Induction of osteoclasts from CD14-positive human peripheral blood mononuclear cells by receptor activator of nuclear factor kappaB ligand (RANKL).

Osteoclasts are bone-resorbing cells that are derived from haemopoietic precursors, including cells present in peripheral blood. The recent identification of RANKL [receptor activator of nuclear factor (NF)-kappaB ligand], a new member of the tumour necrosis factor ligand superfamily that has a key role in osteoclastogenesis, has allowed the in vitro generation of osteoclasts in the absence of cells of the stromal/osteoblast lineage. Human peripheral blood mononuclear cells (PBMC) cultured in vitro with soluble RANKL and human macrophage colony-stimulating factor form osteoclasts. However, PBMC are heterogeneous, consisting of subsets of monocytes and lymphocytes as well as other blood cells. As the CD14 marker is strongly expressed on monocytes, the putative osteoclast precursor in peripheral blood, we have selected CD14(+) cells from PBMC to examine their osteoclastogenic potential and their expression of novel members of the tumour necrosis factor superfamily involved in osteoclastogenesis. Highly purified CD14(+) cells demonstrated mRNA expression of receptor activator of NF-kappaB, but no expression of RANKL or osteoprotegerin, whereas PBMC expressed mRNAs for all three factors. CD14(+) (but not CD14(-)) cells cultured on bone slices for 21 days with human macrophage colony-stimulating factor and soluble RANKL generated osteoclasts and showed extensive bone resorption. Similar numbers of osteoclasts were generated by 10(5) CD14(+) cells and 10(6) PBMC, but there was significantly less intra-assay variability with CD14(+) cells, suggesting the absence of stimulatory/inhibitory factors from these cultures. The ability of highly purified CD14(+) cells to generate osteoclasts will facilitate further characterization of the phenotype of circulating osteoclast precursors and cell interactions in osteoclastogenesis.

Expression of functional RANK on mature rat and human osteoclasts.

Although the important roles of RANK/RANKL in osteoclastogenesis have been established, their roles in the regulation of mature osteoclasts remain uncertain. Microisolation has been used to obtain pure populations of rat and human osteoclasts for RT-PCR analysis. RANK and calcitonin receptor mRNA was detected in all the samples whereas OPG and ALP mRNA was not present in any. RANKL mRNA was detected in two of eight rat and one of four human samples. Treatment of osteoclasts with soluble RANKL resulted in translocation of NF-kappaB to the nucleus and elevation of cytosolic and nuclear calcium levels. We have shown that RANK is highly expressed in mature osteoclasts and that its stimulation by RANKL results in activation of NF-kappaB and calcium signalling.

Osteoclasts from human giant cell tumors of bone lack estrogen receptors.

Although estrogen is important in human skeletal homeostasis, the major target cell in bone is unknown. Estrogen receptors (ER) have been demonstrated in osteoblasts and bone marrow stromal cells, but their presence in osteoclasts remains controversial because completely pure preparations have not been available. We have examined expression of ER-alpha and ER-beta messenger RNA (mRNA) by RT-PCR in samples from human giant cell tumor of bone (GCT), including: whole tumor, cultured mononuclear cells, and a pure osteoclast population obtained by microisolation. Whole tumor expressed both ER-alpha and calcitonin receptor (CTR) mRNA and apparently lower levels of ER-beta mRNA. Passaged cultures of tumor mononuclear stromal cells also expressed ER-alpha and low ER-beta but not CTR mRNA. In pure preparations of microisolated osteoclasts, expression of ER-alpha or ER-beta mRNA was not detected, whereas expression of CTR mRNA was readily identified. Microisolated GCT mononuclear cells expressed ER-alpha, but no detectable CTR mRNA. Fluorescence in situ hybridization (FISH) using an ER-alpha riboprobe demonstrated strong signal in the mononuclear cells but multinucleated osteoclasts showed no detectable signal. In contrast, CTR mRNA was detected in multinucleated osteoclasts but not in stromal-like tumor cells by FISH. 17Beta-estradiol consistently showed no effect on bone resorbing activity of osteoclasts from GCT cultured on cortical bone, although calcitonin was a potent inhibitor. These findings indicate that significant expression of ER does not occur in osteoclasts derived from human GCT and suggest that estrogen effects are mediated by other cells of the bone environment.

