Vitamin D and disease severity in coronavirus disease 19 (COVID-19).

The role of 25-OH-vitamin D in the assessment of coronavirus disease 19 (COVID-19) has not been investigated. We sought to investigate the prevalence of 25-OH-vitamin D deficiency among COVID-19 patients, and to determine the associations between 25-OH-vitamin D status and the severity of the disease. We have conducted a retrospective observational study of COVID-19 patients admitted to the University of Verona Hospital Trust. Demographic, clinical and biochemical parameters were collected at hospital admission, and serum 25-OH-vitamin D levels were measured. The following outcomes were assessed: arterial partial oxygen pressure (PaO2); C-reactive protein (CRP); length of hospitalization; requirement of oxygen therapy; non-invasive ventilation (NIV); mechanical ventilation; and death. Among 61 patients enrolled, 72.1% was 25-OH-vitamin D deficient (<20 ng/mL) and 57.4% had 25-OHvitamin D <15 ng/mL. Patients with arterial PaO2 <60 mmHg had significantly lower mean 25-OH-vitamin D levels compared to patients with PaO2 ≥60 mmHg (13.3 ng/mL vs 20.4 ng/mL respectively, p=0.03). Vitamin D deficiency was associated with 3-fold higher risk of having arterial pO2 <60 mmHg. 25-OH-vitamin D deficiency was associated with increased CRP and dyspnea. 25-OH-vitamin D deficiency was associated with more severe systemic inflammatory response and respiratory failure in COVID-19 patients.

Claimed effects, outcome variables and methods of measurement for health claims proposed under regulation (EC) 1924/2006 and related to cognitive function in adults.

Some food/food components have been the object of request of authorization to the use of health claims related to cognitive function in adults and compliant with the Regulation (EC) 1924/2006. Most of the requests have received a negative opinion by the European Food Safety Authority (EFSA) also because of the choice of not appropriate outcome variables (OVs) and methods of measurement (MMs) selected in the trials used to substantiate the claim. This manuscript referes to the collection, collation and critical analysis of OVs and MMs related to cognitive function in adults. OVs and MMs were collected from the EFSA Guidance document and the applications for authorization of health claims pursuant to the Articles 13(5). The critical analysis of OVs and MMs, performed by a literature review, was aimed at defining their appropriateness in the context of a specific claimed effect. The results highlight the importance of an adequate choice of OVs and MMs for an effective substantiation of the claims related to cognitive functioning. The information provided in this document may serve to EFSA for updating the guidance on the scientific requirements for health claims related to cognitive functions, but also for a better design of randomized controlled trials aimed at substantiating such health claims.

Claimed effects, outcome variables and methods of measurement for health claims proposed under European Community Regulation 1924/2006 in the framework of protection against oxidative damage and cardiovascular health.

BACKGROUND AND AIMS: The high number of negative opinions from the European Food Safety Authority (EFSA) to the requests for authorization of health claims is largely due to the design of human intervention studies, including the inappropriate choice of outcome variables (OVs) and of their methods of measurement (MMs). The present manuscript reports the results of an investigation aimed to collect, collate and critically analyse the information in relation to claimed effects, OVs and MMs, in the context of protection against oxidative damage and cardiovascular health compliant with Regulation 1924/2006. METHODS AND RESULTS: Claimed effects, OVs and the related MMs were collected from EFSA Guidance documents and applications for authorization of health claims under Articles 13.5 and 14. The OVs and their MMs were evaluated only if the claimed effect was sufficiently defined and was considered beneficial by EFSA. The collection, collation and critical analysis of the relevant scientific literature consisted in the definition of the keywords, the PubMed search strategies and the creation of databases of references. The critical analysis of the OVs and their MMs was performed on the basis of the literature review and was aimed at defining the appropriateness of OVs and MMs in the context of the specific claimed effects. CONCLUSIONS: The information provided in this document could serve to EFSA for the development of further guidance on the scientific requirements for health claims, as well as to the stakeholders for the proper design of human intervention studies aimed to substantiate such health claims.

The response of osteoblastic MC3T3-E1 cells to micro- and nano-textured, hydrophilic and bioactive titanium surfaces.

