Locally secreted BiTEs complement CAR T cells by enhancing killing of antigen heterogeneous solid tumors.

Bispecific T cell engagers (BiTEs) are bispecific antibodies that redirect T cells to target antigen-expressing tumors. We hypothesized that BiTE-secreting T cells could be a valuable therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) T cells. Glioblastomas represent a good model for solid tumor heterogeneity, representing a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III, and interleukin-13 receptor alpha 2 (IL13Rα2) on glioma tissues and cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13Rα2-targeting antibody, as the single chain variable fragments to generate new BiTE molecules. Compared with CAR T cells, BiTE T cells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE-secreting bivalent T cells compared with bivalent CAR T cells in a glioma mouse model at early phase, but not in the long term. In summary, BiTEs secreted by mono- or multi-valent T cells have potent anti-tumor activity in vitro and in vivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

Lightning Current Measurement Form and Arrangement Scheme of Transmission Line Based on Point-Type Optical Current Transducer.

Lightning strikes are the main cause of transmission line faults, and the accurate lightning current number is an important basis to guide scientific lightning protection. The use of sensors with excellent sensing performance to carry out lightning current monitoring on transmission lines is beneficial to the accumulation of key parameters of original lightning strikes, so it is necessary to study the lightning current measurement structure of transmission lines. In this paper, an optical current-sensing unit is used to monitor the lightning current on transmission lines. A measuring structure that can monitor key parameters of the lightning current under different types of lightning strikes is proposed. First, establish the lightning current return channel model and the equivalent model of the tower, study the influence of the transmission tower on the current in the lightning channel, and analyze the direct measurement position of the lightning current on the tower; establish the multi-wave impedance model of the tower, and build a multi-base tower. The simulation model of the transmission system analyzes the transmission characteristics of the lightning current on the transmission line and the lightning protection line in the case of different types of faults; from the perspective of the measurement of key parameters of the lightning current, the lightning current measurement structure of the transmission system is constructed to analyze different lightning strikes. The measurement effect of each monitoring position in the case of a lightning strike and the waveform characteristics of the fault current in the case of insulator flashover are analyzed.

Human amniotic epithelial stem cells inhibit microglia activation through downregulation of tumor necrosis factor-α, interleukin-1ß and matrix metalloproteinase-12 in vitro and in a rat model of intracerebral hemorrhage.

BACKGROUND AIMS: The molecular mechanisms by which stem cell transplantation improves functional recovery after intracerebral hemorrhage (ICH) are not well understood. Accumulating evidence suggests that microglia cells are activated shortly after ICH and that this activation contributes to secondary ICH-induced brain injury. We studied the effect of human amniotic epithelial stem cells (HAESCs) on microglia activation. METHODS: To study the effect of HAESCs in vitro, we used thrombin to activate the microglia cells. Twenty-four hours after thrombin treatment, the levels of tumor necrosis factor-α and interleukin-1ß were measured by enzyme-linked immunosorbent assay. In vivo, the HAESCs were transplanted into the rat striatum 1 day after collagenase-induced ICH. The expression levels of matrix metalloproteinase (MMP)-12 and microglia infiltration in the peri-hematoma tissues were determined 7 days after ICH through the use of reverse transcriptase-polymerase chain reaction and immunohistochemical analysis, respectively. RESULTS: Thrombin-activated microglia expression of tumor necrosis factor-α, interleukin-1ß and MMP-12 was significantly reduced through contact-dependent and paracrine mechanisms when the HAESCs were co-cultured with microglia cells. After transplantation of HAESCs in rat brains, the expression levels of MMP-12 and microglia infiltration in the peri-hematoma tissues were significantly reduced. CONCLUSIONS: Our observations suggest that microglia activation could be inhibited by HAESCs both in vitro and in vivo, which may be an important mechanism by which the transplantation of HAESCs reduces brain edema and ameliorates the neurologic deficits after ICH. Therefore, we hypothesize that methods for suppressing the activation of microglia and reducing the inflammatory response can be used for designing effective treatment strategies for ICH.

Inhibition of Extracellular Signal-Regulated Kinase Activity Improves Cognitive Function in Mice Subjected to Myocardial Infarction.

