Cyclosporine A-resistant CAR-T cells mediate antitumour immunity in the presence of allogeneic cells.

Chimeric antigen receptor (CAR)-T therapy requires autologous T lymphocytes from cancer patients, a process that is both costly and complex. Universal CAR-T cell treatment from allogeneic sources can overcome this limitation but is impeded by graft-versus-host disease (GvHD) and host versus-graft rejection (HvGR). Here, we introduce a mutated calcineurin subunit A (CNA) and a CD19-specific CAR into the T cell receptor α constant (TRAC) locus to generate cells that are resistant to the widely used immunosuppressant, cyclosporine A (CsA). These immunosuppressant-resistant universal (IRU) CAR-T cells display improved effector function in vitro and anti-tumour efficacy in a leukemia xenograft mouse model in the presence of CsA, compared with CAR-T cells carrying wild-type CNA. Moreover, IRU CAR-T cells retain effector function in vitro and in vivo in the presence of both allogeneic T cells and CsA. Lastly, CsA withdrawal restores HvGR, acting as a safety switch that can eliminate IRU CAR-T cells. These findings demonstrate the efficacy of CsA-resistant CAR-T cells as a universal, 'off-the-shelf' treatment option.

Adaptive Beam Splitting-Based Broadband Hybrid Precoding for Terahertz Massive MIMO.

Terahertz massive MIMO systems can be used in the local area network (LAN) scene of maritime communication and has great application prospects. To solve the problems of excessive beam training overhead in beam tracking and beam splitting in beam aggregation, a broadband hybrid precoding (HP) is proposed. First, an additional delayer is introduced between each phase shifter and the corresponding antenna in the classical sub-connected HP structure. Then, by precisely designing the time delay of the delayer and the phase shift of the phase shifter, broadband beams with flexible and controllable coverage can be generated. Finally, the simulation results verify that the proposed HP can achieve fast-tracking and high-energy-efficient communication for multiple mobile users.

Effects of different concentrations of metformin on osteoclast differentiation and apoptosis and its mechanism.

This study aimed to investigate the effect of metformin on osteoclast differentiation and apoptosis. Low concentration of metformin inhibited osteoclast differentiation and downregulated the expression of TRAP, RANK, Cathepsink, NFATC-1, MMP-9 and TRAF-6. High concentration of metformin promoted osteoclast apoptosis and upregulated the expression of Bax/Bcl-2 and caspase-3; BV/TV, BS/TV, Tb.N and BMD were increased while Tp.Sp decreased in the group of intraperitoneal metformin+femoral intramedullary osteoclast injection (Met+OC) compared with the control group, 1 nM metformin downregulated Akt, p44/42 MAPK, JNK, p38 MAPK phosphorylation, 5 nM metformin down regulated ERK and Akt phosphorylation. These results suggest that a low concentration of metformin inhibits osteoclast differentiation through PI3K/Akt and MAPK/ERK signaling pathway; high concentrations of metformin promote osteoclast apoptosis through PI3K/Akt and ERK signaling pathway.

Paving the Way Towards Universal Chimeric Antigen Receptor Therapy in Cancer Treatment: Current Landscape and Progress.

Chimeric antigen receptor (CAR) therapy has been proved effective in a stream of clinical trials, especially in hematologic malignancies. However, current CAR therapy is highly personalized as cells used are derived from patients themselves, which can be costly, time-consuming, and sometimes fails to achieve optimal therapeutic results due to poor quality/quantity of patient-derived cells. On the contrary, universal CAR therapy, which is based on healthy individuals' cells, circumvents several limitations of current autologous CAR therapy. To achieve the universality of CAR therapy, the allogeneic cell transplantation related issues, such as graft-versus-host disease (GVHD) and host-versus-graft activities (HVGA), must be addressed. In this review, we focus on current progress regarding GVHD and HVGA in the universal CAR therapy, followed by a universal CAR design that may be applied to allogeneic cells and a summary of key clinical trials in this field. This review may provide valuable insights into the future design of universal CAR products.

