S-Nitrosoglutathione (GSNO)), termed Group 1, and a combination therapeutic, inhibitor-4 (SMYD-3 inhibitor) plus NO (from GSNO), termed Group 2, were evaluated for their efficacy on breast carcinoma cell lines MCF7 and MDA-MB-231, and the normal MCF10A breast cell line, using cellular viability, colony formation capacity, cytotoxicity, and cellular apoptosis analysis. These results indicated that, in Group 1, breast carcinoma lines MCF7 and MDA-MB-231, cells experienced a moderate reduction in cellular viability (~20–25%), a large reduction in colony formation capacity (~80–90%), a moderate increase in the relative number of dead cells, and a moderate increase in cellular apoptosis. Group 2 was significantly more impactful, with a ~50% knockdown in cellular viability, a 100% reduction in colony formation capacity, a large increase in the relative number of dead cells, and a large increase in cellular apoptosis. Additionally, Group 2 induced a very small impact on the normal MCF10A cell line. Cumulatively, this work revealed the exciting impact of this combination therapeutic, indicating its potential for clinical application and further research.Despite enormous advances in the detection and treatment of breast cancer, it still remains the leading cancer diagnosis and has the second highest mortality rate. Thus, breast cancer research is a high priority for academics and clinicians alike. Based on previous research indicating the potential of nitric oxide (NO) and SMYD-3 inhibition, this work sought to expand upon these concepts and combine the two approaches. Both NO (from  Female breast cancer cases in the United States have been continually increasing over the past 20 years . FortunaN-diazeniumdiolates, and S-Nitrosothiols (RSNOs) . Mo. Mo29]. The next method employed to determine therapeutic potential of Group 1 and Group 2 was LIVE/DEAD cytotoxic analysis using fluorescence microscopy. Both breast carcinoma lines, MCF7 and MDA-MB-231 cells, as well as the normal breast cell line, MCF10A, were evaluated and exposed to both treatment groups. In both of the breast carcinoma lines, MCF7 and MDA-MB-231 cells, both treatment groups induced cell death, as shown by the green (dead)-stained cells below. These images also revealed that cell death did occur in MCF10A cells treated by both treatment groups; however, this occurred to a lesser extent than in the breast carcinoma cell lines. Collectively, these images further support the cellular viability and colony formation data analyzed above, highlighting the potential potency and discriminatory capability of the combination treatment approach (Group 2) on breast carcinomas.Group 1Optimistically, the LIVE/DEAD fluorescence microscopic analysis revealed that both breast carcinoma cell lines, MCF 7 and MDA-MB-231 cells, were significantly more impacted by Group 1 than normal MCF10A breast cells. This can be seen in the middle images in , where tGroup 2Encouragingly, the LIVE/DEAD fluorescence microscopic analysis of Group 2 MCF7, MDA-MB-231, and MCF10A cells also reiterated the cell viability and colony formation results from above . SignifiFinally, the last major indicator of therapeutic efficacy assessed in this work was the evaluation of cell apoptosis. The impact of both treatment groups, Group 1 and Group 2, was assessed for the induction of cell apoptosis of MCF7 and MDA-MB-231 breast carcinoma cell lines, as well as normal breast cell line, MCF10A. Two different methods were employed, including a Caspase-Glo 3/7 Assay, to probe for caspase 3/7-mediated induction of apoptosis and Annexin V/FITC and PI staining to display early/late apoptosis and necrosis. Overall, the results showed that Group 2 showed a significantly higher amount of cellular apoptosis in the breast carcinoma cell lines MCF7 (seen via Annexin V/FITC and PI staining) and MDA-MB-231 (seen via increased Caspase 3/7 activity and Annexin V/FITC and PI staining). Annexin V/FITC and PI staining revealed only a very slight increase in apoptosis in the normal MCF10A line. These results support and further reinforce the data collected above, emphasizing the promising potential for this combination therapeutic approach (Group 2) in breast carcinomas.Group 1Caspase 3/7 activation was probed for all three cell lines, MCF7, MDA-MB-231, and MCF10A, using the Caspase-Glo 3/7 assay with luminescence detection for Group 1 . CaspaseGroup 2Caspase 3/7 activation was also evaluated for all three cell lines, MCF7, MDA-MB-231, and MCF10A, using the Caspase-Glo 3/7 assay with luminescence detection for Group 2 . Again, The second method employed to evaluate apoptosis and the final method to determine overall efficacy in NO (Group 1) and the combination therapeutic (Group 2) was Annexin V-FITC and PI staining with fluorescence microscopy. These data showed the occurrence of apoptosis (early or late stage) as well as necrosis in each sample group  for all three cell lines, MCF7, MDA-MB-231, and MCF10A. Untreated PC cells and GSH-treated cells for all three did not show anything of note, so only the images of S cells are included below . OverallGroup 1Apoptosis of NO (Group 1)-treated MCF7, MDA-MB-231, and MCF10A cells was analyzed using Annexin V-FITC and PI staining to observe early  or late-stage apoptosis  as well as necrosis  . ObviousGroup 2Similarly, MCF7, MDA-MB-231, and MCF10A cells consecutively exposed to the combination therapeutic (Group 2), were analyzed for apoptosis using Annexin V-FITC and PI staining . Again, Ultimately, these results highlight the Group 2 combination therapeutic, NO (from GSNO) + SMYD-3 inhibition (from inhibitor-4) as an extremely promising treatment for continued application in breast carcinoma treatment. In particular, this treatment shows discriminatory potential to breast carcinomas while imparting very few negative effects on normal breast cells. Future studies of both NO and NO combinations with SMYD-3 inhibition on breast cancers are necessary to determine their efficacy in clinical applications, such as 3D cell culture platforms and animal models.


The presence of varying groups of metabolites, including aldehydes, sterols, ketones, alcohols, nitrogen-containing compounds, furans, pyridines, acids, vitamins, fatty acids, sulphur-related compounds, esters, terpenes and terpenoids were reported. Bambara groundnut fermented into derived dawadawa products induced either an increase or decrease as well as the formation of some metabolites. The major compounds (with their peak area percentages) identified in Bambara groundnut were furfuryl ether (9.31%), bis (2-(dimethylamino)ethyl) ether (7.95%), 2-monopalmitin (7.88%), hexadecanoic acid, methyl ester (6.98%), 9,12-octadecadienoic acid  and 2-hydroxy-1-(hydroxymethyl)ethyl ester (5.82%). For dehulled dawadawa, the significant compounds were palmitic acid, ethyl ester (17.7%), lauric acid, ethyl ester (10.2%), carbonic acid, 2-dimethylaminoethyl 2-methoxyethyl ester (7.3%), 9,12-octadecadienoic acid -, 2-hydroxy-1-(hydroxymethyl)ethyl ester (5.13%) and maltol (4%), while for undehulled dawadawa, it was indoline, 2-(hydroxydiphenylmethyl) (26.1%), benzoic acid, 4-amino-4-hydroximino-2,2,6,6-tetramethyl-1-piperidinyl ester (8.2%), 2-undecen-4-ol (4.7%), 2-methylbutyl propanoate (4.7%) and ë-tocopherol (4.3%). These observed metabolites reported herein provides an overview of the metabolites in these investigated foods, some of which could be related to nutrition, bioactivity as well as sensory properties. It is important to emphasize that based on some of the metabolites detected, it could be suggested that Bambara groundnut and derived dawadawa might serve as functional foods that are beneficial to health.Metabolite profile provides an overview and avenue for the detection of a vast number of metabolites in food sample at a particular time. Gas chromatography high resolution time-of-flight mass spectrometry (GC-HRTOF-MS) is one of such techniques that can be utilized for profiling known and unknown compounds in a food sample. In this study, the metabolite profiles of Bambara groundnut and  Fermented condiment; GC-HRTOF-MS; Legume; Metabolites; Profiling. These metabolites contribute to the nutritional qualities, taste, shelf life, safety, aroma, health promoting properties and overall composition of fermented foods. Traditionally, these fermented foods are produced from legumes or cereals and undergo various forms of fermentation, such as alkali fermentation, lactic acid fermentation, acetic acid fermentation and alcoholic fermentation . The ferdawadawa, thua-nao, iru, natto and soumbala, are mostly produced from legumes and from Bambara groundnut , kept in sacks or bags and incubated in a plastic bowl/calabash/earthen pot for three to five days   . The moscretion) .Monitoring of these metabolic, biochemical, physicochemical and structural changes occurring during the fermentation process may be somewhat difficult, necessitating the utilization of techniques and robust equipment, which can provide a better overview of these metabolites. Gas chromatography–mass spectrometry is a non-biased, comprehensive and sensitive technological system used for the detection of diverse volatile and semi-volatile metabolites . It has dawadawa (dawadawa (dehulled and unhulled) from Bambara groundnut (BGN) obtained through natural fermentation. Thus, this study was aimed to profile metabolites in BGN and dawadawa (dehulled and unhulled) using GC-HRTOF-MS, envisaging that the metabolites would be beneficial to consumers of these products.While few authors have studied the composition of dawadawa , there i22.1Bambara groundnuts (mixed varieties)  used in this study were procured from a local farmer in Limpopo Province, South Africa. These were subsequently sorted to remove extraneous material or debris and cleaned with water.2.2dawadawa has been previously described in our earlier study dawadawa (UHD), the hulls or seed coats were not removed after soaking and were incubated at 35 °C for 120 h. The fermentation time and temperature conditions  that were used for producing the dawadawa samples was guided by the optimized results earlier achieved and reported in The production of both dehulled and unhulled 2.32O), 80% ACN in H2O, 80% MeOH in H2O, 50% ACN:MeOH (v/v), 1% HCl in MeOH, ACN:MeOH:H2O  and isopropanol:ACN:H2O ] were used to investigate for a possible range of available metabolites in the samples.At the initial stage of sample preparation, different extraction solvents  [100% acetonitrile (ACN), 100% methanol (MeOH), 100% water  at 4 °C for 1 h. The mixture was then centrifuged at 3 500 rpm for 5 min at 4 °C , transferred into a 250 mL round bottom flask and concentrated at 30 °C, under pressure using a rotary evaporator . The extract was reconstituted with 1 mL chromatographic grade MeOH  and filtered into a dark amber vial for analysis. The extraction was done in triplicates for each sample.An informed compromise was reached and extraction using 80% MeOH in H2.4A mass calibration of the instrument was performed prior to analysis on the LECO Pegasus GC-HRTOF-MS system  and subsequent sample analyses done using the method of 2.5m/z fragments obtained from the ChromaTOF-HRT® data station were recorded after which the metabolite class was annotated with corresponding m/z fragments and molecular formula.From the data obtained, peak picking, retention time alignment, peak matching and detection were done on the ChromaTOF-HRT® software . Other data processing parameters adopted included a signal to noise ratio of 100 and a minimum match similarity of >70% prior to when compound name is assigned, using the Mainlib, Feihn and NIST metabolomics database by comparing the molecular formula, retention time and mass spectra data. Percentage peak areas were subsequently calculated, and the respective observed 33.1dawadawa produced (unhulled and dehulled) were analyzed using GC-HRTOF-MS. This is the first report to the best of our knowledge to profile and investigate the metabolites of BGN, unhulled (UHD) and dehulled (DD) dawadawa using GC-HRTOF-MS. In total, 134 metabolites were identified, and their identities presented in dawadawa products, 29 in BGN, 42 in only DD, 17 in only UHD and 12 in all the samples analyzed (dawadawa and 2-methylbutyl propanoate (4.7%), 2-undecen-4-ol (4.7%), benzoic acid,4-amino-4-hydroximino-2,2,6,6-tetramethyl-1-piperidinyl ester (8.2%), ë-tocopherol (4.3%) and indoline, 2-(hydroxydiphenylmethyl)- (26.1%) for unhulled dawadawa samples . Dehulling of the seedcoats exposed fats related components to more oxygen coupled with removal of available antioxidants in the hull. This thus suggests that fat oxidation would likely be higher in the dehulled samples as compared to the unhulled samples  applied during processing   in DD, while none of the fatty acid methyl esters were detected in UHD. This difference might have been due to the cooking process adopted , as heat treatment is known to affect fatty acid constituents of foods . Hexadecr bonds) .4dawadawa samples using GC-HRTOF-MS. From the two fermented samples, dehulled samples had the highest number of metabolites as compared to their unhulled counterparts. Compounds identified included esters, ketones, phenols, flavor related compounds and constituents that could confer organoleptic properties, nutritional and functional benefits of BGN and derived dawadawa. The BGN seeds and dehulled dawadawa possess beneficial components that can potentially be incorporated into human diets for health benefits. Further investigations into the quantification of the metabolites in this study are still needed, particularly for those significant metabolites obtained in all three samples. These would not only provide a better understanding of legume fermentation, but also assist in providing an insight into these significant metabolites that could potentially be biomarkers of dawadawa.A total of 134 metabolites were detected in Bambara groundnuts and derived Janet Adeyinka Adebiyi: Conceived and designed the experiments; Performed the experiments; Wrote the paper.Patrick Berka Njobeh, Eugenie Kayitesi: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.Oluwafemi Ayodeji Adebo: Performed the experiments; Analyzed and interpreted the data.10.13039/501100001321National Research Foundation (NRF) of South Africa (120751) and the NRF of South Africa National Equipment Programme (99047).This work was supported by the Data included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.


