Biomedical subjects

R Testi

Publications and source records attributed to R Testi.

At least 73 records · Page 4Linked to original sources

PubMed1993

A simple method for the evaluation of receptor binding capacity of modified cytokines.

We have developed a flow cytometric method to evaluate the binding of interleukin-2 analogues to receptors. The method relies on competition for binding between a fluorescein-conjugated monoclonal antibody (MoAb) directed against the human interleukin-2 receptor alpha chain (fluorescein isothiocyanate (FITC) anti-IL-2R) and the test protein. IL-2R positive cells are incubated with FITC-anti-IL-2R MoAb in the presence of native IL-2 or IL-2 iodinated by either the chloramine-T or the lactoperoxidase-glucose-oxidase method. The binding of IL-2 is indicated by decreased fluorescence. This method is suitable for measuring the binding capacity of modified IL-2 molecules and avoids the need for radioactive tracers. It provides a simple and reproducible technique, which can be extended readily to the study of the receptor binding capacity of cytokines conjugated with toxins, drugs or other molecules.

Antibodies, Monoclonal↗

PubMed1993

Transcriptional regulation of interleukin-2 gene expression by CD69-generated signals.

The 5' flanking region of the human interleukin (IL)-2 gene was investigated for enhancer activity in response to CD69-generated signals, using a chloramphenicol acetyltransferase (CAT)-driven transient expression system in Jurkat cells. The region extending from -317 to +47 relative to the initiation site of IL-2 gene transcription was shown to contain sequences able to respond to CD69 cross-linking, by enhancing by about 100% a phorbol 12-myristate 13-acetate (PMA)-plus-ionomycin stimulation of CAT activity. A similar increase in CAT activity produced by PMA-plus-anti-CD3 mAb was induced by CD69 cross-linking, while a 200% increase over that obtained by PMA-plus-anti-CD28 mAb stimulation was seen. Analysis of enhancer deletion mutants revealed that proximal AP-1, OCT-1/octamer-associated protein and nuclear factor of activated T cells (NFAT) binding regions were all necessary to allow CD69-mediated enhancement of CAT activity. By gel mobility shift analysis, cyclosporin A-sensitive NFAT-binding induction and enhancement of AP-1 binding activity could be detected in nuclear extracts of both Jurkat and peripheral blood T cells after simultaneous CD69 and protein kinase C stimulation. Finally, CD69-mediated signals could increase NFAT and AP-1 binding activity following PMA and ionomycin stimulation in peripheral blood T cells. Collectively, these data suggest that CD69-generated signals participate in the control of the IL-2 gene expression at the transcriptional level, likely acting through NFAT and AP-1 transcription factor complexes.

Antigens, CD↗

PubMed1993

Continuous in vivo activation and transient hyporesponsiveness to TcR/CD3 triggering of human gut lamina propria lymphocytes.

Three-color immunofluorescence and flow cytometric analysis showed that the vast majority of normal human T-lamina propria lymphocytes (LPL) expressed high levels of the early activation antigen CD69, together with CD45R0, irrespective of their CD4, CD8 or gamma/delta-TcR phenotype, indicating that they are continuously stimulated in vivo. Importantly, measurement of cytoplasmic [Ca2+]i showed that T-LPL had significantly higher basal [Ca2+]i levels, compared to autologous peripheral blood lymphocytes (PBL). Both cytoplasmic [Ca2+]i elevation and inositol (1,4,5) trisphosphate generation following CD3 cross-linking by monoclonal antibodies in vitro were essentially abolished in T-LPL, as compared to autologous T-PBL. Moreover, freshly isolated LPL could be induced to proliferate by CD2- or CD28-mediated signals, but not by CD3-mediated signals. Surprisingly however, impairment in TcR/CD3-mediated early signaling and proliferation in T-LPL could be completely reversed by 24 h incubation of the cells at 37 degrees C in culture medium, a condition which allowed basal intracellular [Ca2+]i to return to levels comparable to peripheral T cells. Our data suggest that selective hyporesponsiveness to TcR/CD3-mediated signaling may represent a transient event during continuous in vivo activation of mucosal lymphocytes.

Calcium↗

PubMed1993

Is binding of vitamin D binding protein related to cell differentiation?