Expressions of IL-12 and its receptors in patients with lumbar disc herniation and their relationship with clinical efficacy.

This study aimed to explore the expressions of interleukin-12 (IL-12) and its receptors IL-23R and IL12RB2 in patients with lumbar disc herniation (LDH) before and after treatment and their relationship with clinical efficacy. A total of 172 LDH patients undergoing surgical treatment in Wuhan Third Hospital, Tongren Hospital of Wuhan University were enrolled as the study group, and 170 healthy subjects as the control group. 5 mL of fasting venous blood was taken before surgery (T0), 1 d (T1), 3 d (T2), 5 d (T3) and 7 d (T4) after treatment respectively. The concentrations of IL-12, IL-23R and IL12RB2 in the two groups were detected, and the correlation between them and the treatment duration and clinical efficacy was analyzed. The study group showed significantly higher serum IL-12, IL-23R and IL12RB2 than the control group before treatment (P < 0.001). In the study group, IL-12, IL-23R and IL-12RB2 were the lowest at T4 (P < 0.001), followed by T3 (P < 0.001). There was no significant difference in IL-23R at T1 and T0 (P > 0.050), and in IL12RB2 at T1 and T2 (P > 0.050). Spearman rank correlation showed that IL-12, IL-23R, IL12RB2 were negatively correlated with treatment duration in the study group (P < 0.001), and were positively correlated with clinical efficacy (P < 0.001). In conclusion, the concentrations of serum IL-12, IL-23R and IL12RB2 in LDH patients are significantly higher than those in normal controls. Moreover, the concentrations are closely related to the rehabilitation of patients and are expected to become therapeutic targets for LDH.

Influence of vitamin D deficiency on T cell subsets and related indices during spinal tuberculosis.

The present study investigated the effects of vitamin D deficiency on T cell subsets in patients with spinal tuberculosis. In addition, the influence of vitamin D deficiency was investigated on the expression of cytokines IL-1ß, IL-6 and TNF-α in intervertebral disc lesions of patients. One hundred and seventeen patients with spinal tuberculosis who received operative treatment in the Department of Orthopedics in Wuhan City Third Hospital from March 2012 to March 2015 were collected. The patients were divided depending upon vitamin D content into the control group (64 cases, vitamin D content <25 nmol/l) and experimental group (53 cases, vitamin D content >50 nmol/l). Immunofluorescence method was applied to determine the content of T cell subsets in both groups of patients. Intervertebral disc lesion tissues of two groups of patients were obtained during surgery then treated with HE staining and immunohistochemical staining. The values of average optical density obtained under light microscope were observed as the expression quantities of IL-1ß, IL-6 and TNF-α, to explore the relationship between vitamin D and the expression of cytokines. When vitamin D is lacking, the expression of T lymphocyte subsets in patients with spinal tuberculosis significantly decreased. Compared with experimental group, the difference was statistically significant (P<0.05). Further, the expression of cytokines IL-1ß, IL-6 and TNF-α in intervertebral disc lesion tissues of patients with spinal tuberculosis were significantly higher than those of patients with spinal tuberculosis whose vitamin D content was normal (P<0.05). In the control group, vitamin D content was negatively correlated with the expression of IL-1ß, IL-6 and TNF-α. The expression of T lymphocyte subsets in patients with vitamin D deficiency was significantly reduced, and the immune function decreased. The expression of IL-1ß, IL-6 and TNF-α in lesions were significantly higher than those of patients with normal vitamin D content. In addition, the lower the content of vitamin D was, the more active the expression of inflammatory factors were, which was not conducive to the recovery of tuberculosis lesions.

Cystathionine- γ-lyase promotes process of breast cancer in association with STAT3 signaling pathway.

