AFM-IR of Electrohydrodynamically Printed PbS Quantum Dots: Quantifying Ligand Exchange at the Nanoscale.

Colloidal quantum dots (cQDs), semiconductor materials with widely tunable properties, can be printed in submicrometer patterns through electrohydrodynamic printing, avoiding aggressive photolithography steps. Postprinting ligand exchange determines the final optoelectronic properties of the cQD structures. However, achieving a complete bulk exchange is challenging, and the conventional vibrational analysis lacks the required spatial resolution. Infrared nanospectroscopy enables quantitative analysis of vibrational signals and structural topography on the nanometer scale upon ligand substitution on lead sulfide cQDs. A solution of ethanedithiol led to rapid (∼60 s) exchange of ≤90% of the ligands, in structures up to ∼750 nm thick. Prolonged exposures (>1 h) caused the degradation of the microstructures, with a systematic removal of cQDs regulated by surface:bulk ratios and solvent interactions. This study establishes a method for the development of devices through a combination of tunable photoactive materials, additive manufacturing of microstructures, and their quantitative nanometer-scale analysis.

Effect of the topical administration of corticosteroids and tuberculin pre-sensitisation on the diagnosis of tuberculosis in goats.

BACKGROUND: Caprine tuberculosis (TB) is a zoonosis caused by members of the Mycobacterium tuberculosis complex (MTBC). Caprine TB control and eradication programmes have traditionally been based on intradermal tuberculin tests and slaughterhouse surveillance. However, this strategy has limitations in terms of sensitivity and specificity. Different factors may affect the performance of the TB diagnostic tests used in goats and, subsequently, the detection of TB-infected animals. In the present study, the effect of two of the factors that may affect the performance of the techniques used to diagnose TB in goats, the topical administration of corticosteroids and a recent pre-sensitisation with tuberculin, was analysed. METHODS: The animals (n = 151) were distributed into three groups: (1) a group topically treated with corticosteroids 48 h after intradermal tuberculin tests (n = 53); (2) a group pre-sensitised with bovine and avian purified protein derivatives (PPDs) 3 days before the intradermal tuberculin test used for TB diagnosis (n = 48); and (3) a control group (n = 50). All the animals were tested using single and comparative intradermal tuberculin (SIT and CIT, respectively) tests, an interferon-gamma release assay (IGRA) and a P22 ELISA. RESULTS: The number of SIT test reactors was significantly lower in the group treated with corticosteroids when compared to the pre-sensitised (p < 0.001) and control (p = 0.036) groups. In contrast, pre-sensitisation with bovine and avian PPDs did not cause a significant reduction in the number of SIT and CIT test reactors compared with the control group. In fact, a higher number of reactors was observed after the prior tuberculin injection in the pre-sensitised group (p > 0.05). No significant effect was observed on IGRA and P22 ELISA due to corticosteroids administration. Nevertheless, a previous PPD injection affected the IGRA performance in some groups. CONCLUSIONS: The application of topical corticosteroid 24 h before reading the SIT and CIT tests can reduce the increase in skin fold thickness and subsequently significantly decrease the number of positive reactors. Corticosteroids used can be detected in hair samples. A previous pre-sensitisation with bovine and avian PPDs does not lead to a significant reduction in the number of intradermal tests reactors. These results are valuable in order to improve diagnosis of caprine TB and detect fraudulent activities in the context of eradication programs.

Tuberculosis caused by Mycobacterium caprae in a camel (Camelus dromedarius).

BACKGROUND: Animal tuberculosis (TB) is distributed worldwide and has a wide range of wild and domestic reservoirs. Few studies concerning TB in camelids have been published in the last decade, particularly as regards Old World Camelids (OWC), but the increase in reports of TB outbreaks in these species in recent years suggests a high susceptibility to the infection. CASE PRESENTATION: We studied a dromedary camel (Camelus dromedarius) herd (n = 24) in which a Mycobacterium caprae infection was detected. The TB infection was confirmed in one animal at necropsy through the detection of TB lesions, mainly in the abdominal organs, and the subsequent isolation of M. caprae (SB0157 spoligotype). The whole herd was additionally tested using cellular and humoral based diagnostic techniques. The intradermal tuberculin test results were compared with those obtained using P22 ELISA for the detection of specific antibodies against the M. tuberculosis complex. The TB infected animal was a positive reactor to both the intradermal tuberculin tests and P22 ELISA, while the others were negative to all the diagnostic tests. CONCLUSION: The present study found M. caprae infection in OWC. This is the first report of M. caprae infection in an OWC not living in a zoo. Since the animal was born in the herd and fed with goat's milk, this practice was suspected to be the potential source of TB infection, which was not confirmed in the other animals present in the herd. Moreover, our results highlight that the intradermal tuberculin test and the P22 ELISA could be valuable tools for the diagnosis of TB in OWC.

