Objective: People living with HIV (PLWH) have an increased cardiovascular risk (CVR) owing to dyslipidemia, insulin resistance, metabolic syndrome, and HIV/combination antiretroviral therapy (cART)-associated lipodystrophy (HALS). Atherosclerosis and inflammation are related to growth differentiation factor-15 (GDF15). The relationship between metabolic disturbances, HALS, and CVR with GDF15 in PLWH is not known. Research design and methods: Circulating GDF15 levels in 152 PLWH  and 34 healthy controls were assessed in a cross-sectional study. Correlations with lipids, glucose homeostasis, fat distribution, and CVR were explored. Results: PLWH had increased circulating GDF15 levels relative to controls. The increase was the largest in cART-treated PLWH. Age, homeostatic model assessment of insulin resistance 1 (HOMA1-IR), HALS, dyslipidemia, C-reactive protein, and CVR estimated with the Framingham score correlated with GDF15 levels. The GDF15-Framingham correlation was lost after age adjustment. No correlation was found between GDF15 and the D:A:D Data Collection on Adverse Effects of Anti-HIV Drugs (D:A:D) score estimated CVR. CVR independent predictors were patient group  and cumulated protease inhibitor or nucleoside reverse transcriptase inhibitor exposure. Conclusions: PLWH, especially when cART-treated, has increased GDF15 levels—this increase is associated with dyslipidemia, insulin resistance, metabolic syndrome, HALS, and inflammation-related parameters. GDF15 is unassociated with CVR when age-adjusted. Combination antiretroviral therapy (cART) has forever changed the landscape for people living with HIV (PLWH) . ConsistA scenario of atherogenic dyslipidemia is depicted in cART-treated PLWH owing to lipid disturbances and insulin resistance, which may translate into increased CVR . BesidesGrowth differentiation factor-15 (GDF15) or macrophage inhibitory cytokine-1, a member of the transforming growth factor-b superfamily, is related to inflammation, chronic vascular diseases, cancer, ischemia, and atherosclerosis, behaving as a lesion-induced factor ,11. GDF-PLWH are prone to accelerated atherosclerosis, metabolic syndrome and increased risk of cardiovascular events, especially when developing HALS. Because GDF15 is suggested as a marker of atherosclerotic burden and metabolic dysregulation, our working hypothesis was that circulating GDF15 levels could be associated with CVR, metabolic alterations and HALS in PLWH.This is a retrospective cross-sectional study see , the priBlood was obtained in the morning, between 8 a.m. and 9 a.m. local time, after a 12 h overnight fast and at least 15 min after placing a peripheral intravenous catheter. Serum samples were split stored as frozen aliquots and when samples recruitment had been completed, a set was delivered to the core laboratory at Hospital Santa Creu i Sant Pau, Barcelona (Spain) for biochemistry laboratory measurements and another to set to the Department of Biochemistry and Molecular Biomedicine of the University of Barcelona (Spain) where GDF15, cytokine and fatty acids were measured. All samples were processed at the same time for analyte quantification. As previously described, all lipid measurements were performed using a Hitachi 911 system  . The diaWeight, body mass index (BMI), and waist circumference were measured as described elsewhere . Whole-bSerum GDF15 levels were determined in duplicate for each sample using an ELISA specific for human GDF15   that detects specific human GDF15. Serum levels of IL-6, IL-8, TNF-a, monocyte chemoattractant protein 1 (MCP-1) and leptin, were detected in duplicate for each sample using an antibody-linked, fluorescently labeled microsphere bead-based multiplex analysis system  and quantified using Luminex 100ISv2 equipment . Serum adiponectin levels were measured, also in duplicate, using a specific ELISA kit for human adiponectin  . The serum composition of fatty acids was determined using the method of Lepage and Roy as previously described  cells/mm3). Nadir CD4 cell count was <100 cells/mm3 in 41 patients (26.9%). There was a strong correlation between cumulated exposure to nucleoside reverse transcriptase inhibitors (NRTI) , especially thymidine analogues , and abacavir  with GDF15 circulating levels, even when age-adjusted. Cumulated exposure to protease inhibitors (PI)  also correlated with GDF15 levels  had undetectable viral load at study entry. The other 20 treated patients presented a median detectable viral load of 2.4 log5 levels . The ass10 pg/mL. There was correlation between GDF15 levels and age . There were no differences in GDF15 levels by gender. GDF15 levels were higher in patients than in controls  (p < 0.0001). GDF15 levels were higher than 3.08 (1200 ng/L) and 3.26 log10 pg/mL (1800 ng/L) in 35% and 24.5% of patients, respectively, compared with 15.4% and 7.7% of controls (p = 0.06 for both). GDF15 levels were not significantly different in smokers, HCV, or HBV co-infected patients, neither regarding their HIV detectability or CD4 count status, but were higher in diabetic (p = 0.001) and hypertensive patients (p = 0.043) (The median serum GDF15 levels for the whole cohort were 2.86 (IQR: 2.56–3.21) log= 0.043) .10 pg/mL for those with or without metabolic syndrome (p = 0.044).The correlation between anthropometric, metabolic, circulating and infection/treatment-related parameters and GDF15 in PLWH is shown in GDF15 levels correlated with LDL cholesterol, PUFA, MUFA, FGF21, and insulin resistance, even after age adjustment, whereas they did not correlate with triglycerides, HDL cholesterol, or non-HDL cholesterol . GDF15 lp < 0.0001). Among PLWH, CVR strata were <10% in 96 patients (63.1%), 10–19% in 34 (22.4%) and >20% in 22 (14.5%). Serum GDF15 levels correlated with Framingham score risk, but this correlation was lost after adjustment for age (p = 0.5984), even without age adjustment. Correlations with CVR scores are shown in 10 pg/mL in patients with prior CVD, whereas they were 2.82 [IQR 2.55–3.15] log10 pg/mL in those without (p = 0.041).Because alterations in GDF15 circulating levels have been associated with increased atherosclerotic burden as mentioned before ,17, we n for age . The medp = 0.001; HOMA1-IR, r = 0.234, p = 0.010; FGF21, r = 0.216, p = 0.011), duration of infection , cumulated NRTI exposure , non-nucleoside reverse transcriptase inhibitors (NNRTI) , and lopinavir/ritonavir (LPV/r) . On removing patients with eGFR <60 mL/min/1.73 m2, glucose homeostasis markers remained correlated to GDF15 levels  as well as cumulated NRTI exposure , NNRTI = 0.212, p= 0.019, and LPV/r All significant correlations with glucose homeostasis and triglycerides disappeared after removal of patients with diabetes mellitus. After removal of patients with previous CVD, GDF15 levels remained correlated to markers of insulin resistance  and PI total exposure (p = 0.082). The analysis using the five-year D:A:D CVR leads to similar results for the risk correlations and for identifying independent predictors: group of patients , NRTI total exposure (p = 0.042) and PI total exposure (p = 0.031).An ordinal logistic regression analysis was performed in order to determine which variables could act as independent predictors of CVD risk. Both risk scores (Framingham and D:A:D) were used as the dependent variables, and duration of infection, patient group, nadir CD4 cell count, current HIV-RNA, BMI, systolic BP, diastolic BP, trunk/appendicular fat ratio, triglycerides, total cholesterol/HDL ratio, cumulative NRTI and PI exposure, AIDS, C reactive protein, and GDF15 levels were used as independent variables. CVR was categorized using the standard cut-off risk levels . The univariate analysis, conducted for all variables, identified the same variables as those statistically significant in analyzing correlations with CVR , except Our study shows that PLWH has increased GDF15 levels compared with uninfected controls, in agreement with previous reports ,33,34. I10 pg/mL) was derived as the upper limit of normal (90th percentile) in healthy European men and women, representing, at the same time, the lower tertile boundary in patients presenting with acute coronary syndromes [10 pg/mL), which was clearly associated with increased mortality [10 pg/mL in a third and a quarter, respectively, suggesting that a substantial proportion of patients in our cohort is at high risk for a cardiovascular event. Despite that, we could not correlate GDF15 levels with estimated CVR by Framingham and D:A:D score when adjusted by age.GDF15 concentrations have been associated with total and cardiovascular mortality and inconsistently with nonfatal myocardial infarction ,37, and yndromes . The upportality ,39,40. AHigh GDF15 circulating levels have been observed in patients with type II diabetes mellitus or obesity related to impaired glucose control . This siEven though the cellular source of GDF15 in diabetic and obese patients has not been established, it is known that GDF15 is expressed in and secreted from human adipocytes and possibly by macrophages infiltrating adipose tissues , and it Dyslipidemia and HALS are contributed by the use of specific antiretroviral drugs, such as thymidine analogs and first-generation PI; others such as abacavir have been associated with an increased risk of myocardial infarction . In addiOur study has the inherent limitation of being a cross-sectional study, and therefore no causal inferences can be made. However, the inclusion in our study of a remarkable number of patients spanning a wide range of cART treatment conditions and extent of HALS development allowed us to obtain substantial conclusions. Our study establishes the strong association of GDF15 levels with metabolic dysregulation and inflammation and highlights also its association with fat distribution alterations in PLWH. However, despite the existing evidence of association of circulating GDF15 levels and cardiovascular event in uninfected individuals and PLWH, we could not find such an association of GDF15 with estimated CVR in PLWH. Despite this, the association of GDF15 with insulin resistance, dyslipidemia and pro-inflammatory cytokines likely reflects an overall state of metabolic derangement and inflammation in PLWH, known to be related with vascular pathologies. In this sense, the possibility that the assessment of CVR by risk scores may underestimate the actual CVR in PLWH—as no direct imaging assessment of atherosclerotic burden was available in the patients—cannot be ruled out. Moreover, some reports indicated that CVR scores may show low sensitivity and specificity in HIV-infected men, with a systematic CVR underestimation ,51, and In conclusion, PLWH show increased levels of GDF15, this rise particularly marked when treated with cART. GDF15 levels correlated positively with parameters indicative of altered metabolism  and inflammation) but did not correlate with currently used indices of CVR  when age-adjusted. Further research is warranted to elucidate the role and potential usefulness as a biomarker of GDF15 in relation to the distinct comorbidities occurring in PLWH.