Here we provide evidences to link cystathionine-γ-lyase (CSE) to the development of breast cancer. CSE expression is up-regulated in both breast cancers and breast cancer cell lines and results in proliferation and migration of breast cancer cells. CSE Function in breast cancer depends on the STAT3 signaling pathway, a regulator of critical cell functions including cell growth in a wide variety of human cancer cells via activating the expression of relative genes. STAT3 positively relates to CSE expression. It activates the CSE promoter via a direct binding to the promoter. Moreover, CSE could reversely regulate STAT3 expression and consequently enhance the effect of STAT3 on CSE. Taken together, these data demonstrate for the first time the roles of CSE in breast cancer leading to breast cancer development in association with STAT3 signaling pathway.

Formalin-induced short- and long-term modulation of Cav currents expressed in Xenopus oocytes: an in vitro cellular model for formalin-induced pain.

Xenopus oocytes expressing high voltage-gated calcium channels (Ca(v)) were exposed to formalin (0.5%, v/v, 5 min.) and the oocyte death and Ca(v) currents were studied for up to 10 days. Ca(v) channels were expressed with alpha(1)beta(1)b and alpha(2)delta sub-units and the currents (I(Ba)) were studied by voltage clamp. None of the oocytes was dead during the exposure to formalin. Oocyte death was significant between day 1 and day 5 after the exposure to formalin and was uniform among the oocytes expressing various Ca(v) channels. Peak I(Ba) of all Ca(v) and A(1), the inactivating current component was decreased whereas the non-inactivated R current was not affected by 5 min. exposure to formalin. On day 1 after the exposure to formalin, Ca(v)1.2c currents were increased, 2.1 and 2.2 currents were decreased and 2.3 currents were unaltered. On day 5, both peak I(Ba) and A(1) currents were increased. Ca(v)1.2c, 2.2 and 2.3 currents were increased and Ca(v)2.1 was unaltered on day 10 after the exposure to formalin. Protein kinase C (PKC) may be involved in formalin-induced increase in Ca(v) currents due to the (i) requirement for Ca(v)beta(1)b sub-units; (ii) decreased phorbol-12-myristate,13-acetate potentiation of Ca(v)2.3 currents; (iii) absence of potentiation of Ca(v)2.3 currents following down-regulation of PKC; and (iv) absence of potentiation of Ca(v)2.2 or 2.3 currents with Ser-->Ala mutation of Ca(v)alpha(1)2.2 or 2.3 sub-units. Increased Ca(v) currents and PKC activation may coincide with changes observed in in vivo pain investigations, and oocytes incubated with formalin may serve as an in vitro model for some cellular mechanisms of pain.

Protein kinase C isozyme-specific potentiation of expressed Ca v 2.3 currents by acetyl-beta-methylcholine and phorbol-12-myristate, 13-acetate.

Protein kinase C (PKC) is implicated in the potentiation of Ca v 2.3 currents by acetyl-beta-methylcholine (MCh), a muscarinic M1 receptor agonist or phorbol-12-myristate, 13-acetate (PMA). The PKC isozymes responsible for the action of MCh and PMA were investigated using translocation as a measure of activation and with isozyme-selective antagonists and siRNA. Ca v channels were expressed with alpha1 2.3, beta1b and alpha2delta subunits and muscarinic M1 receptors in the Xenopus oocytes and the expressed currents (I Ba) were studied using Ba2+ as the charge carrier. Translocation of PKC isozymes to the membrane studied by Western blot revealed that all eleven known PKC isozymes are present in the Xenopus oocytes. Exposure of the oocytes to MCh led to the translocation of PKC alpha whereas PMA activated PKC betaII and epsilon isozymes. The action of MCh was inhibited by Go 6976, an inhibitor of cPKC isozymes or PKC alpha siRNA. PMA-induced potentiation of Ca v 2.3 currents was inhibited by CG533 53, a PKC betaII antagonist, betaIIV5.3, a peptide translocation inhibitor of PKC betaII or PKC betaII siRNA. Similarly, epsilonV1.2, a peptide translocation inhibitor of PKC epsilon or PKC epsilon siRNA inhibited PMA action. The inhibitors of PKC increased the basal I Ba slightly. It is possible that some PKC isozymes have negative control over the I Ba. Our results implicate PKC alpha in the potentiation of Ca v 2.3 currents by MCh and PKC betaII and epsilon in the potentiation of Ca v 2.3 currents by PMA.

