Secondary plant metabolites, e.g., polyphenols, are widely known as health-improving compounds that occur in natural functional foods such as pomegranates. While extracts generated from these fruits inhibit oxidative stress, the allocation of these effects to the different subgroups of substances, e.g., anthocyanins, “copigments” (polyphenols without anthocyanins), or polymeric compounds, is still unknown. Therefore, in the present study, polyphenols from pomegranate juice were extracted and separated into an anthocyanin and copigment fraction using adsorptive membrane chromatography. Phenolic compounds were determined by high performance liquid chromatography with photodiode array (HPLC–PDA) detection and HPLC-PDA electrospray ionization tandem mass spectrometry (HPLC–PDA–ESI–MS/MS), while the free radical scavenging activity of the pomegranate XAD‑7 extract and its fractions was evaluated by the Trolox equivalent antioxidant capacity (TEAC) assay and electron spin resonance (ESR) spectroscopy. Compared to juice, the total phenolic content and free radical scavenging potential was significantly higher in the pomegranate XAD-7 extract and its fractions. In comparison to the anthocyanin and copigment fraction, pomegranate XAD-7 extract showed the highest radical scavenging activity against galvinoxyl and DPPH radicals. Moreover, the enriched XAD-7 extract and its fractions were able to protect human hepatocellular HepG2 cells against oxidative stress induced by hydrogen peroxide. Overall, these results indicated that anthocyanins and copigments act together in reducing oxidative stress. Over recent years, the consumption of so-called “superfoods” has become a new food trend as part of healthier and more conscious nutrition. Although there is no clear definition or food label for the term “superfood” , it genePunica granatum L.), which belongs to the family Punicaceae. Pomegranates are native to Iran and are nowadays cultivated in North Africa as well as in North and South America [One of the most popular superfoods is the subtropical fruit pomegranate  were analyzed. PE showed a significant antioxidant potential, decreased the intracellular ROS formation, and could lead to G2/M cell cycle arrest followed by apoptosis ,19,20. ATherefore, the aim of the present study was to fractionate the phenolic constituents of pomegranates by adsorptive membrane chromatography into the polymeric and monomeric compounds, while the latter group was separated into an anthocyanin and copigment fraction, which consists of various phenolic compounds . In orden-hexane (>95%) were obtained from Fisher Scientific . Formic acid (HPLC grade) was ordered from AppliChem GmbH . Acetic acid (HPLC grade) and hydrogen peroxide  were obtained from VWR International GmbH . Folin–Ciocalteu reagent, dimethyl sulfoxide, p.a. , and Muse Oxidative Stress Kit were purchased from Merck KGaA . Gallic acid monohydrate and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid  were acquired from Fluka . Potassiumperoxodisulfate (K2S2O8) was obtained from Riedel-de-Haën . Sodium carbonate (≥99% p.a.), sodium chloride (≥99% p.a.), and sodium hydroxide (≥99% p.a.) were ordered from Carl Roth GmbH + Co. KG . Galvinoxyl radical and DPPH radical were purchased from Tokyo Chemical Industry . Triton X was obtained from Serva Electrophoresis . Cell Proliferation Kit I -2,5-diphenyltetrazolium bromide) was ordered from Roche . Dulbecco’s phosphate-buffered saline  as well as Hanks’ balanced salt solution  were purchased from PAN Biotech .Double deionized water was obtained by Nanopure resin . Amberlite XAD-7, 2,2′-azino-bis-(3-ethylbenzothiazolin-6-sulfonic acid) , ethanol (HPLC grade), catechin hydrate, and trypsin were purchased from Sigma-Aldrich . Acetonitrile (LC–MS and HPLC grade) was obtained from Honeywell . Formic acid (LC–MS grade) and 3 , 10% (v/v) fetal bovine serum , 100 IU/mL penicillin, and 100 μg/mL streptomycin . For optimal cell cultivation, we kept the cells at 37 °C under a humidified atmosphere of 95% air and 5% CO2 by replacing the medium every 2–3 days. Once a week, the cells were passaged using the cell passages 20–25 for the following in vitro experiments.Human hepatoma cell line HepG2 (DSMZ-No: ACC-180) was purchased from the German Collection of Microorganisms and Cell Cultures . The cells were cultured in RPMI  1640 medium enriched with L-glutamine, 2.0 g/L NaHCOPunica granatum L.) was kindly provided from Haus Rabenhorst O. Lauffs GmbH and Co. KG . In three independent experiments, we generated pomegranate extracts from the juice.A commercially available pomegranate juice NFC . Solvents were evaporated under reduced pressure at <40 °C, and the XAD-7 extract was freeze-dried. About 6 g pomegranate XAD-7 extract was obtained.A scheme comprising the following methodology is shown in v/v) and applied onto the cellulose membrane. First, copigments were eluted with 1 L ethanol/acetic acid . Afterwards, the retained anthocyanins were eluted with 1 L of a mixture of an aqueous NaCl (1 N) solution and ethanol . Both fractions were concentrated in vacuo and the residue was freeze-dried. NaCl from the anthocyanin fraction was removed by Amberlite XAD-7, as described in v/v), and the solvents were evaporated under reduced pressure at <40 °C and freeze-dried. To remove the polymers from the membrane, the membrane was regenerated with 1 N NaOH and the polymers were eluted with a mixture of NaCl (0.1 N) and ethanol . For detailed information, see Ostberg-Potthoff et al. [With reference to Juadjur and Winterhalter  and Ostbf et al. .v/v). After 7 min, 800 µL 7.5% Na2CO3 solution was added, followed by an incubation for 2 h at room temperature. Afterwards, the absorbance was measured at λ = 760 nm using a V-750 UV-visible/NIR (Near-infrared) spectrophotometer . The TPC was calculated by subtraction of the value of the blank sample, followed by quantification with a calibration curve established using 11–66 mg/L gallic acid. The results were expressed as grams of gallic acid equivalents per 100 g extract (g GAE/100 g). The measurements were repeated three times.The TPC was determined using the Folin–Ciocalteu assay according to Singleton and Rossi  with sliv/v) at a flow rate of 0.6 mL/min and 30 °C. The gradient was as follows: 0 min, 0% B; 1 min, 10% B; 25 min, 15% B; 32 min, 20% B; 35 min, 25% B; 60 min, 50% B; 63 min, 100% B; 63 min, 100% B; 66 min, 100% B; 68 min, 0% B; 72 min, 0% B. Anthocyanins were detected at λ = 520 nm, phenolic acids at λ = 280 nm or 320 nm, flavonols at λ = 360 nm, and tannins at λ = 320 nm. The quantification was carried out in triplicate, employing cyanidin-3-O-glucoside  0.13 mg/mL, LOD (limit of detection) 0.001 mg/mL) as an external standard for anthocyanins, chlorogenic acid  as a standard for phenolic acids, quercetin-3-O-glucoside  as standard for flavonols, and punicalin  as standard for tannins.UHPLC–PDA (Ultra high performance liquid chromatography with photodiode array) quantification of anthocyanins, phenolic acids, and flavonols were performed on a Zorbax Eclipse Plus C18  column  using a 1290 Infinity II UHPLC system . The quantification was carried out with solvent systems A (5% formic acid) and B (acetonitrile) at a flow rate of 0.8 mL/min and 30 °C and the following gradient: 0 min 0% B, 12 min 5% B, 17 min 5% B, 23 min 7% B, 25 min 10% B, 27 min 15% B, 28 min 30% B, 29.5 min 95% B, 31 min 95% B, 33 min 0% B, 35 min 0% B. Quantification of tannins was performed on a Synergi Hydro-RP  column . Solvent system A was Nanopure and solvent system B was 0.5% acetic acid/methanol  using a PU-2080 Plus Intelligent HPLC pump, a DG-2080-50 3-line Degasser, a LG-2080-02 Ternary Gradient Unit, an AS-2057 Plus Intelligent Sampler, a CO-2067 Plus Intelligent Column Oven, a MD-2010 Plus Multiwavelength Detector, and a LC-NetII/ADC Transmitter . The quantification was carried out with solvent systems A (5% aqueous formic acid) and B (acetonitrile) at a flow rate of 0.2 and 0.4 mL/min and 40 °C using the following gradient: 0 min, 0% B; 20 min, 0% B; 60 min, 15% B; 65 min, 50% B; 70 min, 50% B; 75 min, 100% B; 80 min, 100% B; 85 min, 0% B; 100 min, 0% B. The quantification was carried out with catechin as standard  at λ = 280 nm.HPLC–PDA–ESI–MS/MS analyses were conducted according to Ostberg-Potthoff et al. . Therefo2S2O8 dissolved in ultrapure water overnight and diluted with ethanol) was mixed with 20 µL of the samples (10 mg/10 mL), a standard, or a blank solution, and incubated for 12 min at room temperature. The radical scavenging effect, which was measured at λ = 734 nm using a V-750 UV-visible/NIR spectrophotometer , was quantified by a Trolox standard curve prepared in ethanol, while all samples were analyzed in triplicate. The results were expressed as millimole of Trolox equivalents per 100 g sample (mmol TE/100 g).The pomegranate XAD-7 extract and its anthocyanin, copigment, and polymeric fractions were analyzed regarding their radical scavenging capacity by the TEAC assay. One milliliter of an ABTS solution , 5.0 × 102 , time constant of 0.3 s, accumulation of 1, and center field of 337.394 mT.Electron spin resonance (ESR) spectroscopy measurements were performed according to Esatbeyoglu et al.  and Nodav/v). This solution was further diluted with water/ethanol  to obtain working solutions with 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, and 0.01 mg/mL concentrations.Test samples: High concentrations of all samples  were dissolved in water/ethanol  solution, followed by adding 60 µL of ethanol solution containing 1.67 mM of DPPH . Then, the solution was vortexed and centrifuged for about 10 s. After centrifugation, the mixed solution was taken up in a capillary tube (around 50 µL). The control experiment was carried out with 40 µL of water/ethanol  solution and ethanol solution containing 1.67 mM of DPPH.DPPH radicals: At first, 10 µL of sample  were mixed with 30 µL of water/ethanol  solution and 50 µL of ethanol solution containing 1 mM of galvinoxyl radical . After mixing of all components, the solution was vortexed and centrifuged for approximately 10 s, followed by transferring the mixture in a capillary tube (around 50 µL). The control experiment was carried out with 50 µL of water/ethanol  and ethanol containing 0.5 mM of galvinoxyl solution.Galvinoxyl radicals: Similar to DPPH, 10 µL of sample was mixed with 40 µL of water/ethanol  to generate stock solutions (100 mg/mL). In the following in vitro assays, DMSO served as solvent control for all test samples.4 HepG2 cells per well and 8 wells per test substance were seeded in 96-well plates . Approximately 24 h later, the medium was removed and replaced by the treatment media containing different concentrations of the test samples . According to the manufacturer’s instructions of the MTT assay, the cell treatment occurred for 24 h, whereas 0.1% DMSO served as solvent and 1% Triton X as positive control. For the cell viability measurement, we washed the cells using a phosphate-buffered saline (PBS), followed by an incubation with a 1:10 (v/v) dilution of the MTT reagent in medium for 4 h at 37 °C. Then, after metabolic MTT reduction, the samples were incubated overnight at room temperature with the solubilization solution. Finally, the cell viability was determined by measuring the absorbance at λ = 595 nm using a microplate absorbance reader  with the included Biorad Microplate Manager Software 6.2 .At the beginning of the in vitro assays, we determined the cytotoxicity of the different fractions using the MTT viability assay. Briefly, 2 × 102O2 as a ROS-inducer and analysis using the specific detection reagent of the Muse Oxidative Stress Kit .For quantification of antioxidant effects, we treated HepG2 cells with PE and its fractions, followed by an incubation with H6 cells per well were seeded with 3 mL medium in a 6-well plate  and cultured for 24 h at 