The relationship between clinical phenotype and kallikrein-kinin bioregulation in different forms of arthritis.

OBJECTIVE: Patients with rheumatoid arthritis (RA) have shown increased levels of neutrophils generating kallikrein-kinin peptides in blood which are potent mediators of inflammation. This study investigated the association between the bioregulation of kinin-mediated inflammation with the clinical, quality of life, and imaging characteristics (e.g. ultrasonography) of different arthritides. METHODS: Patients with osteoarthritis (OA, n = 29), gout (n = 10) and RA (n = 8) were recruited and screened for clinical symptoms, quality of life, and ultrasonographical assessment of arthritis. Blood neutrophils were assessed for the expression of bradykinin receptors (B1R and B2R), kininogens and kallikreins by immunocytochemistry with visualization by bright field microscopy. Levels of plasma biomarkers were measured by ELISA and cytometric bead array. RESULTS: Quality of life (SF-36 domains and summary scores; including pain; and, HAQ) was similar across OA, gout and RA patients; with the exception of worse physical functioning scores between OA and gout patients. Synovial hypertrophy (on ultrasound) differed between groups (p = 0.001), and the dichotomised Power Doppler (PD) score of greater than or equal to 2 (PD-GE2) was marginally significant (p = 0.09). Plasma IL-8 were highest in patients with gout followed by RA and OA (both, P < 0.05). Patients with RA had higher plasma levels of sTNFR1, IL-1ß, IL-12p70, TNF and IL-6, compared to OA and gout patients (all, P < 0.05). Patients with OA had higher expression of K1B and KLK1 on blood neutrophils followed by RA and gout patients (both, P < 0.05). Bodily pain correlated with B1R expression on blood neutrophils (r = 0.334, p = 0.05), and inversely with plasma levels of CRP (r = -0.55), sTNFR1 (r = -0.352) and IL-6 (r = -0.422), all P < 0.05. Expression of B1R on blood neutrophils also correlated with Knee PD (r = 0.403) and PD-GE2 (r = 0.480), both P < 0.05. CONCLUSIONS: Pain levels and quality of life were similar between patients with OA, RA and gout with knee arthritis. Plasma inflammatory biomarkers and B1R expression on blood neutrophils correlated with pain. Targeting B1R to modulate the kinin-kallikrein system may pose as a new therapeutic target in the treatment of arthritis.

Kinin B1 Receptor Signaling in Skin Homeostasis and Wound Healing.

Kinins are proinflammatory peptides that are formed in the skin by the enzymatic action of tissue kallikrein (KLK1) on kininogens. Tissue kallikrein is produced by eccrine sweat glands and also by cells of the stratum granulosum and other skin appendages. Kinin formation may be favored during inflammatory skin disorders when plasma constituents, including kininogens, extravasate from venules and capillaries, which have increased permeability in response to the plethora of inflammatory mediators generated in the course of acute inflammation. By activating either kinin B1 or B2 receptors, kinins modulate keratinocyte differentiation, which relays on activation of several signaling systems that follows receptor stimulation. Participation of the kinin B1 receptor in wound healing is still a matter of controversy though some studies indicate that B1 receptor stimulation regulates keratinocyte migration by controlling metalloproteases 2 and 9 production and by improving wound closure in a mouse model. Development of more stable kinin B1 receptor agonists may be beneficial to modulate wound healing, especially if we take into account that the B1 receptor is up-regulated by inflammation and by cytokines generated in the inflamed microenvironment.

Possible role of phytoestrogens in breast cancer via GPER-1/GPR30 signaling.

Estrogens generated within endocrine organs and the reproductive system act as ligands for at least three types of estrogen receptors. Estrogen receptors α (ERα) and ß (ERß) belong to the so-called classical family of estrogen receptors, whereas the G protein-coupled receptor GPR30, also known as GPER-1, has been described as a novel estrogen receptor sited in the cell membrane of target cells. Furthermore, these receptors are under stimulation of a family of exogenous estrogens, known as phytoestrogens, which are a diverse group of non-steroidal plant compounds derived from plant food consumed by humans and animals. Because phytoestrogens are omnipresent in our daily diet, they are becoming increasingly important in both human health and disease. Recent evidence indicates that in addition to classical estrogen receptors, phytoestrogens also activate GPER-1 a relevant observation since GPER-1 is involved in several physiopathological disorders and especially in estrogen-dependent diseases such as breast cancer.The first estrogen receptors discovered were the classical ERα and ERß, but from an evolutionary point of view G protein-coupled receptors trace their origins in history to over a billion years ago suggesting that estrogen receptors like GPER-1 may have been the targets of choice for ancient phytoestrogens and/or estrogens.This review provides a comprehensive and systematic literature search on phytoestrogens and its relationship with classical estrogen receptors and GPER-1 including its role in breast cancer, an issue still under discussion.

