First, the hemodynamic response of SSPN−/− mice was tested and was similar to SSPN+/+ (wild-type) mice after isoproterenol injection. Using the in situ Langendorff perfusion method, SSPN−/− hearts were subjected to IR injury and found to have increased infarct size and arrhythmia susceptibility compared to SSPN+/+. Ca2+ handling was assessed in single cardiomyocytes and diastolic Ca2+ levels were increased after acute β-AR stimulation in SSPN+/+ but not SSPN−/−. It was also found that SSPN−/− cardiomyocytes had reduced Ca2+ SR content compared to SSPN+/+ but similar SR Ca2+ release. Next, we used qRT-PCR to examine gene expression of Ca2+ handling proteins after acute IR injury. SSPN−/− hearts showed a significant decrease in L-type Ca2+ channels and a significant increase in Ca2+ release channel (RyR2) expression. Interestingly, under oxidizing conditions reminiscent of IR, SSPN−/− cardiomyocytes, had increased H2O2-induced reactive oxygen species production compared to SSPN+/+. Examination of oxidative stress proteins indicated that NADPH oxidase 4 and oxidized CAMKII were increased in SSPN−/− hearts after acute IR injury. These results suggest that increased arrhythmia susceptibility in SSPN−/− hearts post-IR injury may arise from alterations in Ca2+ handling and a reduced capacity to regulate oxidative stress pathways.The protein sarcospan (SSPN) is an integral member of the dystrophin-glycoprotein complex (DGC) and has been shown to be important in the heart during the development and the response to acute stress. In this study, we investigated the role of SSPN in the cardiac response to acute ischemia-reperfusion (IR) injury in SSPN-deficient (SSPN DYS1 gene leading to dystrophin loss and the mdx mouse model was developed to study the disease [Pathogenic variants in the dystrophin-glycoprotein complex (DGC) lead to cardiac and skeletal muscle disorders . The DGC disease ,3. Loss  disease ,5,6,7. SSSPN as a candidate gene for atrial fibrillation (AF) [−/− mice resulted in a blunted cardiac functional response compared to wild type mice [2+ handling. Overall, this study is aimed at understanding the normal functions of SSPN in the developing and adult heart that protect the heart from injury and arrhythmias [The protein SSPN has been shown to be important in maintaining cardiac and skeletal muscle function and integrity ,9. Unlikion (AF) . This stion (AF) ,12,13. Sype mice . Since βhythmias ,16.−/− myocardium, we utilized several arrhythmia-inducing stressors, including β-adrenergic stimulation and ischemia-reperfusion (IR) injury. We also compared the impact of SSPN loss to that of its closely associated proteins. One protein that SSPN makes significant associations with is β1D integrin [To uncover arrhythmogenic triggers for the SSPNintegrin ,17,18 anintegrin . Integriintegrin . Integriintegrin . The losintegrin . Since S2+ handling and response to IR injury. To investigate a role for SSPN in arrhythmia and acute IR injury, we examined ex vivo Langendorff perfused SSPN−/− hearts to assess the intrinsic mechanisms governing electrical activity and conduction independent of autonomic nervous regulation.This is the first study examining the role of SSPN in cardiomyocyte Ca−/− mice compared to age-matched SSPN+/+ controls. In +/+ or SSPN−/− mice. After the administration of the isoproterenol bolus, both SSPN+/+ and SSPN−/− mice exhibited a similar increase in heart rate, recorded as beats per minute (bpm), increasing from baseline values of ~450 bpm to ~550 bpm for SSPN+/+ and ~600 bpm for SSPN−/− as shown in +/+ or SSPN−/− mice before and after the isoproterenol treatment indicate a similar decrease in HRV during stress testing. Next, we assessed the in vivo cardiac contractility in mice using echocardiography under 2% isoflurane with 100% O2. The SSPN−/− mice displayed similar left ventricular fractional shortening  and isovolumic relaxation time  compared to SSPN+/+ mice  injury and the development of arrhythmias, we subjected ex vivo perfused SSPN+/+ and SSPN−/− hearts to IR injury, according to the schematic shown in +/+ and SSPN−/− perfused hearts. Post-IR arrhythmia risk was assessed using an ECG during perfusion and the number of premature ventricular contractions (PVCs) per minute after IR injury were found to be significantly increased in SSPN−/− compared to SSPN+/+ hearts.To determine whether the SSPN−/− myocardium after acute IR injury cardiomyocytes were isolated from SSPN+/+ and SSPN−/− hearts to assess perturbations in cellular Ca2+ handling. Cellular Ca2+ handling was reported in Fura-2 AM-loaded cardiomyocytes after 10 s of electrical stimulation under baseline conditions and after acute exposure to 1 μM isoproterenol (Iso). In +/+ and SSPN−/− cardiomyocytes exhibited similar Fratio, at baseline indicating similar levels of diastolic Ca2+.However, after acute Iso exposure SSPN+/+ cardiomyocytes exhibited a significantly increased Fratio. This suggested that there was an increase in diastolic Ca2+ in SSPN+/+ cardiomyocytes; however, it remained unchanged in SSPN−/− cardiomyocytes. In 2+ transient is shown and reports the peak amplitude of the Ca2+ transient under resting (-Iso) and stimulated conditions (+Iso). Under resting conditions, the Ca2+ T height was significantly lower in SSPN−/− compared to SSPN+/+ cardiomyocytes. Overall, there were no differences in the peak amplitude between SSPN+/+ and SSPN−/− cardiomyocytes upon acute Iso exposure.To investigate the underlying factors contributing to the heightened risk of arrhythmias in the SSPN2+ transient height to decay is reported in 2+ T decay. A delay in Ca2+ T decay suggests action potential prolongation, which were significantly longer under stimulated conditions (+Iso), while there was also a trend toward delayed Ca2+ T decay under resting conditions. This provides important information about the balance between inward ICa and outward Ik currents; therefore, the prolonged Ca2+ transients seen in SSPN−/− may increase the risk of early afterdepolarizations, leading to PVCs under stress conditions. In 2+ T height was measured after caffeine-induced SR Ca2+ release to estimate the SR Ca2+ content. The Fratio increased after Iso treatment, indicating higher diastolic Ca2+ (greater Ca2+ SR release) for both SSPN+/+ and SSPN−/− cardiomyocytes. However, SSPN−/− cardiomyocytes had significantly lower SR Ca2+ T height compared to SSPN+/+ after Iso treatment. After caffeine administration, Iso treatment SR Ca2+ content was significantly higher for both SSPN+/+ and SSPN−/− cardiomyocytes indicated by Fratio. To better understand the functional SR Ca2+ release, the Ca2+ T height was normalized to caffeine induced SR Ca2+ T height, +/+ and SSPN−/− under both conditions tested.The length of time it takes the peak Ca2+ release and cardiac rhythm in response to IR injury. The expression of β-adrenergic receptor 1 (β-AR1) appeared unchanged between the groups after acute IR injury as shown in −/− and SSPN+/+ hearts. Whereas expression of the plasma membrane Ca2+ handling protein L-type Ca2+ channel (LTCC) was increased in SSPN+/+ hearts upon IR injury with a concomitant decrease in in SSPN−/− hearts as indicated in +/Ca2+ exchanger (NCX) and RyR2 expression were similar; however, RyR2 expression was significantly increased while NCX expression trended higher in SSPN−/− hearts as shown in +/+ hearts as shown in 2+ uptake through SERCA2.To assess acute changes in gene expression, qRT-PCR was utilized to assess the rapid regulatory responses of key genes responsible for modulating Ca−/− hearts exhibited larger infarct size in response to acute IR injury, we assessed whether increased reactive oxygen species (ROS) production under oxidative stress could be a contributing factor to heightened myocardial damage in SSPN−/− hearts. To assess this, in +/+ and SSPN−/− cardiomyocytes were loaded with fluorogenic dye DCFDA/H2DCFDA to obtain a quantitative assessment of ROS produced in response to cellular oxidants mimicked by H2O2 exposure. In the left panel of +/+ and SSPN−/− cardiomyocytes exposed to oxidizing conditions for 30 min. In +/+ and SSPN−/− cardiomyocytes due to the increased oxygen species. It is evident from these measurements that SSPN−/− cardiomyocytes produced greater amounts of oxidant-induced excessive oxygen species ROS compared to SSPN+/+.Since perfused SSPN+/+ and SSPN−/− perfused hearts were assessed for alterations in protein expression to better understand immediate alterations in protein stress responsive pathways. In −/− hearts undergo a rapid antioxidant response since NOX4 protein levels increased upon acute IR injury. Furthermore, excessive ROS in the SSPN−/− cytosol after IR injury increased oxidized CAMKII/total CAMKII as shown in 2+ release and uptake channels (RyR2 or SERCA2) [After acute IR injury the SSPN SERCA2) .−/− hearts responsive to an acute β-adrenergic stimulus? (2) Does SSPN-deficiency render the myocardium more susceptible to acute IR injury? (3) Does acute IR injury reveal arrhythmogenesis in SSPN−/− hearts?As a tetraspanin-like protein, SSPN participates in stabilizing protein-protein interactions between important sarcolemma adhesion complexes, including the DGC, UGC and integrin complexes ,23,24. T+/+ and SSPN−/− hearts. SSPN−/− hearts were responsive to acute β-adrenergic challenges with similar increases in heart rate (bpm) and decreases in heart rate variability as the SSPN+/+ hearts  to assess the hemodynamic response of SSPN+ hearts . As SSPNR injury , global R injury , and carR injury . Similar−/− hearts were found to have increased incidence of PVCs, and, therefore, an increased risk of arrhythmias compared to SSPN+/+ hearts. After acute IR injury, the SSPN−/− myocardium exhibited larger infarcted areas and more widespread damage. This suggests that SSPN affords global protection to cells throughout the heart. The increased vulnerability of the SSPN−/− myocardium to acute IR can be attributed to a variety of factors, including reduced localization of dystrophin at the sarcolemma [In rcolemma , alteredrcolemma  and the 2+ handling and redox proteins. Studies in the mdx mouse model are highly informative regarding the impact of complete dystrophin deficiency, destabilization of the DGC and perturbations in Ca2+ handling. Studies in mdx cardiomyocytes show that they have reduced resting sarcomere lengths and increased passive tension, which contributes to poor passive compliance [mdx cardiomyocyte experiments by Viola et al. suggest that the absence of dystrophin disrupts the normal cytoskeletal architecture, affecting LTCC activation and superoxide generation [Perturbations to key pathways can increase cell death in response to acute IR injury; therefore, we examined alterations in key Campliance . Examinimpliance . Additioneration . Whereasneration  and cardneration  muscle s−/− cardiomyocytes may diminish the “shock absorbing” capacity of the sarcolemma by weakening the connection between the ECM and intracellular actin and/or perturb its scaffolding function [2+ handling and redox homeostasis. Relatedly, the absence of dystrophin in DMD leads to mis-localization and abnormal expression/activity of Ca2+ handling and oxidative stress responsive proteins [−/− myocardium does not exhibit overt signs of membrane instability—the increased vulnerability of the SSPN−/− myocardium to IR injury could be attributed to aberrant cardiomyocyte Ca2+ entry or a reduction in submembrane scaffolding orchestrated by dystrophin and/or SSPN.The reduction of dystrophin in SSPNfunction ,34. Thisproteins ,36,37. S2+ handling in SSPN−/− cardiomyocytes to better understand their cellular phenotype compared to the dystrophic cellular phenotype. Our results indicate that mechanisms leading to cytosolic Ca2+ overload in mdx cardiomyocytes may not be substantially activated in SSPN−/− cardiomyocytes. To assess this, we examined intracellular Ca2+ handling in SSPN−/− cardiomyocytes after exposure to acute isoproterenol regimen. We found that in contrast to chronic in vivo β-adrenergic stimulation of SSPN-deficient hearts, isolated SSPN−/− cardiomyocytes remained responsive to acute isoproterenol exposure [In this study, we examine alterations in Caexposure , see Fig2+ entry and efflux across the cell membrane to achieve a given average systolic and diastolic Ca2+ level. Stress induction regimens using acute β-adrenergic stimulation, provide information on the responsive balance of stress and adaptations of excitation-contraction coupling (ECC) [−/− cardiomyocyte Ca2+ handling. Our Ca2+ handling data indicated that SSPN−/− cardiomyocytes maintained responsiveness to acute isoproterenol exposure. Overall, isoproterenol exposure activates PKA through β-adrenergic receptors and increases the amount of SR Ca2+ release through increased Ca2+ current (ICa) and SR Ca2+ contents [2+ height estimates the SR Ca2+ content of cardiomyocytes since caffeine triggers Ca2+ release by reducing the RyR2 activation threshold for luminal but not cytosolic Ca2+ activation [−/− cardiomyocytes had alterations in these two parameters (diastolic Ca2+ and SR Ca2+ content) revealing reduced Ca2+ SR content under two conditions: β-adrenergic and caffeine stimulation. After β-adrenergic stimulation, both the diastolic Ca2+ levels and SR Ca2+ contents were lower, which may be explained by the reduced SR Ca2+ release accompanied by the lower SR Ca2+ content.To maintain normal function, cardiomyocytes undergo infinite adaptations to Cang (ECC)  to uncovcontents . Furthercontents . SR Ca2+tivation . SSPN−/−2+ handling gene expression occur upon acute IR injury [2+ handling gene expression and protein levels. The initial response of healthy cardiac myocytes to electrical stimulation is an increase in intracellular Ca2+. This initially occurs as a small influx of Ca2+ through LTCC that triggers a larger Ca2+ release from the SR through RyR2 [2+ handling proteins essential to ECC we anticipated affected proteins may exhibit transient gene expression changes in response to acute IR injury. The most striking rapid alteration in gene expression in SSPN−/− hearts after acute IR injury was a reduction in LTCC and an increase in RyR2 expression see 2+ homeostasis. Our initial findings suggest that tight transcriptional regulatory mechanisms may compensate for cellular perturbations that exist within SSPN−/− cardiomyocytes.It has been shown that transient alterations in CaR injury . These cR injury . Therefough RyR2 . If loss−/− hearts exhibited widespread damage after acute IR injury, we were interested in whether the antioxidative capacity of the SSPN−/− myocardium was compromised. To assess this, we exposed SSPN−/− cardiomyocytes to oxidizing conditions and measured the production of ROS in response to H2O2, see 2O2-induced ROS production in SSPN−/− cardiomyocytes compared to SSPN+/+ may be a contributing factor to the increased susceptibility of the SSPN−/− myocardium to IR injury. Ca2+ overload also leads to increased ROS in the heart; however, the SSPN−/− cardiomyocyte Ca2+ handling data presented here does not suggest major perturbations in Ca2+ regulation. Therefore, it is more likely that SSPN deficiency has a greater impact on the antioxidant mechanisms within the heart.The scaffold role of dystrophin was demonstrated and the loss of SSPN may potentially influence these processes by altering the activity/sarcolemma localization of proteins regulating redox homeostasis. Excessive ROS production that outpaces the antioxidant capacity of the heart contributes to myocardial damage in ischemic heart disease and heart failure . Since S−/− cardiomyocytes produce higher levels of ROS under oxidative conditions, we examined the protein expression of key oxidative stress markers in SSPN−/− hearts after acute IR injury. This data is shown in 2- or H2O2 and are the major source of ROS. The suppression of either NOX2 or NOX4 has been demonstrated to reduce ROS and protect against IR injury [−/− hearts after IR injury. The previous studies of DMD-associated cardiomyopathy found increased induction of NOX4 in the diseased hearts associated with increased fibrosis and functional decline [2+ regulation in cardiomyocytes and activate CAMKII via oxidation-dependent mechanisms. Remodeling of the cardiac action potential by higher baseline oxidative stress-induced cellular memory may lead to pathological processes, increasing the susceptibility to cardiac arrhythmia.To address the finding that SSPNR injury . Our stu decline . In resp2+ release. These alterations in SSPN−/− hearts can contribute to increased number of premature ventricular contractions and altered propagation of signals throughout the heart leading to an increased risk of arrhythmias.Our findings suggest that SSPN deficiency increases myocardial risk upon IR injury by enhancing pathways that increase oxidative stress. This can cause increased damage to heart tissue under adverse conditions and affect systolic Ca+/+ and SSPN−/−) and their age range was between 4 and 8 months. Differences between the groups were assessed using one-way ANOVA; whereas, the incidence of arrhythmia was compared using the non-parametric Mann-Whitney test.All animal protocols used in this study conformed to the Guiding Principles in the Care and Use of Animals of the American Physiological Society and were approved by The University Animal Care and Use Committee  at Florida State University. The sspntm1Kcam (SSPN-deficient) mice were purchased from The Jackson Laboratory and developed by the Campbell laboratory  these mi2 for 15 min [Echocardiography was performed under 2% of Isoflurane with 100% Or 15 min . The echTotal RNA was isolated from heart tissue using Trizol reagent . For cDNA synthesis the qPCRBIO cDNA Synthesis Kit (PCR Biosystems) was used and each reaction contained 0.4 μg total RNA. The expression of mRNA was analyzed by Real-time quantitative PCR using 2X qPCRBIOSyGreen Mix (PCR BIOsystems) in QuantStudio 3 (Applied Biosystems). β-actin was used to normalize the mRNA. The relative expression was determined using the ΔΔCt method.The sequences of the primers utilized are as follows:β-AR1-F: 5′-CTC ATC GTG GTG GGT AAC GTG-3′ and R: 5′-ACA CAC AGC ACA TCT ACC GAA-3′;LTCC-F: 5′-ATG AAA ACA CGA GGA TGT ACG TT-3′ and R: 5′-ACT GAC GGT AGA GAT GGT TGC-3′;NCX1-F: 5′-AAA GAG TGC AGT TTC TCC CTT G-3′ and R: 5′-TGA AGC CAC CTT TCA ATC CTC-3′;RyR2-F: 5′-ACG GCG ACC ATC CAC AAA G-3′ and R: 5′-AAA GTC TGT TGC CAA ATC CTT CT-3′;SERCA2-F: 5′-GAG AAC GCT CAC ACA CAA AGA CC-3′ and R: 5′-CAA TTC GTT GGA GCC CCA T-3′;PLN-F: 5′-AAA GTG CAA TAC CTC ACT CGC-3′ and R: 5′-GGC ATT TCA ATA GTG GAG GCT C-3′; andβ-actin-F: 5′-GGG TGT ATT CCC CTC CAT CG-3′ and R: 5′-CCA GTT GTT GGT AAC AAT GCC ATG-3′.n = 6 per group. One point was removed from the NCX1 data for SSPN−/− controls as it was identified as an outlier by Prism Graphpad using the ROUT (Q = 1%) method to facilitate plotting but did not alter the results, with nonsignificant trends between the groups. The SSPN−/− IR injury NCX1 consists of n = 6; however, one point is obscured by a similar value.The number of samples used was 2 for 10 min while electrocardiography (ECG) traces were recorded for a period of 5 min while at rest and then after stress induction  for 5 min. The ECG recording and analysis were performed using Powerlab and Lab chart .The mice were anesthetized under 2% Isoflurane with 100% O3, 1.2 NaH2P04, 1 MgCl2, 1.5 CaCl2 and 10 glucose as previously described [2/5% CO2) to achieve a pH of 7.4. The heart was allowed to equilibrate for 10 min prior to initiation of the ischemia-reperfusion (IR) protocol. The baseline ECG measurements were collected for ten minutes prior to exposing the heart to ischemic conditions (no flow) for 30 min and the ECG readings were recorded during continuous reperfusion for an additional 60 min. After completion of the experiment, the hearts were used for protein, mRNA and histological (infarct size) analyses.Mice were euthanized under isoflurane anesthesia and the heart was removed after performing a thoracotomy. The aorta was then cannulated in the Langendorff mode, which involved retrograde perfusion of the aorta at 37 °C with a perfusion buffer containing (mM) 139 NaCl, 4 KCl, 14 NaHCOescribed ,48. The 2-cooled 2-methylbutane [2. For each heart, three adjacent sections were used to calculate the infarct volume, and the number of pixels in the infarct zones were counted to obtain the infarct size in mm2 for each heart.Histological analysis of the heart tissues was performed to measure infarct size. To this end the heart tissues were embedded horizontally in OCT and flash-frozen in liquid Nylbutane . Next, tylbutane . The sel2, 10 glucose, and 10 HEPES (pH 7.4) at room temperature. Intracellular Ca2+ regulation was measured using the previously described protocol [2+ content was then quantified by the application of caffeine . The Ca2+ fluorescence ratios (Fratio) were recorded and normalized with an average vehicle group. The data were analyzed using the commercially available data analysis software IonWizardTM 6.6, IonOptix, Milton, MA, USA.Myocytes were isolated using a modified collagenase/protease method ,52,53, aHeart samples were lysed at 4 °C using a urea-based extraction buffer . After qIsolated cardiomyocytes were loaded with DCFDA/H2DCFDA (3 μM) for 15 min. After they were washed three times with dye. The fluorescent signals were imaged using fluorescence microscopy at baseline for up to 60 min. The fluorescent intensity of the captured images was measured using ImageJ 1.53k.


