Environmental enrichment alleviates hyperalgesia by modulating central sensitization in a nitroglycerin-induced chronic migraine model of mice.

BACKGROUND: Chronic migraine (CM) is a debilitating neurofunctional disorder primarily affecting females, characterized by central sensitization. Central sensitization refers to the enhanced response to sensory stimulation, which involves changes in neuronal excitability, synaptic plasticity, and neurotransmitter release. Environmental enrichment (EE) can increase the movement, exploration, socialization and other behaviors of mice. EE has shown promising effects in various neurological disorders, but its impact on CM and the underlying mechanism remains poorly understood. Therefore, the purpose of this study was to determine whether EE has the potential to serve as a cost-effective intervention strategy for CM. METHODS: A mouse CM model was successfully established by repeated administration of nitroglycerin (NTG). We selected adult female mice around 8 weeks old, exposed them to EE for 2 months, and then induced the CM model. Nociceptive threshold tests were measured using Von Frey filaments and a hot plate. The expression of c-Fos, calcitonin gene-related peptide (CGRP) and inflammatory response were measured using WB and immunofluorescence to evaluate central sensitization. RNA sequencing was used to find differentially expressed genes and signaling pathways. Finally, the expression of the target differential gene was investigated. RESULTS: Repeated administration of NTG can induce hyperalgesia in female mice and increase the expression of c-Fos and CGRP in the trigeminal nucleus caudalis (TNC). Early exposure of mice to EE reduced NTG-induced hyperalgesia in CM mice. WB and immunofluorescence revealed that EE inhibited the overexpression of c-Fos and CGRP in the TNC of CM mice and alleviated the inflammatory response of microglia activation. RNA sequencing analysis identified that several central sensitization-related signaling pathways were altered by EE. VGluT1, a key gene involved in behavior, internal stimulus response, and ion channel activity, was found to be downregulated in mice exposed to EE. CONCLUSION: EE can significantly ameliorate hyperalgesia in the NTG-induced CM model. The mechanisms may be to modulate central sensitization by reducing the expression of CGRP, attenuating the inflammatory response, and downregulating the expression of VGluT1, etc., suggesting that EE can serve as an effective preventive strategy for CM.

BCL2A1 neoepitopes-elicited cytotoxic T lymphocytes are a promising individualized immunotherapy of pancreatic cancer.

Conventional treatments have shown a limited efficacy for pancreatic cancer, and immunotherapy is an emerging option for treatment of this highly fatal malignancy. Neoantigen is critical to improving the efficacy of tumor-specific immunotherapy. The cancer and peripheral blood specimens from human leukocyte antigen (HLA)-A0201 positive pancreatic cancer patient were subjected to next-generation sequencing and bioinformatics analyses were performed to screen high-affinity and highly stable neoepitopes. The activation of cytotoxic T lymphocytes (CTLs) by the mutBCL2A111-20 neoepitope targeting B-cell lymphoma 2-related protein A1 (BCL2A1) mutant epitope was investigated, and the cytotoxicity of mutBCL2A111-20 neoepitope-specific CTLs to pancreatic cancer cells was evaluated. The mutBCL2A111-20 neoepitope was found to present a high immunogenicity and induce CTLs activation and proliferation, and was cytotoxic to mutBCL2A111-20 neoepitope-loaded T2 cells and pancreatic cancer PANC-1-Neo and A2-BxPC-3-Neo cells that overexpressed mutBCL2A111-20 neoepitopes, appearing a targeting neoepitope specificity. In addition, high BCL2A1 expression correlated with a low 5-year progress free interval (PFI) among pancreatic cancer patients. Our findings provide experimental supports to individualized T-cell therapy targeting mutBCL2A111-20 neoepitopes, and provide an option of immunotherapy for pancreatic cancer.

Zero-Shot Fault Diagnosis for Smart Process Manufacturing via Tensor Prototype Alignment.

