An effective regimen of intranasal salmon calcitonin in early postmenopausal bone loss

Summary In order to devise a convenient and effective therapeutic regimen of intranasal salmon calcitonin (sCT) for the treatment of early postmenopausal bone loss, we studied the effects of a 1-year course of sCT nasal spray on vertebral mineral content (VMC), assessed by dual photon densitometry, and bone turnover in 21 early postmenopausal osteoporotic women. Subjects enrolled in the study had a value above the normal average of at least one index of bone turnover: whole body retention (WBR) of ^99mTc-methylenedichloro-bisphosphonate (^99mTc-MDP), serum bone gla protein (BGP), urinary hydroxyproline/creatinine excretion (HOP/Cr). After baseline evaluation, patients were randomized for treatment with either sCT (200 IU every other day) or plabebo. Treatment with sCT significantly increased VMC by 2.7±0.9% at 6 months, and 3.3±0.8% at 1 year, whereas a progressive decline was observed in the placebo group (-2.6±0.5%, and -3.5±0.5% after 6 and 12 months, respectively). These changes were associated with a progressive and significant reduction of all parameters of bone turnover in the sCT-treated patients, whereas no changes were detected in the control group during the study period. The differences between the two groups were significant after 1 year for VMC, BGP, and WBR (P<0.05, one-way analysis of variance). Thus, 200 IU intranasal sCT administered on alternate days is adequate to stop the fast bone loss occurring early after the menopause in women with high bone turnover rates. This therapeutical modality represents an important addition to the available pharmacologic spectrum for the prevention and treatment of postmenopausal osteoporosis.     

Serum calcitonin forms and concentrations in young and elderly healthy females

To further investigate the role of calcitonin (CT) in normal physiology we studied circulating forms and the secretion after calcium clamp in young and elderly healthy females. Heterogeneity of CT in serum was disclosed after immunoextraction, fast protein liquid chromatography, and radioimmunoassay in young (27±3 years; mean±SD, n=6) and elderly females (69±6 years, n=11). Three distinct molecular forms appeared with approximate mol wt of 30, 10, and 3–4 kDa. All young women studied had considerable amounts of circulating monomer-like CT whereas several elderly had undetectable or low levels. The influence of age on basal and calcium stimulated, immunoextracted CT in serum was also studied in young (26±4 years; mean±SD, n=13) and elderly (63±6 years; n=12) healthy females. The calcium stimulation was carried out by means of the standardized calcium clamp method, where calcium was kept on a presettled level at 1.45 mmol/liter (±2%) for 60 minutes. CT was immunoextracted from serum in all series of experiments with a polyclonal antiserum directed against the mid-and carboxyterminal region of the CT molecule, and the amount of extracted CT was determined by radioimmunoassay using another polyclonal antiserum against the carboxyterminal portion. After calcium infusion, the increase in CT was significantly higher in young women than in elderly (P<0.05). At basal conditions, the CT levels were not significantly different but slightly higher in young than in elderly females. In conclusion, several elderly women lack monomer-like calcitonin in serum in contrast to young women. After calcium stimulation of calcitonin, young women had higher serum concentrations than elderly women.     

Can Sporadic Medullary Thyroid Carcinoma Be Biochemically Predicted? Prospective Analysis of 66 Operated Patients with Elevated Serum Calcitonin Levels

Measuring serum calcitonin (CT) in patients with thyroid diseases allows preoperative diagnoses of sporadic medullary thyroid carcinoma (MTC) and C-cell hyperplasia (CCH). The aim of this prospective study was to distinguish biochemically between CCH and MTC. Basal CT (bCT) was determined in 7276 consecutive patients referred for thyroid disease. Patients with recurrent, persistent, or familial MTC were excluded. When bCT was > 10 pg/ml a pentagastrin-stimulated CT (sCT) assay was performed. Patients were routinely operated on when bCT > 30 pg/ml or sCT > 100 pg/ml or when other indications for surgery were present. An extensive search for CCH or microscopic MTC was conducted by immunochemistry. Pathologic findings were correlated with the bCT and sCT values. In this study 66 patients were included. No morphologic alterations of C-cells were observed in 5 patients; 16 patients presented with CCH and 45 with MTC. Statistical analysis revealed a correlation of sCT and overall bCT with tumor size and staging (p <0.001). Considering cutoff values for bCT of ≤ 30 pg/ml and for sCT of ≤ 200 pg/ml, the positive predictive value of the test to detect MTC was 100% and the negative predictive value 63%. No patients with MTC at stage 2 to 4 had bCT <30 pg/ml or sCT <200 pg/ml. A bCT value of ≤ 30 pg/ml or sCT ≤ 200 pg/ml (or both) is highly predictive of MTC, requiring total thyroidectomy with lymph node dissection. Values of bCT <30 pg/ml and sCT <200 pg/ml do not distinguish between CCH and MTC at stage 1. In this case total thyroidectomy at least is recommended, and the role of nodal dissection might be discussed.     