Specificity of serological test for detection of tuberculosis in cattle, goats, sheep and pigs under different epidemiological situations.

BACKGROUND: Serum antibody detection has potential as a complementary diagnostic tool in animal tuberculosis (TB) control, particularly in multi-host systems. The objective of the present study was to assess the specificity (Sp) of an enzyme-linked immunosorbent assay (ELISA) based on the new multiprotein complex P22 for the detection of specific antibodies against the Mycobacterium tuberculosis complex (MTC) in the four most relevant domestic animals acting as MTC hosts: cattle, goat, sheep and pig. We used sera from an officially TB-free (OTF) country, Norway, and from a non-OTF one, Spain. The samples included sera from goats that had been vaccinated against M. avium subsp. paratuberculosis (MAP) and sheep from a herd in which Corynebacterium pseudotuberculosis had been isolated. RESULTS: In cattle, the Sp ranged from 92.5 (IC95% 90.7-94) to 99.4% (IC95% 98.3-99.8) depending on the cut-off used and the origin of the samples (Spain or Norway). Sp in cattle (cut-off point 100) was significantly higher (P < 0.05) for Norwegian samples. By contrast, Sp in goats was consistently low at the 100 cut-off [30.9 (CI95%23.4-39.5)-78% (CI95% 68.9-85)]. A higher cut-off of 150 improved Sp in Norwegian goats [97% (CI95% 91.6-99)], but still yielded a poor Sp of 56.1% (CI95% 47.3-64.6) in Spanish goats. In Norway at the 100 cut-off the Sp was 58.3 (CI95% 42.2-72.9) and 90.6% (CI95% 81-95.6) in MAP vaccinated and non-vaccinated goats, respectively, indicating interference due to MAP vaccination. Sp in sheep was between 94.4 (CI95% 91.7-96.3) and 100% (CI95% 96.3-100) depending on the cut-off and country, and no diagnostic interference due to infection with C. pseudotuberculosis was recorded. Sp in pigs was 100%, regardless the cut-off point applied, and no significant differences were observed between pigs from Norway and from Spain. CONCLUSIONS: Due to its excellent Sp in pigs and acceptable Sp in cattle and sheep, this ELISA may constitute a suitable option for TB screening at herd level, particularly in OTF-countries.

Long-term treatment with raloxifene, but not bisphosphonates, reduces circulating sclerostin levels in postmenopausal women.

UNLABELLED: We determined whether suppression of sclerostin levels by estrogen treatment was mediated by anti-resorptive effect. Raloxifene, but not bisphosphonates, suppressed circulating sclerostin concentration, suggesting that sclerostin may mediate the action of estrogen on bone metabolism, independently of their anti-resorptive effects. INTRODUCTION: Circulating sclerostin concentrations are higher in postmenopausal than in premenopausal women, and estrogen treatment suppresses sclerostin levels in both men and women. We determined whether anti-resorptives may suppress the circulating sclerostin levels. METHODS: We conducted a retrospective observational study. Eighty postmenopausal women were treated with raloxifene for 19.4 ± 7.7 months (n = 16), bisphosphonates for 19.2 ± 6.7 months (n = 32), or were untreated (n = 32) for 17.1 ± 4.6 months. Plasma sclerostin concentrations were measured before and after treatment. RESULTS: Plasma sclerostin levels after treatment were significantly lower in the raloxifene than in the control group (55.8 ± 23.4 pmol/l vs. 92.1 ± 50.4 pmol/l, p = 0.046), but were similar between the bisphosphonate and control groups. Relative to baseline, raloxifene treatment markedly reduced plasma sclerostin concentration (-40.7 ± 22.8%, p < 0.001), with respect to both control (-7.5 ± 29.1%) and bisphosphonate (-3.1 ± 35.2%) groups. Changes in bone-specific alkaline phosphatase and osteocalcin levels showed reverse associations with sclerostin concentration changes in the raloxifene (γ = -0.505, p = 0.017) and control (γ = -0.410, p = 0.020) groups. CONCLUSIONS: Raloxifene, but not bisphosphonates, significantly suppressed circulating sclerostin concentration, suggesting that sclerostin may mediate the action of estrogen on bone metabolism, independently of their anti-resorptive effects.

