Novel targets for immunotherapy associated with exhausted CD8 + T cells in cancer.

In response to prolonged stimulation by tumour antigens, T cells gradually become exhausted. There is growing evidence that exhausted T cells not only lose their potent effector functions but also express multiple inhibitory receptors. Checkpoint blockade (CPB) therapy can improve cancer by reactivating exhausted effector cell function, leading to durable clinical responses, but further improvements are needed given the limited number of patients who benefit from treatment, even with autoimmune complications. Here, we suggest, based on recent advances that tumour antigens are the primary culprits of exhaustion, followed by some immune cells and cytokines that also play an accomplice role in the exhaustion process, and we also propose that chronic stress-induced hypoxia and hormones also play an important role in promoting T-cell exhaustion. Understanding the classification of exhausted CD8+ T-cell subpopulations and their functions is important for the effectiveness of immune checkpoint blockade therapies. We mapped the differentiation of T-cell exhausted subpopulations by changes in transcription factors, indicating that T-cell exhaustion is a dynamic developmental process. Finally, we summarized the novel immune checkpoints associated with depletion in recent years and combined them with bioinformatics to construct a web of exhaustion-related immune checkpoints with the aim of finding novel therapeutic targets associated with T-cell exhaustion in malignant tumours, aiming to revive the killing ability of exhausted T cells and restore anti-tumour immunity through combined targeted immunotherapy.

EphA2 recognizes Dermatophagoidespteronyssinus to mediate airway inflammation in asthma.

Most of the asthma with low Th2 is severe steroid-resistant asthma, the exact pathogenesis of which has not yet been fully elucidated. We found that IL-6 and IL-8 were highly expressed in the sputum supernatant of severe asthma and ephrin type-A receptor 2 (EphA2) was highly expressed on bronchial epithelial cells. So, is there a connection between these two phenomena? To clarify this issue, we stimulated bronchial epithelial cells 16HBE with Dermatophagoides pteronyssinus and its compontents LPS, respectively, and detected the activation of EphA2, activation of downstream pathways and secretion of inflammatory cytokines. A mouse asthma model was established, and the therapeutic effects of inhibiting or blocking EphA2 on mouse asthma were investigated. The results showed that D. pteronyssinus and its component LPS phosphorylated EphA2 on 16HBE, activated downstream signaling pathways STAT3 and p38 MAPK, and promoted the secretion of IL-6 and IL-8. After knockout of EphA2 on 16HBE, the activation of inflammatory pathways was attenuated and the secretion of IL-6 and IL-8 was significantly reduced. Inhibition or blockade of EphA2 on mouse airways resulted in a significant reduction in airway hyperresponsiveness and airway inflammation, and a significant decrease in the expression levels of IL-6, IL-17F, IL-1α, IL-1ß and TNF in bronchoalveolar lavage fluid and lung tissue. Our study uncovers a novel role for EphA2 expressed on airway epithelial cells in the pathogenesis of asthma; EphA2 recognizes D. pteronyssinus or its component LPS and promotes the secretion of IL-6 and IL-8 by airway epithelial cell, thereby mediating airway inflammation. Thus, it is possible to provide a new molecular therapy for severe asthma.

Affinity improvement of the fully human anti­TSLP recombinant antibody.

Thymic stromal lymphopoietin (TSLP) is a potentially important target for the treatment of asthma and malignancies. However, a fully human antibody reactive with TSLP is currently unavailable for clinical use. In a previous study, a human anti­TSLP­single­chain antibody variable fragment (anti­TSLP­scFv) 84 was selected by phage display from a constructed human scFv library. In the present study, a computer simulation method was developed using Discovery Studio 4.5 software, to increase the affinity of anti­TSLP­scFv­84. Specific primers were designed and mutated DNA sequences of anti­TSLP­scFvs were obtained by overlap extension PCR. The mutant scFvs were expressed in pLZ16 and affinity­enhanced anti­TSLP­scFv­M4 was screened using ELISA. However, in general the scFvs have low stability and short half­lives in vivo. Therefore, scFv­84 and scFv­M4 were inserted into eukaryotic expression vectors (pcDNA3.1­sp­Fc and PMH3EN­sp­Fc) and then transfected into 293F cells to express anti­TSLP­scFv­Fc. ELISA and western blotting results indicated the size of the anti­TSLP­scFv­Fc to be ~50 kDa. Binding of anti­TSLP­scFv­Fc­M4 to TSLP was enhanced compared with the pre­mutated scFv­Fc­84. The affinity of the mutated recombinant antibody was determined using the BIAcore technique and found to be ~10­fold greater than the pre­mutated antibody.

Iron-gold alloy nanoparticles serve as a cornerstone in hyperthermia-mediated controlled drug release for cancer therapy.

