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In recent years, Varicocele (VC) has been recognized as a common cause of male infertility that can be treated by surgery or drugs. How to reduce the damage of VC to testicular spermatogenic function has attracted extensive attention in recent years. Among them, overexpressed ROS and high levels of inflammation may play a key role in VC-induced testicular damage. As the key mediated innate immune pathways, cGAS-STING shaft under pathological conditions, such as in cell and tissue damage stress can be cytoplasmic DNA activation, induce the activation of NLRP3 inflammatory corpuscle, triggering downstream of the inflammatory cascade reaction. Chlorogenic acid (CGA), as a natural compound from a wide range of sources, has strong anti-inflammatory and antioxidant activities, and is a potential effective drug for the treatment of varicocele infertility. The aim of this study is to investigate the role of CGA in the spermatogenic dysfunction of the rat testis induced by VC and the potential mechanisms. The results of this study have shown that CGA gavage treatment ameliorated the pathological damage of seminiferous tubules, increased the number of sperm in the lumen, and increased the expression levels of Occludin and ZO-1, which indicated the therapeutic effect of CGA on spermatogenic dysfunction in the testis of VC rats. Meanwhile, the damage of mitochondrial structure was alleviated and the expression levels of ROS, NLRP3 and pro-inflammatory cytokines (IL-1β, IL-6, IL-18) were significantly reduced in the testicular tissues of model rats after CGA treatment. In addition, we demonstrated for the first time the high expression status of cGAS and STING in testicular tissues of VC model rats, and this was ameliorated to varying degrees after CGA treatment. In conclusion, this study suggests that CGA can improve the spermatogenic function of the testis by reducing mitochondrial damage and inhibiting the activation of the cGAS-STING axis, inhibiting the activation of the NLRP3 inflammasome, and improving the inflammatory damage of the testis, highlighting the potential of CGA as a therapeutic agent for varicocele infertility., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Age-related diminished ovarian reserve (DOR) is not absolute. Some advanced maternal age (AMA) still have normal ovarian reserve (NOR) and often show better pregnancy outcomes. Exploring the transcriptomic profile of granulosa cells (GCs) in AMA could lead to new ideas for mitigating age-related diminished ovarian reserve., Aim: This study aimed to analyze the transcriptomic profile of GCs in AMA with different ovarian reserve., Results: In total, 6273 statistically significant differential expression genes (DEGs) (|log2fc|> 1, q < 0.05) were screened from the two groups, among which 3436 genes were upregulated, and 2837 genes were downregulated in the DOR group. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the potential functions of dysregulated genes in AMA with DOR or NOR were predicted. The GO enrichment analysis revealed that the DEGs were mainly enriched in obsolete oxidation-reduction process, mitochondrion, metal ion binding, ATP binding, etc. The KEGG pathway enrichment analysis revealed that the above-mentioned DEGs were mainly enriched in ferroptosis, regulation of actin cytoskeleton, oxidative phosphorylation, etc. Meanwhile, verification of the mRNA expression levels of DEGs revealed the possible involvement of "ferroptosis" in age-related diminished ovarian reserve., Conclusions: From a new clinical perspective, we presented the first data showing the transcriptomic profile in GCs between AMA with different ovarian reserve. At the same time, we identified the role of ferroptosis in the GCs of AMA, providing a new biological basis for studying ovarian aging and improving pregnancy outcomes of AMA., (© 2023. The Author(s).)

Objective: To assess the safety and efficacy of Neuroform Atlas stent used in treatment of unruptured wide-neck intracranial aneurysms., Methods: Clinical data of 62 patients with unruptured wide-neck intracranial aneurysms undergoing Neuroform Atlas stent-assisted coiling from August 2020 to September 2021 were retrospectively analyzed. There were 64 aneurysms in those 62 patients. Among them, 25 aneurysms were located at the bifurcation of M1 segment on middle cerebral artery, 16 at the anterior communicating artery, 10 at the C7 segment of internal carotid artery, 5 at the C6 segment of internal carotid artery, 4 at the apex of basilar artery, 3 at the A3 segment of anterior cerebral artery, and 1 at the M2 segment of middle cerebral artery. All the patients underwent Neuroform Atlas stent-assisted coiling, including 49 patients with single stent assisted coiling and 15 patients with dual stents assisted coiling (14"Y"style and 1"X"style). After the procedure, the immediate DSA was performed to evaluate the status of aneurysm occlusion and the parent artery patency. The clinical follow-up was performed 3 months after the operation and evaluated based on the modified Rankin Scale(mRS).DSA image was reviewed at 6 months after operation and Raymond grading scale was used to assess the status of aneurysm occlusion and the parent artery patency., Results: A total of 62 patients with 64 aneurysms were all achieved technical success(100%).The immediate post-procedural Raymond scale was assessed, including Raymond Ⅰ in 57 aneurysms(89.1%, 57/64), Raymond Ⅱ in 6 aneurysms(9.3%, 6/64) and Raymond Ⅲ in 1 aneurysm(1.6%, 1/64). The peri-procedural complications rate was 4.8%(3/62), 2 patients developed intraoperative thrombosis and 1 patient suffered from local subarachnoid hemorrhage. Among them, 55 patients obtained 3 months clinical follow-up after operation and all the patients had good outcomes (mRS≤2), 50 patients with 52 aneurysms were followed up with DSA 6 months after operation, including Raymond Ⅰ in 45 aneurysms(86.5%, 45/52), Raymond Ⅱ in 4 aneurysms(7.7%, 4/52) and Raymond Ⅲ in 3 aneurysms(5.8%, 3/52)., Conclusion: Neuroform Atlas stent for the treatment of unruptured wide-neck intracranial aneurysms has high safety and good efficacy, and has its advantages over other traditional stents.