Factors affecting the performance of P22 ELISA for the diagnosis of caprine tuberculosis in milk samples.

Caprine tuberculosis (TB) is a zoonosis caused by members of the Mycobacterium tuberculosis complex (MTBC). Caprine TB eradication programmes are based mainly on intradermal tuberculin tests and slaughterhouse surveillance. However, the use of serological test has been extended as a potential diagnostic tool in goats through the use of serum, plasma, or even milk samples. Milk production and the antibodies (Ab) present in milk can vary depending on several circumstances. In the present study, different factors that may affect the performance of humoral TB diagnosis were analysed using goat milk samples: 1) lactation stage, 2) a recent previous skin test (booster effect) and 3) the effect of freeze-thaw cycles on milk samples preserved with azidiol. TB-infected animals (n = 44) were selected to evaluate the evolution of the Ab levels during the 6-month lactation period, along with its potential effect on the P22 ELISA results. In general, no significant changes (p = 0.079) were observed throughout the study as regards Ab levels in milk samples between consecutive analysis although the reactivity to P22 ELISA decreased when samplings were performed at the last two months of the lactation. Regarding the booster effect, the quantitative results showed a significant variation (p < 0.001) for both milk and serum samples when serological tests were carried out 15 days after the skin test. Finally, there were no significant differences (p = 0.99) in the P22 ELISA results when using milk samples preserved with azidiol that had undergone freeze-thaw cycles.

Standoff detection of explosives: critical comparison for ensuing options on Raman spectroscopy-LIBS sensor fusion.

In general, any standoff sensor for the effective detection of explosives must meet two basic requirements: first, a capacity to detect the response generated from only a small amount of material located at a distance of several meters (high sensitivity) and second, the ability to provide easily distinguishable responses for different materials (high specificity). Raman spectroscopy and laser-induced breakdown spectroscopy (LIBS) are two analytical techniques which share similar instrumentation and, at the same time, generate complementary data. These factors have been taken into account recently for the design of sensors used in the detection of explosives. Similarly, research on the proper integration of both techniques has been around for a while. A priori, the different operational conditions required by the two techniques oblige the acquisition of the response for each sensor through sequential analysis, previously necessary to define the proper hierarchy of actuation. However, such an approach does not guarantee that Raman and LIBS responses obtained may relate to each other. Nonetheless, the possible advantages arising from the integration of the molecular and elemental spectroscopic information come with an obvious underlying requirement, simultaneous data acquisition. In the present paper, strong and weak points of Raman spectroscopy and LIBS for solving explosives detection problems, in terms of selectivity, sensitivity, and throughput, are critically examined, discussed, and compared for assessing the ensuing options on the fusion of the responses of both sensing technologies.

Accuracy of tuberculosis diagnostic tests in small ruminants: A systematic review and meta-analysis.

Tuberculosis (TB) in small ruminants is a neglected disease despite its major impact on goat and sheep production and the global public health. The awareness of the role of small ruminants in the epidemiology of animal TB has increased in the last two decades, however, there is a lack of standardization of procedures and robust quantitative estimates on the accuracy of diagnostic TB tests in the scientific literature. To address this knowledge gap, all the available information regarding the use of ante-mortem diagnostic techniques in small ruminants was collected and summarized through a systematic review process. Furthermore, a random-effects meta-analysis was conducted to separately estimate the sensitivity (Se) and specificity (Sp) of cell-based tests among the retrieved studies in goats. Studies included in the meta-analysis were also evaluated using the Quality Assessment of Diagnostic Accuracy Studies included in systematic reviews adapted for animal diagnostic tests (VETQUADAS). Median pooled Se estimates of the single intradermal tuberculin (SIT) test (ranged from 0.51 to 0.59), the comparative intradermal tuberculin (CIT) test (ranged from 0.30 to 0.50) and the interferon-gamma (IFN-γ) release assay (IGRA) (ranged from 0.66 to 0.72) were lower than that reported previously in cattle, regardless the interpretation criteria and the reporting of MAP infection or vaccination. However, the specificity was adequate for all the tests (ranged from 0.95 to 0.99), except for the SIT test in MAP vaccinated herds (ranged from 0.78 to 0.90). This study provides an overview of the accuracy of diagnostic tests for TB in goats, however, the considerable between-study heterogeneity found hampered the conclusive interpretation of the pooled Se and Sp estimates. Therefore, further studies in small ruminants are necessary to optimize the diagnostic Se, which could help to design effective control strategies, accelerate the eradication of TB in these species and harmonize test procedures.

