Antigen-Induced Activation of Hypothalamic Cells (assessed by expression of the c-fos gene)

The aim of the present work was to study the activation of the expression of the c-fos gene (by in situ hybridization) in cells from rat (Sprague Dawley) hypothalamic structures 0.5, 2, 6, and 16 h after i.v. injections of tetanus toxoid (200 g/kg). Tetanus toxoid was selected as the antigen because it does not induce any general non-specific body reactions. Control animals received i.v. doses of apyrogenic physiological saline. The number of c-fos mRNA-positive cells in all the hypothalamic structures studied was insignificant 30 min after injections of tetanus toxoid. c-fos mRNA-positive cells were seen in the posterior, lateral, and anterior hypothalamic fields and in the dorsomedial and ventromedial hypothalamic nuclei 2 h after injections of tetanus toxoid. The intensity of c-fos mRNA expression decreased in the posterior, lateral, and anterior hypothalamic fields 6 h after injections of tetanus toxoid. The maximum number of c-fos mRNA-positive cells in the anterior field and the paraventricular nucleus of the hypothalamic induced by tetanus toxoid, as compared with reactions to administration of physiological saline, were seen at 6 h. Administration of tetanus toxoid and physiological saline did not active the synthesis of c-fos mRNA in the arcuate or supraoptic nuclei at any time point. The number of c-fos mRNA-positive cells returned to baseline by 16 h after tetanus toxoid injections. Thus, this study revealed the temporospatial pattern of activation of hypothalamic structures in response to exposure to an antigen.     

Isolation of Trypanosoma brucei variant specific antigen mRNA by immunoprecipitation of polysomes

The mRNA encoding the variant specific antigen of Trypanosoma brucei has been prepared by immunoprecipitation of polysomes. Polysomes carrying the variant specific antigen account for approx. 3% of the total polysomes. The mRNA thus produced is active in the mRNA dependent rabbit reticulocyte lysate in vitro protein synthesis system and directs the synthesis of a polypeptide of 60 000 daltons which co-migrates both with ¹²⁵I-labelled purified variant specific antigen and with antigen immunoprecipitated from reticulocyte lysate charged with total polyadenylated mRNA from the same clone. The mRNA is being used both to prepare cDNA clones and to prepare high specific radioactivity cDNA to be used to screen a gene bank for clones containing variant specific antigen coding sequences.     

Role of the 2 adenine (g.11293_11294insAA) insertion polymorphism in the 3' untranslated region of the factor VII (FVII) gene: molecular characterization of a patient with severe FVII deficiency

Polymorphic variants in the gene encoding factor VII (F7) affect the plasma levels of this coagulation protein and modify the clinical phenotype of FVII deficiency in some patients. In this study we report the in vitro functional analysis of a novel polymorphic variant located in the 3' untranslated region of F7: g.11293_11294insAA. To determine whether this variant regulates FVII expression, we initially compared an expression vector containing FVII cDNA with g.11293_11294insAA with the FVII wild-type (WT) construct. The kinetics of mRNA production showed that the insertion decreases the steady-state FVII mRNA levels. To assess whether the insertion influences the phenotype of FVII-deficient patients, we evaluated its effect on the expression of FVII in a patient with severe FVII deficiency (undetectable FVII activity and antigen) carrying two additional homozygous missense variations (p.Arg277Cys and p.Arg353Gln). The two substitutions alone reduced the expression of FVII activity and antigen in vitro, but with the insertion polymorphism in our expression vector the patient's phenotype of undetectable plasma FVII was recapitulated. The insertion polymorphism in the 3' untranslated region of F7 is another modifier of FVII expression that might explain the poor genotype-phenotype correlation in some FVII-deficient patients.     

Isolation and characterization of a cDNA coding for a novel human 17.3K myelin basic protein (MBP) variant

Human fetal spinal cord poly A (+) mRNA was found to direct the synthesis of three major myelin basic protein (MBP) variants with molecular weights of 17K, 18.5K, and 21.5K when translated in reticulocyte lysates. In order to investigate the structural relationships between these MBP variants and their corresponding mouse variants, human fetal spinal cord and mouse brain cDNA libraries were constructed and screened for MBP cDNAs. A number of MBP cDNA clones were isolated and characterized. One of these, PP535 contained the entire coding region of the mouse 14K MBP; and another mouse cDNA clone, PP1.85, was almost full-length and coded for either the 21.5K MBP or the 18.5K MBP. A human clone (KK36), 1,173 nucleotides in length, contained the entire coding region of an MBP variant with a molecular weight of 17,342. The structure of this clone within its coding region is significantly different from the corresponding mouse 17K MBP cDNA. It is missing two sequences found in the mouse 17K MBP cDNA (exons 2 and 5); and it contains a sequence (exon 6) that is missing from the mouse 17K MBP cDNA. Thus, this human 17.3K cDNA codes for a "17K" human MBP variant that is quite different from the corresponding mouse variant and is identical to the human 18.5K MBP except for a deletion of a peptide consisting of 11 amino acids that includes the single tryptophan residue of the 18.5K MBP. An analysis of the structure of this 17.3K human MBP cDNA suggests that the major pathway for splicing the primary human MBP gene product may be different from that in the mouse.     