The aim of the present work was to investigate the morphology and activity of the murine osteoblastic cell line MC3T3 on control smooth (Machined), commercially available rough (ZT) titanium discs, and on titanium samples obtained by modifying the ZT treatment protocol, and herein labelled as ZTF, ZTM and ZTFM. Cells were evaluated at SEM and immunofluorescence for morphology and cell-to-cell interactions and by MTT assay and real time PCR for cell growth and function. Microscopy showed that ZT modified protocols could differently affect cell shape and distribution. All the tested surfaces showed good biocompatibility by viability assay. However, cells on smoother surfaces appeared to express higher levels of transcript for Collagen 1a1, the main component of extracellular matrix, by real time PCR. Expression of the early differentiation marker Alkaline Phosphatase was higher on ZTF surfaces and ZTM enhanced the expression of later osteoblastic markers Osteoprotegerin and Osteocalcin. Noteworthy, the expression of Connexin 43, a component of cell-to-cell contacts and hemichannels, followed a similar pattern to differentiation marker genes and was higher in cells on ZTM surfaces, consistently with the microscopic observation of cell clusters. Taken together, this data showed that ZTF and ZTM treatment protocols appeared to improve the basal sand-blasting/acid-etching ZT procedure with ZTM surfaces promoting the most mature stage of differentiation.

[Early detection of autism spectrum disorders]. / Détection précoce des troubles du spectre autistique.

The evaluation of the development of young children had to consider the possible detection of neurodevelopmental disorders in particular autism spectrum disorders. When a child of 18 months has a developmental language delay or a defect in social contact, the hypothesis of autism must be considered through a clinical evaluation. We will point out some clinical guidelines and some early signs to detect the trouble and to propose early treatment interventions. This will help to develop specific skills of the child aiming at influencing positively clinical evolution and reducing mental retardation.

L'évaluation du développement du jeune enfant doit prendre en compte la détection des troubles neurodéveloppementaux et notamment celle des troubles du spectre autistique. Face à un retard de développement du langage à 18 mois ou face à un défaut de contact social, cette hypothèse doit être considérée afin de réaliser une évaluation plus approfondie. Nous donnons quelques repères cliniques ainsi que les signes précurseurs afin de permettre le cas échéant une prise en charge la plus précoce possible. Ceci permettra souvent une évolution plus favorable avec l'objectif de réduire le déficit mental et de permettre le développement des compétences spécifiques de l'enfant.

Pharmacological GSK-3beta inhibition improves osteoblast differentiation on titanium surfaces.

Rough titanium surfaces enhance the activation of Wnt canonical signaling, a pathway required for osteoblast differentiation. The present study investigated the effects of GSK3b-inhibitor (2'Z,3'E)- 6-Bromoindirubin-3'-oxime (BIO) on osteoblastic differentiation on titanium surfaces with different topography and wettability. C2C12 cells were plated on pickled, acid-etched/sand-blasted (SLA), modified hydrophilic SLA titanium discs (modSLA) and stimulated with increasing doses of BIO. Activation of Wnt canonical signaling was measured with a reporter system. Gene expression was measured in the same cell system by Real Time PCR. Osteoblastic MC3T3 cells were then plated on discs with or without BIO and the expression of osteoblast specific genes was assessed by Real Time PCR. One mM BIO activated Wnt canonical signaling in C2C12 cells on all surfaces, and the highest effect was on rough surfaces. BIO markedly increased the expression of Osteoprotegerin and Osteocalcin in MC3T3 cells on rough surfaces at the concentration of 100 nM, and on all surfaces at the concentration of 1 mM. BIO enhances Wnt signaling activation and the expression of osteoblastic genes on rough surfaces and could be a viable approach to improve cell response to implant surfaces.

Characterization of skeletal parameters in a cohort of North Italian rugby players.

