Chemical fertilizers have greatly contributed to the development of agriculture, but alternative fertilizers are needed for the sustainable development of agriculture. 2,3-butanediol  is a promising biological plant growth promoter.R,R-2,3-butanediol  by Paenibacillus polymyxa fermentation. First, gamma-ray mutagenesis was performed to obtain P. polymyxa MDBDO, a strain that grew faster than the parent strain and had high production of R,R-2,3-BDO. The activities of R,R-2,3-butanediol dehydrogenase and diacetyl reductase of the mutant strain were increased by 33% and decreased by 60%, respectively. In addition, it was confirmed that the carbon source depletion of the fermentation broth affects the purity of R,R-2,3-BDO through batch fermentation. Fed-batch fermentation using controlled carbon feeding led to production of 77.3 g/L of R,R-2,3-BDO with high optical purity (> 99% of C4 products) at 48 h. Additionally, fed-batch culture using corn steep liquor as an alternative nitrogen source led to production of 70.3 g/L of R,R-2,3-BDO at 60 h. The fed-batch fermentation broth of P. polymyxa MDBDO, which contained highly pure R,R-2,3-BDO, significantly stimulated the growth of soybean and strawberry seedlings.In this study, we attempted to develop an effective strategy for the biological production of highly pure P. polymyxa MDBDO has potential for use in biological plant growth promoting agent applications. In addition, our fermentation strategy demonstrated that high-purity R,R-2,3-BDO can be produced at high concentrations using P. polymyxa.This study suggests that The online version contains supplementary material available at 10.1186/s12934-023-02133-y. Most of the studies on the plant growth promoting effect of 2,3-BDO have not been done in terms of isomers. Nonetheless, among the three isomers of 2,3-BDO, R,R-2,3-BDO is well known for its plant growth promoting effect [R,R-2,3-BDO and meso-2,3-BDO have been reported to have plant virus resistance effects [An increase in global food demand due to a growing population has led to the indiscriminate use of chemical fertilizers and other agrochemicals to increase agricultural yields . Chemica3CHOH)2 . Several3CHOH)2 , 9, 10. g effect , 6 and ag effect , 12. Dep effects . 2,3-BDO effects , 15. 2,3 effects . Commerc effects .Klebsiella sp. [Enterobacter sp. [Serratia sp. [K. pneumoniae because it can produce the highest 2,3-BDO concentration, about 150 g/L [Klebsiella and its ability to cause opportunistic infections are generally considered obstacles to its use for the large-scale production of 2,3-BD [Bacillus subtilis [B. licheniformis [B. amyloliquefaciens, and Paenibacillus can also produce 2,3-BDO with high efficiency. P. polymyxa in particular has potential for the industrial production of R,R-2,3-BD [P. polymyxa DSM 365 was 111 g/L when using a high concentration organic nitrogen source [R,R-2,3-BDO was produced, the optical purity was 98% (w/w).An alternative approach is the production of 2,3-BDO using microorganisms. Previous studies reported production of microbial 2,3-BDO in various microbial species, including ella sp. , 19, Entcter sp. , 21, andatia sp. , all of  150 g/L . Howeverf 2,3-BD . Certainsubtilis , B. lichniformis , B. amylR-2,3-BD , 28. Pren source . AlthougR,R-2,3-BDO is essential for certain applications, such as high value-added pharmaceuticals and liquid crystals [R,R-2,3-BDO. A recent study reported that a genetically modified strain of P. polymyxa produced R,R-2,3-BDO that had high optical purity [2,3-BDO has a variety of commercial uses. In particular, it is used as an agricultural control agent because it has antimicrobial properties against the bacterial, fungal, and viral pathogens , 13, 30 crystals , 32. Durcrystals . Therefol purity . Howeverl purity .R,R-2,3-BDO by P. polymyxa examined the effect of adding different chemicals  or changing fermentation conditions  [meso-2,3-BDO. Thus, there has been a focus on factors that impact the oxygen transfer coefficient, such as agitation speed and aeration rate. For example, Hassler et al. controlled the agitation speed and aeration rate to prevent the formation of meso-2,3-BDO and increase the optical purity of R,R-2,3-BDO [Previous studies that examined the chiral synthesis of  supply) , 36–38. -2,3-BDO . HoweverPaenibacillus polymyxa converts R-acetoin into R,R-2,3-BDO using acetoin reductase/2,3-butanediol dehydrogenase  with NADH as a coenzyme. Importantly, the AR/2,3-BDH from P. polymyxa is reversible. Thus, Gao et al. reported the Km value of this enzyme from P. polymyxa ZJ-9 was about 38 times higher for R-acetoin than for R,R-2,3-BDO [R,R-2,3-BDO.-2,3-BDO . However-2,3-BDO , 41. It R,R-2,3-BDO using P. polymyxa uses a high-concentration organic nitrogen source [High-concentration production of high-purity n source . Nitrogen source . An apprn source . Howevern source , 45. Then source . TherefoR,R-2,3-BDO by P. polymyxa fermentation. We first performed gamma-ray mutagenesis to obtain a mutant strain of P. polymyxa that had better growth and R,R-2,3-BDO production than the parent strain. We then confirmed the effect of the concentration of the carbon source on the production of acetoin and R,R-2,3-BDO through batch fermentation studies. We also performed fed-batch fermentation studies to determine the most suitable concentration of the carbon source for the production of R,R-2,3-BDO that had high optical purity. We then examined the effect of using CSL as a low-cost nitrogen source on the production of a high concentration of R,R-2,3-BDO. Finally, we examined the effect of the fermentation broth from P. polymyxa on the growth of soybean and strawberry seedlings to confirm its potential as the biological plant growth promoting agent.Our general purpose was to develop an effective strategy for the biological production of highly pure P. polymyxa mutant that had increased cell growth and production of R,R-2,3-BDO relative to the parent strain. The mortality rate of P. polymyxa DSM 365 increased with radiation dose, and was 100% when the dose was 3 kGy  activity. The increased AR/2,3-BDH activity and reduced DAR activity in the mutant were likely responsible for its increased production of R,R-2,3-BDO. Zhang et al. studied the same species and reported that R,R-2,3-BDO with an optical purity of 99.99% was obtained by removal of the DAR gene using genetic engineering [meso-2,3-BDO, because it produces S-2-acetoin  from the diacetyl that is produced by spontaneous decarboxylation. Although neither strain produced detectable meso-2,3-BDO, the decreased DAR activity in the mutant strain could contribute to the production of highly pure R,R-2,3-BDO. These changes in the activity of key enzymes that function in the production of 2,3-BDO demonstrate the potential value of P. polymyxa MDBDO for the industrial production of highly pure R,R-2,3-BDO.To determine the cause of the increased ineering . DAR is R,R-2,3-BDO production in a laboratory scale fermentor [P. polymyxa MDBDO show that both strains consumed all of the supplied carbon source by 12 h, and the cell growth at this time was OD600 nm 28.2 in the parent strain and OD600 nm 30.1 in the mutant  at 12 h and produced no detectable meso-2,3-BDO. The mutant strain produced the most R,R-2,3-BDO (23.5 g/L) at 12 h, and also produced no detectable meso-2,3-BDO. In addition, the maximum productivity of R,R-2,3-BDO was 1.86 g/L/h in the parent strain and 2.39 g/L/h in the mutant strain at 9 h. Thus, R,R-2,3-BDO productivity was about 1.3-times higher in P. polymyxa MDBDO.To examine the potential for microbial production of 2,3-BDO in an industrial setting, it is essential to perform batch fermentation experiments using fermentors. We therefore performed batch fermentation previous reported conditions  that led to the highest ermentor . The batR,R-2,3-BDO gradually decreased in both strains after 12 h, at which time the amount of acetoin gradually increased. Also at 12 h, cell growth entered a stationary phase or death phase due to the depletion of the carbon source. We measured the enzymatic activity of AR/2,3-BDH of P. polymyxa MDBDO on production of acetoin and R,R-2,3-BDO during batch fermentation . Therefore, the DO remains low when the cells are using a carbon source for growth. However, when cell growth slows or stops because of depletion of the carbon source, the DO increases. Aerobic conditions promote the production of meso-2,3-BDO [meso-2,3-BDO was not produced due to low level of oxygen during fermentation, and the increase in DO after carbon source depletion also did not affect meso-2,3-BDO production. It is important to control the DO of the fermentation broth by adjusting agitation speed and aeration rate, but it is also important to supply an appropriate concentration of a carbon source so the cells consume available oxygen. Therefore, an appropriate carbon source is needed to maintain the reducing activity of AR/2,3-BDH and low oxygen concentration in the fermentation broth.The dissolved oxygen (DO) in the fermentation broth also decreases as cell growth increases, but it increases rapidly after depletion of the carbon source. During the fermentation of -2,3-BDO . For thi-2,3-BDO , 36–38. -2,3-BDO , showed R,R-2,3-BDO using two carbon supply strategies: sufficient feeding  . This meing Fig. A and liming Fig. B. Duringssential , 50. Howssential .Fig. 5FeR,R-2,3-BDO production was 77.3 g/L at 48 h, and the amount of acetoin at that time was 0.6 g/L  was 99.2% (w/w). The DO in the fermentation broth declined sharply at the beginning of fermentation, and remained low during the exponential growth period, but it increased during stationary phase and death phase. Thus, as the cells gradually stopped growing, carbon consumption decreased and DO increased. An increased level of DO could have led to the production of meso-2,3-BDO, but P. polymyxa MDBDO did not produce this molecule. Through carbon supply control, P. polymyxa MDBDO produced R,R-2,3-BDO with the highest purity among non-GMO Paenibacillus sp. reported so far .As a facultative anaerobe, 6 h Fig. A, and th at 48 h , and theR,R-2,3-BDO production using the limited feeding strategy was 74.2 g/L at 36 h, and the amount of acetoin at that time was 1.2 g/L  total nitrogen and our CSL contained 4.0% (w/v) total nitrogen [A yeast extract can be used as a nitrogen source to produce nitrogen . Therefo600 nm 27.5 at 24 h, maximal R,R-2,3-BDO production of 72.9 g/L at 54 h an acetoin level of 2.2 g/L 54 h, and no detectable meso-2,3-BDO  (Table We next determined the effect of different doses of the r) Table  A–C. Becr) Table D–G. Simir) Table , Fig. 7HP. polymyxa fermentation broth increased plant growth  in soybean, canola, kiwifruit, pepper, watermelon, rice, maize, potato, cucumber, beans, tomato, barley, Arabidopsis, and other ornamental plants [Arabidopsis thaliana [Bacillus subtilis produced organic volatile compounds that induced systemic resistance in Arabidopsis, and a B. subtilis mutant that had reduced 2,3-BDO production had a weaker effect [R,R-2,3-BDO in promoting plant growth. In agreement, the present study demonstrated that the P. polymyxa MDBDO fermentation broth, which contained highly pure R,R-2,3-BDO, promoted the growth of seedling shoots and roots, and increased plant biomass. Therefore, P. polymyxa MDBDO has potential application as a source for plant growth promoting agent.Previous research also reported that the l plants , 51, 52.l plants , 53. 