Background: serum 25-hydroxyvitamin D (25(OH)D) ”) is the most commonly used indicator of vitamin D status. However, 25(OH)D is mostly bound to the vitamin D binding protein (VDBP) or albumin in blood, and it has been suggested that the remaining bioavailable or free 25(OH)D may be more relevant for vitamin D associated health outcomes. We aimed to explore distributions and determinants of VDBP, total, bioavailable, complementary “non-bioavailable”, and free 25(OH)D in a large cohort of older adults in Germany. Methods: total 25(OH)D, VDBP, and albumin concentrations were measured in blood samples of 5899 men and women aged 50–75 years and used to calculate bioavailable (and complementary “non-bioavailable”) and free 25(OH)D concentrations. Linear regression models were used to evaluate associations of potential determinants of the various vitamin D biomarkers. Results: mean concentrations of VDBP, total, non-bioavailable, bioavailable, and free 25(OH)D were 323.6 µg/mL, 49.8 nmol/L, 43.4 nmol/L, 2.5 ng/mL, and 5.7 pg/mL, respectively. Seasonal variations were observed for all markers, with peak values in spring for VDBP and in summer for total, non-bioavailable, bioavailable, and free 25(OH)D. Consistent inverse associations were seen with age and body mass index for all markers, but divergent associations were seen with C-reactive protein. Strong variations by VDBP genotypes were seen for bioavailable and free 25(OH)D, and, in opposite direction for non-bioavailable 25(OH)D. Conclusion: commonalities and differences in determinants of various markers of vitamin D status were observed, which may help to enable a better understanding of their potential role for various vitamin D related health outcomes. Vitamin D deficiency is common and closely related to various health outcomes among older adults . The mosAlthough a number of studies have addressed distribution and potential determinants of the various vitamin D biomarkers in different populations ,3,6,7,8,Our analysis is based on baseline data from the ESTHER study , a population-based cohort study of older adults conducted statewide in Saarland, a federal state in Southwestern Germany. Details of the study design have been reported elsewhere . BrieflyWe collected information on covariates including age, sex, education, smoking status, physical activity, intake of multivitamin supplements, and fish consumption by using a comprehensive questionnaire at baseline. General practitioners assessed height, weight, and systolic blood pressure, and provided information on presence of hypertension, diabetes, chronic kidney diseases (CKD), and cardiovascular diseases (CVD) at the time of the health check-up. For our analyses, history of CVD was defined as history of coronary heart disease, myocardial infarction, stroke, or stent/balloon catheter operation for revascularization of coronary arteries. We obtained information on previous incident malignant cancer (excluding non-melanoma skin cancer) through record linkage with the long-standing statewide Saarland Cancer Registry.Blood samples were collected at baseline by general practitioners, centrifuged, shipped to the study center, and stored at −80 °C until analysis. Baseline laboratory analyses included total cholesterol (measured by enzymatic chromatography), C-reactive protein , and albumin (measured by fluorescence immunoassay).2 and 25(OH)D3 in the context of two different research projects for women and men. In a first project among women conducted in 2006, total 25(OH)D concentrations were measured by using the DiaSorin–Liaison analyzer . In a subsequent project among men conducted in 2009, the DiaSorin–Liaison method was no longer available, and total 25(OH)D was measured using IDS-iSYS . Later both immunoassays were standardized to the gold standard method of liquid chromatography tandem mass spectrometry. Details of the laboratory methods for measuring total 25(OH)D and their standardization have been reported elsewhere [From the blood samples collected at baseline, we further measured concentrations of total 25(OH)D and VDBP. Total 25(OH)D was measured as the sum of 25(OH)Dlsewhere  and are lsewhere  and are VDBP · Kalb · Dalb + KVDBP; b = KVDBP · DVDBP − KVDBP · Dtotal + Kalb · Dalb + 1; c = −Dtotal, and Dfree indicates free 25(OH)D concentrations; Dalb indicates albumin concentrations; Dtotal indicates total 25(OH)D concentrations; DVDBP indicates VDBP concentrations; Kalb is the affinity constant between vitamin D and albumin ; KVDBP is the affinity constant between vitamin D and VDBP . All concentrations are expressed in mol/L in calculating equations.Bioavailable and free 25(OH)D concentrations were calculated based on the levels of total 25(OH)D, VDBP, albumin, and their affinity constants derived from the VDBP genotypes ,14. We ualb indicates albumin concentrations; Dbioavailable indicates bioavailable 25(OH)D concentrations; Dfree indicates free 25(OH)D concentrations; Kalb is the affinity constant between vitamin D and albumin . All concentrations are expressed in mol/L in calculating equations.We calculated bioavailable 25(OH)D concentrations by using the following equation:We further calculated “non-bioavailable 25(OH)D” concentrations as the difference between total and bioavailable 25(OH)D concentrations.We summarized the baseline characteristics by using descriptive statistics. Additionally, we visualized the distributions of VDBP, total, non-bioavailable, bioavailable, and free 25(OH)D concentrations by sex. We plotted histograms and density curves to show the general distribution of these vitamin D biomarkers, and further plotted bar charts to assess potential variations by season and month of blood draw, and VDBP genotypes. We also calculated Spearman’s rank correlation coefficients and plotted the correlation analysis of VDBP, total, non-bioavailable, bioavailable, and free 25(OH)D.p < 0.05 in two-sided testing.We report mean concentrations of VDBP, total, non-bioavailable, bioavailable, and free 25(OH)D by various demographic, behavioral, and medical characteristics. We used linear regression models to assess individual and independent associations of these factors with vitamin D biomarkers. Using linear regression was judged to be appropriate because all outcomes were approximately normally distributed despite some right skewedness for total and non-bioavailable 25(OH)D among females. Overall, 783 participants had at least one missing value in one or more of the included covariates (13.3%). To minimize potential bias, we applied multiple-imputation and report pooled regression results from 20 imputed databases. All analyses were carried out by R software . Statistical significance was defined by The ESTHER study was approved by the Ethics Committees of the Medical Faculty of the University of Heidelberg and of the Physicians’ Board of Saarland. All participants gave written informed consent.2, 139.9 (SD: 19.6) mmHg, 230.9 (SD: 42.2) mg/dL, and 4.2 (SD: 8.1) mg/L, respectively.Overall mean concentrations of VDBP, total, non-bioavailable, bioavailable, and free 25(OH)D were 323.6 µg/mL, 49.8 nmol/L, 43.4 nmol/L, 2.5 ng/mL, and 5.7 pg/mL, respectively. In this large population-based cohort of older adults from Germany, total 25(OH)D levels were found to be low (mean: 49.8 nmol/L), and to strongly vary by season of blood draw. Levels of non-bioavailable, bioavailable, and free 25(OH)D were strongly correlated with total 25(OH)D levels and showed similar major seasonal variation, with the highest concentrations in summer, and the lowest concentrations in spring. Despite strongly different absolute concentrations, all 25(OH)D measures also showed similar associations with potential non-genetic determinants, with decreasing levels with increasing age and BMI, and higher levels among males, non-smokers, and those reporting regular multivitamin intake. By contrast, VDBP genotypes showed opposite associations with bioavailable and free 25(OH)D , and non-bioavailable 25(OH)D . VDBP concentrations were highest in spring, lower in men than in women and unrelated to smoking, but they also strongly decreased with increasing age and increasing BMI. They showed moderate inverse correlations with bioavailable and free 25(OH)D. In addition, some distinct patterns were seen, with markedly higher VDBP concentrations among those with high cholesterol and high CRP concentrations.The associations of total 25(OH)D with potential determinants observed in our study are consistent with those observed in multiple previous studies and are meanwhile well established. This particularly applies to the strong seasonal variation caused by seasonal variation in ultraviolet-B exposure ,16, whicThe strong genetic determination of VDBP concentrations is well established ,20. SubjTo our knowledge, our study is the first to comprehensively address potential non-genetic determinants of VDBP levels. Despite their strong genetic determination, quite pronounced inverse associations of VDBP levels were also seen with age and BMI, similarly to those seen for 25(OH)D concentrations. In contrast to the latter, however, VDBP concentrations were higher in women, and in participants with high cholesterol and CRP levels. The reasons for these strong associations are unclear and require further study. One possible explanation for the association observed with CRP levels might be an increase of VDBP concentrations in response to inflammation . Our larOur study has several strengths. To our knowledge, our study is the first one to provide a parallel comprehensive comparison of determinants of VDBP, total, bioavailable, and free 25(OH)D concentrations. Our study also has a much larger sample size than most previous studies assessing these various vitamin D related parameters. In addition to a range of genetic, lifestyle, and dietary factors, we simultaneously considered various health conditions reported by physicians in multivariable analyses. Furthermore, due to the population-based nature of our cohort, the generalizability of our findings for the older population may be higher than in studies focusing on specific patient groups.Our study also has a number of limitations. Our analysis was limited to cross-sectional data, which limits any derivation of temporal or causal relationships. Although we have considered a large number of covariates in multivariable analyses, residual confounding might not be ruled out due to the observational nature of the study. We did not directly measure bioavailable and free 25(OH)D, but determined it from total 25(OH)D, VDBP, and albumin concentrations by previously suggested equations. Measured concentrations of free 25(OH)D have been reported to be lower than the calculated ones, especially under physiologic and pathologic conditions (15). Despite these limitations, the quite distinct associations and patterns observed in our study may stimulate further research towards a more complete understanding of the determinants and health relevance of VDBP and the various components of 25(OH)D.In this large population-based study of older adults from Germany, we found high correlations of total 25(OH)D with bioavailable, free, and non-bioavailable 25(OH)D, along with quite consistent associations of these markers of vitamin D status with potential non-genetic determinants, despite quite diverse associations with VDBP genotypes. In addition, apart from their genetic determination, VDBP concentrations were found to strongly vary by a number of non-genetic potential determinants. The biological mechanisms and clinical implications of the observed patterns deserve careful elucidation in further research to better understand a potential role of vitamin D in its various compartments for health at older age.


