scholarly journals Anti-CD3 monoclonal antibody-mediated cytotoxicity occurs through an interleukin-2-independent pathway in CD3+ large granular lymphocytes

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 935-940 ◽  
Author(s):  
TP Jr Loughran ◽  
JA Aprile ◽  
FW Ruscetti
Keyword(s):  
Monoclonal Antibody ◽  
Messenger Rna ◽  
Blot Hybridization ◽  
Interleukin 2 ◽  
Northern Blot Hybridization ◽  
Binding Studies ◽  
Granule Formation ◽  
Receptor Sites ◽  
Gene Transcripts ◽  
Lgl Leukemia

Abstract The mechanism of induction of cytotoxicity produced by anti-CD3 monoclonal antibody (MoAb) was studied in four patients with CD3+ large granular lymphocyte (LGL) leukemia. Anti-CD3 MoAb treatment resulted in increased target cell binding and increased granule formation. After activation, leukemic LGL remained Tac-, with the exception of a patient with CD4+ LGL leukemia. Radiolabeled interleukin-2 (IL-2) binding studies demonstrated that treatment with anti-CD3 MoAb resulted in upregulation of the number of p75 intermediate affinity IL-2 receptor sites per cell. Northern blot hybridization analysis showed expression of gamma-interferon gene transcripts 24 to 48 hours after activation. There was no evidence for expression of IL-2 messenger RNA or secretion of IL-2 after activation. Anti-CD3 MoAb and IL-2 provide different signals for activation of CD3+ LGL. Induction of cytotoxicity produced by anti-CD3 MoAb in leukemic CD3+ LGL is not associated with IL-2 production.

scholarly journals Anti-CD3 monoclonal antibody-mediated cytotoxicity occurs through an interleukin-2-independent pathway in CD3+ large granular lymphocytes

Blood ◽  
1990 ◽  
Vol 75 (4) ◽  
pp. 935-940
Author(s):  
TP Jr Loughran ◽  
JA Aprile ◽  
FW Ruscetti
Keyword(s):  
Monoclonal Antibody ◽  
Messenger Rna ◽  
Blot Hybridization ◽  
Interleukin 2 ◽  
Northern Blot Hybridization ◽  
Binding Studies ◽  
Granule Formation ◽  
Receptor Sites ◽  
Gene Transcripts ◽  
Lgl Leukemia

The mechanism of induction of cytotoxicity produced by anti-CD3 monoclonal antibody (MoAb) was studied in four patients with CD3+ large granular lymphocyte (LGL) leukemia. Anti-CD3 MoAb treatment resulted in increased target cell binding and increased granule formation. After activation, leukemic LGL remained Tac-, with the exception of a patient with CD4+ LGL leukemia. Radiolabeled interleukin-2 (IL-2) binding studies demonstrated that treatment with anti-CD3 MoAb resulted in upregulation of the number of p75 intermediate affinity IL-2 receptor sites per cell. Northern blot hybridization analysis showed expression of gamma-interferon gene transcripts 24 to 48 hours after activation. There was no evidence for expression of IL-2 messenger RNA or secretion of IL-2 after activation. Anti-CD3 MoAb and IL-2 provide different signals for activation of CD3+ LGL. Induction of cytotoxicity produced by anti-CD3 MoAb in leukemic CD3+ LGL is not associated with IL-2 production.

scholarly journals Cloning of cDNA coding for connective tissue activating peptide III from a human platelet-derived lambda gt11 expression library

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1498-1503 ◽  
Author(s):  
RH Wenger ◽  
AN Wicki ◽  
A Walz ◽  
N Kieffer ◽  
KJ Clemetson
Keyword(s):  
Amino Acids ◽  
Connective Tissue ◽  
Cdna Library ◽  
Messenger Rna ◽  
Blot Hybridization ◽  
Single Species ◽  
Leader Sequence ◽  
Northern Blot Hybridization ◽  
Expression Library ◽  
Platelet Factor

Abstract We report here the cloning of the cDNA coding for platelet connective tissue-activating peptide-III (CTAP-III) from a lambda gt11 expression library prepared using messenger RNA (mRNA) isolated from human platelets. The open reading frame of the clone coded for a protein with 128 amino acid residues. Since the precursor of CTAP-III, platelet basic protein (PBP is 94 amino acids long, the 5′-translated region of the cDNA codes for a leader sequence 34 amino acids long. This leader sequence, like the sequence of mature CTAP-III, shows significant homology to the sequence of platelet factor 4 (PF4), the only other platelet specific alpha-granule protein cloned until now, from a human erythroleukemic (HEL) cell line-derived cDNA library. These leader sequences are probably critical for targeting such proteins to the alpha-granule. Northern blot hybridization with platelet and megakaryocyte mRNA shows a single species mRNA of approximately 0.8 kb, suggesting that the corresponding cDNA is full length. The cloning of platelet specific CTAP-III provides additional evidence for the platelet specificity of the cDNA library used.

