Search

A large and steadily growing subfamily of antimicrobially active peptides of animals and plants is formed by the defensins, which are highly disulfide-bonded, cationic peptides with a molecular mass of about 4 kDa. The synthesis of the human β-defensins 1 and 2 (hBD-1, hBD-2) as well as of the novel murine β-defensins 7 and 8 (mBD-7 and mBD-8) is reported. The peptides were synthesized by solid-phase peptide synthesis using fluorenylmethoxycarbonyl chemistry. The linear products were oxidized in the presence of the cysteine/cystine redox system to the biologically active molecules. The correct disulfide connectivity of the resulting cyclic products was partly verified by mass spectrometry and sequence analysis of the fragments obtained after tryptic cleavage. In addition, the recently discovered antimicrobially active human peptide LEAP-1/ hepcidin, which contains four disulfide bonds, was successfully synthesized and subsequently oxidized. For Liver-expressed anti microbial peptide (LEAP)-1/hepcidin and hBD-1, the identity of native and synthetic peptides was demonstrated by highpressure liquid chromatography and capillary electrophoretic analysis. The general synthetic procedure is suitable to rapidly perform the total chemical synthesis of novel fully bioactive defensins, which are expected to be identified soon, as well as of structurally modified analogs. [ABSTRACT FROM AUTHOR]

Background: Modulation of leukocyte recruitment through blocking of chemokine receptors has been proposed as an attractive therapeutic strategy. We have previously demonstrated that n-Nonanoyl-CC chemokine ligand 14 (NNY-CCL14), a modified analog of the naturally occurring chemokine CCL14(9-74) internalizes and desensitizes human CCR3 resulting in the inactivation of eosinophils. However, inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation are assigned to its interaction with CCR1 and CCR5. Aim of the study: As CCL2 and its receptor CCR2 have been shown to play important roles in the development of Th2 inflammation, we further evaluated the effects of NNY-CCL14 treatment on CCL2-mediated activation of CCR2. Methods: Effects of NNY-CCL14 treatment were studied on cell lines transfected with human CCR2 and primary leukocytes. Functional effects were assessed by calcium efflux assays, flow cytometry and chemotaxis. Results: Prestimulation with NNY-CCL14 desensitized CCR2-mediated responses to further stimulation with its selective ligand CCL2. No significant internalization of CCR2 was observed when the cells were stimulated with NNY-CCL14, even at concentrations eliciting maximal [Ca2+]i mobilization. Above all, NNY-CCL14 pretreatment blocked CCL2-induced chemotaxis of monocytes. Conclusions: This study demonstrates that NNY-CCL14 is a partial agonist of CCR2, inhibiting responses of monocytes to the CCR2-selective ligand CCL2. NNY-CCL14 attenuates CCR2-mediated responses by rapidly desensitizing the receptor and preventing chemotaxis, although it is able to induce calcium mobilization but does not lead to CCR2 internalization. Hence this study provides further insights into the possible mechanisms of action of NNY-CCL14, which interacts with multiple chemokine receptors inhibiting the migration and activation of different cell populations involved, thus acting as a potential therapeutic compound to alleviate allergic inflammation. [ABSTRACT FROM AUTHOR]

Background: CC chemokine ligand 11 (CCL11) is the outstanding member of all described CC chemokine receptor 3 (CCR3) ligands and is shown to be selective for this receptor. However, it also activates CCR5 but only in the micromolar range. The in vivo activity of CCL11 is expected to be temporally restricted, as it is degraded by specific proteases such as the dipeptidyl-peptidase IV (DP4), also termed CD26. Based on the approach to inactivate chemokine receptors in allergic disease models as has been demonstrated for DP4-resistant n-nonanoyl (NNY)-CCL14 and for amino-oxypentane (AOP)-CCL5, it is tempting to study similar compounds derived from CCL11. Methods: Synthesis of NNY-CCL11 was performed and it was characterized for biological functions in human and mouse eosinophils as well as in cell lines stably transfected either with human CCR3 or CCR5. Resistance to DP4 treatment was also investigated. Results: The functional activities of NNY-CCL11 mediated via CCR3 show an almost identical pattern to CCL11 with respect to intracellular calcium mobilization and CCR3 internalization. N-terminal cleavage of CCL11 by preincubation with DP4 results in a reduced capacity to internalize CCR3, while preincubation of NNY-CCL11 shows no influence. In contrast to CCL11, NNY-CCL11 also activates CCR5+ cell lines and human monocytes in the nanomolar range, being about 100 times more potent than CCL11. Conclusions: n-Nonanoyl-CCL11 represents a compound with dual activity restricted to CCR3 and CCR5. Because of its receptor-inactivating capacity and stability against DP4 degradation, NNY-CCL11 is a suitable tool for the decoding of the pathophysiological mechanisms of allergic diseases. [ABSTRACT FROM AUTHOR]

