Serum sclerostin levels decline in post-menopausal women with osteoporosis following treatment with intermittent parathyroid hormone.

OBJECTIVE: This study was carried out in order to evaluate the effect of 18-month treatment with PTH (1-34) or PTH (1-84) on serum sclerostin levels in humans. SUBJECTS AND METHODS: We investigated 10 women with severe osteoporosis, previously treated with alendronate and 20 untreated osteoporotic women. Subjects with severe osteoporosis were randomly divided into 2 groups of 5 patients each; the first group was treated with 20 µg of PTH (1-34) and the second one with 100 µg of PTH (1-84) according to an open-label design. Fasting blood samples were collected at baseline and at 2, 4, and 24 h after hormone administration. The same protocol was followed at month 1, 6, 12, 18. Serum sclerostin levels were measured at each time point by a sandwich-type enzyme-linked immunosorbent assay. RESULTS: Basal serum sclerostin levels were not significantly different between patients previously treated with alendronate and those never treated. No significant acute change of serum sclerostin levels was observed after PTH administration. Fitting a mixed effect regression model, we found a significant time effect (p=0.0012) using the sclerostin level as the response variable and the month of drug administration as a single covariate. Treatment with both PTH molecules induced a monthly mean reduction of sclerostin levels of 0.1956 pmol/l. CONCLUSIONS: Our results indicate that long-term therapy with PTH (1-34) or PTH (1-84) in women with osteoporosis previously treated with alendronate is associated with a reduction in circulating sclerostin levels. This is a putative mechanism through which PTH performs its anabolic action.

Serum sclerostin levels positively correlate with lumbar spinal bone mineral density in postmenopausal women--the six-month effect of risedronate and teriparatide.

UNLABELLED: Sclerostin is expressed by osteocytes and inhibits bone formation by osteoblasts. In this study, serum sclerostin was positively correlated with either lumbar spinal bone mineral density or T-score. Furthermore, serum sclerostin was increased after 6 months treatment with risedronate, whereas remained unchanged after 6 months teriparatide treatment. INTRODUCTION: The primary aim of this study was the evaluation of serum sclerostin levels in postmenopausal women and their association with bone mineral density (BMD) and bone turnover markers. The secondary aim was the evaluation of treatment with either teriparatide (TPTD) or risedronate (RIS) on serum sclerostin levels in women with postmenopausal osteoporosis. METHODS: Women with postmenopausal osteoporosis, assigned to receive either TPTD (TPTD group, n = 13) or RIS (RIS group, n = 36) for 6 months, and non-osteoporotic early postmenopausal women (NOEP group, n = 13) were recruited. Main outcome measure was serum sclerostin levels. RESULTS: Serum sclerostin was higher in the NOEP group at baseline compared with either TPTD group (p = 0.007) or RIS group (p = 0.049). Sclerostin was positively correlated with both lumbar spinal (LS) BMD (r = 0.353; p = 0.005) and T-score (r = 0.501; p < 0.001) and negatively correlated with intact parathyroid hormone (r = -0.343; p = 0.024) at baseline. Multiple regression analysis showed that either LS BMD (Beta = 0.653; p = 0.018) or T-score (Beta = 0.711; p = 0.005) were independent predictors of serum sclerostin levels. No significant correlation was observed between serum sclerostin and bone turnover markers or estradiol at baseline. Sclerostin was significantly increased 6 months post-treatment in RIS group (p = 0.002), whereas remained statistically unaffected in the TPTD group. CONCLUSIONS: Serum sclerostin is decreased in women with postmenopausal osteoporosis compared with non-osteoporotic early postmenopausal women and is positively correlated to either LS BMD or LS T-score. Furthermore, serum sclerostin was increased after 6 months treatment with RIS, whereas remained essentially unchanged after 6 months TPTD treatment.

Serum levels of receptor activator of nuclear factor kappaB ligand (RANKL) in healthy women and men.

