To evaluate the in vivo effects of two soluble Aβ species (Aβ25-35 and Aβ1-40) on commissural CA3-to-CA1 (cCA3-to-CA1) synaptic transmission and plasticity, and CA1 oscillatory activity, we used acute intrahippocampal microinjections in adult anaesthetized male Wistar rats. Soluble Aβ microinjection increased cCA3-to-CA1 synaptic variability without significant changes in synaptic efficiency. High-frequency CA3 stimulation was rendered inefficient by soluble Aβ intrahippocampal injection to induce long-term potentiation and to enhance synaptic variability in CA1, contrasting with what was observed in vehicle-injected subjects. Although soluble Aβ microinjection significantly increased the relative power of γ-band and ripple oscillations and significantly shifted the average vector of θ-to-γ phase-amplitude coupling (PAC) in CA1, it prevented θ-to-γ PAC shift induced by high-frequency CA3 stimulation, opposite to what was observed in vehicle-injected animals. These results provide further evidence that soluble Aβ species induce synaptic dysfunction causing abnormal synaptic variability, impaired long-term plasticity, and deviant oscillatory activity, leading to network activity derailment in the hippocampus.Recent evidence indicates that soluble amyloid- Recent D stages –5. In fage of AD , 7. Althge of AD , currentge of AD . New Aβ- benefit ; therefoβ1-40 and Aβ1-42 (Aβ1-42 mainly in the core of early plaques and Aβ1-40 in vascular amyloid deposits) [β fragments, such as Aβ25-35, constitute the biologically active forms of Aβ and are thus responsible for the neurotoxic properties of Aβ1-40 and Aβ1-42 [β25-35 may be expressed in AD brains [β1-40 [β [β25-35 produces more acute toxic effects than Aβ1-42 because of its higher solubility [β1-40 and Aβ1-42 [Senile plaques in AD patients and animal models consist of Aeposits) , 12. It d Aβ1-42 . Aβ25-35D brains –16 possis [β1-40 , 16. Sevβ1-40 [β –15. Howelubility , and it d Aβ1-42 –20.β effects during early AD stages [θ activity, which depends on acetylcholine release from the medial septum [θ and γ activities are associated through inhibitory synapses between GABAergic parvalbumin interneurons and pyramidal neurons [θ and γ activity, is necessary for adequately encoding and storing information in the cortex and hippocampus [In vivo studies have shown the relevance of the CA3-CA1 synapse in associative learning and memory processing [The hippocampus and entorhinal cortex are peculiarly susceptible to deleterious AD stages . Hippocal septum . Moreovel septum , 22. Eacl septum . Moreove neurons . Such ospocampus –27. In vocessing , 29 and ocessing . In a veocessing . Therefoθ-γ oscillations, and hippocampal-dependent memories [β affects θ, δ, and γ bands in different preclinical models of AD [α/θ ratio, γ coherence, θ and δ synchronization) [θ-γ cross-frequency coupling might be used as an early biomarker of AD [β1-42 was related to alterations in oscillatory activity in θ-γ coupling at perforant path-dentate gyrus synapses [β-induced LTP impairment might be associated with oscillatory activity changes in brain structures affected during initial stages of AD.There are tight correlations among long-term potentiation (LTP) mechanisms, memories –34. Evenmemories . Accumulls of AD . Similarization) , 38. Stuer of AD . In anotsynapses . Varioussynapses , 41, 42 β forms and configurations may explain variable effects during AD progression. Considering the huge differences between experimental models (in vivo vs. in vitro), time of exposure to Aβ (acute vs. chronic), and differences in Aβ aggregation states , the reported Aβ effects on neuronal activity have been divergent in terms of excitability, active and passive membrane properties, network activity, and neural plasticity [β forms on oscillatory activity, excitability, or synaptic plasticity [β25-35 and Aβ1-40 effects on hippocampal oscillations  and basal transmission, variability, and long-term plasticity in cCA3-to-CA1 synapses. We found that LTP impairment induced by acute administration of soluble Aβ (Aβ1-40 and Aβ25-35) is associated with abnormal synaptic variability and increased power of γ-band and ripple network oscillations and derailed θ-to-γ phase-to-amplitude coupling in CA1. Such Aβ-induced disruption in synaptic plasticity and network activity may underlie abnormalities in information processing and memory encoding.Several aggregated Aasticity , 43–47. asticity . The aimIn Vivo Experiments (ARRIVE) guidelines [All procedures performed on living animals were performed in conformance with Animal Research: Reporting idelines , followiad libitum. Experiments were performed in the morning. Special care was taken to minimize animal suffering and to reduce the number of animals used.Seventeen 16-20-week-old male Wistar rats, weighing 300 ± 30 g, were used as experimental subjects. Experimental animals were supplied by the Universidad Nacional de Colombia animal facilities. Animals were housed in a sound-attenuated room in polycarbonate cages, in groups of four, under controlled environmental conditions: 20 ± 1°C temperature, 50 ± 10% relative humidity, and 12 h light/dark cycle (lights on from 07:00 to 19:00). Animals had food and water available 2(Z∝/2 + Zβ)2/d2, where  SD = 6.85, Z∝/2 = 1.96 with ∝ = 0.05, Zβ = 0.84 with β = 0.2. Statistical power = 80%, and d = 14. The minimum sample number per group was 5.Sample size was calculated according to the following formula : sample n =6), receiving vehicle microinjection; (2) Aβ25-35 group (n = 6), receiving Aβ25-35 peptide microinjection; and (3) Aβ1-40 group (n = 5), receiving Aβ1-40 peptide microinjection.Animals were randomly assigned to any of three groups: (1) control group  under general anaesthesia, recording and stimulation electrodes were stereotactically inserted in CA1 and contralateral CA3; (2) once stable responses were obtained, the input/output relationship was established; (3) 30 min baseline recording was done to characterize basal synaptic efficiency; (4) the designed solution was microinjected in CA1; (5) the microinjected solution's effect on synaptic transmission was characterized by recording synaptic responses for 30 min after microinjection; (6) high-frequency tetanic stimulation was done to induce long-term plasticity; and (7) the effect of high-frequency stimulation (HFS) on synaptic efficiency was characterized by recording synaptic responses for 60 min after HFS .β effects on NMDA-dependent synaptic plasticity, we chose urethane as the anaesthetic agent, taking into account that it has been reported to have no significant effects on NMDA-type glutamate receptors when given in intermediate doses [Under general anaesthesia induced with 1.5 g/kg of 25% urethane  and 10 mg/kg of xylazine  , the subject was placed in a rat stereotaxic frame . Nociceptive responses  were evaluated during the experiment. In case of slight motor response, an additional injection of urethane at 50% of the initial dose was administered. The respiratory and cardiac rates were monitored during the whole experiment, and a heating blanket was used to avoid hypothermia. Because we were interested in evaluating Ate doses , 52; howte doses , but lefte doses . In ordeΩ impedance enamel-coated wire attached to a 25-G needle), for local field potential (LFP) recording and microinjection, was inserted 2.5 mm from the pial surface, through the skull hole for left CA1 (vide supra for stereotaxic coordinates), using a hydraulic micromanipulator . A stimulating concentric bipolar electrode was lowered 3.5 mm from the pial surface, through the skull hole for right CA3 (vide supra for stereotaxic coordinates), using a micromanipulator . A silver electrode was placed in neck musculature as a reference.A microelectrode cannula (a 5 MCA1 field activity was magnified (100x) using an AC-coupled preamplifier . The preamplified signal was then band-pass filtered (0.1 Hz and 10 kHz cut-off frequencies) and further amplified at 20x . This conditioned signal was digitized using an analogue-to-digital converter  with 10 kHz sampling frequency and stored for offline analysis using commercial  and purpose-designed software.μs monophasic square pulses, delivered at 0.33 Hz frequency, using a stimulus isolation unit , controlled by a pulse generator . Stimulus intensity (100-400 μA) was adjusted so as to obtain stable and reliable CA1 field excitatory postsynaptic potentials (fEPSP). The depth of the stimulating and recording electrodes from the pial surface was finely adjusted so as to obtain short latency fEPSP, with a waveform characteristic of CA1 stratum pyramidale.CA3 was stimulated by applying 100 μA). Stimulus intensity was then successively doubled until fEPSP response saturation was attained . Then, the stimulus intensity required to obtain ~50% of maximal response (I50) was selected to evaluate short- and long-term plasticity from then on. Basal synaptic activity was characterized by recording CA3 stimulation-evoked fEPSP in CA1  for 30 min. Intrahippocampal microinjection effect on basal synaptic activity was characterized by recording such fEPSP for 30 min after microinjection. Long-term potentiation at cCA3-to-CA1 synapses was induced by delivering six trains  at 60 s intertrain intervals (HFS). HFS effect on cCA3-to-CA1 synaptic efficiency was studied by recording left CA1 response to right CA3 stimulation  for 60 min. Deep anaesthetic level was maintained throughout the whole recording session using supplementary anaesthetic doses (about every 4 hours) to attain stable and reliable activity in CA1 [Once a stable CA1 fEPSP was achieved, threshold stimulus intensity was established by decreasing the intensity to one-half the previous one until the fEPSP failure rate was equal to or higher than 50% . The electrodes' position and diffusion trace of microinjection coloured solution were documented by examining the slices with a stereoscope  and taking digital photographs  under oblique back-illumination  was given and the location of stimulating and recording electrode tips was marked by passing continuous current through them delivered by a precision current source . The subject's brain was removed and submerged in 4% paraformaldehyde for 3 days; then, 100 mination .