慢性乙型肝炎患者外周血SOCS3 mRNA的表达及其与Th17的关系

 目的 研究慢性乙型肝炎(CHB)患者外周血辅助性T细胞17(Th17)以及细胞因子信号转导抑制因子3(SOCS3) mRNA的表达状况,探索CHB患者SOCS3 mRNA表达与Th17的关系。方法 选取2021年2—8月于某院门诊就诊的30例CHB患者为研究对象,并选取同期正常体检者15例为对照组。采用流式细胞术(FCM)检测外周血Th17细胞频数,酶联免疫吸附试验(ELISA)检测血清细胞因子IL-17A和IL-23表达水平,实时荧光定量逆转录聚合酶链反应(qRT-PCR)法测定外周血SOCS3、维甲酸相关孤儿核受体γt (RORγt) mRNA表达水平,并比较两组患者的检测结果。结果 CHB患者外周血Th17细胞频数及其效应分子IL-17A、IL-23表达水平高于对照组(均P＜0.05),Th17细胞频数、IL-17A与HBV DNA水平之间存在正相关(r值分别为0.570、0.563,均P＜0.005)。CHB患者外周血SOCS3、RORγt mRNA表达水平高于对照组(均P＜0.05),两者与HBV DNA水平正相关(r值分别为0.662、0.561,均P＜0.05)。CHB患者SOCS3 mRNA与RORγt mRNA、Th17细胞频数、IL-17A之间存在正相关(r值分别为0.552、0.626、0.826,均P＜0.05)。结论 CHB患者外周血SOCS3 mRNA的异常高表达,可能通过调节RORγt mRNA的表达来影响Th17的分化及其效应分子的分泌。     

人食管癌相关基因cDNA片段的克隆、筛选与初步鉴定

目的 :筛选和鉴定人原发性食管癌组织的相关基因 ,揭示食管癌的癌变机理。方法 :用高效、灵敏的荧光mRNA差异显示技术 ,以食管癌及相应的正常食管粘膜组织为对照 ,通过对其基因表达的比较 ,找出差异条带 ,利用ReverseNorthernDotBlot、DNA序列分析和NorthernBlot,并结合mRNA原位杂交技术对被筛片段进行鉴定。结果 :①在实验中分离、鉴定了 74条差异片段 ,其中包括正常组织表达而癌组织中不表达的差异片段 5 4个 ,癌组织中表达而正常组织不表达的差异片段 2 0个。②其中 1个片段C5 7(337bp)在GenBank数据库中没有发现同源的已知基因或片段 ,推测其可能为食管癌相关基因。③经NorthernBlot检测C5 7在癌组织中有表达信号。④原位杂交技术检测C5 7在癌组织中具有较高的阳性表达率 (74% )。结论 :食管癌组织中C5 7可能是新的食管癌相关的候选癌基因 ,它与食管癌的发生发展可能有关     

Discovery and Validation of a Urinary Exosome mRNA Signature for the Diagnosis of Human Kidney Transplant Rejection

BackgroundDeveloping a noninvasive clinical test to accurately diagnose kidney allograft rejection is critical to improve allograft outcomes. Urinary exosomes, tiny vesicles released into the urine that carry parent cells’ proteins and nucleic acids, reflect the biologic function of the parent cells within the kidney, including immune cells. Their stability in urine makes them a potentially powerful tool for liquid biopsy and a noninvasive diagnostic biomarker for kidney-transplant rejection.MethodsUsing 192 of 220 urine samples with matched biopsy samples from 175 patients who underwent a clinically indicated kidney-transplant biopsy, we isolated urinary exosomal mRNAs and developed rejection signatures on the basis of differential gene expression. We used crossvalidation to assess the performance of the signatures on multiple data subsets.ResultsAn exosomal mRNA signature discriminated between biopsy samples from patients with all-cause rejection and those with no rejection, yielding an area under the curve (AUC) of 0.93 (95% CI, 0.87 to 0.98), which is significantly better than the current standard of care (increase in eGFR AUC of 0.57; 95% CI, 0.49 to 0.65). The exosome-based signature’s negative predictive value was 93.3% and its positive predictive value was 86.2%. Using the same approach, we identified an additional gene signature that discriminated patients with T cell–mediated rejection from those with antibody-mediated rejection (with an AUC of 0.87; 95% CI, 0.76 to 0.97). This signature’s negative predictive value was 90.6% and its positive predictive value was 77.8%.ConclusionsOur findings show that mRNA signatures derived from urinary exosomes represent a powerful and noninvasive tool to screen for kidney allograft rejection. This finding has the potential to assist clinicians in therapeutic decision making.