Identifying unseen faults is a crux of the digital transformation of process manufacturing. The ever-changing manufacturing process requires preset models to cope with unseen problems. However, most current works focus on recognizing objects seen during the training phase. Conventional zero-shot recognition methods perform poorly when they are applied directly to these tasks due to the different scenarios and limited generalizability. This article yields a tensor-based zero-shot fault diagnosis framework, termed MetaEvolver, which is dedicated to improving fault diagnosis accuracy and unseen domain generalizability for practical process manufacturing scenarios. MetaEvolver learns to evolve the dual prototype distributions for each uncertain meta-domain from seen faults and then adapt to unseen faults. We first propose the concept of the uncertain meta-domain and then construct corresponding sample prototypes with the guidance of class-level attributes, which produce the sample-attribute alignment at the prototype level. MetaEvolver further collaboratively evolves the uncertain meta-domain dual prototypes by injecting the prototype distribution information of another modality, boosting the sample-attribute alignment at the distribution level. Building on the uncertain meta-domain strategy, MetaEvolver is prone to achieving knowledge transferring and unseen domain generalization with the optimization of several devised loss functions. Comprehensive experimental results on five process manufacturing data groups and five zero-shot benchmarks demonstrate that our MetaEvolver has great superiority and potential to tackle zero-shot fault diagnosis for smart process manufacturing.

TLR2 Mediates Microglial Activation and Contributes to Central Sensitization in a Recurrent Nitroglycerin-induced Chronic Migraine Model.

Central sensitization is an important pathophysiological mechanism underlying chronic migraine (CM). Previous studies have shown that microglial activation and subsequent inflammation in the trigeminal nucleus caudalis (TNC) contribute to central sensitization. Toll-like receptor 2 (TLR2) is a receptor expressed on the membrane of microglia and participates in central sensitization in inflammatory and chronic pain; however, its role in CM is unclear. Therefore, this study investigated TLR2 involvement in CM in detail. Mice treated with recurrent nitroglycerin (NTG) were used as a CM model. Hyperalgesia was assessed using a 50% paw mechanical threshold and a 50% periorbital threshold on a Von Frey filament pain meter. Western blotting and immunofluorescence analyses were used to detect the expression of TLR2, microglia, c-fos and CGRP in TNC. The expression of inflammatory factors (IL-6, IL-1ß、 IL-10、TNF-α and IFN-ß1) was detected using quantitative real-time polymerase chain reaction (qRT-PCR). A selective TLR2 antagonist (C29) was systematically administered to observe its effect on hyperalgesia, microglia activation and the expression of c-fos, CGRP and inflammatory factors. Recurrent administration of NTG resulted in acute and chronic hypersensitivity, accompanied by upregulation of TLR2 expression and microglial activation in TNC. C29 partially inhibited pain hypersensitivity. C29 suppressed microglial activation induced by NTG administration. Inhibition of TLR2 reduced the expression of c-fos and CGRP in TNC after NTG treatment. C29 inhibited the expression of inflammatory mediators in TNC. These data showed that microglial TLR2 plays a critical role in the pathogenesis of CM by regulating microglial activation in TNC.

A c-Fos activation map in nitroglycerin/levcromakalim-induced models of migraine.

BACKGROUND: Chronic migraine is a common and highly disabling disorder. Functional MRI has indicated that abnormal brain region activation is linked with chronic migraine. Drugs targeting the calcitonin gene-related peptide (CGRP) or its receptor have been reported to be efficient for treating chronic migraine. The CGRP signaling was also shared in two types of chronic migraine models (CMMs). However, it remains unclear whether the activation of specific brain regions could contribute to persistent behavioral sensitization, and CGRP receptor antagonists relieve migraine-like pain in CMMs by altering specific brain region activation. Therefore, it's of great interest to investigate brain activation pattern and the effect of olcegepant (a CGRP receptor-specific antagonist) treatment on alleviating hyperalgesia by altering brain activation in two CMMs, and provide a reference for future research on neural circuits. METHODS: Repeated administration of nitroglycerin (NTG) or levcromakalim (LEV) was conducted to stimulate human migraine-like pain and establish two types of CMMs in mice. Mechanical hypersensitivity was evaluated by using the von Frey filament test. Then, we evaluated the activation of different brain regions with c-Fos and NeuN staining. Olcegepant was administered to explore its effect on mechanical hyperalgesia and brain region activation. RESULTS: In two CMMs, acute and basal mechanical hyperalgesia was observed, and olcegepant alleviated mechanical hyperalgesia. In the NTG-induced CMM, the medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), and the caudal part of the spinal trigeminal nucleus (Sp5c) showed a significant increase of c-Fos expression in the NTG group (p < 0.05), while pre-treatment with olcegepant reduced c-Fos expression compared with NTG group (p < 0.05). No significant difference of c-Fos expression was found in the paraventricular thalamic nucleus (PVT) and ventrolateral periaqueductal gray (vlPAG) between the vehicle control and NTG group (p > 0.05). In the LEV-induced CMM, mPFC, PVT, and Sp5c showed a significant increase of c-Fos expression between vehicle control and LEV group, and olcegepant reduced c-Fos expression (p < 0.05). No significant difference in c-Fos expression was found in vlPAG and ACC (p > 0.05). CONCLUSIONS: Our study demonstrated the activation of mPFC and Sp5c in two CMMs. Olcegepant may alleviate hyperalgesia of the hind paw and periorbital area by attenuating brain activation in CMMs.

