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G Carra

Publications and source records attributed to G Carra.

At least 19 recordsLinked to original sources
PubMed2003

Pharmacological profile of the cyclic nociceptin/orphanin FQ analogues c[Cys10,14]N/OFQ(1-14)NH2 and c[Nphe1,Cys10,14]N/OFQ(1-14)NH2.

In this study we describe the activity of two cyclic nociceptin/orphanin FQ (N/OFQ) peptides; c[Cys(10,14)]N/OFQ(1-14)NH(2) (c[Cys(10,14)]) and its [Nphe(1)] derivative c[Nphe(1),Cys(10,14)]N/OFQ(1-14)NH(2) (c[Nphe(1),Cys(10,14)]) in native rat and mouse and recombinant human N/OFQ receptors (NOP). Cyclisation may protect the peptide from metabolic degradation. In competition binding studies of rat, mouse and human NOP the following rank order pK(i) was obtained: N/OFQ(1-13)NH(2)(reference agonist)>N/OFQ=c[Cys(10,14)]>>c[Nphe(1)Cys(10,14)]. In GTPgamma(35)S studies of Chinese hamster ovary cells expressing human NOP (CHO(hNOP)) c[Cys(10,14)] (pEC(50) 8.29) and N/OFQ(1-13)NH(2) (pEC(50) 8.57) were full agonists whilst c[Nphe(1)Cys(10,14)] alone was inactive. Following 30 min pre-incubation c[Nphe(1)Cys(10,14)] competitively antagonised the effects of N/OFQ(1-13)NH(2) with a pA(2) and slope factor of 6.92 and 1.01 respectively. In cAMP assays c[Cys(10,14)] (pEC(50) 9.29, E(max) 102% inhibition of the forskolin stimulated response), N/OFQ(1-13)NH(2) (pEC(50) 10.16, E(max) 103% inhibition) and c[Nphe(1)Cys(10,14)] (~80% inhibition at 10 microM) displayed agonist activity. In the mouse vas deferens c[Cys(10,14)] (pEC(50) 6.82, E(max) 89% inhibition of electrically evoked contractions) and N/OFQ(1-13)NH(2) (pEC(50) 7.47, E(max) 93% inhibition) were full agonists whilst c[Nphe(1)Cys(10,14)] alone was inactive. c[Nphe(1)Cys(10,14)] (10 microM) competitively antagonised the effects of N/OFQ(1-13)NH(2) with a pK(B) of 5.66. In a crude attempt to assess metabolic stability, c[Cys(10,14)] was incubated with rat brain membranes and then the supernatant assayed for remaining peptide. Following 60 min incubation 64% of the 1 nM added peptide was metabolised (compared with 54% for N/OFQ-NH(2)). In summary, we report that c[Cys(10,14)] is a full agonist with a small reduction in potency but no improvement in stability whilst c[Nphe(1)Cys(10,14)] displays tissue (antagonist in the vas deferens) and assay (antagonist in the GTPgamma(35)S assay and agonist in cAMP assay) dependent activity.

Animals↗
PubMed2003

Carotid plaque morphology and cerebrovascular events.

AIM: Beside the degree of stenosis, plaque morphology obtained by the B mode ultrasound technique has been considered to define the plaque at risk for cerebrovascular events, and a subset of patients who deserve more strict surveillance. Our aim was to evaluate the relationship between plaque morphology, progression of stenosis, and the development of new cerebrovascular events. METHODS: We followed up by carotid duplex scan 230 asymptomatic patients, evaluating the degree and progression of internal carotid (ICA) stenoses and plaque morphology of the atherosclerotic lesions. RESULTS: During the follow-up period (median 32 month, range 6-125 months) 7% of patients developed ischemic neurological events of which 1.7% was a stroke. Of these events, only 5.7% correlated with carotid lesions. The new neurological events correlated with the degree and progression of stenoses, with a non homogeneous echographic appearance and irregular surface. The progression of the degree of stenoses was the parameter that correlated the most with the development of new neurologic symptoms. Nevertheless, the lesions that progressed modified their echographic pattern from homogeneous to non homogeneous in 78% of cases. Irregular surface and high degree of stenoses more than the baseline echographic pattern seem to correlate with plaque progression. CONCLUSION: Our follow-up study confirmed that ICA stenosis is a benign condition: very few strokes clearly correlated to the stenosis were observed. Nevertheless, the major predictors of risk for cerebrovascular events, besides the degree of stenoses, are the progression of the degree of stenosis, irregular surface and non-homogeneous echographic appearance.

