scholarly journals Double labeling with [3H]thymidine and [125I]iododeoxyuridine as a method for determining the fate of injected DNA and cells in vivo.

1975 ◽  
Vol 67 (2) ◽  
pp. 484-488 ◽  
Author(s):  
D K Myers ◽  
L E Feinendegen
Keyword(s):  
T Cells ◽  
Human Kidney ◽  
Enzymic Hydrolysis ◽  
Double Labeling ◽  
Exogenous Dna ◽  
Mouse Thymus ◽  
Whole Cells ◽  
Thymus Cells ◽  
Hydrolysis Of

Mice were injected intravenously and intraperitoneally with preparations of intestinal nucleoprotein, spleen nuclei, mouse thymus cells, or human kidney T cells whose DNA had been labeled with both [3H]thymidine (TdR) and [125I]-iododeoxyuridine (IUdR). Since free TdR is reutilized more efficiently than free IUdR produced by enzymic hydrolysis of the exogenous DNA, the ratio of [3H]TdR/[125I]IUdR in the DNA fraction of the tissues of the recipient mice provides a measure of the amount of intact exogenous DNA in the tissue. In most instances, the doubly labeled exogenous DNA was almost completely hydrolyzed within 1 day injection, but survival of the DNA from whole cells could be demonstrated in some cases.

scholarly journals IMMUNOLOGIC REACTIONS TO HAPTENS ON AUTOLOGOUS CARRIERS

1973 ◽  
Vol 137 (2) ◽  
pp. 411-423 ◽  
Author(s):  
John W. Moorhead ◽  
Curla S. Walters ◽  
Henry N. Claman
Keyword(s):  
T Cells ◽  
B Cells ◽  
Aminocaproic Acid ◽  
Single Amino Acid ◽  
Secondary Response ◽  
Spleen Cells ◽  
Activated T Cells ◽  
Thymus Cells

Both thymus-derived (T) and bone marrow-derived (B) lymphocytes participate in the response to a hapten 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP), coupled to a nonimmunogenic isologous carrier, mouse gamma globulin (MGG). Spleen cells from mice immunized with NIP-MGG show increased DNA synthesis in vitro when cultured with NIP-MGG. The participation of and requirement for T cells in the response was demonstrated by treating the spleen cells with anti-θ serum. This treatment resulted in a 77% inhibition of the antigen response. Furthermore, adoptively transferred normal thymus cells could be specifically "activated" by NIP-MGG in vivo and they responded secondarily to the antigen in vitro. The active participation of B cells in the secondary response was demonstrated by passing the immune spleen cells through a column coated with polyvalent anti-MGG serum. Column filtration reduced the number of NIP-specific plaque-forming cells and NIP-specific rosette-forming cells (both functions of B cells) and produced a 47% inhibition of the NIP-MGG response. The ability of the cells to respond to phytohemagglutinin (PHA) was not affected by column filtration showing that T cells were not being selectively removed. The participation of B cells in the in vitro NIP-MGG response was also shown by treatment of the spleen cells with antiserum specific for MGG and MGG determinants. B cells were removed by treatment with anti-IgM or polyvalent anti-MGG serum plus complement, resulting in a respective 46 and 49% inhibition of the response to NIP-MGG. (Treatment with anti-IgM serum had no effect on T cells.) The contribution of the hapten NIP to stimulation of T cells was investigated using NIP-MGG-activated thymus cells. These activated T cells responded in vitro very well to the NIP-MGG complex but not to the MGG carrier alone demonstrating the requirement of the hapten for T cell stimulation. The response was also partially inhibited (41%) by incubating the activated cells with NIP coupled to a single amino acid (epsilon-aminocaproic acid) before addition of NIP-MGG. These results demonstrated that T cells recognize the hapten NIP when it is coupled to the isologous carrier MGG.

