An immunoluminometric assay for cardiotrophin-1: a newly identified cytokine is present in normal human plasma and is increased in heart failure.

Cardiotrophin-1, a member of the interleukin-6 related cytokine family which acts via the glycoprotein 130 signalling pathway, may be involved in the process of ventricular remodelling. Its presence in human plasma has never been reported. We have devised a non-radioactive immunoluminometric sensitive and specific assay for CT-1 based on a competitive ligand binding principle. The chemiluminescent label 4-(2-succinimidyl-oxycarbonylethyl)phenyl-10-methylacridinium 9-carboxylate fluorosulfonate was used to label a peptide representing a domain in the middle section of CT-1. Assay of this domain of CT-1 (amino acids 105-120) in patients with heart failure revealed elevated CT-1 values median 87 [range 74.3-182.8] fmol/ml) compared to normal controls (CT-1 median 29.55 [range 6.9-48.3] fmol/ml, P<0.0005). The molecular weight of human CT-1 was estimated to be 26.7 kD from sodium dodecyl sulphate polyacrylamide gel electrophoresis. This is the first quantitative assessment of CT-1 in humans. Furthermore, this is the first demonstration of significant elevation of plasma CT-1 in patients with heart failure.     

Cytokines are not a requisite part of the pathophysiology leading to cardiac decompensation

An increase in circulating levels of proinflammatory cytokines has been proposed as an important pathogenic factor contributing to cardiac injury during chronic heart failure. To determine whether plasma levels of the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) increase during pacing-induced heart failure, we paced the hearts of seven dogs at 210 beats/min for 3 weeks and at 240 beats/min for an additional week to induce severe clinical signs of cardiac decompensation. Hemodynamic measurements and blood samples from the aorta and coronary sinus (CS) were taken at control, at 3 weeks, and in end-stage failure. Decompensated heart failure occurred at 29 +/- 1.8 days, when left ventricular (LV) end-diastolic pressure was 25 +/- 1.3 mmHg, LV systolic pressure was 92 +/- 4 mmHg, mean arterial pressure was 77 +/- 3 mmHg, and dP/dtmax was 1219 +/- 73 (all P < 0.05 vs control). Arterial concentration of IL-6 was 12 +/- 4.0 U/ml at control, 11 +/- 2.7 U/ml at 3 weeks, and 10 +/- 1.7 U/ml in end-stage failure (NS). At the same time points, IL-6 in CS plasma was 12 +/- 3.5, 13 +/- 2.8 and 11 +/- 2.4 U/ml, respectively (NS vs control and vs arterial concentrations). TNF-alpha did not reach detectable concentrations in arterial or CS blood at any time. TNF-alpha and IL-6 concentrations did not increase in arterial blood, were not released in the CS from the heart during the development of pacing-induced heart failure, and can not universally be implicated in the pathogenesis of all forms of cardiac dysfunction. Our findings are consistent with other data from patients in which severe heart failure was not associated with increased levels of circulating cytokines.     

Atrial natriuretic factor in human plasma

A reproducible and sensitive radioimmunoassay (RIA) was developed to measure ANF in human plasma. Immunoreactive ANF was extracted from plasma with Sep-Pak cartridges, using 0.2% ammonium acetate (pH 4) with acetonitrile. The sensitivity of the assay was 3.9 pg/ml. The coefficient of variance for inter-assay and intra-assay was 16.8% and 6.8%, respectively. In normal healthy subjects (n = 67), ANF content was 11.9 +/- 1.3 pg/ml (mean +/- SEM). Significantly-higher ANF concentrations were found in proximal coronary sinus blood, being 6 to 37 times greater than in the peripheral circulation. Comparison of the prior extraction method with direct RIA revealed a good correlation (r = 91) in samples containing higher than 100 pg/ml ANF. No correlation was observed with lower values. The elution profiles of reverse-phase HPLC of peripheral and coronary sinus plasma extracts were similar but somewhat complex, with the main immunoreactive peak corresponding to a low-molecular-weight peptide.     

