine receptor CXCR5 could migrate into B-cell follicles, which was similar to follicular helper T cells, and the CXCR5+ subset expressed lower amounts of inhibitory receptors and retained more powerful cytotoxicity compared with CXCR5- subset  and Sell (encoding CD62L) compared to WT CD8+ T cells (Prdm1(Blimp1) and Havcr2 (Tim3), which might implicate the repression mechanism for Blimp1 and Tim3  is separately upregulated, while the expression level of AP1 is lower (Pdcd1(PD1), Havcr2(TIM-3), and Ctla4(CTLA-4), whereas the NFAT/IRF4/BATF complex repressed the expression of Tcf7(TCF-1) which was necessary for the maintenance of progenitor-like T cells in Tex, suggesting that NFAT/IRF4/BATF complex promote the development of terminally differentiated Tex cells through repressing TCF-1  and chimeric antigen receptor (CAR) T cells  is one of the most common malignancies and the leading cause of cancer-related mortality worldwide . Unfortu+ T cells exhibited no significant increase of exhaustion levels and expressed low levels of exhaustion markers, including PDCD1, cytotoxic T-lymphocyte antigen 4 (CTLA-4), HAVCR2, LAG-3, and TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domains), which may partly explain the limited benefit of immunotherapy among GC patients (+ T cells reached exhausted state via two exhaustion trajectories. Tissue-resident memory T cells differentiate into IL-17CD8+ T cells (Tc17) and subsequently exhausted T cells with increasing cytolytic scores, while the blood-derived cytolytic CD8+ T cells turn to the exhausted population with lower exhaustion scores and decreasing cytolytic score, which are attributed to distinct transcription programs. Tc17-derived exhausted T cells highly express keratin KRT86, while cytolytic-cell-derived exhausted T cells express high levels of GZMK. As for transcription factors, PRDM1, TOX2, NR3C1, CEBPD, ATF3, and EOMES were expressed in cytolytic-cell-derived exhausted T cells, while BHLHE40, CREM, and RUNX2 were expressed in Tc17-derived exhausted T cells (The advancement and proliferation of single-cell RNA sequencing (scRNA-seq) technology make it possible to exactly map the status and differentiation trajectories of exhausted T cells in the tumor. Recent scRNA-seq research on tumor samples and adjacent non-tumor samples from nine untreated non-metastatic GC patients revealed that CD8patients . The oth T cells . The hig+CD8+ T cells with functional exhaustion occupy a higher proportion. GC tissue and cell lines expressing CD155 deprive CD8+ T cells of glucose, and impair CD8+ T-cell effector functions and IFNγ production, which can be rescued by additional glucose or TIGIT blockade, especially combined with PD-1 blockade  or PD-L2, which is recognized as the main mechanism of tumor cells to escape anti-tumor immune reaction  . The infreatment , 109. Al T cells –112. ThePDCD1 but decreases cytotoxic T cell effector gene expression and inhibits interferon signaling  participate in regulating glucose metabolism and redox state , including Crohn’s disease (CD) and ulcerative colitis (UD), causes intestinal inflammation and mucosal dysplasia, which triggers the development of IBD-related CRC , 146. CDnset IBD , 148. DeWith bioinformatics tools, researchers have identified multiple dysregulated molecules related to TEX and prognosis in GC or CRC tissues, such as CLDN10, C3AR1, ANTXR1, ANXA1, and PDPN –153. How5+ T cells that are associated with the pretreatment tumor burden (PD-1, the key coinhibitory receptor on activated T cells, interacts with overexpressed PD-L1 on cancer cells, tumor-infiltrating lymphocytes, and stroma cells to inhibit T cell proliferation and function . CTLA-4,r burden . These fr burden Figure 56+ T cells in tumor microenvironments have received much attention due to their vital roles in the efficacy of immunotherapies. With the applications of scRNA-seq, exhausted CD8+ T cells are identified with multiple interconnected subpopulations with discernible heterogeneity (The functional states of CD8ogeneity . Nonetheogeneity . Thus, fogeneity . Clinicaogeneity . TherefoAll authors have discussed the proposed scope and content of the article before drafting. J-TD, K-PY, and H-NZ wrote and revised the paper. Y-FH collected literatures. HL and ZZ reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.