Evaluation of a new enzyme-linked immunosorbent assay for the diagnosis of tuberculosis in goat milk.

Caprine tuberculosis (TB) is a zoonosis with sanitary and economic repercussions. Caprine TB control programs are based on a test and cull strategy using the intradermal tuberculin tests and slaughterhouse surveillance. However, this approach is not always feasible and may have a limited sensitivity under specific circumstances. In this study, performance of a new experimental test based on the P22 protein complex (P22 ELISA) was evaluated in two TB-infected herds using milk and serum samples and compared with cell-based diagnostic tests. Samples from a low (n = 62, herd 1) and a high (n = 52, herd 2) TB prevalence herd were selected. Moreover, bulk tank milk samples from both herds were analysed using the P22 ELISA. At the end of the study, a group of animals (n = 21) was euthanized and subjected to post-mortem analysis and bacteriological culture. Significant differences (p < .001) on the qualitative and quantitative (ODs) results were observed between herds using both serum and milk samples in the P22 ELISA. The correlation observed in the quantitative results obtained in serum and milk samples was very strong in animals from flock 2 (rs = 0.91) and moderate in animals from flock 1 (rs = 0.46). Among the slaughtered animals, the P22 ELISA detected a higher proportion of lesion-culture positive animals than cell-based diagnostic tests (61.9 and 66.7% using milk and serum samples, respectively). The P22 ELISA using milk samples demonstrated a similar sensitivity compared with serum samples, suggesting it might be a valuable test for TB control in dairy goats.

Divalent cation ionophores stimulate resorption and inhibit DNA synthesis in cultured fetal rat bone.

Two divalent cation ionophores, A23187 and Ionomycin, which are selective for calcium, stimulated the resorption of fetal rat long bones in organ culture at 0.1 to 1 micromolar but not at higher concentrations. Both agents inhibited DNA synthesis at concentrations that stimulated resorption. These results might explain the differences in ionophore effects on bone previously reported, and they imply that cell replication is not required for osteoclast formation in fetal rat long bone cultures.

A new test to detect antibodies against Mycobacterium tuberculosis complex in red deer serum.

Red deer (Cervus elaphus) farming is a growing economic activity worldwide. However, the capacity of this species to act as reservoir of animal tuberculosis (TB) poses a threat to other wildlife and to livestock. Diagnostic assay accuracy in this species is therefore highly relevant for prevention and control measures. Our aim was to evaluate the diagnostic performance of the protein complex P22, obtained from Mycobacterium bovis derived purified protein derivative (bPPD), as a candidate antigen for the detection of antibodies against Mycobacterium tuberculosis complex (MTC). We assessed the performance of this new antigen in indirect enzyme-linked immunosorbent assays (ELISA) in TB-positive and TB-negative red deer, in comparison with a bPPD-based ELISA. The P22 ELISA achieved a higher specificity (Sp) and similar sensitivity (Se) in comparison with the bPPD ELISA at all the cut-off points considered. The P22 ELISA yielded optimal Sp (99.02%; 95% confidence intervals [CI95%]: 96.5-99.8) and appropriate Se (70.1%; CI95%: 63.6-76) at the selected cut-off point of 100%. These results suggest that P22 can be used as an alternative antigen in the immunodiagnosis of animal TB through the use of an ELISA-type detection of antibodies against MTC in red deer, thus contributing to the diagnosis of animal TB in this species as a measure for further disease prevention and control programs.

Management of hunting waste as control measure for tuberculosis in wild ungulates in south-central Spain.

