Coordinate regulation of estrogen-mediated fibronectin matrix assembly and epidermal growth factor receptor transactivation by the G protein-coupled receptor, GPR30.

Estrogen promotes changes in cytoskeletal architecture not easily attributed to the biological action of estrogen receptors, ERalpha and ERbeta. The Gs protein-coupled transmembrane receptor, GPR30, is linked to specific estrogen binding and rapid estrogen-mediated release of heparin-bound epidermal growth factor. Using marker rescue and dominant interfering mutant strategies, we show that estrogen action via GPR30 promotes fibronectin (FN) matrix assembly by human breast cancer cells. Stimulation with 17beta-estradiol or the ER antagonist, ICI 182, 780, results in the recruitment of FN-engaged integrin alpha5beta1 conformers to fibrillar adhesions and the synthesis of FN fibrils. Concurrent with this cellular response, GPR30 promotes the formation of Src-dependent, Shc-integrin alpha5beta1 complexes. Function-blocking antibodies directed against integrin alpha5beta1 or soluble Arg-Gly-Asp peptide fragments derived from FN specifically inhibited GPR30-mediated epidermal growth factor receptor transactivation. Estrogen-mediated FN matrix assembly and epidermal growth factor receptor transactivation were similarly disrupted in integrin beta1-deficient GE11 cells, whereas reintroduction of integrin beta1 into GE11 cells restored these responses. Mutant Shc (317Y/F) blocked GPR30-induced FN matrix assembly and tyrosyl phosphorylation of erbB1. Interestingly, relative to recombinant wild-type Shc, 317Y/F Shc was more readily retained in GPR30-induced integrin alpha5beta1 complexes, yet this mutant did not prevent endogenous Shc-integrin alpha5beta1 complex formation. Our results suggest that GPR30 coordinates estrogen-mediated FN matrix assembly and growth factor release in human breast cancer cells via a Shc-dependent signaling mechanism that activates integrin alpha5beta1.

Comparison of a solid phase human- versus porcine- thyrotropin receptor-based immunoassay for the measurement of thyrotropin receptor antibodies in patients with thyroid diseases.

Thyrotropin receptor (TSHR) antibody (TRAb) assays based on human or porcine TSHR which are coated to a solid phase are the most commonly used detection methods in clinical practice for patients with thyroid diseases. Yet the difference of the diagnostic values of the two TRAb assays is largely unclear. The aim of our present study was to evaluate the clinical perfomance of a solid phase porcine TRAb assay based on an ELISA technique (TRAb-porcine) and a human TRAb assay based on a chemiluminescence signal detection procedure (TRAb-human). Of 158 patients enrolled in the study, 84 suffered from Graves' disease (GD), 34 had Hashimoto's thyroiditis (HT) and 40 had euthyroid nodular thyroid disease (NTD) without signs of autoimmunity. TRAb measurements were performed according to the manufacturer's instructions. The mean values of TRAb titers detected by the TRAb-human and TRAb-porcine assays in patients with GD were 12.14+/-10.80 IU/L and 15.27+/-13.65 IU/L, respectively. TRAb were detected in 80 and 78 out of 84 GD patients by the TRAb-human and TRAb-porcine assay, respectively. The diagnostic sensitivity of the TRAb-human and TRAb-porcine immunoassay was 95.2 and 92.9% respectively, by 100% specificity of both methods. TRAb values in GD patients detected by the TRAb-human and TRAb-porcine assays were significantly correlated (r=0.929, p<0.0001). Our results indicate that the second generation TRAb-porcine assay based on solid phase technology had a slightly lower diagnostic sensitivity compared to the TRAb-human assay.

Comparison of M22-based ELISA and human-TSH-receptor-based luminescence assay for the measurement of thyrotropin receptor antibodies in patients with thyroid diseases.

Previously, a new procedure for measuring serum TSH receptor autoantibodies (TRAb) was reported in which the autoantibodies inhibit binding of a human monoclonal thyroid stimulating antibody M22 to TSHR-coated ELISA plate wells (TRAb ELISA). The aim of the present study was to evaluate the clinical performance of this assay in comparison to the second generation TRAb assay (TRAb LIA) based on the recombinant human TSH-receptor and chemiluminescence technology (TRAb LIA). Among the 158 patients, 84 patients suffered from Graves' disease (GD), 34 patients had Hashimoto's thyroiditis (HT), and 40 patients had euthyroid nodular thyroid disease (NTD) without signs of autoimmunity. TRAb measurements were performed according to the manufacturer's instructions. Out of 84 GD patients, 80 (95.2%) were TRAb positive as detected by the TRAb LIA. One GD patient had TRAb values within the grey zone (1.0-1.5 IU/l). All patients with HT and NTD were negative except in 6 (8.1%) cases whose TRAb values were within the grey zone. On the basis of the recommended cutoff value (TRAb 1.0 IU/l), the TRAb ELISA found 78 of 84 (92.9%) GD patients to be TRAb positive. None of the patients with HT, but two cases (5.0%) with NTD were TRAb positive. The diagnostic sensitivity of the TRAb LIA and TRAb ELISA assays was 95.2 and 92.9%, while the specificity was 100% and 97.3%, respectively. There was a close correlation (r=0.968, p<0.0001) between both assays in 84 patients with GD. Additionally, the between-run imprecision close to the cutoff limit was assessed. The calculated between-run coefficient of variation (CV) of the TRAb ELISA was 28.2% at the recommended cutoff value of 1.0 IU/l. Due to the evaluated imprecision data we propose a higher cutoff value correlating with a between-run CV of 20% (functional assay sensitivity). Our results indicate that due to a worse imprecision the TRAb ELISA has a slightly lower sensitivity and specificity compared to the TRAb LIA assay. These findings suggest that the M22 monoclonal antibody-based TRAb ELISA is not as reliable as other second generation TRAb assays in the diagnosis of Graves' diseases.