Aged↗
PubMed2000

Biosynthesis and posttranslational regulation of human IL-12.

IL-12 is a heterodimeric proinflammatory cytokine consisting of a light alpha-chain, formerly defined as p35, disulfide-linked to a heavier beta-chain, formerly defined as p40. The beta-chain is also produced in large excess in a free form, and disulfide-linked beta-chain homodimers with anti-inflammatory effects are produced in the mouse. We analyzed the biosynthesis and glycosylation of IL-12 in human monocytes, and in a cell line stably transfected with IL-12 alpha and beta genes (P5-0.1). The IL-12 heterodimer and free beta-chain were immunoprecipitated from supernatants and cell lysates of metabolically labeled cells and resolved in SDS-PAGE. Whereas the beta-chain showed similar pI pattern whether in the free form or associated in the heterodimer, either in the secreted or intracellular form, the alpha-chain in the secreted heterodimer was much more acidic than that present in the intracellular heterodimer. Deglycosylation experiments with neuraminidase and Endo-F combined with two-dimensional PAGE of single bands of the intracellular vs extracellular IL-12 heterodimer revealed that the alpha-chain was extensively modified with sialic acid adducts to N-linked oligosaccharides before secretion. N-glycosylation inhibition by tunicamycin (TM) did not alter free beta-chain secretion, while preventing the IL-12 heterodimer assembling and secretion. Pulse-chase experiments indicated that IL-12 persists intracellularly for a long period as an immature heterodimer, and that glycosylation is the regulatory step that determines its secretion. beta-chain disulfide-linked homodimers were observed in TM-treated P5-0.1 cells, but in neither TM-treated nor untreated monocytes.

Animals↗
PubMed1999

Expression of COX-1, COX-2, and inducible nitric oxide synthase protein in human gastric antrum with Helicobacter pylori infection.

For a better understanding of the regulation of prostaglandin and nitric oxide (NO) synthesis in circumstances in which the gastric mucosa is inflamed, we have examined the ex vivo production of NO and prostaglandin E2 and the protein expression of inducible nitric oxide synthase (iNOS) and 2 cyclo-oxygenase (COX) isoforms in gastric biopsies from nine Helicobacter pylori-infected patients with active gastritis and six Helicobacter pylori (HP)-negative patients. The results indicate a significant increased of NO and PGE2 in patients with HP infection compared with uninfected samples. These findings were paralleled by marked increases in iNOS and in COX-1 and COX-2 protein expression. Expression of iNOS and COX-2 protein was absent in the mucosa of HP-negative controls. We have demonstrated that iNOS protein is expressed in the gastric mucosa of patients with HP infection. It is likely that iNOS expression and the corresponding high release of NO may play an important role in gastric inflammation associated with HP infection. However, the expression of COX-1 and COX-2 and the parallel increase of prostaglandin E2 could imply that these factors could limit the extend of mucosal damage. In previous reports NO has been shown to stimulate the COX activity, so we think that the role of NO could be both in the regulation of normal function and in the genesis of diseases.

Cyclooxygenase 1↗
PubMed1996

The interleukin 12 p40 gene promoter is primed by interferon gamma in monocytic cells.