In recent decades, habitat change and the intensive management of wild ungulates for hunting have led to an increase in their populations in south-central Spain. This implies a higher generation of hunting waste, which can favour the transmission of infectious diseases, including tuberculosis (TB). The aim of this study was to assess the usefulness of the proper disposal of hunting waste as TB control measure in wild boar (Sus scrofa) and red deer (Cervus elaphus) during the 2008/2009 to 2016/2017 hunting seasons. Blood samples from 664 wild boar and 934 red deer were obtained in 14 game estates in two provinces in Andalusia (Area 1), where the disposal of hunting waste was implemented since the 2012/2013 hunting season. Besides, six game estates in the province of Ciudad Real, in Castilla-La Mancha (Area 2), an adjacent region where this management measure was not implemented during the studied period, were used as controls, sampling 277 wild boar and 427 red deer sera. The Mycobacterium tuberculosis complex (MTC), seroprevalence detected in wild boar from Area 1, was significantly higher before the disposal of big game hunting by-products (82.8%; 2008/2009-2012/2013) compared to the second period (61.8%; 2013/2014-2016/2017) (p < .001), after this control measure became established. By contrast, no significant differences between periods were found in wild boar (41.3% versus 44.8%; p = .33) and red deer (14.9% versus 11.6%; p = .19) from Area 2 as well as in red deer (10.8% versus 10.5%; p = .48) from Area 1. The proper disposal of hunting waste contributed to achieve a 25% reduction in MTC seroprevalence in wild boar. These results are of particular relevance regarding wild boar in the current context of re-emerging and emerging diseases such as TB and African Swine Fever in Europe. Further studies are needed to assess the effect of this measure on the health status of livestock and other wildlife species.

DNA synthesis is not necessary for osteoclastic responses to parathyroid hormone in cultured fetal rat long bones.

Osteoclasts, the principal cells mediating bone resorption, are believed to increase their size, number, and resorbing activity in response to parathyroid hormone (PTH) through mechanisms dependent upon the fusion of specific mononuclear precursor cells into either new or existing multinucleated osteoclasts. To address the question of whether these actions of PTH are dependent on the replication of osteoclast precursor cells, we examined the ability of an inhibitor of DNA synthesis, hydroxyurea (HU), to alter bone resorption, osteoclast formation, and DNA synthesis in cultured fetal rat bones treated with PTH. We found that HU significantly reduced [3H]thymidine incorporation into the bones and labeling of osteoclast nuclei by greater than 90%, but did not prevent PTH from stimulating bone resorption, measured as the release of 45Ca, or from increasing the number of osteoclasts in the bones. In bones cultured without PTH, HU decreased the rate of bone resorption, but not the number of osteoclasts per bone. We conclude that in fetal rat bone cultures, PTH can increase osteoclast number and stimulate bone resorption by affecting existing osteoclasts and osteoclast precursors, and that replication of osteoclast precursor cells is not necessary for PTH to stimulate a resorptive response. In unstimulated cultures it appears that HU inhibits bone resorption by affecting mechanisms that are independent of changes in osteoclast number and that may be influenced by cell replication or other unknown factors.

Effects of DNA and prostaglandin synthesis inhibitors on the stimulation of bone resorption by epidermal growth factor in fetal rat long-bone cultures.

We examined two inhibitors of DNA synthesis, hydroxyurea (HU) and aphidicholin (APC), and two inhibitors of prostaglandin cyclooxygenase, indomethacin and flufenamic acid, for their effects on the resorptive responses of fetal rat long-bone cultures to epidermal growth factor (EGF) and parathyroid hormone (PTH). As we have previously found, HU decreased unstimulated 45Ca release but had little effect on the resorptive response to PTH. HU also did not block resorption stimulated by EGF. Addition of the cyclooxygenase inhibitor, indomethacin, did not alter the resorptive responses of unstimulated or PTH-treated cultures in either the presence or absence of HU or the resorptive response of bones cultured with EGF alone. However, indomethacin completely blocked the resorptive response to EGF of bones that were cultured with HU. The effects of indomethacin on EGF-mediated resorption in HU-treated cultures appeared to be related to an inhibition of prostaglandin synthesis since flufenamic acid had similar effects. However, the effects of HU on the resorptive response to EGF may not have resulted solely from its inhibitory action on DNA synthesis since APC, in the absence of cyclooxygenase inhibitors, completely blocked EGF-mediated resorption without significantly affecting the response to PTH. These results demonstrate that the mechanisms regulating PTH- and EGF-mediated resorption in fetal rat long-bone cultures differ, and imply that a component of EGF-mediated resorption in these cultures is dependent on sustained DNA synthesis.

Colony-stimulating factors regulate the development of multinucleated osteoclasts from recently replicated cells in vitro.