BACKGROUND: Vitamin D deficiency is common in the general population and may impair skeletal muscle function. Very few data are available regarding this condition in professional athletes. AIM: To evaluate some skeletal parameters and in particular serum 25-hydroxyvitamin D status in professional rugby players during two different sunlight exposure times (October and early April) and to assess its impact on bone metabolism. MATERIALS AND METHODS: Twenty-one male healthy professional rugby players living in northern Italy at latitude of 44°55'N (age 24.6 ± 4.3 years; height 182.0 ± 0.05 cm; mass 96.3 ± 14.6 kg; BMI 28.9 ± 3.7 kg/m(2)) participated in this observational study. During 2012/2013 Italian rugby season, 25-hydroxyvitamin D, PTH and other related biochemical parameters were monitored. Dietary calcium intake and body composition by DXA were also evaluated. RESULTS: Significant changes were observed between October and April data for 25-hydroxyvitamin D concentration (22.8 ± 5.8 vs. 19.1 ± 5.3 ng/ml; p = 0.001) whereas serum PTH, calcium and phosphorus plasma levels did not change. They presented with an appropriate daily intake of calcium (1,304.8 ± 477.9 mg; max 1,939 mg; min 228 mg). CONCLUSIONS: Professional rugby athletes practicing a sport characterized by intense outdoor training and with good calcium intake are at higher risk of hypovitaminosis D that worsens significantly during times of low cutaneous vitamin D production. Further studies are warranted to evaluate whether an appropriate supplementation with cholecalciferol in professional athletes is needed.

GSK3b-inhibitor lithium chloride enhances activation of Wnt canonical signaling and osteoblast differentiation on hydrophilic titanium surfaces.

AIMS: Promoting bone formation at the tissue interface is an important step to improve implant success. This study investigated whether stimulation of Wnt signaling by GSK3b inhibitor lithium chloride (LiCl) could affect the response of mesenchymal or osteoblastic cells growing on titanium surfaces with different topography and wettability, and improve their differentiation along the osteoblastic lineage. MATERIAL AND METHODS: Murine mesenchymal C2C12 cells were plated on Pickled, acid-etched/sand-blasted (SLA), and hydrophilic SLA titanium disks (modSLA) and stimulated with increasing doses of LiCl. Cell viability was measured using chemiluminescence-based ATP quantitation and activation of Wnt canonical signaling was measured using a Luciferase-based reporter assay. Gene expression was measured using real time PCR in C2C12 cells, murine osteoblastic MC3T3 cells or murine primary bone marrow cells. RESULTS: LiCl stimulated Wnt activation and expression of Wnt markers in C2C12 cells on modSLA. Addition of 1 mM LiCl increased levels for bone marker Osteocalcin in MC3T3 cells on modSLA surfaces. Similarly, LiCl potently enhanced Osteoprotegetrin levels in MC3T3 cells on modSLA. When primary bone marrow cells were stimulated with LiCl, the expression of Wnttarget genes and osteoblastic differentiation markers was increased on modSLA surfaces. CONCLUSIONS: Stimulation of the canonical Wnt pathway promoted osteoblast differentiation on hydrophilic modSLA surfaces. Taken together, these results demonstrate that Wnt activators such as LiCl should be further tested as a possible approach to improve implant osseointegration.

The importance of WNT pathways for bone metabolism and their regulation by implant topography.

Endosseous implants are important tools to replace missing teeth or damaged tissue segments. Their clinical success depends on their integration in bone and, thus, on the response of bone cells to material and surface characteristics. Recent evidence has shown that surface topography and chemistry affect WNT signalling, a pivotal pathway for the commitment of mesenchymal progenitors to the osteoblast lineage and for bone homeostasis. WNT signalling comprises several cascades that, acting through different effectors, modulate several aspects of cell behaviour. It has been shown that cells growing on rough titanium surfaces display a different expression profile for WNT factors, and that surface features can alter the response of bone cells to WNT factors. Although the underlying mechanisms to this regulation are still poorly understood, the present review reports intriguing evidence that that cell cytoskeletal signalling is involved in activating WNT signalling in cells growing on rough implant surfaces.

FoxOs, Wnts and oxidative stress-induced bone loss: new players in the periodontitis arena?