Effect of estrogen and calcitonin on vertebral bone density and vertebral height in osteoporotic women

Estrogen and calcitonin increase bone density of osteoporotic women, particularly on the axial skeleton. To verify whether the effect of these drugs might in part be biased by spurious increases in bone density due to radiologically irrelevant microfractures, and consequent subtle decreases in vertebral height, we followed the changes in vertebral bone density (VBD), assessed by quantitative computed tomography, in relation to vertebral height (VH) in 60 osteoporotic women. VH was measured as the sum of anterior, central and posterior heights of L1–L3 vertebral bodies on lateral radiographs. Patients received either salmon calcitonin (sCT: 50 IU subcutaneously three times per week,n=18), hormonal replacement therapy (HRT: conjugated estrogen 0.625 mg/day, days 1–25, plus medroxyprogesterone acetate 10 mg/day, days 16–25 of each month;n=21) or calcium alone (Ca: 1000 mg/day,n=21). After 1 year, VBD increased in the HRT group (+5.0 ± 1.9%,p=0.010), did not change significantly in the sCT group (+3.3 ± 2.3%,p=0.167), and decreased by 6.1 ± 1.0% (p<0.001) in the Ca group. By analysis of variance, the changes induced by HRT and sCT were significantly different from those observed in the Ca group (F=7.982,p<0.001). VH decreased slightly in all three subsets of patients (–0.9 ± 0.5% in sCT, –1.5 ± 0.3% in HRT, –0.1 ± 0.5% in Ca), but these changes were not significantly different between groups (F=2.545,p=0.081). Correcting for this height decrease by analysis of covariance did not affect the significant effect of sCT and HRT on VBD, compared with that of Ca (F=6.801,p=0.001). Furthermore, no correlation was found between changes in bone VBD and VH in any of the groups. These data indicate that microfractures do not significantly account for the increases in bone density during active treatment of osteoporosis with either estrogen or calcitonin.     

Formation of neutralizing antibodies during intranasal synthetic salmon calcitonin treatment of postmenopausal osteoporosis

Nineteen patients with postmenopausal osteoporosis were treated with 200 u (15 nmol) synthetic salmon calcitonin (sCT) intranasally per day for 15 months. Six months after the start of the nasal administration of sCT, antibodies were recognized in 7, and after 15 months in 10 of the 19 patients studied. The half-maximal dilution of serum binding to 60 pmol/1 [¹²⁵I] sCT (dilution-50) ranged from 2 to 490, and half-maximal inhibition of [¹²⁵I]sCT binding (60 pmol/1) from 91 to 221 pmol/l sCT. In a cultured breast cancer cell line (T47D) cAMP production was stimulated by sCT (EC₅₀ 70 pmol/l). Stimulated cAMP production by sCT (50 pmol/1) was reduced to between 4% and 23% in the presence of serum from patients with antibody dilution-50 of [¹²⁵I]sCT binding exceeding 32. In patients with lower titer antibodies cAMP production was only marginally suppressed. The values of patients with postmenopausal osteoporosis were in the range of those of earlier studied patients with Paget's disease and clinical resistance to sCT. There was a linear relation between the antibody dilution-50 and the serum dilution required for half-maximal inhibition of cAMP production (P<0.01). In conclusion, neutralizing antibodies to sCT may contribute to the decreased responsiveness of bone mineral loss during prolonged treatment with sCT.     