Functional interrelationships between the kallikrein-related peptidases family and the classical kinin system in the human neutrophil.

In the human neutrophil, kallikrein-related peptidases (KLKs) have a significant functional relationship with the classical kinin system as a kinin B1 receptor agonist induces secretion of KLK1, KLK6, KLK10, KLK13 and KLK14 into the medium. Secretion of KLK1, the kinin-forming enzyme, may perpetuate formation of kinin in the inflammatory milieu by hydrolyzing extravasated kininogens present in tissue edema. Secretion of KLKs into the inflammatory milieu, induced by kinins or other proinflammatory mediators, provides the human neutrophil with a wide range of molecular interactions to hydrolyze different cellular and extracellular matrix components, which may be of critical relevance in different mechanisms involving inflammation.

Overview of tissue kallikrein and kallikrein-related peptidases in breast cancer.

The kallikrein family comprises tissue kallikrein and 14 kallikrein-related peptidases (KLKs) recognized as a subgroup of secreted trypsin- or chymotrypsin-like serine proteases. KLKs are expressed in many cellular types where they regulate important physiological activities such as semen liquefaction, immune response, neural development, blood pressure, skin desquamation and tooth enamel formation. Tissue kallikrein, the oldest member and kinin-releasing enzyme, and KLK3/PSA, a tumor biomarker for prostate cancer are the most prominent components of the family. Additionally, other KLKs have shown an abnormal expression in neoplasia, particularly in breast cancer. Thus, increased levels of some KLKs may increase extracellular matrix degradation, invasion and metastasis; other KLKs modulate cell growth, survival and angiogenesis. On the contrary, KLKs can also inhibit angiogenesis and produce tumor suppression. However, there is a lack of knowledge on how KLKs are regulated in tumor microenvironment by molecules present at the site, namely cytokines, inflammatory mediators and growth factors. Little is known about the signaling pathways that control expression/secretion of KLKs in breast cancer, and further how activation of PAR receptors may contribute to functional activity in neoplasia. A better understanding of these molecular events will allow us to consider KLKs as relevant therapeutic targets for breast cancer.

GPER-1/GPR30 a novel estrogen receptor sited in the cell membrane: therapeutic coupling to breast cancer.

INTRODUCTION: Breast cancer is clinically classified as 'estrogen-positive' when at least 1% of cancer cells stain for the estrogen receptor alpha (ERα). However, recent research on both basic and clinical aspects of breast cancer suggests that GPER-1 (G protein-coupled estrogen receptor-1) may have an important role in breast cancer. Areas covered: This review provides a comprehensive and systematic literature search on GPER-1. We have focused on the role of GPER-1 in breast cancer and on resistance to endocrine therapy, an unsolved clinical issue still under discussion. Expert opinion: The discovery of GPER-1 as a novel estrogen receptor is unique and the signaling pathways activated by its stimulation, when compared to the classical nuclear ERα, indicate a potential role of GPER-1 in the genesis and mechanisms of drug resistance in breast cancer. Tumors expressing ERα represent the largest group of breast cancer patients indicating that more women eventually die from ERα-positive breast tumors than from other more malignant breast cancer subtypes such as HER2-positive and the triple negative groups. It is important to develop new strategies on endocrine therapy with regard to ERα and GPER-1 receptors to achieve innovative successful therapeutic tools.

Activation of the human keratinocyte B1 bradykinin receptor induces expression and secretion of metalloproteases 2 and 9 by transactivation of epidermal growth factor receptor.