BACKGROUND AND OBJECTIVE: Chronic periodontitis is a widespread disease affecting tooth-supporting structures that can lead to extensive loss of periodontal ligament and bone, ultimately resulting in tooth loss. Extensive evidence has demonstrated a strong association between age, metabolic disorders such as type II diabetes, oxidative stress and alveolar bone loss. The molecular players controlling bone maintenance and underlying age-related bone loss and its links to the general metabolism are currently the object of intense research. MATERIAL AND METHODS: Recent findings are summarized to elucidate the molecular mechanisms linking oxidative stress, bone loss and metabolic factors. RESULTS: It is well known that reactive oxygen species are an inevitable consequence of cellular respiration and that organisms have developed an efficient array of defenses against them. The core of this complex defense line is a family of transcription factors, known as FoxOs, which can bind to ß-catenin and initiate a transcriptional programme regulating cell apoptosis, DNA repair and degradation of reactive oxygen species. An increase in reactive oxygen species due, for example, to age or insulin resistance, generates a situation in which bone formation is impaired by activation of FoxO, and a decrease in Wnt signaling and bone resorption are promoted. CONCLUSION: The balance between FoxO and the Wnt pathway is finely tuned by systemic and local factors, creating a far-reaching mechanism that dictates the fate of mesenchymal progenitors and regulates the homeostasis of bone, providing a rationale for the impairment of systemic and alveolar bone maintenance clinically observed with age and metabolic diseases.

Assessment of the 10-year risk of fracture in Italian postmenopausal women using FRAX®: a north Italian multicenter study.

The aim of the study was to estimate the absolute risk of fracture in a sample of postmenopausal women with the Italian version of FRAX®, using femoral neck bone mineral density (BMD) and 3 internationally validated clinical risk factors (CRFs) (history of fragility fracture, family history of hip fracture, current smoking). We retrospectively studied 9586 women (mean age 64.1 yr) examined in three osteoporosis centers from Northern Italy over two years (2001-2002). The risk of major osteoporotic (clinical spine, hip, forearm and humerus) and hip fractures was estimated using the online version of the FRAX algorithm adapted for Italy. The median 10-year risk was 7.5% for osteoporotic fracture and 1.7% for hip fracture. 25% of subjects had a 10-year risk ≥ 12.1% for osteoporotic fracture and ≥ 4.1% for hip fracture. The median 10-year risk of fracture increased with the number of prevalent CRFs. For major osteoporotic fractures risk rose from 6.3% to 10.9%, 21.4% and 40.9% with 1, 2 and 3 prevalent CRFs, respectively. For hip fractures the corresponding figures were: 1.3%, 2.7%, 7.0% and 21.9%, respectively. However, it must be emphasized that in 2 out of 3 women, none of the CRFs examined was present and the assessment of risk was limited to age and BMD. Our data provide the first description of the effect of the combination of BMD, age and CRFs on fracture risk stratification in a large sample of Italian postmenopausal women using FRAX®. The results are a useful starting point to define criteria for the application of FRAX® in clinical practice in Italy.

Increased osteoclast development after estrogen loss: mediation by interleukin-6.

Osteoclasts, the cells that resorb bone, develop from hematopoietic precursors of the bone marrow under the control of factors produced in their microenvironment. The cytokine interleukin-6 can promote hematopoiesis and osteoclastogenesis. Interleukin-6 production by bone and marrow stromal cells is suppressed by 17 beta-estradiol in vitro. In mice, estrogen loss (ovariectomy) increased the number of colony-forming units for granulocytes and macrophages, enhanced osteoclast development in ex vivo cultures of marrow, and increased the number of osteoclasts in trabecular bone. These changes were prevented by 17 beta-estradiol or an antibody to interleukin-6. Thus, estrogen loss results in an interleukin-6-mediated stimulation of osteoclastogenesis, which suggests a mechanism for the increased bone resorption in postmenopausal osteoporosis.

The immune system in extreme longevity.