To ascertain the role of inflammation in the response to ocrelizumab in primary-progressive multiple sclerosis (PPMS).Multicenter prospective study including 69 patients with PPMS who initiated ocrelizumab treatment, classified according to baseline presence  or absence  of gadolinium-enhancing lesions in brain MRI. Ten Gd+ (62.5%) and 41 Gd- patients (77.4%) showed non-evidence of disease activity (NEDA) defined as no disability progression or new MRI lesions after 1 year of treatment. Blood immune cell subsets were characterized by flow cytometry, serum immunoglobulins by nephelometry, and serum neurofilament light-chains (sNfL) by SIMOA. Statistical analyses were corrected with the Bonferroni formula.More than 60% of patients reached NEDA after a year of treatment, regardless of their baseline characteristics. In Gd+ patients, it associated with a low repopulation rate of inflammatory B cells accompanied by a reduction of sNfL values 6 months after their first ocrelizumab dose. Patients in Gd- group also had low B cell numbers and sNfL values 6 months after initiating treatment, independent of their treatment response. In these patients, NEDA status was associated with a tolerogenic remodeling of the T and innate immune cell compartments, and with a clear increase of serum IgA levels.Baseline inflammation influences which immunological pathways predominate in patients with PPMS. Inflammatory B cells played a pivotal role in the Gd+ group and inflammatory T and innate immune cells in Gd- patients. B cell depletion can modulate both mechanisms. Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system . It induTherefore, we aimed to explore the mechanisms associated with favorable response to ocrelizumab in inflammatory and non-inflammatory PPMS cases, toward facilitating the early identification of ocrelizumab responders and revealing new putative therapeutic targets in PPMS.This multicenter, prospective longitudinal study included 69 patients with PPMS diagnosed according to the McDonald criteria  who consBlood samples were collected in heparinized tubes immediately before (baseline) and 6 months after (before the second dose) ocrelizumab treatment. In both cases they were obtained the day Ocrelizumab was administered, just before initiating the infusion. Samples were then sent to the Immunology Department at Ramón y Cajal University Hospital (Madrid). Peripheral blood mononuclear cells (PBMCs) were isolated from 20 mL of heparinized whole blood as previously described  and cryoThe monoclonal antibodies used in this study are listed in 6 PBMCs were thawed by a 37°C thermostatic bath and washed twice in RPMI 1640 medium (Thermo Fisher Scientific). Samples were processed and stained as described  in presence of 2 μg/ml Brefeldin A  and 2.1 μM Monensin  during 4 hours at 37°C in 5% CO2 atmosphere.Thawed aliquots to analyze intracellular cytokine production were subdivided in three polypropylene tubes. To study cytokine production by monocytes, an aliquot of 3x105 PBMCs was resuspended and incubated in 1 mL RPMI 1640 medium and stimulated with 50 ng/mL of Phorbol 12‐myristate 13‐acetate  and 750 ng/mL Ionomycin (Merck) in presence of 2 μg/ml Brefeldin A and 2.1 μM Monensin during 4 hours at 37°C in 5% CO2 atmosphere.To study cytokine production by T and B cells (Except IL-10 producing B cells) an aliquot of 3x105 PBMCs was preincubated in 1 mL RPMI 1640 medium with 3 μg/mL of CpG oligonucleotide (In vivoGen) during 20h at 37°C in 5% CO2 atmosphere. After this, it was stimulated with 50 ng/mL of Phorbol 12‐myristate 13‐acetate  and 750 ng/mL Ionomycin (Merck) in presence of 2 μg/ml Brefeldin A and 2.1 μM Monensin during 4 hours at 37°C in 5% CO2 atmosphere.To identify IL-10 producing B cells, an aliquot of 3x10After incubation, the three aliquots were stained with the two-step protocol described previously . PBMCs w4 events were analyzed. We followed the strategy showed in hi CD127-/low. CD56 NK cells were classified as: NKT cells (CD3+ CD56dim), CD56dim NK cells (CD3- CD56dim) and CD56bright NK cells (CD3- CD56br). B cells were classified as: naïve (CD19+ CD38dim CD27-), memory (CD19+ CD27dim CD38dim), plasmablasts (CD19+ CD27hi CD38hi), transitional B cells (CD19+ CD27‐ CD24hi CD38hi) cells or regulatory B cells (Breg) (CD19+ IL-10+) cells. PD‐L1 was explored in monocytes by studying its co-expression with CD14 in PBMCs. We also explored intracellular production of IL-1β, IL-6, IL-10, IL-12 and TNFα by monocytes.IL-1β and TNFα represent innate cell activation, IL-12 induces Th1 responses, IL-6 represent innate cell activation and induces Th17 responses and finally, IL-10 is an anti-inflammatory cytokine. We also explored in CD4 and CD8 T cells the production of IFNγ and TNFα, products of Th1 response; IL-17, a product of the Th17 response; GM-CSF, which induces innate cell activation; and IL-10 that has a regulatory function. Finally, we explored B cells producing IL-6, a pro-inflammatory cytokine that induces Th17 cells; TNFα, an inflammatory cytokine; GM-CSF, inducing innate cell activation; and IL-10 a regulatory cytokine. Representative dot plots showing cytokine production by monocytes, B and T cells are shown in Cells were always analyzed within a maximum period of 1h after staining. Mean autofluorescence values were set using appropriate negative isotype controls. Data analysis was performed using FACSDiva Software V.8.0 (BD Biosciences). A minimum amount of 5x10We recorded for every leukocyte subset total cell counts per mL of blood, calculated by measuring total lymphocyte and monocyte numbers by a coulter counter, and the percentages of every subset over total mononuclear cells. To avoid bias due to B cell depletion, we also recorded the values of every T, B, NK and monocyte subset relative to total T, B, NK and monocyte cells, respectively.® Assay (Quanterix).Serum levels of immunoglobulins  were measured by nephelometry using a DimensionVista analyzer  and serum neurofilament light chain (sNfL) levels were measured using the single molecule array (Simoa) NF-lightP-values were adjusted using the Bonferroni correction and p-values less than 0.05 were considered statistically significant.Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Prism Inc.). Wilcoxon matched pairs test was used to assess differences between the samples collected at baseline and after 6 months from the same patient. Mann-Whitney-U test was used to compare the subgroups of patients. The association between NfL and age has been modelled using a Generalized Additive Model for Location, Scale and Shape (GAMLSS) model and age-normalized measures were obtained for each data point. Z score was used as a continuous measure for the number of standard deviations a given datapoint is above/below the mean in samples of healthy controls of the same age.Written informed consent was obtained from every patient prior to their inclusion in the present study, which was approved by the Ethics Committee of each center participating in this study.Any anonymized data collected for the purpose of this study will be shared with qualified investigators for 3 years from the initial publication of the study upon reasonable request to the corresponding author.Sixty-nine patients with PPMS  treated with ocrelizumab were included in this study. Age and disease duration [median (range)] were, respectively, 52.0 (33.0-71.0) and 9.2 (1.3-24.1) years and basal EDSS score was 5.5 (1.0-8.0). All patients were followed for 1 year. Fifty-one (73.9%) remained NEDA 1 year after ocrelizumab initiation. Using MRI data collected at baseline, we further classified the patients according to their inflammatory status into Gd+ (at least one Gd-enhancing lesion) and Gd- (no Gd-enhancing lesions) groups. Ten Gd+ (62.5%) and 41 Gd- (77.4%) patients were NEDA at the one-year follow-up. We found no significant differences between the four patient subgroups in terms of baseline clinical characteristics except for the MRI data  and Gd- NEDA (p=0.007) groups (-12). The IgG to IgM ratio also increased (-11). Remarkably, this group of patients experienced an increase in the IgA to IgG ratio  suggested that this drug may affect disease progression in younger patients, particularly those with inflammatory lesions . HoweverWe first explored baseline differences in patients classified according to their inflammatory status (presence [Gd+]/absence [Gd-] of gadolinium-enhancing lesions at baseline). We studied different leukocyte subsets  and sNfL values. As expected, Gd+ patients showed high sNfL values. They also showed a trend to higher plasmablast numbers. The association of plasmablasts with inflammation in MS has been widely documented in the CSF . ProbablWe next studied changes associated with NEDA status in both groups of patients by analyzing T, B, NK, and monocyte cell subsets at baseline and 6 months after receiving the first dose of treatment, before receiving the second one. Patients were classified according to their inflammatory condition (Gd+ or Gd-) and to their response to treatment (NEDA or EDA).Although B cells clearly diminished in all groups of patients 6 months after the first ocrelizumab dose, in the EDA inflammatory group, total B cell counts were higher at this point. This increase was mainly due to transitional B cells, thus indicating a higher rate of B cell repopulation for these patients, and to TNF-alpha-producing B cells, thus indicating a rapid differentiation into pro-inflammatory B cells. Another characteristic of EDA Gd+ patients was the increased levels of sNfL levels at baseline compared with the non-inflammatory groups, with a trend also observed for higher values than NEDA Gd+ patients. These data show that baseline Gd enhancing lesions and especially high serum neurofilament levels associate in PPMS with a high rate of B cell repopulation and strongly suggest that these patients should benefit from early re-treatment or dose adjustment. By contrast, in NEDA Gd+ patients, the low B cell counts at 6 months were associated with a significant reduction of the sNfL levels 6 months after the first ocrelizumab dose. Finally, in NEDA and EDA Gd- patients, sNfL levels did not change significantly after ocrelizumab treatment, despite the reduction in B cell counts or response to treatment. This is important, since NfL is being proposed as a biomarker for response to different drugs in MS  and thisT cell activation and cytokine production are also affected by anti-CD20 DMTs , 15. We We next explored the changes in the proportions of the different T cell subsets after 6 months of treatment relative to the total CD4+ and CD8+ T cells. Variations were restricted to the Gd- NEDA group. These patients exhibited a reduction in TD and EM CD4+ T effector cells and increases in naïve CD4+ and CD8+ T cells and in the ratios of naïve/EM and naïve/TD in CD4+ and CD8+ T cells. Likewise, they experienced a decrease in the proportion of CD8+ T cells producing IFNγ. These data show that response to ocrelizumab in Gd- patients is conditioned