37 °C and 5% CO2. Then, the cells were treated for 2 h with the pomegranate XAD-7 extract or its fractions (75 µg/mL pomegranate samples) using HBSS as a medium replacement. Alternatively, for detection of an antioxidant capacity, the cells were incubated for 1 h with the samples or 0.1% DMSO as solvent control, followed by a treatment with a non-cytotoxic concentration of H2O2 , also diluted in HBSS, for another incubation time of 1 h at 37 °C. After removal of the treatment solutions, the cells were washed with PBS, trypsinized with 200 µL trypsin per well for 5 min at 37 °C, and transferred with 400 µL HBSS in a centrifugation tube (1.5 mL Eppendorf). The suspensions were centrifuged for 2 min at 1000 g and 4 °C using the centrifuge Type Micro Star 17R from VWR . The supernatant was removed, and the pellet was re-suspended with 200 µL assay buffer of the Muse Oxidative Stress Kit. Finally, 10 µL of the cell suspension was mixed with 190 µL of a prepared Muse Oxidative Stress working solution, incubated for 30 min at 37 °C. The ROS generation was analyzed by the Muse Cell Analyzer . For each sample, the percentage of ROS-positive and ROS-negative cells was calculated automatically.In more detail, 1 × 10p < 0.05, while data are shown as means ± SD (Standard Deviation) of at least three independent experiments.Statistical analysis was carried out using the software Prism . Data were analyzed for normality of distribution by Shapiro–Wilk test, followed by a one-way ANOVA including Tukey’s multiple comparison test for normally distributed data. Alternatively, the Kruskal–Wallis test followed by Dunn’s multiple comparison post-hoc analysis was performed for detecting significant differences within experimental data, showing no normal distribution. Differences were considered as significant when Punica granatum L.) XAD-7 extract was obtained from 1.56 kg pomegranate juice NFC. The obtained pomegranate XAD-7 extract was composed of 2.06 g/100 g (±0.074 g/100 g) anthocyanins, 53.4 g/100 g (±3.20 g/100 g) copigments (mainly tannins), and 8.47 g/100 g polymeric compounds. After separation of the different fractions by a cellulose membrane adsorber, a relative content of 11% anthocyanins, 14% polymeric compounds, and 75% copigments were obtained from the pomegranate XAD-7 extract (About 6.16 g of a phenolic-enriched pomegranate ( extract A. The di extract B. After m/z 611), delphinidin-3-glucoside (m/z 465), and cyanidin-3-glucoside (m/z 449) are the main pigments. Major copigments in pomegranate are the ellagtannins, with punicalin (m/z 781) being the main compound. HPLC chromatograms of the anthocyanin and copigment fractions at λ = 520 nm and λ = 280 are shown in Compounds of pomegranate anthocyanin and copigment fraction were identified by HPLC–ESI–MS/MS analyses. In pomegranate, cyanidin, delphinidin, and pelargonidin derivates were detected . Cyanidi+∙-radicals; 50 values). In the case of the radical scavenging activity of galvinoxyl radicals, the entire pomegranate XAD-7 extract (IC50 = 28.7 µg/mL) showed the strongest radical scavenging activity compared to pomegranate copigment (IC50 = 38.5 µg/mL) and anthocyanin fraction  was analyzed, whereby no significant effects were documented and could be excluded by the interpretation of the following results. Nevertheless, the non-significant increase in cell viability requires an explanation. The principle of the MTT assay, measuring the mitochondrial dehydrogenase activity, could be influenced by the PE and fractions, leading to the slightly higher cell viability. Wang et al. [In the case of the in vitro assays, the ROS-inducing and cytotoxic potential of the used concentrations of PE, as well as the fractions  of the pomegranate XAD-7 extract. All tested samples  showed radical scavenging activity against glavinoxyl and DPPH radicals using ESR spectroscopy in a dose-dependent manner. However, pomegranate XAD‑7 extract showed the highest activity, suggesting synergistic effects of anthocyanins, hydrolyzable tannins, and/or polymeric compounds inducing the higher radical scavenging activity. Accordingly, an inhibition against H


Hidradenitis suppurativa (HS) is a debilitating chronic inflammatory skin disease characterized by chronic abscess formation and development of multiple draining sinus tracts in the groin, axillae, and perineum. Using proteomic and transcriptomic approaches, we characterized the inflammatory responses in HS in depth, revealing immune responses centered on IFN-γ, IL-36, and TNF, with lesser contribution from IL-17A. We further identified B cells and plasma cells, with associated increases in immunoglobulin production and complement activation, as pivotal players in HS pathogenesis, with Bruton’s tyrosine kinase (BTK) and spleen tyrosine kinase (SYK) pathway activation as a central signal transduction network in HS. These data provide preclinical evidence to accelerate the path toward clinical trials targeting BTK and SYK signaling in moderate-to-severe HS. B-cells and plasma cells are critical pathogenic cell populations in chronic Hidradenitis Suppurativa and are potential therapeutic targets. Hidradenitis suppurativa (HS) is a debilitating chronic inflammatory skin disease characterized by chronic abscess formation and development of multiple draining sinus tracts in the groin, axillae, and perineum . It has The immunopathogenesis of HS is poorly understood, with widely and somewhat discordant pathogenic mechanisms proposed. Complement activation has been implicated in the pathogenesis of HS through elevated concentration of the complement fragment C5a in skin and blood of patients with HS . C5a is Other studies have suggested involvement of the proinflammatory cytokines IL-17A and IFN-γ , 12, as In this manuscript we provide potentially novel insights into the immunopathogenesis of HS, using bulk and single-cell RNA-sequencing and cytometry by time of flight (CyTOF) imaging to outline the major cell types and inflammatory pathways that are dysregulated in HS skin. Our results identify immune complex deposition and complement activation along with B cell and plasma cell activation as critical pathways in HS pathogenesis and provide data suggesting that targeting plasma cell activation could provide a novel therapeutic approach for treating advanced HS.https://doi.org/10.1172/jci.insight.139930DS1) were enrolled, and lesional (HS) and matching control skin samples were obtained and submitted for bulk RNA-Seq. In addition, RNA-Seq of whole blood (WB) cells was performed on 21 HS patients and 10 healthy controls, with 20 of the HS patients and all the healthy controls having both skin and WB samples profiled. Principal components analyses (PCAs) of skin showed nearly complete separation between HS and healthy control skin , CD19 , and CD79a . Other genes included the antimicrobial gene DEFB4A ; CXCL13, a B cell chemoattractant ; and the neutrophil chemokine CXCL1 . In the WB, there were 332 DEGs, of which 230 were increased and 102 decreased  and atopic dermatitis (AD) (n = 32) , IL17A , IL17F , IL36A , and IL36G  compared with healthy controls, whereas IL13 and IL17C expression were overall decreased  and IL-36 (P = 9.3 × 10–4) in HS lesional skin, whereas the effect of Th2 response , IL-17A, or TNF stimulation was absent in HS skin (P < 1 × 10–40), followed by various T cell populations, including Th2, and CD4+ and CD8+ effector memory cells (P < 1 × 10–12) (+ naive cells (P < 1 × 10–20), Th17 cells (P < 1 × 10–15), and Th2 cells (P < 1 × 10–12) (P = 7.64 × 10–84), regulation of immune response (adjusted P = 8.26 × 10–82), complement activation (adjusted P = 2.09 × 10–56), Fc-gamma receptor signaling pathway (FDR = 8.86 × 10–41), innate immune response (FDR = 4.92 × 10–33), B cell receptor signaling (FDR = 2.32 × 10–23), and neutrophil chemotaxis (FDR 8.75 × 10–11). Biological processes enriched among decreased DEGs included PPAR signaling pathway (adjusted P = 3.17 × 10–7) and steroid biosynthesis (adjusted P = 0.008). KEGG pathways among decreased DEGs included cholesterol biosynthetic process (adjusted P = 9.64 × 10–11) and lipid metabolic process (adjusted P = 5.5 × 10–4)   because ecreased . Notably HS skin . These d HS skin . Using b× 10–12) . In cont× 10–12) . Biologi × 10–4) .IL17A- and IFNG-expressing T cells in HS skin , positive regulation of B cell activation (P = 2.8 × 10–10), and antibacterial humoral response (1.6 × 10–5) in the plasma cell population; B cell receptor signaling pathway (P = 4.1 × 10–8) and IFN-γ mediated signaling pathway (P = 0.03) in B cells; inflammatory response (P = 6.2 × 10–15) and neutrophil chemotaxis (P = 3.9 × 10–7) in the myeloid cell population; and T cell receptor signaling pathway (P = 1.8 × 10–14), T cell costimulation (P = 7.0 × 10–11), and regulation of TNF-mediated signaling (P = 8.4 × 10–6) in the T cell compartment (Single-cell RNA-sequencing (scRNA-Seq) was performed on cells isolated from 9 excisional samples from patients with severe HS. We collected 30,636 cells with a median 1974 genes and a median 7342 transcripts. We did unsupervised clustering analysis and grouped the cells into 22 clusters , which w HS skin . Notablyesponses , was pripartment .P < 1 × 10–250), IL-36 responses (adjusted P = 6.4 × 10–77), and TNF responses (adjusted P = 2.7 × 10–9). This cluster was characterized by high expression of antimicrobial genes, such as DEFB4A, S100A7A, and IL36G. In contrast, subclusters 0, 1, 2, 3, and 8 showed enrichment for both type I and type II IFN responses (adjusted P < 1 × 10–250), with IFN-γ additionally showing an enriched response in subcluster 7 (adjusted P < 1 × 10–250). The distribution of each cytokine response can also be observed by plotting the module scores on the uniform manifold approximation and projection (UMAP)  . To addrn (UMAP) , whereasn (UMAP) . These d+ T cells, and neutrophils  . To further evaluate whether the cutaneous B cell repertoire was qualitatively altered in the setting of HS, Jaccard distances were calculated as a measure of dissimilarity. Principal coordinates analysis (PCoA) plots of the beta diversity measures indicated good segregation of samples by disease status for BCR κ and λ light chain  was seen, in terms of both total reads and diversity  and TCR gene segments and complementarity determining region 3–encoding (CDR3-encoding) sequences were mined from RNA-Seq data sets obtained from skin and blood of HS patients and healthy controls. This analysis revealed that based upon BCR CDR3 sequences and BCR gene segment abundances, there was a significant increase in the frequency of B cells in HS skin (ctively) . Next, tctively) . This anht chain . There wht chain . To asseht chain . Complemht chain . To deteht chain . Less priversity .