Recent observations indicate that immunosenescence is not accompanied by an unavoidable and progressive deterioration of the immune function, but is rather the result of a remodeling where some functions are reduced, others remain unchanged or even increased. In addition, it appears that the ancestral/innate compartment of the immune system is relatively preserved during aging in comparison to the more recent and sophisticated adaptive compartment that exhibit more profound modifications. The T-cell branch displays an age-dependent decline of the absolute number of total T-cells (CD3+), involving both CD4+ and CD8+ subsets, accompanied by an increase of NK cells with well-preserved cytotoxic function and by a reduction of B-cells. One of the main characteristics of the immune system during aging is a progressive, age-dependent decline of the virgin T-cells (CD95-), which is particularly profound at the level of the CD8+ subpopulation of the oldest old subjects. The progressive exhaustion of this important T-cell subpopulation dedicated primarily to the defense against new antigenic challenges (viral, neoplastic, bacterial ones), could be a consequence of both the thymic involution and the lifelong chronic antigenic stimulation. The immune function of the elderly, is therefore weakened by the exhaustion of CD95- virgin cells that are replaced by large clonal expansions of CD28- T-cells. The origin of CD28- cells has not been completely clarified yet, but it is assumed that they represent cells in the phase of replicative senescence characterized by shortening telomers and reduced proliferative capacity. A major characteristic of the immune system during aging is the up-regulation of the inflammatory responses which appears to be detrimental for longevity. In this regard, we have recently observed a progressive age-dependent increase of type 1(IL-2, IFN-gamma, TNF-alpha) and type 2 (IL-4, IL-6, IL-10) positive CD8+ T-cells; in particular, type 1 cytokine-positive cells significantly increased, with age, in all CD8+ subsets particularly among effector/cytotoxic and memory cells. A major force able to drive a chronic pro-inflammatory state during aging may be represented by persistent viral infections by EBV and CMV. Therefore, we have determined the frequency and the absolute number of viral antigen-specific CD8+ T-cells in subjects older than 85 years, who were serologically positive for CMV or EBV. In the majority of these subjects we detected the presence of T lymphocytes positive for epitopes of CMV or EBV. In all subjects the absolute number of CMV-positive CD8+ cells outnumbered that of EBV-positive ones. In addition, the majority of CMV+ T cells were included within the CD28- subpopulation, while EBV+ T cells belonged mainly to the CD28+ subset. These data indicate that the chronic antigenic stimulation induced by persistent viral infections during aging bring about important modifications among CD8+ subsets, which are particularly evident in the presence of CMV persistence. The age-dependent expansions of CD8+CD28- T-cells, mostly positive for pro-inflammatory cytokines and including the majority of CMV-epitope-specific cells, underlines the importance of chronic antigenic stimulation in the pathogenesis of the main immunological alterations of aging and may favour the appearance of several pathologies (arteriosclerosis, dementia, osteoporosis, cancer) all of which share an inflammatory pathogenesis.

Interleukin-11: a new cytokine critical for osteoclast development.

Stromal cells of the bone marrow control the development of osteoclasts through the production of cytokines capable of promoting the proliferation and differentiation of hematopoietic progenitors. Moreover, the deregulated production of the cytokine IL-6 in the bone marrow mediates an increase in osteoclastogenesis after estrogen loss. IL-6, however, does not influence osteoclastogenesis in the estrogen-replete state, suggesting that other cytokines might be responsible for osteoclast development under physiologic circumstances. We report here that IL-11, a newly discovered cytokine that is produced by marrow stromal cells, induced the formation of osteoclasts exhibiting an unusually high degree of ploidy in cocultures of murine bone marrow and calvarial cells. Osteoclasts formed in the presence of IL-11 were capable of bone resorption, as evidenced by the formation of resorption pits, as well as the release of 45Ca from prelabeled murine calvaria. Further, an antibody neutralizing IL-11 suppressed osteoclast development induced by either 1,25-dihydroxyvitamin D3, parathyroid hormone, interleukin-1, or tumor necrosis factor; whereas inhibitors of IL-1 or TNF had no effect on IL-11-stimulated osteoclast formation. The effects of IL-11 on osteoclast development were blocked by indomethacin; more important, however, they were independent of the estrogen status of the marrow donors.

17 beta-estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens.