The B1 bradykinin receptor (BDKRB1) is a component of the kinin cascade localized in the human skin. Some of the effects produced by stimulation of BDKRB1 depend on transactivation of epidermal growth factor receptor (EGFR), but the mechanisms involved in this process have not been clarified yet. The primary purpose of this study was to determine the effect of a BDKRB1 agonist on wound healing in a mouse model and the migration and secretion of metalloproteases 2 and 9 from human HaCaT keratinocytes and delineate the signalling pathways that triggered their secretion. Although stimulation of BDKRB1 induces weak chemotactic migration of keratinocytes and wound closure in an in vitro scratch-wound assay, the BDKRB1 agonist improved wound closure in a mouse model. BDKRB1 stimulation triggers synthesis and secretion of both metalloproteases, effects that depend on the activity of EGFR and subsequent phosphorylation of ERK1/2 and p38 mitogen-activated protein kinases and PI3K/Akt. In the mouse model, immunoreactivity for both gelatinases was concentrated around wound borders. EGFR transactivation by BDKRB1 agonist involves Src kinases family and ADAM17. In addition to extracellular matrix degradation, metalloproteases 2 and 9 regulate cell migration and differentiation, cell functions that are associated with the role of BDKRB1 in keratinocyte differentiation. Considering that BDKRB1 is up-regulated by inflammation and/or by cytokines that are abundant in the inflammatory milieu, more stable BDKRB1 agonists may be of therapeutic value to modulate wound healing.

Expression and bioregulation of the kallikrein-related peptidases family in the human neutrophil.

The family of kallikrein-related peptidases (KLKs) has been identified in a variety of immunolabeled human tissue sections, but no previous study has experimentally confirmed their presence in the human neutrophil. We have investigated the expression and bioregulation of particular KLKs in the human neutrophil and, in addition, examined whether stimulation by a kinin B(1) receptor (B1R) agonist or fMet-Leu-Phe (fMLP) induces their secretion. Western blot analysis of neutrophil homogenates indicated that the MM of the KLKs ranged from 27 to 50 kDa. RT-PCR showed that blood neutrophils expressed only KLK1, KLK4, KLK10, KLK13, KLK14 and KLK15 mRNAs, whereas the non-differentiated HL-60 cells expressed most of them, with exception of KLK3 and KLK7. Nevertheless, mRNAs for KLK2, KLK5, KLK6 and KLK9 that were previously undetectable appeared after challenging with a mixture of cytokines. Both kinin B(1)R agonist and fMLP induced secretion of KLK1, KLK6, KLK10, KLK13 and KLK14 into the culture medium in similar amounts, whereas the B(1)R agonist caused the release of lower amounts of KLK2, KLK4 and KLK5. When secreted, the differing proteolytic activity of KLKs provides the human neutrophil with a multifunctional enzymatic capacity supporting a new dimension for its role in human disorders of diverse etiology.

Kinin B1 receptor regulates interactions between neutrophils and endothelial cells by modulating the levels of Mac-1, LFA-1 and intercellular adhesion molecule-1.

Kinins are pro-inflammatory peptides that mimic the cardinal features of inflammation. We examined the concept that expression levels of endothelial intercellular adhesion molecule-1 (ICAM-1) and neutrophil integrins Mac-1 and LFA-1 are modulated by the kinin B1 receptor (B1R) agonist, Lys-des[Arg(9)]bradykinin (LDBK). Stimulation of endothelial cells with LDBK increased the levels of ICAM-1 mRNA transcripts/protein, and also of E-selectin and platelet endothelial adhesion molecule-1. ICAM-1 levels increased in a magnitude comparable with that produced by TNF-α. This stimulatory effect was reduced when endothelial cells, which had been previously transfected with a B1R small interfering RNA, were stimulated with LDBK, under comparable conditions. Similarly, LDBK produced a significant increase in protein levels of LFA-1 and Mac-1 integrins in human neutrophils, an effect that was reversed by pretreatment of cells with 10 µg/ml cycloheximide or a B1R antagonist. Functional experiments performed with post-confluent monolayers of endothelial cells stimulated with LDBK and neutrophils primed with TNF-α, and vice versa, resulted in enhanced adhesiveness between both cells. Neutralizing Abs to ICAM-1 and Mac-1 reduced the adhesion between them. Our results indicate that kinin B1R is a novel modulator that promotes adhesion of leukocytes to endothelial cells, critically enhancing the movement of neutrophils from the circulation to sites of inflammation.