+ T cells, CD20+ B cells, and CD138+ plasma cells mostly concentrated in the deeper aspect of the biopsy, around a sinus tract (P < 1 × 10–25), spleen tyrosine kinase (SYK) signaling (P < 1 × 10–22), and Bruton’s tyrosine kinase (BTK) signaling (P < 1 × 10–13), with enriched signals for IL-17A (P < 1 × 10–7), granzyme B (P < 1 × 10–7), and CCR5 (P < 1 × 10–4) . Its expression was suppressed 19-fold by acalabrutinib, adjusted P = 5.6 × 10–5, and 22.8-fold by ibrutinib, adjusted P = 2.2 × 10–3  . Activat × 10–4) . Network × 10–4) . To dete2 × 10–3 . Less pr2 × 10–3 .NCSTN, PSENEN, and PSEN1) were differentially expressed in our data set, it is possible that our data did not capture changes in gamma-secretase or Notch signaling, as our study was focused on patients with chronic, established inflammatory disease. However, the early sequence of events in HS will need to be addressed in future studies.The etiology of HS is still incompletely understood but appears to be complex, with multiple factors contributing to its pathogenesis, including obesity, sex, hormonal factors , dysreguSeveral studies have implicated complement activation in the pathogenesis of HS. This includes studies showing elevated C5a , which i+ T cells secreting IL-17 and IFN-γ and increased mRNA expression of IL17A and IFNG in HS skin, accompanied by increased IL-17A CD4+ T cells in HS blood, but unchanged frequency of IFN-γ–secreting cells   . Insteadnd IL36G . In thisnd IL36G , 24 and nd IL36G . No clinnd IL36G .MX1, CXCL10, and IFI27. The expression of these was proposed to be driven by NETs interacting with infiltrating pDCs in lesional HS skin  in Hank’s Balanced Saline Solution  at 4°C. Epidermis and dermis were separated. Epidermis was digested in 0.25% Trypsin-EDTA  with 10 U/mL DNase I (Thermo Fisher Scientific) for 1 hour at 37°C, quenched with FBS , and strained through a 70 μM mesh. Dermis was minced, digested in 0.2% Collagenase II  and 0.2% Collagenase V (MilliporeSigma) in plain medium for 1.5 hours at 37°C, and strained through a 70 μM mesh. Epidermal and dermal cells were recombined, and libraries were constructed by the University of Michigan Advanced Genomics Core on the 10x Genomics Chromium system. Libraries were then sequenced on the Illumina NovaSeq 6000 sequencer to generate 151 bp paired-end reads. Data processing, including quality control, read alignment, and gene quantification, was conducted using the 10x Genomics Cell Ranger software. Seurat was used for normalization, data integration, and clustering analysis . Clusterlater , stored overnight at 4°C, and subsequently stored at –80°C until further processing. RNA isolation and sequencing were performed using the Illumina NextSeq platform and sequencing protocols as previously described . The heatmap was constructed using Euclidean distance with complete linkage on centered data using the top clones showing greatest variance. Alpha diversity was assessed by calculating the Shannon diversity index using the vegan software package in R (https://cran.r-project.org/web/packages/vegan/index.html). The Shannon diversity index quantifies diversity through the incorporation of both evenness and richness, with higher values representing more even populations of TCR- or BCR-rich samples. The differences in beta diversity were assessed based on Jaccard distances calculated using the vegan software package in R. To visualize the Jaccard dissimilarities between the samples, PCoA using R package ape was performed.MiXCR software was used to extract TCR and BCR CDR3 sequences from RNA-Seq data. Analysis was performed with the “-p rna-seq” option recommended for analysis of RNA-Seq data , 41. DatFormalin-fixed, paraffin-embedded tissue slides obtained from HS patients and healthy controls were heated for 2 hours at 60°C, deparaffinized, and rehydrated. Slides were placed in pH 9 Tris/EDTA antigen retrieval buffer and heated at 96°C for 30 minutes. After cooling, slides were blocked in 3% BSA and stained with a cocktail of metal-tagged antibodies overnight at 4°C, including CD14 , CD16 , CD68 , CD15 , CD31 , CD45 , E-Cadherin , CD20 , CD8 , collagen , CD27 , CD103 [EPR4166 and converted to.TIFF images. These images were then segmented into individual cells using CellProfiler v3.1.8 for single-cell analysis. The t-SNE dimensionality reduction algorithm and the Phenograph unsupervised clustering algorithm were performed on 12 markers  using HistoCAT v1.75 software. For t-SNE and Phenograph, the data were normalized to the 99th percentile. The heatmap shows 2O2 (5 minutes) and blocked using 10% goat serum (30 minutes). Overnight incubation (4°C) was performed using CR1 (CD35)  and CR2 (CD21) . Staining done in antigen retrieval buffer at pH 9 included IgG1 , phospho-SYK , phospho-BTK , CD3 , C1q , C3b , C4d , LCK , BTK , and SYK . Slides were then washed and treated with appropriate secondary antibodies, peroxidase (30 minutes), and diaminobenzidine substrate.Paraffin-embedded tissue sections from excisional biopsies from patients with hidradenitis and healthy control skin were heated at 60°C for 30 minutes, deparaffinized, and rehydrated. Slides were placed in pH 6 antigen retrieval buffer and heated at 125°C for 30 seconds in a pressure cooker water bath. After cooling, slides were treated with 3% HFormalin-fixed, paraffin-embedded tissue slides obtained from patients and healthy controls were heated for 30 minutes at 60°C, deparaffinized, and rehydrated. Slides were placed in pH 9 antigen retrieval buffer and heated at 125°C for 30 seconds in a pressure cooker water bath. After cooling, slides were blocked using 10% donkey serum (30 minutes). Overnight coincubation (4°C) was then performed using anti–human TNF , anti–human CD20 , and anti–human CD138 . Slides were then washed and treated with relative fluorescence-conjugated secondary antibodies (30 minutes). The secondary antibody used with anti–human TNF was Alexa Fluor 488 AffiniPure donkey anti–rabbit IgG . The secondary antibody used with anti–human CD20 and anti–human CD138 was Alexa Fluor 594 AffiniPure donkey anti-mouse IgG . Slides were prepared in mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) . Images were acquired using a ZEISS Axioskop 2 microscope. Images presented are representative of at least 3 biologic replicates.2 Anti-Human IgM and Goat F(ab′)2 Anti-Human IgG  in the presence of fostamatinib disodium (1 μM), ibrutinib (0.4 μM), acalabrutinib (1 μM), or DMSO control for 6 hours at 37°C. Viability assessed by trypan blue exclusion exceeded 90% for all conditions. Cells were washed in cold PBS, and RNA was isolated using the RNeasy Mini Kit (QIAGEN). Stranded mRNA libraries were prepared using the NEBNext Ultra II RNA Library Prep with poly(A) (New England Biolabs) and sequenced on the Illumina NovaSeq 6000 sequencer by the University of Michigan Advanced Genomics Core. Data were analyzed using the Scientific Data Analysis Platform (SciDAP) (Datirium)  was used to isolate the buffy coat from healthy donor volunteer blood, and the EasySep Human B Cell Isolation Kit (STEMCELL Technologies) was used to isolate B cells. Cells were incubated overnight at 37°C. Stimulation was performed with 5 μg/mL each of Goat F(ab′)atirium) : Trim Gaatirium)  was usedTranscriptomic data sets can be accessed at the National Center for Biotechnology Information’s Gene Expression Omnibus database (GSE154775 and GSE154773).2 fold change| ≥ 1 were used to declare significance in differential expression analysis. Significantly regulated genes were analyzed by creating biological literature-based networks using software  (https://www.genomatix.de). The function-word level was used as the minimum evidence level parameter. We ran CellPhoneDB using all the cell types with the default parameters , Rotterdam, the Netherlands. Patients were off any topical or systemic treatment at least 2 weeks before enrollment. Biopsies from lesional skin and blood samples were collected during routine HS surgery. Ten healthy controls were recruited from patients undergoing surgery for skin cancer or abdominoplasty. All samples were obtained with written informed consent from the participants in accordance with Declaration of Helsinki principles. The study protocol was approved by the Institutional Review Board of the Erasmus MC (MEC-2013-337). The patient characteristics are listed in Design of study was performed by JEG, ASM, JJV, JMK, MDR, EM, RLM, and EPP; subject recruitment and sample collection were performed by KRVS, ARJVV, HHVDZ, PWH, JRCB, JHK, SW, and REB; conducting experiments and data acquirement were done by ACB, KRVS, EX, OP, KCC, SMR, CMY, CZ, XX, YJ, JK, and KCC; data and bioinformatic analyses were performed by LCT, FM, RW, MTP, MML, SMR, and CB; and interpretation of data and writing were performed by JEG, LCT, ACB, KCC, MML, FW, JMK, EM, RLM, and EPP.


A synthetic route to prepare a poly(ethylene glycol) with a single cinnamaldehyde acetal unit in the polymer chain, was successfully established using a newly synthesized cinnamaldehyde acetal diethylene glycol (CADEG) as initiator. This HO-PEG(ca)-OH is non-toxic and would be degraded into a cinnamaldehyde and two PEG diols in acid environment. A whole polyethylene glycol based hydrogel was easily fabricated by thiol-ene “click” reaction in alkalescence aqueous solution using acrylate-PEG(ca)-acrylate and 4-arm PEG-SH as raw materials at room temperature. The gel time was dependent on the pH of the solution and its alkalinity can promote gel. The hydrogel can be degradable in acidic conditions and the stronger the acidity, the faster the degradation. This HO-PEG(ca)-OH also can be used in synthesis of cinnamaldehyde containing PEG derivatives, block copolymers or other acid degradable materials. Mn), in particular when the Mn is higher than 40 kDa, the PEG will accumulate in the liver and result in organ damage due to its non-biodegradable property  (PEG), a prominent synthetic polymer approved by the Food and Drug Administration (FDA) of the United States has been wildly used in material science, biology, cosmetics, drug excipient, typically in PEGylation of protein, drug, or gene owing to its peculiar properties, including non-toxic, chemically inert, extremely low immunogenicity, non-antigenicity, and well soluble in many organic solvents and water  bis(oxy)diethanol or cinnamaldehyde acetal diethylene glycol (CADEG), and its corresponding HO-PEG(ca)-OH, a poly(ethylene glycol) with a single cinnamaldehyde acetal (ca) unit in the polymer chain are described. Acrylate-PEG-acrylate was synthesized as a model of PEG derivatives by post modification of PEG diol with acryloyl chloride. A whole polyethylene glycol based hydrogel was easily fabricated by thiol-ene “click” reaction in alkalescence aqueous solution using acrylate-PEG(ca)-acrylate and 4-arm PEG-SH as raw materials at room temperature. And the hydrogel would be degradable in acidic conditions. Furthermore, a triblock amphiphilic copolymer HO-PCL-b-PEG(ca)-b-PCL-OH was also successfully demonstrated by ring-opening polymerization of ε-caprolactone (ε-CL) using HO-PEG(ca)-OH as initiator.2 under reduced pressure just before use. Ethylene oxide  was purchased from Xiapu Chemical Company and was distilled over CaH2 just before use. Diphenylmethylpotassium (DPMK) was prepared as described elsewhere  was prepared according to a previous protocol  unless otherwise indicated. THF (99%) was refluxed over sodium wire and distilled from sodium naphthalenide solution. DMSO was distilled over CaH1H NMR spectra were obtained on a DMX 400 MHz spectrometer with tetramethylsilane (TMS) as the internal standard and CDCl3 as the solvent. Size exclusion chromatography (SEC) was performed in 0.1 M NaNO3 at 40°C with an elution rate of 1.0 mL/min on a Waters HPLC system with a G1310A pump and a 2414 refractive index (RI) detector, Ultrahydrogel 250 (Waters), and Ultrahydrogel 1000 (Waters) columns in series were calibrated by polyethylene glycol standard. For triblock copolymer PCL-b-PEG-b-PCL, its SEC test was performed in THF at 35°C with an elution rate of 1 ml/min through a 7.8 × 300 mm column (with guard column) and a 2414 RI detector with polystyrene (PS) standard. MALDI-TOF MS spectra were recorded using Bruker REFLEX III. α-Cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix for the ionization operated in the reflection mode. The element analysis of the product was recorded on an Elementar Vario EL-III (Germany). Rheological measurements were performed on a DHR-2 controlled stress rheometer  with a parallel plate geometry (diameter = 8 mm). The strain was kept within the limits of the linear viscoelastic regime at a temperature of 25°C. The rubber-elastic plateau was determined from the frequency independent regime of the storage modulus.p-toluenesulfonic acid monohydrate was added with a stirrer bar under nitrogen atmosphere. The reaction mixture was refluxed at 70°C for 6 h then cooled to room temperature. The resulting crude reaction mixture was concentrated under reduced pressure. The crude product was further purified by column chromatography with EtOAc/petroleum spirit  as eluent to produce a yellowy liquid benzene (compound 2) . 