Interleukin (IL) 12 is a proinflammatory cytokine produced by phagocytic cells, B cells, and other antigen-presenting cells that modulates adaptive immune responses by favoring the generation of T helper type 1 cells. IL-12 mediates some of its physiological activities by acting as a potent inducer of interferon (IFN) gamma production by T and natural killer cells. IFN-gamma enhances the ability of the phagocytic cells to produce IL-12 and other proinflammatory cytokines. Thus, IL-12-induced IFN-gamma acts in a positive feedback loop that represents an important amplifying mechanism in the inflammatory response to infections. We show here that IFN-gamma enhances IL-12 production mostly by priming phagocytic cells for lipopolysaccharide (LPS)-induced transcription of the IL-12 p40 gene, which encodes the heavy chain of the IL-12 heterodimer; furthermore, IFN-gamma directly induces transcription of the IL-12 p35 gene, which encodes the light chain of IL-12, and has at least an additive effect with LPS stimulation in inducing its transcription. The priming effect of IFN-gamma on the LPS-induced p40 gene transcription requires preincubation of the cells with IFN-gamma for at least 8 h to obtain a maximal effect. The priming effect of IFN-gamma for IL-12 production is predominantly at the transcriptional level for both the p40 and the p35 gene, and no evidence for a major role of posttranscriptional or translational mechanisms was found. A 3.3-kb human IL-12 p40 promoter construct transfected into cell lines recapitulated the tissue specificity of the endogenous gene, being silent in two human T cell lines, constitutively active in two human Epstein-Barr virus-positive B lymphoblastoid cell lines, and LPS inducible in the human THP-1 and mouse RAW264.7 monocytic cell lines. Because the RAW264.7 cell line is easily transfectable and regulates the endogenous IL-12 p40 gene in response to IFN-gamma or LPS similarly to human monocytes, it was used for analysis of the regulation of the cloned human IL-12 p40 promoter. A requirement for the region between -222 and -204 in both LPS responsiveness and IFN-gamma priming was established. This region contains an ets consensus sequence that was shown to mediate activation of the promoter by IFN-gamma and LPS, as well as by a cotransfected ets-2. The -222 construct was also regulated in a tissue-specific manner. Two other elements, IRF-1 located at -730 to -719, and NF-IL6 at -520 to -512, were also studied by deletion analysis, which did not result in decreased response to IFN-gamma and LPS stimulation.

Animals↗
PubMed1996

Selective association of a 22-38 kDa glycoprotein with MHC class II DP antigen on activated human lymphocytes at the plasma membrane.

Two-dimensional electrophoretic analysis (2D-PAGE) of cell surface human DP and DR class II antigens identified a glycoprotein, designated pX, that is associated at the cell surface with DP but not DR class II antigen in activated T, B and NK lymphocytes but not in resting B lymphocytes, Raji B lymphoma cells, activated thymic epithelial cells or activated monocytes. pX is a heavily glycosylated protein with an apparent molecular mass spanning between 38 kDa and 22 kDa, that is reduced, after deglycosylation with Endo-F, to 22 kDa. The pX structure appears nonpolymorphic and independent of DP polymorphism, as suggested by 2D-PAGE migrational pattern of 125I-labelled Endo-F deglycosylated DP immunoprecipitates from T cells blasts derived from four donors with different DP allotypes. The apparent absence of polymorphism of pX is further suggested by two-dimensional peptide mapping of a single spot derived from 2D-PAGE of 125I-labelled DP deglycosylated immunoprecipitates from two donors.

B-Lymphocytes↗
PubMed1993

Differential effects of tyrosine kinase inhibition in CD69 antigen expression and lytic activity induced by rIL-2, rIL-12, and rIFN-alpha in human NK cells.

The effect of rIL-12 on induction of CD69 antigen expression and cytolytic activity in purified human NK cells was evaluated in comparison to the effects of rIL-2 and rIFN-alpha. It was found that rIL-12 directly induced CD69 antigen expression in NK cells, although the period of incubation required by rIL-12 was longer than the period required by rIL-2 or by rIFN-alpha. Similarly, the cytolytic activity induced by rIL-12 in NK cells against the NK-resistant target cell line Raji was consistently lower than the cytolytic activity induced by rIL-2 or rIFN-alpha when measured after 6 hr of incubation, and increased during the following 18 hr of incubation. To compare the involvement of tyrosin kinases in activation of NK cells induced by rIL-2, rIL-12, and rIFN-alpha, the effect of the specific inhibitor of tyrosin kinases, genistein, was evaluated on induction of CD69 antigen expression and lytic function mediated by the three cytokines. It was found that genistein inhibited CD69 antigen expression induced by rIL-2 and by rIL-12, but not that induced by rIFN-alpha. Unlike the effect on CD69 antigen expression, the cytolytic activity induced by all three cytokines was inhibited by genistein. These results, together with the finding that CD69 antigen expression induced by rIL-2 but not by rIL-12 or rIFN-alpha was inhibited by addition of rIL-4, strongly suggest that IL-2, IL-12, and IFN-alpha mediate their effects, leading to induction of CD69 antigen expression through different activation pathways.

