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Calcitonin-gene-related peptide (CGRP) and adrenomedullin are related peptides with distinct pharmacological profiles. Here we show that a receptor with seven transmembrane domains, the calcitonin-receptor-like receptor (CRLR), can function as either a CGRP receptor or an adrenomedullin receptor, depending on which members of a new family of single-transmembrane-domain proteins, which we have called receptor-activity-modifying proteins or RAMPs, are expressed. RAMPs are required to transport CRLR to the plasma membrane. RAMP1 presents the receptor at the cell surface as a mature glycoprotein and a CGRP receptor. RAMP2-transported receptors are core-glycosylated and are adrenomedullin receptors.

Background: Acute cutaneous miliary tuberculosis is a rare form of Mycobacterium tuberculosis infection which has been described in only 25 published cases in the last 15 years. The appearance of serious disseminated forms of tuberculosis is enhanced by the severe immunodeficiency which characterizes AIDS., Methods: Four cases are described of disseminated tuberculosis with cutaneous localization clinically described as the acute miliary form. Patients had AIDS and CD4 lymphocyte levels of < 100 cells/mm3. Diagnosis was established in all cases by scraping of cutaneous lesions and direct examination with Ziehl-Neelsen coloration., Results: The 4 patients presented signs and symptoms of disseminated tuberculosis with cutaneous manifestations, without a definite pattern and with localization predominantly on the thorax. Three had BAAR-positive sputum. Multiresistant strains of Mycobacterium tuberculosis were confirmed by antibiogram. One patient infected by a strain sensitive to first-line drugs responded favorably to conventional treatment for disseminated tuberculosis. In the remaining three with multiresistant strains, the evolution of the disease was rapid and unfavorable., Conclusions: Abrupt appearance of cutaneous lesions in a patient with disseminated tuberculosis and advanced HIV-disease should raise the suspicion of its etiology. Scraping and direct examination using Ziehl-Neelsen coloration is practical, economical and non-invasive diagnostic method.

Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5-dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.

The rat basophilic leukaemia (RBL) cell line has been widely used as a convenient model system to study regulated secretion in mast cells. Activation of these cells through the high-affinity receptor for IgE (Fcepsilon-RI) results in degranulation and the extracellular release of mediators. There is good evidence of a role for GTPases in mast cell degranulation, and a number of studies with peptides derived from the Rab3a effector domain have suggested that Rab3a may function in this process. However, in neuroendocrine cells, overexpression of Rab3a can act as a negative regulator of stimulated exocytosis [Holz, Brondyk, Senter, Kuizon and Macara (1994) J. Biol. Chem. 269, 10229-10234; Johanes, Lledo, Roa, Vincent, Henry and Darchen (1994) EMBO J. 13, 2029-2037]. In order to study the function of Rab3a in RBL degranulation, we have generated clones of RBL cells stably expressing Rab3a, and show that in these haematopoietic cells Rab3a can also function as a negative regulator of exocytosis. Overexpression of a mutant form of Rab3a (Asn-135 to Ile), which is predicted to be predominantly GTP-bound, also inhibited degranulation. However, overexpression of a mutant form of Rab3a that was truncated at the C-terminus to remove the sites for geranylgeranylation failed to inhibit degranulation. The effect of Rab3a is specific to secretion, and we observe no effect of Rab3a on receptor-mediated endocytosis. The Rab3a-induced block in degranulation can be bypassed by stimulation of streptolysin-O-permeabilized cells with guanosine 5'-[gamma-thio]triphosphate. We conclude from these studies that Rab3a is implicated in an early stage of granule targeting, whereas fusion of granules with the plasma membrane is regulated by a distinct downstream GTP-binding protein or proteins.

Vesicular transport to and from the lysosome and late endosome is defective in patients with Chediak-Higashi syndrome (CHS) and in mutant beige (bg) mice. CHS and bg cells have giant, perinuclear vesicles with characteristics of late endosomes and lysosomes that arise from dysregulated homotypic fusion. CHS and bg lysosomes also exhibit compartmental missorting of proteins, such as elastase, glucuronidase and cathepsin G. Lyst, a candidate gene for bg, was identified by direct complementary DNA selection from a yeast artificial chromosome (YAC) clone containing a 650-kilobase segment of the bg-critical region on mouse chromosome 13. Lyst is disrupted by a 5-kilobase deletion in bg mice, and Lyst messenger RNA is markedly reduced in bg homozygotes. The homologous human gene, LYST, is highly conserved with mouse Lyst, and contains a frame-shift mutation at nucleotides 117-118 of the coding domain in a CHS patient. Thus bg mice and human CHS patients have homologous disorders associated with Lyst mutations. Lyst encodes a protein with a carboxy-terminal prenylation motif and multiple potential phosphorylation sites. Lyst protein is predicted to form extended helical domains, and has a region of sequence similar to stathmin, a coiled-coil phosphoprotein thought to act as a relay integrating cellular signal response coupling.