by reshaping the T cell compartment to a more tolerogenic profile. The activation of the T cell compartment by B cells may play an important role in the pathology of Gd- PPMS. Furthermore, the beneficial effects of different drugs in patients with MS with low inflammatory contribution to their disease may be reflected by changes in other biomarkers aside from sNfL, especially in those with low baseline levels of this protein.Regarding to Gd- EDA patients, we did not find any clear explanation for the lack of changes in the T cell compartment they showed upon Ocrelizumab treatment. They had no differences on epidemiological, clinical or immune cell subsets at baseline with NEDA group. Exploring antigen presenting B cells and activated dendritic cells in both groups could help to elucidate this conundrum. Future research will demonstrate if antigen presenting B cells could be a biomarker of response to Ocrelizumab in Gd- PPMS patients, as suggested by the remodeling in the T cell compartment showed by NEDA patients.We also explored changes in innate immune cells at baseline and after 6 months of treatment. Again, we only found significant changes in NEDA Gd- patients. They showed a significant increase in total monocyte counts. This increase was mainly due to PD-L1-expressing monocytes. This molecule is a ligand of the PD-1 receptor, which promotes self-tolerance by suppressing T cell inflammatory activity . Its incRegarding serum immunoglobulins in the four groups of patients, ocrelizumab induced a decrease in serum IgM levels, with no changes in IgG values, as previously reported for patients treated with anti-CD20 antibodies , 20. LikIn summary this study shows that baseline inflammation could determine the immunological pathways that drive the response to Ocrelizumab in PPMS and that, regardless of baseline MRI activity, B cell depletion with ocrelizumab can modify both underlying mechanisms, and be effective in more than 60% of patients.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of each participating hospital. The patients/participants provided their written informed consent to participate in this study.JF-V drafted the manuscript, major role in performing experiments, acquisition and analysis of data. PW-D, PL-S, and NV collected samples and performed flow cytometry experiments. JK and AM contributed to sNfL measurement. ER-M and ER supervised flow cytometry studies. EM, VM-L, JM-L, GI, CO-G, FG-G, SS, AS, YB, CD-P, GV-L, JD-D, YA, LB, CÍ, IG-S, LR, JG-D, JS, SL, JM, and LC-F visited MS patients, contributed by sending samples or collected clinical data. LV designed and supervised the study. All authors revised the manuscript and approved the final version.This work was supported by Red Española de Esclerosis Múltiple (REEM)  and PI18/00572 integrated in the Plan Estatal I+D+I and co-funded by ISCIII-Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional .EM received research grants, travel support or honoraria for speaking engagements from Biogen, Merck, Novartis, Roche, and Sanofi-Genzyme; JK received speaker fees, research support, travel support, and/or served on advisory boards by ECTRIMS, Swiss MS Society, Swiss National Research Foundation (320030_189140/1), University of Basel, Bayer, Biogen, Celgene, Merck, Novartis, Roche, Sanofi; VM-L received grants and consulting or speaking fees from Almirall, Biogen, Celgene, Genzyme, Merck, Novartis, Roche and Teva; JM-L has received grants and consulting or speaking fees from Almirall, Biogen, Bristol-Myers-Squibb, Genzyme, Merck, Novartis, Roche and Teva; GI has received consultancy/advice and Conference- travel support from Bayer, Novartis, Sanofi, Merck Serono, Roche, Actelion Celgene and Teva; CO-G has received speaker and consulting fees from Biogen, Celgene, Merck KGaA , Novartis, Roche, Sanofi Genzyme and Teva; FG-G has received funding for research grants, travel support and honoraria for speaking engagements from: Bayer, Biogen, Roche, Merck, Novartis, Almirall, Teva and Genzyme-Sanofi; SS received research grants, travel support, or honoraria for speaking engagements from Almirall, Bayer, Biogen, Merck, Mylan, Novartis, Roche, Sanofi-Genzyme and Teva; AS reports compensation for consulting services and speaker honoraria from Merck-Serono, Biogen-Idec, Sanofi-Aventis, Teva Pharmaceutical Industries Ltd, Novartis, Roche, and Alexion; YB received speaking honoraria from Biogen, Novartis and Genzyme; LB received funding for research projects or in the form of conference fees, mentoring, and assistance for conference attendance from: Bayer, Biogen, Roche, Merk, Novartis, Almirall, Celgen and Sanofi; IG-S received research grants, travel support and honoraria for speaking engagements from Biogen, Merck, Novartis, Roche, Sanofi-Genzyme, TEVA and Alexion; LR received travel support, and honoraria for speaking engagements from Biogen, Merck, Roche and Sanofi-Genzyme; JS received funding for research projects and conference fees, mentoring, and assistance for conference attendance from: Teva, Merck, Biogen, Roche, Novartis, and Sanofi; SL received compensation for consulting services and speaking honoraria from Merck-Serono, Biogen-Idec, Sanofi-Aventis, Teva Pharmaceutical Industries Ltd, Novartis and Roche; LC-F received speaker fees, travel support, and/or served on advisory boards by Biogen, Sanofi, Merck, Bayer, Novartis, Roche, Teva, Celgene, Ipsen, Biopas, Almirall; LV received research grants, travel support or honoraria for speaking engagements from Biogen, Merck, Novartis, Roche, Sanofi-Genzyme and Bristol-Myers.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Inonotus obliquus (Chaga mushroom) is a kind of medicine and health food widely used by folk in China, Russia, Korea, and some occidental countries. Among the extracts from Inonotus obliquus, Inonotus obliquus polysaccharide (IOPS) is supposed to be one of the major bioactive components in Inonotus obliquus, which possesses antitumor, antioxidant, anti-virus, hypoglycemic, and hypolipidemic activities. In this review, the current advancements on extraction, purification, structural characteristics, and biological activities of IOPS were summarized. This review can provide significant insight into the IOPS bioactivities as their in vitro and in vivo data were summarized, and some possible mechanisms were listed. Furthermore, applications of IOPS were reviewed and discussed; IOPS might be a potential candidate for the treatment of cancers and type 2 diabetes. Besides, new perspectives for the future work of IOPS were also proposed. According to World Health Statistics 2020, global life expectancy increased from 66.5 years to 72.0 years during 2000 to 2016 . Beside Inonotus obliquus was used as a folk medicine in Russia, Siberia, and some occidental countries [As early as the 16th century, ountries ,11. It iountries ,13; it iountries . As mostountries ,16,17.Polysaccharides are natural biopolymers that all living organisms have. And the diversity in monosaccharide composition and structure causes them to have different biological activities . Modern Inonotus obliquus, Inonotus obliquus polysaccharides have the broadest biological activity, such as anticancer, hypoglycemic, anti-inflammatory, and anti-oxidation effects [Inonotus obliquus, Inonotus obliquus polysaccharide related articles, a certain degree of upward trend can be seen : moisture 3.5 ± 0.36, crude protein 2.4 ± 0.44, crude fat 1.7 ± 0.25, ash 10.4 ± 0.44, crude fiber 67.5 ± 0.95, reducing sugar 4.2 ± 0.30, polysaccharide 10.3 ± 0.40, respectively [w/v) for material-to-liquid ratio, the yield for IOPS is 2.53% [As shown in ectively . As mostis 2.53% . Despiteis 2.53% ,66.Many people have developed modern physical methods such as microwave assist, ultrasonic heating and air technology, and cellulase-assisted extraction process to improve extraction efficiency ,69,70,71w/v), extraction time of 19 min. Compared with the traditional water extraction method, the ultrasonic-microwave-assisted extraction time was significantly shortened while the yield of polysaccharides increased from 2.12% to 3.25% and the purity increased from 64.03% to 73.16% [Chen used response surface analysis method to determine the optimal process parameters of ultrasonic-microwave-assisted extraction (UMAE), microwave power of 90 W, material-to-liquid ratio of 1:20  -D glycosidic bond on the glucose backbone is necessary for their antitumor effects .For the analysis of the primary structure of IOPS monosaccharide composition and glycoside bond connection mode, there are mainly gas chromatography (GC), periodic acid oxidation method, Smith degradation method, and methylation analysis .Inonotus obliquus liquid fermentation, IOEP1 mainly composed of galactose and mannose with 20 KDa molecular weight, while IOEP2 mainly composed of arabinose and molecular weight was 200 KDa. He found that IOEP1 and IOEP2 were all pyran-type polysaccharides, glycosidic bonds of IOEP1 and IOEP2 were both α-type and β-type via Fourier-transform infrared (FT-IR) and 1H-NMR spectra analysis [According to Chen, the composition of IOPS were rhamnose (Rha), arabinose (Ara), xylose (Xyl), mannose (Man), glucose (Glc), galactose , with molar ratio 10.25:9.38:1:12.45:9.9:11.55. The relative molecular mass of IOPS was 156,611 Da by HPLC gel chromatography column [analysis .Li et al. also studied the monosaccharide composition of IOPS by High performance liquid chromatographic (HPLC) method, he used precolumn-derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) for the detection. The result showed that IOPS was composed of mannose, rhamnose, glucose, galactose, xylose, and arabinose with molar ratio 2.13:1.36:7.01:2.98:1:1.78 .Chen used DEAE-Sepharose CL-6B column for further separation and purification to obtain four polysaccharides: IOPS1, IOPS2, IOPS3, and IOPS4 . The relThere is certain correlation between spatial conformation of polysaccharides and their activities. Large number of studies have shown that polysaccharides in flexed spiral conformation have higher activity than stretchable or wrinkled ribbon-shaped polysaccharides ,81. CongAtomic force microscopy (AFM) was used to observe IOPS spatial structure and surface morphology. The anticancer mechanism of IOPS mainly includes: Decreasing the expression of MMP-2, MMP-7, and MMP-9, increasing the expression of tissue metallopeptidase inhibitor 2 (TIMP-2), and decreasing the expression of NF-κB in cancer cells ,89,90,913--2,5-diphenyltetrazolium bromide (MTT) assay and Fluorescence activated cell sorting (FACS) is commonly used for cellular cytotoxicity studies. MTT assay assesses the effects of different drugs on cell viability, and a flow cytometry experiment can further quantify the extent of cell apoptosis by Annexin-V/PI Apoptosis Detection Kit; nonviable apoptotic or necrotic cells were generally defined to be situated in double positive (PI +/Annexin-V+) quadrant ,93.Researchers found that IOPS have obvious antitumor effects both in vitro and in vivo, despite that some different