Recombinant Mal d 1 facilitates sublingual challenge tests of birch pollen-allergic patients with apple allergy.

It is still unclear whether allergen-specific immunotherapy (AIT) with birch pollen improves birch pollen-related food allergy. One reason for this may be the lack of standardized tests to assess clinical reactions to birch pollen-related foods, for example apple. We tested the applicability of recombinant (r) Mal d 1, the Bet v 1-homolog in apple, for oral challenge tests. Increasing concentrations of rMal d 1 in 0.9% NaCl were sublingually administered to 72 birch pollen-allergic patients with apple allergy. The dose of 1.6 µg induced oral allergy syndromes in 26.4%, 3.2 µg in 15.3%, 6.3 µg in 27.8%, 12.5 µg in 8.3%, 25 µg in 11.1%, and 50 µg in 4.2% of the patients. No severe reactions occurred. None of the patients reacted to 0.9% NaCl alone. Sublingual administration of 50 µg of rMal d 1 induced no reactions in three nonallergic individuals. Our approach allows straight forward, dose-defined sublingual challenge tests in a high number of birch pollen-allergic patients that inter alia can be applied to evaluate the therapeutic efficacy of birch pollen AIT on birch pollen-related food allergy.

Molecular cloning and characterization of NESP55, a novel chromogranin-like precursor of a peptide with 5-HT1B receptor antagonist activity.

The chromogranins comprise a class of acidic proteins that are secreted from large dense core vesicles and expressed in neuronal and endocrine tissues. We describe here the molecular characterization of NESP55 (neuroendocrine secretory protein of Mr 55,000), a novel member of the chromogranins. Several NESP55 cDNA clones were isolated from bovine chromaffin cell libraries. The cDNA sequence of NESP55 totals 1499 nucleotides. All of the clones that were isolated contained in their 3'-untranslated mRNA a sequence that was homologous to exon 2 of the G-protein Gsalpha. The open reading frame encodes for an acidic and hydrophilic protein of 241 amino acids with a predicted molecular mass of 27,494 Da. An antiserum directed against the C terminus of NESP55 labeled a band of Mr 55,000 with an acidic pI ranging from 4.4 to 5.2 in one- and two-dimensional immunoblots of secretory proteins from chromaffin granules. NESP55 is localized within the cell to the large dense secretory vesicles and is expressed, apart from the adrenal medulla, in the anterior and posterior pituitary and various regions of the brain. For the physiological function, one interesting factor has emerged. NESP55 is proteolytically processed within the chromaffin granule to smaller peptides that might be physiologically active. One tetrapeptide, Leu-Ser-Ala-Leu (LSAL), present in the NESP55 sequence and flanked by arginine residues suitable for cleavage by prohormone convertases, has been identified recently as an endogenous antagonist of the serotonergic 5-HT1B receptor subtype. Alterations in the serotonergic system are thought to play an important role in mental disorders, especially depression, and might be related to abnormal ethanol consumption. It is tempting to speculate that increased expression of NESP55 or its proteolytically derived peptide LSAL might contribute to the pathophysiology of the serotonergic transmission.

Limbic seizures induce neuropeptide and chromogranin mRNA expression in rat adrenal medulla.

Rats treated with kainic acid develop limbic seizures and have elevated levels of circulating catecholamines resulting from an extensive stimulation of the adrenal gland. We investigated the levels of several constituents of chromaffin granules in rat adrenal medulla after injection of kainic acid. This treatment increased mRNA steady-state levels of enkephalin, neuropeptide Y and chromogranin B 2-6-fold. Elevated levels of these constituents were found as early as 2 h after treatment and lasted up to 24 h. Chromogranin A and secretogranin II mRNA levels, on the other hand, remained unchanged. Adrenal catecholamine concentrations were reduced by 80%. Pre-treatment of rats with thiopental prior to kainic acid prevented seizures, the decline in catecholamines and the elevation of enkephalin and neuropeptide Y mRNAs but not that of chromogranin B. On the other hand, the peripherally acting ganglionic blocker chlorisondamine did not protect from the kainic acid-induced up-regulation of chromogranin B mRNA, suggesting that chromogranin B mRNA may be regulated by a direct effect of kainic acid on chromaffin cells. The pattern of changes in mRNA expression differed from that seen after insulin hypoglycemia or reserpine treatment. Thus, stimulation of the splanchnic innervation in vivo by various means leads to an individual and independent regulation of granule constituents by quite different mechanisms.

Nicotine and prostaglandin E induce secretogranin II levels in bovine chromaffin cells.

The synthesis regulation of secretogranin II was investigated in bovine chromaffin cells by treatment with various first messengers. Nicotine and prostaglandin E2 elevated secretogranin II mRNA and protein up to three-fold. Angiotensin II, atrial natriuretic peptide, apomorphine, bradykinin and clonidine on the other hand had no effect. The prostaglandin E induced elevation of secretogranin II mRNA was transduced via the calcium/calmodulin pathway but not via the protein kinase A or C pathways as shown by using specific inhibitors. Exposure of chromaffin cells to drugs specifically activating second messenger pathways both elevated and decreased secretogranin II mRNA. The calcium channel agonist Bay K, forskolin and phorbol esters increased secretogranin II mRNA whereas 8-Br-cGMP repressed the secretogranin II message. Thus, although secretogranin II expression can be altered by all major second messenger transduction systems, regulation of secretogranin II in vivo occurs mainly via the calcium/calmodulin pathway. Chromogranin A and B mRNA were not changed by any of the first messengers investigated indicating a differential synthesis regulation of components co-stored in bovine chromaffin granules.