Trypanosoma cruzi, afflicts about 6 million people in Latin America. Here, we investigated the hypothesis that T. cruzi may fuel heart parasitism by activating B1R, a G protein-coupled (brady) kinin receptor whose expression is upregulated in inflamed tissues. Studies in WT and B1R−/− mice showed that T. cruzi DNA levels (15 days post infection—dpi) were sharply reduced in the transgenic heart. FACS analysis revealed that frequencies of proinflammatory neutrophils and monocytes were diminished in B1R−/− hearts whereas CK-MB activity (60 dpi) was exclusively detected in B1R+/+ sera. Since chronic myocarditis and heart fibrosis (90 dpi) were markedly attenuated in the transgenic mice, we sought to determine whether a pharmacological blockade of the des-Arg9-bradykinin (DABK)/B1R pathway might alleviate chagasic cardiomyopathy. Using C57BL/6 mice acutely infected by a myotropic T. cruzi strain (Colombian), we found that daily treatment (15–60 dpi) with R-954 (B1R antagonist) reduced heart parasitism and blunted cardiac injury. Extending R-954 treatment to the chronic phase (120–160 dpi), we verified that B1R targeting (i) decreased mortality indexes, (ii) mitigated chronic myocarditis, and (iii) ameliorated heart conduction disturbances. Collectively, our study suggests that a pharmacological blockade of the proinflammatory KKS/DABK/B1R pathway is cardioprotective in acute and chronic Chagas disease.Chagas disease, the parasitic infection caused by  Trypanosoma cruzi is the etiologic agent of Chagas disease (CD), a chronic parasitic disease that affects approximately 6 million people in Latin America des-Arg9-BK at 1 µM. Host–parasite interactions proceeded for 2 or 24 h at 37 °C in a humidified incubator in a 5% CO2 atmosphere. The involvement of the major cysteine protease of T. cruzi (cruzipain) was tested by pretreating the washed TCTs with K11777 at 10 µM for 20 min versus treatment with DMSO (0.025%) in PBS. After washing, the suspension of TCTs was added to the HUVEC monolayers. At the indicated time point, the cultures were washed three times with HBSS, once with PBS, then fixed with Bouin and stained with Giemsa. The efficiency of infection at 2 h or 24 h was measured as number of intracellular parasites in a total of 100 cells per coverslip. Human primary umbilical vein endothelial cells (HUVECs) were obtained by treatment of umbilical veins with a 0.1% (wt/vol) collagenase IV solution. Primary HUVECs were seeded in 25 cmThe concentrations of IFN-γ in the splenocytes culture were evaluated by enzyme-linked immunosorbent assay (ELISA) DuoSet kits  according to manufacturer’s instructions. −/− mice were infected with Dm28c TCTs . After 15 days, the animals were euthanized, and the spleen and heart were removed. The hearts were perfused with 10 mL of PBS after an incision to the superior vena cava, cut into small pieces and digested in DNAse  and collagenase  solution for 1 h at 37 °C. Splenocytes and cardiac cells were incubated with monensin (500 nM) for 8–10 hat 37 °C in the presence of a soluble extract of T. cruzi epimastigotes (50 μg/mL) plus H-2Kb-restrictedTskb20 peptide . The cells were collected, washed, resuspended in PBS containing 2% fetal calf serum and sodium azide 0.1% (FACS buffer) and then labeled for flow cytometry analysis. After Fc receptors were blocked with anti-CD16/CD32 (BD Biosciences), cells were stained with anti-CD8-FITC (Biolegend) and anti-CD4- PECy7 (Biolegend), fixed for 1 h in paraformaldehyde 1% at RT, and permeabilized for 20 min with FACS buffer containing 0.2% saponin (Sigma-Aldrich). Intracellular staining was performed with anti-IFN-γ-PE (BD Biosciences) and anti-Granzyme B-A647 (Biolegend) antibodies. At least 20,000 gated CD4+ cells or CD8+ cells were acquired.C57BL/6 and B1RT. cruzi-derived for 40 min at 37 °C (target cells) or unloaded (control), as previously described /[%CFSEhigh noninfected/%CFSElow noninfected] × 100%.Splenocytes from naive C57BL/6 mice were divided into two populations and incubated with H-2Kb-restricted TsKb20 peptide escribed . These p8]des-Arg9-BK ; R-954 was supplied by one of the co-authors (PS)  des-Arg9-BK . After being assured that Col TCTs infect HUVECs (24 h) via the cruzipain/B1R pathway, we then challenged C57BL/6 mice (i.p.) with a low inoculum of Col TCTs. Starting at 15 dpi, R-954 was administered daily for 45 days . Although the treatment with R-954 was initiated 45 days earlier, the B1R blocker did not reverse the heart rate alterations induced by the acute infection (p = 0.02). Likewise, the PR interval, which was not significantly increased at 60 dpi  (T. cruzi + R-954 ≈ 42 ms). The QRS duration, which reflects the duration of the depolarization of ventricles, did not differ between T. cruzi-infected mice (60 dpi) and controls . However, R-954 slightly reduced the duration of QRS complex registered in the infected (60 dpi) mice . The duration of the QT interval, corrected by Bazett’s formula (QTc), increased in infected mice (60 dpi) as compared to the NI group  . A second-degree atrioventricular block (AVB2) was observed in 67% of T. cruzi-infected mice (60 dpi.), while the NI animals did not present AVB2, as expected. The incidence of AVB2 in infected animals (60 dpi) was reduced (62%) by treatment with R-954 (T. cruzi-infected mice (60 dpi) was approximately 70% whereas R-954 treatment reduced its frequency to approximately 57% (p = 0.04). Recapitulating the cardioprotective phenotype of B1R−/− mice infected by the Dm28c strain (p = 0.02). In conclusion, R-954 treatment during the acute stage of infection (45 days) reduced the extent of heart parasitism and attenuated cardiac tissue injury. However, the B1R blocker did not reverse heart rates and had modest cardioprotective effects on the incidence of cardiac arrythmias. Next, we interrogated whether R-954 treatment (15–60 dpi) had an impact on cardiac function of the acutely infected mice. ECG records in sedated mice made at 60 dpi showed a reduction in the heart rate of infected mice (≈437 bmp) compared to the non-infected (NI) group (≈530 bmp) (≈ 44 ms) D, did no 0.0003) D, but thth R-954 D. Furthetely 57% D. In linc strain H, the rip = 0.033). Since the B1R antagonist reduced the extent of heart parasitism during the acute stage of infection, we then shifted the time window of R-954 treatment to the chronic stage of infection  and this