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.

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.

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.

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.

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.

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.

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.

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.

Estradiol rapidly inhibits osteoclastogenesis and RANKL expression in bone marrow cultures in postmenopausal women: a pilot study.

UNLABELLED: We examined RANKL expression and OCL formation in cultured bone marrow cells from eight postmenopausal women in response to E(2). E(2) treatment inhibited the ability of hematopoietic cells to form OCLs in response to RANKL, and decreased RANKL production. These changes are likely involved in the ability of E(2) to influence the development of osteoporosis. INTRODUCTION: Estrogen (E(2)) deficiency at menopause increases osteoclast (OCL) formation and bone resorption, predisposing women to osteoporosis. We examined receptor activator of NF-kappa B-ligand (RANKL) expression and in vitro OCL formation in cultured bone marrow cells from eight postmenopausal women before and after 3 weeks of E(2) therapy and three untreated premenopausal women. METHODS: TRAP staining and resorption pit assay determined OCL number and function. Flow cytometry measured the distribution of marrow cell types and expression of RANKL in the macrophage-enriched fraction (R1) and a lymphocyte-enriched fraction (R2). RESULTS: RANKL (3-100 ng/ml) produced a dose-dependent increase in in vitro OC formation and E(2) therapy significantly (p < 0.01) inhibited OCL formation by 33 to 50%. A small proportion of marrow cells bound anti- RANKL Ab (0.2-4.3%). There was no effect of E(2) on the percentage of cells binding the anti-RANKL Ab in the R1 fraction. In the R2 fraction E(2) treatment decreased the percentage of cells binding anti-RANKL Ab by 68 +/- 9% (p < 0.01). CONCLUSION: Three weeks of E(2) treatment had a dual action. It inhibited the ability of hematopoietic cells to form OCLs in response to RANKL, and decreased the production of RANKL in cells of the bone marrow. The observed changes in the osteoclastic potential of bone marrow cells are likely involved in the ability of E(2) to regulate bone mass and influence the development of osteoporosis.

Regulation of receptor activator of nuclear factor-kappa B ligand and osteoprotegerin mRNA expression by parathyroid hormone is predominantly mediated by the protein kinase a pathway in murine bone marrow cultures.

Parathyroid hormone (PTH) stimulates receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and inhibits osteoprotegerin (OPG) mRNA expression in murine bone marrow cultures. To understand the mechanisms influencing these responses, we investigated the role of the protein kinase A (PKA) and protein kinase C (PKC) pathways in the regulation of RANKL and OPG mRNA expression in murine bone marrow cultures. Murine bone marrow cells were stimulated with bovine PTH(1-34) and (1-34) amide, which activate both pathways; PTH(3-34), which more selectively activates the PKC and calcium pathways; and human PTH (1-31), which stimulates adenylyl cyclase, but not protein kinase C. We also examined agents that more directly activate either the PKA pathway (forskolin [FSK] and 8-bromo cAMP [8-Br-cAMP]) or the PKC pathway (phorbol 12-myristate 13-acetate [PMA]) in murine bone marrow cultures. After 1 h, RANKL mRNA expression was stimulated to a similar degree by agents that activate either or both the PKA and PKC pathways. However, this effect was sustained for 24 h only with agents that stimulated PKA. OPG mRNA expression was inhibited by all agents that stimulated PKA at 6 h. In contrast, PKC-specific stimulators [PMA and bPTH(3-34)] had no effect on OPG regulation in this culture system. To determine the involvement of the PKC signaling pathway in responses of RANKL, bone marrow cells were pretreated with PMA for 24 h and then treated with PTH(1-34) or FSK for 2 h. PMA pretreatment did not alter the ability of PTH or FSK to stimulate RANKL or inhibit OPG mRNA expression. Treatment of cells with H-89, a PKA inhibitor, significantly reduced the ability of PTH and FSK to induce RANKL and inhibit OPG mRNA expression. Calphostin C, a PKC inhibitor, significantly reduced PMA-stimulated RANKL mRNA expression without altering PTH- or FSK-mediated effects on RANKL or OPG mRNA. Cycloheximide, an inhibitor for protein synthesis, inhibited PTH-stimulated RANKL mRNA expression by 60% without altering the effect of PTH on OPG mRNA expression. To examine the involvement of prostaglandin in PMA-mediated responses, cells were treated with indomethacin, a nonspecific prostaglandin G/H synthase (PGHS) inhibitor, or NS-398, a selective inhibitor of PGHS-2. Neither PGHS inhibitor altered PMA-induced effects on RANKL and OPG mRNA expression. These results demonstrate that the PKA pathway is predominantly involved in the effects of PTH on RANKL mRNA expression in murine bone marrow cultures, but there is also a PKC-mediated response, which is not sustained. Inhibition of OPG by PTH appears to be a selective PKA response.