Complex shape changes in isolated rat osteoclasts: involvement of protein kinase C in the response to calcitonin.

The cytoplasmic spreading of osteoclasts has been used to assess responsiveness to agents such as calcitonin and associated signal transduction mechanisms. Although cyclic AMP and intracellular calcium are known mediators of calcitonin effects in osteoclasts, the role of protein kinase C (PKC) is less clear. We have used time-lapse videomicroscopy of isolated rat osteoclasts to characterize shape changes induced by calcitonin, forskolin, and phorbol 12-myristate-13-acetate (PMA) in the absence and presence of PKC blockers. Treatment with calcitonin reduced cytoplasmic plan area but increased perimeter length, resulting in a characteristic "stellate" appearance, whereas forskolin produced "nonstellate" contraction. The response of osteoclasts to PMA was dose dependent. High concentrations (10(-7)-10(-6) M) produced biphasic responses with transitory, calcitonin-like "stellate" contraction followed by sustained expansion, whereas low concentrations (10(-11)-10(-9) M) produced expansion only. The effects of low-concentration PMA could be prevented by pretreatment with a PKC blocker, whereas the effects of high concentrations were only partially inhibited. The effects of forskolin were unchanged by pretreatment with the PKC blocker. Treatment with calcitonin in the presence of various PKC blockers resulted in paradoxical transient expansion followed by contraction. These results indicate that calcitonin-induced shape change in osteoclasts is a complex process involving protein kinase C in addition to cyclic AMP-dependent mechanisms and possibly other factors.

Osteoblast-mediated effects of zinc on isolated rat osteoclasts: inhibition of bone resorption and enhancement of osteoclast number.

Zinc is an important element in biology yet little is understood of its role in bone cell metabolism and function. This study examined the effects of zinc on osteoclast (OC) function in cultures derived from neonatal rats and in cocultures of OC and UMR 106-01 osteoblast-like cells (UMR/OC cocultures). Treatment with zinc (10(-12)-10(-4) mol/L) had no effect on either bone resorption or the number of multinucleate cells positive for tartrate-resistant acid phosphatase (TRACP + ve MNC) in OC cultured for 24 h on bone slices. However, in UMR/OC cocultures, 10(-4) mol/L zinc (but not lower concentrations) decreased resorption pit formation by approximately 50% and increased TRACP + ve MNC number by approximately 40%. When osteoblast-like cells were pretreated with zinc prior to, but not during, coculture with OC, effects on TRACP + ve MNC and pit number persisted, although the effect was reduced. Zinc treatment also inhibited resorption and stimulated TRACP and calcitonin receptor (CTR) + ve MNC numbers in long-term (96-120 h) UMR/OC cocultures. Our results indicate that zinc increases TRACP + ve CTR + ve MNC numbers yet inhibits bone-resorbing activity, and that these effects are dependent on the presence of osteoblastic cells. Zinc is abundant in bone and may act as a local regulator of bone cells.

Prospective evaluation of neomycin serum concentrations after direct corpora cavernosa irrigation during penile prosthesis placement.

OBJECTIVES: To determine if toxic serum levels of neomycin are generated after direct corpora cavernosa irrigation during penile prosthesis placement. METHODS: We have used an infection prophylaxis technique that involves directly irrigating the corpora cavernosa tissue (through the corporotomy) with 0.5% neomycin solution. Serum neomycin concentrations were measured at 1 hour and 4 hours after irrigation in 13 patients undergoing penile prosthesis placement. A subset of patients who had preimplant and postimplant serum creatinine concentrations was evaluated for changes in renal function. RESULTS: The mean 1-hour postirrigation serum neomycin level was 1.2 micrograms/mL and the mean 4-hour postirrigation level was 1.2 micrograms/mL. These serum neomycin concentrations are lower than those thought to be necessary to produce nephrotoxicity or ototoxicity. Renal function was not significantly affected by the neomycin irrigation. CONCLUSIONS: Although aminoglycosides are ototoxic and neomycin has the highest nephrotoxic potential of the aminoglycosides, we conclude that direct irrigation of the corpora cavernosa with 0.5% neomycin solution does not produce significant systemic exposure to result in nephrotoxicity or ototoxicity. One-time prophylactic neomycin irrigation remains an effective, safe, and economic adjunct to penile prosthesis placement.