Activation of the novel estrogen receptor G protein-coupled receptor 30 (GPR30) at the plasma membrane.

G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17beta-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [3H]17beta-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK-293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.

Alpha-defensin 1 (human neutrophil protein 1) as an antichemotactic agent for human polymorphonuclear leukocytes.

Medium conditioned by tumor necrosis factor alpha (TNF-alpha)-stimulated polymorphonuclear leukocytes (PMN) (CM-TNF) suppresses PMN migration. Therefore, we wished to identify the agent(s) in CM-TNF that mediated antichemotactic activity. CM-TNF was fractionated by high-performance liquid chromatography, and one fraction with antichemotactic activity contained the bactericidal protein human neutrophil protein 1 (HNP-1). We showed that HNP-1 suppresses PMN migration to formyl-methionyl-leucyl-phenylalanine but not to interleukin 8.

Paracrine suppression of apoptosis by cytokine-stimulated neutrophils involves divergent regulation of NF-kappaB, Bcl-X(L), and Bak.

Dysregulated polymorphonuclear leukocyte (PMN) apoptosis and PMN-mediated organ damage have been associated with several medical conditions such as systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), and ischemia/reperfusion injury. IL-1beta and IL-8 are two cytokines that are elevated under similar conditions. Therefore, we hypothesized that PMN exposed to these cytokines would secrete factors that could affect PMN apoptosis in a cell contact-independent manner. We have previously shown that media conditioned by IL-1beta-stimulated PMN (CM-IL1beta) for 2 h suppressed spontaneous PMN apoptosis. Data presented here demonstrate that media conditioned by IL-8-stimulated PMN (CM-IL8) also have the ability to suppress spontaneous, as well as FasL- and TNF-alpha-induced apoptosis. In contrast, CM-IL1beta was able to suppress FasL-induced, but not TNF-alpha-induced, apoptosis. To elucidate the mechanisms these media use to elicit their effects, we examined the expression and function of several apoptosis-related proteins. Experimental results demonstrate that both CM-IL1beta and CM-IL8 have the ability to delay caspase activation, but have no effect on the expression of their upstream activator, Fas, or its ligand, FasL. Examination of several Bcl-2 family members revealed a selective regulation by each media: CM-IL1beta up-regulated Bcl-X(L), while CM-IL8 down-regulated Bak expression. Additionally, CM-IL1beta, but not CM-IL8, promoted the activation of NF-kappaB, which has anti-apoptotic activity. Together, we can conclude that IL-1beta- and IL-8-stimulated PMN have the ability to suppress PMN apoptosis in a paracrine manner, and that the extent and mechanism of suppression is specific for each.

Regulation of IL-8RA (CXCR1) expression in polymorphonuclear leukocytes by hypoxia/reoxygenation.

Interleukin-8 (IL-8) is an important mediator of neutrophil (PMN) function and the type A IL-8 receptor (IL-8RA) mediates these pro-inflammatory signals. Hypoxia or hypoxia/reoxygenation (H/R) affects the production of IL-8, but no data is available regarding its effect on IL-8RA expression. The purpose of this study was to determine the effects of hypoxia and/or H/R on the expression of IL-8RA in PMN. We demonstrated that IL-8RA mRNA levels were similar under normoxic and hypoxic conditions but H/R resulted in a significant reduction in mRNA expression between 30 and 60 min. IL-8RA protein also decreased with reoxygenation of whole blood, which was altered by the addition of specific antioxidants. Therefore, H/R appears to attenuate the effect of IL-8 by down-regulating IL-8RA in PMN. These data show that changes in oxygen tension within the wound site not only affect the expression of inflammatory cytokines, but also control their actions by regulating their receptors.

Peritonitis caused by Monilia sitophila in a patient undergoing peritoneal dialysis.

Fungi have become an increasingly important cause of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis. The most common cause of fungal peritonitis is Candida. However, in recent years unusual and "nonpathogenic" fungi have been reported as etiologic agents of CAPD-associated peritonitis. We are reporting the first case of CAPD-associated peritonitis caused by Monilia sitophila. This organism had previously been considered to be non-pathogenic, and a troublesome laboratory contaminant. Our patient was successfully managed with intravenous and intraperitoneal amphotericin B, followed by oral itraconazole, without removal of her Tenckhoff catheter.