Cognitive impairment is a commonly observed complication following myocardial infarction; however, the underlying mechanisms are still not well understood. The most recent research suggests that extracellular signal-regulated kinase (ERK) plays a critical role in the development and occurrence of cognitive dysfunction-related diseases. This study aims to explore whether the ERK inhibitor U0126 targets the ERK/Signal Transducer and Activator of Transcription 1 (STAT1) pathway to ameliorate cognitive impairment after myocardial infarction. To establish a mouse model of myocardial infarction, we utilized various techniques including Echocardiography, Hematoxylin-eosin (HE) staining, Elisa, Open field test, Elevated plus maze test, and Western blot analysis to assess mouse cardiac function, cognitive function, and signal transduction pathways. For further investigation into the mechanisms of cognitive function and signal transduction, we administered the ERK inhibitor U0126 via intraperitoneal injection. Reduced total distance and activity range were observed in mice subjected to myocardial infarction during the open field test, along with decreased exploration of the open arms in the elevated plus maze test. However, U0126 treatment exhibited a significant improvement in cognitive decline, indicating a protective effect through the inhibition of the ERK/STAT1 signaling pathway. Hence, this study highlights the involvement of the ERK/STAT1 pathway in regulating cognitive dysfunction following myocardial infarction and establishes U0126 as a promising therapeutic target.

Echinacoside Prevents Sepsis-Induced Myocardial Damage via Targeting SOD2.

Echinacoside (ECH) is a prominent naturally occurring bioactive compound with effects of alleviating myocardial damage. We aimed to explore the beneficial effects of ECH against sepsis-induced myocardial damage and elucidate the potential mechanism. Echocardiography and Masson staining demonstrated that ECH alleviates cardiac function and fibrosis in the cecal ligation and puncture (CLP) model. Transcriptome profiling and network pharmacology analysis showed that there are 51 overlapping targets between sepsis-induced myocardial damage and ECH. Subsequently, chemical carcinogenesis-reactive oxygen species (ROS) were enriched in multiple targets. Wherein, SOD2 may be the potential target of ECH on sepsis-induced myocardial damage. Polymerase chain reaction results showed that ECH administration could markedly increase the expression of SOD2 and reduce the release of ROS. Combined with injecting the inhibitor of SOD2, the beneficial effect of ECH on mortality, cardiac function, and fibrosis was eliminated, and release of ROS was increased after inhibiting SOD2. ECH significantly alleviated myocardial damage in septic mice, and the therapeutic mechanism of ECH is achieved by upregulating SOD2 which decreased the release of ROS.

Reconstruction of the Large Frontal Sinus Defect With the Small Supporting Bone Pieces in Neurosurgical Anterior Skull Base Surgery.

BACKGROUND: Large defects of frontal sinus (FS) might be difficult to manage effectively. Mismanagement of the large defects could lead to serious postoperative complications in anterior skull base surgery. This study introduces a simple and reliable method applying small supporting bone pieces to cover or insert into large FS defects, then the large defect of FS was transformed into the small defects which was sealed by bone wax successfully. METHODS: Eleven patients underwent anterior skull base surgery for lesions or aneurysms, with the reconstruction of large FS defects by small supporting bone pieces. During craniotomy, mild violations of the FS mucosa were spared and sterilized, while severe violations required mucosal removal. Small supporting bone pieces were obtained from the inner plate of the bone flap and carefully covered or inserted into the large defects. The large defects were transformed into some small ones, which could be sealed effectively by bone wax. Demographic, intraoperative, and postoperative complication data were collected. RESULTS: Four patients had severe mucosal violations requiring removal, while 7 had mild violations sparing the mucosa. All underwent effective reconstruction with small supporting bone pieces. Median follow-up was 6 months. All cases showed effective FS morphology reconstruction, with no FS-related complications. CONCLUSIONS: Applying the small supporting bone pieces for large FS defect reconstruction can restore the FS morphology, preserve physiological function, and avoid postoperative complications.

Ultrasound-guided quadratus lumborum block: a powerful way for reducing postoperative pain.

Ultrasound-guided quadratus lumborum block (QLB) has been gradually carried out in clinical practice. However, some clinical evidence is contradictory, and no studies have summarized and described these results. The authors reviewed the anatomical characteristics of QLB and summarized the advantages and disadvantages of four puncture methods, so as to facilitate the clinical application of QLB.

Application of binasal speculum in endoscopic endonasal surgery for lesions in sellar region.