opinions on its mechanism. Some researchers think it stimulates the immune system while the others think the antioxidative ability prevents generation of cancer cells ,94.Zhang used MTT assay to validate IOPS inhibition of hepatic carcinoma cell line (SMMC7721) proliferation. The result showed a concentration correlation, IOPS in the range of 1.0–16.0 μg/mL inhibition rate range from 43.6–69.2% .Lee found that IOPS can effectively inhibit the migration and invasion of human non-small cell lung cancer A549 cells. Its possible mechanism might be that IOPS inhibits NF-κB nuclear translocation and JNK/AKT phosphorylation in A549 cells, and inhibits the AKT/NF-κB signaling pathway that reduces the expression level of matrix metalloproteinases (MMP); therefore, the invasion of human non-small cell lung cancer A549 cells was inhibited ,97.Zhang found that IOPS can induce apoptosis of ovarian cancer cell SKOV3. He speculated that the mechanism might be that IOPS reduces the mRNA expression of Bcl-xl gene by promoting the expression of p53 gene mRNA, thereby regulating the process of cell apoptosis .p < 0.01).Li verified IOPS inhibition effect on human cervical cancer cells (Hela), mouse bone marrow tumor cells (SP2), and human Chang’s liver cells (Chang) through the MTT method . Comparep < 0.01), and it had dose-dependent, and its maximum inhibition rates reached 71.84% and 75.14%, respectively [Chen also used the MTT method to study the inhibitory effect of IOPS on Jurkat and Daudi tumor cells in vitro, and the results showed that IOPS had a significant in vitro inhibitory effect on the proliferation of tumor cells Jurkat and Daudi  [Lin Yan studied the antitumor activity of IOPS by constructing a Kunming mouse S180 tumor-bearing model, and found that the inhibition rate was positively correlated with the IOPS dose. Among them, the inhibition rate of icators) .Besides, Jiang’s research demonstrated that IOPS could significantly inhibit allograft tumor growth of the LLC1 cells with increased apoptosis at 50 mg/kg dosage . Li testp < 0.05) as IOPS could significantly inhibit the activation of the Akt/mTOR and NF-κB signaling pathway (p < 0.05) [IOPS on human osteosarcoma MG-63 cells and U2OS cells’ s proliferation, invasion, migration, and apoptosis was studied in vitro by CCK-8 assays, cell scratch assays, trans-well assays, and flow cytometry. Moreover, 320 μg/mL IOPS significantly inhibited proliferation, decreased migration and invasion ability, and increased apoptosis rate ( < 0.05) .Inonotus obliquus polysaccharide by water extraction and ethanol precipitation. It could increase the mRNA expression of Bax, P21 and Bid genes in human liver cancer cells HepG2, while significantly reducing the expression of Bcl-2 genes. The mechanism of action might be that C-OWC increased the expression of P21 gene mRNA, and blocked the cell cycle of HepG2 cells in the G0/G1 phase, thereby inhibiting cell division and proliferation. Meanwhile it increased the mRNA expression of Bax and Bid genes in cells and reduces Bcl- 2 gene mRNA expression, thereby inducing HepG2 cell apoptosis [Yang obtained C-OWC contained with about 70% poptosis . The diaThe main mechanism of IOPS hypolipidemic activities may be attributed to the increase of lecithin-cholesterol acyl transferase (LCAT) activity, which promotes the synthesis of high-density lipoprotein cholesterol (HDL-C2), and transfers the free cholesterol in peripheral tissues to the liver, catalyzes the hydrolysis of triglycerides (TG) into glycerol and fatty acids, and promotes the conversion of high-density lipoprotein 3-cholesterol (HDL3-C) into HDL-C2, promotes the reverse transport and metabolism of cholesterol, thereby reducing serum total cholesterol (TC) content . The hypI. obliquus extract at 500 mg/kg orally, the blood glucose and insulin resistance of diabetic mice significantly alleviated while the cholesterol transportation in the liver enhanced, liver glycogen content and high-density lipoprotein cholesterol (HDL-C) levels increased with significant decline in total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels. Zhang supposed that the upregulated protein expression levels of phosphatidylinositol-3 kinase (PI3K), p-protein kinase B (Akt), p-adenosine monophosphate activated protein kinase (AMPK) and p-acetyl-CoA carboxylase (ACC) with downregulating in fatty acid synthase (FAS) and sterol regulatory element-binding protein-1c (SREBP-1c) also contributed to the hypoglycemic effects of IOPS [Wang and Zhang investigated the anti-diabetic effects and the potential mechanism of IOPS in vivo and found that IOPS ameliorated insulin resistance and lipid metabolism disorders in streptozotocin-induced type 2 diabetic mice. The possible hypoglycemic and hypolipidemic mechanism might supposedly be via the regulation of the PI3K/Akt and AMPK/ACC signal pathways ,64. As WInonotus obliquus polysaccharide UIOPS-1 in vitro. The digested polysaccharide (UIOPS-1I) after digestion could inhibit α-amylase up to 37.96% at a concentration of 200 μg/mL, which was significantly higher than the initial state of UIOPS-1 (p < 0.001). He speculated that it might be caused by the changes in monosaccharide composition and the decrease in molecular weight [α-Amylase and α-glucosidase are enzymes that promote the breaking of α-1,4-glycosidic bonds in starch, maltose, sucrose and oligosaccharides and hydrolyze them into monosaccharide that are easy to digest and be absorbed by the human body, such as glucose . This car weight ,40,54,55Through in vitro experiments, Xue found that IOEP1 and IOEP2 strongly increased HepG2 cells’ glucose consumption and insulin-resistant HepG2 cells, which proved that IOEP1 and IOEP2 might be suitable anti-diabetes agents in functional foods and natural drugs . With si2, and H2O2 inhibit the progress of the oxidation reaction, thereby blocking the initiation of the lipid peroxidation reaction; complexes of metal ions such as Fe2+ and Cu2+ that are necessary for the production of active oxygen inhibit the production of active oxygen; promote the production of SOD, CAT, GSH-Px and other antioxidant enzymes release or increase their activity, which indirectly exerts antioxidant effects [The human body continuously produces various reactive oxygen free radicals during the oxidative metabolism process of life activities, which may easily cause oxidative damage to DNA and further cause cancer, arteriosclerosis, aging, and other diseases. Polysaccharides and polysaccharide metal ion complexes have excellent antioxidant activity, and their antioxidant mechanisms mainly include: direct capture or removal of active oxygen such as OH, O effects ,116,117.IOPS antioxidant activities were frequently investigated by hydroxyl radical assay, superoxide radical assay, and ferric-reducing antioxidant power assay. Raw 264.7 cell models are often built to assess the in vitro activity ,119,120.2O2-induced oxidative damage in RINm5F pancreatic β cells at 25–100 μg/mL [50 for DPPH scavenging was 0.62 ± 0.01 μg/mL [50 of superoxide anion scavenging was 425 μg/mL [Sim’s study revealed that IOPS protection against H00 μg/mL . Chen fo01 μg/mL ,128. IC525 μg/mL .2O2-induced PC12 cell death. Results indicated that IOPS could scavenge all ROS and rescue PC12 cell viability from 38.6% to 79.8% and 83.0% at a concentration of 20 μg/mL [Mu investigated IOPS antioxidant properties via reactive species (ROS), 1,1’-Diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide anion radical, as well as their protective effects on H20 μg/mL .Inonotus obliquus (PIO-1), polysaccharide fractions 2 of Inonotus obliquus (PIO-2), polysaccharide fractions 3 of Inonotus obliquus (PIO-3) and saline (control group) orally for 30 days, and mice were submitted to the forced-swimming test [The mice swimming model is frequently the test model . Zhong uing test ,129. TotReproduced with permission .A virus is a submicroscopic infectious agent that infects all types of life forms, from animals and plants to microorganisms, including bacteria and archaea . The virInonotus obliquus, and further research is needed. It was reported that IOPS could inhibit the induction of NO and other similar cytokines, a phenomenon that had been associated with Covid-19 [Inonotus obliquus, contains IOPS), and unlike nucleoside analog antiherpetics, it could effectively prevent the entry of HSV-1 by acting on viral glycoproteins and lead to the prevention of membrane fusion [Currently, there are relatively few studies on the antiviral mechanism of Covid-19 ,133. By Covid-19 . Pan inve fusion .Inonotus obliquus by boiling water extraction had significant inhibitory activity on HIV-1 protease. He speculated that the possible mechanism might be that this lignin derivative was adsorbed on the protease and inhibited the reverse transcriptase of HIV, thus, achieving the effect of inhibiting the reproduction of HIV. In contrast, those low-molecular-weight polyphenols and low-molecular-weight lignin could not inhibit the protease [According to Ichimura et al., a high-molecular-weight water-soluble lignin derivative extracted from protease .Fan found that polysaccharide ISP2a significantly enhanced the lymphocyte proliferation and increased the production of TNF-α, demonstrating IOPS to be a potential application as natural antitumor agent with immunomodulatory activity ,46,137.Won investigated the immune stimulating activity of IOPS and the signaling pathway of IOPS-mediated macrophage activation in RAW264.7 macrophage cells . It was p < 0.05) [The activation of NLRP3 inflammasome can cause the overexpression and secretion of inflammation-related cytokines, and mediate the occurrence of inflammation. When the activation of NLRP3 inflammasome is inhibited, the inflammation-related diseases involved in it may be improved. In Lipopolysaccharide (LPS)-induced macrophage inflammation, Zheng found that IOPS can inhibit the expression of IL-18 and IL-1β by inhibiting the phosphorylation of NF-κB-p65 ( < 0.05) ,138,139.V. harveyi infection [By IOPS oral administration, in vivo growth of melanoma tumor was suppressed in tumor-bearing mice. Harikrishnan revealed that IOPS could positively enhance the innate immune system that effectively promote the health status of kelp grouper against nfection . The posHu revealed that IOPS could regulate gut microbiota of chronic pancreatitis in mice . Chen stToxoplasma gondii (T. gondii) infection caused liver injury model in mice are frequently applied for hepatoprotective effects. Xu studied IOPS hepatoprotective effects and mechanism in vivo via this model [is model . The resv/v) chloroform-n-butanol solution, followed by dialyzing with 3500 Da molecular weight dialysis bag in deionized water for 48 h, and then the collected dialysate was freeze-dried. The obtained IOPS could treat atopic dermatitis, food allergy, allergic rhinitis or asthma, especially for systemic allergies caused by OVA in animal experiments; thus, IOPS could be developed into an anti-allergic drug [Chen et al. optimized