β species, Aβ25-35 and Aβ1-40  were chosen for the present work on the basis of their aggregation kinetics, neurotoxicity, and pathogenicity. On the one hand, postmortem examination of AD patients' brains yielded that Aβ1-40 accounts for approximately 90% of total Aβ peptide in senile plaques [β25-35 aggregates more rapidly and displays more neurotoxicity than Aβ1-40 [β25–35 and Aβ1-40 peptides were prepared as previously described [β peptides  at a 1 μL/min rate through the microelectrode cannula inserted in the left hippocampal CA1 region. Aβ25-35 and Aβ1-40 dose (3 nM) and total injection volume (2 μL) were chosen according to previous reports [Once a baseline recording was obtained, a Hamilton syringe connected through 12-G tubing was used to inject 2  reports –63. Stimδ (0.5-3.9 Hz), θ (4-7.9 Hz), α (8-11.9 Hz), β (12-24.9 Hz), γ (25-120 Hz), HFO1 (121-250 Hz), and HFO2 (250-500 Hz) bands, using Welch's method. Time-frequency domain analysis was done by building scalograms for each window, using the Morse wavelet decomposition [γ band scalogram averages were triggered by each θ cycle time window to determine phase-amplitude coupling (PAC) [Electrophysiological data analyses were planned to characterize the effects of both microinjected solutions on basal cCA3-to-CA1 synaptic responses, cCA3-to-CA1 long-term plasticity, and CA1 oscillatory activity. To do so, recordings were divided into 5 min windows around stimulation events and analysed in time, frequency, and time-frequency domains. Time domain analysis was related to fEPSP first component slope measurement. Frequency domain analysis was focused on calculating relative power spectral density (rPSD) in position , 65; theng (PAC)  . Variability of cCA3-to-CA1 synaptic responses from experimental groups was compared using Levene's test . The resH(2) = 0.153, p = 0.797, n = 16) or after intrahippocampal injection  = 1.541, p = 0.251, n = 16: F = 64.898, p < 0.001, n = 16); post hoc analysis showed that Aβ1-40 microinjection significantly increased cCA3-to-CA1 synaptic variability more than the other treatments  = 0.004, p = 0.951, n = 11; control vs. Aβ1-40: F = 71.932, p < 0.001, n = 10; and Aβ1-40vs. Aβ25-35: F = 86.351, p < 0.001, n = 11). Variability progressively increased in control and Aβ25-35 groups after both injection  = 19.232, p < 0.001, n = 5; Aβ25-35: F = 23.761, p < 0.0001, n = 6) and HFS  = 63.522, p < 0.0001, n = 5; Aβ25-35: F = 49.588, p < 0.0001, n = 6). By contrast, even though variability in the Aβ1-40 group increased substantially after being injected  = 299.08, p < 0.0001, n = 5), it did not change after HFS  = 0.63, p = 0.428, n = 5).No significant difference was found between the experimental groups during baseline recording before (H(2) = 9.5, p = 0.009, n = 5). By contrast, both Aβ25-35 and Aβ1-40 impaired such HFS-induced fEPSP slope increase  = 0.504, p = 0.614, n = 6; Aβ1-40: from 100 ± 1.84% to 113.5 ± 25%, F = 0.938, p = 0.418, n = 5; F = 17.741, p < 0.001, n = 16); post hoc analysis (Tukey's test) showed that vehicle-injected subjects displayed fEPSP slope increase significantly greater than Aβ25-35- and Aβ1-40-injected ones , while these later groups were not significantly different to each other . These results show that soluble Aβ microinjection impairs cCA3-to-CA1 long-term synaptic plasticity.HFS induced significant fEPSP slope increase in vehicle-injected subjects  = 8.237, p = 0.007, n = 17) and HFO1 (H(2) = 6.408, p = 0.029, n = 17) bands, but not in other bands. Neither Aβ1-40 nor vehicle injection induced significant changes in relative PSD in any band  = 3.036, p = 0.089; 30 min: H(2) = 1.96, p = 0.403; and 60 min: F = 1.502, p = 0.265), HFO1  = 0.063, p = 0.940; 30 min: F = 0.224, p = 0.803; and 60 min: F = 0.341, p = 0.718), or any other band. HFS did not induce significant changes in relative PSD in any band in vehicle- or Aβ1-40-injected subjects   = 37.220, p < 0.001); neither vehicle  = 1.59, p = 0.254) nor Aβ25-35  = 3.26, p = 0.104) injection induced significant shifts in such modulating phase  = 7.71, p = 0.03) and Aβ25-35 injection-induced  = 4.76, p = 0.05) phase shifts; vehicle and Aβ25-35 injection-induced phase shifts were not significantly different to each other  = 0.43, p = 0.53).Ang phase . PlannedF = 4.47, p = 0.002); this phase shift persisted for up to 1 h after HFS  = 9.25, p = 0.038; 30 min: F = 7.07, p = 0.05; and 60 min: F = 13.11, p = 0.022). In Aβ1-40-injected subjects, HFS induced a smaller phase shift (~45-70°) that reached significance only 60 min later  = 1.21, p = 0.338; 5 min: F = 0.72, p = 0.421; 30 min: F = 0.82, p = 0.392; and 60 min: F = 5.55, p = 0.046). By contrast, HFS did not induce a significant phase shift in Aβ25-35-injected animals  = 1.03, p = 0.4073; 5 min: F = 2.75, p = 0.136; 30 min: F = 0.92, p = 0.366; and 60 min: F = 0.25, p = 0.631).Taking each group injection-induced phase shift as a reference, it was found that HFS induced a significant phase shift (~112-137°) in vehicle-injected subjects  Aβ1-40 enhanced basal synaptic variability significantly more than other treatments did but impaired HFS-induced variability increase; (2) Aβ25-35 injection significantly increased gamma and HFO1 band relative PSD; (3) both soluble amyloid beta peptides (Aβ25-35 and Aβ1-40) impaired HFS-induced LTP; and (4) Aβ1-40 injection induced a significant γ amplitude-modulating θ phase shift (~122°) but, as Aβ25-35 did, impaired the occurrence of a HFS-induced phase shift.This experiment's main findings were that, even though intrahippocampal microinjection of soluble species of Aβ peptides have been repeatedly highlighted as crucial AD pathogenetic initiators. Although the underlying mechanism is not yet fully understood, some studies have indicated that Aβ can impair synaptic transmission and plasticity, leading to changes in spine density and, eventually, synaptic pruning [β in anaesthetized rats affected cCA3-CA1 synapse variability and impaired long-term synaptic plasticity. Overall, such results concur with those of other studies, which have shown that high Aβ oligomer concentration interferes with synaptic efficiency and plasticity [in vivo preparations [in vitro have been reported at concentrations higher than 100 μM (around ten times our preparation) [β oligomerization in a dose-dependent manner (ranging from 0.01 to 445 μM), but that effect is observed only after several days of incubation (4 to 8 days) [β1-40, but not Aβ25-35, induces significant increases in variability in cCA3-to-CA1 synaptic responses without significant changes in fEPSP slope. In agreement with our results, several in vivo studies found that the injection of different amyloid species did not affect hippocampal baseline synaptic potential amplitude or slope [β1-42 has been found to induce spike widening, which would increase synaptic release due to increased calcium influx into presynaptic boutons [β25-35 has also been reported to produce spike broadening, but using doses one order of magnitude higher than the one used in our experiment [β1-42 oligomers depress release probability at CA3-CA1 synapses [β1-40, depending on its concentration and the specific pyramidal cell type affected [β1-40-induced synaptic variability increase, without significant changes in average fEPSP slope. It is plausible that the Aβ25-35 concentration we used was not enough to induce significant changes in synaptic variability .A pruning –71. We hasticity , 62, 72.arations , 74, posaration) , 76. Met 8 days) . We founor slope , 74, 78.or slope . On the  boutons ; Aβ25-35periment . On the synapses ; moreoveβ-intrahippocampal injection nor HFS induced significant CA1 global power spectrum changes  increases in γ and θ spectral power have been recorded in freely behaving adult rats' hippocampus immediately after LTP induction [Although neither Anduction . In the γ oscillations and ripples (140-200 Hz) in the CA1 hippocampal region depend critically on the fast-spiking activity of parvalbumin- (PV-) expressing basket cells [β oligomers have been shown to directly interact with receptor tyrosine-protein kinase erbB-4, increasing its phosphorylation state [β oligomer deposition, which is followed later by PV interneuron dysfunction and more severe cognitive deficits [β25-35 has more affinity than Aβ1-40 for erbB-4.Both et cells . Aβ oligon state ; erbB-4 on state . PV inteon state , 87, seedeficits . It is pγ band (25-120 Hz) activity increases in close association with locomotor behaviour [β species-induced modifications in γ band and ripple oscillations in neural circuits are, therefore, associated with synaptic dysfunction and cognitive impairments [It has been found that ehaviour , 90, worehaviour , and memehaviour . Hippocaehaviour . In factehaviour –97. The airments , 98–100.β species used in our experiment (Aβ25-35 and Aβ1-40) impaired HFS-induced LTP in cCA3-to-CA1 synapses. This deleterious effect has been extensively reported, and many possible underlying mechanisms have been identified; among them, those pointing towards excitatory/inhibitory imbalance are relevant to our findings. Aβ-induced calcium dyshomeostasis underlies distorted synaptic transmission, plasticity, and oscillatory activity in the brain. However, depending on exposure time, brain region, oligomer type, and receptor subunits involved, soluble amyloid has been reported not only to inhibit calcium influx through NMDA receptors in cultured hippocampal [in vivo [β1-42 and Aβ1-40 on NMDA function and LTP are reverted by a specific GLUN2B receptor antagonist [in vivo and ex vivo Aβ1-42 administration deteriorates GABAB-mediated inhibitory transmission in CA3-to-CA1 synapses and impairs HFS-induced LTP of excitatory [β25-35 has been found to act ex vivo as a GirK channel antagonist in CA3 pyramidal neurons [β-induced malfunction of NMDA and GirK channel conductance in pyramidal neurons might contribute, along with PV interneuron dysfunction, to hippocampal network instability, manifested through LTP impairment and aberrant rhythm generation, which may underlie subtle cognitive derailments observed during early AD stages.Both Apocampal  or cortipocampal  but also[in vivo . In additagonist . Acute icitatory  as well citatory , and suc neurons . Aβ-induβ1-40 significantly shifted the resulting angle from the average θ-to-γ PAC vector. In vehicle-injected subjects, HFS induced a significant phase shift of the average θ-to-γ PAC vector persisting up to 60 min. Besides impairing HFS-induced LTP, both Aβ25-35 and Aβ1-40 blocked the phase shift of the average θ-to-γ PAC vector, with the Aβ25-35 effect persisting longer. Increases in θ-to-γ coupling have been described in freely behaving rats after HFS-induced LTP; in that experiment, acute Aβ1-42 treatment not only impaired LTP but also diminished θ-to-γ coupling [θ-nested γ oscillations in CA1 depend on out-of-phase firing sequences of PV interneurons, pyramidal cells, somatostatin-positive (SST) neurons, and CA3-activated feedforward inhibitory interneurons during population θ oscillations, eventually opening windows for synaptic plasticity during specific θ phases [γ band) and SST (θ band) interneurons [θ-to-γ coupling phase shift represents specific variations in such contributions. The observed Aβ injection-induced phase shift, as well as the impairment of HFS-induced phase shifts, may be due to its effect on PV interneurons [β25-35 reduces GABAB-dependent GirK channel activity in pyramidal neurons [θ-to-γ PAC was associated with working memory performance improvement in older adults [Intrahippocampal injection of Aθ phases . Perisomθ phases , 107. Phrneurons ; thereforneurons , 85, 106rneurons . There m neurons ; this mar adults .θ, γ, and ripple oscillatory network activity in the hippocampus are highly correlated with amyloid-induced synaptic plasticity dysfunction in a model of early amyloid-β pathology (similar to what has been reported in early AD stages). Taken together, these results show that intrahippocampal microinjection of soluble forms of Aβ affects synaptic variability and plasticity and modifies neural processing and network activity, changes that might underlie cognitive deficits observed in early AD models. Aβ-induced derailment of the tight functional relationship in hippocampal circuits between θ oscillation  and γ activity (controlled by PV interneurons) implies a dysregulation of the crosstalk of cholinergic, glutamatergic, and GABAergic systems during early AD stages, leading to impaired information processing and encoding. Therefore, the abnormality in θ-to-γ PAC hereby described is worth evaluating as a putative early biomarker of Aβ-induced synaptic dysfunction in AD, long before neurodegeneration is established.The present study results indicate that changes in the functional relationships between θ-to-γ PAC in behavioural models of AD that involve tauopathy and selective chronic neurodegeneration, as well as to test it in further clinical trials through noninvasive electrophysiological methods in patients with mild cognitive impairment and major neurocognitive disorder.AD is a chronic and complex neurological disorder that involves several mechanisms  additional to amyloid pathology. For that reason, it is important to start considering the role of