1H NMR : δ [ppm] 7.47 – 7.19 , 6.72 , 6.15 , 4.96 , 3.36 .To a solution of cinnamaldehyde  in 60 mL dried methanol, trimethyl orthoformate 20.0 g (0.188 mol) and 30 mg (0.015 mmol) 2  in 60 mL dry chloroform, 7.0 g 2-acetoxyethyl vinyl ether (compound 1), 0.05 g hydroquinone (inhibitor), and 0.02 g (0.01 mmol) p-toluenesulfonic acid monohydrate were added at room temperature. The reaction mixture was refluxed at 65°C for 6 h then was concentrated under reduced pressure. The crude product was further purified by column chromatography with EtOAc/petroleum spirit  as eluent to produce a yellowy liquid  bis(oxy)bis diacetate (compound 3) . 1H NMR  δ [ppm] 7.47 – 7.19 , 6.78 , 6.15 , 4.96–4.86 , 4.20 , 3.68 , 2.05 .To a solution of compound 3  were stirred under reflux in a solution of ethanol (10 mL) and water (0.5 mL) for 3 h. After cooling, brine was added and the solution was extracted with DCM three times. After drying over sodium sulfate the organic phase was evaporated to small volume. Pure product innamaldehyde acetal diethylene glycol   was obtained by column chromatography  over silica.1H NMR  δ [ppm] 7.47–7.19 , 6.78 , 6.15 , 4.96 , 3.70 , 3.75–3.50 . Elemental analysis: Calculated: C 65.53, H 7.61; Found: C 65.48, H 7.58.Potassium hydroxide  and compound 4)  dissolved in 40 mL of mixed solvents of DMSO and THF (v/v: 3/2), a solution of DPMK in THF  was slowly introduced. The orange-red color of DPMK was changed to yellow when the alkoxide was formed. Afterward, the homogeneous initiator solution obtained was introduced into the cooled kettle by a syringe, then ethylene oxide (EO)  was added. After the solution was stirred at 60°C for 24 h, the polymerization was terminated by adding of a few drops of acidified methanol (0.1 N HCl in ethanol). Then all the solvents were removed by reduced distillation. The crude product was dissolved in CH2Cl2, filtered, and dried over anhydrous MgSO4, then precipitated in diethyl ether. The white powder HO-PEG(ca)-OH was obtained in the yield of 95% and characterized by 1H NMR and SEC with refractive index detector and MALDI-TOF MS.A 150 mL stainless steel kettle was vacuumed at 90°C for 24 h and cooled to room temperature and then to 0°C under water ice bath. In the anhydrous CADEG -acrylate (compound 6) was obtained in the yield of 92% and characterized by 1H NMR and SEC with refractive index detector. GPC: Mn= 4.13 × 103 g/mol, Mw/Mn= 1.09.One gram dry HO-PEG(ca)-OH (0.26 mmol) and TEA  were dissolved in 20 mL of dry DCM and cooled to 0°C in an ice-bath. To the above solution, acryloyl chloride  was added dropwise over 30 min. The reaction was allowed to slowly warm up to room temperature and then stirred overnight. The reaction mixture was neutralized by 0.1 N HCl and washed with brine (2 × 15 mL), dried over MgSOMn (1H NMR) = 11.2 × 103 g/mol; SEC (polysyrene standard): Mn= 10.2 × 103 g/mol, Mw/Mn= 1.18.The dried HO-PEG(ca)-OH  was weighed into a dry flask and 3.90 g (34.2 mmol) ε-caprolactone was subsequently added. The reaction mixture was stirred for 5 min at 130°C in a preheated oil bath before the catalyst  was added and the polymerization was performed for 12 h. The resulting viscous was cooled to room temperature. The crude polymer was dissolved in dichloromethane and precipitated into diethyl ether twice to afford a white powder . 2Cl2, filtered, and dried over anhydrous MgSO4, then precipitated in diethyl ether. The white powder 4-arm PEG-OH was obtained in the yield of 92%. 1H NMR  δ [ppm]: 3.43 , 3.48-3.92 [-(CH2CH2O)n-]. Mn (1H NMR) = 3.97 × 103 g/mol; SEC: Mn= 3.92 × 103 g/mol, Mw/Mn = 1.17.A 150 mL stainless steel kettle was vacuumed at 90°C for 24 h and cooled to room temperature and then to 0°C under water ice bath. In the anhydrous pentaerythritol  dissolved in 60 mL of mixed solvents of DMSO and THF (v/v: 3/2), a solution of DPMK in THF  was slowly introduced. The orange-red color of DPMK was changed to yellow when the alkoxide was formed. Afterward, the homogeneous initiator solution obtained was introduced into the cooled kettle by a syringe, then ethylene oxide (EO)  was added. After the solution was stirred at 60°C for 24 h, the polymerization was terminated by adding of a few drops of acidified methanol (0.1 N HCl in ethanol). Then all the solvents were removed by reduced distillation. The crude product was dissolved in CHMn = 3.92 × 103 g/mol, 1.28 mmol) was dissolved in 10 mL toluene, then 2.16 g 3-mercaptopropionic acid (MPA) (20.0 mmol) along with HfCl4 ·2THF  were added and stirred at 50°C under nitrogen. The reaction flask was equipped with an azeotropic distillation apparatus and the mixture was refluxed at 110°C under nitrogen for 24 h. Toluene was removed under reduced pressure. The crude product was dissolved in CH2Cl2, dried over anhydrous MgSO4, filtered, and concentrated. The polymer was precipitated from an excess volume of diethyl ether twice to afford a white powder . Mn (1H NMR) = 4.21 × 103 g/mol; SEC: Mn= 4.15 × 103 g/mol, Mw/Mn= 1.18.4-arm PEG-SH was synthesized by the esterification between 4-arm PEG-OH and 3-mercaptopropionic acid. Typically, 5 g of 4-arm PEG-OH . SEC: Mn = 1.90 × 103 g/mol, Mw/Mn= 1.10.1.0 g HO-PEG(ca)-OH with a single cinnamaldehyde acetal unit was dissolved in two buffer solutions at pH 5.0 and 7.4, respectively, and incubated at 37°C for a set time. After hydrolysis, the solution was extracted by dichloromethane three times. After the solvents were removed under reduced pressure, the residue was dissolved in CHMn= 4.13 × 103 g/mol) was dissolved in 3 mL pH = 8.0 PBS buffer to form A solution and 1 g 4-arm PEG-SH (Mn= 4.15 × 103 g/mol) was dissolved in 4 mL pH = 8.0 PBS buffer to form B solution. Then took the same volume of A and B solutions into a vial and vigorously shaking to mix them well. The hydrogel was formed for a period of time according to different pH of PBS buffer or polymer content.Typically, 2 g acrylate-PEG(ca)-acrylate (Mn= 4.13 × 103 g/mol) was dissolved in 8 mL pH=8.0 PBS buffer to form A solution and 1 g 4-arm PEG-SH (Mn= 4.15 × 103 g/mol) was dissolved in 4 mL pH = 8.0 PBS buffer to form B solution. Then took the same volume of A and B solutions into a vial and vigorously shaking to mix them well. Then quickly draw the solution into the mold and place it for a while.Typically, 2 g acrylate-PEG(ca)-acrylate  of HO-PEG(ca)-OH (Mn = 3.88 × 103 g/mol) and HO-PEG-OH (Mn= 1.90 × 103 g/mol) which was recovered from the hydrolytic residue of HO-PEG(ca)-OH in acid, were added and incubated for 24, 48, and 72 h at 37°C. Cells were treated by the MTS-based CellTiter 96® aqueous one solution cell proliferation assay reagent  at 37°C for 3 h according to the manufacture's protocol. The absorbance was read at a wavelength of 490 nm with a SpectraMax Plus 384 Microplate Reader  and the cell viability was calculated.Human NIH 3T3 cells were purchase from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FBS) at 37°C in a 5% COt-test and analysis of variance (ANOVA).All experiments in this investigation were performed at least three times and the data were expressed as mean ± standard deviation (SD). Statistical analysis was performed through 1H NMR  of the polymer was determined by 1H NMR spectrum using group analysis according to the following equation:Poly(ethylene glycol)s were synthesized by the anionic polymerization of ethylene oxide (EO) in a stainless steel kettle using CADEG as initiator with diphenylmethylpotassium (DPMK) as a baseitiators . The polg to SEC . The strspectrum . The sigEO and Am are the peak areas of the sum of protons in the PEO main chain at δ = 3.45–3.80 ppm and methyne protons at δ = 4.96 ppm, respectively, 150.17 and 44.05 are the molecular weight of the cinnamaldehyde acetal moiety plus two protons of terminal hydroxyl group and the ethylene oxide (EO), respectively.Where AMn calculated by 1H NMR is 3.96 × 103 g/mol, which is in close agreement with the measurement by SEC (Mn = 3.88 × 103 g/mol) and by MALDI-TOF mass spectrum s with a single cinnamaldehyde acetal unit has been successfully synthesized using CADEG as the initiator.1H NMR in According to our previous report -b-PCL-OH is 10.2 kDa with 1.18 polydispersity. This amphiphilic triblock can be used in self-assemble for fabricating pH-responsive nanoparticles -OH with two terminal hydroxyl group was used an macroinitiator for synthesis of an amphiphilic triblock copolymer HO-PCL-b-PEG(ca)-b-PCL-OH by ring opening polymerization of ε-caprolactone. The structure of the HO-PCL-b-PEG(ca)-b-PCL-OH was verified by in vitro along with the anticancer ability of HO-PEG(ca)-OH based materials are currently under investigation in other research.The cytotoxicity of this HO-PEG(ca)-OH and its degradable product HO-PEG-OH were then determined for human fibroblast cells using MTS assay. No obvious loss in cell viability  was obsein vivo degradation conditions. After incubation 8 h, the residue was extracted by dichloromethane (DCM) and concentrated by rotary evaporation. The crude product was characterized by 1H NMR and SEC. The result of 1H NMR (2-benzene)-O) disappeared completely and new signals assigned to cinnamaldehyde at δ 9.81 ppm -CHO appeared, indicating the complete degradation of HO-PEG(ca)-OH. This method also can be used to study the degradation kinetics of HO-PEG(ca)-OH. The polymers were incubated at 25 or 37°C in deuterated phosphate buffer at pH 5.0. After a certain time, the solution was neutralized immediately by 0.1 M NaOH and characterized by 1H NMR. As the degradation process going on, the signal of cinnamaldehyde acetal at δ 4.96 ppm will be weaker and weaker, and its integration can be used to calculate the degradation degree. The degradation kinetics was showed in 1H NMR, verified that the HO-PEG(ca)-OH is stable in pH 7.4 PBS buffer solution. To further investigate storage conditions, samples were kept as dry powders at −18°C and in pH 7.4 PBS buffer solution at 4°C. No detectable degradation was found after two months when stored as a dry powder, and no appreciable degradation occurred within 3 days in pH 7.4 PBS buffer at 4°C. These results indicated that this HO-PEG(ca)-OH has the long term stability and the introduction of cinnamaldehyde acetal unit to PEG would enable the cinnamaldehyde delivery as well as the cleavability of PEG.To investigate the degradation behavior of this PEG with a single cinnamaldehyde acetal unit, we compared the rate of hydrolysis in two buffer solutions at pH 5.0 and 7.4 at 37°C to mimic f 1H NMR  showed t1H NMR , and loss modulus (G″) over time. As shown in In conclusion, a poly(ethylene glycol) with a single cinnamaldehyde acetal unit in the polymer chain was synthesized successfully using a novel synthetic cinnamaldehyde acetal diethylene glycol (CADEG) as initiator. This HO-PEG(ca)-OH is non-toxic and would be degraded into a cinnamaldehyde and a PEG diol with around half of the average molecular weight of HO-PEG(ca)-OH in acid environment. A whole polyethylene glycol based hydrogel was easily fabricated by thiol-ene “click” reaction in alkalescence aqueous solution using acrylate-PEG(ca)-acrylate and 4-arm PEG-SH as raw materials at room temperature.All datasets generated for this study are included in the article/ZhiL, ZhoL, and XZ designed the studies. XZ conducted experiments with assistance from PS, DL, and PC for polymer synthesis and characterization, fabricating hydrogel, and cell culture. ZhiL and ZhoL wrote the manuscript. All coauthors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recently, it was demonstrated that TCR itself could become reactive by binding to small molecules free of the pHLA interface. Our work here was to identify a natural ligand binding to an identified pocket on