Background Several skin diseases and atopic disorders including Netherton syndrome and atopic dermatitis have been associated with mutations and deviations of expression of SPINK5, the gene encoding the human 15-domain serine proteinase inhibitor LEKTI. The biochemical mechanisms underlying this phenomenon have not yet been fully clarified. Objectives To identify target proteinases of LEKTI important for processes of desquamation and inflammation of the skin which will enable the development of specific drugs. Methods The inhibitory activities of LEKTI domains 6 and 15 were tested on a number of commercially available serine proteinases and also on the purified kallikreins hK5 and hK7. In addition, recombinant hK5 was used. Results LEKTI domain 6 is a potent inhibitor of hK5 and hK7, whereas LEKTI domain 15 exhibits inhibitory activity on plasmin. hK5 and hK7 in particular are relevant to skin disorders. Conclusions The inhibition of hK5 and hK7 by LEKTI domain 6 indicates an important regulatory role of LEKTI in processes of skin desquamation and inflammation, which may explain the severe pathological symptoms associated with abnormalities of SPINK5 and/or its expression. Thus, LEKTI represents a potential drug for the treatment of these disorders. [ABSTRACT FROM AUTHOR]

Background: Whereas recent studies underlie the fundamental importance of the CC chemokine receptor 3 (CCR3) for the recruitment of eosinophils in allergic diseases, controversial data exist about the relevance of CCR1 on eosinophils. Therefore, the purpose of this study was to investigate the expression and regulation of CCR1 on eosinophils. Methods: Flow cytometric analysis of whole blood eosinophils and CD16-negative selected eosinophils from healthy nonatopic donors and from patients with atopic disorders was performed and CCR1 receptor internalization and re-expression were studied. Results: Flow cytometric analysis of whole blood eosinophils revealed that 17.8% of the donors expressed high levels of CCR1 (CCR1high) and 82.2% low levels of CCR1 (CCR1low). A significant down-regulation of CCR1 was induced by 24 h preincubation of isolated eosinophils from CCR1high donors either with IL-3, CC chemokine ligand 3 (CCL3), CCL5, CCL7, or CCL13. Internalization experiments using eosinophils from CCR1high donors revealed that CCL5 is more effective to induce CCR1 internalization than CCL3. Whereas CCR1 re-expression after stimulation with CCL3 reached prestimulation levels (120 min: 81.3% relative CCR1 surface expression) CCL5 induced a prolonged CCR1 internalization (120 min: 15.7%). Conclusions: This study demonstrates a distinct pattern of CCR1 internalization and re-expression in human eosinophils between CCL3 and CCL5, as CCL5 induces a prolonged CCR1 internalization and the basic value is not reached after 24 h. Since prolonged receptor internalization plays a central role in chemokine-mediated inhibition of receptor function, CCR1 seems to be an attractive target on human eosinophils for chemokine receptor blockade besides CCR3. [ABSTRACT FROM AUTHOR]

Haller C, Meyer M, Scheele T, Koch A, Forssmann W-G and Kübler W (University of Heidelberg; and Lower Saxony Institute for Peptide Research, Hanover; Germany). Radiocontrast-induced natriuresis associated with increased urinary urodilatin excretion (original article). J Intern Med 1998; 243: 155–62. ObjectiveIntravascular radiocontrast agents induce a pronounced diuresis. The aim of the present study was to investigate the (patho-)physiological mechanisms of the radiocontrast-induced diuresis. DesignThe fractional excretion of sodium, the urinary excretion of the renal natriuretic peptide urodilatin and the plasma concentration of atrial natriuretic peptide were measured in 42 unselected patients immediately before and after intravascular radiocontrast administration during coronary angiography. SettingCardiac catheterization laboratory of a university hospital. ResultsAfter angiography both the plasma concentration of atrial natriuretic peptide (median post-pre difference: 3.9 pmol L-1, quartiles -1.2; 7.0) and the urinary excretion of urodilatin (median post-pre difference: 67.0 nmol urodilatin/mol creatinine, quartiles 39.7; 152.1) were increased. The urinary urodilatin excretion was correlated with an increase in the fractional excretion of sodium (median post-pre difference: 1.7%, quartiles 0.6; 3.1). There was no correlation between the serum concentration of atrial natriuretic peptide and urinary sodium excretion. For the radiocontrast-induced increase in both urodilatin and sodium excretion there was no indication for differences between patients without (31) and with (11) intravenous saline infusion. ConclusionThe radiocontrast-induced diuresis is a natriuresis which is associated with an increased urinary excretion of urodilatin. The association between natriuresis and urinary urodilatin excretion irrespective of baseline volume status corroborates the hypothesis that urodilatin contributes to the sodium excretion after radiocontrast administration in a paracrine manner. This finding has pathophysiological and potentially therapeutic implications in radiocontrast-induced nephropathy. [ABSTRACT FROM AUTHOR]

Urodilatin (URO) is a natriuretic peptide isolated from human urine which is thought to be produced by distal tubular cells. We measured urinary URO excretion in 50 healthy children and 23 children with acute (ARF), chronic renal failure (CRF), or hereditary tubular disorders, using a specific radioimmunoassay. The mean URO excreted in these four groups was 56, 45, 94, and 121 fmol/min per 1.73 m², respectively (differences between first three groups not significant). The variation in URO excretion was larger in patients with kidney disease than in controls. There were significant correlations between urinary URO and sodium excretion in controls and CRF, bur not in ARF. URO excretion also correlated with urine flow rate in CRF. Although no correlation was found between URO excretion and creatinine clearance, urinary URO was increased in some patients with advanced CRF, which suggests stimulated tubular production to compensate for reduced sodium excretion. In view of the therapeutic potential of URO in renal insufficiency, further study of the renal handling of URO is warranted. [ABSTRACT FROM AUTHOR]

An abstract of the study "Haemodynamic and neurohumoral effects of the renal natriuretic peptide urodilatin in patients with decompensated congestive heart failure," by V. Mitrovic and colleagues is presented.