寿胎丸靶向miR-374c-5p/ATG12信号轴减轻滋养细胞自噬治疗原因不明复发性自然流产

目的:探讨寿胎丸调控miR-374c-5p/ATG12信号轴减轻滋养细胞自噬治疗原因不明复发性自然流产(URSA)的作用与机制.方法:收集正常早孕妇女(NP)和URSA患者绒毛组织各30例,Western blot检测LC3Ⅱ/Ⅰ、P62及ATG12蛋白表达;qRT-PCR检测ATG12 mRNA及miR-374c-5...     

Tim-3分子表达异常与原因不明复发性流产的关系

目的探究T细胞免疫球蛋白粘蛋白分子3（Tim-3）表达异常与不明原因复发性流产（URSA）的关系。方法选择31例URSA患者作为研究组,并以同期30例正常孕妇作为对照组。采用流式细胞术检测两组外周血单个核细胞（PBMC）中CD4＋Tim-3＋细胞/CD4＋细胞的比例,Western Blot技术检测蜕膜中Tim-3蛋白的相对含量,并以免疫磁珠法分选CD4＋Tim-3＋细胞,荧光定量RT-PCR检测两组外周血PBMC、CD4＋Tim-3＋细胞及蜕膜组织中Tim-3 mRNA的表达。结果 URSA组中,PBMC中CD4＋Tim-3＋细胞/CD4＋细胞为5.23±0.96%,蜕膜中Tim-3蛋白的相对表达量为0.64±0.09,PBMC及蜕膜中Tim-3 mRNA的相对表达量为0.65±0.09及0.50±0.07,均显著高于正常对照组（P〈0.01）。但是,URSA组CD4＋Tim-3＋细胞（Th1细胞）Tim-3 mRNA的表达量低于正常对照组,差异具有显著性（P〈0.01）。结论 Th1细胞Tim-3表达降低,Th1细胞过度活化可能参与了URSA的发生发展过程。     

Cerebrospinal fluid interleukin (IL)-6 in unipolar major depression

BACKGROUND: Previous studies have suggested that regulation of the proinflammatory cytokine interleukin (IL)-6 is abnormal in patients with major depression. This study was undertaken to determine whether IL-6 concentrations in cerebrospinal fluid (CSF) differ between depressed patients and healthy control subjects. METHODS: Lumbar puncture with a standardized procedure was performed on 18 drug-free patients meeting DSM-IV criteria for unipolar major depression and 26 age- and sex-matched healthy volunteers. CSF was assayed for IL-6 using a quantitative 'sandwich' enzyme immunoassay technique. RESULTS: Mean+/-S.D. CSF IL-6 levels did not differ between depressed (2.2+/-1.0 pg/ml) and healthy control (2.4+/-1.9 pg/ml) subjects. LIMITATIONS: This study had adequate power (0.8) to detect a large (d=0.88) effect size at alpha = 0.05. Although sample sizes were comparable to or larger than those of previous CSF studies, it is possible that a less robust difference between depressed and healthy subjects was not detected. CONCLUSIONS: These findings fail to support speculation that immune activation may be causally involved in the pathogenesis of depression.     

TIM-3-Gal-9信号途径在原因不明复发性流产中的研究进展

体内免疫微环境中CD4+Th细胞亚群间平衡对维持正常妊娠的母胎耐受起关键作用,一旦这种格局被打破将导致原因不明复发性流产(URSA)的发生,然而具体机制未明。TIM-3是一个负调控分子,与其配体Gal-9结合诱导T细胞凋亡,抑制Th1和Th17免疫应答,对机体CD4+Th细胞亚群间平衡和免疫耐受调控起重要作用。TIM-3和Gal-9在URSA外周血和脱膜组织中有不同表达,TIM-3-Gal-9信号途径对正常妊娠的免疫平衡调节起重要作用,但机体机制知之甚少。本文就TIM-3-Gal-9信号途径对URSA免疫失衡和母胎耐受调控的研究进展作一综述,为URSA的防治提供新靶点和新思路。     

早期不明原因反复自然流产发生与蜕膜中免疫细胞的研究进展

人类妊娠被认为是一种半同种异体抗原移植，母胎间存在着某种免疫耐受机制来维持妊娠的进行，但目前为止这种免疫耐受机制尚不明确。大量的研究发现不明原因反复自然流产患者蜕膜中调节性T细胞的数量和功能均显著降低，表明调节性T细胞在避免胎儿免疫排斥中发挥着重要的作用。同时NK细胞作为早期妊娠蜕膜中的优势淋巴细胞亦对妊娠的维持起着重要的作用，不明原因反复自然流产患者蜕膜NK细胞数量和活性比正常妊娠妇女明显升高，同时CD56^＋CD16^＋／CD56^＋CD16^-NK细胞比例失衡。由此可见，妊娠早期不明原因反复自然流产的发生与蜕膜中淋巴细胞的异常表达相关，通过对这种复杂机制的研究可以为不明原因反复自然流产的预防和治疗提供依据。     