Bone loss during gonadotropin releasing hormone agonist treatment and use of nasal calcitonin

Gonadotropin releasing hormone (GnRH) agonists have shown to be effective in the treatment of several sex-hormone-dependent conditions. However, their use could be limited by the bone loss they induce. To evaluate the use of nasal salmon calcitonin (sCT) in preventing this bone loss, 40 patients with endometriosis were treated for 6 months with triptoreline (3.75 mg monthly) and calcium (1 g daily), and randomized in three groups — placebo, sCT 100 IU daily and sCT 200 IU daily — in a prospective double-masked study. Dual-energy X-ray absorptiometry and biochemical parameters were used to evaluate the benefit of the treatment. At baseline, there were no statistically significant differences between the groups. After 6 months, estradiol and biochemical markers of bone metabolism were at postmenopausal levels, with no difference between the groups. There was no difference in bone loss in the three groups, at all sites. Mean lumbar bone loss was 4.01±2.59% (mean±SD) in this population. In this study dosages of 100 IU and 200 IU daily of nasal sCT were insufficient to prevent bone loss during GnRH agonist treatment.     

Preoperative and postoperative levels of interleukin-6 in patients with acute appendicitis: comparison between open and laparoscopic appendectomy

Background Cytokine interleukin-6 (IL-6) is an early marker of systemic inflammatory response and tissue damage. This study aimed to evaluate the levels of IL-6 after open and laparoscopic appendectomy to compare the degree of surgical stress associated with these procedures.Methods The levels of IL-6 were measured pre- and postoperatively in the plasma of 37 consecutive patients with a diagnosis of acute appendicitis. After preoperative randomization, 22 patients underwent open appendectomy, and 15 patients underwent laparoscopic appendectomy.Results The preoperative concentrations of IL-6 were 7.2 ± 5.6 pg/ml in the open appendectomy group, as compared with 12.1 ± 9.7 pg/ml in the laparoscopic appendectomy group (p < 0.05). The postoperative levels were 16.9 ± 15.7 and 23.2 ± 19.4 pg/ml, respectively. The mean postoperative to preoperative ratio of IL-6 was slightly higher for open (2.7 ± 2.4) than for laparoscopic (2.3 ± 1.6) appendectomy, but the difference did not reach statistical significance.Conclusion The operative stress in open as compared with laparoscopic appendectomy is not reflected by circulating levels of IL-6.     

Insulin-like growth factor I accelerates recovery of articular cartilage proteoglycan synthesis in culture after inhibition by interleukin 1

Interleukin 1 (IL-1) is a cytokine which induces cartilage proteoglycan (PG) depletion by inhibiting PG synthesis and increasing PG breakdown. Insulin-like growth factor I (IGF-I), in contrast, is known to promote matrix formation. We examined the effects of both mediators in a bovine tissue culture model. IL-1 dose-dependently inhibited PG formation of articular cartilage [half-maximal effect (EC₅₀) at 4 ng/mll, while PG synthesis was increased by IGF-I (EC₅₀) = 15 ng/ml). After inhibition of PG formation with IL-1 for 2 days and subsequent removal of free IL-1, addition of IGF-I dose-dependently accelerated restoration of the original rate of synthesis with a half-maximal effect at 20 ng/ml and a maximal effect at 50 ng/ml. The IGF-I concentration required to elicit a half-maximal effect on cartilage PG synthesis remained constant in the absence or presence of IL-1. We therefore conclude that inhibition of cartilage PG synthesis by IL-1 is not effected by damage to the IGF receptor. Synovial fluid (SF) of 40 patients with rheumatoid arthritis (RA) was found to contain 64 ± 6 ng IGF-I/ml (mean ± SEM). The reported effects of IGF-I in vitro therefore occurred at concentrations comparable to those present in joints in vivo. IL-1 was detectable (> 0.5 pg/ml) in 38 of 40 RA-SF samples (mean 28 ± 6 pg/ml). RA synovial tissue in culture released 330 ± 112 pg IL-1 x g tissue^–1 x d^–1, and this rate could be increased up to 70-fold by the addition of lipopolysaccharides (10 g/ml). The observed accelerated recovery of cartilage PG synthesis by IGF-I after inhibition by IL-1 may be of relevance in rheumatic diseases like RA since IL-1 levels in RA-SF are known to vary considerably with time, and IGFs have been shown previously to be the most important promotors of cartilage PG synthesis in human SF.     