the extraction process and obtained IOPS composed with mannose, glucose, galactose, and xylose of 1.00:4.49:1.00:0.85 mass ratio. Comparing with traditional extraction methods, their patent has shown the modified preparation method: washing the water extract with absolute ethanol and acetone three times and adding 5:1  complex in normal mice. His result confirmed that high dose administration of IOPS had neither an obvious influence on serum profiles levels nor an antioxidant ability, as the organ tissues of the toxicity group maintained organization and integrity after having been given a high dose of IOPS chromium (III) complex [Despite that most polysaccharides are low-toxic with no significant side effects, it is still necessary to evaluate the potential risks and toxicity of polysaccharides in detail before clinical research . Usuallyure IOPS . Cong Wa complex . BrieflyInonotus obliquus (Chaga mushroom) has been used in Kiev to cure lip tumor, and it is a traditional medicine in Siberia [Inonotus obliquus related functional foods are developed. As Liu’s patent CN104366193-A has shown, healthcare food composed of IOPS will be used for preventing and treating diabetes, reducing blood fat, removing free radicals, protecting the heart, and regulating immune function.For centuries,  Siberia . NowadayOwing to its wide range of pharmacological activities, including hypoglycemic activity, improving immunity, anti-fatigue activity, and various beneficial effects, IOPS is used in functional and health products in Russia, China, and the US. Reviewing the existing products on e-commerce platforms such as Amazon and Taobao, we found that most commercial applications are health products, tea, and tincture. The existing and potential IOPS applications are listed in Inonotus obliquus polysaccharides mainly focused on the recent advances in the isolation, purification, physiochemical characterization and bioactivities. By applying new extraction method, the yield of IOPS can be significantly increased. IOPS exhibited significant hypoglycemic, hypolipidemic, antioxidant, and anti-fatigue activity, and cytotoxicity toward multiple cancer cells, including hepatic carcinoma cells, human non-small cell lung cancer cells, ovarian cancer cells, human cervical cancer cells, etc. The low-toxic property and no obvious side effects make it more attractive.As more and more patients tend to find alternative therapies for cancer and diabetes, the awareness towards seeking natural products has increased in both academia and in society. IOPS exhibited enormous potential in immunity enhancement, and antitumor, antioxidant, anti-fatigue, hypoglycemic, and hypolipidemic activity, providing an alternative way for treatment of cancer and diabetes. The present review of Although the development of IOPS has achieved advanced progress in the last decade, some problems still cannot be ignored. Due to the variations of habitat environment and extraction methods, the composition and content of the obtained polysaccharides are not the same. Thus, the establishment of planting and extraction standard is of great importance. Furthermore, despite that more and more remarkable pharmaco-logical advances are being made, the present commercial applications are still limited to several functional foods. On the one hand, deep investigations in precise mechanisms underlying their bioactivities and the structure-activity relationships (SAR) are crucial. On the other hand, further investigations that focus on toxicity and potential risks are also to be conducted to provide solid scientific and theoretical guidance for synthesizing IOPS derivatives with enhanced biological activity and less side effects. Meanwhile, the development of related products, especially commercial applications are also encouraged, in that more and more people can benefit from the multiple bioactivities of natural polysaccharides.


Mycobacterium leprae infection and are the major cause of leprosy-associated pathology. Two types of leprosy reactions with relatively distinct pathogenesis and clinical features can occur: type 1 reaction, also known as reversal reaction, and type 2 reaction, also known as erythema nodosum leprosum. These acute nerve-destructive immune exacerbations often cause irreversible disabilities and deformities, especially when diagnosis is delayed. However, there is no diagnostic test to detect or predict leprosy reactions before the onset of clinical symptoms. Identification of biomarkers for leprosy reactions, which impede the development of symptoms or correlate with early-onset, will allow precise diagnosis and timely interventions to greatly improve the patients' quality of life. Here, we review the progress of research aimed at identifying biomarkers for leprosy reactions, including its correlation with not only immunity but also genetics, transcripts, and metabolites, providing an understanding of the immune dysfunction and inflammation that underly the pathogenesis of leprosy reactions. Nevertheless, no biomarkers that can reliably predict the subsequent occurrence of leprosy reactions from non-reactional patients and distinguish type I reaction from type II have yet been found.Leprosy reactions are acute inflammatory episodes that complicate the course of a  Mycobacterium leprae (M. leprae), an acid-fast, rod-shaped bacillus that preferentially infects macrophages (histiocytes) in the dermis and Schwann cells (SCs) in peripheral nerves  is a comparatively new bacterium which causes severe form of leprosy, namely diffuse lepromatous leprosy (DLL), through nerve invasion and extensive skin ulcerations , borderline tuberculoid (BT), mid-borderline (BB), borderline lepromatous (BL), and lepromatous (LL) .The clinical manifestations of leprosy depend on the magnitude of the host immune response to ous (LL) . LL is crophages . In contimmunity , 9. The immunity . Comparaimmunity . In 1980M. leprae infection and are the major cause of leprosy-associated disabilities. Currently, there is no diagnostic test to detect or predict LRs before the onset of clinical symptoms. Similar inflammatory reactions  occur in other mycobacterial diseases, such as tuberculosis and Mycobacterium ulcerans infection (Buruli ulcer) during the natural course of infection or following antibiotic treatment (Leprosy reactions (LRs) are acute nerve-destructive inflammatory episodes that complicate the course of reatment , 12. Howreatment –16. LRs reatment , 16. Thereatment , 16.Two types of LRs with relatively distinct clinical and pathological features can occur: type 1 reaction  and type 2 reaction . T1R is characterized by acute inflammation in pre-existing leprosy lesions in the skin and peripheral nerves, resulting in edema, which is sometimes accompanied by ulcerative lesions , 17. AltIn contrast to T1R, ENL is a systemic inflammatory response characterized by neutrophil infiltration, activation of the complement system, extravascular deposition of immune complex, and secretion of pro-inflammatory cytokines in both skin lesions and peripheral blood , 19, 20.ENL affects numerous organ systems and is a painful inflammatory complication of leprosy . ImpairmM. leprae components  from November 1, 2020, using keywords including leprosy reactions; reversal reactions; ENL; biomarkers. To be noted, out of numerous published studies of LRs, only a small part have provided scientifically accurate data , such as dendritic cells and macrophages. The APCs then migrate to the regional lymph nodes, where they present the antigen on its surface via major histocompatibility complex (MHC) class II molecules to naïve CD4+ Th cells.T1R is induced by T cell-mediated hypersensitive reactions that predominantly occur in the borderline forms of leprosy . HyperseCD4+ Th cell activation occurs following the binding of the T cell receptor (TCR) and the CD4 co-receptor to the antigen-MHC class II complex of APCs . In addition, CD28 on the surface of CD4+ Th cells binds to B7 molecules (CD80/CD86) on the surface of the APC . Once APCs bind to CD4+ Th cells, they release interleukin (IL)-12 , a cytokine that dictates naïve CD4+ Th cell differentiation into a mature type 1 Th cell (Th1). At this point, the CD4+ Th cell becomes an effector cell and can release the cytokine IL-2, which contributes to the proliferation of both CD4+ Th cells (autocrine) and other cells (paracrine). Interferon-γ (IFN-γ) is also secreted by APCs, inducing Th1 proliferation and macrophage activation . ActivatNaïve CD4+ Th cells can also differentiate into Th17 cells in response to IL-6 and transforming growth factor (TGF)-β secreted by APCs –47. FollM. leprae antigens by APCs. APCs recognize M. leprae molecules via pattern recognition receptors (PRRs), such as toll-like receptors (TLRs)  and lipoarabinomannan (LAM), mediating APC activation  . In leprtivation –52. In ltivation . Furthertivation .M. leprae. In a cohort of Ethiopian patients, a single nucleotide polymorphism (SNP) in TLR2 (597C > T) was associated with protection against T1R, while a 280-bp microsatellite marker was associated with an increased risk of T1R , in the blood, skin, and nerves –64. TherTNFSF15-TNFSF8 locus are associated with excessive inflammatory responses in T1R (LRRK2), which may cause pro-inflammatory responses (The pro-inflammatory cytokine TNF-α belonging to the TNF superfamily (TNFSF) is increased in the skin, serum, and nerves during T1R –71. Genos in T1R , but effs in T1R . GWAS alesponses . Interesesponses .M. leprae clearance and concomitant tissue damage  of a VDR isoform associated with T1R  production, phagosome maturation, and autophagy . Importawith T1R . In addiy of LRs . Activaty of LRs . TGF-β aC-X-C motif chemokine ligand 10  is a pro-inflammatory chemokine that promotes T cell chemotaxis to sites of tissue inflammation . CXCL10 Cyclooxygenase-2 (COX-2) is overexpressed during inflammation, and COX-2 expression is regulated by growth factors and cytokines, such as IL-1β, IL-6, and TNF-α . In skinM. leprae  from patients with T1R and ENL showed significantly higher mRNA levels of IL17A, IL17F, IL23, IL6, and IL21 than those derived from patients with TT and LL  and IL-10 during the onset of T1R , which deactivates cortisol to cortisone, in the skin lesions of patients with T1R  and perilipin and hormone-sensitive lipase (HSL)  . Specifi for LRs .M. leprae-specific inflammatory and immune responses  and the omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid . Omega-3esponses . AA is pesponses . In contesponses , 127. Cuogenesis .4 (LTB4) is released during the acute-phase response, inducing the recruitment and activation of neutrophils, monocytes, and other leukocytes at the site of inflammation and pro-inflammatory cytokine production  is neuroprotective, and higher levels of LXA4 are observed in non-reactional leprosy patients, suggesting that LXA4 preserves nerve function in leprosy , potentially leading to abnormal immune responses due to inadequate immune complex clearance  on macrophages, polymorphonuclear