Natalio Vita

A plasmocyte selective monoclonal antibody (B-B4) recognizes syndecan-1.

We developed a new monoclonal antibody, B‐B4, which specifically identifies human plasma cells. It strongly reacts with all multiple myeloma cell lines and with malignant plasma cells of all tumour samples of the multiple myeloma patients tested. B‐B4 does not react with any peripheral blood, bone marrow or tonsil cells. Cloning of the B‐B4 antigen reveals that the monoclonal antibody recognizes syndecan‐1. It appears that the monoclonal antibody B‐B4 is a suitable marker for human plasmocyte identification among haemopoietic cells and a useful probe for the diagnosis of haematological malignancies. Furthermore, this monoclonal antibody can be used for depletions prior to CD34 grafting.

Cloning and Characterization of a Specific Interleukin (IL)-13 Binding Protein Structurally Related to the IL-5 Receptor α Chain

Interleukin-13 (IL-13) is a cytokine secreted by activated T lymphocytes that shares many, but not all, biological activities with IL-4. These overlapping activities are probably due to the existence of common receptor components. Two proteins have been described as constituents of the IL-4 receptor, a ∼140-kDa glycoprotein (IL-4R) and the γ chain (γc) of the IL-2 receptor, but neither of these proteins binds IL-13. We have cloned a cDNA encoding an IL-13 binding protein (IL-13R) from the Caki-1 human renal carcinoma cell line. The cloned cDNA encodes a 380-amino acid protein with two consensus patterns characteristic of the hematopoietic cytokine receptor family and a short cytoplasmic tail. The IL-13R shows homology with the IL-5 receptor, and to a lesser extent, with the prolactin receptor. COS-7 cells transfected with the IL-13R cDNA bind IL-13 with high affinity but do not bind IL-4. COS-7 cells co-transfected with the cloned IL-13R cDNA and IL-4R cDNA resulted in the reconstitution of a small number of receptors that recognized both IL-4 and IL-13. Reverse transcription-polymerase chain reaction analysis detected the receptor transcript only in cell lines known to bind IL-13.

Primary structure and functional expression of mouse pituitary and human brain corticotrophin releasing factor receptors

Corticotrophin‐releasing factor (CRF) is the principal hypothalamic factor governing the pituitary‐adrenal axis, but the wide extra‐pituitary distribution of CRF and its receptors suggest a major role for this neuropeptide in the integration of the overall physiological and behavioral responses of an organism to stress. We have cloned a CRF receptor complementary DNA (cDNA) by expression in COS‐7 cells of a cDNA library from the AtT20 mouse pituitary tumour cell line. The cloned mouse cDNA was then used as a probe to isolate a human CRF receptor cDNA from a human brain cDNA library. The mouse and human cDNAs both encode 415 amino acid proteins that are 97% identical, containing seven putative transmembrane domains characteristic of G protein‐coupled receptors. The CRF receptor shows homology with the receptors for growth hormone‐releasing factor, vasoactive intestinal peptide, secretin, parathyroid hormone, and calcitonin. COS‐7 cells transfected with the mouse CRF receptor cDNA bind radiolabelled ovine CRF with high affinity and respond specifically to CRF by accumulation of intracellular cAMP. A 2.7 kb mRNA coding for the CRF receptor could be detected in AtT20 cells and human cortex tissue. PCR analysis also detected the receptor transcript in human pituitary, brainstem, and testis.

Molecular cloning of a levocabastine-sensitive neurotensin binding site

A search for sequences homologous to the neurotensin receptor cDNA in a rat hypothalamic library has identified a novel neurotensin receptor (NTR‐2). The 1539 bp cDNA encodes a 416 amino acid protein and shows highest homology to the previously cloned neurotensin receptor (NTR‐1) (64% homology and 43% identity). Binding and pharmacological studies demonstrate that NTR‐2 expressed in COS cells recognizes neurotensin (NT) with high affinity as well as several other agonists and antagonists. However, a fundamental difference was found; unlike NTR‐1, NTR‐2 recognizes, with high affinity, levocabastine, a histamine H1 receptor antagonist previously shown to compete with NT for low‐affinity binding sites in brain.

Cloning and expression of a complementary DNA encoding a high affinity human neurotensin receptor

A human neurotensin receptor (hNTR) cDNA was cloned from the colonic adenocarcinoma cell line HT29. The cloned cDNA encodes a putative peptide of 418 amino acids with 7 transmembrane domains. The amino acid sequence of the hNTR is 84% identical to the rat NTR [Neuron, 4 (1990) 847‐854]. Transfection of this cDNA into COS cells results in the expression of receptors with pharmacological properties similar to those found with HT29 cells. Northern blot analysis using the hNTR cDNA probe indicated a single transcript of 4 kb in the brain, the small intestine and blood mononuclear cells.