Processing of chromogranins in chromaffin cell culture: effects of reserpine and alpha-methyl-p-tyrosine.

Bovine chromaffin cell cultures were treated with either reserpine or alpha-methyl-p-tyrosine for up to 10 days. Afterwards the cells were harvested and the degree of proteolytic processing of secretogranin II, chromogranin A and chromogranin B was determined by immunoblotting and HPLC followed by RIA. There was a significant increase in the proteolysis of all three chromogranins after 4-6 days in the presence of reserpine. The small peptides formed in the presence of reserpine in vitro are also produced in vivo. A similar effect was observed with alpha-methyl-p-tyrosine, an inhibitor of tyrosine hydroxylase, but the response took up to 10 days to develop. Both drugs decreased catecholamine levels but reserpine was more effective, reaching a high degree of depletion after 4 days. In addition, experiments in vitro indicate that low millimolar amounts of either adrenaline (IC50 5.2 mM) or noradrenaline (IC50 2.4 mM) can significantly impair the proteolytic activity of recombinant murine prohormone convertase 1 when assayed with synthetic fluorogenic and/or peptidyl substrates. We conclude that a lowering of catecholamine levels in chromaffin granules leads to a concomitant increase in proteolytic processing of all secretory peptides. Apparently within chromaffin granules the endoproteases are inhibited by catecholamines and thus their removal leads to increased proteolysis.

Large dense-core vesicles in rat adrenal after reserpine: levels of mRNAs of soluble and membrane-bound constituents in chromaffin and ganglion cells indicate a biosynthesis of vesicles with higher secretory quanta.

Rats were injected with a large dose of reserpine known to stimulate the adrenal medulla. Various times after drug treatment the mRNA levels of several constituents of large dense-core vesicles were determined by northern blot analysis and in situ hybridization. The latter method allowed detection of changes in mRNA levels not only in chromaffin cells, but also in the ganglion cells found in adrenal medulla. Levels of the mRNAs of secretory components of large dense-core vesicles (chromogranins A and B, secretogranin II, VGF, and neuropeptide Y) increased in chromaffin cells by 215-857% after 1-3 days of drug treatment. For partly membrane-bound components (dopamine beta-hydroxylase, prohormone convertase 2, carboxypeptidase H, and peptidylglycine alpha-amidating monooxygenase) the changes ranged from 182 to 315%, whereas for glycoprotein III and for intrinsic membrane proteins (cytochrome b561 and vesicle monoamine transporter 2) no change occurred. In ganglion cells the mRNAs that could be detected for VGF, neuropeptide Y, secretogranin II, carboxypeptidase H, and vesicle monoamine transporter 1 showed an analogous pattern of change, with significant increases for the secretory proteins and no change for the membrane components. From these and previous results we suggest the following concept: Long-lasting stimulation of chromaffin cells or neurons does not induce the biosynthesis of a larger number of vesicles but rather leads to the formation of vesicles containing higher secretory quanta of chromogranins and neuropeptides.

Interleukin-1 receptor type I mRNA in mouse brain as affected by peripheral administration of bacterial lipopolysaccharide.

Following administration of endotoxin in vivo, alterations of interleukin-1 (IL-1) receptor binding have been reported. In order to assess in vivo regulation of IL-1 receptor gene expression in brain, mRNA levels for IL-1 receptor type I were measured after peripheral administration of bacterial lipopolysaccharide in mice. When 25 micrograms of lipopolysaccharide were administered intraperitoneally, IL-1 receptor type I mRNA in the brain was increased after 3 h. After 20 h, the level was diminished. This result suggests that bacterial lipopolysaccharide affects regulation of IL-1 receptor type I mRNA expression in mouse brain.

Glycoprotein III (clusterin, sulfated glycoprotein 2) in endocrine, nervous, and other tissues: immunochemical characterization, subcellular localization, and regulation of biosynthesis.

Specific antisera were raised against the A and B chains of glycoprotein III. Immunoblotting revealed that in adrenal medulla both chains migrate very closely together in two-dimensional electrophoresis. Both chains with slightly differing molecular sizes are found in several endocrine tissues and in brain, kidney, liver, and serum. The mRNA has an analogous widespread distribution. In primary cultures of chromaffin cells the level of message becomes significantly increased by treatment with histamine or 12-O-tetradecanoylphorbol 13-acetate/forskolin. However, the increase is small when compared with that of secretogranin II. The subcellular localization of glycoprotein III in endocrine organs and in the posterior pituitary was investigated by subcellular fractionation and immunoelectron microscopy. Glycoprotein III was found to be confined to the large dense-core vesicles of these organs. For a discussion of the function of glycoprotein III, its localization in these organelles has to be taken into account.