2,3P. polymyxa MDBDO, which we obtained by gamma-ray mutagenesis, had increased cell growth and AR/2,3-BDH activity compared to the parent strain. Growth of these cells in fed-batch fermentation with a controlled carbon concentration led to production of highly pure R,R-2,3-BDO, with very little acetoin production. To our best knowledge, we achieved a high concentration of R,R-2,3-BDO production with highest optical purity (> 99%) than previously reported for non-GMO P. polymyxa strains. In addition, the possibility of CSL as an alternative nitrogen source for the production of high-concentration R,R-2,3-BDO was confirmed. The P. polymyxa MDBDO fermentation broth also significantly promoted the growth of soybean and strawberry seedlings. This study suggests that P. polymyxa MDBDO has potential for use in various industrial applications.We demonstrated that R,R-2,3-BDO, meso-2,3-BDO, and acetoin dimer were purchased from Sigma-Aldrich . Acetoin monomers were prepared by incubation of dimers at 95 °C for 1 h. CSL was purchased from Samyang Genex . YPD  medium was used for pre-activating and strain preservation. A previously described growth medium was modified and used as the 2,3-BDO production medium [4, 3 g/L (NH4)2SO4, 1.2 g/L KH2PO4, 0.2 g/L Na2HPO4, and 3 mL of trace element solution. The trace element solution contained 0.4 g/L FeSO4, 5.0 g/L MnCl2·4H2O, 0.1 g/L ZnSO4·7H2O, 0.8 g/L H3BO3, 0.04 g/L CuSO4·5H2O, 0.04 g/L NaMoO4·2H2O, 0.08 g/L Co(NO3)2·6H2O, 1.0 g/L CaCl2·2H2O, 0.01 g/L biotin, and few drops of HCl. All other chemicals were of analytical grade.Analytical standards of n medium . This meP. polymyxa DSM365 stock vial was inoculated in 10 mL of the 2,3-BDO production medium and activated on a shaker at 200 rpm at 37 °C for 36 h. The activated cells were then inoculated (10% v/v) in 40 mL of the 2,3-BDO production medium, then incubated on a shaker at 200 rpm at 37 °C for 12 h. After centrifugation at 4000 rpm at 4 °C for 10 min, the sample was washed with sterile phosphate-buffered saline . For gamma-irradiation, the cells were resuspended in 10 mL of sterile PBS buffer. Gamma-irradiation was performed at the Korea Atomic Energy Research Institute , with gamma-ray doses of 1.0, 2.0, and 3.0 kGy. After irradiation, the cells were diluted, 100 µL was spread on a 2,3-BDO production plate, and the plate was incubated at 37 °C for 2 days. Colonies that were larger than the wild type strain were selected, and were subcultured three times on 2,3-BDO production plates to assure stability and consistency.The 4OH and 2 M HCl.Evaluation of the mutant strains and preparation of seed cells for batch and fed-batch fermentation were performed in 1 L flasks containing 300 mL of the 2,3-BDO production medium. Flasks were incubated on a shaker at 200 rpm at 37 °C for 12 h, and cultures were subsequently inoculated into 5 L fermentors at a concentration of 10% (v/v). Batch fermentation was conducted in a 5 L stirred-vessel system  that contained 3 L of the 2,3-BDO production medium. Unless otherwise stated, fermentation experiments were performed on a shaker at 500 rpm and 0.2 vvm at 37 °C, and the pH was maintained at 6.0 ± 0.1 using 28% (w/v) NH600 nm using a UV–Vis spectrophotometer . All results are averages from two independent experiments.Fed-batch fermentation was conducted in a 5 L stirred-vessel system that contained 3 L of the 2,3-BDO production medium or a modified 2,3-BDO production medium (75 g/L of CSL instead of 30 g/L yeast extract). The fed-batch fermentation used a solution containing 900 g/L glucose as the feed, and feeding was intermittent to maintain the glucose concentration in the fermentation broth. Cells were sampled at different times for enzyme activity assays, and the metabolites in fermentation supernatants were analyzed. Cell growth was monitored by measurement of OD2SO4, the flow rate was 0.6 mL/min, the column temperature was 65 °C, and the cell temperature was 45 °C [Residual substrates and fermentation metabolites were analyzed using a high-performance liquid chromatography system (Agilent 1260) that was equipped with a refractive index detector and an Aminex HPX-87H column . The mobile phase was 2.5 mM Has 45 °C . AnalysiP. polymyxa DSM365 and P. polymyxa MDBDO cells were harvested by centrifugation and washed two times with 33 mM sodium phosphate (pH 6.0). Cells were disrupted using an ultrasonic system for 1 min, using cycles of crushing for 2 s and resting 6 s  in the same buffer. A crude extract was obtained by centrifugation 13,000 rpm for 10 min at 4 °C. Protein concentration of crude extract was determined using the Bradford assay, with bovine serum albumin as the standard. The reducing activity of AR was determined by measuring OD340 nm at 37 °C (corresponding to a decrease of NADH). The activity of the crude extract was measured with 50 mM acetoin and 4 mM NADH in 33 mM sodium phosphate (pH 6.0). The oxidation activity of R,R-2,3-BDH was determined by measuring OD340 nm at 37 °C (corresponding to an increase of NADH). The activity of the crude extract was measured using 50 mM R,R-2,3-BDO and 4 mM NAD+ in 33 mM sodium phosphate (pH 6.0).340 nm at 37 °C (corresponding to a decrease of NADPH). The activity of the crude extract was measured using 50 mM butanedione and 4 mM NADPH in 33 mM sodium phosphate (pH 6.0).The activity of DAR was determined by measuring OD+, NADH, or NADP+) per min, and enzymatic activity was expressed U/µg protein. All results are averages from two independent experiments.One unit of AR/2,3-BDH activity and 1 unit of DAR activity corresponded to the formation of 1 mol of each product (NADP. polymyxa MDBDO fed-batch fermentation broth (48 h), which was established by using sufficient carbon source, on the growth of soybean (cv. Daewon) and strawberry (cv. Sulhyang) seedlings was determined. Two different doses  of the fermentation broth were added to the potted plants at a rate of 1 L/m2 , and control plants received distilled water. For soybean, two doses were applied to the potting soil at one week intervals at 7 days after seed sowing. After 2 weeks of seedling growth, plant height (shoot) and root length were measured using a vernier caliper. There were five seeds per pot, and three pots per treatment. For strawberry, seedlings were collected from a nursery bed and transferred to potting soil. The two doses of the fermentation broth were applied at one week intervals. There was one seedling per pot and three pots per treatment. At two weeks after the final treatment, the number of leaves, leaf surface area, and shoot and root biomass (fresh and dry weight) were measured. Soybean and strawberry seedlings were cultivated at a temperature of 25 °C and under a 16/8 light–dark cycle, and were watered whenever necessary.The effect of the Additional file 1: Table S1. Comparison of production and optical purity of R,R-2,3-butanediol by various Paenibacillus strains.


Cannabis sativa that is increasingly used to manage pain. The potential for CBD to ameliorate dimensional behavior symptoms occurring in multiple psychiatric disorders was suggested, including social interaction impairments. To test this hypothesis, adult male BTBRT+Itpr3tf/J (BTBR) mice, a model of idiopathic autism exhibiting social preference deficits and restrictive repetitive behaviors, were acutely treated with vehicle or 0.1, 1, or 10 mg/kg CBD. Social interaction preference was assessed 50 min after treatment, followed by social novelty preference at 60 min, marble burying at 75 min and social dominance at 120 min. CBD (10 mg/kg) enhanced BTBR social interaction but not social novelty preference, marble burying or dominance, with serum levels = 29 ± 11 ng/mg at 3 h post-injection. Next, acute 10 mg/kg CBD was compared to vehicle treatment in male serotonin transporter (SERT) knock-out mice, since SERT deficiency is an autism risk factor, and in their wildtype background strain controls C57BL/6J mice. CBD treatment generally enhanced social interaction preference and attenuated social novelty preference, yet neither marble burying nor dominance was affected. These findings show acute treatment with as little as 10 mg/kg purified CBD can enhance social interaction preference in male mice that are otherwise socially deficient.Cannabidiol (CBD) is a non-intoxicating phytochemical from   Cannabis sativa (hemp or marijuana) that occurs naturally in several derivative forms (Cannabidiol (CBD) is a bioactive, non-psychotomimetic isolate from ve forms . Most bive forms . Howeverve forms , 4. Yet ve forms . The potve forms , but theve forms , 8.1 and CB2) receptors are complicated: Based on agonist interactions, CBD was initially considered a potent antagonist or inverse agonist of CB1 and CB2 receptors  channel and its related TRPV isoforms to modulate sensations, behavior, and immune function  strain , 18 and Subjects were 44 adult male (4–6 month old) BTBR  with (mean ± SEM) weight = 27 ± 1 g, and 20 C57BL/6J (JAX strain stock #000664) with weight = 29 ± 1 g. They were progeny of mice purchased from Jackson Laboratories  bred in-house for >5 generations. Sixteen male serotonin transporter (SERT) knock-out mice  and water (reverse-osmosis and acidified to pH 2.5–3.0 using HCl) and were housed 4 to 5 per cage with same-sex and strain cage-mates born within a week of each other. All mice were euthanized by cervical dislocation and decapitation by an experienced researcher 3 h after behavior tests. Procedures involving live mice complied with ARRIVE guidelines  on at 0700 h, and temperature of 22°C–25°C. Mice were kept in sterilized JAG75 cages (29 × 18 × 12 cm) with wood-chip bedding , but no enrichment items in ventilated housing racks . Stimulus mice were kept on a separate, non-facing housing rack from the subjects. Mice had idelines , the updidelines  and wereAn hour prior to treatment, mice were moved in home cages from their housing room to the behavior testing room, dimly lit (16 lux) with red compact fluorescent light bulbs. Mice were weighed on a digital scale , and within each cage mice were injected (i.p. volume 0.1 mL/10 g) with either freshly made control vehicle , 0.1, 1, or 10 mg/kg CBD  30 min prior to three chamber test arena conditioning and 50 min before the first phase of social preference tests. The treatment and tissue collection time course of the present study was chosen since the half-life of systemic CBD administration was 3.9 h in mice . Permanevia 3 daily confinements, each lasting 30 min, under wire mesh cups before their use in tests. To acclimate, under