The C-terminal region of interleukin-5 has previously been suggested to be important for biological activity [Mackenzie et al., (1991), Mol. Immunol. 28, 155-158; Kodama et al. (1991), Biochem. Biophys. Res. Commun. 178, 514-519]. We have investigated this region by making a series of truncation mutants. The proteins were expressed in Escherichia coli, purified from inclusion bodies, and were able to refold with the disulfide homodimeric topology typical of interleukin-5. Analysis of the truncated carboxy-terminal proteins in an interleukin-5-dependent proliferation assay on TF-1 cells showed a rapid loss of activity as the C-terminal was shortened by more than two amino acids. This loss of biological activity correlated with a drop in binding affinity to both the alpha chain of the receptor and the high-affinity complex consisting of the alpha and beta subunits. Analysis of the proteins by 1H-NMR showed that the truncated mutants have higher exchange rates with solvent, indicating a less rigid structure. The carboxy-terminal region is therefore necessary to maintain the stability of the four-helix bundle and to orient correctly the important residues of the fourth helix. Inspection of the structure determined by X-ray crystallography shows that Trp-110 acts as the major residue in anchoring the fourth helix.

Since the very beginning of liver transplantation in humans, research in animals has had close relationship with clinical practice. Results obtained in animals have been transferred to the clinics and problems borne in the clinics have been addressed again in animals for to be answered clearly. In this review the authors report their experience of transplantation in the pig model and discuss the significance of a team cooperation in the laboratory as a preparatory step for clinical practice.

The use of yeast as a model system to study mammalian proteins is attractive, because yeast genetic tools can be utilized if a suitable phenotype is created. STE6, the Saccharomyces cerevisiae a-factor mating pheromone transporter, and CFTR, the mammalian cystic fibrosis transmembrane conductance regulator, are both members of the ATP binding cassette (ABC) superfamily. Teem et al. (1993) described a yeast model for studying a mutant form of the cystic fibrosis protein, CFTR delta F508. The model involved expression of a chimeric molecule in which a portion of yeast STE6 was replaced with the corresponding region from mammalian CFTR. The STE6/CFTR chimera complemented a ste6 mutant strain for mating, indicating that it could export a-factor. However, mating efficiency was dramatically reduced upon introduction of delta F508, providing a yeast phenotype for this mutation. In human cells, the delta F508 mutation results in retention of CFTR in the endoplasmic reticulum (ER), and possibly in reduction of its chloride-channel activity. Here we examine the basis for the differences in STE6 activity promoted by the wild-type and mutant STE6/CFTR chimeras. By analysis of protein stability and subcellular localization, we find that the mutant chimera is not ER-retained in yeast. We conclude that the molecular basis for the reduced mating of the STE6/CFTR delta F508 chimera must reflect a reduction in its capacity to transport a-factor, rather than mistrafficking. Thus, STE6/CFTR delta F508 in yeast appears to be a good genetic model to probe certain aspects of protein function, but not to study protein localization.

Interleukin 5 (IL-5) is a T-cell derived cytokine that induces eosinophil growth and differentiation in both mouse and human bone marrow cultures. Elevated levels of IL-5 as well as eosinophils have been detected in the sputum and Bronchoalveolar lavage (BAL) fluids of asthmatics. Since the recruitment of inflammatory cells to tissues requires the participation of adhesion molecules, we have developed a rapid and sensitive assay to examine the effect of IL-5 and other activation stimuli on eosinophil adhesion to recombinant intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Human recombinant IL-5, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), tumour necrosis factor alpha (TNF-alpha), RANTES, MCP-3, C5a, PAF, fMLP, PMA and ConA all induced adhesion of purified eosinophils obtained from normal donors to ICAM-1 and VCAM-1 in a dose and time dependent manner. Adhesion was rapid, within 15 minutes of culture at 37 degrees C, and plateaued within 30 minutes. Activated eosinophils also adhered rapidly to immobilized IgG via the type II Fc gamma receptor (CD32). Analysis of the effect of IL-5 on surface molecule expression by FACS analysis revealed increased expression of CD11b molecules and decreased expression of L-selectin, but no change in the expression of CD11a, CD18, CD29, CD49d and CD32. We also show that Mac-i plays an important role in the regulation of eosinophil activation, since antibodies to CD11b can block IL-5 induced adhesion to IgG and IL-5 induced degranulation.