Antigens, CD↗
PubMed1993

Alpha (p55) and beta (p75) chains of the interleukin-2 receptor are expressed by AML blasts.

In the present study, we have investigated the leukemic cells obtained from 16 patients with acute myeloid leukemia (AML) at diagnosis for the membrane expression of p55 (alpha) and p75 (beta) interleukin-2 receptor (IL-2R) chains using specific monoclonal antibodies (mAbs), as well as for the presence of their transcripts using Northern blot analysis. In addition, immunoprecipitation of the p75 membrane molecule with TU27 and Mik-beta 1 mAbs was carried out in selected cases. The p75 IL-2R beta transcripts were detected in all cases, whereas the membrane p75 molecule was demonstrable by flow cytometry in three cases. However, data from the immunoprecipitation analysis suggest that the lack of the p75 IL-2R detection by flow cytometry might be caused by the low density of molecules per cell rather than the fact that the specific mRNA is not translated into the p75 surface molecule. In addition, a consistent membrane positivity with an anti-p55/CD25 mAb, present on fresh uncultured blasts in 37.5% of the cases, became detectable after short-term culture in 75% of cases. In each individual case, a strict correlation was found between membrane CD25 reactivity and the expression of p55 mRNA. Taken together, our data suggest that the expression of both alpha (p55) and beta (p75) IL-2R molecules is a common feature of leukemic cells in AML, and provide new arguments for reassessing the possible role of IL-2 in leukemic growth.

Acute Disease↗
PubMed1992

Expression and functional role of the p75 interleukin 2 receptor chain on leukemic hairy cells.

Hairy cell leukemia is a chronic lymphoproliferative disorder characterized by the expansion of neoplastic B-cells expressing the p55 chain of the interleukin 2 receptor (IL-2R) system that is recognized by anti-CD25 monoclonal antibodies (mAb) and binds interleukin 2 (IL-2) with low affinity. In the present study we investigated leukemic hairy cells (HC) for the presence of the p75 IL-2R chain which binds IL-2 with intermediate affinity and plays a crucial role in transducing the message to the cell. For this purpose, we tested highly enriched leukemic HC from six hairy cell leukemia patients for the presence of IL-2R transcripts and for the expression of the p55 and p75 IL-2R chains on their surface membrane by flow cytometry and immunoprecipitation analyses. The functional role of IL-2 in the regulation of HC proliferation was also investigated. Our results indicate that freshly isolated HC express detectable messages for both the p75 IL-2R and the p55 IL-2R. Flow cytometry analysis demonstrated detectable levels of p75 IL-2R on the HC from all patients tested. A mixture of two specific mAb was able to immunoprecipitate detectable amounts of p75 IL-2R from leukemic HC. When leukemic HC were cultured in the presence of several concentrations of IL-2 a low proliferative response was observed. Moreover, the IL-2-driven proliferation of HC was markedly inhibited by anti-p75 IL-2R mAb and to a lesser extent by anti-p55 IL-2R mAb. These findings provide direct evidence of the expression of different IL-2 receptors on leukemic HC and suggest that these molecules might play a role in leukemic cell growth.

Adult↗
PubMed1992

Constitutive expression of CD69 in interspecies T-cell hybrids and locus assignment to human chromosome 12.

In this study we describe the generation and characterization of interspecies somatic cell hybrids between human activated mature T cells and mouse BW5147 thymoma cells. A preferential segregation of human chromosomes was observed in the hybrids. Phenotypic analysis of two hybrids and their clones demonstrated coexpression of CD4 and CD69 antigens in the same cells. Segregation analysis of an informative family of hybrids followed by molecular and karyotype studies clearly demonstrated that the locus encoding CD69 antigen mapped to human chromosome 12. Although the expression of CD69 antigen is an early event after T-lymphocyte activation and rapidly declines in absence of exogenous stimuli, in the hybrids described in this study the expression was constitutive, similarly to what was previously found in early thymocyte precursors and mature thymocytes. In this respect it was important to note that the behavior of the hybrids in culture strongly suggested a dominant influence of the thymus-derived mouse tumor cell genome in controlling the constitutive expression of human CD69. These hybrids may thus provide a system to study the genetic and molecular mechanisms controlling the expression and function of this activation antigen.