low red light (16 lux) each subject mouse was placed alone in the middle of a 3-chamber test arena for 10 min. Then interior doors were opened so subjects could explore the full arena for 10 min, and we observed them enter both end chambers at least once as criterion for use in tests. Two BTBR mice treated with 0.1 and 1 mg/kg CBD were dropped from the experiment for failure to enter all three chambers, so sample size in these groups became 9 instead of 10 mice. Social interaction tests began after wire cup cages, one empty (novel object) and the other holding a same-aged stranger male mouse, were randomly placed in opposite end chambers. Mice in arenas were video recorded for 10 min. To measure social novelty preference, subjects were confined in the center chamber as a new stranger mouse was introduced under the empty cage and the “old” stranger in the cage was moved slightly. Doors were opened for subjects and mice were video recorded for another 10 min. Tests took place between 1,300 and 1,500 h, and 6 mice were tested together in different arenas at once.Three chamber tests for social interaction and social novelty preference were performed as previously described , 26. AgeData from mouse videos was collected by treatment blind observers. The cumulative time each mouse spent in chambers with strangers (old and new) or objects (cages) and sniffing them was measured and compared within and between groups. The number of times subject mice entered different chambers, time spent in middle chambers, and the number of fecal boli in the middle chamber were also measured.After sociability tests, subjects were placed individually in 40 cm × 20 cm sterilized cages filled with wood chip bedding, 8 cm deep, on which 15 flat blue glass marbles were put in a 3 × 5 grid. Room lighting was 16 lux. The number of marbles with surface area >2/3 buried with bedding was tallied after 30 min by treatment blind observers.Tube tests for social dominance were performed as previously described  by reseaCBD and its metabolites 7-OH-CBD, and 7-COOH-CBD, along with the internal standard deuterated CBD-D3 were obtained from Sigma-Aldrich Chemical . The CBD metabolite 6-α-OH-CBD was obtained from Cayman Chemical . HPLC grade methanol, isopropanol, acetonitrile and hexane were purchased from Fisher . All other reagents were purchased from Sigma Chemical Company .Milli-Q water was used for preparation of all solutions. CBD, metabolites, and CBD internal standard super stock solutions were prepared in methanol at a concentration of 1 mg/mL and stored in aliquots at −80°C. Working stock solutions were prepared each day from the super stock solutions at concentrations of 0.1, 1, and 10 μg/mL were used to spike the calibrators.CBD, and metabolites, were quantified in mouse serum samples and whole brain homogenates using HPLC with tandem mass spectrographic detection. Briefly, 100 μL of plasma for spiked calibrators and unknown serum samples were mixed with 500 μL of isopropanol by vortexing vigorously with 20 μL of 10 μg/mL of deuterated CBD . Hexane (1 mL) was then added to each sample and vortexed for 30 s. The samples were then centrifuged for 10 min at 32K g, and the supernatants transferred to 1.5 mL microcentrifuge tubes and dried under a nitrogen stream. The residue was resuspended in 50 μL of mobile phase A  and 10 μL were injected into the LC/MS/MS. The ratio of the peak area of CBD and metabolites to CBD-D3 internal standard was compared against a linear regression of calibrator response ratios at concentrations of 0, 5, 10, 50, 100, 500, and 1,000 ng/mL of each in control plasma to quantify CBD and metabolites. The concentration of CBD and metabolites was expressed as ng of each compound/mL serum. Samples were identified as positive if any of the precursor or metabolites were detected above the lower limit of quantitation, determined to be 5 ng/mL.The HPLC system consisted of a Shimadzu CBM-20A Controller, LC-20AD pumps, SIL-20AC autosampler, and an AB Sciex API 4000 tandem mass spectrometer with turbo ion spray using positive mode. The analytical column was an ACE 3 μm C18 75 × 3.0 mm  and was maintained at room temperature (27°C) during the chromatographic runs. The flow rate of the mobile phase was 0.3 mL/min. CBD and metabolites were eluted with a step gradient. The column was equilibrated with 100% mobile phase A. At 6.1 min after injection, the system was switched to 100% mobile phase B (10 mM ammonium formate and 0.1% formic acid dissolved in 90% HPLC grade methanol). At 8.1 min, the system was switched back to 100% mobile phase A. LC-ESIMS/MS analysis using selected reaction monitoring was used to detect and quantify the major fragments of CBD and its metabolites. Analyst 1.1 software (Applied Biosystems) was used to identify the following transitions to quantify CBD and metabolites: CBD 315.315→193.3 Da (CBD), 331→201 (7-OH-CBD), 345→299.3 (7-COOH-CBD), 331.2→271.1 , 318.1→196.3 (CBD-D3).− ± SEM) in the text, unless otherwise noted.Social interaction and social novelty test data from BTBR mice treated with different doses of CBD were compared by two-way analysis of variance (ANOVA) for time in chamber and sniffing choice tests, with Sidak’s post-hoc test to adjust for multiple comparisons of any significant outcomes within and between groups. Preference test time differences, chamber entries, and middle dwelling between groups, mean number of feces, buried marbles, and tube test times were compared by one-way-ANOVA and Sidak’s post-hoc tests. For wins in dominance tube tests, since possible outcomes of 0 = loss, 0.5 = draw or 1 = 1 are non-parametric, a Kruskal-Wallis ANOVA or Mann-Whitney U test was performed. For C57BL/6J and SERT knock-out mice social interaction and novelty test data were compared using a mixed effects model (REML) ANOVA, and all other parameters were compared by unpaired two-tailed t-tests. Prism  was used for all statistics and graphs. Data are shown as mean ±95% confidence interval (95% CI) in graphs and reported as mean ± standard error of the mean  = 3.6, p = 0.003), as shown in p = 0.0001, Sidak’s t (68) = 3.6, p = 0.002), and more time in stranger chambers (t(68) = 3.2, p = 0.006) than controls, as shown in p < 0.0001).The social interaction preference, or difference between stranger and novel object chamber time, for BTBR males was greater than vehicle controls in the 10 mg/kg CBD group  = 4.6, p = 0.037, Sidak’s t(34) = 2.41, p = 0.06). The two-way interaction was not significant  = 2.6, p = 0.06). However, the chamber side effect was significant, and within group comparisons revealed that time spent sniffing stranger mice was longer than the time sniffing novel objects  = 12.8, p = 0.001, Sidak’s t(68) = 4.15, p = 0.0004) only for the 10 mg/kg CBD treated group  = 3.15, ed group .p = 0.89, p = 0.02), but no interaction or CBD dose effect for social novelty preference, based on time in chambers (p = 0.21). The two-way ANOVA also indicated no interactions, or differences among CBD doses or chamber side times for sniffing preferences of any treatment groups  = 0.21, chambers . For soct groups .p = 0.30, pooled X− ± SEM = 86 ± 9 s) or social novelty preference tests  = 1.1, p = 0.34, pooled X− ± SEM = 64 ± 6 s) of the BTBR males. Similarly the number of chamber entries during social interaction  = 1.6, p = 0.2, pooled X− ± SEM = 57 ± 4) and social novelty  = 1.7, p = 0.18, pooled X− ± SEM = 46 ± 4) tests did not differ. However, BTBR males given 10 mg/kg CBD dropped half as many feces (3.4 ± 2 vs. 6 to 7 ± 2.2) as other doses  = 6.19, p = 0.002) in the testing chamber during these two sociability tests.Middle chamber dwelling time was compared across CBD doses, and there were no significant differences during social interaction  = 1.3, p = 0.39). In the REML ANOVA, there was no interaction or CBD effect, but a significant effect of chamber side. This was because CBD treated C57BL/6J mice exhibited preference for interaction as they spent more time in chambers with novel mice than objects  = 7.1, p = 0.01, Sidak’s t(36) = 2.7, p = 0.02, p = 0.86). The two-way ANOVA again revealed no interaction or CBD effect, but a significant chamber side effect F  = 9.35, p = 0.005), as CBD treated C57BL/6J sniffed stranger mice more than novel objects (t(36) = 2.3, p = 0.05, To further assess effects of CBD on social interaction preference at the 10 mg/kg dose, it was tested versus vehicle treatment in adult male C57BL/6J mice, a commonly used strain control for autism-like behaviors of BTBR mice . C57BL/6p = 0.07). While the REML ANOVA indicated a significant interaction  = 6.6, p = 0.014), the Sidak’s post hoc revealed no effect (t(36) = 2.29, p = 0.06) of the 10 mg/kg CBD treatment (p = 0.12). The REML ANOVA likewise revealed no interaction or effect of CBD, but it showed that vehicle control treated mice preferred new over familiar stranger mice  = 13.28, p = 0.002), as shown in p = 0.003), was not seen in the CBD (10 mg/kg) treated C57BL/6J mice (t(18) = 1.41, p = 0.3).In C57BL/6J social novelty preference tests, the novelty preference, or difference in time with novel stranger versus familiar stranger mice, was unaffected by CBD treatment (t(18) = 1.9, reatment . Sniffinp = 0.22, X− ± SEM = vehicle 128 ± 24 s, CBD treated 95 ± 29 s) or novelty (t(18) = 0.1, p = 0.9, X−± SEM = vehicle 109 ± 17 s, CBD treated 111 ± 13 s) tests. Also, the number of chamber entries during interaction (t(18) = 0.8, p = 0.39, X−± SEM = vehicle 49 ± 2, CBD treated 45 ± 4) or novelty (t(18) = 0.3, p = 0.81, X− ± SEM = vehicle 44 ± 4, CBD treated 42 ± 5)) tests did not differ. Also the number of feces dropped (t(18) 0.0, p = 0.99) during tests for vehicle controls (5 ± 0.7) and CBD treated (5 ± 0.8) did not differ for C57BL/6J males.There were no differences among vehicle and CBD (10 mg/kg) treated C57BL/6J male mice in time spent in middle chamber during interaction (t(18) = 1.3, p = 0.31). REML ANOVA of social interaction tests in the male SERT knock-out mice revealed no significant interaction or CBD treatment effects either. Within groups while social interaction preference by CBD treated SERT knock-out mice appeared to be higher, there was no actual significant difference  = 3.9, p = 0.06) based on time in chambers, as shown in Serotonin transporter knock-out (SERT KO) mice were previously reported to exhibit deficits in social interaction preference in three chamber choice tests . For this reason, they were also used to test the effect of the CBD 10 mg/kg dose on social behaviors. In analysis of preference for strangers, no effect of this CBD treatment was evident in the differences in time spent with stranger mice versus novel objects by SERT knock-outs (t(14) = 0.11, p = 0.5) for the male SERT knock-out mice. Likewise, in the REML ANOVA there was no interaction or CBD effect. However, the chamber side effect was significant  = 14.7, p = 0.007) in within group comparisons, since the CBD treated SERT knockouts preferred sniffing strangers versus novel objects (Sidak’s t(28) = 3.2, p = 0.007, p = 0.06).In those same social interaction preference tests, CBD (10 mg/kg) had no effect on sniffing time differences between strangers and novel objects (t(14) = 0.66, p = 0.78). CBD treatment also had no interaction or main effect on SERT knock-out social novelty chamber side dwelling  = 3.4, p = 0.07, p = 0.49). In agreement the REML ANOVA indicated no interaction or CBD effect. However, within groups vehicle treated SERT knock-out mice preferred novel strangers  = 9.8, p = 0.007, Sidak’s t(14) = 2.8, p = 0.03), while CBD treated ones did not (t(14) = 1.7, p = 0.2)  treatment had no effect (t(14) = 0.29, p = 0.2) .p = 0.46, X− ± SEM = 125 ± 33 s control and 172 ± 54s CBD treated) or novelty (t(14) = 0.2, p = 0.9, X− = 133 ± 67 s control and 134 ± 60 s CBD treated) preference tests. Also chamber entries during interaction (t(14) = 1.8, p = 0.09, X− ± SEM = 25 ± 4 in controls, 35 ± in CBD treated) or novelty (t(14) = 1.5, p = 0.2, X−± SEM = 23 ± 4 in controls, 32 ± 4 in CBD treated) preference tests were not significantly different. Also treatment of SERT knock-outs with CBD (10 mg/kg) did not change the number of feces dropped (t((14) = 1.6, p = 0.9, 4.5 ± 1, p = 0.87, X− ± SEM = 5 ± 1) during tests relative to controls (X− ± SEM = 4 ± 1).There were no differences among vehicle and CBD treated SERT knock-out males in time spent in the middle chamber during interaction (t(14) = 0.75, p = 0.7) in male BTBR mice, or at 10 mg/kg in C57BL/6J mice (t(18) = 0.74, p = 0.47). Both strains each buried a pooled X− ± SEM = 5 ± 3 marbles from the time spanning 70−100 min after injection. SERT knock-out marble burying was not reduced by 10 mg/kg CBD treatment relative to controls (t(14) = 1.5, p = 0.2). The SERT knock-outs buried a pooled X− ± SEM of 2 ± 0.4 marbles.Acute CBD treatments at 0.1–10 mg/kg did not significantly change marble burying  = 0.4, p = 0.7), or match duration  = 1.26, p = 0.30, p = 0.5) and SERT knock-outs (Mann Whitney U(14) = 27, p = 0.6), the 10 mg/kg CBD treatment had no effect on wins  = 0.39, p = 0.7, In tube tests no CBD dose tested could increase male BTBR social dominance by measure of wins  = 1.47,  on wins , or matc2 scale in − ± SEM = 3.7 ± 1.2 ng/mg (N = 4 mice), and for 10 mg/kg were 0.1 ± 0.04 ng/mg (N = 5 mice), with a detection limit of 0.05 ng/mg.Serum from 3 male BTBR mice randomly selected for each CBD dose were used to measure CBD and its metabolites 7-OH-CBD, 7-COOH-CBD and 6-α-OH-CBD at 3 h after injection. The detection limit was 0.5 ng/mL and serum CBD was 28.7 ± 14 ng/mL, with lower metabolite levels at 50 mg/kg and at the sociability relevant dose of 10 mg/kg CBD. Levels of CBD are shown in logThe main finding of this study is that acute treatment with cannabidiol CBD) at a dose of 10 mg/kg increased male BTBR mouse social interaction preference relative to the vehicle control treatment, as assessed by difference in time spent in chambers with stranger mice versus novel objects. However, acute CBD (10/mg/kg) effects were not consistent in other measurements of BTBR social interaction preference, such as sniffing times during the social interaction test see . A strenD at a doPrior murine studies at higher CBD doses also show its potential to enhance social behaviors. In a mouse model of traumatic brain injury, CBD treatment ameliorated social interaction preference deficits found in the model . LikewisSocial novelty preference was not enhanced by this regimen of acute CBD treatments in any strain or line of mice examined in the present study. The social novelty preference tests started 60 min and ended 70 min after CBD injection. This appears to be a peak time for CBD to impact mouse social interaction preference behaviors, consistent with findings 1 hour after treatment in other strains, however CBD effects on social novelty preference was not previously examined in mice . HoweverMarble burying behavior was unaffected by 10 mg/kg CBD in the present study at 70–100 min after administration. Prior reports indicate that a dose of 30–120 mg/kg CBD can suppress marble burying in mice acutely or when administered for several days . The 10 This study has three major limitations, the first is the inability to perform additional experiments due to the discontinuation of the commercial formulation of CBD in methanol used. Our use of 10% methanol as a solvent was necessitated by initial use of the United States Drug Enforcement Administration (USDEA) exempt preparation of CBD, of >98 purity that was available at the time of the study from Cayman Chemical Co. . This formulation limited the present study to acute treatments because of the methanol solvent, and subsequently it became unavailable. The equivalent CBD reference is now sold only in an acetone solvent, so we were unable to increase our sample size or expand the study. For future studies, which should include tests of these behaviors after chronic CBD administration, a different solvent, such as 1:1:18 mixture of Tween 80, propylene glycol, and physiologic saline , or a 1:The second major limitation of this study is the lack of female mouse subjects. Our justification for excluding females from this pilot study was that ASD is 4 times more common in males than in females . HoweverThe third major limitation of this preliminary study is that the mechanism of action of CBD to enhance the social behavior of BTBR mice was not elucidated. CBD has more than 65 potential therapeutic targets, and comprehensive studies to discover CBDs role in shaping behaviors through them are just beginning to unfold , 39. StrWe did not find any impact of CBD treatment up to 10 mg/kg on social dominance or marble burying. One possibility is that the effects of CBD acutely administered at 10 mg/kg may have worn off before these tests were performed. Alternatively, the sequence of prior testing may have dampened the effects of CBD treatment in subsequent tests. Randomized controlled clinical trials, are now underway for use of CBD, mixtures of CBD and THC, and structurally related compounds such as cannabidivarin to treat autism symptoms . However1 or CB2 receptors directly  acutely enhanced social interaction in 3 chamber tests of male BTBR mice  underlying CBD’s sociability enhancing properties. Further preclinical studies in laboratory animals are essential to identify which pharmacological action(s) of acute CBD treatment are responsible for the improvements in social interaction preference found in this study.


We hereby report on the immunomodulatory effects induced over time in patients with advanced relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) and B-lineage acute lymphoblastic leukemia (B-ALL) treated with the commercial CAR-T-cell product tisagenlecleucel. We observed that, in addition to its anti-neoplastic activity, tisagenlecleucel was capable of exerting a marked and long-lived in vivo reshaping of the host immune system, in terms of T- and NK-cell expansion and cytokine release. These results may contribute to a further understanding of the complex field of immunotherapy and intercellular crosstalk, suggesting a role for host immune modulation in the long-term control of the disease.Background: Chimeric antigen receptor (CAR)-T cells represent a potentially curative strategy for patients with relapsed or refractory (R/R) B-cell malignancies. To elucidate a possible host immune activation following CAR-T-cell infusion, we investigated the effects of tisagenlecleucel administration on the patients’ immune populations in 25 patients with R/R diffuse large B-cell lymphoma (DLBCL) and B-lineage acute lymphoblastic leukemia (B-ALL). Methods: The modulation of CAR-T cells over time, the numeric changes, as well as the cytokine production capability of different lymphocyte populations and circulating cytokine levels, were analyzed. Results: Our results confirmed the ability of tisagenlecleucel to control the disease, with an overall response observed in 84.6% of DLBCL and in 91.7% of B-ALL patients at 1-month post-infusion, and showed that most patients who subsequently relapsed could undergo further treatment. Interestingly, we could document a significant increase in CD3+, CD4+, CD8+, and NK cells over time, as well as a decrease in Treg cells, and an increased IFNγ and TNFα production by T lymphocytes. Conclusions: Taken together, our results indicate that in patients with DLBCL and B-ALL, the administration of tisagenlecleucel is capable of inducing a marked and prolonged in vivo modulation/reshaping of the host immune system, both in children and adults. Harnessing the host immunity has proven a promising approach to specifically target tumor cells. The well-known anti-tumor activity of T lymphocytes has been indeed at the core of numerous therapeutic strategies, the most recent and encouraging of which is represented by chimeric antigen receptor (CAR)-T cells ,2. CAR-TCAR-T cells have been initially approved for the treatment of relapsed/refractory (R/R) children and young adults with B-lineage acute lymphoblastic leukemia (B-ALL) and then for R/R diffuse large B-cell lymphoma (DLBCL) adult patients ,5,6. TheWhile the clinical application of CAR-T cells is progressively extending to more diseases and indications, very little is known about the immune modulation induced in vivo in patients treated with CAR-T cells. Based on preclinical models, it has been suggested that CAR-T cells, in addition to targeting cancer cells, may also activate the host immune system , and theIn order to further elucidate a possible host immune activation following the infusion of CAR-T cells, in this study, we investigated the changes induced in vivo in 25 patients with B-ALL and DLBCL treated with tisagenlecleucel. To this end, we monitored the modulation of CAR-T cells over time and assessed the changes in T- and NK-cell distribution, as well as the cytokine production capability by different lymphocyte populations and circulating cytokine levels. The obtained results clearly document that, in patients with B-ALL and DLBCL, the administration of tisagenlecleucel is capable of inducing a marked in vivo crosstalk that leads to a profound modulation/reshaping of the host immune system over time, both in children and adults.This observational prospective study was carried out on a cohort of 25 patients admitted to the Hematology Center at the Sapienza University of Rome (15 patients: 13 DLBCL and 2 B-ALL) and the Department of Pediatrics of Monza (10 B-ALL patients). In terms of the inclusion criteria, all DLBCL patients were R/R after a median of 2 previous lines of therapy (range 2–4), 2 of whom had also undergone an autologous stem cell transplant, and all were ineligible for further treatment. B-ALL patients were R/R after a median of 3 lines of treatment (range 2–6), 5 of whom had undergone an allogeneic transplant. For B-ALL patients, indications for treatment comprised an age limit of 25 years (as per indication), while for DLBCL patients, the age limit