Abundant IFN-γ secretion, potent cytotoxicity, and major histocompatibility complex-independent targeting of a large spectrum of tumors make γδ T cells attractive candidates for cancer immunotherapy. Upon tumor recognition through the T-cell receptor (TCR), NK-receptors, or NKG2D, γδ T cells generate the pro-inflammatory cytokines TNF-α and IFN-γ, or granzymes and perforin that mediate cellular apoptosis. Despite these favorable potentials, most clinical trials testing the adoptive transfer of pharmacologically TCR-targeted and expanded γδ T cells resulted in a limited response. Recently, the TCR-independent activation of γδ T cells was identified. However, the modulation of γδ T cell’s effector functions solely by cytokines remains to be elucidated. In the present study, we systematically analyzed the impact of IL-2, IL-12, and IL-18 in parallel with TCR stimulation on proliferation, cytokine production, and anti-tumor activity of γδ T cells. Our results demonstrate that IL-12 and IL-18, when combined, constitute the most potent stimulus to enhance anti-tumor activity and induce proliferation and IFN-γ production by γδ T cells in the absence of TCR signaling. Intriguingly, stimulation with IL-12 and IL-18 without TCR stimulus induces a comparable degree of anti-tumor activity in γδ T cells to TCR crosslinking by killing tumor cells and driving cancer cells into senescence. These findings approve the use of IL-12/IL-18-stimulated γδ T cells for adoptive cell therapy to boost anti-tumor activity by γδ T cells. Known specific stimulators of Vδ21 family ,9,10,11.+ T cells under certain conditions [+ or CD8+ αβ T cells, γδ T cells have the potential of secreting cytokines more abundantly than these cells [In contrast to the kingdom of adaptive immunity where αβ T cells exclusively recognize peptide epitopes presented by MHC or CD1 molecules, the nature of molecules that (may) serve as (co) ligands for the Vδ2 TCR are only gradually deciphered. Recently, TCR signals have been demonstrated to be involved in the acquisition of a regulatory phenotype in human peripheral Vδ2nditions ,13,14,15nditions ,17,18,19nditions ,21,22. Inditions . While tse cells . In thisse cells ,25,26, ase cells .In order to elucidate whether TCR-bypass-stimulated γδ T cells can be a therapeutic tool for cancers, we systematically analyzed the effects of IL-2, IL-12, IL-18 and that of TCR stimulation on γδ T cells and directly compared their impact on proliferation, activation, cytokine production, and anti-tumoral activity in vitro.All procedures performed in this study were in accordance with the ethical standards and recommendations of the institutional ethic committee of the Medical Faculty of the Eberhard-Karls-University and the University Hospital of Tübingen and in accordance with the 1964 Helsinki declaration and its later amendments. The protocol was approved by the ethics committee under the following reference: 105/2017BO2. All subjects included in the study gave written informed consent.Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by Ficoll density gradient centrifugation . Written informed consent was obtained from each blood donor as described above. PBMCs and γδ T cells were cultured in RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin . IL-2 (20 or 50 IU/mL), IL-12, or IL-18 , or in combination, were added in designated experiments. + T cell isolation kit (Miltenyi Biotec), were used. The purity of γδ T cells was ≥ 97%.According to the manufacturer’s protocol, for positive isolation of γδ T cells; FITC-anti-TCRγδ antibody and anti-FITC Microbeads , for untouched isolation of γδ T cells; TCRγ/δ5 cells/well with 20 IU/mL IL-2 in a 96-well plate. As preparation for γδ TCR stimulation, culture plates were coated by overnight incubation with a TCRγδ antibody, IMMU510  at 4 °C. Then the cells were seeded at 5 × 105 cells/well into unmanipulated 96-well plates or those pre-coated with IMMU510, and cultured for 4 days. In addition, IL-12 and or IL-18 , IL-2 (50 U/mL) or combinations of them were added.γδ T cells were negatively or positively isolated and incubated overnight at 5 × 10® Cell Permeabilization Kit  was used. Cells were cultured at 37 °C for 1 h in 96-well plates in 200 μL culture medium in the presence of 1 μg Brefeldin A before staining. Samples were measured using a BD FACS CantoII flow cytometer and analyzed using Flow Jo Software.PE-IL-4, PE-IL-17A, APC-IFN-γ, PE-granzyme B, APC-perforin , PerCP-anti-CD3, PerCP-anti-CD4, APC-H7-CD8, PE-TCRαβ, APC-TNF-α, FITC/PE -TCRγδ and PE-TCRVδ2 , APC-NKG2D , FITC-TCRVγ9 and PE-TCRVδ2 , APC-FasL (Miltenyi Biotec), and FITC-Fas  were used according to the manufacturer’s instructions. For intracellular staining of cells, the FIX & PERMlow cells were calculated as proliferating cells.iIIsolated γδ T cells were labelled with CellTraceTM Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol and stimulated with cytokines and or the IMMU510 antibody. On day4 proliferation of γδ T cells was measured by CTV dye solution using a BD CantoII flow cytometer and analyzed using Flow Jo Software. CTV5 (A673) or 5 × 103 (RH30 and SY5Y) cells/well in a volume of 100 μL and allowed to passively adhere on the electrode surface. Post seeding, the E-Plate was transferred to the RTCA MP instrument inside a cell culture incubator and incubated for the first 24 h without effector cells. Then, γδ T cells untreated or treated with the cytokines and/or IMMU510 were applied onto the grown tumor cell layer, with different effector cell: target cell ratios  in a volume of 100 μL. Changes in impedance, which indicate the attachment and adherence of cells to the plate’s electrode, were reported every 24 h for the following 96 or 120 h. Data analysis was performed using the RTCA Software v1.2.1 (OLS).The xCELLigence RTCA MP instrument  was utilized for cytotoxicity assay. First, 50 μL of culture medium was added to each well of a 96 well E-plate (ACEA Biosciences) and the background impedance was measured. Target cells were seeded at a density of 1 × 103, 1.5 × 104, 9 × 103 cells/well, respectively and on the following day 1 × 104 cells of untouched or TCR-bypass-stimulated or IMMU510/IL-12/IL-18-stimulated γδ T cells were added onto the tumor cells. After three days of co-culturing, EdU-labeling was done for 16 h using a Click-iT EdU Alexa-Fluor 647 Flow Cytometry Kit (ThermoFisher Scientific). Then, cells were harvested and analyzed for cell cycle by detecting incorporated EdU together with staining propidium iodide (PI) by using an FxCycle™ PI/RNase Staining Solution (ThermoFisher Scientific). Where indicated, neutralizing antibodies for IFN-γ and/or TNF-α were included into the culture medium to show these cytokines’ contribution to senescence induction.To neutralize IFN-γ or TNF-α, we used anti-IFN-γ antibody  at a concentration of 50 ng/mL or anti-TNF-α antibody  at a concentration of 10 ng/mL or corresponding isotype controls. T24 (bladder carcinoma), MCF7 (breast cancer) and Wm115 (melanoma) cells were plated on a 48-well plate at the concentration of 3 × 104/well) were seeded to 8-well chambered cell culture slide (ThermoFisher Scientific) for overnight before being co-cultured with untouched or TCR-bypass-stimulated or IMMU510/IL-12/IL-18-stimulated γδ T cells (1.0 × 104/well). After 4 days of incubation, SA-β-galactosidase staining was carried out using the senescence histochemical staining kit  following the manufacturer’s instruction. Cells were incubated at 37 °C for 16 h and pictures were taken.MCF7 cells  and reverse transcription was carried out using Superscript III First Strand Synthesis Super Mix  according to the manufacturer’s protocol.p21): forward; 5′-AGGTGGACCTGGAGACTCTCAG-3′ and reverse; 5′-TCCTCTTGGAGAAGATCAGCCG-3′; CDKN2A (p16): forward; 5′-CTCGTGCTGATGCTACTGAGGA-3′ and reverse; 5′-GGTCGGCGCAGTTGGGCTCC-3′, p53: forward; 5′-CCCAACAACACCAGCTCCT-3′ and reverse; 5′-CTGGGCATCCTTGAGTTCC-3′.For the quantitative analysis of cytokine and transcription factor expression in γδ T cells or of senescence markers in tumor cells, real-time PCR was conducted with SYBR Green kit  in a BioRad C1000 Thermal cycler/CFX96 real-time System . Briefly, 5 ng cDNA was added to a final volume of 10 μL/reaction containing 1 × SYBR Green PCR Master Mix  and 100 nM of each primer. Thermal cycling conditions were: denaturation at 95 °C 10 min, 40 cycles: 95 °C/30 s, 59 °C/30 s and 72 °C, 1 min for elongation. Primers: GAPDH: forward; 5′-CCACATCGCTCAGACACCAT-3′ and reverse; 5′-GGCAACAATATCCACTTTACCAGACT-3′. GranzymeB: forward; 5′-TTCGTGCTGACAGCTGCTCACT-3′ and reverse; 5′-CTCTCCAGCTGCAGTAGCATGA-3′. Perforin: 5′-ACCAGCAATGTGCATGTGTCTG-3′ and reverse; 5′-GCCCTCTTGAAGTCAGGGT-3′, T-bet: forward; 5′-GCCTACCAGAATGCCGAGATTA-3′ and reverse; 5′-ACTCAAAGTTCTCCCGGAATCC-3′, Eomes: forward; 5′-GGCAAAGCGGACAATAACAT-3′ and reverse; 5′-AGCCTCGGTTGGTATTTGTG-3′, CDKN1A  and was 14-fold when simultaneously stimulated via IMMU510 compared to TCR stimulation alone-, which far exceeded the level induced by single IL-12 or IL-18 stimulation both in the absence and presence of TCR stimulus A. TNF-α production by γδ T cells seemed dependent on a combination of IL-2/IL-12 or IL-2/IL-12/IL-18. TNF-α was expressed by a slight proportion of γδ T cells (up to 5%) compared to IFN-γ and was remarkably induced in some donors with high inter-individual variances. In the presence of TCR stimulus, the combination of IL-2, IL-12 and IL-18 induced significant TNF-α production, which increased to about 30-fold of control  B. The frequency of IL-17-producing cells was significantly increased by TCR stimulation in context with IL-2/IL-12. In the absence of TCR stimulus, combinational treatment with IL-2/IL-12/IL-18 increased IL-17-producing cells to about 10-fold of no cytokine treatment to an absolute share of 0.8%. In the presence of a TCR stimulus, IL-2/IL-12 treatment significantly induced IL-17-producing cells to about 7-fold of no cytokine treatment with a share of 2.6% of total γδ T cells C.TCR stimulation significantly elevated the frequency of IL-4-producing γδ T cells in the context of no additional cytokines, or IL-2 alone. However, no significant difference was observed in the IL-4 production induced by the individual cytokine treatments, either with or without simultaneous TCR stimulus. The overall production of IL-4 was about one-tenth of IFN-γ production D. Thus IAs shown in The transcription factor T-bet and Eomes is critical for IFN-γ expression in the mouse ,31 and hTo examine the impact of cytokine- versus TCR-mediated stimulation on the expression of cytotoxic granules involved in tumor cell killing, we analyzed the expression level of granzyme B and perforin in differently stimulated γδ T cells by qPCR. As shown in The NK receptor NKG2D expressed on γδ T cells contributes to their tumor cell recognition as a major killer receptor and serves as a co-stimulatory signal for cytokine production and proliferation ,35,36,37γδ T cells exhibit tumor lysis against various types of tumors . After sAnti-tumor activity against these three tumor cell lines was highly dependent on the E:T ratios. As for A673, independently of the presence of a former TCR stimulus, at a high E:T ratio of 5:1, all cytokine treatment resulted in massive tumor lytic activity in γδ T cells. Interestingly, at the low E:T ratio of 0.5:1, only IL-2/IL-12/IL-18-pretreated γδ T cells exhibited a sustained anti-tumor effect, in fact irrespective of a simultaneous TCR stimulus .Regarding RH30, at a high E:T ratio of 5:1, a significant reduction in tumor growth by γδ T cells was seen after all cytokine treatments with TCR stimulus with the combination of IL-2/IL-12/IL-18 leading to the highest reduction of impedance. Without TCR stimulus, only the cells exposed to combination treatment of IL-2, IL-12 and IL-18 had an effective suppressive effect on tumor growth even at a low E:T ratio, i.e., of 0.5:1. On the other hand, the other cytokine treatments without TCR stimulus showed no tumor lytic effect even at a high E:T ratio of 5:1 .With regard to SH-SY5Y, TCR-stimulated γδ T cells showed no significant tumor growth inhibition even at a high E:T ratio of 5:1 except when combined with IL-2/IL-12/IL-18. In the absence of a TCR stimulus, only IL-2/IL-12/IL-18- treated γδ T cells exerted a clear and lasting anti-tumor effect, even at a low E:T ratio, i.e., of 0.5:1 .