A study of interleukin-8 and defensins in urine and plasma of patients with pyelonephritis and glomerulonephritis

Background: Interleukin-8 (IL-) plays a crucial role in the recruitment and activation of polymorphonucleated leukocytes (PMN) in the site of inflammation. Defensins are specific cationic proteins from azurophil PMN granules which exert antimicrobial, cytotoxic and proinflammatory activities. Methods: Urine and plasma levels of IL-8 and defensins were studied using specific enzyme-linked imunosorbent assays. IL-8 was determined in 107 patients, including 45 with chronic glomerulonephritis (GN) and 62 with chronic pyelonephritis (PN). Urine and plasma levels of defensins were studied simultaneously in 29 patients with GN and 29 with PN. None of the patients examined showed any evidence of renal insufficiency. A group of 24 healthy volunteers was used as a control. Results: Urinary IL-8 was significantly increased in all groups of patients comparing with healthy control (<30 pg/ml). The level of IL-8 in the urine of patients with PN (477±114 pg/ml, mean±SEM) was significantly (P <0.001) higher than in patients with GN (53±7 pg/ml). The concentration of defensins in urine of patients with GN was slightly increased in comparison with the normal level (21±3.5 ng/ml versus 15.7±2.8 ng/ml). Urinary defensins were significantly elevated in patients with PN (134±118 ng/ml, P<0.001),and were significantly higher than in the GN group (P <0.001). A close correlation was observed between IL-8 and defensin concentrations in urine (r=0.62), and between the urinary leukocyte count and IL-8 level (r=0.72). The highest levels of IL-8 were observed in patients with PN, associated with nephrolithiasis 914 patients, 822±219 pg/ml versus 367±72 pg/ml in patients with PN alone, P <0.05). IL-8 and defensin levels increased in older patients with PN, but not in older patients with GN. Conclusions: The levels of IL-8 and defensins in urine were 7-10-fold higher in patients with PN than in patients with GN. Thus, there is a significant difference in IL-8 production between septic and aseptic chronic inflammatory processes in kidney. It is possible to speculate that the timing and progression of kidney inflammation is mediated by an IL-8 dependent mechanism at least in the case of PN.     

Calcitonin metabolism in senile (type II) osteoporosis

The exact role of calcitonin (CT) in the pathogenesis of senile (Type II) osteoporosis remains unknown. Whole plasma calcitonin (iCT) and extracted monomeric calcitonin (eCT) basal levels, metabolic clearance rate (MCR) and production rate (PR) of iCT and eCT were measured in 41 postmenopausal women, including 14 hip fractures (OP II) and 27 healthy controls. No significant difference appeared for basal iCT levels between OP II (mean±SEM: 41.9±3.4 pg/ml) and controls (mean±SEM: 46.2±5 pg/ml). eCT basal levels were similar in OP II (mean±SEM: 5.42±0.5 pg/ml) and in controls (mean±SEM: 7.3±0.7 pg/ml). MCR were similar in the two groups. iCT PR were similar in OP II (mean±SEM: 17.2±1.5 µg/24 h) and controls (mean±SEM: 18.6±1.1 µg/24 h). No difference appeared between eCT PR in OP II (mean±SEM: 2.3±0.2 µg/24 h) and controls (mean±SEM: 3.2±0.3 pg/ml). From these data, no evidence appears that calcitonin might be one of the determinant factors in the pathogenesis of senile osteoporosis.     