in Italy was initially 70 and then 75 years.2 administered daily for 3 days for DLBCL patients and fludarabine for 4 days and cyclophosphamide for 2 days for B-ALL patients, followed by the infusion of CAR-T cells.Treatment consisted of one infusion with the commercial second-generation CD19 CAR-T-cell product tisagenlecleucel (Kymriah™). The dosage of CAR-T cells administered to each patient is reported in B-ALL patients included 4 females and 8 males, with a median age of 10 years (range 3–21), while DLBCL patients included 5 females and 8 males, with a median age of 55 years (range 34–71).The mean percentage of blast cells in B-ALL patients prior to lymphodepleting chemotherapy was 32% ± 50 (range 0–90%). The mean follow-up period at data cutoff after the infusion was 16.9 months (range 2.7–24.5) for B-ALL and 11.5 months (range 3–28.3) for DLBCL patients. Samples from B-ALL and DLBCL patients were obtained with informed consent and ethical approval in accordance with the Declaration of Helsinki and the Ethics Committee of the two institutions (Rome and Monza). Patients’ characteristics are summarized in + cells were assessed following incubation, with the biotinylated CD19 antigen and FITC- or APC-conjugated anti-biotin protein, according to their manufacturers’ instructions . Lymphocyte subsets  were analyzed via staining with a combination of labeled moAbs against the CD3, CD4, CD8, CD16, CD19, CD45, CD56, and CD62L antigens; for Treg cells, moAbs against tCD39, HLADR, CD3, CD45RA, CD127, CD4, CD25, and CD45 were used. All antibodies were from Becton Dickinson . The samples were acquired using the FACSCanto II flow, collecting at least 30,000 events, and analyzed using the Paint-A-Gate software . The representative plots of the flow gating strategy are reported in Peripheral blood (PB) lymphocytes were obtained from whole-blood samples collected on day 0 before CD19 CAR-T-cell infusion and at multiple time points after the infusion , following the EuroFlow Bulk Lysis protocol . Flow cyAbsolute cell counts were determined using the correlation of the percentage of positive populations to WBC. The total WBC counts and lymphocyte percentages for DLBCL and B-ALL patients at the different time points studied (T0–T6M) are reported in Peripheral blood mononuclear cells (PBMCs) were obtained from whole-blood samples collected before the CAR-T-cell infusion and at different time points after the infusion as detailed above, via Ficoll–Paque density centrifugation . To assess cytokine production, fresh, isolated PBMCs were cultured in a complete RPMI 1640 medium  supplemented with 10% heat-inactivated fetal bovine serum , 0.3 mg/mL L-glutamine, and 1% Pen-strep  for 4 h in the presence of phorbol 12-myristate 13-acetate 25 ng/mL  and ionomycin 1 μg/mL  for cell activation, and a GolgiStop™ Protein Transport Inhibitor solution (BD) for cytokine secretion inhibition. Activated T cells were washed twice in PBS + 0.2% FBS, divided into tubes, and then fixed and permeabilized with a Fixation/Permeabilization Kit (BD), according to the manufacturer’s instructions. For T- and NK-cell cytokine production, PBMCs were stained with FITC-, PE-, PerCP-, PE-Cy7-, and APC-labeled moAbs against CD3, CD4, CD16, CD56, IFNγ, and tumor necrosis factor-alpha (TNFα). After washing, the samples were acquired using a FACSCanto I flow cytometer (BD) and analyzed using the FACSDIVA software (BD). The representative plots of the flow gating strategy are reported in Absolute cell counts were determined by correlating the percentage of cytokine-producing populations to the total lymphocyte numbers.For plasma collection before and at different time points after CAR-T-cell infusion, PB samples were centrifuged at 3000 rpm at 20 °C for 10 min, and the supernatants were carefully harvested and stored at −80 °C until cytokine analyses. Concentrations of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IFNγ, and TNFα in the plasma were quantitatively determined using the Cytometric Bead Array (CBA) technique  according to the manufacturer’s instructions. Following the acquisition of the samples on the FACSCanto I flow cytometer (BD), the results were generated in a graphical and tabular format using the BD CBA software FCAP 3.0.1 (BD).t-test was used to evaluate the significance of differences between the groups. The results are expressed as means ± standard errors. Statistical significance was defined for a p value < 0.05.The 2-sided Student’s In the 13 patients with DLBCL, the response to CAR-T cells was assessed via PET according to the Lugano criteria . At 1 moIn the 12 patients with B-ALL, an overall response was observed in 11/12 cases  at 1 month from the infusion, with 1 patient experiencing a non-response. At 3 months, 6/12 patients (50.0%) were still in CR, 1 remained non-responsive, 4 patients showed a molecular relapse, and 1 patient showed disease progression. No neurologic complications were observed. Notably, 5 of the 12 patients who received CAR-T cells suffered from a CRS that was grade 4 in 2 patients, grade 2 in 1 patient, and grade 1 in 2 patients, according to the ASTCT Consensus Grading for CRF and ICANS  Supplem. In addi9/L) were 0.037 ± 0.043; at T7d, the number was 0.034 ± 0.031, whereas at T14d, it was 0.014 ± 0.007; at T28d, it was 0.013 ± 0.009; at T3M, it was 0.07 ± 0.08, and at T6M, it was 0.013 ± 0.014 ; at T7d, it was 0.618 ± 0.315 (p < 0.0001); at T14d, it was 0.846 ± 0.667 (p < 0.001); at T28d, it was 0.918 ± 0.692 (p < 0.001); at T3M, it was 0.966 ± 0.620 (p < 0.0001); and at T6M, it was 0.807 ± 0.354 (p < 0.0001).In DLBCL patients, the number of CD3 lymphocytes at the time of the CAR-T-cell infusion (T0) was 0.052 ± 0.043 × 109/L) cells. At T0, CD3/CD4 cells were 0.022 ± 0.020 and increased to 0.090 ± 0.082 at T3d (p < 0.01), to 0.224 ± 0.127 at T7d (p < 0.0001), to 0.397 ± 0.334 at T14d (p < 0.001), to 0.328 ± 0.132 at T28d (p < 0.0001), to 0.344 ± 0.129 at T3M (p < 0.0001), and to 0.342 ± 0.153 at T6M (p < 0.0001) ; at T7d, they were 0.342 ± 0.200 (p < 0.0001); at T14d, they were 0.449 ± 0.369 (p < 0.001); at T28d, they were 0.580 ± 0.604 (p < 0.01); at T3M, they were 0.586 ± 0.542 (p < 0.01); and at T6M, they were 0.437 ± 0.236 (p < 0.0001)  significantly increased from T14d, and the highest values were observed at T6M. CD3 cells were 0.151 ± 0.164 at T0, 0.492 ± 0.588 at T3d (NS), 0.853 ± 1.232 at T7d (NS), 1.123 ± 0.986 at T14d (p < 0.01), 0.878 ± 0.716 at T28d (p < 0.01), 0.820 ± 0.517 at T3M (p < 0.001) and 1.618 ± 0.899 at T6M (p < 0.001)  of circulating CD3/CD4 cells from B-ALL patients. At T0, they were 0.066 ± 0.057; at T3d, they were 0.119 ± 0.091 (NS); at T7d, they were 0.209 ± 0.207 (p < 0.05); at T14d, they were 0.349 ± 0.308 (p < 0.01); at T28d, they were 0.286 ± 0.245 (p < 0.01); at T3M, they were 0.303 ± 0.245 (p < 0.01); and at T6M, they were 0.556 ± 0.466 (p < 0.01) (A similar pattern was observed for the absolute number (×10p < 0.05); at T28d, they were 0.523 ± 0.537 (p < 0.01); at T3M, they were 0.448 ± 0.344 (p < 0.01); and at T6M, they were 0.901 ± 0.661 (p < 0.001) ; at T7d, they were 0.596 ± 1.069 (NS); at T14d, they were 0.700 ± 0.859 (p < 0.05) at T28d, 1.85 ± 0.81% at T3M (p = 0.05), and 1.20 ± 0.35 at T6M (p < 0.05)  at T3d, 3.19 ± 1.48% (NS) at T7d, 2.56 ± 1.28 (NS) at T14d, 1.76 ± 0.97% ( < 0.05) A. Data of CD3, CD3/CD4, and CD3/CD8, as well as Treg cell monitoring data at each time point of the study, are reported in −/CD56+/CD16+), characterized using flow cytometry, were evaluated as absolute number × 109/L. After CAR-T-cell infusion, NK cells significantly increased over time: at T0, they were 0.003 ± 0.004; at T3d, they were 0.012 ± 0.018 (NS); at T7d, they were 0.025 ± 0.022 (p < 0.01); at T14d, they were 0.051 ± 0.035 (p < 0.001); at T28d, they were 0.081 ± 0.050 (p < 0.0001); at T3M, they were 0.138 ± 0.074 (p < 0.0001); and at T6M, they were 0.170 ± 0.095 (p < 0.0001) (In a subgroup of 15 patients (13 DLBCL and 2 B-ALL) treated at the Hematology Center in Rome, the circulating NK cells (CD3 0.0001) B.p < 0.05) and at T7d for TNFα (p < 0.005), and increased further at later time points , plasma cytokine levels were measured before and after CAR-T-cell infusion. An increase in plasma concentration was observed only for IL-6, IL-8, and IL-10 cytokines in 5/15 patients . In contThe cytokine plasma levels were correlated with the occurrence or not of a CRS. Indeed, a correlation was observed between the increase in the above-mentioned cytokines and the presence of a clinical CRS. In particular, the levels of IL-6 seemed to be the most sensitive marker in view of an evident increase in plasmatic IL-6 observed in five patients  indicated that immune modulation was associated with a decrease in the percent of Treg cells and (vi) an increased capacity of T lymphocytes to produce IFNγ and TNFα. This study aimed to investigate the effect of CAR-T-cell administration in the control of R/R DLBCL and B-ALL and highlight the immunomodulatory effects induced on the host immune system of the treated patients over time in vivo. The results obtained (i) confirmed the ability of tisagenlecleucel to control the disease at least for a limited period of time; (ii) showed that many patients who subsequently relapsed could undergo further treatment; (iii) showed that the in vivo release of cytokines, particularly IL-6, was associated with a clinical CRS; (iv) documented a significant increase in CD3+ B-lineage ALL and DBCL who lack other therapeutic options and an opportunity to obtain a further clinical response and a potential cure [It is well documented that CAR-T cells represent eligible patients with R/R CD19ial cure ,21. Thisial cure ,23,24. ASimilar to previous studies ,26, we a+, CD3/CD4+, and CD3/CD8+, as well as in the NK-cell population (While some studies have focused on the characterization of CAR-T-infused cells ,28, littpulation . Even mopulation , cytokin+ ALL patients treated with the bispecific monoclonal antibody blinatumomab following dasatinib administration in induction. Blinatumomab induced a marked host immune modulation that presumably plays a role in controlling the disease [To the best of our knowledge, these are the first data showing that the infusion of CAR-T cells in patients with DLBCL and B-ALL is capable of exerting a marked and long-lasting modulation of the host immune system, in terms of T- and NK-cell expansion and cytokine release. So far, we had indirect evidence from preclinical models that pointed towards the possible crosstalk between CAR-T cells and the immune system. In a mouse B-cell lymphoma model, Boulch et al.  reported disease ,33,34.In summary, our results suggest that in patients with DLBCL and B-ALL, the administration of tisagenlecleucel is capable of inducing a marked and prolonged in vivo reshaping of the host immune system, both in children and adults, and they provide the basis for a better understanding of the immunologic signatures associated with CAR-T-cell therapy efficacy.