β-galactosidase and cell cycle regulators expressed by the tumor cells were analyzed by qPCR. MCF7 and Wm115 were included since breast cancer and melanoma constitute a major share of adult malignancies. T24 was chosen as in bladder cancer immunotherapeutic strategies have already shown significant effects, i.e., through the instillation of BCG into the bladder of the patient, leading to the activation of γδ T cells [H1 cytokines IFN-γ and TNF-α induces senescence [p21 (CDK inhibitor 1), an inhibitor of cyclin-dependent kinases essential for the control of the cell cycle in mammals, which amongst other functions, suppresses cancer growth when co-incubated with IL-2/IL-12/IL-18-stimulated γδ T cells, regardless of activation via IMMU510 on effector cells. Degree of upregulation in p21 was comparable to the positive control, i.e., pure TH1 effector cytokines IFN-γ/TNF-α  with T24 (bladder carcinoma), MCF7 (breast cancer) or WM115 (melanoma) cancer cells for 96 h and then evaluated the cell cycle status of the tumor cells by EdU-incorporation assay. Additionally we stained senescent cells with SA- T cells . In addinescence , we used-γ/TNF-α , indicat+, IL-17A+, and TNF-α+ cells did not differ between IMMU510 and B1. In addition, cell cycle status of Wm115 cells after co-culture with γδ T cells was almost identical between IMMU510 and B1 (As for the potential difference provoked by different anti- γδ TCR monoclonal antibodies, we directly compared clone IMMU510 and clone B1 in terms of cytokine production and cell cycle arrest induction in tumor cells by stimulated γδ T cells. As shown in 0 and B1 . This suTo reveal whether the senescence induction in tumor cells is dependent on IFN-γ/TNF-α produced by IL-2/IL-12/IL-18-stimulated γδ T cells, we neutralized IFN-γ/TNF-α by anti-IFN-γ antibody or anti-TNF-α antibody or both antibodies. As consistent with γδ T cells infiltrate a variety of cancers and modulate anti-tumor immunity ,42,43,44Corresponding with our findings, a previous study  reportedH1 immune effector cells [Efficient tumor-lytic activity after pharmacologic Vδ2 TCR-stimulation correlates with granzyme B and IFN-γ/TNF-α production in γδ T cells, parameters which we and others found to be enhanced when IL-2, IL-12, and IL-18 are added ,17,18,19or cells . Interesor cells , where hor cells ,50,51,52or cells . In synoTCR-independent, cytokine-instructed γδ T cell function clearly differs from adaptive T cell responses but fits well with γδ T cells sentinel function in the tissues. Arresting the cancer cells paves the way for both arms of the immune system; NK cells in a permanent “ready to kill” mode—wipe out any cell that cannot bind their inhibitory receptors by lack of MHC class I, while engulfment of senescent tumor cells by macrophages facilitates the establishment of adaptive immunity and long-lasting memory. This underpins the macrophages’ key role in the tumor microenvironment as the source of IL-12 and IL-18, thus forming the crystallization point for innate and adaptive immunity. + γδ T cells, so called DOT cells, have been established by Silva-Santos’s group and have already been proven to be safe [In summary, the present study investigates the modulation of γδ T cell effector functions solely by myeloid cytokines, IL-12, IL-18, or the combination of IL-2, IL-12, and IL-18 in the presence and absence of a pharmacological TCR stimulus. This comparative study demonstrates for the first time that TCR bypass stimulation i.e., γδ T cells stimulated solely with combined IL-2/IL-12/IL-18 is enough to induce γδ T cells that exhibit the same efficiency in anti-tumor activity as their counterparts after TCR crosslinking. Moreover, irreversible growth arrest in cancer cells suggests long-lasting tumor control. With regard to cell based adoptive therapy this study shows that in the course of three days the combination of IL-2/IL-12/IL-18 leads to 6-fold expansion of γδ T cells compared to control. These findings open an avenue for the generation of increased numbers of γδ T cells and approve/recommend the adoptive transfer of IL-2/IL-12/IL-18-stimulated γδ T cells for an unbiased clear boost of anti-tumor activity of γδ T cells in clinical settings. Protocols for the generation and transfer of cytokine-activated Vδ1 be safe . Analogo be safe  or donor be safe  can be i+ γδ T cells on αβ T cells is licensed by TCR signaling and correlates with signal strength [It has been known for some time that the TCR is involved in the acquisition of suppressive function of γδ T cells ,13,14 anstrength . Thus TCSince stimulation with IL-12 and IL-18 without simultaneous TCR stimulus induces potent anti-tumoral activity in γδ T cells comparable to that after TCR crosslinking i.e., efficient killing of tumor cells and driving cancer cells into senescence -, the use of IL-12/IL-18-stimulated γδ T cells is highly recommended for adoptive cell therapy to boost anti-tumor activity by γδ T cells.


Sorbus domestica) is a wild fruit tree with immense medicinal and industrial value. This study aimed at determining the four major groups of antioxidants  in rootstocks of Crataegus laevigata (genotypes O-LE-14 and O-LE-21), Aronia melanocarpa (genotypes O-LE-14 and O-LE-21), Chaenomeles japonica (genotype O-LE-9) and Cydonia oblonga (BA 29) (genotypes O-LE-14 and O-LE-21). Hyperoside  was the most abundant flavonoid compound, since its average content in the rootstocks of Crataegus laevigata (O-LE-21) was 180.68 ± 0.04 μg·g−1. Dihydrokaempherol was the least frequently found flavonoid compound, with an average concentration of 0.43 ± 0.01 μg·g−1 in all the rootstocks of plants considered in this study. Among the phenolic compounds, the most represented one was protocatechuic acid, with 955.92 ± 10.25 μg·g−1 in the rootstocks of Aronia melanocarpa (O-LE-14). On the other hand, the least represented p-Coumaric acid exhibited the average concentration of 0.34 ± 0.01 μg·g−1 in the plant rootstocks. Epicatechin was the most abundant catechin compound, with a content of 3196.37 ± 50.10 μg·g−1 in the rootstocks of Aronia melanocarpa (O-LE-14). The lowest represented catechin compound was epigallocatechin, with the average concentration of 0.95 ± 0.08 μg·g−1 in the screened plant rootstocks. From the procyanidin compounds, the most abundant one was procyanidin b2 in the rootstocks of Crataegus laevigata (O-LE-14), with a concentration of 5550.40 ± 99.56 μg·g−1. On the contrary, procyanidin a2, with an average concentration of 40.35 ± 1.61 μg·g−1, represented the least frequent procyanidin compound in all the plant rootstocks screened herein.The service tree ( Plants are known to contain flavonoids, phenolic acids and aldehydes, along with catechin and procyanidin derivatives. These compounds play a significant role in the growth, metabolism and stress tolerance of plants  and are Phenolic acids can be divided into two groups based on their solubility in water: soluble and non-soluble. The water soluble group of phenolic acids are present in vacuoles, whereas the non-soluble ones occur in cell walls . They prGrafting is a method of deliberating vegetative crop reproduction that is commonly used in horticultural practice to maintain and reproduce cultivars of plants that have beneficial properties for growers and for genetic engineering. This method is commonly used in agriculture to increase the yields, modify the industrial production and also in improving stress tolerance. However, the underlying mechanisms fostering the productivity of certain specific combinations of grafts remain largely unknown . When a 2 with epicatechin, evincing the highest interface concentration increase in quince-in-compatible unions [S. domestica.The detection of phenolic compounds at the initial growth stages following grafting is of particular interest, as these may block the vascular connection between the vascular cambium tissues of the rootstock and scion species . Additioe unions . These de unions , who obsSorbus domestica, Crataegus laevigata (genotypes O-LE-14 and O-LE-21), Aronia melanocarpa (genotypes O-LE-14 and O-LE-21), Chaenomeles japonica (genotypes O-LE-9) and Cydonia oblonga (BA 29) (genotypes O-LE-14 and O-LE-21), with one control (S. domestica).Accordingly, this study aimed at determining the selected flavonoid, phenolic acids and aldehydes, catechin and procyanidin compounds in seven grafting-modified kinds of rootstocks of The chemicals used in different procedures during the present study were purchased from Sigma-Aldrich  in ACS purity, unless noted otherwise. S. domestica (as Crataegus laevigata (genotypes O-LE-14 and O-LE-21), Aronia melanocarpa (genotypes O-LE-14 and O-LE-21), Chaenomeles japonica (genotypes O-LE-9) and Cydonia oblonga (BA 29) (genotypes O-LE-14 and O-LE-21)) with the control (seedlings of Sorbus domestica L.) were used. These rootstocks were planted in October 2017 , grafted in February/March 2018 and harvested in July 2018. After the samples had been taken out of the soil, the bark was manually removed from the graft union part and the phloem was instantly deep-frozen in a friction bowl with temperature −80 °C. Subsequently, the samples were crushed by friction in the presence of liquid nitrogen (−196°C).In this study, seven varieties of rootstocks potentially suitable for Sorbus domestica with the control (S. domestica) were used. Methanol  was used in the extraction of the flavonoid, the phenolic acids and aldehydes and the catechin and procyanidin compounds from the rootstocks of S. domestica. The samples were subjected to lyophilization for 24 h, 0.014 mBar vacuum and −55 °C . An equal weight (20 mg) of samples was taken from each of the seven kinds of rootstocks of S. domestica with the control . The samples were homogenized in a friction bowl with 1.0 mL of 80% methanol, and 0.05 to 0.1 g of sea sand, until evaporation. The homogenization was repeated twice. Thereafter, the samples were vortexed  for 1–2 min and were subsequently centrifuged at 25,000 rpm and 16 °C for 15 min . Later, each sample was filtered through a filter . Finally, the samples were pipetted out (400 µL) and analyzed using LC/MS. The results have been recalculated per 1.0 g of plant tissue.In this experiment, seven kinds of rootstocks of To determine the selected flavonoid, phenolic acids and aldehydes, and catechin and procyanidin compounds, a high-performance liquid chromatograph (HPLC Agilent 1200 Series) with a triple quadrupole and the mass detector (6460 Triple Quad) LC/MS equipped with ESI ionization were used. For the separation of the flavonoid, the phenolic acids and aldehydes and the catechin and procyanidin compounds, a Zorbax EC 18 column of 50 × 3.0 mm and a particle size of 2.7 μm was used prior to analyzing the compounds of interest. The measured concentration was the average of three measurements (injections) for each sample of triplicate. The acquired data between triplicates varied within RSD 5%.