alteration was not reversed by the B1R blocker (T. cruzi + R-954 ≈ 696 bpm)  but this response was not reversed by R-954 treatment (T. cruzi + R-954 ≈ 25 ms). Interestingly, however, measurements of the QRS duration (which did not change at 120 dpi), was prolonged at 160 dpi compared to the NI group . However, this ECG abnormality was reversed by R-954 treatment  , and this alteration observed in infected mice was reversed by R-954 treatment   D. Simila696 bpm) B, the PRd group) D. Consisd group) B, the QTd group) D. We thed group) B. The AVd group) D. The ind group) D. Chroniinterval . Importap = 0.0001). Using Picrosirius red staining, we verified that R-954 reduced collagen deposition in the majority (80%) of mice  to evaluate whether R-954 treatment modulated chronic myocarditis and heart fibrosis E–G and f of mice G, but st of mice E, treatm of mice H.T. cruzi has developed overlapping strategies to persistently infect immunocompetent mammalian hosts [Having evolved for millions of years in the sylvatic environment, an hosts ,56. Althan hosts ,16,17,18an hosts ,13,14,31an hosts . Consistan hosts . T. cruzi, in the current study, we provide evidence that the activation of the pro-inflammatory DABK/B1R pathway is detrimental to heart pathology in CD. Briefly recapitulating our main findings, we found that B1R was transcriptionally upregulated in the WT heart at 15 dpi, a time point in which the intracardiac load of Dm28c T. cruzi (15 dpi) was drastically increased as compared to B1R−/− mice. Analyses by flow cytometry revealed that frequencies of pro-inflammatory neutrophils and monocytes were increased in the WT heart. As the infection progressed, T. cruzi DNA levels (60 dpi) remained lower in cardiac tissues of B1R−/− mice. Moreover, CK-MB activity (60 dpi) was exclusively detected in the serum of acutely infected WT mice, implying that cardiac injury was attenuated in the absence of B1R. Finally, histopathological analysis revealed that chronic myocarditis and heart fibrosis (90 dpi) were markedly attenuated in B1R−/− mice, confirming that B1R plays a detrimental role in heart pathology. Using two different models of systemic infection with −/− mice described in our work is reminiscent of findings reported in a mouse model of diabetic cardiomyopathy induced by streptozotocin [At first sight, the cardioprotective phenotype of B1Rozotocin . Admitteozotocin ,42,43,58ozotocin , the assT. cruzi infection in transgenic mice with conditional deficiency of B1R in specific cell types. Since adipocytes constitutively express B1R, the possibility that T. cruzi may exploit this exceptional feature of adipose tissues comes to mind because adipocytes were previously identified as a privileged niche for intracellular outgrowth of T. cruzi [T. cruzi invasions of heart tissues are preceded by intracellular cycles of infection in the pericardial adipose tissue or coronary arteries. There are several limitations in our study, mostly related to the fact that we have not investigated the impact of T. cruzi . In the T. cruzi . Studiesb2r or b1r genes tends to overexpress B1R or B2R in a compensatory manner [T. cruzi-infected B1R−/− mice [−/− mice should have been narrowed if T. cruzi trypomastigotes had profited from compensatory overexpression of B2R in the inflamed heart tissues of the transgenic mice [−/− mice produced similar levels of IFN-γ, whether stimulated by crude T. cruzi antigens or MHC-class I-restricted T. cruzi peptides. Moreover, we did not detect differences in the frequencies of INF-γ-producing T cells nor GzB-expressing CD8+ T cells infiltrating the heart of WT and B1R−/− mice. Future studies using mice with genetic deficiency of B1R limited to the endothelium may clarify whether the activation of the KKS/B1R pathway may enhance TNF/TNFR1-dependent heart infiltration by CCR5+CD8+ effector T cells [Strictly analyzed from the perspective of experimental design, the first part of our study suffered from the fact that global ablation of y manner . Arguabl−/− mice . Howevernic mice . A seconnic mice ,25. In a T cells . Recipro T cells  might co T cells .T. cruzi might have differential ability to upregulate B1R via t-GPI-dependent activation of the TLR2 pathway [Since the egress of trypomastigotes from dying host cells is an asynchronous process ,9, the t pathway ,55. T. cruzi infection , it remains to be determined whether R-954 inhibited DABK-mediated internalization of trypomastigotes by particular subsets of B1-expressing heart cells or inhibited the invasion of a wide range of targets upregulating B1R. Alternatively, the B1R antagonist might have reduced T. cruzi infectivity by limiting plasma leakage and the proteolytic formation of infection-inducing peptides, such as BK and DABK [Involving C57BL/6 mice infected systemically by the Colombian strain, our pharmacological studies revealed that R-954 treatment during the acute phase (15–60 dpi) consistently reduced the number of pseudocysts in heart tissues. Noteworthy, we verified that serum levels of CK-MB were exclusively detected in acutely infected (untreated) mice, implying that the B1R antagonist mitigated cardiac injury caused by the intracellular parasite. Since inflamed heart tissues contain several types of cells that might be susceptible to T. cruzi infection in the spleen during the acute phase, the levels of blood parasitemia are dominantly influenced by the extent of parasite burden in splenic macrophages. Unlike the microcirculation of the heart and gastro-intestinal tissues, which are lined by non-fenestrated endothelium, plasma fluids diffuse freely through the sinusoids of the spleen and liver. As acute infection progresses, the phagocytic uptake of trypomastigotes by splenic macrophages may be vigorously stimulated as a result of free diffusion of plasma-borne opsonins, such as IgG antibodies and complement system factors, through the fenestrated endothelium of splenic sinusoids. Under the circumstances, it is unlikely that pharmacological targeting of B1R might interfere with the phagocytic uptake of T. cruzi by splenic macrophages and this may explain why R-954 failed to reduce blood parasitemia during acute infection. In a seemingly contradictory finding, we verified that R-954 treatment failed to reduce the blood parasitemia during the acute phase of infection. Since macrophages are the main targets of T. cruzi-infected