Objective: This study aims to access the efficacy of the binasal speculum in endoscopic endonasal surgery by evaluating clinical outcomes and examining its utility through process-based performance measures in both surgeons and assistants. Methods: A total of 59 patients with lesions in sellar region who underwent endoscopic endonasal surgery with the binasal speculum between September 2020 and March 2023 were included in this study. We assessed the extent of resection and documented postoperative nasal condition. Both surgeons and assistants completed post-use surveys to exam the utility of the binasal speculum and provide an overall grading. Results: Gross total resection (GTR) was successfully achieved in 94.9% (56/59) of patients, with subtotal resection (STR) observed in 5.1% (4/59) of patients. Intraoperative cerebrospinal fluid (CSF) leakage occurred in 23.7% (14/59) of cases, and nasoseptal flap (NSF) reconstruction was required in 55.9% (33/59) of cases. The nasal airway patency rapidly recovered within 14 days in a significant majority of patients (94.9%, 56/59). Moreover, olfactory function was regained within three months postoperatively by 91.5% (54/59) of patients. The overall post-use survey mean score was 26.4. Specifically, surgeons had a mean score of 26.5, while assistants had a slightly lower mean score of 26.2. The mean overall grading for the binasal speculum was 3. Both surgeons and assistants provided a mean overall grading of 3. Conclusion: The binasal speculum provides nasal mucosa protection and reduces the risk of an endoscopic lens clouded by mucosa or blood. It plays a crucial role in accurate guidance and facilitates the swift delivery of surgical instruments, particularly in left-blinded nasal cavities. The binasal speculum reduces the learning curve, especially for endoscopic surgeons with limited experience, while enhancing collaboration and coordination between surgeons and assistants during surgery. Both surgeons and assistants rated the overall utility of the binasal speculum as "excellent."

Blood vessel assessment using computed tomography : Effects of ephedrine on uterine artery.

BACKGROUND: Ephedrine increased blood pressure due to the contractile properties of resistance vessels. Excessive contraction of the uterine arteries might cause fetal distress. This study was to determine the diameter of the uterine artery of female New Zealand rabbits after the administration of different doses of ephedrine using CT. METHODS: Thirty-two rabbits were randomly divided into a control group (Group C), low dosage group (Group L), medium dosage group (Group M) and high dosage group (Group H). Normal saline and doses corresponding to the human dose of 7.5, 15 and 30 mg of ephedrine were injected respectively. The marginal ear and uterine artery diameters were measured 5, 10, 15, 30, and 45 min after injection using CT, and the hemodynamic changes were recorded. RESULTS: The increase in mean arterial pressure in group M (p = 0.009), and H (p = 0.013) was higher than that in group C. Compared with group C, substantial contraction of the marginal ear artery was observed at the three doses of ephedrine. There were no differences in the uterine artery diameter among groups L, M and C, However, in Group H, a significant contraction of the uterine artery compared with the other groups (p < 0.001) was observed. DISCUSSION: CT can be used to evaluate the effects of drugs on organs and blood vessels. Ephedrine can not only constrict the peripheral blood vessels but also do not affect the uterine artery at a dose of 15 mg or less. However, the dose should not exceed 30 mg, which may cause severe uterine artery depression.

High-Affinity Chimeric Antigen Receptor With Cross-Reactive scFv to Clinically Relevant EGFR Oncogenic Isoforms.

Tumor heterogeneity is a key reason for therapeutic failure and tumor recurrence in glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell (2173 CAR T cells) clinical trial (NCT02209376) against epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood-brain barrier into GBM active tumor sites. However, CAR T cell infiltration was associated only with a selective loss of EGFRvIII+ tumor, demonstrating little to no effect on EGFRvIII- tumor cells. Post-CAR T-treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations, despite loss of EGFRvIII. To address tumor escape, we generated an EGFR-specific CAR by fusing monoclonal antibody (mAb) 806 to a 4-1BB co-stimulatory domain. The resulting construct was compared to 2173 CAR T cells in GBM, using in vitro and in vivo models. 806 CAR T cells specifically lysed tumor cells and secreted cytokines in response to amplified EGFR, EGFRvIII, and EGFR-ECD mutations in U87MG cells, GBM neurosphere-derived cell lines, and patient-derived GBM organoids. 806 CAR T cells did not lyse fetal brain astrocytes or primary keratinocytes to a significant degree. They also exhibited superior antitumor activity in vivo when compared to 2173 CAR T cells. The broad specificity of 806 CAR T cells to EGFR alterations gives us the potential to target multiple clones within a tumor and reduce opportunities for tumor escape via antigen loss.

Molecular and Clinical Characterization of UBE2S in Glioma as a Biomarker for Poor Prognosis and Resistance to Chemo-Radiotherapy.