Effect of IL-6 overexpression on the metastatic potential of rat hepatocellular carcinoma cells

Background: Previous studies demonstrated that excess IL-6 production correlated with the metastatic potential of rat hepatocellular carcinoma cells. In the work reported here a retroviral construct containing the gene for murine IL-6 was introduced into otherwise nonmetastatic tumor cells to directly determine the effect of IL-6 overexpression on tumor metastatic potential. Methods: The clonal cell lines 1682.C.2.9.L0 (L0, poorly metastatic) and 1682.C.2.9.L10 (L10, highly metastatic) were selected from a parental hepatocellular carcinoma induced in ACI rats by feeding an ethionine-containing diet. Viral supernatant was used to infect the PA317 amphotropic cell line, and retrovirus produced from these cells infected the poorly metastatic L0 hepatocellular carcinoma cell line. Neomycin-resistant cells were selected in G418 and designated L0-IL-6. Results: As determined by bioassay, L0 cells produce 10±1.2 U/mL IL-6 in culture, whereas L10 cells release 95±11 U/mL (P<0.01, Student'st-test). Retroviral-mediated IL-6 gene transfer resulted in the production of 1266±48 U/mL IL-6 by L0-IL-6 cells under identical culture conditions. When an inoculum of 5×10⁶ cells is injected subcutaneously, both L0 and L10 cell lines result in primary tumors with equivalent rates of growth; only L10 cells metastasize to the lung, however. A similar inoculation of L0-IL-6 cells produced local tumors in all 24 animals tested. Interestingly, 15 of 24 (62%) animals presented with metastatic nodules in the abdominal cavity, whereas no such tumors were found in animals receiving L10 cells. Conclusion: Overexpression of IL-6 increases metastatic potential of tumor cells, with preferential metastases to the abdominal cavity when compared with tumor cells elaborating endogenous IL-6.Presented at the 50th Annual Cancer Symposium of The Society of Surgical Oncology, Chicago, Illinois, March 20–23, 1997.     

Lymphocyte subpopulations,interleukin-2 and interleukin-2 receptor expression in childhood nephrotic syndrome

Abnormal T lymphocyte function and reduced interleukin-2 (IL-2) production have been implicated in the pathogenesis of the nephrotic syndrome (NS). We investigated: (1) lymphocyte subpopulations and expression of IL-2 receptor (IL-2R) on T cells using two-colour flow cytometry, (2) serum IL-2 and (3) the soluble component of IL-2R (sIL-2R) in serum, using enzyme-linked immunosorbent assay, in 38 children with NS. All children, except those in remission, had marked proteinuria. They were divided into groups according to renal pathology: (1) steroid-sensitive NS (SSNS) not receiving prednisolone therapy, (2) SSNS on prednisolone, (3) focal segmental glomerulosclerosis (FSGS), (4) SSNS in remission and not receiving prednisolone therapy, (5) congenital NS (CNS). Results were compared with 26 age-matched controls. Total T lymphocytes (CD3) were reduced in groups 1 and 2; CD4 count was reduced in groups 1–4; CD8 count increased in groups 2 and 3; CD8 and CD19 (B lymphocytes) were significantly reduced in group 5. Increased IL-2R expression (CD25) on CD4 lymphocytes was noted in groups 1, 2 and 3 implying activation of these cells. In patients with SSNS, increased serum sIL-2R was recorded during relapse (1,273±497 U/l vs. 913±401 U/l in remission,P<0.005) but free serum IL-2 was not detectable at any stage. The specific alterations in lymphocyte subpopulations in SSNS and FSGS would imply an involvement of the immune system distinct from that in CNS.     

Regulation of renal calbindin-D28K: The role of calcitonin

Infusion of calcitonin lowers circulating calcium, but in the distal tubule of the kidney, pharmacological doses of calcitonin increase the active calcium reabsorption. Calbindin-D28k plays a significant role in the calcium reabsorption in the distal convoluted tubule of the kidney. The effect of calcitonin on renal calbindin-D28k in relation to calcium metabolic changes was therefore examined. In study 1, thyroparathyroidectomy followed by autotransplantation of the parathyroid glands (TX) was compared with a sham operation in rats. TX reduced plasma calcitonin from 54±2 to 9±1 pg/ml (P<0.001), whereas ionized calcium and parathyroid hormone were returned to the control value after an initial decrease, indicating a successful implantation of the parathyroid glands. No changes were seen in calbindin-D or plasma 1,25(OH)₂D. In study 2, subcutaneous infusion of salmon calcitonin 2.5 U/kg/hour via osmotic pumps was compared with infusion of vehicle in rats. Ionized calcium was reduced from 1.37±0.01 to 1.33±0.02 mmol/liter (P<0.05), whereas no changes were seen in renal or intestinal calbindin-D or in plasma 1,25(OH)₂D. After TX, only calcitonin decreased whereas the other calcium metabolic parameters showed no change. This indicates that in rats, selective elimination of calcitonin does not influence other parameters of the calcium metabolism and that the effect of calcitonin on calcium transport in the distal tubule is not mediated via an increase in renal calbindin-D28k.     