−1. The compounds were eluted with a linear upward gradient: 0 min (90% B), 2 min (40% B), 4 min (0% B) and 6 min (90% B). The triple quadrupole mass detector was operated in the negative mode. Gas (nitrogen) temperature was kept at 350 °C, the gas flow rate was set to 13 L·min−1, the pressure nebulizer had a value of 50 psi, the temperature of the focusing gas was 400 °C, the flow rate of the focusing gas was set at 12 L·min−1 and the voltage on the capillary tube amounted to 4000 V , 0.17 min (82% B), 0.51 min (70% B), 1.70 min (45% B), 4.00 min (45% B) and 6.00 min (82% B). The triple quadrupole mass detector was operated in negative mode. The gas (nitrogen) temperature was kept at 300 °C, the gas flow rate was set to 12 L·min−1, the pressure nebulizer had a value of 60 psi, the temperature of the focusing gas was 300 °C, the flow rate of the focusing gas was set at 11 L·min−1 and the voltage on the capillary tube amounted to 3500 V , 0.17 min (85% B), 0.51 min (75% B), 1.70 min (70% B), 4.00 min (70% B) and 6.00 min (85% B). The triple quadrupole mass detector was operated in the negative mode. The gas (nitrogen) temperature was 300 °C, the gas flow rate was set to 12 L·min−1, the pressure nebulizer had a value of 45 psi, the temperature of the focusing gas was 250 °C, the flow rate of the focusing gas was set at 11 L·min−1 and the voltage on the capillary tube amounted to 3500 V  and STATISTICA CZ Version 12.0 . The data are expressed as mean ± standard deviation (S.D.), unless otherwise noted (EXCEL). The statistical significance of the measured data was determined using STATISTICA CZ. The Anderson–Darling test was used to test the normality of the data. Differences with p < 0.05 were considered significant and were determined by using a one-way ANOVA test and a post-hoc Dunnett’s test, which was applied as a means of comparison to the control group. Moreover, for the exploratory data analysis (EDA) cluster analysis, the principle component analysis (PCA) and the correlation were done.The data were processed using MICROSOFT EXCELA LC/MS analysis was performed to determine different flavonoid, phenolic, catechin and procyanidin compounds in selected rootstocks of different plants. The determination of the occurrence and content of flavonoid, phenolic acids and aldehydes, catechin, and procyanidin compounds was done using high-performance liquid chromatography with mass detection. Hyperoside  was the most abundant flavonoid compound, followed by isoquercitrin (Q-3-glucoside) and rutin (Q-3-rutinoside) in selected rootstocks of plants studied herein .Crataegus laevigata (O-LE-21), Crataegus laevigata (O-LE-14), the control, Aronia melanocarpa (O-LE-14), Aronia melanocarpa (O-LE-21), Chaenomeles japonica (O-LE-9), BA 29 (O-LE-21) and BA 29 (O-LE-14) exhibited 180.68 ± 0.04 μg·g−1, 107.72 ± 0.08 μg·g−1, 75.30 ± 0.04 μg·g−1, 64.78 ± 0.12 μg·g−1, 57.62 ± 0.03 μg·g−1, 53.75 ± 0.03 μg·g−1, 38.20 ± 0.07 μg·g−1 and 8.60 ± 0.05 μg·g−1 of hyperoside  respectively. A higher concentration of hyperoside compared to the control was recorded with Crataegus laevigata (O-LE-14) and Crataegus laevigata (O-LE-21). Lower concentrations of hyperoside compared to the control were observed in other rootstocks of plants.The most represented flavonoid compound was hyperoside . −1 in Crataegus laevigata (O-LE-21), 98.84 ± 0.04 μg·g−1 in Crataegus laevigata (O-LE-14), 67.97 ± 0.03 μg·g−1 in the control, 62.90 ± 0.04 μg·g−1 in Aronia melanocarpa (O-LE-14), 58.87 ± 0.03 μg·g−1 in Aronia melanocarpa (O-LE-21), 49.46 ± 0.03 μg·g−1 in Chaenomeles japonica (O-LE-9), 33.09 ± 0.01 μg·g−1 in BA 29 (O-LE-21) and 6.58 ± 0.03 μg·g−1 in BA 29 (O-LE-14). A higher concentration of isoquercitrin compared to the control was recorded with Crataegus laevigata (O-LE-14) and Crataegus laevigata (O-LE-21). Lower concentrations of isoquercitrin compared to the control were observed in other rootstocks of plants.The second most represented flavonoid compound was isoquercitrin (Q-3-glucoside), with a concentration of 138.87 ± 0.05 μg·g−1 in the control, 73.19 ± 0.05 μg·g−1 in Crataegus laevigata (O-LE-21), 67.69 ± 0.04 μg·g−1 in Aronia melanocarpa (O-LE-14), 59.87 ± 0.04 μg·g−1 in Chaenomeles japonica (O-LE-9), 57.18 ± 0.03 μg·g−1 in BA 29 (O-LE-21), 42.39 ± 0.04 μg·g−1 in Crataegus laevigata (O-LE-14), 31.21 ± 0.04 μg·g−1 in Aronia melanocarpa (O-LE-21) and 8.10 ± 0.10 μg·g−1 in BA 29 (O-LE-14). Compared to the control, lower contents of rutin were observed in all the rootstocks of plants. The least most represented flavonoid compound was quercitrin (Q-3-rhamnoside). The control, Chaenomeles japonica (O-LE-9), Crataegus laevigata (O-LE-21), Aronia melanocarpa (O-LE-21), Aronia melanocarpa (O-LE-14), BA 29 (O-LE-21), Crataegus laevigata (O-LE-14) and BA 29 (O-LE-14) exhibited, respectively, 52.33 ± 0.04 μg·g−1, 35.22 ± 0.07 μg·g−1, 35.06 ± 0.05 μg·g−1, 29.29 ± 0.03 μg·g−1, 28.59 ± 0.03 μg·g−1, 25.66 ± 0.02 μg·g−1, 19.91 ± 0.04 μg·g−1 and 8.41 ± 0.10 μg·g−1. Lower concentrations of quercitrin compared to the control were observed in all the rootstocks of plants. The third most represented flavonoid compound was rutin (Q-3-rutinoside), with a concentration of 125.69 ± 0.03 μg·gProtocatechuic acid was the most frequent phenolic compound, followed by 3,4-Dihydroxybenzaldehyde, syringic acid, vanilic acid and vanillin in selected rootstocks of plants A. −1 in Aronia melanocarpa (O-LE-14), 872.77 ± 9.88 μg·g−1 in Crataegus laevigata (O-LE-21), 851.34 ± 11.25 μg·g−1 in BA 29 (O-LE-21), 790.29 ± 10.00 μg·g−1 in Aronia melanocarpa (O-LE-21), 735.19 ± 9.99 μg·g−1 in Chaenomeles japonica (O-LE-9), 676.79 ± 8.95 μg·g−1 in the control, 500.65 ± 12.51 μg·g−1 in Crataegus laevigata (O-LE-14) and 403.66 ± 9.85 μg·g−1 in BA 29 (O-LE-14). Compared to the control, a higher concentration of protocatechuic acid was recorded in Aronia melanocarpa (O-LE-14), Crataegus laevigata (O-LE-21), BA 29 (O-LE-21), Aronia melanocarpa (O-LE-21) and Chaenomeles japonica (O-LE-9). A lower concentration of protocatechuic acid occurred in other plant rootstocks when compared to the control.The predominant phenolic compound was protocatechuic acid, occurring with a concentration of 955.92 ± 10.25 μg·g−1 in Aronia melanocarpa (O-LE-21), 330.14 ± 10.01 μg·g−1 in Aronia melanocarpa (O-LE-14), 255.26 ± 12.11 μg·g−1 in Chaenomeles japonica (O-LE-9), 199.20 ± 8.55 μg·g−1 in Crataegus laevigata (O-LE-21), 197.32 ± 8.62 μg·g−1 in Crataegus laevigata (O-LE-14), 166.28 ± 7.99 μg·g−1 in BA 29 (O-LE-21), 146,16 ± 8.23 μg·g−1 in BA 29 (O-LE-14) and 117.58 ± 6.88 μg·g−1 in the control. Notably, compared to the control, lower concentrations of 3,4-Dihydroxybenzaldehyde were observed in all the rootstocks of plants in the present study.The second most represented phenolic compound was 3,4-Dihydroxybenzaldehyde, exhibiting a concentration of 339.96 ± 8.12 μg·gCrataegus laevigata (O-LE-14) rootstocks. In the case of syringic acid, the control exhibited 40.65 ± 1.21 μg·g−1), whereas 243.66 ± 7.93 μg·g−1 of syringic acid occurred in Crataegus laevigata (O-LE-14) rootstocks. The concentration of vanilic acid was very similar to that of syringic acid (238.90 ± 9.68 μg·g−1), and the control showed 47.94 ± 3.41 μg·g−1 of syringic acid. However, the concentration of vanilin, when compared to the control, was surprising. In the extract of rootstocks of Crataegus laevigata (O-LE-14), 242.78 ± 13.21 μg·g−1 of vanillin was observed, whereas its concentration in the control was only 2.73 ± 0.01 μg·g−1. The other flavonoid compounds revealed concentrations not exceeding 189 μg·g−1. Interestingly, syringic acid, vanilic acid and vanilin occurred in higher concentrations in the extract of Catechin and procyanidin compounds were also determined in selected rootstocks of plants. Catechin, epicatechin and epigallocatechin were selected among the catechins for analysis A.a.1. Epicatechin−1, 2806.78 ± 45.12 μg·g−1, 2243.11 ± 60.21 μg·g−1, 2085.88 ± 49.62 μg·g−1, 1800.49 ± 56.88 μg·g−1, 1741.14 ± 66.55 μg·g−1, 1562.60 ± 63.23 μg·g−1 and 1353.07 ± 49.99 μg·g−1 in Aronia melanocarpa (O-LE-14), Aronia melanocarpa (O-LE-21), Chaenomeles japonica (O-LE-9), Crataegus laevigata (O-LE-14), the control, Crataegus laevigata (O-LE-21), BA 29 (O-LE-14) and BA 29 (O-LE-21), respectively. A higher concentration of epicatechin compared to the control was recorded with Aronia melanocarpa (O-LE-14), Aronia melanocarpa (O-LE-21), Chaenomeles japonica (O-LE-9) and Crataegus laevigata (O-LE-14). On the other hand, compared to the control, lower concentrations of epicatechin were observed in other rootstocks of plants.The most often-represented catechin compound in the chosen rootstocks of plants was epicatechin. Epicatechin exhibited concentrations of 3196.37 ± 50.10 μg·ga.2. Catechin−1). However, 1302.59 ± 48.12 μg·g−1, 1093.79 ± 60.89 μg·g−1 and 821.44 ± 33.62 μg·g−1 concentrations of catechin were found in Aronia melanocarpa (O-LE-21), Aronia melanocarpa (O-LE-14) and Chaenomeles japonica (O-LE-9), respectively. For the other rootstocks of plants, the values were almost equal to the average 552.59 ± 35.22 μg·g−1. Catechin is considered to be the main cause of delayed lack of affinity.Catechin was another highly represented compound. The lowest concentration of catechin was measured in the control  (1.23 ± 0.03 μg·g−1) and Crataegus laevigata (O-LE-14) (1.23 ± 0.04 μg·g−1), whereas BA 29 (O-LE-21), compared to the control, exhibited the lowest concentration (0.48 ± 0.01 μg·g−1). The other rootstocks of plants exhibited concentrations lower than 1.00 ± 0.01 μg·g−1.Very low concentrations of epigallocatechin were measured in all the rootstocks of plants tested herein. The control exhibited a concentration value of 0.84 ± 0.01 μg·gb.1. Procyanidin b2Crataegus laevigata (O-LE-14) (5550.40 ± 99.56 μg·g−1), followed by 5452.11 ± 99.10 μg·g−1 of procyanidin b2 in Aronia melanocarpa (O-LE-14), 5238.47 ± 89.92 μg·g−1 in Aronia melanocarpa (O-LE-21), 4690.26 ± 87.55 μg·g−1 in Crataegus laevigata (O-LE-21), 4656.36 ± 88.21 μg·g−1 in Chaenomeles japonica (O-LE-9), 4093.37± 89.99 μg·g−1 in BA 29 (O-LE-21), 3822.64 ± 76.88 μg·g−1 in the control and 1852.16 ± 65.43 μg·g−1 of procyanidin b2 in BA 29 (O-LE-14). All the rootstocks of plants used herein had procyanidin b2 concentrations exceeding that of the control, except for the rootstocks of BA 29 (O-LE-14).Among the procyanidin compounds, procyanidin b2 was the most represented, followed by procyanidin c1 and procyanidin a2 B. The hib.2. Procyanidin c1−1. The concentration of procyanidin c1 has not yet been described in the grafting spot. As for procyanidin b1, the concentration was higher in all the tested rootstocks of plants compared to the control (181.69 ± 21.10 μg·g−1). Procyanidin b1 concentrations of 779.73 ± 29.92 μg·g−1, 740.95 ± 39.10 μg·g−1, 566.55± 37.55 μg·g−1, 475.10 ± 10.21 μg·g−1, 364.93 ± 11.55 μg·g−1, 200.76 ± 9.56 μg·g−1 and 196.42 ± 8.99 μg·g−1 were observed in Aronia melanocarpa (O-LE-21), Aronia melanocarpa (O-LE-14), Crataegus laevigata (O-LE-14), Chaenomeles japonica (O-LE-9), Crataegus laevigata (O-LE-21), BA 29 (O-LE-14) and BA 29 (O-LE-21), respectively.The second most represented compound from the procyanidin group was procyanidin c1. However, there were only statistically insignificant differences between the observed concentrations. The average procyanidin c1 concentration was 2 619.68 ± 101.92 μg·gb.3. Procyanidin a2Crataegus laevigata (O-LE-14) (68.48 ± 4.25 μg·g−1). The concentration of the other rootstocks of plants used was as follows: 56.11 ± 5.88 μg·g−1 in Crataegus laevigata (O-LE-21), 35.61 ± 1.25 μg·g−1 in Aronia melanocarpa (O-LE-14), 32.55 ± 1.99 μg·g−1 in Chaenomeles japonica (O-LE-9), 32.21 ± 1.25 μg·g−1 in BA 29 (O-LE-21), 28.5 ± 0.34 μg·g−1 in Aronia melanocarpa (O-LE-21) and 24.26 ± 1.85 μg·g−1 in BA 29 (O-LE-14). When compared to the control, a higher concentration of procyanidin a2 was recorded with Crataegus laevigata (O-LE-21) and