The effect of 17 beta-estradiol on interleukin-6 (IL-6) synthesis was examined in murine bone marrow-derived stromal cell lines, normal human bone-derived cells, and nontransformed osteoblast cell lines from mice and rats. In all these cell types IL-6 production was stimulated as much as 10,000-fold in response to the combination of recombinant interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). Addition of 17 beta-estradiol in the cultures exerted a dose-dependent inhibition of IL-1-, TNF-, and IL-1 + TNF-induced production of bioassayable IL-6. Testosterone and progesterone (but not 17 alpha-estradiol) also inhibited IL-6, but their effective concentrations were two orders of magnitude higher than 17 beta-estradiol. 17 beta-estradiol also decreased the levels of the IL-6 mRNA. In addition, estradiol inhibited both TNF-induced IL-6 production and osteoclast development in primary bone cell cultures derived from neonatal murine calvaria. The TNF-stimulated osteoclast development was also suppressed by a neutralizing monoclonal anti-IL-6 antibody. This in vitro evidence suggests, for the first time, a mechanistic paradigm by which estrogens might exert at least part of their antiresorptive influence on the skeleton.

Improved scaffold biocompatibility through anti-Fibronectin aptamer functionalization.

UNLABELLED: Protein adsorption is the first and decisive step to define cell-biomaterial interaction. Guiding the adsorption of desired protein species may represent a viable approach to promote cell activities conducive to tissue regeneration. The aim of the present study was to investigate whether immobilized anti-Fibronectin aptamers could promote the attachment and growth of osteoblastic cells. Polyethyleneglycole diacrylate/thiolated Hyaluronic Acid hydrogels (PEGDA/tHA) were coated with anti-Fibronectin aptamers. Hydrogel loading and Fibronectin bonding were investigated, through spectrophotometry and Bradford assay. Subsequently, human osteoblasts (hOBs) were cultured on hydrogels for 10days in 2D and 3D cultures. Cells were monitored through microscopy and stained for focal adhesions, microfilaments and nuclei using fluorescence microscopy. Samples were also included in paraffin and stained with Hematoxylin-Eosin. Cell number on hydrogels was quantitated over time. Cell migration into the hydrogels was also studied through Calcein AM staining. Aptamers increased the number of adherent hOBs and their cytoplasm appeared more spread and richer in adhesion complexes than on control hydrogels. Viability assays confirmed that significantly more cells were present on hydrogels in the presence of aptamers, already after 48h of culture. When hOBs were encapsulated into hydrogels, cells were more numerous on aptamer-containing PEGDA-tHA. Cells migrated deeper in the gel in the presence of DNA aptamers, appearing on different focus planes. Our data demonstrate that anti-Fibronectin aptamers promote scaffold enrichment for this protein, thus improving cell adhesion and scaffold colonization. STATEMENT OF SIGNIFICANCE: We believe aptamer coating of biomaterials is a useful and viable approach to improve the performance of scaffold materials for both research and possibly clinical purposes, because different medical devices could be envisaged able to capture bioactive mediators from the patients' blood and concentrate them where they are needed, on the biomaterial itself. At the same time, this technology could be used to confer 3D cell culture scaffold with the ability to store proteins, such as Fibronectin, taking it from the medium and capture what is produced by cells. This is an improvement of traditional biomaterials that can be enriched with exogenous molecules but are not able to selectively capture a desired molecule.

Acetylphenylhydrazine induced haemoglobin oxidation in erythrocytes studied by Mössbauer spectroscopy.

The oxidative action of acetylphenylhydrazine (APH) on red blood cells obtained from healthy donors and from patients with breast cancer has been investigated by Mössbauer spectroscopy. Whole blood was incubated with APH for different time periods and the Mössbauer spectra of the packed red cells were recorded and compared. The evolution with time of the oxidation products has been followed. The largest difference in red cells analysis between healthy persons and patients was found after about 50 min of treatment where Mössbauer spectra of patient samples show a much broader spectral pattern due to an advanced haemoglobin oxidation.

Estrogen loss upregulates hematopoiesis in the mouse: a mediating role of IL-6.