Inhibitory role of Ser-425 of the alpha1 2.2 subunit in the enhancement of Cav 2.2 currents by phorbol-12-myristate, 13-acetate.

Voltage-gated calcium channels (Ca(v)) 2.2 currents are potentiated by phorbol-12-myristate, 13-acetate (PMA), whereas Ca(v) 2.3 currents are increased by both PMA and acetyl-beta-methylcholine (MCh). MCh-selective sites were identified in the alpha(1) 2.3 subunit, whereas the identified PMA sites responded to both PMA and MCh (Kamatchi, G. L., Franke, R., Lynch, C., III, and Sando, J. J. (2004) J. Biol. Chem. 279, 4102-4109; Fang, H., Franke, R., Patanavanich, S., Lalvani, A., Powell, N. K., Sando, J. J., and Kamatchi, G. L. (2005) J. Biol. Chem. 280, 23559-23565). The hypothesis that PMA sites in the alpha(1) 2.2 subunit are homologous to the PMA-responsive sites in alpha(1) 2.3 subunit was tested with Ser/Thr --> Ala mutations in the alpha(1) 2.2 subunit. WT alpha(1) 2.2 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits. Inward current (I(Ba)) was recorded using Ba(2+) as the charge carrier. T422A, S1757A, S2108A, or S2132A decreased the PMA response. In contrast, S425A increased the response to PMA, and thus, it was considered an inhibitory site. Replacement of each of the identified stimulatory Ser/Thr sites with Asp increased the basal current and decreased the PMA-induced enhancement, consistent with regulation by phosphorylation at these sites. Multiple mutant combinations showed (i) greater inhibition than that caused by the single Ala mutations; (ii) that enhancement observed when Thr-422 and Ser-2108 are available may be inhibited by the presence of Ser-425; and (iii) that the combination of Thr-422, Ser-2108, and either Ser-1757 or Ser-2132 can provide a greater response to PMA when Ser-425 is replaced with Ala. The homologous sites in alpha(1) 2.2 and alpha(1) 2.3 subunits seem to be functionally different. The existence of an inhibitory phosphorylation site in the I-II linker seems to be unique to the alpha(1) 2.2 subunit.

Enhancement of substrate-gated Cl- currents via rat glutamate transporter EAAT4 by PMA.

Glutamate transporters (also called excitatory amino acid transporters, EAAT) are important in extracellular homeostasis of glutamate, a major excitatory neurotransmitter. EAAT4, a neuronally expressed EAAT in cerebellum, has a large portion (approximately 95% of the total L-aspartate-induced currents in human EAAT4) of substrate-gated Cl(-) currents, a distinct feature of this EAAT. We cloned EAAT4 from rat cerebellum. This molecule was predicted to have eight putative transmembrane domains. L-glutamate induced an inward current in oocytes expressing this EAAT4 at a holding potential -60 mV. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, significantly increased the magnitude of L-glutamate-induced currents but did not affect the apparent affinity of EAAT4 for L-glutamate. This PMA-enhanced current had a reversal potential -17 mV at extracellular Cl(-) concentration ([Cl(-)](o)) 104 mM with an approximately 60-mV shift per 10-fold change in [Cl(-)](o), properties consistent with Cl(-)-selective conductance. However, PMA did not change EAAT4 transport activity as measured by [(3)H]-L-glutamate. Thus PMA-enhanced Cl(-) currents via EAAT4 were not thermodynamically coupled to substrate transport. These PMA-enhanced Cl(-) currents were partially blocked by staurosporine, chelerythrine, and calphostin C, the three PKC inhibitors. Ro-31-8425, a PKC inhibitor that inhibits conventional PKC isozymes at low concentrations (nM level), partially inhibited the PMA-enhanced Cl(-) currents only at a high concentration (1 microM). Intracellular injection of BAPTA, a Ca(2+)-chelating agent, did not affect the PMA-enhanced Cl(-) currents. 4alpha-Phorbol-12,13-didecanoate, an inactive analog of PMA, did not enhance glutamate-induced currents. These data suggest that PKC, possibly isozymes other than conventional ones, modulates the substrate-gated Cl(-) currents via rat EAAT4. Our results also suggest that substrate-gated ion channel activity and glutamate transport activity, two EAAT4 properties that could modulate neuronal excitability, can be regulated independently.