Interferon-alpha plus ribavirin for 12 months increases the sustained response rates in chronic hepatitis C relapsers.

BACKGROUND: The effectiveness and tolerability of combination therapy for 12 months have not been evaluated sufficiently in chronic hepatitis C relapsers to interferon. AIMS: To evaluate the sustained response to interferon plus ribavirin for 12 months in chronic hepatitis C relapsers. METHODS: We included 55 chronic hepatitis C relapsers in a 12-month treatment protocol with interferon (3 MU thrice weekly) plus ribavirin (1-1.2 g/day). The effectiveness was evaluated using serum aminotransferase and hepatitis C virus RNA levels, alanine aminotransferase normalization and viraemia clearance after 12 months, defining the end-of-treatment response, and 6 months after completion of therapy, defining the sustained response. Adverse effects were recorded. RESULTS: End-of-treatment response and sustained response were achieved in 47 (85%) and 37 (67%) patients, respectively; there were 10 (21%) relapsers after combination therapy. Predictive factors of sustained response included the genotype (non-1 95% vs. 1 48%; P < 0.001), lower viraemia (503 917 +/- 553 230 vs. 901 393 +/- 548 267 copies/mL; P < 0.005), higher alanine aminotransferase levels (137 +/- 75 vs. 103 +/- 41 IU/L; P < 0.05) and a lower gamma-glutamyl transpeptidase/alanine aminotransferase ratio (0.30 +/- 0.23 vs. 0.49 +/- 0.39; P < 0.05). Tolerance to therapy was good, with no withdrawals. CONCLUSIONS: Interferon plus ribavirin treatment for 12 months in chronic hepatitis C relapsers yields high sustained response rates and is well tolerated. The sustained response is related to a non-1 genotype, lower baseline viraemia, higher alanine aminotransferase level and a lower gamma-glutamyl transpeptidase/alanine aminotransferase ratio.

Cellular and molecular interactions between immune system and bone.

Functional interdependence between immune and bone systems is reflected in a number of regulatory molecules acting on the cells of both systems and common precursors for bone and immune cells. Therefore, the disturbances of the immune system may affect bone metabolism, and vice versa. This review addresses the roles of two major immune cell populations, T and B lymphocytes, in the regulation of bone metabolism. Experimental models and human diseases demonstrated that T lymphocytes may produce many bone cell regulatory cytokines, including two essential stimulators of osteoclastogenesis: receptor for activation of nuclear factor kappa b (NF-kappa B) (RANK) ligand (RANKL) and macrophage colony-stimulating factor. The effect of T lymphocytes on osteoclastogenesis may be both stimulatory and inhibitory, and depends on the activation stage and pattern of cytokine production. We showed that acute removal of T lymphocytes stimulated osteoclast differentiation in vitro and enhanced new cartilage and bone formation at non-osseous sites in vivo. B lymphocytes may be even more closely related to bone cells, as B lymphopoiesis requires an intimate contact with osteoblastic/stromal cells, and estrogens, powerful regulators of bone mass, are also involved in the differentiation of the B lymphocyte lineage. Also, B lymphocyte progenitors may give rise to functional osteoclasts. Both B and T lymphocytes may act through the RANKL/RANK/osteoprotegerin cytokine system, which has been independently discovered within immune and bone systems. These cytokines have crucial roles in the development and function of osteoclasts, dendritic cells, and T and B lymphocytes, as well as in the thymus and lymph node organogenesis. The cytokines produced by immune cells may affect bone cell function and vice versa, but the full complexity of these interactions awaits further investigation.

Role of B lymphocytes in new bone formation.

Although there may be a close relationship between B lymphocytes and osteoclasts, or bone resorbing cells, little is known about the role of B lymphocytes in bone formation. We compared in vivo new bone induction in mice homozygous for the B-cell deficient (microMT) gene knockout, which lack functional B lymphocytes, with bone induction in control wild-type (C57BL/6) mice. Our comparison used two models of new bone induction in vivo: endochondral osteoinduction by subcutaneous implantation of recombinant human bone morphogenetic protein (rhBMP-2) and osteogenic regeneration after tibial bone marrow ablation. The expression of bone-specific proteins (bone sialoprotein, osteopontin, and osteocalcin) and inflammatory/immunomodulatory cytokines (interleukin-1alpha and -1beta, interleukin-6, and tumor necrosis factor-alpha) was assessed by Northern blot analysis or reverse transcription-polymerase chain reaction, respectively. Ossicles induced by rhBMP-2 were larger in volume and mass in microMT knockout mice, but relative volumes of the newly induced bone, cartilage, and bone marrow were similar in the two groups. Six days after tibial bone marrow ablation, microMT knockout mice resorbed the initial blood clot faster and formed more trabecular bone, paralleled by greater levels of bone sialoprotein mRNA than in the wild-type mice. microMT knockout and wild-type mice also differed in the expression pattern of inflammatory/immunomodulatory cytokines during the development of the newly induced bone, suggesting that a genetic lack of B lymphocytes may create a change in the immunological milieu at the site of new bone induction, which stimulates the initial accumulation and proliferation of mesenchymal progenitor.