Osteoblasts mediate thyroid hormone stimulation of osteoclastic bone resorption.

Thyroid hormones increase bone turnover in vivo and stimulate bone resorption in vitro. Clinical states associated with excess circulating thyroid hormone levels are known to produce osteoporosis. To determine the effect of T3 on bone resorption, we used an isolated rat osteoclast bone resorption assay in the absence or presence of added osteoblasts. This makes it possible to distinguish between direct and indirect effects of thyroid hormones on osteoclasts. In short settlement osteoclast cultures, which contain relatively few osteoblasts, 24-h treatment with T3 (10(-10)-10(-8) M) produced no stimulation of bone resorption. However, after 48-h incubation in the presence of T3, an increase in resorption was observed (2.3-fold at 10(-9) M). In cocultures of osteoclasts and osteoblasts (UMR 106-01 osteoblast-like cells or long settlement cultures), T3 stimulated resorption at 24 h. Furthermore, stimulation of resorption occurred when osteoblasts (UMR 106-01 or rat calvarial cells) were pretreated with T3 and the subsequent osteoblast-osteoclast cocultures conducted for 24 h in the absence of T3. Thus, direct exposure of osteoclasts to T3 was not required for the stimulatory effect. Treatment for 48 h with T3 (10(-9) M) or PTH (10(-8) M) had no effect on bone resorption in osteoblast-free cultures derived from human osteoclastoma tumours. T4 was 100-fold less potent than T3 as a stimulator of osteoclast activity, and rT3 had no effect. T3-induced stimulation was inhibited by salmon calcitonin (10(-10) M). These findings indicate that thyroid hormone can act on osteoblasts to indirectly stimulate osteoclastic bone resorption.

Percutaneous decompression: treatment for respiratory distress secondary to multicystic dysplastic kidney.

Multicystic dysplastic kidney is a common renal anomaly in the newborn. Long-term problems, such as pain, infection, hypertension and neoplasm, although infrequent, have been reported. Acute, life-threatening complications resulting from the size of the affected kidney are rare and emergency nephrectomy has been the only reported effective therapy. We present a case of ultrasound-guided percutaneous cyst decompression used as definitive treatment of respiratory failure associated with multicystic dysplastic kidney.

Social memory deficits in adult male rats exposed to cadmium in infancy.

In two experiments infant rats were injected subcutaneously with 0, 1, or 2 mg Cd/kg on Day 5 or 6 after birth. In adulthood (150 days of age) subjects in both experiments who received the 2 mg/kg dose failed to learn the identity of a strange rat in a social recognition test. Cadmium-treated rats investigated familiar and strange rats equally, whereas control subjects investigated familiar rats much less than unfamiliar individuals. Results with rats in the 1 mg/kg group were less consistent; in Experiment 1 they failed to learn the identity of a stranger, in Experiment 2 they behaved like controls. The level of investigation of a strange rat did not differ among the experimental groups, indicating cadmium did not cause a performance deficit. The 2 mg/kg dose of cadmium had no effect on body weight in Experiment 1 and a small (6.98%), but significant, depressant effect on body weight in Experiment 2. Cadmium exposure in infancy appears to affect social memory processes long after the treatment period.

Cadmium exposure in infancy: effects on activity and social behaviors of juvenile rats.

In two experiments male and female infant rats were given single injections of cadmium chloride (0, 1, 2, 3 or 4 mg Cd/kg) on Day 5 or 6. Animals receiving the 3 and 4 mg/kg doses had high mortality rates at weaning; survivors were extremely underweight and were not used in postweaning tests. Male subjects receiving 2 mg/kg in infancy were significantly more active after weaning than littermates who had received 0 or 1 mg/kg doses, and on Day 29 they also engaged in significantly more rough and tumble play with a nontreated partner than did rats in the other groups. This effect of early cadmium exposure was also evident when males were tested with similarly treated subjects on Day 44: rats in the 2 mg/kg group had higher pinning frequencies than rats in the 0 or 1 mg/kg groups. In contrast, females in the 1 and 2 mg/kg groups did not have increased activity or rough and tumble play fighting. These data are consistent with the few correlational studies in human children which suggest changes in social behaviors associated with elevated tissue cadmium levels.