INTRODUCTION: The efficacy of a chemotherapy drug in cancer therapy is highly determined by the ability to control the rate and extent of its release in vivo. However, the lack of techniques to accurately control drug release drastically limits the potency of a chemotherapy drug. MATERIALS AND METHODS: Here, we present a novel strategy to precisely monitor drug release under magnetic stimulation. Methotrexate (MTX), an anticancer drug, was covalently functionalized onto iron-gold alloy magnetic nanoparticles (Fe-Au alloy nanoparticles or NFAs) through 2-aminoethanethiol grafting and the ability of this drug-nanoparticle conjugate (NFA-MTX) in limiting HepG2 (liver carcinoma) cell growth was studied. Well-dispersed NFAs were prepared through pyrolysis. RESULTS AND DISCUSSION: Transmission electron microscopy revealed the average nanoparticle size to be 7.22±2.6 nm, while X-ray diffraction showed distinct 2θ peaks for iron and gold, confirming the presence of iron and gold nanoparticles. Inductively coupled plasma mass spectrometry revealed that the amount of NFA-MTX conjugate ingested by HepG2 cancer cells was 1.5 times higher than that ingested by L929 fibroblasts, thereby proving a higher selective ingestion by cancer cells compared to normal cells. Fourier-transform infrared spectroscopy revealed the breakage of Au-S bonds by the heat generated under magnetic field stimulation to release MTX from the NFA-MTX conjugate, triggering a 95% decrease in cellular viability at 100 µg/mL. CONCLUSION: The ability of NFA-MTX to dissociate under the influence of an applied magnetic field provides a new strategy to induce cancer cell death via hyperthermia. Applications in drug delivery, drug development, and cancer research are expected.

Discovery of potent and selective matrix metalloprotease 12 inhibitors for the potential treatment of chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease associated with irreversible progressive airflow limitation. Matrix metalloproteinase-12 (MMP-12) has been characterized to be one of the major proteolytic enzymes to induce airway remodeling, destruction of elastin and the aberrant remodeling of damaged alveoli in COPD and asthma. The goal of this project is to develop and identify an orally potent and selective small molecule inhibitor of MMP-12 for treatment of COPD and asthma. Syntheses and structure-activity relationship (SAR) studies of a series of dibenzofuran (DBF) sulfonamides as MMP-12 inhibitors are described. Potent inhibitors of MMP-12 with excellent selectivity against other MMPs were identified. Compound 26 (MMP118), which exhibits excellent oral efficacy in the MMP-12 induced ear-swelling inflammation and lung inflammation mouse models, had been successfully advanced into Development Track status.

The role of hypothalamic AMP-activated protein kinase in ovariectomy-induced obesity in rats.

OBJECTIVE: Adenosine monophosphate-activated protein kinase (AMPK) acts as a cellular energy sensor, being activated during states of low energy charge. Hypothalamic AMPK is altered by hormonal and metabolic signals and mediates the feeding response. The aims of this study were to examine whether the phosphorylation of AMPKα in the hypothalamus is affected by ovariectomy (Ovx) and thus would be involved in the development of obesity in rats. METHODS: Body weight, food intake, hypothalamic phosphorylated AMPKα (pAMPKα) protein expression, and plasma leptin and adiponectin levels were measured in female rats after either Ovx or sham operations. These patterns were also observed after treatment with 17ß-estradiol, compound C, and leptin in Ovx rats. RESULTS: Compared with control rats, Ovx led to increased body weight and food intake at 2 to 8 weeks after operation. Meanwhile, plasma leptin and adiponectin levels and hypothalamic pAMPKα expression were significantly increased after Ovx. Replacement of estradiol significantly reversed these effects. Treatment with compound C, an AMPKα inhibitor, for 1 week produced a reduction in food intake, body weight, and plasma leptin and adiponectin levels. Meanwhile, these effects were reversed upon withdrawal of compound C. In addition, central injection of leptin also significantly reduced body weight, food intake, plasma leptin and adiponectin levels, and hypothalamic pAMPKα expression relative to those of the Ovx group. CONCLUSIONS: Increased hypothalamic pAMPKα expression may contribute to hyperphagia during the development of Ovx-induced obesity in rats.

Preliminary experience with anterior interbody titanium cage fusion for treatment of cervical disc disease.

This study evaluated the efficacy and safety of titanium cage implants in cervical reconstruction to treat cervical spondylosis. Surgical data covered a 4-year period from January 1999 to December 2002 and included 34 consecutive patients, 20 men and 14 women, with ages ranging from 27 to 84 years (mean, 57 years). Patients underwent anterior cervical microdiscectomy followed by interbody fusion with a titanium cage implant (rather than an autogenous iliac crest bone graft) at a single level ranging from C3 to C7. Twenty-one patients had a herniated intervertebral disc, nine had degenerative disc disease, and four had previous failed autograft fusion surgery that required revision. At clinical presentation, 26 patients had neck pain, 23 had radiculopathy, and nine had myelopathy. Diagnostic imaging studies included spinal dynamic roentgenography, computerized tomography, and magnetic resonance imaging. Lesions were located at C3-4 in seven cases, C4-5 in 14 cases, C5-6 in nine cases, and C6-7 in four cases. The follow-up period ranged from 7 to 48 months (mean, 26 months). Results revealed that the procedure was technically feasible. There were no intra- or postoperative complications. The most commonly used cage was 9 mm high. Imaging studies showed no cage instability, migration, or pseudarthrosis. Although mild subsidence (< 5 mm) was observed in three cases, these patients preserved adequate postoperative cervical lordosis and the subsidence did not preclude a good clinical result. The advantages of this procedure over a similar operation using traditional tricorticate bone graft are: no graft morbidity; shorter operation time (mean time saved, 35 minutes); reduced blood loss (average blood loss, 75 mL); and early postoperative ambulation (mean, 4.7 hospital days). Nearly all patients rapidly lost their neck pain (92%, 24/26) and radicular symptoms (87%, 20/23) after surgery. The recovery rate from myelopathy was 44% (4/9). Progressive bony shield formation over the anterior/posterior cortex (sentinel sign) indicated fusion in five cases.