After infection of hepatitis B virus (HBV), the virus induces a variety of immune disorders in the host, leading to immune escape and, finally, the chronicity of the disease. This study investigated immune cell defects and functional impairment in patients with chronic hepatitis B (CHB). We analyzed the percentage, function, and phenotypes of various immune cell subpopulations in the peripheral blood along with the concentrations of cytokines in the plasma. We compared the results between patients with CHB and healthy individuals. It was found that in patients with CHB, the cell function was impaired and, there was increased expression of inhibitory receptors, such as NKG2A and PD-1 in both NK and T cells. The impairment of function was mainly in cytokine secretion, and the cytotoxicity was not significantly diminished. We also found that the proportion of dendritic cells (DC) decreased and regulatory B cells (Breg) increased in CHB. In addition, the Breg cells were negatively correlated with T cell cytokine and positively correlated with ALT and HBV viral load. Taken together, various disorders and functional impairments were found in the immune cells of peripheral blood in CHB patients, especially NK and T cells. These cells showed exhaustion and the increase of regulatory B cells may be one of the reasons for this phenomenon. Despite the availability of highly effective preventive vaccines and oral antiviral drugs, chronic infection with hepatitis B virus (HBV) still affects more than 240 million people worldwide and causes 620,000 deaths annually , 2. HBV In patients with chronic hepatitis B (CHB), the host immune response is like a double-edged sword. On one hand, it achieves clearance of the hepatitis B virus by destroying infected hepatocytes, and on the other hand, the immune response causes liver inflammation and aggravates liver damage, leading to liver fibrosis and hepatocellular carcinoma , 7. In aThe immune system is extremely complex, and there is still no clear explanation on the antiviral inflammation of the body after HBV infection and on the immune escape of the virus. This study intends to investigate the possible dysfunction of the immune system in patients with chronic hepatitis B through the analysis of the percentage, phenotype, and function of peripheral immune cells in 48 patients with chronic HBV infection. Our purpose is to provide clinical data for a more in-depth understanding of the mechanisms of immunodeficiency associated with chronic hepatitis B infection and, furthermore, to offer a theoretical basis for immunotherapy in hepatitis B patients.n = 13), immune clearance (IC) phase (n = 12), lower replicative (LR) phase (n = 13), and reactivation (RA) phase (n = 10) according to the Asian Pacific Association for the Study of Liver guidelines  were recruited from the Department of Hepatology in Tongde Hospital of Zhejiang Province. The age of patients ranged from 16 to 72 years old with the mean age of 39 ± 14 years. These patients were HBsAg positive for longer than 6 months, volunteered to participate in the study, and had complete case information. None of the CHB patients had a history of liver surgery, coinfection with another viral hepatitis, autoimmune hepatitis, or human immunodeficiency virus (HIV) infection, and there were also no pregnant or lactating women. The patients were divided into immune tolerant (IT) phase  tubes. The peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation by Ficoll separation. In addition, 5 ml of procoagulated blood was centrifuged at 1,000g for 10 min, the plasma was obtained and stored at −80°C for testing. All treatments were completed within 24 h.All analysis was performed in four protocols. Antigen-presenting cell protocol, CD16-FITC, CD11c-PE, CD56-PC5, CD123-PC7, CD14-APC, CD3-APC750, HLA-DR-PB, CD45-KO, B cell protocol, CD38-FITC, IGD-PE, CD19-PC5, CD CD27-PC7, CD24-APC, T cell protocol, CD45RA-FITC, CD127-PE, CD25-ECD, PD-1-PC5, CD45-PC7, CD4-APC, CD8-APC700, CD3-APC750, CXCR5-PB. NK cell, CD16-FITC, CD8-PE, CD56-PC5, CD45-PC7, NKG2A-APC, CD3-APC750, and NKp30-PB. All antibodies were added with 5 ul. Then each protocol was added 50 ul PBMC, incubated for 20 min in the dark, added 500 ul hemolysin, hemolyzed for 5 min, centrifuged, and discarded supernatant before resuspended. Flow cytometry assay and set up isotype control were performed. All assays were completed within 1 h after isolation of PBMC.Firstly, the cell surface staining was performed by taking 50 ul of PBMC, adding 5 ul to each of CD56-PC5, CD45-PC7, CD4-APC, CD8-APC-700, and CD3-APC-750 antibodies, respectively, and incubating for 20min in the dark. Then, hemolysin, fixative, and membrane breaker were added in order, with 20 min of incubation and centrifugation after the addition of each agent. Finally, 5 ul of perforin -FITC and granzyme-PE antibody was added into each mixture, vortexed, and incubated for 20 min in the dark.2, then stained with surface markers CD45-PC7, CD3-APC-750, CD4-APC, CD8-APC700, and CD56-PC5 while being protected from light for 15 minutes. These were then fixed and broken to stain intracellular cytokines IFN-γ-FITC, IL-21-PE, and IL-17-PB, before finally being detected by flow cytometry.We analyzed intracellular cytokines in 31 of the 48 CHB patients. Cells were stimulated for 3 hours in disposable dry powder tubes precoated with stimulating agents  at 37°C in 5% COPlasma concentration of cytokines was tested in the 31 patients with CHB. Plasma (standard), buffer, mixed capture beads, and detection antibody, each with affinity 25 ul was mixed and incubated for 2 h in the dark. Then, streptavidin with fluorescein was added (SA-PE) and mixed in the dark for half an hour before being centrifuged. The supernatant was then discarded before 100 ul of PBS was added. Finally, the flow cytometer was tested. The results were quantified in the software LEGEND plex V8.0 for analysis.Antibodies: CD279 (PD1) (Clone PD1.3 Beckman Coulter), CD45RA (Clone ALB11 Beckman Coulter), CD185 (CXCR5) (Clone J252D4 Biolegend), CD4 (Clone 13B8.2 Beckman Coulter), CD20 (Clone B9E9 Beckman Coulter), CD19 (Clone J4.119 Beckman Coulter), CD38 (Clone HB-7 Biolegend), CD27 (Clone M-T271 Biolegend), IGD (Clone IA6-2 Biolegend), CD11c (Clone BU15 Beckman Coulter), CD14 (Clone RMO52 Beckman Coulter), CD16 (Clone 3G8 Beckman Coulter), CD24 (Clone ALB9 Beckman Coulter), CD45 (Clone J.33 Beckman Coulter), CD3 (Clone UCHT1 Beckman Coulter), CD8 (Clone B9.11 Beckman Coulter), CD56 (Clone N901 Beckman Coulter), CD57 (Clone NC1 Beckman Coulter), CD25 (Clone B1.49.9 Beckman Coulter), CD127 (Clone R34.34 Beckman Coulter), CD159a (NKG2A)(Clone S19004C Biolegend), CD337 (NKp30) (Clone P30-15 Biolegend), IFN-γ (Clone 45.15 Beckman Coulter), IL-21 (Clone 3A3-N2 Biolegend), IL-17 (Clone BL168 Beckman Coulter), granzyme (Clone QA16A02 Biolegend), perforin (Clone B-D48 Biolegend)Flow Cytometer: Navios, Beckman CoulterCytometric Bead Array: RAISECARE; Analysis Software: Kaluza, LEGEND plex.Alanine aminotransferase activity in serum was measured by an auto biochemical analyzer , with the reference range of 9–50 IU/L. The HBV markers, HBsAg and HBeAg, were determined by commercially available enzyme-linked immunosorbent assays (Shanghai KH Biology). Serum HBV DNA was quantitated by real-time quantitative polymerase chain reaction using a commercially available kit , the lower detection limit was 30 IU/ml.n (%). Student's t-test or One-way ANOVA was conducted to compare two groups and comparisons between three or more means. Correlations between variables were calculated with the Spearman rank correlation test. In our studies, p < 0.05 were considered significant.Statistical analysis was performed using SPSS 22.0 Software . Data were expressed as the mean ± standard deviation and p < 0.001). However, CD16+ and CD16– myeloid dendritic cells (MDC) do not show a significant difference between these two groups. In order to explore whether there are changes between different stages of CHB, we further compared the DC in CHB with different stages, and we found that CD16– MDC in the IT group was lower than that in IC (p = 0.026) group (To determine whether deficiency of antigen-presenting cells (APC) occurs in patients, we investigated the percentage of dendritic cells (DC) in patients with CHB and compared it with the healthy controls. The identification of DC by flow cytometry was shown in 6) group , PDC andp = 0.019) and RA (p = 0.002) stage  of HLA-DR expression in monocyte cells. There was no difference found between CHB and the normal group. However, in different stages of CHB, results show significantly lower MFI expression in IT than that in IC (2) stage .p < 0.001). The CHB patients with abnormal ALT were higher than normal ones (p = 0.040) , and negatively correlated with IL-17 and IFN-γ that was secreted by CD4+ T cells  (p = 0.005), on the contrary, the Memory B cells increased (p = 0.005). There was no significant difference in plasma blast between healthy and CHB groups  . Our dat= 0.040) . In addictively) . In compB groups .p = 0.035). Similarly, in a different stage of CHB, cTfh in IC was higher than in IT patients (p = 0.043) . The cells with high expression of CXCR5 and PD-1 were regarded as circulating T follicular helper cells (cTfh) . Our res= 0.043) .p = 0.019 and p < 0.001, respectively), suggesting increased T cell exhaustion in CHB patients. Interestingly, we did not find differences in PD-1 expression in the comparison of different stages of CHB (We examined the expression of programmed cell death protein 1 (PD-1) in T cells . The ress of CHB .p = 0.038), while the number of CD56 dim subsets did not change (p < 0.001) (p = 0.025) and, in contrast, the activating receptor NKP30 is decreased (p = 0.009) in CHB patients (We measured the percentage of subsets of immune cells defined by CD56 expression levels. We observed that there was an increase in the proportion of NK cells in the group of CD56 positive in CHB patients (t change . The CD5< 0.001) . In ordepatients .p = 0.038 and p = 0.024, respectively). This result suggested enhanced cytotoxic function of T cells in CHB. In contrast, cytokine secretion was impaired in CHB patients, and our results showed that the IFN-γ positivity in CD8+ T cells and the proportion of IFN-γ, IL-21, IL-17 in CD4+ T cells were all significantly lower than those in healthy controls   .p = 0.043), no significant differences were found in IL-6 across the four immune phases (data not shown).The concentrations of cytokine in plasma were measured by Cytometric bead array (CBA). The