Glutamate transporter type 3 attenuates the activation of N-methyl-D-aspartate receptors co-expressed in Xenopus oocytes.

We studied the regulation of N-methy-D-aspartate receptor (NMDAR) current/activation by glutamate transporter type 3 (EAAT3), a neuronal EAAT in vivo, in the restricted extracellular space of a biological model. This model involved co-expressing EAAT3 and NMDAR (composed of NMDAR1-1a and NMDAR2A) in Xenopus oocytes. The NMDAR current was reduced in the co-expression oocytes but not in oocytes expressing NMDAR only when the flow of glutamate-containing superfusate was stopped. The degree of this current reduction was glutamate concentration-dependent. No reduction of NMDAR current was observed in Na+-free solution or when NMDA, a non-substrate for EAATs, was used as the agonist for NMDAR. In the continuous flow experiments, the dose-response curve of glutamate-induced current was shifted to the right-hand side in co-expression oocytes compared with oocytes expressing NMDAR alone. The degree of this shift depended on the abundance of EAAT3 in the co-expression oocytes. Thus, the glutamate concentrations sensed by NMDAR locally were lower than those in the superfusates. These results suggest that EAAT3 regulates the amplitude of NMDAR currents at pre-saturated concentrations of glutamate to EAAT3. Thus, EAATs, by rapidly regulating glutamate concentrations near NMDAR, modulate NMDAR current/activation.

Role of alpha1 2.3 subunit I-II linker sites in the enhancement of Ca(v) 2.3 current by phorbol 12-myristate 13-acetate and acetyl-beta-methylcholine.

Potentiation of Ca(v) 2.3 currents by phorbol 12-myristate 13-acetate (PMA) or acetyl-beta-methylcholine (MCh) may be due to protein kinase C (PKC)-mediated phosphorylation of the alpha1 2.3 subunit. Mutational analysis of potential PKC sites unique to the alpha1 2.3 subunit revealed several sites in the II-III linker that are specific to MCh (Kamatchi, G., Franke, R., Lynch, C., III, and Sando, J. (2004) J. Biol. Chem. 279, 4102-4109). To identify sites responsive to PMA, Ser/Thr --> Ala mutations were made in potential PKC sites homologous to the alpha1 2.3 and 2.2 subunits, both of which respond to PMA. Wild type alpha1 2.3 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits and muscarinic M1 receptors. Inward current (I(Ba)) was recorded using Ba2+ as the charge carrier. Thr-365 of the I-II linker was identified as the primary site of PMA action, and this site also was required, along with the previously identified MCh-selective sites, for the MCh response. Ser-369 and Ser-1995 contributed to current enhancement only if Thr-365 also was available. Mutation of the essential sites to Asp increased the basal I(Ba) and caused a corresponding decrease in the PMA or MCh responses, consistent with possible regulation of these sites by phosphorylation. These results suggest that PMA and MCh both activate a pathway that can regulate the common PMA-sensitive sites in the I-II linker but that MCh also activates an additional pathway required for regulation of the MCh-unique sites, especially in the II-III linker.

Effects of local and intravenous anesthetics on the activity of glutamate transporter type 2.

Glutamate transporters may be important targets for anesthetic action in the central nervous system. The authors investigated the effects of the intravenous anesthetics, thiopental and ketamine, and the local anesthetics, lidocaine and bupivacaine, on the activity of glutamate transporter type 2, EAAT2. EAAT2 was expressed in Xenopus oocytes by injection of its mRNA. By using two-electrode voltage clamping, membrane currents were recorded after the application of L-glutamate (30 microM) in the presence or absence of various concentrations of anesthetics. Lidocaine and bupivacaine did not change glutamate-induced inward currents at the tested concentrations (1-1000 microM). Thiopental and ketamine also did not affect the activity of EAAT2 at the tested concentrations (0.3-300 microM). Our results suggest that the two commonly used local anesthetics (lidocaine and bupivacaine) and intravenous anesthetics (thiopental and ketamine) do not affect the activity of EAAT2 expressed in oocytes. EAAT2 may not be a target for these anesthetics.