the CDR2β loop of these TCR. Using docking of suspected ligands, we were able to show Guanine and Adenine di- and tri-nucleotides readily bind to the identified site. Comparing these with small molecule sites found on other TCR types, we show this interaction is novel. With further molecular dynamic simulations, these sites are shown to be plausible by conducting simple computational based solubility tests as cross validation. Combined with simple proliferative responses, the identified nucleotides are also shown to have functional consequences by inducing T cell proliferation for CD4/Vβ20-1 + T cells, while failing to induce proliferation in other T cell isolates. Merging computational and simple cell assays, this work establishes a role of nucleotides in T cells found to contain this TCR sub-type.Novel aspects of T cells containing TCRV Thesβ20-1 studied here have been isolated from Sjögren’s syndrome patients as a skewed responsive isolate from salivary glands in other studies [T cells positive for the TCRV studies ,23. This studies ,24-26.Nucleotides themselves have proven to be problematic in respect to T cell responses, which show a heterogeneous response ranging from inhibitory expansion, to increased expansion. This difference in response has been identified as being related to P2x or P2y extracellular nucleotide receptors. These immune receptors were shown to be expressed in some CD4+ T cells, dendritic cells and macrophages -29. WithDrawing on model autoimmune disease as a guide, nucleotides as extracellular signaling factors in inflammatory responses and sites found to be relevant on this TCR subtype in ADR models , we use 22.1β20-1, the entire CDR2 loop from residue 52 - 76, was blasted against the model organisms X. leavis, D. rerio, M. musculus, B. tarus, H. sapiens, R. norvegicus, using protein blast (http://blast.ncbi.nlm.nih.gov). Retrieved sequences were then aligned using clustalW . Models of the TCRVα7-1 and 20-1 were generated using Swiss model (http://swissmodel.expasy.org) [http://www.pdb.org). These were further energy minimized in gromacs using force field 53a6, and SPC water at 300K for 2 ns [The functional portion of TCRVasy.org)  and the for 2 ns ,34.2.2β5-1/Vα9-2, TCRVβ20-1/Vα17-1 and TCRVβ9/V α12-2 were isolated as described previously [The clone TCRVeviously ,35, by l2.3β20-1 antibody was purchased from RayBiotech.Inc. Whole blood PBMC were isolated using a ficol gradient by centrifugation at 2000 RPM for 20 minutes, washing 2x in PBS and re-suspension in RPMI with 10% FCS. Immediately following, 100 Million cells added to CD4 attached beads according to MACS protocol, and separated using a bench-top magnet. These were then further enriched with Vβ20-1 (Vβ2 RayBiotech) and plated at 1000 TC to 10,000 irradiated allogeneic PBMC. These were expanded for 2 weeks, as described for cell expansion, again enriched for CD4 and Vβ20-1, and further expanded for 2 weeks before use. Cells were 99% CD4, Vβ20-1 by 4 weeks, as determined by FACS analysis.Unlabeled Microbeads, CD4 labeled Microbeads and magnet were all purchased from MACS®Miltenyl Biotec. Unlabeled mouse anti human V2.4β20-1 containing TCR were generated against PDB 2NTS (β5-1), 3MV8 (Vβ9) or 2IJO (Vβ20-1) using Swiss model (http://swissmodel.expasy.org). Each of these has 96% homology to the respective variable domain. Additionally the CDR3 α or β respectively were α ALSNQAGT, β LLQGNTEA, α AVNFHSGN, β VDADTQYF and α ATDGNQF, β GQGENVY for control TCR models. These models were then energy minimized at 300 K in SPC water, force field 53a6 using gromacs for 1 ns. Resulting models were then utilized with all solvent removed. Small molecules, Sulfamethoxazole, GTP, GDP, cGMP, ATP, ADP, cAMP were generated in ACDlab-sChemsketch , converted to smiles format, and then PDB format generated using pymol  The PyMOL Molecular Graphics System, Version 1.3r1). Bond and angles were checked and corrected further in vacuo using gromacs, to generate final small molecule structures.Models of control or TCRV2.5Docking of Sulfamethoxazole, or derivatives of this compound have been reported previously to these same TCR or control TCR . Nucleot2.6β20-1/Vα17-1 used in docking were further energy minimized in gromacs using force field 53a6, embedded in SPC solvent, 0.14 M NaCl, 0.08 M KCl, 0.06 M MgCl and 0.04 M CaCl at 300 K, 1 ATM for 2 ns, also using initial docked conformations for SDM, SMX and GDP, and restraints. Simulations were then allowed to run for 10 ns simulation time, with V-rescale thermostat at 0.1 ps, and Paranillo-Rahman pressure coupling at 1 ps. The TCR was restrained using default Gromacs positional restraints, while each small molecule was unrestrained. Distances between protein residue TYRβ58 and small molecule centers of mass (COM) were calculated over the entire 10 ns using Gromacs. Means were calculated in qtiplot (Copyright © 2004-2011 Ion Vasilief and Stephen Besch) as a moving window average.Models for TCRV2.73H-thymidine was added after 48 for exactly 12 hrs and proliferation measured via scintillation counting . For Sulfanilamide profiles shown, compounds were used at 250 μg/ml final concentrations [Proliferation assays were conducted as described elsewhere . In shor2.8All Statistics were performed using qtiplot (Copyright © 2004-2011 Ion Vasilief and Stephen Besch). Proliferation data was fed into the software from spread sheets as three independent wells across three different experiments, and standard error, means and standard deviations plotted for individual points in all graphs. One way ANOVA was performed on plotted data, and no significance-scored as P-values > 0.05, significance, <0.05, *, or <0.01, **.33.1β20-1 CDR2 loop, a high level of conservation is observed (α pool. For both of these, mutations highlighted indicate a rapid loss of the site by residues that fill the pocket formed in the CDR2β loop. Together, these alignments suggest a long evolutionary conservation of the loop as it is present across distantly related species with all amino acids found to be important for ligand binding in prior studies [Studies involving TCR evolution have been conducted several times by a number of groups ,39. Compobserved . This isobserved . In the 3.2β20-1, where similar non-activating compounds have only very subtle differences in docking conformations from activating compounds.Initial models were based on prior docking experiments using SMX or related sulfanilamide compounds. These are shown here in 3.3β20-1/Vα17-1/CD4 + T cells with Sjögren’s syndrome tissue damage, we chose to coordinate nucleotide analogues with TCR bearing SMX docked sites. These were limited to GTP, GDP, cGMP, ATP, ADP, and cAMP as a representative pool. Control TCR not containing TCRVβ20-1, but harboring strong affinity for SMX failed to dock any nucleotide analogues. Interestingly the CDR2β loop of the TCRVβ20-1 readily docked GTP, GDP, ATP and ADP, but not either cyclic compound based on a cutoff of –6.5 kcal/mol. For GTP and GDP the affinity was –8.5 kcal/mol, which was much higher than SMX at –6.5 kcal/mol, however the same residues were involved with binding all nucleotides (Based on association with Vleotides . In all 3.4β20-1/Vα17-1 + T cell clone (β20-1 profile, and to add significance to our Vβ20-1/CD4 pull down cell pool, these were also subject to the same proliferation assay (β20-1 pull downs, heterogeneous for CD4/CD8, which contained a significant background. These showed only conclusive response from SMX above a CPM background of 15,000. For the double pull down, a clear pattern matching the original single T cell isolate was determined. This also shows a significant background due to heterogeneity in TCRVα, however demonstrate a uniform response between CD4/Vβ20-1 double positive phenotypes. Additionally, the experimental set up utilized Epstein bar virus immortalized B cells specifically containing HLA DRB10, to make sure the experiments were all conducted exact. These were non-autologous for the pull down CD4/TCRVβ20-1 pool.We previously conducted Sulfanilamide proliferation assays for 12 compounds, inclusive of SMX . This shll clone . To furton assay . Not sho3.5β20-1, were allowed to run for 10 ns (Molecular dynamics is used as a type of solubility test further validating docked small molecules in rational drug design strategies ,31. Thesor 10 ns . This laβ loop, while SDM moves from the pocket almost immediately, however rolls on the surface of the TCR or becomes solvated transiently. Visually, the SDM sulfanilamide-substituted group hinders the SO2 and terminal NH2 of the sulfanilamide core from properly orienting itself within the CDR2β loop, while also allowing greater interactions overall with solvent. This illustrates a very fine specificity for not only the CDR2 loop, but also any site that may show a difference for closely related compounds differing by only small angles or distances at the atomic level. For SMX and GDP, the main interaction is with the sulfanilamide core, or the nucleotide core, also inclusive of the SO2 or first PO2 group and the 3’ O of the sugar ring. Both SMX and GDP slightly reoriented positions, around 0.8 - 1 ns and 5.5 ns. For GDP this is inclusive of HIS 71 interactions with the terminal NH2 of the nucleotide. This secondary computational analysis indicates the docking software properly found docked versus nondocked compounds, even with closely related SDM and SMX. Additionally, a dynamic process is observed with hydrogen bonding within the pocket alternating between closely spaced residues and loop backbone atoms. Through the entire simulation, hydrogen bonding with TYR58 is maintained for both bound molecules.Both SMX and GDP remain in the CDR23.6β5-1/Vα9-2, TCRVβ9/Vα12-2 monocultures and the CDR4/TCRVβ20-1 pull down pool (β5-1/Vα9-2 or TCRVβ9/Vα12-2, had shown a proliferative response to SMX, and sulfanilamide derivatives SMT, STH, and SMP or only SMX respectively but also have differences in background obtained. Significant proliferation was shown only in the CDR4/TCRVβ20-1 pull down pool for any of the nucleotides tested, p-values < 0.01, however the binding affinities from docking did not correlate to strength of proliferative response. The proliferative response itself is well above background, but less than SMX. This indicates adenine and guanine-di or -tri nucleotides themselves can act as ligands, inducing proliferation in T cells containing Vβ20-1. For the control clonotype TCRVβ5-1/Vα9-2, a very weak response is observed for ATP and ADP not mirrored with the TCRVβ9/Vα12-2. For either nucleotide respectively, the p-values were 0.14 and 0.058, but highlight differences observed in the control pool well. These responses together reflect the confounding data represented in literature regarding CD4+ T cell nucleotide responses, with the CD4/Vβ20-1 + pool well above the weak responses reported for cells responding to ATP alone [A simple nucleotide proliferation assay was conducted for TCRVown pool . This laTP alone -29. Over4β20-1 association, along with autoimmune disease found to bear hyper proliferative T cells. From literary review, we found Sjögren’s syndrome overlapped somewhat with prior work. Underlying this, we sought to determine other mitigating aspects of the disease model that could be highlighted by the ADR work already conducted.Work presented here builds on an initial study designed to characterize non-classical TCR mediated drug allergies to sulfanilamides, in particular SMX. For most of these ADR, significant overlap exists with autoimmune disorders, with ADR even being referred to as the imitator of disease . In eithβ20-1 and α17-1, along with control TCR also found to bind to SMX with high affinity. Our initial computer work showed TCRVβ20-1 specificity for guanine and adenine di- and tri-nucleotides, while failing to bind to cyclic mono phosphate analogues. While neither control