capture antibodies for IL-2, IL-6, and IL-10 were coated on 5 um microspheres differentiated by three different levels of APC fluorescence intensity, respectively. IFN-γ, IL-17, IL-4, IL-12, and TNF-α were coated on 7 um microspheres of five APC fluorescence intensity levels in turn, and the PE channel was used for color development before being converted to cytokine concentration on the standard curve . We founNatural Killer cells account for ~5–15% of peripheral blood lymphocytes . It is tNatural Killer cells are activated during acute HBV infection before the onset of adaptive immunity, and the effective immune response of NK cells leads to initial control of the acute infection in the early stages . HoweverThe function of NK cells is closely regulated by activating and inhibiting receptors. The interactions between NK cell receptors and corresponding ligands determine the state of NK cells . In a chInterestingly, it was found that T cells also had “exhaustion” in CHB patients. On one hand, the inhibitory receptor PD-1 of both CD4+ and CD8+ T cells was significantly increased, and on the other hand, the ability of each T cell subpopulation to secrete cytokines was decreased to varying degrees. More importantly, we also found a phenomenon similar to NK cells “functional dichotomy” in T cells, and our results showed the higher cytotoxic function of CTL than healthy controls. Studies have shown that dysfunctional NK CHB cells may exhibit the same molecular characteristics at both transcriptional and protein levels, and it is a kind of signal involved in calcium balance characteristics , which mImmunoinhibitory cytokines and regulatory cells are also involved in the lymphocyte “exhaustion” process of CHB, and negative regulation is an important factor in the induction of CD8+ T and NK cell exhaustion –25. For On the other hand, in Breg analysis, we found that Breg was significantly increased in patients with CHB, which was positively correlated with ALT and DNA levels, and negatively correlated with cytokine secretion of T cells. In previous studies on CHB, studies on B cells mainly focused on antibody production, while other functions such as antigen presentation and immune regulation, which are closely related to immune tolerance and liver injury, were ignored . We specWe also compared the concentrations of plasma cytokines in CHB patients. Among the detected cytokines, the levels of IL-2 and IL-6 in CHB patients were increased, while the widely reported inhibitory factor, IL-10, was not significantly changed , 33, whiWe found no difference in levels of IFN-γ in serum between healthy and CHB, which does not contradict previous studies showing a decrease in the proportion of IFN-γ positive T cells in CHB patients. IFN-γ is low in healthy serum and needs to be stimulated for the cells to secrete it in large quantities, and the results of previous experiments can only suggest an impaired function of cytokine secretion by T cells in CHB patients. Perhaps there is a decrease in the concentration of IFN-γ in inflammatory local tissue when there is a CHB infection compared to the acute infection period, but this needs further experimental.A dendritic cell is the most common antigen presenting cell (APC). Dendritic cells (DCs) are divided into myeloid dendritic cells  and serplike dendritic cells . DCs play important roles in the initiation and maintenance of immune cell function . HoweverInterestingly, IL-21 is the main effector factor of cTfh cells, which has a good positive correlation with the number of cTfh, and cTfh cells can achieve the regulation of B cell development through IL-21 . HoweverIn summary, we found disorders in the immune cells of peripheral blood in CHB patients, especially NK cells and T cells. This phenomenon may be related to the increase of regulatory B cells and the decrease of DC cells in peripheral blood.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committees of the Tongde Hospital of Zhejiang Province (Identification No. 2019KY048). The patients/participants provided their written informed consent to participate in this study.XJ and Z-hY contributed to study concept and design, acquisition of data, analysis and interpretation of data, and drafting of the manuscript. LL contributed to statistical analysis. SL contributed to samples collections. GZ and WL contributed to study concept and design, study supervision, and critical revision of the manuscript. All authors have read and approved the manuscript.This study was supported by the Scientific Research Fund Project of Zhejiang Traditional Chinese Medicine (Grant No.: 2017ZA024) and the Scientific Research Project of Zhejiang Province (Grant No.: 2019KY048).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.


The underlying mechanism of reversal of chemoresistance is correlated with inhibition of drug transporters, induction of apoptosis, and modulation of the tumor microenvironment(TME), as well as the modulation of signaling pathways, such as nuclear factor erythroid-2 related factor 2 (NRF2)/AKT, lncRNA cancer susceptibility candidate 2(CASC2)/ protein tyrosine phosphatase gene (PTEN), AKT/ sirtuin1(SIRT1), epidermal growth factor receptor (EGFR)/ phosphatidylinositol 3-kinase (PI3K)/AKT, PI3K/AKT/ mammalian target of rapamycin(mTOR) and nuclear factor-κB (NF-κB). Since the effects and the mechanisms of ginsenosides on chemoresistance reversal have not yet been reviewed, this review summarized comprehensively experimental data in vivo and in vitro to elucidate the functional roles of ginsenosides in chemoresistance reversal and shed light on the future research of ginsenosides.Chemoresistance has become a prevalent phenomenon in cancer therapy, which alleviates the effect of chemotherapy and makes it difficult to break the bottleneck of the survival rate of tumor patients. Current approaches for reversing chemoresistance are poorly effective and may cause numerous new problems. Therefore, it is urgent to develop novel and efficient drugs derived from natural non-toxic compounds for the reversal of chemoresistance. Researches  For example, Fu et al. found that ginsenosides might facilitate Eca109/DDP cells more sensitive to cisplatin (DDP). And their study showed that co-treatment of ginsenoside Rg3 and DDP decreased expression of P-gp more than treatment of DDP alone, suggesting the decreased expression of P-gp might be responsible for ginsenoside Rg3-mediated reversal of chemoresistance. Similarly, Ma and his coworkers  demonstrα (HIF-1 α), C/-enhancer-binding protein β (C/EBP β), forkhead box-containing protein, O subfamily1(FoxO1), or Y-box binding protein-1(YB-1), which regulated human MDR1 gene transcription or affected the pregnane X receptor (PXR) dependent transcription activity, showing that the effect of Rd on chemoresistance was not related to the transcription of MDR1 gene. Additionally, Rd increased ubiquitination of MDR1 and consequently reversed doxorubicin (DOX)-resistance in MCF-7/Adr cells, which increased MDR1 protein levels. Further study by Zhang et al. -Rh2 in MCF-7/Adr cells. Moreover, inhibiting the MAPK/NF-κB pathway brought about a decrease in the expression of ABCB1 and the cellular pharmacokinetics regulated by Adr was also significantly altered by inhibiting NF-κB. Overall, 20 (S)-Rh2 might down-regulate Adr-induced overexpression of ABCB1 in MCF-7/Adr cells by alleviating the MAPK/NF-κB pathway.Further studies also revealed the underlying mechanism which contributed to the regulation of ginsenosides on overexpression of P-gp. Ginsenosides could also reverse chemoresistance through inhibiting the expression of MDR1 gene and increasing the ubiquitination of MDR1. MDR1 stability is dependent on ubiquitin-dependent protein degradation . A studyg et al.  aimed toIn addition, blocking the binding site of chemotherapeutic drugs and P-gp is efficient to reverse chemoresistance. There are two types of blockers, including competitive and non-competitive ones. The competitive blockers have high affinity and refer to the ones that leave little place for the binding of P-gp to other chemotherapeutic agents, which result in decreased activity of P-gp . In cont3H]-azidopine binding site in DOX resistant AML-2/DX100 cells. Similarly, Gao et al. , DOX, colchicine (COL) and VP-16 in KBV20C were decreased and the intracellular accumulation of drugs was increased in KBV20C cells following Rg3 treatment. Without any effect on MDR1 gene expression or P-gp level, Rg3 resisted vinblastine efflux and reversed chemoresistance to those drugs in KBV20C cells, properly owing to its competition with [3H] azidopine for binding to the P-gp. It was suggested Rg3 could specifically inhibit P-gp-mediated drug accumulation. Thus, reversal of chemoresistance mediated by Rg3 was redounded to its competition with anticancer drugs for binding to P-gp thereby blocking drug efflux. In addition, Rb1, a major component of ginseng, exerted a synergistic effect with vincristine on chemoresistance in HL60/VCR cells but failed to inhibit the expression of MDR1 -Rh2 exerted an inhibitory effect in a noncompetitive instead of a competitive manner. Similarly, Zhao et al. -Rh2, the activity of P-gp immediately returned to the control levels after removal of aPPD and aPPD exerted no effect on ATPase. Another study -Rh2 were not substrates of P-gp, the recovery time of P-gp activity was different when aPPD and 20 (S)-Rh2 was removed due to their different chemical structures.At present, there were lots of studies showing that ginsenosides could decrease drug effluxes through competitively or non-competitively inhibiting drugs from being bound with P-gp. On one hand, ginsenosides always inhibit chemotherapeutic agents from being bound to these proteins by competitively blocking their binding site and therefore resisting drug efflux, without any effects on the mRNA and protein expressions of drug-related proteins. For example, Choi et al.  found tho et al.  found tho et al.  revealed of MDR1 . It was  of MDR1  explored/T cells . The und/T cells . It signo et al.  held thaer study  also indingredient of a lipid bilayer, is responsible for lipid fluidity owing to its function and activity  obtained similar results from the previous findings. Moreover, the nude mice bearing human lung adenocarcinoma that received co-treatment of DDP and Rg3 had obviously reduced tumor volumes and weights compared with animals treated with DDP alone. Moreover, Rg3 treatment enhanced uptake of technetium-99m labeled hexakis-2-methoxyisobutylisonitrile (99mTc-MIBI) suggesting that Rg3 can sensitize chemotherapy in vivo. Consistently, another study  Autophagy usually precedes apoptosis and then activates apoptosis. 