Activated protein C targets immune cells and rheumatoid synovial fibroblasts to prevent inflammatory arthritis in mice

Rheumatology ◽  
2019 ◽  
Vol 58 (10) ◽  
pp. 1850-1860 ◽  
Author(s):  
Meilang Xue ◽  
Suat Dervish ◽  
Kelly J McKelvey ◽  
Lyn March ◽  
Fang Wang ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Synovial Fibroblasts ◽  
Endothelial Protein C Receptor ◽  
Mouse Thymus ◽  
Tnf Α ◽  
Thymus Cells ◽  
Proliferation And Invasion

Abstract Objectives To investigate whether activated protein C (APC), a physiological anticoagulant can inhibit the inflammatory/invasive properties of immune cells and rheumatoid arthritis synovial fibroblasts (RASFs) in vitro and prevent inflammatory arthritis in murine antigen-induced arthritis (AIA) and CIA models. Methods RASFs isolated from synovial tissues of patients with RA, human peripheral blood mononuclear cells (PBMCs) and mouse thymus cells were treated with APC or TNF-α/IL-17 and the following assays were performed: RASF proliferation and invasion by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell invasion assays, respectively; cytokines and signalling molecules using ELISA or western blot; Th1 and Th17 phenotypes in human PBMCs or mouse thymus cells by flow cytometry. The in vivo effect of APC was evaluated in AIA and CIA models. Results In vitro, APC inhibited IL-1β, IL-17 and TNF-α production, IL-17-stimulated cell proliferation and invasion and p21 and nuclear factor κB activation in RASFs. In mouse thymus cells and human PBMCs, APC suppressed Th1 and Th17 phenotypes. In vivo, APC inhibited pannus formation, cartilage destruction and arthritis incidence/severity in both CIA and AIA models. In CIA, serum levels of IL-1β, IL-6, IL-17, TNF-α and soluble endothelial protein C receptor were significantly reduced by APC treatment. Blocking endothelial protein C receptor, the specific receptor for APC, abolished the early or preventative effect of APC in AIA. Conclusion APC prevents the onset and development of arthritis in CIA and AIA models via suppressing inflammation, Th1/Th17 phenotypes and RASF invasion, which is likely mediated via endothelial protein C receptor.

scholarly journals Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane

1988 ◽  
Vol 252 (1) ◽  
pp. 301-304 ◽  
Author(s):  
W H Baricos ◽  
Y Zhou ◽  
R W Mason ◽  
A J Barrett
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Kidney ◽  
Ph Optimum ◽  
Dose Dependent ◽  
Hydrolysis Of

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.

Enzymic Hydrolysis of Stilbestrol Diphosphate in vitro and in vivo.

1961 ◽  
Vol 106 (2) ◽  
pp. 327-330 ◽  
Author(s):  
W. Johnson ◽  
R. Jasmin ◽  
G. Corte
Keyword(s):  
Enzymic Hydrolysis ◽  
Hydrolysis Of

scholarly journals SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO

1932 ◽  
Vol 15 (5) ◽  
pp. 491-495 ◽  
Author(s):  
J. M. Nelson ◽  
Elizabeth T. Palmer ◽  
B. G. Wilkes
Keyword(s):  
Yeast Cells ◽  
Enzymic Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Live Yeast

1. A method is given whereby the course of hydrolysis of sucrose by live yeast cells may be followed with precision equal to that found when invertase solutions prepared from autolyzed yeast are used to cause inversion. 2. The practical value of the equation of Nelson and Hitchcock as a means of following the course of enzymic hydrolysis of sucrose is hereby extended. 3. The inversion of sucrose by live yeast cells and by extracted invertase has been quantitatively compared. 4. The course of hydrolysis of sucrose by the invertase of Fleischmann's yeast has been found to be identical in vivo and in vitro.

scholarly journals Nfil3 Is a Glucocorticoid-Regulated Gene Required for Glucocorticoid-Induced Apoptosis in Male Murine T Cells

Endocrinology ◽  
2013 ◽  
Vol 154 (4) ◽  
pp. 1540-1552 ◽  
Author(s):  
Kirstyn T. Carey ◽  
Kheng H. Tan ◽  
Judy Ng ◽  
Douglas R. Liddicoat ◽  
Dale I. Godfrey ◽  
...  
Keyword(s):  
T Cells ◽  
Natural Killer ◽  
Killer Cell ◽  
Mrna Levels ◽  
Small Interfering Rnas ◽  
Double Positive ◽  
Double Labeling ◽  
Exon 2 ◽  
Protein Levels