Toll-like receptors and their role in neuropathic pain and migraine.

Migraine is a complex neurological disease of unknown etiology involving both genetic and environmental factors. It has previously been reported that persistent pain may be mediated by the immune and inflammatory systems. Toll-like receptors (TLRs) play a significant role in immune and inflammatory responses and are expressed by microglia and astrocytes. One of the fundamental mechanisms of the innate immune system in coordinating inflammatory signal transduction is through TLRs, which protect the host organism by initiating inflammatory signaling cascades in response to tissue damage or stress. TLRs reside at the neuroimmune interface, and accumulating evidence has suggested that the inflammatory consequences of TLR activation on glia (mainly microglia and astrocytes), sensory neurons, and other cell types can influence nociceptive processing and lead to pain. Several studies have shown that TLRs may play a key role in neuropathic pain and migraine etiology by activating the microglia. The pathogenesis of migraine may involve a TLR-mediated crosstalk between neurons and immune cells. Innate responses in the central nervous system (CNS) occur during neuroinflammatory phenomena, including migraine. Antigens found in the environment play a crucial role in the inflammatory response, causing a broad range of diseases, including migraines. These can be recognized by several innate immune cells, including macrophages, microglia, and dendritic cells, and can be activated through TLR signaling. Given the prevalence of migraine and the insufficient efficacy and safety of current treatment options, a deeper understanding of TLRs is expected to provide novel therapies for managing chronic migraine. This review aimed to justify the view that TLRs may be involved in migraine.

Maximizing the peroxidase-like activity of Pd@PtxRu4-x nanocubes by precisely controlling the shell thickness and their application in colorimetric biosensors.

Although the application of nanoscale artificial enzymes in various industries is an attractive way to circumvent the intrinsic drawbacks of natural enzymes, their catalytic constant (Kcat) as a critical reaction parameter is far from satisfactory. Presented here is the rational design and fabrication of a unique peroxidase mimic catalyst based upon Pd@PtxRu4-x (1 ≤ x ≤ 3) prepared by coating PtRu alloy as conformal, ultrathin shells on Pd nanocrystals. Benefiting from an optimal Pt/Ru ratio and well-defined {100} facets, together with confining the Pt-Ru alloy to a shell of averagely 3.3-atomic-layer thick (i.e. Pd@Pt-Ru3.3L), the nanocrystals exhibit the highest catalytic activity and kinetics (1.2 × 106 s-1), resulting in a significant increase of catalytic activity compared with the classical PtRu nanozyme (3.6 × 103 s-1) and horseradish peroxidase (4.0 × 103 s-1), respectively. The following density functional theory calculations demonstrate that the origin of the superior catalytic performance could be attributed to the modulation of the adsorption behavior of the key reaction intermediates on the surface. As a proof of concept, its peroxidase mimicking ability is adopted for sensing glucose and glutathione molecules in human serum, with a long linear range and high selectivity. This work opens new horizons for the future development of advanced catalysts based upon alloy nanocrystals for various applications.

Pannexin1 inhibits autophagy of cisplatin-resistant testicular cancer cells by mediating ATP release.