Objective: To evaluate different starting doses of recombinant human follicle-stimulating hormone (rhFSH) on pregnancy outcomes for patients with normal ovarian reserve during gonadotropin- releasing hormone antagonist (GnRH-ant) protocol-controlled ovarian stimulation of in vitro fertilization (IVF) cycles., Methods: In this retrospective study, a total of 1138 patients undergoing IVF cycles following the GnRH-ant protocol were enrolled. Patients were divided into two groups according to the starting dose of rhFSH. 617 patients received a starting dose of rhFSH of 150 IU, and 521 patients received a starting dose of rhFSH of 225 IU. We compared demographic characteristics, ovarian stimulation and embryological characteristics, and pregnancy and birth outcomes between the two groups. Multivariate logistic regression analysis was performed to examine the possible effects of the known potential confounding factors on pregnancy outcomes., Results: The number of oocytes retrieved in the 150 IU rhFSH group was significantly lower than those in the 225 IU rhFSH group. There was no significant difference between the two groups referring to embryological characteristics. The proportion of fresh embryo transfer in the 150 IU rhFSH group was significantly higher than that in the 225 IU rhFSH group (48.30% vs. 40.90%), and there was no difference in the risk of ovarian hyperstimulation syndrome and pregnancy outcomes between the two groups., Conclusions: In conclusion, the starting dose of rhFSH of 150 IU for ovarian stimulation has a similar pregnancy outcome as starting dose of rhFSH of 225 IU in GnRH-ant protocol for patients with normal ovarian reserve. Considering the potential cost-effectiveness and shorter time to live birth, the starting dose of rhFSH of 150 IU may be more suitable than 225 IU., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jia, Li, Li, Hou, Xia, Yang, Wang, Li, Sun and Guo.)

The gene SYF2 -an RNA splicing factor-can interact with Cyclin D-type binding protein 1 (GICP) in many biological processes, including splicing regulation, cell cycle regulation, and DNA damage repair. In our previous study we performed genome-wide identification and functional analysis of SYF2 in plant species. The phylogenetic relationships and expression profiles of SYF2 have not been systematically studied in animals, however. To this end, the gene structure, genes, and protein conserved motifs of 102 SYF2 homologous genes from 91 different animal species were systematically analyzed, along with conserved splicing sites in 45 representative vertebrate species. A differential comparative analysis of expression patterns in humans and mice was made. Molecular bioinformatics analysis of SYF2 showed the gene was conserved and functional in different animal species. In addition, expression pattern analysis found that SYF2 was highly expressed in hematopoietic stem cells, T cells, and lymphoid progenitor cells; in ovary, lung, and spleen; and in other cells and organs. This suggests that changes in SYF2 expression may be associated with disease development in these cells, tissues, or organs. In conclusion, our study analyzes the SYF2 disease resistance genes of different animal species through bioinformatics, reveals the relationship between the SYF2 genotype and the occurrence of certain diseases, and provides a theoretical basis for follow-up study of the relationship between the SYF2 gene and animal diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Huang, Jia, Yang, Cheng, Liu, Zhang, Chen, Yang and Chen.)

Alternative splicing is an important mechanism for regulating gene expressions at the post-transcriptional level. In eukaryotes, the genes are transcribed in the nucleus to produce pre-mRNAs and alternative splicing can splice a pre-mRNA to eventually form multiple different mature mRNAs, greatly increasing the number of genes and protein diversity. Alternative splicing is involved in the regulation of various plant life activities, especially the response of plants to abiotic stresses and is also an important process of plant growth and development. This review aims to clarify the usefulness of a genome-wide association analysis in the study of alternatively spliced variants by summarizing the application of alternative splicing, genome-wide association analyses and genome-wide association analyses in alternative splicing, as well as summarizing the related research progress.

Objective: To determine the association between serum phosphate level and 1-year clinical outcomes in patients with acute ischemic stroke and transient ischemic attack. Methods: We included 7,353 patients with acute ischemic stroke and transient ischemic attack from the China National Stroke Registry III for analysis. Participants were divided into 4 groups according to serum phosphate quartiles. Composite end point included recurrent stroke, myocardial infarction, other ischemic vascular events, and all-cause mortality. Poor functional outcome is defined as modified Rankin Scale score of 3 to 6. Multivariable Cox regression or logistic regression was used to evaluate the independent association of serum phosphate with 1-year all-cause mortality, recurrent stroke, composite end point and poor functional outcome. Results: The mean age of the included 7,353 patients was 62.5 years, and 68.6% of them were men. Plotting hazard ratios over phosphate levels suggested a U-shaped association especially for recurrent stroke and composite end point, and therefore the third quartile group was set as reference group. Compared with the third quartile of phosphate (1.06-1.20 mmol/L), the adjusted hazard ratios/odds ratios (95% CI) of the lowest quartile (<0.94 mmol/L) were 0.98 (0.67-1.42) for all-cause mortality, 1.31 (1.05-1.64) for stroke recurrence, 1.26 (1.02-1.57) for composite end point, and 1.27 (1.01-1.61) for poor functional outcome, and the adjusted odds ratio of the highest quartile (≥1.2 mmol/L) was 1.40 (1.11-1.77) for poor functional outcome. Conclusions: Serum phosphate may be an independent predictor of stroke recurrence, composite end point and poor functional outcome after ischemic stroke., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhang, Jing, Meng, Pan, Wang, Zhao, Lin, Han, Song, Jia, Wu, Chen, Xue, Anderson, Wu and Wang.)

