Degradation of human kininogens with the release of kinin peptides by extracellular proteinases of Candida spp.

The secretion of proteolytic enzymes by pathogenic microorganisms is one of the most successful strategies used by pathogens to colonize and infect the host organism. The extracellular microbial proteinases can seriously deregulate the homeostatic proteolytic cascades of the host, including the kinin-forming system, repeatedly reported to he activated during bacterial infection. The current study assigns a kinin-releasing activity to secreted proteinases of Candida spp. yeasts, the major fungal pathogens of humans. Of several Candida species studied, C. parapsilosis and C. albicans in their invasive filamentous forms are shown to produce proteinases which most effectively degrade proteinaceous kinin precursors, the kininogens. These enzymes, classified as aspartyl proteinases, have the highest kininogen-degrading activity at low pH (approx. 3.5), but the associated production of bradykinin-related peptides from a small fraction of kininogen molecules is optimal at neutral pH (6.5). The peptides effectively interact with cellular B2-type kinin receptors. Moreover, kinin-related peptides capable of interacting with inflammation-induced B1-type receptors are also formed, but with a reversed pH dependence. The presented variability of the potential extracellular kinin production by secreted aspartyl proteinases of Candida spp. is consistent with the known adaptability of these opportunistic pathogens to different niches in the host organism

Visualisation of tissue kallikrein, kininogen and kinin receptors in human skin following trauma and in dermal diseases

During dermal injury and inflammation the serine proteases kallikreins cleave endogenous, multifunctional substrates (kininogens) to form bradykinin and kallidin. The actions of kinins are mediated by preferential binding to constitutively expressed kinin-B2 receptors or inducible kinin-B1 receptors. A feature of the kinin-B1 receptors is that they show low levels of expression, but are distinctly upregulated following tissue injury and inflammation. Because recent evidence suggested that kinin-B1 receptors may perform a protective role during inflammation, we investigated the specific occurrence of the kallikrein-kinin components in skin biopsies obtained from normal skin, patients undergoing surgery, basalioma, lichenificated atopic eczema, and psoriasis. The tissue was immunolabelled in order to determine the localisation of tissue pro-kallikrein, kallikrein, kininogen and kinin receptors. The kinin components were visualised in normal, diseased and traumatised skin, except that no labelling was observed for kininogen in normal skin. Of the five types of tissue examined, upregulation of kinin-B1 receptors was observed only in skin biopsies obtained following surgery. In essence, the expression of kinin-B1 receptors did not appear to be enhanced in the other biopsies. Within the multiple steps of the inflammatory cascade in wound healing, our results suggest an important regulatory role for kinin-B1 receptors during the first phase of inflammation following injury

SP-Sephadex equilibrium chromatography of bradykinin and related peptides: Application to trypsin-treated human plasma

An analytical method is deseribed for the separation of bradykinin, Lys-bradykinin, and Met-Lys-bradykinin by equilibrium chromatography on SP-Sephadex C-25 eluted in 0.02 Tris-HCl buffer, pH 8.10, 0.12 NaCl. A second elution buffer, 0.02 Tris-HCl buffer, pH 7.70, 0.06 NaCl, serves as a second parameter for the identification of bradykinin and also separates the hormone from plasma bradykinin-potentiating peptides. Ten to one-hundred nanomoles of each peptide can be recovered in high yields, identified by elution position, and measured by bioassay with the isolated guinea pig ileum. The identification of bradykinin as the peptide released by trypsin acting on acid-denatured plasma is documented as an illustration of the method

Furosemide Enhances the Release of Endothelial Kinsis, Nitric Oxide and Prostacyclin

Despite a wealth of data, the mechanism of the direct dilator effect of furosemide on the systemic arterial and venous systems is far from being satisfactorily understood. Therefore, we investigated whether furosemide is capable of stimulating the production of the endogenous vasodilators nitric oxide and prostacyclin in primary cultured bovine aortic endothelial cells by an enhanced synthesis and release of endothelium-derived kinins. Nitric oxide production was assessed in terms of intracellular guanosine cyclic-3',5' monophosphate accumulation; kinin and prostacyclin release were determined by specific radioimmunoassays. Furosemide concentration- and time- dependently increased the formation of nitric oxide and prostacyclin. Maximal increases of both autacoids were already obtained after a 5-min incubation with 3 x 10(-7) to 10(-6) mol/l of furosemide. In the same concentration range, furosemide led to an enhanced release of kinins into the supernatant of the cells. This observation was supported by the inhibitory effect of the specific B2 kinin receptor antagonist icatibant (Hoe 140) on the furosemide-induced increase of nitric oxide and prostacyclin. Thus the hemodynamic effects, and in particular the direct early dilator effect, of furosemide may be explained in part by an enhanced endothelial synthesis and release of bradykinin and related kinins, which in turn stimulates endothelial autacoid formation via B2 kinin receptor activation