Antigens, CD↗
PubMed1992

Different sensitivity to interleukin 4 of interleukin 2- and interferon alpha-induced CD69 antigen expression in human resting NK cells and CD3+, CD4-, CD8- lymphocytes.

The effect of rIL-4 on CD69 antigen expression induced by rIL-2 or by rINF-alpha on human resting NK cells and CD3+, CD4-, CD8- T lymphocytes has been investigated. rIL-4 drastically inhibited CD69 antigen expression induced by rIL-2 in both cell types. In contrast, rIL-4 did not alter rINF-alpha-induced CD69 antigen expression. Consistent results were obtained evaluating the cytolytic activity of NK cells against the Raji target cell line: rINF-alpha-induced lytic activity was not inhibited by rIL-4, while rIL-2-induced lytic activity was drastically inhibited. Proliferative activity of NK cells induced by rIL-2, in contrast, was only slightly reduced by rIL-4. rIL-4 did not alter the expression of the beta chain of IL-2 receptor, evaluated in NK cells by indirect immunofluorescence. Expression of the alpha chain of IL-2 receptor could not be detected in NK cells by indirect immunofluorescence. It can therefore be suggested that the selective inhibitory effect of rIL-4 on rIL-2-induced activation of NK cells is not mediated by downregulation of alpha and beta chains of IL-2 receptor.

Antigens, CD↗
PubMed1988

Heterogeneity of lymphokine-activated killer (LAK) populations at the clonal level: both NK and CD3+, CD4-, CD8- clones efficiently mediate tumor cell killing.

To investigate at the clonal level the phenotypic and functional properties of interleukin 2 (IL-2) activated killer cells (LAK), recombinant IL-2 activated peripheral blood lymphocytes were cultured under limiting conditions. Among 56 clones that lysed P815 in the presence of phytohemagglutinin (PHA) (22% of total proliferating microcultures) 36 clones lysed also the natural killer (NK)-sensitive K562 and the NK-resistant Hu126 glioma cell lines and one clone lysed only the K562 cell line. Several LAK clones were further assayed for both phenotype and functional activity. Of 22 clones, 10 were CD3-, CD4-, CD8-, and expressed the CD16 marker of NK cells; only one clone had the conventional phenotype of cytolytic T cells (CD3+, CD4-, CD8+), while 11 clones were CD3+, CD4-, CD8- and did not express alpha/beta heterodimer of T-cell antigen receptor as identified by WT31 monoclonal antibody. Only one of the latter clones was CD16+. Endogenous production of IL-2 after stimulation with PHA and phorbol myristate acetate was positive in 3/9 CD3- and in 8/8 CD3+, CD4-, CD8- clones. CD3- mediated strong antibody-dependent cellular cytotoxicity, a function exerted also by some CD3+, CD4-, CD8- T-cell clones to a lower extent. CD3+, CD4-, CD8- T-cell clones lysed different major histocompatibility complex unrelated tumor targets; moreover, this lytic activity seems to be CD3 dependent.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1987

Distinct mechanisms regulate MHC class II gene expression in B cells and macrophages.

In a previous series of studies, we had shown that the constitutive Ia expression in an immunoselected Ia-human B cell variant, RJ 2.2.5, could be restored by somatic cell hybridization with mouse B cells. These experiments allowed us to show the existence of a transacting activator factor(s) operating across species barriers and encoded by the aIr-1 locus located on mouse chromosome 16. The aim of the present study was to investigate whether the B cell constitutive Ia expression and the inducible Ia expression, as seen in macrophages treated with IFN-gamma, are controlled by similar intracellular factors. To this purpose, we constructed an interspecies somatic cell hybrid between the human Ia-RJ 2.2.5 B cells and the mouse Ia-P388 D1 macrophage cells. These murine cells transiently express Ia antigens when incubated with IFN-gamma. Our results show that RJ 2.2.5 X P388 D1 cell hybrids do not express either human or mouse class II gene products. Treatment with human recombinant IFN-gamma did not modify the MHC phenotype of either the hybrid cells or the human parental cells. On the other hand, treatment of the hybrid cells with murine recombinant IFN-gamma resulted in de novo expression of mouse Ia mRNA and corresponding cell surface antigens without, however, reinduction of the human class II-positive phenotype. Furthermore, treatment with the mouse lymphokine significantly increased the levels of human HLA class I mRNA and corresponding cell surface antigens in the hybrid cells, further reinforcing the notion of the existence of non-species-specific secondary mediators generated after receptor-ligand interaction in the IFN-gamma system. Together, these results indicate that in macrophages, the intracellular events taking place after binding of IFN-gamma with its own receptor and leading to the expression of a class II-positive phenotype do not operate via an activation of the aIr-1 locus and/or its products. Thus, at least in our experimental system, we can firmly establish a first, relevant distinction between constitutive and inducible class II gene expression. This difference, dictated by the specific differentiation program of each cell type, may be relevant for the understanding of the function of class II gene products.