scholarly journals Induction of hepatic metallothioneins determined at isoprotein and messenger RNA levels in glucocorticoid-treated rats

1988 ◽  
Vol 249 (2) ◽  
pp. 429-433 ◽  
Author(s):  
L D Lehman-McKeeman ◽  
G K Andrews ◽  
C D Klaassen
Keyword(s):  
Detection Limit ◽  
Northern Blot ◽  
Blot Analysis ◽  
Messenger Rna ◽  
Time Course ◽  
Northern Blot Analysis ◽  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
Single Dosage ◽  
Rna Levels

Induction of metallothionein-I (MT-I) and metallothionein-II (MT-II) by glucocorticoids was determined by h.p.l.c. analysis of proteins and Northern-blot analysis of MT mRNAs. Rats were injected with dexamethasone (0.03-10 mumol/kg) and hepatic concentrations of MTs were determined 24 h later. In control rats, only MT-II was detected (9.4 +/- 2.5 micrograms/g of liver), whereas the hepatic concentration of MT-I was below the detection limit (5 micrograms of MT/g). Dexamethasone did not increase MT-I above the detection limit at any dosage tested, but MT-II increased to 2.5 times control values at dosages of 0.30 mumol/kg and higher. Time-course experiments indicated that MT-II reached a maximum at 24 h after a single dosage of dexamethasone and returned to control values by 48 h. To determine whether dexamethasone increased MT-I in liver, samples were saturated with 109Cd, after which the amount of 109Cd in MT-I and MT-II was determined. Results indicated that, by this approach, MT-I and MT-II could be detected in control rats, and there was approx. 1.8 times more 109Cd in MT-II than in MT-I. At 24 h after administration of dexamethasone (1 mumol/kg), there was a small increase in the amount of 109Cd bound to MT-I, whereas the amount of 109Cd bound to MT-II increased to more than 2 times control values. Northern-blot hybridization with mouse cRNA probes indicated that MT-I and MT-II mRNAs increased co-ordinately after administration of dexamethasone. Thus, although glucocorticoids increase both MT-I and MT-II mRNAs, MT-II preferentially accumulates after administration of dexamethasone.

scholarly journals Endothelial cells augment T cell interleukin 2 production by a contact-dependent mechanism involving CD2/LFA-3 interaction.

1990 ◽  
Vol 171 (5) ◽  
pp. 1453-1467 ◽  
Author(s):  
C C Hughes ◽  
C O Savage ◽  
J S Pober
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Blot Hybridization ◽  
Interleukin 2 ◽  
Class Ii ◽  
Cell Contact ◽  
Northern Blot Hybridization ◽  
Ifn Gamma

We have demonstrated that endothelial cells (EC) augment IL-2 production by PHA-stimulated PBMC or purified CD4+ T cells and that the increase is apparent both in the amount of soluble IL-2 secreted and in the level of specific mRNA detectable by Northern blot hybridization. The ability of EC to affect levels of IL-2 cannot be reproduced by soluble factors, including the cytokines IL-1, IL-6, IFN-gamma, or TNF, conditioned medium from resting EC or IL-1, IFN-gamma- or TNF-treated EC, or from resting PBMC + EC cultures. Separation of the EC and PBMC by a Transwell membrane demonstrated that cell contact was required for augmentation of IL-2 synthesis and that this effect was unlikely to be mediated by a short-lived soluble signal. The cell-cell interaction required the ligand pair CD2/LFA-3, since augmentation could be inhibited by antibodies to these structures. Antibodies to ICAM-1, LFA-1, CD4, and MHC class II were without effect. A contact-dependent pathway involving CD2/LFA-3 interactions also may be used by EC to augment IL-2 production from T cells stimulated more specifically through the TCR/CD3 complex with antibody OKT3. This pathway provides a proliferative advantage to T cells stimulated with OKT3 in the presence of EC and may also be involved in the proliferative response of resting T cells to allogeneic class II MHC-expressing EC. We propose that EC augmentation of T cell IL-2 synthesis may be critical in the ability of EC to elicit primary T cell antigen responses and may have consequences for the development of localized cell-mediated immune reactions.

scholarly journals Cloning of cDNA coding for connective tissue activating peptide III from a human platelet-derived lambda gt11 expression library