dogs [Lasting several years, the low-grade intracardiac infection may gradually cause inflammatory lesions that translate into upregulated expression of B1R in cardiac micro vessels. Sporadically, a productive cycle of parasite outgrowth in B1R-expressing endothelial cells may cause microvascular injury, similar to the heart microangiopathy described in ted dogs . Potentited dogs . Strategted dogs ,44. Studted dogs ,58 may cted dogs . T. cruzi [As stated earlier, it is possible that a prolonged destabilization of the endothelial barrier forged via the activation of the KKS/DABK/B1R pathway might translate into reciprocal benefits to the host/parasite relationship ,14. NisiT. cruzi . InteresT. cruzi . Based oT. cruzi . ReciproT. cruzi . Although heart rates were not reversed by R-954 during the acute infection (60 dpi), the clinical benefits of B1R blockade were more persuasive when we shifted the drug treatment to the chronic stage of Col infection (120–160 dpi). During this advanced period of infection ,66,67,68Albeit speculative, it will be interesting to evaluate whether the sudden accumulation of plasma fluids within the intercellular spaces separating heart-conducting fibers might trigger cardiac arrythmia during chronic infection. MRI studies may clarify whether the intracardiac activation of pathogenic subsets of effector CD8 T cells may induce a prolonged myocardial edema via the B1R pathway. For reasons that are unclear, a small number of chronically infected mice were refractory to the anti-fibrotic effects of R-954. Additional studies are required to evaluate whether dysregulated activation of the KKS/DABK/B1R/ACE2 axis might dampen TGF-β-driven cardiac remodeling/fibrosis  via the + T cells (perforin and/or IFN-γ-producing) results in the upregulated expression of ADAM-17/TACE in the inflamed myocardium. By promoting the proteolytic cleavage and shedding of the ectodomain of ACE2 from the surface of cardiovascular cells, ADAM-17/TACE may indirectly (i) drive the hyperactivation of the pro-inflammatory KKS/B1R pathway and (ii) increase the levels of angiotensin II, indirectly stimulating fibrosis via the activation of angiotensin II receptors [From the perspective of disease prognosis, it will be important to determine whether the infiltration of heart tissues by different subsets of effector CD8eceptors ,40. For eceptors  translatT. cruzi infection suggest that the activation of the DABK/B1R pathway might fuel heart parasitism during the acute stage of infection. Beyond the impact of KKS activation on T. cruzi infectivity, we provided evidence that activation of B1R worsens the severity of chronic myocarditis and fibrosis. The findings that R-954 reduced mortality indexes, inhibited chronic myocarditis, and ameliorated disturbances in heart impulse conduction during the late stage of infection suggest that severity of chronic chagasic cardiomyopathy may be attenuated via pharmacological targeting of the KKS/B1R pathway. In summary, the results of studies of KKS function in two mice models of


In particular, by combining the selectivity and high stability properties, which are the most important advantages of molecular imprinted polymers, and the highly sensitive properties of GQDs/B-g-C3N4 nanocomposite, a highly selective and sensitive analytical method was developed for BPA analysis. Firstly, GQDs/B-g-C3N4 nanocomposite was characterized by using microscopic, spectroscopic, and electrochemical techniques. This novel molecular imprinted electrochemical sensor for BPA detection demonstrated a linearity of 1.0 × 10−11–1.0 × 10−9 M and a low detection limit . BPA-imprinted polymer on GQDs/B-g-C3N4 nanocomposite also showed good stability, repeatability and selectivity in food samples.A molecular imprinted electrochemical sensor based on boron-functionalized graphitic carbon nitride (B-g-C BPA is frequently encountered in the stocking and distribution of solid and liquid nutrients. In terms of its endocrinological effects, BPA has potential adverse effects on the receptors of hormones such as estrogen, androgen, and thyroid hormone. It has been shown that BPA has organ toxicity in experimental animals, and it has been reported that the tolerable daily dose of this molecule can be up to a maximum of 4 μg/kg bw. It is absorbed in a short time from the gastrointestinal tract and conjugated with UDP-glucuronic acid in the intestine and liver to form the BPA-glucuronide (BPA-gluc) metabolite, which is excreted through urine and feces . In addi3N4 is a polymeric two-dimensional nanomaterial with superior surface features and quantum properties providing increased sensitivity and performance of the developed electrochemical biosensors/sensor  was obtained on MIP/GQDs/B-g-C3N4/GCE by using BPA amounts and current signals  = 9.8091 × . The electrochemical signals (µA) were obtained for BPA, HDP, DOP, 4-NIT, ETH, and BPS on MIP/GQDs/B-g-C3N4/GCE and NIP/GQDs/B-g-C3N4/GCE  with a low LOD of 3.0 × 10−12 M, providing superior sensitivity and selectivity. This low LOD may be due to the following: (i) the doping treatment of GQDs and the boron element into g-C3N4 resulted in the specific surface area, indicating more interactions with BPA molecules; (ii) the synergistic effect between GQDs and the boron element increased easy electron transfer on the electrode surface. In conclusion, a new and efficient electroanalytical method was constructed for a sensitive bisphenol A sensor to obtain early disease diagnosis and safe food consumption.In conclusion, a novel molecularly imprinted electrochemical sensor based on GQDs/B-g-C