Vitamin D binding protein (Gc) is present on the surface of several blood cells and may interfere with the activity of 1,25(OH)2D3. It has previously been reported that Gc may bind to the U-937 line which is known to differentiate upon exposition to 1,25(OH)2D3. In the present paper, we evaluate the expression of Gc on the surface of U-937 and HL-60 lines. Both cell lines did not express Gc on their surface but U-937 cells were able to bind human purified Gc added to the medium whereas HL-60 were not. After culturing with 1,25(OH)2D3, HL-60 became able to bind Gc. This property seems to be related to the monocytic differentiation induced by 1,25(OH)2D3. Conversely, when present together, 1,25(OH)2D3 reduces binding on U-937.

Calcitriol↗

PubMed1993

Duration of preventive effect of inhaled salbutamol on early airway response to allergen in asthmatic subjects.

Salbutamol is known to effectively prevent early asthmatic response (EAR) after specific bronchial provocation test (sBPT) in asthmatic subjects, but the time-course of this protection is not known. In this study the effects of 200 micrograms salbutamol inhaled 2 h before sBPT were compared with 200 micrograms salbutamol inhaled 10 min before sBPT in eight asthmatic subjects sensitized to Phleum pratensis (PP) or Dermatophagoides pteronyssinus (DP). All subjects showed an EAR (% decrease in FEV1 from baseline value: 30.4 +/- 11%) in a preliminary sBPT performed with cumulated doses of extracts of PP or DP titrated in biological units (BU). Each subject performed, on two different days, in a random, double-blind, cross-over study, two puffs of placebo 2 h before sBPT and two puffs of salbutamol 10 min before sBPT (treatment A) or two puffs of salbutamol 2 h before sBPT and two puffs of placebo 10 min before sBPT (treatment B). Baseline FEV1 were similar in both tests. In treatment B, 10 min, 1 h and 2 h after salbutamol inhalation, FEV1 increased significantly with respect to placebo inhalation in treatment A. Ten minutes after salbutamol inhalation in treatment A, FEV1 became similar to that obtained 2 h after salbutamol inhalation and 10 min after placebo in treatment B. Allergen inhalation induced an EAR in only one out of eight subjects after treatment A, and five of the eight subjects after treatment B.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗

PubMed1993

Effects of therapeutic doses of salbutamol alone and combined with beclomethasone dipropionate on airway responsiveness and cyclic AMP plasma levels in asthmatic patients.

Twelve asthmatic patients received by inhalation for 2 weeks, in a double-blind, cross-over design, beclomethasone dipropionate (BDP; 600 micrograms/day) plus salbutamol (S; 900 micrograms/day), or S (900 micrograms/day) alone. Before and after each treatment course the subjects received intravenous cumulative doses of S up to 200 micrograms. In basal conditions and immediately before the next dose forced expiratory volume in 1 s (FEV1) and plasma cyclic AMP (cAMP) were measured. BDP+S treatment increased FEV1 basal values (p < 0.05) whereas inhaled S resulted in unsignificant improvement of ventilatory parameters. The slopes of the dose-response curves of FEV1 and plasma cAMP to intravenous S were unaffected by the two treatment courses. Our results suggest that DBP+S, differently from S alone, improves ventilatory function in asthmatic patients and that neither S nor S+BDP seem to affect adrenergic function.

Administration, Inhalation↗

PubMed1992

Preferential involvement of a phospholipase A2-dependent pathway in CD69-mediated platelet activation.

CD69 is a signal transducing disulfide-linked homodimer functionally expressed on platelets, CD3bright thymocytes, and activated lymphocytes. In an attempt to investigate early molecular events in CD69-mediated cell activation we studied the relative contribution of phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase C-dependent pathways during platelet activation induced by CD69 stimulation. Thromboxane A2 (TXA2) synthetase inhibitor and TXA2R inhibitor R68070 were able to inhibit platelet aggregation induced by CD69 stimulation, indicating that TXA2 was the main mediator of the response. CD69-induced arachidonic acid release and TXA2 production were essentially PLA2 dependent because they could be blocked by the PLA2 inhibitor quinacrine. Inositol 1,3,4-trisphosphate generation was clearly detectable after CD69 cross-linking, but it was completely abrogated by quinacrine and R68070 and therefore secondary to TXA2 release and TXA2R engagement. Finally, direct measurement of enzymatic activity in vitro using radiolabeled phospholipid vesicles showed that CD69 cross-linking resulted in PLA2-dependent arachidonic acid and lysophosphatidylcholine generation from phosphatidylcholine, which was sensitive to quinacrine but not to R68070. By contrast, CD69-induced 1,2-diacylglycerol release from phosphatidylinositol 4,5-bisphosphate was blocked by both inhibitors. These results indicate a preferential involvement of PLA2 in CD69-dependent signal transduction in platelets and provide evidence for the unique role of PLA2-mediated activation pathways in transmembrane receptor signaling.