BACKGROUND: Osteoporosis is one of the most common conditions associated with aging. It is based on an excess of bone resorption over bone formation, leading to an imbalance of bone turnover. The receptor activator of nuclear factor kappaB ligand (RANKL) is an important regulator of bone metabolism. OBJECTIVE: The aim of this investigation was to evaluate potential age- and gender-related changes in free RANKL and total RANKL (free RANKL+RANKL/osteoprotegerin complexes). METHODS: Two hundred and forty volunteers with a median age of 48 years were included in the study. Serum levels of free RANKL and total RANKL were evaluated. RESULTS: On average, men have a 1.77-fold higher free RANKL level and a 2.12-fold higher free/total RANKL ratio than women of the same age. On average, the RANKL levels decrease by approximately 13% every five years. CONCLUSION: This study showed that serum levels of free RANKL and total RANKL decrease with age, and also revealed some gender-related differences.

Receptor activator of nuclear factor kappaB ligand and osteoprotegerin in men with thyroid cancer.

BACKGROUND: Suppressive thyroid hormone therapy is generally a lifelong treatment for patients with differentiated thyroid cancer (DTC). However, long-standing thyrotropin (TSH) suppression is a risk factor for osteoporosis. Osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) are central regulators of bone turnover. The aim was to analyze the effects of a suppressive thyroid hormone therapy in males with DTC on the OPG/RANKL system and on bone metabolism. PATIENTS AND METHODS: The OPG and soluble RANKL (sRANKL) were determined in 40 men (mean age, 53.2 years) with DTC on suppressive thyroid hormone therapy (TSH; 0.053 +/- 0.037 mU L(-1), duration 5.7 +/- 4.4 years) and 120 healthy controls matched for age. The markers of bone metabolism were C-terminal telopeptide of type I collagen in serum (sCTx) and osteocalcin (OC). RESULTS: The control group had OPG values (mean +/- SD) of 1.9 +/- 1.0 pmol L(-1) and sRANKL values of 0.40 +/- 0.62 pmol L(-1). In patients with DTC, results for OPG were 3.03 +/- 1.04 pmol L(-1) (P < 0.05) and for sRANKL were 0.13 +/- 0.16 pmol L(-1) (P < 0.05). The control group presented values for sCTx of 2669 +/- 1132 pmol L(-1) and for OC of 17.89 +/- 6.5 ng mL(-1). Patients with DTC on suppressive thyroid hormone therapy had increased sCTx values of 3810 +/- 2020 pmol L(-1) (P = 0.03) but comparable OC values of 19.21 +/- 7.67 ng mL(-1) (NS). CONCLUSIONS: Suppressive thyroid hormone therapy in men with DTC increased bone degradation and induced significant changes in the OPG/RANKL system. These changes include, besides the risk of osteoporosis, possible negative effects on the vascular function and an increased risk of cardiovascular disease.

Serum levels of cathepsin K decrease with age in both women and men.

Bone turnover increases with age. In a previous study, we reported on bone metabolism in young and elderly women and men. The aim of the present investigation was to evaluate potential age- and gender-related changes in cathepsin K, a cysteine protease that plays an important role in the degradation of the organic matrix of bone. Twenty-five healthy premenopausal women, 24 young healthy men, 26 elderly women, and 25 elderly men participated in the study. Elderly women and men had significantly lower cathepsin K levels than younger ones. In both men and women, serum levels of cathepsin K were negatively correlated with age. In men there was a statistically significant negative correlation between serum levels of cathepsin K and osteoprotegerin, which inhibits osteoclast differentiation and activation. No association was found between serum levels of cathepsin K and bone-specific alkaline phosphatase, osteocalcin, or 25-hydroxy vitamin D. Thus, the age-related increase in OPG, which markedly inhibits the expression of cathepsin K, may also reduce serum levels of cathepsin K. Despite the age-related increase in bone resorption, this study shows lower cathepsin K values in elderly women and men than in younger subjects. It might be speculated that a different enzyme could compensate for the decline in cathepsin K during old age.

Osteoprotegerin and the receptor activator of NF-kappa B ligand in the serum and synovial fluid. A comparison of patients with longstanding rheumatoid arthritis and osteoarthritis.

We examined OPG and soluble RANKL in the serum (sOPG, sRANKL) and synovial fluid (synOPG, synRANKL) in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). OPG and RANKL were measured in 85 patients (44 with RA, 41 patients with OA) in serum and synovial fluid as well. For measuring of OPG and RANKL ELISA tests were used. The results of OPG and RANKL were compared with clinical and radiological scores. We found a negative correlation for OPG and RANKL in synovial fluids: not only for the whole group of patients (P < 0.003, r = -0.32), but also for the subgroups (RA: P < 0.04, r = -0.28, OA: P < 0.002, r = -0.54). SRANKL and synRANKL were positively correlated in the whole group (P < 0.01, r = 0.25) and in the OA group (P < 0.02, r = 0.35); the RA group was showing a trend (P < 0.063, r = 0.24), however. Serum OPG was lower in RA, synOPG higher in OA. The difference between the two patient groups was only significant for synOPG (P < 0.03, r = 0.056), but not for sOPG (P < 0.09, r = 0.19), sRANKL (P < 0.43, r = 0.85) or synRANKL (P < 0.11, r = 0.22). The synOPG:synRANKL ratio was significantly correlated with the Larsen score (P < 0.004, r = 0.38). Synovial OPG is significantly decreased in rheumatoid joints, whereby synovial RANKL is increased. Lower synOPG could reflect a lower protective effect on bone, thus leading to an earlier and more pronounced bone destruction in RA. However, the effect of different mediators for joint destruction in RA and OA seems not to be important to the pathophysiological changes in the joints. The upregulation of serum OPG might be the result of the inflammation; in contrast, an upregulation of RANKL could not be found in the serum of patients with RA and OA.

Is pro-matrix metalloproteinase-3 a marker for posttraumatic cartilage degradation?

OBJECTIVE: Since the development of posttraumatic osteoarthritis (OA) is a relatively slow process, estimation of OA risk would be of value with regard to chondroprotective measures and medication. In this study we investigated the significance of pro-matrixmetalloproteinase-3 (proMMP-3) for this purpose. DESIGN: Synovial fluid (SF) and serum samples were collected from 259 patients of our trauma clinic at the time of arthroscopy. The extent of cartilage damage was assessed according to the Outerbridge-score. ProMMP-3 levels in SF and serum were determined by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody. Additionally we determined SF and serum levels of total MMP-3 and COMP levels as well as TIMP-1 and -2 concentrations in 40 randomly selected patients by ELISA. RESULTS: Serum proMMP-3 levels of the total cohort were markedly increased compared to healthy controls (P<0.007). The comparison of serum and SF lavage proMMP-3 concentrations showed a significant correlation (r(s)=0.41, P<0.0001), however, only 26% of the investigated samples were increased above normal ranges. The grade of cartilage damage did not correlate with enzyme concentration neither in patients' serum nor in SF samples. ProMMP-3 SF concentration was increased early after trauma. Furthermore, proMMP-3 correlated significantly with total MMP-3 serum and SF levels as well as COMP SF levels. CONCLUSIONS: The measurement of proMMP-3 in serum or SF did not reflect the present cartilage damage and thus appears to have only minor potential for clinical use, but it should be considered for longitudinal studies, since it may reflect a risk for cartilage degradation in a subset of patients.

Serum levels of osteoprotegerin increase with age in a healthy adult population.

Regulation of the balance of osteoblastic and osteoclastic activity is critical for the understanding of normal cell biology and forms the basis of metabolic bone diseases. Our study reports about influences of age and gender on serum levels of osteoprotegerin (OPG) and its association to other clinical parameters of bone metabolism in a precisely determined cohort of 1134 healthy subjects at 17 Austrian outpatient bone clinics, aged between 19 and 96 years (females n = 687, 50 +/- 21 years, 19-94, and males n = 447, 52 +/- 13.5 years, 24-96). Mean OPG serum levels for all participants were 50.83 +/- 51.47 pg/ml (n = 1134; median 36, 2-584) and we observed a sharp increase in females after 60 years and in males after 70 years of age. OPG serum levels increased significantly by age, 2.1 pg/ml in females and 1.9 pg/ml in males for every year (P < 0.0001). Correlation of OPG serum levels and several bone parameters of bone metabolism showed that OPG negatively correlated with serum iPTH (r = -0.14; P < 0.001) and with serum estradiol in females (r = -0.16, P < 0.0001). Bone mineral density measured by DXA method at the spine and at the hip did not correlate with OPG serum levels, except a borderline negative correlation at the trochanteric region (r = -0.1, P < 0.05) in females only. Our results show a significant increase of osteoprotegerin with age in healthy females and males but fluctuations do not predict bone mineral density under in vivo conditions.

[Muscle cell proteins are selectively released into the blood stream by marathon running]. / Marathonlaufen lässt intrazelluläre Proteine aus Muskelzellen selektiv in das Plasma austreten.

In 19 marathon runners of both sexes, plasma concentrations of total creatine kinase (CK) activity, CKMB mass, myoglobin and troponin I were determined before and immediately after the race. Total CK activity and myoglobin increased significantly in all runners and showed neither a correlation with the individual age of the runners nor with the time they needed to reach the goal. In 12 of the runners, CKMB mass increased during the race to a level suggesting myocardial necrosis. However, the runners did not show any detectable deterioration of cardiac function after the race. The appearance of considerable amounts of muscle proteins in plasma precipitated by the muscle strain during the race seems explained by damage of skeletal muscle detected by histological studies. These phenomena may also be a consequence of profoundly disturbed cellular permeability, perhaps due to a kind of local stunning of muscle tissue by prolonged muscular strain.

Assessment of vitamin D and calcium status in healthy adult Austrians.

BACKGROUND: Because there is reason to assume that also in Austria calcium and vitamin D malnutrition is wide-spread, we initiated a comprehensive study on calcium and vitamin D status in relation to bone health in a large group of the normal adult population. SUBJECTS AND METHODS: We assessed dietary calcium and vitamin D intake, serum concentrations of Ca2+, phosphate, alkaline phosphatase, 25(OH)D, 1,25(OH)2D, parathyroid hormone (PTH), follicle-stimulating hormone (FSH), sex hormones and bone mineral density (BMD) by double-energy X-ray absorptiometry at five different skeletal sites in 648 females and 400 males (age 21-76 years). RESULTS: Mean daily intake of vitamin D (101 IU, range 0.2-320) and calcium (569 mg, range 40-2170) was significantly less than the respective recommended dietary allowances. Two hundred and seventy-one (26%) individuals had hypovitaminosis D with serum 25(OH)D < 12 ng mL(-1), while serum Ca2+ was less than normal in 82 (7.8%) subjects. Multiple regression analysis revealed significant correlations between mean calcium intake and BMD in the femoral region in the men (r = 0.13, P < 0.05) though not in the women. No consistent data could be obtained for associations between BMD and vitamin D status, except for 25(OH)D and BMD at the spine in the men (r = 0.10, P < 0.05). 25(OH)D correlated negatively (P < 0.05) with age in the women (r = -0.11) and with PTH in the women (r = -0.11) and men (r = -0.16). Inversely, a significant (P < 0.001) age-related increase in PTH was observed in both sexes (men, r = 0.19; women, r = 0.14). CONCLUSIONS: Prevalence of hypovitaminosis D in adult Austrians is an imminent risk for development of secondary hyperparathyroidism with advancing age, and requires timely correction of nutritional deficits.

Normative data of bone mineral density in an unselected adult Austrian population.

BACKGROUND: There is increasing evidence that correct interpretation of bone mineral density (BMD) measurements by dual energy X-ray absorptiometry (DEXA) requires a population-specific reference range. We therefore collected data on age-related BMD in a random sample of the normal adult Austrian population to establish an appropriate normative database. METHODS: We measured BMD by DEXA at five different skeletal sites in 1089 subjects, i.e. 654 females and 435 males, aged between 21-76 years, who had been recruited by 17 centres across Austria. RESULTS: Age-related bone loss was observed until age 65 years with significant changes at the lumbar spine (r = -0.23), total hip (r = -0.07), trochanter (r = -0.10), femoral neck (r = -0.30) and Ward's triangle (r = -0.40) in the women but only at the femoral neck (r = -0.23) and at Ward's triangle (r = -0.40) in the men. When we calculated T scores from the BMD data of the young normal adult study population and used the T score set points according to the WHO classification of osteopenia and osteoporosis, we found that, depending on the skeletal site measured, 7.6-27.4% of the women and 16-41% of the men in our study group had low bone mass, whereas 0.6-2.7% of the female and 0.2-1.0% of the male study population were osteoporotic. However, osteoporosis was indicated in 4-9-fold more females and 5-15-fold more males when we based our estimates on the normative data provided by the manufacturers of the DEXA systems. CONCLUSION: Our data underscore the importance of using a population-specific reference range for DEXA measurements to avoid overdiagnosis of osteoporosis.

Prediction of outcome by neurohumoral activation, the six-minute walk test and the Minnesota Living with Heart Failure Questionnaire in an outpatient cohort with congestive heart failure.

AIMS: To compare the precursor of atrial and brain natriuretic peptide (N-ANP, N-BNP), brain natriuretic peptide (BNP), big endothelin-1, the 6-min walk test and the Minnesota Living with Heart Failure Questionnaire (LHFQ) with regard to short-term outcome in an ambulatory heart failure population. METHODS AND RESULTS: Ninety-six individuals (left ventricular ejection fraction of 26+/-10%) were included in the study. Within 1 day blood samples of N-ANP, N-BNP, BNP and big endothelin-1 were obtained, and the 6-min walk test and LHFQ were measured. The predictive power of these variables - including renin-angiotensin system antagonist therapy - in respect of 1-year event-free survival were calculated with a Cox regression analysis. All investigated variables had the power to predict outcome in a univariate analysis. Multivariate analysis revealed that N-ANP (chi-square=58 P<0.0001), BNP (chi-square=8 P<0.01), the LHFQ (chi-square=6 P<0.02) and the renin-angiotensin system antagonist (chi-square=4 P<0.05), are independent predictors. CONCLUSION: We conclude that, in an open clinical cohort of patients with large differences in the progression of the disease, N-ANP, BNP and LHFQ are the most reliable predictors of worsening heart failure in the short term. However, the dosage of the ACE inhibitor influenced short-term survival in this population.

Plasma atrial natriuretic peptide (ANP) fragments proANP (1-30) and proANP (31-67) measurements in chronic heart failure: a useful index for heart transplantation?

The family of the atrial natriuretic peptides, proANP fragments and the active alphaANP, is strongly related to heart disease. The aim was to study in CHF subjects the relation of mdANP and NtANP with brain natriuretic peptide (BNP) and with other traditional medical parameters. Sixteen CHF patients (aged 51.9+/-13.7 years) and 16 healthy subjects age matched (50.8+/-5.9 years) were selected. Both NtANP and mdANP were higher in CHF patients than in healthy subjects (1436+/-288 vs. 288+/-22 pmol/l p<0.001 and 2305+/-383 vs. 423+/-65 pmol/l p<0.0001, respectively). BNP in CHF patients was 28.0+/-9 pmol/l (reference values 1.7+/-1.8 pmol/l). Both NtANP and mdANP demonstrated positive correlation with BNP, p<0.0001 and with left atrial end-systolic volume, p<0.05. BNP correlated with left ventricular mass, p<0.03. In conclusion, plasma NtANP and mdANP analyses are useful laboratory markers in CHF patient investigation and follow up. In particular, they could be employed as non-invasive parameters to follow up worsening of systolic dysfunction until heart transplantation is required.

N-terminal fragments of the proatrial natriuretic peptide in plasma and urine of kidney graft recipients.

BACKGROUND: Successful kidney transplantation normalizes elevated proatrial natriuretic peptide (proANP) plasma concentrations of renal failure patients in the early posttransplant period. We evaluated plasma and urinary proANP fragments in the late posttransplant period. METHODS: Immunoreactive proANP(1-30) and proANP(31-67) were determined in 389 renal transplant (Rtx) recipients in the long-term, follow-up period and in 16 healthy controls. RESULTS: Rtx recipients had significantly higher concentrations of proANP(1-30) and proANP(31-67) in both plasma and urine than healthy controls. Although their graft function was normal, all of these long-term Rtx recipients were taking glucocorticoids, which increase proANP(1-30) and proANP(31-67) in the circulation to the extent found in this investigation. Two-thirds of these recipients were also taking cyclosporine, which also increases atrial peptides. Urinary proANP(31-67) was significantly higher than urinary proANP(1-30); 5.5-fold in Rtx patients and 2-fold in controls. Deterioration of renal graft function was associated with a rise of plasma proANP(1-30) from 0.98+/-0.66 to 6.28+/-3.55 nmol/l (P<0.0001) and plasma proANP(31-67) from 1.81+/-1.04 to 7.89+/-3.76 nmol/l (P<0.0001). Urinary excretion of proANP(1-30) increased from 0.27+/-0.34 to 5.96+/-5.07 nmol/24 hr (P<0.0001) and proANP(31-67) from 1.45+/-0.85 to 12.23+/-5.12 nmol/24 hr (P<0.0001). Also proteinuria enhanced plasma and urinary proANP fragments. CONCLUSIONS: ProANP(1-30) and proANP(31-67) of Rtx recipients are affected by immunosuppression, hypertension, renal failure, and proteinuria. One would have expected proANP(1-30) and proANP(31-67) not to normalize because of the glucocorticoids that they were receiving.