2) In sApoptosis, known as programmed death, is one of the important life phenomena in the biological world. Generally, the Fas pathway, mitochondrial passage, adenylyl-cyclase pathway and p53 activation pathway mediate apoptosis. They target Bax and suppress anti-apoptotic proteins such as Bcl-2, finally triggering apoptosis . Data hain vitro and in vivo. For instance, Hu et al.  and protein tyrosine phosphatase gene (PTEN) have been characterized as tumor suppressors and efficient suppressive regulators of cancers. Regulation of CASC2 and PTEN induced by AKT is closely associated with chemoresistance of tumors. For example, Feng et al.  found thsistance . TherefoCo-treatment of Rg3 and gemcitabine (GEM) can inhibit the growth of drug-resistant cells through the CASC2/PTEN/Akt pathway. Zou et al.  found thSirtuin1 (SIRT1) can deacetylate various histone and non-histone substrates, such as p53, c-MYC and FOXO, therefore altering DNA repair, cell cycle, apoptosis and development of chemoresistance . Also, SThere was a study confirming that the combination of ginsenoside Rp1 and ActD exerted a synergistic sensitization on the drug resistant cancer lines by modulating the AKT/SIRT1 pathway . TreatmeEpidermal growth factor receptor (EGFR), known as a key factor in the development of tumor, can bind with a ligand, which leads to autophosphorylation of the receptor, activate downstream signal transduction and activity, involving Ras/MAPK, PI3K/AKT, STAT, and Src family kinases, and promote proliferation, survival, invasion, and migration of tumor cells . Among tin vitro and in vivo. The cell viability, growth and formation of cell colonies in BxPC-3 and AsPC-1 cells were inhibited when exposed to different concentrations of Rg3 and erlotinib. Likewise, apoptosis induced by erlotinib and cleavage of caspase-3, caspase-9 and PARP were obviously promoted by Rg3 in BxPC-3 and AsPC-1 cells. Furthermore, lower expression levels of p-EGFR, p-PI3K and p-AKT were observed when combined with Rg3, which revealed that Rg3 downregulates expressions of p-EGFR, p-PI3K, and p-AKT, thereby sensitizing pancreatic cancer cells to erlotinib. Similarly, the study in vivo showed Rg3 inactivated the EGFR/PI3K/AKT signal pathway. Moreover, the tumor volume was decreased by co-treatment of erlotinib and Rg3 in nude mice implanted with BxPC-3 cells. In conclusion, downregulating the EGFR/PI3K/AKT signal pathway, which induced cell apoptosis, was a vital mechanism related to Rg3-mediated reversal of chemoresistance.Rg3-mediated inactivation of the EGFR/PI3K/AKT pathway is an efficient method to induce cancer cell apoptosis, which has been proved by Jiang and his coworkers . The senActivation of the PI3K/AKT/mTOR pathway is a common feature of a wide range of human cancers . UpregulGinsenoside Rg1 could induce apoptosis in paclitaxel-resistant nasopharyngeal cancer cells which might be correlated to suppression of the mTOR/PI3K/AKT pathway . Rg1 incκB (NF-κB) in cancer treatment induces tumor promotion, angiogenesis, metastasis, apoptosis, and resistance to anticancer drugs , paclitaxel, DDP and Adr, resisted the growth of HCT116 and SW620 colon cancer cells and induced apoptosis via inhibiting NF-κB , especially LNCaP cells. Importantly, co-treatment of Rg3 and TXT caused a significant reduction in the expression of G0/G1 regulators cyclin D1, cyclin E, Cdk2, and Cdk4. Moreover, another study by Yuan et al. -ginsenoside Rg3 was a novel inhibitor of autophagy through increasing LC3II and p62 protein levels in a CHOP and ATG-5 dependent autophagic manner and thus sensitized hepatocellular carcinoma to doxorubicin.In contrast, autophagy inhibition is critical for ginsenosides to reverse chemoresistance. Wang et al.  found thTumor microenvironment (TME), including smooth muscle cells, endothelial cells, fibroblasts of various phenotypes, myofibroblasts, mast cells, T cells, B cells, natural killer, neutrophils, granulocyte, and antigen-presenting cells is a complex and constantly changing matrix surrounding tumor cells. The interaction of these components in TME has affected the biological behavior of tumors including epithelial-mesenchymal transition (EMT), characteristics maintenance of cancer stem cells (CSCs), invasion, metastasis, and chemoresistance . CSCs, tκB pathway thereby retaining the progress of EMT and stemness evidenced as reduced expressions of E-cadherin, N-cadherin, sex determining region Y-box 2 (SOX2), NANOG, OCT4, and CD44. Moreover, consistent results both in vivo and in vitro were observed. Collectively, Rg3 could sensitize hypoxic lung cancer cells to DDP through preventing NF-κB induced EMT and stemness. Also, Rg3 (κB and the ERK pathways. Co-treatment of GPT and PTX inhibited expressions of CSC related genes including octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), NANOG, aldehyde dehydrogenase 1 (ALDH1), and CD44 gene expression, indicating that cell stemness-mediated invasion and tumor growth were inhibited by GPT and PTX. And IRAK1 and S100A7/8/9 formed a feedback loop to drive the malignancy of TNBC cells , IL8, chemokine (C-X-C motif) ligand 1 (CXCL1), and chemokine (C-C motif) ligand 2 (CCL2). Thus, GPD could overcome PTX resistance by mediating stemness of tumor cells which resulted from an inhibition of the IRAK1/NF-κB/ERK pathway.Studies showed that ginsenosides suppressed the ovarian stem cancer cells through inhibiting the process of EMT. For example, Kala et al.  found thlso, Rg3  could talso, Rg3 . Co-trealso, Rg3  sensitizBC cells . The comκB pathways   , a novelin vivo and in vitro and illuminates the mechanisms by which ginsenosides alleviate chemoresistance. The underlying molecular mechanism can be partially elucidated by the roles of the expression and function of P-gp, MDR1, BCRP, LRP, apoptosis, autophagy, and microenvironment as well as CASC2/PTEN/AKT, AKT/SIRT1, EGFR/PI3K/AKT, PI3K/AKT/mTOR and MAPK/NF-κB signaling pathways. Given that ginsenosides can also reduce side effects caused by chemotherapy and elicit anti-cancer activities and little toxicity, they have demonstrated promises as an indispensable strategy for reversing chemoresistance. We look forward to further revealing their reversal mechanisms and widespread clinical applications.This article systematically summarizes the therapeutic potential of ginsenosides in reversing chemoresistance


Background:SCN1A is one of the most common epilepsy genes. About 80% of SCN1A gene mutations cause Dravet syndrome (DS), which is a severe and catastrophic epileptic encephalopathy. More than 1,800 mutations have been identified in SCN1A. Although it is known that SCN1A is the main cause of DS and genetic epilepsy with febrile seizures plus (GEFS+), there is a dearth of information on the other related diseases caused by mutations of SCN1A.Objective: The aim of this study is to systematically review the literature associated with SCN1A and other non-DS-related disorders.Methods: We searched PubMed and SCOPUS for all the published cases related to gene mutations of SCN1A until October 20, 2021. The results reported by each study were summarized narratively.Results: The PubMed and SCOPUS search yielded 2,889 items. A total of 453 studies published between 2005 and 2020 met the final inclusion criteria. Overall, 303 studies on DS, 93 on GEFS+, three on Doose syndrome, nine on the epilepsy of infancy with migrating focal seizures (EIMFS), six on the West syndrome, two on the Lennox–Gastaut syndrome (LGS), one on the Rett syndrome, seven on the nonsyndromic epileptic encephalopathy (NEE), 19 on hemiplegia migraine, six on autism spectrum disorder (ASD), two on nonepileptic SCN1A-related sudden deaths, and two on the arthrogryposis multiplex congenital were included.Conclusion: Aside from DS, SCN1A also causes other epileptic encephalopathies, such as GEFS+, Doose syndrome, EIMFS, West syndrome, LGS, Rett syndrome, and NEE. In addition to epilepsy, hemiplegic migraine, ASD, sudden death, and arthrogryposis multiplex congenital can also be caused by mutations of SCN1A. Figures 2, 3). SCN1A, a Nav1.1 α subunit composed of 26 coding exons and located in the 85-kb gene region, is the most common epileptic gene and the most common pathogenic gene in the Dravet syndrome (DS), a catastrophic and intractable epileptic encephalopathy (EE) . In addition to gene mutations of SCN1A that can cause DS, other genes include PCDH19, SCN2A, SCN8A, SCN1B, GABRA1, GABRG2, GABRB3, STXBP1, HCN1, CHD2, and KCNA2 can also cause DS or DS-like phenotypes (Voltage-gated sodium channel (VGSC) channels play an essential role in normal neurological function , especiaenotypes . They arenotypes .SCN1A” or “scn1a”.A systematic search was performed in PubMed and SCOPUS. The most recent search was performed on October 20, 2021, using the term “SCN1A associated with a particular disease were included in the criteria. We excluded articles not written in English or Chinese, nonoriginal work that has nothing to do with people, such as reviews, meta-analysis, animals or cells, experimental articles not adding information to the question posed in this review, and papers that could not be retrieved via PubMed or SCOPUS. The records were screened by JD and evaluated by XL with respect to the inclusion and exclusion criteria. Disagreements were resolved through a discussion between the two review authors (All the articles with mutations of  authors .n = 331); review, systematic review, and meta-analysis (n = 357); comment, letter, editorial, and erratum (n = 97); reports not in English or Chinese (n = 14); irrelevant exposure (n = 416); full text unavailable (n = 32); or epilepsy type unknown (n = 42). Finally, 453 studies met the inclusion and did not meet the exclusion criteria , the literature search yielded 1,742 articles . After scriteria . It is wSCN1A gene is not only associated with DS and GEFS+, but can also cause other disorders, including epilepsy diseases such as Doose syndrome, epilepsy of infancy with migrating focal seizures (EIMFS), West syndrome, Lennox–Gastaut syndrome (LGS), Rett syndrome, and nonsyndromic epileptic encephalopathy (NEE), as well as nonepileptic diseases such as hemiplegia migraine, autism spectrum disorder (ASD), sudden death (sudden unexpected death in epilepsy [SUDEP] and nonepileptic SCN1A-related sudden deaths), and arthrogryposis multiplex congenita (AMC).The SCN1A are identified predominantly in patients with DS, also known as severe myoclonic epilepsy of