Antigens, CD↗

PubMed1992

FcRIII (CD16) expression on neutrophils from chronic myeloid leukemia. A flow cytometric study.

FcRIII (CD16) expression on neutrophils from 17 patients with chronic myeloid leukemia (CML) was studied by flow cytometry using monoclonal antibodies. A variable proportion of CD16-negative neutrophils were found both in CML patients in chronic phase (3 out of 8 patients) and in CML patients in hematological remission (3 out of 9 patients). Neutrophils with reduced FcRIII expression showed more defective chemiluminescence and phagocytosis than neutrophils with normal FcRIII expression. Circulating myeloid cells from three patients in chronic phase, showing a normal percentage of CD16-positive neutrophils, were isolated and fractionated by discontinuous Percoll gradients. This study showed that CD16 appears at the stage of metamyelocyte, that band cells and segmented neutrophils display an identical pattern of membrane FcRIII, and that the fluorescence intensity shown by metamyelocytes is different from that displayed by more mature cells. The association between low FcRIII expression and function abnormality could be suggestive of a defect in CML neutrophil maturation.

Female↗

PubMed1991

Salbutamol plus beclomethasone dipropionate, but not salbutamol alone, completely prevent early and late asthmatic responses to allergen.

The effect of a week treatment with inhaled salbutamol plus placebo (S+P) vs. salbutamol combined with beclomethasone dipropionate (S+BDP) on early and late asthmatic responses to inhaled allergen was studied in ten atopic patients in a randomized, double-blind, cross-over study. All patients had previously shown a dual type response to the specific bronchial provocative test (sBPT). Each patient performed two periods of treatment for a week, with a 15 day interval between them: (a) salbutamol 0.3 mg, tid + placebo; (b) salbutamol 0.3 mg+BDP 0.2 mg, tid; at the end of each treatment period, sBPT was performed and the last treatments were given 1.5-2 h before and 3-4 h after allergen challenge. S + BDP completely prevented both early and late responses to allergen, while S + P reduced but did not completely inhibit early and late responses. The difference between the two treatments was significant for early and late asthmatic responses. Non-specific bronchial hyperresponsiveness to methacholine was performed before each treatment period, after 6 days of treatment before sBPT and the day after sBPT at the end of the treatment period; there was only a mild increase in PD15FEV1 methacholine after 6 days of treatment with S + BDP in comparison with S + P treatment. These results suggest that salbutamol plus beclomethasone may be used effectively in the prophylaxis of early and late asthmatic reactions induced by allergen in sensitized subjects.

Adolescent↗

PubMed1990

CD69 is expressed on platelets and mediates platelet activation and aggregation.

CD69, a surface dimer so far considered an early activation antigen restricted to lymphocytes, was found constitutively expressed on human platelets. Biochemical analysis revealed that platelet CD69 appears on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad 55-65-kD band, in which three 55-, 60-, and 65-kD components were detectable when nonreduced, and as two 28- and 32-kD bands when reduced, corresponding to the two disulfide-linked chains of the dimer. It therefore closely resembles lymphoid CD69, although the resolution of the three bands under nonreducing conditions is not usually seen in lymphoid cells. Moreover, as CD69 expressed on activated lymphocytes and CD3bright thymocytes, both chains are constitutively phosphorylated. CD69 stimulation by anti-Leu-23 monoclonal antibodies induced platelet aggregation in a dose-dependent fashion. This effect was associated with Ca2+ influx and platelet degranulation, as revealed by adenosine triphosphate release. In addition, CD69 stimulation in platelets induced production of thromboxane B2 and PGE2, suggesting activation of arachidonic acid metabolism by cycloxygenase. As observed for CD69-mediated T cell activation, platelet activation through CD69 requires molecular crosslinking. These results suggest that CD69 may function as an activating molecule on platelets, as on lymphocytes, and point toward a more general role of this surface dimer in signal transduction.

Adenosine Triphosphate↗

PubMed1989

Membrane anchoring of a human IgG Fc receptor (CD16) determined by a single amino acid.