TCR bound any nucleotides tested, other TCR in the entire TCR repertoire may harbor sites not tested. T cells containing the two control TCR, and T cell pools typed only for TCRVβ20-1/CD4 also correlated with the docking. This also demonstrated GTP, GDP, ATP and ADP as ligands capable of causing hyper proliferation with T cells containing variable domain β20-1.Based on this, we conducted nucleotide docking across TCR containing variable domains β subtype, possibly furthered by α subtype as well, has significance [Correlated with Sjögren’s syndrome this particular aspect of T cells containing this ificance ,44. One ificance ,46. Otheificance ,42. Undeβ20-1 that also overlaps with resident skin CD4+ T cells, or Sjögren’s isolated T cells, also marking them as possible similar responsive subsets [In Sjögren’s syndrome, both STAT4 up regulation due to promoter mutations and often similar up regulation of basement membrane proteins such as Lamaninα2 are a pre-defining factor ,26. As a subsets ,12. Thisβ or α repertoire. Already techniques are employed with generalized phenotype approaches, such as modulation of Th17 responses or vaccination in multiple sclerosis against the CDR2 loop of TCRVβ5-1 [While further work would be required to define these aspects in more detail, the findings here illustrate the use of combined computational and direct cellular research techniques to other disease-based models and ADR. As nucleotides were directly shown to be involved with these TCR as a previously undefined mitigating factor, research into ADR pathologies can be shown to open up new possibilities for not only disease models, but also possibly more precise disease treatment strategies. These strategies should foster such techniques when practical, for instance, targeting specific T cell subsets based on only TCRVTCRVβ5-1 ,48. Even5β20-1/CD4 proliferate in the presence of guanine and adenine nucleotides when antigen-presenting cells alone are present. The identified binding site is free of the pHLA interface, and correlates with an SMX determined site previously found to cause hyper proliferation in ADR models. This site itself is an evolutionary conserved CDR2 loop of this variable domain, a site free of TCR hyper variable re-arrangement found in the normal TCR restriction process. Because of this conserved aspect, T cells positive for TCRVβ20-1 have a clear roll in both allergic responses to compounds also found to bind at this site, and also as players in diseases associated with T cell subsets always positive for this variable domain. Additionally, the TCR here adds a further role for free extracellular nucleotides to the entire inflammatory response through this mechanism of proliferative response, which differs from other known nucleotide based immune cell responses.T cells double positive for TCRV


The proliferation of human pulmonary artery smooth muscle cells (HPASMCs) induced by hypoxia was considered as the main cause of pulmonary arterial hypertension (PAH). This study aimed to explore potential genes and long non-coding RNAs (lncRNAs) involved in the mechanism of hypoxia-induced PAH.2 was utilized to induce hypoxia in HPASMCs, and then cell proliferation, apoptosis, and expression of hypoxia-inducible factors (HIF)-1α were determined. Meanwhile, the RNA isolated from CoCl2-treated cells and control cells were sequenced and differentially expressed genes/lncRNA (DEGs/DELs) were screened, followed by protein-protein interaction (PPI) construction, functional enrichment analyses, and lncRNA-target prediction. Finally, the expression of key genes and lncRNAs were validated using quantitative real-time PCR and western blotting.CoCl2 treatment could significantly increase the expression of HIF-1α and the proliferation of HPASMCs. A total of 360 DEGs and 57 DELs were identified between CoCl2 treated and control cells. Functional enrichment analysis showed that up-regulated DEGs and DELs’ targets, including LDHA, PFKP, and VEGFA, were significantly enriched in biological processes related to hypoxia or oxygen levels, and the downregulated DEGs and DELs’ targets were significantly enriched in extracellular-matrix-related biological processes. In addition, LDHA, PFKP, and VEGFA exhibited a strong relationship with miR-100HG and TSPEAR-AS2 in lncRNA-target network. The protein level of LDHA, PFKP, and VEGFA were all increased.CoCl2 hypoxia-induced-HAP, which might serve as promising therapeutic targets for PAH.LDHA, PFKP, VEGFA, and lncRNA miR-100HG and TSPEAR-AS2 probably played crucial roles in the pathogenesis of CoCl Pulmonary hypertension (PH) is a common complicated disorder characterized by a rest mean pulmonary artery pressure of >25 mm Hg. The World Health Organization (WHO) categorized PH into 5 categories according to common clinical parameters, potential pathology, pathophysiology, etiology, and therapies. Specifically, pulmonary arterial hypertension (PAH) belongs to the WHO Group I characterized by an increased vasomotor tone, vascular resistance from abnormal narrowing, and precapillary vasculature remolding associated with abnormalities of proliferation and apoptosis . AlthougHIF-1α has been identified to play a critical role in the regulation of PAH  are a class of transcriptomes with non-protein coding ability and length ≥200 nt , and havHIF-1α on PAH was identified. Meanwhile, CoCl2 treated cells and their corresponding controls were sequenced and analyzed using a bioinformatics method in pulmonary artery smooth muscle cells to further explore the genes and lncRNAs involved in hypoxia-induced PAH pathogenesis, providing some new insights in the understanding and treatment of PAH.To further reveal the pathogenesis of PAH, the effect of This article does not contain any studies with human participants or animals performed by any of the authors.2. HPASMCs were maintained in DMEM supplemented with 15% of FBS at 37°C with 5% CO2 , and human embryonic kidney cells 293T, were used for the following investigations. 293T cell lines were maintained in Dulbecco’s modified eagle medium  supplemented with 10% of fetal bovine serum (FBS) and Penicillin/Streptomycin at 37°C with 5% COh 5% CO2 .2 were determined using a CCK-8 kit . Briefly, HPASMCs were seeded in 96-well plate with 2 × 104 for each well overnight. After adherence, cells were separately treated with 50, 100, or 200 μM CoCl2 for 24 and 48 h. Then, 10 μl CCK-8 solution was added in each well and the cells were cultured at 37°C for 2 h. Following this, the optical density (OD) of each well at 450 nm was measured using an Enzyme-linked immunoassay instrument . Each treatment was repeated six times, and the mean value of OD was calculated as the final result.The proliferation of cells treated with different concentrations of CoClThe BrdU assay was performed according to the protocol of the BrdU Cell Proliferation Assay Kit . Briefly, BrdU labeling solution was added to each well for 4 h. After cells were fixed and DNA denatured, 100 μl BrdU detection antibody were added under a condition of mild vibrating at room temperature for 1 h. Then, the anti- BrdU HRP conjugated antibody was added to the cultures and incubated for 1 h at room temperature. After three washing steps the bound peroxidase was detected by subsequent substrate reaction. This reaction was stopped by adding 100 μl stop buffer and quantified by measuring the optical density (OD) at a wavelength of 450 nm on a microplate reader .2 treatment was also determined in the present study. Briefly, HPASMCs were seeded in six-well plate with 4 × 105 for each well overnight followed by the treatment with 200 μM CoCl2 and DMSO for 48 h. After that, cells were trypsinized and centrifuged at 1500 rpm for 6 min. The cell sediment was washed with phosphate buffer solution and re-suspended with 1 × Binding Buffer. Then, 100 μl cell-binding buffer was collected, and incubated with 5 μl FITC-Annexin V  and 5 μl PI (50 μg/ml) at room temperature in a dark place. Meanwhile, unstaining, single Annexin-V staining, and single PI staining were also performed to minimize error during these procedures. After incubation, solution was mixed with 400 μl 1 × Binding Buffer and measured using a flow cytometry. Each experiment was performed three times, and the mean value was computed.Apoptosis for HPASMCs with CoCl2 for 48 h, and DMSO were used as the control treatment. Each treatment was performed three times. Then, total RNA of cell samples was isolated and sequenced on the Hiseq4000 sequencing platform (Illumina). Based on the recorded data, lower quality of reads and beads were filtered out, and the clean reads were calculated. Following this, clean reads were mapped to human reference genome 19 using tophat , according to the manufacture’s protocol. Then, RNA concentration was quantified using NanoDrop 2000 , and 0.5 μg total RNA was reversed to cDNA using 5 × primeScript RT Master Mix , according to its instructions. Subsequently, gene expression was measured using SYBR Premix EX Taq (2 ×) with the following procedure: 50°C for 3 min, 95°C for 3 min, and 40 cycles of 95°C for 10 s and 60°C for 30 s. The relevant target gene expression levels were calculated with the 2-ΔΔCT method . PrimersCells were lysed in RIPA lysis buffer  coupled with PMSF (Beyotime) at a final concentration of 1 mM, followed by the determination of protein concentration using the BCA Protein Assay Kit (Thermo). Protein from each sample was separated by 10% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Five percent of skim milk (BD) was used to block the non-specific binding for 1–2 h at 37°C, and then incubated with anti-LDHA , anti-VEGFA , and anti-PFKP  primary antibody at 4°C overnight. The membrane was washed with PBST buffer and then incubated with Goat Anti-Rabbit IgG (H + L)  for 2 h at 37°C. After washing with PBST buffer for six times, the membrane was exposed to enhanced chemiluminescence (ECL) reagents (Share-bio) and the Millipore ECL system, and then gray scanning was performed using TanonImage .t-test, and P < 0.05 was considered statistically significant.In the current study, statistical analyses were performed using GraphPad prism . Continuous variables were presented with mean ± standard error of mean. Comparisons between groups were estimated using 2, the expression of HIF-1α in HPASMCs was determined using RT-PCR, and the results showed that the expression levels of HIF-1α were significantly increased in HPASMCs with a dose-dependent manner at 24 and 48 h , monosaccharide metabolic process (P = 4.61 × 10–09), cellular response to oxygen levels (P = 4.61 × 10–09), glycolytic process (P = 6.50 × 10–09), and pyruvate metabolic process (P = 8.32 × 10–09); and the down-regulated DEGs were closely associated with the BP terms of extracellular matrix organization (P = 2.42 × 10–06), collagen catabolic process (P = 3.30 × 10–05), multicellular organismal catabolic process (P = 3.79 × 10–05), extracellular matrix disassembly (P = 3.79 × 10–05), and single-substrate adhesion (P = 1.45 × 10–04) (P = 6.98 × 10–07), Fructose and mannose metabolism (P = 2.16 × 10–07), and metabolic pathways (P = 2.28 × 10–03); the down-regulated DEGs played essential roles in the pathways of systemic lupus erythematosus (P = 1.06 × 10–15), ECM-receptor interaction (P = 3.82 × 10–06), Amoebiasis (P = 1.13 × 10–05), and focal adhesion (P = 4.91 × 10–04)  and 57 DELs (21 up-regulated lncRNAs and 36 down-regulated lncRNAs) were identified in CoCl× 10–04) . Meanwhi× 10–04) .VEGFA (degree = 21), LDHA (degree = 20), HIST1H1B (degree = 17), HIST2H4B (degree = 16), HIST2H4A (degree = 16), HK2 (degree = 15), HIST1H2AI (degree = 15), HIST1H2AE (degree = 15), PFKP (degree = 14), and HIST3H2BB (degree = 14).Based on the selection criteria, PPI among DEGs encoded proteins were predicted and visualized using Cytoscape . The topRP11-486B10.3 (degree = 127), RP11-449P15.2 (degree = 97), RP11-229P13.19 (degree = 93), AF131216.5 (degree = 93), LINC00618 (degree = 93), RP11-429D19.1 (degree = 93), CHRM3-AS1 (degree = 93), CH507-42P11.3 (degree = 88), AP001056.1 (degree = 88), and RP11-488C13.5 (degree = 85). Notably, LDHA, PFKP, and VEGFA as hub genes in PPI network presented a close correlation with miR-100HG and TSPEA-AS2 in lncRNA-target network  and 2855 edges (PPI pairs) . The top network .P = 9.60 × 10–10), cellular response to decreased oxygen levels (P = 9.60 × 10–10), cellular response to oxygen levels (P = 1.57 × 10–09), glycolytic process (P = 2.56 × 10–09), and pyruvate metabolic process (P = 2.84 × 10–09); the down-regulated targets were predominately associated with the GO-BP terms of extracellular matrix organization (P = 2.79 × 10–06), extracellular structure organization (2.79 × 10–06), collagen catabolic process (P = 3.56 × 10–05), multicellular organismal catabolic process (P = 4.13 × 10–05), and extracellular matrix disassembly (P = 4.13 × 10–05) (P = 1.85 × 10–07), fructose and mannose metabolism (P = 7.44 × 10–07), and metabolic pathways (P = 0.0019); the down-regulated targets were significantly enriched in the pathways of systemic lupus erythematosus (P = 1.06 × 10–15), ECM-receptor interaction (P = 3.82 × 10–06), amoebiasis (P = 1.13 × 10–05), and focal adhesion (P = 4.91 × 10–04)  . The KEG× 10–04) .PFKP, miR-100HG, RP11-166d19, VEGFA, LDHA, and AP001065.15 were significantly up-regulated, while expression levels of RP11-568J2, HIST1H1B, HIST2H4A, TSPEAR-AS2, DYX1C1-CCPG1, and LINC00951 were significantly decreased in CoCl2-treated HPASMCs compared with the control groups, which were consistent with the findings based on the bioinformatics analyses  and 57 DELs (21 up-regulated and 36 lncRNAs) were screened in HPASMCs treated with CoCl2 compared with the control group. Interestingly, we found that LDHA, PFKP, and VEGFA, which were regarded as hub genes in PPI network, exhibited a strong relationship with miR-100HG and TSPEAR-AS2 in lncRNA-target network. Additionally, the functional enrichment analysis of these three genes revealed that they probably played pivotal roles in the cellular response to hypoxia and the regulation of glycolysis.Pulmonary arterial hypertension is considered a sub-population of PH, characterized by sustained pulmonary vascular resistance elevation that results in right heart failure and death . Overwhe process . AlthougLDHA, lactate dehydrogenase A  was closely correlated with the development of PH  with hypoxia and the activation of HIF-1α/VEGFA axis acted as a potential driver for vascular remodeling contributing to PH, which was consistent with our results that HIF-1α and VEGFA levels were dramatically increased in hypoxia-induced HPASMCs. Additionally, our functional enrichment analysis indicated that VEGFA played essential roles in cellular response to oxygen levels. Taken together, our results provided support for the hypothesis that the initiation of HIF-1α/VEGFA axis was implicated with HAP under hypoxia exposure.A huge amount of research has found that the vascular endothelial growth factor A (nt of PH . Tiede ePFKP) gene was involved with various cell activities and glucose metabolism by targeting to multiple cell receptors and was reported to be another prominent regulator in the progression of HP  could bind to PFKP to regulate the proliferation and metabolism of PH cells. Interestingly, LDHA, PFKP, and VEGFA all had a close correlation with miR-100HG and TSPEAR-AS2 in our analysis. Unfortunately, few relevant investigations have been undertaken to explore the effect of the interaction between these genes and corresponding mRNAs on PAH occurrence and development. Therefore, the precise underlying mechanisms still need to be further elucidated.Phosphofructokinase  repository with Accession Number TW: conception and design of the research, obtaining funding, and revision of manuscript for important intellectual content. HX: acquisition of data and analysis and interpretation of data. FH: statistical analysis. SC: drafting the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Legionella pneumophila is a facultative intracellular pathogen found in aquatic environments as planktonic cells within biofilms and as intracellular parasites of free-living amoebae such as Acanthamoeba castellanii. This pathogen bypasses the elimination mechanism to replicate within amoebae; however, not all amoeba species support the growth of L. pneumophila. Willaertia magna C2c Maky, a non-pathogenic amoeba, was previously demonstrated to possess the ability to eliminate the L. pneumophila strain Paris. Here, we study the intracellular behaviour of three L. pneumophila strains  within W. magna C2c Maky and compare this strain to A. castellanii and W. magna Z503, which are used as controls. We observe the intracellular growth of strain Lens within W. magna Z503 and A. castellanii at 22 °C and 37 °C. Strain Paris grows within A. castellanii at any temperature, while it only grows at 22 °C within W. magna Z503. Strain Philadelphia proliferates only within A. castellanii at 37 °C. Within W. magna C2c Maky, none of the three legionella strains exhibit intracellular growth. Additionally, the ability of W. magna C2c Maky to decrease the number of internalized L. pneumophila is confirmed. These results support the idea that W. magna C2c Maky possesses unique behaviour in regard to L. pneumophila strains. Legionella pneumophila is an aerobic, Gram-negative bacterium that causes Legionellosis, a severe form of pneumonia, following inoculation with contaminated aerosol [L. pneumophila, serogroup 1 is involved in the majority of infections [L. pneumophila can proliferate [ aerosol . This ba aerosol ,3. Amongfections ,5. This fections . Legionefections . Indeed,liferate ,9,10.L. pneumophila in water cooling towers [L. pneumophila have evolved to avoid the phagolysosome fusion and can multiply within FLA, ultimately killing these amoebae before disseminating into the environment [L. pneumophila with protection against unfavourable conditions and chemical treatments. Therefore, the association between FLA and this pathogen makes the control and monitoring of water-cooling towers difficult and makes eradication of L. pneumophila almost impossible [Free-living amoebae (FLA) are ubiquitous protozoa that inhabit common aquatic environments and are frequently co-isolated with g towers ,12. FLA g towers ,14; howeironment ,15,16,17possible ,19.L. pneumophila strains. While Acanthamoeba sp. and Vermamoeba (formerly Hartmannella) vermiformis support the intracellular growth of L. pneumophila, the Willaertia magna strain C2c Maky has been demonstrated to eliminate the L. pneumophila serogroup 1 strain Paris ATCC 33152 [W. magna C2c Maky is a free-living amoeba that is a member of the Vahlkampfiidae family [http://www.amoeba-biocide.com/en/page/learn-more-about-willaertia-magna-c2c-maky), and it has the capacity to grow at high temperatures (up to 44 °C) in xenic or axenic media. The living forms of this amoeba include a large trophozoite (50–100 µm) and a cyst (18–21 µm) form, and it can produce temporary flagella [W. magna C2c Maky to eliminate L. pneumophila as an alternative to chemical biocides (http://www.amoeba-biocide.com/en/page/revolutionary-biocide). The present study is performed to verify the elimination and the absence of the reservoir effect. Specifically, the decrease in the number of internal L. pneumophila and the absence of internal L. pneumophila multiplication within W. magna C2c Maky, when both microorganisms are co-cultured, is confirmed. The assay is performed by examining adhesion  with three strains of L. pneumophila to assess the consistency of amoeba behaviour toward legionella strains. The assay lasts for one week and includes a daily count of intracellular L. pneumophila and amoebas by culture and Trypan blue staining, respectively. The behaviour of W. magna C2c Maky is compared to that of W. magna Z503 to determine if two amoeba strains of the same species have the same behavior. Moreover, it is compared to A. castellanii, an amoeba known to multiply amoeba-resistant bacteria such as the three L. pneumophila strains studied. Previous studies, however, have demonstrated that all FLAs do not exhibit the same behaviours when they come into contact with CC 33152 , which iCC 33152 . W. magne family . This amflagella ,23. The flagella . AccordiL. pneumophila strains in the calf serum-casein-yeast extract medium (SCYEM) was evaluated at 22 °C and 37 °C . Seen at 37 °C, a mean bacterial uptake of 20 ± 5.5% was observed . The mean initial amount of amoeba-internalized bacteria at 22 °C was 16 ± 0.5% (16% in p < 0.05) in the number of intracellular L. pneumophila Lens per W. magna C2c Maky cell was observed after 24 h , while the level remained nearly constant for A. castellanii at 22 °C and 37 °C and for W. magna Z503 at 22 °C with no significant difference between T0 and T0 + 24 h (p > 0.05)  and A. castellanii . Observed at 37 °C, a small number of A. castellanii cells were still alive (5.6 × 102 ± 5.9 × 102 amoebas/mL), demonstrating that amoeba cell lysis occurred following the intracellular multiplication of L. pneumophila Lens.A significant decrease ( > 0.05) . Occurri0 + 96 h , the perL. pneumophila Lens at 22 °C and 37 °C, a significant increase (p < 0.05) was obtained when the bacterium was co-cultivated with W. magna Z503 and A. castellanii, and this was not observed when L. pneumophila Lens was cultivated alone or in the presence of W. magna C2c Maky . The initial mean amount of cells internalized by amoebas decreased to 14 ± 5.0% at 37 °C . Occurring at 22 °C, we reported a mean L. pneumophila Paris per amoeba cell (p < 0.05) first was observed in the three amoebas after 24 h, with the exception of A. castellanii at 37 °C  (0 + 96 h) only by W. magna C2c Maky, and the percentage of intracellular L. pneumophila Paris per amoeba cell was reduced by 79 ± 2% at 22 °C and 98 ± 0.1% at 37 °C (p < 0.05). The opposite was observed for W. magna Z503 and A. castellanii at 22 °C and 37 °C, as the decrease measured after 24 h was not maintained. Seen at 48 h, the level of intracellular L. pneumophila Paris per amoeba cell began to increase until it reached 4-fold and 3-fold more bacteria per amoeba cell than that observed at T0 for W. magna Z503 and A. castellanii, respectively at 22 °C. Observed at 37 °C for W. magna Z503, the number of intracellular L. pneumophila Paris per amoeba cell at T0 + 96 h was 5-fold the ratio observed at 24 h, but it did not reach the initial ratio. Regarding A. castellanii, a strong increase was observed at both temperatures, and the initial ratio was slightly increased by 3-fold at 22 °C (p > 0.05) and strongly increased by 60,000-fold at 37 °C (p < 0.05). Furthermore, the correlation between the increase in L. pneumophila Paris and the low concentration of viable A. castellanii (5.6 × 102 ± 5.9 × 102 cells/mL) after 96 h indicated that a high intracellular multiplication of L. pneumophila Paris occurred that was followed by a release of bacteria in the medium after A. castellanii death.A significant decrease of the number of intracellular t 37 °C) . This deL. pneumophila Paris at 22 °C, a significant increase (p < 0.05) was obtained when the bacterium was co-cultured with W. magna Z503 and A. castellanii, and this was not observed when L. pneumophila Paris was cultured alone or in the presence of W. magna Z503 at 37 °C and W. magna C2c Maky at both 22 °C and 37 °C  at 22 °C, and the initial amount of internalized cells by amoebas increased to 17 ± 3.8% . The mean bacterial internalization by amoebas was 9 ± 1.1% (9% in p < 0.05) decrease in the number of intracellular L. pneumophila per amoeba cell was observed within 24 h  in the three amoebas (p < 0.05) decrease continued until the death of more than 99% of intracellular L. pneumophila