ABSTRACT
An operationally simple and convenient synthesis method toward a series of diverse spiro[4.4]thiadiazole derivatives via double [3 + 2] 1,3-dipolar cycloaddition of nitrilimines generated in situ from hydrazonyl chlorides with carbon disulfide has been achieved under mild reaction conditions.
ABSTRACT
MicroRNAs (miRNAs) play key roles in the regulation of gene expression during multiple physiological processes, including early development, differentiation, and ageing. However, their involvement in age-related thymic involution is not clear. In this study, we profiled the global transcriptome and miRNAome of thymic epithelial cells in 1- and 3-month-old male and female mice, and predicted the possible transcription factors and target genes of the four most significantly differentially expressed miRNAs (DEMs) (miR-183-5p, miR-199b-5p, miR-205-5p, and miR-200b-3p) by performing bioinformatics analyses. We also evaluated the relationships between the significantly DEMs and mRNAs. We performed quantitative polymerase chain reaction to confirm the changes in the expression of the miRNAs and their predicted target genes. We found that miR-183-5p, miR-199b-5p, miR-205-5p, and miR-200b-3p can be used as a biomarker group for mouse thymus development and involution. In addition, the predicted target genes (Ptpn4, Slc2a9, Pkib, Pecam1, and Prkdc), which were identified by mRNA sequencing analysis, were mainly involved in growth, development, and accelerated senescence. In conclusion, miRNAs and their predicted target genes likely play important roles in thymus development and involution. To the best of our knowledge, this is the first study to systematically analyze the relevance of miRNAs and their targets by mRNA sequencing in mouse thymic epithelial cells. © 2018 IUBMB Life, 70(7):678-690, 2018.
Subject(s)
Aging/genetics , Epithelial Cells/physiology , MicroRNAs/genetics , RNA, Messenger/genetics , Thymus Gland/cytology , Animals , Female , Gene Regulatory Networks , High-Throughput Nucleotide Sequencing , Male , Mice, Inbred BALB C , Protein Interaction Maps/genetics , Real-Time Polymerase Chain Reaction , Reproducibility of Results , Thymus Gland/physiology , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Programmed cell death protein 1 (PD-1), a costimulatory molecule of the CD28 family, has 2 ligands, PD-L1 and PD-L2. Our previous studies showed that the expression of PD-1 and PD-L1 is up-regulated during viral infection in pigs. Extensive studies have shown that blockade of the PD-1/PD-L1 pathways by anti-PD-L1 antibody or soluble PD-1 restores exhausted T-cells in humans and mice. In the present study the extracellular domains of PD-1 and PD-L1 were used to evaluate the binding of PD-1 and PD-L1 with peripheral blood mononuclear cells (PBMCs). We amplified the cDNA encoding the extracellular domains of PD-1 and PD-L1 to construct recombinant expression plasmids and obtain soluble recombinant proteins, which were then labeled with fluorescein isothiocyanate (FITC). The His-ExPD-1 and His-ExPD-L1 recombinant proteins were expressed in the form of inclusion bodies with a relative molecular weight of 33.0 and 45.0 kDa, respectively. We then prepared polyclonal antibodies against the proteins with a multi-antiserum titer of 1:102 400. Binding of the proteins with PBMCs was evaluated by flow cytometry. The fluorescence signals of His-ExPD-1-FITC and His-ExPD-L1-FITC were greater than those for the FITC control. These results suggest that the soluble recombinant proteins may be used to prepare monoclonal antibodies to block the PD-1/PD-L1 pathway.
La protéine de la mort cellulaire programmée (PD-1), une molécule co-stimulatrice de la famille de CD28, a deux ligands, PD-L1 et PD-L2. Nos études antérieures ont montré que l'expression de PD-1 et PD-L1 est régulée à la hausse lors d'une infection virale chez des porcs. Des études exhaustives ont montré chez l'humain et la souris qu'un blocage de la voie PD-1/PD-L1 par des anticorps anti PD-L1 ou du PD-1 soluble permet la régénération des cellules T épuisées. Dans la présente étude les domaines extracellulaires de PD-1 et PD-L1 ont été utilisés afin d'évaluer l'attachement de PD-1 et PD-L1 avec des cellules mononucléaires du sang périphérique (CMSP). Nous avons amplifié l'ADNc codant pour les domaines extracellulaires de PD-1 et PD-L1 pour construire des plasmides d'expression recombinants et obtenir des protéines recombinants solubles, qui ont par la suite été marquées avec de l'isothiocyanate de fluorescéine (ITCF). Les protéines recombinantes His-ExPD-1 et His-ExPD-L1 étaient exprimées sous la forme de corps d'inclusion avec un poids moléculaire relatif de 33,0 et 45,0 kDa, respectivement. Nous avons par la suite préparé des anticorps polyclonaux contre ces protéines avec un antisérum titrant 1:102 400. L'attachement des protéines aux CMSP a été évalué par cytométrie en flux. Les signaux de fluorescence de His-ExPD-1-ITCF et His-ExPD-L1-ITCF étaient supérieurs à ceux pour le témoin ITCF. Ces résultats suggèrent que les protéines recombinantes solubles pourraient être utilisées afin de préparer des anticorps monoclonaux pour bloquer la voie PD-1/PD-L1.(Traduit par Docteur Serge Messier).
Subject(s)
B7-H1 Antigen/metabolism , Leukocytes, Mononuclear/physiology , Programmed Cell Death 1 Receptor/metabolism , Swine , Animals , Antibodies , B7-H1 Antigen/genetics , Cell Adhesion , Cloning, Molecular , Female , Programmed Cell Death 1 Receptor/genetics , Protein Domains , RabbitsABSTRACT
In order to investigate the relationship between the PD-1 pathway and impairment of immune responses with the CSFV infection, the mRNA expression of PD-1 and its ligands were evaluated by quantitative polymerase chain reaction (qPCR) during artificial CSFV infection. Simultaneously, expression of IL-2 and IL-10 mRNA were detected. The T cell proliferation and CSFV load in plasma were also measured. Results showed that the expression of PD-1 and its ligands mRNA were significantly increased (p < 0.01) in PBMC from 3 to 7 days post infection (dpi). Meanwhile the level of IL-10 was up-regulated (p < 0.01). The IL-2 mRNA was not obviously changed but it is significantly increased from 14 dpi. The T cell proliferation was notably decreased at 7 dpi. The CSFV load was also increased in plasma. Overall, our results suggest that the expression of PD-1 and its ligands were up-regulated and probably correlated with immune inhibition during acute CSFV infection.