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1498-1503
Author(s):  
RH Wenger ◽  
AN Wicki ◽  
A Walz ◽  
N Kieffer ◽  
KJ Clemetson
Keyword(s):  
Amino Acids ◽  
Connective Tissue ◽  
Cdna Library ◽  
Messenger Rna ◽  
Blot Hybridization ◽  
Single Species ◽  
Leader Sequence ◽  
Northern Blot Hybridization ◽  
Expression Library ◽  
Platelet Factor

We report here the cloning of the cDNA coding for platelet connective tissue-activating peptide-III (CTAP-III) from a lambda gt11 expression library prepared using messenger RNA (mRNA) isolated from human platelets. The open reading frame of the clone coded for a protein with 128 amino acid residues. Since the precursor of CTAP-III, platelet basic protein (PBP is 94 amino acids long, the 5′-translated region of the cDNA codes for a leader sequence 34 amino acids long. This leader sequence, like the sequence of mature CTAP-III, shows significant homology to the sequence of platelet factor 4 (PF4), the only other platelet specific alpha-granule protein cloned until now, from a human erythroleukemic (HEL) cell line-derived cDNA library. These leader sequences are probably critical for targeting such proteins to the alpha-granule. Northern blot hybridization with platelet and megakaryocyte mRNA shows a single species mRNA of approximately 0.8 kb, suggesting that the corresponding cDNA is full length. The cloning of platelet specific CTAP-III provides additional evidence for the platelet specificity of the cDNA library used.

scholarly journals Construction and partial characterization of a recombinant DNA probe for locust vitellogenin messenger RNA.

1982 ◽  
Vol 205 (3) ◽  
pp. 521-528 ◽  
Author(s):  
T C James ◽  
U M Bond ◽  
C A Maack ◽  
S W Applebaum ◽  
J R Tata
Keyword(s):  
Adult Female ◽  
Messenger Rna ◽  
Recombinant Dna ◽  
Fat Body ◽  
Blot Hybridization ◽  
Northern Blot Hybridization ◽  
Recombinant Plasmids ◽  
Hybrid Molecules ◽  
K 12 ◽  
Vitellogenin Mrna

Double-stranded DNA complementary to poly(A)-containing RNA from the fat body of adult female locusts, Locusta migratoria, was synthesized. Hybrid molecules containing this cDNA was constructed in the PstI site of the plasmid pAT 153 by the technique of dC . dG tailing and amplified in Escherichia coli K-12 strain HB 101. Ten colonies of bacteria were identified as carrying recombinant plasmids containing DNA complementary to locust vitellogenin mRNA by (a) ‘Northern’ blot hybridization analysis and (b) hybrid selection of vitellogenin mRNA and immunological detection of the products of translation of the mRNA. Of the ten recombinant plasmids, one, termed plasmid 4E, containing a cDNA insert of about 650 nucleotides, was characterized in greater detail and a partial restriction map obtained. Using this hybrid plasmid it was possible to derive a value for the average content of vitellogenin mRNA in the adult female locust fat body as 1.5×10(5) molecules/cell, and to establish that the haploid genome of L. migratoria contains only one or two genes coding for vitellogenin.

scholarly journals Distinct modulatory effects of bryostatin 1 and staurosporine on the biosynthesis and expression of the HIV receptor protein (CD4) by T cells.

1991 ◽  
Vol 2 (2) ◽  
pp. 95-103 ◽  
Author(s):  
W M Boto ◽  
L Brown ◽  
J Chrest ◽  
W H Adler
Keyword(s):  
Biological Activities ◽  
Blot Hybridization ◽  
Interleukin 2 ◽  
Culture Conditions ◽  
Receptor Protein ◽  
Northern Blot Hybridization ◽  
Bryostatin 1 ◽  
Marine Animals ◽  
Animal Products ◽  
Normal Peripheral Blood

A family of structurally related macrocyclic lactones, bryostatins, have recently been shown to display several intriguing pharmacologic properties. Bryostatins are biosynthetic products of bryozoa phyllum of marine animals. To extend the analyses of the biological activities of these highly unusual biosynthetic animal products, we have examined the effect of bryostatin 1 (bryo-1) on the steady-state expression of the human immunodeficiency virus receptor, CD4, by normal peripheral blood T lymphocytes. Incubation of the cells with 5 nM bryo-1 caused a substantial loss of CD4 from the cell surface, as analyzed by flow cytometry using anti-CD4 monoclonal antibody. The modulation of CD4 expression by bryo-1 was not due to a cytotoxicity effect: in the culture conditions where it modulated CD4, bryo-1 also stimulated the expression of the interleukin 2 gene, as indicated by northern blot hybridization. In addition, incubation of the lymphocytes with nanomolar amounts of protein kinase C antagonist, staurosporine, resulted in the inhibition of the bryo-1-induced modulation of CD4 expression. The results of radioimmunoprecipitation analysis of detergent lysates of [35S] methionine-labeled lymphocytes strongly suggest that bryo-1 inhibits the glycosylation and expression of CD4 in a manner similar to that of tunicamycin.