Depletion of CD4 and CD8 T lymphocytes in mice in vivo enhances 1,25-dihydroxyvitamin D3-stimulated osteoclast-like cell formation in vitro by a mechanism that is dependent on prostaglandin synthesis.

To investigate the role of T lymphocytes in osteoclastogenesis, we performed in vivo depletion of CD4 and/or CD8 T lymphocyte subsets and evaluated in vitro osteoclast-like cell (OCL) formation. T lymphocyte depletion (TLD) with mAbs was confirmed 24 h later by flow cytometry. OCL formation was stimulated with 1, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) in bone marrow and with recombinant mouse (rm) receptor activator of NF-kappaB ligand (RANK-L) and rmM-CSF in bone marrow and spleen cell cultures. OCL formation was up to 2-fold greater in 1,25-(OH)(2)D(3)-stimulated bone marrow cultures from TLD mice than in those from intact mice. In contrast, TLD did not alter OCL formation in bone marrow or spleen cell cultures that were stimulated with rmRANK-L and rmM-CSF. The effects of TLD seemed to be mediated by enhanced PG synthesis, because the PGE(2) concentration in the medium of 1, 25-(OH)(2)D(3)-stimulated bone marrow cultures from TLD mice was 5-fold higher than that in cultures from intact mice, and indomethacin treatment abolished the stimulatory effect of TLD on OCL formation. There was a 2-fold increase in RANK-L expression and an almost complete suppression of osteoprotegerin expression in 1, 25-(OH)(2)D(3)-stimulated bone marrow cultures from TLD mice compared with those from intact mice. Although there was a small (20%) increase in IL-1alpha expression in 1, 25-(OH)(2)D(3)-stimulated bone marrow cultures from TLD mice, TLD in mice lacking type I IL-1R and wild-type mice produced similar effects on OCL formation. Our data demonstrate that TLD up-regulates OCL formation in vitro by increasing PG production, which, in turn, produces reciprocal changes in RANK-L and osteoprotegerin expression. These results suggest that T lymphocytes influence osteoclastogenesis by altering bone marrow stromal cell function.

Knockout of the murine prostaglandin EP2 receptor impairs osteoclastogenesis in vitro.

Prostaglandin E2 (PGE2) stimulates the formation of osteoclast-like tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNC) in vitro. This effect likely results from stimulation of adenylyl cyclase, which is mediated by two PGE2 receptors, designated EP2 and EP4. We used cells from mice in which the EP2 receptor had been disrupted to test its role in the formation of TRAP + MNC. EP2 heterozygous (+/-) mice in a C57BL/6 x 129/SvEv background were bred to produce homozygous null (EP2 -/-) and wild-type (EP2 +/+) mice. PGE2, PTH, or 1,25 dihydroxyvitamin D increased TRAP+ MNC in 7-day cultures of bone marrow cells from EP2 +/+ mice. In cultures from EP2 -/- animals, responses to PGE2, PTH, and 1,25 dihydroxyvitamin D were reduced by 86%, 58%, and 50%, respectively. A selective EP4 receptor antagonist (EP4RA) further inhibited TRAP+ MNC formation in both EP2 +/+ and EP2 -/- cultures. In cocultures of spleen and calvarial osteoblastic cells, the response to PGE2 or PTH was reduced by 92% or 85% when both osteoblastic cells and spleen cells were from EP2 -/- mice, by 88% or 68% when only osteoblastic cells were from EP2 -/- mice and by 58% or 35% when only spleen cells were from EP2 -/- mice. PGE2 increased receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) messenger RNA expression in osteoblastic and bone marrow cell cultures from EP2 +/+ mice 2-fold but had little effect on cells from EP2 -/- mice. Spleen cells cultured with RANKL and macrophage colony stimulating factor produced TRAP+ MNC. PGE2 increased the number of TRAP+ MNC in spleen cell cultures from EP2 +/+ mice but not in cultures from EP2 -/- mice. EP4RA had no effect on the PGE2 response in spleen cell cultures. PGE2 decreased the expression of messenger RNA for granulocyte-macrophage colony stimulating factor in spleen cell cultures from EP2 +/+ mice but had little effect on cells from EP2 -/- mice. These data demonstrate that the prostaglandin EP2 receptor plays a role in the formation of osteoclast-like cells in vitro. A major defect in EP2 -/- mice appears to be in the capacity of osteoblastic cells to stimulate osteoclast formation. In addition, there appears to be a defect in the response of cells of the osteoclastic lineage to PGE2 in EP2 -/- mice.