Bone turnover markers and sex hormones in men with idiopathic osteoporosis.

BACKGROUND: In contrast to osteoporosis in postmenopausal women, osteoporosis in men has received much less attention. PATIENTS AND METHODS: We determined various biochemical parameters of bone metabolism and sex hormones in 31 men with idiopathic osteoporosis and 35 age matched control subjects. RESULTS: In the men with osteoporosis, a significantly increased urinary excretion of deoxypyridinoline (5.3 +/- 0.2 vs. 4.6 +/- 0.2 nmol mmol-1 creatinine; P = 0.033) in addition to increased serum levels of the c-terminal telopeptide of type I collagen (2677 +/- 230 vs. 2058 +/- 153 pmol; P = 0.037) were found. While parameters of bone formation were not significantly different in the patients and controls, serum bone sialoprotein levels were significantly decreased in the patients (3.7 +/- 0.8 vs. 12.4 +/- 4.0 ng mL-1; P = 0.021). Moreover, in men with idiopathic osteoporosis, lower levels of estradiol (91.3 +/- 5.8 vs. 114.6 +/- 7.8 pmol L-1; P = 0.044), higher levels of sex hormone binding globulin (31.5 +/- 3.1 vs. 24.2 +/- 1.4 nmol L-1; P = 0.034) and a decreased free androgen index (42.6 +/- 5.2 vs. 56.4 +/- 5.9; P = 0.016) were seen. Serum estradiol levels correlated negatively with several parameters of bone resorption. CONCLUSIONS: In men with idiopathic osteoporosis, bone resorption is increased and exceeds bone formation. The excessive bone resorption seen in idiopathic male osteoporosis may be due to decreased estradiol levels and low levels of bioavailable testosterone.

Plasma concentration and urinary excretion of N-terminal proatrial natriuretic peptides in patients with kidney diseases.

BACKGROUND: Biologically active N-terminal fragments such as proANP(1-30), proANP(31-67), and proANP(1-98) derive from the prohormone of alpha-human atrial natriuretic peptide [proANP(99-126) or alpha-ANP]. No systematic data are available for patients with different kidney diseases. METHODS: Specific immunoassays were developed to determine plasma and urine concentrations of these fragments in 121 patients with different degrees of kidney function and urinary protein excretion, respectively. RESULTS: In patients with kidney disease and normal renal function without proteinuria, circulating proANP(1-30) and proANP(31-67) increased 2.8-fold and 6.5-fold, respectively. Urinary excretion of proANP(31-67) increased by a factor of 7.7 in these patients, whereas proANP(1-30) was not affected. Patients with impaired renal function had a dramatic increase of urinary proANP(31-67) excretion even before serum creatinine levels started to rise. The progression of renal failure caused a significant rise of circulating proANP(1-30) (4.3-fold) and proANP(31-67) (3.0-fold) compared with patients with normal renal function. Urinary excretion of proANP peptides significantly increased, particularly when the serum creatinine level was> 5.0 mg/dL [proANP(1-30) 26-fold, proANP(31-67) 8.4-fold]. Urinary excretion of proANP(1-30) increased up to 4.4-fold and urinary excretion of proANP(31-67) increased up to 2.4-fold in patients with proteinuria in excess of 3 g/24 h. CONCLUSIONS: Plasma concentrations and urinary excretion of proANP(1-30) and proANP(31-67) are affected by kidney disease and function, but not by proteinuria per se. It is proposed that the diseased kidney increases early urinary excretion of proANP fragments to participate in the regulation of renal function as well as sodium and water excretion.

Autocrine cell suicide in a Burkitt lymphoma cell line (Daudi) induced by interferon alpha: involvement of tumor necrosis factor as ligand for the CD95 receptor.