Pro-opiomelanocortin messenger RNA levels increase in the fetal sheep pituitary during late gestation

1991 ◽  
Vol 131 (3) ◽  
pp. 483-489 ◽  
Author(s):  
K. Yang ◽  
J. R. G. Challis ◽  
V. K. M. Han ◽  
G. L. Hammond
Keyword(s):  
Messenger Rna ◽  
Blot Hybridization ◽  
Late Gestation ◽  
Northern Blot Hybridization ◽  
Mrna Levels ◽  
Plasma Acth ◽  
Mrna Accumulation ◽  
Fetal Sheep ◽  
Fetal Plasma ◽  
Pomc Mrna

ABSTRACT Plasma levels of ACTH and cortisol in fetal sheep increase progressively during late pregnancy, providing the stimulus for birth. However, little information is available concerning either sources of pro-opiomelanocortin (POMC, the precursor to ACTH) or changes in POMC gene expression, which may be responsible for the elevated fetal plasma ACTH concentrations. We therefore studied the relative amount of POMC mRNA in fetal sheep hypothalami, anterior pituitaries and adrenals at discrete times of pregnancy between day 60 and term (approximately 145 days) and from newborn lambs. Total RNA from these tissues was analysed by Northern blot hybridization using a human POMC DNA probe, and the amount of POMC mRNA was expressed relative to the signal obtained for 18S ribosomal RNA. A single 1·2 kb transcript was detected by day 60 in the anterior pituitary, and its relative amount did not change significantly until after days 125–130. Pituitary POMC mRNA levels increased significantly at days 138–143, remained elevated at term and increased further in newborn lambs. In contrast, POMC mRNA was undetectable in hypothalami and adrenal glands of fetuses at all ages. The results suggested that the prepartum rise in plasma ACTH concentrations in fetal sheep is due to increased POMC biosynthesis in the fetal pituitary. The increase in POMC mRNA occurs at a time when fetal plasma cortisol concentrations are elevated, indicating that the negative feedback effects of circulating glucocorticoids on the fetal hypothalamicpituitary axis may be obscured by other mechanisms that increase pituitary POMC mRNA accumulation during the last week of gestation. Journal of Endocrinology (1991) 131, 483–489

scholarly journals Human blood basophils synthesize interleukin-2 binding sites

Blood ◽  
1990 ◽  
Vol 75 (9) ◽  
pp. 1820-1826 ◽  
Author(s):  
H Stockinger ◽  
P Valent ◽  
O Majdic ◽  
P Bettelheim ◽  
W Knapp
Keyword(s):  
Human Blood ◽  
Binding Sites ◽  
Messenger Rna ◽  
Biochemical Characterization ◽  
Surface Membrane ◽  
Interleukin 2 ◽  
Binding Studies ◽  
Chronic Granulocytic Leukemia ◽  
Plot Analysis

Abstract Recent data suggest that basophils express receptors for a variety of lymphokines. In this study we present the biochemical characterization of the interleukin-2 (IL-2) receptor on the basophil surface membrane. Highly enriched populations (purity: 92% to 99%) of blood basophils were obtained from chronic granulocytic leukemia (CGL) patients (n = 3) by negative selection using monoclonal antibodies (MoAbs) and complement. CGL basophils were found to bind CD25 MoAbs (n = 4) directed against different epitopes of the 55- to 60-Kd subunit of the IL-2 receptor (= Tac peptide). Immunoprecipitation experiments with lysates of purified CGL basophils and CD25 MoAbs showed a protein with a molecular weight of 60 Kd, equivalent to the Tac peptide on human T blasts. Quantitative binding studies and Scatchard plot analysis using radiolabeled recombinant human (rh) IL-2 indicated the presence of 12,000 +/- 4,700 low affinity IL-2 binding sites (kd = 66 nmol/L) per purified CGL basophil. Northern blot analysis with enriched CGL basophils showed two messenger RNA bands of 3.5 and 1.5 kilobases hybridizing to radiolabeled Tac cDNA. Immunoprecipitation of the Tac peptide from enriched basophils metabolically labeled with 35S- methionine showed active synthesis of the IL-2 receptor. Our results show that human blood basophils synthesize and express receptors for IL- 2.

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