Pannexin1 (Panx-1) is a gap junction channel protein that mediates the release of intracellular ATP during autophagy, and thus plays an important role in tumor cell apoptosis and chemo-resistance. However, the role of Panx-1 in cisplatin-resistance of testicular cancer cells remains unclear. We found that cisplatin-resistant I-10 testicular cancer cell lines (I-10/CDDP) autophagy-associated proteins (p62, p-mTOR, mTOR and LC3) exhibited high levels of autophagy in their expression, while LC3-II expression was more significantly in the presence of lysosomal degradation blocked by chloroquine (CQ). Xenograft models using I-10/CDDP cells with knockdown ATG5 and ATG7 were established in mouse models and showed blockade of autophagic flux and inhibition of tumor growth. In addition, inhibition of Panx-1 by carbenoxolone (CBX) and probenecid (PBN), as well as shRNA-mediated knockdown promoted autophagy in the I-10/CDDP cells, which was accompanied by a decrease in the levels of extracellular ATP. In contrast, overexpression of Panx-1 decreased autophagy of I-10/CDDP cells and increased extracellular ATP levels. To further determine the effect of panx-1-mediated ATP release on the autophagy of I-10/CDDP cells, apyrase was used to hydrolyze the extracellular ATP. Apyrase promoted autophagy in I-10/CDDP cells city by decreasing extracellular ATP, regardless of Panx-1 expression. This study demonstrated for the first time that Panx-1-mediated ATP release inhibits autophagy of I-10/CDDP cells, which provides a potential therapeutic strategy for cisplatin-resistant testicular cancer.

Transcriptomics-based analysis of genes related to lead stress and their expression in the roots of Pogonatherum crinitum.

Revealing plants' tolerance and transport genes to heavy metal stress play an important role in exploring the potential of phytoremediation. Taking the heavy metal lead (Pb) hyperaccumulator plant Pogonatherum crinitum (Thunb.) Kunth as the research object, a hydroponic simulation stress experiment was set up to determine the physiological indicators such as antioxidant enzymes and non-enzymatic antioxidants in the roots of P. crinitum under different Pb concentrations (0, 300, 500, 1000, 2000 mg·L-1). RNA-Seq was performed, the Unigenes obtained by transcriptome sequencing were enriched and annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and the differential expression genes (DEGs) of root were screened and verified by quantitative real-time polymerase chain reaction (qRT-PCR). The results are as follows: with the increase of Pb concentration, superoxide dismutase (SOD), catalase (CAT), and ascorbic acid (AsA) content increased. Peroxidase (POD), malondialdehyde (MDA), and ascorbic acid-glutathione (AsA-GSH) cycles showed low promotion with high inhibition. A total of 38.21 Gb of bases were obtained by transcriptome sequencing, and the base quality of each sample reached Q20 and Q30, accounting for 90%, making the sequencing results reliable. Combined with transcriptome sequencing, functional annotation, and qRT-PCR validation results, 17 root Pb-tolerant genes of P. crinitum were screened out, which were related to antioxidation, transportation, and transcription functions. Moreover, qRT-PCR verification results under different Pb stress concentrations were consistent with the transcriptome sequencing results and changes in physiological indicators. In brief, the root of P. crinitum can adapt to the Pb stress environment by up-regulating the expression of related genes to regulate the physiological characteristics.

Detecting Abnormal Neuronal Activity in a Chronic Migraine Model by Egr1-EGFP Transgenic Mice.

Chronic migraine (CM) is a highly disabling neurological disorder characterized by recurrent headache accompanied by a variety of sensory and/or emotional symptoms. However, the mechanisms of migraine onset and its chronicity have not been elucidated. The present study was designed to search for brain regions and neurons that were abnormally activated by CM and might be related to its pathogenesis and different concomitant symptoms. CM models were established here by repeated intraperitoneal injection of nitroglycerin (NTG) every other day for 9 days to early growth response gene 1 (Egr1)-enhanced green fluorescent protein (EGFP) transgenic mice, which allowed monitoring of neuronal activities in the whole brain. CM-related behaviors were recorded through head grooming test and light aversion assay. Elevation of Egr1 expression signals was detected in trigeminal nucleus caudalis (TNC), primary somatosensory cortex (SSp), lateral amygdala nucleus (LA), primary visual area (VISp), and temporal association areas (TEa) 2 h after the last injection of NTG by immunofluorescence and digital slice scanning technology. Meanwhile, no change of Egr1 expression was found in auditory areas (AUD), CA1, ectorhinal area (ECT), piriform (PIR), and anterior cingulate area (ACC). Furthermore, with the strongest support by evidence-based medicine among the current limited oral treatments of CM, topiramate was administrated every day for 11 days from 2 days before the first NTG injection. The results showed that topiramate partially improved the photophobia behavior of CM models in the short-term with gradually weakened efficacy as the course of the disease prolonged. Meanwhile, NTG-induced increase in Egr1 expression was completely reversed in TNC, SSp, and VISp and partially reduced in LA and TEa by topiramate at the same time point mentioned above. In conclusion, the current results suggested that the abnormal hyperactivities in TNC, SSp and VISp were associated with the pathogenesis of CM.