infancy (SMEI), and in the families with GEFS+. However, SCN1A is less common in epileptic and nonepileptic disorders other than DS and GEFS+. Herein, we focus on these rare diseases with the exception of DS and GEFS+.Mutations in the voltage-gated sodium channel subunit gene SCN1A gene  in GEFS+ families , was first discovered by Scheffer and Berkovic in an Australian family in 1997 . Since ind Fgf13 , 26. We se GEFS+ . Variousfamilies  , was previously called myoclonic astatic epilepsy (MAE), a rare childhood EE . First rSCN1A, SCN1B, CACNA1H, SLC2A1, GABRG2, CHD2, SLC6A1, STX1B, GABRB3, SYNGAP1, and WDR45  or infantile malignant migratory partial seizure (MMPSI), is a rare and early-onset developmental EE inherited in an autosomal dominant pattern. It is characterized by onset within 6 months of birth and mainly manifests in the form of frequent, migratory, and varying types of focal seizures. Epileptic seizures are related to the multifocal EEG discharge. Similar to DS, this disease is often associated with severe cognitive impairment and motor impairment. However, unlike DS, the most common causative gene is SCN1A mutation in a female infant diagnosed with MPSI. The female infant, who was delivered to term, developed epilepsy at 10 weeks after birth, accompanied by progressive hemiplegia, apnea, and progression of multifocal migratory partial epileptic seizures, leading to a recurrence of epileptic status and death at 9 months (SCN1A mutation was found in another patient with MPSI (SCN1A mutation-induced MPSI with good therapeutic effect assisted by hypothermia (SCN1A mutation (MPSI)  . In the  infancy . In 2016mutation . Recentlth EIMFS   × 1 (build19)] , is a refractory classic EE characterized by repetitive epileptic spasms (ES) and hypsarrhythmia . The eti disease , 59. Hatuild19)] . In 2003syndrome . Ilona esyndrome , Table 1Lennox–Gastaut syndrome is a childhood EE whose main clinical features include multiple types of drug-resistant seizures, intellectual disability, and abnormal EEG with diffuse spines slow complex wave or paroxysmal fast activity. The etiology of LGS is also complex and varied; about 75% of cases have obvious causes such as cortical malformations, posthypoxic ischemic results, postmeningitis/encephalitis, or metabolic encephalopathy, while about 25% are cryptogenic , transcription regulation genes (CHD2), neurocutaneous syndrome-related genes (TSC1 and TSC2), metabolic genes (Alg13), and others  in the methyl-CpG-binding protein 2 (MECP2) gene , a small fraction of the patients  may carry genetic mutations in other genes, such as the cyclin-dependent kinase-like 5 (CDKL5) and FOXG1 , also known as early onset epileptic encephalopathies, early infantile epileptic encephalopathies (EIEEs), or early infantile-onset developmental and epileptic encephalopathies (EIODEEs) , 51, 66,SCN1A phenotype called early infantile SCN1A encephalopathy, in which the patient had an apparent movement disorder , ATP1A2 (FHM2), and SCN1A (FHM3)  . Familia)  in 20 patients with ASD, and this mutation may be inherited from its parent . Gene sequencing revealed an SCN1A mutation. Microscopic examination of the hippocampus revealed focal bilamination of the dentate gyrus. The other case occurred in a 7-week-old female with two SCN1A mutations (SCN1A mutations are not only closely related to SUDEP but also associated with nonepileptic-related sudden death.Sudden death associated with n et al.  found onn et al.  found anutations  (Figure utations , Table 2SCN1A+/- mice, severe arrhythmias were found to be characterized by prolonged PR interval, increased heart rate variability, and even atrioventricular block, suggesting that changes in the cardiac SCN1A may be related to SUDEP (SCN1A+/− mice; notably, atropine and N-methyl scopolamine were effective in preventing sudden death in mice (SCN1A mutant mice (The exact mechanism of SUDEP remains unclear. In systemically knockout heterozygous to SUDEP . In anot in mice . In addiant mice .Arthrogryposis multiplex congenita refers to an etiologically heterogeneous condition that is characterized by the congenital joint contractures in two or more body areas . AMC is SCN1A, with only two reports documented (SCN1A mutations in three infants with AMC from three different families (SCN1A (Although over 320 genes have been implicated, exemplifying the genetic heterogeneity of the condition , AMC is cumented , 94. Thefamilies . During s (SCN1A , Table 2SCN1A not only causes DS and GEFS+; other epileptic encephalopathies, such as Doose syndrome, EIMFS, West syndrome, LGS, RTT, and NEE, are also directly related to SCN1A. In addition to epilepsy, FHM3, SHM, ASD, sudden death, and AMC can also be caused by SCN1A mutations (utations . This reAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This study was supported by the National Natural Science Foundation of China, Grant/Award Number: 81971085, and the Advantages Discipline Group Project of Ningxia Medical University, Grant/Award Number: XY201511.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.


PNPLA3-rs738409 C>G, TM6SF2-rs58542926 C>T, MBOAT7-rs641738 C>T, and HSD17B13-rs72613567 T>TA have been shown to influence progression to advanced chronic liver disease (ACLD) in patients with chronic hepatitis C (CHC). We aimed to investigate their impact on disease regression , von Willebrand factor (VWF), and VWF/platelet count ratio (VITRO)]) and clinical outcomes after CHC cure in 346 patients with pre-treatment ACLD. Patients carrying the PNPLA3 minor allele had more advanced liver disease prior to antiviral therapy, confirming its impact on liver disease progression. In a subgroup of 88 patients who underwent paired HVPG-measurements and were genotyped for all SNP/indels, PNPLA3/TM6SF2/MBOAT7/HSD17B13 genotypes were not associated with changes in HVPG. In line, changes in non-invasive surrogates of portal hypertension (LSM/VWF/VITRO) were comparable between carriers and non-carriers of the PNPLA3 G-allele in the overall cohort. Finally, carriage of PNPLA3 G-allele was not associated with the development of hepatic decompensation, de-novo hepatocellular carcinoma, or transplant-free mortality during a median follow-up of 42 months after the end of antiviral treatment. Therefore, genetic variants in PNPLA3/TM6SF2/MBOAT7/HSD17B13 do not impact the regression of portal hypertension and clinical outcomes in patients with pre-treatment ACLD after CHC cure.Genetic variants including  Interferon (IFN)-free therapies have revolutionized the treatment of patients with chronic hepatitis C (CHC). Rates of sustained virologic response  have increased to almost 100%, even in previous difficult-to-cure patient populations, such as patients with HIV coinfection  or compeAfter approximately 5 years of follow-up (FU), HCV cure by IFN-based therapies improved the histological stage of fibrosis in 82% of patients and in 61% despite previously established cirrhosis ,8,9,10. Nowadays, liver biopsy—and, to some extent, hepatic venous pressure gradient (HVPG) measurement—have been replaced by non-invasive surrogate markers such as liver stiffness measurement (LSM), von Willebrand factor (VWF) antigen, and von Willebrand factor/platelet count ratio (VITRO) score ,14. AlthA series of single nucleotide polymorphisms (SNP) and indels have been shown to modify the severity of CHC. The rs738409 G-allele encoding the I148M variant of patatin-like phospholipase domain-containing protein 3 (PNPLA3)  has beenAlthough the impact of these genetic variants on liver disease progression is well-defined, their impact on liver disease regression after cure of CHC  has yet to be investigated.Therefore, we aimed to assess their impact on the regression of portal hypertension, as assessed by changes in (I) hepatic venous pressure gradient (HVPG) and (II) non-invasive markers  as well as (III) clinical events in CHC patients with ACLD who achieved SVR to IFN-free therapies.All patients who underwent IFN-free treatment at the Medical University of Vienna between 1 January 2014 and 31 July 2019 and achieved SVR were assessed for eligibility. The study was approved by the ethics committee of the Medical University of Vienna (EK: 1947/2019). Inclusion criteria were: (I) evidence suggestive or confirmative of ACLD  and II.Specifically, data on all investigated genetic variants  were available in 88 thoroughly characterized patients with paired measurements of HVPG as well as LSM and VITRO score (cohort A). Of note, these patients have been previously reported in a study investigating the prognostic value of changes in HVPG  after HCIn addition, we evaluated the influence of PNPLA3 rs738409 C>G  on liver disease regression and post-treatment outcomes in the overall study population of 346 patients (cohort B).https://ldlink.nci.nih.gov/?tab=ldmatrix, accessed on 1 April 2021).DNA was extracted using the QIAamp DNA Blood Mini Kit . Genotyping was performed by StepOnePlus Real-Time PCR System and a TaqMan SNP Genotyping Assay . Linkage disequilibrium was investigated using LDmatrix  in fasting condition. Reliability of liver stiffness measurements was defined by previously established criteria [−1) [Clinical and laboratory parameters were evaluated by chart review. LSM and VITRO were assessed prior to antiviral therapy, as well as a median of 12.9 (24.1) weeks after the end of therapy . LSM was performed by vibration-controlled transient elastography (FibroScancriteria . Controlria [−1) .FU started at end of treatment. In patients with cACLD, first hepatic decompensation was defined by variceal bleeding, incident ascites, or incident hepatic encephalopathy (HE), whereas in patients with decompensated ACLD, further hepatic decompensation was defined by variceal bleeding, requirement of paracentesis, admission for grade 3/4 HE, or development of grade 3/4 HE during admission. Furthermore, de-novo HCC, TIPS placement, LT, and death were recorded.See In 88 patients with data on all four SNP/indels (cohort A), 73 patients (83.0%) exhibited TM6SF2 rs58542926 C/C and 15 (17.0%) the C/T genotype . Moreove−1, median BL-VWF 239 (IQR: 173–321)%, and median BL-VITRO 1.91 (IQR: 1.10–3.38). One hundred and eighty-one patients (52.3%) had clinical evidence of CSPH .Of 346 patients included in the overall cohort (cohort B), 173 patients (50.0%) carried the PNPLA3 rs738409 C/C genotype , 146 patients (42.2%) had the G/C genotype, and 27 patients (7.8%) had the G/G genotype (Hardy–Weinberg equilibrium: Χ² = 0.248). This cohort included 230 (66.5%) males and 116 (33.5%) females, with a mean age of 55.4 ± 10.7 years . Forty-fp = 0.015), a higher BL-MELD score , more patients with evidence of CSPH , and a higher BL-VITRO score  carrying the PNPLA3 rs738409 G-allele to C/C patients, we found a higher prevalence of varices , and BL-VITRO . Moreover, 81.5% of G/G patients had evidence of CSPH compared to 56.8 and 43.9% with G/C and C/C genotype, respectively  and end of treatment  to the second HVPG-assessment, and thus, the time point of the second HVPG measurement did not impact our results.Of note, there was no correlation between changes in HVPG and the time from treatment initiation .