neutrophils and dendritic cells . This biEndothelial cell expression of the leukocyte-endothelial cell adhesion molecule E-selectin is promoted by IL-1β and IFN-γ following activation of TLR2 and Fc receptors, allowing for neutrophil adherence and migration to sites of inflammation , 154. E-in situ neutrophils in ENL, but not non-reactional leprosy, express CD64 (M. leprae after initiating MDT (Resting neutrophils express low levels of the cell surface receptor CD64 (FcγRI); however, stimulation by gram-negative bacteria increases expression . CD64 isess CD64 , 158. CDting MDT , 158. Neting MDT , consistting MDT , 158. Thting MDT .ex vivo, which was blocked by the addition of IL-10 (Neutrophil IL-10 receptor 1 (IL-10R1) was recently proposed as a potential biomarker and target for ENL treatment . A recenof IL-10 . Expressof IL-10 .M. leprae or M. leprae antigens, lesion-derived CD8+ T cells do not proliferate and limit the proliferation and cytokine secretion of bystander T cells , a chemokine produced by monocytes, has also been identified as a potential plasma marker of ENL . CCL11 iα levels .M. leprae are the main players in the humoral immune response involved in leprosy pathogenesis. The role of humoral immune responses in immune defense against intracellular pathogens such as M. leprae is generally thought to be irrelevant. Instead, Ab production at the site of infection may contribute to the immunopathology and tissue injury observed in leprosy, as ENL pathogenesis has been attributed to Abs and immune complex deposition (see section 3.1). ENL patients exhibit elevated IgG1-secreting B cells  against  B cells , with loand IgG3 . As LRs b levels , Ab leveb levels . MB patib levels .M. leprae-specific Abs, and persistently high levels of anti-LID-1 Abs might be a useful tool to predict ENL  is a fusion protein of ML2331 and ML0405 recognized by dict ENL , 192. Indict ENL . In anott of T1R .Negera et al. demonstrated an increase in activated memory B cells in untreated patients with ENL, suggesting a role of memory B cells in the pathology of ENL . UntreatAPPs have also been proposed as potential biomarkers for ENL. Serum pentraxin-3 (PTX-3) levels are higher in MB patients before the onset of acute ENL, persist during LR, and are reduced by thalidomide . PTX-3 bSAA and CRP, systemic markers of inflammation, are elevated in ENL, indicating active inflammatory responses . In addiHemostatic disorders are frequently associated with acute and chronic infections, as exemplified by platelet functions, blood coagulation, and fibrinolysis, and are intimately correlated with immune responses . LeprosyTLR1, rs4833095, which causes a substitution of asparagine to serine (N248S) in the external recognition site, was identified as protective against ENL  is associated with LRs. In a Brazilian study, the presence of the “C” allele on this SNP was a risk factor for T1R and protective against ENL .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Escherichia coli (STEC) are zoonotic pathogens that cause food-borne diseases in humans, where cattle and derived products play a key role as reservoirs and vehicles. We analyzed the genomic data of STEC strains circulating at the livestock-food-human interface in South America, extracting clinically and epidemiologically relevant information . This study included 130 STEC genomes obtained from cattle (n = 51), beef (n = 48), and human (n = 31) samples. The successful expansion of O157:H7 (ST11) and non-O157  clones is highlighted, suggesting common activities, such as multilateral trade and travel. Circulating STEC strains analyzed exhibit high genomic diversity and harbor several genetic determinants associated with severe illness in humans, highlighting the need to establish official surveillance of this pathogen that should be focused on detecting molecular determinants of virulence and clonal relatedness, in the whole beef production chain.Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens responsible for causing food-borne diseases in humans. While South America has the highest incidence of human STEC infections, information about the genomic characteristics of the circulating strains is scarce. The aim of this study was to analyze genomic data of STEC strains isolated in South America from cattle, beef, and humans; predicting the antibiotic resistome, serotypes, sequence types (STs), clonal complexes (CCs) and phylogenomic backgrounds. A total of 130 whole genome sequences of STEC strains were analyzed, where 39.2% were isolated from cattle, 36.9% from beef, and 23.8% from humans. The ST11 was the most predicted (20.8%) and included O-:H7 (10.8%) and O157:H7 (10%) serotypes. The successful expansion of non-O157 clones such as ST16/CC29-O111:H8 and ST21/CC29-O26:H11 is highlighted, suggesting multilateral trade and travel. Virulome analyses showed that the predominant stx subtype was stx2a (54.6%); most strains carried ehaA (96.2%), iha (91.5%) and lpfA (77.7%) genes. We present genomic data that can be used to support the surveillance of STEC strains circulating at the livestock-food-human interface in South America, in order to control the spread of critical clones “from farm to table”.Shiga toxin-producing  Escherichia coli (STEC) is a zoonotic diarrheagenic pathotype of E. coli with the common characteristic of producing cytotoxins of the Shiga toxin family (Stx) [Shiga toxin-producing ly (Stx) . Althougly (Stx) .4 CFU/g feces [Cattle, which lack cellular receptors for Stx , are asy/g feces , increas/g feces ,7,8,9. G/g feces ,11. Ther/g feces . In this/g feces . Neverth/g feces ,15.In humans, STEC infections can cause severe gastroenteritis, hemorrhagic colitis (HC), and life-threatening hemolytic-uremic syndrome (HUS), mainly in children under five years of age . FoodborE. coli strains [Although epidemiological studies on STEC distribution have shown that O157:H7 serotype has been most frequently associated with outbreaks , “big-si strains , making  strains . Therefostx1a, stx1c-e, stx2a-l) [eae gene, which is located in the locus of enterocyte effacement (LEE) [saa gene [The primary virulence factor of STEC is Stx, which is classified into two types, Stx1 and Stx2. Each type is further classified into several subtypes (stx2a-l) . The typstx2a-l) . Additiont (LEE) ; or the saa gene  and the saa gene  in the Lsaa gene ,39,40,41saa gene ,42,43,44E. coli, whole genome sequencing (WGS) is a specific and sensitive method to conduct epidemiological and phylogeographic studies. Several genomic studies have been carried out to determine the virulome, resistome, and the worldwide spread of STEC clones isolated from animals, the environment, food, and humans [Given the high variety of serogroups, virulotypes and resistotypes, and the intrinsic genomic flexibility of d humans ,45,46,47d humans ,48.Genomic surveillance of STEC strains is necessary to improve the knowledge about the dynamic of dissemination, and of the role of cattle as reservoirs and disseminators of STEC lineages associated with severe disease in humans. Thus, the aim of this study was to analyze genomic data of STEC strains isolated from cattle in Chile, and to perform a comparative genomic analysis of STEC strains circulating at the cattle-beef-human interface in South America, extracting clinically and epidemiologically relevant information, including virulome, antimicrobial resistance genes, sequence types, clonal complexes, and phylogenomics, in order to provide relevant epidemiological data to surveillance programs at national and international level, under a One Health approach.http://enterobase.warwick.ac.uk). De novo assemblies that passed quality control with the standard established by Enterobase for E. coli/Shigella were used [Twenty-one STEC strains isolated from cattle were sequenced in this study, of which twenty were isolated in 2018 from cattle at abattoirs in the Región Metropolitana of Chile . Genomicere used . ContigsE. coli strains isolated in any South American country from cattle, beef, and people, whose FASTQ files were available at SRA GenBank (https://www.ncbi.nlm.nih.gov/genbank/) were downloaded on 6 November 2020. These 600 whole genome sequences were mapped using BWA [stx type/subtype. Thus, a total of 109 genomes of stx-positive E. coli strains were selected for comparative analysis with the 21 STEC genomes obtained in this study  and ABRicate (v.0.8.13) (https://github.com/tseemann/abricate/). The assessed virulence genes included aggR , cdtB , eae (intimin), efa1, ehaA , ehxA (enterohemolysin), hlyA (α-hemolysin), hra (intimate attachment to microvilli), iha , lpfA , subA (subtilase cytotoxin), tia , and stx subtypes. In the case of saa and sab  genes, as well as LAA and some of its associated genes, including ag43 , hes , lesP (serine protease), and pagC-like (putative serum resistance), were screened through BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi), according to protocols described in a previous study [https://cge.cbs.dtu.dk/services/ResFinder/) was used to identify the AMR genes. These analyses were performed with a default setting of 90% of identity threshold and 60% minimum gene length overlap. The presence of these genes was confirmed when a coverage and identity >90% was achieved.The prediction of virulence genes was performed on VirulenceFinder 2.0 (us study . ResFindhttp://enterobase.warwick.ac.uk). We have additionally performed a systematic search of scientific literature containing MLST data of STEC strains identified in South American countries. From genome and literature data, we have constructed a map containing the distribution of STs of STEC strains circulating at the livestock-beef-environment-human interface in South America [https://cge.cbs.dtu.dk/services/SerotypeFinder/) was used to determine serotype [http://enterobase.warwick.ac.uk) for phylogenomic analysis of all STEC genomes. The phylogenetic tree was generated with interactive Tree of Life, iTOL v6 (https://itol.embl.de). CSI Phylogeny 1.4 (https://cge.cbs.dtu.dk/services/CSIPhylogeny) was used with default settings to generate an approximately maximum-likelihood phylogenetic tree with the 21 genome assemblies obtained from this study, using E. coli strain K-12 MG1655 (RefSeq accession number NC_000913.3) as reference genome. The percentage of reference genome covered by all isolates was 81.24%, corresponding to 3,771,065 positions found in all genomes. iTOL v6 (https://itol.embl.de) was used to root the tree at midpoint and annotate the tree with data from Enterobase and genomic data.The multilocus sequence typing (MLST) scheme of Achtman was used to identify sequence types (STs) and clonal complexes (CCs) on the Enterobase platform  and the geographic location and isolation source of the strains included in this study (independent variables), where Y (response) can have only two values (Y = 0 or Y = 1) [stx type gene and one for the presence of both stx types at the same time. To ensure adequate convergence of the model and increase the