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.

Parathyroid hormone stimulates TRANCE and inhibits osteoprotegerin messenger ribonucleic acid expression in murine bone marrow cultures: correlation with osteoclast-like cell formation.

We studied the effects of PTH on the expression of tumor necrosis factor-related activation-induced cytokine (TRANCE), osteoprotegerin (OPG), and receptor activator of NF kappaB (RANK) messenger RNA (mRNA) in cultured murine bone marrow, calvaria, and osteoblasts. TRANCE, OPG, and RANK are recently identified regulators of osteoclast formation. Bone marrow cells were cultured with or without PTH(1-34) for 6 days. TRANCE, OPG, and RANK mRNA were measured by RT-PCR. In 6-day cultures, PTH stimulated the number of OCL/well in a dose-dependent manner. A time course showed significant (P < 0.01) increases in OCL/well after 24 h of PTH (100 ng/ml). TRANCE mRNA expression, like OCL formation, increased dose dependently and was maximal, with 10-100 ng/ml PTH. In contrast, OPG mRNA expression was decreased by 0.1 ng/ml PTH (40%) and completely abolished by 1 ng/ml. TRANCE mRNA expression was rapidly stimulated by PTH (maximal response at 1 h, 8.1-fold over control). Expression declined by 40% at 24 h but was still much greater than control at 6 days (4.6-fold) in a time-course study. PTH caused a transient stimulation of OPG mRNA at 1 h (2-fold), which returned to basal levels by 2 h. After 6 h, PTH completely inhibited OPG mRNA. There were only minor effects of PTH on RANK mRNA expression. PTH had less potent effects on TRANCE and OPG mRNA expression in calvaria organ cultures and osteoblasts. In mouse calvaria cultures, TRANCE expression was detectable in controls and was increased 2.9-fold by PTH at 24 h. PTH treatment of calvaria decreased OPG expression by 30% at 6 h. MC3T3 E-1 osteoblastic cells expressed minimal levels of TRANCE mRNA either before or after PTH treatment. OPG mRNA was present in MC3T3 E-1 cells, but levels were not modulated by PTH. In primary osteoblastic cells, PTH stimulated TRANCE mRNA expression 4-fold at 2 h and inhibited OPG mRNA expression by 46%. These results demonstrate a tight correlation between the ability of PTH to stimulate OCL formation in marrow culture and expression of TRANCE (r = 0.87, P < or = 0.05) and OPG mRNA (r = -0.88, P < or = 0.05). Reciprocal regulation of TRANCE and OPG mRNA by PTH preceded its effects on OCL formation by 18-23 h. Hence, it is likely that PTH regulates bone resorption, at least in part, via its effects on TRANCE and OPG expression.

[People accompanying children to pediatric visits: is something changing?]. / Acompañantes de los niños a la consulta de pediatría: algo está cambiando?

OBJECTIVE: To find out if the social father-mother stereotypes as regards the care of children are reproduced in the daily practice of Pediatrics Consultations, in order to do so, this study looks at who accompanies children to the consultations and the factors which led to fathers taking their children. DESIGN: An observational crossover study. SETTING: Otero Health Center, Oviedo. PARTICIPANTS: A sample of 300 visits obtained over a year from the consultations of the Pediatrician. MEASUREMENTS AND MAIN RESULTS: Collected Socioeconomic variables of parents, type of Consultation (Spontaneous or Programmed), who accompanied the child, sufficiency of information brought to the consultation and child management detected. People accompanying children were: Mother only (66.7%; IC 95%: 64.1-74.7), father and mother (14.7%; IC 95%: 11.0-19.3), father only or with others (7.6%; IC 95%: 5.0-11.4). In all cases information was sufficient and child management adequate. Mother came to 70.8% (IC 95%: 60.1-79.7); father to 4.5% (IC 95%: 1.5-11.8) and both to 21.3% (IC 95%: 13.7-31.6) of Programmed Consultations. A Logistic Regression found age of childrens and Education level of father associated with father accompany to consultation. CONCLUSION: Mother normally accompanies child to pediatrician consultations. Father participates in child care, principally in families with a higher Socioeconomic level.