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P Nunes

Publications and source records attributed to P Nunes.

At least 19 recordsLinked to original sources
PubMed2000

Primary cutaneous CD30-positive large cell lymphoma.

Primary cutaneous CD30-positive large cell lymphoma is a recently described entity, recognized by immunohistochemical criteria. Spontaneous regression may occur and although relapses are frequent, most patients have a favourable prognosis. We describe a 83-year-old woman with a large ulcerated tumour on her right forearm, with 2 months evolution. Histological and immunohistochemical examination revealed a CD30-positive large cell lymphoma. Systemic involvement was not detected. The tumour regressed spontaneously. Cutaneous relapses occurred one year later, all with spontaneous regression. After a 2-year follow-up period, the patient is well, without concurrent extracutaneous disease. This case is in keeping with other reports of spontaneous regression and favourable outcome of this type of cutaneous lymphoma.

Aged↗
PubMed1998

IL-1 receptor accessory protein is an essential component of the IL-1 receptor.

The recently described IL-1R accessory protein (IL-1R AcP) interacts with IL-1beta and the IL-1 type-IR (IL-1RI), but an essential requirement for IL-1R AcP in IL-1 signaling in vitro has not been established and its role in vivo has not been examined. In this study, IL-1R AcP-deficient mice and fibroblasts were produced and characterized. All IL-1 agonists bound to IL-1R AcP-deficient cells through the type I IL-1R, but failed to activate gene expression through either the nuclear factor-kappaB or AP-1-dependent signaling pathways. Absence of IL-1R AcP differentially affected the affinity for IL-1 ligands. IL-1R AcP-deficient fibroblasts bound murine IL-1alpha and human IL-1R antagonist protein (IL-1Ra) with only moderately reduced affinity when compared with wild-type cells, whereas murine IL-1beta affinity was reduced by 70-fold. IL-1 also failed to produce a biologic response in vivo in IL-1R AcP-deficient mice. These data demonstrate that a type I IL-1R/IL-1R AcP complex is required for signaling by all IL-1 agonists and for high affinity binding by IL-1beta. Finally, IL-1R AcP is an essential signal transducing component of the functional IL-1R and should represent a novel target for blocking IL-1 function in human disease.

Animals↗
PubMed1997

Absence of IL-1 signaling and reduced inflammatory response in IL-1 type I receptor-deficient mice.

IL-1alpha and IL-1beta are potent inflammatory cytokines that contribute to a number of normal physiologic processes and to the development of a number of inflammatory diseases. Two IL-1R, the type I and type II receptors, have been identified. This work describes the derivation and characterization of mice deficient in expression of the type I IL-1R (IL-1RI). IL-1RI-deficient mice were viable and fertile, but failed to respond to IL-1 in a variety of assays, including IL-1-induced IL-6 and E-selectin expression and IL-1-induced fever. Similar to IL-1beta-deficient mice, IL-1RI-deficient mice had a reduced acute phase response to turpentine. In contrast, IL-1RI-deficient mice had a reduced delayed-type hypersensitivity response and were highly susceptible to infection by Listeria monocytogenes. These data demonstrate that the IL-1RI is essential for all IL-1-mediated signaling events examined, and that both IL-1alpha and IL-1beta are critical to the animals' response to injury and infection. These data also demonstrate that IL-1 function is not required for normal development or homeostasis.

Acute-Phase Reaction↗
PubMed1996

Interaction of phosphorylated FcepsilonRIgamma immunoglobulin receptor tyrosine activation motif-based peptides with dual and single SH2 domains of p72syk. Assessment of binding parameters and real time binding kinetics.

To examine the characteristics of the interaction of the FcepsilonRIgamma ITAM with the SH2 domains of p72(syk), the binding of an 125I-labeled dual phosphorylated FcepsilonRIgamma ITAM-based peptide to the p72(syk) SH2 domains was monitored utilizing a novel scintillation proximity based assay. The Kd for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity binding was reflected in the rapid rate of association for the peptide binding to the SH2 domains. Competition studies utilizing a soluble C-terminal SH2 domain knockout and N-terminal SH2 domain knockouts revealed that both domains contribute cooperatively to the high affinity binding. Unlabeled dual phosphorylated peptide competed with the 125I-labeled peptide for binding to the dual p72(syk) SH2 domains with an IC50 value of 4.8 nM. Monophosphorylated 24-mer FcepsilonRIgamma ITAM peptides, and phosphotyrosine also competed for binding, but with substantially higher IC50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosphorylated and that the preferred binding orientation of the ITAM is such that the N-terminal phosphotyrosine occupies the C-terminal SH2 domain and the C-terminal phosphotyrosine occupies the N-terminal SH2 domain.