Background and Objectives: Milk is healthy and includes several vital nutrients and microbiomes. Probiotics in milk and their derivatives modulate the immune system, fight inflammation, and protect against numerous diseases. The present study aimed to isolate novel bacterial species with probiotic potential for neuroinflammation. Materials and Methods: Six milk samples were collected from lactating dairy cows. Bacterial isolates were obtained using standard methods and were evaluated based on probiotic characteristics such as the catalase test, hemolysis, acid/bile tolerance, cell adhesion, and hydrophobicity, as well as in vitro screening. Results: Nine morphologically diverse bacterial isolates were found in six different types of cow’s milk. Among the isolates, PO3 displayed probiotic characteristics. PO3 was a Gram-positive rod cell that grew in an acidic (pH-2) salty medium containing bile salt and salinity (8% NaCl). PO3 also exhibited substantial hydrophobicity and cell adhesion. The sequencing comparison of the 16S rRNA genes revealed that PO3 was Lactococcus raffinolactis with a similarity score of 99.3%. Furthermore, PO3 was assessed for its neuroanti-inflammatory activity on human oligodendrocyte (HOG) cell lines using four different neuroimmune markers: signal transducer and activator of transcription (STAT-3), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and GLAC in HOG cell lines induced by MOG. Unlike the rest of the evaluated neuroimmune markers, STAT-3 levels were elevated in the MOG-treated HOG cell lines compared to the untreated ones. The expression level of STAT-3 was attenuated in both PO3-MOG-treated and only PO3-treated cell lines. On the contrary, in PO3-treated cell lines, MBP, GFAP, and GLAC were significantly expressed at higher levels when compared with the MOG-treated cell lines. Conclusions: The findings reported in this article are to be used as a foundation for further in vivo research in order to pave the way for the possible use of probiotics in the treatment of neuroinflammatory diseases, including multiple sclerosis. Milk and its products are habits for beneficial bacteria known as probiotics, which have a key role in immunomodulatory, anti-inflammatory, and antimicrobial action against several diseases. They have been part of human food and culture for a long time due to their use in medicine, food manufacturing, and preservation . Their e per day ,3. Certa per day . The comproducts ,5,6. Thelococcus ,9,10,11.lococcus . Hence, Lactococci, which is helpful in developing various cheese products [Lactococcus (38.6%) and Streptococcus quantity increased significantly after 24 h of refrigeration [Lactic acid bacteria are widely used in dairy industries to ferment food products through acidification or low pH to preserve them. Moreover, they contribute proteolytic, lipolytic, and organoleptic characteristics to the fermented foodstuffs, such as flavor and texture ,14. Geneproducts ,17. One geration .Gut microbiota plays a key role in regulating the interactions among the central nervous system, endocrine glands, and inflammatory markers ,20. TheyLactococcus raffinolactis and its other members belong to the family Streptococcaceae, and they are used in different industries for meat preservation, starters, and flavoring purposes [Lactobacillus and Lactococcus. However, few studies correlate the probiotic effect on neurological inflammation. Therefore, this study aims to focus on the probiotic potential of Lactococcus raffinolactis and its role in the modulation of neuroinflammation. It has recently been reported that Lactococcus lactis displayed a significant positive impact in an LPS-induced depression-like model in mice [purposes . There a in mice , allevia in mice , attenua in mice , and pre in mice . DespiteFor the bacterial analysis, 6 samples of lactating dairy cows (one-week lactating milk (Colostrum) and 25th-week lactating milk) were collected from January to March 2023 in the mid-winter season . A total of 400–500 mL of milk was collected in sterilized containers. The samples were labeled and stored at 4 °C until use.Isolation of beneficial bacteria from lactating cow milk was performed using the serial dilution method. A total of 1 mL of cow milk was added to 24 mL of de Man, Rogosa, and Sharpe (MRS) broth, and diluted milk (0.2 mL) was spread and plated on an MRS agar plate for 48 h. The single colonies were selected and stored in 30% glycerol stock with 10% skim milk with consecutive −70 °C storage conditions .2O2 slide drop assay. The hemolytic activity of active isolates was examined on Colombia blood agar  [The Gram-positive isolates from cow milk were screened for catalase activity using the H, India) .w/v) NaCl and at pH 3.0 according to the method described earlier by [Colony morphology, cell shape, Gram staining, catalase activity, and hemolytic activity were investigated after 48 h on MRS agar plates at 30 °C. The isolates were checked for physiological traits, including growth in 8% (rlier by . Based ow/v)) and taurodeoxycholic acid (0.5% (w/v)). Plates were incubated anaerobically at 37 °C for 72 h. Zone forming colonies or white colonies indicated signs of BSH activity. The baseline was MRS agar that had not been changed.The activity of BSH was determined following the method mentioned earlier by , with slThe ability of bacterial cells to adhere to the mucus environment was achieved by using octane solvent in an in vitro environment . The assay was performed according to a previously described method .The hydrophobicity index (HBI) was calculated as follows:−1. The MRS-cholesterol-PEG 600 mixture was incubated at 37 °C for 24 h with each 1% (v/v) inoculum. The turbidity of MRS broth was measured by CFU methods. The assimilation of cholesterol was detected by centrifuging the broth at 4000 rpm for 10 min, and the supernatant was collected for absorbance analysis using a microplate spectrum reader (570 nm). The concentrations of cholesterol were compared to the absorbance of the standard curve, with cholesterol concentrations ranging from 0 to 500 µg/mL in MRS (R2 = 0.987). The efficiencies of beneficial bacteria to cholesterol assimilation in MRS were determined as follows:The ability of beneficial bacteria to assimilate cholesterol was assessed in MRS broth supplemented with cholesterol-polyethylene glycol (PEG) 600  at a final concentration of 100 µg mLg for 10 min at 4 °C and resuspended in sterile physiology saline (0.88%) to adjust the cell culture (OD600 = 1 and 109 CFU/mL).Preparation of bacterial cell suspensions was performed as described by Pithva et al.  to evaluThe simulated gastric acid and intestinal bile fluids were prepared as described previously . The aciThe percentage of bacterial survival was calculated as follows:v/v) fetal bovine serum (FBS) and 100× antimycotic solution under humidified and 5% CO2 conditions.Human oligodendrocyte (HOG) cell lines  were routinely grown at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM) with serum supplemented with 10% (5 cells) were cultivated for 72 h in cell culture inserts (Boyden chamber). After 72 h, the solution containing 5 mM sodium butyrate was replaced, and cells were cultured for 96 h to promote differentiation. HOG cells were aspirated with sterile PBS and bacterial cell suspension and were resuspended in antibiotics-free DMEM. After 90 min in a 37 °C 5% CO2 incubator, each cell suspension was plated on wells. Unbound bacterial cells were removed by sterile PBS aspiration and were subsequently lysed with 0.05% Triton X-100 solution. Spot plate counts on MRS agar were performed after 48 h of incubation at 37 °C to determine the total number of attached bacteria. The following equation was used to determine the adhering abilities of the chosen isolate:Human oligodendrocyte cells were used to study the adherence characteristics of PO3 . HOG cells (1.5 × 104 cells/well) were inoculated in 48-well plates for 24 h. The serum-free DMEM (1% FBS) was stored overnight before treatment. The starved cells were pre-incubated with 1 µg/mL MOG for 4 h, and the active isolate PO3 was inoculated and co-cultured for 24 h. The supernatant was examined for NO production, which was determined using an NO detection kit (Griess reagent kit) in accordance with the manufacturer’s protocol.HOG cell lines induced with MOG at 1 µg/mL  in accordance with the manufacturing guidelines. The amounts of cytokines on the plates were measured using an automated ELISA plate reader at 450 nm . The standard was used for each cytokine marker with Rhttp://pga.mgh.harvard.edu/primerbank (accessed on 15 March 2023).Total RNA was extracted from MOG-induced HOG cell lines in accordance with the instructions provided by the manufacturer . The cDNA synthesis kit was used to generate complementary DNA (cDNA), and the process was carried out in compliance with the guidelines provided by the manufacturer. The qPCR primers that are shown in −ΔΔCt [In order to determine the optimal annealing temperature for each primer set, gradient PCR was used. The quantity of mRNA produced by the genes under investigation was measured using the detection instrument ABI step one plus  and was supplied with SYBR Green master mix manufactured by Amplicon Bio in Denmark. In order to ensure accuracy, all reactions were carried out twice. The relative amount of gene expression was calculated using the formula RQ = 2−ΔΔCt , which wThe isolates of 16S rRNA gene sequences were amplified using PCR following the method mentioned earlier by the authors of . PCR prop < 0.05, Duncan’s Multiple Range Test (DMRT) was used as the appropriate statistical tool. Welch’s t-test, with a significance threshold of p < 0.05, was used to evaluate the immunomodulatory effects of the treatment.All the tests were carried out three times to ensure accurate data, and the findings are presented as the mean along with the standard deviation (SD). The results of tests on acid and bile tolerance and adhesion capacity were analyzed using ANOVA and the SPSS 22.0 program. When comparing mean values at a significance level of 2O2 was added. PN3 and PN4 exhibited hemolytic activities, whereas the remaining isolates did not.Nine morphologically distinct bacterial isolates were obtained from six cow’s milk samples. There were four  and five isolates  obtained from the 1st and 25th week of lactating cows, respectively . As can Four isolates, PN1, PN2, PO2, and PO3, were selected for further screening using bile salt hydrolase (BSH) activity, and the results are presented in The capacity of probiotics to attach to epithelia is assessed in vitro by measuring the cells’ hydrophobicity at the surface for certain solvents (octane). This determines whether or not the probiotics are able to attach to the epithelia. It was discovered