Objective: To explore the technical details and short-term effects of radiofrequency obliteration of varicose veins of lower extremities guided by combined venography and ultrasound., Methods: Thirty-seven patients with varicose veins of lower extremities were treated with radiofrequency obliteration using Olympus Celon RFiTT ® under combined guidance of venography and ultrasound. The indications included varicose veins of lower extremities and reflux of the great saphenous vein confirmed by ultrasound. The contraindications included deep vein thrombosis, cardiac pacemaker, severe cardio- and cerebrovascular diseases or coagulation disorders. Under ultrasound guidance, the saphenous vein around knee level was punctured using a 21G needle, and a 7F sheath was introduced. Through the sheath a venography was made, and an Olympus Celon ProCurve radiofrequency catheter was inserted and advanced to the great saphenous vein under road map, and the catheter tip was positioned at the point 2 cm below the sapheno-femoral junction. The swelling anesthesia was made under ultrasound guidance. Then the radiofrequency obliteration was performed with pressing of the treatment section. The venography was repeated to ensure optimal outcomes. If necessary the radiofrequency obliteration could be repeated once to twice. After that the superficial varicose veins were stripping by small incisions under local anesthesia. After operation, medical decompression stocking was utilized immediately and sustained for three months. The clinical data, intraoperative radiation dose, exposure time and short-term effects were retrospectively analyzed., Results: After the operation, all the patients walked out of the operating room by themselves. The success rate of operation was 100%. The intraoperative radiation dose was 1.78-10.12 mGy (mean 6.56 mGy), and the exposure time was 61-448 s (mean 161 s). By 3 months follow-up, the symptoms were alleviated in all the 37 patients, and the occlusion rate was 100%. No complications such as skin burns, ecchymosis and deep venous thrombosis were found., Conclusion: The short-term effects of radiofrequency obliteration using Olympus Celon RFiTT ® system in a manner of twice fixed point followed by once reciprocating radiofrequency were satisfactory. Radiofrequency obliteration of great saphenous veins guided by venography and ultrasound has not only the advantages of minimal trauma and rapid recovery, but also the advantages of accurate location, exact effect and avoidance of complications.

Aims: Hypertension is a leading cause of cerebral small vessel disease (CSVD). Currently, treatments for CSVD are limited. Nicotinamide riboside (NR) can protect against vascular injury and cognitive impairment in neurodegenerative diseases. In this study, the protective effects of NR against angiotensin - (Ang -)-induced CSVD were evaluated., Methods: To explore the effects of NR in CSVD, C57BL/6 mice were infused with Ang -, and NR was added to the food of the mice for 28 days. Then, short-term memory, blood-brain barrier (BBB) integrity, and endothelial function were detected. Arteriole injury and glial activation were also evaluated., Results: Our data showed that mice infused with Ang - exhibited decreased short-term memory function and BBB leakage due to decreased claudin-5 expression and increased caveolae-mediated endocytosis after 28 days. Furthermore, Ang - decreased the expression of α-smooth muscle actin (α-SMA) and increased the expression of proliferating cell nuclear antigen (PCNA) in arterioles and decreased the expression of neurofilament 200 (NF200) and myelin basic protein (MBP) in the white matter. These CSVD-related damages induced by Ang - were inhibited by NR administration. Moreover, NR administration significantly reduced glial activation around the vessels., Conclusion: Our results indicated that NR administration alleviated Ang --induced CSVD by protecting BBB integrity, vascular remodeling, neuroinflammation, and white matter injury (WMI)-associated cognitive impairment., (© 2020 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.)

Objective: To summarize the preliminary experience of hybrid operation for the treatment of symptomatic long-segment chronic internal carotid artery occlusion (CICAO) without stump., Methods: Clinical data of 12 patients of symptomatic long-segment CICAO without stump undergoing hybrid operation treatment from July 2015 to December 2017 were retrospectively analyzed. The safety and efficacy of hybrid operation for the treatment of symptomatic long-segment CICAO without stump were preliminarily assessed. CICAO was defined as occlusion time being more than 4 weeks. The primary outcome was defined as any stroke (including ischemic or hemorrhagic) or deaths from any cause after hybrid operation within 30 days. The secondary outcome was defined as successful revascularization and occurrence of >50% in-stent restenosis during the follow-up period., Results: In this group, the symptomatic long-segment CICAO of 11 patients were successfully recanalized. Technical success rate was 91.7% (11/12). The main complication rate was 8.3% (1/12). This patient encountered iatrogenic internal carotid artery cavernous sinus fistula caused by micro-guide wire in the midway of the hybrid operation, the proximal segment of this internal carotid artery was ligated and the iatrogenic internal carotid artery cavernous sinus fistula disappeared in the following digital subtraction angiography image. No patient encountered hemorrhagic stroke and ischemic stroke. No death complications occurred. In this group 10 patients of them were followed up. The follow-up period ranged from 10 to 32 months [mean, (19±9) months]. During the follow-up period, 1 patients developed in-stent restenosis and improved after reoperation of percutaneous transluminal angioplasty by the right size balloon without stenting treatment., Conclusion: Hybrid operation for the treatment of highly screened patients with symptomatic long-segment CICAO without stump is safe and effective, could reduce the incidence of complications and improve procedural success rate.