Investigation of salmon calcitonin in regulating fibrosis-related molecule production and cell-substrate adhesion in frozen shoulder synovial/capsular fibroblasts

The purpose of this study was to evaluate the effect of salmon calcitonin (sCT) on improving fibrosis-related indicators in frozen shoulder synovial/capsular fibroblasts (SCFs) and detect the potential downstream pathway. Quantitative real-time polymerase chain reaction and cell-substrate adhesion assays were used to measure alterations in fibrosis-related molecule expression and the cell adhesion ability of frozen shoulder SCFs after treatment with range concentrations of sCT. The presence of calcitonin receptors (CTRs) in shoulder joint synovial/capsular tissue samples was detected by immunohistochemistry (IHC). The downstream pathways of sCT in SCFs were further explored by utilizing three classical pathway inhibitors. With the addition of sCT to the culture medium of frozen shoulder SCFs, the messenger RNA (mRNA) expression of collagen type I (COL1A1), COL3A1, fibronectin 1, laminin 1, transforming growth factor-β1 (TGF-β1), and interleukin-1α (IL-1α) showed a descending trend as the sCT concentration increased. Treatment with sCT increased the expression of vascular endothelial growth factor and IL-6 in a dose-dependent manner. The enhanced adhesion ability of frozen shoulder SCFs gradually diminished with increasing concentrations of sCT. By using IHC, the CTR was detected extensively in the frozen shoulder joint synovium and capsule. Blocking the protein kinase C (PKC) pathway reversed the sCT-mediated suppression of COL1A1 production. Blocking the PKC or protein kinase A (PKA) pathway eliminated the sCT-induced inhibition of TGF-β1 production. This study demonstrated that sCT effectively improved the mRNA expression of fibrosis-related molecules and decreased the enhanced cell-substrate adhesion ability of frozen shoulder SCFs. sCT might achieve these effects by interacting with the CTR that is expressed on the SCF surface and by activating the downstream PKC or PKA pathway.     

Increase of interleukin-6 plasma levels after elective craniotomy: Influence of interleukin-10 and catecholamines

Summary Accidental and operative trauma are able to induce a systemic reaction of the organism characterized by fever, leukocytosis, catabolism, and an activation of the coagulation system. Interleukin-6 (IL-6) has been found to be an important mediator of this acute-phase response. In this study the influence of elective craniotomy on IL-6 plasma levels was evaluated. Blood samples were obtained from 20 patients undergoing elective craniotorny for vascular or tumorous diseases of the brain. IL-6 increased significantly (p < 0.05) from the pre-operative (0 (0–5.4) pg/ml) to the intraoperative (180 min after beginning of surgery) time-point (10.6 (0–18.5) pg/ml). The maximum was reached on the first postoperative morning (13.9 (4.3–45.0) pg/ml). Interleukin-10 (IL-10) is an anti-inflammatory cytokine which suppresses IL-6 synthesis in vitro in various cell lines. IL-10 plasma concentrations showed no alterations throughout the study period. Epinephrine plasma concentrations increased significantly from pre-operative values (15 (0–74) pg/ml) to the postoperative time-point (57 (9–459) pg/ml). A 4.5-fold increase (p < 0.05) of norepinephrine plasma concentrations was found when comparing the data obtained 60 min after beginning of surgery with the data of the first postoperative morning. In monocytes, which are a major source of plasma IL-6, an elevation of intracellular cAMP stimulates the IL-6 synthesis. The postoperative maximum of IL-6 in plasma could be due to a release of catecholamines. In conclusion this study demonstrated an elevation of IL-6 plasma concentrations during and after elective craniotomy. Increased plasma catecholamine concentrations as well as a damage in the blood-brain barrier due to the surgical trauma with a spill-over of IL-6 from brain tissue into plasma could have contributed to this result.     