Amino Acid Sequence↗
PubMed1996

Characterization of anti-mouse IL-12 monoclonal antibodies and measurement of mouse IL-12 by ELISA.

Enzyme-linked immunosorbent assays (ELISAs) capable of quantitatively measuring pg/ml amounts of mouse IL-12 (moIL-12) were developed as an alternative to the current bioassay procedure used for the measurement of moIL-12. A panel of 40 rat anti-moIL-12 monoclonal antibodies were identified and tested for their ability to bind 125I-moIL-12. Two of the MAbs, 2B5 and 9A5, were able to capture 125I-moIL-12 in the presence of unlabelled moIL-12 p35 and moIL-12 p40, suggesting specificity for the moIL-12 p75 heterodimer. Western blot analysis confirmed that MAb 9A5 specifically recognized only moIL-12 p75. Using MAb 9A5, and an additional anti-moIL-12 p40 MAb 5D9, we developed quantitative ELISAs for the specific detection of moIL-12 p75 and p40, respectively. These ELISAs detect moIL-12 with a sensitivity of 40 pg/ml. Whereas the p40 ELISA detected three forms of moIL-12 (p40 monomer, p40 homodimer, and the heterodimer), the p75 ELISA only detected moIL-12 heterodimer. Neither of these assays crossreacted with a panel of additional cytokines. The levels of moIL-12 measured by the p75 ELISA and the bioassay were directly compared and found to correlate well. Therefore, the p75 ELISA represents an alternative to the bioassay for the measurement of moIL-12.

Animals↗
PubMed1995

Molecular cloning and characterization of a second subunit of the interleukin 1 receptor complex.

A monoclonal antibody (mAb) was isolated that blocked the binding and bioactivity of both human and murine interleukin 1 beta (IL-1 beta) on murine IL-1 receptor-bearing cells. This mAb recognized a protein that was distinct from the Type I and Type II IL-1 receptors, suggesting that an additional protein exists that is involved in IL-1 biological responses. By expression cloning in COS-7 cells, we have isolated a cDNA from mouse 3T3-LI cells encoding this putative auxiliary molecule, which we term the IL-1 receptor accessory protein (IL-1R AcP). Sequence analysis of the cDNA predicts an open reading frame that encodes a 570-amino acid protein with a molecular mass of approximately 66 kDa. The IL-1R AcP is a member of the Ig superfamily by analysis of its putative extracellular domain and also bears limited homology throughout the protein to both Type I and Type II IL-1 receptors. Northern analysis reveals that murine IL-1R AcP mRNA is expressed in many tissues and appears to be regulated by IL-1. In mammalian cells expressing natural or recombinant Type I IL-1R and IL-1R AcP, the accessory protein forms a complex with the Type I IL-1R and either IL-1 alpha or IL-1 beta but not IL-1ra. The recombinant accessory protein also increases the binding affinity of the recombinant Type I IL-1R for IL-1 beta when the two receptor proteins are coexpressed. Therefore, the functional IL-1 receptor appears to be a complex composed of at least two subunits.

3T3 Cells↗
PubMed1994

Expression cloning of a human IL-12 receptor component. A new member of the cytokine receptor superfamily with strong homology to gp130.

A cDNA encoding a human IL-12R subunit was isolated by expression cloning. This subunit is a 662 amino acid type I transmembrane protein with an extracellular domain of 516 amino acids and a cytoplasmic domain of 91 amino acids. It is a member of the hemopoietin receptor superfamily and is most closely related over its entire length to gp130 and the receptors for granulocyte-CSF (G-CSF) and leukemia-inhibitory factor. When expressed in COS cells, this IL-12R subunit binds both human and murine IL-12 with an apparent affinity of 2 to 5 nM. The transfected COS cells express both monomers and disulfide-linked dimers or oligomers of the IL-12R subunit on their surface. However, unlike the IL-6-induced dimerization of gp130, the oligomerization of the IL-12R subunit is not dependent on binding of IL-12. Only the IL-12R subunit dimers/oligomers but not the monomers bind IL-12 with an affinity of 2 to 5 nM. A polyclonal antiserum raised against this receptor subunit specifically inhibits IL-12-induced proliferation of PHA-activated PBMC. The data are consistent with the hypotheses that 1) a dimer/oligomer of the cloned IL-12R subunit (IL-12R-beta) represents the low affinity IL-12 binding site identified on human lymphoblasts, 2) the cloned receptor subunit is involved in IL-12 signal transduction, and 3) an additional, as of yet unidentified subunit is required to generate a high affinity IL-12R complex.