Philadelphia in all cases. Even if this decrease could be attributed to the bacterial death in the coculture medium, the experiment demonstrated the absence of intra-amoeba multiplication of L. pneumophila Philadelphia necessary for survival at 22 °C. Occurring at 22 °C, a rapid and significant . Then, differential behaviours were observed depending on the amoeba strains. Regarding W. magna C2c Maky, the significant decrease (p < 0.05) continued until the death of more than 99.99% of the intracellular L. pneumophila Philadelphia per amoeba cell   in intracellular L. pneumophila Philadelphia per amoeba cell appeared after 48 h, demonstrating an intra-amoeba multiplication up to 2600-fold at the end point  d. To connd point c.L. pneumophila Philadelphia at 22 °C, a significant decrease (p < 0.05) was obtained in all cases (p < 0.05) was observed when L. pneumophila Philadelphia was cultured in the presence of A. castellanii  were performed to determine if 0 data obtained in the presence of the three amoebas were not statistically different at 22 °C (p > 0.05); however, at 37 °C, a significant difference in behaviour (p < 0.05) was detected at T0. Concerning the three bacterial strains, TW. magna C2c Maky behaviour was statistically different from that of the two other amoeba strains (W. magna C2c Maky behaved differently compared to W. magna Z503 and A. castellanii cells in the presence of Legionella strains.Pairwise comparisons (Dunn test) established that at 72 h and 96 h at both temperatures and with the three legionella strains,  strains . This siL. pneumophila strains under two temperature conditions (22 °C and 37 °C) that correspond to temperatures found in cooling towers in which L. pneumophila are known to replicate within certain strains of amoebae [W. magna C2c Maky does not multiply L. pneumophila as we aim to propose it as a natural biocide to treat cooling towers.This work explores the permissiveness of three amoeba strains regarding the intracellular multiplication of three pathogenic  amoebae ,25. It iL. pneumophila strains are a representative set of L. pneumophila serogroup 1 that is responsible for 95% of the legionellosis disease world-wide [A. castellanii, A. polyphaga, or A. lenticulate [Legionella in Europe. L. pneumophila Lens was chosen because it was responsible for an outbreak in the north of France between November 2003 and January 2004 where 86 confirmed cases resulted in 17 deaths [L. pneumophila Paris was chosen because, among the endemic strains of L. pneumophila serogroup 1, sequence type 1 (ST1) strains are among the most prevalent, particularly the ST1/Paris pulsotype. This endemic type was responsible for 8.2% of French culture-proven cases of Legionnaire’s disease from 1995 through 2006. ST1/Paris pulsotype isolates also have been detected in clinical and environmental samples taken from several other countries around the world, including Switzerland, Italy, Spain, Sweden, the United States, Japan, Senegal, and Canada [The three ticulate ,27,28,297 deaths . L. pneud Canada ,30.A. castellanii and W. magna Z503, the intracellular L. pneumophila are efficiently eliminated by W. magna C2c Maky at 22 °C and 37 °C. Indeed, the experiments report not only a non-replication, but also an elimination of the intracellular strains Lens, Paris and Philadelphia within W. magna C2c Maky. Furthermore, the coculture medium used in the survey is not adapted to the survival of the legionella bacteria, and they, therefore, must parasitize the amoebae to facilitate their own growth. Indeed, the experiments demonstrate that the three legionella strains were unable to remain at the inoculation level and began to die after 24 h  [L. pneumophila serogroup 1, for example, replicated in A. castellanii at 25 °C but were digested at temperatures below 20 °C [Naegleria sp., Acanthamoeba sp., and Vermamoeba sp. to support the multiplication of L. pneumophila Lens at various temperatures , and they revealed a more efficient intracellular proliferation with increasing temperatures [W. magna strain Z503, but not in W. magna C2c Maky  that was favourable to L. pneumophila survival, so bacteria were not forced to multiply into amoeba to survive. Additionally, Dey and co-workers did not eliminate extracellular bacteria by centrifugation, and the observed increase could be due to extracellular bacterial replication, such as that resulting from necrotrophic growth as previously demonstrated [Our experiments demonstrate differential behaviours among amoeba species infected by the pathogenic bacteria. Compared to ter 24 h . Althougat 37 °C c and doeat 37 °C a within ba (FLA) ,31,32. Leratures . AdditioC2c Maky d. The coadelphia b. The ditesseris ,34. The nd 37 °C c,d. Dey nstrated .W. magna C2c Maky is demonstrated to possess a high efficiency for digesting the intracellular L. pneumophila cells in all strains used in this survey. The growth of L. pneumophila within amoebas is known to enhance the pathogenicity and invasion of L. pneumophila [W. magna C2c Maky with L. pneumophila strains derived from a first co-culture that was thought to be more virulent (unpublished data).umophila ,36; howeL. pneumophila strains and the absence of internal proliferation support the fact that W. magna C2c Maky could be used as a biocide to combat L. pneumophila proliferation in cooling tower water. This observation is consistent with the control of legionella by W. magna C2c Maky observed in real conditions during field trials in functioning cooling towers (http://www.amoeba-biocide.com/sites/default/files/180711_cp_amoeba_us_positive_efficacy_field_test_en_vedf_0.pdf). The traditional method to control bacterial growth in cooling tower water is primarily based on the use of chemical biocides [L. pneumophila proliferation, although some previous studies reported incomplete eradication of legionella from installations and progressive re-colonization within these systems within weeks or months [2O2/Ag) was inadequate for legionella control, and, instead, it caused a rapid increase of one logarithmic unit [L. pneumophila populations, and chemical biocides can promote resistance to biocides and to human health antibiotics [The action on different biocides ,38. Indebiocides . The cher months ,41. Morer months ,43. Descmic unit . Chemicamic unit ,17,45. Fibiotics ,47. W. magna C2c Maky is not associated with any human or animal infection, and this is in agreement with the lack of pathogenicity demonstrated in vivo and suggested by genomic analysis [To conclude, analysis ,48. ThisWillaertia magna C2c Maky (ATCC® PTA-7824), Willaertia magna Z503 (ATCC® 50035), and Acanthamoeba castellanii (ATCC® 30010) were purchased from ATCC and cultivated according to their recommendation into 10 mL of modified PYNFH medium (ATCC medium 1034) in a T-25 tissue culture flask. Amoebae were then grown in cell factories in serum casein yeast extract medium (SCYEM) at 30 °C. SCYEM medium is derived from serum casein glucose yeast extract medium (SCGYEM) medium [−1 casein, 5 g·L−1 yeast extract, 10% foetal calf Serum, 1.325 g·L−1 Na2HPO4, and 0.8 g·L−1 KH2PO4. After 72 h , the cell factories were gently shaken, and the amoeba suspensions were transferred to 50 mL Falcon® tubes. Amoeba populations were then quantified using a Malassez haemocytometer cell counting chamber method  with Trypan blue by mixing 100 µL of Trypan blue with 100 µL of amoeba sample. According to the results, the amoebae concentration in Falcon® tubes was then adjusted to 3 × 105 cells/mL by the addition of SCYEM. The amoebas were then washed twice in SCYEM using centrifugation at 3000× g for 10 min, and the supernatants were then discarded. Amoeba populations were then re-quantified, and the amoeba suspensions were finally adjusted to 3 × 105 cells/mL in 100 mL of SCYEM. A final quantification was performed to verify the concentration. ) medium  and contW. magna C2c Maky, W. magna Z503, and A. castellanii corresponded to working suspensions that were named AWSC2C, AWSZ503, and AWSAC, respectively , L. pneumophila strain Lens (CIP 108280), and L. pneumophila strain Paris (CIP 107629) were grown on buffered charcoal yeast extract (BCYE) agar plates  at 36 °C for 72 hours and then harvested by scraping, suspended in phosphate-buffered saline (PBS), centrifuged at 9500 xg for 10 min, and washed once in PBS. The supernatants were then discarded. The L. pneumophila suspensions were then diluted in PBS to obtain 3 × 107 bacteria/mL. Phila, BWSParis, and BWSLens  in 25 cm3 flasks  and incubated at 22 °C or 37 °C. This operation corresponded to the T0 time point of the bacterial controls. Occurring at T0, T0 + 24 h, T0 + 48 h, T0 + 72 h, and T0 + 96 h, 1 mL was sampled in each flask and then serially 10-fold diluted in SCYEM and plated on buffered charcoal yeast extract plates (BCYE) in triplicate. BCYE plates were incubated at 36 °C, and colony forming units (CFU) were counted after 5 days. Each condition was performed for three independent replicates and repeated three times (n = 9).The three control bacterial conditions were prepared as described in C2C, AWSZ503, or AWSAC) were prepared as described in 3 flasks. Occurring at T0, T0 + 24 h, T0 + 48 h, T0 + 72 h, and T0 + 96 h, the flasks were gently shaken, and the numbers of amoeba cells were quantified using a haemocytometer cell counting chamber method with Trypan blue. Each condition was performed for three independent replicates and repeated three times (n = 9).The three amoeba working solutions  L. pneumophila from the co-culture suspensions. The pellet was resuspended in 10 mL of sterile SCYEM, and the suspension was poured into a new 25 cm3 flask and incubated at 22 °C ± 2 °C or at 37 °C ± 2 °C. This time point corresponded to the T0 time point of the assay. Each condition was performed for three independent replicates and repeated three times (n = 9), with the exception of the co-culture with strain Philadelphia that was repeated four times at 22 °C (n = 15).Amoeba and bacterial working solutions were mixed in 25 cm0, T0 + 24 h, T0 + 48 h, T0 + 72 h, and T0 + 96 h, a washing step was performed. The culture supernatant was removed from each flask and replaced by 10 mL of sterile SCYEM. This step was intended to remove extracellular L. pneumophila to allow for the detection of only intracellular bacteria. Each flask was gently shaken 10 times and an aliquot of 1 mL was sampled. Quantification of amoeba populations was performed using 0.1 mL of each aliquot utilizing a haemocytometer cell counting chamber method with Trypan blue. The remaining 0.9 mL were treated with Triton™ X-100 [L. pneumophila. The sample was then serially 10-fold diluted in SCYEM and plated on BCYE plates in triplicate, with the exception of the undiluted conditions that were spread onto five plates when the number of L. pneumophila was intended to decrease below the detection limit. BCYE plates were incubated at 36 °C, and CFU were counted after 5 days.Occurring at Tn™ X-100  at 0.02%L. pneumophila Philadelphia using the three amoeba strains at 37 °C were sampled from running experiments and stained by the Gimenez technique [0, T0 + 48 h, and T0 + 96 h. Co-cultures (0.1 mL) were deposited onto glass slides by using a Shandon Cytospin 4 cytocentrifuge  at 800× g for 10 min and then stained using the Gimenez technique. Briefly, each of the glass slides were stained with fuchsin solution for 3 min and washed with water. Then, the glass slides were stained with malachite green for 5–10 s and washed, and this step was repeated twice. Finally, the glass slides were allowed to dry at room temperature. Co-cultures of echnique ,51 at T0The observations were performed using a LEICA DM 2500 LED microscope  under an ×100 oil immersion objective.Statistical significance of co-culture studies was determined for 22 °C and 37 °C conditions through the use of analysis of variance (ANOVA) .