Overexpression and biochemical characterization of a truncated endo-α (1 → 3)-fucoidanase from alteromonas sp. SN-1009.

Endo-fucoidanases are important in structural analysis of fucoidans and preparation of fuco-oligosaccharides. However their enzymological properties and analysis of degradation products are scarcely investigated. Truncated endo-α (1 â†’ 3)-fucoidanase Fda1 (tFda1B from Alteromonas sp. was overexpressed and characterized, showing highest activity at pH 7.0, 35 °C, and 1.0 M NaCl. Its Km and kcat were 3.88 ± 0.81 mg/mL and 0.82 ± 0.17 min-1. Fe3+ and Mn2+ enhanced activity by 100% and 19.5% respectively. Co2+ and Cu2+ completely inactivated tFda1B, whereas Ni2+, Mg2+, Zn2+, Pb2+, Ca2+, Ba2+ and Li+ decreased activity by 58.8%, 56.0%, 50.6%, 47.7%, 28.9%, 15.6% and 37.5%, respectively. Catalytic residues were identified through structure and sequence alignment, and confirmed by mutagenesis. Degradation products of Kjellmaniella crassifolia fucoidan by tFda1B were characterized by LC-ESI-MS/MS, confirming tFda1B belongs to endo-(1 â†’ 3)-fucoidanases, and backbone of K. crassifolia fucoidan is 1 â†’ 3 fucoside linkage. This endo-α (1 â†’ 3)-fucoidanase would be useful for elucidating fucoidan structures, and be used as a food enzyme.

Lead contamination alters enzyme activities and microbial composition in the rhizosphere soil of the hyperaccumulator Pogonatherum crinitum.

Pogonatherum crinitum is a promising lead (Pb) hyperaccumulator; however, the effects of Pb contamination on P. crinitum rhizosphere soil enzymatic activities and microbial composition remain largely unexplored. Thus, an indoor experiment was conducted by cultivating P. crinitum seedlings and exposing them to four Pb concentrations (0, 1,000, 2000 and 3000 mg/kg Pb). Protease, urease, acid phosphatase and invertase activities were determined using standard methods while soil bacterial composition was determined by 16 S rDNA sequencing. The results showed that rhizosphere soil acid phosphatase activity significantly increased with increasing Pb concentration, while urease activity was significantly greater in rhizosphere soil contaminated with 1000 and 2000 mg/kg than in the control. There was a clear shift in bacterial composition during phytoremediation by P. crinitum. Compared to the control, Bacteroidetes was more abundant in all Pb-contaminated soils, Actinobacteria was more abundant in 1000 mg/kg Pb-treated soil, and Firmicutes was more abundant in 3000 mg/kg Pb-treated soil. Positive correlations were observed between dominant bacterial phyla and soil enzyme activities. Metabolic pathways, such as ABC transporter, quinine reductase, and ATP-binding protein were significantly increased in rhizosphere soil bacteria with Pb contamination. In conclusion, Pb contamination differentially influenced the activities of rhizosphere soil enzymes, specifically increasing acid phosphatase and urease activities, and alters the dominance of soil bacteria through up-regulation of genes related to some metabolic pathways. The strong correlations between dominant bacterial phyla and enzymatic activities suggest synergetic effects on the growth of P. crinitum during Pb contamination.

[Down-regulation of pannexin 2 channel enhances cisplatin-induced apoptosis in testicular cancer I-10 cells].