Objective: To assess the safety and efficacy of Neuroform EZ stent used in treatment of symptomatic complex severe intracranial atherosclerotic stenosis (ICAS)., Methods: Clinical data of 18 patients with symptomatic complex severe ICAS undergoing Neuroform EZ stent angioplasty from January 2016 to December 2017 were retrospectively analyzed. All the lesions of the patients in this group were considered as complex ICAS, i.e. with severe tortuous access, long (>10 mm) or occlusive or bifurcation lesions, with concurrent aneurysms near the stenotic lesion. The primary outcome was defined as any stroke (including ischemic or hemorrhagic) or deaths from any cause after stenting procedure within 30 days. The secondary outcome was defined as successful revascularization and occurrence of >50% in-stent restenosis during the follow-up period., Results: All the 18 patients achieved technical success (100%) and mean stenosis rate was reduced from 85%±7% to 18%±6%. Of the 18 patients included, the 30-day stroke or death was 5.6% (1/18), which presented as basal ganglia region infarction in a patient with tandem lesions on the left vertebral artery. There was no hemorrhagic and death complications that occurred in the patients of this group. One concurrent aneurysm was embolized with micro coil (stent assisted) by stages after 1 month. In this group 12 patients were followed up with digital subtraction angiography (DSA) after hospital discharge. The follow-up period ranged from 8 months to 26 months [mean: (16±8) months]．During the follow-up period 2 patients in the 12 patients (2/12, 16.7%) developed in-stent restenosis (ISR) confirmed by DSA, and one of them was symptomatic restenosis and restored unobstructed blood flow after balloon angioplasty., Conclusion: Neuroform EZ stent for the treatment of highly screened symptomatic complex severe ICAS is safe and effective. It has its advantages over traditional stent.

Objective: To explore the risk factors, clinical characteristics, precaution and treatment of hyper perfusion syndrome (HPS) after carotid artery stenting (CAS)., Methods: From September 2014 to March 2018, the clinical data of 226 patients with severe carotid stenosis (70%-99%) treated with carotid artery stenting (CAS)at Department of Interventional Radiology and Vascular Surgery, Peking University Third Hospital, were analyzed retrospectively．Five of them developed HPS after CAS．The relationship between the clinical baseline data, imaging characteristics, perioperative management and HPS were assessed., Results: In this group, 5 patients of them (2.21%, 5/226) developed HPS after CAS, and 2 patients of them (0.88%, 2/226) were hyper perfusion induced intracranial hemorrhage (HICH). The 5 patients consisted of 4 men and 1 woman whose age ranged from 58 to 74 years. The symptoms of HPS occurred within 4 hours to 3 days after CAS. Among the 5 cases, the clinical manifestations were that 2 cases with headache, 1 case with delirium,1 case with hemiparesis of left limbs, and 1 case with coma(died ultimately).The main manifestations of case 1 and case 2 were headache in the frontal parietal temporal region of the operative side, accompanied by nausea and vomiting. The symptoms were relieved after blood pressure lowering treatment and mannitol dehydration. The main manifestations of case 3 were excitement and delirium. The symptoms were relieved by a small dose of sedatives, also with blood pressure lowering treatment and mannitol dehydration. The initial symptoms of case 4 were excitement and delirium, accompanied by mild headache of the operative side, and hemiplegia of the contralateral limb occurred within a short time. The main manifestation of case 5 was severe headache and went into deep coma within a short time. This patient died of massive cerebral hemorrhage ultimately., Conclusion: HPS is an uncommon but serious complication after CAS. Improving our understanding and heightening vigilance of HPS is necessary. The earlier diagnosis, the earlier treatment.

Objective: To evaluate the effectiveness and safety of mechanical thrombectomy treatment in patients with acute ischemic stroke (AIS),and to explore influential factors of the clinical prognosis preliminarily., Methods: Clinical data of 26 patients with acute cerebral arterial occlusion treated with mechanical thrombectomy in Peking University Third Hospital from January 2014 to June 2017 were retrospectively collected. The immediate effects of the 26 patients in this group after mechanical thrombectomy treatment were analyzed,The national institutes of health stroke scale (NIHSS) scores between preoperative and at discharge of the 26 patients in this group were compared,and modified Rankin scale (mRS) scores of 90 days post operation were analyzed to assess the prognosis of the 26 patients in this group., Results: (1)In this group, 23 patients (88.5%) achieved vascular recanalization evaluated by thrombolysis in cerebral ischemia scale scores [thrombolysis in cerebral ischemia scale (TICI) scores, 3/2b grades were recognized as vascular recanalization], 19 patients of them reached TICI grade 3 and 4 atients reached TICI grade 2b. In this group 3 patients (11.5%) encountered symptomatic intracranial hemorrhage, 2 patients of them recovered after cerebral hemorrhage absorbed and 1 patient died of massive cerebral hemorrhage. In this group 4 patients (15.4%) died after mechanical thrombectomy treatment,2 patients died of hernia of the brain caused by severe cerebral edema, 1 patient died of symptomatic intracranial hemorrhage and 1 patient died of extensive subarachnoid hemorrhage. (2)The assessment of NIHSS scores at discharge(5.3±2.1)showed significantly lower than those preoperatively(12.6±4.2), P<0.01,and in this group 12 patients (46.2%) achieved favourable prognosis (defined as mRS scores 0-2), 6 patients of them reached mRS 0 score,4 patients reached mRS 1 score and 2 patients reached mRS 2 scores., Conclusion: Mechanical thrombectomy with stent retriever contributed to a high rate of vascular recanalization and favourable prognosis,but some patients had poor prognosis, suggesting that we should screen the enrolled patients strictly.