that the isolate has a significant degree of hydrophobicity . The resp < 0.01). Unlike IL-6, a comparative evaluation of IFN-γ levels in MOG-treated cells was compared to the negative control ones. The presence of PO3 significantly reduced the increased levels of NO seen in MOG-treated cells (The findings for the pro-inflammatory cytokines (IL-6 and IFN-γ) are shown in ed cells A.The impact of PO3 on the induction of neuroimmune markers  in HOG cell lines by MOG (1 µg) was studied and the results are presented in Lactococcus raffinolactis at a similarity level 99.3%. The submitted 16S rDNA sequences received the accession number for PO3 (OQ914364). The neighbor-joining method revealed the active isolate was Lactococcus raffinolactis, with the 0.001 bootstrap closer to Lactococcus raffinolactis NR044359  indicated that AggLr is a member of the SFCBAF protein family [Disturbance of the gut–brain axis, a bidirectional relationship between the microbiota in the digestive tract and the brain, has been linked to neuroinflammatory response and neurological disorders ,30. Cow eactions . It has n family .Limosilactobacillus walteri as a novel probiotic bacterial strain [PO3 was unable to generate the catalase that is required to produce oxygen and water by splitting hydrogen peroxide. This finding also implies that PO3 can live in environments with extremely low concentrations of oxygen. These findings were comparable to those that were published by Saroha et al. (2023) , who desl strain . ScreeniLactococcus lactis has very high hydrophobicity, as measured by microbial adherence to xylene, with a value of 89.3% [Hydrophobicity at the surface of cells is measured in vitro to evaluate the ability of probiotics to attach to epithelia. The ability of the probiotics to bind to the epithelia depends on this. According to the findings, PO3 exhibited the maximum adhesion towards xylene (3%). Selecting probiotic bacteria for use as therapeutic agents requires consideration of their ability to attach to the gut. Colonization of the human gut by probiotics is believed to be needed for them to perform their therapeutic effects, such as the avoidance of enteropathogenic bacteria . The coaof 89.3% ,52.Lactobacillus sp. strain in the earlier investigation [A crucial biological barrier that a probiotic strain has to overcome to reach its target is the acid that is found in the stomach, as well as the bile salts that are found in the intestine. In environments with a low pH, metabolic activity may be restricted, and the growth and survival of probiotics may be hindered. Therefore, the ability of probiotic strains to tolerate acid, gastric juice, and bile is critical to their capacity for survival and proliferation in the gastrointestinal system. In the research that we conducted, we found that PO3 was resistant to bile salt, acidic pH, and stimulated gastric juice. These findings are comparable to those obtained with the probiotic tigation ,54.Lactococcus lactis inhibits experimental autoimmune encephalomyelitis by preventing cell migration into the spinal cord [Bacillus amyloliquifaciens-supplemented camel milk suppress the neuroinflammation of autoimmune encephalomyelitis by regulating inflammatory markers [The modulatory impact of PO3 on the expression of myelin basic protein (MBP) neuroimmune markers , glial fibrillary acidic protein (GFAP), and GLAC) was studied in HOG cell lines induced by MOG. Unlike the rest of the evaluated neuroimmune markers, STAT-3 levels were elevated in the MOG-treated HOG cell lines compared to the untreated ones. The expression level of STAT-3 was attenuated in both PO3-MOG treated cell lines and those only treated with PO3. In contrast, in PO3-treated cell lines, MBP, GFAP, and GLAC were significantly expressed at higher levels when compared with the MOG-treated cell lines. It has been reported that Hsp65-producing nal cord . Further markers , as well markers . STAT-3 H. pylori infection by stimulating phagocytoses, natural killer (NK) cells, DCs, and antibody and anti-inflammatory cytokines [Lactococcus sp. PO3 abrogated STAT-3 and activated the neuroprotective markers to improve cell communication and the functionality of the blood–brain barrier. In support of that, L. rhamnosus and L. acidophilus have been found to block STAT-3 and NF-B activation in dendritic cells via upregulation of certain anti-inflammatory markers (IL-10 and Foxp3+ Treg) [The activation of STAT-3 signaling may be triggered by a variety of stimuli and is crucial for the functions of many distinct types of cells . By maniytokines . STAT-3 3+ Treg) .Lactococcus raffinolactis through 99.3% 16S rRNA gene sequence similarity. PO3 displayed substantial modulatory impacts on the immune markers in the MOG-induced cell lines. The results presented herein are considered a base for further in vivo investigation in order to open the door for potential application of the probiotic to combat neuroinflammation disorders.The six cow’s milk samples yielded nine morphologically distinct bacterial isolates. PO3, from 25th-week lactating cow milk, was a probiotic. PO3 was Gram-positive, and rod cells were developed in highly acidic medium (pH 3), with bile salt, and in a saline environment (8% NaCl). PO3 was hydrophobic and cell-adhesive. The isolate altered cell line proinflammatory markers. PO3 was shown to be


Small cell lung cancer (SCLC) is frequently disseminated and has a dismal prognosis with survival times of approximately two years. This cancer responds well to initial chemotherapy but recurs within a short time as a globally chemoresistant tumor. Circulating tumor cells (CTCs) are held responsible for metastasis, the extremely high numbers of these cells in advanced SCLC allowed us to establish several permanent CTC cell lines. These CTCs are distinguished by the spontaneous formation of large spheroids, termed tumorospheres, in regular tissue culture. These contain quiescent and hypoxic cells in their interior and are associated with high chemoresistance compared to single cell cultures. Nine CTC lines were compared for their expression of 84 proteins associated with cancer either as single cells or in the form of tumorospheres in Western blot arrays. With the exception of the UHGc5 line, all other CTC lines express EpCAM and lack a complete EpCAM-negative, vimentin-positive epithelial-mesenchymal transition (EMT) phenotype. Upon formation of tumorospheres the expression of EpCAM, that mediates cell-cell adhesion is markedly upregulated. Proteins such as E-Cadherin, p27 KIP1, Progranulin, BXclx, Galectin-3, and Survivin showed variable changes for the distinct CTC cell lines. In conclusion, EpCAM presents the most critical marker for individual SCLC CTCs and the assembly of highly chemoresistant tumorospheres. Small cell lung cancer (SCLC) is a neuroendocrine lung tumor representing 15% of lung cancers and shows metastases in most patients at first presentation . Smokersin vitro that grow continuously in tissue culture [The typical overall 5-year survival of 5–10% for extended SCLC requires new treatment modalities and characterization of the mechanism of drug resistance. The dissemination of SCLC seems to be related to the excessively high counts of circulating tumor cells (CTCs) compared to other malignancies ,9. A min culture ,12. ThesAll SCLC CTC lines exhibit the spontaneous formation of large multicellular aggregates under normal cell culture conditions, which grow to diameters of 1–2 mm, designated tumorospheres ,12. Normin vitro as spheroids represent exactly the chemoresistance phenotype of native solid tumors and display pathways of resistance linked to hypoxia, altered chromatin structure, impairment of apoptosis, cell cycle alterations, and decreased drug perfusion [in vivo growing tumors [Cancer cells grown erfusion . Ki-67-eerfusion ,24. Comperfusion . MCTSs cg tumors ,16,26. Tg tumors .Despite high investments to develop new cancer therapeutics, about 90% of drugs finally tested in clinical trials fail ,29. SpheThe mechanisms leading to the spontaneous formation of SCLC CTC tumorospheres are not clear, and therefore, we searched for differences in protein expression between single cells and corresponding spheroids using Western blot arrays. The Oncology XL array assesses the relative expression levels of 84 human cancer-related proteins comprising growth factor receptors, signaling mediators, growth factors, adhesion proteins, proteases, chemokines, hormones, and others. The CTCs in form of single cells or tumorospheres were checked for their chemosensitivity using Topotecan and Epirubicin, which are administered for second-line treatment of SCLC. These spheroids contain quiescent and hypoxic cells and seem to constitute a vital component of the global drug resistance of SCLC tumors.The SCLC CTC cell lines BHGc7, 10, 16, 26, 27, 71, 500, 590, and UHGc5 were established from blood samples of extensive disease-SCLC patients following disease progression under second-line therapy at our institution as described previously . Blood c4 cells in the form of single cells or tumorospheres (TOS) in 100 μl medium were distributed to wells of 96-well microtiter plates . Therafter, ten 2-fold dilutions of the test compounds were added from stock dilutions. Assays were at least performed in triplicate. Calculating CTC cell size and the surface area of the globular tumorospheres, 1 × 104 suspension cells correspond to the same number of cells accessible at the surface of the spheroids. The mean size of the clusters used for the cytotoxicity tests was determined, and the number of clusters/well adjusted accordingly. The plates were incubated for four days under tissue culture conditions, and viable cells were detected using a modified MTT assay . The respective dilutions of the compounds tested are present for the whole incubation period. IC50 values were determined from dose-response curves using Origin Pro, Version 9.9 .1 × 10We determined the relative expression levels of 84 cancer-related proteins in the investigated cell lines by performing a Proteome Profiler Human XL Oncology Array ). The kit consists of 4 nitrocellulose membranes spotted in duplicate with antibodies for cancer-related proteins. Membranes were treated following the enclosed protocol ). Cell culture lysates of 9 cell lines in the form of single cells and their tumorospheres were obtained by removing particulates in samples by centrifugation. Afterward, samples were diluted and incubated with the Human XL Oncology Array overnight, followed by removal of unbound proteins by washing. Subsequently, the array was set with a detection antibody cocktail, followed by applying Streptavidin-HRP and chemiluminescent detection reagents. These reagents produce a signal corresponding to the amount of protein-bound, which