Amino Acid Sequence↗
PubMed1992

IL-12 receptor. I. Characterization of the receptor on phytohemagglutinin-activated human lymphoblasts.

IL-12 is a 75-kDa heterodimeric cytokine composed of disulfide-bonded 35-kDa and 40-kDa subunits. Included among the biologic activities mediated by IL-12 is induction of proliferation of PHA-activated human PBL. The concentration of IL-12 required to stimulate maximum proliferation of PHA-activated lymphoblasts is 50 to 100 pM. In this study, highly purified 125I-labeled IL-12 (7 to 15 microCi/microgram; 50 to 100% bioactive) was used to characterize the receptor for IL-12 on 4-day PHA-activated lymphoblasts. The binding of 125I-labeled IL-12 to PHA-activated lymphoblasts was saturable and specific because the binding of radiolabeled ligand was only inhibited by IL-12 and not by other cytokines. The kinetics of [125I]IL-12 binding to PHA-activated lymphoblasts was rapid at both 4 degrees C and 22 degrees C; reaching equilibrium within 60 min. At 22 degrees C, the rate of dissociation of [125I]IL-12 was slow in the absence of competing IL-12 (t1/2 = 5.9 h) and more rapid in the presence of 25 nM competing IL-12 (t1/2 = 2.5 h). The kinetically derived equilibrium dissociation constant ranged from 10 to 83 pM. Analysis of steady state binding data by the method of Scatchard identified a single binding site with an apparent equilibrium dissociation constant of 100 to 600 pM and 1000 to 9000 sites/lymphoblast. The equilibrium dissociation constant for competing ligands and sites per cell calculated from unlabeled IL-12 competition experiments ranged from 164 to 315 pM and 1067 to 3336, respectively, which is in good agreement with the values determined from steady state binding. The variations in KD and sites per cell were dependent on the individual preparations of lymphoblasts. Although the steady state binding data were consistent with a single class of high affinity binding sites, the kinetic dissociation data indicates a cooperative interaction between receptors on PHA-activated lymphoblasts. Affinity cross-linking of surface bound [125I]IL-12 to PHA-activated lymphoblasts at 4 degrees C identified a major complex of approximately 210 to 280 kDa. Anti-IL-12 antibodies also immunoprecipitated a complex of approximately 210 to 280 kDa that was produced by cross-linking unlabeled IL-12 to 125I-labeled lymphoblast cell-surface proteins. Cleavage of this complex with reducing agent identified one radiolabeled protein of approximately 110 kDa. These data suggest that the IL-12 binding site on PHA-activated lymphoblasts may be composed of a single protein of approximately 110 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells, Cultured↗
PubMed1991

IL-12: monoclonal antibodies specific for the 40-kDa subunit block receptor binding and biologic activity on activated human lymphoblasts.

IL-12, formerly known as cytotoxic lymphocyte maturation factor, is a cytokine that stimulates proliferation of PHA-activated human peripheral blood lymphoblasts and synergizes with low concentrations of IL-2 in the induction of lymphokine-activated killer cells. IL-12 is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. mAb prepared against a partially purified preparation of natural IL-12 have been characterized by 1) immunoprecipitation of 125I-labeled IL-12, 2) immunodepletion of IL-12 bioactivity, 3) Western blotting of IL-12, 4) inhibition of [125I]IL-12 binding to its cellular receptor, and 5) neutralization of IL-12 bioactivity. Twenty antibodies immunoprecipitate 125I-labeled IL-12 and immunodeplete IL-12 bioactivity as assessed in the T cell proliferation and lymphokine-activated killer cell induction assays. Western blot analysis demonstrated that each antibody binds to the 75-kDa heterodimer and to the 40-kDa subunit. An IL-12R-binding assay identified 12 individual antibodies that inhibited the binding of [125I]IL-12 to its cellular receptor. Two inhibitory antibodies, 4A1 and 7B2, were tested in the neutralization assay and found to block IL-12 bioactivity whereas one noninhibitory antibody, 8E3, was shown not to neutralize IL-12 bioactivity. Antibodies 4A1 and 8E3 can simultaneously bind to the 75-kDa heterodimer demonstrating that inhibitory and noninhibitory epitopes are spatially distinct on the 40-kDa protein. The ability of antibodies specific for the 40-kDa subunit of IL-12 to block receptor binding of [125I]IL-12 and to neutralize IL-12 bioactivity suggests that localized determinants on the 40-kDa subunit may be necessary for binding to the IL-12 cellular receptor.

Animals↗
PubMed1991

Involvement of alpha 1- and alpha 2-adrenoceptors in the responses of proximal and distal segments of the canine saphenous vein to exogenous and endogenous noradrenaline.