Objective: This study was designed to evaluate the toxic effects of Atrazine (ATZ) on the reproductive system of male rats., Methods: Male Sprague-Dawley rats were exposed to ATZ by gavage at dosages of 0, 38.5, 77, and 154 mg/kg bw/day for 30 d. The toxic effects of ATZ to rats were assessed through histopathologcal observation, spermatozoa quality evaluation, testicular marker enzyme indicators, antioxidant capacity and reproductive hormone levels., Results: Significant adverse effects on reproductive system were observed in rats exposed to ATZ at different dosages compared with 0 mg/kg group, including an irregular and disordered arrangement of the seminiferous epithelium in 154 mg/kg group; a decreased spermatozoa number and an increased spermatozoa abnormality rate in 77 and 154 mg/kg groups; decreased levels of acid phosphatase (ACP), alkaline phosphatase (AKP), lactic dehydrogenase (LDH), and succinate dehydrogenase (SDH) with the increasing of ATZ concentration; a decreased level of total antioxidant capacity (TAC) in a dose-dependent manner, and a decreased reduced glutathione (GSH) level and an increased malondialdehyde (MDA) content in 154 mg/kg group; and decreased serum levels of testosterone (T) and inhibin-B (INH-B) and an increased serum level of follicle stimulating hormone (FSH) in 77 and 154 mg/kg groups, and an increased serum level of luteinizing hormone (LH) in 154 mg/kg group., Conclusion: These results suggested that relatively high doses of ATZ could exert reproductive toxicity of male rats., (Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Almost all melanoma cells express at least one member of the MAGE-A antigen family, making the cytotoxic T cells (CTLs) epitopes with cross-immunizing potential in this family attractive candidates for the broad spectrum of anti-melanoma immunotherapy. In this study, four highly homologous peptides (P264: FLWGPRALA, P264I9: FLWGPRALI, P264V9: FLWGPRALV, and P264H8: FLWGPRAHA) from the MAGE-A antigens were selected by homologous alignment. All four peptides showed high binding affinity and stability to HLA-A*02:01 molecules, and could prime CTL immune responses in human PBMCs and in HLA-A*02:01/K(b) transgenic mice. CTLs elicited by the four epitope peptides could cross-lyse tumor cells expressing the mutual target antigens, except MAGE-A11 which was not tested. However, CTLs induced by P264V9 and P264I9 showed the strongest target cell lysis capabilities, suggesting both peptides may represent the common CTL epitopes shared by the eight MAGE-A antigens, which could induce more potent and broad-spectrum antitumor responses in immunotherapy.

Background: Regulatory T cells (Tregs) are required for proper maintenance of immunological self-tolerance and immune homeostasis. Folate receptor 4 (FR4) is expressed at high levels in transforming growth factor-beta (TGF-β)-induced Tregs and natural Tregs. Moreover, antibody-mediated targeting of FR4 is sufficient to mediate Treg depletion., Results: In this study, we describe a novel FR4 transcript variant, FR4D3, in which exon 3 is deleted. The mRNA of FR4D3 encodes a FR4 variant truncated by 189 bp. FR4D3 was found to be predominantly expressed in CD4(+)CD25(+) Treg cells. Overexpression of FR4D3 in CD4(+)CD25(+) Treg cells in vitro stimulated proliferation, which may modulate the ability of these cells to bind and incorporate folic acid., Conclusions: Our results suggested that high levels of FR4D3 may be critical to support the substantial proliferative capacity of Treg cells.

The retinoid-related orphan nuclear receptor gamma (ROR γ) plays critical roles in regulation of development, immunity and metabolism. As transcription factor usually forms a protein complex to function, thus capturing and dissecting of the ROR γ protein complex will be helpful for exploring the mechanisms underlying those functions. After construction of the recombinant tandem affinity purification (TAP) plasmid, pMSCVpuro ROR γ-CTAP(SG), the nuclear localization of ROR γ-CTAP(SG) fusion protein was verified. Following isolation of ROR γ protein complex by TAP strategy, seven candidate interacting proteins were identified. Finally, the heat shock protein 90 (HSP90) and receptor-interacting protein 140 (RIP140) were confirmed to interplay with ROR γ by co-immunoprecipitation. Interference of HSP90 or/and RIP140 genes resulted in dramatically decreased expression of CYP2C8 gene, the ROR γ target gene. Data from this study demonstrate that HSP90 and RIP140 proteins interact with ROR γ protein in a complex format and function as co-activators in the ROR γ-mediated regulatory processes of HepG2 cells.