Fat content of control, T1, T2 and T3 were 9.81, 16.34, 25.17 and 31.19%. Fatty acids profile was determined on GC–MS, organic acids and vitamin A and E were determined on HPLC. Free fatty acids, peroxide value and cholesterol were determined. Cheddar-style cheese produced from buttermilk (1% fat) showed that it had softer texture and lacking typical cheese flavor. Gas chromatography–mass spectrometry (GC–MS) analysis showed that long-chain unsaturated fatty acids in control, T1, T2 and T3 samples were 45.88, 45.78, 45.90 and 46.19 mg/100 g. High Performance Liquid Chromatography (HPLC) analysis showed that lactic acid, propionic acid, citric acid and acetic acid gradually and steadily increased during the storage interval of 90 days. At the age of 90 days, lactic acid in control, T1, T2 and T3 was 4,789, 5,487, 6,571 and 8,049 ppm, respectively. At the end of ripening duration of 90 days, free fatty acids in control, T1, T2 and T3 were 0.29, 0.31, 0.35 and 0.42% with no difference in peroxide value. Stability of vitamin A after 90 days storage control, T1, T2 and T3 was 87.0, 80.0, 94.0 and 91.0%. Flavor score of cheddar-type cheese produced from butter milk having 1.0, 2.5 and 3.25% fat content was 81, 89 and 91% of total score (9). Hence, it is concluded that cheddar-type cheese can be produced from buttermilk having 2.5 and 3.25% fat contents with acceptable sensory attributes. Application of buttermilk for the production of other cheese varieties should be studied.Cheddar-type cheese produced from buttermilk had softer texture than standard cheddar cheese due to lower fat content of buttermilk. Fat is extremely important for the functional characteristics and optimum textural attributes of cheese. The effect of different fat contents of buttermilk on chemical characteristics of cheddar-type cheese is not previously investigated. This investigation was conducted to know the effect of different fat contents of buttermilk on fatty acids composition, organic acids, vitamins, lipolysis and sensory characteristics of cheddar-type cheese. Cheddar-type cheese was produced from buttermilk having 1, 1.75, 2.50 and 3.25% fat contents (control, T ChemicaButtermilk is rich in lactic acid bacteria which boost immune system helps body to get rid of pathogens. Its probiotic effect lowers the risk of candida in diabetic women . A generOne biggest challenge is to find ways for efficient utilization and applications of dairy by-products. One of the most significant by-products produced by the dairy industries is butter milk. During the churning process, considerable content of proteins and phospholipids are released from the membrane of fat globules to buttermilk that is perceived to have therapeutic effects . In 2017Fat in cheese is extremely important for the functional characteristics and development of optimum textural attributes. 2.2.1.Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, Starter culture for butter milk (XT-313) Lactococcus lactis ssp. lactis biovar diacetylactis and Leuconostoc mesenteroides ssp. cremoris and rennet were obtained from Christian Hansen .Cheddar cheese starter culture (CH-4219-CN) 2.2.1, T2, T3 respectively). Buttermilk having 1% fat content was used as control (To), pre-acidified for 45 min using 2% bulk starter culture of Lactococcus lactis spp. lactis and Lactococcus lactis spp. cremoris. Calcium chloride 2 mL (35%), rennet 0.02% were added and curd was cut to 1.5–2 cm cubes followed by cooking at 39°C for 45 min, cheddaring (pH 5.2), milling by cheddar cheese mill salting (1.5%), pressing by hydraulic press at 3.5 bar pressure for 16 h, vacuum packaging  and ripening at 6–8°C for 90 days. Analysis was performed in triplicate on days 0, 45 and 90.Sweet buttermilk (having 1% fat content) was heated to 50°C in a water bath  for the separation of cream from butter milk by cream separator  separated cream was used to adjusted the fat content of butter milk to 1.75, 2.5 and 3.25%  in a screw capped test tube and was heated at 100°C for 60 min till fat was fully dissolved. After that, test tube was cooled down to room temperature. Further, 2 mL distilled water and 2 mL n hexane was added in the tubes followed by vortexing (Labnet’s Vortex Mixer VX-200) at 1,500 rpm for 2 min. Then the tube was placed down for 5 min or until two layers were separated. Then supernatant was poured in a tube containing 1 g anhydrous sodium sulphate, that were transferred to GC vials and injected to GC–MS, Agilent Technologies (7890-B), USA using a SP-2560 capillary column . With the split ratio of 50% injectors and 50% detectors were set at 250°C, using FAME-37  standards, flow rate of helium, hydrogen and oxygen were set at 2, 4 and 40 mL/min .2.5.2PO4) was taken in a tube and vortexed at 200 rpm for 1.5 min. Cheese samples were then incubated at 65°C and centrifugation (Beckman-S241.5) was performed at 2958 g for 5 min. By using Whatman filter paper the upper layer and lactic acid, propionic acid, citric acid and acetic acid were analyzed on a ShodexPSpak KC-118 (8 mm × 300 mm i.d.) ion exchange organic column attached with UV detector 214 nm column.Estimation of organic acids was performed on HPLC, 10 g sample with 40 mL dihydrogen phosphate (H2.6.2/kg) and calculated with the help of following formula.As an indicator of lipolysis, free fatty acids (FFA), cholesterol and peroxide values (POV) were determined of all samples and control at 0, 45 and 90 days . For meaWhere as:B: Blank value; EQ: Consumption of titrant at first Equivalence point; T: Actual concentration of the titrant; M: Molecular weight; W: sample weight in g; F1: 1000 Conversion factor; F2: 1 Conversion factor.2.7.A 20 mL buttermilk sample was taken and mixed with 5 mL of ammonia (25%) and 20 mL ethanol (96%). Supernatant was extracted and added with 0.0025% BHT and sol-vent was evaporated with the help of a rotary evaporator at 35°C. With the help of 30 mL potassium hydroxide (5% in ethanol) saponification was performed and extracted with n-hexane. After evaporation of solvent on rotary evaporator, 20 μL sample was injected into HPLC, Waters Corp., Milford, MA, USA/ (Shimadzu) which was equipped with Spherisorb RP-18 column, Supelcogel C-610H, 300 × 7.8 mm  and water 990 detector. In various concentrations, retinyl palmitate was used as standard. Mobile phase was consisted of acetonitrile-methanol 85:15 (v/v) in isocratic system .2.8.n-hexane (HPLC grade) was added to 200 μg of fat. At 1,500 rpm the content was vortexed for 25 s and then injected (10 μL) into HPLC  which was equipped with Spherisorb RP-18 column, Supelcogel C-610H, 300 mm × 7.8 mm, Supelco Inc., Bellefonte, PA, USA). Mobile phase was comprised of 0.5% acetic acid and 0.5% ethyl acetate in n-hexane with a flow rate was adjusted at 1.5 mL/min. Vitamin E were expressed as μg/g  tests were used to express significance difference different versions of cheddar-type cheese. DMR Test were used with the aid of SAS 9.4 statistical software .3.3.1.1, T2 and T3 were 62.52, 54.29, 45.67 and 40.35% , medium-chain  and long chain (C18:0 and above LCFA) are present in milk fat. On an average, ratio of saturated and unsaturated fatty acids in milk is 70:30. Typical aroma, functional properties and nutritional characteristics of milk fat is due to mixture of fatty acids . Palmitih cheese . Most ofh cheese ,b monitoh cheese . Monitorh cheese ; Table 23.3.3 followed by T2, T1 and control (p < 0.05). At the age of 45 days, lactic acid in control, T1, T2 and T3 was 4,571, 4,829, 5,689 and 7,019 ppm  was used, impact of higher fat content in cheese milk was studied on production of organic acids. Concentrations of organic acids were higher when high fat cheese milk was used to produce cheddar cheese as compared to standard cheese . FFA of buttermilk cream were 0.08% and same source of cream was used in T1, T2 and T3 levels. Estimation of FFA in 45 days old cheddar-type cheese samples revealed a significant impact on ripening on production of ripening. Treatments having higher fat content yielded more FFA and were in the order of T3 > T2 > T1 > control . At the end of ripening, POV of control was 0.68 (mEqO2/kg) which was almost similar to cow cheddar cheese . Flavor score of all the treatments was strongly correlated with POV, non-variation in flavor score of control, T2 and T3 was due to minimum variation in peroxide value. Flavor score and POV of 90 days old cheddar cheese and control were strongly correlated . Ripening of 90 days considerably improved flavor and texture score, highest flavor score was obtained by T3 followed by T2. After 90 days, highest texture score was obtained by T3 followed by T2. Texture scores of T1 and control were not different from each other. Flavor score of cheddar-type cheese produced from butter milk having 1, 2.5 and 3.25% fat content was 81, 89 and 91% of total score (9). These results suggested that cheddar-type cheese can be produced from buttermilk having 2.5 and 3.25% fat contents with acceptable sensory attributes  under National Research Program for Universities (NRPU/6978). This work was also supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia [Grant no. 3923]. The publication of this article was funded by the Open Access Fund of Leibniz Universität Hannover.