Interleukin-5 (IL-5) is a key cytokine for the production, differentiation, and activation of eosinophils. IL-5 is a member of the four helical bundle family of cytokines, and in common with many members of the cytokine family it binds to a heterodimeric receptor composed of a ligand binding alpha-chain and a signal-transducing beta-chain. We have established two receptor/ligand binding assays based on the extracellular domain of the receptor alpha-chain which we have produced as a fusion protein. One assay is based on scintillation proximity fluoromicrospheres and radiolabeled ligand and the other on detection of biotinylated ligand binding to immobilized receptor using a chemiluminescent substrate in a 96-well microtiter plate format. Both receptor binding assays have been optimized for high throughput screening for receptor antagonists. These assays were also used for analytical purposes and the binding of ligand to the receptor alpha-chain was compared directly to receptor binding assays performed on TF-1 cells which express the receptor alpha beta-heterodimer. These three assays have been used to study site-directed mutants of IL-5 to determine the important residues for interaction of the cytokine with each chain of the receptor (P. Graber et al. (1995) J. Biol. Chem. 270, 15762-15769).

The ARFs are a family of 21,000 M(r) proteins with biological roles in constitutive secretion and activation of phospholipase D. The structure of ARF-1 complexed to GDP determined from two crystal forms reveals a topology that is similar to that of the protein p21 ras with two differences: an additional amino-terminal helix and an extra beta-strand. The Mg2+ ion in ARF-1 displays a five-coordination sphere; this feature is not seen in p21 ras, due to a shift in the relative position of the DXXG motif between the two proteins. The occurrence of a dimer in one crystal form suggests that ARF-1 may dimerize during its biological function. The dimer interface involves a region of the ARF-1 molecule that is analogous to the effector domain in p21 ras and may mediate interactions with its effectors.

CD40 ligand (CD40L), a surface molecule which can be expressed by T cells, mast cells and basophils, has been shown to be involved in the control of B cell proliferation, immunoglobulin class switching as well as in the activation of monocytes and T cells. We demonstrate that CD40L can also be expressed constitutively by eosinophils from an hypereosinophilic patient or, upon activation, by the eosinophilic cell line EOL-3 and normal blood eosinophils. Eosinophils were able to induce, in conjunction with IL-4, CD40L-dependent B cell proliferation in vitro. These results suggest that CD40L could play a role in the inflammatory processes during which eosinophil infiltration and activation are observed.

We have investigated the polymorphism in intron 2 of the interleukin-1 receptor antagonist gene and identified two new alleles of the system. We have shown that the polymorphism is caused by the variable copy number of an 86-bp sequence, by using the polymerase chain reaction and primers immediately flanking the repeat region, and by direct sequencing. The repeat region contains three potential protein-binding sites and therefore the variable copy number may have functional significance.

The human IL-1 receptor antagonist (IL-1ra) was produced in a high yield E. coli expression system, and was purified in a rapid two-step purification. This recombinant IL-1ra molecule possessed full binding activity to the IL-1 receptor (type I) and totally inhibited IL-1-induced PGE2 production by human dermal fibroblasts. Radioalkylation and analysis of V8-derived IL-1ra peptides indicate that the four cysteines present in the IL-1ra are not disulphide-linked.

In this study we have investigated the effect that interleukin 1 (IL-1) has on cell surface IL-1 receptor expression in the murine thymoma cell line, EL4 6.1. These cells express IL-1 receptors with both high affinity (Kd = 65 pM, 986 receptors/cell) and low affinity (Kd = 14.5 nM, 10,417 receptors/cell). The high- and low-affinity receptors are indistinguishable by crosslinking studies performed at both high and low ligand concentrations. However, the two affinity states could be functionally distinguished on the basis of their internalization of ligand. Receptor-mediated endocytosis was dependent upon the concentration of ligand bound to the cells. In the presence of low IL-1 concentrations receptor-mediated endocytosis was slow, whereas at high IL-1 concentrations, endocytosis was more rapid. Furthermore, receptor-mediated endocytosis of IL-1 did not result in downregulation of surface IL-1 receptors. Indeed, both kinetic and equilibrium binding studies revealed that pre-incubation of cells with IL-1 alpha resulted in an acute upregulation of 125IL-1 alpha binding to high affinity surface receptors in a time and energy dependent manner. Examination of the association kinetics suggested that increased binding was not attributable to positive co-operativity of the high affinity IL-1 receptor, but was due to increasing IL-1 receptor number. This observation was confirmed by equilibrium binding studies. Moreover, receptor numbers were not enhanced by de novo synthesis, nor release of receptors from an intracellular pool. The observed increases in surface ligand binding were most probably due to conversion of the surface pool of low affinity receptors into high affinity receptors.