In pancreatic islet transplantation, the exact contribution of human leukocyte antigen (HLA) matching to graft survival remains unclear. Islets may be exposed to allogenic rejection but also the recurrence of type 1 diabetes (T1D). We evaluated the HLA-DR matching, including the impact of diabetogenic HLA-DR3 or HLA-DR4 matches.We retrospectively examined the HLA profile in 965 transplant recipients and 2327 islet donors. The study population was obtained from patients enrolled in the Collaborative Islet Transplant Registry. We then identified 87 recipients who received a single-islet infusion. Islet-kidney recipients, 2nd islet infusion, and patients with missing data were excluded from the analysis (n=878).HLA-DR3 and HLA-DR4 were present in 29.7% and 32.6% of T1D recipients and 11.6% and 15.8% of the donors, respectively. We identified 52 T1D islet recipients mismatched for HLA-DR (group A), 11 with 1 or 2 HLA-DR-matches but excluding HLA-DR3 and HLA- DR4 (group B), and 24 matched for HLA-DR3 or HLA-DR4 (group C). Insulin-independence was maintained in a significantly higher percentage of group B recipients from year one through five post-transplantation (p<0.01). At five-year post-transplantation, 78% of group B was insulin-independent compared to 24% (group A) and 35% (group C). Insulin-independence correlated with significantly better glycemic control (HbA1c <7%), fasting blood glucose, and reduced severe hypoglycemic events. Matching HLA-A-B-DR (≥3) independently of HLA- DR3 or HLA-DR4 matching did not improve graft survival.This study suggests that matching HLA-DR but excluding the diabetogenic HLA-DR3 and/or 4 is a significant predictor for long-term islet survival. Sincant.org) , 7.On the other hand, HLA antigens are also essential contributors to autoimmune diseases’ pathogenesis. In type 1 diabetes (T1D), specific allele combinations have been associated with >40 increased odds ratio of developing this disease. These include the DRB1*0301-DQA1*0501-DQB1*0201 (DQ2.5) and the DRB1*0401-DQA1*0301-DQB1*0302 DQ8) haplotypes  haplotyp–11. AddiThis study retrospectively analyzed donor and T1D recipient’s HLA profile as reported in the Collaborative Islet Transplant Registry (CITR), the most comprehensive human-to-human islet transplant data collection. We analyzed the impact of HLA-DR matching including the diabetogenic HLA-DR3 and HLA-DR4 on long-term insulin-independence, fasting C-peptide levels, HbA1c %, and severe hypoglycemic events.https://citregistry.org/content/citr-11th-annual-report). The Data Coordinating Center verified participant voluntary informed consent.The CITR is a registry including pancreatic allogeneic islet transplant of Phase I/II/III clinical trials and standard of care since 1999 from North America, European and Australian centers. The CITR is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the Juvenile Diabetes Research Foundation International (2006-2015). T1D islet recipient’s data were previously described  and are Briefly, T1D islet recipients are aged between 18- and 65-year-old and have T1D for at least five years with a documented negative fasting C-peptide (<0.3ng/mL). Additionally, they have impaired hypoglycemia awareness and suffer from repetitive and severe hypoglycemia episodes reflected by glycemic lability and elevated HbA1c levels (>8%). In the first part of the study, HLA antigens  were assessed in 965 islet transplant recipients  and 2327 pancreas donors enrolled from 1999 until 2019. The HLA-matching analysis was then conducted in T1D recipients with single islet infusion (N=87). The outcomes were computed in follow-up post infusion for up to five years or until the next infusion or last available follow-up. One year of follow-up was minimally required. Seven recipients received an islet infusion pooled from two donors. Fasting C-peptide, insulin independence, HbA1c levels and severe hypoglycemic events were analyzed. We defined 5 groups: (A) zero HLA-DR matching; (B) 1 or 2 HLA-DR matching, but excluding HLA-DR3-DR4 matching; (C) HLA-DR3 and/or DR4 matching; (D) <3 HLA-A, -B, -DR matching; (E) 3-6 HLA-A, -B, -DR matching. Of note, Group B could include HLA-DR3 or HLA-DR4 donors if they were mismatched to the recipients (n=2/11).http://hla.alleles.org/antigens/broads_splits.html). Because each individual has two alleles, one HLA can be present one or two times. If only one class I or class II HLA was reported, the individual was considered homozygous for this HLA. T1D islet recipients with missing HLA-DR typing information were excluded from the analysis . HLA A/B/DR matches were defined at the broad level. Finally, HLA-C, HLA-DP and HLA-DQ typing were not examined because this information was not collected.From a total of 1403 allograft islet recipients, 965 had HLA data entered in the CITR. Data on HLA class I  specificities and class II (DR) specificities in islet donors (N=2327) were also retrieved from the CITR registry. This analysis was performed independently of the exclusion criteria used to study the role of HLA-DR matching (see below). HLAs were defined as present or missing information. The classification of HLA changed over time from serological typing to DNA genotyping. As the precision level of HLA typing varied across techniques , time and centers, we regrouped the HLA antigen analysis at the broad and not split level. Split HLA specificities were at first technically undistinguishable at the broad level. As HLAs were reported in the CITR registry either at the broad or split level, we converted split HLA at their broad level to keep the analysis homogenous. Split and broad HLA classification can be found online .via mixed effects models with repeated measures per subject . Primary efficacy outcomes were analyzed following first infusion.Multivariate modeling for each primary efficacy outcome  was done The prevalence of insulin independence is the optimal way to characterize the probability of being insulin independent in follow-up time post islet transplantation Thus, insulin independence can be lost and re-gained, often over periods spanning months or years. Prevalence also reconciles disparities in factors that may predict retention but not achievement or vice versa.All reported p-values are observed, nominal, and outside of any pre-planned Type I error structure. In drawing conclusions, readers should be mindful that the significance levels control for random variance, but not systematic biases in the data nor multiple testing. However, these analyses do provide insight and direction for future questions and analyses.From 1403 allograft recipients enrolled in CITR, 965 had HLA data entered. The analysis of the HLA-A, -B, -DR broad antigens was conducted in 965 pancreatic islet graft recipients and 2327 donors from the CITR. On the broad antigen level, fold change difference for HLA-A  expression comparing T1D islet recipients and pancreas donors ranged between 0.5 (HLA-A11) to 1.4 (HLA-A1); for HLA-B  between 0.4  to 2.2 (HLA-B8); for HLA-DR  between 0.1 (HLA-DR10) to 2.6 (HLA-DR3). Among all, the highest fold change differences were HLA-B8, HLA-DR3 and HLA-DR4, i.e 2.2, 