Previous studies on the frequency of intestinal mast cells and eosinophils in patients with inflammatory bowel disease yielded conflicting results. In the present morphometric study, we quantified mast cells and eosinophils in the lamina propria by histological and immunohistochemical methods in 64 patients suffering from Crohn's disease (33 cases) or ulcerative colitis (31 cases), and in 29 controls. Histological data from 206 biopsies were related to the presence of mucosal inflammation and clinical parameters. The number of eosinophils was increased in patients with inflammatory bowel conditions (mean +/- SE: 331 +/- 44/mm2) as compared to controls (258 +/- 27/mm2), and was dependent on disease activity and drug treatment. Mean mast cell numbers did not differ between patients and controls. However, a reduced mast cell number was found in toluidine blue-stained sections of actively inflamed tissue areas (143 +/- 16/mm2, versus 206 +/- 18/mm2 in non-inflamed tissue). Immunohistochemical studies using antibodies against the granule proteins tryptase and chymase suggest that this decrease in mast cell numbers is due to mast cell degranulation. The present data show that the number of intestinal mast cells and eosinophils is altered in patients with inflammatory bowel diseases, suggesting that both cell types are involved in the pathogenesis of chronic intestinal inflammation.

It is generally accepted that iron homeostasis is mainly controlled in the gastrointestinal tract by absorption of dietary iron. However, recent studies have shown that the kidneys are also involved in iron metabolism. Since the iron-regulatory and antimicrobial peptide hormone hepcidin was originally isolated from human urine we have investigated the expression as well as the zonal and cellular localization of hepcidin in the mammalian kidney and developed an ELISA assay to analyze hepcidin concentrations in serum and urine. The expression of hepcidin was shown by RT-PCR and immunoblot experiments; its cellular localization was studied by immunocytochemistry in human, mouse and rat kidney, which revealed similar patterns of immunoreactivity. Hepcidin expression was absent from the proximal tubule and descending and ascending thin limbs. There was strong expression in the thick ascending limb of the cortex and in connecting tubules. Moderate expression was noted in the thick ascending limb and collecting ducts of the medulla and in collecting ducts of the papilla. Importantly, the cells of the macula densa were unstained. At the cellular level, hepcidin was localized to the apical cell pole of the renal epithelial cells. Based on its presence in urine, hepcidin may be released apically into the urine. Enhanced levels of hepcidin were determined in patients with chronic renal insufficiency (156·8 ng/ml, controls 104·2 ng/ml) indicating that the kidneys may metabolize and/or eliminate the circulating peptide. From the expression of hepcidin in the mammalian kidney, we have concluded that the ironregulatory hormone is an intrinsic renal peptide which is not only eliminated by the kidney but is also synthesized in the kidney tubular system. Localization of hepcidin in the kidney implicates an iron-regulatory role of this peptide hormone in the renal tubular system, possibly in connection with the iron transporter divalent metal transporter-1.

Background and aims: Recently, novel somatostatin receptor (sstr) subtype specific ligand analogues have been developed for medical treatment of neuroendocrine tumours expressing different sstrs (sstr1-5). At present, individual expression patterns of sstr subtypes are based on methods such as in situ hybridisation and polymerase chain reaction at the transcriptional level. Therefore, we generated subtype specific antibodies against sstr1, 2A, 3, and 5 and analysed their presence, cellular localisation, distribution, and expression pattern in 33 gastrinomas, 36 insulinomas, and 35 tumours associated with a carcinoid syndrome by immunohistochemistry at the translational level. Methods: Western blotting experiments were performed in the normal human pancreas used as a reference organ and in tumour tissues; at the cellular level, sstrs were localised by immunohistochemistry in tissue paraffin sections. Results: In western blot analyses, the antibodies identified the respective receptors in their correct molecular range in extracts of the pancreas and neuroendocrine tumours. Using immunohistochemistry and immunofluorescence, the antibodies specifically detected the receptors in islet cells of the normal pancreas. Immunohistochemistry in the tumours revealed that all investigated sstr subtypes were highly expressed in the different tumour types. The frequency and expression pattern of the individual sstr subtypes varied considerably not only between the different tumour types but also in each patient. Conclusions: We conclude that immunohistochemistry with subtype specific antibodies can be used in clinical routine work to analyse sstr expression patterns for each patient before treatment and to facilitate well directed individual medical therapy by administering subtype specific somatostatin analogues.

Guanylin, a bioactive peptide, has recently been isolated from the intet; this peptide activates intestinal guanylate cydase (i.e., guanylate cyclase C) and thus is poten-

(Gut 1997; 40: 745-753)

Body iron is involved in various vital functions. Its uptake in the intestine is regulated by hepcidin, a bioactive peptide originally identified in plasma and urine and subsequently in the liver. In the present study, we provide evidence at the transcriptional and translational levels that hepcidin is also expressed in the pancreas of rat and man. Immunohistochemical studies localized the peptide exclusively to b-cells of the islets of Langerhans. Immunoelectron microscopical analyses revealed that hepcidin is confined to the insulin-storing b-cell secretory granules. As demonstrated in insulinoma-derived RINm5F cells, the expression of hepcidin in b-cells is regulated by iron. Based on the present findings we conclude that pancreatic islets are an additional source of the peptide hepcidin. The localization of this peptide to b-cells suggests that pancreatic b-cells may be involved in iron metabolism in addition to their genuine function in blood glucose regulation. In view of the various linked iron/glucose disorders in the pancreas, the present findings may provide an insight into the phenomenology of intriguing mutual relationships between iron and glucose metabolisms.