原因不明复发性流产患者调节性T细胞数量功能的改变及机制探讨

目的探讨原因不明复发性流产(URSA)发生的免疫机制,了解调节性T细胞在其发病中的作用。方法流式细胞术检测23例原因不明复发性流产(A组)、20例正常妊娠妇女(B组)和15例健康成年女性(C组)中CD4+CD25+T淋巴细胞的比例。免疫磁珠法分离CD4+CD25+细胞,逆转录聚合酶链反应(RT-PCR)法检测FOXP3的表达,酶联免疫吸附试验(ELISA)检测白细胞介素-10的水平。结果三组CD4+CD25+细胞比例无显著差异(P>0.05)。A组CD4+CD25+细胞表达FOXP3及分泌IL-10均低于B组和C组,具有显著性差异(P<0.05),而B组和C组表达FOXP3及分泌IL-10的水平无显著差异(P>0.05)。结论URSA患者调节性T细胞在外周血淋巴细胞中的比例虽无明显改变,但其功能明显减弱,可能是导致其发病的原因之一。     

A case-control study in IL6 and TGFB1 gene polymorphisms and recurrent spontaneous abortion in southern Brazilian patients

PROBLEM: A high proportion of recurrent spontaneous abortions (RSA) remains unexplained. Cytokine genotyping has been investigated. We studied the relationship between unexplained RSA, IL6 (-174 G-->C) and TGFB1 (+869, T-->C; +915, G-->C) gene polymorphisms. METHOD OF STUDY: The case-control study composed of 57 south Brazilian women, with unexplained RSA and 74 controls carefully matched was performed. Cytokine genotyping was performed by the polymerase chain reaction-sequence specific primer method, using the 'Cytokine Genotyping Tray'. RESULTS: The results showed that the genotypic frequencies did not differ from samples for TGFB1 gene. In relation to IL6 gene polymorphism there was a statistical difference in genotypic distribution between samples (P < or = 0.025). The frequency of the C/C genotype was increased in women with RSA in comparison with the frequency observed in controls: 18% versus 4% (P = 0.01). CONCLUSIONS: This result strengthened the importance of IL6 genotypes in the pathogenesis of RSA of unknown cause in the south Brazilian population.     

Cytokine production by maternal lymphocytes during normal human pregnancy and in unexplained recurrent spontaneous abortion

It has been proposed that successful pregnancy is a T helper 2-type phenomenon, and that T helper (Th)1-type reactivity is deleterious to pregnancy. The objective of this study was to compare the concentrations of Th1 and Th2 cytokines produced by peripheral blood mononuclear cells from women undergoing unexplained recurrent spontaneous abortion (RSA) with those produced during normal pregnancy at a similar gestational stage. The control group consisted of 24 women with a history of successful pregnancies and the abortion group comprised of 23 women with a history of unexplained RSA. Blood from the control group was obtained at the end of the first trimester as gestational age controls for the abortion group from whom blood was collected at the time of abortion. Phytohaemagglutinin-stimulated peripheral blood cell culture supernatants were analysed for concentrations of cytokines. Significantly higher concentrations of Th2 cytokines were produced by the first trimester normal group than by the RSA group, while significantly higher concentrations of Th1 cytokines were produced by the abortion group as compared to first trimester normal pregnancy, indicating a distinct Th2-bias in normal pregnancy and a Th1-bias in unexplained RSA.     