Abstract Glucocorticoids (GCs) have essential roles in the regulation of development, integrated metabolism, and immune and neurological responses, and act primarily via the glucocorticoid receptor (GR). In most cells, GC treatment results in down-regulation of GR mRNA and protein levels via negative feedback mechanisms. However, in GC-treated thymocytes, GR protein levels are maintained at a high level, increasing sensitivity of thymocytes to GCs, resulting in apoptosis termed glucocorticoid-induced cell death (GICD). CD4+CD8+ double-positive thymocytes and thymic natural killer T cells in particular are highly sensitive to GICD. Although GICD is exploited via the use of synthetic GC analogues in the treatment of hematopoietic malignancies, the intracellular molecular pathway of GICD is not well understood. To explore GICD in thymocytes, the authors performed whole genome expression microarray analysis in mouse GR exon 2 null vs wild-type thymus RNA 3 hours after dexamethasone treatment. Identified and validated direct GR targets included P21 and Bim, in addition to an important transcriptional regulator Nfil3, which previously has been associated with GICD and is essential for natural killer cell development in vivo. Immunostaining of NFIL3 in whole thymus localized NFIL3 primarily to the medullary region, and double labeling colocalized NFIL3 to apoptotic cells. In silico analysis revealed a putative GC response element 5 kb upstream of the Nfil3 promoter that is strongly conserved in the rat genome and was confirmed to bind GR by chromatin immunoprecipitation. The knockdown of Nfil3 mRNA levels to 20% of normal using specific small interfering RNAs abrogated GICD, indicating that NFIL3 is required for normal GICD in CTLL-2 T cells.

scholarly journals EVIDENCE FOR THE EXPRESSION OF Ia (H-2-ASSOCIATED) ANTIGENS ON THYMUS-DERIVED LYMPHOCYTES

1974 ◽  
Vol 140 (5) ◽  
pp. 1273-1284 ◽  
Author(s):  
Jeffrey A. Frelinger ◽  
John E. Niederhuber ◽  
C. S. David ◽  
Donald C. Shreffler
Keyword(s):  
T Cells ◽  
Lymph Node ◽  
B Cells ◽  
Cytotoxic Activity ◽  
Cell Line ◽  
Titration Curve ◽  
Specific Antibodies ◽  
Highly Sensitive ◽  
Thymus Cells

We have demonstrated in an anti-Ia serum the presence of specific antibodies reacting with T cells, as well as with B cells, using a highly sensitive dye exclusion test. This antiserum reacts with both spleen and lymph node in a characteristic biphasic titration curve killing up to 70% of these cells. It also reacts with cortisone-resistant thymocytes. The A.TH-α-A.TL serum can be absorbed with spleen, lymph node, cortisone-resistant thymus, or normal thymus cells. Further in vivo absorptions in BALB/c nude cannot remove all of the cytotoxic activity for normal BALB lymph node lymphocytes, while completely removing the activity for nude cells. A Thy-1 positive cell line derived from a C57Br leukemia is reactive with this anti-Ia serum.

scholarly journals B CELL ACTIVATION IN VIVO BY NONANTIGEN-SPECIFIC INTERACTION WITH T CELLS

1972 ◽  
Vol 136 (2) ◽  
pp. 381-386 ◽  
Author(s):  
T. Tito ◽  
G. M. Shearer ◽  
D. Trizio ◽  
G. Cudkowicz
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Activation ◽  
Bone Marrow Cells ◽  
Specific Interaction ◽  
The Other ◽  
B Cell Activation ◽  
Bone Marrow Precursor ◽  
Thymus Cells

The number of direct (γM) hemolytic plaque responses of irradiated mice, repopulated with relatively small and limiting numbers of bone marrow and thymus cells, was increased by the simultaneous immunization with two antigen complexes instead of one. Anti-sheep responses were augmented by the following antigen combinations: SRBC + HRBC, SRBC + BRBC, and SRBC + CRBC. Limiting either thymocytes or bone marrow cells indicated that the antigen mixtures acted at the level of T cells increasing severalfold the number of triggered antigen-reactive cells. It was concluded that one of the antigens could have influenced the triggering of antigen-reactive cells specific for the other by promoting synergistic or derepressive T-T cell interactions. Moreover, bone marrow precursor cells could have been activated by the thymic inducers specific for the test antigens as well as by those specific for the second of the priming antigens.

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