Cardiotrophin-1 is expressed in adipose tissue and upregulated in the metabolic syndrome

Adipose tissue is a target for cardiotrophin-1 (CT-1), a cytokine member of the IL-6 family of cytokines that is involved in cardiac growth and dysfunction. However, it is unknown whether adipocytes are a source of CT-1 and whether CT-1 is overexpressed in diseases characterized by increased fat depots [i.e., the metabolic syndrome (MS)]. Thus this work aimed 1) to test whether adipose tissue expresses CT-1 and whether CT-1 expression can be modulated and 2) to compare serum CT-1 levels in subjects with and without MS diagnosed by National Cholesterol Education Program Adult Treatment Panel III criteria. Gene and protein expression of CT-1 was determined by real-time RT-PCR, ELISA, and Western blotting. CT-1 expression progressively increased, along with differentiation time from preadipocyte to mature adipocyte in 3T3-L1 cells. CT-1 expression was enhanced by glucose in a dose-dependent manner in these cells. mRNA and protein CT-1 expression was also demonstrated in human adipose biopsies. Immunostaining showed positive staining in adipocytes. Finally, increased CT-1 serum levels were observed in patients with MS compared with control subjects (127 +/- 9 vs. 106 +/- 4 ng/ml, P < 0.05). Circulating levels of CT-1 were associated with glucose levels (r = 0.2, P < 0.05). Taken together, our data suggest that adipose tissue can be recognized as a source of CT-1, which could account for the high circulating levels of CT-1 in patients with MS.     

Tissue factor, tissue factor pathway inhibitor and cytoadhesive molecules in patients with an acute coronary syndrome

The tissue factor plays a crucial role in initiating blood coagulation after plaque rupture in patients with acute coronary syndrome. It is abundant in atherosclerotic plaques. Moreover, P-selectin, some cytokines, endotoxin and immune complexes can stimulate monocytes and induce the tissue factor expression on their surface. The aim of the study was to compare plasma levels of the tissue factor, tissue factor pathway inhibitor, P-selectin, E-selectin and ICAM-1 in patients with acute myocardial infarction, unstable angina pectoris, stable coronary artery disease and normal control subjects. In addition, plasma levels of the tissue factor, tissue factor pathway inhibitor, P-selectin, E-selectin and ICAM-1 were measured in the blood withdrawn from the coronary sinus in a subgroup of patients with unstable angina pectoris and stable coronary artery disease in which the difference between concentrations in the coronary sinus and systemic blood was calculated. A significant increase in tissue factor pathway inhibitor plasma levels was detected in patients with acute myocardial infarction (373.3+/-135.1 ng/ml, p<0.01) and unstable angina pectoris (119.6+/-86.9 ng/ml, p<0.05) in contrast to the patients with stable coronary artery disease (46.3+/-37.5 ng/ml) and normal subjects (45.1+/-14.3 ng/ml). The plasma levels of tissue factor pathway inhibitor were significantly increased both in the coronary sinus and systemic blood in the patients with unstable angina pectoris. There was only a non-significant trend to higher plasma levels of the tissue factor in patients with acute myocardial infarction and unstable angina pectoris as compared to the patients with stable coronary artery disease and normal subjects, the values being 129.1+/-30.2 pg/ml, 130.5+/-57.8 pg/ml, 120.2+/-45.1 pg/ml and 124.9+/-31.8 pg/ml, respectively. Plasma levels of soluble P-selectin was only slightly, but non-significantly higher in patients with unstable angina pectoris and stable coronary artery disease (184.2+/-85.4 ng/ml and 201.6+/-67.9 ng/ml, respectively) than in patients with the acute myocardial infarction (157.4+/-88.4 ng/ml) or normal subjects (151.4+/-47.1 ng/ml). The difference in plasma levels of soluble ICAM-1 between the blood withdrawn from the coronary sinus and systemic circulation correlated significantly with the corresponding difference in plasma levels of soluble P-selectin and E-selectin. In conclusion, the tissue factor and the tissue factor pathway inhibitor play a crucial role in the initiation of arterial thrombosis. The tissue factor pathway inhibitor levels are increased both in the systemic blood and in the coronary sinus of patients with the acute coronary syndrome.     