Crataegus laevigata (O-LE-14). Compared to the control, a lower concentration of procyanidin a2 was observed in the other plant rootstocks.Compared to the control, the lowest concentration measured in the procyanidin group of compounds was that of procyanidin a2. Compared to the control, its highest concentration was measured in p < 0.001, employing a one-way ANOVA test and a post-hoc Dunnett’s test (p < 0.050), but the control group was still significantly different. Finally, no significant differences were found for quercetin between the control sample and BA 29 (O-LE-14) (p < 0.322); the same is true for dihydrokaempferol between the control sample and BA 29 (O-LE-21) (p < 1.000), Crataegus laevigata (O-LE-14) (p < 0.216) and Aronia melanocarpa (O-LE-14) (p < 0.819).The Anderson–Darling test revealed that all data had a normal distribution. The statistical significance of the differences between the control sample and the other samples, including the majority of anti-oxidative compounds, was tested at t’s test . In two Crataegus laevigata (O-LE-21) and Crataegus laevigata (O-LE-14) were not so similar according to the cluster analysis  revealed the most similar sample to the control sample, based on the cluster analysis A, to be analysis A. Regardanalysis B revealeanalysis B, the moanalysis .1 and chlorogenic acid [Generally, antioxidants have not yet been deeply investigated in connection with the affinity in grafted woods. Hudina et al. reported arbutin as the most abundant phenolic compound in the phloem above and below the graft union, followed by procyanidin Bnic acid . Assuncanic acid . The autnic acid .The research carried out by Canas et al.  on grapeC. leavigata variants  when compared to the control (good affinity). In both C. leavigata variants, the visible incompatibility was not observed, but future disaffinity cannot be ruled out. On the other hand, in variants with visible disaffinity (Chaenomeles japonica (O-LE-9)) and both Aronia melanocarpa (O-LE-14 and O-LE-21), the concentrations of these flavonoids did not exceed those of the control. In quercitine (Q-3-rhamnoside) and rutin (Q-3-rutinoside), none of the variants exceeded the concentration of the control, suggesting that they are not signaling an incompatibility between the service tree and the tested rootstocks.According to Hudina et al. , a higheAronia melanocarpa, the incompatible rootstocks.For the rest of the flavonoids, to the best of our knowledge, no information is available in connection with the disaffinity of incompatible scions and rootstocks. However, among these, the concentrations of naringenin chalocone, quercetin and homoeriodictyol, which are widespread in other species ,46,47, wAronia melanocarpa  and Chaenomeles japonica (O-LE-9)) had the highest concentrations of 3,4-Dihydroxybenzaldehyde, salicylic acid, chlorogenic acid and cryptochlorogenic acid compared to the control. High chlorogenic acid concentration was previously reported as the signal of disaffinity in pear trees [The variants with incompatible rootstocks  and Chaenomeles japonica (O-LE-9), but also in one out of two compatible variants: BA29 (O-LE-21) and Crataegus laevigata (O-LE-21). It seems that protocatechuic acid does not affect or signal the compatibility of the graft and the rootstock. The same could be stated for vanilin, vanilic and syringic acid, where the highest concentrations were observed at compatible rootstocks  may provoke future studies on the subject prior to an elucidation of the other compounds. Additionally, novel biochemical studies aimed at elucidating the biochemical mechanisms of affinities during grafting may also be done based on the clues revealed here in the present study.


The structures of five 4-(4-fluorophenyl)piperazin-1-ium salts of closely related aromatic acids all show different patterns of supramolecular assembly. N-(4-fluorophenyl)piperazine and aromatic acids. In 4-(4-fluorophenyl)piperazin-1-ium 2-fluorobenzoate monohydrate, C10H14FN2+·C7H4FO2−·H2O, (I), the components are linked by a combination of N—H⋯O and O—H⋯O hydrogen bonds to form a chain of alternating R46(12) and R66(16) rings. The ionic components of 4-(4-fluorophenyl)piperazin-1-ium 2-bromobenzoate 0.353-hydrate, C10H14FN2+·C7H4BrO2−·0.353H2O, (II), are linked by N—H⋯O hydrogen bonds to form a centrosymmetric four-ion aggregate containing an R44(12) motif, and these aggregates are linked into a molecular ladder by a single C—H⋯π(arene) hydrogen bond. 4-(4-Fluorophenyl)piperazin-1-ium 2-iodobenzoate, C10H14FN2+·C7H4IO2−, (III), crystallizes with Z′ = 2 in space group PR44(12) motif, and aggregates of this type are linked into a ribbon by a combination of C—H⋯O and C—H⋯π(arene) hydrogen bonds. The anion in 4-(4-fluorophenyl)piperazin-1-ium 2,4,6-trinitrophenolate, C10H14FN2+·C6H2N3O7−, (IV), shows clear evidence of extensive electronic delocalization from the phenolate O atom into the adjacent ring. The ions are linked by a combination of two-centre N—H⋯O and three-centre N—H⋯(O)2 hydrogen bonds to form centrosymmetric four-ion aggregates containing three types of ring. The ions in 4-(4-fluorophenyl)piperazin-1-ium 3,5-dinitrobenzoate, C10H14FN2+·C7H3N2O6−, (V), are again linked by N—H⋯O hydrogen bonds to form centrosymmetric R44(12) aggregates, which are themselves linked by a C—H⋯π(arene) hydrogen bond to form sheets, the stacking of which leads to the formation of narrow channels, containing disordered and/or mobile solvent entities. Comparisons are made with some related structures.The structures are reported for five salts formed by reactions between  N-(4-fluorophenyl)piperazine  has mild psychedelic and euphorigenic properties and, in this respect, it exhibits effects similar to those of the related compound N-(4-methoxyphenyl)piperazine (MeOPP), which has been used as a recreational drug piperazin-1-yl]butan-2-yl}pyridine-3-carboxamide, used in the treatment of autistic disorders -tartaric acids, the last of which crystallizes as a monohydrate. That work was a development from our structural studies –(III) are all different Figs. 1–3 ▸ ▸, In each of the cations in compounds (I)–(V), the piperazine ring adopts an almost perfect chair conformation: in every case the reference cation was selected as one having the ring-puckering angle θ –(III) and (V)et al., 1987Within the anion of compound (IV)et al., 1995n, 0.5, 0.5) alternate with n + 0.5, 0.5, 0.5), where n represents an integer in each case se Fig. 6. A weak se Fig. 6, having n + 0.5, 0.5, 0.5) alternate with rings containing two each of N—H⋯O and C—H⋯π(arene) hydrogen bonds and centred at , where n represents an integer in each case.The ionic components of compound (II)5) Fig. 7. If the 5) Fig. 7, in whicZ′ = 2  2 Fig. 3 and it i2 hydrogen bond within the selected asymmetric unit it Fig. 4, while ad Fig. 10: it is iInversion-related ion pairs in compound (V)f Fig. 11 as previ) Fig. 12. A secon) Fig. 12. As noteThus the cyclic et al., 2019It is of interest briefly to compare the structures reported here with those of some closely related compounds. One obvious comparison is between the monohydrated compound (I)et al., 2020P21/n, they are by no means isomorphous, and in (X) two of the nitro groups exhibit disorder. Whereas the N—H⋯O hydrogen bonds in (IV)et al., 2020et al., 2020et al., 20192 hydrogen bonds, while in (XII) a combination of N—H⋯O and C—H⋯O hydrogen bonds generates a chain of alternating The picrate salt of MeOPP (X) (Kiran Kumar N-(4-fluorophenyl)piperazine  in methanol (10 ml) were mixed with solutions of the appropriate acids (0.55 mol) in methanol (10 ml), viz. 2-fluorobenzoic acid (77.1 mg) for (I)v/v) for (I)v/v for (II)–(IV), and 1:1 v/v for (V)]. M.p. (I)All starting materials were obtained commercially, and all were used as received: solutions of 2), and with Uiso(H) = 1.2Ueq(C). For the H atoms bonded to N atoms, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N) giving the N—H distances shown in Table 1Uiso(H) = 1.5Ueq(O): the water atom O41 was refined isotropically and the refined occupancy of the water molecule was 0.353 (8). The largest maximum and minimum in the final difference map for (III)-3, respectively, were both close to atom I232, at distances of 0.92 and 0.77 Å, respectively. Examination of the difference map for compound (V)−3 lying on a twofold rotation axis, at , and the other, 1.94 e Å−3, lying in a general position at . No plausible solvent model could be developed based upon these two maxima and, accordingly, the data at this stage were subjected to the SQUEEZE procedure  global, I, II, III, IV, V. DOI: 10.1107/S2056989020008749/hb7928Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020008749/hb7928IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989020008749/hb7928IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989020008749/hb7928IVsup5.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2056989020008749/hb7928Vsup6.hklStructure factors: contains datablock(s) V. DOI: Click here for additional data file.10.1107/S2056989020008749/hb7928Isup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020008749/hb7928IIIsup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020008749/hb7928IVsup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020008749/hb7928Vsup10.cmlSupporting information file. DOI: 2012924, 2012923, 2012922, 2012921, 2012920CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Schistosoma japonicum (S. japonicum) infection-induced disease. γδ T cells comprise a small number of innate lymphocytes that quickly respond to foreign materials. In this study, the role of γδ T cells in the lung of S. japonicum-infected C56BL/6 mice was investigated. The results demonstrated that S. japonicum infection induces γδ T cell accumulation in the lung, expressing higher levels of CD25, MHCII, CD80, and PDL1, and lower levels of CD127 and CD62L (P < 0.05). The intracellular cytokines staining results illustrated higher percentages of IL-4-, IL-10-, IL-21-, and IL-6-producing γδ T cells and lower percentages of IFN-γ-expressing γδ T cells in the lung of infected mice (P < 0.05). Moreover, the granuloma size in lung tissue was significantly increased in Vδ−/− mice (P < 0.05). In the lung of S. japonicum-infected Vδ−/− mice, both type 1 and type 2 immune responses were decreased significantly (P < 0.05). In addition, the expression of CD80 and CD69 on B cells was decreased significantly (P < 0.05), and the SEA-specific antibody was markedly decreased (P < 0.05) in the blood of infected Vδ−/− mice. In conclusion, this study indicates that γδ T cells could adjust the Th2 dominant immune response in the lung of S. japonicum-infected mice.Many kinds of lymphocytes are involved in  Schistosoma, is the second most common endemic parasitic disease in the world (Schistosoma japonicum (S. japonicum), which infects both humans and mice, is commonly used to study Schistosoma infection in murine models. After S. japonicum infection, the parasite transitions through a number of tissues, including the lung. Once reaching the adult stage, the fluke lays eggs, which are deposited in the liver, lung, and intestinal wall, inducing granulomatous inflammation, and progressive fibrosis  cells, natural killer (NK) cells, natural killer T (NKT) cells, gamma delta T cells (γδ T cells), myeloid-derived suppressor cells (MDSCs), macrophages, and others , 6. Inteγδ T cells comprise a small number of innate lymphocytes that quickly respond to foreign materials without the need for antigen presentation . γδ T ceStaphylococcus aureus, γδ T cells in the lung are not only beneficial for the removal of bacteria, but also contribute to the repair of damaged tissue (S. japonicum infected mice (S. japonicum infection in C57BL/6 mouse lungs.Dendritic