Purpose: Increased expression of tissue factor (TF) is associated with tumor invasion and metastasis in human colorectal cancer. We have previously observed that TF/FVIIa upregulates matrix metalloproteinase-7 (MMP-7) expression at the transcriptional level in colon cancer cells. MMP-7 overexpression is believed to play an important role in tumor invasion and metastasis. The aim of this study is to elucidate the molecular mechanisms by which TF/FVIIa induced MMP-7 expression and cell invasion in vitro., Methods: Reverse transcription polymerase chain reaction, Western blot, luciferase assay, and chromatin immunoprecipitation (ChIP) were used to determine the potential mechanism and signaling pathways by which TF/FVIIa induced MMP-7 expression and cell invasion in LoVo cells. Small interfering RNA (siRNA) and cell invasion assay was used to examine whether blocking c-Fos expression could abolish FVIIa-mediated upregulation of MMP-7 and cell invasion in vitro., Results: The results showed that FVIIa induced the upregulation of MMP-7 both at the mRNA and protein levels in a time- and dose-dependent manner and increased the invasive behavior of LoVo cells. FVIIa enhanced the promoter activity of MMP-7, and the activator protein-1 (AP-1) binding site was responsible for the activation. Site mutation of the AP-1 binding site in the promoter almost completely abolished FVIIa-mediated response. Furthermore, ChIP assay confirmed that FVIIa promoted the direct binding of c-Fos with the MMP-7 promoter in vivo. FVIIa also induced the expression and nuclear accumulation of the AP-1 subunit c-Fos. siRNA-mediated knockdown of c-Fos eliminated FVIIa-stimulated MMP-7 expression and cell migration in vitro. In addition, selective mitogen-activated protein kinase (MAPK) kinase (MEK1/2) inhibitor (PD98059) and p38 MAPK inhibitor SB203580 suppressed MMP-7 upregulation induced by FVIIa., Conclusions: Our data suggest that a novel TF/FVIIa/MAPK/c-Fos/MMP-7 axis plays an important role in modulating the invasion of colon cancer cells and blockage of this pathway holds promise to treat colon cancer metastasis.

MAGE-A antigens belong to cancer/testis (CT) antigens that are expressed in tumors but not in normal tissues with the exception of testis and placenta. Among MAGE-A antigens, MAGE-A10 is extensively expressed in various histological types of tumors, representing an attractive target for tumor immunotherapy. Cytotoxic T lymphocytes (CTLs) play a key role in anti-tumor immune responses, so the identification of CTL epitopes derived from MAGE-A10 would contribute a lot to the design of epitope-based vaccines for tumor patients. In this study, we predicted HLA-A*0201-restricted CTL epitope peptides of MAGE-A10, followed by peptide/HLA-A*0201 binding affinity and complex stability assays, and induced peptide-specific CTL immune responses. Of the selected three peptides (designated P1, P2 and P3), P1 (MAGE-A10310-318, SLLKFLAKV) could elicit peptide-specific CTLs both in vitro from HLA-A*0201-positive PBMCs and in HLA-A*0201/Kb transgenic mice. And, the induced CTLs could lyse MAGE-A10-expressing tumor cells in a HLA-A*0201-restricted fashion but not MAGE-A10-negative tumor cells. Our results demonstrate that the peptide MAGE-A10310-318 is a HLA-A*0201-restricted CTL epitope of MAGE-A10 and could serve as a target for therapeutic antitumoral vaccination.

Background: Blood coagulation factor VII (FVII) is physiologically synthesized in the liver and released into the blood. Binding of FVII to tissue factor (TF) is related to the metastatic potential of tumor cells, also a significant risk factor in the development of hepatic metastasis in patients with colorectal cancer (CRC). It has been found that some cancer cells can produce FVII extrahepatically. However, little is known about FVII and CRC. We therefore hypothesized that CRC cells may synthese FVII, leading to tumor invasion and metastasis., Methods: We detected the expression of FVII protein in 55 CRC specimens by immunohistochemical staining. The FVII mRNA in 45 of 55 CRC cases, 6 colon cancer cell lines and one hepatoma cell line was measured by real-time reverse transcription-PCR (RT-PCR). Transwell invasion assays were performed to evaluate the changes of cell migration and invasion of LoVo cancer cells in vitro. We further observed the likely effectors regulated by the TF/FVIIa complex Western blotting assay., Results: Extrahepatic synthesis of FVII was detected in the cytoplasm of 32 (58.2%) CRC specimens by immunohistochemistry, but not in normal mucosa. Liver metastasis (P = 0.003) and TNM staging (P = 0.005) were significantly correlated with FVII antigen expression. The positive ratios in stages I, II, III and IV were 33.3%, 40.0%, 52.4% and 87.5%, respectively. The expression of FVII mRNA in CRC with hepatic metastasis was significantly higher than CRC without hepatic metastasis (5.33 ± 2.88 vs. 1.47 ± 0.51, P = 0.03). Ectopic FVIIa induced a slight increase (1.34-fold) in the number of migrating cells, which was inhibited by the specific TF antibody. The formation of TF/FVIIa complex resulted in a marked increase in the expression of matrix metalloproteinases (MMP)-2 (3.5-fold) and MMP-9 (4.7-fold) in a time-dependent and dose-dependent manner., Conclusions: Extrahepatic synthesis of FVII by CRC cells may promote tumor invasion and metastasis. MMPs, as downstream effectors of TF/FVIIa signaling, facilitate the development of metastasis in colon cancer.