The different responses of rat glutamate transporter type 2 and its mutant (tyrosine 403 to histidine) activity to volatile anesthetics and activation of protein kinase C.

Glutamate transporters play an important role in homeostasis of extracellular glutamate, a major excitatory neurotransmitter and a potential neurotoxin. In mammalian brain, glutamate transporter type 2 (EAAT2) is the most abundant form. Studies of molecular structures demonstrated that tyrosine 403 is critical in regulating the ion selectivity and transport mode of EAAT2. We hypothesized that wild type EAAT2 and its mutant at tyrosine 403 have different responses to volatile anesthetics, commonly used anesthetics that have been shown to affect glutamate transporter activity and decrease extracellular glutamate concentrations. We used site-directed mutagenesis and oocyte expression systems to test the hypothesis. Volatile anesthetics did not affect the activity of wild type EAAT2, isolated from rat hippocampus. When tyrosine 403 was replaced by histidine (Y403H), volatile anesthetics (isoflurane or halothane) at clinically relevant concentrations significantly decreased the transporter activity. Okadaic acid, a phosphatase inhibitor, significantly prolonged the isoflurane-induced inhibition. This inhibition was reversed by staurosporine and calphostin C, two protein kinase C (PKC) inhibitors, but not by the third PKC inhibitor, chelerythrine. Phorbol 12-myristate 13-acetate, a PKC activator, inhibited the activity of both wild type and Y403H EAAT2. This inhibition was also reversed by the same two PKC inhibitors but not by the third one. These results suggest that the switch of tyrosine 403 to histidine rendered EAAT2 sensitive to volatile anesthetics, a phenomenon that may require protein phosphorylation. PKC may be involved in the regulation of the activity of both wild type and Y403H EAAT2.

The effects of lidocaine on the activity of glutamate transporter EAAT3: the role of protein kinase C and phosphatidylinositol 3-kinase.

UNLABELLED: Using two electrode voltage clamps, we investigated the effects of lidocaine on one type of glutamate transporter, EAAT3, and the role of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in mediating the lidocaine effects. EAAT3 was expressed in Xenopus oocytes, and membrane currents were recorded after the application of L-glutamate (30 microM). Lidocaine increased glutamate-induced inward currents significantly at 2 concentrations (100 microM and 1 mM), but not at other concentrations. Lidocaine (100 microM) significantly increased the V(max), but not the K(m), of EAAT3 for glutamate compared with control. The action sites of lidocaine on EAAT3 seem to be intracellular, because only intracellularly injected QX314 (permanently charged lidocaine analog) increased the response. The combination of phorbol-12-myrisate-13-acetate, an activator of PKC, and lidocaine did not further increase the responses compared with phorbol-12-myrisate-13-acetate or lidocaine alone, although each of these three groups showed significantly bigger responses than controls. Three PKC inhibitors (staurosporine, calphostin C, and chelerythrine) did not affect the basal EAAT3 activity but abolished lidocaine-enhanced EAAT3 activity. Wortmannin (a specific PI3K inhibitor) inhibited EAAT3 basal activity and lidocaine-enhanced EAAT3 activity. Our results suggest that lidocaine enhances EAAT3 activity at certain concentrations and that PKC and PI3K may mediate these lidocaine effects. IMPLICATIONS: By using the Xenopus oocyte expression system, we investigated the effects of lidocaine on a glutamate transporter (EAAT3). Our findings suggest that lidocaine enhances EAAT3 activity at certain concentrations and that protein kinase C and phosphatidylinositol 3-kinase may mediate these lidocaine effects.