Glioblastoma is the most common and lethal brain cancer globally. Clinically, this cancer has heterogenous molecular and clinical characteristics. Studies have shown that UBE2S is highly expressed in many cancers. But its expression profile in glioma, and the correlation with clinical outcomes is unknown. RNA sequencing data of glioma samples was downloaded from the Chinese Glioma Genome Atlas and The Cancer Genome Atlas. A total of 114 cases of glioma tissue samples (WHO grades II-IV) were used to conduct protein expression assays. The molecular and biological characteristics of UBE2S, and its prognostic value were analyzed. The results showed that high UBE2S expression was associated with a higher grade of glioma and PTEN mutations. In addition, UBE2S affected the degree of malignancy of glioma and the development of chemo-radiotherapy resistance. It was also found to be an independent predictor of worse survival of LGG patients. Furthermore, we identified five UBE2S ubiquitination sites and found that UBE2S was associated with Akt phosphorylation in malignant glioblastoma. The results also revealed that UBE2S expression was negatively correlated with 1p19q loss and IDH1 mutation; positively correlated with epidermal growth factor receptor amplification and PTEN mutation. This study demonstrates that UBE2S expression strongly correlates with glioma malignancy and resistance to chemo-radiotherapy. It is also a crucial biomarker of poor prognosis.

Measuring Chimeric Antigen Receptor T Cells (CAR T Cells) Activation by Coupling Intracellular Cytokine Staining with Flow Cytometry.

Chimeric antigen receptor T cells (CAR T cells) therapy is one kind of immunotherapy that has revolutionally changed the landscape of immunotherapy and been approved by the FDA from 2017 for several blood malignancies. To bring new CAR T cells to clinic, every new CAR need to test in vitro for antigen recognition, tumor cell killing capacity, and off-target cytotoxicity effect. Detecting the secretion of cytokines upon engagement of CAR T cells with tumor antigens is routinely applied to assess these CAR functions. Here we describe coupling of intracellular cytokine staining and multicolor flow cytometry to measure CAR T cells activation upon antigen stimulation.

DNA Methylation Silences Exogenous Gene Expression in Transgenic Birch Progeny.

The genetic stability of exogenous genes in the progeny of transgenic trees is extremely important in forest breeding; however, it remains largely unclear. We selected transgenic birch (Betula platyphylla) and its hybrid F1 progeny to investigate the expression stability and silencing mechanism of exogenous genes. We found that the exogenous genes of transgenic birch could be transmitted to their offspring through sexual reproduction. The exogenous genes were segregated during genetic transmission. The hybrid progeny of transgenic birch WT1×TP22 (184) and WT1×TP23 (212) showed higher Bgt expression and greater insect resistance than their parents. However, the hybrid progeny of transgenic birch TP23×TP49 (196) showed much lower Bgt expression, which was only 13.5% of the expression in its parents. To elucidate the mechanism underlying the variation in gene expression between the parents and progeny, we analyzed the methylation rates of Bgt in its promoter and coding regions. The hybrid progeny with normally expressed exogenous genes showed much lower methylation rates (0-29%) than the hybrid progeny with silenced exogenous genes (32.35-45.95%). These results suggest that transgene silencing in the progeny is mainly due to DNA methylation at cytosine residues. We further demonstrated that methylation in the promoter region, rather than in the coding region, leads to gene silencing. We also investigated the relative expression levels of three methyltransferase genes: BpCMT, BpDRM, and BpMET. The transgenic birch line 196 with a silenced Gus gene showed, respectively, 2.54, 9.92, and 4.54 times higher expression levels of BpCMT, BpDRM, and BpMET than its parents. These trends are consistent with and corroborate the high methylation levels of exogenous genes in the transgenic birch line 196. Therefore, our study suggests that DNA methylation in the promoter region leads to silencing of exogenous genes in transgenic progeny of birch.

CCT128930 induces G1-phase arrest and apoptosis and synergistically enhances the anticancer efficiency of VS5584 in human osteosarcoma cells.