2.6 and 2.1 times more frequent in T1D recipients respectively Table 1.per se as a risk factor for developing T1D   and one with 3-6 HLA matching . Of note 25/87 recipients were excluded, one because of incomplete HLA (A or B) reporting, the other 24 were the DR3/4 matches. Key baseline characteristics of islet transplant recipients and donors were similar between both groups Table 5.This study investigated first the benefit of HLA-DR matching in islet transplantation alone.We not only confirmed that HLA-DR3 and HLA-DR4 prevalence was higher in T1D islet recipients than donors, but also found that long-term insulin independence was significantly better when matching donor islets for HLA-DR but not the diabetogenic HLA-DR3 and HLA-DR4. This correlated with C-peptide production, better glycemic control and reduced severe hypoglycemia.Opportunities for HLA matching in islet transplantation have always been limited and therefore poorly studied given the small numbers of patients on the waiting list and the frequent need for multiple islet infusions expositing patients to multiple HLA mismatches. Interestingly, when reviewing data from pancreas transplantation, results are controversial . Data puEvidence from animal studies suggests that autoantigen presentation is more effective in an MHC matched context increasing the risk of T1D relapse , 26. SucT1D relapse is more studied after pancreas transplantation. In a prospective cohort of 223 recipients of simultaneous pancreas–kidney allografts, the group of Pugliese estimated that recurrence of autoimmunity occurred in 7% of the cases . A selective loss of beta cells was proven histologically . IdentifThis study has several important limitations. First, only a limited number of T1D islet recipients could be included hampering more extensive analysis on the mechanisms driving the loss of insulin-independence and the potential confounding factors. Future sub-analyses comparing outcomes of HLA-DR3 versus HLA-DR4 or HLA-DR3/DR4 double positive (n=14) T1D patients are warranted. Additionally, some protective loci, such as HLA DR15 (DR2 in the old nomenclature) for the development of type I diabetes could represent a significant confounder in our analysis . In futuIn conclusion, our results suggest that matching HLA-DR but excluding the diabetogenic HLA-DR3 and/or 4 is a strong predictor for insulin-independence after single islet infusion. Further studies to confirm and extend the role of diabetogenic HLA DR and associated haplotypes with higher resolution and for multiple islet donors are now needed. Importantly, the feasibility of avoiding the diabetogenic HLA-DR in clinical practice need to be addressed.The raw data that support the findings of this study may be available on request from the Collaborative Islet Transplant Registry Coordinating Center.CITR data are rigorously monitored by the Data Coordinating Center, The EMMES Corporation, Rockville, Maryland, to comply with U.S. Food and Drug Administration Part 21 Code of Federal Regulations requirements. Site participation in the Registry requires local research ethics board approval, strict assurance of patient-identifier confidentiality, and written informed consent by the islet recipients. The CITR Publications and Presentations Committee approved the manuscript.CB and FB researched data and performed the analysis. EP researched data and contributed to discussion. YM, JV, RM and TB designed the original project. YM wrote the manuscript. DB, RA, RM, TE and MK contributed to the discussion and reviewed/edited the manuscript. All authors contributed to the article and approved the submitted version.


Ulcerative colitis (UC) is considered an immune-mediated disease. The disorder of T-lymphocyte subsets plays an important role in the pathogenesis of UC. The aim of this study was to evaluate the significance of peripheral blood T-lymphocyte subsets in assessing disease severity and predicting clinical outcomes in UC patients.t-test, Spearman’s correlation coefficient, multivariable Cox regression analysis, receiver operating characteristic (ROC) curves and cumulative survival analysis were done.The retrospective case-control study was performed in 116 UC patients with active disease and 90 healthy controls (HC). The UC patients included were followed up for 180 days. Analyses of +T cells  and higher proportion of CD8+T cells  than HC. The severely active UC patients had higher proportion of CD3+HLA-DR+ T cells  and CD8+T cells  than mild and moderate group, whereas lower proportion of CD4+CD25+T cells  than mild group and CD4+T cells  than moderate group. The area under the curve (AUC) of CD3+HLA-DR+ T cells for assessing severely active UC was 0.885, with the cut-off value of 5.33%. The sensitivity was 76.32% and specificity was 89.74%. The combination of CD3+HLA-DR+ T cells and CRP had stronger assessment value with AUC of 0.929. The AUC of CD8+T cells, CD4+/CD8+ ratio and CD4+CD25+T cells for assessing disease severity was 0.677, 0.669 and 0.631 respectively. Within the 180 days follow-up, 24 patients (20.69%) had UC-related readmission or surgery, with higher proportion of CD3+HLA-DR+ T cells  and CD8+T cells  than those without readmission and surgery. The proportion of CD3+HLA-DR+ T cells was the independent predictor of UC-related readmission or surgery . The AUC of CD3+HLA-DR+ T cells for predicting readmission or surgery was 0.796 with the cut-off value of 5.38%. UC patients with CD3+HLA-DR+T cells proportion>5.38% had a shorter time to readmission or surgery .The UC patients had lower proportions of CD4+HLA-DR+T cells and CRP may be potential biomarker of disease severity in UC patients. The high proportion of CD3+HLA-DR+T cells may be associated with an increased risk of readmission or surgery in UC patients.The combination of CD3 Ulcerative colitis (UC) is one form of inflammatory bowel disease (IBD) characterized by chronic recurrent inflammation of colon and rectum. UC is considered a progressive disease and approximately 20% UC patients may require hospitalization because of severe disease activity and 10%-15% are at risk of colectomy during the disease course , 4. ColoUC is considered an immune-mediated disease, in particular the widespread and successful use of biological agents targeting immune abnormalities of UC patients in recent years , 11. TheThe