Osteoclasts mediate the process of bone resorption. However, little is known about the mechanisms that regulate the formation of either osteoclasts or osteoclast precursors. In contrast, colony-stimulating factors (CSFs) are well-known to regulate the formation of myeloid cells and their precursors. Because osteoclasts and myeloid cells may originate from a common stem cell, we examined the effects of two CSFs, granulocyte-macrophage CSF (GM-CSF) and interleukin 3 (IL-3), on bone resorption, osteoclast formation, and the incorporation of recently replicated nuclei into the osteoclasts of mouse bone cultures. CSFs had little effect on the formation rate of osteoclasts or their resorptive activity but significantly decreased the percentage of recently replicated osteoclast progenitor cell nuclei present in the osteoclasts of bones treated with parathyroid hormone. GM-CSF also increased the number of myeloid cells in the marrow space of the cultures and the percentage of these cells derived from recently replicated progenitors. These results demonstrate that GM-CSF and IL-3 can regulate the development of osteoclasts from recently replicated precursor cells in cultured fetal mouse long bones. However, the mechanisms by which CSFs influence osteoclast formation are difficult to determine from these studies because markers for the osteoclast progenitor and precursor do not exist. These data also provide evidence that the differentiation of osteoclast progenitors is regulated by different factors at different points in their ontogeny.

Ovariectomy enhances and estrogen replacement inhibits the activity of bone marrow factors that stimulate prostaglandin production in cultured mouse calvariae.

To examine PG production in estrogen deficiency, we studied effects on cultured neonatal mouse calvariae of bone marrow supernatants (MSup) from sham-operated (SHAM), ovariectomized (OVX), or 17 beta-estradiol (OVX+E)-treated mice. MSups were obtained 3 wk after OVX when bone density had decreased significantly. 10-60% MSup increased medium PGE2 and levels of mRNA for inducible and constitutive prostaglandin G/H synthase (PGHS-2 and PGHS-1) and cytosolic phospholipase A2 in calvarial cultures. OVX MSups had twofold greater effects on PGHS-2 and medium PGE2 than other MSups. IL-1 receptor antagonist and anti-IL-1 alpha neutralizing antibody decreased MSup-stimulated PGHS-2 mRNA and PGE2 levels and diminished differences among OVX, sham-operated, and OVX+E groups. In contrast, antibodies to IL-1 beta, IL-6, IL-11, and TNF alpha had little effect. There were no significant differences in IL-1 alpha concentrations or IL-1 alpha mRNA levels in MSups or marrow cells. PGHS-2 mRNA in freshly isolated tibiae from OVX mice was slightly greater than from sham-operated. We conclude that bone marrow factors can increase PG production through stimulation of PGHS-2; that OVX increases and estrogen decreases activity of these factors; and that IL-1 alpha activity, together with additional unknown factors, mediates the differential MSup effects.

Prostaglandin G/H synthase-2 is required for maximal formation of osteoclast-like cells in culture.

We examined the effect on osteoclast formation of disrupting the prostaglandin G/H synthase genes PGHS-1 and-2. Prostaglandin E(2) (PGE(2)) production was significantly reduced in marrow cultures from mice lacking PGHS-2 (PGHS-2(-/-)) compared with wild-type (PGHS-2(+/+)) cultures. Osteoclast formation, whether stimulated by 1,25-dihydroxyvitamin D(3) (1,25-D) or by parathyroid hormone (PTH), was reduced by 60-70% in PGHS-2(-/-) cultures relative to wild-type cultures, an effect that could be reversed by providing exogenous PGE(2). Cultures from heterozygous mice showed an intermediate response. PGHS inhibitors caused a similar drop in osteoclast formation in wild-type cultures. Co-culture experiments showed that supporting osteoblasts, rather than osteoclast precursors, accounted for the blunted response to 1,25-D and PTH. This lack of response appeared to result from reduced expression of RANK ligand (RANKL) in osteoblasts. We cultured spleen cells with exogenous RANKL and found that osteoclast formation was 50% lower in PGHS-2(-/-) than in wild-type cultures, apparently because the former cells expressed high levels of GM-CSF. Injection of PTH above the calvaria caused hypercalcemia in wild-type but not PGHS-2(-/-) mice. Histological examination of bone from 5-week-old PGHS-2(-/-) mice revealed no abnormalities. Mice lacking PGHS-1 were similar to wild-type mice in all of these parameters. These data suggest that PGHS-2 is not necessary for wild-type bone development but plays a critical role in bone resorption stimulated by 1,25-D and PTH.