Animals↗
PubMed1987

Structural analysis of human Ia antigens reveals the existence of a fourth molecular subset distinct from DP, DQ, and DR molecules.

Structural analysis by two-dimensional peptide maps (2D-PM) of the human Ia molecular pool expressed on the cell surface of two distinct lymphoblastoid cell line, LG-2 and Raji, revealed the existence of a novel MHC class II molecular heterodimer that differs at the level of both alpha and beta subunits from the previously described DP, DQ, and DR antigens. These differences were also seen at the level of two-dimensional electrophoresis (2D-PAGE) of biosynthetically labeled intact molecules, although to a lesser extent, due to the intrinsic limitations of this technique in resolving fine structural differences. We have designated this new class II antigen as the fourth Ia subset. The fourth Ia subset seems to represent a small proportion of the human Ia pool. Comparative analysis by 2D-PM of the two cell lines showed the presence of structural variations in the alpha chains of the fourth Ia subset, suggesting the existence of polymorphism for these subunits. Cell surface iodination did not show appreciable labeling of the fourth subset beta chain in LG-2 cells, and this prevented analysis of the structural polymorphism of this subunit. Furthermore, for the first time, we have shown that DP alpha chains display distinct peptide maps in LG-2 and Raji cells, thus suggesting the presence of structural polymorphism for these Ia subunits also. The DQ1 alpha and beta allelic products present in LG-2 cells (DQ homozygous) did not show appreciable structural variation when compared with the homologous allelic products present in Raji cells (DQ heterozygous). Finally, we have confirmed the absence of polymorphism for the DR alpha subunits. By 2D-PM, relatively low structural variation was instead found for the highly polymorphic DR beta subunits expressed in the two cell lines, suggesting that cell surface iodination preferentially labels constant domains of DR beta chains.

Antibodies, Monoclonal↗
PubMed1986

aIr-1, a newly found locus on mouse chromosome 16 encoding a trans-acting activator factor for MHC class II gene expression.

RJ 2.2.5 is a human B cell line that has lost the capacity to express MHC class II genes. The human class II-positive phenotype is restored in somatic cell hybrids between RJ 2.2.5 and mouse spleen cells. By karyotype and molecular studies of an informative family of hybrids we have now shown that the reexpression of human class II gene products, as well as the maintenance of the mouse class II-positive phenotype, correlates with the presence of mouse chromosome 16. Thus, the existence on this mouse chromosome of a newly found locus, designated by us aIr-1, that determines a trans-acting activator function for class II gene expression, is established. Possible implications of this finding are discussed.

Animals↗
PubMed1986

Stable integration of mouse DNA into Ia-negative human B-lymphoma cells causes reexpression of the human Ia-positive phenotype.

RJ 2.2.5, a variant of the human B-lymphoma cell line Raji, does not express the HLA-DR, -DQ, and -DP class II (or Ia) histocompatibility antigens, as a result of a defect in the transcription of the corresponding genes. This defect is corrected after fusion of RJ 2.2.5 cells with mouse Ia-positive cells. Previous work showed that the trans-acting transcriptional activator supplied by the mouse cells is encoded by a locus on mouse chromosome 16. We show here that reexpression of human major histocompatibility complex class II genes by RJ 2.2.5 cells can also be achieved by stable integration of mouse genomic sequences into the RJ 2.2.5 genome after DNA-mediated gene transfer.

Animals↗