CD16 is a low-affinity immunoglobulin G (IgG) Fc receptor that is expressed on natural killer (NK) cells, granulocytes, activated macrophages, and some T lymphocytes. Two similar genes, CD16-I and CD16-II, encode membrane glycoproteins that are anchored by phosphatidylinositol (PI)-glycan and transmembrane polypeptides, respectively. The primary structural requirements for PI-linkage were examined by constructing a series of hybrid cDNA molecules. Although both cDNA's have an identical COOH-terminal hydrophobic segment, CD16-I has Ser203 whereas CD16-II has Phe203. Conversion of Phe to Ser in CD16-II permits expression of a PI-glycan-anchored glycoprotein, whereas conversion of Ser to Phe in CD16-I prevents PI-glycan linkage.

Animals↗

PubMed1989

T cell activation via Leu-23 (CD69).

The CD69 (Leu-23) activation Ag is a phosphorylated 28 to 32-kDa disulfide-linked homodimer that is rapidly induced after lymphocyte activation. CD69 is not present on the surface of peripheral blood resting T cells, but is constitutively expressed by CD3bright thymocytes. Activation of protein kinase C (PKC) by stimulation of the TCR/CD3 or by phorbol esters directly induces CD69 expression on T cells. In the attempt to elucidate the function of CD69 we investigated the ability of the CD69 glycoprotein to transmit an activation signal. Cross-linking of CD69 by mAb induced a prolonged elevation of intracellular [Ca2+], mostly due to an influx of extracellular Ca2+. This signal alone was unable to effectively activate PKC. When PKC was simultaneously activated by PMA, stimulation of CD69 induced IL-2 and IFN-gamma gene expression, enhancement of CD25 expression, and ultimately IL-2-dependent T cell proliferation. Both CD4+ and CD8+ peripheral T cells responded to CD69-mediated activation. Stimulation of CD69 induced proliferation of thymocytes as well as peripheral T cells, but both required independent PKC activation by PMA. Cyclosporin A, which does not prevent PKC-induced CD69 expression, completely suppressed CD69-induced IL-2 and IFN-gamma gene expression. Although the signal delivered by the CD69 initiates T cell proliferation, it is unable to trigger cytotoxicity programs in CD69+-activated T cells or T cell clones.

Antigens, CD↗

PubMed1989

Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule.

Neural cell adhesion molecule (N-CAM) is a membrane glycoprotein expressed on neural and muscle tissues that is involved in homotypic adhesive interactions. We have demonstrated that N-CAM also is expressed on hematopoietic cells, and is recognized by the anti-Leu-19 mAb. Leu-19 is preferentially expressed on NK cells and T lymphocytes that mediate MHC-unrestricted cytotoxicity, but is also present on some myeloid leukemia cell lines. On NK cells, T cells, the KG1a.5 hematopoietic cell line, and a neuroblastoma cell line, Leu-19 is a approximately 140-kD polypeptide with N-linked carbohydrates and abundant sialic acid residues. Sequential immunoprecipitation and peptide mapping demonstrated that the Leu-19 and N-CAM molecules expressed on leukocyte and neuroblastoma cell lines are similar structures. These findings suggest that the Leu-19 antigen on leukocytes may be involved in cell adhesion, analogous to the function on N-CAM on neural cells.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1989

Leu 23 induction as an early marker of functional CD3/T cell antigen receptor triggering. Requirement for receptor cross-linking, prolonged elevation of intracellular [Ca++] and stimulation of protein kinase C.

The Leu 23 Ag is a phosphorylated 28 to 32-kDa disulfide-linked homodimer expressed on the surface of activated T cells, B cells, and NK cells. Resting, unstimulated peripheral blood T cells are Leu 23-, but 20 to 30% of normal thymocytes constitutively express Leu 23. Triggering of the CD3/TCR complex by mAb induced the expression of Leu 23 within 2 h after stimulation. Leu 23 was still detectable on the cell surface 48 h after the stimulus was removed. The induction of Leu 23 strictly correlated with the extent of CD3/TCR cross-linking on the surface of T cells. Anti-CD3-coupled beads, but not soluble IgG or IgM mAb, were able to induce Leu 23 expression on peripheral blood T cells. Soluble anti-CD3 mAb were sufficient to induce Leu 23 on the Jurkat T cell line, but a direct relationship was found between CD3 cross-linking valency and Leu 23 expression. RNA and protein synthesis were both required for Leu 23 induction. Moreover, only CD3/TCR-mediated signals able to stimulate PKC and maintain elevated intracellular calcium ion concentration for greater than 60 min resulted in Leu 23 induction. These findings suggest that the extent of CD3/TCR cross-linking influences the persistence of elevated intracellular calcium ion concentration and PKC activation, and that this in turn determines the commitment of the T cell to activate gene transcription programs necessary for Leu 23 expression. Induction of Leu 23 represented the first detectable cell surface marker after triggering via the CD3/TCR and thus provides an ideal indicator to monitor the very early events in T cell activation.