In the present study the relationship between adrenergic nerve terminals and postjunctional alpha-adrenoceptors mediating the responses to the endogenous transmitter was compared at proximal and distal levels of the canine saphenous vein. Concentration-response curves to noradrenaline and to tyramine as well as frequency-response curves to electrical stimulation were compared at both levels of the vessel, in the absence and presence of either prazosin (100 nmol.l-1) or yohimbine (100 nmol.l-1). The influence of inhibition of neuronal uptake by cocaine (12 mumol.l-1) on the responses to noradrenaline in the presence of prazosin (56 nmol.l-1) or yohimbine (20 nmol.l-1) was compared at the proximal level. The results show that, at the proximal level, the maximal responses to electrical stimulation and tyramine reached 80.1 +/- 2.2 (n = 18) and 74.2 +/- 1.9 (n = 18)%, respectively, of the maximal responses to noradrenaline, and 70.3 +/- 0.8 (n = 15) and 53.1 +/- 1.2 (n = 14)%, respectively, at the distal level. Furthermore, the proximal strips were more sensitive to electrical stimulation than the distal ones. Prazosin had a much greater inhibitory effect on the contractile responses to noradrenaline than on those to electrical stimulation, at both levels. At proximal level, the shifts (to the right) of the concentration (frequency)-response curves (at EC50) amounted to 0.58 +/- 0.02 (n = 16) and 0.18 +/- 0.02 (n = 8) log units, respectively (P less than 0.05), but, at the distal level, to 1.12 +/- 0.03 (n = 16) and 0.28 +/- 0.08 (n = 8) log units, respectively (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Measurement of biologically active interleukin-1 by a soluble receptor binding assay.

A soluble receptor binding assay has been developed for measuring human interleukin-1 alpha (IL-1 alpha), human IL-1 beta, and mouse IL-1 alpha. The assay is based on a competition between unlabeled IL-1 and 125I-labeled mouse recombinant IL-1 alpha for binding to soluble IL-1 receptor prepared from mouse EL-4 cells. The assay measures only biologically active IL-1 folded in its native conformation. The ratio of human IL-1 alpha to human IL-1 beta can be measured in the same sample by a pretreatment step which removes human IL-1 beta from samples prior to assay. This technique has been used to monitor the purification of recombinant IL-1, and may be utilized to specifically and accurately measure bioactive IL-1 in human serum and cell culture supernatants.

Animals↗
PubMed1989

Two high-affinity interleukin 1 receptors represent separate gene products.

Interleukin 1 (IL-1) is a polypeptide hormone that mediates a broad range of biological activities and interacts with surface receptors on numerous cell types. Equilibrium binding studies have identified a class of IL-1 receptors on T cells, fibroblasts, and epithelial cells that have 2- to 5-fold higher affinity than the receptors on bone marrow cells, pre-B cells, and macrophage cell lines. Affinity cross-linking with human 125I-labeled IL-1 alpha (125I-IL-1 alpha) labels an approximately 100-kDa protein on T cells and fibroblasts and an approximately 80-kDa protein on pre-B cells and macrophage cell lines. Monoclonal and polyclonal antibodies specific for the IL-1 receptor on T cells and fibroblasts block human 125I-IL-1 alpha binding to T cells, fibroblasts, and epithelial cells but cannot block IL-1 binding to bone marrow cells, pre-B cells, and macrophages. These antibodies immunoprecipitate the IL-1 receptor-human 125I-IL-1 alpha complex from T cells and fibroblasts but not from pre-B cells and macrophage cell lines. An S1 nuclease protection assay demonstrated that T cells and fibroblasts contain identical IL-1 receptor mRNA but that pre-B cells and macrophages do not contain this receptor mRNA. Taken together, the data demonstrate that mouse T cells, fibroblasts, and epithelial cells express an identical IL-1 receptor, whereas the IL-1 receptor on pre-B cells, macrophages, and bone marrow cells represents a different gene product.

Animals↗
PubMed1985

[New aspects and controversies on the vascularization of the white pulp of the spleen in the rat].

The authors have studied the arterial supply of splenic white pulp of the rat, by means of optic and scanning electron microscopy. This work shows that the marginal zone is a free and interstitial circulatory one that envelopes completely the periarterial lymphocyte sheets. These ones are vascular exclusion areas although crossed by vessels, probably impermeable, that pour their contents at the inner border of the marginal zone. It was impossible to find a so called "marginal sinus" neither a complete nor an incomplete one. These facts can explain and change some poorly understood immunological aspects.

Animals↗