Osteosarcoma is a highly invasive primary malignant bone tumor. PI3K/mTOR pathway plays a key role in tumor progression, and inhibition of PI3K/mTOR pathway represents a novel strategy in therapy of osteosarcoma. CCT128930 and VS5584 are both inhibitors of PI3K/mTOR, but the anticancer mechanism of CCT128930 or/and VS5584 against human osteosarcoma cells remains unclear. Herein, U2OS and MG63 human osteosarcoma cells were cultured, and the anticancer effects of CCT128930 alone and the combined effect of CCT128930 and VS5584 against human osteosarcoma cells were explored. The results showed that CCT128930 as PI3K/mTOR inhibitor effectively inhibited p-p70 and p-AKT expression and dose-dependently inhibited U2OS cells and MG63 human osteosarcoma cells growth. Further studies found that CCT128930 triggered significant G-1 phase arrest and apoptosis, as convinced by the dysfunction of p27, Cyclin B1, Cyclin D1 and Cdc2, and PARP cleavage and caspase-3 activation. Moreover, CCT128930 treatment obviously enhanced VS5584-induced growth inhibition and apoptosis in human osteosarcoma cells, followed by enhanced PARP cleavage and caspase-3 activation. Taken together, CCT128930 alone or combined treatment with CCT128930 and VS5584 both effectively inhibited human osteosarcoma cells growth by induction of G1-phase arrest and apoptosis through regulating PI3K/mTOR and MAPKs pathways.

Natural borneol is a novel chemosensitizer that enhances temozolomide-induced anticancer efficiency against human glioma by triggering mitochondrial dysfunction and reactive oxide species-mediated oxidative damage.

BACKGROUND: Temozolomide (TMZ)-based chemotherapy represents an effective way for treating human glioma. However, its clinical application is limited because of its side effects and resistance to standard chemotherapy. Hence, the search for novel chemosensitizers to augment their anticancer efficiency has attracted much attention. Natural borneol (NB) has been identified as a potential chemosensitizer in treating human cancers. However, the synergistic effect and mechanism of NB and TMZ in human glioma have not been investigated yet. MATERIALS AND METHODS: U251 human glioma cells were cultured, and the cytotoxicity and apoptosis of NB and/or TMZ were examined by MTT assay, flow cytometric analysis and Western blot. Nude mice tumor model was also employed to evaluate the in vivo anticancer effect and mechanism. RESULTS: The results showed that the combined treatment of NB and TMZ more effectively inhibited human glioma growth via triggering mitochondria-mediated apoptosis in vitro, accompanied by the caspase activation. Combined treatment of NB and TMZ also caused mitochondrial dysfunction through disturbing Bcl-2 family expression. Further investigation revealed that NB enhanced TMZ-induced DNA damage through inducing reactive oxide species (ROS) overproduction. Moreover, glioma tumor xenograft growth in vivo was more effectively inhibited by the combined treatment with NB and TMZ through triggering apoptosis and anti-angiogenesis. CONCLUSION: Taken together, our findings validated that the strategy of using NB and TMZ could be a highly efficient way to achieve anticancer synergism.

Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas.

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells' efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.

Transplantation of bone marrow stromal cells enhances nerve regeneration of the corticospinal tract and improves recovery of neurological functions in a collagenase-induced rat model of intracerebral hemorrhage.

The reorganization of brain structures after intracerebral hemorrhage (ICH) insult is crucial to functional outcome. Although the pattern of neuronal rewiring is well-documented after ischemic stroke, the study of brain plasticity after ICH has been focusing on the enhancement of dendritic complexity. Here we hypothesized that functional restoration after ICH involves brain reorganization which may be favorably modulated by stem cell transplantation. In this study, bone marrow stromal cells (BMSCs) were transplanted into the perilesional sites of collagenase-induced ICH in adult rats one day after ICH injury. Forelimb functional recovery was monitored with modified limb placing and vibrissae-elicited forelimb placement tests. Anterograde and retrograde tracing were used to assess the reorganization of bilateral forelimb areas of the sensorimotor cortex. We found that in rats transplanted with BMSCs after ICH injury, axonal sprouting occurred in the contralateral caudal forelimb area of the cortex, and was significantly higher than in ICH rat models that received only the vehicle (P < 0.01). The number of positive neurons in the ipsilateral rostral forelimb area of the cortex of the BMSC group was 1.5-to 4.5-fold greater than in the vehicle group (P < 0.05). No difference was found between the BMSC and vehicle groups in hemispheric atrophy or labeled neurons in the ipsilateral caudal forelimb area (P = 0.193). Scores for improved functional behavior in the BMSC group were in accord with the results from histology. Neuronal plasticity of the denervated corticospinal tract at bilateral forelimb areas of the cortex in the collagenase-induced ICH rat models was significantly enhanced by BMSC transplantation. BMSC transplantation may facilitate functional recovery after ICH injury.