−1, p = 0.045) and FU-PLT . Moreover, PNPLA3 G-allele carriers had higher BL-  and FU-VITRO scores . Importantly, there were no differences in the absolute or relative changes in non-invasive surrogates of portal hypertension between G-allele carriers and non-carriers. Noteworthy, this finding was not affected by accounting for BL values of the respective parameters by an analysis of covariance (ANCOVA).We compared the dynamics of surrogates in portal hypertension  between patients with or without the PNPLA3 rs738409 G-allele in the overall study population . Carrier−1, p = 0.087; relative Δ CAP: 1.5 (IQR: 27.0) % vs. -1.9 (IQR: 25.1) %, p = 0.096). Interestingly, absolute changes in CAP were significantly different between carriers and non-carriers of the PNPLA3 G-allele after adjusting for BMI in ANCOVA (p = 0.046).Additionally, prevalence of hepatic steatosis declined in PNPLA3 rs738409 G-allele carriers (47.3 vs. 41.8%) and non-carriers (57.8 vs. 47.8%). Although BL and FU CAP values were not significantly different between carriers and non-carriers of the PNPLA3 rs738409 G-allele, absolute and relative changes showed a trend towards a discordancy  vs. non-carriers: -6 (IQR: 64) dB × mNext, we investigated the development of clinical events during FU. During a median FU of 42 (IQR: 22–54) months after the end of treatment, 23 patients (6.6%) developed hepatic decompensation. Specifically, nine patients (3.9%) showed development/worsening of ascites, eight patients (2.3%) had development/worsening of HE, and six patients (1.7%) suffered from variceal bleeding as first decompensation event post-treatment. Moreover, 25 patients (7.2%) developed HCC (corresponding to 2.4/100 patient years), one patient (0.3%) underwent TIPS placement, five patients (1.4%) underwent LT, and 20 patients (5.8%) died.p < 0.001), higher BL-LSM , and higher BL-VITRO score .Of note, the distribution of PNPLA3 genotypes was not significantly different between patients with and without hepatic decompensation during FU (p = 0.546) was not independently associated with hepatic decompensation, after correcting for important differences in BL characteristics, i.e., serum albumin level, MELD score, and history of hepatic decompensation  and further decompensation in dACLD patients  yielded no statistically significant differences , as was liver-related transplant-free mortality  did not impact changes in surrogates of portal hypertension  and the development of clinical events  after the removal of the primary etiologic factor in a cohort of 346 patients.Although the genetic variants evaluated in this study have been shown to modulate liver disease progression, their impact on liver disease regression remained unclear. Interestingly, none of these genetic factors had an impact on the changes in HVPG in a thoroughly characterized cohort of 88 patients undergoing paired assessments. Moreover, the Due to the unprecedented advances in the treatment of CHC, the focus of attention has shifted to disease regression and risk stratification after SVR . FurtherPNPLA3, TM6SF2, MBOAT7, and HSD17B13 genotypes impacted the regression of portal hypertension, as assessed by HVPG. Of note, HVPG measurement is not only the diagnostic gold standard for portal hypertension [Firstly, we did not find conclusive evidence that rtension , but alsrtension .PNPLA3) on changes in well-validated surrogates of portal hypertension [PNPLA3 rs738409 G-allele carriage and pre-treatment severity of hepatic dysfunction and portal hypertension, which is in line with the previous literature [PNPLA3 G-allele were considerably younger, which points towards an accelerated progression to ACLD. However, despite the evidence supporting the impact of PNPLA3 rs738409 G-allele carriage on liver disease progression, changes in surrogates of portal hypertension were comparable between carriers and non-carriers of the PNPLA3 rs738409 G-allele, indicating the absence of an impact on liver disease regression.Secondly, to mitigate the risk of type II error due to the limited number of patients undergoing paired invasive HVPG measurements, we evaluated the effect of the presumably most impactful variant , since CTP A cirrhosis could have been more common in those without a G-allele, as observed in our cohort of unselected ACLD patients. In another study, patients who survived the first 90 days after severe alcoholic hepatitis and achieved abstinence had a worse outcome, if they were homozygous for the PNPLA3 rs738409 G-allele [PNPLA3 rs738409 [PNPLA3 rs738409 G/G genotype was only prevalent in 27 patients (7.8%), which did not allow for a comprehensive statistical analysis when applying a recessive model.Finally, carrying the patients . In this disease , which e disease  may haveG-allele . Severalrs738409 ,18,40. Hrs738409 ,20,41, tPNPLA3 rs738409 G-allele in univariate analysis. However, the increased risk was primarily due to more advanced liver disease at BL, as the HR decreased considerably, after adjusting for known differences in BL characteristics.Nevertheless, we found a trend towards a higher risk of hepatic decompensation in patients carrying the PNPLA3 rs738409 genotypes. Importantly, studies reporting an increased incidence of HCC in CHC patients who are homozygous for the PNPLA3 rs738409 G-allele, suggesting a cancerogenic effect of this variant—possibly due to increases in hepatic inflammation [The incidence of HCC after HCV eradication was comparable across ammation —were mosammation . Thus, tammation  ranging ammation ,44,45.PNPLA3 rs738409 G-allele in CLD [PNPLA3, TM6SF2, MBOAT7 have repeatedly been shown to increase the susceptibility for and promote poor outcomes in NAFLD and ALD, while the indel in HSD17B13 assessed in our study was protective in previous analyses. Moreover, there is a growing body of evidence indicating that these genetic variants also impact disease severity in patients with CHC [PNPLA3 rs738409 G-allele . Nevertheless, the diagnostic ability of CAP in patients with ACLD is limited [Following the description of the role of e in CLD , common with CHC ,40,46,47with CHC ,41. Hepawith CHC ,49, whicwith CHC . However limited , and theSince our findings suggest that the negative impact of genetic factors predisposing for progressive liver disease may be overcome by etiological cure, patients with an unfavorable genetic background may be subjected to more aggressive etiological treatment strategies . Although this observation has limited relevance in the context of CHC, which has become easy to cure and should be treated in basically all patients without delay  to achiePNPLA3 rs738409) in the overall cohort, which helped to reduce the probability of type I error due to the investigation of a high number of genetic variants. Although our study aimed to investigate the impact of genetic factors, metabolic factors and medical treatments may also determine clinical outcomes after HCV eradication. In this context, we ruled-out that components of the metabolic syndrome, hepatic steatosis, alcohol consumption, and statin—as well as NSBB use—had a major impact on our findings by demonstrating that all of these factors were well balanced between patients with or without the PNPLA3 rs738409 minor allele. Finally, our study lacked a comparison group of CHC patients who did not achieve SVR; however, the impact of the genetic variants on disease progression has previously been established [PNPLA3 rs738409 G-allele. However, due to a limited number of events, and thus a number of variables that can be included in multivariate models, we were unable to adjust the time-to-event analyses for all differences in BL characteristics. In addition, there may have been differences in unknown factors, that we were unable to account for. Accordingly, our findings clearly require confirmation by further studies.The significance of our findings is limited by the short time frame in which the changes in HVPG and non-invasive surrogates of portal hypertension were evaluated, as previous studies reported even further changes in HVPG if reevaluated at later time points. Accordingly, our data on HVPG and non-invasive surrogates may have primarily captured decreases in the dynamic component of intrahepatic resistance. However, an assessment at a later time point may have increased the number of unevaluable patients and, thus, may have introduced a relevant selection bias. Nevertheless, studies evaluating these parameters at a later time point would be desirable. Moreover, not all patients were evaluated by paired HVPG measurements, and we abstained from including information on paired liver biopsies due to very limited numbers, although the latter may have been particularly informative. However, our cohort of 88 patients is still the second-largest reported in the literature so far, and the largest studies to date did not include genetic information ,10. In aablished ,18,19,47PNPLA3, TM6SF2, MBOAT7, and HSD17B13 do not seem to impact the short-term regression of portal hypertension. Moreover, PNPLA3 genotype does not affect clinical outcomes in patients with pre-treatment ACLD within the first years after HCV cure. Future studies should investigate whether this also applies to long-term outcomes and other etiologies of CLD after the removal of the primary etiologic factor.In conclusion, genetic variants in