cells (DCs), monocytes/macrophages, and B cells are professional antigen presenting cells (APCs), which process and present foreign antigens, activate classic T and B cells, and modulate the type of immune response. Recent reports demonstrated that activated γδ T cells could increase the expression of CD80, CD86, and HLA-DR , acting d tissue . Importad tissue , 22. Theted mice , but the−/− mice  were obtained from JAX Stock (No. 002120). All animal experiments were performed in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (1988.11.1). All protocols for animal use were approved to be appropriate and humane by the institutional animal care and use committee of Guangzhou Medical University (2012-11). Every effort was made to minimize suffering.Six- to eight-weeks old female C57BL/6 mice were purchased from Traditional Chinese Medicine University of Guangzhou Animal Center , and Vδ−/− mice were percutaneously infected with 40 ± 5 S. japonicum cercariae obtained from infected Oncomelania hupensis snails  and euthanized 5 or 6 weeks after infection. Pathogen-free C57BL/6 and Vδ−/− mice were used as controls.C57BL/6 and VδS. japonicum cercariae, SEA, and SWA were obtained from Jiangsu Institute of Parasitic Diseases (China). SEA and SWA were sterile-filtered, and the endotoxin was removed using Polymyxin B agarose beads . A limulus amebocyte lysate assay kit  was used to confirm the removal of the endotoxin from SEA and SWA, as previously described , 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, and 50 μM 2-mercaptoethanol. Single lung cell suspensions were prepared for flow cytometry analysis.Mice were euthanized at week 5 or 6 post-infection. Before obtaining the lung tissue, blood was collected, mice were perfused with sterile saline to remove blood from the body. Excised lung tissue was cut into small pieces and incubated in 5 ml of digestion buffer  for 30 min at 37°C and 5% carbon dioxide. Digested lung tissue was pressed through a 200-gauge stainless-steel mesh and was then suspended in Hank's balanced salt solution (HBSS). Lymphocytes were isolated using Mouse Lymphocyte Separation Medium  density gradient centrifugation. Isolated cells were washed twice in HBSS and re-suspended at 1.5 × 10APC-cy7-conjugated anti-mouse CD3 (145-2C11), FITC-conjugated anti-mouse γδ TCR (GL3), PE-conjugated anti-mouse CD8 (53-6.7), PerCP-cy5.5-conjugated anti-mouse CD4 (RM4-5), PE-conjugated anti-mouse CD25 (3C7), Brilliant Violet 421-conjugated anti-mouse CD274 (MIH5), APC-conjugated anti-mouse CD273 (TY25), PE-conjugated anti-mouse CD183 (CXCR3-173), APC-conjugated anti-mouse CD184 (2B11/CXCR4), PerCP-conjugated anti-mouse CXCR6 (FAB2145C), PE-conjugated anti-mouse IL-4 (11B11), PE-conjugated anti-mouse IL-17 (TC11-18H10), PE-conjugated anti-mouse IL-10 (JES5-16E3), APC-conjugated anti-mouse IL-5 (TRFK5), PE-conjugated anti-mouse IL-2 (554428), and isotype-matched control monoclonal antibodies  were purchased from BD Pharmingen . PE-conjugated anti-mouse CD3 (145-2C11), Brilliant Violet 510-conjugated anti-mouse γδ TCR (GL3), APC-conjugated anti-mouse CD4 (RM4-5), PE-cy5-conjugated anti-mouse CD19 (6D5), PE-conjugated anti-mouse Vγ2 (A7R34), APC-conjugated anti-mouse CD69 (H1.2F3), PE-conjugated anti-mouse CD127 (A7R34), APC-conjugated anti-mouse CD62L (MEL-14), FITC-conjugated anti-mouse CD27 (LG.3A10), APC-conjugated anti-mouse IgD (11-26c.2a), FITC-conjugated anti-mouse MHCII (M5/114.15.2), PE-conjugated anti-mouse CD80 (16-10A1), PE-cy7-conjugated anti-mouse ICOS (C398.4A), APC-conjugated anti-mouse CX3CR1 (SA011F11), APC-conjugated anti-mouse IFN-γ (XMG1.2), Alexa Fluor 647-conjugated anti-mouse IL-21 (BL25168), PerCP-cy5.5-conjugated anti-mouse GM-CSF (MP1-22E9), PE-conjugated anti-mouse IL-1 (ALF-161), and APC-conjugated anti-mouse IL-6 (MP5-20F3) were purchased from BioLegend .Lungs were removed from mice, perfused three times with 0.01 M phosphate-buffered saline (pH = 7.4), fixed in 10% formalin, embedded in paraffin, and sectioned. Sections were then examined by light microscopy after standard hematoxylin-eosin (H&E) staining for visualization of cellular changes under microscopy (Olympus ix71).Paraffin sections of lung tissues from wild type (WT) and infected mice were rehydrated and boiled in Sodium citrate buffer (pH 6.0) for 30 min to induce antigen retrieval. After washing, tissue sections were blocked with 10% goat serum, followed by staining with rabbit anti-mouse CD3 antibody (Abcam) and hamster anti-mouse γδT antibody (Santa Cruz Biotechnology) at 4°C overnight. Sections were washed and incubated with Alexa Fluor 555–conjugated anti-rabbit IgG plus Alexa Fluor 488–conjugated anti-hamster IgG  for 30 min at 37°C in the dark. After a final washing, cover slips were mounted onto slides with fluoroshield mounting medium with DAPI (Abcam). Images were captured with Olympus microscope BX53 and processed with LSM Image Examiner software (Zeiss).6 cells per run) were analyzed using flow cytometry (Beckman CytoFLEX), and the results were analyzed using the CytExpert 1.1 (Beckman Coulter Inc.). The region of single nuclear cells was gated to ensure the dead cell and doublet exclusion. Isotype-matched controls for cell surface markers were included in each staining protocol.Cells were washed in PBS and blocked in PBS buffer containing 1% BSA for 30 min. Cells were then stained for 30 min at 4°C in the dark with conjugated antibodies specific for the cell surface antigens. Expression phenotypes of antibody-labeled lymphocytes (1 × 106/ml. Cells were then stimulated with phorbol 12-myristate 13-acetate (PMA)  and ionomycin  for 5 h . Brefeldin A  was added during the last 4 h of incubation. Cells were washed twice in PBS and stained for 30 min at 4°C in the dark with conjugated antibodies specific for the cell surface antigens CD3 and γδ TCR. Cells were fixed with Fixation and Permeabilization Solution (BD Biosciences) for 20 min at 4°C in the dark and permeabilized overnight at 4°C in PBS buffer containing 0.1% saponin (Sigma), 1% BSA, and 0.05% NaN3. Next, cells were stained with conjugated antibodies that specific for each cytokine. Expression phenotypes of antibody-labeled lymphocytes (1.5 × 106 cells per run) were analyzed using flow cytometry (Beckman CytoFLEX), and the results were analyzed using CytExpert 1.1 (Beckman Coulter Inc.). The region of single nuclear cells was gated to ensure the dead cell and doublet exclusion. Isotype-matched controls for cytokines were included in each staining protocol.Single lymphocyte suspensions were isolated from the lung, and the cell concentration was adjusted to 1.5 × 10−/− mice). Cell concentration was adjusted to 2 × 106/ml, and cells were stimulated with anti-CD3 + anti-CD28, soluble egg antigen (SEA) + anti-CD28, and soluble worm antigen (SWA) + anti-CD28; a non-stimulated group was used as a negative control. After mixing well, 200 μl was added to each well in 96-well-flat-bottomed ELISA plates, with each condition having three replicates. Cells were incubated in a cell incubator at 37°C and 5% CO2 for 72 h. The supernatant was collected and stored at −20°C.Single suspensions were isolated from the lungs of normal and naïve and infected WT and VδLevels of IFN-γ and IL-4 in cell supernatants of cultured cells were analyzed by ELISA according to the manufacturer's instructions . Briefly, the capture antibody was coated on the wells of ELISA plates at 4°C overnight. Wells were washed three times with PBST (0.05% Tween 20 contained PBS) and blocked using 10% fetal calf serum (FCS) in PBS at 37°C for 1 h. Wells were washed three times with PBST again. Supernatants were added and incubated at 37°C for 2 h. After washing, horseradish peroxidase (HRP) linked antibodies were added to wells for 1 h. After further washing, substrates were added and incubated for 20–30 min. Then, wells were read at 450 nm using a microplate reader .−/− mice from the inner canthal vein and cryopreserved at −20°C. SEA and SWA were diluted with sterile PBS solution to 80 μg/ml, and 100 μl of diluted SEA or SWA was added to each well in 96-well ELISA plates at 4°C overnight. The next day, ELISA plates were washed with PBS-T (containing 0.05% Tween) three times. Then, 200 μl of blocking solution  was added to each well and incubated at 37°C for 2 h. ELISA plates were washed with PBST for three times. 100 μl serum diluted with blocking solution  was added to each well. After 1 h incubation at 37°C, ELISA plates were washed with PBS-T three times, and 100 μl HRP-conjugated anti-mouse IgG detection antibody  was added for each well and incubated at 37°C for 1 h. ELISA plates were then washed again with PBS-T for five times before adding 100 μl of TMB to each well. After 30 min incubation at 37°C, 50 μl of 10% H2SO4 was added to each well to terminate the color reaction. Absorbance values of each well were measured by the microplate reader at 450 nm.Sera were extracted from both naïve and infected WT or VδP < 0.05 was considered to be statistically significant.Data were analyzed using SPSS 21.0. Statistical evaluation of the difference between means was assessed using one-way ANOVA. S. japonicum-infected mice, C57BL/6 mice were euthanized, and the lungs were removed 5–6 weeks after S. japonicum infection. Paraffin sections were made and stained with fluorescence-labeled monoclonal antibodies against mouse CD3 and γδ TCR, as well as DAPI, as described in the section Materials and Methods. As shown in +γδTCR+ cells (yellow fluorescence) were present in both naïve and S. japonicum-infected mice.To determine the existence of γδ T cells in the lung of S. japonicum-infected mice. Absolute cell numbers were quantified via microscopy, and the percentage of CD3+γδTCR+ cells was determined by FACS ; however, the absolute number of CD3+γδTCR+ cells after infection was significantly increased (P < 0.01), and the number of γδ T cells in per gram of lung tissue was decreased (P < 0.05).Single cell suspensions were also prepared from the lungs of both naïve and  by FACS . As show+γδTCR+ cells after infection, single lung cells from both normal naïve and infected mice were stained with different fluorescence labeled factors: CD3, γδ TCR, CD4, CD8, Vγ2, CD25, CD69, CD127, CD62L, MHCII, CD80, PDL1, PDL2, CXCR3, CXCR4, CXCR6, and CX3CR1. Cells were detected by flow cytometry, and the differences were compared. As shown in +γδTCR+ cells were gated first, and the expression of cell surface molecules was examined. We observed that the expression of CD25, MHCII, CD80, and PDL1 was significantly increased after infection (P < 0.05). Differences in the expression of chemokine receptors CXCR3, CXCR4, CXCR6, and CX3CR1 were also significant (P < 0.05), but the percentage of positive cells was limited. In contrast, the expression of CD127 and CD62L on γδ T was slightly decreased in infected mice (P < 0.05), while there was no significant difference in the expression of CD4, CD8, Vγ2, CD69, and PDL2 (P > 0.05).To study the characteristics of CD3+γδTCR+ cells to secrete cytokines after infection, pulmonary single cell suspensions were isolated from both naïve and infected mice and stimulated with PMA and ionomycin for 5 h, followed by staining for intracellular cytokines. As shown in +γδTCR+ cells were gated first, and then the expression of IL-4, IFN-γ, IL-10, IL-5, IL-17, IL-21, IL-2, GM-CSF, IL-1, and IL-6 were detected. As shown in +γδTCR+ cells in naïve mouse lung was significantly lower than in infected mouse lung (P < 0.05). However, the percentage of IFN-γ-expressing CD3+γδTCR+ cells in naïve mice was higher than in infected mice (P < 0.05), and the percentage of IL-17-expressing CD3+γδTCR+ cells in naïve mice was higher than in infected mice (P < 0.01). There was no significant