statistical power of the results, epsilon was set at a high level (epsilon = 1 × 10−6). The fit of the models to the data was evaluated with the Hosmer–Lemeshow test [A logistic multivariable regression model was performed to assess the association between r Y = 1) . Three mhow test . The varhow test .n = 88), Argentina (n = 30), Ecuador (n = 7), Paraguay (n = 3), and Uruguay (n = 2). In total 130 STEC genomes were analyzed, of which 21 were obtained in this study. Genomes were representative of STEC isolates recovered from cattle (39.2%), beef (36.9%), and humans (23.8%). Analyzed STEC genomes were from Chile (n = 94) harbored the stx2 gene alone, 20.8% (n = 27) were stx1+stx2 positive, and 6.9% (n = 9) harbored stx1 alone. The stx subtype most frequently detected was stx2a , followed by stx1a , stx2c , stx2d , stx2b and stx2g , and stx1c . Among the other virulence genes assessed, the most frequently detected was ehaA , followed by iha , lpfA , ehxA , hlyA , tia , saa , subA , eae , cdtB , and sab . Additionally, 43.1% (n = 56) harbored LAA, with its associated genes pagC-like and hes , ag43 , and lesP .All strains met the WGS quality parameters of Enterobase, with a total average of filtered reads of 419,230.8 and an average coverage of 28.6 . Among taph(3″)-Ib , aph(6)-Id , and aph(3′)-Ia , which reduce the electrostatic interaction of the drug and the bacterial rRNA [tetA or tetB genes, which encodes for tetracycline efflux proteins [blaTEM-1 β-lactamase encoding genes, which hydrolyze penicillins and narrow-spectrum cephalosporins [sul2 gene, which encodes a drug-resistant dihydropteroate synthase, reducing affinity with sulfonamides [fosA7 gene, encoding a fosfomycin inactivating enzyme [qnrB19 gene, which encodes the Qnr protein that interacts with GyrA/B, preventing the action of fluoroquinolones [blaAmpC [Studies focusing on the AMR of STEC strains isolated from the livestock-beef-human interface in South America are scarce ,61,62,63ial rRNA . Four stproteins . Three sosporins . Two stronamides . Two strg enzyme , and twoinolones . As expe[blaAmpC  (Table Sn = 27), followed by ST297 , and ST58 (CC155) . blaAmpC, STEC strains of ST677 and ST58 isolated from beef samples in Chile carried aph(3″)-Ib, aph(6)-Id and tetB (n = 2), aph(3″)-Ib, aph(6)-Id, sul2 and tetB (n = 1), or qnrB19 (n = 2) resistance genes; whereas human STEC strains belonging to ST16 and ST342, from Uruguay, carried aph(3′)-Ia, aph(3″)-Ib, blaAmpC, blaTEM-1B, sul2 and tetA (n = 1), or blaAmpC and fosA7 (n = 2) resistance genes, respectively. Two human STEC belonging to ST11, from Paraguay, carried blaAmpC and blaTEM-1B genes.MLST typing revealed the presence of 41 STs among the circulating STEC strains, most of them belonging to ST11 (CC11)  and O157:H7 , followed by O113:H21 . Among the 130 STEC genomes analyzed, the most frequently detected serotypes were O-:H7 , ST297/O130:H11 , ST657/O183:h18 (B-61) and ST11238/O183:H18 (B-64) with difference of 124 SNPs, and ST33/O91:H14 and novel ST(-1661)/O91:H14 . Additionally, most clonally related STs shared an identical virulome and resistome .stx1 positivity showed a significant association with the isolation source; stx2 showed no significant association with any of the included variables, while for both stx1 and stx2 positivity showed a significant association with the geographic location of the sample , ST58 155), and ST297 were the most frequently detected. Those STs were isolated worldwide from human and non-human hosts, and are associated with illness both in humans and animals ,87. Whil, and ST2Some specific STs have been identified in two or three countries suggesting common activities, including multilateral trade and travel. The international STEC clone ST223/CC155 (O113:H21) was identified in cattle and beef in Chile (this study) and it has been previously identified in cattle, beef, and hamburgers in Argentina . In factadk-6, fumC-4, gyrB-4, icd-18, mdh-24, purA-8, recA-14), the ST58  is a single-locus variant of the ST155 , which has been designated as the primary founder of the CC155. The ST prediction performed in this study identified the presence of the ST677 (O174:H21) clone in beef samples from Chile and Argentina, whereas ST443 (O178:H19) and ST2388 (O15:H27) were identified in cattle and beef samples from Chile and Uruguay. While ST679 (O163:H19) was predicted in beef and human STEC genomes from Uruguay and Argentina, respectively, previous studies have reported the presence of ST78 and ST106 in Peruvian children and Argentinean cattle [STEC strains of the ST58 have been present in Chile (cattle) and Brazil (humans) ,61, and n cattle ,55. In tn cattle . ST101 an cattle , the ST2n cattle , and in n cattle .Interestingly, the phylogenetic analysis performed to evaluate the genomic relatedness among STEC strains confirmed the presence of clusters that include clones circulating at the livestock-food-human interface, supporting the persistence, adaptation, and successful expansion of specific lineages across and within different countries.stx type was stx2, which has been shown to be associated with more virulent STEC lineages and HUS. In fact, Stx2 presents an LD50 400 times lower than Stx1 in murine models [The most detected e models . Among te models ,95. Addie models  and incre models .eae gene, which has traditionally been recognized as a virulence-marker, but here only 30.8% of strains harbored this gene. These eae-positive strains included ST11 lineages, the “big six”  and other serotypes  considered emergent [lpfA, ehaA, and saa. LpfA correspond to a major fimbrial subunit protein that is able to interact with fibronectin, laminin, and collagen IV [hes and other genes), module II , module III (pagC-like and other genes), and module IV (ag43 and other genes), or as an incomplete structure with some missing modules. Among analyzed strains, this PAI was identified in 43.1% of the strains, mostly as a complete structure. The acquisition of this PAI is probably a recent evolutionary event in STEC [Another important virulence factor of STEC is the emergent . In thislagen IV ; EhaA islagen IV  increasilagen IV . Moreove in STEC , which c in STEC ,103,104.ehxA, hlyA, subA, and cdtB, these last two only being detected in LEE-negative strains. The α-hemolysin (HlyA) and the plasmid-encoded enterohemolysin (EhxA) are widely distributed in STEC strains and have been frequently associated with mild to severe illness in humans [Other toxin-encoding genes detected were n humans ,106. CDTn humans . SubAB in humans , and hasn humans . Given tAlthough antibiotic treatment of STEC infections in humans is not recommended, as they may worsen the disease by inducing toxin-related tissue damage and symptoms in patients , STEC castx1 than those isolated from beef, and that stx2 seems to be a common feature of STEC strains isolated from cattle, beef products, and humans in South America. Additionally, STEC strains from South American countries other than Argentina and Chile present a higher probability of carrying both types of stx. It is important to establish that this study was performed using publicly available genome sequences, which could represent a bias due to underreporting of STEC isolates in this geographical region, highlighting the importance of establishing integrated surveillance programs, both national and regional, ensuring a One Health approach. In summary, these findings support the need for continuous monitoring and surveillance of STEC strains in South America, not only focusing on the detection of O157:H7 serotypes, but also on resistance profiles, virulome, and STs. In this regard, genomic surveillance can be used to rapidly identify and prevent the spread of critical clones “from farm to table”.We report the genomic characteristics of STEC strains circulating in the livestock-beef-human interface in South America, highlighting the successful expansion of O157:H7 (ST11) and non-O157  clones, most likely favored by common activities, such as multilateral trade and travel. Worryingly, some of these STEC clones have been isolated from severe human infections, including HUS, representing a risk for food safety and public health. The logistic regression model indicated that STEC isolates from cattle are more likely to harbor


ATP6V1A), BCL2 interacting protein 3 (BNIP3), calmodulin-dependent protein kinase IV (CAMK4), TOR signaling pathway regulator-like (TIPRL), and the translocase of outer mitochondrial membrane 70 (TOMM70) as upregulated DEGs common to the five datasets. Our analyses revealed that these genes exhibited brain-specific gene co-expression clustering with OPA1, ITFG1, OXCT1, ATP2A2, MAPK1, CDK14, MAP2K4, YWHAB, PARK2, CMAS, HSPA12A, and RGS17. Taking the mean relative expression levels of this geneset in different brain regions into account, we found that the frontal cortex (BA9) exhibited significantly (p < 0.05) higher expression levels of these DEGs, while the hippocampus exhibited the lowest levels. These DEGs are associated with mitochondrial dysfunction, inflammation processes, and various pathways involved in the pathogenesis of AD. Finally, our blood–brain barrier (BBB) predictions using the support vector machine (SVM) and LiCABEDS algorithm and molecular docking analysis suggested that antrocin is permeable to the BBB and exhibits robust ligand–receptor interactions with high binding affinities to CAMK4, TOMM70, and T1PRL. Our results also revealed good predictions for ADMET properties, drug-likeness, adherence to Lipinskís rules, and no alerts for pan-assay interference compounds (PAINS) Conclusions: These results suggest a new molecular signature for AD parthenogenesis and antrocin as a potential therapeutic agent. Further investigation is warranted.Alzheimer’s disease (AD) is the most frequent cause of neurodegenerative dementia and affects nearly 50 million people worldwide. Early stage diagnosis of AD is challenging, and there is presently no effective treatment for AD. The specific genetic alterations and pathological mechanisms of the development and progression of dementia remain poorly understood. Therefore, identifying essential genes and molecular pathways that are associated with this disease’s pathogenesis will help uncover potential treatments. In an attempt to achieve a more comprehensive understanding of the molecular pathogenesis of AD, we integrated the differentially expressed genes (DEGs) from six microarray datasets of AD patients and controls. We identified ATPase H+ transporting V1 subunit A ( Alzheimer’s disease (AD), which is characterized by functional impairment, progressive cognitive dysfunction, and memory loss, is the most frequent cause of neurodegenerative dementia in aging populations, affecting nearly 50 million people worldwide . WorldwiThe pathology of AD is irreversible, and early stage diagnosis is paramount to halting the progression of the disease and avoiding deterioration ,6. MajorIn addition, there is currently no effective treatment for AD, and presently available drugs do not modify the disease because they are unable to halt its progression . The speGene expression microarrays are widely used to comprehensively measure the genome-wide expression profiles of clinical samples, aiding in the identification of disease-related genes . PreviouAntrodia cinnamomea (AC) is a unique fungal species that is found exclusively in Taiwan and is traditionally used by indigenous herbalists to treat various diseases | cut off point were set for DEG selection. The Multiple List Comparator , a web tool, was used to visualize the intersecting DEGs and to generate a Venn diagram to visualize overlapping DEGs. These overlapping DEGs were used for further bioinformatics analysis to uncover the molecular mechanism of AD.DEGs were identified using the LIMMA package of R . The Benhttps://www.networkanalyst.ca/, accessed on 21 September 2021) [Homo sapiens), ID type (gene symbol), tissue-specific co-expression (brain) analysis minimum-order network analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) enrichment analyses of the DEGs were conducted using the Enrich server [http://string-db.org/, accessed on 21 September 2021, v10.5) [p < 0.05.We explored the NetworkAnalyst server (er 2021)  to conduer 2021) ,34, the h server ,36. The , v10.5)  was usedhttps://www.targetvalidation.org/, accessed on 21 September 2021) at a search score of 0.15. The OPENTARGET platform is a bio-web algorithm that integrates genetic, omic, and chemical data to identify the involvement of genes in diseases and aids in systematic drug target identification and prioritization [We analyzed the disease-specific associations of DEGs by exploring the disease/phenotype-specific filters of the OPENTARGET platform  to conduct a functional enrichment analysis of the miRNA targets [p-value adjustment of 0.05 and a minimum required hit of four miR.The miRNA regulatory targets of the DEGs were collected from experimentally verified databases  and predicted databases (miRanda and targetscan). The miRNA regulatory network was visualized using the visNetwork R packages. In addition, we used the miRNA Enrichment Analysis and Annotation (miEAA) tool  [http://way2drug.com/dr, accessed on 21 September 2021) [https://www.cbligand.org/BBB/, accessed on 21 September 2021), which operates based on support vector machine (SVM) and LiCABEDS algorithms on four types of fingerprints from 1593 reported compounds [We analyzed the drug-likeness, pharmacokinetics (PKs), medicinal chemistry, and toxicity of antrocin using SwissADME software (er 2021)  and comper 2021) . We usedompounds  to analyompounds  to furthompounds . The GUSompounds . The GUS2O) molecules were removed [https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index, accessed on 21 September 2021) [The three-dimensional (3D) structures of CAMK4 (PDB:2W4O), ATP6V1A (PDB:6XBY), TIPRL (PDB:5WOW), BNIP3 (PDB:2J5D), and TOMM70 (PDB:3FP3) were downloaded from the protein data bank (PDB). The mol2 file for the 3D structure of antrocin was obtained using the Avogadro molecular builder and visualization tool vers. 1.XX  before s removed . Docking removed . The doc removed  and the er 2021) .p < 0.05 when comparing the AD patients and the control subjects from each dataset (ATP6V1A), BCL2 interacting protein 3 (BNIP3), calmodulin-dependent protein kinase IV (CAMK4), TOR signaling pathway regulator-like (TIPRL), and translocase of outer mitochondrial membrane 70 (TOMM70)  C.ATP6V1A, BNIP3, CAMK4, TIPRL, and TOMM70 were colocalized with the markers of different subcellular localizations: ATP6V1A (cytosol and nucleoplasm), BNIP3 (cytosol), CAMK4 (nucleoplasm), TIPRL (vesicles and cytosol), and TOMM70 (mitochondria) (p < 0.05) higher DEG expression levels. In contrast, the hippocampus exhibited the lowest ATP6V1A, BNIP3, CAMK4, TIPRL, and TOMM70 expression levels compared to the other parts of the brain that were analyzed , and microtubules of A-431 and A549 cells. We found that that hondria) A. In addanalyzed .OXCT1), ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 (ATP2A2), MAPK1, cyclin dependent kinase 14 (CDK14), MAP2K4, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta (YWHAB), parkin RBR E3 ubiquitin protein ligase (PARK2), cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS), heat shock protein family A member 12A (HSPA12A), and regulator of G protein signaling 17 (RGS17). Furthermore, the PPI interaction network yielded 45 nodes and 435 edges, with an average local clustering coefficient of 0.813 and PPI enrichment p-value of <1.0 × 10−16. The PPI network revealed that TOMM70A and ATP6V1A exhibited very high protein family-specific clustering interactions while BNIP3, CAMK4, and TIPRL formed a very minimal cluster  signaling, synaptic vesicle cycling, and peroxisome as major pathways associated with the DEGs A, while 50 values of 618, 26, 804.3, 517.3 g/kg body weight for the i.p., i.v., o.p, and s.c. administrative routes, respectively , BNIP3 (PDB:2J5D), TIPRL (PDB:5WOW), TOMM70 (PDB:3FP3), and ATP6V1A (PDB:6XBY) and demonstrated the respective binding affinities of −6.70, −5.80, −6.60, −6.80, and −5.90 kcal/mol. Our analysis of interactions between the target gene and antrocin revealed that antrocin interacted with the gene targets through several hydrogen bonds, π-alkyl, van der Waals forces , and sevAntrocin bound to CAMK4 with three hydrogen bonds, including THR291, HIS156, and PRO220, with the respective binding distances of 2.72, 1.92, and 3.65 Å. In addition, the antrocin–CAMK4 complex was further stabilized by several van der Waals forces that were formed around the backbone of antrocin with the amino acid residues of MET224, GLU221, THR290, PHE292, and ALA153 in the binding cavity of CAMK4. However, only two hydrophobic contacts (PRO220A and HIS156A) existed between antrocin and the binding cavity of CAMK4 . AntrociNo conventional hydrogen bonds were observed between antrocin and BNIP3, while only a single hydrogen bond was observed between the ligand (antrocin) and the receptors: TOMM70  and ATP6V1A . In addition, several van der Waals forces and hydrophobic contacts were also observed between antrocin and the receptors: BNIP3, TOMM70, and ATP6V1A .AD, one of the most common types of dementia, afflicts millions of people globally. In addition to its negative effects on physical and mental health, AD places a huge burden on both individuals and societies . AlthougIn terms of upregulated genes, our pathway enrichment results included fatty acid biosynthesis, ferroptosis, long-term potentiation, amphetamine addiction, adipocytokine signaling, PPAR signaling, synaptic vesicle cycle, and peroxisome, all of which are commonly accepted components of the pathogenesis and pathological changes of AD ,57,58,59Inflammatory processes play fundamental roles in the pathogenesis of AD . ActivatFurthermore, the enrichment of the regulation of myeloid leukocyte differentiation, the positive regulation of dendritic cell cytokine production, cellular responses to oxygen levels, and the myeloid dendritic cell differentiation in the biological process ontologies of DEGs suggest a mitochondrial dysfunction event, which is an important factor in the pathogenesis of AD and other neurodegenerative disorders such as PD . CollectATP6V1A is a multi-subunit enzyme that is associated with synaptic vesicle proton gradient generation in the brain, energy metabolism, and ATP synthesis. In agreement with a previous study , our comOPA1, ITFG1, OXCT1, ATP2A2, MAPK1, CDK14, MAP2K4, YWHAB, PARK2, CMAS, HSPA12A, and RGS17, all of which were reported to play essential roles in the regulation of cell proliferation and AD pathogenesis. Similar to the results obtained in our analysis, Zhang et al. [ATP6V1A and OXCT1 in the AD cohorts compared to in the healthy cohorts [OPA1 to the pathogenesis of Parkinsonian syndrome and dementia [OXCT1 plays a central role in extrahepatic ketone body catabolism [CAMK4, BNIP3, TIPRL, TOMM70, and ATP6V1A are important DEGs that are associated with the pathogenesis of AD and thus serve as attractive targets in the development of therapeutic intervention.Furthermore, our brain-specific gene co-expression analysis revealed that the DEGs exhibited gene co-expression clustering with g et al.  detected cohorts . Experimdementia , while Otabolism . CollectATP6V1A (PDB:6XBY), with estimated binding affinities of −6.70, −5.80, −6.60, −6.80, and −5.90 kcal/mol, respectively. Our analysis of the protein–antrocin complexes revealed that antrocin interacted with the targets created by several hydrogen bonds, π-alkyl, van der Waals forces, and several hydrophobic contacts. Non-covalent interactions, such as hydrogen, hydrophobic and ionic bondings, and van der Waals forces, play crucial roles in stabilizing the interactions between drug candidates and their protein targets [BNIP3 could be attributed to the absence of H-bonding in the antrocin–BNIP3 complex.Molecular docking is widely employed as an in silico model for elucidating the interactions between proteins and ligands and also in the estimation of the binding affinities of protein–ligand complexes ,69. This targets ,73. The  targets . However2 to 100 Å2 [The BBB is a limiting factor in the therapeutic effects of most medications for treating AD. Although a number of AD drug treatment trials targeting BBB dynamics have been conducted, most of them have failed due to inadequate permeability. In addition, a subset of AD cases with chronic hypoperfusion features is complicated by an inadequate BBB . Therefoo 100 Å2 , which wo 100 Å2 . Antrocio 100 Å2 . No alero 100 Å2 . Our resIntestinal absorption and brain penetration are important factors to consider for therapeutic agents that are intended to treat neurodegenerative diseases in response to oral exposure . Our resTaken together, the present study strengthened our understanding of the molecular pathogenesis of AD, identified disease targets, and suggested the therapeutic potential of antrocin. The main limitation of our study is the lack of the direct confirmation of these genes by reverse-transcription polymerase chain reaction (RT-PCR). However, experimental validation and the detailed therapeutic efficacy of antrocin in an AD animal model is currently ongoing in our laboratory. In addition, the RT-quantitative (q)PCR validation of these identified targets in clinical samples is ongoing.Our findings identified a potential new gene signature, ATP6V1A/BNIP3/CAMK4/ TIPRL/TOMM70, as critical molecular markers in mitochondrial dysfunction, inflammatory processes, and various pathways participating in AD parthenogenesis. Additionally, in silico assays and docking experiments revealed that antrocin interacted well with the crystal structures of the proteins and exhibited a low acute toxicity level based on the in silico modelling of the various administration routes. Our results suggest the potential of antrocin in targeting the DEGs; however, further investigation is warranted for the clinical translation of antrocin to ameliorate and prevent AD.