Relationship of seminal plasma interleukin (IL) -8 and IL-6 with semen quality

The concentration of interleukin (IL) -8 and IL-6 was determined in seminal plasma (SP) samples from 137 randomly chosen subfertile males to evaluate the relationship with other potential parameters of subclinical infection/inflammation such as seminal leukocytes, and with semen quality in a prospective study. All patients were asymptomatic for genital tract infection. A comprehensive semen evaluation included sperm analysis, sperm migration testing, antisperm antibody screening, immunocytochemical round cell differentiation to determine seminal leukocytes counts and the leukocyte ratio, complement fraction C(3) (C(3c)) determination, and semen cultures, in aliquots of the same ejaculates. The SP concentration of IL-8 was inversely related to semen quality, e.g. to the total number of motile spermatozoa or to the outcome of the sperm migration test (motile sperm harvested after a swim-up procedure). IL-8 concentrations were significantly correlated with leukocyte counts per ml (P < 0.0001) and per ejaculate (P < 0.0001), and with the leukocyte ratio (P < 0.001). All leukocytospermic samples had high IL-8 concentrations (< or =2 ng/ml). The SP concentration of IL-6 was much lower, but was significantly correlated with IL-8 (P < 0.0001). Both IL-8 and IL-6 were significantly related with the C(3c). No association of interleukin concentrations with the bacterial colonization of semen samples was found. The results indicate a marked relationship of some pro-inflammatory cytokines with semen quality. The significant association with seminal leukocytes and other potential inflammation markers suggests that IL-8 might be used as sensitive marker for silent male genital tract infection.     

Role of interleukin (IL)-18 in experimental paracoccidioidomycosis.

Interleukin (IL)-18 has been regarded as a Th1 type cytokine involved in many fungal and parasitic infections. Since there have been no studies, as of yet, evaluating the role of this cytokine in paracoccidioidomycosis (PCM), we assessed the function of IL-18 by using an experimental PCM model. Our results showed that IL-18 knockout (IL-18 -/-) BALB/c were more resistant to Paracoccidioides brasiliensis than their littermate controls (WT). In fact, mortality rate was higher in WT mice and in the first month of infection, the number of colony forming units of the etiologic agent recovered from the lungs was greater in WT mice. In histopathological analyses, well-formed granulomas were seen in both WT and IL-18(-/-) mice. However, substantial differences were observed at the second month of infection when epithelioid cells predominated in the lesions of IL-18(-/-) mice, which could infer that IL-18 postpones pulmonary healing. The levels of IL-10 were significantly higher in IL-18 sufficient mice at early stages of infection and therefore account for the delayed fungal clearance observed in WT mice. TNF-alpha augmented later in the infection of WT mice, seemingly to compensate high levels of IL-10. Our results demonstrated that IL-18 has a critical role in protecting BALB/c mice against disseminated PCM.     

Ethnic differences in interleukin 6 (IL-6) and IL6 receptor genes in spontaneous preterm birth and effects on amniotic fluid protein levels

Preterm birth (PTB) is a significant neonatal health problem that is more common in African-Americans (AA) than in European-Americans (EA). Part of this disparity is likely to result from the differing genetic architectures of EA and AA. To begin assessing the role of these differences, patterns of genetic variation in two previously proposed candidate genes, encoding interleukin 6 ( IL6 ) and its receptor ( IL6R ), were analyzed in mothers and fetuses from 496 EA birth-events (149 cases and 347 controls) and 397 birth-events in AA (76 cases and 321 controls). IL-6 levels in amniotic fluid (AF) samples were determined in a subset of these pregnancies. Case-control comparisons revealed a single SNP in IL6R associated with PTB (p=0.04 for allelic and p=0.05 for genotype association). In addition, all of the SNPs studied showed significant frequency differences between AA and EA in at least one comparison, significantly in excess of that expected from general population databases. Higher IL-6 concentrations were associated with the IL6 SNP -661 in EA preterm samples (p=0.0056), and this result seems to be driven by microbial invasion of the amniotic cavity, indicating a gene by infection interaction. These findings indicate that, as a function of IL6 genotype, EA and AA women respond differently to infection with respect to their expression of IL-6. Our data support differential genetic control of levels of IL-6 in amniotic fluid between EA and AA.     

Identification of novel SNPs in the interleukin 6 receptor gene (IL6R)

The human interleukin-6 receptor (IL6R) is responsible for signal transduction of IL6 that might have associations with immune diseases and various infectious diseases. We have sequenced all 10 exons including the putative splicing site to identify single nucleotide polymorphisms in IL6R. Seven novel single nucleotide polymorphisms (SNPs) were identified; one SNP in the promoter region (-183G>A), one synonymous SNP in exon 2 (24013G>A: Ala31Ala), one non-synonymous SNP in exon 9 (48892A>C: Asp358Ala) and four in introns (29753A>C, 42700T>C, 48869T>A and 59818C>T). The frequencies of each SNP in the Korean population (n=300) were 0.48 (-183 G>A), 0.13 (24013 G>A), 0.41 (29753A>C), 0.41 (42700T>C), 0.1 (48869T>A), 0.42 (48892A>C), and 0.07 (59818C>T), respectively. Haplotypes and their frequencies were estimated by the EM algorithm. Linkage disequilibrium coefficients (/D'/) between each SNP pair were also calculated. The information on SNPs and haplotypes in IL6R would be useful for genetic studies of this gene.     