MAGE-A antigens belong to cancer/testis (CT) antigens that are expressed in tumors but not in normal tissues except testis and placenta. MAGE-A antigens and their epitope peptides have been used in tumor immunotherapy trials. MAGE-A4 antigen is extensively expressed in various histological types of tumors, so it represents an attractive target for tumor immunotherapy. In this study, we predicted HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) epitopes of MAGE-A4, followed by peptide/HLA-A*0201 affinity and complex stability assays. Of selected four peptides (designated P1, P2, P3, and P4), P1 (MAGE-A4(286-294), KVLEHVVRV) and P3 (MAGE-A4(272-280), FLWGPRALA) could elicit peptide-specific CTLs both in vitro from HLA-A*0201-positive PBMCs and in HLA-A*0201/K(b) transgenic mice. And the induced CTLs could lyse target cells in an HLA-A*0201-restricted fashion, demonstrating that the two peptides are HLA-A*0201-restricted CTL epitopes and could serve as targets for therapeutic antitumoral vaccination.

Plasmid DNA vaccine is an appealing cancer immunotherapy. However, it is a weak immunogen and immunization with plasmid DNA encoding self-antigens, such as melanoma-associated antigens, could not induce antitumor immunity because of tolerance. In this study, we investigated the feasibility of using a plasmid DNA encoding Xenopus laevis transforming growth factor-beta 5 (aTGF-beta5) as an immunogen to induce neutralizing antibodies against murine TGF-beta1 (mTGF-beta1) and thus enhance the efficacy of plasmid DNA vaccine encoding murine tyrosinase-related protein 2 (mTRP-2) through neutralization of TGF-beta. The results showed that immunization with aTGF-beta5 resulted in the generation of mTGF-beta1-neutralizing antibodies, and immunization with a combination of aTGF-beta5 and mTRP-2 induced specific cytotoxic T lymphocytes (CTLs). On the contrary, immunization with mTRP-2 alone could not elicit the CTL response. Moreover, immunization of C57BL/6 wild-type mice with a combination of aTGF-beta5 and mTRP-2 induced the protective and therapeutic antitumor immunity to B16F10 melanoma, whereas the antitumor activity was abrogated in both CD4-deficient mice and CD8-deficient mice on the C57BL/6 background. Our results indicate that immunization with aTGF-beta5 is capable of breaking immune tolerance and induces mTGF-beta1-neutralizing antibodies. Neutralization of TGF-beta can enhance the efficacy of DNA vaccine encoding mTRP-2 and the induction of antitumor immunity by this immunization strategy is associated with CD4+ and CD8+ T cells.

Aim: To explore how to improve the immunogenicity of HBcAg CTL epitope based polypeptides and to trigger an HBV-specific HLA I-restricted CD8+ T cell response in vitro., Methods: A new panel of mimetic therapeutic peptides based on the immunodominant B cell epitope of HBV PreS2 18-24 region, the CTL epitope of HBcAg18-27 and the universal T helper epitope of tetanus toxoid (TT) 830-843 was designed using computerized molecular design method and synthesized by Merrifield's solid-phase peptide synthesis. Their immunological properties of stimulating activation and proliferation of lymphocytes, of inducing T( H1) polarization, CD8+ T cell magnification and HBV-specific CD8+ CTL mediated cytotoxicity were investigated in vitro using HLA-A2+ human peripheral blood mononuclear cells (PBMCs) from healthy donors and chronic hepatitis B patients., Results: Results demonstrated that the therapeutic polypeptides based on immunodominant HBcAg18-27 CTL, PreS2 B- and universal T(H) epitopes could stimulate the activation and proliferation of lymphocytes, induce specifically and effectively CD8+ T cell expansion and vigorous HBV-specific CTL-mediated cytotoxicity in human PBMCs., Conclusion: It indicated that the introduction of immunodominant T helper plus B-epitopes with short and flexible linkers could dramatically improve the immunogenicity of short CTL epitopes in vitro.

Aim: To explore how to trigger an HLAI-restricted CD8(+) T cell response to exogenously synthesized polypeptides in vivo., Methods: Three mimetic therapeutic polypeptides based on the immunodominant CTL epitope of HBcAg, the B- epitope of HBV PreS(2) region and a common T helper sequence of tetanus toxoid were designed and synthesized with Merrifield's solid-phase peptide synthesis method. Their immunological properties of inducing T( H1) polarization, CD8(+) HBV-specific CTL expansion and CD8(+) T cell mediated cytotoxicity were investigated in HLA-A2 transgenic mice., Results: Results demonstrated that the mimetic polypeptides comprised of the immunodominant CTL, B-, and T helper epitopes could trigger specifically and effectively vigorous CD8(+) HBV-specific CTL-mediated cytotoxicity and T(H1) polarization of T cells in HLA-A2 transgenic mice., Conclusion: A designed universal T helper plus B-epitopes with short and flexible linkers could dramatically improve the immunogenicity of CTL epitopes in vivo. And that the mimetic therapeutic peptides based on the reasonable match of the above CTL, B- and T helper epitopes could be a promising therapeutic peptide vaccine candidate against HBV infection.

Rotaviruses (RV) are a common cause of severe diarrhea in young children, resulting in nearly one million deaths worldwide annually. Rotavirus VP7 was the rotavirus neutralizing protein. Previous study reported that VP7 DNA vaccine can induce high levels of IgG in mice but cannot protect mice against challenge (Choi, A.H., Basu, M., Rae, M.N., McNeal, M.M., Ward, R.L., 1998. Virology 250, 230-240). We found that rotavirus VP7 could maintain its neutralizing immunity when it was transformed into the potato genome. Mice immunized with the transformed tubers successfully elicited serum IgG and mucosal IgA specific for VP7. The mucosal IgA titer was as high as 1000, while serum IgG titer was only 600. Neutralizing assays indicated that IgA could neutralize rotavirus. These results indicate the potential usefulness of plants for production and delivery of edible rotavirus vaccines.