OBJECTIVE: To investigate the effect of down-regulation of pannexin 2 (Panx-2) channels on cisplatin-induced apoptosis in I-10 cells. METHODS: The expression of Panx-2 protein in testicular cancer cells was detected with Western blotting. The testicular cancer cell line I-10 was transfected with two short hairpin RNA (shRNA1 and shRNA2) via Lipofectamine2000, the empty vector (NC group) or Lipofectamine2000 (blank control group), and the changes in the expression of Panx-2 was detected with Western blotting. The effects of transfection with a Panx-2 inhibitor on surviving fraction of the cells treated with cisplatin (16 µmol/L) for 24 h, 48 h and 72 h was assessed with MTT assay, and the clonogenic capacity of the cells was evaluated with colony-forming assay. At 8 h after incubation with 16 µmol/L cisplatin, AnnexinV/PI double staining was used to detect the early apoptosis of the cells. After 24 h of treatment with 16 µmol/L cisplatin, the cells were examined for expressions of caspase-3, Bcl-2 and Bax using Western blotting. RESULTS: The expression of Panx-2 was significantly increased in cisplatin-resistant I-10/DDP (P < 0.001) cells and Tcam-2/DDP (P < 0.01) cells as compared with I-10 cells and Tcam-2 cells. Transfection of I-10 cells with shRNA1 and shRNA2 resulted in significantly decreased Panx-2 expression (P < 0.05) and significantly reduced cell surviving fraction (P < 0.001). In the presence of cisplatin, the cells in NC group showed a higher clonogenic efficiency than those in shRNA1 and shRNA2 groups (P < 0.001). The early-stage apoptosis rate of the cells in shRNA1 and shRNA2 groups were significantly higher than that in NC group (P < 0.01). Panx-2 knockdown in I-10 cells significantly increased caspase-3 and Bax expressions (P < 0.05) and significantly decreased the expression of Bcl-2 (P < 0.01). CONCLUSIONS: Down-regulation of Panx-2 channel enhances cisplatin-induced apoptosis in cultured testicular cancer cells.

[Down-regulation of miR-205-5p enhances pro-apoptotic effect of 3-bromopyruvate on human nasopharyngeal carcinoma CNE2Z cells].

OBJECTIVE: To investigate the effect of down-regulation of miR-205-5p on 3-bromopyruvate-induced apoptosis in human nasopharyngeal carcinoma CNE2Z cells. METHODS: Nasopharyngeal carcinoma CNE2Z cells were transfected with miR- 205-5p-mimic or miR-205-5p-inhibitor, treated with 80 µmol/L 3-bromopyruvate alone, or exposed to both of the treatments. The proliferation of the treated cells was examined with MTT assay, and early apoptosis of the cells was detected using a mitochondrial membrane potential detection kit (JC-1). DAPI fluorescence staining was used to detect morphological changes of the cell nuclei and late cell apoptosis; Annexin V-FITC/PI double staining was employed to detect the cell apoptosis rate. Western blotting was used to detect the expressions of Bcl-2, Bax, Mcl-1 and Bak proteins. RESULTS: Exposure to 3-bromopyruvate significantly inhibited the proliferation of CNE2Z cells, and increasing the drug concentration and extending the treatment time produced stronger inhibitory effects. Treatment with 80 µmol/L 3-bromopyruvate for 24, 48 and 72 h resulted in inhibition rates of (45.7±1.21)%, (64.4±2.02)% and (78.3±1.55)% in non-transfected CNE2Z cells, respectively; the inhibition rates were (27.7±1.04)%, (34.8±2.10)% and (44.3±1.57)% in the cells transfected with miR-205-5p-mimic, and were (80.5 ± 0.94)%, (87.9 ± 0.50)% and (93.8 ± 1.16)% in cells transfected with miR-205-5p-inhibitor, respectively. The results of mitochondrial membrane potential detection showed that the relative proportion of red and green fluorescence decreased significantly in miR-205-5p-inhibitor-transfected cells with 3-bromopyruvate treatment. Combined treatment of the cells with 3-bromopyruvate and miR-205-5p-inhibitor transfection obviously increased nuclear fragmentation and nuclear pyknosis and significantly increased cell apoptotic rate as compared with the two treatments alone (P < 0.01), causing also decreased expressions of Bcl-2 and Mcl-1 proteins and increased expressions of Bax and Bak proteins. CONCLUSIONS: Inhibition of miR-205-5p enhances the proapototic effect of 3-bromopyruvate in CNE2Z cells possibly in relation to the down-regulation of Mcl-1 and Bcl-2 and the up-regulation of Bak and Bax proteins.

The biological functions of LGR5 in promoting non-small cell lung cancer progression.

BACKGROUND: The Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), which is used as a marker of adult stem cells and colorectal cancer stem cells (CSCs), is closely associated with the progression of non-small cell lung cancer (NSCLC). This study aimed to identify the clinical significance and biological function of LGR5 in NSCLC. METHODS: Quantitative reverse transcription polymerase chain reaction (RT-PCR) was applied to detect the expression of LGR5 and stemness-related genes in 22 NSCLC patients, and the clinical significance of LGR5 in NSCLC progression was estimated by statistical analysis. LGR5 overexpressing A549- and H1299-transfected cells were established, and CCK-8 and clone formation assays were used to test the proliferation ability. A wound-healing assay was utilized to clarify the migration ability. The invasion ability was confirmed via the Transwell assay kit. RESULTS: LGR5 expression was markedly higher in NSCLC tissues than in the matched adjacent normal tissues, and had a trend to associate with tumor size, lymph node metastasis, and TNM stage. The proliferation rates, clone formation rates, wound healing rates, number of invasive cells, and the NOTCH1 expression of the LGR5 overexpressing groups, were significantly higher than those of the control groups. CONCLUSIONS: LGR5 plays an essential role in NSCLC tumorigenesis and is closely associated with the proliferation, metastasis, and invasion of NSCLC cells. LGR5 may promote NSCLC progression via NOTCH1 and could be a new target for gene-targeted therapies for NSCLC.

Clinical Significance of B7-H3 Expression During the Progression of Hepatitis B Virus Infection.

B7-H3, one of the costimulatory members participating in checkpoint pathway, has been shown to be upregulated after hepatitis B virus (HBV) infection. To further explore the clinical significance of dynamic B7-H3 expression during the progression of HBV infection, we systematically investigated the expression pattern of B7-H3 and the correlation of B7-H3 expression with the ratio of T lymphocyte subsets and clinical parameters at different stages in the course of the disease. Flow cytometry and enzyme-linked immunosorbent assay data showed that soluble form of B7-H3 (sB7-H3) was positively correlated with the frequency of Treg cells in acute hepatitis B (AHB), chronic hepatitis B (CHB), and hepatocellular carcinoma patients with HBV infection (HBV-HCC). Membrane form of B7-H3 (mB7-H3) expressed on Treg cells and monocytes was positively correlated with the frequency of Treg cells in CHB. SB7-H3 had relationship with mB7-H3 expressed on Treg cells and monocytes at different stages during HBV infection, except for HBV-HCC. MB7-H3 expressed on Treg cells was positively correlated with that on monocytes in AHB, CHB, HBV-liver cirrhosis, and HBV-HCC. The B7-H3 expression was positively correlated with aspartate aminotransferase and alanine aminotransferase levels in CHB and sB7-H3 level was higher in late tumor/node/metastasis (TNM) stage in HCC. Higher mB7-H3 expression was associated with greater tumor size, later TNM stage, and worse prognosis in HBV-HCC indicated by immunohistochemistry. Taken together, these results suggested that B7-H3 might contribute to the progression of HBV infection by triggering inhibitory signals in effector T cells and it was closely associated with the progression and poor prognosis during HBV infection. B7-H3 could be utilized as a potential clinical indicator and a potential target for therapeutic strategies against HBV infection.

Retrospective Analysis of Thyroid Nodules: Thyroid Cancer Risk Factors in Suzhou, China.

BACKGROUND: Thyroid nodules are common, frequently discovered in clinical practice and the incidence has increased in recent decades. They are clinically important primarily due to their malignant potential, because 2 to 5% are malignant. Correct identification of the malignancy in thyroid nodules is a diagnostic challenge, leading to potentially unnecessary surgery in patients for whom final histology is benign. Because there is no accurate preoperative detection, it is very important to predict the risk of malignancy in patients with nodular thyroid disease. METHODS: The medical records of 405 patients who underwent surgery for nodular thyroid disease were retrospectively reviewed. Then clinical parameters and preoperative serum markers were compared between benign thyroid nodular disease and thyroid cancer groups. RESULTS: Younger than 40 years (OR 2.14, 95% CI 1.02 - 4.47, p = 0.044), preoperative TSH levels equal to or higher than 1.79 mIU/L (OR 1.76, 95% CI 1.05 - 2.95, p = 0.033), TgAb positivity (OR 2.59 95% CI 1.25 - 5.37, p = 0.01) and nodules less than or equal to 1 cm (OR 5.51, 95% CI 2.61 - 11.66, p < 0.001) were associated with increased risk of thyroid cancer in patients with thyroid nodules. CONCLUSIONS: The retrospective analysis suggests that younger patients with nodular thyroid disease cannot ignore the small size nodules, especially those with higher TSH levels and TgAb positivity.