Bioregulation of kallikrein-related peptidases 6, 10 and 11 by the kinin B1 receptor in breast cancer cells.

The sera of patients with breast cancer have higher levels of des[Arg(9)]bradykinin, a kinin B1 receptor (B1R) agonist, than that from healthy individuals. Stimulation of breast cancer cells with the analog Lys-des[Arg(9)]bradykinin causes release of metalloproteinases-2 and -9 and increases cell proliferation. We examined the possibility that breast cancer cells, in addition to B1R, express the kinin-forming protease true tissue kallikrein (KLK1) and the endogenous proteins termed kininogens from which kinins are enzymatically released. Furthermore, we investigated whether stimulation of breast cancer cells with a B1R agonist would modify the cellular levels of KLK6, KLK10 and KLK11, three kallikrein-related peptidases with a still poorly-understood biological role in breast cancer. We found that breast cancer cells expressed KLK1 and kininogens, and that stimulation of estrogen-sensitive breast cancer cells with the B1R agonist produced down-regulation of KLK10 (a protease associated with growth suppression) but up-regulation of KLK11 and KLK6 (peptidases related to increased cell proliferation and invasiveness, respectively). Furthermore, we showed that the B1R agonist acts as a functional stimulus for the secretion of KLK1 and KLK6, an event relevant for kinin production and cell invasion, respectively.

Discovery and therapeutic potential of kinin receptor antagonists.

INTRODUCTION: Kinins are bioactive peptide hormones that exert biological effects by activating two types of G protein-coupled receptors namely, B(1) (B(1)R) and B(2) (B(2)R). These modulate normal physiological cellular functions, inflammatory disorders and carcinogenesis. New and novel kinin receptor antagonists have been synthesized and their efficacy evaluated. AREAS COVERED: The authors provide a comprehensive review on the cellular and molecular biology of kinins and their receptors is delineated along with evolution and discovery of selective peptide and non-peptide antagonists. The authors describe the in vitro and in vivo methods used to understand the relative functional roles of B(1)R and B(2)R in physiology and pathohysiology. Furthermore, the authors translate the evaluation of kinin antagonists in selected preclinical models and associated clinical indications. Literature was surveyed from original publications, standard sources, SciFinder, patent applications and clinical trials. EXPERT OPINION: The authors suggest that several key areas of functional biology need consideration, namely: re-evaluation, particularly in vivo, of the mechanism of action and relative functional roles of the B(1)R and B(2)R in physiology and acute and chronic disease in animals and man; need for improved animal models with increased use of humanized and human systems; development of fluorescent probes for use in vivo in animals and man using advanced imaging techniques; combination of kinin receptor antagonists and traditional chemotherapy for various cancers.

Expression of HER2 and bradykinin B1 receptors in precursor lesions of gallbladder carcinoma.

AIM: To determine the expression of HER2 and bradykinin B(1) receptors (B(1)R) in the two pathogenic models of gallbladder cancer: the metaplasia-dysplasia-carcinoma and the adenoma-carcinoma pathways. METHODS: Receptor proteins were visualized by immunohistochemistry on 5-µm sections of paraffin-embedded tissue. Expression of both receptors was studied in biopsy samples from 92 patients (6 males and 86 females; age ranging from 28 to 86 years, mean 56 years). High HER2 expression in specimens was additionally investigated by fluorescence in situ hybridization. Cell proliferation in each sample was assessed by using the Ki-67 proliferation marker. RESULTS: HER2 receptor protein was absent in adenomas and in normal gallbladder epithelium. On the contrary, there was intense staining for HER2 on the basolateral membrane of epithelial cells of intestinal metaplasia (22/24; 91.7%) and carcinoma in situ (9/10; 90%), the lesions that displayed a significantly high proliferation index. Protein up-regulation of HER2 in the epithelium with metaplasia or carcinoma in situ was not accompanied by HER2 gene amplification. A similar result was observed in invasive carcinomas (0/12). The B(1)R distribution pattern mirrored that of HER2 except that B(1)R was additionally observed in the adenomas. The B(1)R appeared either as cytoplasmic dots or labeling on the apical cell membrane of the cells composing the epithelia with intestinal metaplasia (24/24; 100%) and carcinoma in situ (10/10; 100%) and in the epithelial cells of adenomas. In contrast, both HER2 (4/12; 33%) and B(1)R (1/12; 8.3%) showed a low expression in invasive gallbladder carcinomas. CONCLUSION: The up-regulation of HER2 and B(1)R in precursor lesions of gallbladder carcinoma suggests cross-talk between these two receptors that may be of importance in the modulation of cell proliferation in gallbladder carcinogenesis.

Kinin receptors as targets for cancer therapy.

INTRODUCTION: Biological fluids of cancer patients contain increased levels of kinins. Activation of kinin B1 and B2 receptors expressed on cancer cells produce an increase in cell proliferation, migration of tumor cells and release of MMPs, which are cellular and molecular events of primary importance for tumor growth. The effects of kinins on tumor cells may be amplified by stimulation of kinin receptors expressed on other cells, within the tumor microenvironment, which may in turn increase tumor growth. AREAS COVERED: This review provides a comprehensive discourse on kinins and their receptors in human neoplasia. Concepts that view kinin receptors as targets for human cancer are explored, whilst the molecular basis by which the new dimerized kinin receptor antagonists produce arrest of cell proliferation and apoptosis of cancer cells is also examined. Finally, the role of kinin receptor antagonists as therapeutic tools against human neoplasia is analyzed. EXPERT OPINION: At the present time the available potent, dimerized kinin peptide antagonists, are either specific for B1 or B2 receptors, or are effective on both receptor types. The novel approach of using kinin receptor antagonists either alone or in combination therapy will play a definitive role in the treatment of cancer.

Effects of the demethylating agent, 5-azacytidine, on expression of the kallikrein-kinin genes in carcinoma cells of the lung and pleura.

Tissue kallikrein (KLK1) and plasma kallikrein (KLKB1) may regulate the growth and proliferation of tumours of the lung and pleura, through the generation of kinin peptides that signal through the kinin B(1) (BDKRB1) and B(2) (BDKRB2) receptors. The development and progression of cancer results from genetic mutations, as well as epigenetic changes that include methylation of DNA at CpG islands. The aim of this study was to assess whether expression of the kallikrein-kinin genes in lung cancer and mesothelioma cells is regulated by DNA methylation. Quantitative reverse transcriptase-PCR and immunocytochemistry showed differences in the basal expression of the kallikrein-kinin genes and proteins in lung carcinoma and mesothelioma cells, compared with non-malignant lung epithelial and mesothelial cells, respectively. Following treatment with the demethylating agent, 5-azacytidine (5-AZA), KLKB1 mRNA expression was consistently increased in both lung carcinoma and mesothelioma cells, whereas KLK1, BDKRB1 and BDKRB2 mRNA expression was decreased or unchanged. Increased expression of KLKB1 after 5-AZA treatment suggests it may function as a tumour suppressor gene in cancers of the lung and pleura. Studies on DNA methylation of the kallikrein-kinin genes will enhance understanding of their role in carcinogenesis and provide insights into the importance of kallikreins as tumour biomarkers.

Expression and function of retinoblastoma binding protein 6 (RBBP6) in human lung cancer.

Retinoblastoma binding protein 6 (RBBP6) interacts with both p53 and pRb, and has been identified as an E3 ubiquitin ligase due to the presence of a RING finger domain. RBBP6 promotes the degradation of p53, thereby increasing cell proliferation. However it is not known whether RBBP6 is expressed in lung cancer, or interacts with p53 and pRB to modulate the proliferation or apoptosis of lung cancer cells. As assessed by immunohistochemistry, RBBP6 and p53 proteins were overexpressed in lung adenocarcinomas and lung squamous cell carcinomas. Expression of RBBP6 mRNA in lung tumor tissue was demonstrated by quantitative RT-PCR and fluorescence in situ hybridization. Expression of RBBP6 mRNA was low in poorly differentiated tumors but high in well-differentiated tumors. Bronchoalveolar carcinomas showed intense RBBP6 mRNA hybridization in the cytoplasm of cells undergoing mitosis, supporting the association between RBBP6 expression and cell proliferation. By qRT-PCR, RBBP6 mRNA expression was 1.6 fold higher, whereas p53 mRNA expression was 2.9 fold lower in lung tumors compared with normal lung tissue. Transfection of lung adenocarcinoma and squamous cell carcinoma cells with RBBP6 siRNA decreased RBBP6 mRNA expression, whereas transfection with p53 siRNA increased RBBP6 mRNA expression. Treatment with RBBP6 siRNA increased the ratio of Bax/Bcl2 mRNA, suggesting that RBBP6 may have an anti-apoptotic function in lung cancer cells. This is the first report that RBBP6 mRNA and "its protein products" are expressed in subtypes of human lung cancer. RBBP6 may be involved in the degradation of p53, thereby enhancing cell proliferation and inhibiting apoptosis in lung cancer.

Activation of kinin B1 receptor increases the release of metalloproteases-2 and -9 from both estrogen-sensitive and -insensitive breast cancer cells.

The kinin B(1) receptor (B(1)R) agonist Lys-des[Arg(9)]-bradykinin (LDBK) increases proliferation of estrogen-sensitive breast cancer cells by a process involving activation of the epidermal growth factor receptor (EGFR) and downstream signaling via the ERK1/2 mitogen-activated protein kinase pathway. Here, we investigated whether B(1)R stimulation induced release of the extracellular matrix metalloproteases MMP-2 and MMP-9 via ERK-dependent pathway in both estrogen-sensitive MCF-7 and -insensitive MDA-MB-231 breast cancer cells. Cells were stimulated with 1-100nM of the B(1)R agonist for variable time-points. Western blotting and gelatin zymography were used to evaluate the presence of MMP-2 and MMP-9 in the extracellular medium. Stimulation of B(1)R with as little as 1 nM LDBK induced the accumulation of these metalloproteases in the medium within 5-30min of stimulation. In parallel, immunocytochemistry revealed that metalloprotease levels in the breast cancer cells declined after stimulation. This effect was blocked either by pre-treating the cells with a B(1)R antagonist or by transfecting with B(1)R-specific siRNA. Activation of the ERK1/2 pathway and EGFR transactivation was required for release of metalloproteases because both the MEK1 inhibitor, PD98059, and AG1478, an inhibitor of the EGFR-tyrosine kinase activity, blocked this event. The importance of EGFR-dependent signaling was additionally confirmed since transfection of cells with the dominant negative EGFR mutant HERCD533 blocked the release of metalloproteases. Thus, activation of B(1)R is likely to enhance breast cancer cells invasiveness by releasing enzymes that degrade the extracellular matrix and thereby favor metastasis.

Kinin B1 receptor activation turns on exocytosis of matrix metalloprotease-9 and myeloperoxidase in human neutrophils: involvement of mitogen-activated protein kinase family.

During neutrophil activation and degranulation, MMP-9 and MPO are released into the extracellular space to propagate inflammatory disorders. As kinin peptides are major participants in acute inflammatory responses, and the G-protein-coupled B(1)R mediates the chemotaxis of human neutrophils, we examined the release of the neutrophil enzymes MMP-9 and MPO by the B(1)R agonist LDBK and determined the signaling pathways that may regulate this cellular effect. Cytochalasin-treated and -untreated neutrophils were suspended in HBSS and stimulated with a range concentration of LDBK for 5 min. Zymography and Western blotting revealed that LDBK induced the release of MMP-9 and MPO. The use of specific signaling transduction inhibitors showed that release of MMP-9 depended on ERK1/2 and p38 MAPKs, whereas release of MPO involved only the p38 cascade. Inhibition of the key steps in these pathways showed that the release of both enzymes depended on PKC and PI3K. Stimulation of neutrophils with LDBK produced phosphorylation of ERK1/2 and p38 MAPK, which was inhibited by B(1)R antagonists. The phosphorylated ERK1/2 MAPK translocated to the neutrophil nucleus, suggesting that transcription of new genes may follow activation of B(1)R. Our results demonstrate that in human neutrophils, activation of kinin B(1)R by LDBK initiates separate signaling cascades that trigger the release of MMP-9 and MPO from tertiary and primary granules, respectively, suggesting that the B(1)R plays a pivotal role in inflammatory disorders.

Kallikreins, kininogens and kinin receptors on circulating and synovial fluid neutrophils: role in kinin generation in rheumatoid arthritis.

OBJECTIVES: Neutrophils traffic into and have the capacity to generate kinins in SF of RA patients. The aim of this study was to assess the expression of kallikreins, kininogens and kinin receptors in circulating and SF neutrophils, as well as synovial tissue of RA patients, and to assess kinin generation in SF. METHODS: Neutrophils were isolated from blood and SF of RA patients and blood of healthy volunteers. Expression of kallikreins, kininogens and kinin receptors in neutrophils and synovial tissue was assessed by immunocytochemistry using specific antibodies, with visualization by brightfield and confocal microscopy. Levels of basal and generated kinins in SF of RA patients were measured by ELISA. RESULTS: Kinin labelling was significantly reduced, indicating the loss of the kinin moiety from kininogen on circulating (P < 0.001) and SF neutrophils (P < 0.05) of RA patients. Immunolabelling of tissue kallikrein was also decreased, whereas kinin B(1) and B(2) receptor expression was increased in circulating and SF neutrophils of RA patients. Immunolabelling of kallikreins and kinin receptor proteins was similar in RA and normal synovial tissues. The basal kinin level in SF of RA patients was 5.7 +/- 6.1 ng/ml and the mean concentration of kinins generated in vitro was 80.6 +/- 56.3 ng/ml. The capacity for kinin generation was positively correlated with measures of disease activity. CONCLUSIONS: Kallikrein-kinin proteins on neutrophils play an important role in kinin generation and the pathophysiology of RA. Specific kallikrein and kinin receptor antagonists may be useful as IA therapies for inflamed joints.

Expression of kinin receptors on eosinophils: comparison of asthmatic patients and healthy subjects.

Eosinophils contribute to asthmatic airway inflammation by releasing cysteinyl leukotrienes (cysLT) and other inflammatory mediators, and bradykinin (BK) induces bronchoconstriction in asthmatic patients. The aims of this study were to investigate kinin receptor expression on eosinophils of asthmatic and healthy subjects and to assess the effects of kinin stimulation on eosinophils, which were isolated from peripheral blood of asthmatic (n=27) and healthy subjects (n=14). Kinin B(1) and B(2) receptors (B(1)R and B(2)R, respectively) and mRNA expression were investigated by quantitative confocal microscopy, flow cytometry, and RT-PCR. Intracellular Ca(2+) was assessed by live-cell fluorescence confocal microscopy. Production of cysLT and eosinophil migration in response to BK and Lys-des[Arg(9)]-BK were assessed. Eosinophils expressed kinin B(1)R and B(2)R mRNA and proteins. Quantitative immunofluorescence analysis indicated that expression of B(1)R and B(2)R proteins was significantly greater in eosinophils of asthmatic patients compared with those of nonasthmatic subjects. However, kinin B(1)R and B(2)R mRNA expression did not differ significantly between these groups. Expression of kinin B(1)R and mRNA was decreased in patients using high doses of inhaled corticosteroids and in eosinophils treated with a corticosteroid in vitro. Kinin B(1) and B(2) agonists up-regulated expression of their respective receptors but did not increase intracellular Ca(2+) or the production of cysLT or enhance eosinophil migration significantly. Up-regulation of kinin receptor expression in eosinophils of asthmatic patients may be a consequence of inflammation, whereby enhanced release of kinin peptides has a positive-feedback effect on kinin receptor expression. Importantly, anti-inflammatory corticosteroids down-regulated the expression of the kinin B(1)R.

Epigenetic regulation of human epithelial cell cancers.

Cancer is the second leading cause of death in industrialized countries, with epithelial cell cancers (carcinomas) representing approximately 85% of all diagnosed cancers. The 5-year survival rate for many carcinomas remains low, highlighting the requirement for improved diagnosis and more effective therapies. Epigenetic modifications that do not involve changes in the DNA sequence, but result in changes in gene expression, are rapidly being realized as important in carcinogenesis. Evidence is emerging that DNA methylation, histone modification and alternative mRNA splicing are involved in various human epithelial cell cancers, and diagnostic and therapeutic strategies based on these epigenetic phenomena are under investigation. This review provides an overview of studies demonstrating the importance of epigenetic regulation of gene expression in the diagnosis, progression and response to treatment of human carcinomas. The use of therapeutic agents to reverse these epigenetic changes, either as single treatments or in combination with other therapies, is also discussed.