study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (NO. QYFY WZLL 27363). The need for written informed consent was waived by the Ethics Committee of the Affiliated Hospital of Qingdao University due to retrospective nature of the study. The study protocol has been registered in the Chinese Clinical Trial Registry (identifier: ChiCTR2200065103).This retrospective case-control study included 116 patients with confirmed diagnoses of UC and 90 healthy controls (HC) admitted to the Affiliated Hospital of Qingdao University from January 2018 to December 2021. The inclusion criteria of UC group were as follows: (I) A confirmed diagnosis of UC based on the combination of clinical, laboratory, endoscopic and histopathological evaluation ; (II) AlParticipants were followed up for 180 days from the time of T-lymphocyte subsets measurement. The clinical outcomes were defined as UC-related readmission due to recurrence and underwent bowel surgery due to failure of medical therapy.The disease severity of UC patients was assessed by the Mayo score according to stool frequency, rectal bleeding, findings on endoscopy and physician’s global assessment, and further categorized into the mild activity 3-5 scores), moderate activity 6-10 scores) and severe activity (11-12 scores)  scores a. The dis-5 scores+T cells, CD4+T cells, CD8+T cells, CD4+/CD8+ ratio, CD4+CD25+T cells and CD3+HLA-DR+T cells. The demographic and clinical data were collected including sex, age, body mass index (BMI), disease duration, smoking history, alcohol consumption history, history of appendectomy, extraintestinal manifestation and the treatment which the UC patients were taking. The laboratory data were recorded including the counts of white blood cells (WBC), neutrophils (NE), lymphocytes (LYM), platelets (PLT), hemoglobin (Hb), albumin (Alb), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). All demographic, clinical and laboratory data were obtained within 7 days of T-lymphocyte subsets measurement.Peripheral blood T-lymphocyte subsets of participates were recorded containing the proportion of CD3t-test (or Mann-Whitney U test as appropriate), comparisons among mild, moderate and severe subgroups of UC patients were used by one-way analysis of variance with least significant difference post-hoc test for continuous variables and chi-square test (or Fisher’s exact test as appropriate) for categorical variables. The correlations between the proportion of T-lymphocyte subsets and the Mayo score and UCEIS were detected using Spearman’s rank correlation coefficient. The efficacy of T-lymphocyte subsets for assessing severe activity, UC-related readmission and surgery were evaluated by receiver operating characteristic (ROC) curve analysis and the cut-off value were confirmed with maximum sensitivity and specificity. The independent prognostic factors associated with UC-related readmission and surgery were determined using the multivariate Cox proportional hazard regression analysis, with the hazard ratio (HR) and 95% confidence interval (CI) describing the relative effect. The impact of high proportion of CD3+HLA-DR+T cells on the clinical outcomes of UC was evaluated with the Kaplan-Meier (K-M) curves and log-rank test. SPSS 26.0 software (IBM) was used to analyze data and P-value<0.05 was defined as statistical significance.Quantitative variables with normal distribution were presented as mean ± standard deviation, while as median and interquartile range with non-normal distribution. Qualitative variables were presented as numbers and percentages. The Kolmogorov Smirnov test was used to test detect the normality of the data. Comparisons of the clinical baseline data between UC patients and healthy controls were used by P>0.05). The UC patients were divided into mildly active group including 37 patients, moderately active group including 41 patients and severely active group including 38 patients according to Mayo score. The demographic and clinical characteristics of participates were shown in Table A total of 116 UC patients with active disease and 90 HC were included in our study. Among the 116 UC patients, 69 men (59.5%) and 47 women (40.5%) were included with the average age of 46.75±16.47 year-old. There was no statistical difference in gender and age between UC patients and HC (P<0.001), lymphocytes (LYM) count (P=0.005), hemoglobin (Hb) (P<0.001) and albumin (Alb) levels (P<0.001) were lower in UC patients, whereas the white blood cells (WBC) (P<0.001), neutrophils (NE) (P<0.001) and platelets (PLT) (P<0.001) counts were higher. There was no significant difference in smoking history, alcohol consumption and history of appendectomy between UC patients and HC (P>0.05). Compared among three subgroups of UC patients, the severely active UC patients had lower BMI (P<0.001), Hb (P<0.001), and Alb levels (P<0.001), as well as higher PLT counts (P<0.001), CRP (P<0.001) and ESR level (P<0.001) than mildly and moderately active group. Moreover, there were more patients with extensive disease extent in the severely active UC patients compared with the mildly and moderately active group . The severely active UC patients had higher WBC (P<0.001) and NE counts (P<0.001) than the mildly active group.Compared with HC, the body mass index (BMI) (+T cells (P=0.021) and CD4+/CD8+ ratio (P=0.025), as well as higher proportion of CD8+T cells (P=0.008) in UC patients. Compared among three subgroups of UC patients, the difference in T-lymphocyte subsets composition was more pronounced in severely active UC patients, with higher proportions of CD8+T cells  and CD3+HLA-DR+T cells  than mildly and moderately active group. Moreover, the severely active UC patients had lower proportions of CD4+CD25+T cells (P=0.034) than mildly active group and lower proportions of CD4+T cells (P=0.049) than moderately active group. There was no statistical difference of proportions of CD3+T cells, CD4+T cells , CD8+T cells, CD4+CD25+T cells and CD3+HLADR+T cells between mildly and moderately active UC patients , and negative correlation with the proportion of CD4+CD25+T cells   for predicting severe UC was 0.885 (P<0.001). The cut-off value of CD3+HLA-DR+T cells proportion was 5.33%, with the sensitivity of 76.32%, and the specificity of 89.74%. Meanwhile, the efficacy of CD3+HLA-DR+T cells for assessing disease severity was higher than CRP and ESR levels in UC patients, with the