CD8(+) cytotoxic T lymphocytes (CTLs) are now recognized as important mediators of immunity against intracellular pathogens, including human immunodeficiency virus and tumors. How to efficiently evoke antigen-specific CTL responses in vivo has become a crucial problem in the development of modern vaccines. Here, we developed a completely novel CTL vaccine-mimovirus, which is a kind of virus-size particulate antigen delivery system. It was formed by the self-assembly of a cationic peptide containing 18 lysines and a CTL-epitope peptide of HBsAg(28-39), with a plasmid encoding mouse interleukin-12 (IL-12) through electrostatic interactions. We examined the formation of mimovirus by DNA retardation assay, DNase I protection assay, and transmission electron microscopy and demonstrated that mimovirus could efficiently transfer the plasmid encoding IL-12 into mammalian cells such as P815 cells in vitro. Furthermore, it was proved that mimovirus could induce an HBsAg(28-39)-specific CTL response in vivo. Considering its effectiveness, flexibility, and defined composition, mimovirus is potentially a novel system for vaccination against intracellular pathogens and tumors.

Aim: To characterize the biochemical and immunological properties of an experimental ISCOMS vaccine prepared from a novel therapeutic polypeptide based on T cell epitopes of HBsAg, and a heptatis B-ISCOMS was prepared and investigated., Methods: An immunostimulating complexes(ISCOMS)-based vaccine containing a novel therapeutic hepatitis B polypeptide was prepared by dialysis method, and its formation was visualized by electron microscopy and biochemically verified by SDS-polyacrylamide gel electrophoresis. Amount of the peptide within ISCOMS was determined by Bradford assay, and specific CTL response was detected by ELISPOT assay., Results: Typical cage-like structures of submicroparticle with a diameter of about 40nm were observed by electron microscopy. Results from Bradford assay showed that the level of peptide incorporation was about 0.33g.L(-1). At the paralleled position close to the sixth band of the molecular weight marker(3480kDa) a clear band was shown in SDS-PAGE analysis, indicating successful incorporation of polypeptide into ISCOMS. It is suggested that ISCOMS delivery system could efficiently improve the immunogenicity of polypeptide and elicit specific immune responses in vivo by the results of ELISPOT assay, which showed that IFN-gamma producing cells(specific CTL responses) were increased(spots of ISCOMS-treated group: 47+/-5, n =3; control group: 5+/-2, n =3)., Conclusion: ISCOMS-based hepatitis B polypeptide vaccine is successfully constructed and it induces a higher CTL response compared with short polypeptides vaccine in vivo.

To study the influence of different solvent systems on the isomers of melanoma antigen-encoding gene-2(MAGE-2) epitope peptide synthesized by Marrifield's solid synthesis method, MAGE-2(171-179) epitope peptides were pre-treated using ethanol and methanol systems respectively, and then analysed by RP-HPLC/MS, with dimethyl-sulphoxide (DMSO) as control solvent. Results demonstrated that 100% ethanol and 100% methanol solvents could induce the production of isomers of MAGE-2, while 100% DMSO, 50% ethanol and 50% methanol could not. These isomeric peptides could be reversed greatly if treated with trifluoroacetic acid (TFA). The results also showed that the analysis would be distorted and the yield of peptides would decrease dramatically if the peptides were pre-treated with high concentrations of ethanol and methanol before RP-HPLC analysis and preparation. Thus it is suggested that it might be better to decrease the concentration of organic phase in the solvent system for the resolvable hydrophobic peptides, acidified ethanol and methanol might also be better for the highly hydrophobic peptides, and DMSO would be a good solvent for hydrophobic peptides analysis provided that it would not influence the resolving power of chromatography.

Toxic shock syndrome toxin 1 (TSST-1) is a member of the staphylococcal enterotoxin superantigen family. So far, little is known about T-cell epitopes on superantigens. In this study, we developed an improved method for localizing T-cell epitopes on superantigens that involved synthetic peptides plus costimulation by CD28 or phorbol myristate acetate. Using this method, we localized a T-cell epitope to a 34-residue region, TSST-1 (residues 125 to 158), which possessed only two of four TSST-1-targeted beta-chain variable element (Vbeta) specificities of T-cell receptors in humans and mice, human Vbeta2 and murine Vbeta15.

C12H19N3O5, Mr = 285.25, monoclinic, P2(1), a = 7.0180 (6), b = 8.6946 (11), c = 10.7715 (10) A, beta = 91.055 (7) degrees, V = 657.15 A3, Z = 2, Dx = 1.441 g cm-3, lambda(Cu K alpha) = 1.5418 A, mu = 9.63 cm-1, F(000) = 304, T = 287 K, R = 0.039 for 1424 observed reflections. The furanose ring adopts the C(1')-exo envelope conformation (E1), with the glycosyl linkage anti (chi = 193.8 degrees). The pseudo-rotational parameters are P = 130.9 degrees and tau m = 39.4 degrees. In the deoxyribose ring, the side chain on C(5') has the t conformation. In the pyrimidine ring the N4-methyl takes a cis conformation to N(3) and the methoxymethyl side chain is on the same side of the cytidine plane as O(4').