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mass spectrometry-based immunopeptidomics is the only unbiased method to identify naturally presented HLA ligands, which is an indispensable prerequisite for characterizing novel tumor antigens for immunotherapeutic approaches. In recent years, improvements based on devices and methodology have been made to optimize sensitivity and throughput in immunopeptidomics. However, developments in ligand isolation, mass spectrometric analysis, and subsequent data processing can have a marked impact on the quality and quantity of immunopeptidomics data.In this work, we compared the immunopeptidome composition in terms of peptide yields, spectra quality, hydrophobicity, retention time, and immunogenicity of two established immunoprecipitation methods (column-based and 96-well-based) using cell lines as well as primary solid and hematological tumor samples.Although, we identified comparable overall peptide yields, large proportions of method-exclusive peptides were detected with significantly higher hydrophobicity for the column-based method with potential implications for the identification of immunogenic tumor antigens. We showed that column preparation does not lose hydrophilic peptides in the hydrophilic washing step. In contrast, an additional 50% acetonitrile elution could partially regain lost hydrophobic peptides during 96-well preparation, suggesting a reduction of the bias towards the column-based method but not completely equalizing it.Together, this work showed how different immunoprecipitation methods and their adaptions can impact the peptide repertoire of immunopeptidomic analysis and therefore the identification of potential tumor-associated antigens. UtilThus, in this work, we compared cell line- and primary tumor sample-derived immunopeptidome data sets generated either with the column-based , the 96-22.1Peripheral blood mononuclear cells (PBMCs) of a chronic lymphocytic leukemia (CLL) patient and solid tumor tissue of a renal cell carcinoma (RCC) patient were used for HLA ligand isolation by a column- and a 96-well-based preparation method and subsequent MS-based immunopeptidome analysis. Blood of the CLL patient was collected at the CCU Translational Immunology, Department of Internal Medicine, University Hospital Tübingen, Germany and PBMCs were isolated by density gradient centrifugation, snap frozen, and stored at -80°C until further use. Primary RCC tumor tissue was collected at the Department of Urology, University Hospital Tübingen, Germany, and stored at -80°C until further use. Informed consent was obtained according to the Declaration of Helsinki protocol. The study was performed according to the guidelines of the local ethics committees . The Department of Hematology and Oncology, Tübingen, Germany and the Stefan Morsch Stiftung, Birkenfeld, Germany carried out HLA typing.2.28 cells and stored at -80°C until further us.The JY cell line dimethylammonio]-1-propanesulfonate (CHAPS)-based lysis buffer (1.2% (w/v) in PBS (pH 7.2); Panreac AppliChem, Darmstadt, Germany) were used. The masses of the tissue sample were determined and then immediately transferred to a petri dish, covered with lysis buffer, cut into thin slices using a scalpel, and homogenized in a homogenizer. Cell pellets or homogenized tissue samples were incubated in lysis buffer shaking for 1 hour, followed by ultra sonification  and another subsequent incubation of 1 hour. Cell debris was cleared by centrifugation at maximum speed (3100 x g), followed by sterile filtration through a 5 µm filter. The column system consisted of two columns  connected by tubing, where the upper column was used for the mAb W6/32 coupled to cyanobromide-activated sepharose beads ), and the lower column was used for the cyanobromide-activated sepharose beads coupled mAbs Tü-39 and L243 (mixture 1:1). The sample was circulated overnight through the column system containing 1 mg antibody per 1x108 cells or per 0.83 gram tissue. Washing with PBS and double distilled water was performed, followed by transiently drying of the matrix. Four times acid elution were performed afterwards with transiently drying of the matrix in between the elution steps. In the first elution, 150 μl of 0.2% (v/v) trifluoro acetic acid (TFA) and 50 μl of 10% (v/v) TFA were used, followed by 150 μl of 0.2% (v/v) TFA in the last 3 repeats. The incubation time of acidic elution was 15 minutes for each of the four elution steps. All four eluates were combined and then filtered with 3 kDa and 10 kDa ultracentrifuge filters  for HLA class I and HLA class II peptides, respectively. Filtrates were then frozen at -80°C and subsequently concentrated using a lyophilizer, followed by purification and desalting steps using a ZipTip C18 pipette tip . After binding of peptides to the C18 ZipTip, the tip was washed in 0.1% (v/v) TFA, and the peptides were subsequently eluted in 32% (v/v) ACN in 0.2% (v/v) TFA. The 0.1% (v/v) TFA washing solution  was also investigated further to determine a potential loss of peptides during washing. The desalting and final sample volumes were reduced with vacuum centrifugation and filled up to a volume of 25 µl with 1% (v/v) ACN in 0.05% (v/v) TFA and subsequently analyzed by LC-MS/MS.For the cell lysis, 1.25 ml per 1x102.3.2The lyophilized filtrates were desalted with a ZipTip C18 pipette tip during the column-based preparation method. Before the peptides were eluted in 32% (v/v) ACN in 0.2% (v/v) TFA, a washing step was performed in 0.1% (v/v) TFA. The liquid of the washing solution was lyophilized and filled up to a volume of 25 µl with 1% (v/v) ACN in 0.05% (v/v) TFA and separately analyzed by LC-MS/MS.2.3.38 cells or 9 ml per gram tissue of lysis buffer were used. The masses of the tissue sample were determined and immediately transferred to a petri dish, covered with lysis buffer, cut into thin slices using a scalpel, and homogenized in a homogenizer. Cell pellets or homogenized tissue samples were incubated in lysis buffer shaking for 1 hour, followed by ultra sonification  and another subsequent incubation of 1 hour. Cell debris was cleared by centrifugation at maximum speed (3100 x g), followed by sterile filtration through a 5 µm filter. An upper 96-well plate  was filled with the mAb W6/32 crosslinked to protein A sepharose beads ), and a lower 96-well plate with the mAbs Tü-29 and L243 (1:1 mixture) crosslinked to protein A sepharose beads. For the immunoprecipitation-step, the lysates were loaded on both plates by gravity containing 1 mg antibody per 1x108 cells or per 0.83 gram tissue. Washing of samples followed, where several washing steps with different concentrations of Tris-HCl/NaCl (4x 150 mM sodium chloride (NaCl) in 20 mM Tris-HCl pH 8; 4x 400 mM NaCl in 20 mM Tris-HCl pH 8; 4x 150 mM NaCl in 20 mM Tris-HCl pH 8; 2x 20 mM Tris-HCl pH 8) were done. Acidic elution was performed directly with 500 μl 1% TFA (v/v) onto C18 plates , followed by hydrophobic elution with 500 μl 28% (v/v) or 32% (v/v) ACN in 0.1% (v/v) TFA for HLA class I or HLA class II peptides into collection plates, respectively. All eluates were frozen at -80°C, concentrated in a lyophilizer, and filled up to a volume of 25 µl with 1% (v/v) ACN in 0.05% (v/v) TFA and subsequently analyzed by LC-MS/MS.The 96-well-based preparation lysis buffer consisted of sodium deoxycholate (0.25% (w/v); Sigma-Aldrich, Steinheim am Albuch, Germany) and octyl-beta-D glucopyranoside (1% (w/v); Sigma-Aldrich) in PBS (pH 7.2). 1 ml per 102.3.4For the adapted 96-well-based immunopurification of HLA peptides with 50% ACN elution step, another elution from the same C18 plates was performed after the hydrophobic elution of C18-bound peptides with 500 μl 28% (v/v) or 32% (v/v) ACN in 0.1% (v/v) TFA for HLA class I or HLA class II peptides into collection plates. This additional elution was performed with 50% (v/v) ACN in 0.1% (v/v) TFA in a new collection plate for HLA class I or HLA class II, respectively. The sample was frozen at -80°C, concentrated in a lyophilizer, and filled up to a volume of 25 µl with 1% (v/v) ACN in 0.05% (v/v) TFA and subsequently analyzed by LC-MS/MS.2.4Reversed-phase liquid chromatography  was used for peptide separation, followed by an on-line coupled Q Exactive HF mass spectrometer (Thermo Fisher). Samples were analyzed in three technical replicates, where 5 µl with shares of 20% were injected onto a 75 µm x 2 cm trapping column  at 4 µl/min for 5.75 min with 1% (v/v) ACN in 0.05% (v/v) TFA as loading buffer followed by peptide separation at 50°C and a flow rate of 300 nL/min on a 50 µm x 25 cm separation column with 2 µm particle size  applying a gradient ranging from 2.4% to 32.0% of ACN over 90 min. Ionization of eluting peptides was conducted by a nanospray source and analysis occurred in the on-line coupled mass spectrometer by implementing a top 35 HCD (Higher-energy C-trap dissociation) method generating fragment spectra with a resolution of 30,000, a mass range limited to 400-650 m/z for HLA class I peptides and 400-1000 m/z for HLA class II peptides, and positive charge states 2–3 for HLA class I and 2–5 for HLA class II were selected for fragmentation.2.5Data processing was performed as described previously . Integra2.6All figures and statistical analyses were generated using GraphPad Prism 9.4.0 (GraphPad Software). P values of < 0.05 were considered statistically significant. Overlap analyses were performed with InteraciVenn . Grand aTo analyze previously described tumor-associated antigens, datasets from CLL- –31 and RThe Immune Epitope Database (IEDB)  was filtFor the predicted immunogenicity calculation, column- or 96-well-based method-exclusive 9-mer peptides were analyzed with the “Class I immunogenicity” prediction tool on the IEDB2.7via the PRIDE  TFA during the ZipTip C18-based desalting step   were compared with the column preparation. However, up to 24% and 44% of the identified HLA class I (JY 9% (895/10475), CLL 24% (3108/12931), RCC 14% (1817/12948)) and HLA class II (JY 15% (1255/8509), CLL 41% (1968/4821), RCC 44% (1852/4184)) peptides identified in the column-based method remain exclusive even when the 96-well method is supplemented with the 50% ACN elution step  can be found below: The studies involving human participants were reviewed and approved by the Ethics Committee of the faculty of medicine of the University of Tübingen. The patients/participants provided their written informed consent to participate in this study.MW, JB, AN, and JSW conceptualized this study. MW, JB, and LW performed immunopeptidome experiments. MD performed bioinformatic analysis. AN, H-GR, and JSW supervised this study. All authors contributed to the article and approved the submitted version.