Th17/Treg cells imbalance and GITRL profile in patients with Hashimoto's thyroiditis.

Hashimoto's thyroiditis (HT) is an organ-specific immune disease characterized by the presence of lymphocytic infiltration and serum autoantibodies. Previous studies have confirmed the critical role of Th17 cells in the pathopoiesis of HT patients. Additionally, regulatory T cells (Treg) display a dysregulatory function in autoimmune disease. The purpose of this study is to investigate the alteration of Th17 and Treg cells in HT patients and explore contributing factors. We found there was an increased ratio of Th17/Treg in HT patients and a positive correlation with autoantibodies (anti-TgAb). In addition, there was an increased level of GITRL, which has been demonstrated to be correlated with the increassement of Th17 cells in the serum and thyroid glands of HT patients; the upregulated serum level of GITRL has a positive correlation with the percentage of Th17 cells in HT patients. In summary, an increase in GITRL may impair the balance of Th17/Treg, and contribute to the pathopoiesis of Hashimoto's thyroiditis.

Increased frequency of circulating follicular helper T cells in patients with rheumatoid arthritis.

Follicular helper T (Tfh) cells are recognized as a distinct CD4(+) helper T-cell subset, which provides for B-cell activation and production of specific antibody responses, and play a critical role in the development of autoimmune disease. So far, only one study investigated the circulating Tfh cells increased in a subset of SLE patients. Since relatively little is known about the Tfh cells in rheumatoid arthritis (RA) patients, in this study, Tfh-cell frequency, related cytokine IL-21, and transcription factor Bcl-6 were investigated in 53 patients with RA and 31 health controls. Firstly, we found that the frequency of CD4(+)CXCR5(+)ICOS(high) Tfh cells was increased significantly in the peripheral blood of RA patients, compared with that in healthy controls. It is known that Tfh cells are critical for directing the development of an antibody response by germinal centers B cells; secondly, we observed that the Tfh-cell frequency is accompanied by the level of anti-CCP antibody in RA patients. Furthermore, expression of Bcl-6 mRNA and plasma IL-21 concentrations in RA patients was increased. Taken together, these findings have shown that the increased frequency of circulating Tfh cells is correlated with elevated levels of anti-CCP antibody, indicating the possible involvement of Tfh cells in the disease progression of RA.

Increased frequency of follicular helper T cells in patients with autoimmune thyroid disease.

CONTEXT: Follicular helper T (Tfh) cells exert an important role in the autoimmune diseases. AIM: Our study aimed to explore the role of Tfh cells in patients with autoimmune thyroid disease (AITD). DESIGN: Tfh cell is a new subset regulating the antibody production of B cell. Previous studies implicated CD4+CXCR5+ICOShigh or CD4+CXCR5+PD-1high as the markers of circulating Tfh cells. Sixty-five patients with AITD and 30 healthy controls were enrolled in the current study. The percentages of circulating Tfh cells were assessed by flow cytometry. The correlation between the percentages of CD4+CXCR5+ICOShigh T cells and the levels of autoantibodies or hormones was also analyzed. Additionally, polyphasic methods were applied to investigate the status of Tfh cells in thyroid glands of Hashimoto's thyroiditis patients. RESULTS: Increased percentages of circulating Tfh cells in AITD patients were detected, and a positive correlation between the percentages of circulating Tfh cells and the serum concentrations of anti-TSH receptor-Ab/thyroperoxidase-Ab/thyroglobulin-Ab was confirmed. A positive or modest relationship between the percentages of circulating Tfh cells and serum free T3 or free T4 was revealed in Graves' disease patients. Additionally, follow-up analysis indicated that in some Graves' disease patients the percentage of circulating Tfh cells decreased after treatment. Furthermore, a certain number of CD4+CXCR5+ICOShigh T cells together with enhanced expression of IL-21 and Bcl-6 mRNA were detected in thyroid tissues from Hashimoto's thyroiditis patients. CONCLUSION: The current study discovered an increased frequency of Tfh cells in AITD patients, which implies that this cell subset might play an important role in the pathogenesis of AITD.