We have previously demonstrated that ovariectomy causes an increase in the number of colony-forming unit granulocyte/macrophage (CFU-GM) and an upregulation of osteoclastogenesis in mice, both of which are mediated by interleukin-6 (IL-6). IL-6 is involved in the development of several hematopoietic progenitors, including the burst-forming unit-erythroid (BFU-E) and multipotent CFUs (CFU-GEMM). Therefore, we performed studies to examine if other hematopoietic progenitors, besides CFU-GM and their progeny, are affected by estrogen loss. We found that ovariectomy caused an increase in the number of CFU-GEMM and BFU-E, as well as an increase of CFU-GM in marrow cells of the femur. Administration of 17 beta-estradiol or a neutralizing antibody against IL-6 prevented the ovariectomy-induced increase in the number of these progenitors in the marrow. Ovariectomy also caused an increase in the number of circulating lymphocytes, neutrophils, and monocytes, which were suppressed by administration of 17 beta-estradiol or the neutralizing antibody against IL-6; however, the number of circulating platelets was unaffected by loss of ovarian function. These data establish that, in addition to upregulation of osteoclastogenesis, loss of estrogens in the mouse causes widespread effects on hematopoiesis, which are apparently mediated by IL-6.

Inhibition of antigen-presenting cell function by alendronate in vitro.

Bisphosphonates are potent inhibitors of bone resorption in vivo and are emerging as important and widely used drugs for the treatment of a variety of abnormal bone resorptive processes. In the current study we investigated the in vitro effects of 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate), a recently developed, extremely potent bisphosphonate, on the immune functions of human peripheral blood mononuclear cells (PBMCs). PBMC proliferation induced by lectins, alloantigens, and a nominal antigen (tetanus toxoid) was inhibited in a dose-dependent manner by alendronate. Pretreatment of monocytes, but not T cells, with the compound at concentrations ranging from 10(-4) to 10(-8) M was inhibitory, indicating that alendronate acts selectively on antigen-presenting cells (APCs). Alendronate did not affect the viability of monocytes or T cells or the expression of cell surface molecules known to play critical roles in antigen presentation. Alendronate exhibited dose-dependent inhibition of the production of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) by activated monocytes. The inhibitory effect of 10(-6) M alendronate on PBMC proliferation was reversed by 10 U/ml recombinant rIL-1 beta, whereas other cytokines such as IL-6, TNF-alpha, and granulocyte-macrophage colony-stimulating factor (GM-CSF) had no effect. Thus, alendronate acts on monocytes to inhibit their antigen-presenting/accessory cell functions through a mechanism that can be overcome by exogenous IL-1. The inhibitory effect of this agent on cytokine production may contribute to its inhibitory effect on bone resorption.

Increased interleukin-6 production by murine bone marrow and bone cells after estrogen withdrawal.

We have previously shown that cytokine-induced production of interleukin-6 (IL-6) by cultured bone marrow-derived stromal and osteoblastic cells is inhibited by 17 beta-estradiol, and that estrogen withdrawal (ovariectomy) in mice causes an up-regulation of osteoclast development which can be prevented by a neutralizing antibody against IL-6 or estrogen replacement. To directly establish the link between estrogen loss and altered IL-6 production, implied by our earlier studies, we have now compared IL-6 production in ex vivo cultures of bone marrow cells from mice that were sham operated, ovariectomized, or ovariectomized and treated with 17 beta-estradiol. In addition, we have examined the effect of the in vitro withdrawal of estrogens from primary cell cultures of neonatal murine calvaria on IL-6 production. IL-6 production in ex vivo cultures of bone marrow cells maintained in the presence of 1,25-dihydroxyvitamin D3 or PTH was greater in marrow cells from ovariectomized mice than in those from sham-operated animals or ovariectomized animals receiving estrogen replacement. In line with this finding, addition of 17 beta-estradiol to calvaria cell cultures followed by withdrawal of the steroid caused an increase in the amount of IL-6 produced in response to the subsequent stimulation of these cultures with IL-1 or PTH compared to that in cultures that had never been treated with estradiol; when the inactive isomer 17 alpha-estradiol was used, no change in IL-6 production was observed. These results establish that estrogen loss causes an up-regulation of IL-6 production by bone marrow cells and that a similar phenomenon can be elicited in vitro by withdrawal of 17 beta-estradiol from primary cultures of bone cells.