difference in the expression of IL-5, IL-2, GM-CSF, or IL-1 between naïve and S. japonicum-infected lung-resident CD3+γδTCR+ cells (P > 0.05).To investigate the ability of CD3S. japonicum infection, both WT and Vδ−/− mice were infected at the same time. Mice were euthanized 5–6 weeks after infection, and lungs were removed. As shown in −/− mice was similar. However, the color was paler, and there was no visible granuloma in the lungs of naïve mice. Although the weight of the lung from infected mice were higher than that from the uninfected mice (P < 0.05), there was no obvious different in the weight of lung between the infected wild type (WT) and infected Vδ−/− mice (−/− mice (P < 0.05).To elucidate the role of γδ T cells in −/− mice . As show−/− mice, the content, active molecular expression and cytokine producing ability of T cells were compared (+ cells in infected (both WT and Vδ−/−) mice were higher than the uninfected mice .To investigate the modulating role of γδ T cells on T cells in the lung of naïve, infected WT and Vδcompared . The res+, CD3+CD4+, and CD3+CD4− cells in both naïve and infected WT and Vδ−/− mice were measured by FACS , and the expression of CD25 on CD3+ and CD3+CD4− cells was significantly increased in infected Vδ−/− mice compared to the infected WT mice (P < 0.05). The expression of CD62L on CD3+, CD3+CD4+, and CD3+CD4− cells were significantly decreased in the infected mice (P < 0.05). The expression of CD62L on CD3+ cells was significantly decreased in infected WT mice compared to infected Vδ−/− mice (P < 0.05).To examine the role of γδ T cells on activation of T cells, the expression of CD25, CD69, and CD62L on CD3 by FACS . As show−/− mice were prepared, stimulated, and stained as described in the section Materials and Methods. IL-4, IFN-γ, IL-17, and IL-21 were detected on CD3+, CD3+CD4+ and CD3+CD4− cells (+CD4+ cells (P < 0.05), and the expression of IL-4 in CD3+, CD3+CD4+, and CD3+CD4− cells from the infected Vδ−/− mice was higher than that in infected WT mice (P < 0.05). The expression of IFN-γ in the cell from infected mouse were also higher than the uninfected mice, especially in CD3+CD4− cells (P < 0.05), and the percentage of IFN-γ expressing CD3+ and CD3+CD4− cells from infected Vδ−/− mice was lower than in infected WT mice (P < 0.05). There was no difference in the expression of IL-17 and IL-21 on CD3+, CD3+CD4+ or CD3+CD4− cells between infected WT and infected Vδ−/− mice (P > 0.05).To examine the role of γδ T cells on the function of T cells, single cell suspensions from naïve and infected WT and Vδ4− cells . As show−/− mice. Cells were cultured in vitro for 72 h with different stimulators  and compared to non-stimulated negative controls. Levels of IL-4, IFN-γ, and IL-17 in the culture supernatants were detected by ELISA. As shown in P < 0.05) and the infected Vδ−/− mice (P < 0.05). In response to the three stimulation conditions, levels of IFN-γ secreted by lung cells in infected WT mice were significantly higher than in naïve WT mice (P < 0.05). And in the SEA + anti-CD28 and SWA + anti-CD28 stimulated groups, levels of IFN-γ secreted by lung cells in infected WT mice were higher than in infected Vδ−/− mice (P < 0.05).To further explore the effects of γδ T cells on the production of cytokines in other lung cells, single lung cell suspensions were isolated from naïve and infected WT and VδS. japonicum-infected mice, single nuclear cell solutions from both naïve and infected WT and Vδ−/− mice were prepared as described in the section Materials and Methods, and the content of CD19+ B cells was compared by FACS (+ B cells in infected (both WT and Vδ−/−) mice was lower than in naïve mice  mice , and there were no significant differences in the percentage and number of B cells between naïve C57BL/6 and naïve Vδ−/− mice (P > 0.05). Additionally, the number of B cells in per gram lung tissue were calculated and compared. As shown in P < 0.05), and the ratio in the infected Vδ−/− mice was higher than that in infected WT mice (P < 0.05).To investigate the effect of γδ T cells on B cells in the lung of  by FACS . Althoug−/−) mice were significantly higher than in uninfected mice (P < 0.05), and the expression of CD69 and CD80 on B cells from infected Vδ−/− mice was lower than from infected C57BL/6 mice (P < 0.05). In addition, levels of SEA and SWA specific IgG in serum from different experimental groups were detected by ELISA. As shown in −/− mice were significantly decreased compared to infected WT mice (P < 0.05). However, there were no differences in the levels of SWA-specific IgG between infected and Vδ−/− mice . CD25, CD69, CD127, and CD62L are T cell activation-associated molecules that mediate the proliferation and migration of leukocytes at inflammatory sites and the recirculation of lymphocytes between blood and lymphoid tissues (P < 0.01) and CD62L is decreased (P < 0.05) on the surface of infected γδ T cells , suggesting that the γδ T cells might be presented as APCs, which mediate the immune response in the lung of infected mice. PDL1 and PDL2 are inhibitory receptors on APCs that bind to PD-1 molecules expressed on active T cells, inducing T cell functional exhaustion , which could facilitate activation of antigen-specific T cells . It was haustion . We founS. japonicum-infected mice, the profile of inflammatory cytokines was examined and compared between naïve and infected mice. The results indicated that γδ T cells secrete more Th2 cytokines  and fewer Th1 cytokines (IFN-γ) in response to infection (+ T cells (+ γδ T cells decreased significantly after infection (P < 0.05). This is in contrast to the results we observed in the liver of S. japonicum-infected mice (P < 0.05), indicating that γδ T cells could modulate the response of B cells through this pathway.As an important innate lymphocyte, γδ T cells secrete a variety of cytokines to regulate the immune response . To furtnfection . It suggnfection , 34. γδ  T cells . Howeverted mice . This mited mice . In thisS. japonicum infection-induced pulmonary damage, Vδ gene knockout (Vδ−/−) mice were infected with S. japonicum. As shown in −/− mice was significantly increased (P < 0.05), validating the idea that γδ T cells might inhibit the accumulation of inflammatory cells in the lung of S. japonicum-infected mice.To validate the role of γδ T cells in S. japonicum-infected WT and Vδ−/− mice. As shown in +CD4− cells numbers (P < 0.05), percentage of IFN-γ-secreting CD3+CD4− cells (P < 0.05), and increased percentage of IL-4-secreting CD3+CD4+ cells (P < 0.05) could be found in infected Vδ−/− mice, compared to infected WT mice. It suggests that γδ T cells could enhance the differentiation of Th1 cell in this model. This result is in agreement with previous experiments, which demonstrated that γδ T cells have a protective effect in mouse infection and injury models . It implies that γδ T cells could enhance the type 2 immune response in infected mice, which might related to the molecules and cytokines expressing on the infection induced γδ T cells as showed in Furthermore, T cell response was compared between y models , 38. Conted mice . Althoug−/− mice as shown in −/− mice were increased compared to the infected WT mice . These results suggest that γδ T cells can enhance B cell activation. In addition, levels of SEA-specific IgG in the serum of Vδ−/− mice decreased significantly (P < 0.05), whereas SWA-specific IgG levels remained unchanged  demyelinating disease (Additionally, B cell response was investigated in infected Vδ disease .S. japonicum-infected mice, and we found that γδ T cells significantly adjust the type 2 immune response during the course of S. japonicum infection by the expression of specific molecules and production of specific cytokines.In conclusion, this study investigated the phenotypic and functional characteristics of γδ T cells in the lung of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by the institutional animal care and use committee of Guangzhou Medical University.HC, HX, and CJ performed most experiments and analyzed data with the support from JH. CJ, YF, QW, and QY performed animal experiment. AX and SX performed parasite infection experiment. HQ and YQ contributed to scientific planning. ZY and JH oversaw and designed the study. JH and JM wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Charge-assisted C—H⋯π hydrogen bonds along with π–π interactions stabilize the crystalline state. Intermolecular interactions are quantified by Hirshfeld surface analysis. 27H28N2OS, the naphthalene unit is planar to within 0.015 (2) Å and makes a dihedral angle of 14.24 (16)° with the thiazole ring. The anisole ring is inclined to the thiazole ring by a dihedral angle of 13.18 (23)°. The torsion angle between the heptyl chain and the anisole ring is 61.1 (4)°. These dihedral and torsion angles render the molecule non-planar. In the crystal, molecules are linked by C—H⋯π interactions, forming zigzag chains that propagate parallel to the b axis. The roles of the various intermolecular interactions in the crystal packing were clarified by Hirshfeld surface analysis; the most important contributions are from H⋯H (51.5%) and C⋯H/H⋯C (31.8%) contacts.In the title compound, C They are typically synthesized by the condensation of primary amines and active carbonyl groups. The pharmacological activities of Schiff bases include anti-bacterial, anti-fungal, anti-cancer and anti-viral properties -4-(heptyloxy)benzylidene]amino}-N--1,3-thiazol-2-amine, was obtained in crystalline form from the reaction of 2-amino-4-(2-naphthyl)thiazole with 4-N-(heptyloxy)benzaldehyde. We report here the synthesis and the crystal and molecular structures of the title compound, including a Hirshfeld surface analysis to assess the relative importance of the various intermolecular interactions on the crystal packing.One of the most important scaffolds in drug design and heterocyclic chemistry is thiazole, which is widely found in various pharmacologically active substances and in some naturally occurring compounds  −x, y + z + b axis  of the adjacent C5-C10 ring is 3.522 (4) Å.In the crystal, the most important intermolecular contacts are C—H⋯π interactions, which link screw-related molecules is Fig. 2. The diset al., 2016E)-1-(4-(heptyloxy)phenyl)-N-thiazol-2-yl)methanimine fragment revealed three hits. These structures are 4-(pyren-1-yl)-1,3-thiazol-2-amine , C19H12N2S -4-(4-chlorophenyl)-N--5--1,3-thiazol-2-amine, C19H12ClN5O2S  close contacts (Table 2b). The crystal packing is dominated by H⋯H contacts, representing van der Waals interactions (51.5% contribution to the surface), followed by C⋯H/H⋯C and S⋯H/H⋯S interactions, which contribute 31.8% and 7%, respectively benzaldehyde  dropwise to a constantly stirring solution of 2-amino-4-(2-naphthyl)thiazole  in 1-propanol (10 ml). The reaction was catalysed by NaOH (0.1 g) and was stirred for 3 h in a water bath at 278–283 K. The reaction was monitored with thin-layer chromatography (TLC) using a 3:7 ratio of ethyl acetate to n-hexane (Rf = 0.775). The precipitate was filtered, washed with 1-propanol, and dried. The resulting solid was further purified by recrystallization from ethanol and diethyl ether. Single crystals of the title compound suitable for X-ray analysis were obtained by slow evaporation of an acetone solution .The title compound was prepared by adding 4-Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other C-bound H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020007951/pk2631sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020007951/pk2631Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020007951/pk2631Isup3.cmlSupporting information file. DOI: 1979809CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: