Chantal Brunet

Isolation of prostatic kallikrein hK2, also known as hGK-1, in human seminal plasma

To demonstrate the presence of kallikrein hK2 in the human prostate and seminal plasma, we used mouse monoclonal antibodies (MAb) against a recombinant hK2-fusion protein. Using one of these MAb 9D5, we detected the presence of several major immunoreactive spots of 22 kDa and minor ones of 31 and 55 kDa in prostate cytosol and seminal plasma. After ion exchange and immunoaffinity chromatography of seminal plasma proteins, the 22-kDa immunoreactive proteins were isolated along with 55- and 75-kDa proteins. The NH2-terminal amino acid sequencing permitted identification of fragments of hK2 and protein C inhibitor, respectively, in the 22- ad 55-kDa bands. Furthermore, immunoblotting experiments in one and two-D gels with two different anti-hK2 MAbs and one polyclonal anti-PCI antibody suggested that the major 55- and 75-kDa bands were covalent hK2-PCI complexes containing either the full-length hK2 chain or only its carboxyterminal fragment in the presence of mercaptoethanol. These results demonstrate for the first time the existence of kallikrein hK2 and suggest that PCI may regulate its activity in seminal plasma.

Secretion of monocyte chemotactic protein-1 by cytokine-stimulated endometrial cells of women with endometriosis *

OBJECTIVE To evaluate in vitro the production of monocyte chemotactic protein-1 (MCP-1) by endometrial cells of patients with and without endometriosis. DESIGN Primary cultures of stromal and epithelial cells isolated from human endometrium were exposed during 24 hours to different cytokines. Monocyte chemotactic protein-1 secretion was analyzed in the culture medium. SETTING Gynecology clinic and laboratories of endocrinology of reproduction and immunology. PATIENTS Women presenting for infertility or pelvic pain in which endometriosis was diagnosed at laparoscopy (n = 6) and women presenting for tubal ligation without laparoscopic evidence of the disease (n = 6). INTERVENTIONS None. MAIN OUTCOME MEASURES De novo secretion of MCP-1 in the culture supernatant by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis after metabolic labeling with 35S-cysteine. RESULTS The incubation of endometrial epithelial cells of endometriosis women with either interleukin-1 beta or tumor necrosis factor-alpha resulted in the appearance of at least two and sometimes three bands having approximately 15, 13, and 9 kd molecular weights. These bands were identified as three distinct species of MCP-1 as their immunoprecipitation was prevented effectively in presence of an excess of cold MCP-1. In contrast, the endometrial epithelial cells of only one of six normal women produce significant levels of MCP-1 under the same stimulation conditions. The stromal cells of both groups of subjects do not secrete appreciable amounts of MCP-1 or only small quantities in two cases of endometriosis. CONCLUSIONS Monocyte chemotactic protein-1 secretion is upregulated in cytokine-stimulated endometrial epithelial cells of women having endometriosis as compared with normal women without evidence of the disease. Such a difference at the level of eutopic endometrial cell may have a significance in the physiopathology of endometriosis.

Cytokine-induced secretion of monocyte chemotactic protein-1 by human endometriotic cells in culture

OBJECTIVE Local secretion of chemotactic factors could contribute to the attraction of macrophages into the peritoneal cavity of women with endometriosis. The purpose of this study was to investigate the ability of endometriotic cells to produce monocyte chemotactic and activating protein-1 in response to interleukin-1 beta and tumor necrosis factor-alpha, which are found in elevated levels in the peritoneal fluid of patients with endometriosis. STUDY DESIGN Cultures of fibroblast-like and epithelial cells isolated from endometriotic tissue were incubated with different concentrations of cytokines for varying periods of time. The de novo secretion of monocyte chemotactic protein-1 in the culture supernatants was analyzed by immunoprecipitation and electrophoresis after metabolic labeling with sulfur 35-labeled cysteine. RESULTS The incubation of endometriotic fibroblast-like cells with interleukin-1 beta and tumor necrosis factor-alpha resulted in a time- and dose-dependent release of monocyte chemotactic protein-1 into the culture supernatant. Coincubation of the cells with tumor necrosis factor-alpha and interferon gamma resulted in a synergistic and dose-dependent increase of the monocyte chemotactic protein-1 secretion, whereas interferon gamma alone had no significant effect. Preliminary results indicate that monocyte chemotactic protein-1 is also produced by endometriotic epithelial cells in response to the same cytokines. CONCLUSIONS Cytokine-stimulated endometriotic cells synthesize and secrete monocyte chemotactic protein-1 in culture, and they may play a relevant role in the recruitment of macrophages to the peritoneal cavity of patients by the local production of chemotactic factors.

Allergic rhinitis to ragweed pollen: II. Modulation of histamine-releasing factor production by specific immunotherapy

A number of cytokines, including histamine-releasing factors (HRFs), have a role to play in IgE-mediated asthma. However, the influence of HRF in allergic rhinitis without asthma remains to be revealed. This article presents a double-blind, placebo-controlled study on the role of HRF in ragweed-allergic rhinitis and its modulation by natural pollen exposure and specific immunotherapy (IT). Twenty-seven patients allergic to ragweed were randomly assigned to receive either preseasonal alum-precipitated aqueous extracts of ragweed or placebo. Before the onset of therapy and during the ragweed-pollen season, subjects were evaluated for each of the following: clinical scores, ragweed IgE and IgG antibody levels, and spontaneous and allergen-driven HRF production. Thirteen nonatopic volunteers were also studied in the same protocol. First, before the initiation of therapy, more HRF was produced by both unstimulated and ragweed-stimulated mononuclear cells (MNCs) of atopic subjects as compared to MNCs of nonatopic subjects. Second, MNCs of the placebo-treated group produced significantly more spontaneous and ragweed-specific HRF during the pollen season compared to the preseasonal values. Finally, specific IT not only improved the clinical manifestation of allergy but also prevented the seasonal rise of spontaneous and ragweed-driven HRF production, along with a well-known change in other immunologic parameters associated with successful IT.

Increased compound 48/80 induced local histamine release from nonlesional skin of patients with chronic urticaria

Histamine released from skin mast cells in normal skin sites of patients with idiopathic chronic urticaria (CU) and normal volunteers was assessed with the skin chamber technique. Small amounts of histamine were spontaneously and continuously released during the 4-hour observation in both groups but were twofold greater in patients with CU. In addition, histamine levels were significantly more elevated in sites challenged with compound 48/80 than in unstimulated sites. Patients with CU differed from normal volunteers in that histamine release induced by 48/80 compound was significantly greater at 1 and 2 hours after challenge. The number of mast cells and the histamine content of the skin did not differ in the two groups. These observations could suggest a functional defect at the mast cell level rather than a difference in their numbers.

Allergic rhinitis to ragweed pollen: I. Reassessment of the effects of immunotherapy on cellular and humoral responses

This work presents a double-blind, placebo-controlled study of 27 patients with allergic rhinitis to ragweed who received preseasonal desensitization immunotherapy [IT] with alum-precipitated aqueous ragweed extracts. We reassessed the following parameters in relation to clinical responses: clinical scores, nasal reactivity to a provocative dose of ragweed causing a 75% fall in airflow rate (PD75), ragweed IgE and IgG, and ragweed-induced basophil histamine release (BHR). First, the nasal PD75 correlated with the severity of nasal symptoms (p less than 0.05). Second, we confirmed a significant symptomatic improvement in the IT-treated group either by clinical scores (p less than 0.05) or the prevention of the seasonal fall of the PD75 (p less than 0.005). Also, IT reduced the seasonal rise of IgE (p less than 0.02) and induced an increase in IgG (p less than 0.01) and a decrease in BHR (p less than 0.03). There was a significant correlation between IgE and BHR (r = 0.80; p less than 0.01). After selecting out the effects of IgE, the BHR was still higher in the placebo-treated group than in the IT-treated group (p less than 0.02), suggesting the involvement of other modulating factors. Symptomatic improvement after IT correlated only with the summation of both IgE and BHR (PD75; r = 0.64; p less than 0.005). This observation suggests that the severity of clinical symptoms is determined by several interacting factors and not by the antibody response alone.

Chronic idiopathic urticaria: Profiles of skin mast cell histamine release during active disease and remission

Chronic idiopathic urticaria (CIU) is characterized by the increased releasability of histamine by mast cells in normal-appearing skin. In active CIU, this abnormality is consistently present. To determine if this finding subsides when the disease goes into remission phase, we analyzed the histamine secretion in patients with CIU in remission (CIUR) compared with that of patients with CIU and in normal control (NC) subjects, with the skin-chamber technique. The profiles of histamine release in sites challenged with compound 48/80 were significantly different in the groups with CIU and CIUR. Furthermore, patients with CIUR did not differ from NC subjects in terms of histamine releasability under compound 48/80 stimulation (p greater than 0.1). These data suggest that the state of excessive skin mast cell sensitivity is a reversible and transient phenomenon in CIU disease.

Chronic idiopathic urticaria: Possible contribution of histamine-releasing factor to pathogenesis

BACKGROUND Histamine-releasing factor was recently shown to be clinically relevant in allergic rhinitis and asthma. HRF could also be involved in the pathogenicity of chronic idiopathic urticaria (CU). The purpose of this study was to investigate the role of HRF in the pathophysiology of CU. METHODS Blisters were induced on lesional and nonlesional skin of 12 patients with CU and on normal skin of five control subjects. HRF activity and histamine content were measured in all samples recovered from each skin site. RESULTS Significantly more HRF was found in blister fluids from lesional skin of patients with CU as compared with nonlesional skin and skin of control subjects. In addition, histamine content in blister fluids from affected skin of patients with CU was significantly higher in comparison with both nonlesional skin of patients with CU and skin of control subjects. HRF activity was also higher in blister fluids from nonlesional skin of patients with CU than that of control subjects, in spite of equivalent histamine content. CONCLUSION These data suggest that the inflammatory reaction found in CU disease is associated with the cutaneous release of HRF.

Effects of H1-antihistamine drug regimen on histamine release by nonlesional skin mast cells of patients with chronic urticaria

Profiles of compound 48/80-induced histamine release (HR) from mast cells of uninvolved skin from patients with chronic urticaria (CU) and from a normal control (NC) group were compared, and the effects of anti-H1 medications were assessed versus placebo. Then, patients with CU (15) and NC subjects (10) were randomly assigned to take either hydroxyzine (100 mg/day), terfenadine (120 mg/day), or placebo for 28 days. The effects of such treatment on the clinical response and on the profile of compound 48/80-induced HR during a 4-hour period were analyzed. Treatment with hydroxyzine in patients with CU improved the clinical symptoms and modified the profile of HR; more histamine was recovered at 1 hour (p less than 0.05) and 2 hours (p less than 0.05), as compared with baseline. Terfenadine and placebo had no effect on the clinical response or on the profiles of HR. In the NC group, the amounts of histamine recovered at 1 hour after challenge with compound 48/80 were lower than amounts of the pretherapy values (p less than 0.01). It could be concluded that (1) the profile of HR in patients with CU is reproducible during a period of 28 days, (2) only hydroxyzine modifies both the clinical response and the profile of HR, and (3) anti-H1 compounds decrease the HR in the NC group.

Analysis of compound 48 80 -induced skin histamine release and leukotriene production in chronic urticaria

Profiles of histamine release by nonlesional skin mast cells from patients with chronic urticaria (CU) and normal control (NC) subjects were compared on stimulation with a wide range of concentrations of compound 48/80 (0.15 to 4.8 mg/ml). One previous observation demonstrating spontaneous and 48/80-induced release of histamine greater in patients with CU than in NC subjects was confirmed with a concentration of 48/80 believed to produce mast cell activation in most subjects (2.4 mg/ml). In fact, higher concentrations of histamine were released by patients with CU than by NC subjects on stimulation with 48/80 at concentration greater than 0.6 mg/ml. With 0.6 mg/ml of 48/80, the profiles of histamine release were comparable in the two groups, and for concentration less than 0.6 mg/ml, the profiles of release were completely different, being higher in NC subjects than in the group with CU. The peak histamine response was higher in the group of NC subjects (6500 pg/ml) than in the group with CU (5100 pg/ml), and the concentration of 48/80 needed to trigger maximum release was lower in the NC subjects than in the subjects with CU (0.15 versus 0.6 mg/ml). The production of leukotriene B4 and leukotriene C4 was also compared in these two groups after stimulation with 2.4 mg/ml of compound 48/80. No significant amount of leukotriene B4 was found in the collection chambers, but small amounts of leukotriene C4 were measured into skin chamber fluids during a period of 4 hours. No significant difference could then be observed between the two groups.