Characteristics of steroid hormone receptors in cultured MC3T3-E1 osteoblastic cells and effect of steroid hormones on cell proliferation

Summary We examined the binding characteristics of three kinds of steroid hormones—estrogen, androgen, and glucocorticoid—in cultured MC3T3-E1 mouse osteoblastic cells by whole-cell binding assay. The binding studies revealed the presence of a single class of high-affinity binding sites for [³H]17-estradiol, [³H]mibolerone (a synthetic androgen), and [³H]triamcinolone acetonide (a synthetic glucocorticoid). The numbers of binding sites for these steroid hormones were found to be 4534±819, 14312±1884, and 24898±655 sites/cell; and the Kd values were 8.57±0.62 x 10^–10M, 1.12±0.19 x 10^–9 M, and 6.08±1.24 x ^–10M, respectively. We also examined the effects of steroid hormones on the proliferation of MC3T3-E1 cells. 17-estradiol significantly stimulated the proliferation of the cells (130–150% of control). Dihydrotestosterone also significantly stimulated the proliferation of the cells (115% of control); the effect was, however, much less potent than that of 17-estradiol, although the number of binding sites was approximately three times more than that of 17 estradiol. Triamcinolone acetonide and dexamethasone had no effect on cell proliferation. These results suggest that estrogen and androgen act directly on osteoblastic cells through a receptor-mediated mechanism, and that androgen is much less potent than estrogen in stimulating the proliferation of MC3T3-E1 osteoblastic cells.     

Catecholamines modulate growth and differentiation of human preosteoclastic cells

Using a clonal cell line of human osteoclast precursors (FLG 29.1 cells), that after treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) show many functional characteristics of osteoclasts, we demonstrated that catecholamines act as inducers of osteoclast maturation in vitro and as stimulators of osteoclast activity via the binding to ₂ adrenergic receptors. Scatchard analysis of¹²⁵I-labelled iodocyano-pindolol to untreated (undifferentiated) or TPA-treated (differentiated) FLG 29.1 cells revealed the presence of a single high-affinity site with aK _d value around 24 pM and 8 pM respectively and with superimposable binding capacity (1.18 fmol/mg protein). Catecholamines increased in a dose-dependent fashion the intracellular cyclic AMP (cAMP) accumulation in both undifferentiated and TPA-treated FLG 29.1 cells. Pretreatment of untreated and TPA-treated FLG 29.1 cells with propranolol inhibited the catecholamine effect on cAMP accumulation, while pretreatment with clonidine had no effect. Catecholamines also reduced cell proliferation, increased tartrate-resistant acid phosphatase (TRAcP) activity, interleukin 6 (IL-6) production, multinuclearity and response to salmon calcitonin (sCT) in undifferentiated FLG 29.1 cells. In differentiated FLG 29.1 cells only IL-6 release was induced by catecholamine treatment. These findings support a potential role for catecholamines in modulating osteoclast differentiation and mature osteoclast activity.     

Relation of Cytokines to Vasodilation after Coronary Artery Bypass Grafting

Hemodynamic instability is frequent after coronary surgery. The present study tested the hypothesis that inflammation, as determined by circulating cytokine levels, may contribute to the difficulty of controlling arterial blood pressure after coronary artery bypass grafting. A group of 44 male patients undergoing elective coronary artery bypass grafting with cardiopulmonary bypass were studied. Plasma levels of tumor necrosis factor-, interleukin-6 (IL-6), IL-8, and IL-10 were measured before anesthesia induction, 5 minutes and 1 hour after reperfusion to the myocardium, and 2 and 18 hours after arriving in the intensive care unit (ICU). The 29 patients who did not need a vasopressor (norepinephrine) during their ICU stay were designated group I. They were compared to group II, which consisted of 15 patients who required a pressor agent in the ICU. Although no significant differences between groups were found regarding their hemodynamic variables, IL-6 and IL-8 levels were higher in the patients who used a pressor agent in the ICU. The norepinephrine dosage used in the ICU correlated with plasma IL-8 levels 2 hours after arriving in the ICU (r = 0.56, p = 0.031). Circulating IL-6 levels in group II were significantly higher than those in group I 2 hours after arriving in the ICU (126.5 ± 90.5 vs. 66.5 ± 48.2 pg/ml; p < 0.05). The mean IL-8 levels were higher in group II at 5 minutes (34.9 ± 25.7 vs. 17.3 ± 11.3 pg/ml) and 1 hour (38.6 ± 30.5 vs. 22.4 ± 16.7 pg/ml) after reperfusion, and 2 hours (33.0 ± 21.6 vs. 22.8 ± 16.7 pg/ml) after arriving in the ICU (p = 0.036). Postoperative vasodilation was associated with increased circulating IL-8 levels. Strategies that modulate cytokine responses may improve hemodynamic stability after coronary artery bypass grafting.     