Cloning of the human IL-13Rα1 chain and reconstitution with the IL-4Rα of a functional IL-4/IL-13 receptor complex

The human homologue of the recently cloned murine IL‐13 binding protein (IL‐13Rα1) was cloned from a cDNA library derived from the carcinoma cell line CAKI‐1. The cloned cDNA encodes a 427 amino acid protein with two consensus patterns characteristic of the hematopoietic cytokine receptor family and a short cytoplasmic tail. The human protein is 74% identical to the murine IL‐13Rα1, and 27% identical to the human IL‐13Rα2. CHO cells expressing recombinant hIL‐13Rα1 specifically bind IL‐13 (K d≈4 nM) but not IL‐4. Co‐expression of the cloned cDNA with that of IL‐4Rα resulted in a receptor complex that displayed high affinity for IL‐13 (K d≈30 pM), and that allowed cross‐competition of IL‐13 and IL‐4. Electrophoretic mobility shift assay showed that IL‐13 and IL‐4 were able to activate Stat6 in cells expressing both IL‐4Rα and IL‐13Rα1, while no activation was observed in cells expressing either one or the other alone.

Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells

The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.

Soluble CD14 acts as a negative regulator of human T cell activation and function

T cell activation is controlled by the coordination of stimulatory and negative regulatory signals which are not completely defined. In this study we tested for a possible direct effect of CD14 on the regulation of T cell activation and function. We show that soluble CD14 (sCD14) induces inhibition of antigen‐mediated peripheral blood mononuclear cells (PBMC) proliferation and anti‐CD3‐mediated proliferation of CD4+CD8−, CD4−CD8+ and CD4+CD8+ T cell clones. This effect is not due to cell death, but results from a marked inhibition of IL‐2 production. Proliferation of T cell clones due to exogenous IL‐2 is not affected by sCD14. We also found that sCD14 inhibits production of another Th1‐like cytokine, IFN‐γ and a Th2‐like cytokine, IL‐4. Importantly, sCD14 induces a progressive accumulation of the inhibitory protein IκB‐α. We show that sCD14 binds to activated T cells. Following cell activation, biotinylated sCD14 stains CD3+ PBMC, as well as human T cell clones with varying intensity. The binding is saturable, can be inhibited by excess of unlabeled sCD14 and, following binding, sCD14 is internalized. Collectively, these findings reveal a previously unrecognized function of sCD14, namely its capacity to negatively regulate T lymphocyte activation and function by interacting directly with activated T cells.

Anti-inflammatory effects of peripheral benzodiazepine receptor ligands in two mouse models of inflammation.

In vivo treatment of mice with peripheral benzodiazepine receptor ligands exerts an inhibitory effect on the inflammatory response in two models of acute inflammation. In the first model, pretreatment of the animals (24 h) with 1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide (PK11195) and 7-chloro-5-(4-Chlorophenyl)-1, 3-dihydro-1-methyl-2-H-1,4-benzodiazepin-2 (Ro5-4864), at different doses (0.00001-10 mg/kg, i.p.) dose dependently inhibited the formation of mouse paw oedema induced by carrageenan with mean ID(50s) of 0.009 (95% confidence limits=0.0076-0.013) and 0.04 (95% confidence limits=0.025-0.0086) mg/kg, respectively. Both ligands (0. 1 mg/kg, i.p.) inhibited in the same way the mouse paw oedema induced by carrageenan in animals with and without adrenal glands. PK11195 and Ro5-4864 (0.1 mg/kg, i.p.) inhibited the mouse paw oedema induced by several inflammatory mediators. In the second model, the pretreatment (24 h) with peripheral benzodiazepine receptor ligands (0.1 mg/kg, i.p.) exerted an inhibitory effect on neutrophil influx and produce a marked inhibition of carrageenan-produced interleukin-13 and interleukin-6 in pleural exudation. Our results extend previous findings that peripheral benzodiazepine receptor is involved in the inflammatory response, and suggest that this action may be linked to the action of different inflammatory mediators, probably mainly by the inhibition of the release of pro-inflammatory cytokines.

Targeted inactivation of the neurotensin type 1 receptor reveals its role in body temperature control and feeding behavior but not in analgesia.

Three subtypes of neurotensin receptor have been described, two members of the heptahelical transmembrane domain G protein-coupled receptor superfamily NT-1R and NT-2R, and NT-3R unrelated to this family. We have generated NT-1R deficient (NT-1R(-/-)) mice. NT-1R(-/-) mice were born at the expected Mendelian frequency without obvious abnormalities and they were fertile. The NT-induced analgesia on the writhing induced by phenyl-p-benzoquinone administration remained at wild-type levels in the NT-1R(-/-) mice demonstrating that the NT-1R is not implicated in the analgesic effect of NT in this test. The NT-1R(-/-) mice were hyperthermic; their body temperature was not affected by intracerebroventricular (i.c.v.) administration of NT, contrasting with the hypothermia induced in NT-1R(+/+) mice. NT-1R(-/-) mice showed a small significant increase in body weight compared to the NT-1R(+/+) congeners as early as 10 weeks after birth, correlated with a higher food intake. NT-1R(-/-) mice showed similar spontaneous locomotion to the control littermates, but did not respond to i.c.v. NT-induced hypolocomotion. I.c.v. injection of NT inhibited feeding in fasted wild-type mice, but had no effect on feeding of the NT-1R(-/-) mice. I.c.v. administration of the orexigenic neuropeptide Y (NPY) stimulated feeding to the same extent in both wild-type and NT-1R(-/-) mice. This analysis of NT-1R-deficient mice shows that the NT-1R does not play a role in NT-induced analgesia, but that it is clearly implicated in thermal and feeding regulation, weight control, and NT-induced hypolocomotion.