Expression of interleukin (IL)-4 and IL-5 proteins in asthma induced by toluene diisocyanate (TDI)

Background TDI-induced asthma exhibits clinical, functional and morphological similarities with allergen-induced asthma, suggesting that an immunological mechanism is involved in the sensitization to TDL In vitro studies using the technique of cloning lymphocytes demonstrated that a great proportion of T-cell clones derived from bronchial mucosa of subjects with TDI-induced asthma produced IL-5 and interferon-gamma, but not IL-4, upon in vitro stimulation. Objectives To investigate in vivo the role of IL-4 and IL-5 on the inflammatory response of the bronchial mucosa to TDI in sensitized subjects, we performed a quantitative analysis of bronchial biopsies. Methods We obtained bronchial biopsies from six subjects with TDI asthrha 48 h after an asthmatic reaction induced by TDI challenge (challenged group), in six subjects with TDI asthma 1–4 weeks after the last exposure to TDI (chronic group), and in six non-asthmatic controls. The number of eosinophils, mast cells, T-lymphocytes, and IL-4 and IL-5 protein positive cells was determined by immunohistochemistry in the area 100 μn beneath the epithelial basement membrane. Results The characteristic increase of submucosal eosinophils, but not of mast cells and T-lymphocytes, was observed in the subjects with TDI-induced asthma when compared with controls. No differences were detected between the two groups of asthmatics. In the subjects with TDI-induced asthma, cell immunoreactivity for IL-5 was increased when compared with normal controls. There was no difference in the expression of IL-5 protein between challenged and chronic asthmatics. In contrast, the expression of IL-4 protein was increased only in the asthmatic subjects tested after recent exposure to TDI. Conclusions We demonstrated that TDI asthma 48 h after specific bronchial challenge was associated with increased numbers of cells expressing IL-4 and IL-5, whereas chronic TDI asthma was associated with increased expression of IL-5, but not of IL-4. The results suggest that subjects who developed TDI asthma exhibit increased production of IL-5 even in the absence of a recent trigger by the exogenous sensitizer and that production of TH₂-like cytokines in TDI-induced asthma may not always be co-ordinately regulated in vivo.     

绒毛中TRAIL及其受体的表达与不明原因反复自然流产的相关性的探讨

目的探讨肿瘤坏死因子相关凋亡诱导配体TRAIL及其受体在不明原因早期反复自然流产患者(URSA)绒毛中的表达及二者的相关性。方法选择实验组妊娠45～60 d的URSA患者15例,对照组健康人工流产15例。两组分别取绒毛组织采用免疫组化法检测TRAIL及其受体的表达,进行方差分析和t检验比较两组绒毛组织中TRAIL及其受体蛋白表达水平的差异。结果实验组绒毛组织中TRAIL及其受体DR4、DR5蛋白表达较强,而诱骗受体DcR1及DcR2蛋白与正常对照相比表达较少,P值均小于0.05。结论TRAIL及其受体可能是造成自然流产的机制之一。     

T辅助细胞、NK细胞与原因不明复发性自然流产关系的研究进展

目前母-胎界面的免疫耐受机制是国内外的研究热点之一,URSA所表现的母-胎界面免疫耐受异常与T辅助细胞及NK细胞有密切的关系。本文首先对T辅助细胞和NK细胞的免疫状态、免疫耐受机制以及它们与URSA之间的关系进行了阐述,最后对目前与二者有关的URSA的主动免疫治疗研究进展作一综述。     

The role of thrombophilia and thyroid autoimmunity in unexplained infertility, implantation failure and recurrent spontaneous abortion

BACKGROUND: The role of thrombophilia and thyroid autoimmunity in unexplained infertility (UI), implantation failure (IF) and recurrent spontaneous abortion (RSA) is controversial and poorly understood. METHODS: From March, 2004 to January, 2007, 119 women were prospectively included: 32 oocyte donors, 31 patients with UI, 26 with IF and 30 with RSA. The IF and RSA groups presented normal preimplantation genetic screening. Protein C, protein S, antithrombin III, lupus anticoagulant, activated protein C resistance (APCR), immunoglobulin M and G anticardiolipin antibodies, homocystine, Factor V Leiden, prothrombin G20210A mutation, methylentetrahydrofolate reductase C677T mutation, thyroid-stimulating hormone (TSH), free thyroxine, anti-thyroid peroxidase (TPO) and anti-thyroglobulin (TG) antibodies were assessed. RESULTS: The prevalence of thrombophilia was high and similar among groups. In the IF group, the prevalence of APCR (15.4%), lupus anticoagulant (11.5%) and combined thrombophilia (19.2%) was higher, but not significantly different, than the other three groups. The prevalence of thyroid autoimmunity in women with IF (anti-TPO antibodies, P = 0.009; anti-TPO plus anti-TG antibodies,P = 0.04) and UI (anti-TPO, P = 0.002; anti-TG, P = 0.019; anti-TPO plus anti-TG antibodies, P = 0.005) was significantly increased in comparison to those with RSA. There was also a trend towards a higher prevalence of thyroid autoimmunity in the UI and IF groups than in the control group. TSH and free thyroxine levels all remained within a normal range. CONCLUSIONS: When embryo aneuploidy is ruled out, thrombophilia could constitute an etiologic factor in IF. Furthermore, thyroid autoimmunity is strongly related to UI and IF.     