SummaryA novel CC chemokine, HCC-1, was isolated from the hemoflhrate of patients with chronic renal failure. HCC-1 has a relative molecular mass of 8,673 and consists of 74 amino acids including four cysteines linked to disulfide bonds. HCC-1 cDNA was cloned from human bone marrow and shown to code for the mature protein plus a putative 19-residue leader sequence. Mature HCC-1 has sequence identity of 46% with macrophage inflammatory protein (MIP)-lc~ and MIP-][3, and 29-37% with the other human CC chemokines. Unlike MIP-I~x and the other CC chemokines, HCC-1 is expressed constitutively in several normal tissues (spleen, liver, skeletal and heart muscle, gut, and bone marrow), and is present at high concentrations (1-80 nM) in plasma. HCC-1 has weak activities on human monocytes and acts via receptors that also recognize MIP-lot. It induced intracellular Ca 2+ changes and enzyme release, but no chemotaxis, at concentrations of 100-1,000 nM, and was inactive on T lymphocytes, neutrophils, and eosinophil leukocytes. In addition, HCC-1 enhanced the proliferation of CD34 + myeloid progenitor ceils. It was as effective as MIP-lci, but about 100-fold less potent.H emofittration is a routine treatment for patients with chronic renal failure to remove substances that are normally cleared by the kidney. A filter membrane with a molecular mass cut-off of 20 kD is used, which virtually excludes plasma proteins (1, 2). Being available in large quantities, the hemoflltrate is an excellent source of biologically active human peptides that circulate in the blood. During the last 5 yr, a peptide bank was established from >200,000 liters of hemofiltrate, and several peptide hormones were purified (1). During the course of a systematic search for novel bioactive factors (3), we have identified a new member of the recently recognized family of chemotactic cytokines (chemokines). HCC-1 is structurally related to macrophage inflammatory protein (MIP)-loc Unlike MIP-lo~ and the other CC chemokines, HCC-1 is highly expressed in normal tissues and is present at high concentrations in human plasma. Materials and MethodsPurification. Peptides were extracted from batches of 2,000 liters of hemofiltrate by precipitation with 660 g/liter ammonium sulfate as described previously (2). The precipitate ("250 g) was dissolved in water (125 mg/ml). The peptides were precipitated again by addition of 4.5 vol of2-propanol, redissolved in 10 mM phosphate buffer, pH 3.0, and fractionated by cation exchange chromatography (Fractogel TSK SP-650 M, 6 • 20 cm; Merck, Darmstadt, Germany) with a 0-1.0-M NaC1 gradient in the same buffer. Fractions eluting at high salt concentrations were collected and further purified by preparative reverse-phase (lkP) HPLC (Parcosil RP C4, 300 ft,, 20-45 b~m, 3 • 12.5 cm; Biotek, Oestringen, Germany) using a gradient generated from 0.01 M HCI and 50% 2-propanol/30% methanol in 0.01 M HCI. Analytical R.P HPLC (Parcosil RP C4, 300 A, 5 btm, 1 • 12.5 cm, Biotek, Oestringen, Germany) was then performed using an acetonitrile/T...

The Clara cells are nonciliated, nonmucous, secretory cells containing characteristic peptidergic granules; they constitute up to 80% of the epithelial cell population of the distal airways. Despite this exposed histotopology and abundance within the terminal airways where fluid secretion is of pivotal importance, the functional role of the Clara cells remained poorly understood. At the transcriptional, translational, and cellular levels, we provide evidence that the Clara cells are well equipped with the bioactive peptide guanylin and proteins of the cGMP-signaling system including guanylate cyclase C, cGMPdependent protein kinase II, and cystic fibrosis transmembrane conductance regulator (CFTR) together with the two CFTR scaffolding proteins EBP50͞NHERF and E3KARP͞NHERF-2 that are essential for proper function of CFTR. Guanylin was localized to secretory granules underneath the apical membrane of Clara cells and was, in addition, detected in high concentrations in bronchoalveolar lavage fluid, predicting release of the peptide luminally into the bronchiolar airways. On the other hand, the guanylin-receptor guanylate cyclase C, CFTR, and proteins linked to CFTR activation and function were all confined to the adluminal membrane of Clara cells, implicating an intriguing air-side route of action of guanylin. Whole-cell patch-clamp recordings in the Clara cell line H441 revealed that guanylin activates CFTR Cl ؊ conductance via the cGMP but not the cAMP-signaling pathway. Hence, in the critical location of distal airways in situ, the Clara cells may play the outstanding role of CFTR-dependent regulation of epithelial electrolyte͞water secretion through a sophisticated paracrine͞luminocrine mode of guanylin-induced CFTR activation.guanylin ͉ guanylate cyclase c ͉ lung ͉ cystic fibrosis ͉ electrolyte secretion