The SFE composite showed higher mechanical properties with the increase in SiO2 (f), x = 30 to 80, the tensile strength (UTS) was raised from 91.6 MPa to 155.7 MPa, the compression strength (UCS) was increased from 261.1 MPa to 409.6 MPa and the flexural strength was enhanced from 192.3 MPa to 311.9 MPa. Upon further addition of SiO2 (f) to the composite, i.e., x = 90, the mechanical properties were reduced a little, but the dielectric properties were not changed. Increasing SiO2 (f) improved the thermal stability as weight loss was found to be 69% (x = 30) and 24% (x = 90), and average moisture absorption was found to be 1.1 to 1.8%. A silica fiber/epoxy composite, for microelectronics, can be made from a low-cost fiber, and its dielectric properties as well as its mechanical and thermal stability can be tuned or improved by varying fiber fractions.In many electronic applications, the dielectric and structural properties of reinforced composites are vital. In this research work, the influence of fiber proportion on the properties of a silica fiber/epoxy (SFE) composite was investigated. The structure, morphology, dielectric constant and loss factor, mechanical properties, and thermal stability were determined. The increase of wt.% of silica fiber (SiO Silica strength .Polymer-based composites are limited due to their relatively low working temperatures and dielectric instability, which means that they fail to meet increasing demands of industrial applications. Therefore, it is urgent to fabricate polymer-based composites with stable dielectric properties over a wide range of temperature and frequency .Cyanate ester (CE) has excellent dielectric properties, adhesion, mechanical strength, thermal stability, and very low moisture absorption ,17. ElecProduction cost, quality, and shape requirements are important  when sel2~72–75%) and quartz fibers (SiO2 ≥ 99.9) [2 = 95–99.5%) by acid leaching of A-or E-glass fabric. It is commonly being used in fireproofing and industrial insulation applications, has found limited use in aircraft and aerospace applications [Reinforced composites from D-glass (SiO ≥ 99.9)  are used ≥ 99.9) . D-glass ≥ 99.9) . High-siications . High-siications ,34.For various electronic applications, a huge number of dielectric materials have been developed but a single material cannot conform to all requirements along with minimum cost. The basic characteristic property of a very good low dielectric material is that it possesses a very low dielectric constant (ε ≤ 4.5) and dielectric loss (δ ≤ 0.06), however, low cost, good mechanical properties, and environmental resistance are important as well .2~54%) possess excellent mechanical strength and a medium cost but higher ε (5.4~6), S-GFs (SiO2~64–66%) have high strength, ε (5.1), elasticity, and cost. D-GFs have the lowest ε and δ among all GFs and lower mechanical properties with higher cost, Quartz GFs possess low ε and δ close to D-GFs, have the best impact, chemical and temperature resistance, but also have the highest cost [E-GFs  epoxy resin was used due to its good mechanical and electrical characteristics  and low Raw materials used in manufacturing low-dielectric composites  are comp® , density 1.90–1.95 g/cc, viscosity 10,300 cPs, and epoxy content 23%. Commercial-grade silica fabric (BWT260-82) TESCNICA, fiber dia. ~8.0 to 10.0 μm, density ~2.35 g/cc, ply thickness = 0.25 mm, softening point ~1600 °C, was used.SFE composites were prepared from a low-cost commercial liquid DGEBA resin, MagicUnidirectional woven silica fabric was cut into layers of (200 × 200), washed with ethanol and dried. Varying weight proportions of silica fabric (SF) to epoxy (E)  were fabricated as shown in SFE composite specimens were characterized for dielectric constant, loss tangent , density (ASTM D-792) using analytical weighing balance (SHIMADZU-UW 3400 g), structure , morphology by Scanning Electron Microscopy , tensile strength (ASTM D 638), compression strength (ASTM D 6641), three-point bending strength (ASTM D-7264) by universal testing machine AGX-Plus (SHIMADZU Japan), weight loss , and moisture absorption (ASTM-D 570) in both fiber directions using analytical weighing balance (SHIMADZU-UW 3400 g).2 (f) up to x = 70%, but by further adding silica fiber  the density was further raised by 2%, which was said to be insignificant. It was found that a good fiber–matrix adhesion was achieved up to x = 70% by increasing the density to its maximum value.2 (f) (x = 30 to 90) exhibited similar bands at 3441.44 cm−1 and 2924 cm−1, OH stretching (hydroxyl linkage) [−1 corresponded to the C = O double bond (polyamide) in the epoxy linkage. The clear Si-O-Si deformation band was observed at 468 cm−1 [−1 was attributed to the C-N-C stretching of tertiary amine to form an epoxy–ammine network. The bending peaks located at 1097 cm−1 were attributed to the symmetric and asymmetric stretching of C-O-C. The band observed at 800 cm−1 was due to aromatic C-H linkage deformation and was attributed as a possibility of the vibration modes of SiO4 tetrahedron.The presence of organic and inorganic networks in SFE composites was identified by the FTIR spectrum as shown in linkage)  and the 468 cm−1 . The abs2) glass fiber in epoxy resin, as in 2 (f) and curing of the thermoset resin formed an amorphous phase without any additional peaks. The XRD patterns of the fabricated composites were typical X-ray diffractogram patterns of silica wave transparent composites [X-ray diffraction demonstrated the presence of  (x = 70), showed a better fiber–matrix adhesion than SF6E4. The appearance was similar to SF6E4 with a small number of fibers seeming to have been pulled out in some of the areas. There were fewer interfacial separations between fiber–matrix interfaces and small voids despite of higher fraction of reinforcement. Free spaces due to fiber slippage during the compaction of fabric sheets were present.The morphology of the SFE composites  was observed by SEM, as shown in 2 (f) content, a more uniform distribution of fibers was created as compared to the SF6E4 and SF7E3 composites. 2 (f)% which reduced the fiber pull-out. This localized discontinuity and uneven structural morphology are evident in r) as a function of frequency (in S-band) is shown in 2 (f) at x = 30, 40, 50, 60, 70, and 80%, the dielectric constant (εr) at 4GHz was decreased from 3.82, 3.73, 3.57, 3.45, and 3.19 to 3.17, respectively. However, the dielectric constant was unchanged even after further adding SiO2 (f), up to x = 90. Comparatively, higher dielectric constants were observed in the SFE composite with SiO2 (f) x = 30% to 60% than those where SiO2 (f) x = 70%, 80%, and 90%. The constituent (SiO2 (f) and epoxy resin) formed the heterogeneous dielectric structure in which the increased wt.% of SiO2 (f) created space charge polarization with the packed interfacial boundaries. The increased (x) wt.% of SiO2 (f) decreased the hopping electrons and local charge displacement towards the electric field in low electric polarization. The dielectric dispersion was increased in the real and imaginary parts of the dielectric constants and thus the dielectric constants were decreased.In the fabricated SFE composites, the dielectric constant , the movement of free ions was stopped, which decreased the ionic conductivity. The fast growth of large epoxy networks reached a point where the contribution from dipole relaxation was dominant over the ionic conductivity. These all contributed to low dielectric constant values.2 (f) wt.%) at x = 30, 40, 50, 60, 70, and 80, respectively, as shown by 2 (f) and epoxy resin) are non-magnetic, the low dielectric loss was found in the resultant SFE composite. The tangent loss reached its lowest value of 0.036 for SF8E2 (x = 80) and S9E1 (x = 90) and a comparatively higher dielectric loss factor was observed for SF3E7, SF4E6, SF5E5 and SF6E4 than those which contained SiO2 (f) at x = 70, 80, 90. The higher proportion of SiO2 (f) in the composite restricted the local charge displacement towards the electric field. Due to this restriction on the charge migration, polarization was reduced, and tangent loss was decreased. This lowest value of dielectric tan (δ) loss was evidence that charge polarization had been fixed. The lower energy dissipation was evident from the significant reduction (49%) of dielectric loss. Other contributing factors toward the tangent loss value, such as cure conditions, post-curing, resin properties, and plies orientation, were not included in this study.The electromagnetic behavior of dielectric material is primarily described by its relative dielectric constant and tan (δ) loss. When interacting with the electromagnetic fields, the absorption of electromagnetic waves  is due to a high loss tangent (δ) which slows down the transmission of signals. The δ (4GHz) was decreased as 0.071, 0.070, 0.057, 0.047, 0.041, 0.036 and 0.036 corresponds to the increased (SiO2 fiber and Epoxy resin). Due to this insulating nature, the resultant composite has low electrical conductivity. High-silica fibers possess high resistivity of ≥1000 Ω.m [0 × ε′ tan δ, whereω = frequency0 = 8.85 × 10−12εtan δ = ε′′/ε′Dielectric material possesses high resistivity as there are no free electrons to carry current, thus the electrical conductivity of these materials is very low . SFE com1000 Ω.m ,47 and e−20,9.29892 × 10−20, 7.24717 × 10−20, 5.77487 × 10−20, 4.65801 × 10−20, 4.06431 × 10−20, 4.06431 × 10−20, which corresponds to the increased SiO2 (f) (wt.% at x = 30 to 90).The σ was found at 9.65933 × 102 (f) proportion in the composite as shown in 2 (f) (x) increased, UTS was increased from 91.6 MPa (at x = 30), 96.3 MPa (x = 40), 108.5 MPa (x = 50), 115.1 MPa (x = 60), 132.3 MPa (x = 70) and 155.7 MPa (x = 80), which was the highest value. The overall UTS was 41.1% increased, formed a tough composite structure, and resulted in failure at higher loads. Optimum fiber–matrix proportion is key to yielding the higher tensile strength of the composite. However, further addition of SiO2 (f), x = 90, the tensile strength was reduced to 146.3 MPa which attributed that balance between fiber–thermoset resin was changed, and the adhesive bonding was reduced due to weak interfaces. Fibers in the composite were pulled at relatively lower loading due to higher fiber proportion.The ultimate tensile strength (UTS) was increased from 91.6 to 155.7 MPa, corresponding to increased SiO2 (f) from 30 to 80 wt. %. From the initial value of UCS, it was 40.4% increased and reached to its highest value (409.64 MPa) for SF8E2 which revealed that SiO2 (f) enabled it to bear high compressive loads. However, after approaching to maximum value, further addition of SiO2 (f) resulted in a slightly lower UCS to 405.14 MPa (at x = 90). It seemed that stresses due to compressive load were borne by fibers, rather than the epoxy and fiber–matrix interface.Overall, the addition of the silica fibers enhanced the compressive strength (UCS), as shown in 2 (f) from 30 to 80 wt.%, the flexural strength  both contributed to thermal resistance from 200 °C to 250 °C, where all the SFE composites samples remained stable. Above 250 °C, degradation started, and the major degradation was seen from 300 °C to 500 °C, with maximum weight loss. The de-binding in the multilayer fiber-reinforced composite was due to the softening of the organic part (polymeric resin) and the start of the degradation of epoxy. SFE composites with SiO2 (f) at x = 60, 70, 80, and x = 90, offered a better thermal resistance due to higher SiO2 proportion. The degradation of the composite has two regions, from ambient to 250 °C, where these composites  remained stable. From 300 °C to 500 °C the maximum degradation of SF3E7 was 68% (at x = 30). The polymeric part melted and then carbonized completely due to its inherent organic nature. However, in the second region, from 300 °C to 500 °C, the behavior of the samples at  was found to be different from the samples at . It was very clear that the increased proportion of silica fibers (x = 60 to 90) formed a glassy substrate to protect the interface and thus reduced the degradation.Thermo-mechanical behavior of SFE composites against the rising temperature was determined by thermo-gravimetric analysis (TGA) as shown in Average moisture absorption of  was found to be 1.1 to 1.3%  while in traverse fiber directions it was found to be 1.6 to 1.8%. Moisture absorption is significant in determining composite long-term strength and stability as it not only degrades the fiber–matrix bonding, but also mechanical properties (due to plasticizing effect), and electrical properties (due to high ε of water = 70). Moisture was absorbed in the exposed surface as moisture uptake is a function of the shape ratio (specimen surface area and volume) ,42. As dThe properties of the glass fiber composite depend on multiple variables including the nature of the fiber, bonding matrix, their proportion, fiber orientation, direction, mixing ratios, processing methods, curing conditions  are compiled in 2 (f) (w/w%) on properties of reinforced wave transparent composites was studied. FTIR spectra revealed the Si-O peak while –OH, C = O, C-H, and C-N-C peaks were also found. Their amorphous nature was revealed by XRD while the SEM images showed the composite morphology along with fiber–matrix adhesion. Increased proportion of SiO2 (f) (SF = x at 30 to 90 w/w%) resulted in excellent transmission, low εr from 3.85 to 3.12, and low δ from 0.07 to 0.036. With increased SiO2 (f) (at x = 30 to 80), the UTS, UCS, and bending strength was increased, however, with more than optimum addition of SiO2 (f) (x = 90) in the composite, the mechanical properties were reduced by 4.3% (UTS), 1% (UCS), and 1.7% (bend), respectively. SFE composite thermal stability was improved with the addition of SiO2 (f), as 24% weight loss was seen (x = 90) whereas 69% weight was lost (x = 30) while low moisture absorption was found from 1.6 to 1.8%. In conclusion, this silica fabric can be a useful, promising, and cost-effective reinforcement for fabricating a reinforced composite, exhibiting excellent dielectric properties, good mechanical and thermal stability, and low moisture absorption.In this research work, the loading of SiO