By screening a human genomic library with an interleukin-1 receptor antagonist (IL-1ra) cDNA probe, we have isolated a 15 kb clone which contains the entire coding region of the gene as expressed in monocytes, and includes 6 kb of 5'-upstream sequence. The gene contains four exons which code for the secreted form of the IL-1ra, however, our clone does not contain the alternative first exon used to generate an intracellular form of the protein as the protein as found in epithelial cells. Analysis of the sequence reveals a consensus TATA box, and three Alu repeats, two of which are in the upstream region and one in intron 3. The sequence also reveals an 86 bp motif tandomly repeated four times within intron 2, and may reflect the polymorphism known to exist in this region of the gene. By in-situ fluorescence hybridization we have shown that the IL-1ra gene is found on the long arm of chromosome 2 and maps to 2q13-14.1. Previous studies have revealed that IL-1 alpha, and IL-1 beta and both type I and type II forms of the IL-1 receptor all map close to this region of chromosome 2.

In this study we have examined the effect of agents known to perturb certain signal transduction pathways on the biological responses of target cells to stimulation with interleukin-1 (IL-1). In the murine thymoma cell line EL4, IL-1 stimulation results in the secretion of interleukin-2 (IL-2), which was subsequently measured by proliferation of an IL-2-dependent cell line. Agents that elevated intracellular cAMP blocked or partially blocked IL-1 induction of IL-2 secretion, whereas agents that activated protein kinase C (PKC) resulted in a synergistic enhancement. Both pertussis and cholera toxins also inhibited IL-1-induced IL-2 secretion, although probably by acting at different levels. IL-1 simulation of human and murine fibroblasts resulted in release of prostaglandin E2. This response was inhibitable by pertussis toxin but not by cholera toxin, whereas co-stimulation of the fibroblasts with IL-1 and phorbol ester resulted in a synergistic response. Murine fibroblasts could also be stimulated to proliferate by IL-1, and this response was also inhibitable by pertussis toxin. These findings are consistent with coupling of the IL-1 receptor to a signalling pathway via a pertussis toxin substrate.

Non-human primates have been used to study immune function to a much lesser extent than readily available strains of inbred rodents. Nevertheless, in situations where it might be desirable, but impossible, to study human immune responses in vivo, lower primates could provide an acceptable alternative. In order to extent the knowledge of T- and B-lymphocyte function in lower primates, the common marmoset Callithrix jaccus was used as an experimental model. The functional similarities between this species and humans at the level of T-B co-operation in the antibody response were examined, and xenoreactive T-lymphocyte clones were obtained from marmoset spleen cells using Epstein-Barr virus (EBV)-transformed human B cells as stimulators. These clones could act as helper cells when co-cultured with human B lymphocytes, inducing the secretion of both IgM and IgG. Lymphokine production by mitogen-stimulated marmoset T-cell clones was also examined. Interleukins (IL) 2 and 4 activities were detected in clone supernatants using bioassays and interferon-gamma (IFN-gamma) was detected using a solid-phase ELISA system. However, SDS-PAGE analysis of biosynthetically labelled marmoset and human T-cell clone supernatant proteins revealed major differences between the soluble T-cell products of the two species. The proliferative responses of marmoset T and B cells to recombinant human IL-2 and IL-4 were also examined. Stimulation of [3H]thymidine uptake was detected in both T cell- and anti-IgM-stimulated B-cell cultures with both of the lymphokines. These results suggests that the key components of the antibody response are functionally conserved between lower primates and man and that the common marmoset may be useful as an in vivo model of immune function, particularly with regard to the role of interleukins such as IL-2 and IL-4.