Low level lead exposure during lactation increases rough and tumble play fighting of juvenile rats.

Lactating rats were given distilled water or distilled water containing 0.067% lead chloride (500 ppm lead) as their sole source of drinking fluid from Days 1-21 of lactation. Activity, social investigation and rough and tumble play fighting behaviors of the offspring were observed on Day 26 and activity and play solicitation behaviors on Day 36. Although lead treatment reduced the mothers' fluid intake, there were no effects on pup growth and activity or on maternal behaviors. When paired with a group-housed stimulus animal on Day 26, lead-treated subjects had increases in the two measures of play fighting (crossover and pinning), and in social investigation, relative to controls. When tested with a scopolamine-treated, non-playful stimulus on Day 36, increased crossover frequencies were observed in lead-treated subjects when compared with controls. Two-min activity levels on Days 26 and 35 were unaffected by lead exposure. These results indicate that social interactive behaviors of juvenile rats are effective tools in the assessment of exposure to toxic substances early in development.

Social play soliciting by male and female juvenile rats: effects of neonatal androgenization and sex of cagemates.

Male and female juvenile rats were individually exposed to nonplayful juvenile social stimuli in a novel test of play-soliciting behavior to examine hormonal and experiential determinants of sex differences. In Experiment 1, neonatally androgenized females engaged in play soliciting at a level equal to that of male controls and greater than that of nonandrogenized female controls. In Experiment 2, males and females were reared in unisexual and bisexual groups in order to compare long-term sex-related social experience effects on juvenile play soliciting. Males exposed only to other young males engaged in greater play soliciting than males exposed to both sexes; females, in contrast, were unaffected by sex of cagemates. Within rearing conditions, however, males engaged in greater play soliciting than females. The combined results suggest that perinatal gonadal androgen exposure effects on social play are prepotent and contribute essentially to sex differences in the initiation of social play behavior.

Testosterone dependent effects of methylphenidate on social investigatory behavior of the rat.

Previous work suggested a sex difference in social investigatory behavior of a novel conspecific during acute exposure to methylphenidate. In contrast to caffeine, which has been observed to increase social investigatory behavior only in males, methylphenidate decreased social investigatory behavior only in females. Testosterone-treated females were comparable to males and were unaffected by methylphenidate. Castrated males were comparable to females in their sensitivity to methylphenidate. Like caffeine, methylphenidate increased locomotor activity in both males and females, suggesting that drug effects on social investigation cannot be attributed to a nonspecific stimulant effect. The combined results reveal a sex-related interaction of circulating testosterone with acute methylphendiate exposure. Interpretations are discussed including the behavioral contrast following exposure to methylphenidate or to caffeine.

Caffeine and copulatory experience: interactive effects on social investigatory behavior.

Three experiments test the interaction of prior copulatory experience with acute caffeine exposure on social investigatory behavior of the male Norway rat. At a dosage of 20 mg/kg or greater, caffeine counteracts a decrease in social investigation attributed to copulatory experience. At a 10 mg/kg dosage, caffeine increases social investigatory behavior prior to sexual exposure but has no comparable effect after sexual exposure. The results are interpreted as confirming a long-term effect of copulatory exposure on social investigatory behavior and as describing a dose-related interaction of caffeine exposure with prior copulatory experience. Social investigation is viewed as a preliminary component of sexual behavior and the decrease in social investigation following copulatory experience as a gain in efficiency of social discrimination. Acute caffeine exposure apparently interferes with access or retrieval of reference information in long-term memory.

Interactive effects of caffeine, 2-chloroadenosine and haloperidol on activity, social investigation and play fighting of juvenile rats.

The effects of caffeine, 2-chloroadenosine and haloperidol and their interaction on activity, social investigation, and two measures of play fighting (crossover and pinning), were investigated in juvenile male rats. Caffeine (20 mg/kg) increased activity, decreased crossover and pinning, but had no effect on social investigation. Both 2-chloroadenosine (0-10 mg/kg) and haloperidol (0-10 mg/kg) depressed activity, social investigation, crossover and pinning. When given together in varying dosages, caffeine and 2-chloroadenosine had behavioral effects suggestive of a competitive interaction between the two drugs. In contrast, the effects of haloperidol were not appreciably altered by simultaneous caffeine treatment. These results suggest that the influence of caffeine and 2-chloroadenosine on activity, social investigation and play fighting involve interaction with adenosine receptors.