Antigens, CD↗

PubMed1989

Functional expression of CD28 on T cell antigen receptor gamma/delta-bearing T lymphocytes.

T lymphocytes express a T cell antigen receptor (TcR) complex composed of either an TcR alpha/beta or TcR gamma/delta heterodimer in noncovalent association with the CD3 glycoproteins. CD28, a 44-kDa disulfide-linked homodimer, is present on the surface of the majority of TcR alpha/beta-bearing T lymphocytes. Monoclonal antibodies (mAb) directed against CD28 potentiate activation signals delivered through the CD3/TcR alpha/beta complex. Herein, we demonstrate that CD28 is expressed on approximately 40%-60% of TcR gamma/delta-bearing T lymphocytes in most donors. Anti-CD28 mAb substantially augmented proliferative signals delivered through the TcR gamma/delta, demonstrating the presence of functional CD28 molecules on TcR gamma/delta-bearing T lymphocytes. The majority of TcR gamma/delta+ thymocytes also expressed CD28.

Antibodies, Monoclonal↗

PubMed1989

Membrane anchoring and spontaneous release of CD16 (FcR III) by natural killer cells and granulocytes.

CD16 is an IgG Fc receptor that is predominantly expressed on human natural killer (NK) cells and granulocytes. The CD16 antigen expressed on neutrophils is a 50 to 70-kDa glycoprotein attached to the plasma membrane by a phosphatidylinositol-glycan linkage that is susceptible to cleavage by phosphatidylinositol-specific phospholipase C (PIPLC). By contrast, treatment of NK cells with PIPLC did not cleave CD16 from the cell surface. Biochemical analysis of the deglycosylated CD16 revealed a substantial difference in the core polypeptides expressed by NK cells and granulocytes. Culture of radiolabeled NK cells resulted in the spontaneous release of a CD16 molecule, smaller than the membrane form of CD16. These findings demonstrate that structural differences exist in the CD16 antigens expressed on NK cells and granulocytes that may potentially influence their functional activities.

Antigens, Differentiation↗

PubMed1989

CD3Ti+ human thymocyte-derived clones displaying a differential response to activation via CD3Ti and CD2.

Previous studies indicated that, unlike peripheral T-cells, freshly isolated thymocytes show little or no proliferation to activation signals via either the antigen/MHC receptor complex (CD3Ti) or the CD2 structure, unless exogenous IL-2 or phorbol esters are added. To investigate these differences in more detail, we have studied the response of clonal populations of mature thymocyte subsets as well as peripheral T-cell clones to activation via either CD3Ti or CD2. Here we report the characterization of three clones belonging to different subsets of mature thymocytes: CD3+ CD4+ (Ti alpha/beta), CD3+ CD8+ (Ti alpha/beta), and CD3+ CD4- CD8- (Ti gamma/delta). All three clones could be induced to proliferate to insolubilized anti-CD3 mAb. In contrast, activating anti-CD2 mAbs, which induced proliferation in all peripheral T-cell clones tested, did not induce an appreciable proliferation of the thymocyte clones. The latter required additional signals provided by the phorbol ester PMA. However, anti-CD2 mAbs were able to induce early activation events such as phosphoinositide turnover and [Ca2+]i increase to an extent similar to the ones elicited by anti-CD3 mAb. These results further support previous findings suggesting that mature thymocytes are not functionally identical to peripheral T-cells.

Antibodies, Monoclonal↗

PubMed1989

Laminin synthesis by NK cells and modulation of its expression by TPA (12-O-tetradecanoylphorbol-13-acetate).

Natural killer (NK) cells have been suggested to play a major role in resistance against metastatic spread of tumors. This study was aimed at understanding whether laminin (LM), a component of the extracellular matrix involved in the mechanism of tumor invasion and cell interaction, is expressed by NK cells. The results indicate that NK cells can synthesize and display on the cell surface LM and that TPA can modulate its expression. Our findings suggest that the presence of LM on NK cells could be relevant in the control of tumor invasion by NK cells.

Animals↗