Perspectives on the Trypanosoma cruzi-host cell receptor interaction

Chagas disease is caused by the parasite Trypanosoma cruzi. The critical initial event is the interaction of the trypomastigote form of the parasite with host receptors. This review highlights recent observations concerning these interactions. Some of the key receptors considered are those for thromboxane, bradykinin, and for the nerve growth factor TrKA. Other important receptors such as galectin-3, thrombospondin, and laminin are also discussed. Investigation into the molecular biology and cell biology of host receptors for T. cruzi may provide novel therapeutic targets

Extracellular aspartic protease SAP2 of Candida albicans yeast cleaves human kininogens and releases proinflammatory peptides, Met-Lys-bradykinin and des-Arg(9)-Met-Lys-bradykinin

Bradykinin-related peptides, universal mediators of inflammation collectively referred to as the kinins, are often produced in excessive amounts during microbial infections. We have recently shown that the yeast Candida albicans, the major fungal pathogen to humans, can exploit two mechanisms to enhance kinin levels at the sites of candidial infection, one depending on adsorption and activation of the endogenous kinin-generating system of the host on the fungal cell wall and the other relying on cleavage of kinin precursors, the kininogens, by pathogen-secreted proteases. This work aimed at assigning this kininogenase activity to the major secreted aspartic protease of C. albicans (SAP2). The purified SAP2 was shown to cleave human kininogens, preferably the low molecular mass form (LK) and optimally in an acidic environment (pH 3.5-4.0), and to produce two kinins, Met-Lys-bradykinin and its derivative, {[}Hydroxyproline(3)]-Met-Lys-bradykinin, both of which are capable of interacting with cellular bradykinin receptors of the B2 subtype. Additionally, albeit with a lower yield, des-Arg(9)-Met-Lys-bradykinin, an effective agonist of B1-subtype receptors, was released. The pathophysiological potential of these kinins and des-Arg-kinin was also proven by presenting their ability to stimulate human promonocytic cells U937 to release proinflammatory interleukin 1 beta (IL-1 beta) and IL-6

Eluate derived by extracorporal antibody-based immunoadsorption elevates the cytosolic Ca2+ concentration in podocytes via B-2 kinin receptors

Background/Aim: Patients with idiopathic focal segmental glomerulosclerosis (FSGS) often develop a recurrence of the disease after kidney transplantation. In a number of FSGS patients, plasmapheresis and immunoadsorption procedures have been shown to transiently reduce proteinuria and are thought to do this by eliminating a circulating factor. Direct cellular effects of eluates from immunoadsorption procedures on podocytes, the primary target of injury in FSGS, have not yet been reported. Methods: Eluates were derived from antibody-based immunoadsorption of a patient suffering from primary FSGS, a patient with systemic lupus erythematosus, and a healthy volunteer. The cytosolic free Ca2+ concentration ({[}Ca2+](i)) of differentiated podocytes was measured by single-cell fura-2 microfluorescence measurements. Free and total immunoreactive kinin levels were measured by radioimmunoassay. Results: FSGS eluates increased the {[}Ca2+](i) levels concentration dependently (EC50 0.14 mg/ml; n = 3-19). 1 mg/ml eluate increased the {[}Ca2+](i) values reversibly from 82 +/- 12 to 1,462 +/- 370 nmol/l, and then they returned back to 100 16 nmol/l (n = 19). The eluate-induced increase of {[}Ca2+](i) consisted of an initial Ca2+ peak followed by a Ca2+ plateau which depended on the extracellular Ca2+ concentration. The eluate-induced increase of {[}Ca2+](i) was inhibited by the specific B-2 kinin receptor antagonist Hoe 140 in a concentration-dependent manner (IC50 2.47 nmol/l). In addition, prior repetitive application of bradykinin desensitized the effect of eluate on {[}Ca2+](i). A colonic epithelial cell line not reacting to bradykinin did not respond to eluate either (n = 6). Similar to FSGS eluates, the eluate preparations of both the systemic lupus patient and the healthy volunteer led to a biphasic, concentration-dependent {[}Ca2+](i) increase in poclocytes which again was inhibited by Hoe 140. Free kinins were detected in all eluate preparations. Conclusion: The procedure of antibody-based immunoadsorption leads to kinin in the eluate which elevates the {[}Ca2+](i) level of podocytes via B-2 kinin receptors. Copyright (C) 2002 S. Karger AG, Basel