The CD95 receptor, a member of the tumor necrosis factor (TNF) receptor superfamily, mediates signals for cell death on specific ligand or antibody engagement. It was hypothesized that interferon alpha (IFN-alpha) induces apoptosis through activation of the CD95-mediated pathway and that CD95 and ligands of the death domain may belong to the group of IFN-stimulated genes. Therefore, the effect of IFN-alpha on CD95-CD95L expression, on the release of TNF-alpha, and on TNF receptor 1 expression in an IFN-sensitive human Burkitt lymphoma cell line (Daudi) was investigated. After 5 days' incubation, apoptosis in 81% of IFN-alpha-treated Daudi cells was preceded by a release of TNF-alpha and an induction of CD95 receptor expression. Although supernatants of IFN-treated Daudi cells induced apoptosis of CD95-sensitive Jurkat cells, CD95L was undetectable on protein or on messenger RNA levels, and the weak initial expression of TNF receptor 1 increased only slightly during IFN treatment. Surprisingly, binding of TNF-alpha to CD95 was observed and confirmed by 3 different techniques-enzyme-linked immunosorbent assay using immobilized CD95:Fc-immunoglobulin G, immunoprecipitation assay using CD95 receptor precipitates of Daudi cells, and binding of sodium iodide 125-TNF-alpha to Daudi cells, which was strongly stimulated by IFN-alpha and inhibited by CD95L, CD95:Fc, unlabeled TNF-alpha, and anti-TNF-alpha antibody. Preincubation of Daudi cells with antagonists of the CD95-mediated pathway resulted in an inhibition of IFN-alpha-mediated cell death. The present investigation shows that IFN-alpha induces autocrine cell suicide of Daudi cells by a cross-talk between the CD95 receptor and TNF-alpha. The CD95 receptor can be considered a third TNF receptor, in addition to p55 and p75.

Sandwich ELISA for proANP 1-98 facilitates investigation of left ventricular dysfunction.

Proatrial natriuretic peptides are proposed to be sensitive and specific markers for various stages of heart deficiency. Here we present a rapid and specific sandwich immunoassay for the measurement of proANP 1-98 in biological fluids like plasma, serum urine. No sample preparation prior to the assay is necessary. Polyclonal antibodies specific for distinct epitopes of proANP 1-98, predicted to be highly immunogenic, were raised in sheep and purified by immunoaffinity chromatography prior to use in the assay. Antigen binding sites of the antibodies were determined by epitope mapping with a set of peptide fragments. The capture antibody, specific for proANP 16-23, is coated directly onto microtiter plates. Recombinant proANP 1-98 is used as a standard. The biotinylated detection antibody, specific for proANP 80-88, is incubated simultaneously with 20microl of sample for 150 min. Signal is generated with a streptavidin-HRPO-conjugate and TMB as substrate. The detection limit of the assay is 50 pmol/l; intraassay CVs are below 5%, interassay CVs are lower than 10%. Dilution curves show good linearity, recovery of synthetic peptide ranged between 85% and 91%. Reference values from healthy volunteers range from 0 to 2000 pmol/l in human plasma. We conclude this assay is a easy to handle, quick and reliable method for routine measurement of proANP 1-98 and the presented manuscript describes the procedure and performance of this ELISA in detail.

Adsorption of natriuretic factors in uremia.

Patients with end-stage renal disease have a deranged sodium and water homeostasis leading to chronic volume overload. Atrial natriuretic peptides (ANPs) are circulating hormones that are involved in the regulation of volume homeostasis, blood pressure control, and electrolyte balance. In hemodialysis patients plasma ANPs are highly elevated and decrease during the dialysis session when fluid is removed. However, hemodialysis treatment never corrects the defect in the metabolism of these peptides and their circulating concentrations do not return to levels found in healthy controls. Besides uremia and chronic volume overload, other factors such as cardiac dysfunction or hypertension may contribute to the elevated plasma concentrations of ANPs. ProANP fragments which derive from the N-terminus of the ANP prohormone have been also found in the circulation and they have biological functions similar to alpha-ANP (ie, the C-terminus of the prohormone). The proANP peptides proANP(1-30), proANP(31-67), and proANP(1-98) are increased in patients undergoing regular hemodialysis treatment, but their decrease during the dialysis procedure is less pronounced than for alpha-ANP or cyclic GMP. Cellulose triacetate dialyzer membrane material lowered the plasma concentrations of proANP(1-30), proANP(31-67), and proANP(1-98) significantly more than polysulfone, whereas alpha-ANP and cyclic GMP were not differently affected. Aside from a variety of factors that influence circulating natriuretic factors in the uremic patient, there is evidence for differences in dialyzer membrane adsorption of these peptides which speculatively may be linked to dialysis-associated symptoms.