was visualized using a ChemiDoc™ Touch Imaging System .Heatmaps were plotted for nine cell lines in the form of single cells and their tumorospheres using protein expression data of 43 cancer related proteins in the application heat map with dendrogram in Origin Version 9.9 . Thereby a hierarchical cluster analysis was performed. Firstly, clustering rows of 43 genes by group average and Pearson correlation to reveal differences in gene expression via z-score of these genes in 9 cell lines in the form of single cells and their tumorospheres. Secondly, to show the differences in expression of these proteins via z-score within tumorospheres and uncover similarities between the different cell lines by clustering rows and columns by group average and Pearson correlation. Missing values were filled up with column means, while standardization was performed for columns.The Reactome database (Version 3.7) organizes signaling and metabolic molecules and their relations into biological pathways and processes . The resp-value < 0.05 was regarded as statistically significant. The significance of the heatmap data (single cells versus tumorospheres) is presented in Analysis of the data from cytotoxicity assays and heatmap analyses were calculated using the Origin 9.9 software, in particular, the heatmap option with dendrograms. A 50 values of the cell lines for single cells are presented in 2 = 0.11).In p = 3.35E−5), downregulation of ERBB2 signaling (p = 3.35E−5), ERBB2 regulates cell motility (p = 5.03E−6), signaling by ERBB2 KD mutants (p = 3.09E−5), signaling by ERBB2 TMD/JMD mutants (p = 2E−5), ERBB2 activates PTK6 signaling (p = 4.28E−6), GRB2 events in ERBB2 signaling (p = 6.77E−6), SHC1 events in ERBB2 signaling (p = 3.35E−5). Furthermore, pathways involving ERBB4, e.g., signaling by ERBB4 (p = 5.29E−9), nuclear signaling by ERBB4 (p = 1.95E−10), SHC1 events in ERBB4 signaling (p = 6.77E−6).The Reactome pathway analysis of the 14 proteins with reduced expression in tumorospheres  revealed a connection of these genes to signal transduction. In detail, there were several pathways involving ERBB2, e.g., signaling by ERBB2 in cancer (p = 7.01E−9), Interleukin-18 signaling (p = 9.84E−7), Inteleukin-4 and Interleukin-13 signaling (p = 1.33E−6). Furthermore, they are associated with Receptor Tyrosine Kinase signaling (p = 2.36E−9), long-term potentiation (p = 2.15E−5), PI3K/AKT signaling in cancer (p = 5.82E−5), and regulation of PI3K/AKT signaling by PI5P, PP2A, and IER3 (p = 6.78E−5). In summary, these alterations affect cellular signaling, proliferation, and interleukin pathways that are effective in the three CTC cell lines exhibiting the highest proliferative activity.These 14 proteins further influence the signaling of interleukins, which are known for their importance in the function of the immune system, e.g., Interleukin-10 signaling . 3D cultures of tumor cell lines resulting in the formation of different spheroids have been propagated as models of actual tumors that are more representative than traditional 2D cultures. However, as opposed to SCLC CTCs, this buildup of spheroids has to be enforced by nonadherent tissue culture conditions such as hanging drops, agitation, or various scaffolds. The so-called MCTS closely mimics otherapy ,34. Beyootherapy ,36. MCTSotherapy ,38. Compotherapy ,40. The otherapy .The proteins participating in the spontaneous formation of tumorospheres in SCLC CTCs have not been characterized. Chemosensitivity tests demonstrated again that the tumorospheres are more resistant to Topotecan and Epirubicin compared to the corresponding single-cell suspensions . The CTCversus single cells. The formation of spheroids by Caco-2, SW480, and HCT116 colorectal adenocarcinoma cell lines under low-adhesion culture conditions were reported to show a 3-fold higher expression of EpCAM compared to monolayers, but no changes in 19 other markers [For array analysis, the cells and spheroids are lysed, and in the latter case, proteins from proliferative and quiescent cells are mixed in the lysate. Among the proteins analyzed, EpCAM was the single factor that showed consistently higher expression in tumorospheres  markers . The pro markers . Cell cl markers ,45,46. L markers . Drugs t markers .in vivo. Detection of disseminated tumor cells (DTC) in bone marrow (BM) of patients with early-stage NSCLC has been associated with poor outcomes [EpCAM is frequently upregulated in primary tumors and metastases, particularly in adenocarcinomas, certain squamous cell carcinomas, and others . Increasoutcomes . BM aspioutcomes . Antibodoutcomes .In conclusion, the formation of highly chemoresistant tumorospheres by SCLC CTCs is accompanied by elevated expression of EpCAM, a protein involved in cell-cell adhesion. Therefore, EpCAM-positive CTCs seem to be the cell population destined to survive in circulation and lead to chemoresistance and metastasis.


Background: Chronic rhinosinusitis (CRS) is a heterogeneous disease. The pathogenesis of chronic sinusitis is still unclear; however, the nasal cavity and paranasal sinuses are commonly affected by type 2 inflammation, which is caused by Th2 cytokines such as interleukin (IL)-5, IL-4, and IL-13. Previous studies have shown that pendrin promotes local infiltration of neutrophils through the production of human neutrophil elastase (HNE), which is essential for the secretion of mucin 5AC (MUC5AC) in chronic inflammatory diseases of the lower respiratory tract. This study investigated pendrin expression and its relationship to mucin in type 2 inflammation. Methods: A total of 40 patients  were included in this study and were divided into the CRS group and the NC group. A normal nasal mucosa tissue culture model was established in vitro. IL-13 was used to stimulate primary cultures of human nasal epithelial cells (HNECs). Western blot (WB), enzyme-linked immunosorbent assay (ELISA), and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of pendrin, MUC5AC, and MUC5B. After transfecting HNECs with siRNA pendrin or negative control (NC), EGF receptor (EGFR), HNE, MUC5AC, and MUC5B expression were analyzed using qRT-PCR, WB, or ELISA in terms of their relationships with pendrin. Pendrin expression in the tissue was also analyzed. Results: After IL-13 stimulation, pendrin, MUC5AC, and MUC5B expression levels were upregulated; the optimal concentration of IL-13 was 50 ng/mL. The expression levels of HNE, EGFR, MUC5AC, and MUC5B were downregulated after transfection with siRNA pendrin-1650. Pendrin expression in the NC group was lower than in the CRS group. Conclusion: IL-13 is implicated in the inflammation of nasal mucosa, and pendrin is closely related to the excessive secretion of mucin. The expression of mucin is downregulated after transfection with siRNA pendrin. There is a positive relationship between pendrin and EFGR/HNE. Moreover, pendrin plays an important role in type 2 inflammation. Chronic rhinosinusitis (CRS) is a common disease classified as primary chronic rhinosinusitis and secondary chronic rhinosinusitis, which affects 5–12% of the general population . InflammPendrin protein is a soluble transporter. An important part of the family 26A (SLC26A) member, encoded by the SLC26A4 gene, can mediate cell chloride ion, iodine dissociation, and the exchange of various anions such as zinc and bicarbonate ions to maintain a steady state of medium ions in the tissue. Pendrin is primarily localized in the inner ear, thyroid, and kidney ,16,17; hCombining the results of the above studies, we explored that with the stimulation of some inflammatory factors and other pathogenic factors, pendrin may enhance the infiltration of NEU in the nasal cavity and paranasal sinus mucosa, and then promote the secretion and expression of mucin in epithelial and glandular tissues through EGFR signal, which led to the occurrence and development of CRS.In this study, through interleukin (IL)-13 stimulation, and pendrin siRNA in HNECs, we analyzed the expression of HNE, EGF receptor (EGFR), MUC5AC, and MUC5B, and investigated the pendrin–EGFR–MUC pathway in CRS.Samples of nasal mucosa were collected from patients undergoing functional endoscopic surgery. A total of 40 patients  were included in this study from The First Affiliated Hospital of Nanchang University. The 40 patients were divided into the CRS group and the NC (negative control) group. The CRS diagnoses were based on the patients’ clinical histories and anterior rhinoscopy/nasal endoscopy/computed tomography results, in accordance with EPOS 2020 guidelines [The study was approved by the Medical Research Ethics Committee of The First Affiliated Hospital of Nanchang University, and each participant has signed the informed consent form.2), centrifuged, and cultured in bronchial epithelial cell growth medium (BEGM) .HNECs were cultured using a method described in a previous study . Brieflyg, centrifuge for 10 min, and take the supernatant.After the cells reached 80–90% confluence, they were stimulated with different concentrations of recombinant IL-13  over a 24 h period. The cell lysates for protein are as follows. Remove the culture medium and drugs, gently rinse the adherent cells with precooled PBS for 3 times, pay attention to gentleness to prevent the cells from falling off, suck the remaining PBS solution in the culture dish for the last time, and discard it, add protein lysate and phosphorylated protease inhibitor, blow repeatedly, put them on the ice for 10 min, and then use the cell scraper to scrape the cells and lysate, and transfer them to 1.5 mL EP tube, blow repeatedly to avoid gas. Bubble is generated, but attention should be paid to avoid the generation of foam, and it should be placed on ice to continue cracking for 20 min. Put the EP tube into a precooled 4 °C centrifuge, 12,000× The cell lysates for RNA are as follows. The cells were washed by D-hankS solution three times. After trypsin digestion, the cell suspension was collected with a 15 mL centrifuge tube. Total RNA was extracted from cells with Trizol™ reagent .The cell lysates were collected and MUC5AC, MUC5B, and pendrin mRNA expression were determined by qPCR. MUC5AC and MUC5B expression in the cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The protein expression of pendrin was evaluated using Western blot (WB) analysis.FAM NC , NC , Pendrin-homo-373 , Pendrin-homo-920  and Pendrin-homo-1650 .Pendrin (si pendrin) and a non-targeting siRNA (siCTRL) were synthesized by Sangon Biotech  and transfected into target cells using Lipofectamine 2000 . SiNRA are as follows: A qRT-PCR assay was performed as described by Ye . Total R5′-GTAACAGTCCGCCTAGAAGCAC-3′; MUC5AC, forward: 5′-ACCTGTGACAGCAGGATGTG-3′, reverse: 5′-ACTCGCAGTCTCCGTTGAAG-3′; MUC5B, forward: 5′-TCCCACTATTCCACCTTTGACG-3′, reverse: 