Immunohistochemical assessment of melatonin binding in the pineal gland.

Melatonin binding in the pineal gland of albino rats is estimated using an immunohistochemical procedure. Binding is saturable, has relatively high affinity (Apparent KD = 2.7 nM), and competition studies indicate binding of indoleamines possessing an N-acetyl group on the terminus of the side chain (N-acetylserotonin and melatonin). These data are consistent with the interpretation that immunohistochemically determined melatonin in unfixed pineal tissue is assessing binding of N-acetylated indolealkylamines to pineal cell components. In albino rats maintained on 12-hour light: 12-hour dark cycles, melatonin binding exhibits a diurnal rhythm with low levels of saturation (30%) early in the light and saturation by endogenous melatonin near the onset of darkness. An annual rhythm of melatonin binding was observed in albino rats with low levels during the summer and high levels during the winter. Other rats were maintained on 12-hour light:dark cycles and fed for 2 hours either early in the light period or early in the dark period. For both morning- and evening-fed animals, melatonin binding was high prior to feeding and dropped immediately after feeding. Changes in melatonin binding that occur in response to alterations of feeding and time of year suggest the possibility that this binding reflects a functional site for melatonin.

Testosterone dependent effects of caffeine on social investigation by adult male rats.

The testosterone determinants of caffeine induced increases in locomotor activity and social investigation by adult rats was investigated. In all groups where testosterone levels were high (intact or sham castrate adult males or castrate males receiving testosterone replacement) caffeine increased the social investigation of a novel juvenile rat. In condition where androgen levels were low (adult females, castrate males and castrate males receiving oil injections) social investigation was not affected by caffeine. In contrast, locomotor activity was increased by caffeine in all treatment groups. Thus, testosterone has behaviorally specific effects on the caffeine responsiveness of the rat.

Social play in juvenile rats during scopolamine withdrawal.

Scopolamine induced blockade of play fighting in juvenile rats and the rapid induction of behavioral tolerance to an initially effective dosage suggested a rebound in social play following chronic scopolamine exposure. Juvenile rats received daily intraperitoneal scopolamine or saline injections for one week. Play soliciting and play fighting behavior were then measured at 24 and 168 hr after drug withdrawal. Scopolamine treated juveniles engaged in markedly greater play soliciting and play fighting behavior than did controls. Drug-induced increase in muscarinic receptors and supersensitivity to endogenous acetylcholine following scopolamine withdrawal is suggested as the basis for observed differences in social play.

Social play in juvenile rats: a decade of methodological and experimental research.

The social play behavior of juvenile rats was originally described nearly a century ago, but research methods have only recently included the controlled laboratory investigation of psychobiological variables. This review covers the experimental literature of social play or play fighting behavior in juvenile laboratory rats reported during the last decade. Innovative measures for quantifying social play are described; hormonal, pharmacological, and neurological variables are examined; and interpretative concepts of social play are discussed. The current emphasis on measures and procedures as well as the limited scope of current research effort suggests a formative stage of research development.

Acute and chronic caffeine exposure effects on play fighting in the juvenile rat.

Effects of acute and chronic exposure to caffeine on the behavior of juvenile rats were assessed in three experiments. In Experiment 1, two indices of play fighting--pin frequency and duration--were decreased and locomotor activity was increased in dose dependent fashions by caffeine. Social investigation of another juvenile was not affected by the drug. In Experiment 2, juvenile rats were isolated and given caffeine in their drinking fluid for 10-11 days. Play fighting was increased by all except the highest caffeine dose. In Experiment 3, juveniles were housed in groups of four and received either tap water or caffeine in their drinking fluid. Pin frequency of caffeine treated subjects was lower than controls on days 2-4, and higher than controls on days 9-11 after caffeine treatment was initiated. Evidently, caffeine has inhibitory and facilitatory effects on juvenile play fighting behavior, contingent on duration of exposure to the drug.