T and B lymphocyte subsets in patients with unexplained recurrent spontaneous abortion: IVIG versus placebo treatment

PROBLEM: To investigate circulating lymphocyte subsets in women with recurrent spontaneous abortion (RSA) in relation to pregnancy outcome and to treatment with intravenous immunoglobulin (IVIG). METHOD OF STUDY: Forty-one women with a history of unexplained RSA were examined during first trimester of pregnancy before IVIG or placebo treatment and after pregnancy. The results were compared with five healthy, non-pregnant women and five women in the first trimester of normal pregnancy. Circulating lymphocyte subsets with focus on T-cell subpopulations were determined by flow cytometry. RESULTS: The proportions of human leukocyte antigen (HLA)-DR positive T cells (CD3+ HLA-DR+), T-killer/effector cells (CD8+ S6F1+) and B cells (CD19+) were increased, whereas the proportion of T-suppressor/inducer cells (CD4+ CD45RA+) was decreased during first trimester pregnancy of RSA women compared with pregnant normal controls. T and B lymphocyte subsets did not correlate with pregnancy outcome on either IVIG or placebo group. CONCLUSIONS: In RSA patients, the immune system seems to be activated in contrast to the suppression noted in normal pregnancy.     

miR-29c-3p regulates TET2 expression and inhibits autophagy process in Parkinson's disease models

Autophagy in dopamine (DA) neurons is concerned to be associated with Parkinson's disease (PD), but the detailed mechanism remains unknown. Herein, we aimed to investigate the function of microRNA (miR)-29c-3p in autophagy in PD models. Intraperitoneal injection of MPTP (20 mg/kg) was given to C57BL/6 mice to establish PD mouse model. SH-SY5Y cells were treated with MPP⁺ (1 mmol/L) to establish in vitro PD model. The results indicated that in the substantia nigra pars compacta (SNpc) DA neurons of PD mice, autophagy was activated accompanied by down-regulated miR-29c-3p and up-regulated ten-eleven translocation 2 (TET2) expression. Up-regulation of miR-29c-3p inhibited TET2 expression and SNpc (including DA neurons) autophagy in PD mice. In vitro PD model confirmed that MPP⁺ treatment markedly down-regulated miR-29c-3p expression and up-regulated TET2 expression in SH-SY5Y cells in a dose/time-dependent manner. Moreover, miR-29c-3p up-regulation also inhibited autophagy and TET2 expression in vitro. Additionally, TET2 was proved to be targeted and down-regulated by miR-29c-3p. TET2 knockdown inhibited MPP⁺-induced autophagy, whereas TET2 over-expression reversed the effects of miR-29c-3p over-expression on SH-SY5Y cell autophagy. Overall, miR-29c-3p over-expression inhibits autophagy in PD models, which may be mediated by TET2. Our finding may provide new insights for regulating autophagy to improve PD progression.     

利用噬菌体随机肽库筛选IL-5结合的相关多肽

目的：采用噬菌体随机肽库筛选人IL-5，以获得能高亲和力结合IL-5的相关多肽。方法：以重组人IL-5作为筛选分子，应用M13噬菌体PⅢ呈现随机7肽库进行筛选。经过三轮淘筛后，经夹心ELISA、竞争ELISA法进一步鉴定噬菌体克隆与IL-5的亲和力，对阳性克隆进行了扩增和DNA测序，据此推导结合随机多肽的氨基酸序列。结果：经鉴定得到9个阳性噬菌体克隆能与IL-5呈高亲和力结合。通过测序和序列比较分析，发现CX1-2AS为相对保守的结合表位。结论：研究表明通过噬菌体肽库能够筛选到IL-5结合的相关多肽，为进一步研究IL-5结合表位及抑制剂奠定了基础。