5′-CCAGGTAGAGGCTGAGATTCCC-3′; HNE, forward: 5′-GCGTGGCGAATGTAAACGTC-3′, reverse: 5′-ACCCGTTGAGCTGGAGAATC-3′; EGFR, forward: 5′-GCCAAGGCACGAGTAACAAGC-3′, reverse: 5′-GGGCAATGAGGACATAACCAGC-3′.Cell supernatants were collected and subjected to ELISA to determine the levels of human MUC5AC and MUC5B secretion using a specific ELISA assay kit  and  according to the manufacturer’s instructions.WB was performed in accordance with the method described by Luo et al. . Total ct-test. In all analyses, p < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 22.0 . Expression data are presented as dot plots of the median and interquartile range. For the in vitro experiments, cell culture data are presented as means ± standard error of the mean (SEM). Data were analyzed using GraphPad Prism 8 . The differences between the CRS and negative control (NC) groups were examined for significance using Student’s p = 0.0090 and p = 0.0019, respectively); the difference in pendrin expression between the CRSsNP and CRSwNP groups was not statistically significant  stimulation. FAM NC was used to evaluate transfection efficiency. The results showed that after the transfection of siRNA pendrin-1650, both the mRNA and protein levels of pendrin decreased . SiRNA iThe methods of culturing and transfecting HNECs were described earlier. According to qRT-PCR and WB analyses, mRNA and protein levels of HNE and EGFR expression decreased, with pendrin-1650 being the most efficient siRNA . These rCRS is a common and complex disease that affects the quality of life. The main histopathological manifestations of chronic sinusitis are mucosal epithelial thickening, goblet cell proliferation and/or metaplasia, gland hyperplasia, and edema . MUC5AC However, in our previous research, we found that HNE induces the upregulation of MUC5AC in primary HNECs . Zhao etIt has been reported that several inflammatory factors, including IL-4, IL-8, IL-6, IL-13, and IFN-γ, are significantly increased in the mucosa of CRS patients, and that secretion is progressive . Many stPendrin is mainly expressed in the inner ear, thyroid, and kidney; however, recent studies showed that it is also expressed in airway diseases . One stuThe expression of EGFR is low in the normal respiratory epithelium; however, when activated by HNE, EGFR expression is enhanced, which promotes cell metaplasia and mucus secretion, especially the expression of MUC . The secWith IL-13 stimulation, pendrin may promote the secretion and expression of mucin in the epithelium and glands through EGFR signaling, which can lead to the onset of type 2 inflammation.There are still some limitations in our study. First, we did not use the EGFR pathway blocker to explore the relations between EGFR and MUC. Second, we lacked animal models. Experiments in vivo should be carried out to further explore the function of these genes in CRS.In our study, stimulation of HNECs with IL-13 resulted in upregulated MUC5AC, MUC5B, and pendrin expression at the levels of transcription and translation. In addition, the knockdown of pendrin inhibited MUC secretion. Thus, the results of our study indicate that targeting pendrin has the potential as a therapeutic strategy for mucin hypersecretion in CRS patients.


Co-pyrolysis is one possible method to handle different biomass leftovers. The success of the implementation depends on several factors, of which the quality of the produced bio-oil is of the highest importance, together with the throughput and constraints of the feedstock. In this study, the fast co-pyrolysis of palm kernel shell (PKS) and woody biomass was conducted in a micro-pyrolyser connected to a Gas Chromatograph–Mass Spectrometer/Flame Ionisation Detector (GC–MS/FID) at 600 °C and 5 s. Different blend ratios were studied to reveal interactions on the primary products formed from the co-pyrolysis, specifically PKS and two woody biomasses. A comparison of the experimental and predicted yields showed that the co-pyrolysis of the binary blends in equal proportions, PKS with mahogany (MAH) or iroko (IRO) sawdust, resulted in a decrease in the relative yield of the phenols by 19%, while HAA was promoted by 43% for the PKS:IRO-1:1 pyrolysis blend, and the saccharides were strongly inhibited for the PKS:MAH-1:1 pyrolysis blend. However, no difference was observed in the yields for the different groups of compounds when the two woody biomasses (MAH:IRO-1:1) were co-pyrolysed. In contrast to the binary blend, the pyrolysis of the ternary blends showed that the yield of the saccharides was promoted to a large extent, while the acids were inhibited for the PKS:MAH:IRO-1:1:1 pyrolysis blend. However, the relative yield of the saccharides was inhibited to a large extent for the PKS:MAH:IRO-1:2:2 pyrolysis blend, while no major difference was observed in the yields across the different groups of compounds when PKS and the woody biomass were blended in equal amounts and pyrolysed (PKS:MAH:IRO-2:1:1). This study showed evidence of a synergistic interaction when co-pyrolysing different biomasses. It also shows that it is possible to enhance the production of a valuable group of compounds with the right biomass composition and blend ratio. Fast pyrolysis can be used to convert biomass into bio-oil and chemicals. The pyrolysis oil  is composed of compounds such as anhydrosugars, furans, alcohols, ketones, aldehydes, acids, and phenols ,2. HowevThe co-pyrolysis of biomasses is described as the pyrolysis of blends, including two or more different biomasses. Previous studies have shown this to improve the overall quality of the pyrolysis oil, such as by increasing the calorific value and promoting the yield of volatile compounds ,13,14,15A review of most of the previous studies shows that the information is scarce about the primary products’ characteristics and interactions during the fast co-pyrolysis of different native biomass blends comprising an agricultural residue and a woody biomass residue. Moreover, most of the studies were carried out with the use of a fixed-bed and/or thermogravimetric analyser (TGA) ,20,22,23A summary of the proximate analysis in terms of the moisture content, volatile matter, fixed carbon, and ash content, as well as the biomass characterisations for all three biomasses used as blends in this study, are shown in The product distribution from the pyrolysis of the individual biomasses and their blends are found in To investigate the synergistic effects on the product yield of the co-pyrolysed biomass blends, the yield based on pure biomass according to the blend ratio was added  and compared to the measured value . An analysis of the volatile compounds formed for the two biomass blends during pyrolysis, as shown in The relative yields of phenol, 1-hydroxy-2-propanone, and furfural for PKS co-pyrolysed with either MAH or IRO in equal proportions showed no difference. However, the relative content of levoglucosan was strongly inhibited for the pyrolysis of PKS:MAH-1:1. The inhibition of levoglucosan for the PKS:MAH-1:1 blend and the promotion of AA, and HAA when PKS is co-pyrolysed with MAH or IRO, as well as the enhancement of 2,6-dimethoxyphenol and furfural for the co-pyrolysis of the woody biomass, are evidence of a synergistic interaction of the biomass components.2O and CaO decreased, and the decreased yield was most pronounced for the guaiacol and 4-allyl-2-6-dimethoxyphenol compounds. Chang et al. [Nannochloropsis sp. (NC) were co-pyrolysed at 600 °C with a blend ratio of 1:1. The inhibition of the phenols is important for improving the quality of the bio-oil, as they contribute to the instability of the bio-oil during storage and transport [The experimental (Exp.) and predicted values (Pred.) for the different classes of compounds are presented in g et al.  also obsransport . No diffransport . The majransport ,44. Richransport  observedransport . Their rransport , who invransport .Additionally, a comparison of the experimental and predicted values for the woody biomasses (MAH and IRO), co-pyrolysed in equal proportions, indicates that no difference was observed in the relative yield across the different classes of compounds. This could be due to the woody biomasses being composed of similar polysaccharide structures. The result is similar to that presented by Edmunds et al. , who obs4  to 7.53 × 104  for PKS:MAH-1:1, from 3.57 × 104  to 7.36 × 104  for PKS:IRO 1:1, and, finally, from 3.60 × 104  to 6.59 × 104  for MAH:IRO 1:1. This shows that co-pyrolysis could lead to an increase in the amount of volatile compounds formed.Considering the total yields of the volatile compounds formed (count/µg sample) from the pyrolysis of the biomass blends PKS:MAH-1:1, PKS:IRO-1:1, and MAH:IRO-1:1, it can be seen that the experimental values were about two times higher than the predicted values . The yieThe pyrolysis of the three biomass blends was achieved by first blending them in equal proportions (PKS:MAH:IRO-1:1:1 (0.33:0.33:0.33)), and then, secondly, mixing one part of the PKS with two parts each of the woody biomass (PKS:MAH:IRO-1:2:2 (0.20:0.40:0.40)), and thirdly, using two parts of the PKS and one part each of the woody biomass (PKS:MAH:IRO-2:1:1 (0.50:0.25:0.25)).Similar to the two biomass blends, the main chemical compounds formed for each class of the three-biomass blend were as follows: phenol (phenols), 1-hydroxy-2-propanone (ketones), HAA , AA (acids), levoglucosan (saccharides), and furfural (furans) see . A compaThe results from the three biomass blends showed an impact on the primary pyrolysis product. While the yield of the saccharides was promoted when the three biomasses were blended in equal promotion and co-pyrolysed, an increase in the woody biomass composition resulted in a decreased yield of levoglucosan. Additionally, the synergistic interaction was more pronounced when the blend ratio of PKS was lower in the blend, as seen in the PKS:MAH:IRO_1:1:1 and PKS:MAH:IRO-1:2:2  blends, than when they were blended and co-pyrolysed in equal proportions, as seen in the PKS:MAH:IRO-2:1:1  for the ternary blend. An analysis of the volatile compounds formed in response (count/µg sample), shown in Elaeis guineensis), mahogany (Khaya ivorensis) sawdust, and iroko (Chlorophora excelsa) sawdust. These biomasses were selected because of the environmental concerns associated with their disposal, the volume of the produced waste, and their suitability as feedstock for thermochemical conversion. The woody biomasses (MAH and iroko (IRO) sawdust) were collected from sawmills, while the PKS was obtained from a palm oil site, all in the southern parts of Nigeria. The three biomass samples were milled in a cutting mill (Retsch SM 100) with a screen size of 6 mm and sieved to a particle size of 125–250 µm by a Haver (EML 200 pure) test sieve shaker. The