Murine Th1 and Th2 T cell lines differ in their responses to interleukin 1 (IL 1). Therefore, we examined two T-cell lines, D10.G4.1 (Th2) and MTg12B (Th1) in an attempt to correlate IL 1 receptor (IL 1R) expression with their IL 1 responsiveness. D10.G4.1 cells, which respond to IL 1, expressed two forms of the IL 1R, with molecular masses of approximately 80 kDa and approximately 60 kDa. In contrast, MTg12B cells failed to respond to IL 1 and only expressed the approximately 60 kDa receptor form. This suggests that the approximately 80 kDa receptor is essential for signaling. Expression of both IL 1R forms on D10.G4.1 cells could be inhibited by the anti-IL 4 antibody, 11B11. Antigen presentation reversibly upregulated both forms of the IL 1R, whereas stimulation with concanavalin A (ConA) and anti-CD3 only upregulated the approximately 60 kDa moiety. Upregulation of the approximately 80-kDa IL 1R by repeated antigenic stimulation resulted in a marked increase in sensitivity of D10.G4.1 cells to IL 1.

In this study we demonstrate that 125I-labelled interleukin (IL) 1 alpha binds specifically to its receptor on the surface of EL4 6.1 cells and is subsequently endocytosed and translocated from the cell membrane to the nucleus, where it progressively accumulates. Two-dimensional polyacrylamide-gel electrophoresis revealed that the internalized 125I-IL1 alpha associated with the nucleus was intact, with negligible breakdown products present. Specific and saturable binding of 125I-IL1 alpha was demonstrated on purified nuclei isolated from these cells. Binding of the radiolabelled ligand showed similar kinetics to that of the plasma-membrane receptor, and was inhibited by both unlabelled IL1 alpha and IL1 beta. Equilibrium binding studies on isolated nuclei revealed a single high-affinity binding site, with a Kd of 17 +/- 2 pM, and 79 +/- 12 binding sites per nucleus. These studies demonstrate that receptor-mediated endocytosis of IL1 results in its accumulation in the nucleus, and this mechanism may play an important role in mediating some of the actions of IL1.

We have generated a panel of T-lymphocyte clones from a patient suffering from the hyper IgE syndrome, and have attempted to correlate the ability of each to help IgE responses in vitro with the profile of lymphokines secreted after mitogenic stimulation. Clones which showed positive IgE helper activity released larger amounts of interleukin-4 (IL-4) than the non-helpers, which tended to release more interleukin-2 (IL-2). Surprisingly, all clones released moderate amounts of gamma interferon (IFN), which has been shown to inhibit the action of IL-4 on B cells. The clones were analysed by indirect immunofluorescence using monoclonal antibodies to CDw29 and CD45R (4B4 and 2H4 respectively). Those T cells which could provide strong helper activity for all isotypes, expressed high levels of CDw29 and low CD45R. These data suggest that these CD4-positive T cells expressing surface antigen of the 'memory' subset i.e. CDw29, are involved in IgE isotype regulation by virtue of their ability to secrete IL-4 upon antigenic stimulation.

The transcellular transport of polymeric immunoglobulins (pIg) across mucosal epithelia is mediated by a membrane glycoprotein known as the polymeric immunoglobulin receptor (pIgR). The intracellular routing of the pIgR has been used as a model to study protein traffic. Examination of the biosynthesis and processing of the pIgR in mammary gland and liver have led to a working hypothesis for pIgR routing in the cell. These hypotheses are currently being tested in an immortalized cell line derived from rabbit mammary gland alveolar cells.

Peripheral blood mononuclear cells (PBMC) from a patient suffering from the hyper IgE syndrome were used to generate phytohaemagglutinin (PHA)-expanded T-cell clones (all CD4+, CD8-, CD23-). A selection of the clones was tested for their ability to help IgE secretion by culturing with normal B cells in the presence of solid-phase antibody to CD3. Supernatants were harvested on Day 7 and assayed by ELISA for IgE, IgG and IgM. Lymphokine secretion by the clones was assessed by culturing clones for 24 hr with solid-phase antibody to CD3 followed by assay of the supernatants for IL-2, IL-4 and interferon-gamma (IFN-gamma) production. In addition, clones were analysed by flow cytometry for CDw29 and CD45R expression. Initial experiments with seven clones indicated that those clones that could help IgE secretion also stimulated optimal IgG and IgM responses. All clones appeared to secrete IL-2, IL-4 and IFN-gamma, although the amounts of each varied. These results confirm recent findings that human T-cell clones do not fall into Tinf (Th1) and Th (Th2) type subsets as described in the mouse. There was no clear correlation between the lymphokines secreted by the clones and their capacity to help IgE production. However, the helper function of the clones for all isotypes, including IgE, appeared to be related to the level of expression of the surface antigen CDw29.