AUC of 0.878  and 0.797 respectively , 0.669 (P=0.003) and 0.631(P=0.022) respectively. The proportions of CD3+T cells and CD4+T cells did not show the value in assessing the disease severity of UC patients (P>0.05).In order to analyzed the value of T-lymphocyte subsets proportion in assessing disease severity of UC patients, ROC analysis was performed on the UC patients Figure A. On ROC+HLA-DR+T cells combined with CRP level in assessing the disease severity. Binary logistic regression model was performed with disease severity as the dependent variable, CD3+HLA-DR+T cells proportion and CRP level as the independent variables. The combined predicted probability was calculated according to the model, which was used to plot the ROC curve of CD3+HLA-DR+T cells proportion combined with CRP level. The combination of these two indicators had stronger efficacy for predicting severe UC, with the AUC of 0.929, the sensitivity of 89.47%, and the specificity of 85.90% (P<0.001)  and CD8+T cells (P=0.039). Other factors associated with UC-related readmission or surgery included higher levels of Mayo score (P<0.001) and PLT (P<0.001), as well as lower levels of Hb (P<0.001), Alb (P<0.001) and BMI (P<0.001)  and BMI  remained the independent predictors for readmission or surgery .We further explore whether peripheral blood T-lymphocyte subsets were associated with UC-related readmission due to recurrence and surgery. There were 24 (20.69%) patients required readmission or underwent surgery during the follow-up of 180 days. The patients experiencing UC-related readmission or surgery had higher baseline proportion of CD3+HLA-DR+T proportion for predicting clinical outcomes. The data showed CD3+HLA-DR+T cells proportion >5.38% has predicted the UC-related readmission or surgery with the AUC of 0.796    is a late marker of T lymphocyte activation . The actactivity , 22. Morrotectin , 22, 25.rotectin . On the rotectin . That is+HLA-DR+T cells and the poor clinical outcomes in UC patients. We found the proportion of CD3+HLA-DR+T cells was an independent risk factor of readmission and surgery for UC patients. The K-M survival analysis further demonstrated the impact of increased proportion of CD3+HLA-DR+T cells for requirements of readmission or surgery. Patients with the proportion of CD3+HLA-DR+T cells>5.38% were more likely to underwent the UC-related readmission or surgery, which need more frequent follow-up and adjust the treatment timely in order to reduce the risk of relapse and surgery. Dulic et al. [+T cells is associated with the duration of remission in IBD patients. Smids et al. [A further important finding of the present study is the correlation between the proportion of CD3c et al. showed ths et al. found the+T cells to luminal antigen [+T cells in UC patients was related to active disease and more future complications [+T cells than HC. However, the value of CD4+T cells for assessing disease severity and predicting clinical outcomes was not showed in our study although similar results have been reported previously [+T cells, which comprise varying proportions of highly differentiated subsets [+T cells have drawn increased attention in recent years. Several colitis models indicated that CD8+T cells-mediated destruction of intestinal epithelial cells led to the damage of the mucosal barrier, and then activated CD4+T cells and other immune cells, ultimately caused the intestinal inflammation [+T cells was related to disease activity and had prognostic value for disease course in UC patients [+T cells. Although the proportion of CD8+T cells>27.03% predicted the severely active UC, the sensitivity and specificity of this indicator was not high in our study. More studies are needed to explore the potential of CD8+T cells as a biomarker of assessing disease severity for UC patients. The study found that UC patients had a reduced CD4+/CD8+ ratio than HC, which indicated the immune function disorder [+/CD8+ ratio of HC is approximately 2:1, which is consistent with previous studies [The intestinal inflammation in UC has been traditionally contributed to abnormal immune response of CD4 antigen , 29. Preications . The preeviously . The reaammation , 30, 31.patients , 21. In disorder , and the studies .+CD25+ regulatory T (Treg) cells play pivotal roles in the maintenance of normal gut homeostasis, suppressing abnormal immune responses by directly inhibiting the activation and effector function of T cells and inducing various anti-inflammatory cytokines [+CD25+T cells was correlated with the clinical and endoscopic scores and UC patients with the proportion of CD4+CD25+T cells<3.05% were indicative of severe disease activity. However, the evaluation value of CD4+CD25+T cells was not high, which need further studies to explore whether it could be clinically useful as indicator of disease severity. Several studies have also shown that depletion of Treg cells in peripheral blood was correlated with the disease activity of UC, which was consistent with our results, whereas they were increased in the intestinal mucosa [CD4ytokines , 23. Thil mucosa , 35. Thil mucosa , 34.This study has several strengths. To our knowledge, this was the first study exploring the association between peripheral blood proportion of T-lymphocyte subsets and severely active UC, as well as its prognostic value. Moreover, the indicators of peripheral blood T-lymphocyte subsets which we analyzed in this study were easily obtained and tested. This enables T-lymphocyte subsets detection to be applied widely as an evaluation indicator of disease severity in UC patients and may guide clinicians timely adjust the treatment. Nevertheless, there were some limitations. This was a single-center retrospective study that inevitably had a selective bias. And the follow-up time was relatively short. Therefore, a prospective and multi-center research is needed to further explore and confirm these results.+HLA-DR+T cells, CD8+T cells, CD4+/CD8+ ratio and CD4+CD25+T cells may be the promising biomarkers of severe disease activity in UC patients. The combination of CD3+HLA-DR+T cells and CRP levels is more informative in assessing the disease severity. The high proportion of CD3+HLA-DR+T cells may be associated with an increased risk of readmission and surgery in UC patients.This study showed the proportion of specific T-lymphocyte subsets, such as CD3