The role of cytokines in the regulation of local bone resorption.

The skeleton is a complex organ that has a variety of functions. It provides a supportive framework for the body, it is the site of hematopoiesis, and it is the principal storehouse for calcium reserves. Recently, it has become clear that the multiple cell types within bone may interact by producing paracrine factors. These substances were originally recognized as either the products of activated immune cells (cytokines) or as local growth factors. However, they appear to have potent effects on both the cells responsible for the structural integrity of the skeleton and the cells involved in hematopoiesis. In humans the skeleton is constantly remodeling. The cells involved in maintaining the skeleton fall into two broad categories: those responsible for the removal of bone (bone resorption) and those responsible for bone formation. This review concentrates on defining the effects that locally produced factors have on bone-resorbing cells and on the interactions between hematopoietic cells that reside in the skeleton and the cells responsible for maintaining skeletal integrity.

Interleukin-1 alpha and phorbol ester inhibit collagen synthesis in osteoblastic MC3T3-E1 cells by a transcriptional mechanism.

The effects of recombinant human interleukin-1 alpha (IL-1) on procollagen gene expression were examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and 50 micrograms/ml ascorbic acid. Collagen synthesis was assessed as [3H]proline incorporation into collagenase-digestible protein (CDP). Procollagen mRNA levels were determined by Northern blot analysis using a 32P-labeled alpha 1(I) cDNA. Transcription rates were determined by nuclear run-off assay. IL-1 at 1-1000 pg/ml caused a concentration-dependent inhibition of CDP, which was maximally reduced by 75-80%, and a parallel reduction of procollagen alpha 1(I) mRNA levels. The effects of IL-1 were mimicked by the tumor promoter phorbol 12-myristate 13-acetate (PMA) at 1-100 nM, which inhibited CDP and reduced procollagen alpha 1(I) mRNA levels to a similar extent. The effects of IL-1 and PMA were independent of prostaglandin production, since indomethacin did not alter the inhibitory effect of either agent on CDP. Neither IL-1 (up to 10 ng/ml) nor PMA (100 nM) affected adenylate cyclase activity, while forskolin (10 microM), PTH (10 nM) and prostaglandin E2 (1 microM) stimulated adenylate cyclase activity 3- to 5-fold. However, forskolin (10 microM) and (Bu)2cAMP (100 microM) failed to alter CDP or procollagen alpha 1(I) mRNA levels. IL-1 (1 ng/ml) and PMA (100 nM) reduced transcription of the alpha 1(I) procollagen gene by 70% and 80%, respectively, while alpha 2(I) transcription was decreased by 59% and 53%. Neither IL-1 nor PMA affected transcription of the beta-actin or beta-tubulin genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol esters stimulate bone resorption in fetal rat long-bone cultures by mechanisms independent of prostaglandin synthesis.

The phorbol esters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-didecanoate, which activate the enzyme protein kinase C, stimulated resorption in fetal rat long-bone cultures at concentrations of 1 and 10 microM. This effect appeared specific for active phorbol esters, since the inactive analogue 4-alpha-phorbol-12,13-didecanoate was without effect. The resorptive responses of fetal rat long-bone cultures to active phorbol esters differed from those previously described in newborn mouse calvaria cultures, since resorption stimulated by TPA in the rat long bones was not inhibited by either indomethacin (10 microM) or flufenamic acid (10 microM). However, calcitonin, an inhibitor of osteoclastic resorption, did decrease the response to TPA. There were some similarities between the response of fetal rat long-bone cultures to TPA and their response to epidermal growth factor (EGF). Like EGF, TPA stimulated DNA synthesis in the bones (measured as the incorporation of [3H]-thymidine) at concentrations below those necessary to stimulate resorption. TPA also did not stimulate resorption in the presence of aphidicolin (10 microM), an inhibitor of DNA synthesis that has been previously shown to block the resorptive response of these cultures to EGF. However, the responses of the cultures to TPA and EGF were not identical, since, unlike the effects of EGF, the stimulatory effects of TPA on DNA synthesis were biphasic. These results demonstrate that active phorbol esters stimulate bone resorption in fetal rat long-bone cultures through mechanisms that do not require prostaglandin synthesis but do appear to be mediated by osteoclasts.(ABSTRACT TRUNCATED AT 250 WORDS)