Cytokines and adhesion molecules in renal vasculitis and lupus nephritis

Background: Plasma levels of some pro-inflammatory cytokines and soluble adhesion molecules have been suggested to be useful parameters to assess the activity of antineutrophil cytoplasmic antibody (ANCA)-positive vasculitis and lupus nephritis. We hypothesized that the renal activity of these diseases is better reflected by the urinary excretion and fractional excretion of these molecules. Methods: Plasma levels and urinary excretion of tumour necrosis factor-&agr; (TNF-&agr;), interleukin (IL)-6, IL-8, and the soluble cell adhesion molecules sICAM-1 and sVCAM-1 were measured by enzyme-linked immunosorbent assay (ELISA) in 15 patients with ANCA-positive renal vasculitis (eight active, ANCA-A; six in remission, ANCA-R), six patients with active lupus nephritis (LN), 15 patients with IgA nephropathy (IgAN) and nine healthy subjects. Fractional excretion of selected cytokines and adhesion molecules was also calculated. Results: Patients with ANCA-A had increased urinary excretion and fractional excretion of TNF-&agr; (9.27±3.19% vs 0.58±0.02%, P<0.01), IL-6 (120.79±65.83% vs 1.89±0.34%, P<0.01) and increased fractional excretion of IL-8 (23.34±6.38% vs 2.56±1.07%, P<0.01) and sVCAM-1 (0.81±0.33% vs 0.03±0.02%, P<0.01) compared with controls. Urinary excretion of TNF-&agr; and IL-6 and fractional excretion of TNF-&agr;, IL-6 and IL-8 were higher in ANCA-A than in ANCA-R. Patients with LN had increased plasma TNF-&agr; (20.52±2.01 pg/ml vs 12.33±0.23 pg/ml, P<0.05) and sVCAM-1 (1537.88±276.36 ng/ml vs 692.26±44.42 ng/ml, P<0.05) and increased urinary excretion of TNF-&agr; (2.81±0.51 &mgr;g/mol creat vs 0.98±0.05 &mgr;g/mol creat, P<0.01), IL-8 (35.78±14.03 &mgr;g/mol creat vs 12.46±5.19 &mgr;g/mol creat, P<0.05) and sVCAM-1 (48.98±20.20 &mgr;g/mol creat vs 2.92±1.35 &mgr;g/mol creat, P<0.01) compared with controls. Patients with IgAN had, in comparison with controls only increased plasma TNF-&agr; (18.10±0.57 pg/ml vs 12.33±0.23 pg/ml, P<0.05). Conclusions: Urinary excretion and fractional excretion, but not plasma levels of selected proinflammatory cytokines (TNF-&agr;, IL-6 and IL-8) were increased in patients with active ANCA-positive renal vasculitis, but not in ANCA positive vasculitis in remission. These parameters may be useful to monitor the activity of this disease.     

Human calcitonin has the same inhibitory effect on osteoclastic bone resorption by human giant cell tumor cells as salmon calcitonin

Human calcitonin (hCT) has been reported to have a less hypocalcemizing effect on rats and to have a lower binding affinity for the receptor of mouse osteoclasts than salmon CT(sCT). In this study we comparatively examined the effect of hCT and sCT on osteoclastic boneresorbing activity of unfractionated cells obtained from human giant cell tumor of bone and from rabbit and mouse long bones. We found that hCT had the same inhibitory effect as sCT on the bone-resorbing activity of human and rabbit osteoclastic cells, but a different one on that of mouse cells. These results indicate that the activity of drugs should be assayed using human cells if possible.