Alpha-human atrial natriuretic polypeptide is released from the heart and circulates in the body

In order to clarify whether or not atrial natriuretic polypeptides are hormones in man, we have measured plasma alpha-human atrial natriuretic polypeptide (alpha-hANP)-like immunoreactivity (alpha-hANP-LI) with or without extraction procedure. alpha-hANP-LI was detected in plasma extracts from all 5 normal subjects and 7 patients with heart diseases. The alpha-hANP-LI concentration in normal peripheral plasma was 37.7 +/- 7.0 pg/ml (mean +/- SE). Plasma concentrations of alpha-hANP-LI in the coronary sinus obtained by cardiac catheterization were 3 to 10 times higher than those in the peripheral vein, inferior vena cava, right atrium, pulmonary artery and aorta. High performance gel permeation chromatography coupled with a radioimmunoassay (RIA) for alpha-hANP revealed that alpha-hANP-LI in normal peripheral plasma eluted at the position corresponding to that of authentic alpha-hANP without detectable amounts of high molecular weight forms. alpha-hANP-LI extracted from plasma taken from the coronary sinus of two patients also showed a single peak of alpha-hANP-LI co-eluting with alpha-hANP. In contrast, not only alpha-hANP but gamma-hANP and beta-hANP, high molecular weight forms, were present in the human atrial tissue. These results indicate that alpha-hANP is the predominant form of alpha-hANP-LI in human plasma and that this form generated in the atrial cardiocytes is preferentially released from these cells and circulates in the body.     

Presence of immunoreactive endothelin in human plasma

A highly specific and sensitive radioimmunoassay has been established for measurement of human endothelin (hET) in human plasma. After extraction of plasma with an octyl-silica column, this assay allowed for detection of immunoreactive (IR) hET as low as 0.2 fmol/ml. In 16 healthy subjects, the mean concentration of plasma IR-hET was 0.6 fmol/ml. Reverse-phase HPLC coupled with radioimmunoassay revealed two major IR-hET components, one corresponding to authentic hET(1-21) and another with more hydrophilicity than hET(1-21). These data indicate that ET is a circulating vasoconstrictor hormone in man.     

Short-term effect of rate control on plasma endothelin levels of patients with tachyarrhythmias

Radiofrequency catheter ablation or modification of the atrio-ventricular junction is an effective therapy of drug refractory supraventricular tachyarrhythmias (ST). Higher endothelin (ET) levels were observed during nonsustained STs. We aimed to examine the effect of sustained STs and the applied rate-control therapy on plasma ET levels. Twenty-two patients (12 men; mean age, 64.4 +/- 13.2 years; ejection fraction, 41.8 +/- 11.2%; New York Heart Association (NYHA) class I: 3 cases, NYHA II: 11 cases, and NYHA III: 8 cases) suffering of atrial fibrillation (n = 11), atrial flutter (n = 7), atrial paroxysmal tachycardia (n = 3), or sinus tachycardia (n = 1) were studied, having coronary artery disease (n = 8), dilative cardiomyopathy (n = 5), or no underlying diseases (n = 9). All groups went under catheter ablation (same protocol, duration: 35 +/- 10.3 mins; rate before ablation, 100-170/min in every case; after ablation, 70-80/min in Groups I and II and 70-90/min in Group III). A pacemaker (PM) was implanted 2 months before ablation in Group I (n = 9) and during ablation in Group II (n = 7). No PM was implanted in Group III (n = 6). A control group (n = 13; 7 men; mean age, 66.15 +/- 6.7 years) with sinus rhythm got a PM without ST and ablation. Blood samples were collected from the cubital vein immediately before (control), and 5 mins and 24 hrs after ablation. Plasma ET-1 and big ET-1 levels were measured after immunoprecipitation with Western blot analysis. There were no differences between plasma ET-1 levels in the ST groups and the control group (Groups I, II, and III vs. control group: 0.66 +/- 0.04 fmol/ml, 0.93 +/- 0.12 fmol/ml, and 0.68 +/- 0.05 fmol/ml vs. 0.50 +/- 0.05 fmol/ml, respectively; P < 0.05). Comparing the control, 5-min, and 24-hr samples, ET-1 levels decreased significantly after supraventricular tachycardia ablation in Groups I and III (control vs. Group I, 5 mins and 24 hrs: 0.66 +/- 0.04 fmol/ml vs. 0.50 +/- 0.04 fmol/ml and 0.29 +/- 0.05 fmol/ml; control vs. Group III, 24 hrs: 0.68 +/- 0.05 vs. 0.34 +/- 0.05 fmol/ml; P < 0.05). No plasma big ET-1 changes were measured in any of the groups. The rapid decrease of ET levels after catheter ablation suggests that a high ventricular rate can be a trigger of ET production. PM implantation procedure seems to interfere with the ET decrease in ST patients.     

Renal glomerular endothelin receptors in rats with high-output heart failure.

Plasma immunoreactive endothelin (irET) concentration and renal glomerular ET receptors were investigated in rats with chronic high-output heart failure. Plasma irET was higher (0.67 +/- 0.03 fmol/ml vs. 0.52 +/- 0.04 fmol/ml) and the density of glomerular ET receptors lower (Bmax: 420 +/- 20 fmol/mg protein vs. 510 +/- 12 fmol/mg protein) in rats with heart failure than in controls. Our results indicate that circulating ET levels are responsive to changes in cardiovascular hemodynamics and suggest a potential role for ET as a vasoactive regulatory peptide during heart failure.     

Determination of insulin-like growth factor-I in normal subjects and in patients with growth hormone disorders by radioimmunoassay using biosynthetic homologous peptide

A highly sensitive and specific RIA for IGF-I has been developed using recombinant DNA-derived IGF-I of very high purity and specific antiserum to it. This assay system could detect IGF-I at as low concentrations as 20-30 ng/ml. The intra-assay and interassay coefficients of variation at various concentrations of IGF-I were 4.9 to 6.5% and 5.4 to 8.0%, respectively. The recovery rate of pure IGF-I added to plasma was 77.0 +/- 3.7%. The antiserum did not cross-react with porcine insulin, biosynthetic human insulin, hGH, hEGF, the synthetic C-domain of IGF-I or that of IGF-II, but reacted equally with an analog, Thr59-IGF-I. Plasma IGF-I was extracted by the acid-ethanol method before assay to separate IGF-I from its binding protein. When plasma IGF-I was assayed without extraction, the inhibition curves of serial dilution of plasma samples from several individuals were not parallel to the standard curve of IGF-I. The plasma concentration of IGF-I was 147 +/- 49 ng/ml (mean +/- SD) in 156 normal adults aged from 20-59 years. As reported by others, the IGF-I levels were low in cord plasma (41.8 +/- 23.5 ng/ml) and plasma of patients with GH deficiency (64.6 +/- 42.0 ng/ml), while its levels were high in normal children of pubertal ages (12-13 yr, 365 +/- 126 ng/ml) and in patients with active acromegaly (562 +/- 115 ng/ml). This RIA system is a simple and useful method for determining plasma IGF-I in normal and diseased states.     

Outer membrane vesicles from Neisseria meningitidis: effects on cytokine production in human whole blood

The Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine consists of outer membrane proteins (OMPs) as main antigens with significant amounts of lipopolysaccharide (LPS; 5-9% relative to protein). We have studied the ability of this OMV vaccine preparation to induce secretion of pro-inflammatory cytokines, tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), interleukin 6 (IL-6), interleukin 8 (IL-8) and anti-inflammatory cytokines, interleukin 4 (IL-4), interleukin 10 (IL-10) and interleukin 13 (IL-13) in a human whole blood model. Plasma levels of TNF-alpha, IL-1beta, IL-6 and IL-8 were massively increased; mean peak levels of TNF-alpha 44 696+/-7764, IL-1beta 38 043+/-5411, IL-6 10 057+/-1619 and IL-8 30 449+/-5397 pg/ml were obtained with an OMV-LPS concentration of 1 microg/ml; corresponding levels in control plasmas were below the detection limit of the assay. Mean maximal level of IL-10 (2540+/-144 pg/ml) was obtained at OMV-LPS concentration of 10 microg/ml, after 24 h; while the level in control plasma was below detection limit. OMV-LPS did not induce release of IL-4 and IL-13 in doses from 0.001-10 microg/ml. The present results show that OMVs from meningococci have potent pro-inflammatory properties and are likely to contribute to the observed local and systemic inflammatory effects.     

Day-night variations in plasma melatonin and arginine vasotocin concentrations in chronically cannulated flounder (Platichthys flesus).

Chronically catheterised, free swimming flounder (Platichthys flesus) have been used in experiments examining the day-night variations in circulating levels of melatonin (Mel) and arginine vasotocin (AVT). Under normal photoperiod (16 h light/8 h dark) serial blood samples taken from individual fish demonstrated a Mel rhythm with daytime levels at 09.00 and 15.00 h (238+/-14 and 179+/-12 fmol x ml(-1), respectively) lower than those at 23.00 h (1920+/-128 fmol x ml(-1)). Maintenance of fish in 24-h light abolished the light/dark Mel rhythm and circulating levels were comparable to those measured during the day in fish under normal photoperiod illumination. In fish maintained under 24 h dark, although a daily rhythm was still apparent, at the time when it would be normally dark, plasma Mel concentration was reduced and at times when it would be normally light, levels were higher than in fish maintained under normal light/dark illumination. Plasma AVT concentrations were higher in fish during the day (4.4+/-0.8 fmol x ml(-1)) than those at night (1.5+/-0.4 fmol x ml(-1)), the opposite to that seen with Mel. During acute study infusion of AVT resulted in reduced levels of plasma Mel, although this did not achieve statistical significance. Infusion of Mel did not alter circulating AVT concentration.     

A novel IL-18BP ELISA shows elevated serum IL-18BP in sepsis and extensive decrease of free IL-18

IL-18 binding protein (IL-18BP) is a circulating antagonist of the proinflammatory Th1 cytokine IL-18. It effectively blocks IL-18 by forming a 1:1 high affinity (Kd=400 pM) complex, exhibiting a very low dissociation rate. We have developed a sandwich ELISA for IL-18BPa and determined its limit of detection (62 pg/ml). Interference by IL-18 and related cytokines, as well as cross reactivity with other IL-18BP isoforms (b, c, and d) were determined. Using this ELISA, we measured serum IL-18BPa in large cohorts of healthy individuals and in septic patients. Serum IL-18BPa in healthy individuals was 2.15+/-0.15 ng/ml (range 0.5-7 ng/ml). In sepsis, the level rose to 21.9+/-1.44 ng/ml (range 4-132 ng/ml). Total IL-18 was measured in the same sera by an electrochemiluminescence assay and free IL-18 was calculated based on the mass action law. Total IL-18 was low in healthy individuals (64+/-17 pg/ml) and most of it ( approximately 85%) was in its free form. Total IL-18 and IL-18BPa were both elevated in sepsis patients upon admission (1.5+/-0.4 ng/ml and 28.6+/-4.5 ng/ml, respectively). At these levels, most of the IL-18 is bound to IL-18BPa, however the remaining free IL-18 is still higher than in healthy individuals. We conclude that IL-18BPa considerably inhibits circulating IL-18 in sepsis. Yet, exogenous administration of IL-18BPa may further reduce circulating IL-18 activity.     

Brain natriuretic peptide-like immunoreactivity is present in human plasma

A highly specific and sensitive radioimmunoassay (RIA) for a novel porcine brain natriuretic peptide (BNP) has been established to elucidate whether BNP-like immunoreactivity (LI) is present in human plasma. The antibody used was specific for BNP without any crossreactivities with known human atrial natriuretic peptides (hANP). After extraction of human plasma with Sep-Pak cartridge, this assay allowed for detection of BNP-LI as low as 0.1 fmol/tube. In 12 healthy subjects, the mean concentrations of plasma BNP-LI were 1.5 fmol/ml. Reverse-phase HPLC coupled with BNP RIA revealed that the single major component with BNP-LI, corresponding to porcine BNP(1–26), was apparently distinct from that of -hANP. These data indicate that a small molecular mass form with BNP-LI circulates in human blood.     

Effects of cardiotrophin-1 on hemodynamics and endocrine function of the heart

Cardiotrophin-1 (CT-1), a member of the interleukin-6 superfamily of cytokines, possesses hypertrophic actions and atrial natriuretic peptide (ANP)-producing activity in vitro. The goal of our study is to elucidate whether CT-1 affects the cardiovascular system in vivo. Intravenous injection of CT-1 (4-100 microg/kg) in conscious rats evoked significant declines in blood pressure and reflex increases in heart rate (HR) in a dose-dependent manner. CT-1 induced no significant change in cardiac output (from 260.7 +/- 11.0 to 264.7 +/- 26.6 ml. min(-1). kg(-1), P = not significant), which was compatible with the results from isolated perfused rat hearts; HR, change in pressure over time, left ventricular developed pressure, and perfusion pressure were unaffected. Northern blot and RT-PCR analyses revealed that CT-1 increased expression of inducible nitric oxide synthase (iNOS) in lung and aorta but not in heart or liver. Pretreatment with aminoguanidine, a specific iNOS inhibitor, inhibited both iNOS mRNA production and the depressor effect of CT-1. Interestingly, CT-1 increased ventricular expression of ANP and brain natriuretic peptide (BNP). The data demonstrate that CT-1 elicits its hypotensive effect via a nitric oxide-dependent mechanism and that CT-1 induces ANP and BNP mRNA expression in vivo.     

Effect of passive repetitive isokinetic training on cytokines and hormonal changes

It is well known that muscle strength and power are important factors in exercise. Plyometrics is designed to gain muscle strength and power in a shock method. The passive repetitive isokinetic (PRI) machine is developed for plyometrics. The present study aims to understand the effect of ten-week PRI training in different intensities on human plasma concentration cytokines as well as hormonal changes. Thirty young male subjects were enrolled into the ten-week PRI training program and were divided randomly into traditional, low- and high-intensity PRI training groups. Blood samples were obtained before, during, after and 1-, 2-, 3-, 5- and 7-day (D) post-training. The plasma concentrations of cytokines and hormones were measured by an enzyme-linked immunosorbent assay (ELISA). Elevated plasma IL-2 was found in the subjects in all the training programs. Significant increases of proinflammatory cytokines IL-1beta and TNF-alpha were observed at post 7 D in the high-intensity PRI training (29.5 +/- 4.4 and 515.8 +/- 127.1 pg/ml, respectively). No significance in differences in the plasma concentration of IL-6 was observed in the traditional and low-intensity PRI training. Significant elevation of IL-6 was found at post 5 D in high-intensity PRI training. Higher plasma IL-6 concentration was observed at post 3 and 5 D in high-intensity PRI training compared to low-intensity PRI training (P < 0.05). Significant elevation of plasma IL-15 during (week 6) and after (post 0 D) was observed in low-intensity PRI training. Also, there were differences between low-intensity PRI training and traditional training at post 0, 2, 3, and 5 D. The plasma concentration of cortisol was decreased to the lowest value (118.0 +/- 17.3 ng/ml) at post 0 D in traditional training, then returned to the baseline (220.5 +/- 19.1 ng/ml). In the high-intensity PRI training, but not in the low-intensity PRI training, the cortisol level dropped from 224.9 +/- 25.8 ng/ml at post 0 D down to the 123.2 +/- 22.6 ng/ml at post 1 D. Significant differences were found at post 1 and 5 D between low- and high-intensity PRI training, and post 0, 1, 2, and 3 D between traditional and high-intensity PRI training. Significant increased testosterone was found post 0, 1, 2, and 3 D in traditional training. Higher plasma testosterone was observed during and the recovery period in low-intensity, but not in high-intensity, PRI training. In conclusion, high-intensity PRI training could induce the proinflammatory cytokines, i.e., IL-1beta and TNF-alpha, and decrease plasma cortisol in the recovery period.     

Water immersion increases the concentration of the immunoreactive N-terminal fragment of proatrial natriuretic factor in human plasma

Atrial natriuretic factor (ANF) N-terminal (ANF 1-98) and C-terminal (ANF 99-126) fragments were determined by radioimmunoassay in human plasma. Mean basal plasma ANF N-terminal concentrations in 9 healthy subjects were 461 +/- 58 fmol/ml, significantly (p less than 0.0001) higher than ANF C-terminal concentrations (4.8 +/- 0.5 fmol/ml). Central volume stimulation by one hour head-out water immersion (WI) induced a significant (p less than 0.01) increase of the C-terminal peptide levels to 11.6 +/- 2.3 fmol/ml, paralleled by a significant (p less than 0.001) increase of the N-terminal fragment levels to 749 +/- 96 fmol/ml. Increases of plasma concentrations of both fragments upon WI correlated significantly (r = 0.71; p less than 0.05). These data suggest cosecretion of the N-terminal fragment with the C-terminal fragment of pro ANF 1-126 following a physiological stimulus of ANF release in man.