samples were then dried in an oven at 105 °C overnight to a constant weight and stored for further use. The biomasses were then blended into binary and tertiary blends. The binary blends were made from equal amounts of two samples, while the tertiary blends were made from all three samples, but in different proportions. The binary blends correspond to the following weight proportions: PKS:MAH_1:1 (0.50:0.50), PKS:IRO_1:1 (0.50:0.50), and MAH:IRO_1:1 (0.50:0.50), while the tertiary blends correspond to the following weight ratios: PKS:MAH:IRO_1:1:1 (0.33:0.33:0.33), PKS:MAH:IRO_1:2:2 (0.20:0.40:0.40), and PKS:MAH:IRO_2:1:1 (0.50:0.25:0.25).The materials used for this study were palm kernel shell  [0-42618 [The calorific values of the biomass samples were determined with a bomb calorimeter ) by placing 1.0 g of each biomass sample inside a steel vessel containing 500 µL of deionised water. Then, the vessel was filled with oxygen at 30 bar pressure before it was placed inside the equipment in which the sample was ignited.Pyrolysis and analysis of the gaseous product from the raw and blended biomass samples were obtained with a micro-pyrolyser ) connected to a Gas Chromatograph (GC), Mass Spectrometer (MS), and Flame Ionisation Detector (FID), also known as a Py-GC-MS/FID. The biomass samples were blended in different proportions, and close to 500 µg was weighed on an analytical balance (KERN ABT 320-4M) and placed on the platinum filament in the micro-pyrolyser. The platinum filament enables the investigation of primary reactions under isothermal conditions due to its high heating ramp of about 100,000 °C/s and good temperature control. Fast pyrolysis of the raw and blended biomasses was performed at a temperature of 600 °C with a residence time of 5 s. The operating condition was selected in order to obtain the maximum product yield of the released volatile compounds, as reported in previous studies ,29.−1) and a split ratio of 1:7, while the GC oven programme was set at a temperature ramp of 1 min at 60 °C, followed by a ramp of 8 °C/min to 265 °C, and was then held at 265 °C for 20 min. The MS was operated with an ionisation energy of 70 eV, while the MS transfer line and the FID base temperatures were set to 250 °C. Furthermore, the ion-source temperature was kept at 200 °C, and the MS scan was obtained from 25 to 250 m/z. More than 50 chromatography peaks were extracted by the GC-MS Xcalibur software (Version 2.1.0–2.3.0) and identified by the NIST library (NIST MS Search 2.0) and based on previous research [Both the micro-pyrolyser chamber temperature and the transfer tube were set to 150 °C. The low chamber temperature minimises secondary reactions, while it is high enough to keep many of the products in the gas phase. The released pyrolysis vapour was directly transferred to the GC  via a continuous flow of helium gas with a purity of 99.9%. The volatile compounds were identified via the MS  and quantified via an FID (Thermo Scientific). A Zebron™ ZB-5MS (30 m × 0.25 mm × 0.25 µm) capillary column was used with helium carrier gas  decreased the relative yield of phenolic compounds by 19% compared to the pyrolysis of each material individually;The saccharides, mainly levoglucosan, were inhibited to a large extent, while HAA was promoted by 43% for the PKS:IRO-1:1 pyrolysis blend;The relative yields of 2,6-dimethoxyphenol and furfural were also promoted by 21 and 37%, respectively, for the pyrolysis of the MAH:IRO-1:1 blend;No major difference in the relative yield was observed across the different classes of compounds when the woody biomasses were co-pyrolysed together, which is due to their similar chemical structures;The ternary blends showed that the pyrolysis of PKS, MAH, and IRO in equal proportions (PKS:MAH:IRO_1:1:1) led to an increase in the relative yield of the saccharides to a large extent, while an increase in the proportion of the woody biomass in the pyrolysis blend (PKS:MAH:IRO-1:2:2) led to a strong inhibition in the relative yield of the saccharides;Analysis of the individual volatile compounds formed shows that the pyrolysis of PKS:MAH:IRO-1:2:2 resulted in a decreased yield of phenols by 25%, while the relative yields of HAA and levoglucosan were promoted by 34 and 24%, respectively, for PKS:MAH:IRO_1:1:1.


When comparing clinically approved drugs and POMs, better results of POMs in relation to drugs were observed in many cases, since the dose required to have an inhibitory concentration of 50% is 2 to 200 times less, depending on the POMs, highlighting that these compounds could become in the future an alternative to existing drugs in cancer therapy.Polyoxometalates (POMs) are oxoanions of transition metal ions, such as V, Mo, W, Nb, and Pd, forming a variety of structures with a wide range of applications. Herein, we analyzed recent studies on the effects of polyoxometalates as anticancer agents, particularly their effects on the cell cycle. To this end, a literature search was carried out between March and June 2022, using the keywords “polyoxometalates” and “cell cycle”. The effects of POMs on selected cell lines can be diverse, such as their effects in the cell cycle, protein expression, mitochondrial effects, reactive oxygen species (ROS) production, cell death and cell viability. The present study focused on cell viability and cell cycle arrest. Cell viability was analyzed by dividing the POMs into sections according to the constituent compound, namely polyoxovanadates (POVs), polyoxomolybdates (POMos), polyoxopaladates (POPds) and polyoxotungstates (POTs). When comparing and sorting the IC The application of metals such as platin (Pt), lithium (Li), tungsten (W), gold (Au), and vanadium (V), among others, within chemical species, complexes, compounds and/or nanoparticles in biology has been a rapidly growing branch of science ,4,5,6,7.10) is perhaps the most widely studied POV in biology, affecting several biochemical and cellular processes ·6H2O (abbreviation V18) being a vanadium ·6H2O, also designated in the article by compound 1, in order to facilitate [2Na[AsIIIMo6O21(O2CCH2NH3)3]·6H2O (abbreviation POM@SiO2) [2Na2[γ-Mo8O26(O2CCH2NH3)2]·6H2O, designated as compound 2, which are heteropolymolybdates .5H2O [2)2(Mo8O26)]·8H2O .5H2O ; and fin6)]·8H2O .8[Pd13As8O34(OH)6]·42H2O (abbreviation Pd13), Na4[SrPd12O6(OH)3(PhAsO3)6(OAc)3]2NaOAc·32H2O (abbreviation SrPd12), Na6[Pd13(PhAsO3)8]·23H2O (Pd13L), Na12[SnIVO8Pd12(PO4)8]·43H2O (abbreviation SnPd12), Na12[PbIVO8Pd12(PO4)8]·38H2O (abbreviation PbPd12) 20H2O (abbreviation PW9Cu) which has a Dawson structure [6O4(H2O)3[Co(hmta)SbW8O31]3}15− [3GeW9O34]4−·26H2O PAC-320 that has been shown to be an inhibitor of histone deacetylase (HDACi) [2O)4][Na0.7Ni5.3(imi)2(Himi)(H2O)2(SbW9O33)2]} 10H2O, designated as compound 1 and H3[(CH3)4N]4[Na0.7Co5.3(imi)2(Himi)(H2O)2(SbW9O33)2] 12H2O, also known as compound 2 .8H2O, where it was shown to be quite effective with a minimum inhibitory concentration of approximately 135 μg/mL, which is the lowest value reported so far for any octamolybdate-based POM ·8H2O (2Na[AsIIIMo6O21(O2CCH2NH3)3]·6H2O (POM@SiO2) with the same structure as the previous one, but inserted in silica nanoparticles  at 72 h ·8H2O . This sa at 72 h , however13  3}]13− which presents a homochiral, sandwich-like structure 6{CoSb6O4(H2O)3[Co(hmta)SbW8O31]3}]13− being 1.78 µM [2O)6{CoSb6O4(H2O)3[Co(hmta)SbW8O31]3}]13− with a value of 12.75 µM 6H2O 6H2O , represeIn order to analyze in detail which type of POM affected the different phases of the cell cycle in the respectively studied lines, a table was prepared, with the division of the phases of the cell cycle that were blocked by corresponding POMs and respective cell lines .50 previously determined and are described in It should be noted that in the articles where several POMs were studied, cell cycle arrest was only tested for the compound that showed the greatest efficacy. Likewise, of the cell lines analyzed, only those that had presented the best efficacy values were focused on, to verify cell arrest. Therefore, in 2Na[AsIIIMo6O21(O2CCH2NH3 )3]·6H2O (POM@SiO2)), stopping in the S phase [As can be seen, the S phase is predominant under the action of the POMs with 14 of them, followed by the G2/M phase with nine polyoxometalates, and two in the G1 phase. It is also verified that VMOP-31 is the only one that has the ability to stop the cell cycle of SMMC-77221 cells in two distinct cell phases, namely S and G2/M . On the  S phase .Most of the proposed modes of action for antitumor POMs were recently reviewed . Among tIn this review paper, bibliographic research was carried out with the keywords “polyoxometalates” AND “cell cycle”. Thirteen articles were selected on the effect of POMs with anticancer activities, namely in gastric, colon, liver, lung, ovary, breast, prostate, leukemia, osteosarcoma, neuroblastoma and human hepatocellular carcinoma. The types of cancer in which the articles most focused on were lung and breast cancer, with six articles each, followed by liver cancer, covered in four articles. The effects of POMs on cancer cells can be diverse, such as their interaction in the cell cycle, protein expression, mitochondrial effects, ROS production and cell viability. In the present study, we focused mainly on cell viability and cell cycle arrest, since all selected articles analyzed these two effects.50 values were compared and sorted in ascending order, it was found that the POV (VMOP-31) at 24, 48 and 72 h presented, respectively, the lowest values of 1.52, 0.63, and 0.53 µM, in the MCF-7 (human breast cancer cell line). In clinically approved drugs, the lowest IC50 value was found to be 5.78 µM in the BCG-823 lineage at 48h using cisplatin. When comparing drugs and POMs, better results of POMs were observed. In many cases, the POMs dose required to have an inhibitory concentration of 50% is 2- to 200-times lower than the dose that would be necessary with a clinically approved drug. Therefore, POMs are future potential candidates for cancer therapeutic applications.Cell viability was analyzed by dividing the POMs into sections according to the constituent compound, namely POVs, POMos, POPds and POTs. When the ICIn addition to the cell viability, the effect of POMs on cell cycle arrest is highlighted. For the majority of the POMs, cell cycle arrest occurs mostly in the S phase (56%), where DNA synthesis occurs, whereas 36% blocks the G2/M phase. Fewer POMs interfere with the G1 phase (8%), that is, at the beginning of the cell cycle. Although the mechanism of action, directly and/or indirectly, responsible for the POMs cell cycle arrest is still to be deduced and clarified, the scientific evidence described above strengthens the potential use of such metallodrugs in anticancer therapy in the near future.