We describe three pediatric patients between the ages of 10 and 14 years old who were diagnosed with granulomatosis with polyangiitis (GPA) between 2014 and 2019. Each case involves variations in presentation, symptomatology, diagnostics, and induction and maintenance therapy regimens. Patient 1 presented with significant renal involvement, hypertensive emergency, and focal alveolar hemorrhage, a rare presentation of GPA that causes up to 60% mortality.Patient 2 presented with minimal renal involvement and a diffuse petechial rash, which is the most common cutaneous presentation of GPA. Finally, patient 3 presented with significant renal involvement and later on with symptoms of idiopathic intracranial hypertension (IIH), a unique and rare presentation associated with GPA. Despite the heterogeneity of these cases, the similar therapy regimens used in each case successfully achieved induction and maintenance of disease remission, providing an evidentiary basis for these treatment regimens even in severe and unusual pediatric GPA cases. Granulomatosis with polyangiitis (GPA) is a pauci-immune anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) characterized by necrotizing inflammation of small- and medium-sized blood vessels, resulting in a spectrum of phenotypes that range from limited local disease to systemic involvement with organ failure. In GPA, anti-neutrophil cytoplasmic antibodies (ANCA) are formed against neutrophilic proteinase-3, thus stimulating a cascade of unregulated inflammation that commonly affects the small- and medium-sized blood vessels of the kidneys and lungs. GPA is the most common AAV overall, with an incidence of 1.2 per 100,000 and on the rise ,5. The cOver 75% of all AAV patients initially present with rapidly progressive glomerulonephritis, with GPA typically exhibiting more severe renal disease on initial presentation than other ANCA-associated vasculitis -8. With The European League Against Rheumatism (EULAR) released updated consensus guidelines for AAV management in 2016 on induction and maintenance therapies . CurrentCase 1 (patient with hypertensive emergency)A previously healthy 14-year-old male presented to an emergency department of a hospital in the southeastern United States with epistaxis. Six weeks prior to presentation, he was febrile with complaints of sore throat, epistaxis, petechial rash, headache, chest discomfort, and joint and back pain. Two weeks prior to presentation, he reported bleeding of his gums, gross hematuria, and decreased urine output. In the emergency department, his temperature was 38.4°C, pulse was 106 beats per minute, blood pressure was 178/109 mmHg, respiratory rate was 20 breaths per minute, and oxygen saturation was 95% at room air. Notably, on physical examination, the patient had gingival hyperplasia with mucosal bleeding and friability, injected conjunctiva, decreased airy entry on left lung fields, and bilateral lower extremity edema with diffuse purpura Figure . The patLaboratory workup included a serum creatinine (Cr) of 1.25mg/dL , a urinalysis (UA) significant for hematuria and proteinuria with a urine protein-to-creatinine (Up/c) ratio of 2 , erythrocyte sedimentation rate (ESR) 90mm/h , and CRP 15.8mg/L , serum c-ANCA of 1:640 and proteinase-3 (PR3) of 1501U/mL Table .Given serology positive for c-ANCA/PR3, abnormal UA findings, evidence of pulmonary hemorrhage, and renal biopsy consistent with pauci-immune crescentic glomerulonephritis, the patient met the European League Against Rheumatism, Pediatric Rheumatology International Trials Organization, and Pediatric Rheumatology European Society (EULAR/PRINTO/PRES) criteria for granulomatosis with polyangiitis (GPA) .The patient received induction therapy with intravenous (IV) methylprednisolone 1g x3 doses daily, oral (PO) prednisolone 60mg/d, IV cyclophosphamide (Cytoxan) 1000mg x6 doses (750mg/m2 per month), IV rituximab 375mg/m2 x2 doses, therapeutic plasma exchange (TPE) x5 rounds, and IVIG x1 round. Maintenance therapy included daily PO prednisolone with a tapering schedule and azathioprine A previously healthy 13-year-old male presented to an emergency department of a hospital in the southeastern United States with epistaxis, which he reports had been intermittent for the past four weeks. Six weeks prior to presentation, the patient noted joint pain with bilateral upper extremity edema. Three weeks prior to the presentation, he had a purpuric rash on his bilateral ankles. In the emergency department, he was afebrile, normotensive, and saturating adequately on room air without tachypnea or tachycardia. On physical examination, he was noted to have a diffuse, erythematous, papular rash over his bilateral upper and lower extremities, as well as across his anterior trunk. Scattered flesh-colored papules were also noted over his bilateral inner thighs Figure .Laboratory workup included a serum Cr of 0.6mg/dL, a UA significant for hematuria and proteinuria with Up/c of 0.79, ESR 20mm/hr, and CRP 0.28mg/L. Serum c-ANCA of 1:80 and PR-3 of 841U/mL Table .Given serology positive for c-ANCA/PR3, abnormal UA findings, signs of upper airway involvement including epistaxis, and renal biopsy consistent with pauci-immune crescentic glomerulonephritis, the patient met the European League Against Rheumatism, Pediatric Rheumatology International Trials Organization, and Pediatric Rheumatology European Society (EULAR/PRINTO/PRES) criteria for GPA .The patient received induction therapy with IV methylprednisolone 1g x3 doses, PO prednisolone 60mg/d, IV cyclophosphamide 1000mg x6 doses , IV Rituximab 375mg/m2 x4 doses, and TPE x6rounds. IVIG was not given to this patient. Maintenance therapy included daily PO prednisolone with a tapering schedule and azathioprine A previously healthy 10-year-old female, who immigrated from Iraq at the age of five years, presented to an emergency department of a hospital in the northeastern United States with vomiting. One year prior to presentation, she reported epistaxis, hip pain, and abdominal pain with nausea and intermittent vomiting. Six months prior to presentation, she was seen at a local children’s hospital emergency department for hip pain with effusion, which was treated symptomatically and yielded clinical improvement. On her presentation, her temperature was 36.8°C, pulse was 89 beats per minute, blood pressure 108/57, respiratory rate was 17 breaths per minute with oxygen saturation at 100% on room air. Notably, on examination, the patient had a saddle nose deformity, scattered purpura on bilateral upper extremities with right-side predominance, and arthritis of right ankle Figure .On admission, serum Cr was 3.26mg/dL, a UA significant for hematuria and proteinuria with Up/c of 2.8, ESR 127mm/h, and CRP 103mg/L. Serum c-ANCA of 1:80 and PR3 of 188.7U/mL Table . Chest CGiven serology positive for c-ANCA/PR3, abnormal UA findings, signs of upper airway involvement with sinusitis and lung involvement, and renal biopsy consistent with pauci-immune crescentic glomerulonephritis, the patient met the European League Against Rheumatism, Pediatric Rheumatology International Trials Organization, and Pediatric Rheumatology European Society (EULAR/PRINTO/PRES) criteria for GPA .The patient received induction therapy with IV methylprednisolone 1g x3 doses with 500mg x8 doses per week, PO prednisone 2mg/kg daily, IV cyclophosphamide 500-850mg/m2 x7 doses , and IV Rituximab 375mg/m2 x4 doses. TPE and IVIG were not given to this patient. She was also on Enalapril given her HTN and iron for anemia. Maintenance therapy included daily PO prednisone, azathioprine, and Rituximab  and was treated with acetazolamide which was escalated from 5mg/kg to 10mg/kg throughout the course of the admission. The patient’s clinical symptoms improved and she was back at baseline by discharge. The patient continues to be followed up monthly by Pediatric Nephrology and Rheumatology to date.Granulomatosis with polyangiitis (GPA) is an anti-neutrophilic cytoplasmic antibody (ANCA)-associated vasculitis (AAV) that is characterized by systemic granulomatous and necrotizing inflammation affecting the small- and medium-sized blood vessels. AAVs are autoimmune disorders in which autoantibodies are generated against neutrophil granular proteins, namely protease-3 (PR3) and myeloperoxidase (MPO). GPA is most commonly associated with PR-3 ANCAs, which leads to the characteristic clinical manifestations of the disease . While GChildhood-onset GPA is rare and often manifests with non-specific symptoms and variations in the degree of involvement of each system at initial presentation. Ear, nose, and throat (ENT) tract manifestations are the most common features at disease presentation in childhood-onset GPA, followed by constitutional symptoms, nephropathy, and lower respiratory tract involvement . The hetRenal disease associated with GPA is typically characterized by rapidly progressive and crescentic glomerulonephritis, which can ultimately lead to end-stage renal disease ,20. ClinRegarding pulmonary involvement, GPA typically manifests with non-cavitating or cavitating pulmonary nodules on high-resolution computed tomography (HRCT) and alveolar hemorrhage . While tWhile central nervous system (CNS) involvement is not included in the “classic triad” of clinical manifestations of GPA, it occurs in approximately 10-45% of patients . TypicalDespite the different initial presentations, all three patients met the diagnostic criteria for GPA based on the pediatric-specific classification of vasculitis by EULAR/PRES . As suchThe prognosis of GPA has markedly improved with the discovery and appropriate use of immunosuppressive therapy in the past number of decades, with a five-year survival rate currently at approximately 70-80% . ImportaGPA is a serious disease, with a mortality rate that approaches 100% within one year if patients remain untreated. Despite the seriousness and potentially fatal nature of this disease, there is lack of literature regarding the approach to treating pediatric patients with GPA. As such, pediatric nephrologists and rheumatologists have had to extrapolate from adult-based clinical trials to determine the most appropriate management plan for their patients with GPA. The patients in this case series all received similar treatment with induction therapies including IV steroid pulse followed by PO steroids and cyclophosphamide for six months and rituximab. Intensive plasmapheresis was typically reserved for those patients with severe disease, including those requiring dialysis, those with pulmonary hemorrhage, and those patients who fail to respond to therapy. Maintenance therapy in these cases included PO steroids and azathioprine for two years. All three patients responded appropriately and went into remission with undetectable ANCA-antibody levels following induction. The patients in cases 1 and 3 experienced relapse and CKD, respectively, although this might have been expected given the degree of renal disease at onset. Despite these complications and vastly different initial clinical presentations, we postulate that the treatment regimen administered to these patients was appropriate and effective at inducing remission in pediatric patients with GPA.