Dialysate protein losses with bleach processed polysulphone dialyzers.

We measured dialysate protein losses from polysulphone dialyzers undergoing repetitive processing with bleach and formaldehyde. The entire dialysate was collected during the first, fifth and tenth use of F-80 dialyzers. Dialysate protein concentration was 1.5 +/- 0.4 mg/dl N = 11 +/- SEM) during the first use, 2.1 +/- 0.3 mg/dl during the fifth use and 3.6 +/- 0.5 mg/dl (N = 10) during the tenth use. In a follow-up study, dialyzers were evaluated for up to 25 uses. After 12 to 15 uses dialysate protein was 7.9 +/- 0.8 mg/dl (N = 13), after 16 to 20 uses; 12.0 +/- 1.2 mg/dl (N = 13) and after 23 to 25 uses; 19.9 +/- 2.1 mg/dl (N = 5). Mean dialysate volume was 83.9 +/- 1.1 liters (N = 63) yielding total protein losses of up to 20.7 grams per treatment. Dialysate albumin losses, which were unmeasurable during the first use of the dialyzers, revealed a similar increase with reuse resulting in a mean value of 14.4 +/- 3.2 mg/dl after 23 to 25 reuses (N = 5). Dialysate beta-2 microglobulin (beta 2m) levels were 1.05 +/- 0.13 mg/l for dialyzers bleached < 10 times (N = 32) versus 1.54 +/- 0.15 mg/liter for dialyzers bleached > 10 times (N = 31, P < 0.02 vs. < 10 reuses). A random sampling of dialyzers processed without bleach for 8, 14, 15, 24 and 25 reuses revealed minimal protein losses, ranging from 1.4 to 2.7 mg/dl with no relation to reuse number and no measureable albumin.(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of high anion gap metabolic acidosis in severe ethylene glycol poisoning: a potential effect of simultaneous lithium carbonate ingestion.

Ethylene glycol poisoning is accompanied by a high anion gap metabolic acidosis. We describe a case of severe ethylene glycol poisoning in which severe acidosis was not present. This appears to be due to a beneficial effect exerted by simultaneous ingestion of 80 lithium carbonate tablets, each containing approximately 4 mEq carbonate, with a potential bicarbonate load of 320 mEq. We postulate a protective effect of lithium carbonate due to the bicarbonate generated by this substance.

Atrial peptide release after hemorrhage in unanesthetized swine.

Hemorrhage of 14 ml/kg in 5 min was done in two groups of chronically prepared, splenectomized Yorkshire pigs. Group 1 was studied on post-operative day 4 and was conditioned behaviorally with "active restraint", whereas group 2 was studied on postoperative day 6 and was conditioned with behavioral "shaping." The peak decrease in blood volume occurred by 0.25 h after hemorrhage in both groups. However, plasma atrial natriuretic factor (ANF) as measured by radioimmunoassay did not decrease significantly until 2 h in group 1 and 0.5 h in group 2 even though the recovery of blood volume was significantly more rapid in group 1 than in group 2. The responses of ANF differed significantly between groups, suggesting that ANF release after hemorrhage is influenced by prior handling and the time for recovery from surgery. In both groups, some pigs showed increases in ANF during the 1st h after hemorrhage, and changes in ANF were unrelated to decreases in central venous pressure or absolute right atrial volume determined with a conductance catheter. In contrast, changes in ANF after hemorrhage correlated positively with several variables including atrial rate and changes in vasopressin. Multiple regression suggested that the effect of reduced atrial volume on ANF release was opposed by these latter variables or related factors. Furthermore, known actions of ANF do not appear to account for the observed hemodynamic and hormonal responses to hemorrhage.

Mesangial proliferative glomerulonephritis with IgM deposits. Clinicopathologic analysis and evidence for morphologic transitions.

To determine the natural history of mesangial proliferative glomerulonephritis (MesPGN) with IgM deposits and its relationship to minimal change disease (MC) and focal segmental glomerulosclerosis (FGS), we studied the clinical characteristics and outcome in 20 patients with MesPGN, 8 with MC, and 10 with FGS. IgM deposits were present in glomeruli of all MesPGN patients. Progression to FGS was documented in 2 patients with MesPGN, 1 of whom developed renal failure. Transition from MC to MesPGN occurred in 1 patient. 2 MC patients developed FGS, with decline in renal function in 1 of them. These data suggest the possibility of histologic transition from MC to FGS directly or through the stage of MesPGN.

Metoprolol and alopecia.

Beta-adrenoceptor antagonists are very popular agents in the treatment of hypertension. Reversible alopecia of the telogen effluvium variety has been described with propranolol (inderal). We describe a case of reversible alopecia with metoprolol (Lopressor) which also was associated with a telogen effluvium on scalp biopsy, suggesting a similar mechanism for the alopecia associated with these agents.