As these three GVHD prevention/treatment modalities continue their path toward clinical application, it is imperative the field understand both the biological advantages and disadvantages of each approach.The development of graft versus host disease (GVHD) represents a long-standing complication of allogeneic hematopoietic cell transplantation . Different approaches have been used to control the development of GVHD with most relying on variations of chemotherapy drugs to eliminate allo-reactive T cells. While these approaches have proven effective, it is generally accepted that safer, and less toxic GVHD prophylaxis drugs are required to reduce the health burden placed on allo-HCT recipients. In this review, we will summarize the emerging concepts revolving around three biologic-based therapies for GVHD using T regulatory cells (Tregs), myeloid-derived-suppressor-cells (MDSCs) and mesenchymal stromal cell (MSC) exosomes. This review will highlight how each specific modality is unique in its mechanism of action, but also share a common theme in their ability to preferentially activate and expand Treg populations  Graft versus-host disease (GVHD) remains a significant problem following an allogeneic hematopoietic cell transplantation  with GVHD patients experiencing a lower quality of life and a higher risk of death. Our understanding of the cellular and molecular mechanisms of GVHD has provided researchers additional clarity on the etiology of its pathogenesis. This has allowed for a growing number of non-biologic and biologic-based GVHD prophylaxis/treatment therapeutics to be investigated as safer modalities for allo-HCT patients. Specifically, the investigation into the use of biologics for GVHD therapies is still in its infancy but there are several exciting possibilities in the pipeline. In this mini-review, we will highlight three emerging biologic-based therapies that are at different stages of conceptualization and development which include T regulatory cells (Tregs), myeloid-derived-suppressor cells (MDSCs) and mesenchymal stromal cell (MSC) exosomes. We will also emphasize the similarities and differences of each modality in terms of biologic activity and possible clinical use.via CD39/CD73 activity (T regulatory cells (Tregs) represent a T cell polarization state with potent immunosuppressive activity –4. Classactivity Figure 1While the activity of Tregs is predicated on the recognition of self-peptides presented by antigen-presenting-cells (APCs) to prevent the development of an autoimmune disease, Tregs can also develop against non-self-peptides in the periphery . Moreovehigh characteristic of all Treg populations  develop during T cell development and are characterized in mice by the expression of neuropilin-1, the transcription factor Helios and in the expression of CD3, CD4, FOXP3 and CD25ulations –13. Mostulations . This Tr17 polarization. Studies have shown that iTregs can be polarized into TH17 cells and vice versa, highlighting the plasticity of this population and how the balance of iTreg/TH17 polarization can influence immune responses  exit the thymus as conventional T cells but are polarized to iTregs in environments with high levels of TGFβ, retinoic acid, IDO, and other mediators , 14, 15.esponses –20.In both pre-clinical mouse models and clinical studies, it has been established that elevated Tregs frequencies in the graft is correlated with lower rates of GVHD –26. AnotThe TCR repertoire of tTregs and iTregs do not overlap, highlighting the different antigens they recognize (germline self-antigens vs microbiota-derived antigens) , 28, 29.in vitro  may be equally important –35. mTORrization , 37. Add17 cells –37.The polarization of naïve CD4 T cells to iTregs by sirolimus makes it an effective therapeutic candidate that is currently used in the clinic. One multicenter study (BMT-CTN-1501) revealed that sirolimus was equivalent to prednisone for initial GVHD treatment and resulted in a shorter time to immune suppression discontinuation . Anotherin vivo should continue to be prioritized.With so many current GVHD drugs connected with an expansion of the iTreg population, it is logical for a growing number of clinical trials to investigate the direct treatment of Tregs. Unfortunately, iTreg adoptive therapies are hindered by high production costs, the inability to generate/store multiple doses and the need for a patient specific product. Thus, the investigation into new therapies preferentially expand/polarize iTregs Myeloid-derived-suppressor-cells (MDSCs) represent a heterogeneous population of cell types with the general function of dampening immune responses –50. MDSC+, CD33high) but also express several M2 markers  and are CD16-/HLA-DR- while both classical and inflammatory monocytes are all HLA-DR+  but also express the same immunosuppressive markers listed above  but maintain expression of CD33 and the immunosuppressive markers ; however, they have also been shown to prevent GVHD –58. One MDSCs populations have been shown to use a variety of molecular pathways to control T cell responses including indoleamine-pyrrole 2,3-dioxygenase (IDO), cyclooxygenases, arginase, TGF-β, IL-10 and PD-L1 Figure 12 by the receptors EP2 or EP4 have been shown to prevent the polarization of T cells into TH1 cells and directly promote the expression of FOXP3 similar to TGF-β , mRNA and possibly even DNA , 82, 83.As an ever-growing number of studies support the role of exosomes in generating MDSC populations, the mechanism(s) utilized by exosomes to generate these MDSC populations remains largely unexplored Figure 1Compared to standard cellular therapies, exosomes have several fundamental advantages including i) long-term storage, ii) low-immunogenicity, iii) no requirement for HLA-matching and iv) the ability to be mass-produced , 81, 89.Despite the lack of approved exosome-based therapies, there are a growing number of pre-clinical studies using exosomes to treat a variety of diseases. Most of these studies have utilized MSC-based exosomes, which builds upon the growing field of MSC-based cellular therapies, as they contain similar immunosuppressive properties. A series of studies by one team of investigators have sequentially shown that the treatment with human MSCs, or exosomes “educated” myeloid cells were able to prevent lethal acute radiation syndrome (ARS) , 82, 90.The use of MSCs for GVHD prevention and treatment has been explored in a large number of trials with most reporting that MSCs are safe and well tolerated. Unfortunately, the response rates among trials varied considerably and FDA approval is still elusive . RecentlThe field of GVHD research is at an exciting inflection point where the investigation into biologics as novel prophylaxis/treatment modalities may soon become standard. While the three modalities discussed in this review are all poised to make significant advances toward clinical use, these biologic modalities are not without their limitations. Similar to the experiences of CAR manufacturing, the use of Tregs or MDSCs as a cellular therapy will always be challenged with their production times, the manufacturing process and capacity to be used in the allogeneic setting. For these reasons, MSC-exosome treatment represents an exciting and promising modality. Exosome treatments have the potential to be used in the allogeneic setting, mass-produced for repeated injections, and stored for immediate use. Their eventual use in the clinic will ultimately be decided by their ease of use and long-term efficacy compared to direct cellular therapies which boast strong short term activity but are limited to a single infusion and long-term activity.Promoting the expansion and activation of Tregs after allo-HCT remains the most validated method of suppressing GVHD though the optimal approach to achieve this remains under investigation. Exploring the use of MDSCs and MSC-exosomes as novel approaches to activate Tregs should be a focus of investigation in the next decade. The development of these and other biologic-based prophylaxis/treatment modalities will offer allo-HCT recipients an improved quality of life by reducing off-target toxicity and improving overall efficacy by directing their new immune system to control the development of GVHD.NH drafted and performed the literature search. NH, JK and PH revised and edited the manuscript. All authors approved the final version of this manuscript. All authors contributed to the article and approved the submitted version.